Experiment On Fast Construction Of Tissue Engineered Bone By Seletive Cell Retention Technology

Background and objective: Bone defects induced by various kinds of causes are commonly seen in clinic. As a gold standard, autogenous bone transplantation can not satisfy the requirement for bone defects therapy because of limited bone mass and operation complications in donor site. Tissue engineered bone (TEB) constructed by bone marrow stem cells (BMSCs) and biomaterial in treating bone defects in animal studies had obtained certain effects, but the shortcomings were: BMSCs in little resource, long cycle of culture and amplification in vitro, low adhesion rate of cell to scaffold and high specification, thus limited the application in bone defect therapy. Autologous red bone marrow percutaneous injection had obtained some success in treating clinical bone nonunion and bone defect, but the method of direct injection, had the defect of partly stem cell running off. Selective cell retention (SCR) is a technique of bone marrow stem cell enrichment, the mechanism is seletive retenting stem cells and facilitating ossify factors when bone marrow pass through the adequate mesh and favourable surface adhesiveness of scaffolds. The TEB constructed by SCR were replanted immediately for treating bone defect, could obtained the same effect as autogenous bone transplantation. But due to intellectual property rights conservation and enter restriction, enrichment technique and correlated manufactures harded to applicate in our country. Thus, the objectives of our experiment are:⑴Preparation of a new kind of bone marrow stem cells enrichment scaffold, to observe its cellular compatibility in vitro and its histocompatibility in vivo;⑵To explore the enriching effect of new kind of bone marrow stem cells enrichment scaffold constructed by SCR;⑶To study the ectopia osteogenesis effects of tissue engineered bone fast constructed by SCR;⑷To observe the effect of tissue engineered bone fast constructed by SCR on ossify marker and ossify specific activating transcript factor Cbfα1, and explore the mechanism of enrichment scaffold on osteogenesis. Methods:⑴Demineralized bone matrix decorated with poly-L-lysine (PLL-DBM) as a new kind of bone marrow stem cells enrichment scaffold, its surface character such as density, porosity and pore size were determined by liquid displacement method, scanning electron microscope, three dimension video microscope, raman spectroscopy, infrared spectrum and HE staining.⑵New enrichment scaffold and different dense of scaffold leaching liquor were co-cultured with hMSCs, cellular compatibility in vitro were evaluated by morphologic observation, MTT assay, FCM, hemolysis experiment and immunohistochemistry.⑶Nude mouse were accepted intraperitoneal injection of saline leaching liquor extracted from PLL-DBM, back subcutaneous injection of DOC cell suspension after cultured with PLL-DBM leaching liquor, back implantation of PLL-DBM scaffold respectively, histocompatibility in vivo were evaluated by acute toxicity test, carcinogenesis test and subcutaneous implantation test.⑷TEB were constructed quickly after bone marrow enrichment by SCR. PLL-DBM combinated with bone marrow by SCR (SCR group), deminerilized bone matrix ( DBM ) combinated with MSCs (TEB group), DBM combinated with bone marrow (bone marrow group) and DBM (DBM group) were implantated into subcutaneous area of nude mouse, drow the materials at 4, 8, 12, 16 weeks. Image density, histological changes of bone graft area and ossify effects were determined by X ray, CT scanning, scanning electron microscope and HE staining.⑸New enrichment scaffold were handled by SCR, the enriching effects of PLL-DBM to bone marrow nucleated cells (NCs) ,platelets (PLTs) and fibroblast colony forming unit ( CFU-F) were observed before and after enrichment, contents of TGF-β1 and PDGF in bone marrow supernatant were detected by ELISA method before and after enrichment.⑹TEB were constructed fast after bone marrow enrichment by SCR, PLL-DBM combinated with SCR (SCR group), DBM combinated with MSCs (TEB group), DBM combinated with bone marrow (bone marrow group) and DBM (DBM group) were implantated into subcutaneous area of nude mouse, Collagen type I, integrinα2β1, osteocalcin and Cbfα1 mRNA and protein expression at 4,8,12,16 weeks were studied by immunohistochemistry and RT-PCR method.Results:⑴Steady ivory converage were formed at exterior and interior surface by PLL. The density of PLL-DBM is (0.27±0.02) g/ml, the porosity is (73±11)%, the pore size is (412.73±160.29)μm. there are many micropore about 100μm size among pores, a great quantity of pore cross-connected each other in the interior pore, and formed smaller, more steady mesh structure in the natural pore.