Objective To study the result of using nerve conduit coated with chitin and filled with a guide-fiber to repair peripheral nerve defect. Methods Twenty-four female adult SD rats were made the model of 14 mm-gap on bilateral sciatic nerve under sterile condition. The rats were randomly divided into 4 groups(n=6),group A: polymer polyglycolic-lactic acid(PGLA) nerve conduit coated with chitin and filled with a guide-fiber as experimental group to repair 14 mm gap of rat sciatic nerve；group B: PGLA nerve conduit coated with chitin； group C: PGLA nerve conduit； group D: autograft (control group). The repair result was evaluated by normal observation, EMG testing and S-100 histological immunostaining analysis 4 and 12 weeks after operation.Results Four weeks after the operation，there were new regenerated immature fibers in groups A,B and C, 12 weeks after the operation, the regenerated nerve fibers were seen to have bridged the gap. There were myelinated fibers equably distributed and rarely newgenerated nerve fibers in distal parts of group D. The repair result of PGLA nerve conduit coated with a chitin and filled with guide-fiber was better than that of groups B and C（Plt;0.05）. There was significant difference of nerve fiber diameter，thickness of myelin sheath and fiber density in group D from those in groups A, B and C（Plt;0.05），but there were degenerative changes such as vacuoles insheaths and myelin separation in proximal and few new regenerated nerve fibers in distal parts of group D. Conclusion PGLA nerve conduit coated with chitin and filled with a guide-fiber offers a possible substitute for the repair of peripheral nerve defect.
Objective To culture primary parathyroid cells by mean of simulated microgravity, observe their basic morphological characteristics, study survival rate and secretory function of parathyroid cells, and explore more excellent culture mean of parathyroid cells. Methods There were 37 male Wistar rats, the body weight was 150–200 g. The rat was intraperitoneally injected with 1% pentobarbital sodium (50 mg/kg). The parathyroid glands were surgically excised and identified pathologically. The parathyroid gland cells were got and digested them with collagenase Ⅱ, which were divided into three groups: conventional culture group (simple parathyroid cells were cultured), polyglycolic acid (PGA) scaffold culture group (the parathyroid cells were cultured on the PGA scaffold), and simulated microgravity culture group (the parathyroid cells and PGA scaffolds were cultured in simulated microgravity environment). The parathyroid cells were cultured for 1, 3, 5 or 7 days in different culture conditions, then the parathyroid hormone (PTH) was measured, morphological characteristics of the parathyroid cell was observed under phase contrast microscope, survival rate of the parathyroid cells was calculated by acridine orange/propidium iodide staining. Results The parathyroid cell morphologies of most cells were well and center of part of cell mass was necrosis on day 7 in the conventional culture group. The most parathyroid cells were spreading toward the poles along the PGA cell scaffold in the longitudinal direction and the adjacent stents were connected by extracellular matrix on day 7 in the PGA scaffold culture group. The parathyroid cells cultured under the simulated microgravity were got round and formed clusters on day 7 in the simulated microgravity culture group. Compared with the other two groups on day 7, the PTH and the survival rate of the parathyroid cells were significantly higher in the simulated microgravity culture group (P<0.05). Conclusions Parathyroid cells cultured in simulated microgravity environment could maintain better morphology, survival rate is higher, and secretory function is better. Therefore, parathyroid cells cultured in simulated microgravity could be used as good donor cell for treatment of hypoparathyroidism. PGA scaffold could be used as a good carrier for culture of parathyroid cell.
