| Background: Bone defect caused by severe trauma, infection, tumors and so on is very common clinically, and is used to need treatment with bone grafting. At present, as far as treatment for the serious bone defect is concerned, the prostecdtive efficacy has not been ideal yet. And the rise of bone tissue engineering provides a new way to solve the therapeutic problems of large bone defects. Mesenchymal stem cells (MSCs) are considered as the optimal seed cells for bone tissue engineering. MSCs can be derived conveniently because they can be obtained through puncture only with small wounds and fewer complications, and be culturabel with rapid proliferation. Osteogenic induction in vivo and in vitro environment not only can differentiate into bone tissue, but also has more potential to differentiate. In addition, MSCs are also easily separated and cultured. In recent years, autologous osteoblast or MSCs for the individual animal and clinical treatment has produced good results. But MSCs content in the bone marrow is low, and decreasing with age and difficult in rapid amplification in vitro. Moreover, some autoimmune diseases significantly weaken its proliferation ability in MSCs. With increased culturing algebra "anti-differentiation" phenomenon and tumor growth may appear, accompanied with the loss of cell function and the safety. Therefore, autologous MSCs is limited owing to the use of sources and quantitative restrictions and can not meet "off the shelf" requirement. Establishing allogenic seed cell bank of MSCs is the shortcuts to solve these problems. Previous studies in lower-class animals showed that grafting in engineered tendon, cartilage and bone constructed using allogeneic seeding cells elicit no visible immunologic reaction and may influence on repairing bone defect. As a result,allogeneic bone tissue engineering showed a promising approach for bone defect.Objective: (1)To explore MSCs optimal culture methods and condition by which MSCs were isolated and cultured in vitro and induced osteoblast, and its characteristics in cell biology were observed; (2) To investigate effect of osteogenic cells differentiated from mesenchymal stem cells (DOC) under different conditions on peripheral blood mononuclear and cytokine secretion; to investigate DOC immunogenicity and influence of IFN-γon its immunogenicity; to explore MSCs immunomodulatory mechanisms for providing the basis for experiment in vivo; (3) To understand allogeneic bone tissue engineering in vivo immune rejection reaction and the ability to ectopic bone, and to further identify feasibility in MSCs seed cells for allogeneic tissue engineering implantation, tissue-engineered bone constructed with DOC and demineralized bone matrix composite material was implanted under the porcine subcutanenous tissue and compared with demineralized bone matrix (DBM) materials.Methods: 1. The biological characteristics of porcine MSCs. (1) Under aseptic conditions, 2-5m1 bone marrow was aspirated from a minipig iliac crest. MSCs obtained by density gradient centrifugation and purification from bone marrow, were cultured with 5% fetal bovine serum in the culture medium A-DMEM and 10% fetal bovine serum F12-DMEM. the number of CFU-F On 3rd d, 5th d, the largest number of times cell passaged, and the time cell confluence were observed, expression of CD14, CD29, CD44, CD45, SLA-I. SLA-II in the third generation cell are dectcted by FACS. (2) MSCs and DOC cells adherent rate, the growth curve and the growth cycle are detected and compared. (3) MSCs differentiated into osteoblast were identified using alkaline phosphatase staining, Von-Kossa staining, alizarin red and osteocalcin immunohistochemical staining. 2. Experiment on immunogenicity of DOC. (1) Undifferentiated MSCs was used as control group,expression of SLA of MSCs and DOC treated with IFN-γor not. (2) Gene expression of SLA of MSCs and DOC treated with IFN-γwere detected using RT-PCR. (3) By mixed lymphocyte reaction the following were observed:①Effect on PBMC proliferation of different magnitude of the DOC;②Effect on PBMC proliferation of DOC by mitogen-stimulated;③Effect on one-way mixed lymphocyte reaction of DOC pretreated with IFN-γin vitro;④Effect on two-way mixed lymphocyte reaction of DOC pretreated with IFN-γin vitro; (4) MSCs and DOC with pretreated IFN-γor not , expression of TGF-β1 and IL-10 in their culture supernatant was dectcted by ELISA. 