Silk ionomers for encapsulation and differentiation of human MSCs

The response of human bone marrow derived human mesenchymal stem cells (hMSCs) encapsulated in silk ionomer hydrogels was studied. Silk aqueous solutions with silk-poly-l-lysine or silk-poly-l-glutamate were formed into hydrogels via ultrasonication in situ with different net charges. hMSCs were encapsulated within the hydrogels and the impact of matrix charge was assessed over weeks in osteogenic, adipogenic and maintenance growth media. These modified silk charged polymers supported cell viability and proliferative potential, and the hMSCs were able to differentiate toward osteogenic or adipogenic lineages in the corresponding differentiation media. The silk/silk-poly-l-lysine hydrogels exhibited a positive effect on selective osteogenesis of hMSCs, inducing differentiation toward an osteogenic lineage even in the absence of osteogenic supplements, while also inhibiting adipogenesis. In contrast, silk/silk fibroin-poly-l-glutamate hydrogels supported both osteogenic and adipogenic differentiation of hMSCs when cultured under induction conditions. The results demonstrate the potential utility of silk-based ionomers in gel formats for hMSCs encapsulation and for directing hMSCs long term functional differentiation toward specific lineages.     

Silk fibroin/poly(vinyl alcohol) photocrosslinked hydrogels for delivery of macromolecular drugs

Hydrogels are three-dimensional polymer networks widely used in biomedical applications as drug delivery and tissue engineered scaffolds to effectively repair or replace damaged tissue. In this paper we demonstrate a newly synthesized cytocompatible and drug releasing photo-crosslinked hydrogel based on poly(vinyl alcohol) methacrylate and silk fibroin which possesses tailorable structural and biological properties. The initial silk fibroin content was 0%, 10%, 20%, 30%, 40% and 50% with respect to the weight of poly(vinyl alcohol) methacrylate. The prepared hydrogels were characterized with respect to morphology, crystallinity, stability, swelling, mass loss and cytotoxicity. FITC-dextrans of different molecular weights were chosen as model drugs molecules for release studies from the hydrogels. The hydrogels containing different silk fibroin percentages showed differences in pore size and distribution. X-ray diffraction analysis revealed that amorphous silk fibroin in poly(vinyl alcohol) methacrylate is crystallized to β-sheet secondary structure upon gelation. The sol fraction increased with increasing fibroin concentration in the co-polymer gel (from 18% to 45%), although the hydrogel extracts were non-cytotoxic. Similarly, the addition of silk fibroin increased water uptake by the gels (from 7% to 21%). FITC-dextran release from the hydrogels was dependent on the silk fibroin content and the molecular weight of encapsulated molecules. The study outlines a newer type of photo-crosslinked interpenetrating polymer network hydrogel that possess immense potential in drug delivery applications.     

Visible light cured thiol-vinyl hydrogels with tunable degradation for 3D cell culture

We report here a synthetically simple yet highly tunable and diverse visible light mediated thiol-vinyl gelation system for fabricating cell-instructive hydrogels. Gelation was achieved via a mixed-mode step-and-chain-growth photopolymerization using functionalized 4-arm poly(ethylene glycol) as backbone macromer, eosin-Y as photosensitizer, and di-thiol containing molecule as dual purpose co-initiator/cross-linker. N-vinylpyrrolidone (NVP) was used to accelerate gelation kinetics and to adjust the stiffness of the hydrogels. Visible light (wavelength: 400–700 nm) was used to initiate rapid gelation (gel points: ∼20 s) that reached completion within a few minutes. The major differences between current thiol-vinyl gelation and prior visible light mediated photopolymerization are that: (1) the co-initiator triethanolamine (TEA) used in the previous systems was replaced with multifunctional thiols and (2) mixed-mode polymerized gels contain less network heterogeneity. The gelation kinetics and gel properties at the same PEG macromer concentration could be tuned by changing the identity of vinyl groups and di-thiol cross-linkers, as well as concentration of cross-linker and NVP. Specifically, acrylate-modified PEG afforded the fastest gelation rate, followed by acrylamide and methacrylate-functionalized PEG. Increasing NVP concentration also accelerated gelation and led to a higher network cross-linking density. Further, increasing di-thiol peptide concentration in the gel formulation increased hydrogel swelling and decreased gel stiffness. Due to the formation of thiol-ether-ester bonds following thiol-acrylate reaction, the gels degraded hydrolytically following a pseudo first order degradation kinetics. Degradation rate was controlled by adjusting thiol or NVP content in the polymer precursor solution. The cytocompatibility and utility of this hydrogel system were evaluated using in situ encapsulation of human mesenchymal stem cells (hMSC). Encapsulated hMSCs remained alive (>90%) throughout the duration of the study and the cells were differentiated down osteogenic lineage with varying degrees by controlling the rate and mode of gel degradation.     

