0.05).[Conclusion]Porcine articular cartilage extracellular matrix derived scaffold used in heterogenic transplantation or allograft have no immunologic rejection.This research provides a feasibility for application of xenogenic acellular cartilage to clinic.

[Objective]To establish a method for obtaining well-differentiated goat chondrocytes quickly in large scale by RCSS using microcarriers technique. [Method]Articular chondrocytes were harvested from goat by sequential digestion with trypsin and collagenase , and then grown in RCSS bioreactor culture system which contained the Cytodex-3 microcarriers in the culture medium (DMEM) . Growth of chondrocytes on Cytodex-3 microcarriers was observed dynamically under phase contrast microscope . Immunocytochemical analysis was performed for type Ⅰcollagen and type Ⅱcollagen. [Result]The articular chondrocytes attached rapidly to the surface of Cytodex-3 microcarriers. Quick growth of these cells was observed after they fully spread onto the microcarriers. The density of chondrocytes increased by 15～17 times in later stage of culture as compared with the initial density. The harvested chondrocytes had no detectable staining for collagen type Ⅰ, but stained intensively for collagen type Ⅱ. [Conclusion]Microcarrier culture of chondrocytes can yield a large quantity of goat cells within a short time ,which will be of benefit for banking cartilage cells for reconstruction of impaired cartilage by way of tissue engineering.

[Objective]To analyze factors related with surviving rate and to evaluate effectiveness of the adjuvant chemotherapy in the treatment of osteosareoma.[Method]Eighty-four patients aging from 9 to 47 years(averaged,21 years)were analysed respectively:52 of them were male and 32 were female.The tumors were located at the femur in 42,the tibia in 29,the humerus in 13.There were 22 patients classified as stage ⅡA and 62 patients as ⅡB.The pathological study,of subtype of osteosarcoma revealed that 47 were osteoblastic,11 chondroblastic,19 fibroblastic and 7 other subtypes.There were 46 patients who received the chemotherapy;38 patients without chemotherapy,49 of the 84 patients treated surgically had limb salvage procedures,35 had amputations.Multivariate analsis was done by using the proportional hazards model of Cox,categoric data were analyzed by using the chi-square statistic.[Result]All cases were followed up from 6 to 74 months(with an average of 25.5 months).Cox model analysis showed that age,sex,site,and subtype were not significant prognostic variables in this group of patients;the significant affecting prognosis in patients was Enncking staging and chemotherapy.Chi-square showed significant difference in the higher metastasis rates of lung in the group without chemotherapy than in those with chemotherapy group(P

BACKGROUND:Soft tissue engineering mainly includes seed cells,scaffolds,cytokines and bioreactors,among which,the scaffolds are the key link in the construction of tissue-engineered cartilage.OBJECTIVE:To prepare an articular cartilage extracellular matrix/human umbilical cord Wharton gel porous scaffold,and to evaluate its physicochemical properties and biocompatibility.METHODS:The articular cartilage extracellular matrix/human umbilical cord Wharton gel porous scaffold was prepared by freeze thawing drying method using porcine articular cartilage extracellular matrix and human umbilical cord Wharton glue as raw materials.The porosity,water absorption,tissue composition and longitudinal compressive elastic modulus of the scaffold were measured and histologically stained.Rabbit chondrocytes were co-cultured with the articular cartilage extracellular matrix/human umbilical cord Wharton gel porous scaffold for 7 days.Then,scanning electron microscopy,live-dead cell staining and hematoxylin-eosin staining were performed.In addition,rabbit chondrocytes were cultured in the extract of the articular cartilage extracellular matrix/human umbilical cord Wharton gel porous scaffold and cell culture medium for 6 days,respectively;and MTT assay was used to detect cell proliferation.RESULTS AND CONCLUSION:The articular cartilage extracellular matrix/human umbilical cord Wharton gel porous scaffolds had a cross-section of uniform porous network structure and a vertical cross-section of the vertical tubular structure,and the pore wall was densely covered with cartilage fibers.The composite porous scaffold was positive for hematoxylin-eosin staining,safranin O staining and toluidine blue staining,and contained collagen and glycosaminoglycan ingredients.The water absorption,porosity and longitudinal compressive elastic modulus of the scaffolds were (17.418 8±0.909 0)％,(81.495 1±6.621 0)％ and (2.833 3±0.456 4) kPa,respectively.After 7 days of co-culture,rabbit chondrocytes adhered to the scaffold and proliferated,and further grew into the pores of the scaffold.Moreover,the scaffold was non-toxic to the rabbit chondrocytes.To conclude,the physiochemical properties and biochemical components of articular cartilage extracellular matrix/human umbilical cord Wharton gel porous scaffolds are similar to those of natural cartilage,and the scaffold has good biocompatibility.

