ObjectiveTo observe the bladder regeneration by collagen membrane scaffolds for bladder construction to find a new alternative scaffold material. MethodsTwelve healthy adult male Sprague Dawley rats, weighing 300-350 g, were randomly divided into collagen membrane scaffold group (experimental group, n=6), and sham operated group (control group, n=6). Upper hemicystectomy was performed and collagen scaffold was used for reconstruction in experimental group, while the bladder was turned over without bladder resection in control group. At 30 days after operation, the animals were sacrificed and grafts were harvested;HE staining and Masson staining were used to evaluate the bladder regeneration, immunohistochemical staining was performed with α-smooth muscleactin (α-SMA) and von Willebrand factor (vWF) markers to evaluate the percentage of α-SMA positive area and capillary number. ResultsThe rats of 2 groups survived to the end of the experiment, and no urine leakage or infection was observed in experimental group. Histologically, control group presented a pattern of normal bladder structure, experimental group presented a pattern of almost normal urothelium with a small amount of smooth muscle cells and a thin layer of undegraded collagen fibers. Immunohistochemically, experimental group showed ingrowth of smooth muscle fibers and new capillary formation along the collagen membrane scaffolds. The percentage of α-SMA positive area and capillary number in experimental group were significantly lower than those in control group (6.49%±2.14% vs. 52.42%±1.78% and 4.83±0.75 vs. 14.83±1.17, respectively)(t=40.40, P=0.00; t=17.62, P=0.00). ConclusionThe collagen membrane scaffolds could be an effective scaffold material for bladder reconstruction.
ObjectiveTo summarize the research progress of several three-dimensional (3-D) printing scaffold materials in bone tissue engineering. MethodThe recent domestic and international articles about 3-D printing scaffold materials were reviewed and summarized. ResultsCompared with conventional manufacturing methods, 3-D printing has distinctive advantages, such as enhancing the controllability of the structure and increasing the productivity. In addition to the traditional metal and ceramic scaffolds, 3-D printing scaffolds carrying seeding cells and tissue factors as well as scaffolds filling particular drugs for special need have been paid more and more attention. ConclusionsThe development of 3-D printing porous scaffolds have revealed new perspectives in bone repairing. But it is still at the initial stage, more basic and clinical researches are still needed.
Objective To review the research status of the neovascularization of adi pose tissue engineering in the past decade so as to provide theoretical references for the development of the rapid revascularization of tissue engineered adi pose. Methods The l iterature about the revascularization of adi pose tissue engineering was extensively reviewed andanalyzed, centering on 5 elements: specificity of histological structures and blood supply, revascularization mechanism, coculture of different seed cells, modification of scaffold, and microenvironment. Results Adi pose tissue engineering offers a new solution for soft tissue defects. However, there is still the unfulfilled need in the size of engineered adipose tissue (less than 1 mL), which was determined by the degree of neovascularization in engineered tissue. Overall, rapid neovascularization in engineering tissue is a key l ink of experimental study changing into cl inical appl ication. Conclusion Providing a sufficient supply with nutrients and oxygen by means of a sufficient and rapid neovascularization will be at the heart of any attempts to obtain bigger tissue engineered adipose to meet the demand of repairing large soft tissue defect.
