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The biocompatibility and chemical stability of implantable devices are crucial for their long-term success. CarboSil® is a silicon polycarbonate polyurethane copolymer with good biocompatibility and biostability properties. Here, we explored the possibility to improve these characteristics by introducing 30% of extra-chain cross-linkable poly(dimethyl siloxane) (PDMS). Patches made of CarboSil and CarboSil-30% PDMS were manufactured by spray, phase-inversion technique and subjected to a heating-pressure treatment. Both materials showed good biocompatibility, either in viability and proliferation of cell-based experiments both with mouse fibroblasts and subcutaneous implant in rats. Fourier-transform infrared spectroscopy showed a significant decrease in soft segment loss in CarboSil-30% PDMS samples with respect to CarboSil in in vitro accelerated oxidative treatments with CoCl

Rare earth elements (REEs) are critical raw materials with a wide range of industrial applications. As a result, the recovery of REEs via adsorption from REE-rich matrices, such as water streams from processed electric and electronic waste, has gained increased attention for its simplicity, cost-effectiveness and high efficacy. In this work, the potential of nanometric cerium oxide-based materials as adsorbents for selected REEs is investigated. Ultra-small cerium oxide nanoparticles (CNPs, mean size diameter ≈ 3 nm) were produced via a precipitation-hydrothermal procedure and incorporated into woven–non-woven polyvinyl alcohol (PVA) nanofibres (d ≈ 280 nm) via electrospinning, to a final loading of ≈34 wt%. CNPs, CNP–PVA and the benchmark material CeO2 NM-212 (JRCNM02102, mean size diameter ≈ 28 nm) were tested as adsorbents for aqueous solutions of the REEs Eu3+, Gd3+ and Yb3+ at pH 5.8. Equilibrium adsorption data were interpreted by means of Langmuir and Freundlich data models. The maximum adsorption capacities ranged between 16 and 322 mgREE gCeO2−1, with the larger value found for the adsorption of Yb3+ by CNP. The trend of maximum adsorption capacity was CNPs > NM-212 > CNP–PVA, which was ascribed to different agglomeration and surface area available for adsorption. Langmuir equilibrium constants KL were substantially larger for CNP–PVA, suggesting a potential higher affinity of REEs for CNPs due to a synergistic effect of PVA on adsorption. CNP–PVA were effectively used in repeated adsorption cycles under static and dynamic configurations and retained the vast majority of adsorptive material (>98% of CeO2 retained after 10 adsorption cycles). The small loss was attributed to partial solubilisation of fibre components with change in membrane morphology. The findings of this study pave the way for the application of CNP–PVA nanocomposites in the recovery of strategically important REEs from electrical and electronic waste.

Intrinsic properties of a compound (e.g., electronic structure, crystallographic structure, optical and magnetic properties) define notably its chemical and physical behavior. In the case of nanomaterials, these fundamental properties depend on the occurrence of quantum mechanical size effects and on the considerable increase of the surface to bulk ratio. Here, we explore the size dependence of both crystal and electronic properties of CeO2 nanoparticles (NPs) with different sizes by state-of-the art spectroscopic techniques. X-ray diffraction, X-ray photoelectron spectroscopy, and high-energy resolution fluorescence-detection hard X-ray absorption near-edge structure (HERFD-XANES) spectroscopy demonstrate that the as-synthesized NPs crystallize in the fluorite structure and they are predominantly composed of CeIV ions. The strong dependence of the lattice parameter with the NPs size was attributed to the presence of adsorbed species at the NPs surface thanks to Fourier transform infrared spectroscopy and thermogravimetric analysis measurements. In addition, the size dependence of the t2g states in the Ce LIII XANES spectra was experimentally observed by HERFD-XANES and confirmed by theoretical calculations., Comment: Inorganic Chemistry (2020)

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., This work was supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, for the Ph.D grant of Catarina Silva (UMINHO/BD/33/2016; NORTE-08-5369-FSE-000012), and by the Portuguese Science and Technology Foundation (FCT) for the cells project Cells4_ID (PTDC/BTM-SAL/28882/2017).

Vascular diseases arc among the primary causes of death worldwide. In serious conditions, replacement of the damaged vessel is required. Autologous grafts arc 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 arc 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 (Ca-2(+)). 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 MRG5 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., This work was supported by the Portuguese Foundation for Science and Technology (FCT) by the project Cells4_ID (PTDC/BTM-SAL/ 28882/2017) and the NORTE 2020 Structured Project, cofunded by Norte2020 (NORTE-01-0145-FEDER-000021).

