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• Circulatory HAPLN1 level decreased with aging. • Restoking HAPLN1 in aged mice elevates skin collagen and hyaluronic acid levels. • rhHAPLN1 prevents TGF-β R2 degradation in dermal fibroblast thus up-regulates collagen and HA production. • rhHAPLN1 down-regulates endocytosis frequency and promotes CD44 clustering. • rhHAPLN1 rejuvenates skin aging by up-regulating HA-CD44-mediated TGF-β and NRF2 signaling. The skin seems to rejuvenate upon exposure to factors within the circulation of young organisms. Intrinsic factors that modulate skin aging are poorly understood. We used heterochronic parabiosis and aptamer-based proteomics to identify serum-derived rejuvenating factors. We discovered a novel extracellular function of hyaluronan and proteoglycan link protein 1 (HAPLN1). Its serum levels decreased with age, disturbing the integrity of the skin extracellular matrix, which is predominantly composed of collagen I and hyaluronan; levels of various markers, which decrease in aged skin, were significantly restored in vivo and in vitro by the administration of recombinant human HAPLN1 (rhHAPLN1). rhHAPLN1 protected transforming growth factor beta receptor 2 on the cell surface from endocytic degradation via mechanisms such as regulation of viscoelasticity, CD44 clustering. Moreover, rhHAPLN1 regulated the levels of nuclear factor erythroid 2–related factor 2, phosphorylated nuclear factor kappa B, and some cyclin-dependent kinase inhibitors such as p16 and p21. Therefore, rhHAPLN1 may act as a novel biomechanical signaling protein to rejuvenate aged skin. [ABSTRACT FROM AUTHOR]

Purposes Osteoarthritis (OA) is a degenerative joint disease that commonly occurs in middle-aged and elderly people. Mechanical microenvironment is one of the most significant factors in the development of OA. However, when the mechanical microenvironment changes, the inflammatory response of chondrocyte is elusive. Methods By adopting polydimethylsiloxane (PDMS) substrates with varying stiffness which can mimic the physiological stiffness of chondrocyte pericellular matrix (PCM), influences of co-regulated substrate stiffness and inflammatory factors interleukin-1β (IL-1β) on chondrocyte morphology, inflammatory mediators, and PCM remodeling protein expression are quantitatively analyzed. First, prostaglandin E2 (PGE2) and nitric oxide (NO) released in different stiffness substrates and IL-1β stimulated substrates with chondrocytes are detected. Second, the changes in different stiffness substrates and IL-1β stimulated substrates through immunofluorescence technique are observed and recorded. Third, the protein expressions of type Ⅱ collagen (COLII) and matrix metalloproteinase-13 (MMP13) are measured by Western blot assay. Findings The experimental results identify that substrate stiffness regulates the response of chondrocyte to inflammatory signals. Soft substrate dramatically enhances the release of PGE2 and NO (P

The pericellular matrix (PCM) surrounding chondrocytes is essential for articular cartilage tissue engineering. As the current isolation methods to obtain chondrocytes with their PCM (chondrons) result in a heterogeneous mixture of chondrocytes and chondrons, regenerating the PCM using a tissue engineering approach could prove beneficial. In this study, we aimed to discern the behavior of articular chondrocytes (ACs) in regenerating the PCM in such an approach and whether this would also be true for articular cartilage-derived progenitor cells (ACPCs), as an alternative cell source. Bovine ACs and ACPCs were encapsulated in agarose microgels using droplet-based microfluidics. ACs were stimulated with TGF-β1 and dexamethasone and ACPCs were sequentially stimulated with BMP-9 followed by TGF-β1 and dexamethasone. After 0, 3, 5, and 10 days of culture, PCM components, type-VI collagen and perlecan, and ECM component, type-II collagen, were assessed using flow cytometry and fluorescence microscopy. Both ACs and ACPCs synthesized the PCM before the ECM. It was seen for the first time that synthesis of type-VI collagen always preceded perlecan. While the PCM synthesized by ACs resembled native chondrons after only 5 days of culture, ACPCs often made less well-structured PCMs. Both cell types showed variations between individual cells and donors. On one hand, this was more prominent in ACPCs, but also a subset of ACPCs showed superior PCM and ECM regeneration, suggesting that isolating these cells may potentially improve cartilage repair strategies.

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 50 µm 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 9.0 and 38.0 kPa. 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. [ABSTRACT FROM AUTHOR]

The utility of bioprosthetic heart valves (BHVs) is limited to certain patient populations because of their poor durability compared to mechanical prosthetic valves. Histological analysis of failed porcine BHVs suggests that degeneration of the tissue extracellular matrix (ECM), specifically the loss of proteoglycans and their glycosaminoglycans (GAGs), may lead to impaired mechanical performance, resulting in nucleation and propagation of tears and ultimately failure of the prosthetic. Several strategies have been proposed to address this deterioration, including novel chemical fixatives to stabilize ECM constituents and incorporation of small molecule inhibitors of catabolic enzymes implicated in the degeneration of the BHV ECM. Here, biomimetic proteoglycans (BPGs) were introduced into porcine aortic valves ex vivo and were shown to distribute throughout the valve leaflets. Incorporation of BPGs into the heart valve leaflet increased tissue overall GAG content. The presence of BPGs also significantly increased the micromodulus of the spongiosa layer within the BHV without compromising the chemical fixation process used to sterilize and strengthen the tissue prior to implantation. These findings suggest that a targeted approach for molecularly engineering valve leaflet ECM through the use of BPGs may be a viable way to improve the mechanical behavior and potential durability of BHVs. [ABSTRACT FROM AUTHOR]

Mechanosensing at the interface of a cell and its surrounding microenvironment is an essential driving force of physiological processes. Understanding molecular activities at the cell-matrix interface has the potential to provide novel targets for improving tissue regeneration and early disease intervention. In the past few decades, the advancement of atomic force microscopy (AFM) has offered a unique platform for probing mechanobiology at this crucial microdomain. In this review, we describe key advances under this topic through the use of an integrated system of AFM (as a biomechanical testing tool) with complementary immunofluorescence (IF) imaging (as an in situ navigation system). We first describe the body of work investigating the micromechanics of the pericellular matrix (PCM), the immediate cell micro-niche, in healthy, diseased, and genetically modified tissues, with a focus on articular cartilage. We then summarize the key findings in understanding cellular biomechanics and mechanotransduction, in which, molecular mechanisms governing transmembrane ion channel-mediated mechanosensing, cytoskeleton remodeling, and nucleus remodeling have been studied in various cell and tissue types. Lastly, we provide an overview of major technical advances that have enabled more in-depth studies of mechanobiology, including the integration of AFM with a side-view microscope, multiple optomicroscopy, a fluorescence recovery after photobleaching (FRAP) module, and a tensile stretching device. The innovations described here have contributed greatly to advancing the fundamental knowledge of extracellular matrix biomechanics and cell mechanobiology for improved understanding, detection, and intervention of various diseases. [ABSTRACT FROM AUTHOR]

