Osteoblast-oriented differentiation of BMSCs by co-culturing with composite scaffolds constructed using silicon-substituted calcium phosphate, autogenous fine particulate bone powder and alginate in vitro.

Ye Tian,Shou-Yang Xiang,Yu-Fu Wang,De-Chun Chen,Xin-Tao Wang,Rui Fu,Chang-Long Zhou,Wen-Xiao Xu,Li-Huang Cui,Xiao-Qi Liu

doi:10.18632/oncotarget.19015

Ye Tian, Shou-Yang Xiang + Show 8 more

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https://doi.org/10.18632/oncotarget.19015

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Abstract

Autogenous bone graft is the best for spinal fusion in clinics, however, lacking sources, bleeding and infection are limited its practice. Seeking alternative materials are urgent for orthopaedic surgeon. Here, we evaluated osteoblast-oriented differentiation of rabbit BMSCs by co-culturing with composite scaffolds constructed using silicon-substituted-CaP-fine particulate bone powder-alginate. Using CCk8-kit, biocompatibility was evaluated by testing BMSCs proliferation; morphology and survival of osteoblasts within scaffolds were observed using EM and HE staining; growth factors and related genes were detected using RT-PCR. HE staining showed spindle-shaped BMSCs after the 3rd passage; EM data showed that uneven surface and longitudinal section were observed with scattered distribution of 5-100 mm interspaces, which leave enough space for BMSCs adhesion and growth. Interestingly, at 14-day culture with HE staining, osteocytes within the scaffolds grew well with regular shape and integrate structure. RT-PCR results showed that expression levels of BMP2, TGF-b and COL-I, ALP, OPN were increased significantly and time-dependently. Collectively, all mentioned effects were more obvious in co-culture BMSCs with scaffolds than those with other components. Immunohistochemistry showed that positive OPN expression was detected at 7-day co-culturing BMSCs with scaffold, rather than other situations. These results suggest that composite scaffolds constructed with Si-CaP-fine particulate bone powder-alginate have a certain degree of biocompatibility and bioactivity to promote osteoblast-oriented BMSCs differentiation.

Highlights

Spinal fusion is commonly used in those patients with spinal diseases causing from spinal degenerative diseases,trauma, inflammation, congenital deformity, and tumor and autogenous bone transplantation would be the gold standard for these patients, the clinical practice of this therapy has been largely limited by lacking source of autogenous bone and significant complication including infection, bleeding, pain, fracture and so on even though the bone transplantation has obvious advantages such as biocompatibility and no immunogenicity [1, 2]
Www.impactjournals.com/oncotarget cells passage (P) culture was carrierd our according to 1:2 ratio at 12 days after primary culture as P0 and at the day-3 (d3) of P3 cultured, spindle-shaped Bone mesenchymal stem cells (BMSCs) covered ~100% flask with mono-layer (Figure 1D), suggesting BMSCs were in a good condition and the survival rate was calculated by ramdomly counting ~200 cells stained using trypan blue
For identification of BMSCs in the current expeirmental condition, BMSCs were identified through HE staining with Eosin staining, by which the majority of BMSCs presented as typical spindle-shape and some of them were lolygon-shape, the nucleus were stained at deep blue color and some of nucleus were under the division phase (Figure 3)

Summary

Introduction

Spinal fusion is commonly used in those patients with spinal diseases causing from spinal degenerative diseases,trauma, inflammation, congenital deformity, and tumor and autogenous bone transplantation would be the gold standard for these patients, the clinical practice of this therapy has been largely limited by lacking source of autogenous bone and significant complication including infection, bleeding, pain, fracture and so on even though the bone transplantation has obvious advantages such as biocompatibility and no immunogenicity [1, 2]. Bone mesenchymal stem cells (BMSCs) have been demonstrated its differentiation of osteogenetic potential [8,9,10,11], the capability of BMSCs differentiated towards to bone and cartilage are more potent compared with adipose-derived stem cells (ADSCs) [12] with significant bone morphological protein-2 (BMP-2), collegen type-I (Col-I), alkaline phosphatase (ALP), and osteocalcin (OCN). Another in vitro study has shown that the osteogenetic capability of BMSCs in restoration of experimental bone defect is obviously dramatic compared with ADSCs [13]. The aim of the present study was to elucidate the biocpmpatibility and bioactivity of the scaffolds constructed using Si-CaP, fine particulate bone powder, and alginate, as well as the diffentiation potential of BMSCs towards osteoblast by co-culturing them with the scaffolds using state-of-the-art techniques

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