AFM study of morphology and mechanical properties of a chimeric spider silk and bone sialoprotein protein for bone regeneration.

Atomic force microscopy (AFM) was used to assess a new chimeric protein consisting of a fusion protein of the consensus repeat for Nephila clavipes spider dragline protein and bone sialoprotein (6mer+BSP). The elastic modulus of this protein in film form was assessed through force curves, and film surface roughness was also determined. The results showed a significant difference among the elastic modulus of the chimeric silk protein, 6mer+BSP, and control films consisting of only the silk component (6mer). The behavior of the 6mer+BSP and 6mer proteins in aqueous solution in the presence of calcium (Ca) ions was also assessed to determine interactions between the inorganic and organic components related to bone interactions, anchoring, and biomaterial network formation. The results demonstrated the formation of protein networks in the presence of Ca(2+) ions, characteristics that may be important in the context of controlling materials assembly and properties related to bone formation with this new chimeric silk-BSP protein.

Antimicrobial coating of spider silk to prevent bacterial attachment on silk surgical sutures.

Microbial infections from post-surgery or other medical-related procedure is a serious health problem. Nowadays, the research is focused on the development of new drug-free materials with antibacterial properties to prevent or minimize the risk of infections. Spider silk is known for its unique biomechanical properties allied with biocompatibility. Recombinant DNA technology allows to bioengineering spider silk with antimicrobial peptides (AMP). Thus, our goal was to bioengineered spider silk proteins with AMP (6mer-HNP1) as an antibacterial drug-free coating for commercial silk sutures (Perma-Hand®) for decreasing bacterial infections. Perma-Hand® sutures were coated with 6mer-HNP1 by dip coating. In vitro tests, using human fetal lung fibroblasts (MRC5), showed that coated sutures sustained cell viability, and also, the contact with red blood cells (RBCs) demonstrate blood compatibility. Also, the coatings inhibited significantly the adherence and formation of biofilm, where sutures coated with 6mer-HNP1 produced a 1.5 log reduction of Methicillin-Resistant Staphylococcus aureus (MRSA) and a 2 log reduction of Escherichia coli (E. coli) compared to the uncoated Perma-Hand® suture. The mechanical properties of Perma-Hand® sutures were not affected by the presence of bioengineered spider silk proteins. Thus, the present work demonstrated that using spider silk drug-free coatings it is possible to improve the antibacterial properties of the commercial sutures. Furthermore, a new class of drug-free sutures for reducing post-implantation infections can be developed. STATEMENT OF SIGNIFICANCE: Microbial infections from post-surgery or other medical-related procedure is a serious health problem. Developing new drug-free materials with antibacterial properties is an approach to prevent or minimize the risk of infections. Spider silk is known for its unique biomechanical properties allied with biocompatibility. Recombinant DNA technology allow to bioengineering spider silk with antimicrobial peptides (AMP). Our goal is bioengineered spider silk proteins with AMP as an antibacterial coating for silk sutures. The coatings showed exceptional antibacterial properties and maintained intrinsic mechanical features. In vitro studies showed a positive effect of the coated sutures on the cell behavior. With this new drug-free bioengineered spider silk coating is possible to develop a new class of drug-free sutures for reducing post-implantation infections.

Silk-Based Antimicrobial Polymers as a New Platform to Design Drug-Free Materials to Impede Microbial Infections.

Surgical site infections (SSI) represent a serious health problem that occur after invasive surgery, thus new antimicrobial biomaterials able to prevent SSI are needed. Silks are natural biopolymers with excellent biocompatibility, low immunogenicity and controllable biodegradability. Spider silk-based materials can be bioengineered and functionalized with specific peptides, such as antimicrobial peptides, creating innovative polymers. Herein, we explored new drug-free multifunctional silk films with antimicrobial properties, specifically tailored to hamper microbial infections. Different spider silk domains derived from the dragline sequence of the spider Nephila clavipes (6mer and 15mer, 27 and 41 kDa proteins, respectively) were fused with the two antimicrobial peptides, Hepcidin (Hep) and Human Neutrophil peptide 1 (HNP1). The self-assembly features of the spider silk domains (ß-sheets) were maintained after functionalization. The bioengineered 6mer-HNP1 protein demonstrated inhibitory effects against microbial pathogens. Silk-based films with 6mer-HNP1 and different contents of silk fibroin (SF) significantly reduced bacterial adhesion and biofilm formation, whereas higher bacterial counts were found on the films prepared with 6mer or SF alone. The silk-based films showed no cytotoxic effects on human foreskin fibroblasts. The positive cellular response, together with structural and antimicrobial properties, highlight the potential of these multifunctional silk-based films as new materials for preventing SSI.

