Silk-Based Matrices and c-Kit-Positive Cardiac Progenitor Cells for a Cellularized Silk Fibroin Scaffold: Study of an in vivo Model.

The production of a cellularized silk fibroin scaffold is very difficult because it is actually impossible to differentiate cells into a well-organized cardiac tissue. Without vascularization, not only do cell masses fail to grow, but they may also exhibit an area of necrosis, indicating a lack of oxygen and nutrients. In the present study, we used the so-called tyrosine protein kinase kit (c-Kit)-positive cardiac progenitor cells (CPCs) to generate cardiac cellularized silk fibroin scaffolds, multipotent cells isolated from the adult heart to date that can show some degree of differentiation toward the cardiac phenotype. To test their ability to differentiate into the cardiac phenotype in vivo as well, CPC and collagen organoid-like masses were implanted into nude mice and their behavior observed. Since the 3-dimensional structure of cardiac tissue can be preserved by scaffolds, we prepared in parallel different silk fibroin scaffolds with 3 different geometries and tested their behavior in 3 different models of immunosuppressed animals. Unfortunately, CPC cellularized silk fibroin scaffolds cannot be used in vivo. CPCs implanted alone or in collagen type I gel were destroyed by CD3+ lymphocyte aggregates, whereas the porous and partially oriented scaffolds elicited a consistent foreign body response characterized by giant cells. Only the electrospun meshes were resistant to the foreign body reaction. In conclusion, c-Kit-positive CPCs, although expressing a good level of cardiac differentiation markers in vitro with or without fibroin meshes, are not suitable for an in vivo model of cardiac organoids because they are degraded by a T-cell-mediated immune response. Even scaffolds which may preserve the survival of these cells in vivo also induced a host response. However, among the tested scaffolds, the electrospun meshes (F-scaffold) induced a lower response compared to all the other tested structures.

Enthesis Healing Is Dependent on Scaffold Interphase Morphology-Results from a Rodent Patellar Model.

The use of multiphasic scaffolds to treat injured tendon-to-bone entheses has shown promising results in vitro. Here, we used two versions of a biphasic silk fibroin scaffold to treat an enthesis defect created in a rat patellar model in vivo. One version presented a mixed transition between the bony and the tendon end of the construct (S-MT) while this transition was abrupt in the second version (S-AT). At 12 weeks after surgery, the S-MT scaffold promoted better healing of the injured enthesis, with minimal undesired ossification of the insertion area. The expression of tenogenic and chondrogenic markers was sustained for longer in the S-MT-treated group and the tangent modulus of the S-MT-treated samples was similar to the native tissue at 12 weeks while that of the S-AT-treated enthesis was lower. Our study highlights the important role of the transition zone of multiphasic scaffolds in the treatment of complex interphase tissues such as the tendon-to-bone enthesis.

Development of alginate-based hydrogels for blood vessel engineering.

Vascular diseases are among the primary causes of death worldwide. In serious conditions, replacement of the damaged vessel is required. Autologous grafts are preferred, but their limited availability and difficulty of the harvesting procedures favour synthetic alternatives' use. However, as synthetic grafts may present significant drawbacks, tissue engineering-based solutions are proposed. Herein, tubular hydrogels of alginate combined with collagen type I and/or silk fibroin were prepared by ionotropic gelation using gelatin hydrogel sacrificial moulds loaded with calcium ions (Ca2+). The time of exposure of alginate solutions to Ca2+-loaded gelatin was used to control the wall thickness of the hydrogels (0.47 ± 0.10 mm-1.41 ± 0.21 mm). A second crosslinking step with barium chloride prevented their degradation for a 14 day period and improved mechanical properties by two-fold. Protein leaching tests showed that collagen type I, unlike silk fibroin, was strongly incorporated in the hydrogels. The presence of silk fibroin in the alginate matrix, containing or not collagen, did not significantly improve hydrogels' properties. Conversely, hydrogels enriched only with collagen were able to better support EA.hy926 and MRC-5 cells' growth and characteristic phenotype. These results suggest that a two-step crosslinking procedure combined with the use of collagen type I allow for producing freestanding vascular substitutes with tuneable properties in terms of size, shape and wall thickness.

