Crosslinked hyaluronan scaffolds as a biologically active carrier for valvular interstitial cells

Hyaluronic acid (HA), a major component of the cardiac jelly during heart morphogenesis, is a polysaccharide that upon modification can be photopolymerized into hydrogels. Previous work in our lab has found that photopolymerizable HA hydrogels are suitable scaffolds for the culture and proliferation of valvular interstitial cells (VICs), the most prevalent cell type in native heart valves. The physical properties of HA gels are easily modified through alteration in material crosslink density or by copolymerizing with other reactive macromolecules. Degradation products of HA gels and the starting macromers significantly increased VIC proliferation when added to cell cultures. With low molecular weight HA (<6700 Da) exhibiting greatest stimulation of VIC proliferation. Low molecular weight HA degradation products added to VIC cultures also resulted in a four-fold increase in total matrix production and a two-fold increase in elastin production over untreated controls. VIC internalization of HA, as shown by cellular uptake of fluorescently labeled HA, likely activates signaling cascades resulting in the biological responses seen here. Lastly, VICs encapsulated within HA hydrogels remained viable, and significant elastin production was observed after 6 weeks of culture. This work shows promise for the creation of a tissue-engineered heart valve utilizing the synergistic relationship between hyaluronic acid and VICs.     

Controlling cartilaginous matrix evolution in hydrogels with degradation triggered by exogenous addition of an enzyme

Crosslinked hydrogels provide an accommodating environment for cartilage regeneration. However, degradation of the crosslinked network is necessary to create gels with an initially desirable mechanical stiffness and long-term distribution of properly assembled matrix molecules. In this study, chondrocytes were encapsulated in crosslinked poly(ethylene glycol) (PEG) hydrogels with caprolactone blocks that enabled an exogenously controlled, enzymatic degradation mechanism. At different stages of in vitro culture, a lipase enzyme was added to culture media to trigger degradation of the gel network. In gel constructs that never received lipase, the large cartilage matrix molecule, type II collagen, was localized to the pericellular region. Constructs that received lipase in the media for at least 1 week degraded enough to allow some distribution of collagen, but the timing and duration of lipase administration affected the outcome of regenerated tissue after 8 weeks of in vitro culture. Degradation that was triggered too early resulted in more significant defects in the cartilaginous matrix. The hydrogels applied in this study allow explicit control over degradation, and therefore provide a useful tool for investigating the effects of specific mass loss profiles on the evolution of neocartilage in vitro.     

Designing scaffolds for valvular interstitial cells: cell adhesion and function on naturally derived materials

Valvular interstitial cells (VICs) possess many properties that make them attractive for use in the construction of a tissue-engineered valve; however, we have found that the surfaces to which VICs will adhere and spread are limited. For example, VICs adhere and spread on collagen and laminin-coated surfaces, but display altered morphology and do not proliferate. Interestingly, fibronectin (FN) was one adhesion protein that facilitated VIC adhesion and proliferation. Yet VICs did not spread on surfaces modified with RGD, a ubiquitous cell-adhesive peptide, nor with other FN-specific peptide sequences such as EILDV and PHSRN. Hyaluronic acid (HA) is a highly elastic polysaccharide that is involved in natural valve morphogenesis and possesses binding interactions with FN. Hyaluronic acid was modified to form photopolymerizable hydrogels, and VICs were found to spread and proliferate on HA-based gels, forming a confluent monolayer on the gels within 4 days. Modified HA retained its ability to specifically bind FN, allowing for the formation of gels containing both HA and FN. Valvular interstital cells cultured on HA surfaces displayed significantly increased production of extracellular matrix proteins, indicating that HA-based scaffolds may provide useful biological cues to stimulate heart valve tissue formation.     

Semi-interpenetrating networks of hyaluronic acid in degradable PEG hydrogels for cartilage tissue engineering

