Processing silk hydrogel and its applications in biomedical materials

This review mainly introduces the types of silk hydrogels, their processing methods, and applications. There are various methods for hydrogel preparation, and many new processes are being developed for various applications. Silk hydrogels can be used in cartilage tissue engineering, drug release materials, 3D scaffolds for cells, and artificial skin, among other applications because of their porous structure and high porosity and the large surface area for growth, migration, adhesion and proliferation of cells that the hydrogels provide. All of these advantages have made silk hydrogels increasingly attractive. In addition, silk hydrogels have wide prospects for application in the field of biomedical materials. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:630–640, 2015     

Highly extensible, tough, and elastomeric nanocomposite hydrogels from poly(ethylene glycol) and hydroxyapatite nanoparticles

Unique combinations of hard and soft components found in biological tissues have inspired researchers to design and develop synthetic nanocomposite gels and hydrogels with elastomeric properties. These elastic materials can potentially be used as synthetic mimics for diverse tissue engineering applications. Here we present a set of elastomeric nanocomposite hydrogels made from poly(ethylene glycol) (PEG) and hydroxyapatite nanoparticles (nHAp). The aqueous nanocomposite PEG-nHAp precursor solutions can be injected and then covalently cross-linked via photopolymerization. The resulting PEG-nHAp hydrogels have interconnected pore sizes ranging from 100 to 300 nm. They have higher extensibilities, fracture stresses, compressive strengths, and toughness when compared with conventional PEO hydrogels. The enhanced mechanical properties are a result of polymer nanoparticle interactions that interfere with the permanent cross-linking of PEG during photopolymerization. The effect of nHAp concentration and temperature on hydrogel swelling kinetics was evaluated under physiological conditions. An increase in nHAp concentration decreased the hydrogel saturated swelling degree. The combination of PEG and nHAp nanoparticles significantly improved the physical and chemical hydrogel properties as well as some biological characteristics such as osteoblast cell adhesion. Further development of these elastomeric materials can potentially lead to use as a matrix for drug delivery and tissue repair especially for orthopedic applications.     

Structure and properties of silk hydrogels

Control of silk fibroin concentration in aqueous solutions via osmotic stress was studied to assess relationships to gel formation and structural, morphological, and functional (mechanical) changes associated with this process. Environmental factors potentially important in the in vivo processing of aqueous silk fibroin were also studied to determine their contributions to this process. Gelation of silk fibroin aqueous solutions was affected by temperature, Ca(2+), pH, and poly(ethylene oxide) (PEO). Gelation time decreased with increase in protein concentration, decrease in pH, increase in temperature, addition of Ca(2+), and addition of PEO. No change of gelation time was observed with the addition of K(+). Upon gelation, a random coil structure of the silk fibroin was transformed into a beta-sheet structure. Hydrogels with fibroin concentrations >4 wt % exhibited network and spongelike structures on the basis of scanning electron microscopy. Pore sizes of the freeze-dried hydrogels were smaller as the silk fibroin concentration or gelation temperature was increased. Freeze-dried hydrogels formed in the presence of Ca(2+) exhibited larger pores as the concentration of this ion was increased. Mechanical compressive strength and modulus of the hydrogels increased with increase in protein concentration and gelation temperature. The results of these studies provide insight into the sol-gel transitions that silk fibroin undergoes in glands during aqueous processing while also providing important insight in the in vitro processing of these proteins into useful new materials.     

Controlled hydrogel formation of a recombinant spider silk protein

Due to their biocompatibility, biodegradability, and low immunogenicity, recombinant spider silk proteins have a high potential for a variety of applications when processed into morphologies such as films, capsules, beads, or hydrogels. Here, hydrogels made of the engineered and recombinantly produced spider silk protein eADF4(C16) were analyzed in detail. It has previously been shown that eADF4(C16) nanofibrils self-assemble by a mechanism of nucleation-aggregation, providing the basis of silk hydrogels. We focused on establishing a reproducible gelation process by employing different protein concentrations, chemical crosslinking, and functionalization of eADF4(C16) with fluorescein. Fluorescein strongly influenced assembly as well as the properties of the hydrogels, such as pore sizes and mechanical behavior, possibly due to its interference with packing of silk nanofibrils during hydrogel formation.     

