Objective To explore the osteogenic potential of cervical intervertebral disc fibroblasts in vitro, to investigate the regulatory factors of recombinant human bone morphogenetic protein 2(rhBMP-2) and tumor necrosis factor α(TNF-α) on osteogenic phenotype of fibroblasts and to discuss the condition that facilitates osteogenesis of fibroblasts. Methods Theannulus fibroblasts cell lines of experiment goats were established in vitro and the biologicspecificity was found. According to different medias, 4 groups were included in this experiment: control group, TNF-α group ( 50 U/ml TNF-α), rhBMP-2 group (0.1 μg/ml rhBMP-2) and TNF-α+rhBMP-2 group (50 U/ml TNF-α+0.1 μg/ml rhBMP-2). Thefibroblasts were incubated in the media for about 3 weeks,and then the markers for osteogenic features were investigated by biochemistry, histochemistry observations. Results rhBMP-2 and TNF-α had no effect on the proliferation of fibroblasts from the experiment goats. rhBMP-2 or TNF-α could stimulate fibroblasts to secrete alkaline phosphatase and collagen type Ⅰ. The combined use of rhBMP-2 and TNF-α or the single use of rhBMP-2 could make fibroblasts to secrete osteocalin and the morphological changes of the fibroblasts were very obvious. Histochemical study of the nodules with specific new bone labeler(Alizarin red S) revealed positive reaction, denoting that the nodules produced by the fibroblasts werebone tissues. There was statistically significant difference(Plt;0.05) inALP activity between 3 experimental groups and control group and in secretion of osteocalcin between rhBMP-2 group, TNF-α+rhBMP-2 group and control group. Conclusion The results point out clearly that rhBMP-2 can induce theosteogenic potential of annulus fibroblasts in vitro.
Objective To investigate the effectiveness of mosaicplasty in repair of large-sized osteochondral compound defects and the integrity of transplanted tissue with recipient sites so as to lay a foundation for clinical application. Methods Twenty-four adult goats were divided into 3 groups randomly. The diameters of defect were 6 mm for the medium-sized defects and 9 mm for the large-sized defects, which were created by a trepan. All of the defects were repaired with osteochondral plugs in diameters of 2 mm(the mediumsized defects) or 3 mm(the large-sized defects). The osteochondral plugs were harvested around the intercondylar fossa or intertrochlea groove, and pressed into the recipient sites by specialized instruments in a mosaic mode. No internal fixation was needed and the animal wereallowed to move freely after operation. From 4 to 24 weeks postoperatively, thespecimens were observed in gross and under electromicroscopy. X-ray detection and glycosaminoglycan(GAG) analysis were also performed to testify the healing processand the integrity of the cartilage and subchondral bone. Results The transplanted subchondral bone was integrated firmly with each other or with recipient sites in both mosaicplasty groups. But 24 weeks postoperatively, transplanted cartilage was not integrate with each other apparently. Obvious cleavage between cartilage plugs could be seen. But in the largesized defect groups, some of the osteochondral plugs were relapsed into the defects leaving the recipient sites some steps, leading to some degree of abrasion in the opposing articular cartilage. There was no significant difference in the GAG content between the transplanted cartilage and normal cartilage. X-ray analysis also demonstrated the healing process between the subchondral bone. Conclusion Mosaicplasty can repair the medium or small-sized osteochondral defects efficiently.
Objective To study the vascularization of the compositeof bio-derived bone and marrow stromal stem cells(MSCs) in repairing goat tibial shaft defect.Methods Bio-derived bone was processed as scaffold material. MSCs were harvested and cultured in vitro. The multiplied and induced cells were seeded onto the scaffold to construct tissue engineered bone. A 20 mm segmental bone defect inlength was made in the middle of the tibia shaft in 20 mature goats and fixed with plate. The right tibia defect was repaired by tissue engineered bone (experimental side), and the left one was repaired by scaffold material (control side).The vascularization and osteogenesis of the implants were evaluated by transparent thick slide, image analysis of the vessels, and histology with Chinese ink perfusion 2, 4, 6, and 8 weeks after operation.Results More new vessels were found in control side than in experimental side 2 and 4 weeks after implantation (Plt;0.05). After 8 weeks, there was no significant difference in number of vessels between two sides(Pgt;0.05), and the implants were vascularized completely. New bone tissue was formed gradually as the time and the scaffold material degraded quickly after 6 and 8 weeks in the experimental side. However, no new bone tissue was formed andthe scaffold degraded slowly in control side 8 weeks after operation.Conclusion Bio-derived bone has good quality of vascularization. The ability of tissue-engineered bone to repair bone defect is better than that of bio-derived bone alone.
