Objective To investigate the influence of collagen on the biomechanics strength of tissue engineering tendon. Methods All of 75 nude mice were madethe defect models of calcaneous tendons, and were divided into 5 groups randomly. Five different materials including human hair, carbon fibre (CF), polyglycolic acid (PGA), human hair and PGA, and CF and PGA with exogenous collagen were cocultured with exogenous tenocytes to construct the tissue engineering tendons.These tendons were implanted to repair defect of calcaneous tendons of right hind limb in nude mice as experimental groups, while the materials without collagenwere implanted to repair the contralateral calcaneous tendons as control groups. In the 2nd, 4th, 6th, 8th and 12th weeks after implantation, the biomechanicalcharacteristics of the tissue engineering tendon was measured, meanwhile, the changes of the biomechanics strength were observed and compared. Results From the 2nd week to the 4th week after implantation, the experimental groups were ber than the control groups in biomechanics, there was statistically significantdifference (Plt;0.05). From the 6th to 12th weeks, there was no statisticallysignificant difference between the experiment and control groups (Pgt;0.05). Positivecorrelation existed between time and intensity, there was statistically significant difference (Plt;0.05). The strength of materials was good in human hair,followed by CF, and PGA was poor. Conclusion Exogenous collagen can enhance the mechanics strength of tissue engineering tendon, and is of a certain effect on affected limb rehabilitation in early repair stages.
ObjectiveTo investigate the changes of fibrinogen and classical markers of collagen metabolism [carboxy-terminal propeptide of type Ⅰ procollagen (PICP) and carboxy-terminal cross-linked peptide of type Ⅰ collagen (ICTP)] in peripheral blood and pericardial drainage after coronary artery bypass grafting (CABG) and/or heart valve replacement (VR), and to evaluate their relationship with postoperative atrial fibrillation (POAF) after cardiac surgery. MethodsPatients who underwent CABG and/or VR in the Heart Center of Beijing Chao-Yang Hospital from March to June 2021 were included. Peripheral blood and pericardial drainage fluid samples were collected before surgery and at 0 h, 6 h, 24 h and 48 h after surgery to detect PICP, ICTP and fibrinogen levels, and preoperative, intraoperative and postoperative confounding factors were also collected. PICP, ICTP and fibrinogen levels were measured by enzyme-linked immunosorbent assay (ELISA). ResultsA total of 26 patients with 125 blood samples and 78 drainage samples were collected. There were 18 males and 8 females with an average age of 64.04±7.27 years. The incidence rate of POAF was 34.6%. Among the factors, the fibrinogen level in pericardial drainage showed two peaks within 48 h after operation (0 hand 24 h after operation) in the POAF group, while it showed a continuous downward trend in the sinus rhythm (SR) group, and the change trend of fibrinogen in pericardial drainage was significantly different over time between the two groups (P=0.022). Fibrinogen in blood, PICP and ICTP in blood and drainage showed an overall decreasing trend, and their trends over time were not significantly different between the two groups of patients (P>0.05). Univariate analysis showed that fibrinogen at 24 h and 48 h after pericardial drainage, fibrinogen in preoperative blood, PICP immediately after surgery and right atrial long axis diameter were significantly higher or longer in the POAF group than those in the SR group. Multiple regression showed that fibrinogen≥11.47 ng/mL in pericardial drainage 24 h after surgery (OR=14.911, 95%CI 1.371-162.122, P=0.026), right atrial long axis diameter≥46 mm (OR=10.801, 95%CI 1.011-115.391, P=0.049) were independent predictors of POAF. ConclusionThis study finds the regularity of changes in fibrinogen and collagen metabolic markers after CABG and/or VR surgery, and to find that fibrinogen in pericardial drainage 24 h after surgery is a potential novel and predictive factor for POAF. The results provide a new idea for exploring the mechanism of POAF, and provide a research basis for the accurate prediction and prevention of clinical POAF.