Microfluidic-assisted electrospinning, an alternative to coaxial, as a controlled dual drug release system to treat inflammatory arthritic diseases.

Inflammatory arthritic diseases are characterized by a persistent inflammation of the synovial tissues where tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) pro-inflammatory cytokines are over-expressed, leading to progressive musculoskeletal disability. Methotrexate (MTX), a disease-modifying-anti-rheumatic drug (DMARD) commonly applied in their treatment, can be used in combination with biological-DMARDs as anti-TNFα antibody to improve the treatments efficacy. However, their systemic administration comes with severe side-effects and limited therapeutic efficacy due to their off-target distribution and short half-life. To overcome such limitations, encapsulation of clinically relevant concentrations of MTX and anti-TNFα antibody into polycaprolactone (PCL) or poly(vinyl-alcohol) (PVA) microfluidic-assisted or coaxial electrospun fibrous meshes is proposed as local controlled dual drug release systems. Release studies show that microfluidic-assisted electrospinning meshes encapsulating both drugs achieved higher concentrations than coaxials. Biological assays using human articular chondrocytes (hACs) and monocytic cells (THP-1 cell line) demonstrate that fibrous meshes encapsulating the drugs are non-toxic. The systems' efficacy is proved by a significant decrease of TNFα and IL-6 concentrations in conditioned medium of lipopolysaccharide (LPS)-stimulated THP-1 cells, especially in the presence of microfluidic-assisted electrospun meshes, when compared with THP-1 conditioned medium (59.5% and 83.9% less, respectively). Therefore, microfluidic-assisted electrospinning fibrous meshes with encapsulating drugs represent an alternative to coaxial, as a local therapy for inflammatory arthritis diseases.

Preparation and evaluation of gellan gum hydrogel reinforced with silk fibers with enhanced mechanical and biological properties for cartilage tissue engineering.

Various research about cartilage regeneration using biomaterials has been done recently. Particularly, gellan gum hydrogel (GG) is reported to be suitable as a biomaterial for cartilage tissue engineering (TE) for its water uptaking ability, producibility, and environmental resemblance of native cartilage. Despite these advantages, mechanical and cell adhesion properties are still difficult to modulate. Reinforcement is essential to overcome these problems. Herein, GG was modified by physically blending with different lengths of silk fiber (SF). As SF is expected to improve such disadvantages of GG, mechanical and biological properties were characterized to confirm its reinforcement ability. Mechanical properties such as degradation rate, swelling rate, compression strength, and viscosity were studied and it was confirmed that SF significantly reinforces the mechanical properties of GG. Furthermore, in vitro study was carried out to confirm morphology, biocompatibility, proliferation, and chondrogenesis of chondrocytes encapsulated in the hydrogels. Overall, chondrocytes in the GG blended with SF (SF/GG) showed enhanced cell viability and growth. According to this study, SF/GG can be a promising biomaterial for cartilage TE biomaterial.

Multinucleated Giant Cells Induced by a Silk Fibroin Construct Express Proinflammatory Agents: An Immunohistological Study.

Multinucleated giant cells (MNGCs) are frequently observed in the implantation areas of different biomaterials. The main aim of the present study was to analyze the long-term polarization pattern of the pro- and anti-inflammatory phenotypes of macrophages and MNGCs for 180 days to better understand their role in the success or failure of biomaterials. For this purpose, silk fibroin (SF) was implanted in a subcutaneous implantation model of Wistar rats as a model for biomaterial-induced MNGCs. A sham operation was used as a control for physiological wound healing. The expression of different inflammatory markers (proinflammatory M1: CCR-7, iNos; anti-inflammatory M2: CD-206, CD-163) and tartrate-resistant acid phosphatase (TRAP) and CD-68 were identified using immunohistochemical staining. The results showed significantly higher numbers of macrophages and MNGCs within the implantation bed of SF-expressed M1 markers, compared to M2 markers. Interestingly, the expression of proinflammatory markers was sustained over the long observation period of 180 days. By contrast, the control group showed a peak of M1 macrophages only on day 3. Thereafter, the inflammatory pattern shifted to M2 macrophages. No MNGCs were observed in the control group. To the best of our knowledge, this is study is the first to outline the persistence of pro-inflammatory MNGCs within the implantation bed of SF and to describe their long-term kinetics over 180 days. Clinically, these results are highly relevant to understand the role of biomaterial-induced MNGCs in the long term. These findings suggest that tailored physicochemical properties may be a key to avoiding extensive inflammatory reactions and achieving clinical success. Therefore, further research is needed to elucidate the correlation between proinflammatory MNGCs and the physicochemical characteristics of the implanted biomaterial.

