In Vitro and In Vivo Tests of Hydroxyapatite-Gleatin Nanocomposites For Bone Regeneration: A Preliminary Report.
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In Vitro And In Vivo Tests Of Hydroxyapatite-Gleatin Nanocomposites For Bone Regeneration: A Preliminary Report. Ko CC1, Wu Y-L1, Douglas WH1, Narayanan R2, Hu W-S2 1
Minnesota Dental Research Center for Biomaterials and Biomechanics Department of Oral Science, Minneapolis, MN 55455 2 Department of Chemical Engineering & Materials Sciences, University of Minnesota, Minneapolis, MN 55455 ABSTRACT A biomimetic process has been developed to fabricate hydroxyapatite-gelatin (HAP-GEL) nanocomposites for bone regeneration (Chang and Ko et al. 2003). We hypothesize that this newly developed HAP-GEL is osteoconductive and is suitable for tissue engineered scaffolds. This preliminary study is aimed to characterize cell affinity and osseous regeneration of the HAP-GEL. The HAP-GEL was synthesized according to the procedures described in the previous publication. The attachment and proliferation of human fetal osteoblasts on HAP-GEL discs were evaluated using three different gelatin contents (2g, 3g, and 4g). The cells were seeded onto each disc and incubated at 34 degrees Celsius in 5% CO2 air atmosphere. At different time points of cultivation, cells were stained with fluorescein diacetate (FDA) and ethidium bromide (EB) to determine their viability and morphology. To assess the cell proliferation, cells were detached at Days 1, 4, and 7 by trypsinzation and counted. For in vivo tests, HAP-GEL rods were implanted into the proximal femur of Sprague-Dawley rats. One month after the implantation, the femurs were harvested and the undecalcified HAP-GEL-bone sections were stained for histopathology. Four hours after attachment, most cells appeared round in all discs; cell spreading was observed after 24 hours. The highest gelatin content supported a significantly higher cell growth than the others at 7 days. Thus all compositions support satisfactory attachment, spreading and growth. In vivo results showed excellent interfacial bone regeneration. No necrotic tissues were found. In conclusion, the HAP-GEL not only mimics the biochemistry and nanostructures of bone but also supports the attachment, proliferation and differentiation (bone formation) of osteoblasts. The HAP-GEL we developed provides a suitable surface for regeneration. INTRODUCTION Bone regeneration is challenging and various biomaterials have been demonstrated to successfully carry cells to form reparative constructs in cell cultures or in in vivo subcutaneous transplantation (Yoshikawa et al. 1996, Ellis & Yannas 1996, Behravesh & Mikos 2003). Such tissue engineering approaches represent an emerging area of regenerative medicine in hard tissue replacement. However, restoring the complex morphologic features and function for the large bony defects remains a difficult task, due to the limited available biomaterials.
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A hydroxyapatite-gelatin nanocomposite (HAP-GEL), fabricated via a biomimetic process (Chang and Ko et al., 2003), possesses elastic properties and compressive strengths equivalent to those of natural bone. The physical properties of
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