Innovative Antibacterial, Photocatalytic, Titanium Dioxide Microstructured Surfaces Based on Bacterial Adhesion Enhancement.

Bacterial colonization and biofilm formation are found on nearly all wet surfaces, representing a serious problem for both human healthcare and industrial applications, where traditional treatments may not be effective. Herein, we describe a synergistic approach for improving the performance of antibacterial surfaces based on microstructured surfaces that embed titanium dioxide nanoparticles (TiO2 NPs). The surfaces were designed to enhance bacteria entrapment, facilitating their subsequent eradication by a combination of UVC disinfection and TiO2 NPs photocatalysis. The efficacy of the engineered TiO2-modified microtopographic surfaces was evaluated using three different designs, and it was found that S2-lozenge and S3-square patterns had a higher concentration of trapped bacteria, with increases of 70 and 76%, respectively, compared to flat surfaces. Importantly, these surfaces showed a significant reduction (99%) of viable bacteria after just 30 min of irradiation with UVC 254 nm light at low intensity, being sixfold more effective than flat surfaces. Overall, our results showed that the synergistic effect of combining microstructured capturing surfaces with the chemical functionality of TiO2 NPs paves the way for developing innovative and efficient antibacterial surfaces with numerous potential applications in the healthcare and biotechnology market.

Natural and Genetically Engineered Proteins for Tissue Engineering.

To overcome the limitations of traditionally used autografts, allografts and, to a lesser extent, synthetic materials, there is the need to develop a new generation of scaffolds with adequate mechanical and structural support, control of cell attachment, migration, proliferation and differentiation and with bio-resorbable features. This suite of properties would allow the body to heal itself at the same rate as implant degradation. Genetic engineering offers a route to this level of control of biomaterial systems. The possibility of expressing biological components in nature and to modify or bioengineer them further, offers a path towards multifunctional biomaterial systems. This includes opportunities to generate new protein sequences, new self-assembling peptides or fusions of different bioactive domains or protein motifs. New protein sequences with tunable properties can be generated that can be used as new biomaterials. In this review we address some of the most frequently used proteins for tissue engineering and biomedical applications and describe the techniques most commonly used to functionalize protein-based biomaterials by combining them with bioactive molecules to enhance biological performance. We also highlight the use of genetic engineering, for protein heterologous expression and the synthesis of new protein-based biopolymers, focusing the advantages of these functionalized biopolymers when compared with their counterparts extracted directly from nature and modified by techniques such as physical adsorption or chemical modification.

Untangling the biological and inflammatory behavior of silk-like sutures In vivo.

Recombinant spider silk materials with antimicrobial peptides are a promising new class of drug-free antimicrobial materials capable of preventing surgical site infections (SSI), but their potential to impede infections is unclear. Herein, we aimed to unravel the biological and inflammatory potential of bioengineered spider silk materials to prevent SSI using an infection animal model. Silk-like fibers made of silk fibroin and spider silk proteins with antimicrobial peptides (6mer-HNP1) held improved stiffness (2.9 GPa) and had a slow biodegradation profile while inhibiting bacterial adherence in vitro by 5-log and 6-log reduction on Methicillin-Resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli), respectively. In vivo studies showed that fibers with 6mer-HNP1 elicited a short-term low to mild local inflammatory response, similar to implanted commercial sutures. In the presence of a bacterial infection, the mediators related to infection and inflammation were downregulated suggesting that the fibers maintained a low but active response to bacterial infection. Thus, the presence of 6mer-HNP1 helped the host maintain an active response to bacterial infection, impairing the development of an acute infection. Our findings further support the use of bioengineered spider silk proteins to develop drug-free antimicrobial sutures capable to impair SSI.

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.

A Graded, Porous Composite of Natural Biopolymers and Octacalcium Phosphate Guides Osteochondral Differentiation of Stem Cells.

Lesions involving the osteochondral unit are difficult to treat. Biomimetic scaffolds are previously shown as promising alternative. Such devices often lack multiple functional layers that mimic bone, cartilage, and the interface. In this study, multilayered scaffolds are developed based on the use of natural extracellular matrix (ECM)-like biopolymers. Particular attention is paid to obtain a complex matrix that mimics the native osteochondral transition. Porous, sponge-like chitosan-collagen-octacalcium phosphate (OCP) scaffolds are obtained. Collagen content increases while the amount of OCP particles decreases toward the cartilage layer. The scaffolds are bioactive as a mineral layer is deposited containing hydroxyapatite at the bony side. The scaffolds stimulate proliferation of human adipose-derived mesenchymal stem cells, but the degree of proliferation depends on the cell seeding density. The scaffolds give rise to a zone-specific gene expression. RUNX2, COL1A1, BGLAP, and SPP1 are upregulated in the bony layer of the scaffold. SOX9 is upregulated concomitant with COL2A1 expression in the cartilage zone. Mineralization in presence of the cells is prominent in the bone area with Ca and P steadily increasing over time. These results are encouraging for the fabrication of biomimetic scaffolds using ECM-like materials and featuring gradients that mimic native tissues and their interface.