A novel and selective silk fibroin fragmentation method.

In the tissue-engineering field silk fibroin can be tailored to the target applications by modifying its secondary structure and molecular weight, and functionalizing the molecule with specific active groups linked to the amino acid side chains. To better tune the silk fibroin molecular weight and structural properties, we propose the creation of a lower molecular weight fibroin-derived material through a selective and tunable enzymatic attack on the fibroin chain. Cleavage at specific amino acid sites leads to precise silk fibroin fragmentation and, thus, lower molecular weight materials whose length and properties can be tuned with the enzyme concentration. The cleavage increased the presence of free amino groups, hence reactivity, and aqueous solutions of the resulting polymer remained stable for up to seven days. Films of fragmented fibroin were prepared and characterized, demonstrating that the fragmentation did not affect ß-sheet formation after methanol treatment, but differences were detected after the water-vapor annealing process, confirmed by structural and thermal analyses. The adopted fragmentation method is fast, controllable and precise, allowing the creation of a silk-derived material class that is stable in water, with a tunable molecular weight and secondary structure rearrangements, and is thus a versatile tool for the further tunability and modulation of bioengineered constructs.

Injectable Scaffold-Systems for the Regeneration of Spinal Cord: Advances of the Past Decade.

Nowadays, whenever is possible and as an alternative to open spine surgery, minimally invasive procedures are preferred to treat spinal cord injuries (SCI), with percutaneous injections or small incisions, that are faster, less traumatic, and require less recovery time. Injectable repair systems are based on materials that can be injected in the lesion site, can eventually be loaded with drugs or even cells, and act as scaffolds for the lesion repair. The review analyzes papers written from 2010 onward on injectable materials/systems used/proposed for the regenerative and combinatorial therapies of SCI and discusses the in vivo models that have been used to validate them.

A Bio-inspired Multifunctionalized Silk Fibroin.

A bio-inspired multifunctionalized silk fibroin (BMS) was synthesized in order to mimic the interaction of nidogen with the type IV collagen and laminin of basement membranes. The designed BMS consists of a motif of laminin α-chain-derived, called IK peptide, and type IV collagen covalently bound to the silk fibroin (SF) by using EDC/NHS coupling and a Cu-free click chemistry reaction, respectively. Silk fibroin was chosen as the main component of the BMS because it is versatile and biocompatible, induces an in vivo favorable bioresponse, and moreover can be functionalized with different methods. The chemical structure of BMS was analyzed by using X-ray photoelectron spectroscopy, attenuated total reflection-Fourier transform infrared, cross-polarization magic angle spinning nuclear magnetic resonance techniques, and colorimetric assay. The SF and BMS solutions were cross-linked by sonication to form hydrogels or casted to make films in order to evaluate and compare the early adhesion and viability of MRC5 cells. BMS hydrogels were also characterized by rheological and thermal analyses.

Natural Fibrous Protein for Advanced Tissue Engineering Applications: Focusing on Silk Fibroin and Keratin.

As one of the important branches of natural biopolymer, natural fibrous protein has a lot of advantages including good mechanical properties, excellent biocompatibility, controllable biodegradability, renewability, abundant sources, and so on. Moreover, natural fibrous protein is also a protein that could only be used for structure supporting without any bioactivities, which attracts a lot of attentions in the field of tissue engineering scaffold. This chapter is taking silk fibroin and keratin as model materials of natural fibrous protein, focusing on their protein structure, chemical compositions, processing and extraction methods, chemical modification methods, and their applications in tissue engineering through advanced manufacturing.

Spider (Linothele megatheloides) and silkworm (Bombyx mori) silks: Comparative physical and biological evaluation.