Analogous to articular cartilage, changes in spatial chondrocyte organisation have been proposed to be a strong indicator for local tissue degeneration in the intervertebral disc (IVD). While a progressive structural and functional degradation of the extracellular (ECM) and pericellular (PCM) matrix occurs in osteoarthritic cartilage, these processes have not yet been biomechanically elucidated in the IVD. We aimed to evaluate the local stiffness of the ECM and PCM in the anulus fibrosus of the IVD on the basis of local chondrocyte spatial organisation. Using atomic force microscopy, we measured the Young’s modulus of the local ECM and PCM in human and bovine disc samples using the spatial chondrocyte patterns as an image-based biomarker. By measuring tissue from 31 patients and six bovine samples, we found a significant difference in the elastic moduli (E) of the PCM in clusters when compared to the healthy patterns single cells (p = 0.029), pairs (p = 0.016), and string-formations (p = 0.010). The ECM/PCM ratio ranged from 0.62–0.89. Interestingly, in the bovine IVD, the ECM/PCM ratio of the E significantly varied (p = 0.002) depending on the tissue origin. Overall the reduced E in clusters demonstrates that cluster formation is not only a morphological phenomenon describing disc degeneration, but it marks a compromised biomechanical functioning. Immunohistochemical analyses indicate that collagen type III degradation might be involved. This study is the first to describe and quantify the differences in the E of the ECM in relation to the PCM in the anulus fibrosus of the IVD by means of atomic force microscopy on the basis of spatial chondrocyte organisation.

The extracellular matrix (ECM) has long been regarded as a packing material; supporting cells within the tissue and providing tensile strength and protection from mechanical stress. There is little surprise when one considers the dynamic nature of many of the individual proteins that contribute to the ECM, that we are beginning to appreciate a more nuanced role for the ECM in tissue homeostasis and disease. Articular cartilage is adapted to be able to perceive and respond to mechanical load. Indeed, physiological loads are essential to maintain cartilage thickness in a healthy joint and excessive mechanical stress is associated with the breakdown of the matrix that is seen in osteoarthritis (OA). Although the trigger by which increased mechanical stress drives catabolic pathways remains unknown, one mechanism by which cartilage responds to increased compressive load is by the release of growth factors that are sequestered in the pericellular matrix. These are heparan sulfate-bound growth factors that appear to be largely chondroprotective and displaced by an aggrecan-dependent sodium flux. Emerging evidence suggests that the released growth factors act in a coordinated fashion to drive cartilage repair. Thus, we are beginning to appreciate that the ECM is the key mechano-sensor and mechano-effector in cartilage, responsible for directing subsequent cellular events of relevance to joint health and disease. [ABSTRACT FROM AUTHOR]

Background: Daily physiologic loading causes fluctuations in hydration of the intervertebral disc (IVD); thus, the embedded cells experience cyclic alterations to their osmotic environment. These osmotic fluctuations have been described as a mechanism linking mechanics and biology, and have previously been shown to promote biosynthesis in chondrocytes. However, this phenomenon has yet to be fully interrogated in the IVD. Additionally, the specialized extracellular matrix surrounding the cells, the pericellular matrix (PCM), transduces the biophysical signals that cells ultimately experience. While it is known that the PCM is altered in disc degeneration, whether it disrupts normal osmotic mechanotransduction has yet to be determined. Thus, our objectives were to assess: (1) whether dynamic osmotic conditions stimulate biosynthesis in nucleus pulposus cells, and (2) whether pericellular heparan sulfate proteoglycans (HSPGs) modulate the biosynthetic response to osmotic loading. Methods: Bovine nucleus pulposus cells isolated with retained PCM were encapsulated in 1.5% alginate beads and treated with or without heparinase III, an enzyme that degrades the pericellular HSPGs. Beads were subjected to 1 h of daily iso‐osmotic, hyper‐osmotic, or hypo‐osmotic loading for 1, 2, or 4 weeks. At each timepoint the total amount of extracellular and pericellular sGAG/DNA were quantified. Additionally, whether osmotic loading triggered alterations to HSPG sulfation was assessed via immunohistochemistry for the heparan sulfate 6‐O‐sulfertransferase 1 (HS6ST1) enzyme. Results: Osmotic loading significantly influenced sGAG/DNA accumulation with a hyper‐osmotic change promoting the greatest sGAG/DNA accumulation in the pericellular region compared with iso‐osmotic conditions. Heparanase‐III treatment significantly reduced extracellular sGAG/DNA but pericellular sGAG was not affected. HS6ST1 expression was not affected by osmotic loading. Conclusion: Results suggest that hyper‐osmotic loading promotes matrix synthesis and that modifications to HSPGs directly influence the metabolic responses of cells to osmotic fluctuations. Collectively, results suggest degeneration‐associated modifications to pericellular HSPGs may contribute to the altered mechanobiology observed in disease. [ABSTRACT FROM AUTHOR]

Abstract Background Daily physiologic loading causes fluctuations in hydration of the intervertebral disc (IVD); thus, the embedded cells experience cyclic alterations to their osmotic environment. These osmotic fluctuations have been described as a mechanism linking mechanics and biology, and have previously been shown to promote biosynthesis in chondrocytes. However, this phenomenon has yet to be fully interrogated in the IVD. Additionally, the specialized extracellular matrix surrounding the cells, the pericellular matrix (PCM), transduces the biophysical signals that cells ultimately experience. While it is known that the PCM is altered in disc degeneration, whether it disrupts normal osmotic mechanotransduction has yet to be determined. Thus, our objectives were to assess: (1) whether dynamic osmotic conditions stimulate biosynthesis in nucleus pulposus cells, and (2) whether pericellular heparan sulfate proteoglycans (HSPGs) modulate the biosynthetic response to osmotic loading. Methods Bovine nucleus pulposus cells isolated with retained PCM were encapsulated in 1.5% alginate beads and treated with or without heparinase III, an enzyme that degrades the pericellular HSPGs. Beads were subjected to 1 h of daily iso‐osmotic, hyper‐osmotic, or hypo‐osmotic loading for 1, 2, or 4 weeks. At each timepoint the total amount of extracellular and pericellular sGAG/DNA were quantified. Additionally, whether osmotic loading triggered alterations to HSPG sulfation was assessed via immunohistochemistry for the heparan sulfate 6‐O‐sulfertransferase 1 (HS6ST1) enzyme. Results Osmotic loading significantly influenced sGAG/DNA accumulation with a hyper‐osmotic change promoting the greatest sGAG/DNA accumulation in the pericellular region compared with iso‐osmotic conditions. Heparanase‐III treatment significantly reduced extracellular sGAG/DNA but pericellular sGAG was not affected. HS6ST1 expression was not affected by osmotic loading. Conclusion Results suggest that hyper‐osmotic loading promotes matrix synthesis and that modifications to HSPGs directly influence the metabolic responses of cells to osmotic fluctuations. Collectively, results suggest degeneration‐associated modifications to pericellular HSPGs may contribute to the altered mechanobiology observed in disease.