Sodium oleate induced rapid gelation of silk fibroin

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

Sodium dodecyl sulfate-induced rapid gelation of silk fibroin

The in situ formation of injectable silk fibroin (SF) hydrogels have potential advantages over various other biomaterials due to the minimal invasiveness during application. Biomaterials need to gel rapidly under physiological conditions after injection. In the current paper, a novel way to accelerate SF gelation using an anionic surfactant, sodium dodecyl sulfate (SDS), as a gelling agent is reported. The mechanism of SDS-induced rapid gelation was determined. At low surfactant concentrations, hydrophobic interactions among the SF chains played a dominant role in the association, leading to decreased gelation time. At higher concentrations of surfactant, electrostatic repulsive forces among micellar aggregates gradually became dominant and gelation was hindered. Gel formation involves the connection of clusters formed by the accumulation of nanoparticles. This process is accompanied by the rapid formation of β-sheet structures due to hydrophobic and electrostatic interactions. It is expected that the silk hydrogel with short gelation time will be used as an injectable hydrogel in drug delivery or cartilage tissue engineering.     

壳聚糖改性丝素合金膜的制备及其相关性质研究

以无毒氧化葡萄糖醛作交联剂 ,采用溶液共混交联法制备壳聚糖改性丝素合金膜。用 FTIR、DSC表征其结构 ,测定其等电点、力学性能、不同 p H条件下的溶胀率和对模型药物 5 - Fu的渗透性。结果表明 :改性丝素合金膜中丝素和壳聚糖分子间存在着强烈的氢键相互作用及良好的相容性。改性膜的等电点对应的 p H值是 5 .35 ,而丝素膜的等电点是 4 .5。改性膜的力学性能优于单组分膜 ,当壳聚糖含量为 4 0 %～ 6 0 %时 ,具有最大的抗张强度和拉伸率 ,分别为 71.4～ 72 .7MPa和 2 .96～ 3.82 %。改性丝素合金膜对 5 - Fu的渗透量与壳聚糖的含量和时间成正相关关系 ,渗透系数随 p H值增大 (5→ 9)先逐渐减小然后略有增大 ,在 p H=7时最小。     

Potential of biocompatible regenerated silk fibroin/sodium N-lauroyl sarcosinate hydrogels

Hydrogels are becoming widely used in biomaterial applications. The available methods for the preparation of these materials are continually growing. The gelation time (GT) of silk protein fibroin is difficult to control by physical methods. The cross-linkers used in available chemical techniques are likely to impact the biocompatibility of the resultant materials. In this paper, we demonstrate that the addition of sodium N-lauroyl sarcosinate (an amino-acid-based surfactant) accelerates the formation of hydrogels from fibroin. GT, turbidity variations, changes of viscoelasticity during the gelation process, and the mechanical properties of the products are measured. The secondary structure was probed by Fourier transform infrared spectroscopy, X-ray diffraction and the morphologies of the products were investigated by scanning electron microscopy. Transformations in the β-sheet content were monitored by the fluorescence of Thioflavine T and circular dichroism measurements. The relationship between the surface tension of sodium N-lauroyl sarcosinate and the GT was also explained. To investigate cell compatibility, fibroblast cells were seeded onto the surface of the hydrogels. The results indicate that the sodium N-lauroyl sarcosinate/fibroin GT can be controlled. This blend-hydrogel demonstrates excellent cell compatibility, good compression strength, and outstanding compression-recovery characteristics. Sodium N-lauroyl sarcosinate/silk fibroin hydrogels containing β-sheets have considerable potential as replacement materials in addressing the tissue defects involved with repair surgery.     

