| Background:Bone and cartilage defects, injuries or other types of damage is one of the most devastating and costly problems in human health care. Some surgical strategies have been developed to address these problems, including autografts, allografts, and xenografts have been used to restore damaged bone or cartilage. However, each type of surgical strategies comes with its own set of limitations. To fulfill various clinical applications in bone healing and cartilage reconstruction, many engineers, doctors and biomaterial specialists have attempted for decades to reproduce in the laboratory, hoping to achieve a strategies capable of truly mimicking real bone healing or cartilage reconstruction.Our research mainly about the bone and cartilage tissue engineering, for the different tissue with the different strategies of problem solving.Conventional TE approaches are based on combining stem cells with a synthetic or biological scaffold which provides a specific location to culture a substitute graft in vitro suited for promoting the repair and regeneration of the targeted tissue. Major components of such approaches include an ECM-mimicking scaffold, locally releasable growth factors. One limit of utilizing these methods is the opportunity to create constructs of clinically relevant vascularization. Although extremely limited by the lack of complex hierarchical structure in engineered constructs which need big sizes and special shapes. Therefore, tissue engineers and material scientists need create new technologies inspiration from new discoveries and understandings for promising outcomes.To meet these problem, bottom-up or modular assembly tissue engineering have emerged as means to engineer controlled architectures precisely. These approaches use various microfabrication to drive the aggregation of microscale building blocks to generate complex architectures. Modular method allows for the formation of complex 3D constructs by hierarchical assemblies of multiple cell and building blocks types. Major components of such assembly system were multiscale (from nanometer-scale to macro-scale) hydrogels which with specific shape and geometry and mixed with cells. Currently electrospinning is one of major ways to fabricate nanofibrous scaffolds for tissue engineering. And also microscale nanofibrous scaffolds recently showed great promise in bottom-up tissue engineering as building blocks.In the first port of this paper, three kind of modified microscale nanofibrous scaffolds that were assembled into in vascularized bone as the repeating units by magnetic assembly.In order to truly mimicking hierarchical structures of bone, we build three kind of modules, the first one is for osteogenesis, and second one is for endothelialization, third one is for magnetic assembly. The PCL/gelatin nanofiber add nHA were preparated for osteogensis. The alined PCL/gelatin with lots platelets adhered after coating dopamine were applied to mimic the structure of endothelial cell ECM. These physico-chemical properties can promote the differentiation of ADSCs into endothelial cells. We have fabricated magnetic nanoparticle (MNP) loaded microscale nanofibrous scaffolds, tight stacked layers can formed very quickly after magent were applied. Finally, these three kind modules were well-organized assembled to enhance vascularization in bone tissue engineering and generate hierarchical structures.Hydrogel scaffold plays a pivotal role in the cartilage regeneration. Recently, new non-invasive way to deliver cells and bioactive molecules to the defect become preferable approach. Injectable hydrogels are good candidates that would be able to deal with the irregularly shaped defect. They can transfer the required agents to the specific location while minimizing the risk of open surgery. Hydrogels can support the cells and maintain their original round shape, and their porous structure provides them the ability to transport nutrient and waste. So here we report a new injectable hydrogel based on amino-diethoxypropane modified alginate and chitosan.Concrete research content including following several parts:1. Osteogenesis moduls Purposes:The second level of bone hierarchical structures is formed by the mineralization of collagen fibrils. Here we add inorganic hydroxyapatite nanoplatelets into the organic PCL/gelatin nanofiber to mimicking the collagen fibrils, and get the woven arrangements by electrospining. Materials and methods:Preparation of polymer solution, use the 10% PCL in TEF,10% gelatin in TFE, mixed with 1:1 ratio, magnetic stirring to make them blending. Preparation 2% F-127 to disperse nHA in the solution, after added into the F-127 ultrasonic dispersion were utilized for 30min, after added to the polymer solution for ultrasonic dispersion were utilized for 2h. Ajust