Objective To summarize the influence of microstructure on performance of triply-periodic minimal surface (TPMS) bone scaffolds. Methods The relevant literature on the microstructure of TPMS bone scaffolds both domestically and internationally in recent years was widely reviewed, and the research progress in the imfluence of microstructure on the performance of bone scaffolds was summarized. Results The microstructure characteristics of TPMS bone scaffolds, such as pore shape, porosity, pore size, curvature, specific surface area, and tortuosity, exert a profound influence on bone scaffold performance. By finely adjusting the above parameters, it becomes feasible to substantially optimize the structural mechanical characteristics of the scaffold, thereby effectively preempting the occurrence of stress shielding phenomena. Concurrently, the manipulation of these parameters can also optimize the scaffold’s biological performance, facilitating cell adhesion, proliferation, and growth, while facilitating the ingrowth and permeation of bone tissue. Ultimately, the ideal bone fusion results will obtain. Conclusion The microstructure significantly and substantially influences the performance of TPMS bone scaffolds. By deeply exploring the characteristics of these microstructure effects on the performance of bone scaffolds, the design of bone scaffolds can be further optimized to better match specific implantation regions.
ObjectiveTo explore the effect of vascular endothelial growth factor 165 (VEGF165)-loaded porous poly (ε-caprolactone) (PCL) scaffolds on the osteogenic differentiation of adipose-derived stem cells (ADSCs).MethodsThe VEGF165-loaded porous PCL scaffolds (written, Sf-g/VEGF) were fabricated through a combination of solvent casting/salt leaching and a thermal-induced phase separation technique and then observed under scanning electron microscope (SEM). The release kinetics was determined by ELISA kit. The ADSCs were isolated from inguinal fat pads of 15 Sprague Dawley rats and cultured. The passage 3-4 ADSCs were seeded into the scaffolds, and then cultured in vitro for 7 days. The passage 3-4 ADSCs were seeded into the porous PCL scaffolds (written, Sf-g) as control. The alizarin red S (ARS) staining, ARS activity assay, and real-time quantitative PCR (RT-PCR) were performed to measure the osteogenic differentiation of ADSCs in vitro. Six Sprague Dawley rats were recruited to prepare the bilateral calvarial bone defects models (n=12). The 12 calvarial bone defects were randomly divided into 3 group (n=4). The defects of negative control group were not treated; the defects of Sf-g group and Sf-g/VEGF group were repaired with ADSCs-Sf-g scaffold complex and ADSCs-Sf-g scaffold complex, respectively. At 8 weeks after transplantation, the Micro-CT and HE staining were conducted to evaluate the osteogenic effects in vivo.ResultsThe morphology of the Sf-g/VEGF scaffolds were porous and well-connected, and the cumulative release rate was approximately 80% in 120 hours. The ARS staining showed that the ARS activity of Sf-g/VEGF group were stronger than that of Sf-g group (t=10.761, P=0.000). The mRNA expressions of osteogenic specific markers [special AT-rich sequence protein 2 (Satb2), alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN)] were significantly higher in Sf-g/VEGF group than in Sf-g group (P<0.05). The results of Micro-CT and HE staining also confirmed the promotion effect of Sf-g/VEGF scaffolds. All defects of 2 groups were partially repaired by new bone tissue, especially in Sf-g/VEGF group. The volume and area of new bone tissue were significantly higher in Sf-g/VEGF group than in Sf-g group (P<0.05).ConclusionThe VEGF165-loaded scaffolds can significantly improve the osteogenic differentiation of ADSCs both in vitro and in vivo.
