Structurally and Functionally Optimized Silk‐Fibroin–Gelatin Scaffold Using 3D Printing to Repair Cartilage Injury In Vitro and In Vivo

Articular cartilage repair remains a great challenge for clinicians and researchers. Recently, there emerges a promising way to achieve one‐step cartilage repair in situ by combining endogenic bone marrow stem cells (BMSCs) with suitable biomaterials using a tissue engineering technique. To meet the increasing demand for cartilage tissue engineering, a structurally and functionally optimized scaffold is designed, by integrating silk fibroin with gelatin in combination with BMSC‐specific‐affinity peptide using 3D printing (3DP) technology. The combination ratio of silk fibroin and gelatin greatly balances the mechanical properties and degradation rate to match the newly formed cartilage. This dually optimized scaffold has shown superior performance for cartilage repair in a knee joint because it not only retains adequate BMSCs, due to efficient recruiting ability, and acts as a physical barrier for blood clots, but also provides a mechanical protection before neocartilage formation and a suitable 3D microenvironment for BMSC proliferation, differentiation, and extracellular matrix production. It appears to be a promising biomaterial for knee cartilage repair and is worthy of further investigation in large animal studies and preclinical applications. Beyond knee cartilage, this dually optimized scaffold may also serve as an ideal biomaterial for the regeneration of other joint cartilages.     

Injectable silk/hydroxyapatite nanocomposite hydrogels with vascularization capacity for bone regeneration

Localized and sustained osteogenic-angiogenic stimulation to bone defects is critical for effective bone repair.Here,desferrioxamine(DFO)was loaded on silk fibroin nanofibers and blended with hydroxyapatite nanorods(HA),forming injectable DFO-loaded silk fibroin-HA nanocomposite hydrogels.The composite hydrogels remained homogeneous distribution of HA with high ratio(60%)and also higher stiffness than that of pure silk fibroin nanofiber hydrogels,which provided stable osteogenic stimulation niches for tissue regeneration.Without the scarify of injectability,the hydrogels achieved slow delivery of DFO for above 60 days,resulting in suitable angiogenesis in vitro and in vivo and better osteogenesis than DFO-free systems.Compared to previous injectable silk fibroin-HA hydrogels,the introduction of vascularization capacity further stimulated the osteogenic differentiation of stem cells and accelerated new bone formation.Quicker and better bone healing were detected at defect sites after the injection of DFO-loaded nanocomposite hydrogels,indicating the effective synergistic effect of osteogenic and angiogenic cues.This work provides a simple and effective strategy of introducing angiogenic cues to bone matrices.We believe that the injectable nanocomposite hydrogels are suitable for the regeneration of bone tissues.     

Enhanced mechanical properties of thermosensitive chitosan hydrogel by silk fibers for cartilage tissue engineering

Articular cartilage has limited repair capability following traumatic injuries and current methods of treatment remain inefficient. Reconstructing cartilage provides a new way for cartilage repair and natural polymers are often used as scaffold because of their biocompatibility and biofunctionality. In this study, we added degummed chopped silk fibers and electrospun silk fibers to the thermosensitive chitosan/glycerophosphate hydrogels to reinforce two hydrogel constructs which were used as scaffold for hyaline cartilage regeneration. The gelation temperature and gelation time of hydrogel were analyzed by the rheometer and vial tilting method. Mechanical characterization was measured by uniaxial compression, indentation and dynamic mechanical analysis assay. Chondrocytes were then harvested from the knee joint of the New Zealand white rabbits and cultured in constructs. The cell proliferation, viability, production of glycosaminoglycans and collagen type II were assessed. The results showed that mechanical properties of the hydrogel were significantly enhanced when a hybrid with two layers of electrospun silk fibers was made. The results of GAG and collagen type II in cell-seeded scaffolds indicate support of the chondrogenic phenotype for chondrocytes with a significant increase in degummed silk fiber–hydrogel composite for GAG content and in two-layer electrospun fiber–hydrogel composite for Col II. It was concluded that these two modified scaffolds could be employed for cartilage tissue engineering.     