Towards a fully synthetic substitute of alginate: optimization of a thermal gelation/chemical cross-linking scheme ("tandem" gelation) for the production of beads and liquid-core capsules

Fully synthetic polymers were used for the preparation of hydrogel beads and capsules, in a processing scheme that, originally designed for calcium alginate, was adapted to a "tandem" process, that is the combination a physical gelation with a chemical cross-linking.The polymers feature a Tetronic backbone (tetra armed Pluronics), which exhibits a reverse thermal gelation in water solutions within a physiological range of temperatures and pHs. The polymers bear terminal reactive groups that allow for a mild, but effective chemical cross-linking. Given an appropriate temperature jump, the thermal gelation provides a hardening kinetics similar to that of alginate. With slower kinetics, the chemical cross-linking then develops an irreversible and elastic gel structure, and determines its transport properties. In the present article this process has been optimized for the production of monodisperse, high elastic, hydrogel microbeads, and liquid-core microcapsules. We also show the feasibility of the use of liquid-core microcapsules in cell encapsulation. In preliminary experiments, CHO cells have been successfully encapsulated preserving their viability during the process and after incubation. The advantages of this process are mainly in the use of synthetic polymers, which provide great flexibility in the molecular design. This, in principle, allows for a precise tailoring of mechanical and transport properties and of bioactivity of the hydrogels, and also for a precise control in material purification.     

Biocompatible and biodegradable dual-drug release system based on silk hydrogel containing silk nanoparticles

We developed a facile and quick ethanol-based method for preparing silk nanoparticles and then fabricated a biodegradable and biocompatible dual-drug release system based on silk nanoparticles and the molecular networks of silk hydrogels. Model drugs incorporated in the silk nanoparticles and silk hydrogels showed fast and constant release, respectively, indicating successful dual-drug release from silk hydrogel containing silk nanoparticles. The release behaviors achieved by this dual-drug release system suggest to be regulated by physical properties (e.g., β-sheet contents and size of the silk nanoparticles and network size of the silk hydrogels), which is an important advantage for biomedical applications. The present silk-based system for dual-drug release also demonstrated no significant cytotoxicity against human mesenchymal stem cells (hMSCs), and thus, this silk-based dual-drug release system has potential as a versatile and useful new platform of polymeric materials for various types of dual delivery of bioactive molecules.     

Application of a dense gas technique for sterilizing soft biomaterials

Sterilization of soft biomaterials such as hydrogels is challenging because existing methods such as gamma irradiation, steam sterilization, or ethylene oxide sterilization, while effective at achieving high sterility assurance levels (SAL), may compromise their physicochemical properties and biocompatibility. New methods that effectively sterilize soft biomaterials without compromising their properties are therefore required. In this report, a dense-carbon dioxide (CO(2) )-based technique was used to sterilize soft polyethylene glycol (PEG)-based hydrogels while retaining their structure and physicochemical properties. Conventional sterilization methods such as gamma irradiation and steam sterilization severely compromised the structure of the hydrogels. PEG hydrogels with high water content and low elastic shear modulus (a measure of stiffness) were deliberately inoculated with bacteria and spores and then subjected to dense CO(2) . The dense CO(2) -based methods effectively sterilized the hydrogels achieving a SAL of 10(-7) without compromising the viscoelastic properties, pH, water-content, and structure of the gels. Furthermore, dense CO(2) -treated gels were biocompatible and non-toxic when implanted subcutaneously in ferrets. The application of novel dense CO(2) -based methods to sterilize soft biomaterials has implications in developing safe sterilization methods for soft biomedical implants such as dermal fillers and viscosupplements.     