BACKGROUND:Accumulative evidence supports that co-culture technology can be applied to construct the tissue-engineered cartilage with excellent biological characters. OBJECTIVE:To elaborate the co-culture concept and conclude and analyze seed cell sources, cel mixed ratio, spatial y-defined co-culture models and biomaterials in co-culture systems to conclude and analyze the biological characters of tissue-engineered cartilage, and to prospect progression of co-culture systems in cartilage tissue engineering. METHODS:The first author retrieved the databases of PubMed, Web of Science, and CNKI for relative papers published from January 1976 to May 2016 using the keywords ofco-culture, co-culture systems;articular cartilage, chondrocytes, mesenchymal stem cells;tissue engineering, articular cartilage tissue engineeringin English and Chinese, respectively. Finally 60 literatures were included in result analysis, including 1 Chinese and 59 English articles. RESULTS AND CONCLUSION:Co-culture technology emphasizes the role of microenvironment in terms of various physical, chemical and biological factors in the cell processing. In cartilage tissue engineering, co-culture systems contribute to maintain the viability and natural cell phenotype of chondrocytes and induce cartilage differentiation of mesenchymal stem cells. In addition, co-culture technology provides a novel way for cartilage tissue engineering to overcome the shortage of chondrocytes and repair injury to the cartilage-subchondral bone. However, the mechanisms of cell-cell interaction in co-culture systems still need to be explored in depth, so as to optimize the co-culturing conditions and construct perfect tissue-engineered cartilage.

BACKGROUND:At present, a variety of extracel ular matrix-derived scaffolds have been successful y applied for cartilage tissue engineering in experiment and clinical practice. OBJECTIVE:To summarize the application and research status of extracel ular matrix-derived scaffolds in cartilage tissue engineering. METHODS:A computer-based online search in PubMed, CNKI, CqVip and WanFang databases was performed using the keywords of“tissue engineering, cartilage, extracel ular matrix, scaffolds”in English and Chinese, respectively. A total of 1 140 literatures were retrieved, and final y 65 eligible literatures were included. RESULTS AND CONCLUSION:In terms of the components, extracel ular matrix-derived scaffolds are divided into monomeric natural polymers, mixed natural polymers, natural polymers compositing with synthetic polymers as wel as acel ular extracel ular matrix-derived materials. Extracel ular matrix-derived scaffolds hold good biocompatibility and degradability, and can promote proliferation and differentiation of choncrodytes;therefore, they as good bionic scaffolds have been applied for cartilage tissue engineering in clinical practice, However, poor mechanical properties and difficulty to molding should never be ignored. Further research should focus on improving the preparation technology by combining synthetic materials with extracel ular matrix-derived scaffolds for cartilage tissue engineering.

BACKGROUND:Choose an ideal vector for amplified chondrocytes arouses more and more attention during the construction of epiphyseal cartilage tissue engineering. OBJECTIVE:To explore the feasibility and performance of epiphyseal cartilage tissue engineering scaffold constructed by chitosan and extracellular matrix of cartilage. DESIGN,TIME AND SETTING:An in vitro study was performed at Department of Orthopedics,General Hospital of Chinese PLA from December 2007 to March 2003. MATERIALS:Chitosan(deacetylation:90%;Mr10?105) was provided by Haihui Bioengineering Company,Qingdao;articular cartilage of swine was collected from market. METHODS:Fresh porcine articular cartilages were obtained and shattered in the iso-osmia liquid. After pulverization and gradient centrifugation,3% artilage microfilament suspension was equally mixed with 2% chitosan. Three-dimensional porous scaffolds were fabricated using a simple freeze-drying method. MAIN OUTCOME MEASURES:After the second gradient ethanol treatment,the scaffolds were investigated by histological staining and scanning electron microscopy to measure aperture,porosity,and water absorption rate. MTT test was also done to assess cytotoxicity of the scaffolds. After induced by transforming growth factor-?1(TGF-?1) ,bone marrow mesenchymal stem cells(BMSCs) of rabbits were incubated onto the scaffolds. Cell proliferation and differentiation were analyzed using inverted microscopy and scanning electron microscopy. RESULTS:The three-dimensional porous scaffold had good pore interconnectivity with pore diameter(161?31) ?m,(90.1?1.6) % porosity and(2 361?132) % water absorption rate. The histological staining showed that toluidine blue,safranin O and anti-collagen II immunohistochemistry staining were positive. The intrinsic cytotoxicity assessment of the scaffolds using MTT test showed that the scaffolds had no cytotoxic effect on BMSCs. Most of the BMSCs attached and covered the surface of the scaffolds with matrix secretion. CONCLUSION:The three-dimensional porous scaffold constructed by extracellular matrix of cartilage and chitosan has good pore diameter and porosity,non-toxicity and good biocompatibility,so it is a suitable scaffold for epiphyseal cartilage tissue engineering.

