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This study investigated osteogenesis of human mesenchymal stem cells encapsulated in matrix‐metalloproteinase (MMP)‐sensitive poly(ethylene glycol) (PEG) hydrogels in chemically defined medium (10 ng/ml bone morphogenic factor‐2). Thiol‐norbornene photoclick hydrogels were formed with CRGDS and crosslinkers of PEG dithiol (nondegradable), CVPLS‐LYSGC (P1) or CRGRIGF‐LRTDC (P2; dash indicates cleavage site) at two crosslink densities. Exogenous MMP‐2 degraded P1 and P2 hydrogels similarly. MMP‐14 degraded P1 hydrogels more rapidly than P2 hydrogels. Cell spreading was greatest in P1 low crosslinked hydrogels and to a lesser degree in P2 low crosslinked hydrogels, but not evident in nondegradable and high crosslinked MMP‐sensitive hydrogels. Early osteogenesis (Alkaline phosphatase [ALP] activity) was accelerated in hydrogels that facilitated cell spreading. Contrarily, late osteogenesis (mineralization) was independent of cell spreading. Mineralized matrix was present in P1 hydrogels, but only present in P2 high crosslinked hydrogels and not yet present in nondegradable hydrogels. Overall, the low crosslinked P1 hydrogels exhibited an accelerated early and late osteogenesis with the highest ALP activity (Day 7), greatest calcium content (Day 14), and greatest collagen content (Day 28), concomitant with increased compressive modulus over time. Collectively, this study demonstrates that in chemically defined medium, hydrogel degradability is critical to accelerating early osteogenesis, but other factors are important in late osteogenesis. [ABSTRACT FROM AUTHOR]

Background: In this study, we investigate the in vitro and in vivo chondrogenic capacity of a novel photopolymerizable cartilage mimetic hydrogel, enhanced with extracellular matrix analogs, for cartilage regeneration. Purpose: To (1) determine whether mesenchymal stem cells (MSCs) embedded in a novel cartilage mimetic hydrogel support in vitro chondrogenesis, (2) demonstrate that the proposed hydrogel can be delivered in situ in a critical chondral defect in a rabbit model, and (3) determine whether the hydrogel with or without MSCs supports in vivo chondrogenesis in a critical chondral defect. Study Design: Controlled laboratory study. Methods: Rabbit bone marrow–derived MSCs were isolated, expanded, encapsulated in the hydrogel, and cultured in chondrogenic differentiation medium for 9 weeks. Compressive modulus was evaluated at day 1 and at weeks 3, 6, and 9. Chondrogenic differentiation was investigated via quantitative polymerase reaction, safranin-O staining, and immunofluorescence. In vivo, a 3 mm–wide × 2-mm-deep chondral defect was created bilaterally on the knee trochlea of 10 rabbits. Each animal had 1 defect randomly assigned to be treated with hydrogel with or without MSCs, and the contralateral knee was left untreated. Hence, each rabbit served as its own matched control. Three groups were established: group A, hydrogel (n = 5); group B, hydrogel with MSCs (n = 5); and group C, control (n = 10). Repair tissue was evaluated at 6 months after intervention. Results: In vitro, chondrogenesis and the degradable behavior of the hydrogel by MSCs were confirmed. In vivo, the hydrogel could be delivered intraoperatively in a sterile manner. Overall, the hydrogel group had the highest scores on the modified O'Driscoll scoring system (group A, 17.4 ± 4.7; group B, 13 ± 3; group C, 16.7 ± 2.9) (P = .11) and showed higher safranin-O staining (group A, 49.4% ± 20%; group B, 25.8% ± 16.4%; group C, 36.9% ± 25.2%) (P = .27), although significance was not detected for either parameter. Conclusion: This study provides the first evidence of the ability to photopolymerize this novel hydrogel in situ and assess its ability to provide chondrogenic cues for cartilage repair in a small animal model. In vitro chondrogenesis was evident when MSCs were encapsulated in the hydrogel. Clinical Relevance: Cartilage mimetic hydrogel may offer a tissue engineering approach for the treatment of osteochondral lesions. [ABSTRACT FROM AUTHOR]

