Mechanical stimulation of TMJ condylar chondrocytes encapsulated in PEG hydrogels

Temporomandibular joint (TMJ) disorders are most commonly associated with TMJ disc dislocation and osteoarthritis, which can cause erosion of the articular cartilage on the head of the mandibular condyle. There has been little attention focused on treating the damaged condylar cartilage. Therefore, the overall goal of this research is to create a tissue engineering therapy for resurfacing the damaged cartilage of the condylar process with healthy living tissue. Initially, bovine condylar cartilage explants were studied to understand the tissue structure, composition, and gene expression of the native tissue. The cell response of isolated condylar chondrocytes encapsulated in photopolymerized poly(ethylene glycol) hydrogels as a tissue engineering scaffold was examined in the presence and absence of dynamic loading for up to three days of culture. Condylar chondrocyte viability was maintained within the PEG hydrogel constructs over the culture period and loading conditions. Cell response was examined through real-time RTPCR for collagen types I and II and aggrecan, nitric oxide production, cell proliferation, proteoglycan (PG) synthesis, and spatial distribution of extracellular matrix through histology. This study demonstrates that PEG hydrogel constructs are suitable for condylar chondrocyte encapsulation in the absence of loading. However, dynamic compressive strains resulted in inhibition of gene expression, cell proliferation, and PG synthesis.     

Cell–matrix interactions and dynamic mechanical loading influence chondrocyte gene expression and bioactivity in PEG-RGD hydrogels

The pericellular matrix (PCM) surrounding chondrocytes is thought to play an important role in transmitting biochemical and biomechanical signals to the cells, which regulates many cellular functions including tissue homeostasis. To better understand chondrocytes interactions with their PCM, three-dimensional poly(ethylene glycol) (PEG) hydrogels containing Arg–Gly–Asp (RGD), the cell-adhesion sequence found in fibronectin and which is present in the PCM of cartilage, were employed. RGD was incorporated into PEG hydrogels via tethers at 0.1, 0.4 and 0.8 mM concentrations. Bovine chondrocytes were encapsulated in the hydrogels and subjected to dynamic compressive strains (0.3 Hz, 18% amplitude strain) for 48 h, and their response assessed by cell morphology, ECM gene expression, cell proliferation and matrix synthesis. Incorporation of RGD did not influence cell morphology under free swelling conditions. However, the level of cell deformation upon an applied strain was greater in the presence of RGD. In the absence of dynamic loading, RGD appears to have a negative effect on chondrocyte phenotype, as seen by a 4.7-fold decrease in collagen II/collagen I expressions in 0.8 mM RGD constructs. However, RGD had little effect on early responses of chondrocytes (i.e. cell proliferation and matrix synthesis/deposition). When isolating RGD as a biomechanical cue, cellular response was very different. Chondrocyte phenotype (collagen II/collagen I ratio) and proteoglycan synthesis were enhanced with higher concentrations of RGD. Overall, our findings demonstrate that RGD ligands enhance cartilage-specific gene expression and matrix synthesis, but only when mechanically stimulated, suggesting that cell–matrix interactions mediate chondrocyte response to mechanical stimulation.     

Incorporation of tissue-specific molecules alters chondrocyte metabolism and gene expression in photocrosslinked hydrogels

Hydrogels are highly swollen, insoluble networks which can entrap chondrocytes and provide a 3-D environment necessary for the re-growth of cartilaginous tissue. In this study, hydrogels were formulated with a synthetic poly(ethylene glycol) (PEG) component to provide control over the macroscopic gel properties and from a cartilage specific compound, chondroitin sulfate (ChSA), to capture features of the chondrocytes' native environment. PEG was chosen as the base hydrogel chemistry, because it forms a 3-D environment that maintains chondrocyte function. ChSA, a highly negatively charged main component of proteoglycans, was then selectively incorporated into the PEG gel. Macroscopic gel properties were manipulated to obtain high compressive moduli coupled with a high degree of swelling by formulating copolymer gels with these chemistries. The gel compressive modulus of cell-free PEG gels increased from 34 to 140 kPa with the incorporation of ChSA for similar degrees of swelling. When chondrocytes were encapsulated in pure ChSA gels, synthesis of collagen and glycosaminoglycans was inhibited. However, when PEG was introduced into the copolymer gels, both extracellular matrix components were stimulated. Total collagen content increased from non-detectable in the pure ChSA gels to 0.48+/-0.05 mg/g wet weight in the copolymer gels (40/60 ChSA/PEG). Gene expression for collagen type II was also enhanced by the incorporation of PEG into the gel, illustrating an important influence of gel chemistry on chondrocyte function; however, aggrecan gene expression was unaffected. This study demonstrates that the macroscopic properties of chondrocyte gel carriers can be controlled through the incorporation of charge into networks by ChSA, but the neutral, non-interactive base PEG chemistry facilitates extracellular matrix deposition.     

