Cartilage is one of the earliest reconstructed tissues used in tissue engineering. Due to the lack of appropriate seeding cells, cartilage tissue engineering is, however, relatively lagged behind. With the emergence of stem cell research, adipose stem cells(ASCs) are introduced as seeding cells into tissue engineering for possessing many advantages such as wide spreading, large amount of cells available and easy to obtain. However, the outcome of tissue engineered cartilage construction by ASCs is not as ideal as that by bone marrow stem cells (BMSCs) yet. Low efficiency of ASC chondrogenesis is considered the major cause. This review summarizes the purification of adipose-derived cells, maintenance of sternness and optimization of ehondrogenie induction, which play vital roles in improving ASC s chondrogenesis.

Autologous cells of bone, cartilage, intervertebral disc and tendon, are hard to be cultured and proliferate in vitro and thus have difficulty to be introduced in the repair of damaged tissue. Stem cells have the ability to self-renew and differentiate into many tissue types. Recent progress in stem cell research has led to an enthusiastic effort to utilize stem cells for orthopaedic tissue regeneration. Due to the abundance and easiness of harvest, adipose-derived mesenchymal celI(ASC) is an attractive, readily available aduh stem cell that has become increasingly popular for use in stem cell and tissue engineering applications. This review focuses on the use of ASC in orthopaedic tissue repair. Recent results from in vivo defect repair utilizing ASC suggested the great potential of ACS in clinical orthopaedic tissue regeneration.

Objective This experiment aimed to confirm whether man made porous chitosan scaffold is a appropriate scaffold for chondrocyte culutre of tissue engineering Methods Chondrocytes were seeded onto porous chitosan and chitosan collagen complex scaffolds for culture in a three dimensional environment The scaffolds in hydrophilia and adhesion to chondrocytes were observed with light microscope and scanning electron microscope The number of the cells attached to the scaffolds and the function of the cells were detected with MTT automated colormetric microassay Result Chondrocytes can multiple and secrete the matrix on the poros chitosan and chitosan collagensc scaffolds The cell adhesion rates were 81 25% and 87 50% respectively Conclusion Chitosan can be fabricated into a suitable three dimensional porous scaffold Porous chitosan collagen complex scafflold may be a more suitable scaffold for chondrocyte culutre of tissue engineering

