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Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_XXXVIII | Pages 3 - 3
1 Sep 2012
Sarfati D Gao C Waly F Henderson J
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Purpose

Up to 70% of the differences in human bone mass have been attributed to genetic background. These differences are associated with alterations in the biomechanical properties, micro-architecture and remodeling of bone as well as its susceptibility to fracture and its capacity for repair. In previous work it was shown that C57Bl6 mice carrying one copy of the parathyroid hormone related protein (PTHrP+/−) gene developed osteopenia by four months of age. The current study was designed to determine if the haploinsufficient phenotype was maintained on a C3H background.

Method

PTHrP+/+ and PTHrP+/− mice on C57Bl6 and C3H backgrounds were euthanised between 6 and 18 months of age. The femurs were harvested, fixed in 4% paraformaldehyde overnight and scanned on a Skyscan 1172 equipped with a 10kV X-ray source and a 10 megapixel camera at a resolution 5μm. The amount and quality of cortical and trabecular bone was quantified from 2D images and 3D reconstructions using CTAn, CTvol and CTVox software. The undecalcified specimens were embedded at low temperature in MMA, sectioned at 5 μm and stained with Von Kossa and Toluidine Blue to distinguish mineralized from unmineralized tissue.


Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_XXXVIII | Pages 5 - 5
1 Sep 2012
Carli A Gao C Khayyat-Kholghi M Wang H Li A Ladel C Harvey EJ Henderson J
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Purpose

Internal fixation of fractures in the presence of osteopenia has been associated with a failure rate as high as 25%. Enhancing bone formation and osseointegration of orthopaedic hardware is a priority when treating patients with impaired bone regenerative capacity. Fibroblast Growth Factor (FGF) 18 regulates skeletal development and could therefore have applications in implant integration. This study was designed to determine if FGF 18 promotes bone formation and osseointegration in the osteopenic FGFR3−/− mouse and to examine its effect on bone marrow derived mesenchymal stem cells (MSCs).

Method

In Vivo: Intramedullary implants were fabricated from 0.4 × 10mm nylon rods coated with 300nm of titanium by physical vapour deposition. Skeletally mature, age matched female FGFR3−/− and wild type mice received bilateral intramedullary femoral implants. Left femurs received an intramedullary injection of 0.1μg of FGF 18 (Merck Serono), and right femurs received saline only. Six weeks later, femurs were harvested, radiographed, scanned by micro CT, and processed for undecalcified for histology. In Vitro: MSCs were harvested from femurs and tibiae of skeletally mature age matched FGFR3−/− and wild type mice. Cells were cultured in Alpha Modified Eagles Medium (αMEM) to monitor proliferation or αMEM supplemented with ascorbic acid and sodium beta-glycerophosphate to monitor differentiation. Proliferation was assessed through cell counts and metabolic activity at days 3, 6 and 9. Differentiation was assessed through staining for osteoblasts and mineral deposition at days 6, 9 and 12.


Orthopaedic Proceedings
Vol. 93-B, Issue SUPP_IV | Pages 553 - 553
1 Nov 2011
Gao C Nguyen O Serpooshan V Eichaarani B Nazhat SN Harvey EJ Henderson JE
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Purpose: Poor bone quality is a common challenge to orthopaedic surgeons and frequently leads to complications such as non union and implant failure, particularly the elderly whose capacity for tissue repair is significantly reduced. The current study was designed to determine if bone marrow derived mesenchymal stem cells (MSC) seeded in dense collagen scaffolds and delivered to a surgically-induced femoral defect will expedite bone healing.

Method: Ex Vivo: MSC isolated from four month old donor mice were expanded ex vivo, seeded into hydrated type I collagen, which was subjected to unconfined compression to generate dense collagen scaffolds. The cell-seeded scaffolds were then cultured for up to 21 days. MSC viability was monitored using the AlamarBlue® metabolic assay and differentiation into osteoblasts using alkaline phosphatase (ALP) and von Kossa stain. In Vivo: A 3mm x 1mm window defect was drilled in the femur of elderly recipient C57Bl6 and C3H mice. The C3H mice were assigned to one of two study groups:

LEFT femur drill hole alone; RIGHT femur acellular scaffold.

LEFT femur acellular scaffold; RIGHT femur cell-seeded scaffold.

The quantity and quality of bone regeneration was assessed after 2 and 4 weeks using micro computed tomography (mCT) and histology.

Results: Ex Vivo: The dense collagen scaffold had superior mechanical properties and supported the survival and differentiation of MSC into osteoblasts up to 21 days in culture. Cells in uncompressed gels and those in compressed gels in non-osteogenic medium, had fewer ALP-positive cells at early time point and less mineral deposited at later times compared with those in compressed gels in osteogenic medium. In Vivo: A high incidence of postoperative fracture was seen in C57Bl6 mice compared with age matched C3H mice in the first study group. Furthermore, the empty surgical defect healed more rapidly than that containing the dense collagen scaffold, in which bone volume compared with tissue volume (BV/TV), trabecular number (Tb.N.) and connectivity were lower. In study group two, bone regeneration was evident at 2 weeks post operative and transplantation of MSC-seeded dense collagen scaffolds resulted in higher BV/TV, Tb.N. and trabecular connectivity compared with the acellular dense collagen scaffold.

Conclusion: Bone fragility in elderly C57Bl6 mice led to post operative fracture after generation of a non-critical sized drill hole defect in the proximal femur whereas age-matched C3H mice with higher bone mass sustained no fractures. Dense collagen scaffolds supported the survival and osteoblast differentiation of bone marrow derived MSC in 3D culture. Their superior mechanical properties allowed for transplantation into non-critical sized femoral defects, suggesting the approach shows promise as adjunct therapy for use with bone grafts and implants in patients with poor quality bone.