ABSTRACT: Focal chondral lesions and early osteoarthritis (OA) are responsible for progressive joint pain and disability in millions of people worldwide, yet there is currently no surgical joint preservation treatment available to fully restore the long term functionality of cartilage. Limitations of current treatments for cartilage defects have prompted the field of cartilage tissue engineering, which seeks to integrate engineering and biological principles to promote the growth of new cartilage to replace damaged tissue. Toward improving cartilage repair, hydrogel design has advanced in recent years to improve their utility. Injectable hydrogels have emerged as a promising scaffold due to their wide range of properties, the ability to encapsulate cells within the material, and their ability to provide cues for cell differentiation. Some of these advances include the development of improved control over in situ gelation (e.g., light), new techniques to process hydrogels (e.g., multi‐layers), and better incorporation of biological signals (e.g., immobilization, controlled release, and tethering). This review summarises the innovative approaches to engineer injectable hydrogels toward cartilage repair. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:64–75, 2018. [ABSTRACT FROM AUTHOR]

ABSTRACT Poly(ethylene glycol) (PEG) hydrogels are highly tunable platforms that are promising cell delivery vehicles for chondrocytes and cartilage tissue engineering. In addition to characterizing the type of extracellular matrix (ECM) that forms, understanding the types of proteins that are secreted by encapsulated cells may be important. Thus, the objectives for this study were to characterize the secretome of chondrocytes encapsulated in PEG hydrogels and determine whether the secretome varies as a function of hydrogel stiffness and culture condition. Bovine chondrocytes were encapsulated in photoclickable PEG hydrogels with a compressive modulus of 8 and 46 kPa and cultured under free swelling or dynamic compressive loading conditions. Cartilage ECM deposition was assessed by biochemical assays and immunohistochemistry. The conditioned medium was analyzed by liquid chromatography-tandem mass spectrometry. Chondrocytes maintained their phenotype within the hydrogels and deposited cartilage-specific ECM that increased over time and included aggrecan and collagens II and VI. Analysis of the secretome revealed a total of 64 proteins, which were largely similar among all experimental conditions. The identified proteins have diverse functions such as biological regulation, response to stress, and collagen fibril organization. Notably, many of the proteins important to the assembly of a collagen-rich cartilage ECM were identified and included collagen types II(α1), VI (α1, α2, and α3), IX (α1), XI (α1 and α2), and biglycan. In addition, many of the other identified proteins have been reported to be present within cell-secreted exosomes. In summary, chondrocytes encapsulated within photoclickable PEG hydrogels secrete many types of proteins that diffuse out of the hydrogel and which have diverse functions, but which are largely preserved across different hydrogel culture environments. Biotechnol. Bioeng. 2017;114: 2096-2108. © 2017 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Photopolymerizable and hydrolytically labile poly(ethylene glycol) (PEG) hydrogels formed from photo-clickable reactions were investigated as cell delivery platforms for cartilage tissue engineering (TE). PEG hydrogels were formed from thiol-norbornene PEG macromers whereby the crosslinks contained caprolactone segments with hydrolytically labile ester linkages. Juvenile bovine chondrocytes encapsulated in the hydrogels were cultured for up to four weeks and assessed biochemically and histologically, using standard destructive assays, and for mechanical and ultrasound properties, as nondestructive assays. Bulk degradation of acellular hydrogels was confirmed by a decrease in compressive modulus and an increase in mass swelling ratio over time. Chondrocytes deposited increasing amounts of sulfated glycosaminoglycans and collagens in the hydrogels with time. Spatially, collagen type II and aggrecan were present in the neotissue with formation of a territorial matrix beginning at day 21. Nondestructive measurements revealed an 8-fold increase in compressive modulus from days 7 to 28, which correlated with total collagen content. Ultrasound measurements revealed changes in the constructs over time, which differed from the mechanical properties, and appeared to correlate with ECM structure and organization shown by immunohistochemical analysis. Overall, non-destructive and destructive measurements show that this new hydrolytically degradable PEG hydrogel is promising for cartilage TE. Statement of Significance Designing synthetic hydrogels whose degradation matches tissue growth is critical to maintaining mechanical integrity as the hydrogel degrades and new tissue forms, but is challenging due to the nature of the hydrogel crosslinks that inhibit diffusion of tissue matrix molecules. This study details a promising, new, photo-clickable and synthetic hydrogel whose degradation supports cartilaginous tissue matrix growth leading to the formation of a territorial matrix, concomitant with an increase in mechanical properties. Nondestructive assays based on mechanical and ultrasonic properties were also investigated using a novel instrument and found to correlate with matrix deposition and evolution. In sum, this study presents a new hydrogel platform combined with nondestructive assessments, which together have potential for in vitro cartilage tissue engineering. [ABSTRACT FROM AUTHOR]

