Yunzhi Peter Yang

All Publications

• In-situ stable injectable collagen-based hydrogels for cell and growth factor delivery. Materialia Moeinzadeh, S., Park, Y., Lin, S., Yang, Y. P. 2021; 15

  Abstract

  Here we report development of in-situ stable injectable hydrogels for delivery of cells and growth factors based on two precursors, alginate, and collagen/calcium sulfate (CaSO4). The alg/col hydrogels were shear-thinning, injectable through commercially available needles and stable right after injection. Rheological measurements revealed that pre-crosslinked alg/col hydrogels fully crosslinked at 37°C and that the storage modulus of alg/col hydrogels increased with increasing the collagen content or the concentration of CaSO4. The viscoelastic characteristics and injectability of the alg/col hydrogels were not significantly impacted by the storage of precursor solutions for 28 days. An osteoinductive bone morphogenic protein-2 (BMP-2) loaded into alg/col hydrogels was released in 14 days. Human mesenchymal stem cells (hMSCs) encapsulated in alg/col hydrogels had over 90% viability over 7 days after injection. The DNA content of hMSC-laden alg/col hydrogels increased by 6-37 folds for 28 days, depending on the initial cell density. In addition, hMSCs encapsulated in alg/col hydrogels and incubated in osteogenic medium were osteogenically differentiated and formed a mineralized matrix. Finally, a BMP-2 loaded alg/col hydrogel was used to heal a critical size calvarial bone defect in rats after 8 weeks of injection. The alg/col hydrogel holds great promise in tissue engineering and bioprinting applications.

  View details for DOI 10.1016/j.mtla.2020.100954

  View details for PubMedID 33367226

• Regenerative Approaches for the Treatment of Large Bone Defects. Tissue engineering. Part B, Reviews Stahl, A., Yang, Y. P. 2020

  Abstract

  A variety of engineered materials have gained acceptance in orthopaedic practice as substitutes for autologous bone grafts, though the regenerative efficacy of these engineered grafts is still limited compared with that of transplanted native tissues. For bone defects greater than 4 to 5 cm, however, common bone grafting procedures are insufficient and more complicated surgical interventions are required to repair and regenerate the damaged or missing bone. In this review, we describe current grafting materials and surgical techniques for the reconstruction of large bone defects, followed by tissue engineering efforts to develop improved therapies. Particular emphasis is placed on graft vascularization, since for both autologous bone and engineered alternatives, achieving adequate vascular development within the regenerating bone tissues remains a significant challenge in the context of large bone defects. To this end, tissue engineering and surgical strategies to induce development of a vasculature within bone grafts are discussed.

  View details for DOI 10.1089/ten.TEB.2020.0281

  View details for PubMedID 33138705

• The effects of tubular structure on biomaterial aided bone regeneration in distraction osteogenesis JOURNAL OF ORTHOPAEDIC TRANSLATION Pan, Q., Li, Y., Xu, J., Kang, Y., Li, Y., Wang, B., Yang, Y., Lin, S., Li, G. 2020; 25: 80?86

  View details for DOI 10.1016/j.jot.2020.09.009

  View details for Web of Science ID 000598640000015

• Development of PLGA-PEG-COOH and Gelatin-Based Microparticles Dual Delivery System and E-Beam Sterilization Effects for Controlled Release of BMP-2 and IGF-1 PARTICLE & PARTICLE SYSTEMS CHARACTERIZATION Bai, Y., Moeinzadeh, S., Kim, S., Park, Y., Lui, E., Tan, H., Zhao, W., Zhou, X., Yang, Y. 2020

  View details for DOI 10.1002/ppsc.202000180

  View details for Web of Science ID 000560347300001

• Development of a Dual Hydrogel Model System for Vascularization. Macromolecular bioscience Kim, S., Pan, C., Yang, Y. P. 2020: e2000204

  Abstract

  Numerous hydrogel-based culture systems are used to create in vitro model for prevascularization. Hydrogels used to induce a microenvironment conducive to microvessel formation are typically soft and fast degradable, but often suffer from maintaining a lasting perfusable channel in vitro. Here, a dual hydrogel system that consists of photo-crosslinkable gelatin methacrylate (GelMA) and polyethylene glycol dimethacrylate (PEGDMA) is reported. GelMA hydrogels present soft and rapidly degradable properties and show microporous structures while PEGDMA is relatively stiff, almost nondegradable in vitro, and less porous. The dual hydrogel system is sequentially photo-crosslinked to construct an endothelial cell (EC)-lined perfusable PEGDMA channel and surrounding GelMA for endothelial vascular networks. Such dual hydrogel system exhibits seamless integration of the stiff PEGDMA channel and the surrounding soft GelMA, and facilitates rapid EC sprouting and extensive microvessel formation from a stable endothelium on the PEGDMA channel into the GelMA. Furthermore, diffusivity of biomolecules in the perfusable dual hydrogel system is affected by both the structural and physicochemical properties of the hydrogel system and the microvascular networks formed in the system. The establishment of the dual hydrogel system for vascularization holds great promise as an in vitro angiogenesis model and prevascularization strategy of large tissue constructs.

  View details for DOI 10.1002/mabi.202000204

  View details for PubMedID 32790230

• The Influence of Electron Beam Sterilization on In Vivo Degradation of beta-TCP/PCL of Different Composite Ratios for Bone Tissue Engineering. Micromachines Kang, J., Kaneda, J., Jang, J., Sakthiabirami, K., Lui, E., Kim, C., Wang, A., Park, S., Yang, Y. P. 2020; 11 (3)

  Abstract

  We evaluated the effect of electron beam (E-beam) sterilization (25 kGy, ISO 11137) on the degradation of beta-tricalcium phosphate/polycaprolactone (beta-TCP/PCL) composite filaments of various ratios (0:100, 20:80, 40:60, and 60:40 TCP:PCL by mass) in a rat subcutaneous model for 24 weeks. Volumes of the samples before implantation and after explantation were measured using micro-computed tomography (micro-CT). The filament volume changes before sacrifice were also measured using a live micro-CT. In our micro-CT analyses, there was no significant difference in volume change between the E-beam treated groups and non-E-beam treated groups of the same beta-TCP to PCL ratios, except for the 0% beta-TCP group. However, the average volume reduction differences between the E-beam and non-E-beam groups in the same-ratio samples were 0.76% (0% TCP), 3.30% (20% TCP), 4.65% (40% TCP), and 3.67% (60% TCP). The E-beam samples generally had more volume reduction in all experimental groups. Therefore, E-beam treatment may accelerate degradation. In our live micro-CT analyses, most volume reduction arose in the first four weeks after implantation and slowed between 4 and 20 weeks in all groups. E-beam groups showed greater volume reduction at every time point, which is consistent with the results by micro-CT analysis. Histology results suggest the biocompatibility of TCP/PCL composite filaments.

  View details for DOI 10.3390/mi11030273

  View details for PubMedID 32155781

• Acoustic Patterning of Growth Factor for 3D Tissue Engineering. Tissue engineering. Part A Shanjani, Y., Siebert, S. M., Ker, D. F., Mercado-Pagan, A., Yang, Y. P. 2020

  Abstract

  Temporal and spatial presentations of biological cues are critical for tissue engineering. There is a great need in improving the incorporation of bioagent(s) (specifically growth factor(s)) onto three-dimensional (3D) scaffolds. In this study, we developed a process to combine additive manufacturing technology with acoustic droplet ejection (ADE) technology to control growth factor distribution. More specifically, we implemented ADE to control the distribution of recombinant human Bone Morphogenetic Protein-2 (rhBMP-2) onto polycaprolactone (PCL)-based tissue engineering constructs. Three substrates were used in this study: (1) succinimide-terminated PCL (PCL-NHS) as model material, (2)alkali-treated PCL (PCL-NaOH) as first control material, and (3) fibrin-coated PCL (PCL-Fibrin) as second control material. It was shown that our process enables a pattern of BMP-2 spots of ~250 m in diameter with ~700 m center-to-center spacing. An initial concentration of BMP-2 higher than 300 g/L was required to retain a detectable amount of growth factor on the substrate after a wash with phosphate buffer solution. However, to obtain detectable osteogenic differentiation of C2C12 cells, the initial concentration of BMP-2 higher than 750 g/L was needed. The cells on PCL-NHS samples showed spatial alkaline phosphatase staining correlating with local patterns of BMP-2 although the intensity was lower than the controls (PCL-NaOH and PCL-Fibrin). Our results have demonstrated that the developed AM-ADE process holds great promise in creating tissue engineering constructs with highly controlled growth factor patterning.

  View details for DOI 10.1089/ten.TEA.2019.0271

  View details for PubMedID 31950880

• The efficacy of core decompression for steroidassociated osteonecrosisof the femoral head in rabbits. Journal of orthopaedic research : official publication of the Orthopaedic Research Society Maruyama, M., Lin, T., Kaminow, N. I., Thio, T., Storaci, H. W., Pan, C. C., Yao, Z., Takagi, M., Goodman, S. B., Yang, Y. P. 2020

  Abstract

  Although core decompression (CD) is often performed in the early stages of osteonecrosis of the femoral head (ONFH), the procedure does not always prevent subsequent deterioration and the effects of CD are not fully clarified.The aim of this study was to evaluate the efficacy of CD for steroid associated ONFH in rabbits.Twelve male and twelve female New Zealand rabbits were injected intramuscularly 20 mg/kg of methylprednisolone once and were divided into the disease control and CD groups. In the disease control group, rabbits had no treatment and were euthanized at 12 weeks post-injection. In the CD group, rabbits underwent left femoral CD at 4 weeks post-injection and were euthanized 8 weeks postoperatively. The left femurs were collected to perform morphological, biomechanical and histological analysis.Bone mineral density and bone volume fraction in the femoral head in the CD group were significantly higher than in the disease control group. However, no difference in the mechanical strength was observed between the two groups. Histological analysis showed thatalkaline phosphatase and CD31 positive cells significantly increased in the males after CD treatment. The number of empty lacunae in the surrounding trabecular bone was significantly higher in the CD group.The current study indicated that CD improved the morphological properties, but did not improve the mechanical strength in the femoral headat early stage ONFH.These data suggest the need for additional biological, mechanical strategies,and therapeutic windows to improve the outcome of early stage steroid associated ONFH. This article is protected by copyright. All rights reserved.

  View details for DOI 10.1002/jor.24888

  View details for PubMedID 33095462

• Administration of allogeneic mesenchymal stem cells in lengthening phase accelerates early bone consolidation in rat distraction osteogenesis model. Stem cell research & therapy Yang, Y., Pan, Q., Zou, K., Wang, H., Zhang, X., Yang, Z., Lee, W. Y., Wei, B., Gu, W., Yang, Y. P., Lin, S., Li, G. 2020; 11 (1): 129

  Abstract

  Distraction osteogenesis (DO) is a surgical technique to promote bone regeneration which may require long duration for bone consolidation. Bone marrow-derived mesenchymal stem cells (MSCs) have been applied to accelerate bone formation in DO. However, the optimal time point for cell therapy in DO remains unknown. This study sought to determine the optimal time point of cell administration to achieve early bone consolidation in DO. We hypothesized that the ratio of circulating MSCs to peripheral mononuclear cells and the level of cytokines in serum might be indicators for cell administration in DO.Unilateral tibial osteotomy with an external fixator was performed in adult Sprague Dawley rats. Three days after osteotomy, the tibia was lengthened at 0.5?mm/12?h for 5?days. At first, 5 rats were used to analyze the blood components at 6 different time points (3?days before lengthening, on the day lengthening began, or 3, 6, 10, or 14?days after lengthening began) by sorting circulating MSCs and measuring serum levels of stromal cell-derived factor 1 (SDF-1) and interleukin 1?. Then, 40 rats were used for cell therapy study. A single dose of 5?×?105 allogeneic MSCs was locally injected at the lengthening site on day 3, 6, or 10 after lengthening began, or 3 doses of MSCs were injected at the three time points. Sequential X-ray radiographs were taken weekly. Endpoint examinations included micro-computed tomography analysis, mechanical testing, histomorphometry, and histology.The number of circulating MSCs and serum level of SDF-1 were significantly increased during lengthening, and then decreased afterwards. Single injection of MSCs during lengthening phase (on day 3, but not day 6 or 10) significantly increased bone volume fraction, mechanical maximum loading, and bone mineralization of the regenerate. Triple injections of MSCs at three time points also significantly increased bone volume and maximum loading of the regenerates.This study demonstrated that bone consolidation could be accelerated by a single injection of MSCs during lengthening when the ratio of peripheral MSCs to mononuclear cells and the serum SDF-1 presented at peak levels concurrently, suggesting that day 3 after lengthening began may be the optimal time point for cell therapy to promote early bone consolidation.

  View details for DOI 10.1186/s13287-020-01635-5

  View details for PubMedID 32197646

• Development of PLGA-PEG-COOH and gelatin-based microparticles dual delivery system and E-beam sterilization effects for controlled release of BMP-2 and IGF-1. Particle & particle systems characterization : measurement and description of particle properties and behavior in powders and other disperse systems Bai, Y., Moeinzadeh, S., Kim, S., Park, Y., Lui, E., Tan, H., Zhao, W., Zhou, X., Yang, Y. P. 2020; 37 (10)

  Abstract

  The purpose of this study was to develop a PLGA-PEG-COOH- and gelatin-based microparticles (MPs) dual delivery system for release of BMP-2 and IGF-1. We made and characterized the delivery system based on its morphology, loading capacity, Encapsulation efficiency and release kinetics. Second, we examined the effects of electron beam (EB) sterilization on BMP-2 and IGF-1 loaded MPs and their biological effects. Third, we evaluated the synergistic effect of a controlled dual release of BMP-2 and IGF-1 on osteogenesis of MSCs. Encapsulation efficiency of growth factors into gelatin and PLGA-PEG-COOH MPs are in the range of 64.78% to 76.11%. E-beam sterilized growth factor delivery systems were effective in significantly promoting osteogenesis of MSCs, although E-beam sterilization decreased the bioactivity of growth factors in MPs by approximately 22%. BMP-2 release behavior from gelatin MPs/PEG hydrogel shows a faster release (52.7%) than that of IGF-1 from the PLGA-PEG-COOH MPs/PEG hydrogel (27.3%). The results demonstrate that the gelatin and PLGA-PEG-COOH MPs based delivery system could realize temporal release of therapeutic biomolecules by incorporating different growth factors into distinct microparticles. EB sterilization was an accessible method for sterilizing growth factors loaded carriers, which could pave the way for implementing growth factor delivery in clinical applications.

  View details for DOI 10.1002/ppsc.202000180

  View details for PubMedID 33384477

  View details for PubMedCentralID PMC7771709

• Cell-Based and Scaffold-Based Therapies for Joint Preservation in Early-Stage Osteonecrosis of the Femoral Head: A Review of Basic Research. JBJS reviews Maruyama, M., Lin, T., Pan, C., Moeinzadeh, S., Takagi, M., Yang, Y. P., Goodman, S. B. 2019

  View details for DOI 10.2106/JBJS.RVW.18.00202

  View details for PubMedID 31503099

• Ruminants: Evolutionary past and future impact. Science (New York, N.Y.) Ker, D. F., Yang, Y. P. 2019; 364 (6446): 1130?31

  View details for DOI 10.1126/science.aax5182

  View details for PubMedID 31221843

• A simple layer-stacking technique to generate biomolecular and mechanical gradients in photocrosslinkable hydrogels. Biofabrication Ko, H., Suthiwanich, K., Mary, H., Zanganeh, S., Hu, S., Ahadian, S., Yang, Y. P., Choi, G., Fetah, K., Niu, Y., Mao, J., Khademhosseini, A. 2019

  Abstract

  Physicochemical and biological gradients are desirable features for hydrogels to enhance their relevance to biological environments for three-dimensional (3D) cell culture. Therefore, simple and efficient techniques to generate chemical, physical and biological gradients within hydrogels are highly desirable. This work demonstrates a technique to generate biomolecular and mechanical gradients in photocrosslinkable hydrogels by stacking and crosslinking prehydrogel solution in a layer by layer manner. Partial crosslinking of the hydrogel allows mixing of prehydrogel solution with the previous hydrogel layer, which makes a smooth gradient profile, rather than discrete layers. This technique enables the generation of concentration gradients of bovine serum albumin in both gelatin methacryloyl (GelMA) and poly(ethylene glycol) diacrylate hydrogels, as well as mechanical gradients across a hydrogel containing varying gel concentrations. Fluorescence microscopy, mechanical testing, and scanning electron microscopy show that the gradient profiles can be controlled by changing both the volume and concentration of each layer as well as intensity of UV exposure. GelMA hydrogel gradients with different Young's moduli were successfully used to culture human fibroblasts. The fibroblasts migrated along the gradient axis and showed different morphologies. In general, the proposed technique provides a rapid and simple approach to design and fabricate 3D hydrogel gradients for in vitro biological studies and potentially for in vivo tissue engineering applications.

  View details for DOI 10.1088/1758-5090/ab08b5

  View details for PubMedID 30786263

• The effects of a functionally-graded scaffold and bone marrow-derived mononuclear cells on steroid-induced femoral head osteonecrosis BIOMATERIALS Maruyama, M., Nabeshima, A., Pan, C., Behn, A. W., Thio, T., Lin, T., Pajarinen, J., Kawai, T., Takagi, M., Goodman, S. B., Yang, Y. 2018; 187: 39?46

  View details for DOI 10.1016/j.biomaterials.2018.09.030

  View details for Web of Science ID 000449446000005

• Effect of Plasma Treatment and Its Post Process Duration on Shear Bonding Strength and Antibacterial Effect of Dental Zirconia. Materials (Basel, Switzerland) Park, C., Park, S., Yun, K., Ji, M., Kim, S., Yang, Y. P., Lim, H. 2018; 11 (11)

  Abstract

  We have investigated the effect of non-thermal atmospheric pressure plasma (NTAPP) treatment and the post process time on the bonding strength and surface sterilization of dental zirconia. Presintered zirconia specimens were manufactured as discs, and then subjected to a 30-min argon treatment (Ar, 99.999%; 10 L/min) using an NTAPP device. Five post-treatment durations were evaluated: control (no treatment), P0 (immediate), P1 (24 h), P2 (48 h), and P3 (72 h). The surface characteristics, shear bonding strength (SBS) with two resin cements, and Streptococcus mutans biofilm formation of these plasma-treated dental zirconia were tested. Plasma did not change the roughness, and caused surface element changes and surface energy increase. Due to this increase in surface energy, SBS increased significantly (p < 0.05) within 48 h when RelyXTM U200 was used. However, the increase of surface oxygen significantly decreased (p < 0.05) the SBS of Panavia F 2.0 when using plasma immediately (P0). S. mutans adhesion decreased significantly (p < 0.05) for the P0, P1, and P2 groups compared to the control. The P0 group exhibited lower biofilm thickness than the other experimental groups due to the increased hydrophilicity (p < 0.05). Our study suggests that there is a suitable time window for the post NTAPP treatment regarding bonding strength and antimicrobial growth persist.

