Objective To investigate the biocompatibility of acellular urinary bladder submucosa (AUBS). Methods The acellular collagen matrix of human urinary bladder submucosa was developed using freeze-thawed enzymatic treatment and freeze-drying technique. Human oral keratinocytes were cultured and seeded on AUBS at a density of 2×106/ml in vitro.The proliferation of the cells were observed. Pockets were created in the abdominal muscle wall of 18 SD rats. AUBS in size 1 cm×1 cm was implanted into the pocket. The grafts were observed by light microscope 3, 6, 10, 14, 21 and 28 days after operation. Results AUBSmainly consisted of collagen fibers with a three-dimensional network structure. After the oral keratinocytes were seeded, continous oral epithelium layer was formed on the surface of AUBS after 10 days in vitro. Histological observation of the grafted AUBS showed progressive cell infiltration at 6 days. New capillaries formed at 14 days. The collagen fibers arranged regularly at 28 days after implantation. Conclusion Freeze-dried AUBS may be used as a suitable scaffold for tissue regeneration, which can induce cell proliferation both in vivo and in vitro and has good biocompatibilty.
Objective To explore the effective autologous bone marrow stem cell dosage for treatment of severe lower limb ischemia. Methods From December 2003 to December 2004, 22 cases of bilateral lower limb ischemia were treated with autologous bone morrow cell transplantation. All the patients were randomly divided into two groups according to ischemia degree. In group A(severe ischemia side), the amount of transplanted autologous bone marrow cells was more than 1×108, and ingroup B(mild ischemia side), the amount was less than 1×105. A series of subjective indexes, such as improvement of pain, cold sensation and numbness, and objective indexes, such as increase of ankle/brachial index (ABI) and transcutaneous oxygen pressure (TcPO2), angiography, amputation rate, and improvement of foot wound healing were used to evaluate the effect of autologous bone marrow stem cells implantation. Results The rates of pain relief were 90.0% in group A and 16.7% in group B (Plt;0.01); the rates of cold sensation relief were 90.5% in group A and 5.3% in group B（Plt;0.01）;the improvement of numbness was 62.5% in group A and 9.1% in group B（Plt;0.01）. Increase of ABI was 31.8% and 0 in groups A and B respectively（Plt;0.01） at 4 weeks after implantation. Increase of TcPO2was 94.4% and 11.1% in groups A and B respectively（Plt;0.01） at 4 weeks after implantation. Twelve cases of angiography showed rich new collateral vessels in 100% of the limbs in group A while no remarkable new collateral vessel in group B. The amputation rates were 4.5% in group A and 27.3% in group B（Plt;0.05） at 4 weeks after implantation. The rate of improvement of foot wound healing was 75% in group A and there was no changein wound healing in group B after 4 weeks of implantation. Conclusion The effectiveness of autologous bone marrow stem cell implantation depends on the number of implanted stem cells. Effectiveness is expected in most patients if the implanted stem cell is more than 1×108, whereas there would be little effect if the cell number is less than 1×105.
The aim of this experiment was to study the osteogenesis in vivo of allogenic osteoblast combined culture with calcium phosphate composites. The osteoblasts were obtained by enzymatic digestion of periosteum from fibula subcultured to 13 generations, the cells were combined culture with hydroxyapatite and biphasic calcium phosphate. Subseguently, the composite was implanted into rabbits subcutaneously or intramuscularly. The blank material was implanted in the contralateral side as control. Four weeks later, all animals were sacrificed. All the implants were examined by gross observation, histological examination and EDXA. The results showed: 1. obvious ingrowth of connective tissue with very little inflammatory reaction; 2. new bone formation in the composites with deposit of Ca and P on the surface of osteoblast, but none in the blank materials; 3. no significant difference of new bone formation between the different sites of implantation or different materials, but those implanted intramuscularly had lamellae form of new bone while those implanted subcutaneously had only mineralization of extracellular matrix. The conclusion were: 1. the composites are biocompatible with prior osteogenesis property; 2. periosteal-derived allogenic osteoblasts obatined by enzymatic digestion could survive following implantation with bioactivity; 3. rich blood supply might be advantageous to new bone formation and its maturation.