Objective To investigate effects of the autologous bone mesenchymal stem cells (MSCs) enriched by the small intestinal submucosa (SIS) film implantation on the myocardial structure, cardiac function, and compensator y circulation after myocardial infarction in the goats. Methods Sixteen black goats were selected and divided randomly into the control group (n=8)and the experimental group (n=8). The chronic myocardial infarction models were made by the ligation of the far end of the left anterior desc ending coronary artery. At the same time, MSCs were aspired from the thigh bone of the goats in the experimental group. MSCs were isolated by the centrifu gation through a percoll step gradient and purified by the plating culture and depletion of the non-adherent cells. Primary MSCs were cultured in the DMEM me dium supplemented with the fetal bovine serum in vitro. After that, the cultures were labeled by 5- BrdU. The active cells were transplanted into the SIS film. Six weeks after the ligation, the MSCs-SIS film was implanted by its being sutured onto the infarction area; whereas, the control group underwent a shamoperation. In both groups, echocardiographic measurements were performed before infarction, 6 weeks after infarction and 6 weeks after the MSC-collagen mplantion, respectively, to assess the myocardial structure and ca rdiac function. The left coronary artery angiography was performed with the digi tal subtraction angiography. Results In an assessment of the left ventricular function, at 6 weeks after operation, t he stroke volume and the ejection fraction of the control group and the experim ental group were 42.81±4.91, 37.06±4.75 ml and 59.20%±5.41%, 44.56%±4.23%, respectively (Plt;0.05). The enddisatolic volume and the endsystolic volume of the control group and the experimental group were 72.55±8.13, 83.31±8.61 ml and 29.75±5.98, 46.25±6.68 ml, respectively (Plt;0.05). The maximal velocity of peak E of contral group and experimental group were 54.8 5±6.35 cm/s and 43.14±4.81cm/s (Plt;0.01); and the maximal velocity of peak A o f control group and experimental grouop were 52.33±6.65 cm/s and 56.91±6.34 cm/s (Pgt;0.05). Echocowdiogr aphy sho wing a distinctly dilatation of left ventricle with the ventricular dyskinesia i n contral group, but without the ventricular dyskinesia in experimental group. T he selective-coronary evngiography revealed that the obvious compensatory circu l ation established between the anterior descending branch and the left circumflex branch in the experimental group. Conclusion Implantation of the autologus MSCs enriched by the SIS film can prevent dilatation of the left ventricular chamber and can improve the contractile ability of the myocardium, cardiac function, and collateral perfusion.
Objective To investigate the effect of cleft palate on the development of the mid-part of the face so as to provide an optimum animal model for the fetal cleft repair. Methods Twenty female Boer hybrid goats were selected, aging from 8 to 12 months and weighing from 35 to 55 kg. The mating day was identified as 0 day of pregnancy. The goats werediagnosed with pregnancy by the B-ultrasound examination at 30 days, and were allocated into experimental group (n=14) and control group (n=6). In experimental group, uterine cavitory operation was performed at 65 days of pregnancy to form cleft palate which was a fissure between oral and nasal cavity; no treatment was given as the control group. At 120 days of pregnancy, and after 1 month and 3 months of birth, the gross observation and 3-dimensional skull CT reconstruction were performed; and the maxillary bone width named as PPMM and the maxillary bone length named as APMM were measured. Results After operation, 2 goats died of infection, miscarriage occurred in 3 goats; 9 goats were included into the experiment. The operation success rate was 64.3%. In experimental group, maxillary dysplasia occurred in all the fetal goats at 120 days of pregnancy, and more obvious maxillary dysplasia was observed at 1 month and 3 months after birth; no maxillary dysplasia occurred in control group. There were significant differences in PPMM and APMM between 2 groups at different time points (P lt; 0.05). In experimental group, the lambs had poor chewing function, and died of pulmonary infection after aspiration at 1-4 months after birth. Conclusion The surgical procedure for partial ablation of secondary primitive palate in the midl ine could make the model of cleft palate.
