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 evaluate the feasibil ity of intrauterine abdominal wall defect repair of fetal lamb at late pregnancy. Methods Eight healthy pregnant ewes at 110-115 days of gestation (weighing 14-22 kg) were randomly divided into 2 groups. In group A (n=3), the abdominal wall defect of 5 cm × 1 cm was made in the fetal lambs, then was closed by strengthening suture; in group B (n=5), the abdominal wall defect of 5 cm × 2 cm was made in the fetal lambs, then was repairedby 2 layers of biological patches. After the lambs del ivered naturally, the lambs and their wounds were observed; at 10th day after birth, the scars were harvested for biomechanical and histological observations. Results One ewe of group A and 2 ewes of group B aborted, while the others were successfully del ivered. In group A, the abdominal incisions of 2 lambs healed well with a l ine-l ike scar and mild intra-abdominal adhesion, and the scar thickness was 4-5 mm. In group B, the abdominal incisions of 3 lambs did not heal completely with minor intra-abdominal adhesions, and the scar thickness was 3-4 mm. The wound breaking strength was 16, 20 N in group A and 10, 14, and 18 N in group B, respectively. A sl ight scar was seen in group A; skin ulcer and underlying fibrous connective tissue with inflammatory cell infiltration were seen in group B. Conclusion It was feasible to repair the abdominal wall defect of fetal lamb at late pregnancy in uterine. Small abdominal wall defect can be sutured directly; biological patch can be used to repair larger abdominal wall defect.
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.
ObjectiveTo evaluate the effect of poly-amino acid/nano-hydroxyapatite/calcium sulfate (PHC) Cage in lumbar interbody fusion of the goat. MethodsEighteen mature female goats (weighing 29-33 kg) were divided into 3 groups randomly: PHC Cage group (group A), titanium Cage group (group B), and ilium group (group C). A left extraperitoneal approach was used to establish the animal model of discectomy and interbody fusion with Cage or ilium. The general situation was observed for 24 weeks after operation. X-ray films were taken to measure disc space height (DSH) before operation and at 4, 12, and 24 weeks after operation. CT three dimensional reconstuction was performed at 24 weeks after operation to evaluate the interbody fusion according to modified Brantigan grading. The specimens of L3, 4 were harvested for mechanical test, histological, and scanning electron microscope (SEM) observation at 24 weeks after operation. ResultsAll goats survived to the end of experiment. DSH at 4 weeks after operation increased when compared with preoperative one in each group, and then decreased;DSH was significantly lower at 12 and 24 weeks after operation than preoperative one in group C (P<0.05). There was no significant difference in DSH among 3 groups at preoperation and 4 weeks after operation (P>0.05);at 12 and 24 weeks after operation, DSH of groups A and B was significantly higher than that of group C (P<0.05), but no significant difference was found between groups A and B (P>0.05). CT three dimensional reconstuction showed that bony fusion was obtained in all goats of groups A and C, and in 3 goats of group B;according to modified Brantigan grading, the scores of groups A and C were significantlly higher than that of group B (P<0.05), but no significant difference between groups A and C (P>0.05). The biomechanical test showed that there was no significant difference in range of motion between group A and group B (P>0.05), which were significantly lower than that of group C (P<0.05). Microscopy and SEM observations showed that the interface between the Cage and vertebral body in group A was compact without obvious gap, and most conjunctive region was filled with osseous tissue;the interface was filled with soft tissue, and the connection was slack with obvious gap in some region in group B;the interface connection was compact, most region was filled with osseous tissue in group C. ConclusionThe interbody fusion with PHC Cage is effective in goat lumbar interbody fusion model. The interface connection is compact between the Cage and the host bone followed by micro-degradation of PHC Cage, but the long-term degradation need further observation.
Objective To investigate the ability to repair goat tibia defect with marrow stromal stem cells (MSCs) and bio-derived bone, and the feasibility of the compounds as bone substitute material. Methods MSCs were cultured with the bioderived bone in vitro, and the 20 mm tibia defect of goat was made and fixedwith plate. Eighteen goats were divided into experimental group, control group and blankgroup. The defects were not filled with anything in blank group, with tissue engineering bone in experimental group and bio-derived bone in control group. Therepair capability was assessed by physical, X-ray and bone mineral density examinations8,12,16, and 24 weeks after operation. Results In experimental group, the defects were partially repaired 8 weeks, and completely repaired12 and 16 weeks; there was significant difference in bone density between experimental group and control group (P＜0.05) 8,12 and 16 weeks, but no significant difference 24 weeks. The defects of blank group were not repaired 24weeks. Conclusion The tissue engineering bone can efficiently repair bone defect, and its repair capability is better than that of bio-derived bone alone both in quantity and quality of boneformation.