3. Experimental research on immunological reaction the and ability to ectopic bone of allogeneic tissue engineered bone implanted under the porcine subcutanenous tissue. (1) DBM materials were preparated from porcine fresh tibia by defatting , decalcifying and deproteinizating, and the morphological and histological structure were observed under inverted phase contrast microscope and electron microscopy; (2) TEB were constructed in vitro, and inverted phase contrast microscope and electron microscopy viewed the cell attachment, cell growth and secretion of matrix. (3) DBM materials were used as control, allo-TEB were implanted porcine subcutaneous tissue next to the left dorsal spine in 15 minipigs with healthy immune function used as experiment Group, and contralateral subcutaneous tissue was only implanted DBM material. The ectopic bone formation was detected in the 1stw, 2ndw , 4thw, 8thw and 12thw using hematoxylin and eosin and Masson stain. The level of TNF-αand IL-2 in local tissue and peripheral blood in1w, 2w, 4w, 8w and 12w were respectively dected using ELISAResults:1.The biological characteristics of porcine MSCs.(1)The characteristics of MSCs cultured using A-DMEM and F12-DMEM medium is the same, cells appearance presented cambiform or triangular-shaped, fibroblasts-like and vortex-like. The third generation was showed by FACS that expression of CD29, CD44, and SLA-Iwas strong positive in the cultured cells, and expression of CD14, CD34 and SLA-II is negative. Compared with MSCs cultured with F12-DMEM medium, MSCs cultured with A-DMEM medium on 3rdd and 5thd of primary culture growed adherently and more cloned, growed to 80-100% in a relatively more short time, with more number of times cell largest passaged. (2) There was no obvious difference in adherence of DOC and MSCs. Doubling time in Growth curve of MSCs was 36.8h, and DOC was 38.9h. Compared with the cell cycle of DOC, MSCs accounted for larger proportion in S+G2 phase of cell cycle, relatively small proportion in G1 phase. These suggested that MSCs proliferated and growed faster than DOC. (3) On 14thd after osteogenic induction, there were a lot of calcium deposition in intercellular substance with Von-Kossa staining; there were 85% positive cells with ALP staining; there were a lot of calcium deposition in the massive cells with Alizarin red staining; there were osteocalcin-positive cells by immunocytochemical staining. 2. Experiment on immunogenicity of DOC. (1) Results by FACS showed that expression of SLA-I increased in MSCs+IFN-γgroup and DOC+IFN-γgroup(P<0.05); morever, expression of SLA-II increased significantly (P <0.01).(2) Results by RT-PCR showed that expression of SLA-I(P1, P14) increased expression in DOC group, MSCs+ IFN-γgroup and DOC+IFN-group (P<0.05); and expression of SLA-II(DRA, DRB,DQA,DQB) significantly increased(P<0.01). (3)①Above 1×104 magnitude of DOC failed to stimulate PBMC proliferation, less than 1×104 magnitude of DOC promoted PBMC proliferation. Inhibition effects correlated with cell quantity positively.②DOC inhibit PBMC proliferation by the PHA-stimulated.③DOC with IFN-γpretreatment still inhibit PBMC proliferation by Con-A and PHA-stimulated.④DOC with IFN-γpretreatment inhibit PBMC proliferation in vitro two-way mixed lymphocyte reaction (pPBMC+hPBMC). (4) DOC and MSCs could secrete TGF-β1 and IL-10, and secretion of the IL-10 levels in DOC higher than in the MSCs (P <0.01). However, TGF-β1 levels secreted from MSCs pretreaed with IFN-γis significantly higher than those without the stimulation of IFN-γ, and TGF-β1 secretion levels of DOC pretreaed with IFN-γsignificantly lower than without the treatment with IFN-γ. 3. Experimental research on immunological reaction the and ability to ectopic bone of allogeneic tissue engineered bone implanted under the porcine subcutanenous tissue. (1) DBM materials maintained natural reticular structure.(2) On 7thd, afte DOC were constructed with DBM materials, cell adhesion in the surface and inner porous wall of material was shown, and the number of mitosis doubled. SEM viewed cells alignment with the rules surrounding a secretion of extracellular matrix. (3) All minipig failed to show off postoperative fever, chills and other systemic reactions. The minor tissue reaction were only seen around bilateral implants after 1, 2 and 4 weeks , but in the 8th and 12th weeks gradually disappear. Ectopic bone formation mostly depended on os endochondrale through histological observation.Conclusions:(1) Porcine MSCs are strongly osteogenic ability which can be differentiated into osteoblast, and express alkaline phosphatase and osteocalcin under the inducible conditions, therby are with the potential as seed cells.(2) The in vitro experiments show the progeny of porcine MSCs differentiated into osteoblast remains low immuogenicity, but the proinflammatory cytokine stimulation may enhance the immunogenicity, which can regulate immune responses by probably secreting cytokines for immune regulatory role. (3) The in vivo experiments show allo-TEB graft has good ectopic bone effects, but which can trigger early mild immune reaction to a host which is gradually disappearing with the extended time.In short, it may be good and effective therapeutic choice for tissue engineeried bone in vivo that allogeneic MSCs used as seed cells and DBM materials are co-cultured.
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