Objective To fabricate cartilage extracellular matrix (ECM) oriented scaffolds and investigate the attachment, proliferation, distribution and orientation of bone marrow mesenchymal stem cells (BMSCs) cultured within the scaffolds in vitro. Methods Cartilage slices were shattered in sterile phosphate-buffered saline (PBS) and the suspension were differentially centrifugated untill the micro- fiber of the cartilage extracellular matrix was disassociated from the residue cartilage fragments. At last the supernatant were centrifugated, the precipitation were collected and were made into 2%-3% suspension. Using unidirectional solidification as a freezing process and freeze-dried method, the cartilage extracellular matrix derived oriented scaffolds was fabricated. The scaffolds were then cross-linked by exposure to ultraviolet radiation and immersion in a carbodiimide solution. By light microscope and scan electron microscope (SEM) observation, histological staining, and biomechanical test, the traits of scaffolds were studied. After being labelled with PKH26 fluorescent dye, rabbit BMSCs were seeded onto the scaffolds. The attachment, proliferation and differentiation of the cells were analyzed using inverted fluorescent microscope. Results The histological staining showed that toluidine blue, safranin O, alcian blue and anti-collagen Ⅱ immunohistochemistry staining of the scaffolds were positive. A perpendicular pore-channel structures which has a diameter of 100 μm were verified by light microscope and SEM analysis. The cell-free scaffolds showed the compression moduli were (2.02±0.02) MPa in the mechanical testing. Inverted fluorescent microscope showed that most of the cells attached to the scaffold. Cells were found to be widely distributed within the scaffold, which acted as a columnar arrangement. The formation of a surface cells layer was found on the surface of the scaffolds which resembled natural cartilage. Coclusion The cartilage extracellular matrix derived oriented scaffolds have promising biological, structural, and mechanical properties.

Objective To probe the immunological traits of mesenchymal stem cells derived from umbilical cord Wharton's jelly (WJMSCs). Methods The diced Wharton's jelly which was from healthy fullterm birth human umbilical cord was cultured. The mesenchymal stem cells were identified with mesenchymal stem cells markers expression by flow cytometry and multiple differentiation ability. The expression of MHC- Ⅰ / Ⅱ, costimulatory molecules (CD40, CD80 and CD86) was detected with flow cytomctry, immunocytochemistry, and RT-PCR. The expression of immune inhibitors like HLA-G, IDO, and PGE2 was detected by immunocytochemistry and RT-PCR. The expression of immune-related molecules as IL-10, TGF-β, FGF and VEGF was detected with antibody microarray and western blot. Further more, to clarify the in vivo immune reaction of hWJMSCs, we fabricated the hWJMSC-scaffold constructs and implanted them into the rabbit backs. The lymphocyte infiltration and implanted cell survival observed with immunofluorescence. Results After culturinge of diced Wharton's jelly tissue, we obtained spindle-shaped cells. With differentiation medium, the cells can differentiate into osteoblasts, chongdrocytes, adipose cells and schwann cells. Expression of MHC, costimulatory molecules, and a series of immune suppressive-related molecules was found. Immune inhibitors as HLA-G, 1DO, PGE2, and immune suppressive related molecules as HGF, VEGF, TGFand IL-10 were positively expressed. But the cells did not express MHC-Ⅱ. No immune rejection was observed in vivo after implantation of hWJMSC-scaffold constructs. Conclusion It can be concluded that hWJMSCs have very low immunogenicity, which means the cells have potential to induce immune tolerance.The hWJMSCs do not provoke immune rejection in vivo.

BACKGROUND:Cartilage tissue engineering has been widely used to achieve cartilage regeneration in vitro and repair cartilage defects. Tissue-engineered cartilage mainly consists of chondrocytes, cartilage scaffold and in vitro environment. OBJECTIVE:To mimic the environment of articular cartilage development in vivo, in order to increase the bionic features of tissue-engineered cartilage scaffold and effectiveness of cartilage repair. METHODS: Knee joint chondrocytes were isolated from New Zealand white rabbits, 2 months old, and expanded in vitro. The chondrocytes at passage 2 were seeded onto a scaffold of articular cartilage extracelular matrix in the concentration of 1×106/L to prepare cel-scaffold composites. Cel-scaffold composites were cultivated in an Instron bioreactor with mechanical compression (1 Hz, 3 hours per day, 10% compression) as experimental group for 7, 14, 24, 28 days or cultured staticaly for 1 day as control group. RESULTS AND CONCLUSION:Morphological observations demonstrated that the thickness, elastic modulus and maximum load of the composite in the experimental group were significantly higher than those in the control group, which were positively related to time (P < 0.05). Histological staining showed the proliferation of chondrocytes, formation of cartilage lacuna and synthesis of proteoglycan in the experimental group through hematoxylin-eosin staining and safranin-O staining, which were increased gradualy with mechanical stimulation time. These results were consistent with the findings of proteoglycan kit. Real-time quantitative PCR revealed that mRNA expressions of colagen type I and colagen type II were significantly higher in the experimental group than the control group (P < 0.05). The experimental group showed the highest mRNA expression of colagen type I and colagen type II at 21 and 28 days of mechanical stimulation, respectively (P < 0.05). With the mechanical stimulation of bioreactor, the cel-scaffold composite can produce more extracelular matrix, such as colagen and proteoglycan, strengthen the mechanical properties to be more coincident with thein vivo environment of cartilage development, and increase the bionic features. With the progress of tissue engineering, the clinical bioregeneration of damaged cartilage wil be achieved.