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.
Objective To investigate the biocompatibility of type I collagen scaffold with rat bone marrow mesenchymal stem cell (BMSCs) and its role on proliferation and differentiation of BMSCs so as to explore the feasibility of collagen scaffold as neural tissue engineering scaffold. Methods Type I collagen was used fabricate collagen scaffold. BMSCs were isolated by density gradient centrifugation. The 5th passage cells were used to prepare the collagen scaffold-BMSCs complex. The morphology of collagen scaffold and BMSCs was observed by scanning electron microscope (SEM) and HE staining. The cell proliferation was measured by MTT assay at 1, 3, 5, and 7 days after culturein vitro. After cultured on collagen scaffold for 24 hours, the growth and adhesion of green fluorescent protein positive (GFP+) BMSCs were observed by confocal microscopy and live cell imaging. Results The confocal microscopy and live cell imaging results showed that GFP+ BMSCs uniformly distributed in the collagen scaffold; cells were fusiform shaped, and cell process or junctions between the cells formed in some cells, indicating good cell growth in the collagen scaffold. Collagen scoffold had porous fiber structure under SEM; BMSCs could adhered to the scaffold, with good cell morphology. The absorbance (A) value of BMSCs on collagen scaffold at 5 and 7 days after culture was significantly higher than that of purely-cultured BMSCs (t=4.472,P=0.011;t=4.819,P=0.009). HE staining showed that collagen scaffold presented a homogeneous, light-pink filament like structure under light microscope. BMSCs on the collagen scaffold distributed uniformly at 24 hours; cell displayed various forms, and some cells extended multiple processes at 7 days, showing neuron-like cell morphology. Conclusion Gelatinous collagen scaffold is easy to prepare and has superior biocompatibility. It is a promising scaffold for neural tissue engineering.
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.
The establishing of myocardial tissue engineering techniques not only solve a series of issues that generate in cell and tissue transplantation after myocardial infarction, but also create a platform for selecting better materials and transplantation techniques. However, both experimental animal studies and recent clinical trials indicate that current transplantation techniques still have many defects, mainly including lack of suitable seed cells, low survival rate and low differentiation rate after transplantation. In this context, extracellular matrix （ECM）, as myocardial tissue engineering scaffold materials, has gained increasing attention and become a frontier and focus of medical research in recent years. ECM is no longer merely regarded as a scaffold or a tissue, but plays an important role in providing essential signals to influence major intracellular pathways such as cell proliferation, differentiation and metabolism. The involved models of ECM can be classified into following types：natural biological scaffold materials, synthetic polymer scaffold materials and composite scaffold materials with more balanced physical and biological properties. This review mainly introduces research progress of ECM in myocardial tissue engineering and ECM materials.
Objective To construct polyhydroxyalkanoate (PHA) microspheres loaded with bone morphogenetic protein 2 (BMP-2) and human β-defensin 3 (HBD3), and evaluate the antibacterial activity of microspheres and the effect of promoting osteogenic differentiation, aiming to provide a new option of material for bone tissue engineering. Methods The soybean lecithin (SL)-BMP-2 and SL-HBD3 were prepared by SL-mediated introduction of growth factors into polyesters technology, and the functional microsphere (f-PMS) containing BMP-2 and HBD3 were prepared by microfluidic technology, while pure microsphere (p-PMS) was prepared by the same method as the control. The morphology of microspheres was observed by scanning electron microscopy and the water absorption was detected; the release curves of BMP-2 and HBD3 in f-PMS were detected by ELISA kit. The antibacterial effect of microspheres in Staphylococcus aureus and Escherichia coli was tested with the LIVE/DEADTM BacLightTM bacterial staining kit; the biocompatibility of microspheres was tested using Transwell and cell counting kit 8 (CCK-8). The effect of microspheres on osteogenic differentiation was determined by collagen type Ⅰ (COL-1) immunofluorescence staining and alkaline phosphatase (ALP) concentration. Results In this experiment, the f-PMS and p-PMS were successfully constructed. Morphological characteristics showed that p-PMS surface was rough and distributed with micropores of 1-3 μm, while f-PMS surface was smooth and existed white granular material. There was no significant difference in water absorption between the two groups (P>0.05). The release curves of BMP-2 and HBD3 in the f-PMS and p-PMS were basically the same, showing both early sudden release and late slow release. The antibacterial activity of f-PMS was significantly higher than that of p-PMS in the test that against Staphylococcus aureus and Escherichia coli (P<0.05), but there was no significant difference in biocompatibility between the two groups (P>0.05). The results of osteogenic differentiation of human BMSCs showed that the fluorescence intensity of osteogenic specific protein COL-1 of f-PMS was significantly higher than that in p-PMS, and the activity of ALP in f-PMS was also significantly higher than that in p-PMS (P<0.05). Conclusion The p-PHA have good antibacterial activity and biocompatibility, and can effectively promote the osteogenic differentiation of human BMSCs, which is expected to be applied to bone tissue engineering in the future.
