Objective To investigate the feasibility of a dual-crosslinked injectable hydrogel derived from acellular musclar matrix (AMM) for promoting myoblasts proliferation and myogenic differentiation. Methods Firstly, hyaluronic acid was oxidized with NaIO4 and methylated to prepare methacrylamidated oxidized hyaluronic acid (MOHA). Then, AMM obtained by washing enzymatically treated muscle tissue was aminolyzed to prepare aminated AMM (AAMM). MOHA hydrogel and AAMM were crosslinked using Schiff based reaction and UV radiation to prepare a dual-crosslinked MOHA/AAMM injectable hydrogel. Fourier transform infrared spectroscopy (FTIR) was used to characterize MOHA, AAMM, and MOHA/AAMM hydrogels. The injectability of MOHA/AAMM hydrogel were evaluated by manual injection, and the gelation performance was assessed by UV crosslinking. The rheological properties and Young’s modulus of the hydrogel were examined through mechanical tests. The degradation rate of the hydrogel was assessed by immersing it in PBS. The active components of the hydrogel were verified using immunofluorescence staining and ELISA assay kits. The promotion of cell proliferation by the hydrogel was tested using live/dead staining and cell counting kit 8 (CCK-8) assays after co-culturing with C2C12 myoblasts for 9 days. The effect of the hydrogel on myogenic differentiation was evaluated by immunofluorescence staining and real time quantitative polymerase chain reaction (RT-qPCR). ResultsFTIR spectra confirmed the successful preparation of MOHA/AAMM hydrogel. The hydrogel exhibited good injectability and gelation ability. Compared to MOHA hydrogel, MOHA/AAMM hydrogel exhibited higher viscosity and Young’s modulus, a reduced degradation rate, and contained a higher amount of collagen (including collagen type Ⅰ and collagen type Ⅲ) as well as bioactive factors (including epidermal growth factor, fibroblast growth factor 2, vascular endothelial growth factor, and insulin-like growth factor 1). The live/dead cell staining and CCK-8 assay indicated that with prolonged incubation time, there was a significant increase in viable cells and a decrease in dead cells in the C2C12 myoblasts within the MOHA/AAMM hydrogel. Compared with MOHA hydrogel, the difference was significant at each time point (P<0.05). Immunofluorescence staining and RT-qPCR analysis demonstrated that the deposition of IGF-1 and expression levels of myogenic-related genes (including Myogenin, Troponin T, and myosin heavy chain) in the MOHA/AAMM group were significantly higher than those in the MOHA group (P<0.05). ConclusionThe MOHA/AAMM hydrogel prepared based on AMM can promote myoblasts proliferation and myogenic differentiation, providing a novel dual-crosslinked injectable hydrogel for muscle tissue engineering.
ObjectiveTo study the feasibility of acellular matrix materials prepared from deer antler cartilage and its biological compatibility so as to search for a new member of the extracellular matrix family for cartilage regeneration. MethodsThe deer antler mesenchymal (M) layer tissue was harvested and treated through decellular process to prepare M layer acellular matrix; histologic observation and detection of M layer acellular matrix DNA content were carried out. The antler stem cells [antlerogenic periosteum (AP) cells] at 2nd passage were labelled by fluorescent stains and by PKH26. Subsequently, the M layer acellular matrix and the AP cells at 2nd passage were co-cultured for 7 days; then the samples were transplanted into nude mice to study the tissue compatibility of M layer acellular matrix in the living animals. ResultsHE and DAPI staining confirmed that the M layer acellular matrix did not contain nucleus; the DNA content of the M layer acellular matrix was (19.367±5.254) ng/mg, which was significantly lower than that of the normal M layer tissue [(3 805.500±519.119) ng/mg](t=12.630, P=0.000). In vitro co-culture experiments showed that AP cells could adhere to or even embedded in the M layer acellular matrix. Nude mice transplantation experiments showed that the introduced AP cells could proliferate and induce angiogenesis in the M layer acellular matrix. ConclusionThe deer antler cartilage acellular matrix is successfully prepared. The M layer acellular matrix is suitable for adhesion and proliferation of AP cells in vitro and in vivo, and it has the function of stimulating angiogenesis. This model for deer antler cartilage acellular matrix can be applied in cartilage tissue engineering in the future.