Objective To discuss the stabil ity and practical ity of temporomandibular joint replacement by establ ishing goats artificial temporomandibular joint replacement model. Methods Six healthy mature goats were selected, the male and female being half and weighing 35.3-37.0 kg. According to the parameters from X-ray films of goat’ s temporomandibular joint and the shape of the same kind goat’s skull, the total temporomandibular joint prosthesis was prepared. The one side temporomandibular joints of six goats were replaced by prosthesis randomly as the experimental group (n=6, fossa and condyle according to replacement location) and the other side by titanium plate as the control group (n=6). At 4,8, and 12 weeks, the histological observation, scanning electron microscope (SEM) observation were carried out for observing structural changes in the interface. The mechanical test and histochemistry test were used for observing the combination degree of interface and the alkal ine phosphatase (ALP) activity. Results All animals were al ive to the end of experiment with normal open mouth, good recovery of masticatory function, and normal eating. At 4, 8, and 12 weeks, implants were stable in 2 groups without loosening. The histological observation and SEM observation showed the amount of osteoblasts in interface increased over times. There were significant differences in the shearing force and the ALP activity between fossa in experimental group and control group at 4 weeks (P lt; 0.05), but there was no significant difference between other groups (P gt; 0.05). Conclusion The total temporomandibular prosthesis has good stabil ity in temporomandibular joint reconstruction of goat after replacement.
Objective To evaluate the influence of PKH26 labeling on the biological function of the goat nucleus pulposus cells and the biological function of seeded cells in nude mice by in vivo imaging techonology. Methods Primary nucleus pulposus cells were isolated by enzymatic digestion from the nucleus pulposus tissue of the 1-year-old goat disc. The nucleus pulposus cells at passage 1 were labeled with PKH26 and the fluorescent intensity was observed under the fluorescence microscopy. The labeled cells were stained with toluidine blue and collagen type II immunocytochemistry. The cells viability and proliferation characteristics were assessed by trypan blue staining and MTT assay, respectively. Real-time fluorescent quantitative PCR was used to detect the gene expressions of collagen types I and II, and aggrecan. The fluorescent intensity and scope of the nucleus pulposus cells-scaffold composite in vivo for 6 weeks after implanting into 5 6-week-old male nude mice were measured by in vivo imaging technology. Results Primary nucleus pulposus cells were ovoid in cell shape, showing cluster growth, and the cells at passage 1 showed chondrocyte-like morphology under the inverted phase contrast microscope. The results of toluidine blue and collagen type II immunocytochemistry staining for nucleus pulposus cells at passage 1 were positive. The fluorescent intensity was even after labeling, and the cell viability was more than 95% before and after PKH26 labeling. There was no significant difference in cell growth curve between before and after labeling (P gt; 0.05). The real-time fluorescent quantitative PCR showed that there was no significant difference in gene expressions of collagen types I and II, and aggrecan between before and after labeling (P gt; 0.05). Strong fluorescence in nucleus pulposus cells-scaffold composite was detected and by in vivo imaging technology. Conclusion The PKH26 labeling has no effect on the activity, proliferation, and cell phenotype gene expression of the nucleus pulposus cells. A combination of PKH26 labeling and in vivo imaging technology can track the biological behavior of the cells in vivo.