Characteristics of platelet gels combined with silk

Platelet gel, a fibrin network containing activated platelets, is widely used in regenerative medicine due the capacity of platelet-derived growth factors to accelerate and direct healing processes. However, limitations to this approach include poor mechanical properties, relatively rapid degradation, and the lack of control of release of growth factors at the site of injection. These issues compromise the ability of platelet gels for sustained function in regenerative medicine. In the present study, a combination of platelet gels with silk fibroin gel was studied to address the above limitations. Mixing sonicated silk gels with platelet gels extended the release of growth factors without inhibiting gel-forming ability. The released growth factors were biologically active and their delivery was modified further by manipulation of the charge of the silk protein. Moreover, the silk gel augmented both the rheological properties and compressive stiffness of the platelet gel, tuned by the silk concentration and/or silk/platelet gel ratio. Silk-platelet gel injections in nude rats supported enhanced cell infiltration and blood vessel formation representing a step towards new platelet gel formulations with enhanced therapeutic impact.     

丝素材料的药物吸附释放性能与调控研究

为了了解丝素材料对药物等的吸附释放性能 ,并探讨对丝素材料的吸附释放性能的调控 ,用不同离子型化合物作为药物模型 ,比较了分别用未修饰和经羧基进行酰胺化修饰后的丝素制作成的多孔丝素凝胶对不同离子型的化合物的吸附释放行为。结果表明 :经修饰后丝素蛋白质的等电点为 pH6 .0左右 ,而天然的为pH4.0左右。未修饰和经修饰的多孔丝素凝胶都随着溶液 pH的上升 ,对阳离子化合物的吸附量增加 ,释放速度减慢 ;对阴离子化合物吸附量减少 ,释放速度加快。但在相同 pH下与未修饰相比 ,经修饰的多孔丝素凝胶所吸附的阳离子化合物的释放速度加快 ,释放量也增加 ;所吸附的阴离子化合物的释放速度和释放量则明显降低。用羧基酰胺化修饰的方法 ,可在一定程度上改变丝素材料对离子型化合物的吸附释放行为     

Time-Dependent Alginate/Polyvinyl Alcohol Hydrogels as Injectable Cell Carriers

Alginate hydrogels have been widely utilized as cell carriers due to their simplicity for fabricating cell-immobilized gel beads or 3-dimentional porous scaffolds, biocompatibility and non-toxicity to cells. Generally alginate hydrogels have been produced by contacting alginate solution with CaCl₂ as a cross-linking agent. However, the major disadvantages of this system are that the gelation rate is too fast and hard to control, and the prepared alginate gels cannot be injected. Injectable alginates have been prepared by using CaSO₄ or CaCO₃ as a cross-linking agent. However, the gelation rate of alginate with CaCO₃ is slow owing to the low solubility of CaCO₃ in water, while that with CaSO₄ is too fast to form uniform gels. In this study, we prepared injectable alginate/polyvinyl alcohol (PVA) blend hydrogels with controllable gelation rate by using CaSO₄ as a cross-linking agent and Na₂HPO₄ as a cross-linking retardation agent. The gelation rate could be controlled by adjusting CaSO₄/Na₂HPO₄ ratio in the solution. The alginate and PVA showed good compatibility in aqueous solutions or gels. The gelation rate of alginate increased with increasing Na₂HPO₄ and decreasing CaSO₄ concentrations, as expected. The PVA itself in the alginate/PVA blend did not affect the gelation rate. All alginate/PVA hydrogels demonstrated some extraction of PVA, but the extraction extent was not much even after 7 days immersion in water. The alginate/PVA hydrogels were examined for their in vitro cell compatibility by the culture of chondrocytes (human chondrocyte cell line) in the gels up to 28 days. The cells were grown almost linearly in the alginate/PVA hydrogels with higher PVA compositions showing better cell growth. The GAG contents from the cells in the hydrogels did not show dramatic changes with culture time, however, they also increased gradually in the alginate/PVA hydrogels with higher PVA composition. The PVA in the hydrogels seemed to play positive roles for the growth and activity of chondrocytes. The alginate/PVA hydrogels with controllable gelation rate are expected to be applicable as injectable cell carriers.     