Thermoreversible hydrogel scaffolds for articular cartilage engineering

Articular cartilage has limited potential for repair. Current clinical treatments for articular cartilage damage often result in fibrocartilage and are associated with joint pain and stiffness. To address these concerns, researchers have turned to the engineering of cartilage grafts. Tissue engineering, an emerging field for the functional restoration of articular cartilage and other tissues, is based on the utilization of morphogens, scaffolds, and responding progenitor/stem cells. Because articular cartilage is a water-laden tissue and contains within its matrix hydrophilic proteoglycans, an engineered cartilage graft may be based on synthetic hydrogels to mimic these properties. To this end, we have developed a polymer system based on the hydrophilic copolymer poly(propylene fumarate-co-ethylene glycol) [P(PF-co-EG)]. Solutions of this polymer are liquid below 25 degrees C and gel above 35 degrees C, allowing an aqueous solution containing cells at room temperature to form a hydrogel with encapsulated cells at physiological body temperature. The objective of this work was to determine the effects of the hydrogel components on the phenotype of encapsulated chondrocytes. Bovine articular chondrocytes were used as an experimental model. Results demonstrated that the components required for hydrogel fabrication did not significantly reduce the proteoglycan synthesis of chondrocytes, a phenotypic marker of chondrocyte function. In addition, chondrocyte viability, proteoglycan synthesis, and type II collagen synthesis within P(PF-co-EG) hydrogels were investigated. The addition of bone morphogenetic protein-7 increased chondrocyte proliferation with the P(PF-co-EG) hydrogels, but did not increase proteoglycan synthesis by the chondrocytes. These results indicate that the temperature-responsive P(PF-co-EG) hydrogels are suitable for chondrocyte delivery for articular cartilage repair.     

Attachment and spreading of fibroblasts on an RGD peptide-modified injectable hyaluronan hydrogel

Hyaluronan (HA) hydrogels resist attachment and spreading of fibroblasts and most other mammalian cell types. A thiol-modified HA (3,3'-dithiobis(propanoic dihydrazide) [HA-DTPH]) was modified with peptides containing the Arg-Gly-Asp (RGD) sequence and then crosslinked with polyethylene glycol (PEG) diacrylate (PEGDA) to create a biomaterial that supported cell attachment, spreading, and proliferation. The hydrogels were evaluated in vitro and in vivo in three assay systems. First, the behavior of human and murine fibroblasts on the surface of the hydrogels was evaluated. The concentration and structure of the RGD peptides and the length of the PEG spacer influenced cell attachment and spreading. Second, murine fibroblasts were seeded into HA-DTPH solutions and encapsulated via in situ crosslinking with or without bound RGD peptides. Cells remained viable and proliferated within the hydrogel for 15 days in vitro. Although the RGD peptides significantly enhanced cell proliferation on the hydrogel surface, the cell proliferation inside the hydrogel in vitro was increased only modestly. Third, HA-DTPH/PEGDA/peptide hydrogels were evaluated as injectable tissue engineering materials in vivo. A suspension of murine fibroblasts in HA-DTPH was crosslinked using PEGDA plus PEGDA peptide, and the viscous, gelling mixture was injected subcutaneously into the flanks of nude mice; gels formed in vivo following injection. After 4 weeks, growth of new fibrous tissue had been accelerated by the sense RGD peptides. Thus, attachment, spreading, and proliferation of cells is dramatically enhanced on RGD-modified surfaces but only modestly accelerated in vivo tissue formation.     

