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BACKGROUND:Artificial knee joint replacement in older patients often combines with basic diseases, such as hypertension and diabetes. Perioperative blood loss is an important factor affecting the safety of replacement. OBJECTIVE: To explore the effect of the early closure of drainage tube on blood loss after primary total knee arthroplasty. METHODS: We randomly selected 90 patients with osteoarthritis of the knee who underwent primary total knee arthroplasty in the Affiliated Hospital of Qingdao University from January 2014 to July 2015. The patients were randomly divided into three groups (n=30). In the 4-hour occlusion group, the drainage tube was closed for 4 hours in early stage of replacement. In the 2-hour occlusion group, the drainage tube was closed for 2 hours in early stage of replacement. In the control group, the drainage tube was not closed. Because of the use of tourniquet during surgery, the amount of intraoperative blood loss was considered as 0 mL. Drainage blood loss after surgery was recorded. Total blood loss was calculated according to Gross formula through patient height, weight and preoperative and postoperative hematocrit. Hidden blood loss was gotten by subtracting the visible blood loss from total loss. Under the observation of postoperative joint sweling and subcutaneous ecchymosis, knee Hospital for Special Surgery score was recorded at 6 weeks after replacement, and compared among groups. RESULTS AND CONCLUSION:Statistical analysis indicated that significant differences in total blood loss and dominant blood loss were detected among the three groups (P 0.05). The incidence of joint sweling and subcutaneous ecchymosis was increased in the 4-hour occlusion group (P < 0.05). Above results confirmed that drainage tube occlusion can decrease total blood loss and dominant blood loss after total knee arthroplasty, but cannot reduce hidden blood loss. 2-hour occlusion after total knee arthroplasty is an ideal choice, but the amount of hidden blood loss should be carefuly considered.
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BACKGROUND:Mesenchymal stem cel s are the seed cel s for tendon tissue engineering which can be obtained in large quantities, but how to induce in vitro is a key technology. OBJECTIVE:To explore the effect of platelet-rich plasma on the proliferation and col agen production of in vitro cultured mesenchymal stem cel s. METHODS:The rabbit mesenchymal stem cel s were separated ad cultured. The high-dose platelet-rich plasma group, middle-dose platelet-rich plasma group and low-dose platelet-rich plasma group were set to induce the mesenchymal stem cel s, and the blank control group was set as control. RESULTS AND CONCLUSION:The proliferation of mesenchymal stem cel s in the high-dose platelet-rich plasma group, middle-dose platelet-rich plasma group and low-dose platelet-rich plasma group was high, and in rapid growth with big increase amplitude, and there was no significant difference in the proliferation when compared with the blank control group (P<0.05). The effect was positively correlated with the culture time, and after cultured for a certain time, the effect was in dose-dependent manner, as higher dose platelet-rich plasma had more significant effect on the proliferation of the cel s. The results indicate that platelet-rich plasma can significantly promote the synthesis of col agen type Ⅰ and Ⅲ of mesenchymal stem cel s, the higher the dose, the more significant the effect on the col agen.
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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.
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BACKGROUND: Up to now, there are few reports addressing the biological properties and differentiation potential of umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs). OBJECTIVE: To observe the biological characteristics, osteogenic and adipogenic differentiation potential of UCB-MSCs. METHODS: MSCs were harvested and cultured from UCB at various gestational ages (GA). Harvested UCB-MSCs were cultured primarily and subcultured, and then induced to differentiate into osteoblasts and adipocytes. RESULTS AND CONCLUSION: Under the inverted phase contrast microscope, UCB-MSCs adhered to the wall, showing fibroblast like morphology and whirlpool like growth alignment. Observation of the ultramicrostructure under transmission electron microscope showed that UCB-MSCs had a big cellnucleus, fewer cellular organelles and big karyoplasmic ratio.Allofthe growth curves of primary and passaged UCB-MSCs presented S-shaped. The 3rd and 5th passages of MSCs showed the strongest proliferation activity. The count of colony forming unit fibroblasts varied with GA, significant difference was found among the three GA groups (P < 0.05), and the lower GA group had a higher count of colony forming unit fibroblasts than that in the older GA group. Flow cytometry showed that these cells expressed CD29, CD44 and CD90 positively, but they failed to express hematopoiesis related molecules such as CD34 and CD45. When the MSCs were induced to osteogenic and adipogenic differentiation for 3 weeks, strong expression of alkaline phosphatase was found and the formation of a mineral extracellular matrix was detected by alizarin red staining were detected; and neutral lipid vacuoles were detected by oil red O staining. UCB-MSCs have similarmorphologicaland biological characteristics and cell surface molecule markers with MSCs derived from bone marrow, both of which have great capability of proliferation and regeneration. UCB-MSCs can be induced to osteoblasts and adipocytes in a suitable condition in vitro.
