OBJECTIVE: To explore the possibility of prefabricate a vascularized artificial bone-muscular flap, a compound of xenogenic inorganic bone and auto-marrow, with capability of osteogenesis. METHODS: Twelve 6-month New Zealand rabbits were divided into two groups, compound of xenogenic inorganic bone and auto-marrow implanted into the muscle near to radia and ulna of left forefoot as experimental group. Simple xenogenic inorganic bone implanted into the same site of right side as control group. After 2, 8, 12 weeks, x-ray examination, gross evaluation and histological observation were carried out. RESULTS: X-ray film showed that the implant had a cancellus-like density, and no variance with time. Since 2 weeks after implantation, gross observation showed a complete fusion formed between the implant and the host muscular tissue, with obvious blood vessels on the surface of the muscular flap in which the compound was encapsulated. No necrosis was observed in the following 30 minutes after the compound was dissected from its surrounding tissues. The histological investigation showed a contact connection between implant and its surroundings. In the experimental group, no new bone formed but in-growth of blood vessels was observed at the end of the 2nd week, and a little new bone formed along the edge of xenogenic inorganic bone at the end of the 8th week, while at the end of 12th week, more new bone formed in the compound with osteocytes in bone lacuna, plenty blood vessels in bone matrix, and lots of osteoblasts surrounded by un-differentiated mesenchymal cells at the fringe of the new bone. While in the control group, there were only loose connective tissue with blood vessels grew into xenogenic inorganic bone with no new bone formation until 12 weeks. CONCLUSION: The compound of xenogenic inorganic bone and auto-marrow can promote the formation of vascularized myo-bone flap with new bone formation.
From January 1984 to November 1997, the boiling xenogenic bone (porcine and bovine bone) was applied in 65 patients. The xenogenic bone was used to promote bone healing in 24 cases, intramedullary bone graft in 37 and osteomyelitis in 4. All of the patients were followed up for 2 to 35 months. The results showed that in five cases there was infection after operation, and all other the wounds had primary healing. The xenogenic bone seemed to induce rejection in vivo. In the sevious cases immunosuppressive treatment was often needed. The volume of the bone grafted and the extent of the periosteum being stripped seemed to be important whether xenogenic bone graft would be successful or. There were lots of problem needed investigation.
OBJECTIVE: To investigate the expression and distribution of xenoantigen in intervertebral disk of Chinese banna minipig inbred line, and to study the availability of xenograft transplantation of intervertebral disk. METHODS: Samples of intervertebral disk were collected from six Banna pigs of 8 to 11-month-old. The fixation, embedment and slice were performed. α-Gal specific binding lection (BSI-B4) were used as affinity reagents and affinity-immunohistochemistry assays (SABC methods and DAB stain) were conducted to detect the expression and distribution of xenoantigen (α-Gal). RESULTS: alpha-Gal was found in chondrocyte cell and chondrocyte-like cell in intervertebral disk which have the positive yellow-stained particulate aggradation. There was no stain in the matrix, elastic fiber and collagen fiber. CONCLUSIONS: The distribution of xenoantigen is locally in the tissue of intervertebral disk and its expression is weak. This suggests that the intervertebral disk of Banna pig may be alternative donor for xenotransplantation.