Objective: To fabricate and investigate the properties of fibroin and polyvinyl alcohol (PVA) hydrogels containing sericin prepared using high-pressure carbon dioxide (CO(2)). Approach: In this study, fibroin/PVA hydrogels with and without sericin were prepared using the high-pressure CO(2) method. The physical and mechanical properties of the hydrogels were investigated using field-emission scanning electron microscopy, Fourier-transform infrared spectroscopy, thermogravimetric analysis, and differential scanning calorimetry, and the swelling, water retention, and compressive properties were assessed. Results: The hydrogels obtained from the combination of fibroin and PVA presented a compositional gradient along the hydrogel thickness and structure. The upper layer of the hydrogel consisted of a fibroin-based hydrogel blended with PVA, whereas the lower layer contained only fibroin. The mechanical properties regarding compression of the fibroin/PVA hydrogel were significantly better than those of the pure fibroin hydrogel, for hydrogels with and without sericin. Moreover, the mechanical properties of the hydrogels with sericin were significantly better than those without sericin. The water contents of all samples were >90%. Innovation: This study assessed a new combination of a wound healing agent and a biomaterial dressing. Moreover, this hydrogel production technique used a clean method without the need for a chemical crosslinking agent. Conclusion: The combination of the fibroin and PVA hydrogel and sericin prepared using the high-pressure CO(2) method led to good physical properties. This material may be a candidate for medical applications.

In this work, novel electrically conductive cyclic olefin copolymer/exfoliated graphite nanoplatelets foams were prepared through a supercritical carbon dioxide treatment starting from the corresponding unfoamed materials prepared by melt compounding, in order to investigate their thermo-electrical properties. For both unfoamed and foamed samples, the exfoliated graphite nanoplatelets introduction led to a systematic enhancement of the thermal degradation temperature. Dynamic-mechanical thermal analysis revealed that the nanofiller addition promoted an enhancement of the storage modulus and of the glass transition temperature over the whole range of the applied foaming pressures. While for unfoamed materials exfoliated graphite nanoplatelets introduction determined an important decrease of the electrical resistivity, the foaming process induced the breakage of the conductive path, with a consequent increase of electrical resistivity. Evaluation of the surface heating upon voltage application showed that the surface temperature of unfoamed materials could be noticeably increased at relatively low voltage levels, while a less pronounced surface heating could be obtained with the corresponding nanocomposite foams.

Tissue regeneration requires scaffolds that exhibit mechanical properties similar to the tissues to be replaced while allowing cell infiltration and extracellular matrix production. Ideally, the scaffolds’ porous architecture and physico-chemical properties can be precisely defined to address regenerative needs. We thus developed techniques to produce hybrid fibers coaxially structured with a polycaprolactone core and a 4-arm, polyethylene glycol thiol-norbornene sheath. We assessed the respective effects of crosslink density and sheath polymer size on the scaffold architecture, physical and mechanical properties, as well as cell-scaffold interactions in vitro and in vivo. All scaffolds displayed high elasticity, swelling and strength, mimicking soft tissue properties. Importantly, the thiol-ene hydrogel sheath enabled tunable softness and peptide tethering for cellular activities. With increased photopolymerization, stiffening and reduced swelling of scaffolds were found due to intra- and inter- fiber crosslinking. More polymerized scaffolds also enhanced the cell-scaffold interaction in vitro and induced spontaneous, deep cell infiltration to produce collagen and elastin for tissue regeneration in vivo. The molecular weight of sheath polymer provides an additional mechanism to alter the physical properties and biological activities of scaffolds. Overall, these robust scaffolds with tunable elasticity and regenerative cues offered a versatile and effective platform for tissue regeneration.

Rare earth elements (REEs) are critical raw materials with a wide range of industrial applications. As a result, the recovery of REEs

Hydrogen sulfide (H2S) is a physiological gasotransmitter known to possess a regulatory role in several tissues, including bone. The exogenous administration by injection of solutions of H2S-releasing compounds (e.g., GYY4137) has been previously investigated as a novel therapeutic approach for the treatment of bone diseases. Here, GYY4137 was embedded into fibroin sponges, previously shown to be suitable as scaffolds for bone, thanks to their biocompatibility, scalable porous structure, and biodegradability rate. Fibroin porous scaffolds were produced by solvent casting and the particulate leaching method, and GYY4137 was successively incorporated by using dimethyl sulfoxide (DMSO) as vehicle. The process used to produce GYY4137-loaded scaffolds allowed the incorporation of different controlled amounts of GYY4137 into fibroin matrices. The loading process preserved the properties of the system components in the final products, as assessed by SEM, FT-IR, NMR, and different thermal analyses techniques. Rel...