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 review the literature on the research status of vascularization of tissue engineered peripheral nerve so as to provide the theoretical basis for the vascularization of tissue engineered peripheral nerve.MethodsThe literature related to the vascularization of peripheral nerve tissue engineering in recent years was reviewed and summarized according to the five aspects of promoting vascularization: local microenvironment and blood supply characteristics of peripheral nerve regeneration, scaffold material modification, seed cells, autologous vascular bundle implantation, and pro-vascular factors.ResultsTissue engineered peripheral nerve has brought a new hope for the repair of peripheral nerve injury, but the repair effect of large nerve defects is not good, which is mainly related to the degree of vascularization of the nerve grafts. So it is particularly important to promote the early vascularization of tissue engineered peripheral nerve. Previous studies have mainly focused on the four aspects of scaffold material modification, seed cells, autologous vascular bundle implantation, and angiogenesis related factors. Recent studies show that the combination of the above two or more factors in the tissue engineered peripheral nerves can better promote the vascularization of tissue engineered peripheral nerves.ConclusionPromoting early vascularization of tissue engineered peripheral nerves can provide timely nutritional support for seed cells on the scaffold, promote axon growth and nerve regeneration, and facilitate the repair of large peripheral nerve defects in clinical practice.
ObjectiveTo observe the feasibility of acellular cartilage extracellular matrix (ACECM) oriented scaffold combined with chondrocytes to construct tissue engineered cartilage.MethodsChondrocytes from the healthy articular cartilage tissue of pig were isolated, cultured, and passaged. The 3rd passage chondrocytes were labeled by PKH26. After MTT demonstrated that PKH26 had no influence on the biological activity of chondrocytes, labeled and unlabeled chondrocytes were seeded on ACECM oriented scaffold and cultivated. The adhesion, growth, and distribution were evaluated by gross observation, inverted microscope, and fluorescence microscope. Scanning electron microscope was used to observe the cellular morphology after cultivation for 3 days. Type Ⅱ collagen immunofluorescent staining was used to check the secretion of extracellular matrix. In addition, the complex of labeled chondrocytes and ACECM oriented scaffold (cell-scaffold complex) was transplanted into the subcutaneous tissue of nude mouse. After transplantation, general physical conditions of nude mouse were observed, and the growth of cell-scaffold complex was observed by molecular fluorescent living imaging system. After 4 weeks, the neotissue was harvested to analyze the properties of articular cartilage tissue by gross morphology and histological staining (Safranin O staining, toluidine blue staining, and typeⅡcollagen immunohistochemical staining).ResultsAfter chondrocytes that were mainly polygon and cobblestone like shape were seeded and cultured on ACECM oriented scaffold for 7 days, the neotissue was translucency and tenacious and cells grew along the oriented scaffold well by inverted microscope and fluorescence microscope. In the subcutaneous microenvironment, the cell-scaffold complex was cartilage-like tissue and abundant cartilage extracellular matrix (typeⅡcollagen) was observed by histological staining and typeⅡcollagen immunohistochemical staining.ConclusionACECM oriented scaffold is benefit to the cell adhesion, proliferation, and oriented growth and successfully constructes the tissue engineered cartilage in nude mouse model, which demonstrates that the ACECM oriented scaffold is promise to be applied in cartilage tissue engineering.
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.
