Objective To study the feasibil ity of preparation of the poly-D, L-lactide acid (PDLLA) scaffolds treated by ammonia plasma and subsequent conjugation of Gly-Arg-Gly-Asp-Ser (GRGDS) peptides via amide l inkage formation. Methods PDLLA scaffolds (8 mm diameter, 1 mm thickness) were prepared by solvent casting/particulate leaching procedure and then treated by ammonia plasma. The consequent scaffolds were labeled as aminated PDLLA (A/ PDLLA). The pore size, porosity, and surface water contact angle of groups 0 (un-treated control), 5, 10, and 20 minutes A/ PDLLA were measured. A/PDLLA scaffolds in groups above were immersed into the FITC labelled GRGDS aqueous solutionwhich contain 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide hydrochloride (EDC.HCl) and N-hydroxysuccinimide(NHS), the molar ratio of peptides/EDC.HCL /NHS was 1.5 ∶ 1.5 ∶ 1.0, then brachytely sloshed for 24 hours in roomtemperature. The consequent scaffolds were labelled as peptides conjugated A/PDLLA (PA/PDLLA). The scaffolds in groups 0, 5, 10, and 20 minutes A/PDLLA and groups correspondingly conjugation of peptides were detected using X-ray photoelectron spectroscopy (XPS). The scaffolds in groups of conjugation of peptides were measured by confocal laser scanning microscope and high performance l iquid chromatography (HPLC), un-treated and un-conjugated scaffolds employed as control. Bone marrow mesenchymal stem cells (BMSCs) from SD rats were isolated and cultured by whole bone marrow adherent culture method. BMSCs at the 3rd–6th passages were seeded to the scaffolds as follows: 20 minutes ammonia plasma treatment (group A/PDLLA), 20 minutes ammonia plasma treatment and conjugation of GRGDS (group PA/PDLLA), and untreated PDLLA control (group PDLLA). After 16 hours of culture, the adhesive cells on scaffolds and the adhesive rate were calculated. After 4 and 8 days of culture, the BMSCs/scaffold composites was observed by scanning electron micorscope (SEM). Results No significant difference in pore size and porosity of PDLLA were observed between before and after ammonia plasma treatments (P gt; 0.05). With increased time of ammonia plasma treatment, the water contact angle of A/PDLLA scaffolds surface was decreased, and the hydrophil icity in the treated scaffolds was improved gradually, showing significant differences when these groups were compared with each other (P lt; 0.001). XPS results indicated that element nitrogen appeared on the surface of PDLLA treated by ammonia plasma. With time passing, the peak N1s became more visible, and the ratio of N/C increased more obviously. AfterPDLLA scaffolds treated for 0, 5, 10, and 20 minutes with ammonia plasma and subsequent conjugation of peptides, the ratio of N/C increased and the peak of S2p appeared on the surface. The confocal laser scanning microscope observation showed that the fluorescence intensity of PA/PDLLA scaffolds increased obviously with treatment time. The amount of peptides conjugated for 10 minutes and 20 minutes PA/PDLLA was detected by HPLC successfully, showing significant differences between 10 minutes and 20 minutes groups (P lt; 0.001). However, the amount of peptides conjugated in un-treated control and 0, 5 minutes PA/PDLLA scaffolds was too small to detect. After 16 hours co-culture of BMSCs/scaffolds, the adhesive cells and the adhesive rates of A/PDLLA and PA/PDLLA scaffolds were higher than those of PDLLA scaffolds, showing significant difference between every 2 groups (P lt; 0.01). Also, SEM observation confirmed that BMSCs proliferation in A/PDLLA and PA/PDLLA groups was more detectable than that in PDLLA group, especially in PA/PDLLA group. Conclusion Ammonia plasma treatment will significantly increase the amount of FITC-GRGDS peptides conjugated to surface of PDLLA via amide l inkage formation. This new type of biomimetic bone has stablized bioactivities and has proved to promote the adhesion and proliferation of BMSCs in PDLLA.
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.
ObjectiveTo investigate the effect of repairing radial bone defect with scaffold material of attapulgite/collagen type I/poly (caprolactone) (ATP/Col I/PCL) in rabbits and the possibility as bone graft substitutes. MethodsATP/Col I/PCL materials were prepared via adding ATP to hexafluoroisopropanol after dissolved Col I/PCL (3∶2), and Col I/PCL materials via dissolving Col I/PCL (3∶2) in hexafluoroisopropanol served as control. The structure of scaffolds was observed under scanning electron microscope (SEM). Twenty-four Japanese white rabbits (male, 2 months old) were used to establish the bilateral radius defect model of 15 mm in length, and randomly divided into group A (6 rabbits, 12 defects), group B (9 rabbits, 18 defects), and group C (9 rabbits, 18 defects); then the Col I/PCL scaffold was implanted in the bone defect area in group B, the ATP/Col I/PCL scaffold in group C, no treatment was done in group A as control. The general condition of rabbits was observed after operation, and bone defect repair was evaluated by X-ray at 4, 8, and 12 weeks. At 12 weeks, the tissue of defect area was harvested for the general, SEM, Micro-CT, histological, and immunohistochemical staining to observe defect repair and material degradation. ResultsSEM observation showed that two kinds of materials were porous structure, ATP/Col I/PCL structure was more dense than Col I/PCL. All animals survived to the end of experiment, and no incision infection occurred during repair process.X-ray films showed that the bone marrow cavity was re-opened in defect area of group C with time, the repair effect was superior to that of groups A and B. At 12 weeks after operation, general observation showed that scaffold material had good fusion with the surrounding tissue in groups B and C, defect was filled with connective tissue in group A. SEM indicated that the surface and pore of the scaffold were covered with a large number of cells and tissues in groups B and C. Micro-CT demonstrated that the new bone volume, bone mineral content, tissue mineral content, and connectivity density of group C were significantly higher than those of groups A and B (P<0.05). The observation of histology and immunohistochemical staining indicated that there were lots of connective tissues in defect area of group A, and ALP, Col I, and OPN were weakly expressed; there were many collagen fibers in scaffold degradation area in group B, and the expression levels of ALP, Col I, and OPN were higher than those of group A; there was few new bone in group C, the degradation rate of the scaffold was slower than that of group B, and the expression of Col I and OPN were enhanced, while ALP was weakened when compared with groups A and B. ConclusionATP/Col I/PCL composite scaffold material can degrade in vivo, and has dense three-dimensional porous structure, good biocompatibility, and high potentiality of bone repair, so it can be used as bone substitute material.