ObjectiveTo investigate the early effects of acellular xenogeneic nerve combined with adipose-derived stem cells (ADSCs) and platelet rich plasma (PRP) in repairing facial nerve injury in rabbits.MethodsThe bilateral sciatic nerves of 15 3-month-old male Sprague-Dawley rats were harvested and decellularized as xenografts. The allogeneic ADSCs were extracted from the neck and back fat pad of healthy adult New Zealand rabbits with a method of digestion by collagenase type Ⅰ and the autologous PRP was prepared by two step centrifugation. The 3rd generation ADSCs with good growth were labelled with CM-Dil living cell stain, and the labelling and fluorescence attenuation of the cells were observed by fluorescence microscope. Another 32 New Zealand rabbits were randomly divided into 4 groups and established the left facial nerve defect in length of 1 cm (n=8). The nerve defects of groups A, B, C, and D were repaired with CM-Dil-ADSCs composite xenogeneic nerve+autologous PRP, CM-Dil-ADSCs composite xenogeneic nerve, xenogeneic nerve, and autologous nerve, respectively. At 1 and 8 weeks after operation, the angle between the upper lip and the median line of the face (angle θ) was measured. At 4 and 8 weeks after operation, the nerve conduction velocity was recorded by electrophysiological examination. At 8 weeks after operation, the CM-Dil-ADSCs at the distal and proximal ends of regenerative nerve graft segment in groups A and B were observed by fluorescence microscopy; after toluidine blue staining, the number of myelinated nerve fibers in regenerated nerve was calculated; the structure of regenerated nerve fibers was observed by transmission electron microscope.ResultsADSCs labelled by CM-Dil showed that the labelling rate of cells was more than 90% under fluorescence microscope, and the labelled cells proliferated well, and the fluorescence attenuated slightly after passage. All the animals survived after operation, the incision healed well and no infection occurred. At 1 week after operation, all the animals in each group had different degrees of dysfunction. The angle θ of the left side in groups A, B, C, and D were (53.4±2.5), (54.0±2.6), (53.7±2.4), and (53.0±2.1)°, respectively; showing significant differences when compared with the healthy sides (P<0.05). At 8 weeks after operation, the angle θ of the left side in groups A, B, C, and D were (61.9±4.7), (56.8±4.2), (54.6±3.8), and (63.8±5.8)°, respectively; showing significant differences when compared with the healthy sides and with the values at 1 week (P<0.05). Gross observation showed that the integrity and continuity of regenerated nerve in 4 groups were good, and no neuroma and obvious enlargement was found. At 4 and 8 weeks after operation, the electrophysiological examination results showed that the nerve conduction velocity was significantly faster in groups A and D than in groups B and C (P<0.05), and in group B than in group C (P<0.05); no significant difference was found between groups A and D (P>0.05). At 8 weeks after operation, the fluorescence microscopy observation showed a large number of CM-Dil-ADSCs passing through the distal and proximal transplants in group A, and relatively few cells passing in group B. Toluidine blue staining showed that the density of myelinated nerve fibers in groups A and D were significantly higher than those in groups B and C (P<0.05), and in group B than in group C (P<0.05); no significant difference was found between groups A and D (P>0.05). Transmission electron microscope observation showed that the myelinated nerve sheath in group D was large in diameter and thickness in wall. The morphology of myelin sheath in group A was irregular and smaller than that in group D, and there was no significant difference between groups B and C.ConclusionADSCs can survive as a seed cell in vivo, and can be differentiated into Schwann-like cells under PRP induction. It can achieve better results when combined with acellular xenogeneic nerve to repair peripheral nerve injury in rabbits.
Limitation of donor source for allograft makes the research on xenograft progress. Pig is regarded as one of the ideal donor animals. The major obstacle in xenograft is hyperacute rejection, which is caused by complements after they are activated by xenogeneic antigens combined with natural antibodies. It has been confirmed that alpha-Gal is the major target antigen, whose expression is incharged by alpha-1,3 galactosyltransferase (alpha-GT). The approaches to overcome hyperacute rejection against alpha-Gal included: immunoadsorption of xenogeneic natural antibodies, lysis of antigen by enzyme and genetic manupilation to obtain animal lack of alpha-GT. Besides alpha-Gal, there were other antigens binding to human serum antibody, such as gp65 and gp100, which was expressed on PAEC after induced by TNF, the A-like antigen. But their function was still unknown. It was debatable on the role of MHC in xenograft. Both direct and indirect pathway were involved in cellular response in xenograft.
OBJECTIVE: To explore the kidney anatomic structure of banna minipig inbred-lines, and to provide data for kidney xenotransplantation. METHODS: The fresh and infused kidneys of banna minipig (including the vessel and the ureter) were checked by anatomic microscope and vernier caliper in original location and away body. The tissue structure was observed by HE stain. RESULTS: The structure of kidney of banna minipig inbred-lines (including the vessel and the ureter) are similar to that of human being. The fascia propria of kidney is divided into three layers including capsula fibrosa, capsula adipose and fascia renalis. The thickness of cortex renalis is (20.0 +/- 2.4) mm. The average diameter of renal artery is 5.1 mm and is similar to that of human being. All the kidneys of banna minipig inbred-lines have a single branch renal artery. The diameters of left and right ureters are 5.1 mm and 4.7 mm, respectively. CONCLUSION: The kidney of banna minipig inbred-lines is an ideal replacement of human kidney for xenotransplantation.