  View details for PubMedID 30423984

• Identifying deer antler uhrf1 proliferation and s100a10 mineralization genes using comparative RNA-seq. Stem cell research & therapy Ker, D. F., Wang, D., Sharma, R., Zhang, B., Passarelli, B., Neff, N., Li, C., Maloney, W., Quake, S., Yang, Y. P. 2018; 9 (1): 292

  Abstract

  BACKGROUND: Deer antlers are bony structures that re-grow at very high rates, making them an attractive model for studying rapid bone regeneration.METHODS: To identify the genes that are involved in this fast pace of bone growth, an in vitro RNA-seq model that paralleled the sharp differences in bone growth between deer antlers and humans was established. Subsequently, RNA-seq (>60 million reads per library) was used to compare transcriptomic profiles. Uniquely expressed deer antler proliferation as well as mineralization genes were identified via a combination of differential gene expression and subtraction analysis. Thereafter, the physiological relevance as well as contributions of these identified genes were determined by immunofluorescence, gene overexpression, and gene knockdown studies.RESULTS: Cell characterization studies showed that in vitro-cultured deer antler-derived reserve mesenchyme (RM) cells exhibited high osteogenic capabilities and cell surface markers similar to in vivo counterparts. Under identical culture conditions, deer antler RM cells proliferated faster (8.6-11.7-fold increase in cell numbers) and exhibited increased osteogenic differentiation (17.4-fold increase in calcium mineralization) compared to human mesenchymal stem cells (hMSCs), paralleling in vivo conditions. Comparative RNA-seq identified 40 and 91 previously unknown and uniquely expressed fallow deer (FD) proliferation and mineralization genes, respectively, including uhrf1 and s100a10. Immunofluorescence studies showed that uhrf1 and s100a10 were expressed in regenerating deer antlers while gene overexpression and gene knockdown studies demonstrated the proliferation contributions of uhrf1 and mineralization capabilities of s100a10.CONCLUSION: Using a simple, in vitro comparative RNA-seq approach, novel genes pertinent to fast bony antler regeneration were identified and their proliferative/osteogenic function was verified via gene overexpression, knockdown, and immunostaining. This combinatorial approach may be applicable to discover unique gene contributions between any two organisms for a given phenomenon-of-interest.

  View details for PubMedID 30376879

• Effect of Electron Beam Sterilization on Three-Dimensional-Printed Polycaprolactone/Beta-Tricalcium Phosphate Scaffolds for Bone Tissue Engineering TISSUE ENGINEERING PART A Bruyas, A., Moeinzadeh, S., Kim, S., Lowenberg, D. W., Yang, Y. 2019; 25 (3-4): 248?56

  View details for DOI 10.1089/ten.tea.2018.0130

  View details for Web of Science ID 000448564600001

• The effects of a functionally-graded scaffold and bone marrow-derived mononuclear cells on steroid-induced femoral head osteonecrosis. Biomaterials Maruyama, M., Nabeshima, A., Pan, C., Behn, A. W., Thio, T., Lin, T., Pajarinen, J., Kawai, T., Takagi, M., Goodman, S. B., Yang, Y. P. 2018; 187: 39?46

  Abstract

  Osteonecrosis of the femoral head (ONFH) is a debilitating disease that may progress to femoral head collapse and subsequently, degenerative arthritis. Although injection of bone marrow-derived mononuclear cells (BMMCs) is often performed with core decompression (CD) in the early stage of ONFH, these treatments are not always effective in prevention of disease progression and femoral head collapse. We previously described a novel 3D printed, customized functionally-graded scaffold (FGS) that improved bone growth in the femoral head after CD in a normal healthy rabbit, by providing structural and mechanical guidance. The present study demonstrates similar results of the FGS in a rabbit steroid-induced osteonecrosis model. Furthermore, the injection of BMMCs into the CD decreased the osteonecrotic area in the femoral head. Thus, the combination of FGS and BMMC provides a new therapy modality that may improve the outcome of CD for early stage of ONFH by providing both enhanced biological and biomechanical cues to promote bone regeneration in the osteonecrotic area.

  View details for PubMedID 30292940

• Effect of electron beam sterilization on 3D printed PCL/beta-TCP scaffolds for bone tissue engineering. Tissue engineering. Part A Bruyas, A., Moeinzadeh, S., Kim, S., Lowenberg, D. W., Yang, Y. P. 2018

  Abstract

  3D printing of composite materials such as polycaprolactone/beta-tricalcium phosphate (PCL/beta-TCP) enables the design and manufacturing of scaffolds with advanced geometries, along with improved physical and biological properties for large bone defect repair. Terminal sterilization of the scaffolds is inevitable for clinical applications. Electron beam (E-beam) is non-toxic, and can be used for sterilizing heat sensitive scaffolds by the use of high radiation dose in a short period of time. In this paper, we assessed the influence of E-beam sterilization on the properties of 3D printed PCL/beta-TCP scaffolds, focusing on the key physical and biological properties for bone tissue engineering. More specifically, we characterized the effect of a single dose E-beam sterilization (25kGy, ISO 11137) on surface morphology, hydrophilicity, degradation and mechanical properties of the scaffolds as well as in vitro biological responses. The results showed that E-beam irradiation did not alter the surface properties of scaffolds. A 14% increase in initial mechanical stiffness and strength of the scaffolds was observed after E-beam treatment. In addition, the E-beam treated scaffolds had 25% faster degradation. The PCL chains within the scaffolds had larger polydispersity after the E-beam irradiation that was indicative of a concurrent crosslinking and chain scission. Moreover, in vitro cell studies showed no influence of E-beam sterilization on viability, attachment, proliferation, and osteogenic differentiation of cells seeded on the PCL/beta-TCP scaffolds.

  View details for PubMedID 30234441

• Systematic characterization of 3D-printed PCL/beta-TCP scaffolds for biomedical devices and bone tissue engineering: Influence of composition and porosity JOURNAL OF MATERIALS RESEARCH Bruyas, A., Lou, F., Stahl, A. M., Gardner, M., Maloney, W., Goodman, S., Yang, Y. 2018; 33 (14): 1948?59

  View details for DOI 10.1557/jmr.2018.112

  View details for Web of Science ID 000440179300003

• Tunable Elastomers with an Antithrombotic Component for Cardiovascular Applications. Advanced healthcare materials Stahl, A. M., Yang, Y. P. 2018: e1800222

  Abstract

  This study reports the development of a novel family of biodegradable polyurethanes for use as tissue engineered cardiovascular scaffolds or blood-contacting medical devices. Covalent incorporation of the antiplatelet agent dipyridamole into biodegradable polycaprolactone-based polyurethanes yields biocompatible materials with improved thromboresistance and tunable mechanical strength and elasticity. Altering the ratio of the dipyridamole to the diisocyanate linking unit and the polycaprolactone macromer enables control over both the drug content and the polymer cross-link density. Covalent cross-linking in the materials achieves significant elasticity and a tunable range of elastic moduli similar to that of native cardiovascular tissues. Interestingly, the cross-link density of the polyurethanes is inversely related to the elastic modulus, an effect attributed to decreasing crystallinity in the more cross-linked polymers. In vitro characterization shows that the antiplatelet agent is homogeneously distributed in the materials and is released slowly throughout the polymer degradation process. The drug-containing polyurethanes support endothelial cell and vascular smooth muscle cell proliferation, while demonstrating reduced levels of platelet adhesion and activation, supporting their candidacy as promising substrates for cardiovascular tissue engineering.

  View details for PubMedID 29855176

• Functionally Graded, Bone- and Tendon-Like Polyurethane for Rotator Cuff Repair ADVANCED FUNCTIONAL MATERIALS Ker, D., Wang, D., Behn, A., Wang, E., Zhang, X., Zhou, B., Mercado-Pagan, A., Kim, S., Kleimeyer, J., Gharaibeh, B., Shanjani, Y., Nelson, D., Safran, M., Cheung, E., Campbell, P., Yang, Y. 2018; 28 (20)

  Abstract

  Critical considerations in engineering biomaterials for rotator cuff repair include bone-tendon-like mechanical properties to support physiological loading and biophysicochemical attributes that stabilize the repair site over the long-term. In this study, UV-crosslinkable polyurethane based on quadrol (Q), hexamethylene diisocyante (H), and methacrylic anhydride (M; QHM polymers), which are free of solvent, catalyst, and photoinitiator, is developed. Mechanical characterization studies demonstrate that QHM polymers possesses phototunable bone- and tendon-like tensile and compressive properties (12-74 MPa tensile strength, 0.6-2.7 GPa tensile modulus, 58-121 MPa compressive strength, and 1.5-3.0 GPa compressive modulus), including the capability to withstand 10 000 cycles of physiological tensile loading and reduce stress concentrations via stiffness gradients. Biophysicochemical studies demonstrate that QHM polymers have clinically favorable attributes vital to rotator cuff repair stability, including slow degradation profiles (5-30% mass loss after 8 weeks) with little-to-no cytotoxicity in vitro, exceptional suture retention ex vivo (2.79-3.56-fold less suture migration relative to a clinically available graft), and competent tensile properties (similar ultimate load but higher normalized tensile stiffness relative to a clinically available graft) as well as good biocompatibility for augmenting rat supraspinatus tendon repair in vivo. This work demonstrates functionally graded, bone-tendon-like biomaterials for interfacial tissue engineering.

  View details for PubMedID 29785178

• Systematic characterization of 3D-printed PCL/?-TCP scaffolds for biomedical devices and bone tissue engineering: influence of composition and porosity. Journal of materials research Bruyas, A., Lou, F., Stahl, A. M., Gardner, M., Maloney, W., Goodman, S., Yang, Y. P. 2018; 33 (14): 1948?59

  Abstract

  This work aims at providing guidance through systematic experimental characterization, for the design of 3D printed scaffolds for potential orthopaedic applications, focusing on fused deposition modeling (FDM) with a composite of clinically available polycaprolactone (PCL) and ?-tricalcium phosphate (?-TCP). First, we studied the effect of the chemical composition (0% to 60% ?-TCP/PCL) on the scaffold's properties. We showed that surface roughness and contact angle were respectively proportional and inversely proportional to the amount of ?-TCP, and that degradation rate increased with the amount of ceramic. Biologically, the addition of ?-TCP enhanced proliferation and osteogenic differentiation of C3H10. Secondly, we systematically investigated the effect of the composition and the porosity on the 3D printed scaffold mechanical properties. Both an increasing amount of ?-TCP and a decreasing porosity augmented the apparent Young's modulus of the 3D printed scaffolds. Third, as a proof-of-concept, a novel multi-material biomimetic implant was designed and fabricated for potential disk replacement.

  View details for PubMedID 30364693

• Investigating Regeneration DEVELOPMENTAL CELL Marshall, W. F., Alvarado, A., Shaw, T., Tanaka, E. M., Unguez, G. A., Poss, K., Kusumi, K., Amaya, E., Seifert, A. W., Yang, Y., Dev Cell Editorial Team 2017; 43 (4): 373?76

  View details for Web of Science ID 000415808100001

  View details for PubMedID 29161584

• Functional Outcomes of Heparin Binding - Epidermal Growth Factor Like Growth Factor for Regeneration of Chronic Tympanic Membrane Perforations in Mice. Tissue engineering. Part A Santa Maria, P. L., Gotlieb, P., Santa Maria, C., Puria, S., Kim, S., Yang, Y. P. 2017

  Abstract

  We aim to demonstrate that regeneration of chronic tympanic perforations with heparin-binding epidermal growth factor-like growth factor (HB-EGF) delivered by an injectable hydrogel restored hearing to levels similar to that of nonperforated tympanic membranes. Chronic tympanic membrane perforation is currently managed as an outpatient surgery with tympanoplasty. Due to the costs of this procedure in the developed world and a lack of accessibility and resources in developing countries, there is a great need for a new treatment that does not require surgery. In this study, we show in a mouse model through measurement of auditory brainstem response and distortion product otoacoustic emissions that tympanic perforations lead to hearing loss and this can be predominantly recovered with HB-EGF treatment (5??g/mL). Our animal model suggests a return to function between 2 and 6 months after treatment. Auditory brainstem response thresholds had returned to the control levels at 2 months, but the distortion product otoacoustic emissions returned between 2 and 6 months. We also show how the vibration characteristics of the regenerated tympanic membrane, as measured by laser Doppler vibrometry, can be similar to that of an unperforated tympanic membrane. Using the best available methods for preclinical evaluation in animal models, it is likely that HB-EGF-like growth factor treatment leads to regeneration of chronic tympanic membrane perforations and restoration of the tympanic membrane to normal function, suggesting a potential route for nonsurgical treatment.

  View details for DOI 10.1089/ten.TEA.2016.0395

  View details for PubMedID 28142401

• Endothelial pattern formation in hybrid constructs of additive manufactured porous rigid scaffolds and cell-laden hydrogels for orthopedic applications JOURNAL OF THE MECHANICAL BEHAVIOR OF BIOMEDICAL MATERIALS Shanjani, Y., Kang, Y., Zarnescu, L., Bowden, A. K., Koh, J., Ker, D. F., Yang, Y. 2017; 65: 356-372

  Abstract

  Vascularization of tissue engineering constructs (TECs) in vitro is of critical importance for ensuring effective and satisfactory clinical outcomes upon implantation of TECs. Biomechanical properties of TECs have remarkable influence on the in vitro vascularization of TECs. This work utilized in vitro experiments and finite element analysis to investigate endothelial patterns in hybrid constructs of soft collagen gels and rigid macroporous poly(?-caprolactone)-?-tricalcium phosphate (PCL-?-TCP) scaffold seeded/embedded with human umbilical vein endothelial cells (HUVECs) for bone tissue engineering applications. We first fabricated and characterized well-defined porous PCL-?-TCP scaffolds with identical pore size (500µm) but different strut sizes (200 and 400µm) using additive manufacturing (AM) technology, and then assessed the HUVEC?s proliferation and morphogenesis within collagen, PCL-?-TCP scaffold, and the collagen-scaffold hybrid construct. Results showed that, in the hybrid construct, the cell population in the collagen component dropped by day 7 but then increased by day 14. Also, cells migrated onto the struts of the scaffold component, proliferated over time, and formed networks on the thinner struts (i.e., 200µm). Also, the thinner struts resulted in formation of long linear cellular cords structures within the pores. Finite element simulation demonstrated principal stress patterns similar to the observed cell-network pattern. It is probable that the scaffold component modulated patterns of principal stresses in the collagen component as biomechanical cues for reorganization of cell network patterns. Also, the scaffold component significantly improved the mechanical integrity of hydrogel component in the hybrid construct for weight-bearing applications. These results have collectively indicated that the manipulation of micro-architecture of scaffold could be an effective means to further regulate and guide desired cellular response in hybrid constructs.

  View details for DOI 10.1016/j.jmbbm.2016.08.037

  View details for Web of Science ID 000390625100032

  View details for PubMedID 27631173

• Additive Manufacturing of Vascular Grafts and Vascularized Tissue Constructs. Tissue engineering. Part B, Reviews Elomaa, L., Yang, Y. P. 2017; 23 (5): 436?50

  Abstract

  There is a great need for engineered vascular grafts among patients with cardiovascular diseases who are in need of bypass therapy and lack autologous healthy blood vessels. In addition, because of the severe worldwide shortage of organ donors, there is an increasing need for engineered vascularized tissue constructs as an alternative to organ transplants. Additive manufacturing (AM) offers great advantages and flexibility of fabrication of cell-laden, multimaterial, and anatomically shaped vascular grafts and vascularized tissue constructs. Various inkjet-, extrusion-, and photocrosslinking-based AM techniques have been applied to the fabrication of both self-standing vascular grafts and porous, vascularized tissue constructs. This review discusses the state-of-the-art research on the use of AM for vascular applications and the key criteria for biomaterials in the AM of both acellular and cellular constructs. We envision that new smart printing materials that can adapt to their environment and encourage rapid endothelialization and remodeling will be the key factor in the future for the successful AM of personalized and dynamic vascular tissue applications.

  View details for PubMedID 27981886

  View details for PubMedCentralID PMC5652978

• Single Administration of a Sustained-Release Formulation of KB-R7785 Inhibits Tympanic Membrane Regeneration in an Animal Model JOURNAL OF INTERNATIONAL ADVANCED OTOLOGY Maria, P. L., Maria, C. S., Kim, S., Yang, Y. P. 2016; 12 (3): 237-240

  Abstract

  A pressure equalization tube placed within the tympanic membrane is the only clinically available method for inhibiting tympanic membrane regeneration. Problems associated with this include associated otorrhea, biofilm formation, medial migration of the tube, tube retention, induction of granulation tissue, and a small but significant rate of cholesteatoma. We aimed to demonstrate that a single administration of a sustained-release polymer formulation of KB-R7785 maintains tympanic membrane perforation for at least 6 months.Sustained-release KB-R7785 was delivered within a novel polymer hydrogel to 20 mice with bilateral acute tympanic membrane perforations (a total of 40 perforations). The perforations were monitored at 3-month intervals until 9 months.At 3 months, 90% of perforations were open (n=36/40). At 6 months, 75% of perforations were open (total n=30/40). At 9 months, 22.5% of perforations were open (total n=6/40). The majority of tympanic membrane perforations (75%) were open (not healed) beyond 6 months and close (fully healed) prior to 9 months (77.5%). Once healed, tympanic membranes resembled their normal histological appearance.This study demonstrates that a single administration of a sustained-release polymer formulation of KB-R7785 inhibits tympanic membrane regeneration for 6-9 months.

  View details for DOI 10.5152/iao.2016.3124

  View details for PubMedID 28031154

• No systemic exposure of transtympanic heparin-binding epidermal growth factor like growth factor. Drug and chemical toxicology Santa Maria, P. L., Kim, S., Yang, Y. P. 2016; 39 (4): 451-454

  Abstract

  Heparin-binding epidermal growth factor like growth factor (HB-EGF) is an emerging therapeutic for the regeneration of the tympanic membrane (TM).Our aim was to determine whether the doses of HB-EGF delivered in a sustained release hydrogel into a middle ear mouse model, would be measurable in the systemic circulation. We also aimed to observe, in the scenario that the intended dose was absorbed directly into the circulation, whether these levels could be measured above the background levels of HB-EGF in the circulation.A total of 12 mice had transtympanic injections of 5??g/ml of HB-EGF contained within a previously described novel hydrogel vehicle, while another 12 mice had intravenous delivery of 10??g/kg of HB-EGF. Intravenous blood samples were collected at 0-, 3-, 24-, 168-, 288- and 720-h post-injection. A double-antibody sandwich one-step process enzyme-linked immunosorbent assay (ELISA) was used to determine the level of HB-EGF in the serum.No mice in the transtympanic administration group and no mice in the intravenous administration group were found to have blood level measured above that in the controls.The inability of the positive control to measure levels above background, suggest the total dose used in our studies, even if 100% absorbed into the system circulation is insignificant.HB-EGF at the doses and delivery method proposed for treatment of chronic TM perforation in a mouse model are likely to have no measurable systemic effect.

