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.
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 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 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 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.
目的 研究組織工程骨結合帶鎖髓內釘修復成年山羊大段負重骨缺損的可行性，探索更可行的技術路徑。 方法 將24只成年山羊，通過骨髓穿刺法獲取山羊骨髓間充質干細胞（BMSC），將體外擴增及成骨定向誘導的第2代BMSC與同種異體脫鈣骨基質（DBM）通過雙相接種法構建組織工程骨。24只成年山羊，以帶鎖髓內釘構建股骨中段3 cm骨缺損模型。隨機分為3組，每組8只。實驗組以組織工程骨修復骨缺損，對照組單獨使用DBM和空白組曠置。術后1、12、24周行X線片觀察及評分，12、24周每組各處死4只動物行組織學觀察和生物力學檢測。 結果 標本大體觀察示實驗組和對照組術后12周骨缺損部位被骨痂連接，髓腔貫通，24周全部愈合；實驗組24周恢復正常解剖形態，對照組外形仍然粗糙、不規則；空白組術后12周及24周缺損部位均為纖維組織充填。術后1周各組X線評分無明顯差異（P＞0.05），實驗組術后12周及24周X線評分均優于對照組和空白組，對照組優于空白組，各組24周X線評分均高于12周時，差異均有統計學意義（P＜0.05）。實驗組術后12、24周的最大抗扭強度分別達正常側的47.07% ± 5.05%和83.73% ± 2.33%，顯著高于對照組和空白組（P＜0.05）；空白組2個時間點最大抗扭強度均不超過正常的15%，與骨不連時的纖維連接相符。組織學檢查示術后12周實驗組和對照組骨缺損區DBM支架材料基本被吸收，有典型的同心圓排列的哈弗系統形成，周圍偶見淋巴細胞；術后24周，實驗組和對照組股骨缺損均被修復，但實驗組較對照組的新骨更多、骨塑形更好；空白組術后24周骨缺損區中央仍為纖維組織填充。 結論 組織工程骨結合帶鎖髓內釘能夠更有效修復成年山羊負重骨大段骨缺損，滿足負重骨的生物力學要求。
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 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.
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 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.