A bioinspired multi-layer hydrogel was developed for the encapsulation of human mesenchymal stem cells (hMSCs) as a platform for osteochondral tissue engineering. The spatial presentation of biochemical cues, via incorporation of extracellular matrix analogs, and mechanical cues, via both hydrogel crosslink density and externally applied mechanical loads, were characterized in each layer. A simple sequential photopolymerization method was employed to form stable poly(ethylene glycol)-based hydrogels with a soft cartilage-like layer of chondroitin sulfate and low RGD concentrations, a stiff bone-like layer with high RGD concentrations, and an intermediate interfacial layer. Under a compressive load, the variation in hydrogel stiffness within each layer produced high strains in the soft cartilage-like layer, low strains in the stiff bone-like layer, and moderate strains in the interfacial layer. When hMSC-laden hydrogels were cultured statically in osteochondral differentiation media, the local biochemical and matrix stiffness cues were not sufficient to spatially guide hMSC differentiation after 21 days. However dynamic mechanical stimulation led to differentially high expression of collagens with collagen II in the cartilage-like layer, collagen X in the interfacial layer and collagen I in the bone-like layer and mineral deposits localized to the bone layer. Overall, these findings point to external mechanical stimulation as a potent regulator of hMSC differentiation toward osteochondral cellular phenotypes. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Damage to cartilage caused by injury or disease can lead to pain and loss of mobility, diminishing one''s quality of life. Because cartilage has a limited capacity for self-repair, tissue engineering strategies, such as cells encapsulated in synthetic hydrogels, are being investigated as a means to restore the damaged cartilage. However, strategies to date are suboptimal in part because designing degradable hydrogels is complicated by structural and temporal complexities of the gel and evolving tissue along multiple length scales. To address this problem, this study proposes a multi-scale mechanical model using a triphasic formulation (solid, fluid, unbound matrix molecules) based on a single chondrocyte releasing extracellular matrix molecules within a degrading hydrogel. This model describes the key players (cells, proteoglycans, collagen) of the biological system within the hydrogel encompassing different length scales. Two mechanisms are included: temporal changes of bulk properties due to hydrogel degradation, and matrix transport. Numerical results demonstrate that the temporal change of bulk properties is a decisive factor in the diffusion of unbound matrix molecules through the hydrogel. Transport of matrix molecules in the hydrogel contributes both to the development of the pericellular matrix and the extracellular matrix and is dependent on the relative size of matrix molecules and the hydrogel mesh. The numerical results also demonstrate that osmotic pressure, which leads to changes in mesh size, is a key parameter for achieving a larger diffusivity for matrix molecules in the hydrogel. The numerical model is confirmed with experimental results of matrix synthesis by chondrocytes in biodegradable poly(ethylene glycol)-based hydrogels. This model may ultimately be used to predict key hydrogel design parameters towards achieving optimal cartilage growth. [Copyright &y& Elsevier]

Abstract: This work describes the development and testing of poly(ethylene glycol) (PEG) hydrogels with independently controlled dimensions of wide and deep macrochannels for their ability to promote alignment of skeletal myoblasts and myoblast differentiation. A UV-photopatterned thiol-ene mold was employed to produce long channels, which ranged from ∼40 to 200μm in width and from ∼100 to 200μm in depth, within a PEG–RGD hydrogel. Skeletal myoblasts (C2C12) were successfully cultured multiple cell layers deep within the channels. Decreasing channel width, increasing channel depth and, interestingly, increasing cell layer away from the channel base all contributed to a decreased interquartile range of cell angle relative to the long axis of the channel wall, indicating improved cell alignment. Differentiation of skeletal myoblasts into myotubes was confirmed by gene expression for myoD, myogenin and MCH IIb, and myotube formation for all channel geometries, but was not dependent on channel size. Qualitatively, myotubes were characteristically different, as myotubes were larger and had more nuclei in larger channels. Overall, our findings demonstrate that relatively large features, which do not readily facilitate cell alignment in two dimensions, promote cell alignment when presented in three dimensions, suggesting an important role for three-dimensional spatial cues. [Copyright &y& Elsevier]

