Objective To study the mechanism of ectopic osteogenesis of nacre/Polylactic acid (N/P) artificial bone combined with allogenic osteoblasts, and to explore the possibility as a scaffold material of bone tissue engineering. Methods The allogenic- osteoblasts seeded onto N/P artificial bone were co-cultured in vivo 1 week.The N/P artificial bone with allogenic osteoblasts were implanted subcutaneously into the left back sites of the New Zealand white rabbits in the experimental group and the simple N/P artificial bone into the right ones in the control group. The complexes were harvested and examined by gross observation, histologic analysis and immunohistochemical investigation 2, 4 and 8 weeks after implantation respectively.Results In experimental group, the osteoid formed after 4 weeks, and the mature bone tissue withbone medullary cavities formed after 8 weeks; but in control group there was nonew bone formation instead of abundant fibrous tissue after 4 weeks, and more fibrous tissue after 8 weeks.Conclusion N/P artificial bone can be used as an optical scaffold material of bone tissue engineering.
Objective To explore a novel nanometer biomaterial which could induce the regeneration of tooth tissues intell igently, and to evaluate the feasibil ity of using this kind of biomaterial as the scaffold for tooth tissue engineering by investigating the role it plays in tooth tissue engineering. Methods The scaffold for tooth tissue engineering containing recombinant human bone morphogenetic protein 2 (rhBMP-2) was prepared by mixing nanoscale β tricalcium phosphate (β-TCP)/collagen particles. Forty-six 8-10 weeks old specific pathogen free Sprague Dawley (SD)rats, including 34 females and 12 males, weighing 250-300 g, were involved in this study. Tooth germs were removed under a stereomicroscope from the mandible of newborn SD rat, then digested and suspended. Scanning electronic microscope (SEM), adhesion rate of cells, and MTT assay were used to evaluate the effects of the scaffold on the tooth germ cells cultured in vitro. The tissue engineered tooth germ which was constructed by tooth germ cells and scaffold was transplanted under SD rat’s kidney capsule as the experimental group (n=12); the tooth germ cells (cell-control group, n=12) or scaffold without cells (material-control group, n=4) were transplanted separately as control groups Specimens were harvested to perform general and histological observations at 4 and 8 weeks after transplantation. Results β-TCP/collagen showed a loose and porous appearance with soft texture and excellent hydrophil icity. Tooth germ cells grew well and could attach to the scaffold tightly 3 days after coculture. The adhesion rates of tooth germ cells were 27.20% ± 2.37%, 44.52% ± 1.87%, and 73.81% ± 4.15% when cocultured with scaffold for 4, 8, and 12 hours, respectively. MTT assay showed that the cell prol iferation status of experimental group was similar to that of the control group, showing no significant difference (P gt; 0.05). Some white calcified specimens could be harvested at 4-8 weeks after transplantation. At 4 weeks after transplantation some typical structures of dental cusp and enamel-dentin l ike tissues could be seen in the experimental group. Enamel-dentin l ike tissues also formed in some specimens of cell-control group, but they arranged irregularly. At 8 weeks after transplantation the enamel-dentin l ike tissue of experimental group exhibited a mature appearance and organized structure in comparison with that at 4 weeks. And mature enamel or dentin l ike tissue also could be seen in cell-control group. In contrast, there was no enamel or dentin l ike tissue in material-control group at 4 or 8 weeks after transplantation. Conclusion rhBMP-2 decorated β-TCP/collagen scaffold has good biocompatibil ity and can be used as a novel nanometer biomaterial, so it is a good choice in scaffolds for tooth tissue engineering.
Objective To review the appl ication of electrospinning in preparation of tendon tissue engineered scaffolds, to describe its appl ication effect and prospects. Methods Recent l iterature was extensively reviewed and summarized from various aspects, concerning the appl ication of electrospinning in preparing tendon tissue engineered scaffolds. Results Because of its huge surface and high porosity, the electrospun fibers prepared by electrospinning technology have been widely used in the manufacture of tendon tissue engineered scaffolds in recent years. A variety of materials, including polylactic acid, have been successfully electrospun into various types of tendon tissue engineered scaffolds, and goodresults in the repair of tendon defect were achieved. Conclusion The electrospinning technology has provide a new way for the preparation of the tendon tissue engineered scaffolds, with the perfection of the technology they will have broad application prospects in the field of tendon tissue engineering.