  View details for DOI 10.3109/01480545.2016.1143482

  View details for PubMedID 26887920

• Engineering a Dual-Layer Chitosan-Lactide Hydrogel To Create Endothelial Cell Aggregate-Induced Microvascular Networks In Vitro and Increase Blood Perfusion In Vivo ACS APPLIED MATERIALS & INTERFACES Kim, S., Kawai, T., Wang, D., Yang, Y. 2016; 8 (30): 19245-19255

  Abstract

  Here, we report the use of chemically cross-linked and photo-cross-linked hydrogels to engineer human umbilical vein endothelial cell (HUVEC) aggregate-induced microvascular networks to increase blood perfusion in vivo. First, we studied the effect of chemically cross-linked and photo-cross-linked chitosan-lactide hydrogels on stiffness, degradation rates, and HUVEC behaviors. The photo-cross-linked hydrogel was relatively stiff (E = ?15 kPa) and possessed more compact networks, denser surface texture, and lower enzymatic degradation rates than the relatively soft, chemically cross-linked hydrogel (E = ?2 kPa). While both hydrogels exhibited nontoxicity, the soft chemically cross-linked hydrogels expedited the formation of cell aggregates compared to the photo-cross-linked hydrogels. Cells on the less stiff, chemically cross-linked hydrogels expressed more matrix metalloproteinase (MMP) activity than the stiffer, photo-cross-linked hydrogel. This difference in MMP activity resulted in a more dramatic decrease in mechanical stiffness after 3 days of incubation for the chemically cross-linked hydrogel, as compared to the photo-cross-linked one. After determining the physical and biological properties of each hydrogel, we accordingly engineered a dual-layer hydrogel construct consisting of the relatively soft, chemically cross-linked hydrogel layer for HUVEC encapsulation, and the relatively stiff, acellular, photo-cross-linked hydrogel for retention of cell-laden microvasculature above. This dual-layer hydrogel construct enabled a lasting HUVEC aggregate-induced microvascular network due to the combination of stable substrate, enriched cell adhesion molecules, and extracellular matrix proteins. We tested the dual-layer hydrogel construct in a mouse model of hind-limb ischemia, where the HUVEC aggregate-induced microvascular networks significantly enhanced blood perfusion rate to ischemic legs and decreased tissue necrosis compared with both no treatment and nonaggregated HUVEC-loaded hydrogels within 2 weeks. This study suggests an effective means for regulating hydrogel properties to facilitate a stable, HUVEC aggregate-induced microvascular network for a variety of vascularized tissue applications.

  View details for DOI 10.1021/acsami.6b04431

  View details for Web of Science ID 000380968300009

  View details for PubMedID 27399928

• Synthesis and characterization of polycaprolactone urethane hollow fiber membranes as small diameter vascular grafts MATERIALS SCIENCE & ENGINEERING C-MATERIALS FOR BIOLOGICAL APPLICATIONS Mercado-Pagan, A. E., Stahl, A. M., Ramseier, M. L., Behn, A. W., Yang, Y. 2016; 64: 61-73

  Abstract

  The design of bioresorbable synthetic small diameter (<6mm) vascular grafts (SDVGs) capable of sustaining long-term patency and endothelialization is a daunting challenge in vascular tissue engineering. Here, we synthesized a family of biocompatible and biodegradable polycaprolactone (PCL) urethane macromers to fabricate hollow fiber membranes (HFMs) as SDVG candidates, and characterized their mechanical properties, degradability, hemocompatibility, and endothelial development. The HFMs had smooth surfaces and porous internal structures. Their tensile stiffness ranged from 0.09 to 0.11N/mm and their maximum tensile force from 0.86 to 1.03N, with minimum failure strains of approximately 130%. Permeability varied from 1 to 14×10(-6)cm/s, burst pressures from 1158 to 1468mmHg, and compliance from 0.52 to 1.48%/100mmHg. The suture retention forces ranged from 0.55 to 0.81N. HFMs had slow degradation profiles, with 15 to 30% degradation after 8weeks. Human endothelial cells proliferated well on the HFMs, creating stable cell layer coverage. Hemocompatibility studies demonstrated low hemolysis (<2%), platelet activation, and protein adsorption. There were no significant differences in the hemocompatibility of HFMs in the absence and presence of endothelial layers. These encouraging results suggest great promise of our newly developed materials and biodegradable elastomeric HFMs as SDVG candidates.

  View details for DOI 10.1016/j.msec.2016.03.068

  View details for Web of Science ID 000376547700008

  View details for PubMedID 27127029

• In Response to the Letter to the Editor Regarding: Heparin Binding-Epidermal Growth Factor-Like Growth Factor for the Regeneration of Chronic Tympanic Membrane Perforations in Mice. Tissue engineering. Part A Santa Maria, P. L., Kim, S., Varsak, Y. K., Yang, Y. P. 2016; 22 (5-6): 570-571

  View details for DOI 10.1089/ten.TEA.2016.0059

  View details for PubMedID 26908042

• A novel bioprinting method and system for forming hybrid tissue engineering constructs BIOFABRICATION Shanjani, Y., Pan, C. C., Elomaa, L., Yang, Y. 2015; 7 (4)

  View details for DOI 10.1088/1758-5090/7/4/045008

  View details for Web of Science ID 000366896900014

  View details for PubMedID 26685102

• Geometrical versus Random beta-TCP Scaffolds: Exploring the Effects on Schwann Cell Growth and Behavior PLOS ONE Sweet, L., Kang, Y., Czisch, C., Witek, L., Shi, Y., Smay, J., Plant, G. W., Yang, Y. 2015; 10 (10)

  Abstract

  Numerous studies have demonstrated that Schwann cells (SCs) play a role in nerve regeneration; however, their role in innervating a bioceramic scaffold for potential application in bone regeneration is still unknown. Here we report the cell growth and functional behavior of SCs on ?-tricalcium phosphate (?-TCP) scaffolds arranged in 3D printed-lattice (P-?-TCP) and randomly-porous, template-casted (N-?-TCP) structures. Our results indicate that SCs proliferated well and expressed the phenotypic markers p75LNGFR and the S100-? subunit of SCs as well as displayed growth morphology on both scaffolds, but SCs showed spindle-shaped morphology with a significant degree of SCs alignment on the P-?-TCP scaffolds, seen to a lesser degree in the N-?-TCP scaffold. The gene expressions of nerve growth factor (?-ngf), neutrophin-3 (nt-3), platelet-derived growth factor (pdgf-bb), and vascular endothelial growth factor (vegf-a) were higher at day 7 than at day 14. While no significant differences in protein secretion were measured between these last two time points, the scaffolds promoted the protein secretion at day 3 compared to that on the cell culture plates. These results together imply that the ?-TCP scaffolds can support SC cell growth and that the 3D-printed scaffold appeared to significantly promote the alignment of SCs along the struts. Further studies are needed to investigate the early and late stage relationship between gene expression and protein secretion of SCs on the scaffolds with refined characteristics, thus better exploring the potential of SCs to support vascularization and innervation in synthetic bone grafts.

  View details for DOI 10.1371/journal.pone.0139820

  View details for Web of Science ID 000362510600085

  View details for PubMedID 26444999

  View details for PubMedCentralID PMC4596809

• Development of mRuby2-Transfected C3H10T1/2 Fibroblasts for Musculoskeletal Tissue Engineering PLOS ONE Ker, D. F., Sharma, R., Wang, E. T., Yang, Y. P. 2015; 10 (9)

  View details for DOI 10.1371/journal.pone.0139054

  View details for PubMedID 26407291

• Heparin Binding Epidermal Growth Factor-Like Growth Factor Heals Chronic Tympanic Membrane Perforations With Advantage Over Fibroblast Growth Factor 2 and Epidermal Growth Factor in an Animal Model OTOLOGY & NEUROTOLOGY Maria, P. L., Weierich, K., Kim, S., Yang, Y. P. 2015; 36 (7): 1279-1283

  Abstract

  That heparin binding epidermal growth factor-like growth factor (HB-EGF) heals chronic tympanic membrane (TM) perforations at higher rates than fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF) in an animal model.A nonsurgical treatment for chronic TM perforation would benefit those unable to access surgery or those unable to have surgery, as well as reducing the cost of tympanoplasty. Growth factor (GF) treatments have been reported in the literature with variable success with the lack of a suitable animal providing a major obstacle.The GFs were tested in a validated mouse model of chronic TM perforation. A bioabsorbable hydrogel polymer was used to deliver the GF at a steady concentration as it dissolved over 4 weeks. A control (polymer only, n = 18) was compared to polymer loaded with HB-EGF (5 ?g/ml, n = 18), FGF2 (100 ?g/ml, n = 19), and EGF (250 ?g/ml, n = 19). Perforations were inspected at 4 weeks.The healing rates, as defined as 100% perforation closure, were control (5/18, 27.8%), HB-EGF (15/18, 83.3%), FGF2 (6/19, 31.6%), and EGF (3/19, 15.8%). There were no differences between FGF2 (p = 0.80) and EGF (p = 0.31) with control healing rates. HB-EGF (p = 0.000001) showed a significant difference for healing. The HB-EGF healed TMs showed layers similar to a normal TM, whereas the other groups showed a lack of epithelial migration.This study confirms the advantage of HB-EGF over two other commonly used growth factors and is a promising nonsurgical treatment of chronic TM perforations.

  View details for DOI 10.1097/MAO.0000000000000795

  View details for Web of Science ID 000358409500021

• Heparin Binding-Epidermal Growth Factor-Like Growth Factor for the Regeneration of Chronic Tympanic Membrane Perforations in Mice TISSUE ENGINEERING PART A Maria, P. L., Kim, S., Varsak, Y. K., Yang, Y. P. 2015; 21 (9-10): 1483-1494

  Abstract

  We aim to explore the role of epidermal growth factor (EGF) ligand shedding in tympanic membrane wound healing and to investigate the translation of its modulation in tissue engineering of chronic tympanic membrane perforations. Chronic suppurative otitis media (CSOM) is an infected chronic tympanic membrane perforation. Up to 200 million suffer from its associated hearing loss and it is the most common cause of pediatric hearing loss in developing countries. There is a need for nonsurgical treatment due to a worldwide lack of resources. In this study, we show that EGF ligand shedding is essential for tympanic membrane healing as it's inhibition, with KB-R7785, leads to chronic perforation in 87.9% (n=58) compared with 0% (n=20) of controls. We then show that heparin binding-EGF-like growth factor (5??g/mL), which acts to shed EGF ligands, can regenerate chronic perforations in mouse models with 92% (22 of 24) compared with 38% (10 of 26), also with eustachian tube occlusion with 94% (18 of 19) compared with 9% (2 of 23) and with CSOM 100% (16 of 16) compared with 41% (7 of 17). We also show the nonototoxicity of this treatment and its hydrogel delivery vehicle. This provides preliminary data for a clinical trial where it could be delivered by nonspecialist trained healthcare workers and fulfill the clinical need for a nonsurgical treatment for chronic tympanic membrane perforation and CSOM.

  View details for DOI 10.1089/ten.tea.2014.0474

  View details for Web of Science ID 000353952300002

  View details for PubMedID 25567607

  View details for PubMedCentralID PMC4426296

• Development and evaluation of elastomeric hollow fiber membranes as small diameter vascular graft substitutes. Materials science & engineering. C, Materials for biological applications Mercado-Pagán, Á. E., Kang, Y., Findlay, M. W., Yang, Y. 2015; 49: 541-548

  Abstract

  Engineering of small diameter (<6mm) vascular grafts (SDVGs) for clinical use remains a significant challenge. Here, elastomeric polyester urethane (PEU)-based hollow fiber membranes (HFMs) are presented as an SDVG candidate to target the limitations of current technologies and improve tissue engineering designs. HFMs are fabricated by a simple phase inversion method. HFM dimensions are tailored through adjustments to fabrication parameters. The walls of HFMs are highly porous. The HFMs are very elastic, with moduli ranging from 1-4MPa, strengths from 1-5MPa, and max strains from 300-500%. Permeability of the HFMs varies from 0.5-3.5×10(-6)cm/s, while burst pressure varies from 25 to 35psi. The suture retention forces of HFMs are in the range of 0.8 to 1.2N. These properties match those of blood vessels. A slow degradation profile is observed for all HFMs, with 71 to 78% of the original mass remaining after 8weeks, providing a suitable profile for potential cellular incorporation and tissue replacement. Both human endothelial cells and human mesenchymal stem cells proliferate well in the presence of HFMs up to 7days. These results demonstrate a promising customizable PEU HFMs for small diameter vascular repair and tissue engineering applications.

  View details for DOI 10.1016/j.msec.2015.01.051

  View details for PubMedID 25686982

• Vascularization in Bone Tissue Engineering Constructs ANNALS OF BIOMEDICAL ENGINEERING Mercado-Pagan, A. E., Stahl, A. M., Shanjani, Y., Yang, Y. 2015; 43 (3): 718-729

  Abstract

  Vascularization of large bone grafts is one of the main challenges of bone tissue engineering (BTE), and has held back the clinical translation of engineered bone constructs for two decades so far. The ultimate goal of vascularized BTE constructs is to provide a bone environment rich in functional vascular networks to achieve efficient osseointegration and accelerate restoration of function after implantation. To attain both structural and vascular integration of the grafts, a large number of biomaterials, cells, and biological cues have been evaluated. This review will present biological considerations for bone function restoration, contemporary approaches for clinical salvage of large bone defects and their limitations, state-of-the-art research on the development of vascularized bone constructs, and perspectives on evaluating and implementing novel BTE grafts in clinical practice. Success will depend on achieving full graft integration at multiple hierarchical levels, both between the individual graft components as well as between the implanted constructs and their surrounding host tissues. The paradigm of vascularized tissue constructs could not only revolutionize the progress of BTE, but could also be readily applied to other fields in regenerative medicine for the development of new innovative vascularized tissue designs.

  View details for DOI 10.1007/s10439-015-1253-3

  View details for Web of Science ID 000351742500018

  View details for PubMedID 25616591

• Engineering a vascularized collagen-beta-tricalcium phosphate graft using an electrochemical approach ACTA BIOMATERIALIA Kang, Y., Mochizuki, N., Khademhosseini, A., Fukuda, J., Yang, Y. 2015; 11: 449-458

  Abstract

  Vascularization of three-dimensional large synthetic grafts for tissue regeneration remains a significant challenge. Here we demonstrate an electrochemical approach, named the cell electrochemical detachment (CED) technique, to form an integral endothelium and use it to prevascularize a collagen-?-tricalcium phosphate (?-TCP) graft. The CED technique electrochemically detached an integral endothelium from a gold-coated glass rod to a collagen-infiltrated, channeled, macroporous ?-TCP scaffold, forming an endothelium-lined microchannel containing graft upon removal of the rod. The in vitro results from static and perfusion culture showed that the endothelium robustly emanated microvascular sprouting and prevascularized the entire collagen/?-TCP integrated graft. The in vivo subcutaneous implantation studies showed that the prevascularized collagen/?-TCP grafts established blood flow originating from the endothelium-lined microchannel within a week, and the blood flow covered more areas in the graft over time. In addition, many blood vessels invaded the prevascularized collagen/?-TCP graft and the in vitro preformed microvascular networks anastomosed with the host vasculature, while collagen alone without the support of rigid ceramic scaffold showed less blood vessel invasion and anastomosis. These results suggest a promising strategy for effectively vascularizing large tissue-engineered grafts by integrating multiple hydrogel-based CED-engineered endothelium-lined microchannels into a rigid channeled macroporous scaffold.

  View details for DOI 10.1016/j.actbio.2014.09.035

  View details for Web of Science ID 000347747900042

  View details for PubMedID 25263031

• Heparin Binding Epidermal Growth Factor-Like Growth Factor Heals Chronic Tympanic Membrane Perforations With Advantage Over Fibroblast Growth Factor 2 and Epidermal Growth Factor in an Animal Model. Otology & neurotology : official publication of the American Otological Society, American Neurotology Society [and] European Academy of Otology and Neurotology Santa Maria, P. L., Weierich, K., Kim, S., Yang, Y. P. 2015; 36 (7): 1279?83

  Abstract

  That heparin binding epidermal growth factor-like growth factor (HB-EGF) heals chronic tympanic membrane (TM) perforations at higher rates than fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF) in an animal model.A nonsurgical treatment for chronic TM perforation would benefit those unable to access surgery or those unable to have surgery, as well as reducing the cost of tympanoplasty. Growth factor (GF) treatments have been reported in the literature with variable success with the lack of a suitable animal providing a major obstacle.The GFs were tested in a validated mouse model of chronic TM perforation. A bioabsorbable hydrogel polymer was used to deliver the GF at a steady concentration as it dissolved over 4 weeks. A control (polymer only, n = 18) was compared to polymer loaded with HB-EGF (5 ?g/ml, n = 18), FGF2 (100 ?g/ml, n = 19), and EGF (250 ?g/ml, n = 19). Perforations were inspected at 4 weeks.The healing rates, as defined as 100% perforation closure, were control (5/18, 27.8%), HB-EGF (15/18, 83.3%), FGF2 (6/19, 31.6%), and EGF (3/19, 15.8%). There were no differences between FGF2 (p = 0.80) and EGF (p = 0.31) with control healing rates. HB-EGF (p = 0.000001) showed a significant difference for healing. The HB-EGF healed TMs showed layers similar to a normal TM, whereas the other groups showed a lack of epithelial migration.This study confirms the advantage of HB-EGF over two other commonly used growth factors and is a promising nonsurgical treatment of chronic TM perforations.