Objective To evaluate the feasibility of poly-L-lactide(PLLA)/porcinederived xenogeneic bone(PDXB) composite as a scaffold for the bone tissue engineering. Methods The film and the scaffold of the PLLA-PDXB composite were respectively prepared by a solution casting method and a solution casting-particle leaching method. The composite film and scaffold were further treated by the surface alkaline hydrolysis. The surface morphology of the composite was observed by the scanning electron microscopy, and hydrophilicity degree of the composite was measured. The OCT-1 osteoblastlike cells were cultured and amplified in vitro as the seeding cells, which werethen implanted on the film and scaffold. The adherence rate, adherence shape,proliferating activity, and growing morphology of the OCT-1 osteoblastlikecells were observed on the film. Results The PDXB particle 50 μm in diameter on average had a similar phase structure to that of hydroxyapatite. But its Ca/P ratio was lower than that of hydroxyapatite. After the surface alkaline hydrolysis, the PDXB particle could be exposed on the surface of the PLLA-PDXB composite. The surface roughness and hydrophilicity of the PLLAPDXB composite were obviously enhanced. The cell adherence rate and the cell proliferation activity of the PLLAPDXB composite were higher than those of the pure PLLA material. The cells tended to grow on the exposed surface of the PDXB particles. The cells seeded on the composite scaffold could migrate to the inside of the composite scaffold and grew well. Conclusion The PLLA-PDXB composite has a good cell affinity, and this kind of composite can hopefullybecome a new scaffold material to be used in the bone tissue engineering.
Dental pulp stem cells(DPSCs) are adult stem cells with strong proliferative ability, self-renewal ability and multidirectional differentiation potential. DPSCs have abundant source are easy to obtain, and do not have ethical problems. As seed cells, they played an important role and showed great potential in tissue engineering and regenerative medicine, making them potential ideal seed cells for repairation and regeneration of tissue and organ. Clinical application of DPSCs in bone regeneration has already been achieved, and studies on differentiation of DPSCs into other tissues are still at different levels of basic stage. In this paper, the research and application of directional differentiation potential such as tooth formation, osteogenesis, and nerve formation are reviewed in order to provide clues and ideas for further study on DPSCs in the field of tissue engineering and regenerative medicine.
Objective To review the research progress of in-situ three dimensional (3D) bio-printing technology in the repair of bone and cartilage injuries. Methods Literature on the application of in-situ 3D bio-printing technology to repair bone and cartilage injuries at home and abroad in recent years was reviewed, analyzed, and summarized. Results As a new tissue engineering technology, in-situ 3D bio-printing technology is mainly applied to repair bone, cartilage, and skin tissue injuries. By combining biomaterials, bioactive substances, and cells, tissue is printed directly at the site of injury or defect. At present, the research on the technology mainly focuses on printing mode, bio-ink, and printing technology; the application research in the field of bone and cartilage mainly focuses on pre-vascularization, adjusting the composition of bio-ink, improving scaffold structure, printing technology, loading drugs, cells, and bioactive factors, so as to promote tissue injury repair. Conclusion Multiple animal experiments have confirmed that in-situ 3D bio-printing technology can construct bone and cartilage tissue grafts in a real-time, rapid, and minimally invasive manner. In the future, it is necessary to continue to develop bio-inks suitable for specific tissue grafts, as well as combine with robotics, fusion imaging, and computer-aided medicine to improve printing efficiency.
ObjectiveTo review the application of silk fibroin scaffold in bone tissue engineering. MethodsThe related literature about the application of silk fibroin scaffold in bone tissue engineering was reviewed, analyzed, and summarized. ResultsSilk fibroin can be manufactured into many types, such as hydrogel, film, nano-fiber, and three-dimensional scaffold, which have superior biocompatibility, slow biodegradability, nontoxic degradation products, and excellent mechanical strength. Meanwhile these silk fibroin biomaterials can be chemically modified and can be used to carry stem cells, growth factors, and compound inorganic matter. ConclusionSilk fibroin scaffolds can be widely used in bone tissue engineering. But it still needs further study to prepare the scaffold in accordance with the requirement of tissue engineering.
