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Objective To investigate the feasibility of bone marrow mesenchymal stem cells (BMSCs) combined with type Ⅱ collagen-hyaluronic acid-oxidized chondroitin sulfate (Col Ⅱ-HA-OCS) biomimetic hydrogel to repair articular cartilage defect in porcine and the role of the transplanted cells played in the process of cartilage repair.Methods A articular cartilage defect model which remaining cartilage calcified zone was created in the knee of Bama minipigs,the autologous BMSCs was used as seeds for transplantation and was labeled by the 5,6-carboxyfluorescein diacetate succinimidyl ester (CFDA SE).Animals were randomly divided into three groups:Group A (blank group) was left untreated,group B (cell-free biomimetic hydrogel group) was filled with biomimetic hydrogel and group C (BMSCs combined with biomimetic hydrogel group) was filled with the CFDA SE labeled autologous BMSCs combined with biomimetic hydrogel.One month after the operation,BrdU labeled liquid was injected intravenously into the animals 24 h and 48 h before specimens were taken from the executed animals.Partial cartilage repaired tissue in Group C was taken,cryosectioned and stained with DAPI and BrdU immunofluorescence.Confocal laser scanning microscope was used to observe and count the cells.Specimens of the three groups were analyzed through gross observation and histological staining,and scored according to the international cartilage repair society (ICRS) gross morphological score and ICRS histological score.Results Laser scanning confocal microscopy showed (97.3 ± 2.6) ％ of the cells were derived from the implanted BMSCs in repaired tissue and that the ratio of these cells with proliferative capacity was (76.6 ± 2.5) ％.Gross observation suggested most of the cartilage defect areas in Group C were filled with ivory tissue,but those in Group A and B were still obvious depression.Histological staining showed the cartilage defect areas in Group C were filled with cartilage like tissue,which was well integrated with the surrounding normal cartilage,presented a few cartilage lacunas could be seen,and had contents of Col Ⅱ and glycosaminoglycan similar with the adjacent normal cartilage.There was almost no filler in the defect area in Group A.There was little fibrous tissue in the defect area in Group B.ICRS gross score was (8.3 ± 1.0) points in Group C,higher than that in Group A [(0.5 ± 0.6) points] and Group B [(2.3 ± 0.5) points] (P ＜ 0.05).ICRS histological score was (10.3 ± 2.4) points in Group C,higher than that in Group A [(0.5 ± 0.6) points] and Group B [(4.5 ± 1.0) points] (P ＜ 0.05).Conclusions BMSCs combined with Col Ⅱ-HA-OCS biomimetic hydrogel for repairing porcine articular cartilage defects can achieve satisfactory results.Implanted BMSCs are the main component of the cell composition in the repaired tissue and gradually differentiated into chondrocytes.
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Objective To compare the stress distributions on the anterior horn,body part and posterior horn of menisci between the normal and the injured knees with anterior cruciate ligament (ACL) deficiency using the three-dimensional finite element analysis.Methods A three-dimensional finite element model of tibiofemoral joint was created to simulate the motion states of the normal and ACL-deficiency knees at extension,as well as 15° and 30° flexions.Meanwhile,700N axial load and 134N posterior load were applied to the femur.Then,the stress on the anterior horn,body part and posterior horn of medial and lateral menisci were compared between the normal and ACL-deficient knees.Results With ACL deficiency,when stretching the knees straightly,the stress on the anterior horn of medial meniscus increased to 100.7％ of the normal knees,bigger than that of the affected lateral meniscus (30.7％).At 15° and 30° flexions,the stress on the posterior horn of the medial meniscus in ACL-deficiency knees increased by 36.4％ and 59.7％ respectively when compared to normal knees,while the stress on that of the lateral meniscus did not increase significantly.Apart from the stress on the body part of the lateral meniscus increasing by 39.5％ at extension in ACL-deficiency knees,no obvious changes were observed in the stress on the body part of the medial and lateral menisci.Conclusion ACL deficiency has different effect on the stress of different parts of the meniscus.It mainly increases the stress on the anterior horn of the medial meniscus at extension and that of the posterior horn of the medial meniscus at flexion.