Objective To explore the effect of cyclopamine （Cyc） which is the inhibitor of the Hedgehog signaling pathway on portal venous pressure of normal and liver cirrhosis rats, and it’s possible mechanisms. Moreover, to provide the experimental basis of drug efficacy and clinical treatment. Methods Thirty two healthy male SD rats were randomly average divided into four groups：normal control group, normal treatment group, liver cirrhosis control group, and liver cirrhosis treatment group. The liver cirrhosis models of rat were established by using the thioacetamide （TAA） method, which made 0.03% of TAA as the initial water concentration, and then the concentration of TAA in drinking water was adjusted according to the changes of the weekly body weight of rats lasting for twelve weeks. In thirteenth week, intraperitoneal injection of corn oil （0.1 ml/100 g body weight, 1 time/d） were performed lasting for a week in rats of the normal control group and liver cirrhosis control group; intraperitoneal injection of Cyc 〔1 mg （0.1 ml）/100 g body weight, 1 time/d〕were performed lasting for a week in rats of the normal treatment group and liver cirrhosis treatment group. In fourteenth week, the liver function, portal venous pressure （PVP）, and the ration of liver or spleen weight to body weight were detected, the expressions of α-smooth muscle actin （α-SMA） and typeⅠcollagen α1 （Col1α1） of hepatic stellate cell were detected by using immunohistochemistry. Results PVP were （10.7±0.9） and （12.3±1.3） cm H2O （1 cm H2O=0.098 kPa） in normal control group and normal treatment group, respectivly, the latter was higher than the former （t=-2.918，P=0.011）. PVP were （21.8±0.7） and （14.3±1.4） cm H2O in liver cirrhosis control group and liver cirrhosis treatment group, respectivly, the latter was lower than the former（t=13.602，P=0.000）. The expressions of α-SMA and Col1α1 in liver cirrhosis treatment group was lower than the liver cirrhosis control group. There were no significant difference of the liver function and ration of liver or spleen weight to body weight between the treatment group and the control group （P＞0.05）. Conclusion Cyclopamine could signally reduce the PVP of liver cirrhosis rats through reducing the expressions of α-SMA and Col1α1.
Objective To analyze the contents of collagen type Ⅰ, type Ⅲ and the ratio of collagen type Ⅰ to collagen type Ⅲ in posterior rectus sheath of different person. Methods One hundred and four tissues specimen of posterior rectus sheath were obtained during patients’ abdominal operation. The contents of collagen type Ⅰand type Ⅲ were detected by using immunohistochemistry methods. The differences of collagen contents between male and female, physical work group and non-physical work group, smoking group and non-smoking group were observed. The relationships between the contents of collagen and age, body mass index (BMI), and height were analyzed, respectively. Results ① The content of collagen typeⅠand the ratio of collagen type Ⅰ／Ⅲ were both lower in male than those in female （Plt;0.01）； there were no obvious differences in the content of collagen type Ⅲ and the total amount of collagen （Pgt;0.05）. ② There were no differences between physical work group and non-physical work group with the amount and the ratio of collagens （Pgt;0.05）. ③ When compared with non-smoking group, less collagen typeⅠ(Plt;0.01) and lower ratio of collagen Ⅰ／Ⅲ （Plt;0.05） were found in smoking group； but there was no difference with content of collagen Ⅲ（Pgt;0.05）, as well as the total amount of collagen （Pgt;0.05）. ④ The total amount of collagen, the content of collagen type Ⅰand the ratio of collagen Ⅰ／Ⅲ all decreased as age increases （r=0.341, 0.392, 0.212， P＜0.001, Plt;0.05）； no obvious change was observed in the content of collagen Ⅲ （r=0.089， Pgt;0.05）. ⑤ The content and ratio of collagen had no obvious relationships with BMI and height （Pgt;0.05）. Conclusion Smoking, gender and age are all influential factors of the content and ratio of collagens in the tissue.
