Extracellular Matrix Macromolecules Research Articles

The interplay between tumor and nodal microenvironments for the formation of nodal premetastatic niche in head and neck cancer.

Head and neck cancer (HNC) encompasses a number of malignancies originating in the head and neck area. In patients with HNC, cervical lymph nodes constitute metastatic sites for cancer cells that escape primary tumors. The premetastatic niche (PMN) is a crucial concept in understanding metastatic disease. PMN refers to the microenvironment resulting mainly from primary tumor cells to foster metastatic tumor cell growth at a distant organ. Tumor microenvironment (TME) plays an important part in the pathogenesis of PMN. A significant prognostic factor is the close association between metastases of lymph nodes and organ dissemination in many different malignancies. The nodal premetastatic niche (NPMN) is a particular type of PMN located within the lymph nodes. NPMN formation is specifically important in HNC as regional lymph node metastasis commonly occurs. The formation happens when tumor cells create a supportive microenvironment within lymph nodes, facilitating their survival, growth, spread, and invasion. This complex mechanism involves multiple steps and cellular interactions between the primary tumor and tumor microenvironment. Several extracellular matrix (ECM) macromolecules, cytokines, and growth factors are implicated in this process. The aim of this article is to present the most recent data on the regulation of the lymph node PMN at molecular and cellular levels in HNC, as well as insights with respect to the relationship between primary tumor cells and the microenvironment of lymph nodes, and the formation of NPMN. We also critically discuss on potential targets for preventing or disrupting nodal metastases and identify potential biomarkers for predicting HNC outcomes.

Endogenously produced hyaluronan contributes to the regulation of peritoneal adhesion development.

Peritoneal adhesions are postsurgical fibrotic complications connected to peritoneal inflammation. The exact mechanism of development is unknown; however, an important role is attributed to activated mesothelial cells (MCs) overproducing macromolecules of extracellular matrix (ECM), including hyaluronic acid (HA). It was suggested that endogenously-produced HA contributes to the regulation of different fibrosis-related pathologies. However, little is known about the role of altered HA production in peritoneal fibrosis. We focused on the consequences of the increased turnover of HA in the murine model of peritoneal adhesions. Changes of HA metabolism were observed in early phases of peritoneal adhesion development in vivo. To study the mechanism, human MCs MeT-5A and murine MCs isolated from the peritoneum of healthy mice were pro-fibrotically activated by transforming growth factor β (TGFβ), and the production of HA was attenuated by two modulators of carbohydrate metabolism, 4-methylumbelliferone (4-MU) and 2-deoxyglucose (2-DG). The attenuation of HA production was mediated by upregulation of HAS2 and downregulation of HYAL2 and connected to the lower expression of pro-fibrotic markers, including fibronectin and α-smooth muscle actin (αSMA). Moreover, the inclination of MCs to form fibrotic clusters was also downregulated, particularly in 2-DG-treated cells. The effects of 2-DG, but not 4-MU, were connected to changes in cellular metabolism. Importantly, the inhibition of AKT phosphorylation was observed after the use of both HA production inhibitors. In summary, we identified endogenous HA as an important regulator of peritoneal fibrosis, not just a passive player during this pathological process.

Fabrication of Collagen-Hyaluronic Acid Cryogels by Directional Freezing Mimicking Cartilage Arcade-like Structure.

The internal architecture of tissue-like constructs is fundamental to their structural and biological functions. Here, we introduce a simple and robust method to fabricate cryogels based on derivatized extracellular matrix (ECM) macromolecules with porosity arranged according to the typical Benninghoff zonal architecture of articular cartilage. To obtain this arcade-like structure, the technique used the growth of ice crystals from copper pins at cryogenic temperatures. The directional cryogel formation enabled the organized growth of ice crystals over a large distance (>4 mm). The compositional properties were achieved by forming double networks (DNs) of hyaluronic acid and collagen derivatives (MeHA and CollGTA, respectively), which also served to improve the mechanical properties of the otherwise weak collagen scaffolds. Compositionally biomimetic and more resilient MeHA-CollGTA DNs (Young's modulus ≈ 200 kilopascals) were therefore produced. The technique presented expands the fabrication methods available for providing ECM macromolecules with architectural elements mimicking cartilage complexity.