Silks, in particular silkworm silks, have been studied for decades as possible candidate materials for biomedical applications. Recently, great attentions have been paid to spider silks, mainly due to their unique and remarkable mechanical properties. Both materials express singular interactions with cells through specific biorecognition moieties on the core proteins making up the two silks. In this work, the silk from a Colombian spider, Linothele megatheloides (LM), which produces a single type of silk in a relatively large amount, was studied in comparison with silk from Bombyx mori silkworm, before and after degumming, with the evaluation of their chemical, mechanical and biological properties. Unexpected biological features in cell culture tests were found for the LM silk already at very early stage, so suggesting further investigation to explore its use for tailored biomedical applications.

Injectable taurine-loaded alginate hydrogels for retinal pigment epithelium (RPE) regeneration.

The purpose of this study is to produce injectable taurine (Tr)-loaded alginate (Agn) hydrogel for age-related macular degeneration (AMD) treatment by inducing the regeneration of RPE (retinal pigment epithelium) cells. Porosity and swelling ratio were measured to evaluate the mechanical properties of the hydrogels, and Fourier transform infrared spectroscopy (FTIR) was used to evaluate the physical and chemical properties. RPE cells extracted from the pigmented epithelium of rabbits were encapsulated in the Tr/Agn hydrogels. Cells proliferation and migration were improved in Tr/Agn hydrogels with an enhanced expression of RPE-specific genes including RPE65, CRALBP, NPR-A, MITF and collagen type I and II. In vivo tests demonstrated the excellent biocompatibility and biodegradability without inflammatory response by the host when implanted with the hydrogel. Moreover, when the Tr/Agn hydrogels were injected into the sub-retinal space, high adhesion of RPE cells and retinal regeneration were confirmed. These results demonstrated a potential role of injectable Tr/Agn hydrogels as potential therapeutic tools for the treatment of retinal diseases, including AMD.

Synthesis of Gold Nanoparticles Decorated with Multiwalled Carbon Nanotubes (Au-MWCNTs) via Cysteaminium Chloride Functionalization.

Gold nanoparticles (AuNPs) decorated CNTs are promising materials for photocatalytics and biosensors. However, the synthesis of AuNPs chemically linked to the walls of MWCNTs is challenging and toxic products such as thionylchloride (SOCl2) or [1-ethyl-3(dimethyl-amino) propyl] carbodiimide hydrochloride (EDAC) need to be used. This work reports a new approach to prepare gold nanoparticles decorated multiwalled carbon nanotubes (MWCNTs) by using cysteaminium chloride via the formation of a Zwitterionic acide-base bond. The grafting process consists of 3 mains steps: oxidation, thiolation and decoration of AuNPs on the surface of MWCNTs. The completion of each step has been verified out by both spectroscopic (Raman, UV-Vis, FT-IR) and Scanning Electron Miscroscopy (SEM). The chemical bonding states of synthesized products have been proven by X-ray photoelectron spectroscopy (XPS).

Breath Figures decorated silicon oxinitride ceramic surfaces with controlled Si ions release for enhanced osteoinduction.

Bioactive coatings are usually applied to bone and dental prostheses to enhance the integration and their stability in the bone. Recently, silicon (Si) oxynitride ceramics have been demonstrated to possess osteoconductive properties due to the release of Si ions, particularly important in the early stage of bone formation. In addition, the pattern of the bone contacting surface has been reported to affect cells' differentiation and metabolic activity. In this work, we propose the Breath Figure (BF) process combined with a pyrolysis step, starting from a photo-crosslinkable alkoxy silicone precursor, as a method to realize bioactive patterned coating on metal bone and dental prostheses. Four different surface patterned coatings were applied to Ti4Al6V disks starting from solutions with different precursor concentrations. Morphology, chemical composition, and Si ions' release were evaluated and compared. Moreover, all samples underwent to biological in vitro testing with human mesenchymal stem cells (hMSCs) in comparison with the uncoated titanium alloy. The results indicated that the Si released from the coatings determined an increase in the cellular activity with the BF pattern influencing the hMSCs' initial adhesion and proliferation. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1284-1294, 2019.

Sodium oleate induced rapid gelation of silk fibroin.