Objective To investigate the application potential of alginate-strontium (Sr) hydrogel as an injectable scaffold material in bone tissue engineering. Methods The alginate-Sr/-calcium (Ca) hydrogel beads were fabricated by adding 2.0wt% alginate sodium to 0.2 mol/L SrCl2/CaCl2 solution dropwise. Microstructure, modulus of compression, swelling rate, and degradability of alginate-Sr/-Ca hydrogels were tested. Bone marrow mesenchymal stem cells (BMSCs) were isolated from femoral bones of rabbits by flushing of marrow cavity. BMSCs at passage 5 were seeded onto the alginate-Sr hydrogel (experimental group) and alginate-Ca hydrogel (control group), and the viability and proliferation of BMSCs in 2 alginate hydrogels were assessed. The osteogenic differentiation of cells embeded in 2 alginate hydrogels was evaluated by alkaline phosphate (ALP) activity, osteoblast specific gene [Osterix (OSX), collagen type I, and Runx2] expression level and calcium deposition by fluorescent quantitative RT-PCR and alizarin red staining, Von Kossa staining. The BMSCs which were embeded in alginate-Ca hydrogel and cultured with common growth medium were harvested as blank control group. Results The micromorphology of alginate-Sr hydrogel was similar to that of the alginate-Ca hydrogel, with homogeneous pore structure; the modulus of compression of alginate-Sr hydrogel and alginate-Ca hydrogel was (186.53 ± 8.37) and (152.14 ± 7.45) kPa respectively, showing significant difference (t=6.853, P=0.002); there was no significant difference (t=0.737, P=0.502) in swelling rate between alginate-Sr hydrogel (14.32% ± 1.53%) and alginate-Ca hydrogel (15.25% ± 1.64%). The degradabilities of 2 alginate hydrogels were good; the degradation rate of alginate-Sr hydrogel was significantly lower than that of alginate-Ca hydrogel on the 20th, 25th, and 30th days (P lt; 0.05). At 1-4 days, the morphology of cells on 2 alginate hydrogels was spherical and then the shape was spindle or stellate. When three-dimensional cultured for 21 days, the DNA content of BMSCs in experimental group [(4.38 ± 0.24) g] was significantly higher than that in control group [(3.25 ± 0.21) g ] (t=8.108, P=0.001). On the 12th day after osteogenic differentiation, the ALP activity in experimental group was (15.28 ± 1.26) U/L, which was significantly higher than that in control group [(12.07 ± 1.12) U/L] (P lt; 0.05). Likewise, the mRNA expressions of OSX, collagen type I, and Runx2 in experimental group were significantly higher than those in control group (P lt; 0.05). On the 21th day after osteogenic differentiation, alizarin red staining and Von Kossa staining showed calcium deposition in 2 groups; the calcium nodules and phosphate deposition in experimental group were significantly higher than those in control group (P lt; 0.05). Conclusion Alginate-Sr hydrogel has good physicochemical properties and can promote the proliferation and osteogenic differentiation of BMSCs, so it is an excellent injectable scaffold material for bone tissue engineering.
Objective To explore the method of preparing spongy and porous scaffold materials with swine articular cartilage acellular matrix and to investigate its appl icabil ity for tissue engineered articular cartilage scaffold. Methods Fresh swine articular cartilage was freeze-dried and freeze-ground into microparticles. The microparticles with diameter of less than 90 μm were sieved and treated sequentially with TNE, pepsin and hypotonic solution for decellularization at cryogenic temperatures. Colloidal suspension with a mass/volume ratio of 2% was prepared by dissolving the microparticles into 1.5% HAc, and then was lyophil ized for molding and cross-l inked by UV radiation to prepare the decellularized cartilage matrix sponge. Physicochemical property detection was performed to identify aperture, porosity and water absorption rate. Histology and scanning electron microscope observations were conducted. The prepared acellular cartilage matrix sponge was implanted into the bilateral area of spine in 24 SD rats subcutaneously (experimental group), and the implantation of Col I sponge served as control group. The rats were killed 1, 2, 4, and 8 weeks after operation to receive histology observation, and the absorption and degeneration conditions of the sponge in vivo were analyzed. BMSCsobtained from femoral marrow of 1-week-old New Zealand white rabbits were cultured. The cells at passage 3 were cultured with acellular cartilage matrix sponge l ixivium at 50% (group A), acellular cartilage matrix sponge l ixivium at 100% (group B), and DMEM culture medium (group C), respectively. Cell prol iferation was detected by MTT method 2, 4, and 6 days after culture. Results The prepared acellular cartilage matrix sponge was white and porous. Histology observation suggested that the sponge scaffold consisted primarily of collagen without chondrocyte fragments. Scanning electron microscope demonstrated that the scaffold had porous and honeycomb-shaped structure, the pores were interconnected and even in size. The water absorption rate was 20.29% ± 25.30%, the aperture was (90.66 ± 21.26) μm, and the porosity of the scaffold was 90.10% ± 2.42%. The tissue grew into the scaffold after the subcutaneous implantation of scaffold into the SD rats, angiogenesis was observed, inflammatory reaction was mild compared with the control group, and the scaffold was degraded and absorbed at a certain rate. MTT detection suggested that there were no significant differences among three groups in terms of absorbance (A) value 2 and 4 days after culturing with the l ixivium (P gt; 0.05), but significant differences were evident among three groups 6 days after culturing with the l ixivium (P lt; 0.05). Conclusion With modified treatment and processing, the cartilage acellular matrix sponge scaffold reserves the main components of cartilage extracellular matrix after thorough decellularization, has appropriate aperture and porosity, and provides even distribution of pores and good biocompatibil ity without cytotoxicity. It can be used as an ideal scaffold for cartilage tissue engineering.