The aim of this article is to study how andrographolide-releasing collagen scaffolds influence rabbit articular chondrocytes in maintaining their specific phenotype under inflammatory environment. Physical blending combined with vacuum freeze-drying method was utilized to prepare the andrographolide-releasing collagen scaffold. The characteristics of scaffold including its surface morphology and porosity were detected with environmental scanning electron microscope (ESEM) and a density instrument. Then, the release of andrographolide from prepared scaffolds was measured by UV-visible spectroscopy. Rabbit chondrocytes were isolated and cultured in vitro and seeded on andrographolide-releasing collagen scaffolds. Following culture with normal medium for 3 d, seeded chondrocytes were cultured with medium containing interleukin-1 beta (IL-1β) to stimulate inflammation in vitro for 7 d. The proliferation, morphology and gene transcription of tested chondrocytes were detected with Alamar Blue assay, fluorescein diacetate (FDA) staining and reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) test respectively. The results showed that the collagen scaffolds prepared by vacuum freeze-dry possess a high porosity close to 96%, and well-interconnected chambers around (120.7±17.8) μm. The andrographolide-releasing collagen scaffold continuously released andrographolide to the PBS solution within 15 d, and collagen scaffolds containing 2.22% andrographolide significantly inhibit the proliferation of chondrocytes. Compared with collagen scaffolds, 0.44% andrographolide-containing collagen scaffolds facilitate chondrocytes to keep specific normal morphologies following 7 d IL-1β induction. The results obtained by RT-qPCR confirmed this effect by enhancing the transcription of tissue inhibitor of metalloproteinase-1 (TIMP-1), collagen II (COL II), aggrecan (Aggrecan) and the ratio of COL II/ collagen I(COL I), meanwhile, reversing the promoted transcription of matrix metalloproteinase-1 (MMP-1) and matrix metalloproteinase-13 (MMP-13). In conclusion, our research reveals that andrographolide-releasing (0.44%) collagen scaffolds enhance the ability of chondrocytes to maintain their specific morphologies by up-regulating the transcription of genes like COL II, Aggrecan and TIMP-1, while down-regulating the transcription of genes like MMP-1 and MMP-13 which are bad for phenotypic maintenance under IL-1β simulated inflammatory environment. These results implied the potential use of andrographolide-releasing collagen scaffold in osteoarthritic cartilage repair.
ObjectiveTo prepare of a novel functional self-assembling peptide nanofiber hydrogel scaffold RADKPS designed with linking the short functional motif of bone morphogenetic protein 7 (BMP-7) and to evaluate its biocompatibility so as to provide the experimental basis for in vivo studies on regeneration of degenerated nucleus pulposus tissue. MethodA functional self-assembling peptide RADA-KPSS was designed by linking the short functional motif of BMP-7 to the self-assembling peptide RADA16-I. And the novel functional self-assembling peptide RADKPS was finally prepared by isometric mixing RADA16-I with RADA-KPSS. The structure characteristic of the functional self-assembling peptide nanofiber hydrogel scaffold RADKPS was evaluated by general observation and atomic force microscopy. Bone marrow mesenchymal stem cells (BMSCs) were isolated from 3-month-old New Zealand white rabbits and cultured. After the 3rd generation BMSCs were seeded on the peptide nanofiber hydrogel scaffold RADKPS for 7 days, the cellular compatibility of RADKPS was evaluated through scanning electron microscopy assay, cellular fluorescein diacetate/propidium iodide staining, and MTT assay. 1%RADKPS was injected into isolated intervertebral disc organs from 6-month-old New Zealand white rabbits, then the organs were cultured and the cellular activity of the intervertebral disc organs was observed. The blood compatibility of RADKPS was evaluated with hemolytic assay. After RADKPS was implanted into subcutaneous part of Kunming mice (aged 6-8 weeks) for 28 days, general observation and HE staining were carried out to evaluate the tissue compatibility. ResultsThe functional self-assembling peptide solution RADKPS presented a homogeneous transparent hydrogel-like. Atomic force microscopy revealed that the RADKPS could self-assemble into three-dimensional nanofiber hydrogel scaffolds; the fibre diameter was (25.68±4.62) nm, and the fibre length was (512.42±32.22) nm. After BMSCs cultured on RADKPS for 7 days, scanning electron microscopy showed that BMSCs adhered to the scaffolds. And cell viability was maintained over 90%. MTT assay revealed that RADKPS of 0.1%, 0.05%, and 0.025% could increase the proliferation of BMSCs. The result of hemolytic assay revealed that the hemolysis rates of the RADKPS solutions with different concentrations were less than 5%, indicating that it met the requirement of hemolytic assay standard for medical biomaterials. After subcutaneous implantation, no vesicle, erythema, and eschar formation around injection site were observed. Meanwhile, HE staining showed inflammatory cells infiltration (lymphocytes), substitution of hydrogel scaffold by fibrous tissue, and good tissue compatibility. ConclusionsThe novel functional self-assembling peptide nanofiber hydrogel scaffold RADKPS has good biocompatibility and biological reliability, which would be suitable for tissue engineering repair and regeneration of nucleus pulposus tissue.