Silkworm and spider silk scaffolds for chondrocyte support

Objective To create scaffolds with silkworm cocoon, spider egg sac and spider dragline silk fibres and examine their use for chondrocyte attachment and support. Methods Three different kinds of scaffolds were developed with Bombyx mori cocoon, Araneus diadematus egg sac and dragline silk fibres. The attachment of human articular cartilage cells were investigated on these bioprotein matrices. The chondrocytes produced an extracellular matrix which was studied by immunostaining. Moreover, the compression behaviour in relation to the porosity was studied. Results The compression modulus of a silkworm silk scaffold was related to its porosity. Chondrocytes were able to attach and to grow on the different fibres and in the scaffolds for several weeks while producing extracellular matrix products. Conclusion Porous scaffolds can be made out of silkworm and spider silk for cartilage regeneration. Mechanical properties are related to porosity and pore size of the construct. Cell spreading and cell expression depended on the porosity and pore-size.     

负载BMP-2和BSA的丝素蛋白/聚左旋乳酸-己内酯三维纤维屏障膜的制备及性能

利用骨形态发生蛋白-2(BMP-2)的骨诱导作用和牛血清白蛋白(BSA)的稳定作用,结合同轴静电纺丝技术和共混静电纺丝技术,制备了三组纤维膜,包括同时携载BMP-2和BSA的丝素蛋白(SF)/聚左旋乳酸-己内酯(PLCL)同轴载药纤维膜(BMP-2-BSA@SF/PLCL,Mat A);同时携载BMP-2和BSA的SF/PLCL共混载药纤维膜(BMP-2-BSA-SF/PLCL,Mat B)和非载药SF/PLCL纤维膜(SF/PLCL,Mat C)。通过研究纤维膜的表观形态,得出三种纤维膜均呈均匀网状结构,其中Mat A组具有稳定的核层结构;理化性能研究证实三组纤维的接触角和力学性能依次增大;体外细胞实验和膜屏障实验表明,三组纤维膜均具有良好的生物相容性和屏障功能,且具有药物缓释作用的组纤Mat A维膜能够更加有效地刺激细胞生长和早期骨向分化。所制备的BMP-2-BSA@SF/PLCL能够满足引导性骨组织再生(GBR)生物膜的基本要求,是促进骨缺损修复的理想载体。     

Formulation of chitosan-TPP-pDNA nanocapsules for gene therapy applications

The encapsulation of DNA inside nanoparticles meant for gene delivery applications is a challenging process where several parameters need to be modulated in order to design nanocapsules with specific tailored characteristics. The purpose of this study was to investigate and improve the formulation parameters of plasmid DNA (pDNA) loaded in chitosan nanocapsules using tripolyphosphate (TPP) as polyanionic crosslinker. Nanocapsule morphology and encapsulation efficiency were analyzed as a function of chitosan degree of deacetylation and chitosan-TPP ratio. The manipulation of these parameters influenced not only the particle size but also the encapsulation and release of pDNA. Consequently the transfection efficiency of the nanoparticulated systems was also enhanced with the optimization of the particle characteristics. Overall, the differently formulated nanoparticulated systems possess singular properties that can be employed according to the desired gene delivery application.     