  View details for PubMedID 26075672

• Three-dimensional fabrication of cell-laden biodegradable poly(ethylene glycol-co-depsipeptide) hydrogels by visible light stereolithographye JOURNAL OF MATERIALS CHEMISTRY B Elomaa, L., Pan, C., Shanjani, Y., Malkovskiy, A., Seppala, J. V., Yang, Y. 2015; 3 (42): 8348-8358

  View details for DOI 10.1039/c5tb01468a

  View details for Web of Science ID 000363868000011

• Biodegradable Photocrosslinkable Poly(depsipeptide-co-epsilon-caprolactone) for Tissue Engineering: Synthesis, Characterization, and In Vitro Evaluation JOURNAL OF POLYMER SCIENCE PART A-POLYMER CHEMISTRY Elomaa, L., Kang, Y., Seppala, J. V., Yang, Y. 2014; 52 (23): 3307-3315

  View details for DOI 10.1002/pola.27400

  View details for Web of Science ID 000344359500002

• Novel osteoinductive photo-cross-linkable chitosan-lactide-fibrinogen hydrogels enhance bone regeneration in critical size segmental bone defects ACTA BIOMATERIALIA Kim, S., Bedigrew, K., Guda, T., Maloney, W. J., Park, S., Wenke, J. C., Yang, Y. P. 2014; 10 (12): 5021-5033

  View details for DOI 10.1016/j.actbio.2014.08.028

  View details for Web of Science ID 000345468300011

• Novel osteoinductive photo-cross-linkable chitosan-lactide-fibrinogen hydrogels enhance bone regeneration in critical size segmental bone defects. Acta biomaterialia Kim, S., Bedigrew, K., Guda, T., Maloney, W. J., Park, S., Wenke, J. C., Yang, Y. P. 2014; 10 (12): 5021-5033

  Abstract

  The purpose of this study was to develop and characterize a novel photo-cross-linkable chitosan-lactide-fibrinogen (CLF) hydrogel and evaluate the efficacy of bone morphogenetic protein-2 (BMP-2) containing a CLF hydrogel for osteogenesis in vitro and in vivo. We synthesized the CLF hydrogels and characterized their chemical structure, degradation rate, compressive modulus and in vitro BMP-2 release kinetics. We evaluated bioactivities of the BMP-2 containing CLF hydrogels (0, 50, 100 and 500ngml(-1)) in vitro using W-20-17 preosteoblast mouse bone marrow stromal cells and C2C12 mouse myoblast cells. The effect of BMP-2 containing CLF gels (0, 0.5, 1, 2 and 5?g) on bone formation was evaluated using rat critical size segmental bone defects for 4weeks. Fourier transform infrared spectroscopy spectra and scanning electron microscopy images showed chemical and structural changes by the addition of fibrinogen into the chitosan-lactide copolymer. The incorporation of fibrinogen molecules significantly increased the compressive modulus of the hydrogels. The in vitro BMP-2 release study showed initial burst releases from the CLF hydrogels followed by sustained releases, regardless of the concentration of the BMP-2 over 4weeks. Cells in all groups were viable in the presence of the hydrogels regardless of BMP-2 doses, indicating non-cytotoxicity of hydrogels. Alkaline phosphate activity and mineralization of cells exhibited dose dependence on BMP-2 containing CLF hydrogels. Radiography, microcomputed tomography and histology confirmed that the BMP-2 containing CLF hydrogels prompted neo-osteogenesis and accelerated healing of the defects in a dose-dependent manner. Thus the CLF hydrogel is a promising delivery system of growth factors for bone regeneration.

  View details for DOI 10.1016/j.actbio.2014.08.028

  View details for PubMedID 25174669

• Hemocompatibility evaluation of small elastomeric hollow fiber membranes as vascular substitutes. Journal of biomaterials applications Mercado-Pagán, Á. E., Ker, D. F., Yang, Y. 2014; 29 (4): 557-565

  Abstract

  One of the main challenges for clinical implementation of small diameter vascular grafts (SDVGs) is their limited hemocompatibility. Important design specifications for such grafts include features that minimize the long-term risks of restenosis, fouling, and thrombus formation. In our lab, we have developed elastomeric hollow fiber membranes (HFMs), using a phase inversion method, as candidates for SDVGs. Here, we present our results for in vitro hemocompatibility testing of our HFM under flow and static conditions. Our results showed that the polymer-based HFMs do not damage the integrity of human red blood cells (RBCs) as shown by their low hemolytic extent (less than 2%). When analyzed for blood cell lysis using lactate dehydrogenase (LDH) activity as an indicator, no significant differences were observed between blood exposed to our HFMs and uncoagulated blood. Analysis of protein adsorption showed a low concentration of proteins deposited on the surfaces of HFM after 24?h. Platelet adhesion profiles using human platelet-rich plasma (PRP) showed that a low level of platelets adhered to the HFMs after 24?h, indicating minimal thrombotic potential. Under the majority of conditions, no significant differences were observed between medical-grade polymers and our HFMs. Eventual optimization of hemocompatible elastomeric HFM vessel grafts could lead to improved tissue vascularization as well as vascularized, tissue-engineered scaffolds for organ repair.

  View details for DOI 10.1177/0885328214537541

  View details for PubMedID 24913612

• In vitro evaluation of photo-crosslinkable chitosan-lactide hydrogels for bone tissue engineering JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART B-APPLIED BIOMATERIALS Kim, S., Kang, Y., Mercado-Pagan, A. E., Maloney, W. J., Yang, Y. 2014; 102 (7): 1393-1406

  View details for DOI 10.1002/jbm.b.33118

  View details for Web of Science ID 000342963000004

• Hemocompatibility evaluation of small elastomeric hollow fiber membranes as vascular substitutes JOURNAL OF BIOMATERIALS APPLICATIONS Mercado-Pagan, A. E., Ker, D. F., Yang, Y. 2014; 29 (4): 557-565

  View details for DOI 10.1177/0885328214537541

  View details for Web of Science ID 000342807300007

• Fabrication, vascularization and osteogenic properties of a novel synthetic biomimetic induced membrane for the treatment of large bone defects. Bone Ren, L., Kang, Y., Browne, C., Bishop, J., Yang, Y. 2014; 64: 173-182

  Abstract

  The induced membrane has been widely used in the treatment of large bone defects but continues to be limited by a relatively lengthy healing process and a requisite two stage surgical procedure. Here we report the development and characterization of a synthetic biomimetic induced membrane (BIM) consisting of an inner highly pre-vascularized cell sheet and an outer osteogenic layer using cell sheet engineering. The pre-vascularized inner layer was formed by seeding human umbilical vein endothelial cells (HUVECs) on a cell sheet comprised of a layer of undifferentiated human bone marrow-derived mesenchymal stem cells (hMSCs). The outer osteogenic layer was formed by inducing osteogenic differentiation of hMSCs. In vitro results indicated that the undifferentiated hMSC cell sheet facilitated the alignment of HUVECs and significantly promoted the formation of vascular-like networks. Furthermore, seeded HUVECs rearranged the extracellular matrix produced by hMSC sheet. After subcutaneous implantation, the composite constructs showed rapid vascularization and anastomosis with the host vascular system, forming functional blood vessels in vivo. Osteogenic potential of the BIM was evidenced by immunohistochemistry staining of osteocalcin, tartrate-resistant acid phosphatase (TRAP) staining, and alizarin red staining. In summary, the synthetic BIM showed rapid vascularization, significant anastomoses, and osteogenic potential in vivo. This synthetic BIM has the potential for treatment of large bone defects in the absence of infection.

  View details for DOI 10.1016/j.bone.2014.04.011

  View details for PubMedID 24747351

• Fabrication, vascularization and osteogenic properties of a novel synthetic biomimetic induced membrane for the treatment of large bone defects. Bone Ren, L., Kang, Y., Browne, C., Bishop, J., Yang, Y. 2014; 64: 173-182

  View details for DOI 10.1016/j.bone.2014.04.011

  View details for PubMedID 24747351

• Engineering Vascularized Bone Grafts by Integrating a Biomimetic Periosteum and beta-TCP Scaffold ACS APPLIED MATERIALS & INTERFACES Kang, Y., Ren, L., Yang, Y. 2014; 6 (12): 9622-9633

  Abstract

  Treatment of large bone defects using synthetic scaffolds remain a challenge mainly due to insufficient vascularization. This study is to engineer a vascularized bone graft by integrating a vascularized biomimetic cell-sheet-engineered periosteum (CSEP) and a biodegradable macroporous beta-tricalcium phosphate (?-TCP) scaffold. We first cultured human mesenchymal stem cells (hMSCs) to form cell sheet and human umbilical vascular endothelial cells (HUVECs) were then seeded on the undifferentiated hMSCs sheet to form vascularized cell sheet for mimicking the fibrous layer of native periosteum. A mineralized hMSCs sheet was cultured to mimic the cambium layer of native periosteum. This mineralized hMSCs sheet was first wrapped onto a cylindrical ?-TCP scaffold followed by wrapping the vascularized HUVEC/hMSC sheet, thus generating a biomimetic CSEP on the ?-TCP scaffold. A nonperiosteum structural cell sheets-covered ?-TCP and plain ?-TCP were used as controls. In vitro studies indicate that the undifferentiated hMSCs sheet facilitated HUVECs to form rich capillary-like networks. In vivo studies indicate that the biomimetic CSEP enhanced angiogenesis and functional anastomosis between the in vitro preformed human capillary networks and the mouse host vasculature. MicroCT analysis and osteocalcin staining show that the biomimetic CSEP/?-TCP graft formed more bone matrix compared to the other groups. These results suggest that the CSEP that mimics the cellular components and spatial configuration of periosteum plays a critical role in vascularization and osteogenesis. Our studies suggest that a biomimetic periosteum-covered ?-TCP graft is a promising approach for bone regeneration.

  View details for DOI 10.1021/am502056q

  View details for Web of Science ID 000338184500088

  View details for PubMedID 24858072

  View details for PubMedCentralID PMC4075998

• Hydrogel bioprinted microchannel networks for vascularization of tissue engineering constructs LAB ON A CHIP Bertassoni, L. E., Cecconi, M., Manoharan, V., Nikkhah, M., Hjortnaes, J., Cristino, A. L., Barabaschi, G., Demarchi, D., Dokmeci, M. R., Yang, Y., Khademhosseini, A. 2014; 14 (13): 2202-2211

  Abstract

  Vascularization remains a critical challenge in tissue engineering. The development of vascular networks within densely populated and metabolically functional tissues facilitate transport of nutrients and removal of waste products, thus preserving cellular viability over a long period of time. Despite tremendous progress in fabricating complex tissue constructs in the past few years, approaches for controlled vascularization within hydrogel based engineered tissue constructs have remained limited. Here, we report a three dimensional (3D) micromolding technique utilizing bioprinted agarose template fibers to fabricate microchannel networks with various architectural features within photocrosslinkable hydrogel constructs. Using the proposed approach, we were able to successfully embed functional and perfusable microchannels inside methacrylated gelatin (GelMA), star poly(ethylene glycol-co-lactide) acrylate (SPELA), poly(ethylene glycol) dimethacrylate (PEGDMA) and poly(ethylene glycol) diacrylate (PEGDA) hydrogels at different concentrations. In particular, GelMA hydrogels were used as a model to demonstrate the functionality of the fabricated vascular networks in improving mass transport, cellular viability and differentiation within the cell-laden tissue constructs. In addition, successful formation of endothelial monolayers within the fabricated channels was confirmed. Overall, our proposed strategy represents an effective technique for vascularization of hydrogel constructs with useful applications in tissue engineering and organs on a chip.

  View details for DOI 10.1039/c4lc00030g

  View details for Web of Science ID 000337096800008

  View details for PubMedID 24860845

• Biodegradable Photocrosslinkable Poly(depsipeptide-co-?-caprolactone) for Tissue Engineering: Synthesis, Characterization, and In Vitro Evaluation Journal of Polymer Science, Part A Elomma, L., Kang, Y., Seppälä, J. V., Yang, Y. 2014; 52 (23): 3307-3315
• Radiation combined injury models to study the effects of interventions and wound biomechanics. Radiation research Zawaski, J. A., Yates, C. R., Miller, D. D., Kaffes, C. C., Sabek, O. M., Afshar, S. F., Young, D. A., Yang, Y., Gaber, M. W. 2014; 182 (6): 640?52

  Abstract

  In the event of a nuclear detonation, a considerable number of projected casualties will suffer from combined radiation exposure and burn and/or wound injury. Countermeasure assessment in the setting of radiation exposure combined with dermal injury is hampered by a lack of animal models in which the effects of interventions have been characterized. To address this need, we used two separate models to characterize wound closure. The first was an open wound model in mice to study the effect of wound size in combination with whole-body 6 Gy irradiation on the rate of wound closure, animal weight and survival (morbidity). In this model the addition of interventions, wound closure, subcutaneous vehicle injection, topical antiseptic and topical antibiotics were studied to measure their effect on healing and survival. The second was a rat closed wound model to study the biomechanical properties of a healed wound at 10 days postirradiation (irradiated with 6 or 7.5 Gy). In addition, complete blood counts were performed and wound pathology by staining with hematoxylin and eosin, trichrome, CD68 and Ki67. In the mouse open wound model, we found that wound size and morbidity were positively correlated, while wound size and survival were negatively correlated. Regardless of the wound size, the addition of radiation exposure delayed the healing of the wound by approximately 5-6 days. The addition of interventions caused, at a minimum, a 30% increase in survival and improved mean survival by ?9 days. In the rat closed wound model we found that radiation exposure significantly decreased all wound biomechanical measurements as well as white blood cell, platelet and red blood cell counts at 10 days post wounding. Also, pathological changes showed a loss of dermal structure, thickening of dermis, loss of collagen/epithelial hyperplasia and an increased density of macrophages. In conclusion, we have characterized the effect of a changing wound size in combination with radiation exposure. We also demonstrated that the most effective interventions mitigated insensible fluid loss, which could help to define the most appropriate requirements of a successful countermeasure.

  View details for DOI 10.1667/RR13751.1

  View details for PubMedID 25409125

• Deletion of the Transforming Growth Factor beta Receptor Type II Gene in Articular Chondrocytes Leads to a Progressive Osteoarthritis-like Phenotype in Mice ARTHRITIS AND RHEUMATISM Shen, J., Li, J., Wang, B., Jin, H., Wang, M., Zhang, Y., Yang, Y., Im, H., O'Keefe, R., Chen, D. 2013; 65 (12): 3107-3119

  Abstract

  While transforming growth factor ? (TGF?) signaling plays a critical role in chondrocyte metabolism, the TGF? signaling pathways and target genes involved in cartilage homeostasis and the development of osteoarthritis (OA) remain unclear. Using an in vitro cell culture method and an in vivo mouse genetic approach, we undertook this study to investigate TGF? signaling in chondrocytes and to determine whether Mmp13 and Adamts5 are critical downstream target genes of TGF? signaling.TGF? receptor type II (TGF?RII)-conditional knockout (KO) (TGF?RII(Col2ER)) mice were generated by breeding TGF?RII(flox/flox) mice with Col2-CreER-transgenic mice. Histologic, histomorphometric, and gene expression analyses were performed. In vitro TGF? signaling studies were performed using chondrogenic rat chondrosarcoma cells. To determine whether Mmp13 and Adamts5 are critical downstream target genes of TGF? signaling, TGF?RII/matrix metalloproteinase 13 (MMP-13)- and TGF?RII/ADAMTS-5-double-KO mice were generated and analyzed.Inhibition of TGF? signaling (deletion of the Tgfbr2 gene in chondrocytes) resulted in up-regulation of Runx2, Mmp13, and Adamts5 expression in articular cartilage tissue and progressive OA development in TGF?RII(Col2ER) mice. Deletion of the Mmp13 or Adamts5 gene significantly ameliorated the OA-like phenotype induced by the loss of TGF? signaling. Treatment of TGF?RII(Col2ER) mice with an MMP-13 inhibitor also slowed OA progression.Mmp13 and Adamts5 are critical downstream target genes involved in the TGF? signaling pathway during the development of OA.

  View details for DOI 10.1002/art.38122

  View details for Web of Science ID 000327692600014

  View details for PubMedID 23982761

• Synthesis and characterization of novel elastomeric poly(D,L-lactide urethane) maleate composites for bone tissue engineering EUROPEAN POLYMER JOURNAL Mercado-Pagan, A. E., Kang, Y., Ker, D. F., Park, S., Yao, J., Bishop, J., Yang, Y. P. 2013; 49 (10): 3337-3349

  Abstract

  Here, we report the synthesis and characterization of a novel 4-arm poly(lactic acid urethane)-maleate (4PLAUMA) elastomer and its composites with nano-hydroxyapatite (nHA) as potential weight-bearing composite. The 4PLAUMA/nHA ratios of the composites were 1:3, 2:5, 1:2 and 1:1. FTIR and NMR characterization showed urethane and maleate units integrated into the PLA matrix. Energy dispersion and Auger electron spectroscopy confirmed homogeneous distribution of nHA in the polymer matrix. Maximum moduli and strength of the composites of 4PLAUMA/nHA, respectively, are 1973.31 ± 298.53 MPa and 78.10 ± 3.82 MPa for compression, 3630.46 ± 528.32 MPa and 6.23 ± 1.44 MPa for tension, 1810.42 ± 86.10 MPa and 13.00 ± 0.72 for bending, and 282.46 ± 24.91 MPa and 5.20 ± 0.85 MPa for torsion. The maximum tensile strains of the polymer and composites are in the range of 5% to 93%, and their maximum torsional strains vary from 0.26 to 0.90. The composites exhibited very slow degradation rates in aqueous solution, from approximately 50% mass remaining for the pure polymer to 75% mass remaining for composites with high nHA contents, after a period of 8 weeks. Increase in ceramic content increased mechanical properties, but decreased maximum strain, degradation rate, and swelling of the composites. Human bone marrow stem cells and human endothelial cells adhered and proliferated on 4PLAUMA films and degradation products of the composites showed little-to-no toxicity. These results demonstrate that novel 4-arm poly(lactic acid urethane)-maleate (4PLAUMA) elastomer and its nHA composites may have potential applications in regenerative medicine.

  View details for DOI 10.1016/j.eurpolymj.2013.07.004

  View details for Web of Science ID 000325233800049

  View details for PubMedCentralID PMC4012890

• The effect of rhBMP-2 and PRP delivery by biodegradable beta-tricalcium phosphate scaffolds on new bone formation in a non-through rabbit cranial defect model JOURNAL OF MATERIALS SCIENCE-MATERIALS IN MEDICINE Lim, H., Mercado-Pagan, A. E., Yun, K., Kang, S., Choi, T., Bishop, J., Koh, J., Maloney, W., Lee, K., Yang, Y. P., Park, S. 2013; 24 (8): 1895-1903

  Abstract

  This study evaluated whether the combination of biodegradable ?-tricalcium phosphate (?-TCP) scaffolds with recombinant human bone morphogenetic protein-2 (rhBMP-2) or platelet-rich plasma (PRP) could accelerate bone formation and increase bone height using a rabbit non-through cranial bone defect model. Four non-through cylindrical bone defects with a diameter of 8-mm were surgically created on the cranium of rabbits. ?-TCP scaffolds in the presence and absence of impregnated rhBMP-2 or PRP were placed into the defects. At 8 and 16 weeks after implantation, samples were dissected and fixed for analysis by microcomputed tomography and histology. Only defects with rhBMP-2 impregnated ?-TCP scaffolds showed significantly enhanced bone formation compared to non-impregnated ?-TCP scaffolds (P < 0.05). Although new bone was higher than adjacent bone at 8 weeks after implantation, vertical bone augmentation was not observed at 16 weeks after implantation, probably due to scaffold resorption occurring concurrently with new bone formation.