In osteoarthritis (OA), degradation of cartilage pericellular matrix (PCM), the proteoglycan-rich immediate cell microniche, is a leading event of disease initiation. This study demonstrated that biomimetic proteoglycans (BPGs) can diffuse into human cartilage from both normal and osteoarthritic donors and are preferentially localized within the PCM. Applying immunofluorescence (IF)-guided AFM nanomechanical mapping, we show that this localization of BPGs increases the PCM micromodulus of both normal and OA specimens. These results illustrate the capability of BPGs to integrate with degenerative tissues and support the translational potential of BPGs for treating human OA and other diseases associated with proteoglycan degradation.

Mechanical stress to the temporomandibular joint (TMJ) is an important factor in cartilage degeneration, with both clinical and preclinical studies suggesting that repeated TMJ overloading could contribute to pain, inflammation, and/or structural damage in the joint. However, the relationship between pain severity and early signs of cartilage matrix microstructural dysregulation is not understood, limiting the advancement of diagnoses and treatments for temporomandibular joint‐osteoarthritis (TMJ‐OA). Changes in the pericellular matrix (PCM) surrounding chondrocytes may be early indicators of OA. A rat model of TMJ pain induced by repeated jaw loading (1 h/day for 7 days) was used to compare the extent of PCM modulation for different loading magnitudes with distinct pain profiles (3.5N—persistent pain, 2N—resolving pain, or unloaded controls—no pain) and macrostructural changes previously indicated by Mankin scoring. Expression of PCM structural molecules, collagen VI and aggrecan NITEGE neo‐epitope, were evaluated at Day 15 by immunohistochemistry within TMJ fibrocartilage and compared between pain conditions. Pericellular collagen VI levels increased at Day 15 in both the 2N (p = 0.003) and 3.5N (p = 0.042) conditions compared to unloaded controls. PCM width expanded to a similar extent for both loading conditions at Day 15 (2N, p < 0.001; 3.5N, p = 0.002). Neo‐epitope expression increased in the 3.5N group over levels in the 2N group (p = 0.041), indicating pericellular changes that were not identified in the same groups by Mankin scoring of the pericellular region. Although remodeling occurs in both pain conditions, the presence of pericellular catabolic neo‐epitopes may be involved in the macrostructural changes and behavioral sensitivity observed in persistent TMJ pain. [ABSTRACT FROM AUTHOR]

The specialized pericellular matrix (PCM) surrounding chondrocytes within articular cartilage is critical to the tissue's health and longevity. Growing evidence suggests that PCM alterations are ubiquitous across all trajectories of osteoarthritis, a crippling and prevalent joint disease. The PCM geometry is of particular interest as it influences the cellular mechanical environment. Observations of asymmetrical PCM thickness have been reported, but a quantified characterization is lacking. To this end, a novel microscopy protocol was developed and applied to acquire images of the PCM surrounding live cells. Morphometric analysis indicated a statistical bias towards thicker PCM on the inferior cellular surface. The mechanical effects of this bias were investigated with multiscale modelling, which revealed potentially damaging, high tensile strains in the direction perpendicular to the membrane and localized on the inferior surface. These strains varied substantially between PCM asymmetry cases. Simulations with a thicker inferior PCM, representative of the observed geometry, resulted in strain magnitudes approximately half of those calculated for a symmetric geometry, and a third of those with a thin inferior PCM. This strain attenuation suggests that synthesis of a thicker inferior PCM may be a protective adaptation. PCM asymmetry may thus be important in cartilage development, pathology, and engineering. [ABSTRACT FROM AUTHOR]

As an indispensable component of the extracellular matrix, perlecan (Pln) plays an essential role in cartilaginous tissue function. Although there exist studies suggesting that Pln expressed by cartilaginous tissues is critical for chondrogenesis, few papers have discussed the potential impact Pln may have on cartilage regeneration. In this review, we delineate Pln structure, biomechanical properties, and interactive ligands—which together contribute to the effect Pln has on cartilaginous tissue development. We also review how the signaling pathways of Pln affect cartilage development and scrutinize the potential application of Pln to divisions of cartilage regeneration, spanning vascularization, stem cell differentiation, and biomaterial improvement. The aim of this review is to deepen our understanding of the spatial and temporal interactions that occur between Pln and cartilaginous tissue and ultimately apply Pln in scaffold design to improve cell-based cartilage engineering and regeneration. As a key component of the basement membrane, Pln plays a critical role in tissue development and repair. Recent findings suggest that Pln existing in the pericellular matrix surrounding mature chondrocytes is actively involved in cartilage regeneration and functionality. We propose that Pln is essential to developing an in vitro matrix niche within biological scaffolds for cartilage tissue engineering. Perlecan's potential application in cartilage regeneration. [Display omitted] [ABSTRACT FROM AUTHOR]