Poly(ethylene glycol) (PEG) hydrogels, modified with RGD, are promising platforms for cell encapsulation and tissue engineering. While these hydrogels offer tunable mechanical properties, the extent of the host response may limit their in vivo applicability. The overall objective was to characterize the effects of hydrogel stiffness on the in vitro macrophage response and in vivo host response. We hypothesized that stiffer substrates induce better attachment, adhesion, and increased cell spreading, which elevates the macrophage classically activated phenotype and leads to a more severe foreign body reaction (FBR). PEG-RGD hydrogels were fabricated with compressive moduli of 130, 240, and 840 kPa, and the same RGD concentration. Hydrogel stiffness did not impact macrophage attachment, but elicited differences in cell morphology. Cells retained a round morphology on 130 kPa substrates, with localized and dense F-actin and localized αV integrin stainings. Contrarily, cells on stiffer substrates were more spread, with filopodia protruding from the cell, a more defined F-actin, and greater αV integrin staining. When stimulated with lipopolysaccharide, macrophages had a classical activation phenotype, with increased expression of TNF-α, IL-1β, and IL-6, however the degree of activation was significantly reduced with the softest hydrogels. A FBR ensued in response to all hydrogels when implanted subcutaneously in mice, but 28 days postimplantation the layer of macrophages at the implant surface was significantly lower in the softest hydrogels. In conclusion, hydrogels with lower stiffness led to reduced macrophage activation and a less severe and more typical FBR, and therefore are more suited for in vivo tissue engineering applications. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012. [ABSTRACT FROM AUTHOR]

Abstract: Biochemical and biomechanical cues are known to influence the differentiation of stem cells. Biomechanical cues arise from cellular interactions with their surrounding matrix and from applied forces. This study investigates the role of biomechanical cues in chondrogenic and osteogenic differentiation of human marrow stromal cells (hMSC) when encapsulated in synthetic hydrogels. Poly(ethylene glycol) hydrogels were fabricated with tethered cell adhesion moieties, RGD. Cell-laden hydrogels were subjected to 4h daily intermittent dynamic compressive loading (0.3Hz, 15% amplitude strain) for up to 14days and the cell response evaluated by gene expression and matrix deposition for chondrogenic and osteogenic markers. The three-dimensional hydrogel supported chondrogenesis and osteogenesis under free swelling conditions, as shown by the up-regulation of cartilage-related markers (SOX9, Col II, Col X, and aggrecan) and staining for type II collagen and aggrecan and osteogenically by up-regulation of ALP and staining for type I collagen and for mineralization. However, under dynamic loading the expression of cartilage-related markers SOX9, Col II, Col X, and aggrecan were down-regulated, along with reduced aggrecan staining and no positive staining for type II collagen. Additionally, the bone-related markers RUNX2, Col I, and ALP were down-regulated and positive staining for type I collagen and mineralization was reduced. In conclusion, the selected loading regime appears to have an inhibitory effect on chondrogenesis and osteogenesis of hMSC encapsulated in PEG–RGD hydrogels after 14days in culture, potentially due to overloading of the differentiating hMSC before sufficient pericellular matrix is produced and/or due to large strains, particularly for osteogenically differentiating hMSC. [Copyright &y& Elsevier]

Abstract: Hydrogels that solidify in response to a dual, physical and chemical, mechanism upon temperature increase were fabricated and characterized. The hydrogels were based on N-isopropylacrylamide, which renders them thermoresponsive, and contained covalently cross-linkable moieties in the macromers. The effects of the macromer end group, acrylate or methacrylate, and the fabrication conditions on the degradative and swelling properties of the hydrogels were investigated. The hydrogels exhibited higher swelling below their lower critical solution temperature (LCST). When immersed in cell culture medium at physiological temperature, which was above their LCST, hydrogels showed constant swelling and no degradation over 8weeks, with the methacrylated hydrogels showing greater swelling than their acrylated analogs. In addition, hydrogels immersed in cell culture medium under the same conditions showed lower swelling compared with phosphate-buffered saline. The interplay between chemical cross-linking and thermally induced phase separation affected the swelling characteristics of the hydrogels in different media. Mesenchymal stem cells encapsulated in the hydrogels in vitro were viable over 3weeks and markers of osteogenic differentiation were detected when the cells were cultured with osteogenic supplements. Hydrogel mineralization in the absence of cells was observed in cell culture medium with the addition of fetal bovine serum and β-glycerol phosphate. The results suggest that these hydrogels may be suitable as carriers for cell delivery in tissue engineering. [Copyright &y& Elsevier]