Objective To review the research progress of cell-scaffold complex in the tendon tissue engineering. Methods Recent literature concerning cell-scaffold complex in the tendon tissue engineering was reviewed, the research situation of the cell-scaffold complex was elaborated in the aspects of seed cells, scaffolds, cell culture, and application. Results In tendon tissue engineering, a cell-scaffold complex is built by appropriate seed cells and engineered scaffolds. Experiments showed that modified seed cells had better therapeutic effects. Further, scaffold functionality could be improved through surface modification, growth factor cure, mechanical stimulation, and contact guidance. Among these methods, mechanical stimulation revealed the most significant results in promoting cell proliferation and function. Through a variety of defect models, it is demonstrated that the use of cell-scaffold complex could achieve satisfactory results for tendon regeneration. Conclusion The cell-scaffold complex for tendon tissue engineering is a popular research topic. Although it has not yet met the requirement of clinical use, it has broad application prospects.
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 observe the ability to repair bilateralradius bone defect with the composite of β-tricalciumphosphate(βTCP),hyaluronic acid(HA),type I collagen(COL-Ⅰ) and induced marrow stromal cells(MSCs), and to investigate the feasibility of the composite as a bone substitute material.Methods The MSCs of the New Zealand white rabbits were induced into ostoblasts, then combined with β-TCP, HA and COL-Ⅰ. Thirty New Zealand white rabbits were made the bilateral radius bone defects of 2 cm and divided into groups A, B and C. After 8 weeks, β-TCP-HA-COL-Ⅰ-MSCs （group A, n=27 sides）, autograft （group B, n=27 sides）andno implant（group C as control, n=6 sides）were implanted into the areas ofbilateral radius bone defects, respectively. The structure of the composite was observed by scanning electron microscope. The repairing effect was observed by gross, histomorphology, X-ray examination, and the degradation rate of inorganic substance at 4, 8 and 12 weeks. The ostogenic area and biomechanics ofgroup A were compared with those of group B at 12 weeks.Results The MSCs could stably grow in vitro, relatively rapidly proliferated, and could be induced into the ostoblasts.The composite was porous. The results of gross, histomorphology and X-ray showed that the bone defects were perfectly repaired in group A and group B, but not in group C. The ostogenic area or biomechanics had no statistically significant difference between groups A and B(Pgt;0.05). The weight of inorganic substance in group A were 75% ,57% and 42% at 4,8,12 weeks, respectively.Conclusion MSCs can be used as seedcells in the bone tissue engineering. The composite has porous structure, no reactions of toxicity to the tissue and rapid degradation, and it is an ideal carrier of seed cells.The β-TCP-HA-COL-Ⅰ-MSCs composite has the high ability of repairing bone defect and can serve as an autograft substitute material.