Objective To design a novel stentless porcine aortic bioprosthesis and test the feasibility and its function in vitro after the valve was implanted by a modified method. Methods Six stentless porcine aortic bioprosthesis were divided into two groups according to different implantation, single layer suture group: new improvement stentless porcine aortic bioprosthesis sutured with single layer was implanted; double layer suture group: stentless porcine aortic bioprosthesis developmented by our laboratory used double layer suture was implanted. Each group contained three scales: 23 mm ,25 mm and 27 mm. Analogue ex vivo aortic valve replacement was performed , the feasibility of the new implantation was detected. Effective orifice area, transvalvular pressure gradient and regurgitation ratio were recorded at the cardiac output of 2.0 L/min, 3.5 L/min, 5.0 L/min and 7.0 L/min under the guideline of International Organization for tandardization （ISO）5840. Results The average aortic valve implantation time used for single layer suture and tradition double layer suture were 50 min and 70 min respectively. The transvalvular pressure gradient in the single layer suture group were significantly lower than those in double layer suture group under the flow of 5.0 L/min in 23 mm valve and 27 mm valve （13.51±0.51 mm Hg vs. 14.44±0.99 mm Hg, 7.36±0.19 mm Hg vs. 7.53±0.28 mm Hg；P＜0.01）;and the effective orifice area in the single layer suture group were larger than those in double layer suture group in the same case(1.87±0.06 cm2 vs. 1.76±0.08 cm2, 2.26±0.07 cm2 vs. 2.16±0.05 cm2；P＜0.01). There was no statistically difference in other parameters between both groups. Conclusion The novel design of new improvement stentless porcine aortic bioprosthesis used single layer suture has good hemodynamic characteristics as the nature structure . The modified suture method decrease the implantation time.Nemerical data of the evaluation in vitro show that the difference between single layer suture group and double layer suture group in effective orifice area,transvalvular pressure gradient and regurgitation ratio haveno statistical significance. This experiment is the foundation of the animal and clinical experiment in the future.
A mechanical study on the bones of 29 rabbits following implantation of carbontendon was carried out. The rabbits were divided into seven groups according to the observation time (2,4,6,8,12,20 and 30 weeks after operation). A bundle of artificial tendon composed of 7,000 carbon fibers was passedthrough a tunnel in the tibia, and both ends of the artificial tendon were ligated to the muscle fibers. The mechanical strength and histological structure of the carbonbone junction and their relationship were studied in each group. Carbon fiberwas split and degradated in six to eight weeks after operation. The tensile strength of carbontendon in the soft tissue was decreased from 82±4.6N in the second week to 27±5.31N and6.3±1.81N in the sixth and eighth week respectively. The tensile strength of carbontendon increased from 3.01±1.2N to 6.1±2.01N at the carbon -tendon-bone junction in the bone. The tensile strength of carbon-tendon was unsatisfactory for implantation into bone. The carbon-tendon was split and degradated and the tensile strength was not b enough to cope with the early functional exercises.
Objective To investigate the influence of infarcted myocardial construction by umbilical cord blood mesenehymal stem cells（MSC） with induced to myo-derived stem cells and implanted into infarcted myocardium. Methods Thirty-six adult mongrel canines were randomly divided into MSC transplant group and control group （18 each group）. Transplant group： the umbilical cord blood MSC differented to myo-derived stem cells induced by 5-azacytidine（5-aza） were implanted into the acute myocardial infarct site via the ligated left anterior descending （LAD） artery. Control group： administer the same volume of IMDM culture medium containing 0. 02% 4,6- diamidino-2-phenylindone （DAPI）. At 2, 4, 8 weeks after implantation, the change of basic myocardial construction and the distribution of desmin were observed by using Nagar-Olsen staining and immunohistology respectively. Results With less fusing, the arrangement of gelatine fibers and elastic fibers were in order in transplant group,and they were partly fused in control group by Nagar-Olsen staining. The expression of desmin of infarcted myocardial cell in transplant group was much higher than those in control group （P〈0. 01）. No significant difference was detected in the expression of desmin in normal site of both groups （P〉0. 05）. Conclusion There is an protective effect on the basic myocardial construction in infarcted myocardium after the umbilical cord blood MSC was differented to myo-derived stem cell by induced with 5-aza in vitro and implanted into the acute myocardial infarction.
In order to investigate the possibility of repairing injuried tendon with living artificial tendon, after combining culture, subcultured autogenous tendon cells with carbon fibers were implanted into the calcaneous tendon of rabbits. In different stages, the synthesis of type I collagen and their relevant morphological changes were observed. The results showed as follows: after implantation, tendon cells continued proliferating. Four weeks after implantation, tendon cells were detached from the carbon fibers and proliferated and produced collagen among the carbon fibers. The collagen fibrils were linked with each other to formed a dense structure. In the linkage site, the collagen fibrils originated from the implants joined to that from the ruptured end of the tendon, which meaned that the implant was healed with the recipient tendon. Observed under scanning electronic microscope, the tendon cells were lined among the carbon fibers evenly and in order, the collagen fibrils joined each other and formed an network, the fibrils were lined parallel to the carbon fibers. Under transparent electron microscope, the nucleolus were clear and organelle were abundant.