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BACKGROUND: Mesenchymal stem cells (MSCs) exist in umbilical cord blood (UCB), currently, there is not a method to in vitro separate, culture and amplificate human UCB-MSCs effectively. OBJECTIVE: To explore factors that influence yields of UCB-MSCs. METHODS: The relationship between the success rate of yielding UCB-MSCs and several factors, such as gestational ages (≥40 weeks, 37 weeks and ≤32 weeks), the number of mononuclear cells (MNCs) in UCB (≥2.5×109/L, <2.5×109/L), the inoculum density of MNCs (1×107, 1×109, 1×1011/L), the concentration of fetal bovine serum (FBS, 5%, 10%, 15%, 20%) in culture medium, and whether the culture flask being coated with FBS or not beforehand, as well as relationships among these factors were investigated. RESULTS AND CONCLUSION: The success rate of yielding UCB-MSCs was up to 58.3%. The success rate decreased as the gestational ages increasing (P < 0.01). The success rate could be enhanced to 76.9% when the MNCs count was more than 2.5×109/L, and there was significant difference when comparing to that of the group (36.4%) with MNCs count less than 2.5×109/L (x2=8.07, P=0.005). There was a negative correlation between the MNCs count and the gestational ages in the specimens with the same volume of UCB (r=-0.95, P < 0.01). In the group with the cell inoculum density of 1×1011/L, the growth and proliferation of primary and subculturing MSCs were better than that of the groups with the cell inoculum density lower than 1×1011/L. The adherence of MSCs in the group with the culture medium containing 5% FBS happened much later than other 3 groups, while the purity of MSCs in this group was much higher. When comparing the passage rate, there was no significant difference among the 4 groups with different concentration of FBS. In the group of culture flask being coated with FBS beforehand, the purity and proliferation ability of MSCs was higher than that in the groups with culture flask not being coated. It is suggested that culture of UCB-MSCs was influenced by several factors. The success rate could be increased by choosing the fetus with relative lower gestational ages, collecting enough volume of UCB, inoculating cells with a higher density, choosing the medium with lower concentration of FBS, and coating the culture flask with FBS beforehand.
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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.
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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.
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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.
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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.
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BACKGROUND: Studies have showed that transforming growth factor-β1 (TGF-β1) could yield to the collagen synthesis and adhesion formation of tendon cells at the process of healing. OBJECTIVE: To investigate the preventive effect of TGF-β1 neutralizing antibody on the collagen production and adhesion formation of flexor tendon. DESIGN, TIME AND SETTING: Randomized grouping observational experiments were performed in the Experimental Animal Center of Tongji Medical College between September 2005 and June 2006. MATERIALS: New Zealand white rabbits aged 2-5 months, weighing 3.5-4.5 kg. TGF was offered by Santa Cruz Biotechnology, USA. METHODS: Sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes were obtained from rabbit flexor tendons. Cells were divided into two groups at random. In the experiment group, each cell culture was supplemented with 1 μg/L of TGF-β at increasing dose (0.1, 0.5, 1.0 mg/L) of TGF-β1 neutralizing antibody. No reagents were given in the control group. Collagen Ⅰ production was measured by enzyme-linked immunoabsorbent assay. Eighty-four adult New Zealand white rabbit forepaws underwent sharp transection of middle toe flexor digitorum profundus, followed by immediate repair. Thirty-six adult New Zealand white rabbit were divided into three groups randomly (n=12), injecting with the saline, 1.0 mg/L TGF-β1 neutralizing antibody and 2.0 mg/L TGF-β1 neutralizing antibody into tendon sheath respectively. Tendons were harvested at 4 and 8 weeks to conduct adhesion detection, biomechanical testing, histological evaluation and scanning electron microscopy observation. The remaining 48 New Zealand white rabbits were divided into two groups randomly (n=24), undergoing the saline and 1,0 mg/L TGF-β1 neutralizing antibody injection in tendon sheath respectively. Tendons were harvested at an increasing time interval (1, 2, 4, 8 weeks) and analyzed by in situ hybridization to determine the mRNA expression of TGF-β1 and collagen Ⅰ. MAIN OUTCOME MEASURES: Collagen production and adhesion of rabbit tendon cells. RESULTS: ELISA exhibited that TGF-β1 increased collagen Ⅰ production and the addition of neutralizing antibody significantly reduced TGF-β-induced collagen Ⅰ production in all cell cultures. The effect between antibody and collagen Ⅰ was dose dependent. At 4 and 8 weeks after operation, the gliding excursion ratio of the tendon was shortened and the simulated active flexion ratio were less in saline group compared with 1.0 and 2.0 mg/L TGF-β1 groups (P < 0.05). The tendon anastomosis breaking strength was shown no significant differences among 3 groups (P > 0.05). Scanning electron microscopy and histological observation showed that collagen fibers arranged irregularly in saline group, but arranged regularly in 1.0 and 2.0 mg/L TGF-β1 groups at 4 and 8 weeks after operation. The in situ hybridization examination revealed that TGF-β1 and collagen Ⅰ mRNA expression in 1.0 mg/L TGF-β1 group was lower than that in saline group at each time (P < 0.05). CONCLUSION: TGF-β1 neutralizing antibody can inhibit the function of the TGF-β1 effectively following the flexor tendon injury and repair, and can prevent adhesion formation.