The enhancement of chondrogenic differentiation of human mesenchymal stem cells by enzymatically regulated RGD functionalities

A thiol-acrylate photopolymerization was used to incorporate enzymatically cleavable peptide sequences into PEG hydrogels to induce chondrogenic differentiation of encapsulated human mesenchymal stem cells (hMSCs). An adhesive sequence, RGD, was designed with an MMP-13 specific cleavable linker. RGD promotes survival of hMSCs encapsulated in PEG gels and has shown to induce early stages of chondrogenesis, while its persistence can limit complete differentiation. Therefore, an MMP-13 cleavage site was incorporated into the peptide sequence to release RGD mimicking the native differentiation timeline. Active MMP-13 production of encapsulated hMSCs was seen to increase from day 9 to 14 and only in chondrogenic differentiating cultures. Seeded hMSCs attached to the material prior to enzymatic cleavage, but a significant population of the cells detach after cleavage and release of RGD. Finally, hMSCs encapsulated in RGD-releasing gels produce 10 times as much glycosaminoglycan as cells with uncleavable RGD functionalities, by day 21 of culture. Furthermore, 75% of the cells stain positive for collagen type II deposition where RGD is cleavable, as compared to 19% for cultures where RGD persists. Collectively, these data provide evidence that temporal regulation of integrin-binding peptides is important in the design of niches in differentiating hMSCs to chondrocytes.     

Lyophilized silk fibroin hydrogels for the sustained local delivery of therapeutic monoclonal antibodies

The development of sustained delivery systems compatible with protein therapeutics continues to be a significant unmet need. A lyophilized silk fibroin hydrogel matrix (lyogel) for the sustained release of pharmaceutically relevant monoclonal antibodies is described. Sonication of silk fibroin prior to antibody incorporation avoids exposing the antibody to the sol-gel transition inducing shear stress. Fourier Transform Infrared (FTIR) analysis showed no change in silk structural composition between hydrogel and lyogel or with increasing silk fibroin concentration. Antibody release from hydrogels occurred rapidly over 10 days regardless of silk concentration. Upon lyophilization, sustained antibody release was observed over 38 days from lyogels containing 6.2% (w/w) silk fibroin and above. In 3.2% (w/w) silk lyogels, antibody release was comparable to hydrogels. Swelling properties of lyogels followed a similar threshold behavior. Lyogels at 3.2% (w/w) silk recovered approximately 90% of their fluid mass upon rehydration, while approximately 50% fluid recovery was observed at 6.2% (w/w) silk and above. Antibody release was primarily governed by hydrophobic/hydrophilic silk-antibody interactions and secondarily altered by the hydration resistance of the lyogel. Hydration resistance was controlled by altering β-sheet (crystalline) density of the matrix. The antibody released from lyogels maintained biological activity. Silk lyogels offer an advantage as a delivery matrix over other hydrogel materials for the slow release of the loaded protein, making lyogels suitable for long-term sustained release applications.     

Fibroin hydrogels for biomedical applications: preparation, characterization and in vitro cell culture studies

Silk fibroin hydrogels prepared either by treating a 2% (w/v) silk fibroin aqeuous solution at 4 degrees C (thermgel) or by adding 30% (v/v) of glycerol (glygel), were characterized by using Environmental Scanning Electron Microscopy (ESEM), Fourier Transform Infrared Spectroscopy (FT-IR), Differential Scanning Calorimetry (DSC), Thermogravimetrical Analysis (TGA) and molecular weight determination. The preparation procedure affected morphology and molecular weight of hydrogels, with no or negligible differences being displayed by FT-IR and DSC analyses. While thermgel presented a well uniform porous structure, the morphology of glygel appeared to be non-porous and heterogeneous. Glygel presented lower water content and lower degradation temperatures, associated with the presence of glycerol but likely also to less-organized protein structures. Cytoxicity tests with human osteoblast-like cells indicated that both gels were not cytoxic, while cell cultures pointed out a faster cell proliferation on glygel and a higher cell activation and differentiation on thermgel. These gels could be used as scaffolds able to promote in situ bone regeneration.     