State of water, molecular structure, and cytotoxicity of silk hydrogels

A novel technique was developed to regulate the bulk water content of silk hydrogels by adjusting the concentrations of silk proteins, which is helpful to investigate the effects of the state of water in polymeric hydrogel on its biological functions, such as cytotoxicity. Gelation of the silk hydrogel was induced with ethanol and its gelation behavior was analyzed by rheometry. The silk hydrogels prepared at various silk concentrations were characterized with respect to their water content, molecular and network structures, state of water, mechanical properties, and cytotoxicity to human mesenchymal stem cells. The network structure of silk hydrogel was heterogeneous with β-sheet and fibrillar structures. The influence of the state of water in the silk hydrogel on the cytotoxicity was recognized by means of differential scanning calorimetry and cell proliferation assay, which revealed that the bound water will support cell-adhesion proteins in the cellular matrix to interact with the surface of the silk hydrogels.     

Characterization of photo-cross-linked oligo[poly(ethylene glycol) fumarate] hydrogels for cartilage tissue engineering

Photo-cross-linkable oligo[poly(ethylene glycol) fumarate] (OPF) hydrogels have been developed for use in tissue engineering applications. We demonstrated that compressive modulus of these hydrogels increased with increasing polymer concentration, and hydrogels with different mechanical properties were formed by altering the ratio of cross-linker/polymer in precursor solution. Conversely, swelling of hydrogels decreased with increasing polymer concentration and cross-linker/polymer ratio. These hydrogels are degradable and degradation rates vary with the change in cross-linking level. Chondrocyte attachment was quantified as a method for evaluating adhesion of cells to the hydrogels. These data revealed that cross-linking density affects cell behavior on the hydrogel surfaces. Cell attachment was greater on the samples with increased cross-linking density. Chondrocytes on these samples exhibited spread morphology with distinct actin stress fibers, whereas they maintained their rounded morphology on the samples with lower cross-linking density. Moreover, chondrocytes were photoencapsulated within various hydrogel networks. Our results revealed that cells encapsulated within 2-mm thick OPF hydrogel disks remained viable throughout the 3-week culture period, with no difference in viability across the thickness of hydrogels. Photoencapsulated chondrocytes expressed the mRNA of type II collagen and produced cartilaginous matrix within the hydrogel constructs after three weeks. These findings suggest that photo-cross-linkable OPF hydrogels may be useful for cartilage tissue engineering and cell delivery applications.     

Morphology and gelation of thermosensitive xyloglucan hydrogels

Galactose modified xyloglucan is a thermally reversible hydrogel that is increasingly used in the biomedical field due to the ease of altering the gelation time and temperature by modifying the galactose removal ratio. However there is little information concerning the morphology and rheological properties of the hydrogel under physiological conditions. Differential scanning microcalorimetry (DSmicroC) showed the thermal gelation process to occur over a broad temperature range (5-50 degrees C). The rheological properties of the hydrogels were investigated as a function of concentration, temperature and ionic strength. The final elastic moduli of the hydrogels increased with increases in concentration. Isothermal rheology suggests that the gelation occurred in two distinct stages, which was influenced by the solution media. Scanning electron microscopy (SEM) was used to characterize the morphology of the xyloglucan which were thermally gelled at 37 degrees C. The resultant morphology was strongly dependent on the concentration of the hydrogel. Strong hydrogels were only obtained at 3 wt.% at 37 degrees C, and the morphology characterized by an open 3-dimensional network, comprised of thin membranes. It is proposed that the first stage of the isothermal gelation is the formation and growth of the thin membranes, followed by the formation of a three dimensional network.     