A wide variety of hydrogels have been proposed for tissue engineering applications, cell encapsulation, and bioinks for bioprinting applications. Cell-laden hydrogel constructs rely on natural hydrogels such as alginate, agarose, chitosan, collagen, gelatin, fibroin, and hyaluronic acid (HA), as well as on synthetic hydrogels such as poloxamers (Pluronics

Intrinsic properties of a compound (e.g., electronic structure, crystallographic structure, optical and magnetic properties) define notably its chemical and physical behavior. In the case of nanomaterials, these fundamental properties depend on the occurrence of quantum mechanical size effects and on the considerable increase of the surface to bulk ratio. Here, we explore the size dependence of both crystal and electronic properties of CeO

A wide variety of hydrogels have been proposed for tissue engineering applications, cell encapsulation, and bioinks for bioprinting applications. Cell-laden hydrogel constructs rely on natural hydrogels such as alginate, agarose, chitosan, collagen, gelatin, fibroin, and hyaluronic acid (HA), as well as on synthetic hydrogels such as poloxamers (Pluronics®) and polyethylene glycol (PEG). Alginate has become more and more important in the last years, thanks to the possibility to prepare alginate hydrogels suitable for cell encapsulation mainly because of the mild and reversible cross-linking conditions. In this paper alginate will be described in detail with respect to its chemistry, cross-linking behavior, biocompatibility, manufacturing capacity, and possible modifications.

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.

Dissolution and gelation procedures have a great influence on gelation time, microstructure and mechanical properties of reconstituted collagen products. We have investigated the dissolution of atelocollagen in CO2/water solutions at low temperature (4 degrees C) at different CO2 pressures (0.3-0.9 MPa), as well as gelation kinetics and physico-chemical properties of the hydrogel obtained after CO2 removal. Compared to conventional methods, the CO2-assisted technique resulted in faster soluble collagen dissolution and faster gelation into transparent gels characterized by thin 10 nm fibrils. Electrophoresis and CD spectroscopy demonstrated that the process did not denature the soluble collagen. The possibility to obtain collagen dissolution and gelation without the use of chemical agent other than water and CO2 makes this process particularly appealing for biomedical 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.

The regeneration of bone tissue via tissue engineering procedures is a challenging task that requires the selection of materials/scaffolds, with tailored biological and physical properties, and possibly the use of osteogenic additives that can stimulate cells to produce and mineralize collagen. Apart from growth factors, whose use is restricted by their high cost, inorganic compounds, such as calcium phosphates or silicates, have demonstrated intrinsic ability to facilitate the collagen mineralization and to address the differentiation of cells towards osteoblast lineage. Silicon, in particular, is considered to have a great relevance in the early stages of bone formation. In the past, we investigated the use of silk fibroin scaffolds, in form of sponges or gels, for bone tissue engineering. In this paper we have combined silk fibroin with diatoms, that are silica-based organisms, and we have evaluated the biological response of human mesenchymal stem cells (hMSCs) cultured on diatom loaded fibroin sponges. We found proofs of the improved osteogenic activity of the diatoms loaded scaffolds in the increase of alkaline phosphatase activity (ALP) and in the early fibronectin and collagen type I formation.

Silicon plays an important role in bone formation and maintenance, improving osteoblast cell function and inducing mineralization. Often, bone deformation and long bone abnormalities have been associated with silica/silicon deficiency. Diatomite, a natural deposit of diatom skeleton, is a cheap and abundant source of biogenic silica. The aim of the present study is to validate the potential of diatom particles derived from diatom skeletons as silicon-donor materials for bone tissue engineering applications. Raw diatomite (RD) and calcined diatomite (CD) powders were purified by acid treatments, and diatom microparticles (MPs) and nanoparticles (NPs) were produced by fragmentation of purified diatoms under alkaline conditions. The influence of processing on the surface chemical composition of purified diatomites was evaluated by X-ray photoelectron spectroscopy (XPS). Diatoms NPs were also characterized in terms of morphology and size distribution by transmission electron microscopy (TEM) and Dynamic light scattering (DLS), while diatom MPs morphology was analyzed by scanning electron microscopy (SEM). Surface area and microporosity of the diatom particles were evaluated by nitrogen physisorption methods. Release of silicon ions from diatom-derived particles was demonstrated using inductively coupled plasma optical emission spectrometry (ICP/OES); furthermore, silicon release kinetic was found to be influenced by diatomite purification method and particle size. Diatom-derived microparticles (MPs) and nanoparticles (NPs) showed limited or no cytotoxic effect in vitro depending on the administration conditions.