Nanostructured interfacial self-assembled peptide-polymer membranes for enhanced mineralization and cell adhesion.

Soft interfacial materials, such as self-assembled polymer membranes, are gaining increasing interest as biomaterials since they can provide selective barriers and/or controlled affinity interactions important to regulate cellular processes. Herein, we report the design and fabrication of multiscale structured membranes integrating selective molecular functionalities for potential applications in bone regeneration. The membranes were obtained by interfacial self-assembly of miscible aqueous solutions of hyaluronan and multi-domain peptides (MDPs) incorporating distinct biochemical motifs, including mineralizing (EE), integrin-binding (RGDS) and osteogenic (YGFGG) peptide sequences. Circular dichroism and Fourier transform infrared spectroscopy analyses of the MDPs revealed a predominant ß-sheet conformation, while transmission electron microscopy (TEM) showed the formation of fibre-like nanostructures with different lengths. Scanning electron microscopy (SEM) of the membranes showed an anisotropic structure and surfaces with different nanotopographies, reflecting the morphological differences observed under TEM. All the membranes were able to promote the deposition of a calcium-phosphate mineral on their surface when incubated in a mineralizing solution. The ability of the MDPs, coated on coverslips or presented within the membranes, to support cell adhesion was investigated using primary adult periosteum-derived cells (PDCs) under serum-free conditions. Cells on the membranes lacking RGDS remained round, while in the presence of RGDS they appear to be more elongated and anchored to the membrane. These observations were confirmed by SEM analysis that showed cells attached to the membrane and exhibiting an extended morphology with close interactions with the membrane surface. We anticipate that these molecularly designed interfacial membranes can both provide relevant biochemical signals and structural biomimetic components for stem cell growth and differentiation and ultimately promote bone regeneration.

Silk-based biomaterials functionalized with fibronectin type II promotes cell adhesion.

The objective of this work was to exploit the fibronectin type II (FNII) module from human matrix metalloproteinase-2 as a functional domain for the development of silk-based biopolymer blends that display enhanced cell adhesion properties. The DNA sequence of spider dragline silk protein (6mer) was genetically fused with the FNII coding sequence and expressed in Escherichia coli. The chimeric protein 6mer+FNII was purified by non-chromatographic methods. Films prepared from 6mer+FNII by solvent casting promoted only limited cell adhesion of human skin fibroblasts. However, the performance of the material in terms of cell adhesion was significantly improved when 6mer+FNII was combined with a silk-elastin-like protein in a concentration-dependent behavior. With this work we describe a novel class of biopolymer that promote cell adhesion and potentially useful as biomaterials for tissue engineering and regenerative medicine. STATEMENT OF SIGNIFICANCE: This work reports the development of biocompatible silk-based composites with enhanced cell adhesion properties suitable for biomedical applications in regenerative medicine. The biocomposites were produced by combining a genetically engineered silk-elastin-like protein with a genetically engineered spider-silk-based polypeptide carrying the three domains of the fibronectin type II module from human metalloproteinase-2. These composites were processed into free-standing films by solvent casting and characterized for their biological behavior. To our knowledge this is the first report of the exploitation of all three FNII domains as a functional domain for the development of bioinspired materials with improved biological performance. The present study highlights the potential of using genetically engineered protein-based composites as a platform for the development of new bioinspired biomaterials.

Design and characterization of a biodegradable double-layer scaffold aimed at periodontal tissue-engineering applications.

