BACKGROUND: Self-repairing capability of articular cartilage tissue is poor, due to lack of the distribution of vessels and lymph.OBJECTIVE: To concisely describe the research progress of autologous chondrocyte implantation (ACI), including matrix-induced autologous chondrocyte implantation (MACI), in vivo scaffolds, and related tissue engineering technologies, and to prospect the future developments.METHODS: A search across the databases of ISI Web of Knowledge and PubMed (1979 to February 2009) was performed, with key words of "articular cartilage, transplantation, stern cells, tissue engineering". As well, a search in the database of CNKI (1979 to Febraruy 2009) was performed with the key words of "articular cartilage, repair, tissue engineering". Contents referring to ACI,MACI, in vivo scaffolds and related tissue engineering technologies were included, while contents regarding to the clinical imaging of articular cartilage defects, intracellular signaling pathways in chondrocytes, or gene therapy for articular cartilage defects were excluded.RESULTS AND CONCLUSION: 824 articles were obtained from the preliminary search across the databases. Based on the nominated evaluation criterions to the outcome, analysis focusing on ACI, MACI, in vivo scaffolds and related tissue engineering technologies was performed. As the most successful treatment for articular cartilage defects in the past decade, ACI has undergone a significant development. Recent improvements of ACI include MACI, in vivo scaffolds and related tissue engineering technologies, which exhibit relatively more success in engineering and clinical practice. Nonetheless, limitations still exist and therefore, further researches are required. As a promising alternative of ACI, MACI is more and more widely used in clinical practice for treating articular cartilage defects these years. The long-term curative effect of MACI, however, requires further clinical data to confirm. In addition, other improvements of ACI, in terms of material science, cytology and molecular biology, have been also provided by the developments of in vivo scaffolds and related tissue engineering technologies.

In order to evaluate the efficacy of low intensity ultrasound and tissue engineering technique to repair segmental bone defects, the rabbit models of 1.5-cm long rabbit radial segmental osteoperiosteum defects were established and randomly divided into 2 groups. All defects were implanted with the composite of calcium phosphate cement and bone mesenchymal stem cells, and additionally those in experimental group were subjected to low intensity ultrasound exposure, while those in control group to sham exposure. The animals were killed on the postoperative week 4, 8 and 12 respectively, and specimens were harvested. By using radiography and the methods of biomechanics, histomorphology and bone density detection, new bone formation and material degradation were observed. The results showed that with the prolongation of time after operation, serum alkaline phosphatase (AKP) levels in both groups were gradually increased, especially in experimental group, reached the peak at 6th week (experimental group: 1.26 mmol/L; control group: 0.58 mmol/L), suggesting the new bone formation in both two group, but the amount of new bone formation was greater and bone repairing capacity stronger in experimental group than in control group. On the 4th week in experimental group, chondrocytes differentiated into woven bone, and on the 12th week, remodeling of new lamellar bone and absorption of the composite material were observed. The mechanical strength of composite material and new born density in experimental group were significantly higher than in control group, indicating that low intensity ultrasound could not only effectively increase the formation of new bone, but also accelerate the calcification of new bone. It was concluded that low intensity ultrasound could evidently accelerate the healing of bone defects repaired by bone tissue engineering.

0.05) Lactate produced more type Ⅰ,Ⅱ,and Ⅲ collagen tissue compared to not adding lactate(P