Objective To investigate the in vivo degradable properties of new calcium phosphate cement (CPC) containing poly lactic-co-glycolic acid (PLGA) so as to lay a foundation for the future clinical application. Methods A novel CPC containing PLGA (CPC/PLGA) was prepared according to a ratio of 45% dicalcium phosphate anhydrous ∶ 45% partially crystallized calcium phosphates ∶ 10% PLGA. Thirty-two adult New Zealand rabbits (weighing 2.2-3.0 kg, male or female in half) were divided into the experimental group (n=17) and the control group (n=15). The bone defect models of the bilateral femoral condyles (4.5 mm in diameter and 1.5 cm in depth) were made by drilling hole. Defect at the right side was repaired with CPC/ PLGA in the experimental group and with CPC in the control group, while defect at the left side was not treated as blank control. The general condition of rabbits was observed after operation; the histological observation and bone histomorphometric analysis were performed at 2, 4, 8, 16, and 24 weeks; and scanning electronic microscope (SEM) observation was performed at 8 and 16 weeks after operation. Results All rabbits survived to the end of experiment. The histological observation showed: CPC/PLGA degraded gradually, and the new-born bone trabecula ingrew; bone trabeculae became rough and b; and CPC/PLGA almost biodegraded at 24 weeks in the experimental group. The CPC degradation was much slower in the control group than in the experimental group. The total bone tissue percentage was 44.9% ± 23.7% in the experimental group, and 25.7% ± 10.9% in the control group, showing significant difference between 2 groups (t=3.302, P=0.001); and the bone tissue percentage showed significant difference between 2 groups at 8, 16, and 24 weeks (P lt; 0.05). The results of SEM observation showed that the pore size was 100-300 μm at 8 weeks after operation, new-born bone trabecula grew into the pores and combined bly with residual cement in the experimental group. Conclusion Novel CPC/PLGA has good in vivo degradable properties, and it can be an ideal bone substitute in future clinical application.
Objective To evaluate the feasibility of reconstructionof urothelium tissue in vivo using tissue-engineering technique. Methods The urothelium cells were obtained from young rabbit, bladder by mechanical and enzyme digested methods. After expanded in vitro, the 4th to 5th generation urothelium cells were seeded onto the surface of 8 Polylatical／glycolic acid copolymer polymer，the polymer matrix without seeding cells served as control group. A total of 8 cell-polymer scaffolds and 4 simply scaffolds were separately implanted into subcutaneous pockets of athymic mice. Theexperiment groups included cell-polymer scaffolds 4 weeks and cell-polymer scaffolds 8 weeks. The control group included simply scaffold 4 weeks and simply scaffold 8 weeks.After 4 and 8 weeks, the specimens were obtained and examined by gross inspection, histologically and immunohistochemically. Results The results of HE and Masson staining showed that the polymer were covered by urothelium cells layers and cells layers increased markly in experimental group. Immuocytochemical studies revealed that the cells were stained positively for anti-cytokeratins (AE1/AE3) in experimental group. Fiber tissue deposition were found on the surface of polymers in control group by HE and Masson staining. Immunocytochemical staining of implants showed the negative result for cytokeratins in control group. Conclusion It is feasibility that reconstruction of urothelium tissue using tissue-engineering -technique,whichprovides basic understandings for further development of the bladder and ureteral tissue engineered research.
