Objective To investigate effects of the autologous bone mesenchymal stem cells (MSCs) enriched by the small intestinal submucosa (SIS) film implantation on the myocardial structure, cardiac function, and compensator y circulation after myocardial infarction in the goats. Methods Sixteen black goats were selected and divided randomly into the control group (n=8)and the experimental group (n=8). The chronic myocardial infarction models were made by the ligation of the far end of the left anterior desc ending coronary artery. At the same time, MSCs were aspired from the thigh bone of the goats in the experimental group. MSCs were isolated by the centrifu gation through a percoll step gradient and purified by the plating culture and depletion of the non-adherent cells. Primary MSCs were cultured in the DMEM me dium supplemented with the fetal bovine serum in vitro. After that, the cultures were labeled by 5- BrdU. The active cells were transplanted into the SIS film. Six weeks after the ligation, the MSCs-SIS film was implanted by its being sutured onto the infarction area; whereas, the control group underwent a shamoperation. In both groups, echocardiographic measurements were performed before infarction, 6 weeks after infarction and 6 weeks after the MSC-collagen mplantion, respectively, to assess the myocardial structure and ca rdiac function. The left coronary artery angiography was performed with the digi tal subtraction angiography. Results In an assessment of the left ventricular function, at 6 weeks after operation, t he stroke volume and the ejection fraction of the control group and the experim ental group were 42.81±4.91, 37.06±4.75 ml and 59.20%±5.41%, 44.56%±4.23%, respectively (Plt;0.05). The enddisatolic volume and the endsystolic volume of the control group and the experimental group were 72.55±8.13, 83.31±8.61 ml and 29.75±5.98, 46.25±6.68 ml, respectively (Plt;0.05). The maximal velocity of peak E of contral group and experimental group were 54.8 5±6.35 cm/s and 43.14±4.81cm/s (Plt;0.01); and the maximal velocity of peak A o f control group and experimental grouop were 52.33±6.65 cm/s and 56.91±6.34 cm/s (Pgt;0.05). Echocowdiogr aphy sho wing a distinctly dilatation of left ventricle with the ventricular dyskinesia i n contral group, but without the ventricular dyskinesia in experimental group. T he selective-coronary evngiography revealed that the obvious compensatory circu l ation established between the anterior descending branch and the left circumflex branch in the experimental group. Conclusion Implantation of the autologus MSCs enriched by the SIS film can prevent dilatation of the left ventricular chamber and can improve the contractile ability of the myocardium, cardiac function, and collateral perfusion.
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 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 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 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 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.
Objective To evaluate the application of artificial lamina of multi-amino-acid copolymer (MAACP)/nano-hydroxyapatite (n-HA) in prevention of epidural adhesion and compression of scar tissue after posterior cervical laminectomy. Methods Fifteen 2-year-old male goats [weighing, (30 ± 2) kg] were randomly divided into experimental group (n=9) and control group (n=6). In the experimental group, C4 laminectomy was performed, followed by MAACP/n-HA artificial lamina implantations; in the control group, only C4 laminectomy was performed. At 4, 12, and 24 weeks after operation, 2, 2, and 5 goats in the experimental group and 2, 2, and 2 goats in the control group were selected for observation of wound infection, artificial laminar fragmentation and displacement, and its shape; Rydell’s degree of adhesion criteria was used to evaluate the adhesion degree between 2 groups. X-ray and CT images were observed; at 24 weeks after operation, CT scan was used to measure the spinal canal area and the sagittal diameter of C3, C4, and C5 vertebrea, 2 normal goats served as normal group; and MRI was used to assess adhesion and compression of scar tissue on the dura and the nerve root. Then goats were sacrificed and histological observation was carried out. Results After operation, the wound healed well; no toxicity or elimination reaction was observed. According to Rydell’s degree of adhesion criteria, adhesion in the experimental group was significantly slighter than that in the control group (Z= — 2.52, P=0.00). X-ray and CT scan showed that no dislocation of artificial lamina occurred, new cervical bone formed in the defect, and bony spinal canal was rebuilt in the experimental group. Defects of C4 vertebral plate and spinous process were observed in the control group. At 24 weeks, the spinal canal area and sagittal diameter of C4 in the experimental group and normal group were significantly larger than those in the control group (P lt; 0.05), but no significant difference was found between experimental group and normal group (P gt; 0.05). MRI showed cerebrospinal fluid signal was unobstructed and no soft tissue projected into the spinal canal in the experimental group; scar tissue projected into the spinal canal and the dura were compressed by scar tissue in the control group. HE staining and Masson trichrome staining showed that artificial lamina had no obvious degradation with high integrity, some new bone formed at interface between the artificial material and bone in the experimental group; fibrous tissue grew into defect in the control group. Conclusion The MAACP/n-HA artificial lamina could maintaine good biomechanical properties for a long time in vivo and could effectively prevent the epidural scar from growing in the lamina defect area.
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 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 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.