| Backgroud:Repair of large segmental bone defects caused by tumor resection, trauma, congenital osteoarthropathy, bone infection and osteoarthritis remains a major challenge in clinic. Tissue-engineering approaches, which incorporate mesenchymal stem cells (MSCs) and biodegradable three-dimensional (3D) porous scaffolds to construct tissue-engineered bones (TEB) and deliver TEB to the defect sites, have been proved of efficacy and security and widely documented as one of the most promising strategies. During the reparative process mediated by TEB, seeded MSCs play crucial roles. However, the underlying mechanism remains largely elusive, constraining the further application and development of TEB. Currently, abundant evidence from the fields of tissue engineering and regenerative medicine has shown that seeded MSCs can recruit osteogenesis-related cells of host origin to the defect site to accelerate bone repair. During culture in vitro, MSCs secrete multiple chemokines involving with cell migration spontaneously, and a set of chemokine receptors are expressed by MSCs. Against this background, we propose the possible mechanism for MSCs-induced osteogenesis:During the early stage of bone defect repair, inflammatory microenvironment incited by local trauma stimulates MSCs to secrete various chemokines and cytokines, such as stromal cell-derived factor-1 (SDF-1) and monocyte chemotactic protein-1 (MCP-1). As a result, host osteogenic cells are recruited to the bone defect site and contribute to the bone repair.Objective:(1) Isolate and culture human bone marrow-derived mesenchymal stem cells (hBMSCs) and mouse bone marrow-derived mesenchymal stem cells (mBMSCs) to serve as research objects and experimental materials.(2) Explore the effects of the inflammatory microenvironment, which is mimicked in vitro, on the secretory profile and host-hBMSCs-recruiting capability of exogenous hBMSCs. Screen the chemokines with important roles in the process of donor-hBMSCs-induced host-hBMSCs recruitment.(3) Incorporate mBMSCs into partially demineralized bone matrix (pDBM) scaffolds to construct TEB. Establish a reliable, reproducible, bilateral, femoral critical-size defect mouse model which is applicable to the basic research of bone tissue engineering. Evaluate the osteogenic effacicy of TEB and compare the proportions of exogenous and endogenous osteogenic cells/osteocytes in the newly-formed bones.(4) Observe the recruitment of host cells, identify the recruited cells and preliminarily explore the mechanism underlying seed-cells-induced host cell recruitment.Methods:(1) hBMSCs were isolated by density gradient centrifugation and adherent method and mBMSCs were isolated by the method of whole bone marrow cells attachment. Both hBMSCs and mBMSCs were characterized by morphology, immunophenotypic analysis, and multi-lineage differentiation potential.(2) Pro-inflammatory factors, including interleukin-1? (IL-10), interleukin-6 (IL-6) and tumor necrosis factor-a (TNF-a), were added into the culture of hBMSCs to mimic the inflammatory microenvironment in vitro. The resulting conditioned media were collected. The effects of inflammatory microenvironment on the host-hBMSCs-recruiting capability of different conditioned media were revealed by transwell migration assays. Employing semi-quantitative and quantitative cytokine antibody assays, chemokines with important roles in the process of donor-hBMSCs-induced host-hBMSCs recruitment were screened.(3) Using a two-side static seeding method, TEB were fabricated with wild-type mBMSCs and pDBM scaffolds, and the constructing result was confirmed by environmental scanning electron microscopy (ESEM). With the use of green fluorescent protein (GFP)+ transgenic mouse and the self-designed internal fixation steel plate, a bilateral, femoral critical-size defect mouse model was established. TEB and pDBM were implanted to the left and right femoral defects, respectively. After implantation, the reparative effects of TEB and pDBM scaffolds were compared by Micro-CT, hematoxylin-eosin (HE) staining and osteocalcin (OCN) mRNA expression. The proportions of exogenous and endogenous osteogenic cells/osteocytes in the newly-formed bones were calculated and compared using a confocal laser scan microscope (CLSM).(4) At designated time points postoperatively, bilateral femurs or implants were collected. The recruitment of host cells was preliminarily observed with a small-animal in-vivo fluorescence imaging system. Then, tissue sections were prepared and the host cell recruitment induced by seed cells was evaluated by pathological staining and cell count. The recruited host cells were identified by immunofluorescence. The differences of SDF-1, C-X-C motif chemokine receptor-4 (CXCR4), MCP-1, C-C motif chemokine receptor-2 (CCR2), CD34, CD 14 and CD31 mRNA expression between TEB and pDBM were compared by RT-PCR to preliminarily explore the mechanism of seed-cells-induced host cell recruitment.Results:(1) hBMSCs (passage 4) isolated and culture by density gradient centrifugation and adherent method were fibroblast-like and grew with adherence. Cells had a uniform fusiform shape. Immunophenotypic analysis showed that cells were homogeneously CD29+, CD44+ CD73+, CD90+, CD105+, CD31-, CD34- and CD45-. After induction, the osteogenic and adipogenic differentiation potentials of cells at passage 4 were identified by alizarin red S and Oil Red O staining, respectively. mBMSCs (passage 4) obtained by the method of whole bone marrow cells attachment had a uniform spindle-shaped morphology and grew in whirl manner. Cells were homogeneously CD73+, CD90+, CD105+, CD31-, and CD45-. The osteogenic and adipogenic differentiation potentials of cells at passage 4 were identified by pathological staining.