Radiation synthesis of poly(N-vinyl-2-pyrrolidone)-kappa-carrageenan hydrogels and their use in wound dressing applications. I. Preliminary laboratory tests

Poly(N-vinyl-2-pyrrolidone)-kappa-carrageenan hydrogels (PVP-KC) were prepared by irradiating the mixtures of aqueous solutions of PVP, KC, potassium chloride, and poly(ethylene glycol) by gamma-rays at different doses. Their preliminary laboratory tests were evaluated to identify their usability in wound dressing applications. For investigation of the effect of components on the gelation of PVP, sol-gel analyses were made and gel fractions of the hydrogels were determined. Mechanical experiments were conducted for both unirradiated and irradiated samples. For investigation of the fluid uptake capacity of the hydrogels, swelling experiments were performed in pseudo-extracellular fluid solution at various temperatures. Acidity/alkalinity (pH) and electrical conductivity tests were achieved from aqueous extracts of hydrogels, and bioadhesion strength of the hydrogels was investigated on human skin.     

Silk fibroin electrogelation mechanisms

A silk fibroin gel system (e-gel), formed with weak electric fields, has potential utility in medical materials and devices. The mechanism of silk e-gel formation was studied to gain additional insight into the process and control of the material properties. Silk fibroin nanoparticles with sizes of tens of nanometers, composed of metastable conformations, were involved in e-gel formation. Under electric fields the nanoparticles rapidly assembled into larger nano- or microspheres with size range from tens of nanometers to several microns. Repulsive forces from the negative surface charge of the acidic groups on the protein were screened by the local decrease in solution pH in the vicinity of the positive electrode. By controlling the formation and content of silk fibroin nanoparticles e-gels could be formed even from low concentration silk fibroin solutions (1%). When e-gel formation was reversed to the solution state the aggregated nano- and microspheres dispersed into solution, a significant observation related to future applications for this process, such as drug delivery.     

Human bone marrow stromal cell responses on electrospun silk fibroin mats

Fibers with nanoscale diameters provide benefits due to high surface area for biomaterial scaffolds. In this study electrospun silk fibroin-based fibers with average diameter 700+/-50 nm were prepared from aqueous regenerated silkworm silk solutions. Adhesion, spreading and proliferation of human bone marrow stromal cells (BMSCs) on these silk matrices was studied. Scanning electron microscopy (SEM) and MTT analyses demonstrated that the electrospun silk matrices supported BMSC attachment and proliferation over 14 days in culture similar to native silk fibroin (approximately 15 microm fiber diameter) matrices. The ability of electrospun silk matrices to support BMSC attachment, spreading and growth in vitro, combined with a biocompatibility and biodegradable properties of the silk protein matrix, suggest potential use of these biomaterial matrices as scaffolds for tissue engineering.     

Ethoxysilane-capped PEO-PPO-PEO triblocks: a new family of reverse thermo-responsive polymers

New reverse thermo-responsive polymers systems combining reverse thermal gelation behavior and a gradual increase in the mechanical properties, were created by crosslinking ethoxysilane-capped poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblocks in aqueous solutions at physiological conditions. Pluronic F127 (PEO(99)-PPO(67)-PEO(99)) was functionalized with (3-isocyanatopropyl) triethoxysilane (IPTS) by reacting its terminal hydroxyl groups with the isocyanate. The silane-capped PEO-PPO-PEO triblock was characterized by (1)H-NMR, GPC, FT-IR and DSC and the rheological behavior of its aqueous solutions were studied. The silane-containing triblock retained the reverse thermo-responsive characteristics displayed by the original Pluronic. Over time, the ethoxysilane groups hydrolysed and created silanol moieties that subsequently condensated, crosslinking the material and generating hydrogels that exhibited gradually increasing mechanical properties. It was found that the higher the pH, the faster the process and the higher the viscosity levels attained. Finally, the ability of these gels to perform as matrices for drug delivery was exemplified by releasing metronidazole and methylene blue. Findings showed that while a 30% F127 gel at 37 degrees C delivered all the drug within less than 3 days, F127di-IPTS gels completed the process at a much slower rate (up to 15 days).