Pericellular Matrix Formation Research Articles

Pericellular Matrix Formation and Atomic Force Microscopy of Single Primary Human Chondrocytes Cultured in Alginate Microgels.

One of the main components of articular cartilage is the chondrocyte's pericellular matrix (PCM), which is critical for regulating mechanotransduction, biochemical cues, and healthy cartilage development. Here, individual primary human chondrocytes (PHC) are encapsulated and cultured in 50µm diameter alginate microgels using drop-based microfluidics. This unique culturing method enables PCM formation and manipulation of individual cells. Over ten days, matrix formation is observed using autofluorescence imaging, and the elastic moduli of isolated cells are measured using AFM. Matrix production and elastic modulus increase are observed for the chondrons cultured in microgels. Furthermore, the elastic modulus of cells grown in microgels increases ≈ten-fold over ten days, nearly reaching the elastic modulus of in vivo PCM. The AFM data is further analyzed using a Gaussian mixture model and shows that the population of PHCs grown in microgels exhibit two distinct populations with elastic moduli averaging 9.0and 38.0kPa. Overall, this work shows that microgels provide an excellent culture platform for the growth and isolation of PHCs, enabling PCM formation that is mechanically similar to native PCM. The microgel culture platform presented here has the potential to revolutionize cartilage regeneration procedures through the inclusion of in vitro developed PCM.

Mechanical stimulation is a key component in the development of pericellular matrix around human chondrocytes

Purpose: Articular cartilage within the joint experience multi-directional mechanical stimulation. These stimuli are transferred to the chondrocytes through the pericellular matrix, a small shell surrounding the cells. The signal is then translated by the cell, which subsequently responds by releasing catabolic or anabolic markers. Our aim was to be able to determine if, depending on the mechanical stimulus applied onto the chondrocytes, there would be a variation in extracellular matrix production. To address this, we used our cartilage-on-chip and studied the effects of mechanical stimulation on human articular chondrocytes. The cartilage-on-chip system includes a 3D cell-hydrogel compartment, a perfusion channel to provide nutrients and a mechanical actuation unit. The mechanical actuation unit, which is separated from the rest of the system by a thin elastic polydimethylsiloxane membrane, comprises 3 individually addressable chambers were positive or negative pressure can be applied (Fig. 1). We report here how different mechanical stimuli: 1) affect IL-6 cytokine production; and 2) the chondrocyte’s phenotype compared to static conditions; 3) influence the directionality in extracellular matrix deposition; 4) stimulate pericellular matrix formation, and; 5) differentially affect overall protein expression. Methods: The cartilage-on-chip was fabricated using soft-lithography with polydimethylsiloxane. Application of positive and/or negative pressure resulted in different sets of stimulations onto the 3D hydrogel (compression or compression & shear strain). Low melting temperature agarose (2% w/v) was used to embed human primary chondrocytes (hCHs), which were cultured in the platform for one day with proliferation medium followed by chondrocyte differentiation medium. First, the cell-laden constructs were stimulated for 7 days (1 h a day at 1 Hz) with either compression, or compression and shear strain (wave form) or without mechanical stimuli. Medium was collected from the device at day 1, 2, 3, 5 and 7 and IL-6 concentration quantified using enhanced SPRi. qPCR was performed at day 7 for Collagen type I and type II. Next, cell-laden hydrogels were cultured for 7 days in static condition and subsequently stimulated for 7 days with the two aforementioned mechanical actuation patterns, or in static conditions for a total of 14 days. Samples were fixed and stained for aggrecan, collagen type II and collagen type VI. Results: The expression of COL2A1 mRNA was higher in the mechanically stimulated samples in particular when using a combination of compression and shear strain. Interestingly, COL1A1 mRNA expression was inversely correlated with the COL2A1 mRNA with highest expression in the static conditions. Consequently, these results suggest the involvement of mechanical stimulation on the transition of human chondrocytes towards a more hyaline like cartilage phenotype. IL-6 secretion decreased over time with no detectable signal at day 7 for the static conditions indicating chondrocyte adaptation to the 3D environment. Moreover, IL-6 was higher in the constructs exposed to mechanical stimuli, indicating a role of mechanical stimulation in IL-6 production. Aggrecan, collagen type II and collagen type VI immunostaining revealed higher pericellular matrix production in dynamic conditions. The pericellular matrix formed was mainly present in the direction of the mechanical stimulation suggesting cell adaptation to the applied stimuli (Fig. 3). Furthermore, the combination of compression and shear strain additionally enhanced both aggrecan and collagen production. Conclusions: Our cartilage-on-a-chip platform allows exposure of hydrogel embedded chondrocytes to multi-modal mechanical stimuli. Mechanical actuation supported the creation of a pericellular matrix surrounding the cells. Here, chondrocytes responded differently both at the gene and protein levels depending on the stimuli applied. Interestingly, chondrocytes produced their matrix in the direction of the stimuli suggesting a cell adaptation or “self-protection” mechanism. We are currently investigating if, by applying different biochemical stimuli, it is possible to influence the composition of the pericellular matrix.View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Perlecan Knockdown Significantly Alters Extracellular Matrix Composition and Organization During Cartilage Development