The inefficacy of the currently used therapies in achieving the regeneration ad integrum of the periodontium stimulates the search for alternative approaches, such as tissue-engineering strategies. Therefore, the core objective of this study was to develop a biodegradable double-layer scaffold for periodontal tissue engineering. The design philosophy was based on a double-layered construct obtained from a blend of starch and poly-ε-caprolactone (30:70 wt%; SPCL). A SPCL fibre mesh functionalized with silanol groups to promote osteogenesis was combined with a SPCL solvent casting membrane aiming at acting as a barrier against the migration of gingival epithelium into the periodontal defect. Each layer of the double-layer scaffolds was characterized in terms of morphology, surface chemical composition, degradation behaviour and mechanical properties. Moreover, the behaviour of seeded/cultured canine adipose-derived stem cells (cASCs) was assessed. In general, the developed double-layered scaffolds demonstrated adequate degradation and mechanical behaviour for the target application. Furthermore, the biological assays revealed that both layers of the scaffold allow adhesion and proliferation of the seeded undifferentiated cASCs, and the incorporation of silanol groups into the fibre-mesh layer enhance the expression of a typical osteogenic marker. This study allowed an innovative construct to be developed, combining a three-dimensional (3D) scaffold with osteoconductive properties and with potential to assist periodontal regeneration, carrying new possible solutions to current clinical needs. Copyright © 2013 John Wiley & Sons, Ltd.

Evaluation of a starch-based double layer scaffold for bone regeneration in a rat model.

Damages in the maxillofacial bones are frequent in humans following trauma, metabolic diseases, neoplasia, or inflammatory processes. Many of the available treatments to regenerate bone are often ineffective. The goal of this work was to assess the in vivo behavior of an innovative double-layered scaffold based on a blend of starch and polycaprolactone (SPCL) that comprises a membrane obtained by solvent casting, which aims to act as a guided tissue regeneration membrane, and a wet-spun fiber mesh (in some cases functionalized with osteoconductive silanol groups) targeting bone regeneration. The behavior of the double layer scaffold, functionalized with silanol groups (SPCL-Si) or without (SPCL), was assessed in a mandibular rodent model and compared to a commercial collagen membrane (positive control) and to empty defects (negative control). After 8 weeks of implantation, the micro-computed tomography and the histomorphometric analysis revealed that the SPCL-Si scaffolds induced significantly higher new bone formation compared to the collagen membrane and to the empty defects, although they had a similar performance when compared to the SPCL scaffolds.

A tissue engineering approach for periodontal regeneration based on a biodegradable double-layer scaffold and adipose-derived stem cells.

Human and canine periodontium are often affected by an inflammatory pathology called periodontitis, which is associated with severe damages across tissues, namely, in the periodontal ligament, cementum, and alveolar bone. However, the therapies used in the routine dental practice, often consisting in a combination of different techniques, do not allow to fully restore the functionality of the periodontium. Tissue Engineering (TE) appears as a valuable alternative approach to regenerate periodontal defects, but for this purpose, it is essential to develop supportive biomaterial and stem cell sourcing/culturing methodologies that address the complexity of the various tissues affected by this condition. The main aim of this work was to study the in vitro functionality of a newly developed double-layer scaffold for periodontal TE. The scaffold design was based on a combination of a three-dimensional (3D) fiber mesh functionalized with silanol groups and a membrane, both made of a blend of starch and poly-ɛ-(caprolactone). Adipose-derived stem cells (canine adipose stem cells [cASCs]) were seeded and cultured onto such scaffolds, and the obtained constructs were evaluated in terms of cellular morphology, metabolic activity, and proliferation. The osteogenic potential of the fiber mesh layer functionalized with silanol groups was further assessed concerning the osteogenic differentiation of the seeded and cultured ASCs. The obtained results showed that the proposed double-layer scaffold supports the proliferation and selectively promotes the osteogenic differentiation of cASCs seeded onto the functionalized mesh. These findings suggest that the 3D structure and asymmetric composition of the scaffold in combination with stem cells may provide the basis for developing alternative therapies to treat periodontal defects more efficiently.

Undifferentiated human adipose-derived stromal/stem cells loaded onto wet-spun starch-polycaprolactone scaffolds enhance bone regeneration: nude mice calvarial defect in vivo study.