Objective To explore the method of preparing the electrospinning of synthesized triblock copolymers of ε-caprolactone and L-lactide (PCLA) for the biodegradable vascular tissue engineering scaffold and to investigateits biocompatibil ity in vitro. Methods The biodegradable vascular tissue engineering scaffold was made by the electrospinning process of PCLA. A series of biocompatibil ity tests were performed. Cytotoxicity test: the L929 cells were cultured in 96-wellflat-bottomed plates with extraction media of PCLA in the experimental group and with the complete DMEM in control group, and MTT method was used to detect absorbance (A) value (570 nm) every day after culture. Acute general toxicity test: the extraction media and sal ine were injected into the mice’s abdominal cavity of experimental and control groups, respectively, and the toxicity effects on the mice were observed within 72 hours. Hemolysis test: anticoagulated blood of rabbit was added into the extracting solution, sal ine, and distilled water in 3 groups, and MTT method was used to detect A value in 3 groups. Cell attachment test: the L929 cells were seeded on the PCLA material and scanning electron microscope (SEM) observation was performed 4 hours and 3 days after culture. Subcutaneous implantation test: the PCLA material was implanted subcutaneously in rats and the histology observation was performed at 1 and 8 weeks. Results Scaffolds had the characteristics of white color, uniform texture, good elasticity, and tenacity. The SEM showed that the PCLA ultrafine fibers had a smooth surface and proper porosity; the fiber diameter was 1-5 μm and the pore diameter was in the range of 10-30 μm. MTT detection suggested that there was no significant difference in A value among 3 groups every day after culturing (P gt; 0.05). The mice in 2 groups were in good physical condition and had no respiratory depression, paralysis, convulsion, and death. The hemolysis rate was 1.18% and was lower than the normal level (5%). The SEM showed a large number of attached L929 cells were visible on the surface of the PCLA material at 4 hours after implantation and the cells grew well after 3 days. The PCLA material was infiltrated by the inflammatory cells after 1 week. The inflammatory cells reduced significantly and the fiber began abruption after 8 weeks. Conclusion The biodegradable vascular tissue engineering scaffold material made by the electrospinning process of PCLA has good microstructure without cytotoxicity and has good biocompatibil ity. It can be used as an ideal scaffold for vascular tissue engineering.
Objective To review the current research status and clinical application progress of extracellular matrix (ECM) material in tissue engineering. Methods The literature about the latest progress in the preparation, biocompatibility, mechanical property, degradability, and clinical application of ECM material was extensively reviewed. Results The improvement of the ECM preparation method and thorough understanding of the immunological properties have laid the foundation for the repair and reconstruction of the tissue. Moreover, a series of animal studies also confirm that the feasibility and effectiveness of the ECM such as small intestinal submucosa, bladder ECM grift, and acellular dermis which have been applied to the repair and reconstruction of the urethra, bladder, arteries, and skin tissue. It shows a wide prospect of clinical application in the future. Conclusion ECM material is a good bio-derived scaffold, which is expected to become an important source of alternative materials for the repair and reconstruction of the tissue.
ObjectiveTo summarize the research progress of tissue-engineered bile duct in recent years. MethodsThe related literatures about the tissue-engineered bile duct were reviewed. ResultsIn recent years, the research of tissue-engineered bile duct has made a breakthrough in scaffold materials, seed cells, growth factors etc. However, the tissue-engineered bile duct is still in the research stage of animal experiments, which can not be directly applied to clinical practice. ConclusionsThe research of tissue-engineered bile duct becomes popular at present. With the rapid development of materials science and cell biology, the basic research and clinical application of tissue-engineered duct will be more in-depth research and extension, which might bring new ideas and therapeutic measures for patients with biliary defect or stenosis.
