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 compare the effect of mosaicplasty, mosaicplasty with gene enhanced tissue engineering and mosaicplasty with the gels of non-gene transduced BMSCs in alginate on the treatment of acute osteochondral defects. Methods Western blot test was conducted to detect the expression of hTGF-β1, Col II and Aggrecan in 3 groups, namely hTGF-β1 transduction group, Adv-βgal transduction group and blank control group without transduction. Eighteen 6-month-old Shanghai mascul ine goats weighing 22-25 kg were randomized into groups A, B and C (n=6). BMSCs were isolatedfrom the autologous bone marrow of groups B and C, and were subcultured to get the cells at passage 3. In group B, the BMSCs were transduced with hTGF-β1. For the animals of 3 groups, acute cyl indrical defects 5 mm in diameter and 3 mm in depth were created in the weight bearing area of the medial femoral condyle of hind l imbs. In group A, the autologous osteochondral mosaicplasty was performed to repair the defect; in group B, besides the mosaicplasty, the dead space between the cyl indrical grafts and the host cartilage were injected with the suspension of hTGF-β1 gene transduced autogenous BMSCs in sodium alginate, and CaCl2 was dropped into it to form calcium alginate gels; in group C, the method was the same as the group B, but the BMSCs were not transduced. General condition of the goats after operation was observed, the goats were killed 12 and 24 weeks after operation to receive gross and histology observation, which was evaluated by the histological grading scale of O’Driscoll, Keeley and Salter. Immunohistochemistry and TEM observation were performed 24 weeks after operation. Results Western blot test showed the expression of the hTGF-β1, Col II and the Aggrecan in the hTGF-β1 transduction group were significantly higher than that of the Adv-βgal transduction and the blank control groups. All the goats survived until the end of experiment and all the wounds healed by first intention. Gross observation revealed the boundaries of the reparative tissue in group B were indistinct, with smooth and continuous surfaces of the whole repaired area; while there were gaps in the cartilage spaces of groups A and C. Histology observation showed the dead space between the cyl indrical grafts in group A had fibrocartilage-l ike repair tissue, fill ing of fibrous tissue or overgrowth of the adjacent cartilage; the chondrocytes in group B had regular arrangements, with favorable integrations; while the dead space between the cyl indrical grafts in group C had fibrocartilage-l ike repair tissue, with the existence of gaps. The histology scores of group B at different time points were significantly higher than that of groups A and C, and group C was better than group A (P lt; 0.05); for group B, significant difference was detected between 12 weeks and 24 weeks in the histology score (P lt; 0.05）. Immunohistochemistry staining for Col II 24 weeks after operation showed the chondrocytes and lacuna of the reparative tissue in group B was obviously stained, while groups A and C presented l ight staining. TEM observation showed there were typical chondrocytes in the reparative tissue in group B, while parallel or interlaced arrangement collagen fiber existed in groups A and C. Conclusion Combining mosaicplasty with tissue engineering methods can solve theproblem caused by single use of mosaicplasty, including the poor concrescence of the remnant defect and poor integration with host cartilages.
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 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 study the method to prepare the animal model of goat cleft palate by injection of anabasine and the effect of the malformation on the development of the facial mid-part. Methods A total of 40 female boer hybrid goats were selected, aging 8-12 months and weighing 35-55 kg. The mating day was 0 day, and at 30 days the goats assured pregnant byB type ultrasonic test were divided into 4 groups (n=10) according to intramuscular injection of 10 (experimental group 1), 15 (experimental group 2), 20 (experimental group 3) mg/ d, and no injection (control group), respectively, from the 31st to 42nd day. At pregnant 120 days and 1 month after birth, 5 fetal goats of each group were used for three dimensional reconstruction ofskull with CT scan. The maxillary bone width named as PPMM and the maxillary bone length named as APMM were measured then the hard palate general observation was performed and dry skull of goats was harvested to observe the development of maxillary. Results After injection, all pregnant lambs aborted in experimental group 3; 2 pregnant lambs aborted and 8lambs maintained pregnancy in experimental group 2. At 120 days of pregnant, no cleft palate was observed in 5 fetal lambs of experimental group 1 and control group, respectively; cleft palate and maxillary dysplasia occurred in 3 fetal lambs of experimental group 2. Among 11 newborn lambs of experimental group 1 and 8 newborn lambs of control group, no cleft palate was observed;among 7 newborn lambs of experimental group 2, cleft palate occurred in 5 with obvious maxillary dysplasia and eating difficultly. General observation of hard palate and dry skull showed obvious hypoplasia of maxillary in experimental group 2. There were significant differences in PPMM and APMM between the experimental group 2 and the control group at pregnant 120 days and 1 month after birth (P lt; 0.05). Five lambs with cleft palates of experimental group 2 survived for 1-2 months. Conclusion The animal models of goat cleft palate can established by intramuscular injection of anabasine at a dose of 15 mg/d from the 31st to 42nd day of pregnant. The facial character of the induced cleft palate goat is similar to that of human cleft palate.
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.
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 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.
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.