The repair of large bony defects remains challenging in the clinical setting. Human adipose-derived stromal/stem cells (hASCs) have been reported to differentiate along different cell lineages, including the osteogenic. The objective of the present study was to assess the bone regeneration potential of undifferentiated hASCs loaded in starch-polycaprolactone (SPCL) scaffolds, in a critical-sized nude mice calvarial defect. Human ASCs were isolated from lipoaspirate of five female donors, cryopreserved, and pooled together. Critical-sized (4 mm) calvarial defects were created in the parietal bone of adult male nude mice. Defects were either left empty, treated with an SPCL scaffold alone, or SPCL scaffold with human ASCs. Histological analysis and Micro-CT imaging of the retrieved implants were performed. Improved new bone deposition and osseointegration was observed in SPCL loaded with hASC engrafted calvarial defects as compared to control groups that showed little healing. Nondifferentiated human ASCs enhance ossification of nonhealing nude mice calvarial defects, and wet-spun SPCL confirmed its suitability for bone tissue engineering. This study supports the potential translation for ASC use in the treatment of human skeletal defects.

Bone marrow stromal cells on a three-dimensional bioactive fiber mesh undergo osteogenic differentiation in the absence of osteogenic media supplements: the effect of silanol groups.

Osteogenic differentiation is a tightly regulated process dependent on the stimuli provided by the micro-environment. Silicon-substituted materials are known to have an influence on the osteogenic phenotype of undifferentiated and bone-derived cells. This study aims to investigate the bioactivity profile as well as the mechanical properties of a blend of starch and poly-caprolactone (SPCL) polymeric fiber mesh scaffolds functionalized with silanol (Si-OH) groups as key features for bone tissue engineering strategies. The scaffolds were made from SPCL by a wet spinning technique. A calcium silicate solution was used as a non-solvent to develop an in situ functionalization with Si-OH groups in a single-step approach. We also explored the relevance of silicon incorporated in SPCL-Si scaffolds to the in vitro osteogenic process of goat bone marrow stromal cells (gBMSCs) with and without osteogenic supplements in the culture medium. We hypothesized that SPCL-Si scaffolds could act as physical and chemical millieus to induce per se the osteogenic differentiation of gBMSCs. Results show that osteogenic differentiation of gBMSCs and the production of a mineralized extracellular matrix on bioactive SPCL-Si scaffolds occur for up to 2weeks, even in the absence of osteogenic supplements in the culture medium. The omission of media supplements to induce osteogenic differentiation is a promising feature towards simplified and cost-effective cell culturing procedures of a potential bioengineered product, and concomitant translation into the clinical field. Thus, the present work demonstrates that SPCL-Si scaffolds and their intrinsic properties sustain gBMSC osteogenic features in vitro, even in the absence of osteogenic supplements to the culture medium, and show great potential for bone regeneration strategies.

In vivo biological responses to silk proteins functionalized with bone sialoprotein.

Recombinant 6mer + BSP protein, combining six repeats of the consensus sequence for Nephila clavipes dragline (6mer) and bone sialoprotein sequence (BSP), shows good support for cell viability and induces the nucleation of hydroxyapatite and tricalcium phosphate during osteoblast in vitro culture. The present study is conducted to characterize this bioengineered protein-based biomaterial further for in vivo behavior related to biocompatibility. 6mer + BSP protein films are implanted in subcutaneous pouches in the back of mice and responses are evaluated by flow cytometry and histology. The results show no major differences between the inflammatory responses induced by 6mer + BSP films and the responses observed for the controls. Thus, this new chimeric protein could represent an alternative for bone regeneration applications.

In situ functionalization of wet-spun fibre meshes for bone tissue engineering.

Bone tissue engineering success strongly depends on our ability to develop new materials combining osteoconductive, osteoinductive and osteogenic properties. Recent studies suggest that biomaterials incorporating silanol (Si-OH) groups promote and maintain osteogenesis. The purpose of the present research work was to provide evidence that using wet-spinning technologies and a calcium silicate solution as a coagulation bath, it was possible to develop an in situ functionalization methodology to obtain 3D wet-spun fibre meshes with Si-OH groups, through a simple, economic and reliable process. SPCL (blend of starch with polycaprolactone) fibre meshes were produced by wet-spinning, using a calcium silicate solution as a non-solvent and functionalized in situ with Si-OH groups. In vitro tests, using goat bone marrow stromal cells (GBMSCs), showed that SPCL-Si scaffolds sustained cell viability and proliferation. Furthermore, high ALP activity and matrix production indicated that Si-OH groups improve cellular functionality towards the osteoblastic phenotype. Using this methodology, and assembling several wet-spun fibre meshes, 3D meshes can be developed, aiming at designing osteoconductive/osteoinductive 3D structures capable of stimulating bone ingrowth in vivo.