ObjectiveTo explore the biocompatibility of the poly-lactide-co-glycolide (PLGA)/collagen type I scaffold with rat vaginal epithelial cells, and the feasibility of using PLGA/collagen type I as scaffold to reconstruct vagina by the tissue engineering. MethodsPLGA/collagen type I scaffold was prepared with PLGA covered polylysine and collagen type I. The vaginal epithelial cells of Sprague Dawley rat of 10-12 weeks old were cultured by enzyme digestion method. The vaginal epithelial cells of passage 2 were cultured in the leaching liquor of scaffold for 48 hours to detect its cytotoxicity by MTT. The vaginal epithelial cells were inoculated on the PLGA/collagen type I scaffold (experimental group) and PLGA scaffold (control group) to calculate the cell adhesion rate. Epithelial cells-scaffold complexes were implanted subcutaneously on the rat back. At 2, 4, and 8 weeks after implantation, the epithelial cells-scaffold complexes were harvested to observe the cell growth by HE staining and immunohistochemical analysis. The epithelial cells-scaffold complexes were transplanted to reconstruct vagina in 6 rats with vaginal defect. After 3 and 6 months, the vaginal length was measured and the appearance was observed. The neovagina tissues were harvested for histological evaluation after 6 months. ResultsThe epithelial cells grew and proliferated well in the leaching liquor of PLGA/collagen type I scaffold, and the cytotoxicity was at grade 1. The cell adhesion rate on the PLGA/collagen type I scaffold was 71.8%±9.2%, which significantly higher than that on the PLGA scaffold (63.4%±5.7%) (t=2.195, P=0.005). The epithelial cells could grow and adhere to the PLGA/collagen type I scaffolds. At 2 weeks after implanted subcutaneously, the epithelial cells grew and proliferated in the pores of scaffolds, and the fibroblasts were observed. At 4 weeks, 1-3 layers epithelium formed on the surface of scaffold. At 8 weeks, the epithelial cells increased and arranged regularly, which formed the membrane-like layer on the scaffold. The keratin expression of the epithelium was positive. At 3 months after transplantation in situ, the vaginal mucosa showed pink and lustrous epithelialization, and the majority of scaffold degraded. After 6 months, the neovagina length was 1.2 cm, without obvious stenosis; the vaginal mucosa had similar appearance and epithelial layer to normal vagina, but it had less duplicature; there were nail-like processes in the basal layer, but the number was less than that of normal vagina. The immunohistochemistry staining for keratin was positive. ConclusionThe PLGA/collagen type I scaffolds have good cytocompatibility with the epithelial cells, and can be used as the biodegradable polymer scaffold of the vaginal tissue engineering.
Objective Platelet-rich plasma (PRP) can promote wound heal ing. To observe the effect of PRP injection on the early heal ing of rat’s Achilles tendon rupture so as to provide the experimental basis for cl inical practice. Methods Forty-six Sprague Dawley rats were included in this experiment, female or male and weighing 190-240 g. PRP and platelet-poor plasma (PPP) were prepared from the heart arterial blood of 10 rats; other 36 rats were made the models of Achilles tendon rupture, and were randomly divided into 3 groups (control group, PPP group, and PRP group), 12 rats for each group. In PPP and PRP groups, PPP and PRP of 100 μL were injected around the tendons once a week, respectively; in the control group, nothing was injected. The tendon tissue sample was harvested at 1, 2, 3, and 4 weeks after operation for morphology, histology, and immunohistochemistry observations. The content of collagen type I fibers also was measured. Specimens of each group were obtained for biomechanical test at 4 weeks. Results All the animals survived till the end of the experiment. Tendon edema gradually decreased and sliding improved with time. The tendon adhesion increased steadily from 1 week to 3 weeks postoperatively, and it was relieved at 4 weeks in 3 groups. There was no significant ifference in the grading of tendon adhesion among 3 groups at 1 week and at 4 weeks (P gt; 0.05), respectively. The inflammatory cell infiltration, angiogenesis, and collagen fibers were more in PRP group than in PPP group and control group at 1 week; with time, inflammatory cell infiltration and angiogenesis gradually decreased. Positive staining of collagen type I fibers was observed at 1-4 weeks postoperatively in 3 groups. The positive density of collagen type I fibers in group PRP was significantly higher than that in control group and PPP group at 1, 2, and 3 weeks (P lt; 0.05), but no significant difference was found among 3 groups at 4 weeks (P gt; 0.05). The biomechanical tests showed that there was no significant difference in the maximal gl iding excursion among 3 groups at 4 weeks postoperatively (P gt; 0.05); the elasticity modulus and the ultimate tensile strength of PRP group were significantly higher than those of control group and PPP group at 4 weeks (P lt; 0.05). Conclusion PRP injection can improve the healing of Achilles tendon in early repair of rat’s Achilles tendon rupture.