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.
Objective To review the current status and problems in the developing scaffolds for the myocardial tissue engineering appl ication. Methods The l iterature concerning the myocardial tissue engineering scaffold in recent years was reviewed extensively and summarized. Results As one of three elements for tissue engineering, a proper scafold is veryimportant for the prol iferation and differentiation of the seeding cells. The naturally derived and synthetic extracellular matrix (ECM) materials aim to closely resemble the in vivo microenvironment by acting as an active component of the developing tissue construct in myocardial tissue engineering. With the advent and continuous refinement of cell removal techniques, a new class of native ECM has emerged with some striking advantages. Conclusion Through using the principle of composite scaffold, computers and other high-technology nano-polymer technology, surface modification of traditional biological materials in myocardial tissue engineering are expected to provide ideal myocardial scaffolds.
ObjectiveTo summarize the research progress of tissue engineered bile duct in recent years.MethodsThis paper summarized recently-published papers related to tissue-engineered bile duct on in vitro test platform, scaffold materials, acquisition methods of seed cells, and in vivo repair effectiveness after the fusion of seed cells and materials, in an attempt to review the basic and clinical application studies of tissue-engineered bile duct.ResultsTissue-engineered bile duct had been developing rapidly. At present, great progress had been made in the fields of in vitro test platform, scaffold materials, seed cells, and repair effectiveness in animal models. However, further study was still needed in terms of its clinical application. The external bile duct platform included 3D printing and biological simulation; in the aspect of scaffold material, apart from the progress of various artificial materials, acellular matrix was introduced; the selection of seed cells included the induction and differentiation of bile duct-derived stem cells, human bone marrow mesenchymal stem cells (hMSCs), hepatic oval cell (HOC), pluripotent stem cells (PSCs), and other stem cells; animal models of tissue-engineered bile ducts had also achieved good results in animals such as pigs and dogs.ConclusionThe development of tissue-engineered bile duct will promote the progress of fundamental in vitro studies on extrahepatic biliary tract diseases, thus introducing new options to the clinical treatment of extrahepatic biliary tract injuries.
ObjectiveTo analyze the progress in biological tissue engineering scaffold materials and the clinical application, as well as product development status. MethodsBased on extensive investigation in the status of research and application of biological tissue engineering scaffold materials, a comprehensive analysis was made. Meanwhile, a detailed analysis of research and product development was presented. ResultsConsiderable progress has been achieved in research, products transformation, clinical application, and supervision of biological scaffold for tissue engineering. New directions, new technology, and new products are constantly emerging. With the continuous progress of science and technology and continuous improvement of life sciences theory, the new direction and new focus still need to be continuously adjusted in order to meet the clinical needs. ConclusionFrom the aspect of industrial transformation feasibility, acellular scaffolds and extracellular matrix are the most promising new growth of both research and product development in this field.
Objective To summarize and analyze the research progress of scaffold materials used in tissue engineered meniscus. Methods The classification and bionics design of scaffold materials were summarized by consulting domestic and foreign literature related to the research of tissue engineered meniscus in recent years. Results Tissue engineered meniscus scaffolds can be roughly classified into synthetic polymers, hydrogels, extracellular matrix components, and tissue derived materials. These different materials have different characteristics, so the use of a single material has its unique disadvantages, and the use of a variety of materials composite scaffolds can learn from each other, which is a hot research area at present. In addition to material selection, material processing methods are also the focus of research. At the same time, according to the morphological structure and mechanical characteristics of the meniscus, the bionic design of tissue engineered meniscus scaffolds has great potential. Conclusion At present, there are many kinds of scaffold materials for tissue engineered meniscus. However, there is no material that can completely simulate the natural meniscus, and further research of scaffold materials is still needed.
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.
ObjectiveTo review the methods of improving the mechanical properties of hydrogels and the research progress in bone tissue engineering. MethodsThe recent domestic and foreign literature on hydrogels in bone tissue engineering was reviewed, and the methods of improving the mechanical properties of hydrogels and the effect of bone repair in vivo and in vitro were summarized. ResultsHydrogels are widely used in bone tissue engineering, but their mechanical properties are poor. Improving the mechanical properties of hydrogels can enhance bone repair. The methods of improving the mechanical properties of hydrogels include the construction of dual network structures, inorganic nanoparticle composites, introduction of conductive materials, and fiber network reinforcement. These methods can improve the mechanical properties of hydrogels to various degrees while also demonstrating a significant bone repair impact. ConclusionThe mechanical properties of hydrogels can be effectively improved by modifying the system, components, and fiber structure, and bone repair can be effectively promoted.