Objective To investigate whether the implanted myoblasts with the soluble carriers can improve the repairing efficiency for theseverelycryodamaged tibialis anterior muscles. Methods The skeletal myoblasts were isolated from the newborn SD rats by the use of the enzyme digestion. They were purified and serially subcultivated; the subcultivated myoblasts of the 3rd generation were marked with BrdU. The severelycryodamaged tibialis anterior muscle models were established from 84 SD rats aged 5 months. They were randomly divided into 4 groups, including Group A1 (the implanted myoblasts with the carriersF12 containing 0.1% sodium hyaluronate), Group A2 (the implanted myoblasts, with the carriersF12 that did not contain 0.1% sodiumhyaluronate), Group B1 (the implanted carrier solution containing 0.1% sodium hyaluronate, but with no myoblasts), and Group B2 (with no carrier solution or myoblasts). Six rats were killed at the following time points: at 2, 5 and 9 days,and 2, 4, 8 and 12 weeks after operation; the immunohistochemical and the Mallory staining studies were performed for an evaluation on the repairing efficiencyfor the severelycryodamaged tibialis anterior muscles. By the imaging analysis, the number of the survived cells in each group was compared at 2 days, and the area ratio of the collagen fiber in each group was also compared at 8 weeks. Results The BrdU immunohistochemical staining showed that the number of the remaining implanted cells was significantly greater in Groups A1 than in Group A2, the migrating area of the myoblasts was greater, the distribution of the cells was more uniform, the cell differentiating potential was undestroyed, the repairing efficiency for the severelycryodamaged tibialis anterior muscles was significantly improved. There was no bluestained nucleus at each time point in Group B. The Mallory staining showed that the fibrous degeneration inthe tissue repairing process was significantly inhibited in Groups A1, A2 and B1; the inhibition was most obvious in Group A1, and next in Group A2. The imaging analysis indicated that at 2 days after operation, the number of the survived cells was significantly-greater in Group A1 than in Group A2 (Plt;0.05). At 8 weeks after operation, the collagen fiber was the least in Group A1, less in Group A2, more in Group B1,and the most in Group B2 (Plt;0.05). Conclusion The implanted myoblasts can significantly improve the repairing efficiency for the severelycryodamaged muscle tissues, and the implanted carrier solution containing 0.1% sodium hyaluronate can improve the implanting efficiency for the myoblasts.
Objective To study the immunological rejection occurred in different period after the in vivo implantation of vitreous-cryopreservation tissue engineered tendons for the repair of tendon defect and investigate its influences on the hepatic, renal, and cardiovascular function of rats. Methods Tenocytes obtained from tail tendon of one-weekold SD rats were cultured in vitro. The tenocytes at passage 2-4 (5 × 106 cells/mL) were co-cultured with 1.5 cm bio-derived tendon material to reconstruct tissue engineered tendon. The 21% DMSO was used as cryopreservation protection solution andthe Eurocoll ins solution served as basic solution for pre-frozen solution (4 ) and eluent. The cell-scaffold composites were vitreous-cryopreserved by self-designed method. Seventy-two healthy SD rats (male and/or female) weighing 210-230 g were randomly divided into three groups: group A (n=32), group B (n=32), and group C (n=8). The 0.5 cm tendon defect model was establ ished in the middle part of Achilles tendon in groups A and B. The defect in group A and B was repaired by the transplantation of tissue engineered tendon with and without vitreous-cryopreservation, respectively. At 2, 4, 6, and 8 weeks after transplantation, the general observation and the detection of hepatic function, renal function, and cardiovascular function were conducted. At 2, 4, and 6 weeks after transplantation, serum immunology test was conducted. Results There were no tissue necrosis, hydrops, and suppurative infection in groups A and B. The adhesion was evident in groups A and B 2 weeks after transplantation, improved gradually during 4-6 weeks, and disappeared at 8 weeks. The neonatal tissue had full integration and continuity, and the bridging region of the tendon healed and was similar to the normal tendon. For serum IgG and IgM content, there was no significant difference when group A or B was compared with group C, and between group A and group B 2, 4, and 6 weeks after transplantation (P gt; 0.05). Hepatic function: aspartate aminotransferase (AST) content of group A was less than that of group C 4 weeks after transplantation (P lt; 0.05); AST content of group B was less than that of group C 4 and 6 weeks after transplantation (P lt; 0.05); but there was no significant difference when group A or B was compared with group C in terms of other indexes 8 weeks after transplantation (P gt; 0.05). Renal function: serum albumin and creatinine in groups A and B were decreased obviously, and significant difference was evident when compared with group C (P lt; 0.05). Cardiovascular function: there was no significant difference between group A and group C in terms of blood glucose, triglyceride, and cholesterol (P gt; 0.05);there was a significant difference between group B and group C in terms of triglyceride 8 weeks after transplantation (P lt; 0.05). Conclusion Repairing tendon defect with the implantation of vitreous-cryopreservation tissue engineered tendons results in no obvious immunological rejection and exerts no obvious influences on hepatic, renal, and cardiovascular function.
OBJECTIVE: To evaluate the clinical effect of drilling procedure following the hydroxyapatite orbital implantation. METHODS: From February 1996 to April 2000, 146 consecutive patients who received hydroxyapatite orbital implant were drilled and inserted a motility peg 6 to 16 months after hydroxyapatite implantation. Among them, there were 97 males and 49 females, aged from 18 to 60 years old, of the 146 motility pegs, 36 were sleeved pegs and 110 were nonsleeved. Goldman visual field analyzer was applied to measure the degree of artificial eye’s movement before and after drilling. RESULTS: Followed up for 1 to 40 months, no secondary infection occurred. The mobility of the prosthesis increased from (18.7 +/- 3.8) degrees preoperatively to (42.3 +/- 3.7) degrees postoperatively. CONCLUSION: The delayed drilling procedure and motility peg insertion improve the range of movement and the sensitivity of the artificial eye with a low rate of complications.