Objective To investigate the effect of anti-infective reconstitutedbone xenograft (ARBX) as primary grafting on repair of a segmental contaminateddefect in canine radius. Methods The contaminated segmentaldefects of 1.5 cm were made in both radius of 8 canine and 1 ml of staphylococal suspension was injected into the defect region at a concentration of 5×106 CFU/ml. ARBX(experimental side) or RBX(control side) was implanted into the two sides of the defects respectively as primary grafting followed by internal fixation. The results were compared between the two grafting materials in repairing the contaminated segmental defect. Results In ARBX side, the defects were repaired completely in 5 cases and partially in 1 case, and there existed no osteomyelitis in all cases; while in RBX side, the defects were repaired partially in 1 case and were not repaired in 5 cases after 6 months of operation, and there existed osteomyelitis in all cases. Conclusion Besides its b osteoinductive and osteoconductive activity, ARBX is highly antibacterial and can be used as primary grafting in repairing contaminated segmental defects.
Objective To study efficiency of vascularized bone graft combining with reconstituted bone xenograft (RBX) in repairing bone defect and the expression of the vascular endothelial growth factor (VEGF) in serum. Methods From January 1998 to December 2002, 27 cases of bones defects were treated and randomly divided into 3 groups according to different repair materials: group A (the vascularized bone graft-RBX group, n=9), group B (the vascularized bone graft group, n=10)and group C(the RBX group, n=8). The bone defect repair, the bone healing time and the bone graft resorption were observed by radiograph after 3 months, 6 months and 12 months of operation, and the expression of VEGF in serum was assayed with lumino-enzyme immunoassay before operation and after operative 2 weeks, 4 weeks, 6 weeks and 8 weeks respectively. Results The X-ray films showed that the bonehealing was achieved in 8 cases of group A, in 6 cases of group B and in 3 cases of group C after 3 months; in 1 case of group A, respectively in 3 cases of both group B and group C after 6 months. The bone graft resorption was observed in1 case of group B and in 2 cases of group C after 12 months. The serum VEGF values after operative 2 weeks and 4 weeks were higher than those before operation in all of 3 groups(Plt;0.05), and the VEGF values of groups A and B were higher than that group C(Plt;0.05) after 4 weeks. There were no significant differences (Pgt;0.05) in serum VEGF level between postoperative 6, 8 weeks and preoperation in 3 groups. Conclusion The expression of serum VEGF obviously increase in the early period of bone transplanting, it is value of clinical evaluation of reparative efficiency of bone defect.
Objective To develop a new small-caliber vascular xenograft and evaluate the feasibility of xenogenic artery for coronary artery bypass grafting. Methods Canine carotid arteries were decellularized by detergent and enzymatic extraction. All decellularized xenografts were randomly divided into two groups. Heparin-linked group （n=24）： grafts were then covalently linked with heparin. Non-heparin-linked group （n=24）： as control. Xenografts in two groups were implanted in rabbits＇ left and right carotid artery respectively as bypass grafts. Graft patency was checked by ultrasonography after 3 weeks, 3 and 6 months. Grafts were harvested after 3 and 6 months. Microscopic observation and immunohistochemical staining were performed. Results All the cells were removed while the extracellular matrix were well preserved observed. Heparin was successfully linked to the grafts through their whole thickness. There was no obstruction at both sides after implantation of the grafts, while less thrombus was found in the decellularized heparin-linked grafts than in the other side. Smooth muscle cells densely populated the graft wall and endothelial cells covered the lumen at 3 months after implantation. Conclusion Canine common carotid artery treated by detergent and enzymatic extraction and heparin linkage may be a new small-caliber vascular xenograft for coronary artery bypass grafting.
OBJECTIVE: To observe the heart anatomic and histological structure of the Banna mini-pig inbred-lined and to provide the morphological data for heart xenotransplantation and breeding transgens pig. METHODS: Ten Banna mini-pigs (12-18 months old) were affused and fixed by common coratid artery. The heart were observed and measured by gross anatomy and histology. RESULTS: There were many similarities between the Banna pig heart and the human heart in anatomy and histology. However, the following differences were observed in the Banna pig heart: 1. Azygos vein directly drew into right atrium cordis. 2. The intercalated disk of cardiac muscle was less than that of human. 3. The Purkinje’s fibre was bigger than that of human. CONCLUSION: On the morphology and histology, the structure of Banna pig heart is similar to the heart of human being. It is possible that Banna minipig heart becomes organ donors for xenotransplantation.