Objective Mechanical stimulation and inductive factors are both crucial aspects in tissue engineered cartilage. To evaluate the effects of mechanical stimulation combined with inductive factors on the differentiation of tissue engineered cartilage. Methods Bone marrow mesenchymal stem cells (BMSCs) were isolated from newborn porcine (aged7 days and weighing 3-6 kg) and expanded in vitro. The BMSCs at passage 2 were seeded onto a scaffold of poly (lactic-coglycol ic acid) (PLGA) in the concentration of 5 × 107/mL to prepare cell-scaffold composite. Cell-scaffold composites were cultivated in a medium with chondrocyte-inducted factors (group A), in a vessel with mechanic stimulating only (group B), or mechanic stimulating combined with chondrocyte-inducted factors (group C) (parameters of mechanics: 1 Hz, 0.5 MPa, and 4 hours/day). Cell-scaffold composite and auto-cartilage served as positive control (group D) and negative control (group E), respectively. After 4 weeks of cultivation, the thickness, elastic modulus, and glycosaminoglycan (GAG) content of composites were measured. Additionally, BMSCs chondrogenic differentiation was assessed via real-time fluorescent quantitative PCR, immunohistochemistry, and histological staining. Results The thickness, elastic modulus, and maximum load in group C were significantly higher than those in groups A and B (P lt; 0.05). In groups A, B, and C, cartilage lacuna formation, GAG expression, and positive results for collagen type II were obsersed through HE staining, Safranin-O staining, and immunohistochemistry staining. The dyeing depth was deeper in group A than in group B, and in group C than in groups A and B; group C was close to group E. The GAG content in group C was significantly higher than that in groups A and B (P lt; 0.05). Real-time fluorescent quantitative PCR revealed that mRNA expressions of collagen type I, collagen type II, and GAG in group C were significantly higher than those in groups A and B (P lt; 0.05), and in group A than in group B (P lt; 0.05). Conclusion Mechanical stimulation combined with chondrocyte inductive factors can enhance the mechanical properties of the composite and induce higher expression of collagen and GAG of BMSCs.
Objective To introduce the research progress on the technique of improving cell infiltration in electrospun scaffold. Methods The recent original articles about improving cell infiltration in electrospun scaffold were extensively reviewed and analyzed. Results The technique includes regulation of the electrospun parameters, modification of electrospun scaffold, and dynamic culture of scaffold-cells composite etc. The effect is limited and most of them need further optimization. Conclusion Cell infiltration in electrospun scaffold is of great significance in tissue engineering application. The relatively high compressed density and small pore size have become the bottleneck problem that prevents cell infiltration and tissue ingrowth into the scaffold. The combination of different techniques will be more effective to overcome this problem.
Objective To observe the adhesion and prol iferation of late endothel ial progenitor cells (EPCs) planted on nanoporous PLLA scaffold in vitro and to provide a new approach that optimizes tissue engineered material. Methods Male and female New Zealand rabbits (weight 2.5-3.0 kg) were used. Isolated late EPCs from rabbit peri pheral blood were cultured. Electrostatic spinning technique was adopted to prepare misal igned nanofibers, al igned nanofibers and super-al igned nanofibers, and low temperature plasma technique was appl ied to prepare misal igned membrane, al igned membrane and super-al igned membrane. After being divided into group A (cells only), B (misal igned membrane), C (normal membrane), D (al igned membrane) and E (super-al igned membrane), the primary late EPCs (1 × 105/mL) werecultured on scaffolds and MTT method was used to detect cell prol iferation abil ity at 3, 5, 7, 9, 11, 13, 15 and 17 days afterculture. After being divided into group A (misal igned membrane), B (normal membrane), C (al igned membrane) and D (superal igned membrane), precipitation method was appl ied to detect cell adhesion rate at 4, 12 and 24 hours after compound culture, and the morphologic changes of cells were observed at 4, 24 and 72 hours after compound culture. Results Fiber diameters in nanofibrous PLLA scaffolds were 300-400 nm, with a porosity rate of above 90%. At 3, 5, 7, 9, 11, 13, 15 and 17 days after culture, A value of each group was increased with time and the cells in each group grew well, showing there was no significant difference between group A and group B at each time point (P gt; 0.05 ); during the period of 7-15 days after culture, the difference between groups C, D and E and groups A and B was significant (P lt; 0.05). At 4 hours after compound culture, the adhesion rate of group A was superior to that of groups B, C and D (P lt; 0.05); at 12 and 24 hours after compound culture, the adhesion rate of groups B, C and D was remarkably higher than that of group A (P lt; 0.05); significant difference was noted in each group between the time point of 4 hours and the time point of 12 and 24 hours after compound culture (P lt; 0.05), but no significant difference between 12 hours and 24 hours was detected (P gt; 0.05). Morphology observation demonstrated that cells grew well on the scaffolds, the cells in groups A and B grew sporadically and disorderly, while the cells in groups C and D attached and al igned along fiber and prol iferated, with an excretion of ECM. Group D was better at maintaining cell morphology. Conclusion Al igned and superal igned nanofibers of PLLA scaffold can promote the adhesion and prol iferation of seed cells on the scaffold and maintain good cell morphology, which is an appropriate candidate scaffold material for blood vessel tissue engineering. Late EPCs is an ideal cell source for blood vessel tissue engineering.