Optimized electro- and wet-spinning techniques for the production of polymeric fibrous scaffolds loaded with bisphosphonate and hydroxyapatite.

This research activity was aimed at the development of composite bioactive scaffolds made of biodegradable three-arm branched-star poly(ε-caprolactone) (*PCL), hydroxyapatite nanoparticles (HNPs) and clodronate (CD), a bisphosphonate that has demonstrated efficacy in the treatment of various bone diseases and as an anti-inflammatory drug. During the experimental work, the processing conditions for the fabrication of fibrous meshes, by either electrospinning or wet-spinning, were optimized. Stemming from a previous research activity on electrospinning of *PCL, *PCL/HNPs 3D meshes were developed, evaluating the influence of fabrication parameters on the fibres' morphology. By exploiting the binding affinity of bisphosphonates for hydroxyapatite, a methodology was set up for obtaining a physical linkage between CD and HNPs, with the aim of having a dual bioactive system loaded into *PCL fibrous mats. Fibres loaded with either CD or CD-HNP particles were thus produced and analysed by scanning electron microscopy for their morphology and by energy dispersive X-ray spectroscopy for their elemental composition.

Chitosan scaffolds incorporating lysozyme into CaP coatings produced by a biomimetic route: a novel concept for tissue engineering combining a self-regulated degradation system with in situ pore formation.

This study describes an innovative self-regulated degrading material with gradual in situ pore formation ability for bone tissue engineering applications. This approach is based on the incorporation of the lysozyme enzyme into calcium phosphate (CaP) coatings, prepared on the surface of chitosan scaffolds by means of a biomimetic coating technique with the aim of controlling their degradation rate and subsequent formation of pores. However, because lysozyme has antibacterial properties, these coatings may act as a carrier for its sustained release, preventing infection upon implantation. In order to prove the concept of in situ pore formation, the coated scaffolds (with and without lysozyme) were incubated in two different solutions at different pH to simulate normal physiological conditions (pH 7.4) and inflammatory response (pH 5). The weight loss and morphology of the scaffolds was monitored over time. At pH 7.4, the scaffolds remained more stable than at pH 5. The scaffolds incubated at pH 5 showed a rapid decrease in their initial weight, and scanning electron microscopy imaging revealed the formation of a highly porous structure. Furthermore, evaluation of the activity of the incorporated lysozyme revealed that the enzyme was able to hydrolyse the peptidoglycan of the bacteria cell walls (as detected by the decrease in optical density with time), indicating that the enzyme remained active after being incorporated into the CaP coating.

Biomimetic apatite deposition on polymeric microspheres treated with a calcium silicate solution.

Bioactive polymeric microspheres can be prepared by means of coating them with a calcium silicate solution and subsequently soaking in a simulated body fluid (SBF). Such combination should allow for the development of bioactive microspheres for several applications in the medical field including tissue engineering carriers. Four types of polymeric microspheres, with different sizes, were used in this work: (i) ethylene-vinyl alcohol copolymer (20-30 mum), (ii) polyamide 12 with 10% magnetite (100 mum), (iii) polyamide 12 (10-30 mum) and (iv) polyamide 12 (300 mum). These microspheres were soaked in a calcium silicate solution at 36.5 degrees C for various periods of time under different conditions. Afterwards, they were dried in air at 60 and 100 degrees C for 24 hr. Then, the samples were soaked in SBF for 1, 3, and 7 days. Fourier transformed infrared spectroscopy, thin-film X-ray diffraction, and scanning electron microscopy showed that after the calcium silicate treatment and the subsequent soaking in SBF, the microspheres successfully formed an apatite layer on their surfaces in SBF within 7 days due to the formation of silanol groups, which are effective for apatite formation.