ObjectiveTo explore the degradation of AZ31 magnesium alloy and poly (lactic-co-glycolic acid) (PLGA) in the femoral condyle, and then evaluate the laws of degradation of AZ31 magnesium alloy by Micro-CT images and data. MethodsForty 3-month-old male New Zealand white rabbits (weighing, 2.5 kg) were randomly divided into 4 groups, 10 rabbits each group. Forty micro-arc-oxidized AZ31 magnesium alloy pins and 40 PLGA pins were implanted into the right and left femoral condyle, respectively. Micro-CT images and data analysis were used to evaluate the degradation at 4, 8, 12, and 16 weeks after operation (n=10). Degradation was evaluated by weight difference between pre-and post-implantation. The inflammatory response was observed around the implants by HE staining. The weight loss of magnesium alloy and Micro-CT results were compared. ResultsThe Micro-CT images showed that PLGA pins had gray low signal, which was similar to the soft tissue around. At 4 weeks after operation, no signs of degradation were observed, and there were little corrosion pitting on the magnesium alloy. At 8 weeks, corrosion pitting gradually expanded, the boundary between the longitudinal axis and the cross section became blurred; at 16 weeks, corrosion pitting became bigger, and the boundary was discontinuous. Micro-CT quantitative analysis showed that the volume fraction of magnesium pins decreased slowly at 4 and 8 weeks; it was significantly lower at 12 and 16 weeks than 4 and 8 weeks (P < 0.05). The magnesium cylinder mineral density continuously decreased during the study period, it had a rapidly speed from 12 to 16 weeks (P < 0.05). However, the magnesium CT image density showed a slight change (P>0.05). The surface-to-volume ratio of the pins constantly increased, and the ratio was significantly larger at 12 and 16 weeks than 4 and 8 weeks, and at 16 weeks than 12 weeks (P < 0.05). There was more and more corrosion pitting on the surface with time, which resulted in a decrease in the radius that mean trabecular thickness gradually decreased, showing significant difference between different time points after 8 weeks (P < 0.05). The weight loss detection showed that the degradation of magnesium pin and PLGA gradually increased with time (P < 0.05), and the degradation rate of magnesium pin was significantly lower than that of PLGA at 8-12 weeks (P < 0.05), but the degradation rate of magnesium pin was higher than that of PLGA at 16 weeks. At each time point, the weight loss of magnesium alloy was similar to that by Micro-CT, but mass fraction was lower than volume fraction and had significant differences at 8, 12, and 16 weeks (P < 0.05). HE staining revealed that slight inflammatory response was observed around the magnesium pins at 4 weeks, and inflammatory reaction gradually reduced with time and disappeared at 16 weeks, but no inflammatory reaction was seen around PLGA. ConclusionMicro-CT has the advantages of non-trauma, in vivo detection, quantitative analysis, and precise data in evaluating the degradation of AZ31 magnesium alloy. Regarding the degradation of the magnesium alloy and PLGA in vivo, the degradation rate is slow in the early stage, and then increases with time. The degradation of PLGA is faster and earlier but it is then overtaken by AZ31 magnesium alloy at 16 weeks. During the degradation, the density of the magnesium has almost no change. The biomaterials can not firmly attach to the surrounding tissues due to inadequate holding forces.
Objective To investigate the effect of a porous calcium phosphate/bone matrix gelatin (BMG) composite cement (hereinafter referred to as the " porous composite cement”) for repairing lumbar vertebral bone defect in a rabbit model. Methods BMG was extracted from adult New Zealand rabbits according to the Urist’s method. Poly (lactic-co-glycolic) acid (PLGA) microsphere was prepared by W/O/W double emulsion method. The porous composite cement was developed by using calcium phosphate cement (CPC) composited with BMG and PLGA microsphere. The physicochemical characterizations of the porous composite cement were assessed by anti-washout property, porosity, and biomechanical experiment, also compared with the CPC. Thirty 2-month-old New Zealand rabbits were used to construct vertebral bone defect at L3 in size of 4 mm×3 mm×3 mm. Then, the bone defect was repaired with porous composite cement (experimental group, n=15) or CPC (control group, n=15). At 4, 8, and 12 weeks after implantation, each bone specimen was assessed by X-ray films for bone fusion, micro-CT for bone mineral density (BMD), bone volume fraction (BVF), trabecular thickness (Tb. Th.), trabecular number (Tb.N.), and trabecular spacing (Tb. Sp.), and histological section with toluidine blue staining for new-born bone formation. Results The study demonstrated well anti-washout property in 2 groups. The porous composite cement has 55.06%±1.18% of porosity and (51.63±6.73) MPa of compressive strength. The CPC has 49.38%±1.75% of porosity and (63.34±3.27) MPa of compressive strength. There were significant differences in porosity and compressive strength between different cements (t=4.254, P=0.006; t=2.476, P=0.034). X-ray films revealed that the zone between the cement and host bone gradually blurred with the time extending. At 12 weeks after implantation, the zone was disappeared in the experimental group, but clear in the control group. There were significant differences in BMD, BVF, Tb. Th., Tb. N., and Tb. Sp. between 2 groups at each time point (P<0.05). Histological observation revealed that there was new-born bone in the cement with the time extending in 2 groups. Among them, bony connection was observed between the new-born bone and the host in the experimental group, which was prior to the control group. Conclusion The porous composite cement has dual bioactivity of osteoinductivity and osteoconductivity, which are effective to promote bone defect healing and reconstruction.