  View details for DOI 10.1007/s10856-013-4939-9

  View details for Web of Science ID 000321915300008

  View details for PubMedID 23779152

• Modeling vascularized bone regeneration within a porous biodegradable CaP scaffold loaded with growth factors BIOMATERIALS Sun, X., Kang, Y., Bao, J., Zhang, Y., Yang, Y., Zhou, X. 2013; 34 (21): 4971-4981

  Abstract

  Osteogenetic microenvironment is a complex constitution in which extracellular matrix (ECM) molecules, stem cells and growth factors each interact to direct the coordinate regulation of bone tissue development. Importantly, angiogenesis improvement and revascularization are critical for osteogenesis during bone tissue regeneration processes. In this study, we developed a three-dimensional (3D) multi-scale system model to study cell response to growth factors released from a 3D biodegradable porous calcium phosphate (CaP) scaffold. Our model reconstructed the 3D bone regeneration system and examined the effects of pore size and porosity on bone formation and angiogenesis. The results suggested that scaffold porosity played a more dominant role in affecting bone formation and angiogenesis compared with pore size, while the pore size could be controlled to tailor the growth factor release rate and release fraction. Furthermore, a combination of gradient VEGF with BMP2 and Wnt released from the multi-layer scaffold promoted angiogenesis and bone formation more readily than single growth factors. These results demonstrated that the developed model can be potentially applied to predict vascularized bone regeneration with specific scaffold and growth factors.

  View details for DOI 10.1016/j.biomaterials.2013.03.015

  View details for Web of Science ID 000319630000008

  View details for PubMedID 23566802

• Osteogenic and angiogenic potentials of monocultured and co-cultured human-bone-marrow-derived mesenchymal stem cells and human-umbilical-vein endothelial cells on three-dimensional porous beta-tricalcium phosphate scaffold ACTA BIOMATERIALIA Kang, Y., Kim, S., Fahrenholtz, M., Khademhosseini, A., Yang, Y. 2013; 9 (1): 4906-4915

  Abstract

  The use of biodegradable beta-tricalcium phosphate (?-TCP) scaffolds holds great promise for bone tissue engineering. However, the effects of ?-TCP on bone and endothelial cells are not fully understood. This study aimed to investigate cell proliferation and differentiation of mono- or co-cultured human-bone-marrow-derived mesenchymal stem cells (hBMSCs) and human-umbilical-vein endothelial cells (HUVECs) on a three-dimensional porous, biodegradable ?-TCP scaffold. In co-culture studies, the ratios of hBMSCs:HUVECs were 5:1, 1:1 and 1:5. Cellular morphologies of HUVECs, hBMSCs and co-cultured HUVECs/hBMSCs on the ?-TCP scaffolds were monitored using confocal and scanning electron microscopy. Cell proliferation was monitored by measuring the amount of double-stranded DNA (dsDNA) whereas hBMSC and HUVEC differentiation was assessed using the osteogenic and angiogenic markers, alkaline phosphatase (ALP) and PECAM-1 (CD31), respectively. Results show that HUVECs, hBMSCs and hBMSCs/HUVECs adhered to and proliferated well on the ?-TCP scaffolds. In monoculture, hBMSCs grew faster than HUVECs on the ?-TCP scaffolds after 7 days, but HUVECs reached similar levels of proliferation after 14 days. In monoculture, ?-TCP scaffolds promoted ALP activities of both hBMSCs and HUVECs when compared to those grown on tissue culture well plates. ALP activity of cells in co-culture was higher than that of hBMSCs in monoculture. Real-time polymerase chain reaction results indicate that runx2 and alp gene expression in monocultured hBMSCs remained unchanged at days 7 and 14, but alp gene expression was significantly increased in hBMSC co-cultures when the contribution of individual cell types was not distinguished.

  View details for DOI 10.1016/j.actbio.2012.08.008

  View details for Web of Science ID 000313376900047

  View details for PubMedID 22902820

  View details for PubMedCentralID PMC3508299

• Directed endothelial cell morphogenesis in micropatterned gelatin methacrylate hydrogels BIOMATERIALS Nikkhah, M., Eshak, N., Zorlutuna, P., Annabi, N., Castello, M., Kim, K., Dolatshahi-Pirouz, A., Edalat, F., Bae, H., Yang, Y., Khademhosseini, A. 2012; 33 (35): 9009-9018

  Abstract

  Engineering of organized vasculature is a crucial step in the development of functional and clinically relevant tissue constructs. A number of previous techniques have been proposed to spatially regulate the distribution of angiogenic biomolecules and vascular cells within biomaterial matrices to promote vascularization. Most of these approaches have been limited to two-dimensional (2D) micropatterned features or have resulted in formation of random vasculature within three-dimensional (3D) microenvironments. In this study, we investigate 3D endothelial cord formation within micropatterned gelatin methacrylate (GelMA) hydrogels with varying geometrical features (50-150 ?m height). We demonstrated the significant dependence of endothelial cells proliferation, alignment and cord formation on geometrical dimensions of the patterned features. The cells were able to align and organize within the micropatterned constructs and assemble to form cord structures with organized actin fibers and circular/elliptical cross-sections. The inner layer of the cord structure was filled with gel showing that the micropatterned hydrogel constructs guided the assembly of endothelial cells into cord structures. Notably, the endothelial cords were retained within the hydrogel microconstructs for all geometries after two weeks of culture; however, only the 100 ?m-high constructs provided the optimal microenvironment for the formation of circular and stable cord structures. Our findings suggest that endothelial cord formation is a preceding step to tubulogenesis and the proposed system can be used to develop organized vasculature for engineered tissue constructs.

  View details for DOI 10.1016/j.biomaterials.2012.08.068

  View details for Web of Science ID 000310721900011

  View details for PubMedID 23018132

• Cytokine combination therapy prediction for bone remodeling in tissue engineering based on the intracellular signaling pathway BIOMATERIALS Sun, X., Su, J., Bao, J., Peng, T., Zhang, L., Zhang, Y., Yang, Y., Zhou, X. 2012; 33 (33): 8265-8276

  Abstract

  The long-term performance of tissue-engineered bone grafts is determined by a dynamic balance between bone regeneration and resorption. We proposed using embedded cytokine slow-releasing hydrogels to tune this balance toward a desirable final bone density. In this study we established a systems biology model, and quantitatively explored the combinatorial effects of delivered cytokines from hydrogels on final bone density. We hypothesized that: 1) bone regeneration was driven by transcription factors Runx2 and Osterix, which responded to released cytokines, such as Wnt, BMP2, and TGF?, drove the development of osteoblast lineage, and contributed to bone mass generation; and 2) the osteoclast lineage, on the other hand, governed the bone resorption, and communications between these two lineages determined the dynamics of bone remodeling. In our model, Intracellular signaling pathways were represented by ordinary differential equations, while the intercellular communications and cellular population dynamics were modeled by stochastic differential equations. Effects of synergistic cytokine combinations were evaluated by Loewe index and Bliss index. Simulation results revealed that the Wnt/BMP2 combinations released from hydrogels showed best control of bone regeneration and synergistic effects, and suggested optimal dose ratios of given cytokine combinations released from hydrogels to most efficiently control the long-term bone remodeling. We revealed the characteristics of cytokine combinations of Wnt/BMP2 which could be used to guide the design of in vivo bone scaffolds and the clinical treatment of some diseases such as osteoporosis.

  View details for DOI 10.1016/j.biomaterials.2012.07.041

  View details for Web of Science ID 000310401000008

  View details for PubMedID 22910219

• The osteogenic differentiation of human bone marrow MSCs on HUVEC-derived ECM and beta-TCP scaffold BIOMATERIALS Kang, Y., Kim, S., Bishop, J., Khademhosseini, A., Yang, Y. 2012; 33 (29): 6998-7007

  Abstract

  Extracellular matrix (ECM) serves a key role in cell migration, attachment, and cell development. Here we report that ECM derived from human umbilical vein endothelial cells (HUVEC) promoted osteogenic differentiation of human bone marrow mesenchymal stem cells (hMSC). We first produced an HUVEC-derived ECM on a three-dimensional (3D) beta-tricalcium phosphate (?-TCP) scaffold by HUVEC seeding, incubation, and decellularization. The HUVEC-derived ECM was then characterized by SEM, FTIR, XPS, and immunofluorescence staining. The effect of HUVEC-derived ECM-containing ?-TCP scaffold on hMSC osteogenic differentiation was subsequently examined. SEM images indicate a dense matrix layer deposited on the surface of struts and pore walls. FTIR and XPS measurements show the presence of new functional groups (amide and hydroxyl groups) and elements (C and N) in the ECM/?-TCP scaffold when compared to the ?-TCP scaffold alone. Immunofluorescence images indicate that high levels of fibronectin and collagen IV and low level of laminin were present on the scaffold. ECM-containing ?-TCP scaffolds significantly increased alkaline phosphatase (ALP) specific activity and up-regulated expression of osteogenesis-related genes such as runx2, alkaline phosphatase, osteopontin and osteocalcin in hMSC, compared to ?-TCP scaffolds alone. This increased effect was due to the activation of MAPK/ERK signaling pathway since disruption of this pathway using an ERK inhibitor PD98059 results in down-regulation of these osteogenic genes. Cell-derived ECM-containing calcium phosphate scaffolds is a promising osteogenic-promoting bone void filler in bone tissue regeneration.

  View details for DOI 10.1016/j.biomaterials.2012.06.061

  View details for Web of Science ID 000308269600010

  View details for PubMedID 22795852

  View details for PubMedCentralID PMC3427692

• Vascularized Bone Tissue Engineering: Approaches for Potential Improvement TISSUE ENGINEERING PART B-REVIEWS Nguyen, L. H., Annabi, N., Nikkhah, M., Bae, H., Binan, L., Park, S., Kang, Y., Yang, Y., Khademhosseini, A. 2012; 18 (5): 363-382

  Abstract

  Significant advances have been made in bone tissue engineering (TE) in the past decade. However, classical bone TE strategies have been hampered mainly due to the lack of vascularization within the engineered bone constructs, resulting in poor implant survival and integration. In an effort toward clinical success of engineered constructs, new TE concepts have arisen to develop bone substitutes that potentially mimic native bone tissue structure and function. Large tissue replacements have failed in the past due to the slow penetration of the host vasculature, leading to necrosis at the central region of the engineered tissues. For this reason, multiple microscale strategies have been developed to induce and incorporate vascular networks within engineered bone constructs before implantation in order to achieve successful integration with the host tissue. Previous attempts to engineer vascularized bone tissue only focused on the effect of a single component among the three main components of TE (scaffold, cells, or signaling cues) and have only achieved limited success. However, with efforts to improve the engineered bone tissue substitutes, bone TE approaches have become more complex by combining multiple strategies simultaneously. The driving force behind combining various TE strategies is to produce bone replacements that more closely recapitulate human physiology. Here, we review and discuss the limitations of current bone TE approaches and possible strategies to improve vascularization in bone tissue substitutes.

  View details for DOI 10.1089/ten.teb.2012.0012

  View details for Web of Science ID 000309516500003

  View details for PubMedID 22765012

• Effect of Coadministration of Vancomycin and BMP-2 on Cocultured Staphylococcus aureus and W-20-17 Mouse Bone Marrow Stromal Cells In Vitro ANTIMICROBIAL AGENTS AND CHEMOTHERAPY Nguyen, A. H., Kim, S., Maloney, W. J., Wenke, J. C., Yang, Y. 2012; 56 (7): 3776-3784

  Abstract

  In this study, we aimed to establish an in vitro bacterium/bone cell coculture model system and to use this model for dose dependence studies of dual administration of antibiotics and growth factors in vitro. We examined the effect of single or dual administration of the antibiotic vancomycin (VAN) at 0 to 16 ?g/ml and bone morphogenetic protein-2 (BMP-2) at 0 or 100 ng/ml on both methicillin-sensitive Staphylococcus aureus and mouse bone marrow stromal cells (W-20-17) under both mono- and coculture conditions. Cell metabolic activity, Live/Dead staining, double-stranded DNA (dsDNA) amounts, and alkaline phosphatase activity were measured to assess cell viability, proliferation, and differentiation. An interleukin-6 (IL-6) enzyme-linked immunosorbent assay (ELISA) kit was used to test the bone cell inflammation response in the presence of bacteria. Our results suggest that, when delivered together in coculture, VAN and BMP-2 maintain their primary functions as an antibiotic and a growth factor, respectively. Most interestingly, this dual-delivery type of approach has shown itself to be effective at lower concentrations of VAN than those required for an approach relying strictly on the antibiotic. It may be that BMP-2 enhances cell proliferation and differentiation before the cells become infected. In coculture, a dosage of VAN higher than that used for treatment in monoculture may be necessary to effectively inhibit growth of Staphylococcus aureus. This could mean that the coculture environment may be limiting the efficacy of VAN, possibly by way of bacterial invasion of the bone cells. This report of a coculture study demonstrates a potential beneficial effect of the coadministration of antibiotics and growth factors compared to treatment with antibiotic alone.

  View details for DOI 10.1128/AAC.00114-12

  View details for Web of Science ID 000305673000042

  View details for PubMedID 22564844

  View details for PubMedCentralID PMC3393469

• Sequential delivery of BMP-2 and IGF-1 using a chitosan gel with gelatin microspheres enhances early osteoblastic differentiation ACTA BIOMATERIALIA Kim, S., Kang, Y., Krueger, C. A., Sen, M., Holcomb, J. B., Chen, D., Wenke, J. C., Yang, Y. 2012; 8 (5): 1768-1777

  Abstract

  The purpose of this study was to develop and characterize a chitosan gel/gelatin microsphere (MSs) dual delivery system for sequential release of bone morphogenetic protein-2 (BMP-2) and insulin-like growth factor-1 (IGF-1) to enhance osteoblast differentiation in vitro. We made and characterized the delivery system based on its degree of cross-linking, degradation, and release kinetics. We also evaluated the cytotoxicity of the delivery system and the effect of growth factors on cell response using pre-osteoblast W-20-17 mouse bone marrow stromal cells. IGF-1 was first loaded into MSs, and then the IGF-1-containing MSs were encapsulated into the chitosan gel which contained BMP-2. Cross-linking of gelatin with glyoxal via Schiff bases significantly increased thermal stability and decreased the solubility of the MSs, leading to a significant decrease in the initial release of IGF-1. Encapsulation of the MSs into the chitosan gel generated polyelectrolyte complexes by intermolecular interactions, which further affected the release kinetics of IGF-1. This combinational delivery system provided an initial release of BMP-2 followed by a slow and sustained release of IGF-1. Significantly greater alkaline phosphatase activity was found in W-20-17 cells treated with the sequential delivery system compared with other treatments (P<0.05) after a week of culture.

  View details for DOI 10.1016/j.actbio.2012.01.009

  View details for Web of Science ID 000302989700012

  View details for PubMedID 22293583

  View details for PubMedCentralID PMC3314097

• Effect of Coadministration of Vancomycin and BMP-2 on Cocultured Staphylococcus aureus and W-20-17 Mouse Bone Marrow Stromal Cells In Vitro Antimicrob. Agents Chemother A. H. Nguyen, S. Kim, W. J. Maloney, J. C. Wenke, Y. Yang 2012; 56 (7): 3776-3784
• In vitro evaluation of an injectable chitosan gel for sustained local delivery of BMP-2 for osteoblastic differentiation. Journal of biomedical materials research. Part B, Applied biomaterials Kim, S., Tsao, H., Kang, Y., Young, D. A., Sen, M., Wenke, J. C., Yang, Y. 2011; 99 (2): 380-390

  Abstract

  We investigated the effect of sustained release of bone morphogenetic protein-2 (BMP-2) from an injectable chitosan gel on osteoblastic differentiation in vitro. We first characterized the release profile of BMP-2 from the gels, and then examined the cellular responses of preosteoblast mouse stromal cells (W-20-17) and human embryonic palatal mesenchymal (HEPM) cells to BMP-2. The release profiles of different concentrations of BMP-2 exhibited sustained releases (41% for 2 ng/mL and 48% for 20 ng/mL, respectively) from the chitosan gels over a three-week period. Both cell types cultured in the chitosan gels were viable and significantly proliferated for 3 days (p < 0.05). Chitosan gels loaded with BMP-2 enhanced ALP activity of W-20-17 by 3.6-fold, and increased calcium mineral deposition of HEPM by 2.8-fold at 14 days of incubation, compared to control groups initially containing the same amount of BMP-2. In addition, schitosan gels loaded with BMP-2 exhibited significantly greater osteocalcin synthesis of W-20-17 at seven days, and of HEPM at both 7 and 14 days compared with the control groups (p<0.05). This study suggests that the enhanced effects of BMP-2 released from chitosan gels on cell differentiation and mineralization are species and cell type dependent.

  View details for DOI 10.1002/jbm.b.31909

  View details for PubMedID 21905214

• Creation of bony microenvironment with CaP and cell-derived ECM to enhance human bone-marrow MSC behavior and delivery of BMP-2 BIOMATERIALS Kang, Y., Kim, S., Khademhosseini, A., Yang, Y. 2011; 32 (26): 6119-6130

  Abstract

  Extracellular matrix (ECM) comprises a rich meshwork of proteins and proteoglycans, which not only contains biological cues for cell behavior, but is also a reservoir for binding growth factors and controlling their release. Here we aimed to create a suitable bony microenvironment with cell-derived ECM and biodegradable ?-tricalcium phosphate (?-TCP). More specifically, we investigated whether the ECM produced by bone marrow-derived mesenchymal stem cells (hBMSC) on a ?-TCP scaffold can bind bone morphogenetic protein-2 (BMP-2) and control its release in a sustained manner, and further examined the effect of ECM and the BMP-2 released from ECM on cell behaviors. The ECM was obtained through culturing the hBMSC on a ?-TCP porous scaffold and performing decellularization and sterilization. SEM, XPS, FTIR, and immunofluorescent staining results indicated the presence of ECM on the ?-TCP and the amount of ECM increased with the incubation time. BMP-2 was loaded onto the ?-TCP with and without ECM by immersing the scaffolds in the BMP-2 solution. The loading and release kinetics of the BMP-2 on the ?-TCP/ECM were significantly slower than those on the ?-TCP. The ?-TCP/ECM exhibited a sustained release profile of the BMP-2, which was also affected by the amount of ECM. This is probably because the ?-TCP/ECM has different binding mechanisms with BMP-2. The ?-TCP/ECM promoted cell proliferation. Furthermore, the BMP-2-loaded ?-TCP/ECM stimulated reorganization of the actin cytoskeleton, increased expression of alkaline phosphatase and calcium deposition by the cells compared to those without BMP-2 loading and the ?-TCP with BMP-2 loading.

