Objective To observe the release pattern of the microcysts and the effect of ectopic osteogenesis of combined micromorselized bone by optimized preparation of microcysts. Methods Optimized poly-DLlactide-co-glycolide (PLGA) microcysts manufacturing method was performed with the orthogonal design, and the accumulated release amount of microcysts was calculated at 2 h, 4 h, 8 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, 96 h, 120 h, 144 h, 168 h, 192 h, 216 h, 240 h and 264 h. Twentyfour Wistar rats were divided into 4 groups (n=6) and 1 cm length incision was cut in their bilateral thighs skin, forming 48 gluteus maximus muscle sackmodels. In group A,collagen was implanted to bilateral muscle sacks respectively. In group B, collagen and autologous morselized bone were implanted to bilateral muscle sacks. Ingroup C, collagen and rhBMP-2/PLGA delayed release microcysts were implanted to bilateralmuscle sacks respectively. In group D, collagen and morselized bone/rhBMP-2/PLGA delayed release microcysts were implanted to bilateral muscle sacks. Gross and histologic observations were made at 3, 4 and 5 weeks postoperatively.Results Every optimized variance had an effect on particle diameter of microcyst and its encapsulating rate. The microcyst’s surface was smooth and had a fine spheroplast, which released slowly within 11 days in vitro. In thethird week postoperatively, the graft in group A could not be touched, while the graft in all other 3 groups was still found. After 3 weeks, collagen was absorbed completely in group A, the residual collagen could be seen in groups B, C andD. After 4 weeks, collagen could be seen in group A; micromorselized bone continued to be absorbed and became smaller in group B; microsphere became smaller, osteoblasts increased in group C; micromorselized bone and microsphere continuedto be absorbed, oteoblasts and chondroblasts increased. After 5 weeks, implantsbecame small, microsphere was absorbed, osteoblasts and chondroblasts became more in groups B, C and D. Microcysts presented with white granuloshape and were packaged in tissue pieces. Histologic observation showed that the PLGA microcysts in 3 weeks and 4 weeks could be absorbed gradually as the time in vivo, if combining with morselzed bone they could produce abundant induced osteoblasts and chondroblasts. Conclusion Optimizing the preparation technology of microcysts has delayed their release during a long period in vitro. Autologous micromorselized bone can be ectopicly induced to produce large amount of osteoblasts in gluteus maximus muscle sack, where PLGA microcysts can combine organically and bring about the bone formation with less amount of growth factors.
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 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 manufacture a poly (lactic-co-glycolic acid) (PLGA) scaffold by low temperature deposition three-dimensional (3D) printing technology, prepare a PLGA/decellularized articular cartilage extracellular matrix (DACECM) cartilage tissue engineered scaffold by combining DACECM, and further investigate its physicochemical properties. Methods PLGA scaffolds were prepared by low temperature deposition 3D printing technology, and DACECM suspensions was prepared by modified physical and chemical decellularization methods. DACECM oriented scaffolds were prepared by using freeze-drying and physicochemical cross-linking techniques. PLGA/DACECM oriented scaffolds were prepared by combining DACECM slurry with PLGA scaffolds. The macroscopic and microscopic structures of the three kinds of scaffolds were observed by general observation and scanning electron microscope. The chemical composition of DACECM oriented scaffold was analyzed by histological and immunohistochemical stainings. The compression modulus of the three kinds of scaffolds were measured by biomechanical test. Three kinds of scaffolds were embedded subcutaneously in Sprague Dawley rats, and HE staining was used to observe immune response. The chondrocytes of New Zealand white rabbits were isolated and cultured, and the three kinds of cell-scaffold complexes were prepared. The growth adhesion of the cells on the scaffolds was observed by scanning electron microscope. Three kinds of scaffold extracts were cultured with L-929 cells, the cells were cultured in DMEM culture medium as control group, and cell counting kit 8 (CCK-8) was used to detect cell proliferation. Results General observation and scanning electron microscope showed that the PLGA scaffold had a smooth surface and large pores; the surface of the DACECM oriented