BACKGROUND: The transplantation of allogeneic cartilage has local immunological rejection, and it is necessary to further reduce the rejection to promote its application in clinic, thus it is significant to perform a series of experiments to induce local immune privilege.OBJECTIVE: To observe the in vivo growth of tissue engineered allogeneic cartilage reconstructed by chondrocytes transfected with recombinant retroviral vector pLNCX2-FasL.DESIGN: A randomized controlled observation.SETTING: Shanghai Jiao Tong University.MATERIALS: Thirty-six allogeneic New Zealand rabbits as recipients and 45 1-week-old chinchillas as donors, either sex,were purchased by the experimental animal center of Chinese Academy of Sciences. Amphotropic recombinant retrovirus coated cell line PT67 was purchased from Clontech Company; Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS), G418 and Polybrene were bought from GIBCO BRL.METHODS: The experiment was carried out in original Shanghai Second Medical University from January 2000 to July 2005. The New Zealand rabbits were randomly divided into three groups: FasL-transfected group (n =12), untransfected group (n =12) and blank control group (n =12). The rabbit allogeneic cartilages were constructed by the compound of pLNCX2-FasL transfected chondrocytes and tissue engineered material of pluronic F-127. ① Gross observation and mass changes of the grafts: Corresponding materials were infused subcutaneously, the grafts were removed at 1, 2 and 3 months after transplantation for gross observation and the mass changes. ② Staining observation: The grafts were removed at 1, 2 and 3 months after transplantation, then prepared into sections, and observed by hematoxylin and eosin (HE), Safranin'O and Masson's trichrome stainings. ③ Antibody detection: Blood samples (1 mL) were collected at 1 and 2 months after transplantation, the chondrocytes of the chinchillas were lysed freezingly with lysis antigen as the mixed antigen, and separated by electrophoresis in agarose medium, then acted with serum of recipient to observe whether corresponding antibody generated. ④ Complement dependent cytotoxicity (CDC) test: The chondrocytes of chinchillas were prepared into cell suspension (2×109/L), and then seeded into 96-well plate, attached grew for 24 hours, then recipient serum was added for the CDC test, and the percentage of apoptotic cells was counted under microscope.MAIN OUTCOME MEASURES: ① Gross observation and mass changes of the grafts;② Histological changes; ③ Results of the antibody detection; ④ Percentage of apoptotic cells.RESULTS: All the 81 rabbits were involved in the analysis of results. ① Gross observation and mass changes of the grafts: Two weeks after inoculation, there were obvious nod formations at the inoculated sites, but no nod formed in the blank control group. The new cartilage tissues became smaller gradually and completely disappeared at 4 months in the untransfected group, whereas those in the FasL-transfected group became smaller, but still existed after 7 months. The masses of grafts in the FasL-transfected group were higher than those in the untransfected group (P ＜ 0.05). ②Histological observation: Plenty of lymphocytic infiltrations around cartilage tissue could be observed in the untransfected group, and obviously decreased in the FasL-transfected group. No lymphocyte was observed inside the chondrocytes.Masson's trichrome staining was performed, and it was observed under light microscope that the small white parts in the middle were immature chondrocytes, and there were green collagen around most of the mature chondrocytes. Safranin O staining showed strong positive reaction, suggested that there were rich glycosaminoglycan in matrix. ③ Antibody detection: The chondrocytes of the chinchillas were lysed freezingly with lysis antigen as the mixed antigen, then acted with serum of recipient, and the results showed that no corresponding antibody generated. ④ Percentage of apoptotic cells: The percentages of serum CDC apoptotic cells in the FasL can ransfected group, untransfected group and blank control group were 5%, 6% and 1%, which were all negative.CONCLUSION: Rabbit allogeneic chondrocytes transfected with recombinant retroviral vector pLNCX2-FasL can reconstruct tissue engineered cartilage, and can postpone the degeneration by 3 months.

OBJECTIVETo compare the tissue engineered cartilage constructed with chondrocytes derived from auricular and articular cartilage.

METHODSChondrocytes were isolated from porcine auricular and articular cartilage, and BMSCs were obtained from bone marrow aspirate and cultured. Each kind of chondrocytes were resuspended alone or mixed with BMSCs at a ratio of 1:1, and seeded onto PGA/PLA scaffolds to construct tissue engineered cartilage (n = 4). The constructs were cultured for 8 weeks in vitro and then subcutaneously implanted into nude mice for 6 weeks. The differences between chondrocytes monoculture from articular and auricular cartilage or between each of them co-cultured with BMSCs were evaluated by gross view, measurement of thickness and wet weight, histological examinations including H&E, Safranin O, type II collagen, and Ponceau's & Victoria blue staining, and gene expression analysis of cartilage related genes.

RESULTSNo obvious differences were found histologically among the complexes constructed in vitro 8 weeks except for few elastic fibers secreted in the auricular chondrocytes + BMSCs co-culture group. Neo-cartilage is thicker in the groups of articular chondrocytes (38. 1% than the group of auricular chondrocytes, P < 0.05) and articular chondrocytes + BMSCs co-culture (19.3% than the group of auricular chondrocytes + BMSCs, P < 0.05). However, after 6 weeks in vivo the elastic fibers were found positive in the complexes constructed by auricular chondrocytes, and its staining was even stronger and more homogenous in the group of auricular chondrocytes + BMSCs co-culture. The tissues generated by articular chondrocytes alone and co-cultured with BMSCs both formed the characteristic features of three-layer structure of hyaline cartilage and ossified in vivo with significant up-regulation of COL10A1 and MMP-13. To summarize, auricular chondrocytes formed the elastic cartilage while articular chondrocytes formed the hyaline cartilage during the development of tissue engineered cartilage either by monoculture or the co-culture with BMSCs.

