OBJECTIVE: To build the trestle of tissue engineering for skin with the collagen. METHODS: The collagen was obtained from the baby cattle hide pretreated by Na2S and elastinase and Protease M, then the collagen was dissolved in 0.5 mol/L acetic acid solution. The collagen was treated with Protease N to minimize its immunogenicity. The resulting collagen could be used to build the trestle of tissue engineering for skin because of good biocompatibility. The collagen molecular weight and structure were analyzed by SDS-PAGE. The bioactivity of trestle was tested in the experiment of the mice wound healing and the cell implantation. RESULTS: The SDS-PAGE result of the collagen treated by Protease M showed the typical spectrum of type I collagen. The built trestle was a collagen sponge matrix in which micropore size was 50-200 microns. It could accelerate wound healing and the implanted fibroblasts could proliferate well. CONCLUSION: The collagen treated by Protease N can get good biocompatibilily and is suitable for building the trestles of tissue engineering for skin with good bioactivity.
Objective To review the latest development in the research on the application of the electrostatic spinning technology in preparation of the nanometer high polymer scaffold. Methods The related articles published at home and abroad during the recent years were extensively reviewed and comprehensively analyzed. Results Micro/nano-structure and space topology on the surfaces of the scaffold materials, especially the weaving structure, were considered to have an important effect on the cell adhesion, proliferation, directional growth, and biological activation. The electrospun scaffold was reported to have a resemblance to the structure of the extracellular matrix and could be used as a promising scaffold for the tissue engineeringapplication. The electrospun scaffolds were applied to the cartilage, bone, blood vessel, heart, and nerve tissue engineering fields. Conclusion The nanostructured polymer scaffold can support the cell adhesion, proliferation, location, and differentiation，and this kind of scaffold has a considerable value in the tissue engineering field.
Objective To review the recent advances in the application of graphene oxide (GO) for bone tissue engineering. Methods The latest literature at home and abroad on the GO used in the bone regeneration and repair was reviewed, including general properties of GO, degradation performance, biocompatibility, and application in bone tissue engineering. Results GO has an abundance of oxygen-containing functionalities, high surface area, and good biocompatibility. In addition, it can promote stem cell adhesion, proliferation, and differentiation. Moreover, GO has many advantages in the construction of new composite scaffolds and improvement of the performance of traditional scaffolds. Conclusion GO has been a hot topic in the field of bone tissue engineering due to its excellent physical and chemical properties. And many problems still need to be solved.
ObjectiveTo investigate the influences of lactic acid (LA), the final degradation product of polylactic acid (PLA) on the prol iferation and osteoblastic phenotype of osteoblast-l ike cells so as to provide theoretical basis for bone tissue engineering. MethodsRos17/2.8 osteoblast-l ike cells were harvested and divided into 3 groups. In groups A and B, the cells were cultured with the medium containing 4, 8, 16, 22, and 27 mmol/L L-LA and D, L-LA, respectively. In group C, the cells were cultured with normal medium (pH7.4). The cell prol iferation was determined with MTT method after 1, 3, and 5 days. The relative growth ratio (RGR) was calculated, and the cytotoxicity was evaluated according to national standard of China. In addition, the alkal ine phosphatase (ALP) activity of cells cultured with medium containing 4 mmol/L L-LA (group A), 4 mmol/ L D, L-LA (group B), and normal medium (group C) after 1 and 5 days were detected with ALP kits, and the relative ALP ratio (RAR) was calculated; after 21 days, the calcium nodules were tested with von Kossa staining method, and were quantitatively analyzed. ResultsWhen LA concentration was 4 mmol/L, the mean RGR of both groups A and B were all above 80%, and the cytotoxic grades were grade 0 or 1, which meant non-cytotoxicity. When LA concentration was 8 mmol/L and 16 mmol/ L, groups A and B showed cytotoxicity after 5 days and 3 days, respectively. When LA concentration was above 22 mmol/L, cell prol iferations of groups A and B were inhibited evidently after 1-day culture. At each LA concentration, RGR of group A was significantly higher than that of group B at the same culture time (P<0.05) except those at 4 mmol/L after 1-day and 3-day culture. After 1 day, the RAR of group A was significantly higher than that of group B on 1 day (144.1%±3.2% vs. 115.2%±9.8%, P<0.05) and on 5 days (129.6%±9.8% vs. 78.2%±6.9%, P<0.05). The results of von Kossa staining showed that the black gobbets in group A were obviously more than those of groups B and C. The staining area of group A (91.2%±8.2%) was significantly higher than that of groups B (50.3%±7.9%) and C (54.2%±8.6%) (P<0.05). ConclusionThe concentration and composition of LA have significant effects on the cell proliferation and osteoblastic phenotype of osteoblast-l ike cells.