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Objective To compare the clinical outcomes of irradiated versus non-irradiated deepfrozen bone-patellar tendon-bone (B-PF-B) ullograft in anterior cruciate ligament (ACL) reconstruction. Methods A total of 66 patients undergoing arthroecopic ACL reconstruction were prospectively random-ized consecutively into two groups, ie, Group A ( irradiated deep-frozen allograft, n = 34) and Group B ( non-irradiated deep-frozen allograft, n = 32). All ACL reconstructions were done by the same senior surgeon with the same arthroscopic technique. Before and after surgery, the clinical results were compared in aspects of general conditions, range of motion ( ROM), Pivot shift test, Lachman and Anterior Drawer Test (ADT), Daniel's one-leg hop and Harner's vertical jump tests, overall international knee docu-mentation committee (IKDC) rating and KT-2000 arthrometer testing. Results Of all, 63 patients in-eluding 32 patients in Group A and 31 in Group B were available for a follow-up of average 38 months ( mean 38.3 months in Group A and mean 37.7 months in Group B) and three lost follow-up. There was one patient with late septic infection. While there was no statistical difference in aspects of general condi-tions including hospital stay, duration of fever and complications ( P > 0. 05 ), but there was a trend that the patients in Group A had a longer postoperative duration of fever ( mean 8.9 days ) than Group B (mean 7.8 days). Physical examinations showed no statistical difference upon ROM in both groups ( P > 0. 05), while there was statistical difference between Lachman test and ADT ( P < 0.05 ). The positive Pivot shift test was found in 12 patients in Group A and 3 in Group B, with statistical difference ( P < 0. 05 ). KT-2000 testing showed a side-to-side difference of less than 3 mm in 26 patients in Group B and only 8 in Group A, and a side-to-side difference of more than 5 mm in two patients in Group B and 12 in Group A, with statistical difference (P < 0.05 ). The anterior and rotational stability was decreased sig-nificantly in Group A. No statistical difference was found between two groups in overall IKDC rating, Daniel' s one-leg hop and Harner' s vertical jump tests ( P > 0.05 ). However, the function and IKDC score tended to decrease in Group A. Conclusion The short term clinical outcomes of the ACL reconstruction with irradiated B-PT-B allograft are adversely affected and unsatisfactory, indicating a cautious use.
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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.
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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.
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0.05) Lactate produced more type Ⅰ,Ⅱ,and Ⅲ collagen tissue compared to not adding lactate(P
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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.
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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.
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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.
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BACKGROUND: Ischemic osteonecrosis of femoral head can cause dysfunction of the patients and the most frequently advocated treatment for advanced osteonecrosis of femoral head is total hip arthroplasty. But the reason is unknown. A potential cause in patients with osteonecrosis of femoral head is abnormal cancellous bone in the proximal femur which is short of further investigation.OBJECTIVE: To probe into proximal femoral change of patients with ischemic osteonecrosis of the femoral head.DESIGN: A non-randomized concurrent controlled study based on the patients.SETTING: Department of Traumatic Surgery, Affiliated Hospital of Medical College of Qingdao UniversityPARTICIPANTS: This study was done at the Department of Traumatic Surgery, Affiliated Hospital of Medical College of Qingdao University from September 1999 to May 2003. Eighteen patients with osteonecrosis of the femoral head including 12 males and 6 females with the average age of (50 ± 5) years and eighteen patients with osteoarthritis of the hip attended including 10 males and 8 female with the average age of(62 ±3)years were involved in this study.METHODS: Histological examination of the femoral heads and cancellous bone biopsies from four regions of the proximal femur in 18 patients with osteonecrosis of the femoral head following total hip arthroplasty was perforned. Eighteen patients with osteoarthritis were set as control group. All histological specimens were examined in a double-blinded fashion.MAIN OUTCOME MEASURES: The femoral neck, the greater trochanter, the lesser trochanter, the necrosis scoring and necrosis positive rate of shaft of femur of the patients in the two groups.RESULTS: Extensive osteonecrosis was found in the proximal femur up to 4 cm below the lesser trochanter in the group with osteonecrosis. The osteonecrosis scoring was 82 and 16 in the osteonecrosis group and control group respectively and the osteonecrotic positive rate was 63.89% and 19.44% respectively, There was an overall statistically significant difference in the extent of osteonecrosis death in the proximal femur between the two groups ( P ＜ 0. 001 ).CONCLUSION: Bone change of proximal femur might be one of the reasons for the early failure of total hip replacement reported in patients with osteonecrosis of the femoral head.