Hydrogel properties influence ECM production by chondrocytes photoencapsulated in poly(ethylene glycol) hydrogels.

When using hydrogel scaffolds for cartilage tissue engineering, two gel properties are particularly important: the equilibrium water content (q, equilibrium swelling ratio) and the compressive modulus, K. In this work, chondrocytes were photoencapsulated in degrading and nondegrading poly(ethylene glycol)-based hydrogels to assess extracellular matrix (ECM) formation as a function of these gel properties. In nondegrading gels, the glycosaminoglycan (GAG) content was not significantly different in gels when q was varied from 4.2 to 9.3 after 2 and 4 weeks in vitro. However, gels with a q of 9.3 allowed GAGs to diffuse throughout the gels homogenously, but a q < or = 5.2 resulted in localization of GAGs pericellularly. Interestingly, in the moderately crosslinked gels with a K of 360 kPa, an increase in type II collagen synthesis was observed compared with gels with a higher (960 kPa) and lower (30 kPa) K after 4 weeks. With the incorporation of degradable linkages into the network, gel properties with an initially high K (350 kPa) and final high q (7.9) were obtained, which allowed for increased type II collagen synthesis coupled with a homogenous distribution of GAGs. Thus, a critical balance exists between gel swelling, mechanics, and degradation in forming a functional ECM.     

The effects of varying poly(ethylene glycol) hydrogel crosslinking density and the crosslinking mechanism on protein accumulation in three-dimensional hydrogels

Matrix stiffness has been shown to play an important role in modulating various cell fate processes such as differentiation and cell cycle. Given that the stiffness can be easily tuned by varying the crosslinking density, poly(ethylene glycol) (PEG) hydrogels have been widely used as an artificial cell niche. However, little is known about how changes in the hydrogel crosslinking density may affect the accumulation of exogenous growth factors within 3-D hydrogel scaffolds formed by different crosslinking mechanisms. To address such shortcomings, we measured protein diffusivity and accumulation within PEG hydrogels with varying PEG molecular weight, concentration and crosslinking mechanism. We found that protein accumulation increased substantially above a critical mesh size, which was distinct from the protein diffusivity trend, highlighting the importance of using protein accumulation as a parameter to better predict the cell fates in addition to protein diffusivity, a parameter commonly reported by researchers studying protein diffusion in hydrogels. Furthermore, we found that chain-growth-polymerized gels allowed more protein accumulation than step-growth-polymerized gels, which may be the result of network heterogeneity. The strategy used here can help quantify the effects of varying the hydrogel crosslinking density and crosslinking mechanism on protein diffusion in different types of hydrogel. Such tools could be broadly useful for interpreting cellular responses in hydrogels of varying stiffness for various tissue engineering applications.     

The effect of bioactive hydrogels on the secretion of extracellular matrix molecules by valvular interstitial cells

Valvular interstitial cells (VICs) were encapsulated in enzymatically degradable, crosslinked hydrogels formed from hyaluronic acid (HA) and poly(ethylene glycol) (PEG) macromolecular monomers. Titration of PEG with HA allowed for the synthesis of gels with a broad compositional spectrum, leading to a range of degradation behavior upon exposure to bovine testes hyaluronidase. The rate of mass loss and release of HA fragments from the copolymer gels depended on the PEG content of the network. These hydrogels were shown to have the dual function of permitting the diffusion of ECM elaborated by 3D cultured VICs and promoting the development of a specific matrix composition. Initial cleavage of hydrogel crosslinks, prior to network mass loss, permit the diffusion of collagen, while later stages of degradation promote elastin elaboration and suppress collagen production due to HA fragment release. Exogenous HA delivery through the cell culture media further demonstrated the utility of delivered HA on manipulating the secretory properties of encapsulated VICs.     

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.     