ObjectiveTo investigate the feasibility of animal model of the reconstruction of right ventricular outflow tract in rats.MethodsA total of 15 female Sprague-Dawley (SD) rats underwent right ventricular outflow tract reconstruction surgery. Before the operation, the collagen scaffolds were treated with g 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride chemistry (EDC), and seeded with human bone marrow stem cells (h-MSCs). Three days after the surgery, 3 rats were randomly sacrificed to evaluate the transmural resection of right ventricular outflow tract. One or 3 months later, other 3 rats at each timepoint were sacrificed, stained with Masson’s Trichrome to observe the degradation of scaffold. Furthermore, 4 weeks after the surgery, 4 rats were sacrificed and the hearts were sliced. Anti-human mitochondria staining was used to identify the survival of seeding cells.ResultsThe transmural resection of right ventricular outflow tract was feasible in rats at an acceptable mortality (13.3%). After EDC treatment, the degradation rate of collagen scaffold was extended greatly. The seeding cells were detected by anti-mitochandria immunofluorescent staining in all patches 4 weeks after the operation.ConclusionRat model of right ventricular outflow tract reconstruction could be a stable, reliable and economical screening model for engineered heart tissue research.
ObjectiveTo review the research progress of natural biomaterial polyhydroxyalkanoate (PHA) in orthopedics. Methods The literature concerning PHA devices for bone defects, bone repair, and bone neoplasms, respectively, in recent years was extensively consulted. The three aspects of the advantages of PHA in bone repair, the preparation of PHA medical devices for bone repair and their application in orthopedics were discussed. ResultsDue to excellent biodegradability, biocompatibility, and potential osteoinduction, PHA is a kind of good bone repair material. In addition to the traditional PHA medical implants, the use of electrostatic spinning and three-dimensional printing can be designed to various functional PHA medical devices, in order to meet the orthopedic clinical demands, including the bone regeneration, minimally invasive bone tissue repair by injection, antibacterial bone repair, auxiliary establishment of three-dimensional bone tumor model, directed osteogenic differentiation of stem cells, etc. ConclusionAt present, PHA is a hotspot of biomaterials for translational medicine in orthopedics. Although they have not completely applied in the clinic, the advantages of repair in bone defects have been gradually reflected in tissue engineering, showing an application prospect in orthopedics.
Objective To review the latest progress of seeding cells for articular cartilage tissue engineering. Methods The recent original l iteratures on seeding cells for articular cartilage tissue engineering were extensively reviewed. Results The chondrocytes derived from BMSCs’ differentiation would be a main source of seeding cells articular cartilage for tissue engineering. Three-dimensional scaffolds and cultivation surroundings played important roles in the field of articular cartilage tissue engineering. Conclusion The util ization of cytokine and transgenic technology as well as improvements of three-dimensional scaffolds and cultivation surroundings will promote the development of articular cartilage tissue engineering.
Objective To construct the recombinant adeno-associated virus vector with human bone morphogenetic protein 4 gene（AAV-hBMP4）. Methods The hBMP-4 gene primer was designed basing on the corresponding gene sequence in GenBank. EcoR I site was introduced into the upstream of the primer and Sal Ⅰ site into downstream. The hBMP-4 gene was amplifiedwith the template of EX-A0242-M01-hBMP-4, then was cloned into pUC18 vectorto construct recombinant plasmid pUC18-hBMP-4. The plasmids pUC18-hBMP-4 and plasmid pSNAV cut by EcoR Ⅰ and Sal Ⅰenzyme, the fragments were collected and linked with T4 DNA ligase at 16℃ over night, recombinant plasmid pSNAVhBMP-4 was obtained. The recombinant plasmid was then transfected into BHK21 cells using Lipofectamine TM2000. The G418 resistant cells were obtained consequently. Thesecells were infected with HSV1-rc/△UL2 which has the function of packaging andcopying the recombinant AAV. After purification, the construction of recombinant AAV-hBMP-4 was completed. Results The construction of the recombinant pSNAV-hBMP-4 was confirmed by PCR electrophoresis and digestion with restriction enzyme. The gene sequence in the recombinant pSNAV-hBMP-4 wascorrect. The virus titer was about 1.5×1012 μg/ml.The purity of the virus was more than 95% using the SDSPAGE method. Conclusion With this method, high virus titers and purity of AAV-hBMP-4 can be acquired successfully and it is useful to bone tissue engineering.