Hydrolytically biodegradable poly(ethylene glycol) (PEG) hydrogels offer a promising platform for chondrocyte encapsulation and tuning degradation for cartilage tissue engineering, but offer no bioactive cues to encapsulated cells. This study tests the hypothesis that a semi-interpenetrating network of entrapped hyaluronic acid (HA), a bioactive molecule that binds cell surface receptors on chondrocytes, and crosslinked degradable PEG improves matrix synthesis by encapsulated chondrocytes. Degradation was achieved by incorporating oligo (lactic acid) segments into the crosslinks. The effects of HA molecular weight (MW) (2.9 × 10⁴ and 2 × 10⁶ Da) and concentration (0.5 and 5 mg g⁻¹) were investigated. Bovine chondrocytes were encapsulated in semi-interpenetrating networks and cultured for 4 weeks. A steady release of HA was observed over the course of the study with 90% released by 4 weeks. Incorporation of HA led to significantly higher cell numbers throughout the culture period. After 8 days, HA increased collagen content per cell, increased aggrecan-positive cells, while decreasing the deposition of hypertrophic collagen X, but these effects were not sustained long term. Measuring total sulfated glycosaminoglycan (sGAG) and collagen content within the constructs and released to the culture medium after 4 weeks revealed that total matrix synthesis was elevated by high concentrations of HA, indicating that HA stimulated matrix production although this matrix was not retained within the hydrogels. Matrix-degrading enzymes were elevated in the low-, but not the high-MW HA. Overall, incorporating high-MW HA into degrading hydrogels increased chondrocyte number and sGAG and collagen production, warranting further investigations to improve retention of newly synthesized matrix molecules.     

Dynamic stiffening of poly(ethylene glycol)-based hydrogels to direct valvular interstitial cell phenotype in a three-dimensional environment

Valvular interstitial cells (VICs) are active regulators of valve homeostasis and disease, responsible for secreting and remodeling the valve tissue matrix. As a result of VIC activity, the valve modulus can substantially change during development, injury and repair, and disease progression. While two-dimensional biomaterial substrates have been used to study mechanosensing and its influence on VIC phenotype, less is known about how these cells respond to matrix modulus in a three-dimensional environment. Here, we synthesized MMP-degradable poly(ethylene glycol) (PEG) hydrogels with elastic moduli ranging from 0.24 kPa to 12 kPa and observed that cell morphology was constrained in stiffer gels. To vary gel stiffness without substantially changing cell morphology, cell-laden hydrogels were cultured in the 0.24 kPa gels for 3 days to allow VIC spreading, and then stiffened in situ via a second, photoinitiated thiol-ene polymerization such that the gel modulus increased from 0.24 kPa to 1.2 kPa or 13 kPa. VICs encapsulated within soft gels exhibited αSMA stress fibers (∼40%), a hallmark of the myofibroblast phenotype. Interestingly, in stiffened gels, VICs became deactivated to a quiescent fibroblast phenotype, suggesting that matrix stiffness directs VIC phenotype independent of morphology, but in a manner that depends on the dimensionality of the culture platform. Collectively, these studies present a versatile method for dynamic stiffening of hydrogels and demonstrate the significant effects of matrix modulus on VIC myofibroblast properties in three-dimensional environments.     

Effects of PEG hydrogel crosslinking density on protein diffusion and encapsulated islet survival and function

The rational design of immunoprotective hydrogel barriers for transplanting insulin-producing cells requires an understanding of protein diffusion within the hydrogel network and how alterations to the network structure affect protein diffusion. Hydrogels of varying crosslinking density were formed via the chain polymerization of dimethacrylated PEG macromers of varying molecular weight, and the diffusion of six model proteins with molecular weights ranging from 5700 to 67,000 g/mol was observed in these hydrogel networks. Protein release profiles were used to estimate diffusion coefficients for each protein/gel system that exhibited Fickian diffusion. Diffusion coefficients were on the order of 10(-6)-10(-7) cm(2)/s, such that protein diffusion time scales (t(d) = L(2)/D) from 0.5-mm thick gels vary from 5 min to 24 h. Adult murine islets were encapsulated in PEG hydrogels of varying crosslinking density, and islet survival and insulin release was maintained after two weeks of culture in each gel condition. While the total insulin released during a 1 h glucose stimulation period was the same from islets in each sample, increasing hydrogel crosslinking density contributed to delays in insulin release from hydrogel samples within the 1 h stimulation period.     

