| According to statistics, there are about one thousand and three hundred million people suffer from bone fracture in China every year,10-15%of which won't be able to be cured.Moreover, the number of orthopedical operation from replacement arthroplasty, trauma,abnormal development of skeleton will be one million or so,about half of which need bone transplantation, but the materials of bone transplantation are insufficient whatever in number or quality. The question that orthopedists often face in most time is to deal with bone fracture in clinic. Most patients suffering from bone fracture will be cured if they are treated in time, but in defined time they aren't healed which are called delayed union. The reason of poor heal include oversize of interspace of fracture fragments, hypopexia, inadequate blood supply of fracture fragments, open fractures combined with widespread damage of soft tissue and so on. Methods to deal with these things are to keep continuous immobilization, but sometimes the state of patients suffering immobilization being not cured are called disunion. Ways of curing disunion maybe physical stimulation or bone transplantation. So far, most bone transplants originate from autograft, namely get a bone from himself or herself to transplant some required site in himself or herself. Generally speaking, autograft has a best effect on bone transplantation and has no disease transmission in clinic. But the number of autograft is inadequate to supply strong and gross bone mass. So the apply of autograft is limited. While allograft that getting bones from other donors need special handing(e.g. cryogenic freezing, freeze dehydration, irradiation treatment) to reduce the antigen to lower reject reaction.The most disadvantage of allograft is that it possible spread hepatitis virus and human immunodeficiency virus, which deserve paying attention.Therefore, the emphasis of this paper focus on xenograft.Namely, bones of other species are handled by Triton X-100 to get rid of antigen of these bones and leave minerals and some organic principle to apply as bone transplant mass, which are able to supply degradated bone scaffold, which are cultured in vitro with autoallergic bone marrow stromal stem cells(BMSCs) by ways of tissue engineering to be transplanted into the defect of animal bones finally to assess the repair effect. Meezan first used chemical detergents, to isolate morphological and chemical pure basement membranes from several tissues, Then, Wilson use Triton X-100 to get rid of cells, antigen, fat, resoluble protein and glucosamine to produce acellular matrix and apply SEM and microscopic observation to proved that no cells existed in the prepared acellular matrix of small caliber vascular prostheses of dog.Most collegen and plastic protein existed in this kind of matrix.American Lifecell Company used the same method to made a success in business. Dahms'results about acellular gallbladder indicated that type I and III collegen and plastic proteins would existed in the acellular matrx. Therefore, current method kept most bioactive components to promote the growth of cells and provided an effect resolution to repair tissue defect.We believed that used Triton X-100 and other associated drug to make bone scaffolds for tissue engineering to repair animal bone defect with own's bone marrow mesenchymal stem cells in vitro.Main methods and results:1. Preparation of acellular bone matrixComparing with flesh spongy bone, appearance of ANECM is more shallow and white. We also use different concentration of TritonX-100 and time of cell extraction and Prove 30 days of cell extraction and 3%TritonX-100 to be most effective. Cell component isn't found under light and electron microscope. Under light microscope, collagen fibers of arrange uniformly. It's blankness in ellipse bone lacunas and there is no nucleus and other configuration. Osteocytes are in focus under scanning electron miscroscope of flesh spongy bone. No osteocyte and remnent is observed in ANECM. The wall of bone lacuna is smooth and regular. We surveyed the density and other parameters of histomorphology of bone lacunas. Those parameters have important significance to measure the bulk of osteocytes. We may estimate the cell number per volumn of bone tissue and provide the foundation for construction of engineering bone.2. Immunohistochemical stainingLN and FN dyeing submit bolt and line shape in bone matrix around bone lacuna. Distinct brown provides negative. Negative comparisons have not brown granules.There are positive markers around bone lacuna and because high electron density distribution of DAB reaction production submits block and reticulation.3. SEM observationScanning electron microscope displayed that different layers of acellular natural bone matrix connected loosely and presented amounts of pore space between layers.4. TEM observationTransmission electron microscope showed that no high density image appeared in lacuna area.5. Residul test of chemical detergentsApexes of comparition sample in result are 2.495,4.008,4.009min, and apex areas are 946825,1652336,1645545. Sample of bone acellular extra-cell matrix has not apex about 3min. The lowest examination limit is 0.5μl/ml.6. Transplantation immunological analysisLymphocyte stimulation assay showed that immunological rejection of flesh bone was much greater than that of acellular natural bone matrix (p< 0.01).7. Culture of bone marrow mesenchymal stem cellsThe primary cells and the passage cells were mostly fusiform in shape, to be similar to fibroblast cell.8. Analysis of flow cytometrThe flow cytometer analysis showed that the membrane mark CD44, CD90,CD106 were positive, CD34,CD45,CDllb were negative.9. Observation of the induced cellsMost induced cells changed into round cells and square cells in shape,and their body became larger and many minite black pellet present in the cytoplasm of these cells.10. Identity of induced cellsAfter BMSCs were induced, cell alizarin carmine staining showed that it was positive in osteogenetic cell, and ALP staining showed that black granules existed in induced cells. Immunohistochemisty staining showed that type I collagen existed in cytoplasm.11. SEM observation of acellular extra-cell matrix with induced cellsScanning electron microscopy displayed that osteoblast-like cells on the surface of acellular natural bone matrix aggregated together and grew as three-dimensional style.12. ALP detectionAlkaline phosphatase detection kit analysis indicated that acellular natural bone matrix prepared with TritonX-100 were able to promote the growth of osteoblast-like cells compared with calcium hydroxyapatite after 48 hours of culture.13. Experiment on bone acelluar extra-cell matrix with induced BMSCs repair-ing aminal bone defectBoth bone acelluar extra-cell matrix and calcium hydroxyapatite combined with osteoblast-like cells from BMSCs were implanted into the femural defect of rats after the defect models were made successfully. At the 4th week, fluorescence microscope showed that bar-like calcium hydroxyapatite and new bone with green fluorescence appear in scope of eyes in control group. Compared with the control group, experimental bone acelluar extra-cell matrix fused with peripheral new bone and we couldn't tell the differences with each other under flurescence microscope. At the 8th week, toluidine blue staining showed that osteon in new bone have formed whatever in control or test group.Conclusions:1. Prepare bone acelluar extra-cell matrix successfully.2. BMSCs were able to differentiate into osteoblast-like cells after the conditonal fluids was added.3. Prepared bone acelluar extra-cell matrix fit for the induced cells in vitro.4. Bone acelluar extra-cell matrix with osteoblast-like cells are able to repair femural defect of rats successfully. |
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