Perlecan is a critical proteoglycan found in the extracellular matrix (ECM) of cartilage. In healthy cartilage, perlecan regulates cartilage biomechanics and we previously demonstrated perlecan deficiency leads to reduced cellular and ECM stiffness in vivo This change in mechanics may lead to the early onset osteoarthritis seen in disorders resulting from perlecan knockdown such as Schwartz-Jampel syndrome (SJS). To identify how perlecan knockdown affects the material properties of developing cartilage, we used imaging and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to study the ECM in a murine model of SJS, Hspg2C1532Y-Neo Perlecan knockdown led to defective pericellular matrix formation, whereas the abundance of bulk ECM proteins, including many collagens, increased. Post-translational modifications and ultrastructure of collagens were not significantly different; however, LC-MS/MS analysis showed more protein was secreted by Hspg2C1532Y-Neo cartilage in vitro, suggesting that the incorporation of newly synthesized ECM was impaired. In addition, glycosaminoglycan deposition was atypical, which may explain the previously observed decrease in mechanics. Overall, these findings provide insight into the influence of perlecan on functional cartilage assembly and the progression of osteoarthritis in SJS.

3D models of chondrocytes within biomimetic scaffolds: Effects of cell deformation from loading regimens

BackgroundMechanical conditioning has been widely used to attempt to enhance chondrocyte metabolism for the evolution of functionally competent cartilage. However, although upregulation of proteoglycans have been reported through the application of uniaxial compression, minimal collagen has been produced. The study is designed to examine whether alternative loading regimens, equivalent to physiological conditions, involving shear in addition to compression can enhance collagen production. MethodsFinite element models were developed to determine how the local chondrocyte environments within agarose constructs were influenced by a range of static and dynamic loading regimens. 3-D poro-viscoelastic models were validated against experimental data. In particular, these models were used to characterise chondrocyte deformation in compression with and without shear superimposed, with special reference to the formation of pericellular matrix around the cells. FindingsThe models of the hydrogel constructs under stress relaxation and dynamic cyclic compression conditions were highly correlated with the experimental data. The cell deformation (y/z) in the constructs was greatest in the centre of the constructs, increasing with magnitude of compression up to 25%. The superposition of shear however did not produce significant additional changes in deformation, with the presence of PCM reducing the chondrocyte deformation. InterpretationThe use of FE models can prove important in the definition of appropriate, optimised mechanical conditioning regimens for the synthesis and organisation of mature extra cellular matrix by chondrocyte-seeded constructs. They will also provide insight into the mechanisms relating cell deformation to mechanotransduction pathways, thereby progressing the development of functionally competent tissue engineered cartilage.

Complementary techniques to analyse pericellular matrix formation by human MSC within hyaluronic acid hydrogels

Hydrogels are used widely for cell encapsulation to mimic the native ECM. Here, we characterise and visualise the matrix secreted by encapsulated cells by combining fluorescent non-canonical amino acid tagging with confocal Raman spectral imaging.

Maintenance and Acceleration of Pericellular Matrix Formation within 3D Cartilage Cell Culture Models.

