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 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 further investigate the possible mechanism of the correction of scol iosis with Staple by quantifying the effect of Staple on growth rate of vertebral growth plates in goat scol iosis. Methods Experimental scol iosis was created in 10 juvenile female goats by using unilateral pedicle screws asymmetric tethering. After 8-10 weeks, goats were divided randomly into Staple treated group (n=5) and control group (n=5). All tethers were removed in both groups and Staplegroup underwent anterior vertebral stapl ing with 4-5 shape memory alloy Staples along the convexity of the maximal curvature after posterior tether being removed. All goats were observed for an additional 8-13 weeks, the Cobb angle were measured to observe the correction of scol iosis. The fluorochromes Oxytetracycl ine and Calcein were administered respectively 18 and 3 days before death to label the ossifying front under the growth plates. Superior intervertebral disc of apical vertebra and two adjacent growth plates were completely harvested in all goats. All specimens were embedded with polymethyl methacrylate and sl iced undecalcified. The growth rates of the vertebral growth plates were calculated by measuring the distance between the two fluorescent l ines with fluorescence microscope. Results Nine (5 in Staple treated group and 4 in control group) of 10 tethered goats had progressive scol iotic curves of significant magnitude after 8-10 weeks of tethering. In Staple treated group, the Cobb angles were (34.8 ± 12.4)° at the instant after treatment , and (15.6 ± 11.7)° 8-13 weeks after treatment; showing statistically significant difference (P lt; 0.05). In the control group, the Cobb angles were (49.3 ± 18.0)° at the instant after treatment, and(49.0 ± 17.6)° 8-13 weeks after treatment; showing no statistically significant difference (P gt; 0.05). In Staple treated group, the growth rate of growth plate in the concavity (3.27 ± 0.96) μm/d was higher than that in convexity (1.84 ± 0.52) μm/d (P lt; 0.05), while the growth rate of the concavity did not differ significantly from that of the convexity in control group (P gt; 0.05). Conclusion Staple can significantly alter the growth rates of two sides of vertebrae in scol iosis with the growth rate of concavity exceeding the one of convexity, which results in correction of deformity.
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.
ObjectiveTo discuss the effect of three different ways of annulus fibrosus incision on the biomechanical strength of intervertebral disc. MethodsA total of 30 goats underwent intervertebral disc nucleus pulposus extraction at L3, 4 and L4, 5 by the working channel in group A (n=10), by circular incision in group B (n=10), and by square incision in group C (n=10). The body weight, male and female ratio, age, intraoperative blood loss, and wound healing time were recorded and compared among 3 groups. The survival rate and wound healing situation were observed after operation. At 24 weeks after operation, the goats were sacrificed, MRI images were taken to observe the signal intensity of nucleus pulposus. The disc height of L3, 4 and L4, 5 was measured to calculate the loss of disc height; biomechanical test was used to assess the strength of the disc and anulus. Histological staining was also conducted to observe the repair effect at L4, 5. ResultsThere was no significant difference in body weight, male to female ratio, age, intraoperative blood loss, and wound healing time among groups (P>0.05). All goats survived to the end of the experiment. MRI examination showed decreased signal intensity in 3 groups, indicating intervertebral disc degeneration. According to modified Thompson classification method, the degree of intervertebral disc degeneration of group A was significantly higher than that of groups B and C (P<0.05), but no significant difference was found between groups B and C (P>0.05). Difference was not significant in intervertebral space height before operation among 3 groups (P>0.05). But after 24 weeks, the intervertebral space height in group A was significantly higher than that in groups B and C (P<0.05), and the intervertebral space height loss in group A was significantly lower than that in groups B and C (P<0.05). The biomechanical strength in group A was also significantly higher than that in groups B and C (P<0.05), but no significant difference was found between group B and group C (P>0.05). HE and Masson staining showed good continuity of annulus fibrosus and clear layers in group A; poor continuity of annulus fibrosus and obvious scar tissues were observed in groups B and C. ConclusionApplication of working channel may have less destruction of annulus fibrosus, it plays a positive role in the maintenance of biomechanical strength and repair of annulus fibrosus.
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 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 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.
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.
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.