BACKGROUND: Under special conditions, bone marrow mesenchymal stem cells (BMSCs) can differentiate into osteoblasts and chondroblasts. However, MSCs are few in bone marrow. How to harvest, purity and rapidly proliferate in vitro is a foundation of application in tissue engineering technique. OBJECTIVE: To optimize, collect, purity, assess rabbit BMSCs and to observe the biological character of BMSCs. DESIGN, TIME AND SETTING: The observational study was performed at the Animal Experimental Center of Tongji Medical College from September 2005 to July 2006. MATERIALS: One female New Zealand rabbits aged 2 months were used for MSC collection and primary culture. METHODS: Bone marrow solution was purified by density gradient centrifugation and adherence screening method. Culture solution was obtained. BMSCs were incubated in phosphate buffered solution (PBS), supplemented with 2.5 g/L trypsin (3.0 mL), and placed in an incubator at 37 ℃ for two or three minutes. Cell morphology was observed using an inverted microscope. The digestion was stopped when cytoplasm recovery, long and thin cells with large intercellular space, and few round cells appeared. Subsequently, BMSCs were incubated in serum-free L-DMEM, and placed in a plastic culture flask at 1.0×108/L. MAIN OUTCOME MEASURES: MSC morphology, ultrastructura and surface marker; Proliferation of the first, third, fifth, eighth and tenth passages of BMSCs; Cell growth curve was drawn. RESULTS: BMSCs was pure following density gradient centrifugation and adherence screening method. The third and fifth passage of cells had typical whirlpool-shape. Transmission electron microscope demonstrated that round or oval MSCs possessed large nuclei, big nucleus proportion, a few cellular organ. These were low-differentiated cells. Growth curve of cultured MSCs was "S" shape. The first, third and fifth passage cells had strong reproductive capability. The eighth and tenth passage of cells had significantly reduced proliferation. Cells isolated were positive for CD44 and CD90, but negative for CD34. These were low-differentiated cells under the electron microscope. CONCLUSION: Isolated cells are MSCs, with the property of stem cells. The third and fifth passage cells are pure, with strong reproductive capability.

BACKGROUND: Tendon cells possess collagen-secreting function, which plays an important role in the wound healing and adhesion. Little is known about the biological effects of lactate on tendon cells. OBJECTIVE: This study was designed to study the proliferation and collagen production of tendon sheath fibroblasts,epitenon tenocytes, and endotenon tenocytes and investigate the effect of lactate on cell proliferation, collage production and secretion of transforming growth factor- β1(TGF- β 1),basic fibroblast growth factor (bFGF), and interleukin-8 (IL-8) by each of these 3 cell types in rabbit flexor tendon.DESIGN, TIME AND SETTING: This study, a randomized controlled animal experiment, was performed at the Animal Laboratory Center, Affiliated Hospital of Qingdao University Medical College between September 2005 and July 2006.MATERIALS: Six adult New Zealand clean rabbits of either gender were included in the present study.METHODS: Tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes were isolated from rabbit flexor tendon and cultured. Each cell type was identified by observing morphological structure through methylene blue staining.Three types of cells were cultured with media supplemented with 25 mol/L lactate to obtain cell number. The collagen production and secretion of TGF- β 1, b-FGF and FL-8 were compared after adding culture media supplemented with 25, 50,100, and 200 mmol/L lactate, respectively. At the same time, the above-mentioned indices measured after adding medium were compared with those measured without adding lactate.MAIN OUTCOME MEASURES: Type Ⅰ, Ⅱ, and Ⅲ collagen production was detected by immunohistochemistry; The effects of different concentrations of lactate on type Ⅰ collagen production as well as secretion of TGF- β 1,b-FGF, and IL-8by an enzyme linked immunosorbent assay (ELISA).RESULTS: 25 mmol/L lactate reduced 3 types of cultured cells. There was no significant difference in the cell number among the 3 types of cells (P > 0.05) Lactate produced more type Ⅰ, Ⅱ ,and Ⅲ collagen tissue compared to not adding lactate (P < 0.05) When lactate concentration increased to 50 mmol/L, type Ⅰ collagen reached its peak level in the 3 types of cells. There was significant difference in type Ⅰ collagen compared with lactate concentration was 0 mmol/L(t = 4.58, 3.97,3.16, P < 0.01 ) . When lactate concentration increased to 100 mmol/L and 200 mmol/L, type Ⅰ collagen was noticeably decreased. When lactate concentration was 25 mmol/L in the 3 types of cells, the secretion of TGF- β 1 and bFGF was increased and the secretion of IL-8 was decreased. There was significant difference compared with not adding lactate (P <0.05).CONCLUSION: Lactate can increase the collagen production of tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes. The increasing degree of collagen production is related to lactate concentration, in particular at 50 mmol/L. Such a stimulation may be related to the increase of TGF- β 1 and bFGF and the decrease of IL-8 secretion.