Influence of glycosaminoglycan identity on vocal fold fibroblast behavior

Poly(ethylene glycol) (PEG) hydrogels have recently begun to be studied for the treatment of scarred vocal fold lamina propria due, in part, to their tunable mechanical properties, resistance to fibroblast-mediated contraction, and ability to be polymerized in situ. However, pure PEG gels lack intrinsic biochemical signals to guide cell behavior and generally fail to mimic the frequency-dependent viscoelastic response critical to normal superficial lamina propria function. Recent results suggest that incorporation of viscoelastic bioactive substances, such as glycosaminoglycans (GAGs), into PEG networks may allow these gels to more closely approach the mechanical responses of normal vocal fold lamina propria while also stimulating desired vocal fold fibroblast behaviors. Although a number of vocal fold studies have examined the influence of hyaluronan (HA) on implant mechanics and vocal fold fibroblast responses, the effects of other GAG types have been relatively unexplored. This is significant, since recent studies have suggested that chondroitin sulfate C (CSC) and heparan sulfate (HS) are substantially altered in scarred lamina propria. The present study was therefore designed to evaluate the effects of CSC and HS incorporation on the mechanical response of PEG gels and vocal fold fibroblast behavior relative to HA. As with PEG-HA, the viscoelasticity of PEG-CSC and PEG-HS gels more closely approached that of the normal vocal fold lamina propria than pure PEG hydrogels. In addition, collagen I deposition and fibronectin production were significantly higher in CSC than in HA gels, and levels of the myofibroblast marker smooth muscle α-actin (SM α-actin) were greater in CSC and HS gels than in HA gels. Since collagen I, fibronectin, and SM α-actin are generally elevated in scarred lamina propria these results suggest that CSC and HS may be undesirable for vocal fold implants relative to HA. Investigation of various signaling intermediates indicated that alterations in NFκB-p50, NFκB-p65, or pERK1/2 levels may underlie the observed differences among the PEG-GAG gels.     

Network connectivity, mechanical properties and cell adhesion for hyaluronic acid/PEG hydrogels

The study aimed to explore the influence of the network architecture on the mechanical properties and degradability of HA/PEG gels, and to highlight the relationship between Young's modulus and cell colonization with a selected architecture. Three different families of hyaluronic acid (HA)-based photopolymerized PEG diacrylate (PEGDA) hydrogels were compared, using different concentrations and molecular weights (64 and 234?kDa) of HA: semi-IPNs containing native HA in a PEG network (type I gels); co-networks obtained using thiolated HA as chain transfer agent during PEGDA polymerization (type II gels); co-networks obtained from the in situ preparation of a macromonomer derived from the Michael-type addition of thiolated HA on PEGDA (type III gels). From a comparative study of rheological properties and enzymatic degradability, type II gels were selected for a further study aiming to link their mechanical properties to cell spreading. Employing RGD-functionalized materials, Young's moduli were measured via AFM nanoindentation while the cell spreading behavior was quantitatively evaluated by monitoring morphology and metabolic activity (MTS assay) of L929 fibroblasts. By revealing a clear relation between increasing modulus and increasing cell spreading/proliferation, the study showed the possibility to fine tune the cell/material interactions with appropriate reactive processing techniques.     

Screening of hyaluronic acid–poly(ethylene glycol) composite hydrogels to support intervertebral disc cell biosynthesis using artificial neural network analysis

Hyaluronic acid (HA)–poly(ethylene glycol) (PEG) composite hydrogels have been widely studied for both cell delivery and soft tissue regeneration applications. A very broad range of physical and biological properties have been engineered into HA–PEG hydrogels that may differentially affect cellular “outcomes” of survival, synthesis and metabolism. The objective of this study was to rapidly screen multiple HA–PEG composite hydrogel formulations for an effect on matrix synthesis and behaviors of nucleus pulposus (NP) and annulus fibrosus (AF) cells of the intervertebral disc (IVD). A secondary objective was to apply artificial neural network analysis to identify relationships between HA–PEG composite hydrogel formulation parameters and biological outcome measures for each cell type of the IVD. Eight different hydrogels were developed from preparations of thiolated HA (HA–SH) and PEG vinylsulfone (PEG–VS) macromers, and used as substrates for NP and AF cell culture in vitro. Hydrogel mechanical properties ranged from 70 to 489 kPa depending on HA molecular weight, and measures of matrix synthesis, metabolite consumption and production and cell morphology were obtained to study relationships to hydrogel parameters. Results showed that NP and AF cell numbers were highest upon the HA–PEG hydrogels formed from the lower-molecular-weight HA, with evidence of higher sulfated glycosaminoglycan production also upon lower-HA-molecular-weight composite gels. All cells formed more multi-cell clusters upon any HA–PEG composite hydrogel as compared to gelatin substrates. Formulations were clustered into neurons based largely on their HA molecular weight, with few effects of PEG molecular weight observed on any measured parameters.     