⑵New enrichment scaffold and different density scaffold leaching liquor were co-cultured with hMSCs, morphologic observation showed that MSCs growth well, cytotoxicity showed that MSCs proliferate well and toxity range from 0 to 1 grade. FCM showed that hMSCs and DOC had no abnormal DNA ploid, the marker of CD29 and CD105 are positive, CD34 and CD45 are negative, scaffold leaching liquor had no affect on hMSCs surface molecule, and could promote its proliferation by transform it into multiplication period. After ossify induction, DOC could form calcium nodule and express Collagen type I, and the ALP activity had not been affected. Hemolysis experiment showed that hemolysis rate was 2.617% which in accordance with the standard of biomaterial that the rate must less than 5%.⑶In acute toxicity test, nude mouse growth well; in carcinogenesis test, nude mouse survival better, tumour had not been seen after 8 months, tumour cells had not been in the organization such as heart, liver, brain, lung, kidney and spleen. In subcutaneous implantation test, histological observation showed that subcutaneous tissue tighted the scaffold surface with a thin fiber tissue around it, lymphocyte/macrophage infiltration, cytolysis and necrosis had not been seen.⑷The density of each composite implantation increase gradually followed the time, and had significant difference at each time point when compared with single DBM; the density in SCR group was similar to that in TEB group, but was superior to in bone marrow group and DBM group. Scanning electron microscope and HE staining showed that, PLL-DBM scaffold degradated gradually as time passed, bone trabecula and small vessels formed gradually into the scaffold. DBM started to degradation at 8 weeks, a lot of cells and a few small vessels formed, and part of newly formed bone generated into the scaffold. DBM partly degraded at 12 weeks, smaller vessels entered scaffold and new bone formation was found; rigid bone tissue were seen at 16 weeks, and medullary tissue and vessels were seen in some region of implantation site. Scanning electron microscope and HE staining in TEB group were similar to SCR group, but superior to bone marrow group and DBM group.⑸The enriching effects of PLL-DBM increased gradually with the PLL dnsity. The concentration enrichment multiple rate of enrichment scaffold decorated with 0.1% PLL of bone marrow NCs was 3.18±0.31, the adhesion rate was 53%±12%; The concentration enrichment multiple rate of platelet was 3.88±0.68, the adhesion rate was 34%±10%, The concentration enrichment multiple rate of CFU-F was 5.25±1.40, the adhesion rate was 73%±13%, the selective rate was 1.41±0.34. Compared with high density PLL, 0.1% PLL-DBM had the same effect, showed that PLL-DBM had saturability to NCs adhesion. concentration enrichment multiple rate of TGF-β1 was 42.327±4.561 and concentration enrichment multiple rate of PDGF was 9.618±1.251 in the enrichment scaffold, and enrichment technique could increase the content of TGF-β1 and PDGF significantly.⑹Expression of Collagen type I, integrinα2β1, osteocalcin mRNA and protein were increased gradually followed the time, and reached the peak at 16 weeks, the expression in SCR group was similar to that in TEB group, superior to bone marrow group and DBM group. Immunohistochemistry and RT-PCR showed that, Cbfα1 mRNA and protein expression increased significantly at ossify earlier period, but decreased gradually followed the bone maturation. The expression of Cbfα1in SCR group was similar to in TEB group, superior to in bone marrow group and DBM group.Conclusion:⑴PLL-DBM possess three dimensional space as autogenous bone and PLL for cell adhesion, and have favourable cellular compatibility, histocompatibility and biodegradability, can promote the adhesion and proliferation of hMSCs, but not affect the ossify activity of DOC, is an ideal enrichment scaffold for bone marrow stem cells.⑵TEB constructed fast by SCR have favourable bone formation, and the effect of ossify is similar to MSCs combined with DBM.⑶Enrichment scaffold can increase the concentration enrichment multiple rate and the adhesion rate of bone marrow NCs, platelet and CFU-F significantly, and increase the contents of TGF-β1 and PDGF in TEB, it is said that the local high concentration of bone marrow stem cells and facilitate ossify growth factor TGF-β1 and PDGF, maybe the one of the mechanism of high ossify activity of TEB constructed fast by SCR.⑷Increased Cbfα1 expression in earlier period of osteogenesis and increased expression of Collagen type I, integrinα2β1 and osteocalcin in middle-advanced stage maybe the one of the molecule mechanism of high ossify activity of TEB constructed fast by SCR.