scaffold was rough, which was a 3D structure with loose pores and interconnected; and the PLGA/DACECM oriented scaffold had a rough surface, and the large hole and the small hole were connected to each other to construct a vertical 3D structure. Histological and immunohistochemical qualitative analysis demonstrated that DACECM was completely decellularized, retaining the glycosaminoglycans and collagen typeⅡ. Biomechanical examination showed that the compression modulus of DACECM oriented scaffold was significantly lower than those of the other two scaffolds (P<0.05). There was no significant difference between PLGA scaffold and PLGA/DACECM oriented scaffold (P>0.05). Subcutaneously embedded HE staining of the three scaffolds showed that the immunological rejections of DACECM and PLGA/DACECM oriented scaffolds were significantly weaker than that of the PLGA scaffold. Scanning electron microscope observation of the cell-scaffold complex showed that chondrocytes did not obviously adhere to PLGA scaffold, and a large number of chondrocytes adhered and grew on PLGA/DACECM oriented scaffold and DACECM oriented scaffold. CCK-8 assay showed that with the extension of culture time, the number of cells cultured in the three kinds of scaffold extracts and the control group increased. There was no significant difference in the absorbance (A) value between the groups at each time point (P>0.05). Conclusion The PLGA/DACECM oriented scaffolds have no cytotoxicity, have excellent physicochemical properties, and may become a promising scaffold material of tissue engineered cartilage.
OBJECTIVE: To investigate the feasibility to seed vascular endothelial cell(VEC) and vascular smooth muscle cell (VSMC) into tissue engineered blood vessel scaffold material. METHODS: 1. A blood vessel scaffold with a combined polymer was designed, which mainly is composed of rabbit VSMC and collagen with reinforcement by a non-spinning fabric mesh made of polyglycolic acid (PGA). 2. VEC were isolated from rabbit thoracic aorta by enzyme digestion methods and subcultured and purified. Then the cells were seeded into scaffold material. The morphological characteristics of tissue engineered blood vessel was analyzed by scanning electron microscopy. RESULTS: VEC could adhere well to the inner surface of the tissue engineered tubular scaffold material with a tenacity and elasticity. VSMC could sustain bioactivity of cell. CONCLUSION: Non-spinning PGA porous biodegradable materials coated with collagen is benefit for cells to adhere and grow. It will lay a foundation of a laminated structure of tissue engineered blood vessel.
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 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.
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 investigate the feasibility and characteristic of tissue engineered testicular prosthesis with highdensity polyethylene(HDPE，trade name: Medpor) and polyglycolic acid(PGA). Methods The chondrocytes were isolated from the swine articular.The PGA scaffold was incorporated with medpor which semidiameters were 6mmand 4mm respectively.Then, the chondrocytes (5×10 7/ml) were seeded onto Medpor-PGA scaffold and cultured for 2 weeks. The ten BALB／C mice were divided into two groups randomly(n=5). In the experimental group, the cell-scaffold construct was implanted into subcutaneous pockets on the back of nude mice. In the control group, the Medpor-PGA scaffold was implanted. The mice of two groups were sacrificed to harvest the newly formed cartilage prosthesis after 8 weeks. Macroscopy, histology and immunohistochemistry observations were made. Results The gross observation showed that on changes were in shape and at size, the color and elasticity were similar to that of normal cartilage and that the cartilage integrated with Medpor in the experimental group; no cartilage formed and fiberlike tissue was found in the control group. HE staining showed that many mature cartilage lacuna formed without blood vessel and some PGA did not degradated completely. Toluidine blue staining showed extracellular matrix had metachromia. Safranin O-fast green staining showed that many proteoglycan deposited and collagen type Ⅱ expression was bly positive. In the control group, Medpor was encapsulated by fiber tissue with rich blood vessel. Conclusion The newly formed complex of Medpor-PGA and cells was very similar to testicle in gross view and to normal cartilage in histology. This pilot technique of creating testicular prosthesis by incorporating tissue-engineered cartilage with Medpor demonstrated success.