Evaluation of the in vitro degradation of macroporous hydrogels using gravimetry,confined compression testing,and microcomputed tomography

This study investigated the in vitro degradation characteristics of macroporous hydrogels based on poly(propylene fumarate-co-ethylene glycol) (P(PF-co-EG)). Four formulations were fabricated to test the effect of porosity and cross-linking density on the degradation of the resulting macroporous hydrogels. Macroporosity was introduced by the addition of sodium bicarbonate and ascorbic acid, the precursors of the carbon dioxide porogen, in the initiation system for the hydrogel cross-linking. Macroporous hydrogels with porosities of 0.80 +/- 0.03 and 0.89 +/- 0.03 were synthesized by the addition of sodium bicarbonate of concentrations 40 and 80 mg/mL and ascorbic acid of concentrations 0.05 and 0.1 mol/L, respectively. Poly(ethylene glycol) diacrylate (PEG-DA) was utilized as a cross-linker. The molecular weight between cross-links had a significant effect on weight loss after 12 weeks, where samples with M(C) of 1,880 +/- 320 synthesized with a P(PF-co-EG):PEG-DA ratio of 3:1 had a significantly greater mass loss due to degradation than those with M(C) of 1,000 +/- 100 synthesized with a P(PF-co-EG):PEG-DA ratio of 1:1. In contrast, porosity played a minimal role in determining the weight loss. Mechanical testing of the hydrogels under confined compression showed a decrease in compressive modulus over the degradation time for all formulations. In addition, an increase in hydrogel equilibrium water content and pore wall thickness was observed with degradation time, whereas the hydrogel porosity and surface area density remained invariant. The results from microcomputed tomography corroborated with the rest of the measurements and indicated a bulk degradation mechanism of the macroporous hydrogels.     

Photopolymerized thermosensitive hydrogels: synthesis, degradation, and cytocompatibility

In situ forming hydrogels based on thermosensitive polymers have attractive properties for tissue engineering. However, the physical interactions in these hydrogels are not strong enough to yield gels with sufficient stability for many of the proposed applications. In this study, additional covalent cross-links were introduced by photopolymerization to improve the mechanical properties and the stability of thermosensitive hydrogels. Methacrylate groups were coupled to the side chains of triblock copolymers (ABA) with thermosensitive poly( N-(2-hydroxypropyl) methacrylamide lactate) A blocks and a hydrophilic poly(ethylene glycol) B block. These polymers exhibit lower critical solution temperature (LCST) behavior in aqueous solution and the cloud point decreased with increasing amounts of methacrylate groups. These methacrylate groups were photopolymerized above the LCST to render covalent cross-links within the hydrophobic domains. The mechanical properties of photopolymerized hydrogels were substantially improved and their stability was prolonged significantly compared to nonphotopolymerized hydrogels. Whereas non-UV-cured gels disintegrated within 2 days at physiological pH and temperature, the photopolymerized gels degraded in 10 to 25 days depending on the degree of cross-linking. To assess biocompatibility, goat mesenchymal stem cells were seeded on the hydrogel surface or encapsulated within the gel and they remained viable as demonstrated by a LIVE/DEAD cell viability/cytotoxicity assay. Expression of alkaline phosphatase and production of collagen I demonstrated the functionality of the mesenchymal stem cells and their ability to differentiate upon encapsulation. Due to the improved mechanical properties, stability, and adequate cytocompatibility, the photopolymerized thermosensitive hydrogels can be regarded as highly potential materials for applications in tissue engineering.     