Silk fibroin has acquired increasing interest in the last years for application in medicine and namely in tissue engineering. Several methods have been developed to process fibroin and for the fabrication of nets, sponges, films and gels. This paper deals with the fabrication and characterization of fibroin hydrogels obtained by using sodium oleate as gelation agent. Gels have been prepared by mixing Silk fibroin (SF) and Sodium oleate (SO) water solutions in different concentrations, and a quite wide frame of compositions have been explored. Rheological tests have been performed to determine the gelation times, scanning electron microscopies have been made to evaluate morphologies, FTIR analysis has been done to determine the conformation of the starting materials and of the resulting gels, water content has been measured and cytotoxicity tests have been performed to validate the potential biomedical use of the hydrogels. Depending on the SF and SO different gelation times have been obtained thanks to the formation of intermolecular bonds between the fibroin chains. The obtained fastest gelation of about 80 s could make this specific formulation compatible with in situ gelation. By changing composition, gels with different morphologies, rheological properties and water contents have been prepared.

Heparin functionalization increases retention of TGF-ß2 and GDF5 on biphasic silk fibroin scaffolds for tendon/ligament-to-bone tissue engineering.

The tendon/ligament-to-bone transition (enthesis) is a highly specialized interphase tissue with structural gradients of extracellular matrix composition, collagen molecule alignment and mineralization. These structural features are essential for enthesis function, but are often not regenerated after injury. Tissue engineering is a promising strategy for enthesis repair. Engineering of complex tissue interphases such as the enthesis is likely to require a combination of biophysical, biological and chemical cues to achieve functional tissue regeneration. In this study, we cultured human primary adipose-derived mesenchymal stem cells (AdMCs) on biphasic silk fibroin scaffolds with integrated anisotropic (tendon/ligament-like) and isotropic (bone/cartilage like) pore alignment. We functionalized those scaffolds with heparin and explored their ability to deliver transforming growth factor ß2 (TGF-ß2) and growth/differentiation factor 5 (GDF5). Heparin functionalization increased the amount of TGF-ß2 and GDF5 remaining attached to the scaffold matrix and resulted in biological effects at low growth factor doses. We analyzed the combined impact of pore alignment and growth factors on AdMSCs. TGF-ß2 and pore anisotropy synergistically increased the expression of tendon/ligament markers and collagen I protein content. In addition, the combined delivery of TGF-ß2 and GDF5 enhanced the expression of cartilage markers and collagen II protein content on substrates with isotropic porosity, whereas enthesis markers were enhanced in areas of mixed anisotropic/isotropic porosity. Altogether, the data obtained in this study improves current understanding on the combined effects of biological and structural cues on stem cell fate and presents a promising strategy for tendon/ligament-to-bone regeneration. STATEMENT OF SIGNIFICANCE: Regeneration of the tendon/ligament-to-bone interphase (enthesis) is of significance in the repair of ruptured tendons/ligaments to bone to improve implant integration and clinical outcome. This study proposes a novel approach for enthesis regeneration based on a biomimetic and integrated tendon/ligament-to-bone construct, stem cells and heparin-based delivery of growth factors. We show that heparin can keep growth factors local and biologically active at low doses, which is critical to avoid supraphysiological doses and associated side effects. In addition, we identify synergistic effects of biological (growth factors) and structural (pore alignment) cues on stem cells. These results improve current understanding on the combined impact of biological and structural cues on the multi-lineage differentiation capacity of stem cells for regenerating complex tissue interphases.

Silk fibroin porous scaffolds by N2O foaming.

Silk fibroin has acquired increasing interest for biomedical applications, and namely for the fabrication of scaffolds for tissue engineering, because of its highly positive biological interaction and the possibility to adapt the material to several application requirements by adopting different fabrication methods, in order to make films, sponges, fibers, nets or gels with predictable degradation times. For tissue engineering, in most cases porous scaffolds are required, in some cases possibly in situ forming and therefore fabricated in mild body-compatible conditions. In this work, we present a novel one-step method for the preparation of silk fibroin foams starting from water solutions and using low-pressure nitrous oxide gas as foaming agent. This foaming technique allows preparing fibroin porous scaffolds with easily tunable porosity, in mild processing conditions with the use of a relatively inert foaming agent saturating a fibroin water solution, that could be occasionally injected through a thin needle in the implantation site where expansion and foaming would occur. Optimal foaming processing conditions have been investigated, and the prepared foams have been characterized with Fourier Transform Infrared Spectroscopy (FTIR) compressive mechanical and rheological properties measurements, and by scanning electron microscopy and microCT.

