Multiscale design and synthesis of biomimetic gradient protein/biosilica composites for interfacial tissue engineering.
ABSTRACT: Continuous gradients present at tissue interfaces such as osteochondral systems, reflect complex tissue functions and involve changes in extracellular matrix compositions, cell types and mechanical properties. New and versatile biomaterial strategies are needed to create suitable biomimetic engineered grafts for interfacial tissue engineering. Silk protein-based composites, coupled with selective peptides with mineralization domains, were utilized to mimic the soft-to-hard transition in osteochondral interfaces. The gradient composites supported tunable mineralization and mechanical properties corresponding to the spatial concentration gradient of the mineralization domains (R5 peptide). The composite system exhibited continuous transitions in terms of composition, structure and mechanical properties, as well as cytocompatibility and biodegradability. The gradient silicified silk/R5 composites promoted and regulated osteogenic differentiation of human mesenchymal stem cells in an osteoinductive environment in vitro. The cells differentiated along the composites in a manner consistent with the R5-gradient profile. This novel biomimetic gradient biomaterial design offers a useful approach to meet a broad range of needs in regenerative medicine.
Project description:Chitosan (CH), silk fibroin (SF), and hydroxyapatite (HA) were used to prepare CH/SF/HA composites and the resulting composites were electrospun into nanofibrous membrane units with gradient compositional and structural features. The optimal membrane unit was used together with CH/HA and CH/SF composites to fabricate a type of three-layer scaffold that is intended for osteochondral repair. The bottom layer of the scaffold was built with CH/HA composites and it served as a subchondral layer, the integrated nanofibrous membrane unit functioned as the middle layer for mimicking the calcified layer and the top layer was constructed using CH/SF composites for acting as a chondral layer. The nanofibrous membrane unit was found to be permeable to some molecules with limited molecular weight and was able to prevent the seeded cells from migrating cross the unit, functioning approximately like the calcified layer in the osteochondral matrix. Layered scaffolds showed abilities to promote the growth of both chondrocytes and osteoblasts that were seeded in their chondral layer and bony layer, respectively, and they were also able to support the phenotype preservation of seeded chondrocytes and the mineralization of neotissue in the bony layer. Results suggest that this type of layered scaffolds can function as an analogue of the osteochondral matrix and it has potential in osteochondral repair.
Project description:Biomimetic mineralization can lead to advanced crystalline composites with common chemicals under ambient conditions. An exceptional example is biomimetic nacre with its superior fracture toughness. The synthesis of the prismatic layer with stiffness and wear resistance nonetheless remains an elusive goal. Herein, we apply a biomimetic mineralization method to grow prismatic-type CaCO3 thin films, mimicking their biogenic counterparts found in mollusk shells with a three-step pathway: coating a polymer substrate, deposition of a granular transition layer, and mineralization of a prismatic overlayer. The synthetic prismatic overlayers exhibit structural similarity and comparable hardness and Young's modulus to their biogenic counterparts. Furthermore, employment of a biomacromolecular soluble additive, silk fibroin, in fabrication of the prismatic thin films leads to micro-/nano-textures with enhanced toughness and emerging under-water superoleophobicity. This study highlights the crucial role of the granular transition layer in promoting competition growth of the prismatic layer.
Project description:Biomaterial design via genetic engineering can be utilized for the rational functionalization of proteins to promote biomaterial integration and tissue regeneration. Spider silk has been extensively studied for its biocompatibility, biodegradability and extraordinary material properties. As a protein-based biomaterial, recombinant DNA derived derivatives of spider silks have been modified with biomineralization domains which lead to silica deposition and potentially accelerated bone regeneration. However, the influence of the location of the R5 (SSKKSGSYSGSKGSKRRIL) silicifying domain fused with the spider silk protein sequence on the biosilicification process remains to be determined. Here we designed two silk-R5 fusion proteins that differed in the location of the R5 peptide, C- vs. N-terminus, where the spider silk domain consisted of a 15mer repeat of a 33 amino acid consensus sequence of the major ampullate dragline Spidroin 1 from Nephila clavipes (SGRGGLGGQG AGAAAAAGGA GQGGYGGLGSQGT). The chemical, physical and silica deposition properties of these recombinant proteins were assessed and compared to a silk 15mer control without the R5 present. The location of the R5 peptide did not have a significant effect on wettability and surface energies, while the C-terminal location of the R5 promoted more controlled silica precipitation, suggesting differences in protein folding and possibly different access to charged amino acids that drive the silicification process. Further, cell compatibility in vitro, as well as the ability to promote human bone marrow derived mesenchymal stem cell (hMSC) differentiation were demonstrated for both variants of the fusion proteins.