Dynamic compressive loading influences degradation behavior of PEG-PLA hydrogels

Biodegradable hydrogels are attractive 3D environments for cell and tissue growth. In cartilage tissue engineering, mechanical stimulation has been shown to be an important regulator in promoting cartilage development. However, the impact of mechanical loading on the gel degradation kinetics has not been studied. In this study, we examined hydrolytically labile gels synthesized from poly(lactic acid)-b-poly(ethylene glycol)-b-poly-(lactic acid) dimethacrylate macromers, which have been used for cartilage tissue engineering. The gels were subject to physiological loading conditions in order to examine the effects of loading on hydrogel degradation. Initially, hydrogels were formed with two different cross-linking densities and subject to a dynamic compressive strain of 15% at 0.3, 1, or 3 Hz. Degradation behavior was assessed by mass loss, equilibrium swelling and compressive modulus as a function of degradation time. From equilibrium swelling, the pseudo-first-order reaction rate constants were determined as an indication of degradation kinetics. The application of dynamic loading significantly enhanced the degradation time for the low cross-linked gels (P < 0.01) while frequency showed no statistical differences in degradation rates or bulk erosion profiles. In the higher cross-linked gels, a 3 Hz dynamic strain significantly increased the degradation kinetics resulting in an overall faster degradation time by 6 days compared to gels subject to the 0.3 and 1 Hz loads (P < 0.0001). The bioreactor set-up also influenced overall degradation behavior where the use of impermeable versus permeable platens resulted in significantly lower degradation rate constants for both cross-linked gels (P < 0.001). The compressive modulus exponentially decreased with degradation time under dynamic loading. Together, our findings indicate that both loading regime and the bioreactor setup influence degradation and should be considered when designing and tuning a biodegradable hydrogel where mechanical stimulation is employed.     

Simultaneously physically and chemically gelling polymer system utilizing a poly(NIPAAm-co-cysteamine)-based copolymer

The objective of this work was to create an in situ physically and chemically cross-linking hydrogel for in vivo applications. N-Isopropylacrylamide (NIPAAm) was copolymerized with N-acryloxysuccinimide (NASI) via free radical polymerization. Poly(NIPAAm-co-NASI) was further modified to obtain poly(NIPAAm-co-cysteamine) through a nucleophilic attack on the carbonyl group of the NASI by the amine group of the cysteamine. Modification was verified by nuclear magnetic resonance. In addition to thermoresponsive physical gelling due to the presence of NIPAAm, this system also chemically gels via a Michael-type addition reaction when mixed with poly(ethylene glycol) diacrylate. The presence of both physical and chemical gelation resulted in material properties that are much improved compared to purely physical gels. The chemical gelation time of the copolymers was not significantly affected by the amount of thiol present due to the increased pKa of the copolymer containing more thiols. In addition, the swelling of the copolymers was highly dependent on the temperature and thiol content. Last, the rate of nucleophilic attack in the Michael-type addition reaction was shown to be highly dependent on pH and on the mole ratio of thiol to acrylate. Due to the improved mechanical properties, this material may be better suited for long-term functional replacement applications than other thermosensitive physical gels. With further development and biocompatibility testing, this material could potentially be applied as a temperature-responsive injectable biomaterial for functional embolization.     

Poly(ethylene glycol) methacrylate/dimethacrylate hydrogels for controlled release of hydrophobic drugs

Hydrogels have been successfully used to entrap hydrophilic drugs and release them in a controlled fashion; however, the entrapment and release of hydrophobic drugs has not been well studied. We report on the release characteristics of a model hydrophobic drug, the steroid hormone estradiol, entrapped in low (MW 360/MW 550) and high (MW 526/MW 1000) molecular weight poly(ethylene glycol) methacrylate (PEG-MA)/dimethacrylate (PEG-DMA) hydrogels. The cross-linking ratio, temperature, and pH ranged from 10:1 to 10:3, from 33 to 41 degrees C, and from 2 to 12, respectively. The gelation of the PEG-MA/PEG-DMA hydrogel was initiated with UV irradiation. The absence of poly(glutamic acid) in the hydrogel formulation resulted in a loss of pH sensitivity in the acidic range, which was displayed by the hydrogels' similarities in swelling ratios in the pH buffers of pH 2, 4, and 7. Use of high molecular weight polymers resulted in a higher hydrogel swelling (300%) in comparison to the low molecular weight polymers. Drug size was found to be a significant factor. In comparison to 100% estradiol (MW 272) release, the fractional release of insulin (MW 5733) was 12 and 24% in low and high molecular weight gels at pH 2, respectively, and 17% in low molecular weight gels at pH 7. On the release kinetics of the estradiol drug, the hydrogels displayed a non-Fickian diffusion mechanism, which indicated that the media penetration rate is in the same range as the drug diffusion. The synthesis, entrapment, and release of estradiol by the PEG-MA/PEG-DMA hydrogels proved to be successful, but the use of ethanol in the buffers to promote the hydrophobic release of the estradiol in the in vitro environment caused complications, attributed to the process of transesterification.     