A cycloolefin copolymer matrix was melt mixed with exfoliated graphite nanoplatelets (xGnP) and the resulting nanocomposites were foamed by supercritical carbon dioxide. The density of the obtained foams decreased with the foaming pressure. Moreover, xGnP limited the cell growth during the expansion process thus reducing the cell diameter (from 1.08 to 0.22 mm with an XGnP amount of 10 wt% at 150 bar) and increasing the cell density (from 12 to 45 cells/mm 2 with a nanofiller content of 10 wt% at 150 bar). Electron microscopy observations of foams evidenced exfoliation and orientation of the nanoplatelets along the cell walls. Quasi-static compressive tests and tensile creep tests on foams clearly indicated that xGnP improved the modulus (up to a factor of 10 for a xGnP content of 10 wt%) and the creep stability.

Hydrogen sulfide (H2S)-based therapy is a promising therapeutic strategy for several biomedical applications. Following the observation that endogenous and exogenous H2S plays a prominent role as a bone anabolic agent, we recently developed a silk fibroin (SF) porous scaffold loaded with GYY4137 (GYY), an H2S donor, for applications in bone tissue engineering. Here, we assayed whether the combination of SF with H2S potentiates the osteoconductive properties of SF. Biocompatibility and osteoanabolic activity were assayed in vitro using human bone marrow mesenchymal stromal cells. Cell cultures were performed on a perfusion bioreactor to obtain results closer to the in vivo microenvironment. Cytotoxicity was excluded by lactate dehydrogenase and live/dead assays. Cell colonization and mineral apposition were evaluated by Haematoxylin & Eosin and Von Kossa/Alizarin Red-S stainings respectively. PCR array for human osteogenesis and immunohistochemical analyses were performed to identify pathways and targets involved. Our findings show that H2S-releasing SF scaffolds supported cell adhesion, proliferation and viability. Moreover, H2S activated genes and proteins involved in ossification, osteoblast differentiation, bone mineral metabolism and angiogenesis allowing a high and early mineralization. Based on these properties, we suggest the use of H2S-releasing SF scaffolds for bone healing and regeneration applications.

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 β2 (TGF-β2) and growth/differentiation factor 5 (GDF5). Heparin functionalization increased the amount of TGF-β2 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-β2 and pore anisotropy synergistically increased the expression of tendon/ligament markers and collagen I protein content. In addition, the combined delivery of TGF-β2 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.

Materials selection is a critical aspect for the production of scaffolds for osteochondral tissue engineering. Synthetic materials are the result of man-made operations and have been investigated for a variety of tissue engineering applications. Instead, the products of physiological processes and the metabolic activity of living organisms are identified as natural materials. Over the recent decades, a number of natural materials, namely, biopolymers and bioceramics, have been proposed as the main constituent of osteochondral scaffolds, but also as cell carriers and signaling molecules. Overall, natural materials have been investigated both in the bone and in the cartilage compartment, sometimes alone, but often in combination with other biopolymers or synthetic materials. Biopolymers and bioceramics possess unique advantages over their synthetic counterparts due similarity with natural extracellular matrix, the presence of cell recognition sites and tunable chemistry. However, the characteristics of natural origin materials can vary considerably depending on the specific source and extraction process. A deeper understanding of the relationship between material variability and biological activity and the definition of standardized manufacturing procedures will be crucial for the future of natural materials in tissue engineering.

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.

Materials selection is a critical aspect for the production of scaffolds for osteochondral tissue engineering. Synthetic materials are the result of man-made operations and have been investigated for a variety of tissue engineering applications. Instead, the products of physiological processes and the metabolic activity of living organisms are identified as natural materials. Over the recent decades, a number of natural materials, namely, biopolymers and bioceramics, have been proposed as the main constituent of osteochondral scaffolds, but also as cell carriers and signaling molecules. Overall, natural materials have been investigated both in the bone and in the cartilage compartment, sometimes alone, but often in combination with other biopolymers or synthetic materials. Biopolymers and bioceramics possess unique advantages over their synthetic counterparts due similarity with natural extracellular matrix, the presence of cell recognition sites and tunable chemistry. However, the characteristics of natural origin materials can vary considerably depending on the specific source and extraction process. A deeper understanding of the relationship between material variability and biological activity and the definition of standardized manufacturing procedures will be crucial for the future of natural materials in tissue engineering.

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.

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.

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 ], β-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.