In native articular cartilage, chondrocytes are surrounded by a thin pericellular matrix (PCM) forming chondrons. The PCM is exclusively rich in type VI collagen. The retention of the PCM has a significant influence on the metabolic activity of the chondrocytes. This study investigated the influence of 2 hydrogels (hyaluronic acid [HA] and agarose) and 2 media compositions (basal and chondrogenic) on the preservation/maintenance and acceleration of PCM formation over a 21-day time course. Different combinations of chondrocytes, chondrons, and mesenchymal stem cells (MSCs) were studied. Both hydrogels preserved chondrons PCM from day 1 up to 21-day culture regardless of media composition. Type VI collagen immunostaining of the cultured chondrons appeared both dense and homogenous. The presence of MSCs did not influence this outcome. At day 1, type VI collagen was not present around chondrocytes alone or their co-culture with MSCs. In the HA hydrogel, type VI collagen was located within the PCM after 7 days in both mono- and co-cultures. In the agarose hydrogel, collagen VI was located within the PCM at 7 days (co-cultures) and 14 days (monocultures). In both hydrogel systems, chondrogenic media enhanced the production of key extracellular matrix components in both mono- and co-cultures in comparison to basal media (11.5% and 14% more in glycosaminoglycans and type II collagen for chondrocytes samples at day 21 culture samples, respectively). However, the media types did not enhance type VI collagen synthesis. Altogether, a 3D chondrogenic hydrogel environment is the primary condition for maintenance and acceleration of PCM formation.

Formononetin Antagonizes the Interleukin-1β-Induced Catabolic Effects Through Suppressing Inflammation in Primary Rat Chondrocytes.

In the present study, we demonstrated the anti-catabolic effects of formononetin, a phytoestrogen derived from herbal plants, against interleukin-1β (IL-1β)-induced severe catabolic effects in primary rat chondrocytes and articular cartilage. Formononetin did not affect the viability of primary rat chondrocytes in both short- (24h) and long-term (21days) treatment periods. Furthermore, formononetin effectively antagonized the IL-1β-induced catabolic effects including the decrease in proteoglycan content, suppression of pericellular matrix formation, and loss of proteoglycan through the decreased expression of cartilage-degrading enzymes like matrix metalloproteinase (MMP)-13, MMP-1, and MMP-3 in primary rat chondrocytes. Moreover, catabolic oxidative stress mediators like nitric oxide, inducible nitric oxide synthase, cyclooxygenase-2, and prostaglandin E2 were significantly downregulated by formononetin in primary rat chondrocytes treated with IL-1β. Sequentially, the upregulation of pro-inflammatory cytokines (like IL-1α, IL-1β, IL-6, and tumor necrosis factor α), chemokines (like fractalkine, monocyte chemoattractant protein-1, and macrophage inflammatory protein-3α), and vascular endothelial growth factor were significantly downregulated by formononetin in primary rat chondrocytes treated with IL-1β. These data suggest that formononetin may suppress IL-1β-induced severe catabolic effects and osteoarthritic condition. Furthermore, formononetin may be a promising candidate for the treatment and prevention of osteoarthritis.

Neighboring cells override 3D hydrogel matrix cues to drive human MSC quiescence

Physical properties of modifiable hydrogels can be tuned to direct stem cell differentiation in a role akin to that played by the extracellular matrix in native stem cell niches. However, stem cells do not respond to matrix cues in isolation, but rather integrate soluble and non-soluble signals to balance quiescence, self-renewal and differentiation. Here, we encapsulated single cell suspensions of human mesenchymal stem cells (hMSC) in hyaluronic acid-based hydrogels at high and low densities to unravel the contributions of matrix- and non-matrix-mediated cues in directing stem cell response. We show that in high-density (HD) cultures, hMSC do not rely on hydrogel cues to guide their fate. Instead, they take on characteristics of quiescent cells and secrete a glycoprotein-rich pericellular matrix (PCM) in response to signaling from neighboring cells. Preventing quiescence precluded the formation of a glycoprotein-rich PCM and forced HD cultures to differentiate in response to hydrogel composition. Our observations may have important implications for tissue engineering as neighboring cells may act counter to matrix cues provided by scaffolds. Moreover, as stem cells are most regenerative if activated from a quiescent state, our results suggest that ex vivo native-like niches that incorporate signaling from neighboring cells may enable the production of clinically relevant, highly regenerative cells.

Vibrational spectroscopic monitoring and biochemical analysis of pericellular matrix formation and maturation in a 3-dimensional chondrocyte culture model.