Influence of cross-linker chemistry on release kinetics of PEG-co-PGA hydrogels

An investigation of encapsulated plasmid DNA release from degradable poly(ethylene glycol)-co-poly(glycolic acid) hydrogels (PEG-co-PGA) is presented. We determined by varying the chemistry of the cross-linker group, significant variations in hydrogel degradation kinetics could be achieved to control the release profiles of plasmid DNA. We prepared three analogues of PEG-co-PGA hydrogels by a photopolymerization process and measured variation in degradation rates by monitoring mechanical properties and release of plasmid DNA. 1H 1D DOSY NMR (one-dimensional diffusion ordered nuclear magnetic resonance spectroscopy) was used to measure conversion of vinyl groups after photocross-linking. Nearly full vinyl conversion was reached after 10 min exposure under ultraviolet light. Gel electrophoresis analysis confirmed that plasmid DNA remained structurally intact after photoencapsulation and release from the gels. This approach provides an additional strategy for controlling the release of biologically active compounds from hydrogels.     

The effect of hyaluronic acid incorporation on fibroblast spreading and proliferation within PEG-diacrylate based semi-interpenetrating networks

The nanometer-scale mesh size of many synthetic crosslinked hydrogel networks restricts encapsulated cells to a rounded morphology that can inhibit cellular processes such as proliferation and migration that are essential for the early stages of remodeling and tissue formation. The objective of these studies was to investigate an approach for accelerating cellular remodeling based on the creation of semi-interpenetrating networks (IPNs) composed of hydrolytically degradable poly(ethylene glycol) (PEG) diacrylate macromers and native, enzymatically degradable extracellular matrix (ECM) components (collagen, gelatin and hyaluronic acid (HA)). Among the three ECM components investigated, addition of HA at concentrations of 0.12% w/v and greater supported fibroblast spreading throughout the three-dimensional network and significantly increased proliferation relative to control hydrogels without HA. Incorporation of HA resulted in relatively small changes in hydrogel physical/chemical properties such as swelling, degradation rate, and elastic modulus. Fibroblast spreading was eliminated by the addition of hyaluronidase inhibitors, demonstrating that cell-mediated enzymatic degradation of HA is a necessary mechanism responsible for the observed increases in fibroblast activity. By accelerating early cellular remodeling and growth, these semi-IPNs may be useful vehicles for cell transplantation in a variety of tissue engineering applications.     

Synthesis and characterization of hyaluronic acid–poly(ethylene glycol) hydrogels via Michael addition: An injectable biomaterial for cartilage repair

Injectable hydrogels based on hyaluronic acid (HA) and poly(ethylene glycol) (PEG) were designed as biodegradable matrices for cartilage tissue engineering. Solutions of HA conjugates containing thiol functional groups (HA-SH) and PEG vinylsulfone (PEG-VS) macromers were cross-linked via Michael addition to form a three-dimensional network under physiological conditions. Gelation times varied from 14 min to less than 1 min, depending on the molecular weights of HA-SH and PEG-VS, degree of substitution (DS) of HA-SH and total polymer concentration. When the polymer concentration was increased from 2% to 6% (w/v) in the presence of 100 U ml^?1 hyaluronidase the degradation time increased from 3 to 15 days. Hydrogels with a homogeneous distribution of cells were obtained when chondrocytes were mixed with the precursor solutions. Culturing cell–hydrogel constructs prepared from HA185k-SH with a DS of 28 and cross-linked with PEG5k-4VS for 3 weeks in vitro revealed that the cells were viable and that cell division took place. Gel–cell matrices degraded in approximately 3 weeks, as shown by a significant decrease in dry gel mass. At day 21 glycosaminoglycans and collagen type II were found to have accumulated in hydrogels. These results indicate that these injectable hydrogels have a high potential for cartilage tissue engineering.     

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.     

Early tissue response to citric acid-based micro- and nanocomposites

A polymerizable superoxide dismutase mimetic (SODm) was incorporated into poly(ethylene glycol) (PEG) hydrogels to protect encapsulated cells from superoxide-mediated damage. Superoxide and other small reactive oxygen species (ROS) can cause oxidative damage to donor tissue encapsulated within size exclusion barrier materials. To enzymatically breakdown ROS within biomaterial cell encapsulation systems, Mn(III) Tetrakis[1-(3-acryloxy-propyl)-4-pyridyl] porphyrin (MnTTPyP-acryl), a polymerizable manganese metalloporphyrin SOD mimetic, was photopolymerized with PEG diacrylate (PEGDA) to create functional gels. In unmodified PEG hydrogels, a significant reduction in metabolic activity was observed when encapsulated Min6 β-cells were challenged with chemically generated superoxide. Cells encapsulated within MnTPPyP-co-PEG hydrogels, however, demonstrated greatly improved metabolic activity following various superoxide challenges. Further, cells were encapsulated and cultured for 10 days within MnTPPyP-co-PEG hydrogels and challenged with superoxide on days 4, 6, and 8. At the conclusion of this study, cells in blank PEG hydrogels had no observable metabolic activity but when encapsulated in MnTPPyP-functionalized hydrogels, cells retained 60 ± 5% of the metabolic activity compared to untreated controls.     