Development of a hybrid dextrin hydrogel encapsulating dextrin nanogel as protein delivery system

Dextrin, a glucose polymer with low molecular weight, was used to develop a fully resorbable hydrogel, without using chemical initiators. Dextrin was first oxidized (oDex) with sodium periodate and then cross-linked with adipic acid dihidrazide, a nontoxic cross-linking molecule. Furthermore, a new bidimensional composite hydrogel, made of oxidized dextrin incorporating dextrin nanogels (oDex-nanogel), was also developed. The oDex hydrogels showed good mechanical properties and biocompatibility, allowing the proliferation of mouse embryo fibroblasts 3T3 cultured on top of the gel. The gelation time may be controlled selecting the concentrations of the polymer and reticulating agent. Both the oDex and oDex-nanogel hydrogels are biodegradable and present a 3-D network with a continuous porous structure. The obtained hybrid hydrogel enables the release of the dextrin nanogel over an extended period of time, paralleling the mass loss curve due to the degradation of the material. The dextrin nanogel allowed the efficient incorporation of interleukin-10 and insulin in the oDex hydrogel, providing a sophisticated system of controlled release. The new hydrogels present promising properties as an injectable carrier of bioactive molecules. Both proteins and poorly water-soluble low-molecular-weight drugs are efficiently encapsulated in the nanogel, which performs as a controlled release system entrapped in the hydrogel matrix.     

Structural and mechanical properties of UV-photo-cross-linked poly(N-vinyl-2-pyrrolidone) hydrogels

Biocompatible poly( N-vinyl-2-pyrrolidone) (PVP) hydrogels have been produced by UV irradiation of aqueous polymer mixtures, using a high-pressure mercury lamp. The resulting materials have been characterized by a combination of experimental techniques, including rheology, small-angle neutron scattering (SANS), electron paramagnetic resonance (EPR), and pulsed gradient spin-echo nuclear magnetic resonance (PGSE-NMR), to put in evidence the relationship between the microstructural properties and the macrofunctional behavior of the gels. Viscoelastic measurements showed that UV photo-cross-linked PVP hydrogels present a strong gel mechanical behavior and viscoelastic moduli values similar to those of biological gels. The average distance between the cross-linking points of the polymer network was estimated from the hydrogels elastic modulus. However, SANS measurements showed that the network microstructure is highly inhomogeneous, presenting polymer-rich regions more densely cross-linked, surrounded by a water-rich environment. EPR and PGSE-NMR data further support the existence of these water-rich domains. Inclusion of a third component, such as glycerol, in the PVP aqueous mixture to be irradiated has been also investigated. A small amount of glycerol (<3% w/w) can be added keeping satisfactory properties of the hydrogel, while higher amounts significantly affect the cross-linking process.     

Characterization of the spontaneously forming hydrogels composed of water-soluble phospholipid polymers