Abstract: This study assessed the role of immobilized cell adhesion moieties on controlling the cellular attachment, adhesion and phenotype of cardiac muscle cells towards developing scaffolds for cardiac muscle tissue engineering. Collagen I, laminin and the cell-adhesive oligopeptide, arginine-glycine-aspartic acid (RGD) at concentrations of 0.5 and 5mM were covalently bound to flexible two-dimensional hydrogels. A robust skeletal myoblast cell line demonstrated good bioactivity for the modified hydrogels, resulting in myoblast attachment and development of an intracellular contractile network after 1 day. Primary neonatal rat ventricular myocytes cultured for up to 7 days, however, were more sensitive to the different modified substrates. Although total cardiomyocyte DNA content did not vary significantly with surface modification, immunostaining for the contractile protein Troponin I and focal adhesion protein vinculin revealed marked improvements in spreading and intracellular contractile protein deposition for cells attached to protein-modified hydrogels over those modified with RGD, regardless of RGD concentration. On the RGD-modified surfaces, cardiomyocytes self-associated, forming aggregates that exhibited a disorganized cytoarchitecture. Cardiomyocyte maturation was assessed through the fetal gene program where expression for atrial natriuretic peptide decreased and sarco(endo)plasmic reticulum Ca2+ increased with culture time for the protein-modified surfaces, indicating a trend towards maturation, while the α/β-myosin heavy-chain ratio remained near fetal expression levels for all surfaces. Overall, our findings suggest that whole proteins, collagen and laminin, are effective in promoting cardiomyocyte interaction with hydrogels and cardiomyocyte maturation while RGD does not provide adequate extracellular matrix cues for cardiomyocytes. [Copyright &y& Elsevier]

Abstract: 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 0.8mM concentrations. Bovine chondrocytes were encapsulated in the hydrogels and subjected to dynamic compressive strains (0.3Hz, 18% amplitude strain) for 48h, 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 0.8mM 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. [Copyright &y& Elsevier]

Abstract: Since pore size and geometry strongly impact cell behavior and in vivo reaction, the ability to create scaffolds with a wide range of pore geometries that can be tailored to suit a particular cell type addresses a key need in tissue engineering. In this contribution, we describe a novel and simple technique to design porous, degradable poly(2-hydroxyethyl methacrylate) hydrogel scaffolds with well-defined architectures using a unique photolithography process and optimized polymer chemistry. A sphere-template was used to produce a highly uniform, monodisperse porous structure. To create a patterned and porous hydrogel scaffold, a photomask and initiating light were employed. Open, vertical channels ranging in size from 360±25 to 730±70μm were patterned into ∼700μm thick hydrogels with pore diameters of 62±8 or 147±15μm. Collagen type I was immobilized onto the scaffolds to facilitate cell adhesion. To assess the potential of these novel scaffolds for tissue engineering, a skeletal myoblast cell line (C2C12) was seeded onto scaffolds with 147μm pores and 730μm diameter channels, and analyzed by histology and digital volumetric imaging. Cell elongation, cell spreading and fibrillar formation were observed on these novel scaffolds. In summary, 3D architectures can be patterned into porous hydrogels in one step to create a wide range of tissue engineering scaffolds that may be tailored for specific applications. [Copyright &y& Elsevier]