OBJECTIVE: To explore the distribution and effect of endogenic bone morphogenetic protein (BMP) in repairing rabbit skull with tissue engineered bone. METHODS: The autologous osteoblast-like cells were instantly implanted onto polyglycolic acid (PGA) matrix coated with collagen. The rabbit skull defect models were established by resection of bilateral 1.5 cm x 1.0 cm full-thickness parietal bone in 18 New Zealand rabbits, which were randomly divided into two groups. In one group, the composite of osteoblast- like cells and PGA matrix were grafted into the defect on one side of the skull as experimental group I, leaving the same defect area on the other side as control group without any graft implanted. In the other group, simply PGA was done in the same way as experimental group II. The tissue samples were harvested at 3, 8 and 14 days postoperatively and examined by histological and immunohistochemistry methods. The concentrations of BMP in different regions of the samples were measured using computer image analysis system. RESULTS: After 3 days of operation, the BMP positive cells were found in the matrix of experimental group I. At 8 days postoperatively, the formation of new bone on experimental group I was prior to that of experimental group II and control group. On the 14th day, bone trabecula was formed on the experimental group I, but there was only fibrous tissue on control group. The concentration of BMP on the experimental group I and II were higher than that of corresponding region on control side. CONCLUSION: The osteoblast-like cells instantly implanted onto PGA matrix can synthesize and secrete BMP. It may be one of the reasons of tissue engineered bone inducing new bone regeneration that localizing endogenic BMP in bone defect area, increasing the concentration of endogenic BMP and improving its distribution by tissue engineering technique.
Objective To study the influence of in vitro force-vascularization on in vivo vascularization of porous polylactic glycolic acid copolymer(PLGA) scaffolds with internal network channels (PPSINC). Methods After the in vitro forcevascula ization of PPSINCs covered with microvessel endothelial cells (MVEC) of mice, they were divided into two groups: the force-vascularization group (group A) and the control group with only PSINCs (group B). All the PPSINCs were planted in the mesentery of 12 mice for 2 and 4 weeks, the PPSINCs were cut out, the vascular ization of PPSINCs was investigated by histology and immunohistochemistry, and the vascularization area of the histologic section of the PPSINCswas measured with the computer-assistant image analysis system. Result After the in vitro forcevascularization of PPSINCs, the MVEC of the mice sticking on the channel wall could be seen. After the scaffold was im planted into the mice for 2 weeks, the vascularization area of the histologic section of PPSINCs (VA) in group A (2 260.91±242.35 μm2) was compared with that in group B (823.64±81.29 μm2)，and the difference was sig nificant in statistics（P＜0.01).The VA for 4 weeks in group A (17 284.36 ±72.67 μm2) was compared with that in group B (17 041.14±81.51 μm2), and the difference was not significant in statistics（P＞0.05）.The area of the actin positivestaining (AA) in the histologi c section of PPSINCs for 2 weeks’ implantation in group A (565.22±60.58 μm2) was compared with that in group B (205.91±16.25 μm2), and the difference was signi ficant in statistics（P＜0.01). After the implantation for 4 weeks, the VA in group A (4 321.09±19.82 μm2) was compared with group B (4 260.28±27.17 μm2), and the difference was not significant in statistics（P＞0.05）. Conclusion The PPSINC is a good simple scaffold model of vasculariazation. The in vitro force-vascularization can increase the in vivo vascularization of PPSINCs in the early stage.