The lateral resolution of infrared spectroscopy has been inadequate for accurate biochemical characterization of the cell microenvironment, a region regulating biochemical and biomechanical signals to cells. In this study, we demonstrate the capacity of a high-resolution Fourier transform infrared microspectroscopy (HR-FTIR-MS) to characterize the collagen content of this region. Specifically, we focus on the collagen content in the cartilage cell (chondrocyte) microenvironment of healthy and osteoarthritic (OA) cartilage. Human tibial cartilage samples (N = 28) were harvested from 7 cadaveric donors and graded for OA severity (healthy, early OA, advanced OA). HR-FTIR-MS was used to analyze the collagen content of the chondrocyte microenvironment of five distinct zones across the tissue depth. HR-FTIR-MS successfully showed collagen content distribution across chondrocytes and their environment. In zones 2 and 3 (10 - 50% of the tissue thickness), we observed that collagen content was smaller (P < 0.05) in early OA compared to the healthy tissue in the vicinity of cells (pericellular region). The collagen content loss was extended to the extracellular matrix in advanced OA tissue. No significant differences in the collagen content of the chondrocyte microenvironment were observed between the groups in the most superficial (0–10%) and deep zones (50–100%). HR-FTIR-MS revealed collagen loss in the early OA cartilage pericellular region before detectable changes in the extracellular matrix in advanced OA. HR-FTIR-MS-based compositional assessment enables a better understanding of OA-related changes in tissues. This technique can be used to identify new disease mechanisms enabling better intervention strategies. Osteoarthritis (OA) is the most common degenerative joint disease causing pain and disability. While significant progress has been made in OA research, OA pathogenesis is still poorly understood and current OA treatments are mainly palliative. This study demonstrates that high-resolution FTIR microspectroscopy (HR-FTIR-MS) can characterize OA-induced compositional changes in the cell microenvironment (pericellular matrix) during the early disease stages before tissue changes in the extracellular matrix become apparent. This technique may further enable the identification of new OA mechanisms and improve our current understanding of OA pathogenesis, thus, enabling the development of better treatment methods. [Display omitted] [ABSTRACT FROM AUTHOR]

The pericellular matrix (PCM) is the immediate microniche surrounding resident cells in various tissue types, regulating matrix turnover, cell-matrix cross-talk and disease initiation. This study elucidated the structure-mechanical properties and mechanobiological functions of the PCM in fibrocartilage, a family of connective tissues that sustain complex tensile and compressive loads in vivo. Studying the murine meniscus as the model tissue, we showed that fibrocartilage PCM contains thinner, random collagen fibrillar networks that entrap proteoglycans, a structure distinct from the densely packed, highly aligned collagen fibers in the bulk extracellular matrix (ECM). In comparison to the ECM, the PCM has a lower modulus and greater isotropy, but similar relative viscoelastic properties. In Col5a1 +/- menisci, the reduction of collagen V, a minor collagen localized in the PCM, resulted in aberrant fibril thickening with increased heterogeneity. Consequently, the PCM exhibited a reduced modulus, loss of isotropy and faster viscoelastic relaxation. This disrupted PCM contributes to perturbed mechanotransduction of resident meniscal cells, as illustrated by reduced intracellular calcium signaling, as well as upregulated biosynthesis of lysyl oxidase and tenascin C. When cultured in vitro, Col5a1 +/- meniscal cells synthesized a weakened nascent PCM, which had inferior properties towards protecting resident cells against applied tensile stretch. These findings underscore the PCM as a distinctive microstructure that governs fibrocartilage mechanobiology, and highlight the pivotal role of collagen V in PCM function. Targeting the PCM or its molecular constituents holds promise for enhancing not only meniscus regeneration and osteoarthritis intervention, but also addressing diseases across various fibrocartilaginous tissues.

Articular chondrocytes synthesize and maintain the avascular and aneural articular cartilage. In vivo these cells are surrounded by a 3D pericellular matrix (PCM) containing predominantly collagen VI. The PCM protects chondrocytes and facilitates mechanotransduction, and PCM stiffness is critical in transmitting biomechanical signals to chondrocytes. Various culture systems with different hydrogels have been used to encapsulate chondrocytes for 3D culture, but many lack either the PCM or the in vivo stiffness of the cartilage matrix. Here, we demonstrate that primary chondrocytes cultured in alginate will form a pericellular matrix and display a phenotype similar to in vivo conditions. We found that primary human and bovine chondrocytes, when cultured in alginate beads with addition of sodium L-ascorbate for 7 days, had a pronounced PCM, retained their phenotype, and synthesized both collagens VI and II. This novel culture system enables alginate-encapsulated chondrocytes to develop a robust PCM thereby creating a model system to study mechanotransduction. We also observed distinct compression-induced changes in metabolomic profiles between the monolayer-agarose and alginate-released agarose-embedded chondrocytes indicating physiological changes in cell metabolism. Our data suggest that 3D preculture of chondrocytes in alginate before encapsulation in physiologically-stiff agarose leads to a pronounced development of pericellular matrix that is sustained in the presence of ascorbate. This novel model can be useful in studying the mechanism by which chondrocytes respond to cyclical compression and other types of loading simulating in vivo physiological conditions.

Articular cartilage is comprised of two main components, the extracellular matrix (ECM) and the pericellular matrix (PCM). The PCM helps to protect chondrocytes in the cartilage from mechanical loads, but in patients with osteoarthritis, the PCM is weakened, resulting in increased chondrocyte stress. As chondrocytes are responsible for matrix synthesis and maintenance, it is important to understand how mechanical loads affect the cellular responses of chondrocytes. Many studies have examined chondrocyte responses to in vitro mechanical loading by embedding chondrocytes in 3-D hydrogels. However, these experiments are mostly performed in the absence of PCM, which may obscure important responses to mechanotransduction. Here, drop-based microfluidics is used to culture single chondrocytes in alginate microgels for cell-directed PCM synthesis that closely mimics the in vivo microenvironment. Chondrocytes formed PCM over 10 days in these single-cell 3-D microenvironments. Mechanotransduction studies were performed, in which single-cell microgels mimicking the cartilage PCM were embedded in high-stiffness agarose. After physiological dynamic compression in a custom-built bioreactor, microgels exhibited distinct metabolomic profiles from both uncompressed and monolayer controls. These results demonstrate the potential of single cell encapsulation in alginate microgels to advance cartilage tissue engineering and basic chondrocyte mechanobiology.

• Decorin regulates the aggrecan network integrity and micromechanics of cartilage pericellular matrix. • The highly negative charged osmotic microenvironment of pericellular matrix is required for normal chondrocyte mechanotransduction in situ. • Decorin affects the intracellular calcium signaling of chondrocytes by mediating the aggrecan-endowed osmotic microenvironment of pericellular matrix. • The impact of decorin loss on the disruption of chondrocyte mechanobiology is increasingly aggravated during maturation. In cartilage tissue engineering, one key challenge is for regenerative tissue to recapitulate the biomechanical functions of native cartilage while maintaining normal mechanosensitive activities of chondrocytes. Thus, it is imperative to discern the micromechanobiological functions of the pericellular matrix, the ~ 2–4 µm-thick domain that is in immediate contact with chondrocytes. In this study, we discovered that decorin, a small leucine-rich proteoglycan, is a key determinant of cartilage pericellular matrix micromechanics and chondrocyte mechanotransduction in vivo. The pericellular matrix of decorin-null murine cartilage developed reduced content of aggrecan, the major chondroitin sulfate proteoglycan of cartilage and a mild increase in collagen II fibril diameter vis-à-vis wild-type controls. As a result, decorin-null pericellular matrix showed a significant reduction in micromodulus, which became progressively more pronounced with maturation. In alignment with the defects of pericellular matrix, decorin-null chondrocytes exhibited decreased intracellular calcium activities, [Ca2+ i , in both physiologic and osmotically evoked fluidic environments in situ , illustrating impaired chondrocyte mechanotransduction. Next, we compared [Ca2+ i activities of wild-type and decorin-null chondrocytes following enzymatic removal of chondroitin sulfate glycosaminoglycans. The results showed that decorin mediates chondrocyte mechanotransduction primarily through regulating the integrity of aggrecan network, and thus, aggrecan-endowed negative charge microenvironment in the pericellular matrix. Collectively, our results provide robust genetic and biomechanical evidence that decorin is an essential constituent of the native cartilage matrix, and suggest that modulating decorin activities could improve cartilage regeneration. [ABSTRACT FROM AUTHOR]