Preparation of emulsifying wax/glyceryl monooleate nanoparticles and evaluation as a delivery system for repurposing simvastatin in bone regeneration

Simvastatin (Sim) is a widely known drug in the treatment of hyperlipidemia, which has attracted so much attention in bone regeneration due to its potential osteoanabolic effect. However, repurposing of Sim in bone regeneration will require suitable delivery systems that can negate undesirable off-target/side effects. In this study, we have investigated a new lipid nanoparticle (NP) platform that was fabricated using a binary blend of emulsifying wax (Ewax) and glyceryl monooleate (GMO). Using the binary matrix materials, NPs loaded with Sim (0–500?µg/mL) were prepared and showed an average particle size of about 150?nm. NP size stability was dependent on Sim concentration loaded in NPs. The suitability of NPs prepared with the binary matrix materials in Sim delivery for potential application in bone regeneration was supported by biocompatibility in pre-osteoclastic and pre-osteoblastic cells. Additional data demonstrated that biofunctional Sim was released from NPs that facilitated differentiation of osteoblasts (cells that form bones) while inhibiting differentiation of osteoclasts (cells that resorb bones). The overall work demonstrated the preparation of NPs from Ewax/GMO blends and characterization to ascertain potential suitability in Sim delivery for bone regeneration. Additional studies on osteoblast and osteoclast functions are warranted to fully evaluate the efficacy of Sim-loaded Ewax/GMO NPs using in-vitro and in-vivo approaches.     

Magnetic responsive scaffolds and magnetic fields in bone repair and regeneration

Increasing evidence shows that magnetic fields and magnetic responsive scaffolds can play unique roles in promoting bone repair and regeneration. This article addresses the synergistic effects of magnetic scaffolds in response to external magnetic fields on the bone regeneration in situ. Additionally, the exploration of using magnetic scaffolds as tools in the bone implant fixation, local drug delivery and mimicking microenvironment of stem cell differentiation are introduced. We also discussed possible underlying mechanisms and perspectives of magnetic responsive scaffolds in the bone repair and regeneration.     

生物玻璃在组织修复中的研究进展

生物玻璃最早被应用于硬骨组织修复,近些年越来越多的研究证明其还可用于修复软骨和软组织,如皮肤、角膜、肺、神经组织等。具有代表性的生物玻璃包括硅酸盐生物玻璃和硼酸盐生物玻璃,这类生物玻璃在体内环境中能够快速释放大量的离子,发挥特殊的生物学效应,如抑菌活性,细胞附着、繁殖和迁移、血管生成等。掺杂元素对生物玻璃的性能起到重要的作用,进而调控组织修复中的不同效果。并且,生物玻璃的结构也对细胞的生长及组织修复有着重要的作用。由于生物玻璃韧性低、脆性高,常与金属、多聚物等制备成复合材料应用于组织修复。综述了生物玻璃物理化学性质与生物学活性的关系,同时介绍和探讨了基于生物玻璃衍生出来的复合材料及其应用,为开发新型的组织修复材料提供参考。     

Design of novel polysaccharidic nanostructures for gene delivery

The goal of the present work was to develop a new synthetic nanosystem for gene delivery. For this purpose, we chose two polysaccharides, hyaluronic acid (HA) and chitosan (CS), as the main components of the nanocarrier. Nanoparticles with different hyaluronate:chitosan (HA:CS) mass ratios (0.5:1 and 1:1) and different polymer molecular weights (hyaluronate 170 (HA) or <10?kDa (HAO) and chitosan 125 (CS) or 10-12?(CSO)?kDa) could be obtained using an ionic crosslinking method. These nanoparticles were loaded with pDNA and characterized for their size, zeta potential and pDNA association efficiency. Moreover, their toxicity and ability to transfect the model plasmid pEGFP-C1 were evaluated in the cell line HEK 293, as well as their intracellular fate. The results showed that HA:CS nanoparticles have a small size in the range of 110-230?nm, a positive zeta potential of +10 to +32?mV and a very high pDNA association efficiency of 87-99% (w/w). On the other hand, nanoparticles exhibited low cell toxicity and transfection levels up to 25% GFP expressing HEK?293 cells, lasting for the whole observation period of 10 days. We also provide basic information about the role of both polymers, HA and CS, and the effect of their molecular weight on the effectiveness of the resulting DNA nanocarrier, being the highest transfection levels observed with HAO:CSO 1:1 nanoparticles. In?conclusion, HA:CS nanoparticles are promising carriers for gene delivery.     