Prostaglandin E 2 (PGE 2) is a key signaling molecule produced by osteocytes in response to mechanical loading, but its effect on osteocytes is less understood. This work examined the effect of PGE 2 on IDG-SW3-derived osteocytes in standard 2D culture (collagen-coated tissue culture polystyrene) and in a 3D degradable poly(ethylene glycol) hydrogel. IDG-SW3 cells were differentiated for 35 days into osteocytes in 2D and 3D cultures. 3D culture led to a more mature osteocyte phenotype with 100-fold higher Sost expression. IDG-SW3-derived osteocytes were treated with PGE 2 and assessed for expression of genes involved in PGE 2 , anabolic, and catabolic signaling. In 2D, PGE 2 had a rapid (1 h) and sustained (24 h) effect on many PGE 2 signaling genes, a rapid stimulatory effect on Il6 , and a sustained inhibitory effect on Tnfrsf11b and Bglap. Comparing culture environment without PGE 2 , osteocytes had higher expression of all four EP receptors and Sost but lower expression of Tnfrsf11b , Bglap , and Gja1 in 3D. Osteocytes were more responsive to PGE 2 in 3D. With increasing PGE 2 , 3D led to increased Gja1 and decreased Sost expressions and a higher Tnfrsf11b / Tnfsf11 ratio, indicating an anabolic response. Further analysis in 3D revealed that EP4, the receptor implicated in PGE 2 signaling in bone, was not responsible for the PGE 2 -induced gene expression changes in osteocytes. In summary, osteocytes are highly responsive to PGE 2 when cultured in an in vitro 3D hydrogel model suggesting that autocrine and paracrine PGE 2 signaling in osteocytes may play a role in bone homeostasis. • A 3D degradable hydrogel promotes a mature osteocyte phenotype in IDG-SW3 cells. • IDG-SW3-derived osteocytes are highly sensitive to PGE 2 in 3D hydrogel culture. • In 3D, increasing PGE 2 concentration leads to an anabolic response in osteocytes. • The EP4 receptor does not mediate PGE 2 -induced changes in osteocyte gene expression. [ABSTRACT FROM AUTHOR]

Focal defects in articular cartilage are unable to self-repair and, if left untreated, are a leading risk factor for osteoarthritis. This study examined cartilage degeneration surrounding a defect and then assessed whether infilling the defect prevents degeneration. We created a focal chondral defect in porcine osteochondral explants and cultured them ex vivo with and without dynamic compressive loading to decouple the role of loading. When compared to a defect in a porcine knee four weeks post-injury, this model captured loss in sulfated glycosaminoglycans (sGAGs) along the defect's edge that was observed in vivo , but this loss was not load dependent. Loading, however, reduced the indentation modulus of the surrounding cartilage. After infilling with in situ polymerized hydrogels that were soft (100 kPa) or stiff (1 MPa) and which produced swelling pressures of 13 and 310 kPa, respectively, sGAG loss was reduced. This reduction correlated with increased hydrogel stiffness and swelling pressure, but was not affected by loading. This ex vivo model recapitulates sGAG loss surrounding a defect and, when infilled with a mechanically supportive hydrogel, degeneration is minimized. • An ex vivo chondral defect recapitulated proteoglycan loss observed in vivo. • Infilling the defect with an in situ polymerized hydrogel reduces degeneration. • Cartilage-matched stiffness and exerted swelling pressures are beneficial. [ABSTRACT FROM AUTHOR]

The implantation of non-biological materials, including scaffolds for tissue engineering, ubiquitously leads to a foreign body response (FBR). We recently reported that this response negatively impacts fibroblasts encapsulated within a synthetic hydrogel and in turn leads to a more severe FBR, suggesting a cross-talk between encapsulated cells and inflammatory cells. Given the promise of mesenchymal stem cells (MSCs) in tissue engineering and recent evidence of their immunomodulatory properties, we hypothesized that MSCs encapsulated within poly(ethylene glycol) (PEG) hydrogels will attenuate the FBR. In vitro , murine MSCs encapsulated within PEG hydrogels attenuated classically activated primary murine macrophages by reducing gene expression and protein secretion of pro-inflammatory cytokines, most notably tumor necrosis factor-α. Using a COX2 inhibitor, prostaglandin E2 (PGE2) was identified as a mediator of MSC immunomodulation of macrophages. In vivo , hydrogels laden with MSCs, osteogenically differentiating MSCs, or no cells were implanted subcutaneously into C57BL/6 mice for 28 days to assess the impact of MSCs on the fibrotic response of the FBR. The presence of encapsulated MSCs reduced fibrous capsule thickness compared to acellular hydrogels, but this effect diminished with osteogenic differentiation. The use of MSCs prior to differentiation in tissue engineering may therefore serve as a dynamic approach, through continuous cross-talk between MSCs and the inflammatory cells, to modulate macrophage activation and attenuate the FBR to implanted synthetic scaffolds thus improving the long-term tissue engineering outcome. [ABSTRACT FROM AUTHOR]