During osteoarthritis, chondrocytes change their spatial arrangement from single to double strings, then to small and big clusters. This change in pattern has recently been established as an image‐based biomarker for osteoarthritis. The pericellular matrix (PCM) appears to degrade together alongside cellular reorganization. The aim of this study was to characterize this PCM‐degradation based on different cellular patterns. We additionally wanted to identify the earliest time point of PCM‐breakdown in this physiopathological model. To this end, cartilage samples were selected according to their predominant cellular pattern. Qualitative analysis of PCM degradation was performed immunohistochemically by analysing five main PCM components: collagen type VI, perlecan, collagen type III, biglycan, and fibrillin‐1 (n = 6 patients). Their protein content was quantified by enzyme‐linked immunosorbent assay (127 patients). Accompanying spatial cellular rearrangement, the PCM is progressively destroyed, with a pericellular signal loss in fluorescence microscopy for collagen type VI, perlecan, and biglycan. This loss in protein signal is accompanied by a reduction in total protein content from single strings to big clusters (P <.001 for collagen type VI, P =.003 for perlecan, and P <.001 for biglycan). As a result of an increase in the number of cells from single strings to big clusters, the amount of protein available per cell also decreases for collagen type III and fibrillin‐1, where total protein levels remain constant. Biochemical changes of the PCM and cellular rearrangement are thus highly interconnected hallmarks of osteoarthritis. Interestingly, the earliest point in time for a relevant PCM impairment appears to be at the transition to small clusters. [ABSTRACT FROM AUTHOR]

The pericellular matrix (PCM) of cartilage is a structurally distinctive microdomain surrounding each chondrocyte, and is pivotal to cell homeostasis and cell-matrix interactions in healthy tissue. This study queried if the PCM is the initiation point for disease or a casualty of more widespread matrix degeneration. To address this question, we queried the mechanical properties of the PCM and chondrocyte mechanoresponsivity with the development of post-traumatic osteoarthritis (PTOA). To do so, we integrated Kawamoto's film-assisted cryo-sectioning with immunofluorescence-guided AFM nanomechanical mapping, and quantified the microscale modulus of murine cartilage PCM and further-removed extracellular matrix. Using the destabilization of the medial meniscus (DMM) murine model of PTOA, we show that decreases in PCM micromechanics are apparent as early as 3 days after injury, and that this precedes changes in the bulk ECM properties and overt indications of cartilage damage. We also show that, as a consequence of altered PCM properties, calcium mobilization by chondrocytes in response to mechanical challenge (hypo-osmotic stress) is significantly disrupted. These aberrant changes in chondrocyte micromechanobiology as a consequence of DMM could be partially blocked by early inhibition of PCM remodeling. Collectively, these results suggest that changes in PCM micromechanobiology are leading indicators of the initiation of PTOA, and that disease originates in the cartilage PCM. This insight will direct the development of early detection methods, as well as small molecule-based therapies that can stop early aberrant remodeling in this critical cartilage microdomain to slow or reverse disease progression. Post-traumatic osteoarthritis (PTOA) is one prevalent musculoskeletal disease that afflicts young adults, and there are no effective strategies for early detection or intervention. This study identifies that the reduction of cartilage pericellular matrix (PCM) micromodulus is one of the earliest events in the initiation of PTOA, which, in turn, impairs the mechanosensitive activities of chondrocytes, contributing to the vicious loop of cartilage degeneration. Rescuing the integrity of PCM has the potential to restore normal chondrocyte mechanosensitive homeostasis and to prevent further degradation of cartilage. Our findings enable the development of early OA detection methods targeting changes in the PCM, and treatment strategies that can stop early aberrant remodeling in this critical microdomain to slow or reverse disease progression. Image, graphical abstract [ABSTRACT FROM AUTHOR]

Despite the fact that type III collagen is the second most abundant collagen type in the body, its contribution to the physiologic maintenance and repair of skeletal tissues remains poorly understood. This study queried the role of type III collagen in the structure and biomechanical functions of two structurally distinctive tissues in the knee joint, type II collagen-rich articular cartilage and type I collagen-dominated meniscus. Integrating outcomes from atomic force microscopy-based nanomechanical tests, collagen fibril nanostructural analysis, collagen cross-link analysis and histology, we elucidated the impact of type III collagen haplodeficiency on the morphology, nanostructure and biomechanical properties of articular cartilage and meniscus in Col3a1 +/− mice. Reduction of type III collagen leads to increased heterogeneity and mean thickness of collagen fibril diameter, as well as reduced modulus in both tissues, and these effects became more pronounced with skeletal maturation. These data suggest a crucial role of type III collagen in mediating fibril assembly and biomechanical functions of both articular cartilage and meniscus during post-natal growth. In articular cartilage, type III collagen has a marked contribution to the micromechanics of the pericellular matrix, indicating a potential role in mediating the early stage of type II collagen fibrillogenesis and chondrocyte mechanotransduction. In both tissues, reduction of type III collagen leads to decrease in tissue modulus despite the increase in collagen cross-linking. This suggests that the disruption of matrix structure due to type III collagen deficiency outweighs the stiffening of collagen fibrils by increased cross-linking, leading to a net negative impact on tissue modulus. Collectively, this study is the first to highlight the crucial structural role of type III collagen in both articular cartilage and meniscus extracellular matrices. We expect these results to expand our understanding of type III collagen across various tissue types, and to uncover critical molecular components of the microniche for regenerative strategies targeting articular cartilage and meniscus repair. • Collagen III regulates fibril structure and mechanics in both collagen II-rich cartilage and collagen I-dominated meniscus. • Collagen III affects the aggrecan network compression possibly by mediating the aggrecan and collagen network integration. • Collagen III regulates cartilage pericellular matrix micromechanics, potentially mediating chondrocyte mechanotransduction. • Disruption of structure due to reduced collagen III outweighs increase in collagen cross-linking, causing reduced modulus. [ABSTRACT FROM AUTHOR]