微胶囊型自愈合聚合物基复合材料研究进展

自愈合材料是一种可以模仿生物体自行愈合的新型智能材料,具有广泛的应用前景。微胶囊型自愈合聚合物基复合材料是近年来复合材料自愈合方法领域内的研究热点之一。本文对目前聚合物基复合材料自愈合方法进行了综述,着重介绍了微胶囊型自愈合聚合物基复合材料的自愈合概念和机制、微胶囊结构及其技术发展状况,并详细介绍了微胶囊的芯材、壁材、自愈合基体材料及微胶囊临界应力等因素对复合材料自愈合性能的影响,以及自愈合效率的评估方法。最后讨论了微胶囊型自愈合聚合物基复合材料的发展趋势和面临挑战。     

Recent Progress in Cartilage Tissue Engineering—Our Experience and Future Directions

Given the limited spontaneous repair that follows cartilage injury, demand is growing for tissue engineering approaches for cartilage regeneration. There are two major applications for tissue-engineered cartilage. One is in orthopedic surgery, in which the engineered cartilage is usually used to repair cartilage defects or loss in an articular joint or meniscus in order to restore the joint function. The other is for head and neck reconstruction, in which the engineered cartilage is usually applied to repair cartilage defects or loss in an auricle, trachea, nose, larynx, or eyelid. The challenges faced by the engineered cartilage for one application are quite different from those faced by the engineered cartilage for the other application. As a result, the emphases of the engineering strategies to generate cartilage are usually quite different for each application. The statuses of preclinical animal investigations and of the clinical translation of engineered cartilage are also at different levels for each application. The aim of this review is to provide an opinion piece on the challenges, current developments, and future directions for cartilage engineering for both applications.     

Aligned collagen–GAG matrix as a 3D substrate for Schwann cell migration and dendrimer-based gene delivery

The development of artificial off-the-shelf conduits that facilitate effective nerve regeneration and recovery after repair of traumatic nerve injury gaps is of fundamental importance. Collagen–glycosaminoglycan (GAG) matrix mimicking Schwann cell (SC) basal lamina has been proposed as a suitable and biologically rational substrate for nerve regeneration. In the present study, we have focused on the permissiveness of this matrix type for SC migration and repopulation, as these events play an essential role in nerve remodeling. We have also demonstrated that SCs cultured within collagen–GAG matrix are compatible with non-viral dendrimer-based gene delivery, that may allow conditioning of matrix-embedded cells for future gene therapy applications.     

微胶囊埋植型自修复涂层研究进展

微胶囊埋植型自修复涂层是自修复复合材料的研究热点之一,它是模拟生物体损伤自愈合的原理,实现对涂层微裂纹、划痕等缺陷的自我修复,可有效提升涂层的耐蚀性能和使用寿命。从修复机理进行分类,微胶囊埋植型自修复涂层可分为腐蚀抑制型和反应修复型。腐蚀抑制型是通过微胶囊释放缓蚀剂延缓基体的腐蚀进程,反应修复型则是通过微胶囊释放的修复剂实现对涂层损坏处的修补。系统地回顾了微胶囊埋植型自修复涂层的典型研究成果和近期动态,详细阐述了自修复涂层的设计和修复机制,着重对不同涂层修复体系的优缺点和适用范围进行了讨论。最后,根据微胶囊埋植型自修复涂层的研究现状,提出了该领域存在的缺陷以及未来的发展方向。     

Remodeling Microenvironment for Endogenous Repair through Precise Modulation of Chondroitin Sulfate Proteoglycans Following Spinal Cord Injury