CONCLUSIONSThe chondrogenic response of chondrocytes from different cartilage origins demonstrates that an initial chondrocyte and cartilage type recapitulates the same in later tissue-engineered development.

Animals ; Bone Marrow Cells ; cytology ; Cartilage, Articular ; cytology ; Cells, Cultured ; Chondrocytes ; Coculture Techniques ; Ear Auricle ; cytology ; Mesenchymal Stromal Cells ; cytology ; Mice, Nude ; Swine ; Tissue Engineering ; methods ; Tissue Scaffolds

0.05),between the two groups,but in body mass index [(24.451?3.752) vs(22.468?2.434),P=0.030],previous history of pancreatitis(23.8% vs.7.5%,P=0.046) and bloody ascites(95.24% vs.46.24%,P0.05).MODS rate was higher in HL-group(52.4% vs.29%,P=0.04).The two groups had similar operation rates(66.7% vs 80.6%,P=0.178),hospitalization days [(25.476?14.383) vs(22.796?7.191),P=0.216] and mortality rate(28.6% vs.11.8%,P=0.069).Conclusions Hyperlipidemic severe acute pancreatitis has the characters of heavier weight habitus,history of frequent recurrence,high incidence of bloody ascites and prone to develop MOF.

BACKGROUND: Classic isolation method of bone marrow mesenchymal stem cells (BMSCs) is Percoll density gradient centrifugation. Blood cell component was removed. However, this method is complicated. Preparation density was needed when isolating dog bone marrow. Moreover, centrifugation was frequent, which had a great damage to cells. OBJECTIVE: To establish methods of the isolation, proliferation, culture and osteoinduction of canine BMSCs, and observe the in vitro proliferation and ability to osteoinduction differentiation. METHODS: 10 mL bone marrow was extracted from dog posterior superior iliac spine, heparin anticoagulation, diluted using Hanks juice, treated with 1.077 g/mL Ficoll solution 3 mL, and centrifuged at 2 000 r/min for 20 minutes. Karyocytes were absorbed to form white cloudlike layering interface, and then centrifuged twice using DMEM supplemented with fetal bovine serum, incubated at 12×10~4/cm~2 at 37 ℃ in a 5% CO_2 incubator. Following subculture, cells were incubated in DMEM containing dexamethasone, β-sodium phosphoglycerol and ascorbic acid 2-phosphate. Immunocytochemical staining and immunofluorescence staining were utilized to detect osteocalcin, osteopontin and type Ⅰ collagen expression in osteoblasts. Alkaline phosphatase staining and alizarin red staining were performed. RESULTS AND CONCLUSION: 1.077 g/mL Ficoll density gradient centrifugation was used to isolate karyocytes that were significant compared with Percoll solution. Obtained BMSCs had high purity, good growth and the mean doubling time was 24 hours. Following in vitro osteogenic incubation of dog BMSCs, osteocalcin, osteopontin and type Ⅰ collagen showed positive expression. Alkaline phosphatase staining demonstrated bluish-green cytoplasm. Alizarin red staining showed red nodes in extracellular matrix, and could differentiate into osteoblasts in vitro.