Extracellular matrix (ECM) determines the physiological function of all tissues, including musculoskeletal tissues. In tendon, ECM provides overall tissue architecture, which is tailored to match the biomechanical requirements of their physiological function, that is, force transmission from muscle to bone. Tendon ECM also constitutes the microenvironment that allows tendon‐resident cells to maintain their phenotype and that transmits biomechanical forces from the macro‐level to the micro‐level. The structure and function of adult tendons is largely determined by the hierarchical organization of collagen type I fibrils. However, non‐collagenous ECM proteins such as small leucine‐rich proteoglycans (SLRPs), ADAMTS proteases, and cross‐linking enzymes play critical roles in collagen fibrillogenesis and guide the hierarchical bundling of collagen fibrils into tendon fascicles. Other non‐collagenous ECM proteins such as the less abundant collagens, fibrillins, or elastin, contribute to tendon formation or determine some of their biomechanical properties. The interfascicular matrix or endotenon and the outer layer of tendons, the epi‐ and paratenon, includes collagens and non‐collagenous ECM proteins, but their function is less well understood. The ECM proteins in the epi‐ and paratenon may provide the appropriate microenvironment to maintain the identity of distinct tendon cell populations that are thought to play a role during repair processes after injury. The aim of this review is to provide an overview of the role of non‐collagenous ECM proteins and less abundant collagens in tendon development and homeostasis. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:23–35, 2020 [ABSTRACT FROM AUTHOR]

Biomimetic proteoglycan (BPG) diffusion into articular cartilage has the potential to restore the lost proteoglycan content in osteoarthritic cartilage given these molecules mimic the structure and properties of natural proteoglycans. We examined the diffusion characteristics of BPGs through cartilage with the use of a custom‐made in vitro cartilage diffusion model in both normal bovine and human osteoarthritic cartilage explants. BPGs were introduced into the cartilage through essentially one‐dimensional diffusion using osteochondral plugs. The molecular diffusion was shown to be size and concentration dependent. Diffusion profiles were found over different diffusion time intervals and the profiles were fit to a nonlinear Fickian diffusion model. Steady state 011012‐7diffusion coefficients for BPGs were found to be 4.01 and 3.53 μm2/s for 180 and 1600 kDa BPGs, respectfully, and these values are similar to other large molecule diffusion in cartilage. In both bovine and osteoarthritic human cartilage, BPGs were found localized around the chondrocytes. BPG localization was examined by labeling collagen type VI and soaking 5 μm thick sections of cartilage with BPG solutions demonstrating that the BPGs diffused into the cartilage and preferentially localized alongside collagen type VI in the pericellular matrix. [ABSTRACT FROM AUTHOR]

Cell-cell adhesion strength, measured as tissue surface tension, spans an enormous 1000-fold range when different cell types are compared. However, the examination of basic mechanical principles of cell adhesion indicates that cadherin-based and related mechanisms are not able to promote the high-strength adhesion experimentally observed in many late embryonic or malignant tissues. Therefore, the hypothesis is explored that the interaction of the pericellular matrices of cells generates strong adhesion by a mechanism akin to the self-adhesion/self-healing of dynamically cross-linked hydrogels. Quantitative data from biofilm matrices support this model. The mechanism links tissue surface tension to pericellular matrix stiffness. Moreover, it explains the wide, matrixfilled spaces around cells in liquid-like, yet highly cohesive, tissues, and it rehabilitates aspects of the original interpretation of classical cell sorting experiments, as expressed in Steinberg's differential adhesion hypothesis: that quantitative differences in adhesion energies between cells are sufficient to drive sorting. [ABSTRACT FROM AUTHOR]

The present manuscript provides a numerical study on the influence of the pericellular matrix (PCM) and elastin fibers in the multiscale mechanics of tendon fascicles. To this aim, a three-dimensional finite element representative volume element (RVE) of a tendon fascicle, designed in view of several experimental observations, is proposed for the numerical investigations. Three material phases (collagen fibers, cells and PCM) comprising five finite strain viscoelastic models (collagen fibrils, collagen fibers, cells, PCM and elastin fibers) compose the proposed RVE. A set of numerical results concerning microscopic stress and strain fields and macroscopic (homogenized) stress-strain curves support the following hypotheses regarding the PCM and elastin fibers in the multiscale mechanics of tendon fascicles: neither the elastin fibers nor the PCM contributions affect the macroscopic viscoelastic responses of tendon fascicles; elastin fibers do not significantly influence the cellular stress and strain fields; the PCM stiffness influences strain localization on cells, which may play a role in cellular mecanotransduction mechanisms. [ABSTRACT FROM AUTHOR]

Objective: During osteoarthritis (OA), chondrocytes seem to change their spatial arrangement from single to double strings, small and big clusters. Since the pericellular matrix (PCM) appears to degrade alongside this reorganisation, it has been suggested that spatial patterns act as an image-based biomarker for OA. The aim of this study was to establish the functional relevance of spatial organisation in articular cartilage.Method: Cartilage samples were selected according to their predominant spatial cellular pattern. Young's modulus of their PCM was measured by atomic force microscopy (AFM) (∼500 measurements/pattern). The distribution of two major PCM components (collagen type VI and perlecan) was analysed by immunohistochemistry (8 patients) and protein content quantified by enzyme-linked immunosorbent assay (ELISA) (58 patients).Results: PCM stiffness significantly decreased with the development from single to double strings (p = 0.030), from double strings to small clusters (p = 0.015), and from small clusters to big clusters (p < 0.001). At the same time, the initially compact collagen type VI and perlecan staining progressively weakened and was less focalised. The earliest point with a significant reduction in protein content as shown by ELISA was the transition from single strings to small clusters for collagen type VI (p = 0.016) and from double strings to small clusters for perlecan (p = 0.008), with the lowest amounts for both proteins seen in big clusters.Conclusions: This study demonstrates the functional relevance of spatial chondrocyte organisation as an image-based biomarker. At the transition from single to double strings PCM stiffness decreases, followed by protein degradation from double strings to small clusters. [ABSTRACT FROM AUTHOR]