The differential effect of scaffold composition and architecture on chondrocyte response to mechanical stimulation

This study aims to explore the differential effect of scaffold composition and architecture on chondrogenic response to dynamic strain stimulation using encapsulating PEG-based hydrogels and primary bovine chondrocytes. Proteins and proteoglycans were conjugated to functionalized poly(ethylene glycol) (PEG) and immobilized in PEG hydrogels to create bio-synthetic materials to be used as scaffolds. Four different compositions were tested, including: PEG-Proteoglycan (PP), PEG-Fibrinogen (PF), PEG-Albumin (PA), and PEG only. Primary articular chondrocytes were encapsulated in the hydrogel scaffolds and subjected to 15% dynamic compressive strain stimulation at 1-Hz frequency for 28 days. Stimulation of PP, PF, PA and PEG constructs resulted in a respective increase in the unconfined true compressive modulus by 32%, 45.4%, 33.6%, and 28.2%, compared to their static controls. The PF showed a significantly larger relative increase in the modulus in comparison to all other scaffolds tested. These results support the hypothesis that mechanical stimulation and material bioactivity have a significant effect on the reported chondrocyte response. Similar trends were observed with the swelling ratio of the constructs. These findings indicate that while stimulation causes metabolic changes in chondrocytes seeded in PEG hydrogels, the matrix bioactivity has a significant role in enhancing chondrocyte mechanotransduction in encapsulating scaffolds subjected to physical deformations.     

3D cell entrapment in crosslinked thiolated gelatin-poly(ethylene glycol) diacrylate hydrogels

The combined use of natural ECM components and synthetic materials offers an attractive alternative to fabricate hydrogel-based tissue engineering scaffolds to study cell-matrix interactions in three-dimensions (3D). A facile method was developed to modify gelatin with cysteine via a bifunctional PEG linker, thus introducing free thiol groups to gelatin chains. A covalently crosslinked gelatin hydrogel was fabricated using thiolated gelatin and poly(ethylene glycol) diacrylate (PEGdA) via thiol-ene reaction. Unmodified gelatin was physically incorporated in a PEGdA-only matrix for comparison. We sought to understand the effect of crosslinking modality on hydrogel physicochemical properties and the impact on 3D cell entrapment. Compared to physically incorporated gelatin hydrogels, covalently crosslinked gelatin hydrogels displayed higher maximum weight swelling ratio (Qmax), higher water content, significantly lower cumulative gelatin dissolution up to 7 days, and lower gel stiffness. Furthermore, fibroblasts encapsulated within covalently crosslinked gelatin hydrogels showed extensive cytoplasmic spreading and the formation of cellular networks over 28 days. In contrast, fibroblasts encapsulated in the physically incorporated gelatin hydrogels remained spheroidal. Hence, crosslinking ECM protein with synthetic matrix creates a stable scaffold with tunable mechanical properties and with long-term cell anchorage points, thus supporting cell attachment and growth in the 3D environment.     

Maintaining dimensions and mechanical properties of ionically crosslinked alginate hydrogel scaffolds in vitro

Ionically crosslinked alginate hydrogels are attractive scaffolds because of their biocompatibility and mild gelation reaction that allows for gentle cell incorporation. However, the instability of ionically crosslinked hydrogels in an aqueous environment is a challenge that limits their application. This report presents a novel method to control the dimensions and mechanical properties of ionically crosslinked hydrogels via control of the ionic concentration of the medium. Homogeneous calcium-alginate gels were incubated in physiological saline baths adjusted to specific calcium ion concentrations. Swelling and shrinking occurred at low and high ionic concentrations of the medium, respectively, while an "optimal" intermediate calcium ion concentration of the medium was found to maintain original size and shape of the hydrogel. This optimal calcium ion concentration was found to be a function of crosslinking density and polymer concentration of the hydrogel and chemical composition of the alginate. The effects of optimal and high calcium ion concentrations of the medium on swelling behavior, calcium content, dry weight, and mechanical properties of the immersed hydrogels were investigated. It was found that the resulting hydrogel composition and mechanical properties depended on not only the calcium concentration of the medium, but also the crosslinking density and polymer concentration of the gel. In an 8-week experiment, controlled dimensions and mechanical properties of alginate gels in an aqueous environment were demonstrated. This new technique significantly enhances the potential of alginate hydrogels for tissue engineering and other biomedical applications.     