ratio of nHA to polymer to be 10wt%,20wt% and 40wt%, get three gradients. Add the mixture into the syringe, install the syringe to a pump, the spinning speed was lml/h, the voltage was 15kv and the receive distance was 15cm. After 1h, stop spinning and peel off the nanofiber mesh. Put the material in the glutaraldehyde vapor to crosslink 1h, air dry, irradiation sterilization brefore cell culture.CharacterizationSEM, TEM, XRD, TGA test were did to explore the physical properties of nanofiber. Biocompatibility test and osteogenesis testLive/dead assasy were did after 24h,3-day culture, Alizarin Red staining, immunocytochemical analysis of OCN and RUNX2 were did after a 14-day culture under a osteogenic medium, as the control was the 3-day cultured.Result:According the SEM image, the PCL/gelatin composite fibers have a diameter of ca. 500 nm, however, the diameter of nHA/PCL/gelatin nanofiber decreased to ca.200 nm. The nHA/PCL/gelatin nanofiber membrane showed a uniform structure with interconnected macro-scale pores. The microstructures of both PCL/gelatin nanofibers and different nHA concentration nHA/PCL/gelatin nanofibers were further investigated by TEM. Clearly, nHA nanoparticles were encapsulated in the polymer matrix, little particles were sintered together in 10wt%,20wt% degree, but in 40wt% the morphology of the nanofiber were not good as the low degree one. The crystalline structures of the samples were investigated by XRD, showed peaks which are characteristic of nHA, the nHA/PCL/gelatin nanofibers keep the original crystalline structure of nHA nanoparticles. TGA analysis scaffolds showed the real nHA ration in the nanofiber were cosle to the desigen. Based on these characterization we have chosen the 20wt% nHA/PCL/gelatin nanofibers as the scaffold to do the biocompatibility test and osteogenesis test.The ADSCs used were cultured on nanofibrous scaffolds composed of PCL/gelatin composites with a nHA content of 20wt% to assess the basic cytocompatibility of these nanofibous scaffolds and to evaluate their potential for use in bone tissue engineering applications. LIVE/DEAD viability/cytotoxicity kit (Invitrogen) were used for the qualitative viability analyses of ADSCs on nanofibrous scaffolds. Live cells (stained green) cultured 3-day on the nanofibrous scaffolds were obviously more numerous than those 24h cultured. The SEM image showed a spindle-like morphology of the cell and many cell-matrix interactions, after 14-day in osteogenic medium the mineralized nodules were seen. Alizarin Red staining of the ADSCs showed more mineralized modules were tested after 14-day culture in osteogenic medium than 3-day culture. Further immunocytochemical analysis indicates that cells culture 14-day on the nanofibrous scaffolds are characterized by an increase in the RuNX2 and an enhanced production of osteo-related proteins, like osteocalcin (OCN) compare the 3day. The results indicate that nanofibrous scaffolds are cytocompatible and strongly interact with the cells, suitable for osteogenic differentiation of ADSCs.Conclusion:This strategy develops a smart nanofibous matrix composed of two key components nanofiber and nHA, suitable for activate osteoblastic differentiation of ADSCs, can be used as osteogenesis moduls for the formation of vascularized bone.2. Angiogenesis modulsPurposes:Engineering of angiogenesis moduls by highly aligned nanofibers and platelets to induce endothelial differentiation of ADSCs. Materials and methods:Preparation of polymer solution, use the 10% PCL in TEF,10% gelatin in TFE, mixed with 1:1 ratio, magnetic stirring to make them blending. Add the mixture into the syringe, install the syringe to a pump, the spinning speed was lml/h, the voltage was 15kv, a drum rotating at a high speed was used as a collector, and the receive distance was 15cm. After 30min, stop spinning and peel off the nanofiber mesh. Put the material in the glutaraldehyde vapor to crosslink 1h, air dry, irradiation sterilization brefore cell culture. To achieve a polydopamine film on substrates, the nanofiber mesh were placed in a pillbox containing 20mL of dopamine (2 mg/mL) solution in a 50 mM Trizma buffer adjusted to pH 8.5. The reaction was performed at room temperature for 16h. The PDA treated nanofiber mesh were rinsed with deionized water, dried at RT. Platelets were prepared from the blood of healthy rat by centrifugation at 150 g for 15 min, then the supernatant was centrifuged at 500 g for 10 min, and the pellet was suspended in DMEM. The platelets were washed with DMEM and then resuspended in DMEM. The nanofiber mesh were placed in a cell culture dish containing 5mL platelets solution, and keeped in 37℃ for 2h, then washed with DMEM. Characterization:SEM of the nanofiber mesh were did to character alignment of the nanofibers, effects of