The aim of this study was to immunolocalise type VI collagen and perlecan and determine their interactive properties in the intervertebral disc (IVD). Confocal laser scanning microscopy co-localised perlecan with type VI collagen as pericellular components of IVD cells and translamellar cross-bridges in ovine and murine IVDs. These cross-bridges were significantly less abundant in the heparin sulphate deficient Hspg2 exon 3 null mouse IVD than in wild type. This association of type VI collagen with elastic components provides clues as to its roles in conveying elastic recoil properties to annular tissues. Perlecan and type VI collagen were highly interactive in plasmon resonance studies. Pericellular colocalisation of perlecan and type VI collagen provides matrix stabilisation and cell-matrix communication which allows IVD cells to perceive and respond to perturbations in their biomechanical microenvironment. Perlecan, at the cell surface, provides an adhesive interface between the cell and its surrounding extracellular matrix. Elastic microfibrillar structures regulate tensional connective tissue development and function. The 2010 Global Burden of Disease study examined 291 disorders and identified disc degeneration and associated low back pain as the leading global musculoskeletal disorder emphasising its massive socioeconomic impact and the need for more effective treatment strategies. A greater understanding of how the IVD achieves its unique biomechanical functional properties is of great importance in the development of such therapeutic measures.

Abstract Site-specific and depth-dependent properties of cartilage were implemented within a finite element (FE) model to determine if compositional or structural changes in the tissue could explain site-specific alterations of chondrocyte deformations due to cartilage loading in rabbit knee joints 3 days after a partial meniscectomy (PM). Depth-dependent proteoglycan (PG) content, collagen content and collagen orientation in the cartilage extracellular matrix (ECM), and PG content in the pericellular matrix (PCM) were assessed with microscopic and spectroscopic methods. Patellar, femoral groove and samples from both the lateral and medial compartments of the femoral condyle and tibial plateau were extracted from healthy controls and from the partial meniscectomy group. For both groups and each knee joint site, axisymmetric FE models with measured properties were generated. Experimental cartilage loading was applied in the simulations and chondrocyte volumes were compared to the experimental values. ECM and PCM PG loss occurred within the superficial cartilage layer in the PM group at all locations, except in the lateral tibial plateau. Collagen content and orientation were not significantly altered due to the PM. The FE simulations predicted similar chondrocyte volume changes and group differences as obtained experimentally. Loss of PCM fixed charge density (FCD) decreased cell volume loss, as observed in the medial femur and medial tibia, whereas loss of ECM FCD increased cell volume loss, as seen in the patella, femoral groove and lateral femur. The model outcome, cell volume change, was also sensitive to applied tissue geometry, collagen fibril orientation and loading conditions. [ABSTRACT FROM AUTHOR]

Study Design: Human intervertebral disc (hIVD) cells were isolated from 41 surgically excised samples and assessed for their phenotypic alterations with age.Objective: Toward the design of novel anti-aging strategies to overcome degenerative disc disease (DDD), we investigated age-correlated phenotypic alterations that occur on primary hIVD cells.Summary Of Background Data: Although regenerative medicine holds great hope, much is still to be unveiled on IVD cell biology and its intrinsic signaling pathways, which can lead the way to successful therapies for IDD. A greater focus on age-related phenotypic changes at the cell level would contribute to establish more effective anti-aging/degeneration targets.Methods: The study was subdivided in four main steps: i) optimization of primary cells isolation technique; ii) high-throughput cell morphology analysis, by imaging flow cytometry (FC) and subsequent validation by histological analysis; iii) analysis of progenitor cell surface markers expression, by conventional FC; and iv) statistical analysis and correlation of cells morphology and phenotype with donor age.Results: Three subsets of cells were identified on the basis of their diameter: small cell (SC), large cell (LC), and super LC (SLC). The frequency of SCs decreased nearly 50% with age, whereas that of LCs increased nearly 30%. Interestingly, the increased cells size was due to an enlargement of the pericellular matrix (PCM). Moreover, the expression pattern for CD90 and CD73 was a reflexion of age, where older individuals show reduced frequencies of positive cells for those markers. Nevertheless, the elevated percentages of primary positive cells for the mesenchymal stem cells (MSCs) marker CD146 found, even in some older donors, refreshed hope for the hypothetical activation of the self-renewal potential of the IVD.Conclusion: These findings highlight the remarkable morphological alterations that occur on hIVD cells with aging and degeneration, while reinforcing previous reports on the gradual disappearance of an endogenous progenitor cell population.Level Of Evidence: N/A. [ABSTRACT FROM AUTHOR]

Mutations in collagen VI genes cause two major clinical myopathies, Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD), and the rarer myosclerosis myopathy. In addition to congenital muscle weakness, patients affected by collagen VI-related myopathies show axial and proximal joint contractures, and distal joint hypermobility, which suggest the involvement of tendon function. To gain further insight into the role of collagen VI in human tendon structure and function, we performed ultrastructural, biochemical, and RT-PCR analysis on tendon biopsies and on cell cultures derived from two patients affected with BM and UCMD. In vitro studies revealed striking alterations in the collagen VI network, associated with disruption of the collagen VI-NG2 (Collagen VI-neural/glial antigen 2) axis and defects in cell polarization and migration. The organization of extracellular matrix (ECM) components, as regards collagens I and XII, was also affected, along with an increase in the active form of metalloproteinase 2 (MMP2). In agreement with the in vitro alterations, tendon biopsies from collagen VI-related myopathy patients displayed striking changes in collagen fibril morphology and cell death. These data point to a critical role of collagen VI in tendon matrix organization and cell behavior. The remodeling of the tendon matrix may contribute to the muscle dysfunction observed in BM and UCMD patients.

Human mesenchymal stem cells (hMSCs) induced towards chondrogenesis develop a pericellular matrix (PCM), rich in type VI collagen (ColVI) and proteoglycans such as decorin (DCN). Individual PCM protein functions still need to be elucidated to fully understand the mechanobiological role of this matrix. In this study we identified ColVI and DCN as important contributors in the mechanical function of the PCM and as biochemical modulators during chondrogenesis through targeted knockdown using shRNA lentiviral vectors. Gene expression, western blotting, immunofluorescence and cell deformation analysis were examined at 7, 14 and 28 days post chondrogenic induction. ColVI and DCN knockdown each affected gene expression of acan, bgn, and sox9 during chondrogenesis. ColVI was found to be of central importance in resisting applied strains, while DCN knockdown had strain dependent effects on deformation. We demonstrate that by using genetic engineering to control the biophysical microenvironment created by differentiating cells, it may be possible to guide cellular mechanotransduction.