The fluid-filled cystic cavity sealed by a dense scar developed following traumatic spinal cord injury (SCI) has been a major obstacle to neural regeneration and functional recovery. Here the transected lesion is bridged using a functional self-assembling peptide (F-SAP) hydrogel loaded with membrane-permeable intracellular sigma peptide (ISP) and intracellular LAR peptide (ILP), targeted at perturbing chondroitin sulfate proteoglycan (CSPG) inhibitory signaling. As compared to F-SAP hydrogel loaded with chondroitinase ABC, the F-SAP+ISP/ILP promotes a beneficial anti-inflammatory response via manipulation of microglia/macrophages infiltration and assembly of extracellular matrix (ECM) molecules into fibrotic matrix rather than scarring tissues. The remodeled ECM creates a permissive environment that supports axon regrowth and the formation of synaptic connections with neurons derived from endogenous neural stem cells. The remodeled networks contribute to functional recovery, as demonstrated by improved hind limb movements and electrophysiological properties. This work proposes a unique mechanism that ECM remodeling induced by CSPG-manipulation-based anti-inflammation can construct a permissive environment for neural regeneration, and shed light on the advancement of manipulation of cascading cellular and molecular events potential for endogenous repair of SCI.     

Plasmid DNA-loaded asymmetrically porous membrane for guided bone regeneration

Although bone defects can be restored spontaneously,bone reconstruction with sufficient strength and volume continues to be a challenge in clinical practices.In recent years,the use of a variety of biomaterials with bioactivity has been attempted to compensate for this limitation.Herein,we fabricated a pDNA(encoding for BMP-2)-loaded asymmetrically porous polycaprolactone(PCL)/Pluronic F127 membrane as a bioactive guided bone regeneration(GBR)membrane,using a modified immersion-precipitation method.It was observed that the GBR membrane allows continuous release of pDNA for more than20 weeks.The pDNA was sufficiently transfected into human bone marrow stem cells(h BMSCs)without significant cytotoxicity and the gene-transfected cells showed prolonged synthesis of BMP-2.From in vitro osteogenic differentiation and in vivo animal studies,the effective induction of osteogenic differentiation of h BMSCs and enhanced bone regeneration by the pDNA-loaded asymmetrically porous PCL/Pluronic F127 membrane was observed,suggesting that the pDNA-loaded membrane as a bioactive GBR membrane can be an alternative therapeutic technique for effective bone regeneration.     

Chitosan/hyaluronic acid/plasmid-DNA nanoparticles encoding interleukin-1 receptor antagonist attenuate inflammation in synoviocytes induced by interleukin-1 beta

Synovial inflammation mainly resulting from interleukin-1 beta (IL-1β) plays a crucial role in the early and late stage of osteoarthritis. Recent progress in therapeutic gene delivery systems has led to promising strategies for local sustained target gene expression. The aim of this study was to design a nanoparticle made of chitosan (CS)/hyaluronic acid (HA)/plasmid-DNA (pDNA) encoding IL-1 receptor antagonist gene (pIL-1Ra) and furtherly use it to transfect the primary synoviocytes, and then investigate whether CS/HA/pIL-1Ra nanoparticles could make the synoviocytes overexpress functional IL-1Ra to attenuate inflammation induced by IL-1β. In this study, CS was modified with HA to generate CS/HA nanoparticles and then combined with pIL-1Ra to form CS/HA/pIL-1Ra nanoparticles. The physicochemical characteristics results showed that CS/HA nanoparticles exhibited an appropriate particle size (144.9?±?2.8?nm) and positive zeta potential (?+?28?mV). The gel retardation assay revealed that pDNA was effectively protected and released in a sustained manner more than 15 days. Cytotoxicity results showed that CS/HA/pIL-1Ra nanoparticles had a safe range (0-80?μg/ml) for the application to synoviocytes. RT-qPCR and western blot analysis demonstrated that CS/HA/pIL-1Ra nanoparticles were able to increase IL-1Ra expression in primary synoviocytes, and reduce the mRNA and protein levels of matrix metalloproteinase-3 (MMP-3), matrix metalloproteinase-13 (MMP-13), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in IL-1β-induced synoviocytes. Our findings indicated that CS/HA/pIL-1Ra nanoparticles efficiently transfected synoviocytes and attenuated synovitis induced by IL-1β, which will provide a potential strategy for OA synovitis.