Tissue engineering is an attractive strategy for tendon/ligament-to-bone interface repair. The structure and extracellular matrix composition of the interface are complex and allow for a gradual mechanical stress transfer between tendons/ligaments and bone. Thus, scaffolds mimicking the structural features of the native interface may be able to better support functional tissue regeneration. In this study, we fabricated biphasic silk fibroin scaffolds designed to mimic the gradient in collagen molecule alignment present at the interface. The scaffolds had two different pore alignments: anisotropic at the tendon/ligament side and isotropic at the bone side. Total porosity ranged from 50% to 80% and the majority of pores (80-90%) were

Injectable drug-loaded matrices and controlled release technology offer numerous advantages over conventional dosages. Cross-linkable alginate hydrogels have been proposed for in vivo injection, but their large initial burst release of encapsulated drugs represents a limitation for the transition to the clinical phase. To reduce this effect, a new drug delivery system was prepared by combining uncross-linked, drug-loaded gelatin microbeads with cross-linkable alginate solution. Gelatin microbeads ranging from 5 to 50 µm were obtained depending on gelatin concentration, stirring rate, and emulsifying time. The release behavior of drug-loaded gelatin microbeads encapsulated within cross-linked alginate gel was characterized both at room temperature and 37°C and compared with the release from gelatin microbeads and cross-linked alginate gel alone. Gelatin microbeads reduced the initial burst release of fluorescein from cross-linked alginate matrix, with a corresponding decrease in the release efficiency. Burst release in the first 2 h was reduced from 30% to about 5%, while cumulative release at 37°C declined from about 95% to 50% after 7 days. This system represents a promising approach for the development of novel and versatile injectable drug delivery systems.

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

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 WileySons, Ltd.

Cell-matrix and cell-biomolecule interactions play critical roles in a diversity of biological events including cell adhesion, growth, differentiation, and apoptosis. Evidence suggests that a concise crosstalk of these environmental factors may be required to direct stem cell differentiation toward matured cell type and function. However, the culmination of these complex interactions to direct stem cells into highly specific phenotypes in vitro is still widely unknown, particularly in the context of implantable biomaterials. In this study, we utilized tunable hydrogels based on a simple high pressure CO2 method and silk fibroin (SF) the structural protein of Bombyx mori silk fibers. Modification of SF protein starting water solution concentration results in hydrogels of variable stiffness while retaining key structural parameters such as matrix pore size and β-sheet crystallinity. To further resolve the complex crosstalk of chemical signals with matrix properties, we chose to investigate the role of 3D hydrogel stiffness and transforming growth factor (TGF-β1), with the aim of correlating the effects on the vascular commitment of human mesenchymal stem cells. Our data revealed the potential to upregulate matured vascular smooth muscle cell phenotype (myosin heavy chain expression) of hMSCs by employing appropriate matrix stiffness and growth factor (within 72 h). Overall, our observations suggest that chemical and physical stimuli within the cellular microenvironment are tightly coupled systems involved in the fate decisions of hMSCs. The production of tunable scaffold materials that are biocompatible and further specialized to mimic tissue-specific niche environments will be of considerable value to future tissue engineering platforms. Statement of Significance This article investigates the role of silk fibroin hydrogel stiffness and transforming growth factor (TGF-β1), with the aim of correlating the effects on the vascular commitment of human mesenchymal stem cells. Specifically, we demonstrate the upregulation of mature vascular smooth muscle cell phenotype (myosin heavy chain expression) of hMSCs by employing appropriate matrix stiffness and growth factor (within 72 h). Moreover, we demonstrate the potential to direct specialized hMSC differentiation by modulating stiffness and growth factor using silk fibroin, a well-tolerated and -defined biomaterial with an impressive portfolio of tissue engineering applications. Altogether, our study reinforce the fact that complex differentiation protocols may be simplified by engineering the cellular microenvironment on multiple scales, i.e. matrix stiffness with growth factor.

Acid-Solubilized Collagen (ASC) and Pepsin-Solubilized Collagen (PSC) were extracted from the mantle of the common European squid, and were comparatively characterized. ASC and PSC were isolated with an extraction yield of 5.1 and 24.2% (on dry weight basis), respectively. SDS-PAGE showed that the ASC was mostly comprised of α1- and α2-chains; while the PSC presented relevant β- and γ-components. GPC analysis confirmed that both the ASC and the PSC consisted of fractions characterized by different molecular weight. Thermal denaturation behavior of ASC and PSC were followed by calorimetric and rheological analyses; denaturation temperature was estimated to be 22°C for ASC and 21°C for PSC. Amino acid composition and solubility of collagen were also investigated. Finally, the cytotoxicity of the isolated collagen was evaluated in vitro and no cytotoxic activity caused by the collagen extracts was observed. This study demonstrated that squid mantle has potential as an alternative source of collagen-derived materials.