Isolated and monolayer expanded chondrocytes are not the ideal cell form to produce a cartilage matrix. In articular cartilage, each chondrocyte is surrounded by a 2-4 μm thick collagen VI-rich pericellular matrix (PCM) forming a chondron. Freshly extracted chondrons form a more cartilage-like extracellular matrix (ECM) than chondrocytes and their surrounding PCM is thought to maintain the chondrocyte phenotype. To regenerate articular cartilage, preserving and/or regenerating a functional PCM is essential. In this study, a highly biomimicking hyaluronic acid (HA) hydrogel was used as a 3-dimensional system to culture freshly isolated bovine chondrons (with an intact PCM) and chondrocytes (without a PCM) for up to 21 days. We assessed the HA hydrogel's capacity to maintain and potentially re-generate PCM formation by both biochemical and immunological analyses of the key components of the PCM. For the first time, synchrotron based Fourier transform infrared (SR-FTIR) microspectroscopy was utilised to reveal the dynamic process of PCM re-generation. At day 1, highly specific collagen VI staining was visible within chondron containing HA hydrogels. In contrast, collagen VI was absent at day 1 but punctate, focal staining increased during the culture period of chondrocyte containing HA hydrogels. Chondron containing HA hydrogels produced more collagen II and GAGs than the chondrocyte containing HA hydrogels. Principal component analysis (PCA) of spectra in fingerprint regions of the chondrocyte-containing constructs at day 7, 14 and 21 culturing showed clear spectral differences. The clusters of day 14 and day 21 samples were closer to the chondron samples, while the day 7 samples were closer to chondrocytes. PCA scores in the lipid region revealed no major differences between chondrocyte and chondron samples, but showed that the cultured chondrocyte samples at day 7, day 14 and day 21 clustered together. These data would indicate that SR-FTIR microspectroscopy can help to better understand the PCM formation and maturation in tissue engineered models, which involves subtle changes in collagen and aggrecan.

Characterization and Standardization of Cultured Cardiac Fibroblasts for Ex Vivo Models of Heart Fibrosis and Heart Ischemia.

A full understanding of cardiac fibroblast (cFB) biology is essential to study the adverse cardiac remodeling and recovery of myocardium infarction. However, compared to cardiac myocytes, cFBs are less well characterized. Important questions, including the variability introduced by cell age (neonatal vs. adult), culture conditions (passage, plate coating, and culture medium), and responses to stimuli (e.g., hypoxia and drug treatments), have not been well addressed and standardization of techniques is lacking. This variability invites inconsistency and the confounding of study conclusions. Thus, we here focus on characterizing cell responses and standardizing procedures for cFB isolation and culture conditions to provide reliable platforms to address important questions about cFB proliferation, activation, collagen matrix formation, and responses to relevant stimuli. Thirty litters of 1-3-day pups and 30 female (240-330 g) Sprague-Dawley rats were used to isolate neonatal and adult cFBs. We detail and validate procedures to isolate cFBs for the use of culture or direct analysis. We characterize the differences between neonatal and adult cFBs, define the changes of cFBs during serial passage, and identify the response of cFBs to different culture conditions. We have also established models for the functional screening of profibrotic and antifibrotic drugs based on cFB proliferation, myofibroblast activation, and pericellular collagen matrix formation, and models of hypoxia/reoxygenation with appropriate time course and media conditions to achieve consistent cell injury. Our standardized procedures will ensure consistency in assessing cFB function. This original contribution provides a valid platform for the ex vivo investigation of the role of cFBs in cardiac ischemia and fibrosis.

4-Methylumbelliferone Suppresses Hyaluronan Synthesis and Tumor Progression in SCID Mice Intra-abdominally Inoculated With Pancreatic Cancer Cells.

ObjectivesPancreatic ductal adenocarcinoma contains large amounts of the glycosaminoglycan hyaluronan (HA), which is involved in various physiological processes. Here, we aimed to clarify the anticancer mechanisms of 4-methylumbelliferone (MU), a well-known HA synthesis inhibitor.MethodsMIA PaCa-2 human pancreatic cancer cells were used. We evaluated cellular proliferation, migration, and invasion in the presence of MU, exogenous HA, and an anti-CD44 antibody. We also analyzed apoptosis, CD44 expression, and HA-binding ability using flow cytometry. The HA content in tumor tissue was quantified and histopathologically investigated in mice who had been inoculated with cancer cells.ResultsIn vitro, MU inhibited pericellular HA matrix formation; however, HAS3 mRNA was up-regulated. Treatment with 0.5 mM MU suppressed cellular proliferation by 26.4%, migration by 14.7%, and invasion by 22.7%. Moreover, MU also significantly increased apoptosis. CD44 expression and HA-binding ability were not altered by MU. In vivo, MU suppressed HA accumulation in pancreatic tumors and improved survival times in tumor-bearing mice.Conclusions4-Methylumbelliferone indirectly caused apoptosis in pancreatic cancer cells by inhibiting HA production. 4-Methylumbelliferone may be a promising agent in the treatment of pancreatic cancer.