  View details for DOI 10.1016/j.biomaterials.2011.05.015

  View details for Web of Science ID 000292904100018

  View details for PubMedID 21632105

• SAM-based Cell Transfer to Photopatterned Hydrogels for Microengineering Vascular-Like Structures Biomaterials N. Sadr, M. Zhu, T. Osaki, T. Kakegawa, Y. Yang, M. Moretti, J. Fukuda, A. Khademhosseini 2011; 32: 7479-7490
• Enhanced mechanical performance and biological evaluation of a PLGA coated ?-TCP composite scaffold for load-bearing applications. European polymer journal Kang, Y., Scully, A., Young, D. A., Kim, S., Tsao, H., Sen, M., Yang, Y. 2011; 47 (8): 1569?77

  Abstract

  Porous ?-tricalcium phosphate (?-TCP) has been used for bone repair and replacement in clinics due to its excellent biocompatibility, osteoconductivity, and biodegradability. However, the application of ?-TCP has been limited by its brittleness. Here, we demonstrated that an interconnected porous ?-TCP scaffold infiltrated with a thin layer of poly (lactic-co-glycolic acid) (PLGA) polymer showed improved mechanical performance compared to an uncoated ?-TCP scaffold while retaining its excellent interconnectivity and biocompatibility. The infiltration of PLGA significantly increased the compressive strength of ?-TCP scaffolds from 2.90 MPa to 4.19 MPa, bending strength from 1.46 MPa to 2.41 MPa, and toughness from 0.17 MPa to 1.44 MPa, while retaining an interconnected porous structure with a porosity of 80.65%. These remarkable improvements in the mechanical properties of PLGA-coated ?-TCP scaffolds are due to the combination of the systematic coating of struts, interpenetrating structural characteristics, and crack bridging. The in vitro biological evaluation demonstrated that rat bone marrow stromal cells (rBMSCs) adhered well, proliferated, and expressed alkaline phosphatase (ALP) activity on both the PLGA-coated ?-TCP and the ?-TCP. These results suggest a new strategy for fabricating interconnected macroporous scaffolds with significantly enhanced mechanical strength for potential load-bearing bone tissue regeneration.

  View details for DOI 10.1016/j.eurpolymj.2011.05.004

  View details for PubMedID 21892228

  View details for PubMedCentralID PMC3164545

• A chitosan/beta-glycerophosphate thermo-sensitive gel for the delivery of ellagic acid for the treatment of brain cancer BIOMATERIALS Kim, S., Nishimoto, S. K., Bumgardner, J. D., Haggard, W. O., Gaber, M. W., Yang, Y. 2010; 31 (14): 4157-4166

  Abstract

  We report here the development of a chitosan/beta-glycerophosphate(Ch/beta-GP) thermo-sensitive gel to deliver ellagic acid (EA) for cancer treatment. The properties of the Ch/beta-GP gels were characterized regarding chemical structure, surface morphology, and viscoelasticity. In vitro EA release rate from the EA loaded Ch/beta-GP gel and chitosan degradation rate were investigated. The anti-tumor effect of the EA loaded Ch/beta-GP gel on brain cancer cells (human U87 glioblastomas and rat C6 glioma cells) was evaluated by examining cell viability. Cell number and activity were monitored by the MTS assay. The Ch/beta-GP solution formed a heat-induced gel at body temperature, and the gelation temperature and time were affected by the final pH of the Ch/beta-GP solution. The lysozyme increased the EA release rate by 2.5 times higher than that in the absence of lysozyme. Dialyzed chitosan solution with final pH 6.3 greatly reduced the beta-GP needed for gelation, thereby significantly improving the biocompatibility of gel (p < 0.001). The chitosan gels containing 1% (w/v) of ellagic acid significantly reduced viability of U87 cells and C6 cells compared with the chitosan gels at 3 days incubation (p < 0.01, and p < 0.001, respectively).

  View details for DOI 10.1016/j.biomaterials.2010.01.139

  View details for Web of Science ID 000276541300045

  View details for PubMedID 20185170

• Novel template-casting technique for fabricating beta-tricalcium phosphate scaffolds with high interconnectivity and mechanical strength and in vitro cell responses. Journal of biomedical materials research. Part A Liu, Y., Kim, J., Young, D., Kim, S., Nishimoto, S. K., Yang, Y. 2010; 92 (3): 997-1006

  Abstract

  A novel template-casting method was developed to produce completely interconnected, macroporous biodegradable beta-tricalcium phosphate (beta-TCP) scaffolds, whose architecture and chemistry can be fully manipulated by varying the templates and casting materials. The processing route includes preparation of beta-TCP slurry; casting and shaping into preformed templates comprised of paraffin beads; solidifying, drying; and sintering. Structural, chemical, and mechanical properties of the prepared macroporous scaffolds were characterized using micro computed tomography, scanning electron microscopy, x-ray diffractometry, Fourier transform infrared spectroscopy, and mechanical testing. Human embryonic palatal mesenchymal cells were used to evaluate cell proliferation within the scaffolds in vitro. The scaffolds consisted of interconnected macropores and solid struts, leading to a reticular network. Two groups of scaffolds with larger pores, approximately 600-800 microm and smaller pores approximately 350-500 microm, were demonstrated. The interconnected windows between neighboring macropores were 440 +/- 57 microm in diameter for the larger-pored scaffolds, and 330 +/- 50 microm for the smaller-pored scaffolds. The scaffolds were highly crystallized and composed dominantly of beta-tricalcium phosphate (beta-TCP) accompanied by minor phase of hydroxyapatite (HA). The hydroxyl group was clearly detected by FTIR on the scaffolds. High mechanical strength (9.3 MPa) was demonstrated by the completely interconnected scaffolds with approximately 79% porosity. The human embryonic palatal mesenchymal (HEPM) cells proliferated well on the smaller-pored and larger-pored scaffolds, exhibiting a significantly higher level of proliferation in the first 11 days of culture on the smaller pored scaffolds. High levels of differentiation were also evidenced in both pore sizes of scaffolds.

  View details for DOI 10.1002/jbm.a.32443

  View details for PubMedID 19296544

• Anodic Oxidized Nanotubular Titanium Implants Enhance Bone Morphogenetic Protein-2 Delivery J Biomed Mater Res In-Ho Bae, Kwi-Dug Yun, Hyun-Seung Kim, Byung-Chul Jeong, Hyun-Pil Lim, Sang-Won Park, Kwang-Min Lee, Young-Chai Lim, Kyung-Ku Lee, Yun-Zhi Yang, Jeong-Tae Koh. 2010; 93B: 484-491
• Effect of growth factors in combination with injectable silicone resin particles on the biological activity of dermal fibroblasts: a preliminary in vitro study. Journal of biomedical materials research. Part B, Applied biomaterials Jennings, J. A., Crews, R. M., Robinson, J., Richelsoph, K., Cole, J. A., Bumgardner, J. D., Yang, Y., Haggard, W. O. 2010; 92 (1): 255-260

  Abstract

  Injections of silicone fluid have been clinically evaluated to treat and prevent foot ulcers due to diminished plantar fat-pad in neuropathic diabetics. The objective of this study was to determine preliminary in vitro effects of an injectable form of silicone resin particles in combination with growth factors to determine the suitability of this potential therapy for prevention of diabetic foot ulcers. Basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF-BB) were added to monolayer culture along with silicone resin particles (12 microm average diameter). Growth factors were also combined as follows: bFGF+PDGF-BB, EGF+PDGF-BB, and bFGF+EGF. Growth factors alone and in combination increased fibroblast proliferation, but the presence of particles did not significantly affect cellular proliferation. The addition of particles significantly increased fibronectin production 117% in the control group and 151% in the PDGF only group. Collagen production was increased with exposure to EGF and growth factor combinations, but the presence of particles did not lead to any significant differences, except an 81% increase in the bFGF group. These preliminary results suggest that a combination of PDGF and bFGF may be effective in stimulating proliferation and matrix production around injectable silicone resin particles to generate a fibrous tissue pad to alleviate the abnormal distribution of high pressures that contribute to diabetic foot ulcer formation.

  View details for DOI 10.1002/jbm.b.31512

  View details for PubMedID 19904740

• Effect of nanotubular-micro-roughened titanium surface on cell response in vitro and osseointegration in vivo Materials Science and Engineering Yun, K., Yang, Y., Lim, H., Oh, G., Koh, J., Bae, I., Kim J., Lee, K., Park, S. 2010; 30C: 27-33
• The inhibition of glioma growth in vitro and in vivo by a chitosan/ellagic acid composite biomaterial BIOMATERIALS Kim, S., Gaber, M. W., Zawaski, J. A., Zhang, F., Richardson, M., Zhang, X. A., Yang, Y. 2009; 30 (27): 4743-4751

  Abstract

  This study has developed a chitosan-based delivery system to locally administer ellagic acid for brain cancer treatment. We fabricated chitosan/ellagic acid composite films with various concentrations of ellagic acid. In vitro release study was performed by using a UV spectrophotometer, and enzymatic degradation rate was determined by analyzing the increased free amino groups. Viability of brain cancer cells (human U87 glioblastomas and rat C6 glioma cells) was measured via direct and indirect cell culture on the films by MTS assay. Caspase-3 activation, Western blot for p53, and anti-angiogenesis assays were also examined. In the in vivo study, GFP-tagged rat C6 glioma cells were implanted subcutaneously at the right flank region of nude mice and treatments were initiated by implanting the films subcutaneously. Tumor growth was evaluated by measuring tumor volume using a caliper, an ultrasound machine, and an optical imaging system. The chitosan/ellagic acid composite films were enzymatically degradable and exhibited a sustained slow release of ellagic acid. These materials could inhibit the cancer cell growth in an ellagic acid concentration-dependent manner by inducing apoptosis of cancer cells as well as suppressing angiogenesis. These materials also significantly suppressed tumor tissue growth in vivo.

  View details for DOI 10.1016/j.biomaterials.2009.05.010

  View details for Web of Science ID 000269330400036

  View details for PubMedID 19501395

• Development of chitosan-ellagic acid films as a local drug delivery system to induce apoptotic death of human melanoma cells. Journal of biomedical materials research. Part B, Applied biomaterials Kim, S., Liu, Y., Gaber, M. W., Bumgardner, J. D., Haggard, W. O., Yang, Y. 2009; 90 (1): 145-155

  Abstract

  This study was designed to develop a local chemotherapy device using chitosan as a local drug carrier and ellagic acid (EA) as an anticancer drug. We fabricated chitosan-ellagic acid (Ch-EA) films with concentrations of 0, 0.05, 0.1, 0.5, and 1% (w/v) of EA and examined the films using Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), and contact angle measurement. The WM115 human melanoma cell line as a skin cancer model was used to evaluate cell response to the films with the MTS assay and apoptosis assay, and HS68 human newborn fibroblast cell line as a control. With the increase in the concentration of the EA, the composite films exhibit increasing amide and ester groups and diffraction peaks of the crystallized EA and greater surface roughness and hydrophilicity. The chitosan films with 0.5 and 1% (w/v) of EA were found to have a potent antiproliferative effect on the melanoma cells by inducing apoptotic cell death. Localized effect of composites on cell behaviors has been clearly demonstrated. Our study demonstrated that the novel Ch-EA film can be potentially used in local chemotherapy.

  View details for DOI 10.1002/jbm.b.31266

  View details for PubMedID 18985785

• Lyophilization to improve drug delivery for chitosan-calcium phosphate bone scaffold construct: a preliminary investigation. Journal of biomedical materials research. Part B, Applied biomaterials Reves, B. T., Bumgardner, J. D., Cole, J. A., Yang, Y., Haggard, W. O. 2009; 90 (1): 1-10

  Abstract

  Lyophilization was evaluated in chitosan-calcium phosphate microspheres and scaffolds to improve drug delivery of growth factors and antibiotics for orthopedic applications. The dual delivery of an antibiotic and a growth factor from a composite scaffold would be beneficial for treatment of complex fracture sites, such as comminuted fractures and segmental bone defects. The aim of this investigation was to increase the loading capacity of the composite by taking advantage of the increased porosity, due to lyophilization, and to produce an extended elution profile using a secondary chitosan-bead coating. The physiochemical properties of the composite were investigated, and loading and elution studies were performed with alkaline phosphatase (ALP), bone morphogenetic protein-2 (BMP-2), and amikacin. Lyophilization was found to increase the surface area of scaffolds by over 400% and the porosity of scaffolds by 50%. Using ALP as a model protein, the loading capacity was increased by lyophilization from 4.3 +/- 2.5 to 24.6 +/- 3.6 microg ALP/mg microspheres, and the elution profile was extended by a supplemental chitosan coating. The loading capacity of BMP-2 for composite microspheres was increased from 74.4 +/- 3.7 to 102.1 +/- 8.0 microg BMP-2/g microspheres with lyophilization compared with nonlyophilized microspheres. The elution profiles of BMP-2 and the antibiotic amikacin were not extended with the supplemental coating. Additional investigations are planned to improve these elution characteristics for growth factors and antibiotics.

  View details for DOI 10.1002/jbm.b.31390

  View details for PubMedID 19441116

• Design and characterization of a novel chitosan/nanocrystalline calcium phosphate composite scaffold for bone regeneration. Journal of biomedical materials research. Part A Chesnutt, B. M., Viano, A. M., Yuan, Y., Yang, Y., Guda, T., Appleford, M. R., Ong, J. L., Haggard, W. O., Bumgardner, J. D. 2009; 88 (2): 491-502

  Abstract

  To meet the challenge of regenerating bone lost to disease or trauma, biodegradable scaffolds are being investigated as a way to regenerate bone without the need for an auto- or allograft. Here, we have developed a novel microsphere-based chitosan/nanocrystalline calcium phosphate (CaP) composite scaffold and investigated its potential compared to plain chitosan scaffolds to be used as a bone graft substitute. Composite and chitosan scaffolds were prepared by fusing microspheres of 500-900 microm in diameter, and porosity, degradation, compressive strength, and cell growth were examined. Both scaffolds had porosities of 33-35% and pore sizes between 100 and 800 . However, composite scaffolds were much rougher and, as a result, had 20 times more surface area/unit mass than chitosan scaffolds. The compressive modulus of hydrated composite scaffolds was significantly higher than chitosan scaffolds (9.29 +/- 0.8 MPa vs. 3.26 +/- 2.5 MPa), and composite scaffolds were tougher and more flexible than what has been reported for other chitosan-CaP composites or CaP scaffolds alone. Using X-ray diffraction, scaffolds were shown to contain partially crystalline hydroxyapatite with a crystallinity of 16.7% +/- 6.8% and crystallite size of 128 +/- 55 nm. Fibronection adsorption was increased on composite scaffolds, and cell attachment was higher on composite scaffolds after 30 min, although attachment rates were similar after 1 h. Osteoblast proliferation (based on dsDNA measurements) was significantly increased after 1 week of culture. These studies have demonstrated that composite scaffolds have mechanical properties and porosity sufficient to support ingrowth of new bone tissue, and cell attachment and proliferation data indicate composite scaffolds are promising for bone regeneration.

  View details for DOI 10.1002/jbm.a.31878

  View details for PubMedID 18306307

• Fabrication and characterization of Functionally Graded Nano-Micro Porous Titanium Surface by Anodizing. J Biomed Mater Res HS Kim, Y Yang, JT Koh, KK Lee, DJ Lee, KM Lee, SW park 2009; 88B: 427-435
• Development of Sputtered Nanoscale Dense Titanium Oxide Coating on Osseointegrated Implant Devices and Biological Evaluation Vacuum Y Yang, Y Liu, Park S, Lee K, Kim, H Koh, H Ji, X Wang, J Ong. 2009; 83: 569-574
• Development of sputtered nanoscale titanium oxide coating on osseointegrated implant devices and their biological evaluation 9th International Symposium on Sputtering and Plasma Processes Yang, Y., Park, S., Liu, Y., Lee, K., Kim, H., Koh, J., Menge, X., Kim, K., Ji, H., Wang, X., Ong, J. L. PERGAMON-ELSEVIER SCIENCE LTD. 2008: 569?74

  View details for DOI 10.1016/j.vacuum.2008.04.062

  View details for Web of Science ID 000262883200028

• Plasma surface modification of poly(D,L-lactic acid) as a tool to enhance protein adsorption and the attachment of different cell types. Journal of biomedical materials research. Part B, Applied biomaterials Alves, C. M., Yang, Y., Marton, D., Carnes, D. L., Ong, J. L., Sylvia, V. L., Dean, D. D., Reis, R. L., Agrawal, C. M. 2008; 87 (1): 59-66

  Abstract

  We have studied the influence of oxygen radio frequency glow discharge (RfGD) on the surface and bulk properties of poly(D,L-lactic acid) (PDLLA) and the effect of this surface modification on both protein adsorption and bone cell behavior. PDLLA films were characterized before and after plasma surface modification by water contact angle, surface energy, and adhesion tension of water as well as by scanning electron microscopy (SEM), X-ray electron spectroscopy (XPS), and Fourier transform infra-red (FTIR) spectroscopy. RfGD-films showed an increase in hydrophilicity and surface energy when compared with untreated films. Surface morphological changes were observed by SEM. Chemical analysis indicated significant differences in both atomic percentages and oxygen functional group. Protein adsorption was evaluated by combining solute depletion and spectroscopic techniques. Bovine serum albumin (BSA), fibronectin (FN), vitronectin (VN), and fetal bovine serum (FBS) were used in this study. RfGD-treated surfaces adsorbed more BSA and FN from single specie solutions than FBS that is a more complex, multi-specie solution. MG63 osteoblast-like cells and primary cultures of fetal rat calvarial (FRC) cells were used to assess both the effect of RfGD treatment and protein adsorption on cell attachment and proliferation. In the absence of preadsorbed proteins, cells could not distinguish between treated and untreated surfaces, with the exception of MG63 cells cultured for longer periods of time. In contrast, the adsorption of proteins increased the cells' preference for treated surfaces, thus indicating a crucial role for adsorbed proteins in mediating the response of osteogenic cells to the RfGD-treated PDLLA surface.