Objective To establish a rapid, simple, and economic method to prepare osteoporosis (OP) in vitro model. Methods Eighty pairs of fresh goat femur were collected from 18-month-old female goats and were randomly divided into 4 groups (20 pairs in each group). The femur was immersed decalcifying solution (18% EDTA) for 1-5 days (group B), 6-10 days (group C), and 11-15 days (group D), while group A had no treatment as control. Four pairs of femur were taken out every day. Quantitative computed tomography was used to scan the medial and lateral femoral condyles, and the bone mineral density (BMD) was calculated. Electronic universal testing machine was used to do three-point bending test and compress and tensile ultimate strenght test, and the mechanical parameters for femur were calculated. Results With demineralized time passing, BMD of the medial and lateral femoral condyles were downtrend in groups A, B, C, and D, showing significant differences among 4 groups (P lt; 0.05); BMD of the lateral femoral condyle was significantly higher than that of the medial femoral condyle in each group (P lt; 0.05). The three-point bending test showed that broken load, ultimate strength, and elastic modulus of groups A and B were significantly higher than those of groups C and D (P lt; 0.05); but no significant difference was found between groups A and B, and between groups C and D (P gt; 0.05). Compress and tensile ultimate strength test showed that the compress and tensile ultimate strengths were significantly higher in group A than in groups C and D (P lt; 0.05), and in group B than in group D (P lt; 0.05), but no significant difference was found between groups A and B, between groups B and C, and between groups C and D (P gt; 0.05). Conclusion The 18% EDTA immersing for 6-15 days is a fast, simple, economical method to prepare an OP in vitro model of goat femur.
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 Currently, there are few researches on lordosis associated with scol iosis. To explore the effects of nickel-titanium memory alloy staple (Staple) on the growth of thoracic lordosis by observing the histological changes of cartilage cells in the osteoepiphysis of the thoracic vertebrates in goats. Methods Eighteen 2-3 months old female goats, weighing 8-12 kg, were randomly divided into long staple group (n=6), short staple group (n=6), and blank control group (n=6). Long staple (7 mm) and short staple (4 mm) were implanted into T6-11 segments of goats in long and short staplegroups by anterior approach, respectively. The blank control group was not treated. The X-ray examination was performedpre-operatively and at 3 months post-operatively to observe the changes of Cobb angle. Then the growth plates and inferior facet processes of the apex vertebral body were harvested to observe the histological grades of cartilage by HE staining, and to observe prol iferation and apoptosis of chondrocytes through immunohistochemistry double label ing staining with poly-ADPribose- polymerase-p85 and prol iferating cell nuclear antigen. Results At 3 months after operation, the T6-11 Cobb angles were significantly higher than those of pre-operation in short staple group and long staple group, which were significantly higher than those in blank control group (P lt; 0.05), but there was no significant difference between short staple group and long staple group (P gt; 0.05). The results of HE staining and immunohistochemistry double staining showed that the number of chondrocytes were reduced obviously with irregular columnar arrangement and increased volume ratio of surrounding extracellular matrix in prol iferative zone and hypertrophic zone of growth plate and inferior articular process in both long and short staple groups, and this tendency was more noticeable in long staple group. There were significant differences in the grades of prol iferation viabil ity of chondrocytes between 2 staple groups and blank control group (P lt; 0.05), but there was no significant difference tewteen long staple group and short staple group (P gt; 0.05). The prol iferation viabil ities of chondrocytes in growth plate and inferior articular process were significantly higher in blank control group than in 2 staple groups (P lt; 0.01), but there was no significant difference between long staple group and short staple group (P gt; 0.05). Conclusion The histological evidences prove that the Staple implantation by anterior approach can reduce prol iferation viabil ity of chondrocytes in growth plate and inferior articular process of the thoracic vertebrates in goats, which conduces the growth direction of thoracic vertebrates to kyphosis.
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.
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.