ObjectiveTo manufacture a polycaprolactone (PCL)/type Ⅰ collagen (COL Ⅰ) tissue engineered meniscus scaffold (hereinafter referred to as PCL/COL Ⅰ meniscus scaffold) by three-dimensional (3D) printing with low temperature deposition technique and to study its physicochemical properties.MethodsFirst, the 15% PCL/4% COLⅠ composite solution and 15% PCL simple solution were prepared. Then, 15% PCL/4% COL Ⅰmeniscus scaffold and 15% PCL meniscal scaffold were prepared by using 3D printing with low temperature deposition techniques. The morphology and microstructure of the scaffolds were observed by gross observation and scanning electron microscope. The compression modulus and tensile modulus of the scaffolds were measured by biomechanical test. The components of the scaffolds were analyzed by Fourier transform infrared spectroscopy (FTIR). The contact angle of the scaffold surface was measured. The meniscus cells of rabbits were cultured with the two scaffold extracts and scaffolds, respectively. After cultured, the cell proliferations were detected by cell counting kit 8 (CCK-8), and the normal cultured cells were used as controls. Cell adhesion and growth of scaffold-cell complex were observed by scanning electron microscope.ResultsAccording to the gross and scanning electron microscope observations, two scaffolds had orientated 3D microstructures and pores, but the surface of the PCL/COLⅠ meniscus scaffold was rougher than the PCL meniscus scaffold. Biomechanical analysis showed that the tensile modulus and compression modulus of the PCL/COL Ⅰ meniscus scaffold were not significantly different from those of the PCL meniscus scaffold (P>0.05). FTIR analysis results showed that COL Ⅰ and PCL were successful mixed in PCL/ COL Ⅰ meniscus scaffolds. The contact angle of PCL/COLⅠ meniscus scaffold [(83.19±7.49)°] was significantly lower than that of PCL meniscus scaffold [(111.13±5.70)°] (t=6.638, P=0.000). The results of the CCK-8 assay indicated that with time, the number of cells cultured in two scaffold extracts showed an increasing trend, and there was no significant difference when compared with the control group (P>0.05). Scanning electron microscope observation showed that the cells attached on the PCL/ COL Ⅰ meniscus scaffold more than that on the PCL scaffold.ConclusionPCL/COLⅠmeniscus scaffolds are prepared by 3D printing with low temperature deposition technique, which has excellent physicochemical properties without cytotoxicity. PCL/COLⅠmeniscus scaffold is expected to be used as the material for meniscus tissue engineering.
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 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 observe the biocompatibility of the acellular corneal stroma materials prepared by three different methods. Methods Three different serial digestion methods were used to produce the acellular corneal stroma materials. The biocompatibility of the materials was investigated by the cell seeding and the materials were implanted into the rabbit corneal stroma layer. Results The cells in the materials 1 and 2 were not decellularized completely. The rabbit corneal fibroblasts died on the materials 1 and 2 after the cell seeding for 3-4 days. An obvious rejection could be observed after the implantation. The cells in material 3 were decellularized completely and the collagen fibers or elastic fibers were reserved integrally，showing a typical three-dimensional net work. The rabbit corneal fibroblasts could expand on the materials in vitro. No obvious rejection could be observed and the materials were gradually absorbed. Conclusion The acellular porcine cornea stroma materials prepared by trypsin-Dnase-Rnase are suitable for reconstruction of the tissue engineered cornea.
To summarize the medium-term cl inical result of bio-derived bone transplantation in orthopedics with tissue engineering technique. Methods From December 2000 to June 2001, 10 cases of various types of bone defect were treated with tissue engineered bone, which was constructed in vitro by allogenous osteoblasts from periosteum (1 × 106/ mL) with bio-derived bone scaffold following 3 to 7 days co-culture. Six men and 4 women were involved in this study, aged from 14 to 70 years with a median of 42 years. Among them, there were 2 cases of bone cyst, 1 case of non-union of old fracture, 6 cases of fresh comminuted fracture with bone defect, and 1 case of chronic suppurative ostemyel itis. The total weight of tissue engineered bone was 3-15 g in all the cases, averaged 7.3 g in each case. Results The wound in all the case healed by first intention. For 7 year follow up, bone union was completed within 3.0 to 4.5 months in 9 cases, but loosening occurred and the graft was taken out 1 year after operation in 1 case. The X-ray films showed that 9 cases achieved union except one who received resection of the head of humerus. No obvious abnormities were observed, and the function of affected l imbs met daily l ife and work. Conclusion Bio-derived tissue engineered bone has good osteogenesis. No obvious rejection and other compl ications are observed in the cl inical appl ication.
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.