  View details for DOI 10.1002/jbm.b.31068

  View details for PubMedID 18360882

• The effect of titanium surface roughening on protein absorption, cell attachment, and cell spreading INTERNATIONAL JOURNAL OF ORAL & MAXILLOFACIAL IMPLANTS Nishimoto, S. K., Nishimoto, M., Park, S., Lee, K., Kim, H., Koh, J., Ong, J. L., Liu, Y., Yang, Y. 2008; 23 (4): 675-680

  Abstract

  The purpose of this study was to compare properties of roughened and polished titanium with respect to their ability to attach to cells and bind to protein as well as their cell spreading behavior.Three different titanium surface treatments were compared for their ability to support cell attachment and spreading: sandblasted and acid-etched, resorbable blast media, and machine-polished titanium. The surface of the materials was characterized for surface roughness, surface energy, and surface chemistry. Osteoblast-like MG-63 cells were tested for in vitro attachment and spreading in the presence of serum proteins. Cell attachment was assessed by direct counting, dye binding, and microculture titanium assays. Cell spreading was determined by measuring area/cell in phalloidin-AlexaFluor 488 stained cells. Absorption of bovine serum albumin was determined by assay.Scanning electron micrography and x-ray diffractometry confirmed increased surface roughness of the roughened materials. All 3 materials had similar albumin binding kinetics. Three different methods confirmed that roughened surfaces enhance early cell attachment to titanium in the presence of serum. Cells spread better on smoother machined surfaces than on the roughened surfaces.Roughened titanium surfaces exhibited better early cell attachment than smooth surfaces in the presence of serum. The cells attached to roughened titanium were less spread than those attached to machined titanium. Although albumin binding was not different for roughened surfaces, it is possible that roughened surfaces preferentially bound to serum adhesive proteins to promote early cell attachment.

  View details for Web of Science ID 000258914700012

  View details for PubMedID 18807564

• Chitosan-coated stainless steel screws for fixation in contaminated fractures CLINICAL ORTHOPAEDICS AND RELATED RESEARCH Greene, A. H., Bumgardner, J. D., Yang, Y., Moseley, J., Haggard, W. O. 2008; 466 (7): 1699-1704

  Abstract

  Stainless steel screws and other internal fixation devices are used routinely to stabilize bacteria-contaminated bone fractures from multiple injury mechanisms. In this preliminary study, we hypothesize that a chitosan coating either unloaded or loaded with an antibiotic, gentamicin, could lessen or prevent these devices from becoming an initial nidus for infection. The questions investigated for this hypothesis were: (1) how much of the sterilized coating remains on the screw with simulated functional use; (2) is the unloaded or loaded chitosan coating bacteriostatic and biocompatible; and (3) what amount and rate does an antibiotic elute from the coating? In this study, the gentamicin eluted from the coating at a detectable level during 72 to 96 hours. The coating was retained at the 90% level in simulated bone screw fixation and the unloaded and loaded chitosan coatings had encouraging in vitro biocompatibility with fibroblasts and stem cells and were bacteriostatic against at least one strain of Staphylococcus aureus. The use of an antibiotic-loaded chitosan coating on stainless steel bone screws and internal fixation devices in contaminated bone fracture fixation may be considered after optimization of antibiotic loading and elution and more expanded in vitro and in vivo investigations with other organisms and antibiotics.

  View details for DOI 10.1007/s11999-008-0269-5

  View details for Web of Science ID 000256658900027

  View details for PubMedID 18443893

• Novel fabrication of nano-rod array structures on titanium and in vitro cell responses 5th Asian-Australian Conference on Composite Materials (ACCM-5_ Liu, Y., Chen, W., Yang, Y., Ong, J. L., Tsuru, K., Hayakawa, S., Osaka, A. SPRINGER. 2008: 2735?41

  Abstract

  Nano-scale rod arrays of titania were fabricated on titanium surface by a glass phase topotaxy growth (GPT) method, which was featured by an interfacial reaction between sodium tetraborate coating and the preheated metallic titanium at elevated temperature. The samples were characterized by thin-film X-ray diffraction (XRD), scanning electron microscope (SEM), profilometer and contact angle measurement. Thin-film XRD analysis indicated that the nano-rod arrays were composed of pure rutile titania phase. SEM images showed that these rutile rods were 100-200 nm wide and 1-2 microm long. The nano-rod arrays had significantly higher average roughness (P < 0.05) and greater hydrophilicity (P < 0.05) compared to the control. Human embryonic palatal mesenchymal (HEPM) cells were grown to evaluate in vitro cell responses to the nano-rod array structures in terms of cell attachment and proliferation. An equivalent high attachment rate of 94% was observed after 4-h incubation, but a lower proliferation rate was observed on the nano-rod array after 12-day culture compared to the control (P < 0.05).

  View details for DOI 10.1007/s10856-008-3396-3

  View details for Web of Science ID 000255879100027

  View details for PubMedID 18305905

• Characterization of biomimetic calcium phosphate on phosphorylated chitosan films JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A Chesnutt, B. M., Yuan, Y., Brahmandam, N., Yang, Y., Ong, J. L., Haggard, W. O., Bumgardner, J. D. 2007; 82A (2): 343-353

  Abstract

  This study examined the effect of chitosan degree of deacetylation (DDA), concentration of simulated body fluid (SBF), and mineralization time on the composition, structure, and crystallinity of calcium phosphate (CaP) biomimetically deposited on chitosan and on osteoblast cell growth. Phosphorylated chitosan films of 92.3%, 87.4%, and 80.6% DDA were soaked in SBF (1.0x or 1.5x) for 7, 14, or 21 days. Scanning electron microscopy revealed that CaP precipitated from 1.5x SBF had a porous, granular morphology; while the coatings precipitated in 1.0x SBF were smoother and more uniform. X-ray diffraction showed that films mineralized in 1.0x SBF were amorphous, while films mineralized in 1.5x SBF for 21 days exhibited crystalline peaks similar to hydroxyapatite, with the most crystalline peaks seen on 92.3% DDA chitosan. When mineralized films were placed in cell media for 14 days, more calcium phosphate precipitated onto all films, and the most calcium phosphate was found on 92.3% DDA films mineralized in 1.5x SBF. After seven days of osteoblast culture, there were approximately three times as many cells (based on DNA measurements, p < 0.05) on 92.3% DDA films soaked in 1.0x SBF for seven or 21 days than on 80.6% DDA films soaked in 1.0x SBF for any length of time or any films soaked in 1.5x SBF. The DDA of chitosan, concentration of SBF and mineralization time affect the structure of and biological response to chitosan/biomimetic CaP films, and these factors must be considered when designing new materials to be used in orthopaedic and dental/craniofacial implant applications.

  View details for DOI 10.1002/jbm.a.31070

  View details for Web of Science ID 000247836600010

• Microstructure, mechanical properties, and biological response to functionally graded HA coatings Symposium on Next Generation Biomaterials Rabiei, A., Blalock, T., Thomas, B., Cuomo, J., Yang, Y., Ong, J. ELSEVIER SCIENCE BV. 2007: 529?33

  View details for DOI 10.1016/j.msec.2006.05.036

  View details for Web of Science ID 000245130000024

• The integration of chitosan-coated titanium in bone: An in vivo study in rabbits IMPLANT DENTISTRY Bumgardner, J. D., Chesnutt, B. M., Yuan, Y., Yang, Y., Appleford, M., Oh, S., McLaughlin, R., Elder, S. H., Ong, J. L. 2007; 16 (1): 66-79

  Abstract

  Much research is directed at surface modifications to enhance osseointegration of implants. A new potential coating is the biopolymer, chitosan, the deacetylated derivative of the natural polysaccharide, chitin. Chitosan is biocompatible, degradable, nontoxic, and exhibits osteogenic properties. The aim of this research was to investigate the hypothesis that chitosan-coated titanium supports bone formation and osseointegration.Chitosan (1 wt% of 92.3% deacetylated chitosan in 1% acetic acid) was solution cast and bonded to rough ground titanium pins (2-mm diameterx4-mm long) via silane reactions. Calcium phosphate sputter-coated titanium and uncoated titanium pins were used as controls. Two chitosan-coated pins, and 1 each of calcium phosphate coated and uncoated pins were implanted unilaterally in the tibia of 16 adult male New Zealand white rabbits. At 2, 4, 8, and 12 weeks, undecalcified sections were histologically evaluated for healing and bone formation.Histological evaluations of tissues in contact with the chitosan-coated pins indicated minimal inflammatory response and a typical healing sequence of fibrous, woven bone formation, followed by development of lamellar bone. These observations were similar to those for tissues interfacing the control calcium phosphate-coated and uncoated titanium implants. Quantitative comparisons of the bone-implant interface were not possible since 31% of the implants migrated into the tibial marrow space after implantation due to insufficient cortical bone thickness to hold pins in place during healing.These data support the hypothesis that chitosan-coatings are able to develop a close bony apposition or the osseointegration of dental/craniofacial and orthopedic implants.

  View details for DOI 10.1097/ID.0b013e3180312011

  View details for Web of Science ID 000249965300010

  View details for PubMedID 17356373

• Effect of genipen crosslinking on degradation and protein release from chitosan microspheres Carbohydrate polymers Y Yuan, BC Chesnutt, G Utturkar, J Ong, Y Yang, W haggard, J Bumgardner 2007; 68: 561-567
• Modulating bone cells response onto starch-based biomaterials by surface plasma treatment and protein adsorption BIOMATERIALS Alves, C. M., Yang, Y., Carnes, D. L., Ong, J. L., Sylvia, V. L., Dean, D. D., Agrawal, C. M., Reis, R. L. 2007; 28 (2): 307-315

  Abstract

  The effect of oxygen-based radio frequency glow discharge (rfGD) on the surface of different starch-based biomaterials (SBB) and the influence of proteins adsorption on modulating bone-cells behavior was studied. Bovine serum albumin, fibronectin and vitronectin were used in single and complex protein systems. RfGD-treated surfaces showed to increase in hydrophilicity and surface energy when compared to non-modified SBB. Biodegradable polymeric blends of cornstarch with cellulose acetate (SCA; 50/50wt%), ethylene vinyl alcohol (SEVA-C; 50/50wt%) and polycaprolactone (SPCL; 30/70wt%) were studied. SCA and SCA reinforced with 10% hydroxyapatite (HA) showed the highest degree of modification as result of the rfGD treatment. Protein and control solutions were used to incubate with the characterized SBB and, following this, MG63 osteoblast-like osteosarcoma cells were seeded over the surfaces. Cell adhesion and proliferation onto SCA was found to be enhanced for non-treated surfaces and on SCA+10%HA no alteration was brought up by the plasma modification. Onto SCA surfaces, BSA, FN and VN single solutions improved cell adhesion, and this same effect was found upscaled for ternary systems. In addition, plasma treated SEVA-C directed an increase in both adhesion and proliferation comparing to non-treated surfaces. Even though adhesion onto treated and untreated SPCL was quite similar, plasma modification clearly promoted MG63 cells proliferation. Regarding MG63 cells morphology it was shown that onto SEVA-C surfaces the variation of cell shape was primarily defined by the protein system, while onto SPCL it was mainly affected by the plasma treatment.

  View details for DOI 10.1016/j.biomaterials.2006.09.010

  View details for Web of Science ID 000242310500018

  View details for PubMedID 17011619

• Bone cell attachment and growth on well-characterized chitosan films Polymer International V Hamilton, Y Yuan, DA Rigney, BM Chesnutt, AD Puckett, JL Ong, Y Yang, WO Haggard, SH Elder, JD Bumgardner 2007; 56: 641-647
• Microstructure, mechanical properties, and biological response to functionally graded HA coatings Mater Sci and Eng Afsaneh Rabiei, Travis Blalock, Brent Thomas, Jerry Cuomo, Y. Yang, Joo Ong 2007; 27C: 529-533
• Characterization of chitosan films and effects on fibroblast cell attachment and proliferation 10th International Conference on Polymers in Medicine and Surgery (PIMS2004) Hamilton, V., Yuan, Y., Rigney, D. A., Puckett, A. D., Ong, J. L., Yang, Y., Elder, S. H., Bumgardner, J. D. SPRINGER. 2006: 1373?81

  Abstract

  Chitosan has been researched for implant and wound healing applications. However, there are inconsistencies in reports on the tissue and fibroblast responses to chitosan materials. These inconsistencies may be due to variations in chitosan material characteristics. The aim of this study was to correlate fibroblast responses with known chitosan material characteristics. To achieve this aim, chitosan was characterized for degree of deacetylation (DDA), molecular weight (MW), residual protein and ash contents, and then solution cast into films and characterized for hydrophilicity by water contact angle. The films were seeded with normal human dermal fibroblasts and the number of attached cells was evaluated for after 30 min. Cell proliferation was evaluated over 5 days. This study found no relationship between DDA, contact angle, cell attachment, and or proliferation. General trends were observed for increasing proliferation with increasing residual ash content and decreasing residual protein. These data indicate that chitosan characteristics other than DDA may be important to their biological performance.

  View details for DOI 10.1007/s10856-006-0613-9

  View details for Web of Science ID 000242511100023

  View details for PubMedID 17143770

• Enhancement of osseointegration using surface modified titanium implants Journal of Minerals, Metals, and Materials Yang, Y., N. Oh, Y. Liu, W. Chen, S. Oh, M. Appleford, S. Kim, K. Kim, S. Park, J. Bumgardner, W. Haggard, J Ong 2006; 58: 71-76
• Preparation and characterization of anodized titanium surfaces and their effect on osteoblast responses. journal of oral implantology Kim, K., Kwon, T., Kim, S., Kang, I., Kim, S., Yang, Y., Ong, J. L. 2006; 32 (1): 8-13

  Abstract

  In this study, titanium (Ti) surface was modified by anodizing with a mixture of beta-glycerophosphate sodium and calcium (Ca) acetate, and the anodized surfaces were characterized by scanning electron microscopy, X-ray diffraction, and electron probe microanalysis. In vitro osteoblast response to anodized oxide was also evaluated. The anodic oxide produced was observed to have interconnected pores (0.5-2 microm in diameter) and intermediate roughness (0.60-1.00 microm). In addition, anodic oxide was observed to have amorphous and anatase oxide. Calcium and phosphorus ions were deposited on the Ti oxide during anodization. Osteoblast differentiation, as indicated by alkaline phosphatase production, was enhanced on anodized surfaces. It was thus concluded from this study that Ca phosphate can be deposited on Ti surfaces by anodization. It was also concluded that the phenotypic expression of osteoblast was enhanced by the presence of Ca phosphate and higher roughness on anodized Ti surfaces.

  View details for PubMedID 16526576

• Characterization and development of bioactive hydroxyapatite coatings. J ournal of Minerals, Metals, and Materials J. Ong, M. Appleford, S. Oh, Yang, Y., W. Chen, J.D. Bumgardner, W. Haggard. 2006; 58: 67-70
• Enhancement of osseointegration using surface modified titanium implants Journal of Minerals, Metals, and Materials Yang, Y., N. Oh, Y. Liu, W. Chen, S. Oh, M. Appleford, S. Kim, K. Kim, S. Park, J. Bumgardner, W. Haggard, J Ong 2006; 58: 71-76
• A study on functionally graded HA coatings processed using ion beam assisted deposition with in-situ heat treatment Surface and coating technology Rabiei A, Narayan TR, Cuomo, J, Yang, Y, Ong, J.L. 2006; 200: 6111-6116
• Effects of applied voltages on hydroxyapatite coating on titanium by electrophoretic deposition J Biomed Mater Res Meng X.,, Kwon, T.; Yang, Y.; Ong, J.L.; Kim, K. 2006; 78B: 373-377
• Enhancement of osseointegration using surface modified titanium implants Journal of Minerals, Metals, and Materials Yang, Y., N. Oh, Y. Liu, W. Chen, S. Oh, M. Appleford, S. Kim, K. Kim, S. Park, J. Bumgardner, W. Haggard, J Ong 2006; 58: 71-76
• The effect of sputtered calcium phosphate coatings of difference crystallinity on osteoblast differentiation JOURNAL OF PERIODONTOLOGY Berube, P., Yang, Y. Z., Carnes, D. L., Stover, R. E., Boland, E. J., Ong, J. L. 2005; 76 (10): 1697-1709

  Abstract

  Coating titanium implants with hydroxyapatite (HA) has been suggested to increase osseointegration by stimulating early osteoblast function. The goal of this study was to determine the extent to which the crystalline content of the HA surface affected osteoblast function in vitro.Osteoblasts were isolated from fetal rat calvaria. Titanium coupons were sputter coated and analyzed. Mineralized nodule formation on plastic using von Kossa staining was compared to tetracycline and procion dye labeling. Cell proliferation, adhesion, alkaline phosphatase activity, morphology and spreading, and cytoskeletal arrangement were analyzed. Reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the expression of mRNA for specific proteins.The percent crystallinity of coatings was 0% (HA1), 1.9% +/- 0.4% (HA2), and 66.4% +/- 2.8% (HA3). The nodule formation and cell number were greatest on titanium and HA3 compared to HA1 and HA2 (P < 0.01). At weeks 2 to 4, all samples showed strong alkaline phosphatase, osteocalcin, monocyte-colony stimulating factor (M-CSF), and receptor activator of nuclear factor kappa B ligand (RANKL) expression, but the specific activity of alkaline phosphatase decreased. Cell adherence was greater than 60% of applied cells for all surfaces except HA3. The cells were significantly more elongated on titanium, with no difference on the HA-coated surfaces. Actin filaments were arranged peripherally at 5 hours but arranged parallel to the long axis of the cell at 20 hours.Procion labeling is a valid method for evaluating mineralized nodule formation on opaque surfaces. There were no major differences in osteoblast function using titanium or high-crystalline coatings, and most functions were decreased on amorphous or low-crystalline coatings.

  View details for Web of Science ID 000232511500011

  View details for PubMedID 16253092

• In vivo evaluation of hydroxyapatite coatings of different crystallinities INTERNATIONAL JOURNAL OF ORAL & MAXILLOFACIAL IMPLANTS Oh, S., Tobin, E., Yang, Y. Z., Carnes, P. L., Ong, J. L. 2005; 20 (5): 726-731

  Abstract

  The influence of calcium phosphate (CaP) and hydroxyapatite (HA) crystallinity on bone-implant osseointegration is not well established. In this study, the effect of HA crystallinity and coating method on bone-implant osseointegration was investigated using a rat tibia model.HA coatings 1 to 5 microm thick were produced using a supersonic particle acceleration (SPA) technology. The HA crystallinities used for this study were weight ratios of 30%, 50%, 70%, and 90%. A total of 128 HA-coated implants were placed into the tibiae of 64 male Sprague-Dawley rats. Bone-implant interfaces were evaluated using histology and push-out strength testing at 3 and 9 weeks after implantation.The 70% crystalline coatings exhibited significantly greater interfacial strength (5 implants/time point/treatment) than the 30%, 50%, and 90% crystalline coatings at 3 and 9 weeks following implantation. The implants with coatings of 70% crystallinity also had the greatest bone contact length. In addition, the HA coatings produced with SPA demonstrated greater interfacial strength and bone contact length than plasma-sprayed HA coatings (except for the HA coating with 30% crystallinity).HA coatings of different crystallinities exhibited different dissolution and re-precipitation properties which may enhance early bone formation and bone bonding.This study suggested that coating crystallinity and coating methods can influence the bone-implant interface.