目的 研究组织工程骨结合带锁髓内钉修复成年山羊大段负重骨缺损的可行性，探索更可行的技术路径。 方法 将24只成年山羊，通过骨髓穿刺法获取山羊骨髓间充质干细胞（BMSC），将体外扩增及成骨定向诱导的第2代BMSC与同种异体脱钙骨基质（DBM）通过双相接种法构建组织工程骨。24只成年山羊，以带锁髓内钉构建股骨中段3 cm骨缺损模型。随机分为3组，每组8只。实验组以组织工程骨修复骨缺损，对照组单独使用DBM和空白组旷置。术后1、12、24周行X线片观察及评分，12、24周每组各处死4只动物行组织学观察和生物力学检测。 结果 标本大体观察示实验组和对照组术后12周骨缺损部位被骨痂连接，髓腔贯通，24周全部愈合；实验组24周恢复正常解剖形态，对照组外形仍然粗糙、不规则；空白组术后12周及24周缺损部位均为纤维组织充填。术后1周各组X线评分无明显差异（P＞0.05），实验组术后12周及24周X线评分均优于对照组和空白组，对照组优于空白组，各组24周X线评分均高于12周时，差异均有统计学意义（P＜0.05）。实验组术后12、24周的最大抗扭强度分别达正常侧的47.07% ± 5.05%和83.73% ± 2.33%，显著高于对照组和空白组（P＜0.05）；空白组2个时间点最大抗扭强度均不超过正常的15%，与骨不连时的纤维连接相符。组织学检查示术后12周实验组和对照组骨缺损区DBM支架材料基本被吸收，有典型的同心圆排列的哈弗系统形成，周围偶见淋巴细胞；术后24周，实验组和对照组股骨缺损均被修复，但实验组较对照组的新骨更多、骨塑形更好；空白组术后24周骨缺损区中央仍为纤维组织填充。 结论 组织工程骨结合带锁髓内钉能够更有效修复成年山羊负重骨大段骨缺损，满足负重骨的生物力学要求。
Objective To establish an effective model of myocardial infarction in black goat so as to provide a safe, convenient and credible model of myocardial infarction for treatment and research. Methods Sixteen black goats were made chronic myocardial infarction by ligation of far end of left anterior descending coronary artery through incision below xiphoidprocess. Electrocardiogram(ECG) and serum myocardial enzymes were investigated before and after occlusion. Echocardiographic measurements were performed, and left coronary artery angiography was performed with digital subtraction angiography (DSA) before infarction and 6 weeks after infarction. The myocardial ultrastructure were observed. Results All goats survived more than 6 weeks. ECG showed ambulatory change, ST-segment elevated half an hour after occlusion and pathologic Q waves 6 weeks after infarction, CK-MB significantly increased. Echocardiographic indexes showed significant decrease of maximal peak A, percent wall thickening(WHT) and ejecting fraction (EF), increase ofend-systolic volume (ESV), end-diastolic volume (EDV), and dilation of left ventricle. DSA showed block or decrease of perfusion of far end of left anterior descending coronary artery. Conclusion It is safe, convenient and credible to establish model of myocardial infarction by ligation of far end of left anterior descending coronary artery through incision below xiphoidprocess in black goat.
Objective To investigate the effect of hydrostatic pressure and insulin-like growth factor 1 (IGF-1) on the expression of filamentous actin (F-actin) of temporomandibular joint disc cells in goats, and to analyze the F-actin changes of temporomandibular joint disc cells in vitro under hydrostatic pressure and IGF-1 stimulation. Methods The bilateral temporomandibular joint discs were harvested from 4 1-month-old goats, and temporomandibular joint disc cells were isolated with collagenase. Immunohistochemical staining for collagen type I and collagen type II was performed for identification. The cells at passages 2-3 were used; the experiment was divided into 4 groups according to different interventions: the cells were cultivated with complete medium in group A as control; the cells were intervened by hydrostatic pressure (0.2 MPa and 1 Hz for 3 hours) in group B, by complete medium containing IGF-1 (10 ng/mL) in group C, and by a combination of hydrostatic pressure (0.2 MPa and 1 Hz for 3 hours) and complete medium containing IGF-1 (10 ng/mL) in group D. The changes of F-actin at 24 and 72 hours after cultivation were observed by immunofluorescence staining. The cell fluorescence intensity was measured. Results The cultivated cells were identified to be temporomandibular joint disc cells by morphological observation and immunohistochemical staining. At 24 hours, fluorescence intensity of groups A and C was b and clear, with normal morphology of temporomandibular joint disc cells; F-actin arranged in disorder in group B, and F-actin was thinner with arrangement disorder in group D. At 72 hours, the F-actin arranged regularly in groups A and C; however, some F-actin became blurry with irregular arrangement, breakage, and pseudopodia in group B; and F-actin was thinner and ruptured formed in group D. With time passing, the fluorescence intensity of F-actin in groups A, B, and D had an increasing trend, showing significant differences between 24 hours and 72 hours (P lt; 0.05); but there was no significant difference between 24 and 72 hours in group C (t=0.284, P=0.781). At 24 hours, fluorescence intensity of F-actin was highest in group C and was lowest in group B, showing significant difference when compared with groups A and D (P lt; 0.05). At 72 hours, fluorescence intensity in groups B and D was significantly lower than that in groups A and C (P lt; 0.05), but there was no significant difference between groups B and D, and between groups A and C (P gt; 0.05). Conclusion Hydrostatic pressure may cause the F-actin breakage and rearrangement of temporomandibular joint disc cells, and IGF-1 can up-regulate the F-actin expression. Such effects may be correlated with the biological behavior of the temporomandibular joint disc cells.