BACKGROUND: There are plentful studies about bioreactor of tissue engineering of blood vessel, tendon, cartilage, heart valve,trachea, bladder and stern cell. OBJECTIVE: To construct small-sized tissue-engineerad blood vessels with human bone marrow stromal stem cells (hBMSCs) in improved bioreactor system.DESIGN, TIME AND SETTING: The single sample observation stuay was performed at the School of Mechanical and Power Engineering, East China University of Science&Technology, and Shanghai Tissue Engineering Research & Development Center from June 2005 to March 2008.MATERIALS: Vessel bioreactor was self-made by East China University of Science&Technology. hBMSCs were harvested from healthy volunteers. METHODS: A set of support bracket constructing tissue engineered blood vessels with the diameter of 2 mm was designed with the application of Finite Element Methods as an analysis method analyzing support bracket of tissue engineered small-sized blood vessel. Primary hBMSCs were first Collected and further cultivated exvivo. The third passage cultured cells were then seeded on the polyglycolic acid (PGA) to fabricate the cell-scaffold composite. Subsequently, this composite was subjected to dynamical culture in the blood vessel bioreactor. After cultured for 4 weeks, the composite was removed from the bioreactor.MAIN OUTCOME MEASURES: The following mentioned references were measured: composite growth; other correlation detection of the hBMSCs-PGA composite, RESULTS: Gross observation and scanning electron microscope were used at4 weeks after hBMSCs-PGA composite culture. It was observed that the tissue-engineered blood vessel had a bright color and certain elasticity. The blood vessel could rebound to its odginal shape after repeated press by the forceps.The secreted collagen matrix arrayed orderly around the cells and smooth muscle elastic acttn could also be detected in the formed tissues using immunohistochemistry. CONCLUSION: The in vivo mechanics conditions of blood vessels can be simulated using the current blood vessel bioreactor system. Using hBMSCs, the construction of tissue engineered small-sized blood vessels can be successfully achieved.

Objective To investigate the interferences of the variations in the blood pressure and vasoreactivity to the parameters of arterial elasticity via cold pressor test(CPT) in healthy young men. Methods The wave intensities(WI) of the right common carotid artery(RCCA) in twenty-two healthy young men during CPT were measured by echo-tracking technique. The parameters of arterial elasticity, including stiffness parameter (β),pressure-strain etastic modulus(Ep), arlerial compliance(AC),augument index(AI) , pulse wave veloctiy-β (PWVβ),and pulse wave velocity_WI(PWV_WI) from WI analysis during CPT were recorded and statistically compared with the baseline data. Results During CPT, the heart rates increased slightly ( P <0. 05) and the mean arterial pressures increased obviously ( P <0. 05); As to the parameters of arterial elasticity, the Ep,AI, PWVβ,and PWV_WI increased obviously ( P <0. 05), AC decreased ( P <0. 05), while the (3 didn't change. Conclusions The β is affected little by the blood pressure and vasoreactivity, and can reflect the actual arterial elasticity; while the Ep, AC, AI, PWVβ, and PWV_WI are affected much by blood pressure and vasoreactivity. When the latter parameters are used to evaluate the arterial elasticity, the interferences of variations in blood pressure and vasoreactivity must be excluded.

Objective To investigate the effects of cryopreservation on the growth and osteogenesis capa- bility of human bone marrow stromal cells(BMSCs)on demineralized bene matrix(DBM).Methods Bone marrow aspirates were obtained from the lilac crests of three donors.The BMSCs were isolated from the bone marrow by density gradient centrifugation.Cells of passage 3 were cryopreserved in liquid nitrogen for 24 hours,and then re- covered.The non-cryopreserved BMSCs were used as the control,The cryopreserved and control BMSCs were cul- tured in osteogenic media,collected and labeled with Dil to be seeded onto the DBM when cells were confluent.The percentage of BMSCs adhered to the DMB was detected.The cell morphology and matrices secreted by BMSCs on the DBM were observed by the inverted phase-contrasted microscope,fluorescence microscope and scanning electron microscope(SEM).The growth and viability of BMSCs on the DBM were determined using the modified MTT ashy. The osteogenesis ability of BMSCs on the DBM was determined by assessment of the alkaline phosphatase(ALP) activity and osteocalcin(OCN)content.Results The percentages of the cryopreserved and control cells adhered to DBM were(97.25?1.17)% and(97.00?1.09)% respectively.The cells adhered well to the DBM and grew rapidly.Large amounts of matrices on the DBM were observed by the light microscope and SEM.The cells embedded in the matrices could be observed by fluorescence microscope.There were no significant differences in the assay values of MTT,ALP and OCN between the cryopreserved and control BMSCs on the DBM.Conclusion Since cryopreservation does not affect the growth and osteogenesis capability of BMSCs on DBM,the cryopreserved BMSCs can be used as a cell source in bone tissue engineering.