The effect of protein structure on their controlled release from an injectable peptide hydrogel

Hydrogel materials are promising vehicles for the delivery of protein therapeutics. Proteins can impart physical interactions, both steric and electrostatic in nature, that influence their release from a given gel network. Here, model proteins of varying hydrodynamic diameter and charge are directly encapsulated and their release studied from electropositive fibrillar hydrogels prepared from the self-assembling peptide, MAX8. Hydrogelation of MAX8 can be triggered in the presence of proteins for their direct encapsulation with neither effect on protein structure nor the hydrogel's mechanical properties. Bulk release of the encapsulated proteins from the hydrogels was assessed for a month time period at 37 °C before and after syringe delivery of the loaded gels to determine the influence of the protein structure on release. Release of positively charged and neutral proteins was largely governed by the sterics imposed by the network. Conversely, negatively charged proteins interacted strongly with the positively charged fibrillar network, greatly restricting their release to <10% of the initial protein load. Partition and retention studies indicated that electrostatic interactions dictate the amount of protein available for release. Importantly, when protein encapsulated gels were delivered via syringe, the release profiles of the macromolecules show the similar trends as those observed for non-sheared gels. This study demonstrates that proteins can be directly encapsulated in self assembled MAX8 hydrogels, which can then be syringe delivered to a site where subsequent release is controlled by protein structure.     

Bioresponsive phosphoester hydrogels for bone tissue engineering

Bioresponsive and intelligent biomaterials are a vehicle for manipulating cell function to promote tissue development and/or tissue engineering. A photopolymerized hydrogel based on a phosphoester- poly(ethylene glycol) polymer (PhosPEG) was synthesized for application to marrow-derived mesenchymal stem cell (MSC) encapsulation and tissue engineering of bone. The phosphor-containing hydrogels were hydrolytically degradable and the rate of degradation increased in the presence of a bone-derived enzyme, alkaline phosphatase. Gene expression and protein analysis of encapsulated MSCs demonstrated that PhosPEG-PEG cogels containing an intermediate concentration of phosphorus promoted the gene expression of bone-specific markers including type I collagen, alkaline phosphatase, and osteonectin, without the addition of growth factors or other biological agents, compared with pure poly(ethylene glycol)-based gels. Secretion of alkaline phosphatase, osteocalcin, and osteonectin protein was also increased in the PhosPEG cogels. Mineralization of gels increased in the presence of phosphorus in both cellular and acellular constructs compared with PEG gels. In summary, phosphate-PEG-derived hydrogels increase gene expression of bone-specific markers, secretion of bone-related matrix, and mineralization and may have a potential impact on bone-engineering therapies.     

Controlled release of bioactive TGF-beta 1 from microspheres embedded within biodegradable hydrogels

Transforming growth factor-beta1 (TGF-beta1) is of great relevance to cartilage development and regeneration. A delivery system for controlled release of growth factors such as TGF-beta1 may be therapeutic for cartilage repair. We have encapsulated TGF-beta1 into poly(DL-lactide-co-glycolide) (PLGA) microspheres, and subsequently incorporated the microspheres into biodegradable hydrogels. The hydrogels are poly(ethylene glycol) based, and the degradation rate of the hydrogels is controlled by the non-toxic cross-linking reagent, genipin. Release kinetics of TGF-beta1 were assessed using ELISA and the bioactivity of the released TGF-beta1 was evaluated using a mink lung cell growth inhibition assay. The controlled release of TGF-beta1 encapsulated within microspheres embedded in scaffolds is better controlled when compared to delivery from microspheres alone. ELISA results indicated that TGF-beta1 was released over 21 days from the delivery system, and the burst release was decreased when the microspheres were embedded in the hydrogels. The concentration of TGF-beta1 released from the gels can be controlled by both the mass of microspheres embedded in the gel, and by the concentration of genipin. Additionally, the scaffold permits containment and conformation of the spheres to the defect shape. Based on these in vitro observations, we predict that we can develop a microsphere-loaded hydrogel for controlled release of TGF-beta1 to a cartilage wound site.     