There is always a considerable clinical need for vascular grafts. Considering the availability, physical and mechanical properties, and regenerative potential, we have developed and characterized readily available, strong, and compliant multilayer grafts that support cell culture and ingrowth. The grafts were made from heterogeneous materials and structures, including a thin, dense, nanofibrous core composed of poly-ε-caprolactone (PCL), and a thick, porous, hydrogel sleeve composed of genipin-crosslinked collagen-chitosan (GCC). Because the difference in physicochemical properties between PCL and GCC caused layer separation, the layer adhesion was identified as a determinant to graft property and integrity under physiological conditions. Thus, strategies to modify the layer interface, including increasing porosity of the PCL surface, decreasing hydrophobicity, and increasing interlayer crosslinking, were developed. Results from microscopic images showed that increasing PCL porosity was characterized by improved layer adhesion. The resultant graft was characterized by high compliance (4.5%), and desired permeability (528 mL/cm(2)/min), burst strength (695 mmHg), and suture strength (2.38 N) for readily grafting. Results also showed that PCL mainly contributed to the graft mechanical properties, whereas GCC reduced the water permeability. In addition to their complementary contributions to physical and mechanical properties, the distinct graft layers also provided layer-specific structures for seeding and culture of vascular endothelial and smooth muscle cells in vitro. Acellular graft constructs were readily used to replace abdominal aorta of rabbits, resulting in rapid cell ingrowth and flow reperfusion. The multilayer constructs capable of sustaining physiological conditions and promoting cellular activities could serve as a platform for future development of regenerative vascular grafts.

Controlled molecule release from scaffolds can dramatically increase the scaffold ability of directing tissue regeneration in vitro and in vivo. Crucial to the regeneration is precise regulation over release direction and kinetics of multiple molecules (small genes, peptides, or larger proteins). To this end, we developed gradient micropatterns of electrospun nanofibers along the scaffold thickness through programming the deposition of heterogeneous nanofibers of poly(e-caprolactone) (PCL) and poly(D,L-lactide-co-glycolide) acid (PLGA). Confocal images of the scaffolds containing fluorophore-impregnated nanofibers demonstrated close matching of actual and designed gradient fiber patterns; thermal analyses further showed their matching in the composition. Using acid-terminated PLGA (PLGAac) and ester-terminated PLGA (PLGAes) to impregnate molecules in the PCL-PLGA scaffolds, we demonstrated for the first time their differences in nanofiber degeneration and molecular weight change during degradation. PLGAac nanofibers were more stable with gradual and steady increase in the fiber diameter during degradation, resulting in more spatially confined molecule delivery from PCL-PLGA scaffolds. Thus, patterns of PCL-PLGAac nanofibers were used to design versatile controlled delivery scaffolds. To test the hypothesis that molecule-impregnated PLGAac in the gradient-patterned PCL-PLGAac scaffolds can program various modalities of molecule release, model molecules, including small fluorophores and larger proteins, were respectively used for time-lapse release studies. Gradient-patterns were used as building blocks in the scaffolds to program simultaneous release of one or multiple proteins to one side or, respectively, to the opposite sides of scaffolds for up to 50 days. Results showed that the separation efficiency of molecule delivery from all the scaffolds with a thickness of 200 μm achieved >88% for proteins and >82% for small molecules. In addition to versatile spatially controlled delivery, micropatterns were designed to program sequential release of proteins. The hierarchically structured materials presented here may enable development of novel multifunctional scaffolds with defined 3D dynamic microenvironments for tissue regeneration.

Silk fibroin, one of the most promising natural materials for tissue engineering, has positive interactions with the biological environment, particularly in the field of bone and cartilage regeneration. A new approach was developed to create hydrogels from water-based fibroin solutions by applying an electric field to effect protein migration and coagulation at the anode (Aluminium or Ti6Al4V alloy) of an electrochemical cell. The process was easily controlled by the voltage applied to the electrodes (3, 10, and 30 V), solution concentration (1%, 2%, 2.6% w/v), time (up to 100 s) and electrode distance (1—6 mm). The hydrogel thickness can be increased up to 60 μm and, depending on processing conditions, porous coatings or compact films can be obtained. The ability of electrodeposited fibroin hydrogels to coat metal objects with complex shape and surface morphology, together with the acclaimed properties of fibroin, makes it a promising technique to enhance the osteointegration of dental or orthopedic prostheses.

Sericin was extracted from three strains of Thai Bombyx mori silk cocoons (white shell Chul1/1, greenish shell Chul3/2, and yellow shell Chul4/2) by a high-pressure and high-temperature technique. The characteristics of sericin extracted from different fractions (15, 45, and 60 min extraction process) were compared. No differences in amino acid composition were observed among the three fractions. For all silk strains, sericin extracted from a 15-min process presented the highest molecular weight. The biological potential of the different sericin samples as a bioadditive for 3T3 fibroblast cells was assessed. When comparing sericin extracted from three silk strains, sericin fractions extracted from Chul4/2 improved cell proliferation, while sericin from Chul 1/1 activated Type I collagen production to the highest extent. This study allows the natural variability of sericin obtained from different sources and extraction conditions to be addressed and provides clues for the selection of sericin sources.