the PDA coatings, and quantitative analysis of the number of adherent platelets on the various surfaces. The surface wettability of the PDA coated nanofiber mesh was examined by water contact angle (WCA) measurements. Biocompatibility test and endothelial differentiation test We have designed four kind of scaffolds for cell culture:PCL/gelatin, PCL/gelatin+ platelets, PCL/gelatin+PDA+platelets, PCL/gelatin+EGM2. LIVE/DEAD viability/cytotoxicity kit were used for the qualitative viability analyses of ADSCs on nanofibrous scaffolds with or without platelets. Immunocytochemical analysis of CD31 and vWF were did after a 14-day culture to compare four group to illustrate endothelial differentiation.Result:Nanofiber meshs were obtained in a highly aligned conformation by depositing the nanofiber on the edge of a rotating drum (2800 rpm) instead of a static collector. The fiber diameter of PCL/gelatin nanofiber is about 500 nm-lum. The thickness of the polydopamine film were showed by the morphology of single nanofiber around ten nm. Original PCL/gelatin nanofiber mesh has a much higher water contact angle than PDA coating mesh. More adherent platelets on the surfaces of PDA coating observed by SEM, and morphology of adherent platelets were round. Little platelets on the surfaces of PCL/gelatin nanofiber mesh, many platelets were spreading observed using SEM.In all four groups live cells (stained green) cultured 3-day on the nanofibrous scaffolds were obviously more numerous than those 24h cultured. The addition of platelets to PCL/gelatin+PDA+platelets constructs dramatically promoted ADSCs survival, attachment and elongation over the subsequent 2 days of culture. Cells with the bipolar morphology of ADSCs with evidence of cell proliferation on PCL/gelatin+PDA+platelets group could be clearly observed from 3-day. Immunohistochemical staining for CD31 and vWF were chosen for the basal characterization of endothelial-like cells. Undifferentiated ADSCs showed almost no specific staining for CD31 and vWF, after 14 days of cultivation in PCL/gelatin group and PCL/gelatin+platelets group. But the overall fluorescence intensity of the differentiated MSCs was markedly enhanced in PCL/gelatin+PDA+platelets, PCL/gelatin+EGM2 group. The different was evaluated by direct positively stained cell counting, the positively stained cell ratio of PCL/gelatin+PDA+platelets group is more big than the PCL/gelatin group(p<0.05) and PCL/gelatin+platelets group(p<0.05), and have no different with PCL/gelatin+EGM2 group(p>0.05).Conclusion:The ADSCs can be differentiated into endothelial Cells when co-cultured with lots platelets on a parallel array of aligned nanofibers. Micron-sized constructs made by PCL/gelatin+PDA+platelets can be as one kind of prevascularised moduls for the formation of vascularized bone.3. Magnetic modulsPurposes:Fabricated superparamagnetic nanoparticle loaded microscale nanofiber mesh. Apply these moduls to develop practical solution which can assembled nanofiber mesh into 3D multilayer constructs. Materials and methodsSPIONs were prepared by the chemical coprecipitation technique. Preparation of polymer solution, use the 10% PCL in TEF,10% gelatin in TFE, mixed with 1:1 ratio, magnetic stirring to make them blending. Preparation 2% F-127 to disperse SPIONs in the solution, after added into the F-127 ultrasonic dispersion were utilized for 30min, after added to the polymer solution for ultrasonic dispersion were utilized for 2h. Ajust ratio of SPIONs to polymer to be 2.5wt%,5wt% and 10wt%, get three gradients. Add the mixture into the syringe, install the syringe to a pump, the spinning speed was lml/h, the voltage was 15kv and the receive distance was 15cm. After 1h, stop spinning and peel off the nanofiber mesh. Put the material in the glutaraldehyde vapor to crosslink 1h, air dry, irradiation sterilization brefore cell culture.SEM, TEM, XRD, FTIR test were did to explore the physical properties of nanofiber.Biocompatibility test and MRI testLIVE/DEAD viability/cytotoxicity kit (Invitrogen) were used for the qualitative viability analyses of ADSCs on nanofibrous scaffolds with or without SPIONs. The magntic scaffolds were implanted into the rat abdominal subcutaneous tissue. And tested by MRI after 3-day.Result:According the SEM image, the diameter of SPIONs/PCL/gelatin nanofiber decreased to about 200 nm. The SPIONs/PCL/gelatin nanofiber membrane showed a uniform structure with interconnected macro-scale pores. The microstructures of both PCL/gelatin nanofibers and different SPIONs concentration SPIONs/PCL/gelatin nanofibers were further investigated by TEM. Clearly, SPIONs nanoparticles were encapsulated in the polymer matrix, no particles were sintered together in 2.5wt%, 5wt% and 10wt%, the morphology of the nanofiber were good. The crystalline