Spontaneously forming hydrogels composed of 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymers, poly(MPC-co-methacrylic acid) (PMA), and poly(MPC-co-n-butyl methacrylate) (PMB) were examined. The MPC copolymer hydrogel was observed to have a spontaneous gelation property. To determine the properties of the hydrogels and why the gelation takes place, we have studied the properties of the hydrogels by scanning electron microscopy, X-ray photoelectron spectroscopy (XPS), and differential scanning calorimetry (DSC). The morphologies of the hydrogels were spongelike with a homogeneous structure. By XPS analysis in terms of the molecular distributions in the hydrogels, it was observed that a stabilization time was required for the hydrogel to undergo chain rearrangement. DSC thermograms of the hydrogels were different from their components, PMA and PMB. For the hydrogel, a crystallization peak around -30 degrees C was observed. This result indicated that some ordered structures existed in the hydrogels. To determine the role of the MPC groups, aqueous solutions of poly(methacrylic acid) (PMAc) and PMB were mixed. The mixture of PMAc-PMB turned into a sol state, and the sol state remained for a week. When the mixture was cooled, a very weak hydrogel was prepared. This result suggested that the MPC groups were the dominant unit for spontaneously forming the hydrogels.     

Fabrication of elastomeric silk fibers

Methods to generate fibers from hydrogels, with control over mechanical properties, fiber diameter, and crystallinity, while retaining cytocompatibility and degradability, would expand options for biomaterials. Here, we exploited features of silk fibroin protein for the formation of tunable silk hydrogel fibers. The biological, chemical, and morphological features inherent to silk were combined with elastomeric properties gained through enzymatic crosslinking of the protein. Postprocessing via methanol and autoclaving provided tunable control of fiber features. Mechanical, optical, and chemical analyses demonstrated control of fiber properties by exploiting the physical cross‐links, and generating double network hydrogels consisting of chemical and physical cross‐links. Structure and chemical analyses revealed crystallinity from 30 to 50%, modulus from 0.5 to 4 MPa, and ultimate strength 1–5 MPa depending on the processing method. Fabrication and postprocessing combined provided fibers with extensibility from 100 to 400% ultimate strain. Fibers strained to 100% exhibited fourth order birefringence, revealing macroscopic orientation driven by chain mobility. The physical cross‐links were influenced in part by the drying rate of fabricated materials, where bound water, packing density, and microstructural homogeneity influenced cross‐linking efficiency. The ability to generate robust and versatile hydrogel microfibers is desirable for bottom‐up assembly of biological tissues and for broader biomaterial applications.     

Biodegradable and biocompatible synthetic saccharide-Peptide hydrogels for three-dimensional stem cell culture

Saccharide-peptide hydrogels have been developed in our laboratory as new synthetic extracellular matrices for regenerative medicine applications. In this work, we have expanded on our previously reported system and applied copolymerization of cysteine (Cys) and vinyl sulfone (VS)-functionalized saccharide-peptide polymers via Michael-type addition for encapsulation and 3D culture of cells. Specifically, our aims were to (1) develop a novel hydrogel platform, which could be applied for encapsulating and culturing mesenchymal stem cells (MSCs) in a 3D environment, (2) characterize the tunable properties of the hydrogel, specifically, degradation, mechanical, and gel network properties, and (3) determine the biocompatibility of the saccharide-peptide hydrogel material with MSCs. Hydrogel mechanical properties were tunable by varying the VS:Cys ratio (= 0.5, 1, or 2) as well as the pH (6, 7, or 8) of the cross-linking components. Stiffer gels were formed at VS:Cys = 1 and pH 6 or 7. Gels formed at pH 8 or with excess Cys (VS:Cys = 0.5) or VS (VS:Cys = 2) were significantly softer. Cross-linking pH and VS:Cys ratio also had an effect on the degradation behavior of the VS:Cys gels, with higher cross-linking pH resulting in an accelerated loss of mass. On the basis of environmental scanning electron microscopy (ESEM) analysis and fluorescence microscopy, all hydrogels appeared to exhibit porous gel networks. MSCs cultured in monolayer and exposed to soluble Cys or VS copolymers (0.1-5 mg/mL) did not exhibit measurable cytotoxicity. In addition, MSCs were cultured in 3D for up to 14 days in vitro without deleterious effects on cell viability. In summary, we have established and characterized a tunable 3D saccharide-peptide hybrid copolymer hydrogel platform for culturing MSCs. Future studies will focus on utilizing the hydrogel system for controlling the differentiation of MSCs.