Lactoferricin mediates anti‐inflammatory and anti‐catabolic effects via inhibition of IL‐1 and LPS activity in the intervertebral disc

The catabolic cytokine interleukin-1 (IL-1) and endotoxin lipopolysaccharide (LPS) are well-known inflammatory mediators involved in degenerative disc disease, and inhibitors of IL-1 and LPS may potentially be used to slow or prevent disc degeneration in vivo. Here, we elucidate the striking anti-catabolic and anti-inflammatory effects of bovine lactoferricin (LfcinB) in the intervertebral disc (IVD) via antagonism of both IL-1 and LPS-mediated catabolic activity using in vitro and ex vivo analyses. Specifically, we demonstrate the biological counteraction of LfcinB against IL-1 and LPS-mediated proteoglycan (PG) depletion, matrix-degrading enzyme production, and enzyme activity in long-term (alginate beads) and short-term (monolayer) culture models using bovine and human nucleus pulposus (NP) cells. LfcinB significantly attenuates the IL-1 and LPS-mediated suppression of PG production and synthesis, and thus restores PG accumulation and pericellular matrix formation. Simultaneously, LfcinB antagonizes catabolic factor mediated induction of multiple cartilage-degrading enzymes, including MMP-1, MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5, in bovine NP cells at both mRNA and protein levels. LfcinB also suppresses the catabolic factor-induced stimulation of oxidative and inflammatory factors such as iNOS, IL-6, and toll-like receptor-2 (TLR-2) and TLR-4. Finally, the ability of LfcinB to antagonize IL-1 and LPS-mediated suppression of PG is upheld in an en bloc intradiscal microinjection model followed by ex vivo organ culture using both mouse and rabbit IVD tissue, suggesting a potential therapeutic benefit of LfcinB on degenerative disc disease in the future.

Novel effects of adenosine receptors on pericellular hyaluronan matrix: implications for human smooth muscle cell phenotype and interactions with monocytes during atherosclerosis

Hyaluronan (HA) is responsive to pro-atherosclerotic growth factors and cytokines and is thought to contribute to neointimal hyperplasia and atherosclerosis. However, the specific function of the pericellular HA matrix is likely depend on the respective stimuli. Adenosine plays an important role in the phenotypic regulation of vascular smooth muscle cells (VSMC) and is thought to inhibit inflammatory responses during atherosclerosis. The aim of this study was to examine the regulation and function of HA matrix in response to adenosine in human coronary artery SMC (HCASMC). The adenosine receptor agonist NECA (10 μM) caused a strong induction of HA synthase (HAS)1 at 6 h and a weaker induction again after 24 h. Use of selective adenosine receptor antagonists revealed that adenosine A2(B) receptors (A2(B)R) mediate the early HAS1 induction, whereas late HAS1 induction was mediated via A2(A)R and A3R. The strong response after 6 h was mediated in part via phosphoinositide-3 kinase- and mitogen-activated protein kinase pathways and was inhibited by Epac. Functionally, NECA increased cell migration, which was abolished by shRNA-mediated knock down of HAS1. In addition to HA secretion, NECA also stimulated the formation of pronounced pericellular HA matrix in HCASMC and increased the adhesion of monocytes. The adenosine-induced monocyte adhesion was sensitive to hyaluronidase. In conclusion, the current data suggest that adenosine via adenosine A2(B)R and A2(A)R/A3R induces HAS1. In turn a HA-rich matrix is formed by HCASMC which likely supports the migratory HCASMC phenotype and traps monocytes/macrophages in the interstitial matrix.