Project description:In the present study, an artificial spider silk gene, 6mer, derived from the consensus sequence of Nephila clavipes dragline silk gene, was fused with different silica-binding peptides (SiBPs), A1, A3 and R5, to study the impact of the fusion protein sequence chemistry on silica formation and the ability to generate a silk-silica composite in two different bioinspired silicification systems: solution-solution and solution-solid. Condensed silica nanoscale particles (600-800 nm) were formed in the presence of the recombinant silk and chimeras, which were smaller than those formed by 15mer-SiBP chimeras, revealing that the molecular weight of the silk domain correlated to the sizes of the condensed silica particles in the solution system. In addition, the chimeras (6mer-A1/A3/R5) produced smaller condensed silica particles than the control (6mer), revealing that the silica particle size formed in the solution system is controlled by the size of protein assemblies in solution. In the solution-solid interface system, silicification reactions were performed on the surface of films fabricated from the recombinant silk proteins and chimeras and then treated to induce ?-sheet formation. A higher density of condensed silica formed on the films containing the lowest ?-sheet content while the films with the highest ?-sheet content precipitated the lowest density of silica, revealing an inverse correlation between the ?-sheet secondary structure and the silica content formed on the films. Intriguingly, the 6mer-A3 showed the highest rate of silica condensation but the lowest density of silica deposition on the films, compared with 6mer-A1 and -R5, revealing antagonistic crosstalk between the silk and the SiBP domains in terms of protein assembly. These findings offer a path forward in the tailoring of biopolymer-silica composites for biomaterial related needs.
Project description:OBJECTIVES:Articular cartilage plays a vital role in bearing and buffering. Injured cartilage and subchondral bone repair is a crucial challenge in cartilage tissue engineering due to the peculiar structure of osteochondral unit and the requirement of osteogenic/chondrogenic bi-directional differentiation. Based on the bionics principle, a nanotextured silk fibroin (SF)-chondroitin sulphate (CS)/hydroxyapatite (HAp) nanowire tough bilayer structure was prepared for osteochondral repair. METHODS:The SF-CS/HAp membrane was constructed by alcohol-induced ?-sheet formation serving as the physical crosslink. Its osteochondral repairing capacity was evaluated by culturing bone marrow mesenchymal stem cells (BMSCs) in vitro and constructing a rat osteochondral defect model in vivo. RESULTS:The bilayer SF-CS/HAp membrane with satisfactory mechanical properties similar to natural cartilage imitated the natural osteochondral unit structural layers and exerted the function of bearing and buffering timely after in vivo implantation. SF-CS layer upregulated the expression of chondrogenesis-related genes of BMSCs by surface nanotopography and sustained release CS. Meanwhile, nanotextured HAp layer assembled with nanowire endowed the membrane with an osteogenic differentiation tendency for BMSCs. In vivo results proved that the biomimetic bilayer structure dramatically promoted new cartilage formation and subchondral bone remodelling for osteochondral defect model after implantation. CONCLUSIONS:The SF-CS/HAp biomimetic bilayer membrane provides a promising strategy for precise osteochondral repair.
Project description:Major ampullate (MA) dragline silk supports spider orb webs, combining strength and extensibility in the toughest biomaterial. MA silk evolved ~376?MYA and identifying how evolutionary changes in proteins influenced silk mechanics is crucial for biomimetics, but is hindered by high spinning plasticity. We use supercontraction to remove that variation and characterize MA silk across the spider phylogeny. We show that mechanical performance is conserved within, but divergent among, major lineages, evolving in correlation with discrete changes in proteins. Early MA silk tensile strength improved rapidly with the origin of GGX amino acid motifs and increased repetitiveness. Tensile strength then maximized in basal entelegyne spiders, ~230?MYA. Toughness subsequently improved through increased extensibility within orb spiders, coupled with the origin of a novel protein (MaSp2). Key changes in MA silk proteins therefore correlate with the sequential evolution high performance orb spider silk and could aid design of biomimetic fibers.
Project description:Orthopedic surgical procedures based on the use of conventional biological graft tissues are often associated with serious post-operative complications such as immune rejection, bacterial infection, and poor osseointegration. Bioresorbable bone graft substitutes have emerged as attractive alternatives to conventional strategies because they can mimic the composition and mechanical properties of the native bone. Among these, bioactive glasses (BGs) hold great potential to be used as biomaterials for bone tissue engineering owing to their biomimetic composition and high biocompatibility and osteoinductivity. Here, we report the development of a novel composite biomaterial for bone tissue engineering based on the incorporation of a modified strontium- and lithium-doped 58S BG (i.e., BG-5/5) into gelatin methacryloyl (GelMA) hydrogels. We characterized the physicochemical properties of the BG formulation via different analytical techniques. Composite hydrogels were then prepared by directly adding BG-5/5 to the GelMA hydrogel precursor, followed by photocrosslinking of the polymeric network via visible light. We characterized the physical, mechanical, and adhesive properties of GelMA/BG-5/5 composites, as well as their in vitro cytocompatibility and osteoinductivity. In addition, we evaluated the antimicrobial properties of these composites in vitro, using a strain of methicillin-resistant Staphylococcus Aureus. GelMA/BG-5/5 composites combined the functional characteristics of the inorganic BG component, with the biocompatibility, biodegradability, and biomimetic composition of the hydrogel network. This novel biomaterial could be used for developing osteoinductive scaffolds or implant surface coatings with intrinsic antimicrobial properties and higher therapeutic efficacy.