Objective To prepare the silk fibroin (SF)-chitosan (CS) scaffolds by adjusting the mass ratio between CS and SF, and test and compare the properties of the scaffolds at different mass ratios. Methods According to the mass ratios of 6 ∶ 4 (group A), 6 ∶ 8 (group B), and 6 ∶ 16 (group C) between SF and CS, CS-SF scaffolds were prepared by freeze-drying method, respectively. The material properties, porosity, the dissolubility in hot water, the modulus elasticity, and the water absorption expansion rate were measured; the aperture size and shape of scaffolds were observed by scanning electron microscope (SEM). Density gradient centrifugation method was used to isolate the bone marrow mesenchymal stell cells (BMSCs) of 4-week-old male Sprague Dawley rats. The BMSCs at passage 3 were seeded onto 3 scaffolds respectively, and then the proliferation of cells on the scaffolds was detected by MTS method. Results The results of fourier transform infrared spectroscopy proved that with the increased content of CS, the absorption peak of random coil/α helix structure (1 654 cm-1 and 1 540 cm-1) constantly decreased, but the absorption peak of corresponding to β-fold structure (1 628 cm-1 and 1 516 cm- 1) increased. The porosity was 87.36% ± 2.15% in group A, 77.82% ± 1.37% in group B, and 72.22% ± 1.37% in group C; the porosity of group A was significantly higher than that of groups B and C (P lt; 0.05), and the porosity of group B was significantly higher than that of group C (P lt; 0.05). The dissolubility in hot water was 0 in groups A and B, and was 3.12% ± 1.26% in group C. The scaffolds had good viscoelasticity in 3 groups; the modulus elasticity of 3 groups were consistent with the range of normal articular cartilage (4-15 kPa); no significant difference was found among 3 groups (F=5.523, P=0.054). The water absorption expansion rate was 1 528.52% ± 194.63% in group A, 1 078.22% ± 100.52% in group B, and 1 320.05% ± 179.97% in group C; the rate of group A was significantly higher than that of group B (P=0.05), but there was no significant difference between groups A and C and between groups B and C (P gt; 0.05). SEM results showed the aperture size of group A was between 50-250 μm, with good connectivity of pores; however, groups B and C had structure disturbance, with non-uniform aperture size and poor connectivity of pores. The growth curve results showed the number of living cells of group A was significantly higher than that of groups B and C at 1, 3, 5, and 7 days (P lt; 0.05); and there were significant differences between groups B and C at 3, 5, and 7 days (P lt; 0.05). Conclusion The CS-SF scaffold at a mass ratio of 6 ∶ 4 is applicable for cartilage tissue engineering.
ObjectiveTo study the preparation method of acellular dermal matrix (ADM) for cartilage tissue engineering and analyze its biocompatibility. MethodsThe dermal tissues of the calf back were harvested, and decelluarized with 0.5% SDS, and the ADM was reconstructed with 0.5% trypsin, cross-linked with formaldehyde, and modified with 0.5% chondroitin sulfate which can promote the proliferation of chondrocytes. And the porosity, cytotoxicity, and biocompatibility were determined. Co-cultured 2nd passage chondrocytes and bone marrow stromal cells in a proportion of 3 to 7 were used as seed cells. The cells were seeded on ADM (experimental group) for 48 hours to observe the cell adhesion. The expressions of mRNA and protein of collagen type Ⅱ were tested by RT-PCR and Western blot methods, respectively. And the expressions were compared between the cells seeded on the scaffold and cultured in monolayer (control group). ResultsAfter modification of 0.5% trypsin, the surface of ADM was smooth and had uniform pores; the porosity (85.4%±2.8%) was significantly higher than that without modification (72.8%±5.8%) (t=-4.384, P=0.005). The cell toxicity was grade 1, which accords to the requirements for cartilage tissue engineering scaffolds. With time passing, the number of inflammatory cells decreased after implanted in the back of the rats (P<0.05). The scanning electron microscope observation showed that lots of seed cells adhered to the scaffold, the cells were well stacked, displaying surface microvilli and secretion. The expressions of mRNA and protein of collagen type Ⅱ were not significantly different between experimental and control groups (t=1.265, P=0.235;t=0.935, P=0.372). ConclusionThe ADM prepared by acellular treatment, reconstruction, cross-linking, and modification shows perfect characters. And the seed cells maintain chondrogenic phenotype on the scaffold. So it is a proper choice for cartilage tissue engineering.