Abstract
Demineralized bone allografts and mesenchymal stromal cells have been used to promote bone regeneration. However, the degree to which cortical bone should be demineralized for use in combination with adipose-derived mesenchymal stromal cells (Ad-MSCs) remains to be clarified. In this study, the in vitro osteogenic ability of Ad-MSCs on allografts was investigated in relation to the extent of demineralization. Three treatment groups were established by varying exposure time to 0.6 N HCL: partially demineralized (PDB; 12 h), fully demineralized (FDB; 48 h), and non-demineralized bone (NDB; 0 h, as a control). Allografts were prepared as discs 6 mm in diameter for in vitro evaluation, and their demineralization and structure were evaluated by micro-computed tomography and scanning electron microscopy. Ad-MSC adhesion and proliferation were measured by MTS assay, and osteogenesis-related gene expression was assessed by quantitative reverse transcription polymerase chain reaction. PDB and FDB demineralization rates were 57.13 and 92.30%, respectively. Moreover, Ad-MSC adhesion rates on NDB, PDB, and FDB were 53.41, 60.65, and 61.32%, respectively. Proliferation of these cells on FDB increased significantly after 2 days of culture compared to the other groups (P < 0.05). Furthermore, expression of the osteogenic genes ALP, BMP-7, and TGF-β in the FDB group on culture day 3 was significantly elevated in comparison to the other treatments. Given its biocompatibility and promotion of the osteogenic differentiation of Ad-MSCs, our results suggest that FDB may be a suitable scaffold for use in the repair of bone defects.
Highlights
Bone allografts have been successfully used as an alternative tissue engineering material for bone regeneration [1, 2]
Given its biocompatibility and promotion of the osteogenic differentiation of Ad-Mesenchymal stromal cells (MSCs), our results suggest that fully demineralized bone (FDB) may be a suitable scaffold for use in the repair of bone defects
Osteoclasts or macrophages degrade an allograft at the implantation site, digesting the calcium component, before blood vessels and osteoblasts move into the demineralized construct, resulting in new bone formation [4]
Summary
Bone allografts have been successfully used as an alternative tissue engineering material for bone regeneration [1, 2]. Cortical bone contains bone morphogenetic proteins (BMPs) and matrix proteins. These former are osteoinductive glycoproteins that participate in bone formation [3] and can be activated by demineralization of cortical bone. MSCs derived from adipose and placental tissue, bone marrow, muscle, and umbilical cord blood are capable of osteoblastic lineage differentiation under osteogenic culture conditions [6]. Demineralization by hydrochloric acid exposes type I collagen fibers in bone through the removal of mineral components. Certain synthetic scaffolds, such as those composed of tricalcium phosphate, hydroxyapatite, and poly (methyl methacrylate), exhibit a high level of mechanical stiffness but achieve relatively poor cell seeding efficiency and inadequate cell distribution [7]. Demineralized bone allografts consist of a natural biomaterial, but are osteoinductive and accelerate bone regeneration by eliminating the time needed to decalcify nondemineralized bone allografts in vivo
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