Interleukin-10 and Hyaluronan are Essential to the Fetal Fibroblast Functional Phenotype

Mid-gestational fetal wounds heal regeneratively with high levels of hyaluronan (HA) produced by fetal fibroblasts (FFb). FFb have distinct cellular functions compared to adult fibroblasts (AFb). We have previously shown an important role for IL-10 in fetal regenerative tissue repair with elevated levels in fetal skin, the ability to induce fetal HA-rich pericellular matrix (PCM) deposition in AFB, and the ability to induce regenerative wound healing in postnatal wounds. We hypothesize that IL-10 mediated HA-rich PCM formation is essential to the FFb functional phenotype and that this profile can be recapitulated in AFb by IL-10.

Essential Role of the Anti-Inflammatory Cytokine IL-10 in the Fetal Regenerative Phenotype is Mediated Via Stat3 and Hyaluronan Synthase: Implications for Scarless Wound Healing

Introduction: Mid-gestation fetal skin heals without scar with high levels of hyaluronan (HA) in the extracellular matrix (ECM) and an attenuated inflammatory response. We have previously demonstrated an essential role of interleukin-10 (IL-10) in fetal wound healing and the ability of fetal fibroblasts (FFb) to produce a large HA-rich pericellular matrix (PCM). These data include 1)elevated IL-10 levels in fetal skin, 2)IL-10-/- fetal wounds heal with scar, and 3)IL-10 induces postnatal wounds to heal scarlessly. Combining these data with accepted IL-10 signaling pathways, we hypothesize that the regenerative effects of IL-10 in the fetus are partially mediated through HA-rich PCM formation dependent on IL-10 receptor 1 (IL-10R1), STAT3 and hyaluronan synthase (HAS1-3) function. We further hypothesize IL-10 can recapitulate this fetal regenerative PCM phenotype in adult fibroblasts (AFb). Methods: to determine the role of IL-10 in FFb PCM production, murine IL-10-/- (E14.5) fibroblasts were cultured for 24 hours and PCM assessed. to determine if FFb produced PCM is HA rich, FFB were treated with hyaluronidase. Next in a sequential series of loss-of-function experiments, murine FFb (E14.5) were treated with inhibitors of IL-10R1 (IL-10R1 neutralizing Ab), STAT3 (STA-21) and HAS 1-3 (4-methyumbelliferone, 4-MU) or PBS and analyzed for PCM production. AFb were cultured with IL-10 (200ng/ml) and PCM assessed. PCM was quantified using erythrocyte particle exclusion assay and computer assisted morphometric analysis which generated a PCM area/cell area ratio. Data expressed as mean±SEM. Results: FFb produce a robust PCM (2.82+1.02). Treatment with hyaluronidase results in a significantly attenuated PCM in FFb (FFb with treatment 1.60±0.11 vs FFb 2.79±0.35;p<0.01). FFB produce a significantly greater PCM than IL10-/- FFb (FFb 2.82±1.02 vs IL10-/- 1.58±0.15;p<0.01). Blockade of IL-10R1 significantly reduces PCM formation compared to FFb (FFb with IL-10R1Ab 1.41±0.05 vs FFb 2.82±1.02;p=0.01). Inhibition of STAT3 by STA-21 results in decreased PCM formation (FFb with STA21 1.24±0.08 vs FFb 2.82±1.02;p<0.001). Attenuation of HAS1-3 function by 4-MU results in decreased PCM formation (FFb with 4-MU 1.63±0.16 vs FFb 2.82±1.02;p<0.01). Treatment with IL-10 significantly increases PCM production in adult Fb (AFb 1.84±0.40 vs AFb+IL-10 2.77±0.72;p<0.01) to levels which recapitulate the fetal regenerative PCM phenotype (AFb+IL-10 2.77±0.72vs FFb 2.82±1.02;p=NS). Conclusions: IL-10 is essential to the fetal fibroblast's ability to produce an HA-rich PCM. FFb production of the PCM is dependent on IL-10R1, STAT3 and HA synthase function. IL-10 can recapitulate PCM component of the fetal regenerative phenotype in adult fibroblasts. These data suggest that IL-10 plays a unique role in the fetal regenerative response, not only as an immunomodulatory agent, but as a regulator of the ECM.