Objective To review new progress of related research of peri pheral nerve defect treatment with tissue engineering in recent years. Methods Domestic and internationl l iterature concerning peri pheral nerve defect treatment with tissue engineering was reviewed and analyzed. Results Releasing neurotrophic factors with sustained release technology included molecular biology techniques, poly (lactic-co-glycol ic acid) microspheres, and polyphosphate microspheres. The mixture of neurotrophic factors and ductus was implanted to the neural tube wall which could be degraded then releasing factors slowly. Seed cells which were the major source of active ingredients played an important role in the repair and reconstruction of tissue engineering products. The neural tube of Schwann cells made long nerve repair and the quality of nerve regeneration was improved. Nerve scaffold materials included natural and synthetic biodegradable materials. Tube structure usually was adopted for nerve scaffold, which performance would affect the nerve repair effects directly. Conclusion With the further research of tissue engineering, the treatment of peripheral nerve defects with tissue engineering has made significant progress.
Objective To fabricate a novel composite scaffold with acellular demineralized bone matrix/acellular nucleus pulposus matrix and to verify the feasibility of using it as a scaffold for intervertebral disc tissue engineering through detecting physical and chemical properties. Methods Pig proximal femoral cancellous bone rings (10 mm in external diameter, 5 mm in internal diameter, and 3 mm in thickness) were fabricated, and were dealed with degreasing, decalcification, and decellularization to prepare the annulus fibrosus phase of scaffold. Nucleus pulposus was taken from pig tails, decellularized with Triton X-100 and deoxycholic acid, crushed and centrifugalized to prepare nucleus pulposus extracellular mtrtix which was injected into the center of annulus fibrosus phase. Then the composite scaffold was freeze-dryed, cross-linked with ultraviolet radiation/carbodiimide and disinfected for use. The scaffold was investigated by general observation, HE staining, and scanning electron microscopy, as well as porosity measurement, water absorption rate, and compressive elastic modulus. Adipose-derived stem cells (ADSCs) were cultured with different concentrations of scaffold extract (25%, 50%, and 100%) to assess cytotoxicity of the scaffold. The cell viability of ADSCs seeded on the scaffold was detected by Live/Dead staining. Results The scaffold was white by general observation. The HE staining revealed that there was no cell fragments on the scaffold, and the dye homogeneously distributed. The scanning electron microscopy showed that the pore of the annulus fibrosus phase interconnected and the pore size was uniform; acellular nucleus pulposus matrix microfilament interconnected forming a uniform network structure, and the junction of the scaffold was closely connected. The novel porous scaffold had a good pore interconnectivity with (343.00 ± 88.25) μm pore diameter of the annulus fibrosus phase, 82.98% ± 7.02% porosity and 621.53% ± 53.31% water absorption rate. The biomechanical test showed that the compressive modulus of elasticity was (89.07 ± 8.73) kPa. The MTT test indicated that scaffold extract had no influence on cell proliferation. Live/Dead staining showed that ADSCs had a good proliferation on the scaffold and there was no dead cell. Conclusion Novel composite scaffold made of acellular demineralized bone matrix/acellular nucleus pulposus matrix has good pore diameter and porosity, biomechanical properties close to natural intervertebral disc, non-toxicity, and good biocompatibility, so it is a suitable scaffold for intervertebral disc tissue engineering.