Open image in new window

     

High-strength mineralized collagen artificial bone

Mineralized collagen （MC） is a biomimetic material that mimics natural bone matrix in terms of both chemical composition and microstructure. The biomimetic MC possesses good biocompatibility and osteogenic activity, and is capable of guiding bone regeneration as being used for bone defect repair. However, mechanical strength of existing MC artificial bone is too low to provide effective support at human load-bearing sites, so it can only be used for the repair at non-load-bearing sites, such as bone defect filling, bone graft augmentation, and so on. In the present study, a high strength MC artificial bone material was developed by using collagen as the template for the biomimetic mineralization of the calcium phosphate, and then followed by a cold compression molding process with a certain pressure. The appearance and density of the dense MC were similar to those of natural cortical bone, and the phase composition was in conformity with that of animal＇s cortical bone demonstrated by XRD. Mechanical properties were tested and results showed that the compressive strength was comparable to human cortical bone, while the compressive modulus was as low as human cancellous bone. Such high strength was able to provide effective mechanical support for bone defect repair at human load-bearing sites, and the low compressive modulus can help avoid stress shielding in the application of bone regeneration. Both in vitro cell experiments and in v/vo implantation assay demonstrated good biocompatibility of the material, and in v/vo stability evaluation indicated that this high-strength MC artificial bone could provide long-term effective mechanical support at human load- bearing sites.     

Hybrid polymer-grafted multiwalled carbon nanotubes for in vitro gene delivery

Carbon nanotubes (CNTs) consist of carbon atoms arranged in sheets of graphene rolled up into cylindrical shapes. This class of nanomaterials has attracted attention because of their extraordinary properties, such as high electrical and thermal conductivity. In addition, development in CNT functionalization chemistry has led to an enhanced dispersibility in aqueous physiological media which indeed broadens the spectrum for their potential biological applications including gene delivery. The aim of this study is to determine the capability of different cationic polymer-grafted multiwalled carbon nanotubes (MWNTs) (polymer-g-MWNTs) to efficiently complex and transfer plasmid DNA (pCMV-βGal) in vitro without promoting cytotoxicity. Carboxylated MWNT is chemically conjugated to the cationic polymers polyethylenimine (PEI), polyallylamine (PAA), or a mixture of the two polymers. In order to explore the potential of these polymer-g-MWNTs as gene delivery systems, we first study their capacity to complex plasmid DNA (pDNA) using agarose gel electrophoresis. Gel migration studies confirm pDNA binding to polymer-g-MWNT with different affinities, highest for PEI-g-MWNT and PEI/PAA-g-CNT constructs. β-galactosidase expression is assessed in human lung epithelial (A549) cells, and the cytotoxicity is determined by modified LDH assay after 24 h incubation period. Additionally, PEI-g-MWNT and/or PEI/PAA-g-MWNT reveal an improvement in gene expression when compared to the naked pDNA or to the equivalent amounts of PEI polymer alone. Mechanistically, pDNA was delivered by the polymer-g-MWNT constructs via a different pathway compared to those used by polyplexes. In conclusion, polymer-g-MWNTs may be considered in the future as a versatile tool for efficient gene transfer in cancer cells in vitro, provided their toxicological profile is established.     

丝素蛋白柔性电子器件材料的研究进展

丝素蛋白材料凭借良好的生物相容性、可控生物降解性、再生形貌多样性等已被制成柔性电子器件在电子领域进行了应用研究.本文首先综述不同溶解方法对蚕丝再生材料制备的影响,同时对丝素蛋白材料的(微球、膜、纤维、凝胶、支架等)制备方法、材料性能进行分析,最后总结了近年来丝素蛋白基柔性电子材料的应用研究进展.尽管已有研究表明可获得各...