Objective To evaluate the feasibility and validity of chondrogenic differentiation of marrow clot after microfracture of bone marrow stimulation combined with bone marrow mesenchymal stem cells (BMSCs)-derived extracellular matrix (ECM) scaffold in vitro. Methods BMSCs were obtained and isolated from 20 New Zealand white rabbits (5-6 months old). The 3rd passage cells were cultured and induced to osteoblasts, chondrocytes, and adipocytes in vitro, respectively. ECM scaffold was manufactured using the 3rd passage cells via a freeze-dying method. Microstructure was observed by scanning electron microscope (SEM). A full-thickness cartilage defect (6 mm in diameter) was established and 5 microholes (1 mm in diameter and 3 mm in depth) were created with a syringe needle in the trochlear groove of the femur of rabbits to get the marrow clots. Another 20 rabbits which were not punctured were randomly divided into groups A (n=10) and B (n=10): culture of the marrow clot alone (group A) and culture of the marrow clot with transforming growth factor β3 (TGF-β3) (group B). Twenty rabbits which were punctured were randomly divided into groups C (n=10) and D (n=10): culture of the ECM scaffold and marrow clot composite (group C) and culture of the ECM scaffold and marrow clot composite with TGF-β3 (group D). The cultured tissues were observed and evaluated by gross morphology, histology, immunohistochemistry, and biochemical composition at 1, 2, 4, and 8 weeks after culture. Results Cells were successfully induced into osteoblasts, chondrocytes, and adipocytes in vitro. Highly porous microstructure of the ECM scaffold was observed by SEM. The cultured tissue gradually reduced in size with time and disappeared at 8 weeks in group A. Soft and loose structure developed in group C during culturing. Chondroid tissue with smooth surface developed in groups B and D with time. The cultured tissue size of groups C and D were significantly larger than that of group B at 4 and 8 weeks (P lt; 0.05); group D was significantly larger than group C in size (P lt; 0.05). Few cells were seen, and no glycosaminoglycan (GAG) and collagen type II accumulated in groups A and C; many cartilage lacunas containing cells were observed and more GAG and collagen type II were synthesized in groups B and D. The contents of GAG and collagen increased gradually with time in groups B and D, especially in group D, and significant difference was found between groups B and D at 4 and 8 weeks (P lt; 0.05). Conclusion The BMSCs-derived ECM scaffold combined with the marrow clot after microfracture of bone marrow stimulation is effective in TGF-β3-induced chondrogenic differentiation in vitro.
ObjectiveTo observe the feasibility of acellular cartilage extracellular matrix (ACECM) oriented scaffold combined with chondrocytes to construct tissue engineered cartilage.MethodsChondrocytes from the healthy articular cartilage tissue of pig were isolated, cultured, and passaged. The 3rd passage chondrocytes were labeled by PKH26. After MTT demonstrated that PKH26 had no influence on the biological activity of chondrocytes, labeled and unlabeled chondrocytes were seeded on ACECM oriented scaffold and cultivated. The adhesion, growth, and distribution were evaluated by gross observation, inverted microscope, and fluorescence microscope. Scanning electron microscope was used to observe the cellular morphology after cultivation for 3 days. Type Ⅱ collagen immunofluorescent staining was used to check the secretion of extracellular matrix. In addition, the complex of labeled chondrocytes and ACECM oriented scaffold (cell-scaffold complex) was transplanted into the subcutaneous tissue of nude mouse. After transplantation, general physical conditions of nude mouse were observed, and the growth of cell-scaffold complex was observed by molecular fluorescent living imaging system. After 4 weeks, the neotissue was harvested to analyze the properties of articular cartilage tissue by gross morphology and histological staining (Safranin O staining, toluidine blue staining, and typeⅡcollagen immunohistochemical staining).ResultsAfter chondrocytes that were mainly polygon and cobblestone like shape were seeded and cultured on ACECM oriented scaffold for 7 days, the neotissue was translucency and tenacious and cells grew along the oriented scaffold well by inverted microscope and fluorescence microscope. In the subcutaneous microenvironment, the cell-scaffold complex was cartilage-like tissue and abundant cartilage extracellular matrix (typeⅡcollagen) was observed by histological staining and typeⅡcollagen immunohistochemical staining.ConclusionACECM oriented scaffold is benefit to the cell adhesion, proliferation, and oriented growth and successfully constructes the tissue engineered cartilage in nude mouse model, which demonstrates that the ACECM oriented scaffold is promise to be applied in cartilage tissue engineering.