Development of an injectable,in situ crosslinkable,degradable polymeric carrier for osteogenic cell populations. Part 2. Viability of encapsulated marrow stromal osteoblasts cultured on crosslinking poly(propylene fumarate)

The effect of temporary encapsulation of rat marrow stromal osteoblasts in crosslinked gelatin microparticles on cell viability and proliferation was investigated in this study for microparticles placed on a crosslinking poly(propylene fumarate) (PPF) composite over a 7 day time period. Encapsulated cells were seeded on crosslinking PPF composites at times up to 10 min following initiation of the crosslinking reaction, and also on fully crosslinked PPF composites and tissue culture polystyrene controls, with a cell seeding density of 5.3 x 10(4) cells/cm2. The crosslinked PPF composite exhibited an average gel point of 10.3 min and an average maximum crosslinking temperature of 47.5 degrees C. Cell viability and proliferation were assessed by DNA and 3H-thymidine assays and the results were compared with those for nonencapsulated cells. The results showed that the addition time of cells to a crosslinking PPF composite had a large effect on cell viability and proliferation for both encapsulated and nonencapsulated cells with more surviving cells added at later time points. Most importantly, the temporary encapsulation of cells significantly enhanced cell viability at earlier time points. The data indicate that the presence of gelatin microparticles does not affect the crosslinking of a PPF composite. They further suggest that the temporary encapsulation of cells in crosslinked gelatin microparticles may preserve the viability of cells contained in an actively crosslinking PPF composite used as an injectable polymeric scaffold serving also as a carrier for osteogenic cell populations.     

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.     

Visible light crosslinkable chitosan hydrogels for tissue engineering

In situ gelling constructs, which form a hydrogel at the site of injection, offer the advantage of delivering cells and growth factors to the complex structure of the defect area for tissue engineering. In the present study, visible light crosslinkable hydrogel systems were presented using methacrylated glycol chitosan (MeGC) and three blue light initiators: camphorquinone (CQ), fluorescein (FR) and riboflavin (RF). A minimal irradiation time of 120 s was required to produce MeGC gels able to encapsulate cells with CQ or FR. Although prolonged irradiation up to 600 s improved the mechanical strength of CQ- or FR-initiated gels (compressive modulus 2.8 or 4.4 kPa, respectively), these conditions drastically reduced encapsulated chondrocyte viability to 5% and 25% for CQ and FR, respectively. Stable gels with 80-90% cell viability could be constructed using radiofrequency (RF) with only 40s irradiation time. Increasing irradiation time up to 300s significantly improved the compressive modulus of the RF-initiated MeGC gels up to 8.5 kPa without reducing cell viability. The swelling ratio and degradation rate were smaller at higher irradiation time. RF-photoinitiated hydrogels supported proliferation of encapsulated chondrocytes and extracellular matrix deposition. The feasibility of this photoinitiating system as in situ gelling hydrogels was further demonstrated in osteochondral and chondral defect models for potential cartilage tissue engineering. The MeGC hydrogels using RF offer a promising photoinitiating system in tissue engineering applications.     

Comparison of photopolymerizable thiol-ene PEG and acrylate-based PEG hydrogels for cartilage development

When designing hydrogels for tissue regeneration, differences in polymerization mechanism and network structure have the potential to impact cellular behavior. Poly(ethylene glycol) hydrogels were formed by free-radical photopolymerization of acrylates (chain-growth) or thiol-norbornenes (step-growth) to investigate the impact of hydrogel system (polymerization mechanism and network structure) on the development of engineered tissue. Bovine chondrocytes were encapsulated in hydrogels and cultured under free swelling or dynamic compressive loading. In the acrylate system immediately after encapsulation chondrocytes exhibited high levels of intracellular ROS concomitant with a reduction in hydrogel compressive modulus and higher variability in cell deformation upon compressive strain; findings that were not observed in the thiol-norbornene system. Long-term the quantity of sulfated glycosaminoglycans and total collagen was greater in the acrylate system, but the quality resembled that of hypertrophic cartilage with positive staining for aggrecan, collagens I, II, and X and collagen catabolism. The thiol-norbornene system led to hyaline-like cartilage production especially under mechanical loading with positive staining for aggrecan and collagen II and minimal staining for collagens I and X and collagen catabolism. Findings from this study confirm that the polymerization mechanism and network structure have long-term effects on the quality of engineered cartilage, especially under mechanical loading.     