ObjectiveTo study the effect and feasibility of poly-lactide-co-glycolide (PLGA) loaded with recombinant human bone morphogenetic protein 2 (rhBMP-2) on repairing articular cartilage defect in rabbits. Methods PLGA was made into cylinders which were 4 mm in diameter and 3 mm in thickness. rhBMP-2 was fully homogenated before used. PLGA combined with 0.5 mg rhBMP-2 under the condition of vacuum(700 mmHg),and then lyophilized, packed ,sterilized with ethylene oxide and reserved. Defects of 4 mm in diameter and reaching medullary cavity were made in femoral condyles of 72 two-month-old New Zealand white rabbits. The 36 right defects were repaired with PLGA-rhBMP-2 composites as the experimental group, the 36 left defects with PLGA only as PLGA group, the other 36 left defects were left untreated as control group, and the other 36 right defects with PLGA-MSCs composites as cell group. At 4, 8, 12, 24, 36 and 48 weeks after operation, macroscopical and microscopical observations were made, and the histological grade wasdone.Results After 4 weeks of operation: In the experimental group and cell group, defects were filled with white translucent tissue which appeared smooth and soft; the matrix around chondrocytes was weakly metachromatic, the newly formed cartilage tissue was thicker than normal cartilage tissue; there was no formed tissue in the PLGA group and the blank control group. After 8 weeks of operation: In the experimental group and cell group, the new tissue was white, translucent, tenacious and smooth. The boundary with normal cartilage became vague. New cartilage cells distributed evenly. The cells of the surface layerparalleled, but the deeper layer lost directivity. The matrix dyed weakly. The new cartilage gradually became thinner, but it still thicker than the normal cartilage ones. The PLGA degraded besides some drops.In the blank control group and PLGA group, a little white membrane formed at the bottom of the defect. After 1224 weeks of operation: In the experimental group and cell group, defects were filled with new tissues which were white, translucent, tenacious and smooth. The boundary disappeared.The thickness of the new cartilage was similar to that of the normal ones. The cells of the surface layer paralleled to each other,but the cells of the deeper layer tended to arrange vertically. The matrix around chondrocytes was metachromatic,but the color was lighter than that of the normal cartilage. Bone under the cartilage and the tide mark recovered. The new cartilage linked with nomal cartilage finely.In the blank control group and PLGA group, there was a little fibrous tissue at the bottom of the defect withe obvious boundary. After 36 weeks and 48 weeks of operation：in the experimental group and the cell group, the new cartilage was slightly white，continuous and less smooth. The boundary disappeared. There was no proliferated synovial membrane.The thickenss of the new cartilage was thinner than that of the normal ones. The matrix around chondrocytes was weakly metachromatic. In the blank control group and PLGA group, the defect still existed, but became smaller.At the bottom of the defect, fibrous tissues formed. Some cartilage denudated and became less smooth.Some bone under cartilage exposed,and the synovial membrane became thick. The histologic grade of the repair tissue at 12 weeks and 24 weeks of operation in experimental group and cell group was significantly different from that at 4, 8 and 48 weeks of operation(Plt;0.01). There was also significant difference in the experimental group and cell group compared with the blank control group and PLGA group at each time after operation(Plt;0.01). But there was no significant difference between the experimental group and the cell group. Conclusion In the course of degradation。。。。。。.