Project description:Protein polymers provide a unique opportunity for tunable designs of material systems due to the genetic basis of sequence control. To address the challenge of biomineralization interfaces with protein based materials, we genetically engineered spider silks to design organic-inorganic hybrid systems. The spider silk inspired domain (SGRGGLGGQG AGAAAAAGGA GQGGYGGLGSQGT)15 served as an organic scaffold to control material stability and to allow multiple modes of processing, whereas the hydroxyapatite binding domain VTKHLNQISQSY (VTK), provided control over osteogenesis. The VTK domain was fused either to the N-, C- or both terminals of the spider silk domain to understand the effect of position on material properties and mineralization. The addition of the VTK domain to silk did not affect the physical properties of the silk recombinant constructs, but it had a critical role in the induction of biomineralization. When the VTK domain was placed on both the C- and N-termini the formation of crystalline hydroxyapatite was significantly increased. In addition, all of the recombinant proteins in film format supported the growth and proliferation of human mesenchymal stem cells (hMSCs). Importantly, the presence of the VTK domain enhanced osteoinductive properties up to 3-fold compared to the control (silk alone without VTK). Therefore, silk-VTK fusion proteins have been shown suitable for mineralization and functionalization for specific biomedical applications.Organic-inorganic interfaces are integral to biomaterial functions in many areas of repair and regeneration. Several protein polymers have been investigated for this purpose. Despite their success the limited options to fine-tune their material properties, degradation patterns and functionalize them for each specific biomedical application limits their application. Various studies have shown that the biological performance of such proteins can be improved by genetic engineering. The present study provides data relating protein design parameters and functional outcome quantified by biomineralization and human mesenchymal stem cell differentiation. As such, it helps the design of osteoinductive recombinant biomaterials for bone regeneration.
Project description:Silk fibers have many inherent properties that are suitable for their use in biomaterials. In this study, the silk fibroin was genetically modified by including a Ca-binding sequence, [(AGSGAG)6ASEYDYDDDSDDDDEWD]2 from shell nacreous matrix protein. It can be produced as fibers by transgenic silkworm. The Ca-binding activity and mineralization of the transgenic silk fibroin were examined in vitro. The results showed that this transgenic silk fibroin had relatively higher Ca-binding activity than unmodified silk fibroin. The increased Ca-binding activity could promote the usage of silk fibroin as a biomaterial in the pharmaceutical industry. This study shows the possibility of using silk fibroin as a mineralization accelerating medical material by generating genetically modified transgenic silkworm.
Project description:Temporally and spatially controlled delivery of growth factors in polymeric scaffolds is crucial for engineering composite tissue structures, such as osteochondral constructs. In the present study, microsphere-mediated growth factor delivery in polymer scaffolds and its impact on osteochondral differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) was evaluated. Two growth factors, bone morphogenetic protein 2 (rhBMP-2) and insulin-like growth factor I (rhIGF-I), were incorporated as a single concentration gradient or reverse gradient combining two factors in the scaffolds. To assess the gradient making system and the delivery efficiency of polylactic-co-glycolic acid (PLGA) and silk fibroin microspheres, initially an alginate gel was fabricated into a cylinder shape with microspheres incorporated as gradients. Compared to PLGA microspheres, silk microspheres were more efficient in delivering rhBMP-2, probably due to sustained release of the growth factor, while less efficient in delivering rhIGF-I, likely due to loading efficiency. The growth factor gradients formed were shallow, inducing non-gradient trends in hMSC osteochondral differentiation. Aqueous-derived silk porous scaffolds were used to incorporate silk microspheres using the same gradient process. Both growth factors formed deep and linear concentration gradients in the scaffold, as shown by enzyme-linked immunosorbent assay (ELISA). After seeding with hMSCs and culturing for 5 weeks in a medium containing osteogenic and chondrogenic components, hMSCs exhibited osteogenic and chondrogenic differentiation along the concentration gradients of rhBMP-2 in the single gradient of rhBMP-2 and reverse gradient of rhBMP-2/rhIGF-I, but not the rhIGF-I gradient system, confirming that silk microspheres were more efficient in delivering rhBMP-2 than rhIGF-I for hMSCs osteochondrogenesis. This novel silk microsphere/scaffold system offers a new option for the delivery of multiple growth factors with spatial control in a 3D culture environment for both understanding natural tissue growth process and in vitro engineering complex tissue constructs.