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.     

Injectable chitosan hyaluronic acid hydrogels for cartilage tissue engineering

Injectable cartilaginous constructs that can form gels in tissue defects have many advantages in tissue engineering applications. In this study we created an injectable hydrogel consisting of methacrylated glycol chitosan (MeGC) and hyaluronic acid (HA) by photocrosslinking with a riboflavin photoinitiator under visible light. A minimum irradiation time of 40 s was required to produce stable gels for cell encapsulation with 87–90% encapsulated chondrocyte viability. Although increasing the irradiation time from 40 to 600 s significantly enhanced the compressive modulus of the hydrogels up to 11 or 17 kPa for MeGC or MeGC/HA, respectively, these conditions reduced the encapsulated cell viability to 60–65%. The majority of chondrocytes encapsulated in MeGC hydrogels after 300 s irradiation maintained a rounded shape with a high cell viability of ～80–87% over a 21 day culture period. The incorporation of HA in MeGC hydrogels increased the proliferation and deposition of cartilaginous extracellular matrix by encapsulated chondrocytes. These findings demonstrate that MeGC/HA composite hydrogels have the potential for cartilage repair.     

Anticancer efficacy of tannic acid is dependent on the stiffness of the underlying matrix

Tannic acid (TA) has been previously shown to have anticancer potential for breast cancer but its effects on melanoma have not yet been investigated. Similarly, stiffness of the tumor microenvironment is known to have a profound effect on breast cancer metastasis, but little is known about its role on melanoma. The goal of the current study is to investigate the synergistic effects of TA and matrix stiffness on melanoma progression. A375 melanoma cells with metastatic potential were cultured on TA crosslinked uncompacted (UC; soft) and electrochemically compacted (ECC; stiff) collagen gels and the effects of TA on gel morphology, mechanical properties, and cellular response (i.e. morphology and proliferation) were evaluated. SEM results showed that TA crosslinking induced merging of collagen fibrils that resulted in decrease in pore size of both UC and ECC collagen gels. Tensile testing showed that TA crosslinking significantly (p < 0.05) improved the mechanical properties of ECC collagen gels. Results from Alamar blue assay showed that TA preferentially inhibited the proliferation of A375 melanoma cells compared to the non-cancerous NIH 3T3 fibroblasts on UC collagen gels. However, on ECC collagen gels, preferential effect of TA was not prevalent as proliferation of both cell types was inhibited to a similar extent. When comparing the two gel types, inhibition of A375 melanoma cell proliferation was more pronounced on TA crosslinked UC collagen gels compared to TA crosslinked ECC collagen gels. Overall, these results suggest that TA incorporated into UC collagen gels may more selectively inhibit the proliferation of melanoma cells, and that matrix stiffness is an important driver of tumor proliferation and progression.     

Oligo(trimethylene carbonate)-poly(ethylene glycol)-oligo(trimethylene carbonate) triblock-based hydrogels for cartilage tissue engineering

A triblock co-polymer of oligo(trimethylene carbonate)-block-poly(ethylene glycol) 20000-block-oligo(trimethylene carbonate) diacrylate (TMC20) was used as a photo-polymerizable precursor for the encapsulation of primary articular chondrocytes. The efficacy of TMC20 as a biodegradable scaffold for cartilage tissue engineering was compared with non-degradable poly(ethylene glycol) 20000 diacrylate (PEG20) hydrogel. Chondrocytes encapsulated in PEG hydrogels containing oligo(trimethylene carbonate) (OTMC) moieties underwent spontaneous aggregation during in vitro culture, which was not observed in the PEG hydrogel counterparts. The aggregation of cells was found to be dependent on the initial cell density, as well as the mesh size of the hydrogels. Similarly, cell aggregation was also found in biodegradable PEG hydrogels containing caprolactone moieties. The aggregation of cells in TMC20 hydrogels resulted in enhanced cartilage matrix production compared with their PEG20 counterparts over 3 weeks of culture. Taken together, these results indicate that PEG hydrogels containing degradable OTMC moieties promote the aggregation and biosynthetic activity of encapsulated chondrocytes, indicating their potential as scaffolds for the repair of cartilage tissue.