Chitosan/ß-TCP composites scaffolds coated with silk fibroin: a bone tissue engineering approach.

Bone regeneration and natural repair are long-standing processes that can lead to uneven new tissue growth. By introducing scaffolds that can be autografts and/or allografts, tissue engineering provides new approaches to manage the major burdens involved in this process. Polymeric scaffolds allow the incorporation of bioactive agents that improve their biological and mechanical performance, making them suitable materials for bone regeneration solutions. The present work aimed to create chitosan/beta-tricalcium phosphate-based scaffolds coated with silk fibroin and evaluate their potential for bone tissue engineering. Results showed that the obtained scaffolds have porosities up to 86%, interconnectivity up to 96%, pore sizes in the range of 60-170 µm, and a stiffness ranging from 1 to 2 MPa. Furthermore, when cultured with MC3T3 cells, the scaffolds were able to form apatite crystals after 21 d; and they were able to support cell growth and proliferation up to 14 d of culture. Besides, cellular proliferation was higher on the scaffolds coated with silk. These outcomes further demonstrate that the developed structures are suitable candidates to enhance bone tissue engineering.

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.

The effects of platelet lysate patches on the activity of tendon-derived cells.

Platelet-derived biomaterials are widely explored as cost-effective sources of therapeutic factors, holding a strong potential for endogenous regenerative medicine. Particularly for tendon repair, treatment approaches that shift the injury environment are explored to accelerate tendon regeneration. Herein, genipin-crosslinked platelet lysate (PL) patches are proposed for the delivery of human-derived therapeutic factors in patch augmentation strategies aiming at tendon repair. Developed PL patches exhibited a controlled release profile of PL proteins, including bFGF and PDGF-BB. Additionally, PL patches exhibited an antibacterial effect by preventing the adhesion, proliferation and biofilm formation by S. aureus, a common pathogen in orthopaedic surgical site infections. Furthermore, these patches supported the activity of human tendon-derived cells (hTDCs). Cells were able to proliferate over time and an up-regulation of tenogenic genes (SCX, COL1A1 and TNC) was observed, suggesting that PL patches may modify the behavior of hTDCs. Accordingly, hTDCs deposited tendon-related extracellular matrix proteins, namely collagen type I and tenascin C. In summary, PL patches can act as a reservoir of biomolecules derived from PL and support the activity of native tendon cells, being proposed as bioinstructive patches for tendon regeneration. STATEMENT OF SIGNIFICANCE: Platelet-derived biomaterials hold great interest for the delivery of therapeutic factors for applications in endogenous regenerative medicine. In the particular case of tendon repair, patch augmentation strategies aiming at shifting the injury environment are explored to improve tendon regeneration. In this study, PL patches were developed with remarkable features, including the controlled release of growth factors and antibacterial efficacy. Remarkably, PL patches supported the activity of native tendon cells by up-regulating tenogenic genes and enabling the deposition of ECM proteins. This patch holds great potential towards simultaneously reducing post-implantation surgical site infections and promoting tendon regeneration for prospective in vivo applications.

Calcium phosphates and silicon: exploring methods of incorporation.

BACKGROUND: Bioinorganics have been explored as additives to ceramic bone graft substitutes with the aim to improve their performance in repair and regeneration of large bone defects. Silicon (Si), an essential trace element involved in the processes related to bone formation and remodeling, was shown not only to enhance osteoblasts proliferation but also to stimulate the differentiation of mesenchymal stem cells (MSCs) and preosteoblasts into the osteogenic lineage. In this study, the added value of Si to calcium phosphate (CaP) coatings was evaluated. METHODS: Tissue culture plastic well plates were coated with a thin CaP layer to which traces amounts of Si were added, either by adsorption or by incorporation through coprecipitation. The physicochemical and structural properties of the coatings were characterized and the dissolution behavior was evaluated. The adsorption/incorporation of Si was successfully achieved and incorporated ions were released from the CaP coatings. Human MSCs were cultured on the coatings to examine the effects of Si on cell proliferation and osteogenic differentiation. For the statistical analysis, a one-way ANOVA with Bonferroni post-hoc test was performed. RESULTS: The results showed that human MSCs (hMSCs) responded to the presence of Si in the CaP coatings, in a dose-dependent manner. An increase in the expression of markers of osteogenic differentiation by human MSCs was observed as a result of the increase in Si concentration. CONCLUSIONS: The incorporation/adsorption of Si into CaP coatings was successfully achieved and hMSCs responded with an increase in osteogenic genes expression with the increase of Si concentration. Furthermore, hMSCs cultured on CaP-I coatings expressed higher levels of ALP and OP, indicating that this may be the preferred method of incorporation of bioinorganics into CaPs.