BACKGROUND: We have paid more attention on the effects of growth factors on tendon healing and adhesion formation, especially on the correlation of transforming growth factor with tissue adhesion and scar formation. OBJECTIVE: To investigate the expression of transforming growth factor beta-1 mRNA in the zone Ⅱ flexor tendon of wound-healing rabbit models. DESIGN: Randomized controlled animal study. SETTING: Department of Orthopaedics, Affiliated Hospital of Medical College, Qingdao University. MATERIALS: Sixty clean adult New Zealand white rabbits weighting 4.0-4.5 kg, of either sex, were provided by Qingdao Animal Experimental Center. Left forelimbs of each animal were as experimental side, and right forelimbs of each animal were as control. There were 6 time points, namely at days 1, 7, 14, 21, 28 and 56, 10 rabbits in each time point. Of the 10 rabbits, 6 rabbits received the in situ hybridization and 4 rabbits received the immunohistochemical staining. Animal intervention met the animal ethical standard. METHODS: Experiments were performed at the Animal Experimental Center of Hospital Affiliated to Medical College of Qingdao University from September 2005 to July 2006. After anesthesia, each rabbit underwent complete transection of the profundus middle flexor tendon in zone Ⅱ, and then the tendon was repaired by the Kessler method. Rabbits in the control group did not receive any intervention. Rabbits were anesthetized and killed 1, 7, 14, 21, 28 and 56 days after the surgery. Skin was incised along the original incision at the experimental sides to obtain tendons and tendon sheaths. The same measurements were performed in the control group. MAIN OUTCOME MEASURES: Tenocytes and tendon sheath cells were detected with the in situ hybridization and the immunohistochemical staining to observe the expression of transforming growth factor beta-1. RESULTS: Sixty rabbits were involved in the result analysis. ①The in situ hybridization results: Expression of transforming growth factor beta-1 mRNA was increased at day 1 after tendon injury in the experimental group, reached a peak at days 14-21 after tendon injury, reduced at day 28 and was still in a high level at day 56. Expression of transforming growth factor beta-1 mRNA was high in tendon sheath cells around the repaired region. At the same time point, the expression of transforming growth factor beta-1 mRNA was higher in tendon sheath cells than in tenocytes. Low expression of transforming growth factor beta-1 mRNA was found in tenocytes and tendon sheath cells in the control group. The expression of transforming growth factor beta-1 mRNA in tenocytes and tendon sheath cells was higher in the experimental group than in the control group at each time point (P ＜ 0.05). ②Immunohistochemical staining results: Expression of transforming growth factor beta-1 protein was elevated at day 1 after the surgery, reached the peak at days 14-21 and was still in a high level at day 56 in the experimental group. Low expression of transforming growth factor beta-1 protein was seen in the control group. CONCLUSION: The normal uninjured tenocytes and tendon sheath cells produce transforming growth factor beta-1. The cytokine is activated in the injured tendon. The increase of this cytokine in both tenocytes and tendon sheath fibroblasts are coincidence with both extrinsic and intrinsic mechanisms for tendon repair.