Objective To investigate the construction of a novel tissue engineered meniscus scaffold based on low temperature deposition three-dimenisonal (3D) printing technology and evaluate its biocompatibility. Methods The fresh pig meniscus was decellularized by improved physicochemical method to obtain decellularized meniscus matrix homogenate. Gross observation, HE staining, and DAPI staining were used to observe the decellularization effect. Toluidine blue staining, safranin O staining, and sirius red staining were used to evaluate the retention of mucopolysaccharide and collagen. Then, the decellularized meniscus matrix bioink was prepared, and the new tissue engineered meniscus scaffold was prepared by low temperature deposition 3D printing technology. Scanning electron microscopy was used to observe the microstructure. After co-culture with adipose-derived stem cells, the cell compatibility of the scaffolds was observed by cell counting kit 8 (CCK-8), and the cell activity and morphology were observed by dead/live cell staining and cytoskeleton staining. The inflammatory cell infiltration and degradation of the scaffolds were evaluated by subcutaneous experiment in rats. Results The decellularized meniscus matrix homogenate appeared as a transparent gel. DAPI and histological staining showed that the immunogenic nucleic acids were effectively removed and the active components of mucopolysaccharide and collagen were remained. The new tissue engineered meniscus scaffolds was constructed by low temperature deposition 3D printing technology and it had macroporous-microporous microstructures under scanning electron microscopy. CCK-8 test showed that the scaffolds had good cell compatibility. Dead/live cell staining showed that the scaffold could effectively maintain cell viability (>90%). Cytoskeleton staining showed that the scaffolds were benefit for cell adhesion and spreading. After 1 week of subcutaneous implantation of the scaffolds in rats, there was a mild inflammatory response, but no significant inflammatory response was observed after 3 weeks, and the scaffolds gradually degraded. Conclusion The novel tissue engineered meniscus scaffold constructed by low temperature deposition 3D printing technology has a graded macroporous-microporous microstructure and good cytocompatibility, which is conducive to cell adhesion and growth, laying the foundation for the in vivo research of tissue engineered meniscus scaffolds in the next step.
ObjectiveTo improve the poor mechanical strength of porous ceramic scaffold, an integrated method based on three-dimensional (3-D) printing technique is developed to incorporate the controlled double-channel porous structure into the polylactic acid/β-tricalcium phosphate (PLA/β-TCP) reinforced composite scaffolds (double-channel composite scaffold) to improve their tissue regeneration capability and the mechanical properties. MethodsThe designed double-channel structure inside the ceramic scaffold consisted of both primary and secondary micropipes, which parallel but un-connected. The set of primary channels was used for cell ingrowth, while the set of secondary channels was used for the PLA perfusion. Integration technology of 3-D printing technique and gel-casting was firstly used to fabricate the double-channel ceramic scaffolds. PLA/β-TCP composite scaffolds were obtained by the polymer gravity perfusion process to pour PLA solution into the double-channel ceramic scaffolds through the secondary channel set. Microscope, porosity, and mechanical experiments for the standard samples were used to evaluate the composite properties. The ceramic scaffold with only the primary channel (single-channel scaffold) was also prepared as a control. ResultsMorphology observation results showed that there was no PLA inside the primary channels of the double-channel composite scaffolds but a dense interface layer between PLA and β-TCP obviously formed on the inner wall of the secondary channels by the PLA penetration during the perfusion process. Finite element simulation found that the compressive strength of the double-channel composite scaffold was less than that of the single-channel scaffold; however, mechanical tests found that the maximum compressive strength of the double-channel composite scaffold[(21.25±1.15) MPa] was higher than that of the single-channel scaffold[(9.76±0.64) MPa]. ConclusionThe double-channel composite scaffolds fabricated by 3-D printing technique have controlled complex micropipes and can significantly enhance mechanical properties, which is a promising strategy to solve the contradiction of strength and high-porosity of the ceramic scaffolds for the bone tissue engineering application.
Objective To review the decellularized methods for obtaining extracellular matrix (ECM) and the applications of decellularized ECM scaffold in tissue engineering. Methods Recent and related literature was extensively and comprehensively reviewed. The decellularized methods were summarized and classified. The effects of different sterilization methods on decellularized scaffolds were analyzed; the evaluation criterion of extent of decellularization was put forward; and the application of decellularized ECM scaffold in different tissues and organs engineering field was summarized. Results The decellularized methods mainly include physical methods, chemical methods, and biological methods, and different decellularization methods have different effects on the extent of cell removal and ECM composition and structure. Therefore, the best decellularization method will be chosen according to the characteristics of the tissues and decellularization methods to achieve the ideal result. Conclusion It is very important to choose the appropriate decellularized method for preparing the biological materials desired by tissue engineering. The biological scaffolds prepared by decellularized methods will play an important role in tissue engineering and regenerative medicine.
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.