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Chemical Cross-linking and Incorporation of Minerals in Collagen-GAG Scaffolds Enhance In Vitro Osteogenesis
Clifford Thomas Pereira, MD; Weibiao Huang, PhD; David Bischoff, PhD; George Rudkin, MD; Dean Yamaguchi, MD, PhD; Timothy Miller, MD; UCLA
UCLA, Redondo Beach, CA, USA

Introduction:Tissue-engineered bone is emerging as an exciting alternative to heal critical bone defects in post-traumatic and post-oncologic bone resection in hand surgery without the inherent limitations of currently used autologous bone grafts. Development of an ideal tissue engineered bone graft requires efficient bioactivity screening of biomaterials in clinically relevant three-dimensional systems. We evaluated the structural strength and bone formation of chemical cross linkage and mineralized stabilization of collagen scaffolds.

Methods:Human mesenchymal stem cells (HMSCs) were seeded onto three-dimensional non-mineralized (C-GAG) and mineralized (MC-GAG), non-mineralized cross-linked (XC-GAG) and mineralized cross-linked (XMC-GAG) type I collagen scaffolds; incubated in osteogenic media; and harvested at 1, 4, 7, and 14 days. Some cultures were maintained for 7 weeks to study long-term in vitro osteogenesis. Messenger RNA expression was analyzed using quantitative real-time reverse-transcriptase polymerase chain reaction for osteogenic markers i.e. osteocalcin (OCN) and bone sialoprotein (BSP). Structural integrity was measured by the ability of the scaffolds to maintain their original dimensions. Mineralization in long term cultures was detected by alizarin red staining and micro-computed tomographic imaging at 7 weeks. Data was analyzed using Microsoft Office Excel 2007.

Results:We noted a significant temporal upregulation of OCN (207% and 509% respectively) and BSP (162% and 223% respectively) in both MC-GAG and X-MC-GAG compared to their C-GAG counterparts (p<0.05). X-linking of C-GAG and MC-GAG greatly enhanced structural integrity of both scaffolds, decreasing contraction to 68.8% in XC-GAG and 71.9% in XMC-GAG compared to original size (p<0.05). XMC-GAG was the most favorable matrix for in vitro osteogenesis in hMSCs as detected by microCT (250 fold increase, p<0.05) and Alizarin Red staining. It allowed hMSCs to evenly deposit minerals within a structure that was well maintained throughout the entire test period.

Conclusions:Our current data validates previous studies proving improved structural integrity with chemical cross-linking. Mineralization on its own is not sufficient to maintain scaffold configuration. However in conjunction with cross-linking, mineralization acts synergistically to cause an additive increase in maintaining 3D scaffold integrity, and may be the best combination for enhanced cell adhesion, differentiation and mineralization.

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