Objective To investigate the expression of connective tissue growth factor（CTGF）in human proliferative membranes of proliferative vitreoretinopathy（PVR），and the relationship among CTGF，transforming growth factor-beta; receptor（TGF-beta;R）and extracellular matrix（ECM）. Methods Immunohistochemistry method of streptavidin-biotin-peroxidase complex（SABC）was used to detect the expression of CTGF，TGF-beta;RⅡ，fibronectin（FN），collagen Ⅰ，and collagen Ⅲ protein in43periretinal membranes（PRM）of PVR obtained by vitrectomy，and the correlations of the expression of CTGF，TGF-beta;RⅡ and ECM were analyzed by statistics. Results CTGF and TGF-beta;RⅡ protein highly expressed in PRM of PVR and most of the CTGF-positive cells were epithelial cells.The result of immunohistochemistry showed that the positive rates of CTGF and TGF-beta;RⅡ protein were 70.6% and 76.5%in PVR C membranes，and 73.9% and 69.6%in PVR D membranes respectively.Relationship between positive expression and membranesprime; grades appeared no statistical correlation(P＞0.05).Statistical analysis showed that there was a correlation between the expression of CTGF and TGF-beta;RⅡ，FN，and collagen Ⅰ and Ⅲ protein，respectively. Conclusions The expression of CTGF and TGFbeta;RⅡ protein is up-regulated in PRM of PVR，which suggests that the activation of TGF-beta;RⅡ is involved in the production of CTGF，and CTGF is closely related to the production of ECM and play an important role in the pathogenesis of PVR. (Chin J Ocul Fundus Dis, 2006, 22: 192-195)
ObjectiveTo study the feasibility of human adipose-derived stem cells (hADSCs) combined with small intestinal submucosa powder (SISP)/chitosan chloride (CSCl)-β-glycerol phosphate disodium (GP)-hydroxyethyl cellulose (HEC) for adipose tissue engineering. MethodshADSCs were isolated from human breast fat with collagenase type I digestion, and the third passage hADSCs were mixed with SISP/CSCl-GP-HEC at a density of 1×106 cells/mL. Twenty-four healthy female nude mice of 5 weeks old were randomly divided into experimental group (n=12) and control group (n=12), and the mice were subcutaneously injected with 1 mL hADSCs+SISP/CSCl-GP-HEC or SISP/CSCl-GP-HEC respectively at the neck. The degradation rate was evaluated by implant volume measurement at 0, 1, 2, 4, and 8 weeks. Three mice were euthanized at 1, 2, 4, and 8 weeks respectively for general, histological, and immunohistochemical observations. The ability of adipogenesis (Oil O staining), angiopoiesis (CD31), and localized the hADSCs (immunostaining for human Vimentin) were identified. ResultsThe volume of implants of both groups decreased with time, but it was greater in experimental group than the control group, showing significant difference at 8 weeks (t=3.348, P=0.029). The general observation showed that the border of implants was clear with no adhesion at each time point;fat-liked new tissues were observed with capillaries on the surface at 8 weeks in 2 groups. The histological examinations showed that the structure of implants got compact gradually after injection, and SISP gradually degraded with slower degradation speed in experimental group;adipose tissue began to form, and some mature adipose tissue was observed at 8 weeks in the experimental group. The Oil O staining positive area of experimental group was greater than that of the control group at each time point, showing significant difference at 8 weeks (t=3.411, P=0.027). Immunohistochemical staining for Vemintin showed that hADSCs could survive at each time point in the experimental group;angiogenesis was most remarkable at 2 weeks, showing no significant differences in CD31 possitive area between 2 groups (P>0.05), but angiogenesis was more homogeneous in experimental group. ConclusionSISP/CSCl-GP-HEC can use as scaffolds for hADSCs to reconstruct tissue engineered adipose.
