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.
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 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 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.
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 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 explore the effect of tissue engineered cartilage reconstructed by using sodium alginate hydrogel and SIS complex as scaffold material and chondrocyte as seed cell on the repair of full-thickness articular cartilage defects. Methods SIS was prepared by custom-made machine and detergent-enzyme treatment. Full-thickness articularcartilage of loading surface of the humeral head and the femoral condyle obtained from 8 New Zealand white rabbits (2-3weeks old) was used to culture chondrocytes in vitro. Rabbit chondrocytes at passage 4 cultured by conventional multipl ication method were diluted by sodium alginate to (5-7) × 107 cells/mL, and then were coated on SIS to prepare chondrocyte-sodium alginate hydrogel-SIS complex. Forty 6-month-old clean grade New Zealand white rabbits weighing 3.0-3.5 kg were randomized into two groups according to different operative methods (n=20 rabbits per group), and full-thickness cartilage defect model of the unilateral knee joint (right or left) was establ ished in every rabbit. In experimental group, the complex was implanted into the defect layer by layer to construct tissue engineered cartilage, and SIS membrane was coated on the surface to fill the defect completely. While in control group, the cartilage defect was filled by sodium alginate hydrogel and was sutured after being coated with SIS membrane without seeding of chondrocyte. General condition of the rabbits after operation was observed. The rabbits in two groups were killed 1, 3, 5, 7, and 9 months after operation, and underwent gross and histology observation. Results Eight rabbits were excluded due to anesthesia death, wound infection and diarrhea death. Sixteen rabbits per group were included in the experiment, and 3, 3, 3, 3, and 4 rabbits from each group were randomly selected and killed 1, 3, 5, 7, and 9 months after operation, respectively. Gross observation and histology Masson trichrome staining: in the experimental group, SIS on the surface of the implant was fused with the host tissue, and the inferface between them disappeared 1 month after operation; part of the implant was chondrified and the interface between the implant and the host tissue was fused 3 months after operation; the implant turned into fibrocartilage 5 months after operation; fiber arrangement of the cartilage in theimplant was close to that of the host tissue 7 months after operation; cartilage fiber in the implant arranged disorderly andactive cell metabol ism and prol iferation were evident 9 months after operation. While in the control group, no repair of thedefect was observed 9 months after operation. No obvious repair was evident in the defects of the control group within 9months after operation. Histomorphometric evaluation demonstrated that the staining intensity per unit area of the reparative tissue in the defect of the experimental group was significant higher than that of the control group at each time point (P lt; 0.05), the chondrification in the experimental group was increased gradually within 3, 5, and 7 months after operation (P lt; 0.05), and it was decreased 9 months after operation comparing with the value at 7 months after operation (P lt; 0.05). Conclusion Constructed by chondrocyte-sodium alginate hydrogel-SIS in complex with surficial suturing of SIS membrane, the tissue engineered cartilage can in-situ repair cartilage defect, promote the regeneration of cartilage tissue, and is in l ine with physiological repair process of articular cartilage.
OBJECTIVE: To sum up the clinical results of bio-derived bone transplantation in orthopedics with tissue engineering technique. METHODS: From January 2000 to May 2002, 52 cases with various types of bone defect were treated with tissue engineered bone, which was constructed in vitro by allogeneous osteoblasts from periosteum (1 x 10(6)/ml) with bio-derived bone scaffold following 3 to 7 days co-culture. Among them, there were 7 cases of bone cyst, 22 cases of non-union or malunion of old fracture, 15 cases of fresh comminuted fracture of bone defect, 4 cases of spinal fracture and posterior route spinal fusion, 3 cases of bone implant of alveolar bone, 1 case of fusion of tarsotarsal joint. The total weight of tissue engineered bone was 349 g in all the cases, averaged 6.7 g in each case. RESULTS: All the cases were followed up after operation, averaged in 18.5 months. The wound in all the case healed by first intention, but 1 case with second intention. Bone union was completed within 3 to 4.5 months in 50 cases, but 2 cases of delayed union. Six cases were performed analysis of CD3, CD4, CD8, ICAM-1 and VCAM-1 before and after operation, and no obvious abnormities were observed. CONCLUSION: Bio-derived tissue engineered bone has good osteogenesis. No obvious rejection and other complications are observed in the clinical application.