Effect of Cryopreservation on Cell-Laden Hydrogels: Comparison of Different Cryoprotectants.

Cell encapsulation in hydrogels is a technique that offers a variety of applications, ranging from drug delivery to biofabrication of three-dimensional scaffolds. The assembly of cell-laden hydrogel building blocks aims to generate complex biological constructs by manipulating microscale units. An important issue for the clinical implementation of this technique is the long-term storage of a large stock of cell/hydrogel building blocks. In this work, the impact of cryopreservation on the viability and functionality of cells encapsulated in alginate matrices is presented comparing different cryoprotective agents (CPAs). Human osteosarcoma MG63 cells were encapsulated in sodium alginate fiber constructs with wetspinning method and exposed to different formulations of cryopreservation media, containing dimethyl sulfoxide (DMSO), glycerol, and trehalose. The cell-laden fibers were subsequently slow-cooled down to -80°C and stored in liquid nitrogen. After thawing, viability and death pathway of encapsulated cells were investigated, and metabolic activity and proliferative capacity of cells released from the alginate matrix were evaluated. The viability of MG63 cells encapsulated in alginate matrix ranged from 71% ± 4% to 85% ± 2%, depending on the cryoprotective media formulation with no protracted harmful effects from the CPAs. On the other side, cells cryopreserved in encapsulated conditions and released from the hydrogel showed larger metabolic activity and proliferative capacity in tissue culture plate compared to cells cryopreserved in suspension, in particular when DMSO and glycerol were used as CPAs. Results have been correlated with the viscoelastic properties and water content changes of the alginate constructs loaded with the different CPAs.

Bioactivity and mineralization of natural hydroxyapatite from cuttlefish bone and Bioglass® co-sintered bioceramics.

In this study, bioactive hydroxyapatite (HAP)-based bioceramics starting from cuttlefish bone powders have been prepared and characterized. In particular, fragmented cuttlefish bone was co-sintered with 30 wt% of Bioglass® -45S5 to synthesize HAP-based powders with enhanced mechanical properties and bioactivity. Commercial synthetic HAP was treated following the same procedure and used as a reference. The structure and composition of the bioceramics formulations were characterized using Fourier transform infrared spectroscopy, X-ray diffraction and scanning electron microscopy. After the thermal treatment of cuttlefish bone powder added with 30 wt% Bioglass, new phases with compositions of sodium calcium phosphate [Na3 Ca6 (PO4 )5 ], ß-tricalcium phosphate [Ca3 (PO4 )] and amorphous silica were detected. In vitro cell culture studies were performed by evaluating proliferation, metabolic activity and differentiation of human osteoblast-like cells (MG63). Scaffolds made with cuttlefish bone powder exhibited increased apatite deposition, alkaline phosphatase activity and cell proliferation compared with commercial synthetic HAP. In addition, the ceramic compositions obtained after the combination with Bioglass® further enhanced the metabolic activity of MG63 cell and promoted the formation of a well-developed apatite layer after 7 days of incubation in Dulbecco's modified Eagle's medium.

Enhancing bioactive properties of silk fibroin with diatom particles for bone tissue engineering applications.

Many studies have highlighted the role of silicon in human bone formation and maintenance. Silicon, in fact, is considered to nucleate the precipitation of hydroxyapatite and to reduce the bone resorption. For this reason, we have combined silk fibroin (SF) with silicon-releasing diatom particles (DPs), as potential material for bone tissue engineering applications. Sponges of fibroin loaded with different amounts and sizes of DPs were prepared by solvent casting-particulate leaching method, and their morphology, porosity and mechanical properties were evaluated. The biological effect of diatom addition was assessed on human osteosarcoma cell line MG63, a suitable osteoblast-like model, through cell adhesion, metabolic activity and proliferation assays. In addition, alkaline phosphatase activity, osterix and collagen type I production in MG63 cell line were assessed as markers of early bone formation to demonstrate a pro-mineralization potential of scaffolds. Results of the studies showed that addition to fibroin of diatoms particles improved the osteogenic properties of osteoblast-like cells compared with the pure SF. Copyright © 2016 John Wiley & Sons, Ltd.