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Objective: The aim of the present study was to develop a more realistic finite element [FE] model of the human anterior cruciate ligament [ACL] tibial insertion and to analyze the stress distribution in the ACL internal fibers under load. Methods: The ACL tibial insertions were processed histologically. With Photoshop software, digital images taken from the histological slides were collaged, contour lines were drawn, and different gray values were filled based on the structure. The data were exported to Amira software and saved as ".hmascii" file. This document was imported into HyperMesh software. The solid mesh model generated using HyperMesh software was imported into Abaqus software. The material properties were introduced, boundary conditions were set, and load was added to carry out the FE analysis. Results: The stress distribution of the ACL internal fibers was uneven. The lowest stress could be observed in the ACL lateral fibers under tensile and shear load. Conclusion: The establishment of ACL tibial insertion FE model and mechanical analysis could reveal the stress distribution in the ACL internal fibers under load. There was greater load carrying capacity in the ACL lateral fibers which could sustain greater tensile and shear forces
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Objective To establish osteochondral scaffolds with remained calcified cartilage zone for finding an ideal scaffold for tissue engineered repair of osteochondral defect.Methods Cartilage zone was harvested from fresh adult porcine knee to fabricate type Ⅱ collagen hydrogel.Bone blocks measuring 8 mm in diameter with calcified cartilage zone were prepared by trephine and acellular treatment was performed.Histological staining was used to identify complete removal of cells.To fabricate the osteochondral models containing calcified cartilage zone,type Ⅱ collagen was seeded onto the acellular bone blocks with calcified cartilage zone,lyophilized,and cross-linked with 10 g/L of genipin ethanol solution.Then cell seeding and scanning electron microscope were performed after the models were established.Results HE staining,toluidine blue staining,solid green staining,safranin O staining,and DAPI staining showed cells were completely removed from bone blocks by decellularized process.Porosity of type Ⅱ collagen sponge was (91.1 ±3.8) ％ and pore size was (79.7 ± 17.1) μm.Porosity of acellular bone blocks was (73.5 ±2.6)％ and pore size was (470.2 ± 158.8) μm.Cells seeded onto osteochondral scaffolds grew well by scanning electron microscope.Conclusion Osteochondral scaffolds with calcified cartilage zone provide good biocompatibility and suitable pore size and porosity and may be an ideal material for repairing osteochondral defect in tissue engineering.
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The aim of this study was to label rabbit bone derived mesenchymal stem cells (BMSCs) with superparamagnetic iron oxide particles (SPIO) and to study the effects of magnetic labeling on the multi-differentiation of BMSCs. Rabbit BMSCs were isolated, purified, expanded, then coincubated with SPIO(25 microg/ml) complexed to protamine sulfate (Pro) transfection agents overnight. Prussian blue staining and transmission electron microscopy were performed to show intracellular iron. Cell differentiation was evaluated. Both labeled and unlabeled BMSCs were subjected to osteogenic, adipogenic and chondrogenic differentiation to assess their differentiation capacity for 21 d. Osteogenic cells were stained with alizarin red to reveal calcium deposition, adipogenic cells were stained with oil redO' respectively. Chondrogenic cells stained with Safranin-O, glycosamino glycans, and type II collagen production was assessed by standard immunohistochemistry. Cell with immunohistochemistry staining were detected by polarized light microscopy and analysed by Image-Pro Plus software. The results showed that intracytoplasmic nanoparticles were stained with Prussian blue and observed by transmission electron microscopy clearly except the unlabeled control. As compared with the nonlabeled cells, it showed no statistically significant difference on the differentiation of the labeled BMSCs. And the differentiation of the labeled cells were unaffected by the endosomal incorporation of SPIO. In summary, BMSCs can be labeled with SPIO without significant change in cell multi-differentiation capacity.
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Animals , Rabbits , Adipocytes , Cell Biology , Bone Marrow Cells , Cell Biology , Cell Differentiation , Physiology , Cell Proliferation , Cell Tracking , Cells, Cultured , Chondrocytes , Cell Biology , Dextrans , Ferric Compounds , Magnetite Nanoparticles , Mesenchymal Stem Cells , Cell Biology , Osteoblasts , Cell Biology , Staining and LabelingABSTRACT
