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 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 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 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 performance, safety, and precision of the Yuanhua robotic-assisted total knee arthroplasty system (YUANHUA-TKA) through animal experiments, which will provide reference data for human clinical trials.MethodsSix 18-month-old goats, weighing 30-35 kg, were used in this study. The experimental study was divided into two parts: the preoperative planning and intraoperative bone resection. CT scans of the goats’ lower extremities were firstly performed before the experiments. Then the CT scans were segmented to generate the femoral and tibial three-dimensional (3D) models in the YUANHUA-TKA system. The volumes and angles of each resection plane on the femur and tibia were planned. The bone resection was finally implemented under the assistance of the YUANHUA-TKA system. After completing all bone resections, the lower extremities of each goat were taken to have CT scans. By comparing the femoral and tibial 3D models before and after the experiments, the actual bone resection volumes and angles were calculated and compared with the preoperative values.ResultsDuring the experiments, no abnormal bleeding was found; the YUANHUA-TKA system ran smoothly and stably and was able to stop moving and keep the osteotomy in the safe zone all the time. After the experiment, the resection planes were observed immediately and found to be quite flat. There was no significant difference between the planned and actual osteotomy thickness and osteotomy angle (P>0.05); the error of the osteotomy thickness was less than 1 mm, and the error of the osteotomy angle was less than 2°.ConclusionThe YUANHUA-TKA system can assist the surgeons to perform osteotomy following the planned thickness and angle values. It is expected to assist surgeons to implement more accurate and efficient osteotomy in the future clinical applications.
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 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 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 study the preparation method of acellular vascular matrix and to evaluate its biocompatibil ity and safety so as to afford an ideal scaffold for tissue engineered blood vessel. Methods Fresh caprine carotids (length, 50 mm) were harvested and treated with repeated frozen (—80 )/thawing (37℃), cold isostatic pressing (506 MPa, 4 ), and 0.125% sodium dodecyl sulfate separately for preparation of acellular vascular matrix. Fluorescence staining and DNA remain test were used to assess the cell extracting results. Biological characteristics were compared with the raw caprine carotids using HE staining, Masson staining, scanning electron microscope (SEM), and mechanical test. Biocompatibil ity wasdetected using cell adhesion test, MTT assay, and subcutaneously embedding test. Ten SD rats were divided into 2 groups (n=5). In experimental group, acellular vascular matrix preserved by the combination of repeated frozen/thawing, ultrahigh pressure treatment and chemical detergent was subcutaneously embedded; and in control group, acellular vascular matrix preserved only by repeated frozen/thawing and ultrahigh pressure treatment was subcutaneously embedded. Results HE staining and Masson staining revealed that no nucleus was detected in the acellular vascular matrix. SEM demonstrated that a lot of collagen fibers were preserved which were beneficial for cell adhesion. Fluorescence staining and DNA remain test showed that the cells were removed completely. There was no significant difference in stress and strain under the maximum load between before and after treatment. Mechanical test revealed that the acellular vascular matrix reserved mechanical properties of the raw caprine carotids. Cell adhesion test and MTT assay confirmed that cytotoxicity was grade 0-1, and the acellular vascular matrix had good compatibil ity to endothel ial cells. After subcutaneously embedding for 8 weeks, negl igible lymphocyte infiltration was observed in experimental group but obvious lymphocyte infiltration in control group. Conclusion The acellular vascular matrix, which is well-preserved by the combination of repeated frozen/thawing, ultrahigh pressure treatment, and chemical detergent, is an ideal scaffold for tissue engineered blood vessel.
Objective To analyze the distribution of stress in the upper and lower plates of the prosthesis-bone interface, and the effect of interface pressure on osseointegration. Methods CT scanning was performed on goats at 1 week after artificial cervical disc replacement to establish the finite element model of C3, 4. The stress distribution of the upper and lower plates of the interface was observed. At 6 and 12 months after replacement, Micro-CT scan and three dimensional reconstruction were performed to measure the bone volume fraction (BVF), trabecular number (Tb. N), trabecular thickness (Tb. Th), trabecular separation (Tb. Sp), bone mineral density (BMD), bone surface/bone volume (BS/BV), and trabecular pattern factor (Tb. Pf). The C3 lower plate and C4 upper plate of 4 normal goat were chosen to made the cylinder of the diameter of 2 mm. The gene expressions of receptor activator for nuclear factor κB ligand (RANKL), osteoprotegerin (OPG), transforming growth factor β (TGF-β), and macrophage colony-stimulating factor (M-CSF) were detected by real time fluorescent quantitative PCR at immediate after cutting and at 24 and 48 hours after culture. The samples of appropriate culture time were selected to made mechanical loading, and the gene expressions of RANKL, OPG, M-CSF, and TGF-β were detected by real time fluorescent quantitative PCR; no mechanical loading samples were used as normal controls. Results Under 25 N axial loading, the stress of the upper plate of C3, 4 was concentrated to post median region, and the stress of the lower plate to middle-front region and two orbits. According to stress, the plate was divided into 5 regions. The Micro-CT scan showed that BMD, Tb.Th, BVF, and Tb.N significantly increased, and BS/BV, Tb.Sp, and Tb.Pf significantly decreased at 12 months after replacement when compared with ones at 6 months (P<0.05). At 24 and 48 hours after culture, the gene expressions of RANKL, OPG, and TGF-β were signifi-cantly higher than those at immediate (P<0.05), but no significant difference was found between at 24 and 48 hours after culture (P>0.05). The mechanical loading test results at 24 hours after culture showed that the RANKL and OPG gene expressions and OPG/RANKL ratio in C3 lower plate and C4 upper plate were significantly up-regulated when compared with controls (P<0.05), but no significant difference was shown in TGF-β and M-CSF gene expressions (P>0.05). Conclusion Domestic artificial cervical disc endplate has different pressure distribution, the stress of lower plate is higher than that of upper plate. Pressure has important effect on local osseointegration; the higher pressure area is, the osseointegration is better. Under the maximum pressure in interface, the osteoblast proliferation will increase, which is advantageous to the local osseointegration.