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BACKGROUND: Bone defect is commonly treated in clinic with auto graft, allograft and artificial bone graft, but with the disadvantages to various extents. Therefore, how to better repair bone tissue in bone defect becomes the hot spot in surgical field.OBJECTIVE: The complex of marrow stromal stem cell (MSC) and calcium-phosphates artificial bone (CPAB) was prepared and the repair of bone defect was observed in animal experiment.DESIGN: Randomized controlled experiment was designed.SETTING: Department of Traumatic Surgery, Affiliated Hospital of Qingdao University Medical College.MATERIALS: The experiment was performed in Animal Experimental Center of Affiliated Hospital of Qingdao University Medical College from May 2002 to September 2003, in which, 24 New Zealand health normal big white adult rabbits were employed, aged varied from 6 to 14 months, mass weighted from 9 to 3.5 kg, of either sex.METHODS: (① Rabbit MSCs were differentiated and proliferated and CPAB was taken as carrier to prepare the complex of CPAB and MSCs. ②24 rabbits (48 laterals) were prepared as model of bone defect, divided into 3 groups, named complex group (24 laterals), in which, unilateral bone defect of 24 rabbits were implanted with cell + carrier complex; The rtificial bone group (12 laterals), in which, CPAB was implanted contralaterally;and blank control (12 laterals), in which, no any management was done contralaterally in bone defect of 12 rabbits. In 8, 16 and 24 weeks after surgery, 4 animals were collected at each time spot in each group for radionuclide monitoring; and the animals were sacrificed under anesthesia for X-ray picturing and histological staining analysis.MAIN OUTCOME MEASURES: ① Gross observation of bone defect in each group. ② Results of X-ray examination. ③ Results of histomorphologic observation. ④ Results of radionuclide monitoring.RESULTS: 24 rabbits all entered result analysis. ① Gross observation of bone defect in each group and results of X-ray examination: In complex group of 24 hours, the fractutred ends were connected partially, the bounder was not clear between the regenerated bone and material and reopened medullary cavity was not visible. In simple artificial bone group, the implanted material was integrated with the cross end and material volume did not change obviously compared with the initial implanting stage. In bland control, ununion fracture presented and medullary cavity was closed. ②Results of histomorphological observation: In 24 hours, the bone defect was smaller in size and the union happened on the defect area in complex group. In artificial bone group, the regenerated bone was integrated tightly with the material and grew inside partially. In blank control group,medullary cavity was closed and bones were not connected. ③ Results of radionuclide monitoring: It was remarkably distinguished with naked eye that radioactive concentration was in tendency of obvious increasing in both complex group and artificial bone group in 8-24 weeks.CONCLUSION: The complex of CPAB and MSCs regenerates bone tissue and repair radial defect in whole. In addition, the repair ability is superior remarkably to simple CPAB.
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Objective To study the prevalence of cholelithiasis among spinal cord injured (SCI)male patients, and the correlation of cholelithiasis with the SCI patients′ age, weight,and the level of injury, severity and duration of the SCI. Methods One hundred male SCI patients including 58 patients with ASIA class A and B and 42 with ASIA class C and D,with aged 20-65 years old(average 46.5 years) were followed up.They suffered from a spinal cord injury more than one year. The control group consisted of 100 male volunteers without both SCI and biliary diseases, with aged 20-68 year old(average 42.6 years). All patients in the two groups underwent ultrasonographic imaging to evaluate the gallbladder and the biliary tract. Results The prevalence of cholelithiasis was 26.0% in the SCI group patients and 10.0% in the control group,the difference was statistical difference (P 0.05). Conclusions SCI represents a major risk factor for the development of cholelithiasis . There are no correlation of cholelithiasis with the SCI patients′ age , weight,SCI level,severity and duration of SCI.