BACKGROUND: Chitosan-disodium β-glycerol phosphate (C/GP) gel has been shown to be compatible with the entrapment of viable chondrocytes, and bone marrow mesenchymal stem cells (BMSCs) are considered to be the potential cells used in tissue engineering. This experiment is aimed to observe the cytocompatibility of BMSCs with C/GP gel.OBJECTIVE: To study the effect of C/GP gel on the growth, proliferation and chondrogenic differentiation in vitro cultured BMSCs and explore a new carrier for the application of cartilage tissue engineering.DESIGN: Completely randomized controlled experiment.SETTING: Department of Bone and Joint Surgery, Affiliated Hospital of Luzhou Medical College; Center Laboratory of Southwest Hospital Affiliated to the Third Military Medical University of Chinese PLA.MATERIALS: The experiment was performed in Center Laboratory of Southwest Hospital Affiliated to the Third Military Medical University of Chinese PLA between October 2005 and April 2006, Six adult female mini-pigss were employed. C/GP gel is prepared by mixture the HCI solution of chitosan with salt solution of β-glycerol phosphate, allowed gel at 37 ℃ in incubator for about 5 to 10 minutes.METHODS: ① BMSCs culture: 4-6 mL of bone marrow harvested from the posterior superior lilac crest were plated at 20 ×10<'6>/100 mm dish and then grown for 14 days in complete media, consisting of DMEM/F-12 supplemented with 10% fetal ovine serum. Cells were harvested and re-seeded for subculture. ② BMSCs differentiation assays: Osteogenic differentiation was assessed by histologic detection of alkaline phosphatase activity and calcium in cultures under osteogenic conditions. Chondrogenic differentiation was evaluated by histology for toluidine blue and immunohistochemistry for type Ⅱ collagen in cultures under chondrogenic conditions. ③ In vitro assays, expanded BMSCs were suspended in C/GP solution and allowed gel at 37 ℃ in incubator for about 5 to 10 minutes, then cultured under chondrogenic conditions for 3 weeks. Cells attached to and viability in C/GP gel was monitored with the aid of an inverted light microscope. Chondrogenic differentiation of cell in C/GP gel were assessed by histological and immunohistocbemistry. The cell proliferated was monitored by MTT after 2, 5, 8 days seeding.MAIN OUTCOME MEASURES: ① Characterization of mini-pigs' BMSCs; ② BMSCs attached to and viability in C/GP gel; ③ Chondrogenic differentiation of BMSCs in C/GP gel; ④ BMSCs proliferated in C/GP gel.RESULTS: ① Characterization of mini-pigs' BMSCs: Cultured BMSCs showed fibroblastic morphology and were able to differentiate to chondrocytes or osteogenic cells under chondrgenic or osteogenic cultured condition respectively. ②BMSCs attached to and viability in C/GP gel: BMSCs attached to and remained > 90% viable in C/GP gels immediately post-casting, and throughout the 21 days, using MTT staining. ③ Chondrogenic differentiation of BMSCs in C/GP gel: During 21 days culture period in vitro, chondrogenic induced BMSCs produced amounts of de novo cartilage matrix in the chitosan, as assessed by histological and biochemical criteria. ④ BMSCs proliferated in C/GP gel: Chondrogenic induced BMSCs cultured in C/GP gels continued to proliferate. There was a significant difference among the values of optical density in the cells-gel constructs compared to the controls without cells after 2, 5, and 8 days of culture (P<0.05).CONCLUSION: It is confirmed that C/GP gel shows good cytocompatibility with BMSCs and contributes to the growth, proliferation and chondrogenic differentiation for BMSCs in vitro culture. C/GP gel can be a potential cell-carrier for tissue engineering of articular cartilage.
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[Objective]Study on the Pathology shape structure of calcified cartilage one in Osteoarthritis knee joint and provide the theory for diagnosis and treatment of correlated disease.[Method]Osteoarthritis condyles of femur(n=21) were collected from knee joint replacement and the paraffin sections were prepared after fixation and decalcification.After stained with Safranin O/fast green,the shape structure of calcified cartilage zone was observed by microscope.[Result]Mean age of osteoarthritis patient was 65.57?7.43 yearsold;mean stature was(155.38+5.32)cm;mean weight was(59.95 ?8.99)kg;mean pathogenesis was(13.04?9.66) years.After stained with Safranin O/fast green,the pathological changes of calcified cartilage zone were defined as follow.Tidemark drift and thickening accompany with blood vessel invasion.The gap of tidemark increased width.Calcified cartilage zone desmoid changed or defect.[Conclusion]Calcified cartilage zone in Osteoarthritis knee joint taken place specific pathological changes.
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Objective To investigate the effect of goat autologous serum at different concentrations on the proliferation of bone marrow-derived mesenchymal stem cells(bMSCs) for optimal serum dose.Methods Autologous serum was prepared from 6 goats at 12 months old,and their bMSCs were isolated from bone marrow.Then the obtain bMSCs were cultured in DMEM/F12 medium with supplement of 5%,10%,or 15% of corresponding autologous serum,and 10% fetal bovine serum served as control.The morphology of bMSCs,cells counting and MTT assay were observed or carried out.Results Morphology of the bMSCs in the different groups were similar.The proliferation of 5% autologous serum group was slower compared with that of 10% fetal bovine serum group(P
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Objective To study the effects of hypoxia on repair of radiation damage and the possible mechanism. Methods Rat jejunal epithelium cell was taken as study object and divided into control group, irradiation group and irradiation-hypoxia group. The irradiation group was irradiated only once by 60 Co-? ray at the dose of 12 Gy and the irradiation-hypoxia group was treated with hypoxia (5% oxygen) for 6 h after irradiation. MTT was used to detect the survival rate of the irradiated cells, 3H-TdR incorporation was used to detect cell proliferation, and unscheduled DNA synthesis and single cell gel electrophoresis were used to detect DNA repair and flow cytometry was used to detect cell cycle. Results As compared with the irradiation group, the cells in the irradiation-hypoxia group repaired rapidly, proliferated obviously and the survival rate was increased about 10% (P