Degradation Improves Tissue Formation in (Un)Loaded Chondrocyte-laden Hydrogels

Photopolymerizable poly(ethylene glycol) (PEG) hydrogels offer a platform to deliver cells in vivo and support three-dimensional cell culture but should be designed to degrade in sync with neotissue development and endure the physiologic environment. We asked whether (1) incorporation of degradation into PEG hydrogels facilitates tissue development comprised of essential cartilage macromolecules; (2) with early loading before pericellular matrix formation, the duration of load affects matrix production; and (3) dynamic loading in general influences macroscopic tissue development. Primary bovine chondrocytes were encapsulated in hydrogels (n = 3 for each condition). The independent variables were hydrogel degradation (nondegrading PEG and degrading oligo(lactic acid)-b-PEG-b-oligo(lactic acid) [PEG-LA]), culture condition (free swelling, unconfined dynamic compressive loading applied intermittently for 1 or 4 weeks), and time (up to 28 days). The dependent variables were neotissue deposition through biochemical contents, immunohistochemistry, and compressive modulus. Degradation led to 2.3- and 2.9-fold greater glycosaminoglycan and collagen contents, respectively; macroscopic cartilage-like tissue formation comprised of aggrecan, collagen II and VI, link protein, and decorin; but decreased moduli. Loading, applied early or throughout culture, did not affect neotissue content in either hydrogel but affected neotissue spatial distribution in degrading hydrogels where 4 weeks of loading appeared to enhance hydrogel degradation resulting in tissue defects. PEG-LA hydrogels led to macroscopic tissue development comprised of key cartilage macromolecules under loading, but hydrogel degradation requires further tuning. PEG-LA hydrogels have potential for delivering chondrocytes in vivo to replace damaged cartilage with a tissue-engineered native equivalent, overcoming many limitations associated with current clinical treatments.

Regulation of UDP‐glucose dehydrogenase is sufficient to modulate hyaluronan production and release, control sulfated GAG synthesis, and promote chondrogenesis

Glycosaminoglycans (GAGs) are critical for extracellular matrix (ECM) integrity in cartilage but mechanisms regulating their synthesis are not defined. UDP-glucose dehydrogenase (UGDH) catalyses UDP-glucose oxidation to UDP-glucuronic acid, an essential monosaccharide in many GAGs. Our previous studies in articular surface (AS) cells from embryonic joints have established pivotal roles for mitogen-activated protein kinases (MAPK) in synthesis of the unsulfated GAG, hyaluronan (HA). We investigated the functional significance of UGDH in GAG production and chondrogenesis, and determined roles for MEK-ERK and p38MAPK pathways in regulating UGDH expression and function. Inhibitors of MEK and p38MAPK reduced UGDH protein in AS cells. Treatment with TGF-β (archetypal growth factor) increased UGDH expression, sulfated (s)-GAG/HA release and pericellular matrix formation in a p38MAPK-dependent manner. Retroviral overexpression of UGDH augmented HA/sGAG release and pericellular matrix elaboration, which were blocked by inhibiting MEK but not p38MAPK. UGDH overexpression increased cartilage nodule size in bone marrow culture, promoted chondrogenesis in limb bud micromass culture and selectively suppressed medium HA levels and modified GAG sulfation, as assessed by FACE analysis. Our data provide evidence that: (i) TGF-β regulates UGDH expression via p38MAPK to modulate sGAG/HA secretion, (ii) MEK-ERK, but not p38MAPK facilitates UGDH-induced HA and sGAG release, and (iii) increased UGDH expression promotes chondrogenesis directly and differential modifies GAG levels and sulfation. These results indicate a more diverse role for UGDH in the support of selective GAG production than previously described. Factors regulating UGDH may provide novel candidates for restoring ECM integrity in degenerative cartilage diseases, such as osteoarthritis.Arthritis Research Campaign.

Versican induces a pro-metastatic ovarian cancer cell behavior which can be inhibited by small hyaluronan oligosaccharides