Objective To investigate the modulating effect of transforming growth factor beta;2 (TGFbeta;2) and extracellular matrix (ECM) on the transdifferentiation of human fetal RPE (hfRPE) cells into myofibroblast-like cells , and to determine the mechanism of signal transduction. Methods hfRPE cells were cultured on ECM coated or uncoated petri dish with or witho ut TGFbeta;2 in the medium. The expression of alpha;-smooth muscle actin (alpha;-SMA) were detected by immunocytochemistry examination, flow cytometry and Western blotting via calphostin C, genistein, PD98059, and Wortmannin. Results After cultured on ECM coated petri dish with TGFbeta;2 in the medium,there were obvious morphological changes of hfRPE cells including cellular elongating and appearing of actin microfilaments. The results of flow cytometry and immunocytochemistry examination showed that expression of alpha;-SMA obviously increased after TGFbeta;2 was added in the medium in a dose-dependent manner. Compared with which of hfRPE cells cultured on the uncoated surface of culture plates, the total mean fluore scence intensity (TMFI) of hfRPE cells cultured on FN-coated surface increased (38.01plusmn;1.14)% when the stimulation concentration of TGFbeta;2 was 50ng/ml(Plt;0.05). Western blotting further confirmed the effects. The changes mentioned above could be inhibited mostly by protein kinase C (PKC) and calphostin C (10 nmol/L)(Plt;0.01). Conclusion TGFbeta;2 may induce the transdifferentiation of hfRPE cells into myofibroblast-like cells in a dose dependent manner, which could be intensified by FN. These mediated effects of TGFbeta;2 and ECM may act via the PKC signal transduction pathway. (Chin J Ocul Fundus Dis, 2006, 22: 328-332)
OBJECTIVE To investigate the methods to fabricate repair materials of tissue engineered peripheral nerve with bioactivity of Schwann cells (SC). METHODS 1. The materials were made by dry-wet spinning process to fabricate PLA hollow fiber canal with external diameter of 2.3 mm, internal diameter of 1.9 mm, thickness of 0.4 mm, pore size of 20 to 40 microns, pore ratio of 70% and non-spinning fiber net with pore size of 100 to 200 microns, pore ratio of 85%. 2. SC were implanted into excellular matrix (ECM) gel to observe the growth of SC. 3. SC/ECM complex were implanted into non-spinning PLA fiber net to observe the growth of SC. 4. SC, SC/ECM and SC/ECM/PLA were implanted into PLA hollow fiber canal to bridge 10 mm defect of rat sciatic nerve. RESULTS 1. SC were recovered bipolar shape at 1 day after implantation, and could be survived 14 days in ECM gel. 2. After SC/ECM complex was implanted into PLA net, most of SC were retained in the pore of PLA net with the formation of ECM gel. SC could be adhered and grown on PLA fiber. 3. Most of SC in ECM gel could be survived to 21 days after transplantation. Survival cell numbers of SC/ECM and SC/ECM/PLA groups were obviously higher than SC suspension group. CONCLUSION Non-spinning PLA porous biodegradable materials with ECM is benefit for SC to be adhered and grown.
ObjectiveTo understand the research progress of the matrix metalloproteinases (MMPs) family in regulating the development of hepatocellular carcinoma (HCC) and its mechanism, in order to provide a reference for the basic research and clinical diagnosis and treatment of HCC. MethodThe relevant literature on the regulation of HCC occurrence, development, and mechanisms by MMPs both domestically and internationally in recent years was reviewed. ResultsThe extracellular matrix (ECM) microenvironment of HCC cells determined the invasiveness and degree of metastasis of tumor cells. The degradation and remodeling of ECM during epithelial mesenchymal transition (EMT) were the main factors contributing to the invasion and metastasis of HCC. The abnormal expression of most members of the MMPs family could lead to ECM breakdown, cell invasion and attachment, and markedly accelerate the process of EMT, thereby promoting the invasion and metastasis of HCC cells. At present, there were many MMPs related to the development of HCC, including MMP-1, 2, 3, 7, 9, 12, 13, 14. The relevant research on the relation between MMP-8, 10, 11, 15, 16, 20, 21, 26 or 28 and the development of HCC was relatively limited, while the exact research on the relationship between the MMP-17, 19, 23, 24, 25 or 27 and HCC development had not been retrievaled. ConclusionsThe MMPs family members (especially MMP-2, 3, 7, 9, 10, 12) play a crucial role in the progression of HCC, including proliferation, invasion, and metastasis. Further exploration of the potential intrinsic relation between all members of the MMPs family members and the development of HCC is crucial for predicting HCC metastasis potentiality and prognosis, as well as developing new or improved targeted anti-cancer therapies for HCC.
