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• W2113863126 abstract "Introduction: Bioengineering osseous tissue requires recapitulating the cellular, matrix, and lacunocanalicular components of bone. A construct must have a microvascular network which requires simultaneous co-culture of endothelial and osteogenic cells. Recreation of the matrix requires optimization of composition and microarchitecture. Engineering of constructs large enough to solve actual clinical problems requires novel strategies that address chemotransportative requirements by replicating lacunocanalicular flow. Methods: Cells: Adipose-derived mesenchymal stem cells (MSCs) were isolated and expanded from human lipoaspirate and differentiated into osteoprogenitor-rich (OPC) and endothelioprogenitor-rich (EPC), confirmed by RT-PCR. Normal human osteoblasts (NHOst) and human umbilical vein endothelial cells (HUVEC) served as terminally differentiated cell lines. The effects of co-culture (e.g OPC+HUVEC, OPC+EPC etc) on capacity for bone formation was evaluated by von Kossa assay. Matrix: Murine alveolar defects were created. Scaffolds composed of either absorbable collagen sponge (ACS) or biphasic hydroxyapatite/tri-calcium phosphate (HA-TCP) in a 15/85 ratio were constructed and implanted. HA-TCP scaffolds were further investigated, comparing 15/85 and 60/40 HA/TCP in a rabbit calvarial model. Scaffold pore size (380/180 microns) and strut size (250/180 microns) were also investigated. New bone formation was analyzed histomorphometrically using micro-CT. Lacunocanalicular flow: We have developed a novel flow perfusion bioreactor designed to mimic lacunocanalicular flow. To validate, murine femurs were explanted to the bioreactor for 14 days. Viability and function were evaluated using thiazolyl blue tetrazolium bromide (MTT), DNA quantification, alkaline phosphatase (ALP) assay, and tetracycline labelling. Furthermore, optimal culture conditions were tested with MSC-seeded custom thick 3D HA-TCP scaffolds cultured in static conditions or in flow perfusion. Cellularity was assessed by SEM, DNA quantification and ALP assay. Results: Cells: Proliferation and function were greatest when more-differentiated vasculogenic cells (i.e. HUVECs) were co-cultured with less-differentiated osteogenic cells (i.e. OPCs). For example, co-cultured HUVEC/OPC formed more bone nodules (263,945μm2) than HUVEC/NHOst (179,840μm2) or NHOst alone (89,608μm2). Matrix: HA-TCP showed more bone growth (86±5.8%) than ACS (69±12.4%). With larger pore size, new bone formation pattern resembled cancellous bone whereas smaller pore sizes generated cortical appearing bone. Similarly, larger strut sizes remodeled more slowly than smaller struts. Lacunocanalicular flow: MTT staining confirmed osteocyte viability in flow perfused femur explants, DNA quantification demonstrated up to 100% preservation, ALP activity was upregulated, and fluorochrome labelled mineralizing surfaces were seen throughout. In cell-seeded scaffolds, cellular function was enhanced in flow perfusion eg ALP activity was greater (3.86±0.320 mM/g) than static culture (0.909±0.460 mM/g, p=0.002). SEM demonstrated MSCs were only viable on the periphery static scaffolds but were viable throughout in flow perfusion. Conclusions: Recapitulation of native bone cells, matrix and lacunocanalicular flow can recreate bone constructs for replacement and repair avoiding the limitations associated with traditional autografts or allogeneic transplantation." @default.
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• W2113863126 date "2010-02-01" @default.
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• W2113863126 title "In vitro Biomimicry for Vascularized Bone Engineering" @default.
• W2113863126 doi "https://doi.org/10.1016/j.jss.2009.11.413" @default.
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