Abstract: Poly(ethylene glycol) (PEG)-based hydrogels are promising in situ cell carriers for tissue engineering. However, their success in vivo will in part depend upon the foreign body reaction (FBR). This study tests the hypothesis that the FBR affects cells encapsulated within PEG hydrogels, and in turn influences the severity of the FBR. Fibroblasts were encapsulated within PEG hydrogels containing RGD to support cell attachment. Macrophages were seeded on top of cell-laden hydrogels to mimic in vivo macrophage interrogation and treated with lipopolysaccharide to induce an inflammatory phenotype. The presence of activated macrophages reduced fibroblast gene expression for extracellular matrix molecules and remodeling, but stimulated VEGF and IL-1β gene expression. Fibroblasts impacted macrophage phenotype leading to increased iNOS, IL-1β and TNF-α expressions. Syngeneic cell-laden and acellular hydrogels were also implanted subcutaneously into C57bl/6 mice for 2, 7 and 28 days. Encapsulated fibroblasts secreted collagen type I during the first week, but tissue deposition and cellularity decreased by 28 days. The presence of encapsulated fibroblasts led to greater acute inflammation, but did not influence the fibrotic response. In summary, this work emphasizes the importance of the host response in tissue engineering, and the potentially deleterious impact it may have on cell-laden synthetic scaffolds. [Copyright &y& Elsevier]

Abstract: Scanning electrochemical microscopy has been used to examine the molecular transport properties of photopolymerized poly(ethylene glycol) (PEG) hydrogels having different mesh sizes. Both the molecular weight (508 Da or 3000 Da) of the PEG diacrylate macromer and its weight percent (20 wt%, 40 wt%, or 60 wt%) in solution prior to photopolymerization were varied. Mesh size was estimated from equilibrium swelling measurements and a thermodynamic model. Estimated mesh sizes ranged from ca. 10 Å for 60 wt% PEG 508 gels to ca. 100 Å for 20 wt% PEG 3000 gels. The electrochemically active diffusing species, ferrocenemethanol, was detected via oxidation at a platinum microelectrode. For a given hydrogel, multiple approach curves showed a consistent relationship between current and distance. Electrochemically estimated diffusivities followed the same trend as predictions based on mesh size and ranged from 25% to 80% of the diffusivity in aqueous solution. As a proof of concept, scanning electrochemical microscopy was successfully used to map the topography of hydrogels with complex architecture, which are being designed as cell scaffolds. [Copyright &y& Elsevier]

Abstract: This study aimed to elucidate the role of charge in mediating chondrocyte response to loading by employing synthetic 3D hydrogels. Specifically, neutral poly(ethylene glycol) (PEG) hydrogels were employed where negatively charged chondroitin sulfate (ChS), one of the main extracellular matrix components of cartilage, was systematically incorporated into the PEG network at 0%, 20% or 40% to control the fixed charge density. PEG hydrogels were employed as a control environment for extracellular events which occur as a result of loading, but which are not associated with a charged matrix (e.g., cell deformation and fluid flow). Freshly isolated bovine articular chondrocytes were embedded in the hydrogels and subject to dynamic mechanical stimulation (0.3Hz, 15% amplitude strains, 6h) and assayed for nitric oxide production, cell proliferation, proteoglycan synthesis, and collagen deposition. In the absence of loading, incorporation of charge inhibited cell proliferation by ~75%, proteoglycan synthesis by ~22–50% depending on ChS content, but had no affect on collagen deposition. Dynamic loading had no effect on cellular responses in PEG hydrogels. However, dynamically loading 20% ChS gels inhibited nitrite production by 50%, cell proliferation by 40%, but stimulated proteoglycan and collagen deposition by 162% and 565%, respectively. Dynamic loading of 40% ChS hydrogels stimulated nitrite production by 62% and proteoglycan synthesis by 123%, but inhibited cell proliferation by 54% and collagen deposition by 52%. Upon removing the load and culturing under free-swelling conditions for 36h, the enhanced matrix synthesis observed in the 20% ChS gels was not maintained suggesting that loading is necessary to stimulate matrix production. In conclusion, extracellular events associated with a charged matrix have a dramatic affect on how chondrocytes respond to mechanical stimulation within these artificial 3D matrices suggesting that streaming potentials and/or dynamic changes in osmolarity may be important regulators of chondrocytes while cell deformation and fluid flow appear to have less of an effect. [Copyright &y& Elsevier]