The assembly of pericellular matrix containing hyaluronan (HA) and versican has been shown to be a pre-requisite for proliferation and migration of mesenchymal cells. In this study, we investigated whether treatment with recombinant versican could induce the formation of a pericellular matrix by ovarian cancer cells (OVCAR-3, OVCAR-5, and SKOV-3) and promote their motility, invasion, and adhesion to peritoneal cells in vitro. We also determined whether versican-induced pericellular matrix formation and metastatic cancer cell behavior could be blocked by small HA oligosaccharides. Only combined treatment with recombinant versican and HA resulted in pericellular matrix formation by OVCAR-5 and SKOV-3 but not by OVCAR-3 cells, which lack the HA receptor, CD44. The motility of OVCAR-5 and SKOV-3 cells was significantly increased in scratch wound and chemotaxis assays following treatment with recombinant versican and HA. Versican and HA also promoted invasion of SKOV-3 and OVCAR-5 cells but had no effect on OVCAR-3 cells. We have demonstrated that exogenous HA significantly increased OVCAR-5 and SKOV-3 adhesion to peritoneal cells but adhesion was not further increased by versican treatment. Small HA oligomers (6-10 disaccharides) were able to significantly block formation of pericellular matrix by OVCAR-5 cells, as well as the increased motility and invasion induced by recombinant versican. HA oligomers also significantly blocked OVCAR-5 adhesion to peritoneal cells both in the presence and absence of exogenous HA. The dependence of CD44 for the versican and HA mediated effects were demonstrated by the inhibition of pericellular matrix formation as well as motility and invasion of OVCAR-5 cells following treatment with CD44 neutralizing antibody in the presence of versican and HA. We conclude that the acquisition of a HA/versican pericellular matrix by ovarian cancer cells increases their metastatic potential. HA oligomers can block this mechanism and are promising inhibitors of ovarian cancer dissemination.

Effect of pericellular matrix formation by chondrocytes cultured in agarose gel on the viscoelastic properties of agarose gel matrix

The viscoelasticity of chondrocyte-seeded agarose gel (AGC0) and that of chondrocyte-seeded agarose gel after 21 days of cultivation (AGC3) were investigated. In AGC3, pericellular matrix (PCM)-like material around each chondrocyte was found to be constructed, which was confirmed by an optical micrograph in conjunction with toluidine blue staining. The relaxation modulus of each of the chondrocyte-agarose gel composite systems was measured by a non-constrained indentation method. Stress-strain curves for all of the specimens examined had a toe region followed by a linear region terminated by specimen fracture. The slope of the linear region of AGC0 was smaller than that of AG, while the SS curve of AGC0 was indistinguishable from that of AGC3. All of the relaxation curves studied were typical of gels, having a fast relaxation process up to 103 s followed by a plateau. The relaxation modulus of AGC0 was smaller than that of agarose gel (AG), the decrement in relaxation modulus from AG to AGC0 being attributed to the seeding of chondrocytes that have a smaller modulus than that of agarose gels. However, the relaxation modulus of AGC3 was increased at the early viscoelastic region in particular, as compared with that of AGC0. The increments in the relaxation modulus in AGC3 were attributed to the PCM-like material produced by chondrocytes, where the produced material may provide crosslink points and reinforce the agarose gel.

Pericellular Matrix Formation Alters the Efficiency of Intracellular Uptake of Oligonucleotides in Osteosarcoma Cells

One of the crucial roles of tumor extracellular matrix is to act as a barrier to drug delivery. In this study, we analyzed the relationship between the formation of tumor extracellular matrix and the efficiency of intracellular uptake of oligonucleotides in human osteosarcoma cell lines, HOS, and MG-63. Oligonucleotides used in this study were nuclear factor-kappa B (NF-kappaB) decoy, which might be a therapeutic tool for neoplasms. Pericellular matrix formation was examined by particle exclusion assay. Cellular uptake of fluorescein isothiocyanate-labeled NF-kappaB decoy was evaluated by fluorescent microscopy and flow cytometry. Effects of NF-kappaB decoy on cell viability and cell cycle arrest in MG-63 cells were determined by MTT assay and flow cytometry, respectively. MG-63 cells exhibited abundant pericellular matrix with time compared with HOS cells. Uptake of fluorescein isothiocyanate-labeled NF-kappaB decoy decreased in MG-63 cells with time but not in HOS cells in both monolayer and three-dimensional culture using matrigel. However, after enzymatic removal of pericellular matrix, the uptake markedly recovered in MG-63 cells. NF-kappaB decoy inhibited cell proliferation and induced G0/G1 cell cycle arrest in MG-63 cells. These results suggest that abundant pericellular matrix might disturb the uptake of NF-kappaB decoy, and modification of pericellular matrix composition would increase the efficacy of exogenous oligonucleotides treatment for neoplasms.