| BackgroundRecently, nacre was paid much attention as a new kind of biological bone-substitute. Nacre is the inner layer of the shell of the bivalve marine mollusks, such as pearl shell, oyster, abalone and fan mussel which is mainly composed of aragonite calcium carbonate but a minor part of organic matrix and microelements which is supposed to be the most important factors in promoting osteogenesis. Our studies in the past few years showed that the nacre artificial bone (NAB) made of nacre powder of Pteria martensii and Poly-DL-lactic acid displayed the characters of good biocompatibility, rare immunogenicity, degradability, osteoconduction and osteogenesis in bone defect repairing in vivo. The nacre is considered to be better than other inorganic biological materials in animal research as supposed to be the attribution of the organic elements in nacre which may be effective during the osteoinductive and osteogenic processes. In this study, the water soluble matrix (WSM) extracted from nacre was used on culturing human bone marrow derived stromal cells (hBMSCs) in vitro, by which the roles and mechanisms of hBMSCs osteogenic differentiation induced by WSM is expected to be clarified. Meanwhile, co-effects of osteoinduction of WSM and PDGF-BB and biocompatibility of NPAB and hBMSCs will be investigated.Objectives1. To investigate the roles and mechanisms of hBMSCs osteogenic differentiation induced by WSM in vitro and the co-effects of osteoinduction of WSM and PDGF-BB.2. To establish a co-culture system for hBMSCs-osteoblasts so as to investigate the features of both kinds of cells in one system and the affected outcomes after the WSM was added into the culture media.3. To investigate the allotopic bone formation of the rBMSCs-NAB compounds in SD rats in vivo.4. To detect the biocompatibility of NAB and hBMSCs.Methods1. The human bone marrow derived stromal cells were harvested, cultured, expanded in vitro and then identified by ways of cell morphology and flow cytometry.2. The water soluble matrix (WSM) extracted from nacre powder of Pteria martensii was condensed by vacuum freeze drying. The hBMSCs were cultured in the media including WSM with different concentrations. A dose-effect curve of the cell AKP activity was drew by MTT assay.3. The cell cycles of hBMSCs were detected by means of flow cytometry after the induction with WSM and PDGF-BB to the third passage of hBMSCs while with osteo-induced medium as the control group. AKP Gomori stain and alizarin red nodus stain were used to evaluate the osteogenesis differentiation effects of hBMSCs induced by WSM and/or PDGF-BB. At the same time, the expression of BMP-2, TGF-β1, Cbfa- 1 in hBMSCs were detected with One-step RT-PCR.4. hBMSCs and OBs were cultured in a Transwell system to establish a BMSCs-OBs co-culture system. The proliferation, apoptosis and other features of the BMSCs and OBs were detected by means of MTT assay, acridine orange stain and AKP activity detection. Bone formation gene marker such as BMP-2, AKP, TGF-β1, Cbf-α1, Osteonection, Osteocalcin and Osteopontin were detected by One-step RT-PCR after WSM was added into the co-culture system for 7 days, by which the osteogenesis differentiation roles of hBMSCs induced by WSM is to be clarified.5. BMSCs of SD rats (rBMSCs) were isolated and cultured in vitro and induced with WSM for 10 days, then the rBMSCs were seeded onto NAB. The composites of rBMSCs and NAB were embed into the musculi dorsi of matured SD rats, while two other control groups NAB group with rBMSCs un-induced and NAB group only. X-ray photographs were used to evaluate the osteogenesis of the NAB with rBMSCs in vivo after the engraftments were embedded ld, 4w, 8w and 12w. The composites harvested at 4, 8 and 12 weeks postoperatively were examined by gross observation, histological and immunohistochemical investigation.6. The hBMSCs were cultured in vitro with NAB, and the proliferation of the hBMSCs were detected by MTT assay, observation of scanning electron microscope. Results1. Under the phase contrast microscope, the human marrow stromal cells were irregular such as round, spindle or triangle like when they were just adherence. The hBMSCs were growing Whirlpool-like after they were passaged and proliferated and the growth curve of hBMSCs was S-shape. The flow cytometry results showed that the CD29, CD44 and CD105 of hBMSCs were positive while the CD34 and CD45 negative.2. The WSM was condensed to 30mg/ml by vacuum freeze-drying and then was added into the media of hBMSCs and the AKP activity was detected. The results showed that the WSM 150/μg/ml could enhanced the AKP activity of hBMSCs significantly. No dose-depending effect was observed, so the WSM 150/zg/ml was adopted in this study.3. The hBMSCs in WSM group, PDGF-BB+WSM group and mineralization medium group showed AKP positive when stained using Gormori stain after hBMSCs were treated 3 days, and the tendency was more obvious on the 7th day. But AKP stain of the hBMSCs in the control group and PDGF-BB group were negative or weakly positive at both time intervals. Typical red calcification nodus were seen in the mineralization medium group after the hBMSCs were induced 18 days, and immature typical calcification nodus were seen in the WSM group. More small calcification nodus were seen in the PDGF-BB+WSM group, while little calcification nodus were seen in control and PDGF-BB group.4. BMSCs-OBs co-culture system could promote the proliferation and AKP activity of OBs and WSM can enhance the effects during the process. BMSCs-OBs co-culture system lowered the apoptosis of the BMSCs, promoted the aggregation of BMSCs. BMSCs-OBs co-culture system can promote BMP-2 and AKP expression of the BMSCs (P＜0.05) and WSM enhanced the expression of the two factors higher(P＜0.05), but WSM had no effects on the expression of Cbfal,TGF-B1, Collage-1 and Osteonectin in the BMSCs-OBs co-culture system (P＞0.05).5. Allotopic ossification experiments in vivo results: in WSM+rBMSCs+NAB group, the osteoid was formed after 8 weeks, and mature bone tissue with trabecular bone was formed after 12 weeks, but in NAB group there was no evidence of new bone formation instead of abundant fibrous tissue after 4 weeks, and among the materials more fibrous tissue than before was formed after 8 and 12 weeks. Small quantity irregular trabecular bone was found in rBMSCs+NAB group. The X-ray photograph showed that the density of NAB in WSM+rBMSCs+NAB group was higher than that of other groups after 12 weeks (P＜0.05), which was coincident to histological examination results.6. MTT results indicated that the NAB had rare affect on the growth and proliferation of hBMSCs (P＞0.05) on 2nd, 4th, 6th and 8th day after co-cultured together. The adhesion, growth and proliferation of hBMSCs were seen on the surface and pore of the material by observation of scanning electron microscope in vitro. WSM could promote total protein expression of hBMSCs.Conclusions1. WSM could promote hBMSCs osteogenic differentiation in vitro and PDGF-BB showed cooperativity in this process, But no evidence proved that PDGF-BB had osteoinductive to hBMSCs in vitro.2. In BMSCs-OBs co-culture system, hBMSCs could enhance ossification ability of OBs by accelerating its proliferation and AKP activity, at the same time, OBs lowered the apoptosis of hBMSCs which implied that hBMSCs and OBs could promote functions between. 3. In BMSCs-OBs co-culture system, WSM could promote OBs grow and reinforce hBMSCs' BMP-2 and AKP expression. WSM has ossifying effects on both hBMSCs and OBs.4. Allotopic bone formation was found in the grafts of NAB and rBMSCs induced by WSM in vivo, which indicated that WSM could promote rBMSCs osteogenic differentiation.5. hBMSCs displayed good biocompatibility with NAB, which can benefit to further clinical trial. |
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