  View details for Web of Science ID 000232455400008

  View details for PubMedID 16274146

• Effect of heat-treated titanium surfaces on protein adsorption and osteoblast precursor cell initial attachment. Implant dentistry Kern, T., Yang, Y., Glover, R., Ong, J. L. 2005; 14 (1): 70-76

  Abstract

  The clinical success of dental implants is governed in part by surface properties of implants and their interactions with the surrounding tissues. The objective of this study was to investigate the effect of heat-treated titanium surfaces on protein adsorption and osteoblast precursor cell attachment in vitro. Passivated titanium samples used in this study were either non heat treated or heat treated at 750 degrees C for 90 minutes. It was observed that the contact angle on heat-treated titanium surfaces was statistically lower compared with the non-heat-treated titanium surfaces. The non-heat-treated titanium surface was also observed to be amorphous oxide, whereas heat treatment of titanium resulted in the conversion of amorphous oxide to crystalline anatase oxide. No significant difference in albumin and fibronectin adsorption was observed between the heat-treated and non-heat-treated titanium surfaces. In addition, no significant difference in initial cell attachment was observed between the two groups. It was concluded that heat treatment of titanium resulted in significantly more hydrophilic surfaces compared to non-heat-treated titanium surfaces. However, differences in oxide crystallinity and wettability were not observed to affect protein adsorption and initial osteoblast precursor cell attachment.

  View details for PubMedID 15764948

• Protein adsorption and osteoblast precursor cell attachment to hydroxyapatite of different crystallinities INTERNATIONAL JOURNAL OF ORAL & MAXILLOFACIAL IMPLANTS Yang, Y. Z., Dennison, D., Ong, J. L. 2005; 20 (2): 187-192

  Abstract

  The effect of hydroxyapatite (HA) crystallinity on protein adsorption and osteoblast precursor cell attachment to HA was investigated.Different weight ratios of 100% crystalline HA and 100% amorphous calcium phosphate powders were mixed and pressed into disks (0.5 g) of different crystallinities--either 0% (HAO), 30% (HA30), 50% (HA50), 70% (HA70), or 100% (HA100).X-ray diffraction indicated differences in HA crystallinities. In addition, dissolution of the HA was dependent on its crystallinity, with an increase in phosphorus dissolution as the degree of crystallinity was decreased. No significant difference in albumin adsorption and initial osteoblast precursor cell attachment was observed in the range of HA0 to HA70 surfaces. However, a significantly lower albumin adsorption and initial osteoblast precursor cell attachment were observed on HA100.It was suggested that changes in ionic interactions as a result of a change in crystallinity affect the amount of calcium ion ligands readily available to electrostatically bind to proteins.It was thus concluded from this study that HA crystallinity affects the amount of albumin adsorbed and initial osteoblast attachment.

  View details for Web of Science ID 000228312500003

  View details for PubMedID 15839111

• Review on calcium phosphate coatings produced using a sputtering process - an alternative to plasma spraying BIOMATERIALS Yang, Y. Z., Kim, K. H., Ong, J. L. 2005; 26 (3): 327-337

  Abstract

  New promising techniques for depositing hydroxyapatite (HA) and calcium phosphate (CaP) coatings on medical devices are continuously being investigated. Given the vast number of experimental deposition process currently available, this review will focus only on CaP and/or HA coatings produced using the sputtering process. This review will discuss the characterization of sputtered CaP coatings before and after post-deposition treatments and tissue responses to some of the characterized coating surfaces. From the studies observed in the literature, current research on sputtered CaP coatings has shown some promises that may eliminate some of the problems associated with the plasma-spraying process. It has been generally accepted that sputtered HA and CaP coatings improve bone strength and initial osseointegration rate. However, optimal coating properties required to achieve maximal bone response are yet to be reported. As such, the use of well-characterized sputtered CaP and/or HA surfaces in the evaluation of biological responses should be well documented to avoid controversial results. In addition, future investigations of the sputtering process should include clinical trials, to continue the understanding of bone responses to coated-implant surfaces of different properties, and the possibility of coupling sputtered HA and CaP coatings with growth factors.

  View details for DOI 10.1016/j.biomaterials.2004.02.029

  View details for Web of Science ID 000224014400010

  View details for PubMedID 15262475

• Development of hydroxyapatite thin film on titanium substrate by electrophoretic deposition 17th International Symposium on Ceramics in Medicine You, C. K., Meng, X. W., Kwon, T. Y., Yang, Y. Z., Ong, J. L., Kim, S., Kim, K. H. TRANS TECH PUBLICATIONS LTD. 2005: 901?904

  View details for Web of Science ID 000228359500220

• Ectopic osteoinduction and eraly degradation of recombinant human bone morphogenetic protein-2 loaded porous beta-tricalcium phosphate in mice Biomaterials Liang, G., Yang, Y. Oh, S, Ong, J.L., Zheng, C, Ran, J, Yin, G, Zhou, D. 2005; 26 (20): 4265-4271
• Effects of dissolved calcium and phosphorous on osteoblast responses. journal of oral implantology Ma, S., Yang, Y., Carnes, D. L., Kim, K., Park, S., Oh, S. H., Ong, J. L. 2005; 31 (2): 61-67

  Abstract

  The dissolution behavior of hydroxyapatite (HA) and its effect on the initial cellular response is of both fundamental and clinical importance. In this study, plasma-sprayed HA coatings were characterized by X-ray diffraction and Fourier transform infrared spectroscopy (FTIR). Calcium (Ca) and inorganic phosphorous (Pi) ions released from plasma-sprayed HA coatings within 3 weeks were measured by flame atomic absorption and colorimetrically molybdenum blue complex, respectively. To investigate the effect of dissolution of HA coatings on osteoblast response, additional Ca and Pi were added into the cell culture media to simulate the dissolution concentrations. Human embryonic palatal mesenchyme cells, an osteoblast precursor cell line, were used to evaluate the biological responses to enhanced Ca and Pi media over 2 weeks. Osteoblast differentiation and mineralization were measured by alkaline phosphatase-specific assay and 1,25 (OH)2 vitamin D3 stimulated osteocalcin production. The coatings exhibited an HA-type structure. FTIR indicated the possible presence of carbonates on the coatings. A dissolution study indicated a continual increase in Ca and Pi over time. In the cell culture study, enhanced osteoblast differentiation occurred in the presence of additional Ca concentration in the cell culture media. However, additional Pi concentration in the cell culture media was suggested to slow down osteoblast differentiation and mineralization.

  View details for PubMedID 15871524

• Interaction of hydroxyapatite-titanium at elevated temperature in vacuum environment BIOMATERIALS Yang, Y. Z., Kim, K. H., Agrawal, C. M., Ong, J. L. 2004; 25 (15): 2927-2932

  Abstract

  In this study, the interaction between hydroxyapatite (HA) and titanium (Ti) at elevated temperature in vacuum environment was investigated. The 80 wt% HA-20 wt% Ti powder mixtures and 90 wt% HA-10 wt% Ti powder mixtures were dry pressed and heat-treated at 1100 degrees C in vacuum environment. HA powders and the commercially pure Ti powders were used as controls. The heat-treated samples were characterized using X-ray diffraction (XRD), Fourier transform infrared spectroscopy, scanning electron microscope (SEM) and energy disperse spectra. XRD and SEM indicated densification of metallic Ti specimens during the in-vacuum heat treatment. Heat treatment of HA specimens in vacuum resulted in the loss of hydroxyl groups as well the formation of a secondary beta-tricalcium phosphate phase. Metallic Ti was not observed in the in-vacuum heat-treated HA-Ti specimens. However, alpha-tricalcium phosphate, tetracalcium phosphate and calcium titanium oxide were observed for the in-vacuum heat-treated HA-Ti specimens. It was concluded that the in-vacuum heat-treatment process completely converted the metal-ceramics composites to ceramic composites.

  View details for DOI 10.1016/j.biomaterials.2003.09.072

  View details for Web of Science ID 000189221600003

  View details for PubMedID 14967524

• Protein adsorption on titanium surfaces and their effect on osteoblast attachment. Journal of biomedical materials research. Part A Yang, Y., Cavin, R., Ong, J. L. 2003; 67 (1): 344-349

  Abstract

  The objective of this study was to investigate the adsorption of albumin and fibronectin on titanium (Ti) surfaces and the effect of preadsorbed albumin and fibronectin on osteoblast attachment in vitro. Bovine serum albumin and bovine fibronectin were used in this study. Maximum adsorption of bovine serum albumin and fibronectin on Ti surfaces was observed to occur after 180-min incubation. In the presence of preadsorbed proteins, osteoblast attachment on Ti surfaces was observed to be enhanced compared to control Ti surfaces. However, cell attachment was affected by the types of protein adsorbed. Preadsorbed albumin was observed to have no significant effect on the amount of osteoblast cells attached. In comparison to control Ti surface and Ti surfaces preadsorbed with albumin, Ti surfaces preadsorbed with fibronectin for 15 min was observed to significantly increase osteoblast cell attachment, whereas Ti surfaces preadsorbed with fibronectin for 180 min did not affect cell attachment. In addition, cell morphology of the attached cells on protein preadsorbed Ti surfaces was not affected by the type of protein used in this study. It was concluded from this study that the concentration of fibronectin adsorbed on Ti surfaces was higher compared to albumin. In addition, it was also concluded that the concentration of fibronectin on Ti surfaces plays a role in governing cell attachment.

  View details for PubMedID 14517894

• Influence of post-deposition heating time and the presence of water vapor on sputter-coated calcium phosphate crystallinity JOURNAL OF DENTAL RESEARCH Yang, Y., Kim, K. H., Agrawal, C. M., Ong, J. L. 2003; 82 (10): 833-837

  Abstract

  Extensive research suggested that calcium phosphate (CaP) coatings on titanium implants are essential for early bone response. However, the characterization of CaP crystallinity and the means to control coating crystallinity are not well-established. In this study, the effect of a 400 degrees C heat treatment for 1, 2, or 4 hours, and in the presence or absence of water vapor, on CaP crystallinity was investigated. Scanning electron microscopy indicated dense as-sputtered coatings. Increase in coating crystallinity was observed to be consistent with the increasing number of PO(4) peaks observed as a result of different heat treatments. In addition, x-ray diffraction analyses indicated amorphous as-sputtered coatings, whereas crystalline CaP coatings in the range of 0-85% were observed after different post-deposition heat treatments. It was concluded that the presence of water vapor and post-deposition heat treatment time significantly affect the crystallinity of CaP coatings, which may ultimately affect bone healing.

  View details for Web of Science ID 000185608500014

  View details for PubMedID 14514766

• Osteoblast precursor cell attachment on heat-treated calcium phosphate coatings JOURNAL OF DENTAL RESEARCH Yang, Y., Bumgardner, J. D., Cavin, R., Carnes, D. L., Ong, J. L. 2003; 82 (6): 449-453

  Abstract

  The influence of properties of calcium phosphate (CaP) coatings on bone cell activity and bone-implant osseointegration is not well-established. This study investigated the effects of characterized CaP coatings of various heat treatments on osteoblast response. It was hypothesized that heat treatments of CaP coatings alter the initial osteoblast attachment. The 400 degrees C heat-treated coatings were observed to exhibit poor crystallinity and significantly greater phosphate or apatite species compared with as-sputtered and 600 degrees C heat-treated coatings. Similarly, human embryonic palatal mesenchyme (HEPM) cells, an osteoblast precursor cell line, seeded on 400 degrees C heat-treated coatings, exhibited significantly greater cell attachment compared with Ti surfaces, as-sputtered coatings, and 600 degrees C heat-treated coatings. The HEPM cells on Ti surfaces and heat-treated coatings were observed to attach through filopodia, and underwent cell division, whereas the cells on as-sputtered coatings displayed fewer filopodia extensions and cell damage. Analysis of the data suggested that heat treatment of CaP coatings affects cell attachment.

  View details for Web of Science ID 000183066700009

  View details for PubMedID 12766197

• Deposition of highly adhesive ZrO2 coating on Ti and CoCrMo implant materials using plasma spraying BIOMATERIALS Yang, Y. Z., Ong, J. L., Tian, J. 2003; 24 (4): 619-627

  Abstract

  ZrO(2) (4% CeO(2)) and ZrO(2) (3% Y(2)O(3)) coatings were deposited on titanium (Ti) and CoCrMo implants using plasma spraying and the adhesive, morphological and structural properties of the plasma-sprayed coatings were evaluated. Characterization of these coatings was performed using X-ray diffraction (XRD), scanning electron microscopy (SEM), surface roughness, hardness, and adhesive strength. XRD patterns showed that both the coatings appeared to be primitive tetragonal phase. SEM observations showed that both the ZrO(2) coatings appeared to be rough, porous and melted. The cross-section surface morphology of the coatings, coating-substrate interfaces and substrates without acid etching was very dense and smooth. After acid etching, as compared to the dense ZrO(2) coating-CoCrMo substrate interfaces, the thin gaps appeared within the ZrO(2) coating-Ti substrate interfaces. It is suggested that plasma spraying probably formed an amorphous Ti layer in the coating-Ti substrate interface that can be removed by acid etching. The average surface roughness of ZrO(2) (3% Y(2)O(3)) and ZrO(2) (4% CeO(2)) coatings was correlated to the starting powder size and substrates. No significant difference between the hardness of all coatings and substrates was observed. The adhesive strengths of ZrO(2) (4% CeO(2)) coating to Ti and CoCrMo substrates were higher than 68MPa and significantly greater than that of ZrO(2) (3% Y(2)O(3)) coatings.

  View details for Web of Science ID 000179561600010

  View details for PubMedID 12437956

• Contact angle, protein adsorption, and osteoblast attachment to chitosan coatings bonded to titanium J Biomater Sci: Polymer Edition JD Bumgardner, R Jouett, Y Yang, JL Ong 2003; 14: 1401-1410
• Fibronectin adsorption on titanium surfaces and its effect on osteoblast precursor cell attachment Colloids and Surfaces B: Interfaces Yang, Y., Glover, R., Ong, J.L. 2003; 30B: 291-297
• Bond strength, compositional and structural properties of HA coating on Ti substrate, ZrO2-coated Ti substrate, and TPS-coated Ti substrate J Biomed Mater Res Yang, Y., Ong, J.L. 2003; 64A: 509-516
• In vivo evaluation of modified titanium implant surfaces produced using a hybrid plasma spraying processing International Conference on Materials for Advanced Technologies (ICMAT2001) Yang, Y. Z., Ong, J. L., Tian, J. M. ELSEVIER SCIENCE BV. 2002: 117?24

  View details for Web of Science ID 000176088200017

• Morphological behavior of osteoblast-like cells on surface-modified titanium in vitro BIOMATERIALS Yang, Y. Z., Tian, J. M., Deng, L., Ong, J. L. 2002; 23 (5): 1383-1389

  Abstract

  In recent papers, we reported the results of a study on the graded porous titanium coatings on titanium by plasma spraying and amino-group ion implantation. The paper is to preliminarily evaluate the biocompatibility of surface-modified titanium through 2, 5 and 7 days cell culture in vitro. Cell morphology was observed by a scanning electron microscope. Cell proliferation and type I collagen synthesis were measured by 3(4.5-dimethyl-thiazole-2-yl)2,5-diphenyl tetrazolium bromide (MTT) and enzyme-linked immunosorbent assay (ELISA), respectively. Our experimental results showed that osteoblast-like cells attached and spread well on surface-modified titanium. Cells were observed to grow into the pores and form extracellular matrix. MTT and ELISA results showed no detrimental effect on the development of cell. These studies support the biocompatibility of surface-modified titanium.

  View details for Web of Science ID 000173220100013

  View details for PubMedID 11804294

• Rapid sintering of hydroxyapatite by microwave processing Journal of Materials Science Letter Yang, Y., J.L. Ong, J. Tian 2002; 21: 67-69
• In vivo evaluation of modified titanium implant surfaces using a hybrid plasma sparying processing Mater Sci and Eng Yang, Y., J.L. Ong, J. Tian 2002; 120C: 117-124
• Surface modification of titanium through amino group implantation JOURNAL OF BIOMEDICAL MATERIALS RESEARCH Yang, Y. Z., Tian, J. M., Tian, J. T., Chen, Z. Q. 2001; 55 (3): 442-444

  Abstract

  We modified Ti surfaces by implantation of amino (NH(2+)) groups at 10(16) and 10(17) cm(-2). The implanted surfaces were characterized by X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), scanning Auger electron spectroscopy (AES), and second ion mass spectroscopy (SIMS). The experimental results showed that the implanted Ti specimens were covered by a dominant hydrocarbon overlayer due to contamination and the surface oxide layer of implanted specimens became thicker. XPS, AES, and SIMS depth profiles showed that implanted elements had a typical ion implantation distribution and that titanium nitride (TiN) was formed.

  View details for Web of Science ID 000167677200022

  View details for PubMedID 11255199

• Preparation of graded porous titanium coatings on titanium implant materials by plasma spraying JOURNAL OF BIOMEDICAL MATERIALS RESEARCH Yang, Y. Z., Tian, J. M., Tian, J. T., Chen, Z. Q., Deng, X. J., Zhang, D. H. 2000; 52 (2): 333-337

  Abstract

  Graded porous titanium coatings have been deposited on titanium substrates for dental implants by plasma spraying in an argon atmosphere. X-ray diffraction (XRD), scanning electron microscopy (SEM), surface roughness measurement, and tensile strength tests were performed on graded porous coatings. The results showed that Ti(3)O(5) was formed in the outermost surface of the porous coatings due to oxidation. The graded porous coatings consisted of three layers. The outer layer was full of macropores with a surface roughness of approximately 100 microm. The diameter of many macropores reached and even surpassed 150 microm, which could be beneficial for tissue to grow into the coating. The middle layer consisted of a mixture of micropores and macropores. The inner layer was a very dense and tight interface layer that included mechanical, physical, and metallurgical bonding. In tensile strength tests, testing bars peeled off the coatings, because the adhesive agent fractured, but the coatings remained intact.

  View details for Web of Science ID 000088812900012

  View details for PubMedID 10951372