Catering the hydrogel manufacturing process toward defined viscoelastic properties for intended biomedical use is important to hydrogel scaffolding function and cell differentiation. Silk fibroin hydrogels may undergo “physical” cross-linking through β-sheet crystallization during high pressure carbon dioxide treatment, or covalent “chemical” cross-linking by genipin. We demonstrate here that time-dependent mechanical properties are tunable in silk fibroin hydrogels by altering the chronological order of genipin cross-linking with β-sheet formation. Genipin cross-linking before β-sheet formation affects gelation mechanics through increased molecular weight, affecting gel morphology, and decreasing stiffness response. Alternately, genipin cross-linking after gelation anchored amorphous regions of the protein chain, and increasing stiffness. These differences are highlighted and validated through large amplitude oscillatory strain near physiologic levels, after incorporation of material characterization at molecular and micron length scales.

Autologous saphenous vein is still the conduit of choice for peripheral by-pass. Synthetic vascular grafts in polyethylene terephthalate and expanded polytetrafluoroethylene are used if vein access cannot be obtained. However they are successfully used to replace large diameter vessels, but they fail in small diameters (

The importance of mesenchymal stem cells (MSC) in vascular regeneration is becoming increasingly recognized. However, few in vitro studies have been performed to identify the effects of environmental elasticity on the differentiation of MSC into vascular cell types. Electrospinning and photopolymerization techniques were used to fabricate a three-dimensional (3-D) polyethylene glycol dimethacrylate nanofiber hydrogel matrix with tunable elasticity for use as a cellular substrate. Compression testing demonstrated that the elastic modulus of the hydrated 3-D matrices ranged from 2 to 15 kPa, similar to the in vivo elasticity of the intima basement membrane and media layer. MSC seeded on rigid matrices (8-15 kPa) showed an increase in cell area compared with those seeded on soft matrices (2-5 kPa). Furthermore, the matrix elasticity guided the cells to express different vascular-specific phenotypes with high differentiation efficiency. Around 95% of MSC seeded on the 3-D matrices with an elasticity of 3 kPa showed Flk-1 endothelial markers within 24h, while only 20% of MSC seeded on the matrices with elasticity8 kPa demonstrated Flk-1 marker. In contrast, ∼80% of MSC seeded on 3-D matrices with elasticity8 kPa demonstrated smooth muscle α-actin marker within 24h, while fewer than 10% of MSC seeded on 3-D matrices with elasticity5 kPa showed α-actin markers. The ability to control MSC differentiation into either endothelial or smooth muscle-like cells based purely on the local elasticity of the substrate could be a powerful tool for vascular tissue regeneration.

Vascular grafts are often used as blood vessel substitutes. Until now, synthetic materials have not matched the efficacy of native tissues, particularly in the applications of small-diameter vascular grafts (

Essential to growing or regenerating 3-dimensional tissues is the formation of functional microcirculation that provides nutrients, oxygen and signal molecules for tissue survival and function regeneration. In the past decade, molecule-based microvascular formation has been achieved in vitro and in vivo. However, direct delivery of angiogenic molecules often results in malformed hyperpermeable microvessels, microvessels with low density. This can be attributed to the lack of effective molecule mechanisms that regulate vascular formation. More recent studies utilize biodegradable materials to control the delivery of biomolecules for vascularization of engineered or ischemic tissues, and exciting results have shown the importance of molecule kinetics to the vascular formation. Molecule delivery mechanisms that mimic precisely-regulated spatiotemporal signaling events during natural vascularization may be a possible way to improve or optimize the process. Hence, this study is designed to develop a new release system capable of degrading in the body and releasing biomolecules in a spatiotemporally controlled manner.Copyright © 2011 by ASME

Autologous vessels are the gold standard for small-diameter (

Synthetic implant materials often lack of the anisotropic mechanical properties and cell-interactive surface which are shown by natural tissues. For example, engineered vascular grafts need to be developed to address the mechanical and biological problems associated with the graft materials. This study has demonstrated a double-electrospinning fabrication process to produce a poly(e-caprolactone)-fibroin multilayer composite which shows well-integrated nanofibrous structure, endothelial-conducive surface and anisotropic mechanical property, suitable as engineered vascular constructs. Electrospinning parameters such as voltage, solution concentration, feed rate, and relative humidity were optimized to obtain defect-free, uniform nanofibers. To mimic the different mechanical properties of natural vessels in the circumferential and longitudinal directions, a rotating cylinder was used as collector, resulting in the production of constructs with anisotropic properties. The combination of the collector shape and the collector rotation allows us to produce a tubular structure with tunable anisotropic mechanical properties. Fourier transform infrared spectroscopy, differential scanning calorimetry, and uniaxial tensile tests were used to characterize the electrospun constructs. Cell cultures with primary endothelial cells demonstrated that cells showed spread morphology and strong adhesion on fibroin richer surfaces. The platform for producing robust multilayer scaffolds with intermixing nanofiber structure, tunable anisotropy ratio, and surface with specific compositions may hold great potential in tissue engineering applications. © 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2011.