Objective Choose polylactide-co-glycolide/hydroxyapatite (PLGA/HA) and porous phosphate calcium (PPC) as the object that we will study, compare their degradabality and choose one as a suitable scaffold for rib reconstruction. Methods All the experiments were divided into PLGA/HA group and CPC group. Degradabality experiment in exvivo: put the two scaffold which have the same size into 0.9% NaCl, keep sterile, then put the container into warm cage，get out and weigh them in 2, 4, 8, 12 and 24 weeks, compare the different speed of the two scaffold. Degradability experiment in vivo: put the two scaffold which have the same size under the skin of the rabbit, and weigh them in 2, 4, 8, 12 and 24 weeks, the tissue around the scaffold was examinzed by HE and the scaffold was examined by electron scanning microscope. Results Micro-CT and Scanning electron microscopy shows that CPC group had better structure （1101.2228±0.6184 mg/ccm vs. 1072.5523±0.7442 mg/ccm）and porosity（70.26％±0.45％ vs.72.82％±0.51％）than PLGA/HA group; The result of degradabality experiment in vitro shows that the speed of the two scaffolds was slow. It is at 24 weeks that the degradability is obvious,and the PLGA/HA group degraded a lot which was 60%. The result of degradabality experiment in vivo shows that the speed of degradabality of PLGA/HA group was faster than that is in the 0.9% Nacl, also was faster than that of CPC group which was 96%.The reponse of tissue around the PLGA/HA was more sever than that of CPC group which is in favour of the growth of cells. Conclusion As for the reconstruction of large defect of rib, CPC is more suitable than PLGA/HA.
ObjectiveTo explore the biocompatibility of the poly-lactide-co-glycolide (PLGA)/collagen type I scaffold with rat vaginal epithelial cells, and the feasibility of using PLGA/collagen type I as scaffold to reconstruct vagina by the tissue engineering. MethodsPLGA/collagen type I scaffold was prepared with PLGA covered polylysine and collagen type I. The vaginal epithelial cells of Sprague Dawley rat of 10-12 weeks old were cultured by enzyme digestion method. The vaginal epithelial cells of passage 2 were cultured in the leaching liquor of scaffold for 48 hours to detect its cytotoxicity by MTT. The vaginal epithelial cells were inoculated on the PLGA/collagen type I scaffold (experimental group) and PLGA scaffold (control group) to calculate the cell adhesion rate. Epithelial cells-scaffold complexes were implanted subcutaneously on the rat back. At 2, 4, and 8 weeks after implantation, the epithelial cells-scaffold complexes were harvested to observe the cell growth by HE staining and immunohistochemical analysis. The epithelial cells-scaffold complexes were transplanted to reconstruct vagina in 6 rats with vaginal defect. After 3 and 6 months, the vaginal length was measured and the appearance was observed. The neovagina tissues were harvested for histological evaluation after 6 months. ResultsThe epithelial cells grew and proliferated well in the leaching liquor of PLGA/collagen type I scaffold, and the cytotoxicity was at grade 1. The cell adhesion rate on the PLGA/collagen type I scaffold was 71.8%±9.2%, which significantly higher than that on the PLGA scaffold (63.4%±5.7%) (t=2.195, P=0.005). The epithelial cells could grow and adhere to the PLGA/collagen type I scaffolds. At 2 weeks after implanted subcutaneously, the epithelial cells grew and proliferated in the pores of scaffolds, and the fibroblasts were observed. At 4 weeks, 1-3 layers epithelium formed on the surface of scaffold. At 8 weeks, the epithelial cells increased and arranged regularly, which formed the membrane-like layer on the scaffold. The keratin expression of the epithelium was positive. At 3 months after transplantation in situ, the vaginal mucosa showed pink and lustrous epithelialization, and the majority of scaffold degraded. After 6 months, the neovagina length was 1.2 cm, without obvious stenosis; the vaginal mucosa had similar appearance and epithelial layer to normal vagina, but it had less duplicature; there were nail-like processes in the basal layer, but the number was less than that of normal vagina. The immunohistochemistry staining for keratin was positive. ConclusionThe PLGA/collagen type I scaffolds have good cytocompatibility with the epithelial cells, and can be used as the biodegradable polymer scaffold of the vaginal tissue engineering.