Bovine primary chondrocyte culture in synthetic matrix metalloproteinase-sensitive poly(ethylene glycol)-based hydrogels as a scaffold for cartilage repair

A poly(ethylene glycol) (PEG)-based hydrogel was used as a scaffold for chondrocyte culture. Branched PEG-vinylsulfone macromers were end-linked with thiol-bearing matrix metalloproteinase (MMP)-sensitive peptides (GCRDGPQGIWGQDRCG) to form a three-dimensional network in situ under physiologic conditions. Both four- and eight-armed PEG macromer building blocks were examined. Increasing the number of PEG arms increased the elastic modulus of the hydrogels from 4.5 to 13.5 kPa. PEG-dithiol was used to prepare hydrogels that were not sensitive to degradation by cell-derived MMPs. Primary bovine calf chondrocytes were cultured in both MMP-sensitive and MMP-insensitive hydrogels, formed from either four- or eight-armed PEG. Most (>90%) of the cells inside the gels were viable after 1 month of culture and formed cell clusters. Gel matrices with lower elastic modulus and sensitivity to MMP-based matrix remodeling demonstrated larger clusters and more diffuse, less cell surface-constrained cell-derived matrix in the chondron, as determined by light and electron microscopy. Gene expression experiments by real-time RT-PCR showed that the expression of type II collagen and aggrecan was increased in the MMP-sensitive hydrogels, whereas the expression level of MMP-13 was increased in the MMP-insensitive hydrogels. These results indicate that cellular activity can be modulated by the composition of the hydrogel. This study represents one of the first examples of chondrocyte culture in a bioactive synthetic material that can be remodeled by cellular protease activity.     

Controlling the spatial distribution of ECM components in degradable PEG hydrogels for tissue engineering cartilage

In developing a scaffold to support new tissue growth, the degradation rate and mass loss profiles of the scaffold are important design parameters. In this study, hydrogels were prepared by copolymerizing a degradable macromer, poly(lactic acid)-b-poly(ethylene glycol)-b-poly(lactic acid) endcapped with acrylate groups (PEG-LA-DA) with a nondegradable macromer, poly(ethylene glycol) dimethacrylate (PEGDM). The resulting hydrogels exhibited a range of degradation behavior and mass loss profiles. Chondrocytes were photoencapsulated in gels formulated with 50:50, 25:75, and 15:85 (mol % PEGDM: mol % PEG-LA-DA) and cultured for 6 weeks in vitro. The neocartilaginous tissue formed was examined biochemically and histologically. After 6 weeks, the DNA content in gels with 75 and 85% degradable crosslinks was nearly twice that of the DNA content in the 50% gels. The total collagen content was significantly higher in the 85% gel [2.4 +/- 0.8% wet weight (ww)] compared to the 50% gel (0.22 +/- 0.29% ww). In examining the neocartilaginous tissue with immunohistochemistry, type II collagen was localized in the pericellular region in the 50% gel; however, when increased degradation was incorporated into the gel, type II collagen was found throughout the neotissue. In summary, the important role of hydrogel degradation in controlling and influencing the deposition and distribution of extracellular matrix molecules was demonstrated and quantified.     

Development of porous PEG hydrogels that enable efficient,uniform cell-seeding and permit early neural process extension

Three-dimensional polymer scaffolds are useful culture systems for neural cell growth and can provide permissive substrates that support neural processes as they extend across lesions in the brain and spinal cord. Degradable poly(ethylene) glycol (PEG) gels have been identified as a particularly promising scaffold material for this purpose; however, process extension within PEG gels is limited to late stages of hydrogel degradation. Here we demonstrate that earlier process extension can be achieved from primary neural cells encapsulated within PEG gels by creating a network of interconnected pores throughout the gel. Our method of incorporating these pores involves co-encapsulating a cell solution and a fibrin network within a PEG gel. The fibrin is subsequently enzymatically degraded under cytocompatible conditions, leaving behind a network of interconnected pores within the PEG gel. The primary neural cell population encapsulated in the gel is of mixed composition, containing differentiated neurons, and multipotent neuronal and glial precursor cells. We demonstrate that the initial presence of fibrin does not influence the cell-fate decisions of the encapsulated precursor cells. We also demonstrate that this fabrication approach enables simple, efficient and uniform seeding of viable cells throughout the entire porous scaffold.