Pressure-sensitive biological response is simulated in "rotating-cup" bioreactors with unidirectional modulations in compressive stress at the cylindrical wall that stimulate bone-tissue growth. Anchorage-dependent mammalian cells (i) adhere to a protein coating, (ii) receive nutrients and oxygen from an aqueous medium via radial diffusion toward the active surface, and (iii) respond to physiological modulations in centrifual-force-induced fluid pressure at the cell/aqueous-medium interface. This process is modeled by the classic diffusion equation (i.e., Fick's second law), with a time-dependent reaction/diffusion boundary condition at the wall. Non-reversing angular velocity modulations resemble pulsations at physiological frequencies. Computer simulations of nutrient consumption profiles suggest that rotational bioreactor designs should consider the effects of normal stress when the pressure-sensitive Damköhler number (i.e., ratio of the pressure-dependent zeroth-order rate of nutrient consumption relative to the rate of nutrient diffusion toward active cells adhered to the cylindrical wall), evaluated under steady rotation, is greater than approximately 10-20% of the stress-free Damköhler number (i.e., beta(0,1st-order)=0.025) for simple 1st-order stress-free kinetics, and approximately 1% of the stress-free Damköhler number (i.e., beta(0,2nd-order)=0.40) for complex 2nd-order stress-free nutrient consumption. When the peak-to-peak amplitude of angular velocity modulations of the cylindrical wall is the same as or larger than the angular velocity for steady rotation, the effect of non-reversing centrifugal-force-induced dynamic normal stress in rotational bioreactors, superimposed on steady rotation, can be significant when one is below the critical value of the pressure-sensitive Damköhler number that has been identified under steady rotation.

Stress-sensitive biological response is simulated in a modified parallel-disk viscometer that implements steady and unidirectional dynamic shear under physiological conditions. Anchorage-dependent mammalian cells adhere to a protein coating on the surface of the rotating plate, receiving nutrients and oxygen from an aqueous medium that flows radially and tangentially, accompanied by transverse diffusion in the z -direction toward the active surface. This process is modeled as radial convection and axial diffusion with angular symmetry in cylindrical coordinates. The reaction/diffusion boundary condition on the surface of the rotating plate includes position-dependent stress-sensitive nutrient consumption via the zr - and zΘ -elements of the velocity gradient tensor at the cell/aqueous-medium interface. Linear transport laws in chemically reactive systems that obey Curie's theorem predict the existence of cross-phenomena between scalar reaction rates and the magnitude of the second-rank velocity gradient tensor, selecting only those elements of ∇ v experienced by anchorage-dependent cells that are bound to protein-active sites. Stress sensitivity via the formalism of irreversible thermodynamics introduces a zeroth-order contribution to heterogeneous reaction rates that must be quenched when nutrients, oxygen, chemically anchored cells, or vacant active protein sites are not present on the surface of the rotating plate. Computer simulations of nutrient consumption profiles via simple n th-order kinetics (i.e., n = 1,2) suggest that rotational bioreactor designs should consider stress-sensitivity when the shear-rate-based Damkohler number (i.e., ratio of the stress-dependent zeroth-order rate of nutrient consumption relative to the rate of nutrient diffusion toward active cells adhered to the rotating plate) is greater than ≈ 25% of the stress-free Damkohler number. Rotational bioreactor simulations are presented for simple 1st-order, simple 2nd-order, and complex stress-free kinetics, where the latter includes a 4th-order rate expression that considers adsorption/desorption equilibria via the Fowler–Guggenheim modification of the Langmuir isotherm for receptor-mediated cell–protein binding, accompanied by the formation of receptor complexes. Dimensionless parameters are identified to obtain equivalent stress-free nutrient consumption in the exit streams of 2-dimensional creeping-flow rotational bioreactors and 1-dimensional laminar-flow tubular bioreactors. Modulated rotation of the active plate at physiological frequencies mimics pulsatile cardiovascular flow and demonstrates that these rotational bioreactors must operate above the critical stress-sensitive Damkohler number, identified under steady shear conditions, before dynamic shear has a distinguishable effect on bioreactor performance.