Objective To investigate the effect of vanadate on prol iferation and collagen type I production of rat medial collateral l igament (MCL)fibroblasts. Methods A total of 12 adult male SD rats were included, weighing 350-375 g. MCL was cut into small pieces (1 mm × 1 mm × 1 mm) in aseptic conditions, and then placed and cultured in culture chambers. Fibroblasts were passaged with 0.25% trypsin. The vanadate (0, 1.0, 2.5, 5.0 ng/mL) was added in the 3rd passage MCL fibroblasts, respectively, and the samples were divided into 4 groups (0, 1.0, 2.5, 5.0 ng/mL groups). MTT was used to measure the cell prol iferation. The production of collagen type I was measured by RT-PCR and ELISA. Twelve samples in each group were measured. Results In fibroblast prol iferation, the absorbency values of the 1.0, 2.5, 5.0 ng/mL groups were significantly different from that of the 0 ng/mL group (P lt; 0.05). The absorbency values of the 0, 1.0, 2.5, and 5.0 ng/mL groups were 0.213 ± 0.016, 0.327 ± 0.023, 0.449 ± 0.137, and 0.561 ± 0.028, respectively. In collagen secretion, vanadate in 1.0, 2.5, 5.0 ng/mL groups could significantly induce the production of collagen type I (P lt; 0.05) ina dose-dependent manner. The expressions of collagen type I of 0-5 ng/mL groups were 0.47 ± 0.02, 0.51 ± 0.03, 0.60 ± 0.01, and 0.72 ± 0.02, respectively. There was significant difference between the 1.0, 2.5, 5.0 ng/mL groups and 0 ng mL group (P lt; 0.05). RT-PCR displayed a dramatic increase of band density. The ratio of band density in the 0-5 ng mLgroups was 1.37 ± 0.76, 1.97 ± 0.53, 2.41 ± 0.94, and 2.73 ± 0.82, respectively. The gene expression of collagen type I in the 1.0, 2.5 and 5.0 ng/mL groups was higher than that in the 0 ng/mL group, and there was significant difference (P lt;0.05). There were statistical significant differences among 1.0, 2.5 and 5.0 ng/mL groups in each index mentioned above.Conclusion Vanadate can effectively induce the prol iferation of fibroblasts and the production of collagen type I in vitro, which may provide a new approach to the treatment of MCL injury.
【摘要】 目的 探讨微弧氧化（microarc oxidation，MAO）结合应用于纯钛种植体表面处理的可行性。 方法 根据对纯钛钛片处理的不同将实验分为对照组（A组，不作处理）、MAO组（B组，纯钛片上进行MAO处理）及MAO加Ⅰ型胶原组（C组，纯钛片上MAO处理后吸附Ⅰ型胶原）。将成骨细胞培养于各组钛片上，通过扫描电镜、MTT法检测不同时间点各组钛片表面的细胞生长及增殖情况，并检测碱性磷酸酶（alkaline phosphatase，ALP）活性。 结果 扫描电镜显示成骨细胞在C组钛片上细胞黏附情况优于A、B组；MTT法及ALP活性检测示培养3、6 d，成骨细胞在C组钛片上的增殖及ALP活性与A、B组比较差异均有统计学意义（Plt;0.05）。 结论 MAO结合Ⅰ型胶原处理的钛片可更有效提高成骨细胞表面附着、增殖，且具有较高的ALP活性。【Abstract】 Objective To study the feasibility of applying microarc oxidation (MAO) with collagen Ⅰ in surface modification of pure titanium. Methods According to different processing methods, the pure titanium was divided into three groups: the control group (without surface modification, group A), MAO group (with microarc oxidation applied in pure titanium surface modification, group B), MAO+Ⅰ group (with microarc oxidation and collagen Ⅰ applied in pure titanium treatment, group C). Osteoblasts were cultured on the surface of titanium in each group, and the cell proliferation in each group was detected at different time points by scanning electron microscopy and MTT method. Moreover, the activity of alkaline phosphatase (ALP) was also detected. Results Scanning electron microscopy showed that adhesion of osteoblasts for group C was better than group A and group B. MTT method and ALP activity detection indicated that there was a significant difference between group C and group A, B in cell proliferation and ALP activity on the third and sixth day of cultivation (Plt;0.05). Conclusion MAO with collagen Ⅰ applied in surface modification of pure titanium may increase osteoblast attachment, and promote its proliferation and ALP activity.