BACKGROUND:Transforming growth factor beta-1(TGF-β1)is a cytokine having variously biological effects in repair and renew of tissue injuries; meanwhile, tendon sheath fibroblasts and collagen Ⅰ play important roles in healing and desmoplasia of tendon.OBJECIVE:To study the effects of TGF-β1 on the proliferation and collagen production of tendon sheath fibroblasts.epitenon tenocytes and endotenon tenocytes in the three cell types of rabbit fexor tendon.DESIGN:Contrast observation study.SETTING:Department of Trauma Surgery,Affiliated Hospital of Medical College,Qingdao University.MATERIALS:The experiment was carried out in the Animal Laboratory,Affiliated Hospital of Medical College,Qingdao University from July 2004 to September 2005.A total of 6 adult New Zealand rabbits,of either gender,weighing 3.5-4.5 kg,were selected from Qingdao Experimental Animal Center.Collagenase was provided by Sigma Company;collagen Ⅰ,Ⅱand Ⅲ antibody by Sigma Company;TGF-β1 by Wuhan Boster Biology Company.METHODS: Three cell lines of tendon sheath,epitenon and endotenon were isolated from rabbit flexor tendon and cultured in serum culture media and then in serum-free culture media.In addition,the cells in the experimental group were added with 5 μg/L TGF-β1 in each well,but they were not added with any additive in the control group.MAIN OUTCOME MEASURES:①Proliferation in the two groups was measured with cytometry at 1,2,3 and 4 days after culture.②Preduction of collagens Ⅰ,Ⅱ and Ⅲ was measured with immunohistochemical staining at 4 days after culture.③Collagen contents of the three types were measured with enzyme linked immunosorbent assay(ELISA)in the two groups;expressJon of collagen Ⅰ gene was detected with reverse transcription polymerase chain reaction(RT-PCR).④Contents of collagen Ⅰ induced by TGF-β1 in various dosages of 0,5.10,15 and 20 μg/L were detected with ELISA technique.RESULTS:①Proliferated rates were similar in the two groups at 1 day after culture;however,proliferated rate of tendon sheath fibroblasts was rapidly increased, and there was significant difference as compared with that of epitenontenocytes and endotenon tenocytes(P＜0.05).②Expressions of collagens Ⅰ, Ⅱ and Ⅲ:Immunocytochemical stain demonstrated that three kinds of cells could produce collagens Ⅰ, Ⅱ and Ⅲ;while ELISA indicated that the contents of collagens in three types produced by tendon sheath fibroblasts were the most;in addition,content of collage Ⅰ was higher in the experimental group than that in the control group(P＜0.05-0.01).③Expression of collage Ⅰ gene of tendon sheath fibroblasts was increased as 1.3 times in the experimental group as that in the control group and there was signiflcant difierence(P＜0.01);meanwhile,expressions in epitenon tenocytes and endotenon tenocytes were also higher in the experimental group than those in the control group(P＜0.05).④TGF-β1 in the dosage of 5-10 μg/L had obvious effects on increasing production of collagen;however,production of collagen was not obviously changed when it was affeCted by TGF-β1 in the dosage of 10-20 μg/L.CONCLUSION: TGF-β1 can increase the production of collagen in tendon sheath fibroblasts,epitenon tenocytes and endotenon tenocytes and the expression of collagen Ⅰ gene. In addition, it is important for regulating level of TGF-β1 after tendon injury to prevent adhesion of tendon.