Objective To investigate the feasibil ity of using thermo-sensitive chitosan hydrogen as a scaffold to construct tissue engineered injectable nucleus pulposus (NP). Methods Three-month-old neonatal New Zealand rabbits (male or female) weighing 150-200 g were selected to isolate and culture NP cells. The thermo-sensitive chitosan hydrogel scaffold wasmade of chitosan, disodium β-glycerophosphate and hydroxyethyl cellulose. Its physical properties and gross condition were observed. The tissue engineered NP was constructed by compounding the scaffold and rabbit NP cells. Then, the viabil ity of NP cells in the chitosan hydrogel was observed 2 days after compound culture and the growth condition of NP cells on the scaffold was observed by SEM 7 days after compound culture. NP cells went through histology and immunohistochemistry detection and their secretion of aggrecan and expression of Col II mRNA were analyzed by RT-PCR 21 days after compound culture. Results The thermo-sensitive chitosan hydrogel was l iquid at room temperature and sol idified into gel at 37 (15 minutes) due to crossl inking reaction. Acridine orange-propidiumiodide staining showed that the viabil ity rate of NP cells in chitosan hydrogel was above 90%. Scanning electron microscope observation demonstrated that the NP cells were distributed in the reticulate scaffold, with ECM on their surfaces. The results of HE, toluidine blue, safranin O and histology and immunohistochemistry staining confirmed that the NP cells in chitosan hydrogel were capable of producing ECM. RT-PCR results showed that the secretion of Col II and aggrecan mRNA in NP cells cultured three-dimensionally by chitosan hydrogen scaffold were 0.631 ± 0.064 and 0.832 ± 0.052, respectively,showing more strengths of producing matrix than that of monolayer culture (0.528 ± 0.039, 0.773 ± 0.046) with a significant difference (P lt; 0.05). Conclusion With good cellular compatibilities, the thermo-sensitive chitosan hydrogel makes it possible for NP cells to maintain their normal morphology and secretion after compound culture, and may be a potential NP cells carrier for tissue engineered NP.
Objective Extracellular matrix is one of the focus researches of the adi pose tissue engineering. To investigate the appropriate method to prepare the porcine skeletal muscle acellular matrix and to evaluate the biocompatibility of the matrix. Methods The fresh skeletal muscle tissues were harvested from healthy adult porcine and were sl iced into2-3 mm thick sheets, which were treated by hypotonic-detergent method to remove the cells from the tissue. The matrix was then examined by histology, immunohistochemistry, and scanning electron microscopy. The toxic effects of the matrix were tested by MTT. Human adi pose-derived stem cells (hADSCs) were isolated from adi pose tissue donated by patients with breast cancer, and identified by morphology, flow cytometry, and differentiation abil ity. Then, hADSCs of passage 3 were seeded into the skeletal muscle acellular matrix, and cultured in the medium. The cellular behavior was assessed by calcein-AM (CA) and propidium iodide (PI) staining at 1st, 3rd, 5th, and 7th days after culturing. Results Histology, immunohistochemistry, and scanning electron microscopy showed that the muscle fibers were removed completely with the basement membrane structure; a large number of collagenous matrix presented as regular network, porous-like structure. The cytotoxicity score of the matrix was grade 1, which meant that the matrix had good cytocompatibil ity. The CA and PI staining showed the seeded hADSCs had the potential of spread and prol iferation on the matrix. Conclusion Porcine skeletal muscle acellular matrix has good biocompatibility and a potential to be used as an ideal biomaterial scaffold for adi pose tissue engineering.
