Surfactant-assisted photo-crosslinked silk fibroin sponges: A versatile platform for the design of bone scaffolds.

Critical size bone defects cannot heal without aid and current clinical approaches exhibit some limitations, underling the need for novel solutions. Silk fibroin, derived from silkworms, is widely utilized in tissue engineering and regenerative medicine due to its remarkable properties, making it a promising candidate for bone tissue regeneration in vitro and in vivo. However, the clinical translation of silk-based materials requires refinements in 3D architecture, stability, and biomechanical properties. In earlier research, improved mechanical resistance and stability of chemically crosslinked methacrylate silk fibroin (Sil-Ma) sponges over physically crosslinked counterparts were highlighted. Furthermore, the influence of photo-initiator and surfactant concentrations on silk properties was investigated. However, the characterization of sponges with Sil-Ma solution concentrations above 10 % (w/V) was hindered by production optimization challenges, with only cell viability assessed. This study focuses on the evaluation of methacrylate sponges' suitability as temporal bone tissue regeneration scaffolds. Sil-Ma sponge fabrication at a fixed concentration of 20 % (w/V) was optimized and the impact of photo-initiator (LAP) concentrations and surfactant (Tween 80) presence/absence was studied. Their effects on pore formation, silk secondary structure, mechanical properties, and osteogenic differentiation of hBM-MSCs were investigated. We demonstrated that, by tuning silk sponges' composition, the optimal combination boosted osteogenic gene expression, offering a strategy to tailor biomechanical properties for effective bone regeneration. Utilizing Design of Experiment (DoE), correlations between sponge composition, porosity, and mechanical properties are established, guiding tailored material outcomes. Additionally, correlation matrices elucidate the microstructure's influence on gene expressions, providing insights for personalized approaches in bone tissue regeneration.

Nanocomposite Methacrylated Silk Fibroin-Based Scaffolds for Bone Tissue Engineering.

The treatment of bone defects is a clinical challenge. Bone tissue engineering is gaining interest as an alternative to current treatments, with the development of 3D porous structures (scaffolds) helpful in promoting bone regeneration by ensuring temporary functional support. In this work, methacrylated silk fibroin (SilMA) sponges were investigated as scaffolds for bone tissue engineering by exploiting the combination of physical (induced by NaCl salt during particulate leaching) and chemical crosslinking (induced by UV-light exposure) techniques. A biomimetic approach was adopted to better simulate the extracellular matrix of the bone by introducing either natural (mussel shell-derived) or synthetic-origin hydroxyapatite nanoparticles into the SilMA sponges. The obtained materials were characterized in terms of pore size, water absorption capability and mechanical properties to understand both the effect of the inclusion of the two different types of nanoparticles and the effect of the photocrosslinking. Moreover, the SilMA sponges were tested for their bioactivity and suitability for bone tissue engineering purposes by using osteosarcoma cells, studying their metabolism by an AlamarBlue assay and their morphology by scanning electron microscopy. Results indicate that photocrosslinking helps in obtaining more regular structures with bimodal pore size distributions and in enhancing the stability of the constructs in water. Moreover, the addition of naturally derived hydroxyapatite was observed to be more effective at activating osteosarcoma cell metabolism than synthetic hydroxyapatite, showing a statistically significant difference in the AlamarBlue measurement on day 7 after seeding. The methacrylated silk fibroin/hydroxyapatite nanocomposite sponges developed in this work were found to be promising tools for targeting bone regeneration with a sustainable approach.

Silk fibroin films with embedded magnetic nanoparticles: evaluation of the magneto-mechanical stimulation effect on osteogenic differentiation of stem cells.

We report about a biomaterial in the form of film ∼10 µm thick, consisting of a silk fibroin matrix with embedded iron oxide superparamagnetic nanoparticles, for prospective applications as bioactive coating in regenerative medicine. Films with different load of magnetic nanoparticles are produced (nanoparticles/silk fibroin nominal ratio = 5, 0.5 and 0 wt%) and the structural, mechanical and magnetic properties are studied. The nanoparticles form aggregates in the silk fibroin matrix and the film stiffness, as tested by nanoindentation, is spatially inhomogeneous, but the protein structure is not altered. In vitro biological tests are carried out on human bone marrow-derived mesenchymal stem cells cultured on the films up to 21 days, with and without an applied static uniform magnetic field. The sample with the highest nanoparticles/silk fibroin ratio shows the best performance in terms of cell proliferation and adhesion. Moreover, it promotes a faster and better osteogenic differentiation, particularly under magnetic field, as indicated by the gene expression level of typical osteogenic markers. These findings are explained in light of the results of the physical characterization, combined with numerical calculations. It is established that the applied magnetic field triggers a virtuous magneto-mechanical mechanism in which dipolar magnetic forces between the nanoparticle aggregates give rise to a spatial distribution of mechanical stresses in the silk fibroin matrix. The film with the largest nanoparticle load, under cell culture conditions (i.e. in aqueous environment), undergoes matrix deformations large enough to be sensed by the seeded cells as mechanical stimuli favoring the osteogenic differentiation.

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.