The purpose of this study was to investigate the effect of biaxial tensile strain on the osteogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs) in vitro. The rBMSCs were isolated from tibia and femur of 4 weeks-old Sprague-Dawley (SD) rats. The rBMSCs were cultured in DMEM-LG complete culture medium and grew to subconfluence in the cell culture device for loading tensile strain. The biaxial tensile strain was applied to the rBMSCs for periods of 2, 4 and 6 hours every day, respectively, lasting 3 days. The amplitude of biaxial tensile strain applied to the rBMSCs were 1%, 2% and 5% respectively, at a frequency of 1 Hz. Unstrained rBMSCs were used as blank control (control group). The rBMSCs cultured with DMEM-LG complete culture medium containing 100 nmol/L β-Estradiol (E2) were used as positive control. The mRNA expression of alkaline phosphatase (ALP), collagen typeⅠ (ColⅠ), Runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) was examined with real-time quantitative PCR and the protein expression of ALP, ColⅠ, Runx2 and OCN was detected with Western blot method. The results showed as follws: (1) The mRNA and protein expression of the ALP, ColⅠ, Runx2, OCN were significantly higher in rBMSCs of the E2 group than those in the control group (P＜0.05). (2) The mRNA and protein expression level of the ALP, Runx2 were higher markedly in the 1% tensile strain groups than those in the control group (P＜0.05), but lower than those in the E2 group (P＜0.05). (3) The mRNA and protein expression level of the ALP, ColⅠ, Runx2, OCN were significantly higher in the 2% tensile strain groups than those in the control group (P＜0.05), and the mRNA and protein expression level of ColⅠ and Runx2 in the group applied with 2% amplitude of tensile strain for 4 h/d was significantly higher than those in E2 group (P＜0.05). (4) The mRNA and protein expression level of the ALP, ColⅠ, Runx2 were significantly higher in the groups applied with 5% amplitude of tensile strain for 2 h/d or for 4 h/d than those in the control group (P＜0.05). In our study, E2 and mechanical stimulation played an important role in the regulation of differentiation of rBMSCs into osteoblasts, and the manner applied with the 2% amplitude of tensile strain for 4 h/d, lasting 3 days was an optimal stimulus for up-regulating the mRNA and protein expression of ALP, ColⅠ, Runx2, OCN of rBMSCs.
Objective To investigate the preventive effect of carbachol on the formation of postoperative intra-abdominal adhesion. Methods Forty-four Wistar rats were randomly divided into sham operation group (SO group, n=12), operation group (n=16) and carbachol treated group (carbachol group, n=16, carbachol 50 μg/kg). Animal model of abdominal adhesion was established by rubbing the procussus vermiformis of cecum with dry sterile gauze, and by clamping and scuffing abdominal wall. Half of rats were separately killed on day 7 and day 14 after surgery, respectively. The degree of adhesion was evaluated according to Phillips 5-scale grade and the feature of this model. The histopathological changes of adhesive tissues were observed and the content of collagen type Ⅰ in the tissues was detected by immunohistochemistry. Results The scores of intra-abdominal adhesion were significantly lower in the carbachol group than those in operation group both on 7 d and 14 d (P＜0.01). Mild inflammatory changes and less fibrous proliferation were observed in carbachol group microscopically. The contents of collagen type Ⅰ detected by immunohistochemistry were significantly lower in the carbachol group than those in operation group both on 7 d and 14 d (P＜0.01). There was no significant difference of the score of abdominal adhesion and content of collagen type Ⅰ in the same group between 7 d and 14 d (Pgt;0.05). Conclusion Carbachol may take a significant role in the prevention of postoperative abdominal adhesion in rat.