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.
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 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.
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 investigate the feasibility oftissue engineered intervertebral disc for regeneration of discs. Methods A three-dimensional porous poly(L-lactic-co-glycolic acid) (PLGA) scaffold was fabricated by temperature induced phase separation method. Human fetal disc cells were isolated and cultured in vitro. The disc cells labeledwith a PKH-26 fluorescent dye were seeded into a threedimensional porous scaffold. The proliferation of disc cells with PKH-26 fluorescent labels was assessed by using MTT uptake, laser fluorescence microscopy and SEM. Results Human fetal disc cells displayed a polygonal shape in primary monolayer culture. A regular arrangement and microtubules orientationstructure scaffold with 50-300 μm in diameter was fabricated by thermal-induced phase separation technique. MTT uptake and fluorescent microscopy examination indicated that the seeded disc cells were viable and showed proliferation activity within a porous scaffold. Conclusion The above findings support potential applications of tissue engineered disc in treatment of disc degenerative diseases.
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 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.
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 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 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.