Stapled fascial suture: ex vivo modeling and clinical implications.

BACKGROUND: Recently, in the field of abdominal wall repair surgery, some minimally invasive procedures introduced the use of staplers to provide a retromuscular prosthetic repair. However, to the knowledge of the authors, there are little data in the literature about the outcomes of stapled sutures adoption for midline reconstruction. This study aims to investigate the biomechanics of stapled sutures, simple (stapled), or oversewn (hybrid), in comparison with handsewn suture. From the results obtained, we tried to draw indications for their use in a clinical context. METHODS: Human cadaver fascia lata specimens, sutured (handsewn, stapled, or hybrid) or not, underwent tensile tests. The data on strength (maximal stress), ultimate strain (deformability), Young's modulus (rigidity), and dissipated specific energy (ability to absorb mechanical energy up to the breaking point) were recorded for each type of specimens and analyzed. RESULTS: Stapled and hybrid suture showed a significantly higher strength (handsewn 0.83 MPa, stapled 2.10 MPa, hybrid 2.68 MPa) and a trend toward a lower ultimate strain as compared to manual sutures (handsewn 344%, stapled 249%, hybrid 280%). Stapled and hybrid sutures had fourfold higher Young's modulus as compared to handsewn sutures (handsewn 1.779 MPa, stapled 7.374 MPa, hybrid 6.964 MPa). Handsewn and hybrid sutures showed significantly higher dissipated specific energy (handsewn 0.99 mJ-mm3, stapled 0.73 mJ-mm3, hybrid 1.35 mJ-mm3). CONCLUSION: Stapled sutures can resist high loads, but are less deformable and rigid than handsewn suture. This suggests a safer employment in case of small defects or diastasis (< W1 in accord to EHS classification), where the presumed tissutal displacement is minimal. Oversewing a stapled suture improves its efficiency, becoming crucial in case of larger defects (> W1 in accord to EHS classification) where the expected tissutal displacement is maximal. Hybrid sutures seem to be a good compromise.

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.

3D Printed SiOC(N) Ceramic Scaffolds for Bone Tissue Regeneration: Improved Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells.

Bone tissue engineering has developed significantly in recent years as there has been increasing demand for bone substitutes due to trauma, cancer, arthritis, and infections. The scaffolds for bone regeneration need to be mechanically stable and have a 3D architecture with interconnected pores. With the advances in additive manufacturing technology, these requirements can be fulfilled by 3D printing scaffolds with controlled geometry and porosity using a low-cost multistep process. The scaffolds, however, must also be bioactive to promote the environment for the cells to regenerate into bone tissue. To determine if a low-cost 3D printing method for bespoke SiOC(N) porous structures can regenerate bone, these structures were tested for osteointegration potential by using human mesenchymal stem cells (hMSCs). This includes checking the general biocompatibilities under the osteogenic differentiation environment (cell proliferation and metabolism). Moreover, cell morphology was observed by confocal microscopy, and gene expressions on typical osteogenic markers at different stages for bone formation were determined by real-time PCR. The results of the study showed the pore size of the scaffolds had a significant impact on differentiation. A certain range of pore size could stimulate osteogenic differentiation, thus promoting bone regrowth and regeneration.

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.

Horseradish Peroxidase-Crosslinked Calcium-Containing Silk Fibroin Hydrogels as Artificial Matrices for Bone Cancer Research.

Hydrogels, being capable of mimicking the extracellular matrix composition of tissues, are greatly used as artificial matrices in tissue engineering applications. In this study, the generation of horseradish peroxidase (HRP)-crosslinked silk fibroin (SF) hydrogels, using calcium peroxide as oxidizer is reported. The proposed fast forming calcium-containing SF hydrogels spontaneously undergo SF conformational changes from random coil to ß-sheet during time, exhibiting ionic, and pH stimuli responsiveness. In vitro response shows calcium-containing SF hydrogels' encapsulation properties and their ability to promote SaOs-2 tumor cells death after 10 days of culturing, upon complete ß-sheet conformation transition. Calcium-containing SF hydrogels' angiogenic potential investigated in an in ovo chick chorioallantoic membrane (CAM) assay, show a high number of converging blood vessels as compared to the negative control, although no endothelial cells infiltration is observed. The in vivo response evaluated in subcutaneous implantation in CD1 and nude NCD1 mice shows that calcium-containing SF hydrogels are stable up to 6 weeks after implantation. However, an increased number of dead cells are also present in the surrounding tissue. The results suggest the potential of calcium-containing SF hydrogels to be used as novel in situ therapeutics for bone cancer treatment applications, particularly to osteosarcoma.

Hydrogen sulfide-releasing silk fibroin scaffold for bone tissue engineering.

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.

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.

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.

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.

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.

Homeostasis maintenance of encapsulated cells.