(2) The chemotactic activity of conditioned media from non-treated hBMSCs was markedly higher than blank media, while slightly higher than conditioned media containing pro-inflammatory factors. Moreover, conditioned media from inflammation-stimulated hBMSCs induced the significantly greater chemotaxis as compared with conditioned media from non-treated hBMSCs. Furthermore, culturing hBMSCs with pro-inflammatory factors induced significant increases in secretion of 11 cytokines, as revealed by semi-quantitative cytokine antibody assays. These cytokines included CXC chemokine ligand 16 (CXCL16), osteoprotegerin, growth-regulated oncogene (GRO), epithelial neutrophil-activating peptide-78 (ENA78), monocyte chemoattractant protein-1? (MIP-1?), MCP-1,2,3, granulocyte chemotactic protein-2 (GCP-2), IL-6 and interleukin-2 receptor ? (IL-2R?). Results from quantitative cytokine antibody assays further confirmed that the inflammatory microenvironment could increase chemokine production from mBMSCs, and 21 chemokines were identified with higher concentration.(3) TEB fabricated with the two-side static seeding method showed a high seeding efficacy. As revealed by ESEM, mBMSCs tightly attached on the pore surface of scaffold after 10 days, with abundant extracellular matrix deposited. The whole operative time for the establishment of bilateral femoral critical-size defects in one mouse was short and postoperatively, the normal daily activity was recovered soon. Successful modeling was confirmed by mammography X-ray. The significant osteogenic superiority of TEB over pDBM scaffolds was also observed and further validated by Micro-CT, HE staining and OCN mRNA expression. With the use of GFP+ transgenic mice, we found that in the newly-formed bones induced by TEB, the proportion of host cells was approximately 90.42%, which was greatly higher than that of exogenous cells.(4) As revealed by the small-animal in-vivo fluorescence imaging system, the mean fluorescence intensities of both TEB and pDBM scaffolds significantly increased over time. At 3 days postoperatively, no significant difference in the mean value of fluorescence signal was observed between TEB and pDBM scaffold. However, as time extended to 7 or 10 days, the fluorescence signal of TEB was significantly brighter than that of pDBM scaffold. This was further confirmed by CLSM. At day 7, the amount of host cells was significantly greater within TEBs than pDBM scaffolds, and such difference still existed at day 10. Immunofluorescence staining showed that the amounts of CD73+ and CD90+ host cells in TEB increased over time and were significantly larger than that in pDBM scaffolds. For CD14+host cells, the amount in TEB was reduced at postoperative day 3, as compared with day 1, and smaller than that in pDBM scaffolds. At day 7, abundant CD14+ host cells emerged in TEB and the amount was significantly greater than that in pDBM scaffolds. Such difference was also present at day 10. As terms with CD31+host cells, the amount in TEB was significantly greater than that in pDBM scaffolds all the time. The amount of CD31+host cells in TEB increased to peak at day 10 and gradually decreased afterthat. At 7 and 10 days postoperatively, the expressions of SDF-1 and CXCR4 mRNA in TEB were significantly higher than those in pDBM scaffolds. Consistently, the expression of CD34 mRNA exhibited a similar pattern. Moreover, the MCP-1, CCR2 and CD 14 mRNA levels were significantly higher in TEB at day 7. While at day 10, only MCP-1 and CD14 showed higher mRNA expression in TEB.Conclusion:(1) hBMSCs isolated and culture with the density gradient centrifugation and adherent method and mBMSCs obtained by the method of whole bone marrow cells attachment were all fibroblast-like with excellent proliferative and multi-lineage differentiation potential, thus being suitable to serve as research subjects and experimental materials.(2) During expansion in vitro, hBMSCs could secrete various cytokines which promoted the migration of host hBMSCs. Such secretory and host-hBMSCs-recruiting capacity could be enhanced by the presence of inflammatory microenvironment. Among the chemokines secreted by inflammatory microenvironment-stimulated hBMSCs, the maximal concentration occurred in GRO, which might affect the migration of host hBMSCs to some extent.(3) TEB fabricated with the two-side static seeding method could meet the present study requirement. The bilateral femoral critical-size defect model was reliable, reproducible, and the radiographic, molecular biological and histomorphological examinations were accessible. Thus the present model was applicable to the basic research of bone tissue engineering. TEB were significantly superior to blank scaffolds in repairing bone defects, indicating the pivotal roles of seed cells. However, the proportion of exogenous cells in the newly-formed bones was very low, suggesting that the osteogenesis could be mainly attributed to the contribution of host osteogenic cells.(4) During the early inflammatory phase of bone defect repair, seeded MSCs recruited more host cells, including MSCs, monocytes and endothelial progenitor cells, to the defect site to accelerate bone repair. The SDF-1/CXCR4 and MCP-1/CCR2 signal pathways played crucial roles in the host cell recruitment. |
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