Retinal detachments create two pathological surfaces, the surface of the outer neural retinal, and an apical retinal-pigmented epithelium (RPE) surface. The physicochemical properties of these two new surfaces are poorly understood. At a molecular level little is known how detachments form, how to optimize reattachment, or prevent extension of the detachment. A major limitation is lack of information about the biophysical consequences of the retina–RPE separation. The primary challenge is determining the molecular properties of the pathological interface surfaces. Here, using detached bovine retina, we show that this hurdle can be overcome through a combination of biophysical and ultrastructural approaches. The outer surface of freshly detached bovine neural retina, and isolated molecular components of the outer retina were subjected to: 1) Contact angle goniometry to determine the critical surface tension of the outer retinal surface, isolated insoluble interphotoreceptor matrix (IPM) and purified interphotoreceptor retinoid binding protein (IRBP); 2) Multiple attenuated internal reflectance infrared (MAIR-IR) spectroscopy was used to characterize the molecular composition of the retinal surface. MAIR-IR depth penetration was established through ellipsometric measurement of barium-stearate films. Light microscopy, immunohistochemistry and electron microscopy defined the structures probed spectroscopically. Furthermore, the data were correlated to IR spectra of docosahexaenoic acid, hyaluronan, chondroitin-6-sulfate and IRBP, and imaging by IR-microscopy. We found that the retinal critical surface tension is 24 mN/m, similar to isolated insoluble IPM and lower than IRBP. Barium-stearate calibration studies established that the MAIR-IR spectroscopy penetration depth was 0.2 μm. Ultrastructural observations and MAIR-IR studies of isolated outer retina components determined that the pericellular IPM coating the outer retinal surface is primarily responsible for these surface properties. The critical surface tension of detached bovine retina is dictated not by the outer segments, but by a pericellular IPM covering the outer segment tips. [ABSTRACT FROM AUTHOR]

Purpose of Review: Solute transport in the lacunar-canalicular system (LCS) plays important roles in osteocyte metabolism and cell-cell signaling. This review will summarize recent studies that establish pericellular matrix (PCM), discovered inside the LCS, as a crucial regulator of solute transport in bone.Recent Findings: Utilizing confocal imaging and mathematical modeling, recent studies successfully quantified molecular diffusion and convection in the LCS as well as the size-dependent sieving effects of the PCM, leading to the quantification of the effective PCM fiber spacing (10 to 17 nm) in murine adult bones. Perlecan/HSPG2, a large linear proteoglycan, was identified to be an essential PCM component.Summary: The PCM-filled LCS is bone’s chromatographic column, where fluid/solute transport to and from the osteocytes is regulated. The chemical composition, deposition rate, and turnover rate of the osteocyte PCM should be further defined to better understand osteocyte physiology and bone metabolism. [ABSTRACT FROM AUTHOR]

Extracellular matrix microfibrils are critical components of connective tissues with a wide range of mechanical and cellular signalling functions. Collagen VI is a heteromeric network-forming collagen which is expressed in tissues such as skin, lung, blood vessels and articular cartilage where it anchors cells into the matrix allowing for transduction of biochemical and mechanical signals. It is not understood how collagen VI is arranged into microfibrils or how these microfibrils are arranged into tissues. Therefore we have characterised the hierarchical organisation of collagen VI across multiple length scales. The frozen hydrated nanostructure of purified collagen VI microfibrils was reconstructed using cryo-TEM. The bead region has a compact hollow head and flexible tail regions linked by the collagenous interbead region. Serial block face SEM imaging coupled with electron tomography of the pericellular matrix (PCM) of murine articular cartilage revealed that the PCM has a meshwork-like organisation formed from globular densities ∼30 nm in diameter. These approaches can characterise structures spanning nanometer to millimeter length scales to define the nanostructure of individual collagen VI microfibrils and the micro-structural organisation of these fibrils within tissues to help in the future design of better mimetics for tissue engineering. Statement of Significance Cartilage is a connective tissue rich in extracellular matrix molecules and is tough and compressive to cushion the bones of joints. However, in adults cartilage is poorly repaired after injury and so this is an important target for tissue engineering. Many connective tissues contain collagen VI, which forms microfibrils and networks but we understand very little about these assemblies or the tissue structures they form. Therefore, we have use complementary imaging techniques to image collagen VI microfibrils from the nano-scale to the micro-scale in order to understand the structure and the assemblies it forms. These findings will help to inform the future design of scaffolds to mimic connective tissues in regenerative medicine applications. [ABSTRACT FROM AUTHOR]

Cells in solid tissues sense and respond to mechanical signals that are transmitted through extracellular matrix (ECM) over distances that are many times their size. This long-range force transmission is known to arise from strain-stiffening and buckling in the collagen fiber ECM network, but must also pass through the denser pericellular matrix (PCM) that cells form by secreting and compacting nearby collagen. However, the role of the PCM in the transmission of mechanical signals is still unclear. We therefore studied an idealized computational model of cells embedded within fibrous collagen ECM and PCM. Our results suggest that the smaller network pore sizes associated with PCM attenuates tension-driven collagen-fiber alignment, undermining long-range force transmission and shielding cells from mechanical stress. However, elongation of the cell body or anisotropic cell contraction can compensate for these effects to enable long distance force transmission. Results are consistent with recent experiments that highlight an effect of PCM on shielding cells from high stresses. Results have implications for the transmission of mechanical signaling in development, wound healing, and fibrosis.

Cells are surrounded by a hyaluronan-rich coat called ‘pericellular matrix’ (PCM), mainly constituted by hyaluronan, a long-chain linear polysaccharide which is secreted and resorbed by the cell, depending on its activity. Cell attachment to a surface is mediated by PCM before integrins and focal adhesions are involved. As hyaluronan is known to bear a negative charge at physiological pH, the relevance of its electrical properties in driving the early cell adhesion steps has been studied, exploring how PCM mediates cell adhesion to charged surfaces, such as polyelectrolyte multilayer (PEM) films. Poly(ethylene imine) (PEI) and poly(sodium 4-styrene sulphonate) (PSS), assembled as PEI/PSS and PEI/PSS/PEI layers, were used. The nanoscale morphology of such layers was analysed by atomic force microscopy, and the detailed surface structure was analysed by X-ray photoemission spectroscopy. PCM-coated and PCM-depleted MG63 osteoblast-like cells were used, and cell density, morphology and adhesive structures were analysed during early steps of cell attachment to the PEM surfaces (1-6 h). The present study demonstrates that the pericellular matrix is involved in cell adhesion to material surfaces, and its arrangement depends on the cell interaction with the surface. Moreover, the PCM/surface interaction is not simply driven by electrostatic effects, as the cell response may be affected by specific chemical groups at the material surface. In the development of biomimetic surfaces promoting cell adhesion and function, the role of this unrecognised outer cell structure has to be taken into account