Interaction of Bone Cells With Biomimetic Hydroxyapatite Gelatin Nanocomposites Towards Developing Bone Tissue Engineering

SIGNIFICANCE: Bone tissue engineering provides an alternative to restore the complex morphologic features and functions of bone during repair of large bone defects. The biomaterials investigated in bone tissue engineering to date have their inherent disadvantages. An ideal scaffold biomaterial remains to be developed.

OBJECTIVES: A novel biomimetic hydroxyapatite/gelatin nanocomposite (HAP/GEL) was developed to be a potential scaffold material. The objective is; therefore, to investigate the interaction between bone cells and the HAP/GEL in vitro and in vivo. This dissertation lays the groundwork and provides the protocols for the future development of this HAP/GEL nanocomposite as a scaffold for bone tissue engineering.

MATERIALS AND METHODS: The human fetal osteoblastic cell line was used to assess cell morphology, cell spreading, cell growth and cell differentiation on HAP/GEL in vitro by using techniques of confocal laser scanning microscopy, PicoGreen DNA assay, and real-time PCR. The drill-hole defects were created in rat femurs to examine the biocompatibility, osteoconductivity, and biodegradation of HAP/GEL by using the techniques of fluorochrome labeling, histology, histomorphometry, and immunohstochemistry.

RESULTS: In vitro: Cells adhered on HAP/GEL appeared more elongated spindle shape but exhibited same degree of cell spreading compared to cells adhered on hydroxyapatite (HA) or glass. The number of cells grown on the HAP/GEL or HA increased over time, but at a slower rate than that increased on glass. The gene expression of alkaline phosphatase, bone sialoprotein, osteopontin, or osteocalcin was similar for cells grown on HAP/GEL, HA, or glass. Type I collagen expression was lower for cells grown on HAP/GEL. In vivo: Osteoblastic activity and new bone formation occurred on the surface of HAP/GEL. There was a trend that HAP/GEL may enhance bone formation in defect walls at two weeks postopertiavely. The osteoconductivity of HAP/GEL increased over time during eight weeks of healing period. Foreign body reaction was found at the early stage. Osteoclasts were observed on the surface of HAP/GEL.

CONCLUSIONS: HAP/GEL provides a satisfactory surface for cell spreading, cell growth, and cell differentiation in vitro. HAP/GEL supports the recruitment of the osteogenic cells and osteogenesis in vivo. Osteoclasts may participate in the biodegradation of HAP/GEL.

Year	Entry
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Year

Entry

1998

Young RG, Butler DL, Weber W, Caplan AI, Gordon SL, Fink DJ. Use of mesenchymal stem cells in a collagen matrix for achilles tendon repair. J Orthop Res. July 1998;16(4):406-413.

2004

Simmons CA, Alsberg E, Hsiong S, Kim WJ, Mooney DJ. Dual growth factor delivery and controlled scaffold degradation enhance in vivo bone formation by transplanted bone marrow stromal cells. Bone. August 2004;35(2):562-569.

1994

Roach HI. Why does bone matrix contain non‐collagenous proteins? the possible roles of osteocalcin, osteonectin, osteopontin and bone sialoprotein in bone mineralisation and resorption. Cell Biol Int. 1994;18(6):617-628.

2002

Zhang M, Xuan S, Bouxsein ML, von Stechow D, Akeno N, Faugere MC, Malluche H, Zhao G, Rosen CJ, Efstratiadis A, Clemens TL. Osteoblast-specific knockout of the insulin-like growth factor (IGF) receptor gene reveals an essential role of IGF signaling in bone matrix mineralization. J Biol Chem. November 15, 2002;277(46):44005-44012.

2002

Lieberman JR, Daluiski A, Einhorn TA. The role of growth factors in the repair of bone: biology and clinical applications. J Bone Joint Surg. June 2002;84A(6):1032-1044.