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.

Redox activity of melanin from the ink sac of Sepia officinalis by means of colorimetric oxidative assay.

The redox properties of natural extract from cuttlefish ink sac (Sepia officinalis) and synthetic melanin used as a biomimetic in melanin structural investigation were determined by comparison of this phenol-based heterogeneous pigment with gallic acid used as a standard in Folin-Ciocalteu colorimetric assay widely employed for characterisation of oxidative properties of biomaterials. Reactivity of sepia melanin reported here is much higher than previously indicated and this protocol should allow the redox characterisation of all melanins irrespective of their origin and composition.

Unveiling the effect of three-dimensional bioactive fibre mesh scaffolds functionalized with silanol groups on bacteria growth.

The need to replace or repair deteriorating bones and simultaneously prevent the formation of bacteria biofilm without impairing local tissue integration has pushed scientists to look for new designs and processing methods to develop innovative biomaterials. Silicon-based biomaterials, widely studied for application in bone regeneration, have demonstrated antibacterial properties. Herein, the aim of this work is to investigate the potential of the functionalization of biomaterials surfaces with silanol groups to prevent the bacterial biofilm formation. For that, we evaluated the adherence and biofilm formation of Escherichia coli (E. coli, Gram negative) and Staphylococcus aureus (S. aureus, Gram positive) on starch-based scaffolds. Three-dimensional fibre meshes scaffolds were developed by wet-spinning and functionalized with silanol (Si-OH) groups using a calcium silicate solution as a nonsolvent. The functionalization of the scaffolds was confirmed by X-ray photoelectron spectroscopy. The developed scaffolds showed no biocide activity against the bacterial tested, although the colony-forming units (CFU) mL(-1) counts were significant lower between 4 and 12 h of incubation for both bacteria. The adherence of E. coli and S. aureus to the scaffolds was also investigated. After a growth period of 12 h, the SPCL scaffolds functionalized with Si-OH groups showed a reduced bacterial adherence of E. coli and S. aureus. The functionalized scaffolds showed a positive effect in preventing the formation of biofilm in the case of S. aureus, however, in the case of E. coli this was not observed, suggesting that silanol groups may only have a positive effect in preventing the proliferation of gram-positive bacteria. The in vitro biological assessment of the functionalized materials showed that these materials sustained cell proliferation and induced their osteogenic differentiation. The outcome of this work suggests that the presence of Si-OH groups in SPCL scaffolds maintained bactericidal activity against S. aureus. Further research is still needed in order to understand the full antibacterial potential of Si-OH groups. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2189-2199, 2016.

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.

Combinatorial Effect of Silicon and Calcium Release from Starch-Based Scaffolds on Osteogenic Differentiation of Human Adipose Stem Cells.

Instructive materials able to drive cells, in particular the differentiation of stem cells toward osteoblastic lineages, have been investigated as a promising strategy for bone tissue engineering. Inorganic ions, such as phosphorus, calcium, silicon, and strontium, have been used in bone regeneration strategies as instructive ions for material-based approaches. The use of effective inorganic ions is being investigated as a promising approach for bone regeneration applications, mainly because they are highly available and cost-effective and thus reducing the need to use expensive and less-stable growth factors. The aim of the present study is to investigate the effect of the release of silicon (Si) and calcium (Ca) ions from a blend of starch and poly caprolactone (SPCL) scaffolds on the osteogenic behavior of human adipose stem cells (hASCs). The scaffolds were developed by a wet-spinning technique and two different solutions were used as coagulation bath, one containing Ca and Si ions and other one containing only Si ions. The composition of the scaffolds as well as their mechanical properties was also evaluated. Our study showed that both scaffolds were able to sustain cell attachment and induce their differentiation into the osteogenic lineage in basal medium, i.e., in the absence of osteogenic factors. The scaffolds containing both ions, Si and Ca, had a stronger influence on the osteogenic differentiation of hASCs than the scaffolds containing only Si ion. Thus, the present work highlights the importance of combining Si and Ca ions in the control of cellular response, namely, cell differentiation and/or in stem cells recruitment upon implantation of a cell-free scaffold, and thus, avoiding the use of costly growth factors.

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.