structures of the samples were investigated by XRD, showed peaks which are characteristic of SPIONs, the SPIONs/PCL/gelatin nanofibers keep the original crystalline structure of SPIONs nanoparticles. FTIR analysis scaffolds showed the SPIONs in the nanofiber. The saturation magnetization (Ms) values of the magnetic nanofibrous membranes containing different concentrations of SPIONs were 6.3 emu g-1 with 2.5 wt% SPIONs,11.8 emu g-1 with 5wt% SPIONs, and 19.2 emu g-1 with 10 wt% SPIONs, higher SPIONs content led to a higher Ms value of the scaffolds, but all of which are much lower than 65.2 emu g-1 of bare SPIONsLive cells (stained green) cultured 3-day on the four kind of nanofibrous scaffolds almost 90%. Based on these characterization we have chosen the 10wt% SPIONs/PCL/gelatin nanofibers as the scaffold to do the magnetic assembly test and MRI test.In this study, we present a rapid and easy post-processing strategy to create complex multilayered 3D structures using an externally applied magnetic field. Our SPIONs/PCL/gelatin membranes were easily transferred and stacked via placement of a magnet under the glass slide. We implanted 3D multilayer scaffolds composed of nanofibrous membranes, and MR images of the coronal and axial sections of T2-weighted images of rats taken 3 days post-implantation. Dark images were obtained in the anterior abdominal subcutaneous fascia due to the inherent high magnetic susceptibility of SPIONs. Conclusion:We present a magnetic moduls that offers rapid and easy layer-by-layer assembly of a biomimetic system for bottom-up tissue engineering applications as well as multifunctional structures with theranostic functionality.4. Build hierarchically bone tissue Purposes:Magnetic directing the assembly of spatially organized of three kind modules to enhance vascularization in bone tissue engineering and generate hierarchical structures.Materials and methodsPrepare three kinds modules be mentioned by 1,2and3 sections. The multilayer constructions were designed according hierarchical structures of bone. The angiogenesis moduls was put as interlayer, then were the osteogenesis moduls as outside, finally magnetic moduls were used to assembly at the outermost layer. The magnet was put under the cell culture dish to manipulate magnetic moduls assembly together. After culture for 7-day, stability of big multilayer constructions formed by the single kind of moduls or the combined three kinds of moduls were tested. HE staining, actin and CD31 immunofluorescence staining of single layer and multilayer were tested.Result:The magentic assembly technique controlled architectures precisely. After 7-day cell culture process. We observed that all layers easily separated for the nonmagnetic control PCL/gelatin membranes.3D multilayer structures fabricated with two layers containing SPIONs in them keep a perfect steady state as one whole structure. HE staining showed the structure different from single layer and multilayer. The actin and CD31 IF showed the capillary structure in the multilayer. Conclusion:Dense and highly organized 3D bone constructs can be achieved by utilizing the magentic assembly technique. A great benefit of utilizing these three kinds of moduls to create 3D scaffolds is well-controlled cellular type and location. This reaseach show a possible strategies to improve clinically relevant bone regeneration by enhancement of vascularization in engineered constructs.5. BMSCs laden hydrogel for cartilage reconstructionPurposes:We want develop an injectable hydrogel for cartilage reconstruction.Materials and methods:Different from the traditional oxidizing method, we first introduced an amino-diethoxypropane to the alginate and formed hemiacetal group on it. The hydrogel was formed when the modified alginate met with chitosan. We characterized the chemical and rheological properties of the hydrogel and investigated the growth of BMSC in hydrogel. For further testing, we implanted BMSCs laden hydrogel to a rabbit knee cartilage defect model.Result:After 12 weeks, the BMSCs laden hydrogel group presented a better repair than other groups by the result of the histology analysis of HE, Alcian blue and Masson’s trichrome staining. And result of real-time qPCR showed that the gene expression of aggrecan, type II collagen, proteoglycans, and SOX9 were significantly higher in the BMSCs loaded hydrogel treated group than in defect group (p<0.05) and the hydrogel treated group (p<0.05), but the type I collagen gene expression was lower in BMSCs loaded hydrogel treated group than in defect group (p<0.05) and the hydrogel treated group (p<0.05).Conclusion:Our study showed that the developed injectable hydrogel can be loaded with BMSCs, and it can promote cartilage regeneration. |
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