Objective To develop a new method for a tissue engineered vascular graft by combining endothelial cells and an acelluarized allogenic matrix. Methods Acellularized matrix tubes were obtained by a 0.1% trypsin and 0 02% EDTA solution for 24 hours and 1% Triton X 100 for 176 hours, respectively. Endothelial cells were isolated from alloaorta and expanded in vitro. Finally, the inner surface of acellularized matrix was reseeded with endothelial cells. Acellularity and reseeding were analysed by light microscopy and scanning electron microscopy. Results The acellularization procedure resulted in an almost complete removal of the original cells and the loose three-dimensional (3D) matrix. The acellular matrix could be reseeded with expanded endothelial cells in vitro, and endothelial cells had the potential of spread and proliferation. Conclusion Acellular matrix produces by Tritoon X-100 and trypsin possesses satisfactory biocompatibility for allogenic endothelial cell. Vascular grafts can be generated in vitro by a combination of endothelial cells and allogenic acelluarized matrix.
To study how to repair the cartilage defect according to the principles of tissue engineering with acellular cartilage matrix as scaffold material. Methods The ear cartilage was obtained from a New Zealand white rabbit(weighing 2.4 kg )and then treated by a modified Courtman’s four-step method to produce the acellular cartilage matrix. Eighteen New Zealand white rabbits (aged 6 months, weighing 2.4-2.6 kg) with no sex l imit were divided into three groups. Forevery rabbit, two pieces of ear cartilage measured 1 cm × 1 cm were excised in each ear. Defects were repaired as follows: group A with the combined graft of acellular cartilage matrix and perichondium, group B with acellular cartilage matrix and group C with perichondium. Three animals in each group were killed 4 and 12 weeks postoperatively, respectively. Tissue samples obtained were analyzed with gross observation, hematoxyl in-eosin stain, Safranine O-alcian blue stain and type II collagen messenger RNA in situ hybridization respectively. Results In gross observation, the repaired sites in groups A and B were not change meaningfully in their shape 4 weeks postoperatively; but they felt a bit of thicker and harder 12 weeks postoperatively. In group C two repaired sites formed scabs at 2 weeks and perforated at 5 weeks. In histological observation, there was a sl ight inflammatory reaction surrounding the acellular cartilage matrix 4 weeks after it was implanted in groups A and B. The inflammatory cells were mainly lymphocytes. The perichondrium graft in group C was collapsed in the defects in HE stain. The defect sites were negative for Safranine O-alcian blue stain and type II collagen mRNA in situ hybridization in all groups. At 12 weeks cells were found in the acellular matrix which arranged in irregular manner in group A in HE stain and was positive for Safranine O-alcian blue stain and type II collagen mRNA in site hybridization. In groups B and C, no new cell was found in HE stain and the repaired sites were negative for Safranine O-alcian blue stain and type II collagen mRNA in situ hybridization. Conclusion Acellular
Objective To introduce the latest advances of research on repair of the degenerative intervertebral disc with gene transduction.Methods The recentlypublished articles about the treatment of degenerative disc with gene transduction were reviewed, especially the articles published during the recent 5 years about the application of this therapy to regulating the synthesisand degradation of the extracellular matrix of the degenerative intervertebral disc.Results The shape and function of the normal intervertebral disc were reported to be closely related to the synthesis and degradation of the extracellular matrix of the intervertebral disc. The extracellular matrix of the intervertebral disc was a target for the gene transduction to repair the degenerative intervertebral disc. There was a great development of the treatment with gene transduction, especially in vector choice, target gene transduction, and transgene regulation and safety. Conclusion The advances of the research have indicated that repair of the degenerative intervertebral disc with gene transduction is a keyto curing the disease of the degenerative intervertebral disc.