Cell niche homeostasis plays a critical role in many bodily functions including tissue functionality, stem cell maintenance and differentiation, wound healing, cancer development and propagation, and many others. Many tissue engineering approaches overlook the importance of engineered constructs homeostasis, in particular for transplantation purposes. Here, we present a study of the effect of encapsulation duration on engineered tissue maturation and provide a protocol for the evaluation of critical conditions for transplantation purposes. In brief, SHSY5Y human neuroblastoma cells were encapsulated in 2% alginate by electrohydrodynamic jetting method for up to 4 weeks. We evaluated extracellular matrix niche formation and tissue maturation in situ through COL1A1 expression. In in vitro conditions, we studied the ability of cells to maintain their critical functions after being released from alginate beads. Cellular viability was evaluated via an apoptosis/necrosis detection kit and AlamarBlue assay, and functionality via immunocytochemistry. We proved the importance of engineered tissue homeostasis stabilization for future cell recovery, in particular, for our system cells encapsulated for 28 days met all critical requirements for successful tissue transplantation. Maturation of engineered tissue constructs could be accelerated by enriching alginate with growth factors or extracellular matrix molecules.

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.

* Fabrication and Characterization of Biphasic Silk Fibroin Scaffolds for Tendon/Ligament-to-Bone Tissue Engineering.

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 <100-300 µm. Young's modulus varied from 689 to 1322 kPa depending on the type of construct. In addition, human adipose-derived mesenchymal stem cells were cultured on the scaffolds to evaluate the effect of pore morphology on cell proliferation and gene expression. Biphasic scaffolds supported cell attachment and influenced cytoskeleton organization depending on pore alignment. In addition, the gene expression of tendon/ligament, enthesis, and cartilage markers significantly changed depending on pore alignment in each region of the scaffolds. In conclusion, the biphasic scaffolds fabricated in this study show promising features for tendon/ligament-to-bone tissue engineering.

Co-culture of outgrowth endothelial cells with human mesenchymal stem cells in silk fibroin hydrogels promotes angiogenesis.

Sufficient vascularization of the implant construct is required for tissue regeneration to ensure the supply of oxygen and nutrients. In our previous work, we established sonication-induced silk fibroin hydrogel to load neural stem cells for brain tissue engineering applications. In this study, we explored the application of silk fibroin as an injectable hydrogel for vascularization of soft tissues. We investigated the ability of outgrowth endothelial cells (OECs) in mono-culture or in co-culture with human bone marrow-derived mesenchymal stem cells (BM-MSCs) to form capillary networks in silk fibroin hydrogels. Furthermore, the silk fibroin hydrogel was modified with IKVAV peptide revealing a sequence derived from the extracellular matrix component laminin-1 to test its effects on angiogenesis, using unmodified and VVIAK modified silk fibroin hydrogel as controls. In monocultures of OECs, no angiogenic structures were observed in silk fibroin hydrogels. In contrast, vascular structures were abundant and increased in co-culture, as confirmed by immunocytochemistry and scanning electron microscopy (SEM) over 10 d of culture in silk fibroin-based hydrogels. Although no significant differences in angiogenic activity seem to be caused by the IKVAV peptide in our experimental settings, these results indicate that sonication-induced silk fibroin-based hydrogels support the formation of functional endothelial tubes and vascularization networks in the presence of mesenchymal cells supporting the vascular sprouting of endothelial cells.

Human mesenchymal stem cells cultured on silk hydrogels with variable stiffness and growth factor differentiate into mature smooth muscle cell phenotype.

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 ß-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-ß1), 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 72h). 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-ß1), 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 72h). 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.

Development of Injectable Hyaluronic Acid/Cellulose Nanocrystals Bionanocomposite Hydrogels for Tissue Engineering Applications.

Injectable hyaluronic acid (HA)-based hydrogels compose a promising class of materials for tissue engineering and regenerative medicine applications. However, their limited mechanical properties restrict the potential range of application. In this study, cellulose nanocrystals (CNCs) were employed as nanofillers in a fully biobased strategy for the production of reinforced HA nanocomposite hydrogels. Herein we report the development of a new class of injectable hydrogels composed of adipic acid dihydrazide-modified HA (ADH-HA) and aldehyde-modified HA (a-HA) reinforced with varying contents of aldehyde-modified CNCs (a-CNCs). The obtained hydrogels were characterized in terms of internal morphology, mechanical properties, swelling, and degradation behavior in the presence of hyaluronidase. Our findings suggest that the incorporation of a-CNCs in the hydrogel resulted in a more organized and compact network structure and led to stiffer hydrogels (maximum storage modulus, E', of 152.4 kPa for 0.25 wt % a-CNCs content) with improvements of E' up to 135% in comparison to unfilled hydrogels. In general, increased amounts of a-CNCs led to lower equilibrium swelling ratios and higher resistance to degradation. The biological performance of the developed nanocomposites was assessed toward human adipose derived stem cells (hASCs). HA-CNCs nanocomposite hydrogels exhibited preferential cell supportive properties in in vitro culture conditions due to higher structural integrity and potential interaction of microenvironmental cues with CNC's sulfate groups. hASCs encapsulated in HA-CNCs hydrogels demonstrated the ability to spread within the volume of gels and exhibited pronounced proliferative activity. Together, these results demonstrate that the proposed strategy is a valuable toolbox for fine-tuning the structural, biomechanical, and biochemical properties of injectable HA hydrogels, expanding their potential range of application in the biomedical field.