BACKGROUND: Due to lack of the distribution of vessels and nerve, self-repairing capability of articular cartilage tissue is poor after inflammatory erosion.OBJECTIVE: To evaluate the effects of melatonin combined with compound betamethasone on the articular cartilage of osteoarthritis (OA) in rats.METHODS: Thirty Sprague-Dawley rats received intra-articular injection of papain solution for establishing knee OA models.Meanwhile, 20 of them underwent constant intensive light condition for establishing pinealectomy models. Ten rats that under pinealectomy were administered melatonin combined with compound betamethasone. Another 10 normal control rats receiving no treatment served as controls. After 4 weeks of treatment, serum melatonin concentrations at 2 a.m. (highest melatonin concentration within circadian rhythms) and 2 p.m. (lowest melatonin concentration within circadian rhythms) were detected by ELISA. At the same time, all rats were sacrificed to collect femoral condyle cartilage for gross observation.After decalcification and toluidine blue staining, articular cartilage lesions were evaluated based on Mankin scores.RESULTS AND CONCLUSION: After OA model was created, cartilage surface was uneven, lost their luster, the chondrocytes were poorly arranged, severe loss of staining was observed, serum level of melatonin was decreased, and circadian change was unobvious. Constant intensive light condition further aggravated cartilage damage. After treatment by melatonin combined with compound betamethasone, softened cartilage disappeared, there were more regular chondrocytes arrangement, and dispersed chondrocytes and loss of staining were gradually decreased. In addition, there was significant difference in Mankin scores of toludine blue staining among groups (P ＜ 0.05). These findings indicate that melatonin combined with compound betamethasone can restrain the progression of cartilage damage.

BACKGROUND: Transforming growth factor beta (TGF-β) has an important role in tendon healing and adhesion formation.Inhibiting TGF-β and its receptor expression may prevent adhesions after tendon open.OBJECTIVE: To study the effects of mannose-6-phosphate, a natural inhibitor of TGF-β, on TGF-β and its receptor production in tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes of rabbit flexor toes.METHODS: Tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes were isolated from rabbit flexor tendon and cultured separately. All these cells were divided into 2 groups at random, experiment group supplemented with mannose-6-phosphate and control group without mannose-6-phosphate. The expression of TGF-β and TGF-β receptor was quantified with enzyme-linked immunosorbent assay. The expression of TGF-β1 was also assessed with in situ hybridization and immunohistochemistry.RESULTS AND CONCLUSION: The expression of TGF-β and TGF-β receptor in experiment group was significantly lower than that in control group (P ＜ 0.05). In experimental group, the positive expression of TGF-β1 mRNA and the expression level of intracellular TGF-β1 mRNA in all tendon cells demonstrated significantly lower than those in the control group (P ＜ 0.05).Immunohistochemical staining showed expression of TGF-β1 were significantly lower in all three types of tendon cell cultured with mannose-6-phosphate.

BACKGROUND: Bone morphogenetic protein 2 and transforming growth factor β are important factors in bone regeneration, increasing the expressions of bone morphogenetic protein 2 and transforming growth factor β can promote the osteogenic differentiation of bone marrow mesenchymal stem cells. OBJECTIVE: To construct the lentivirus vector carrying bone morphogenetic protein 2 and transforming growth factor β3, and to observe the expression of lentivirus vector in bone marrow mesenchymal stem cells. METHODS: The recombinant lentiviral vectors carrying transforming growth factor β3, bone morphogenetic protein 2 and green fluorescent protein were constructed with recombinant lentiviral technology, and then the recombinant lentiviral vectors were used to transfect the passage 3 rabbit bone marrow mesenchymal stem cells in vitro cultured (transfection group). The bone marrow mesenchymal stem cells transfected with single gene lentivirals (single gene transfection group) carrying transforming growth factor β3 and bone morphogenetic protein 2 or single lentivirals were as control (control group). At 1 week after trasfection, the total RNA and protein were extracted from each group for detection. RESULTS AND CONCLUSION: The green fluorescence bone marrow mesenchymal stem cells transfected with transforming growth factor β3 and bone morphogenetic protein 2 gene for 3 days could be observed under fluorescence microscope, and the transfection efficiency was over 90%. Reverse transcription-PCR and Western blot results showed the mRNA and protein expressions of transforming growth factor β3 and bone morphogenetic protein 2 in the transfection group were higher than those in the single gene transfection group and the control group. The results indicate that lentivirus can successful y transfect transforming growth factor β3 and bone morphogenetic protein 2 into the bone marrow msenchymal stem cells and achieve its high expression, and these two genes have the synergistic effect of promoting expression.