Arthrofibrosis - a myth or true joint disorder?

Authors, mostly specialists on rehabilitation and orthopedic surgery prove that arthrofibrosis is a commonly overlooked phenomenon, which may lead to serious limitation in the range of movement, leading to limitation in patients quality of functioning. The main goal of this article is to emphasize the importance of understanding a such complex condition. Non typical patomechanism, lack of biomarkers dedicated to this dysfunction and general lack of understanding in this pathology causes that risk factors and the most effective strategies remain vastly unknown. Pathophysiology of the arthrofibrosis in the joints is definitely multifactorial, but intense production of collagen seems to be the main factor. Most modern pharmacological methods concentrate on the regulation of collagen fiber production and reducing the inflammation. Inflammation from joint contractures stimulates the proliferation of activated cells that results in the production of extracellular matrix macromolecules to form fibrotic tissue that is deposited into the capsule, thereby resulting in fibrosis. Lack of unified classification scale is caused by relatively high variation of the functions fulfilled by particular joints and each treatment plan should be constructed individually. Quality of surgical treatment and physical therapy play a major role in both prevention and treatment of such complex condition as arthrofibrosis. Both iatrogenic mistakes and overly aggressive manual therapy are some of main factors increasing the risk of this pathological condition. Introducing properly conducted physical therapy treatment in the early stage is crucial to main the range of movement and preventing this significant problem.

Mechanical characterization of articular cartilage degraded combinedly with MMP-1 and MMP-9

Matrix metalloproteinases (MMPs) degrade the extracellular matrix (ECM) subsequently damaging cartilage and altering biomechanical properties. Collectively, MMPs cleave every ECM macromolecule. However, MMPs present complex substrate interactions and digest differing ECM components making it difficult to understand the individual role each MMP plays in cartilage degradation. To understand the combined impact MMPs have on cartilage biomechanical properties, MMPs from two subfamilies: collagenase and gelatinase were investigated. Three ratios of MMP-1 (c) and MMP-9 (g), c1:g1, c1:g0 and c0:g1 were considered. Cartilage plugs (n = 30) were collected from the femoral condyles of 3 bovine stifle joints. In groups of 10, samples were treated with MMP-1, MMP-9, or a combination. Samples were subjected to indentation loading up to 20% bulk strain and were assessed mechanically and histologically to determine the degradative impact. Young's modulus and peak load were compared between the control and degraded explants. In comparison to samples degraded by MMP-1 or MMP-9 individually, cartilage degraded with both enzymes resulted in a 9–15% greater reduction in stiffness and peak load. Individually, MMP-1 and MMP-9 have a minimum effect on cartilage micromechanical properties, but synergistically the two enzymes digest ECM components and a much greater degradative effect is observed.

The Role of Matrix Metalloproteinases (MMP-8, MMP-9, MMP-13) in Periodontal and Peri-Implant Pathological Processes.

Severe periodontitis, a destructive inflammatory disease of the supporting tissues of the teeth, ranks sixth in terms of global spread, affecting about 11% of the population. Metalloproteinases (MMPs) are extracellular matrix (ECM) macromolecules that are important in cellular development and morphogenesis, and they are capable of activating growth factors in their proximity, cell surface receptors, and adhesion molecules. MMPs are part of a major family of zinc-dependent endopeptidases, and their activity is modulated and regulated by certain inhibitors known as tissue metalloproteinase inhibitors (TIMPs). Because type I collagen is the major component of the periodontal extracellular matrix, special attention has been paid to the role of collagenases, especially MMP-8 and MMP-13 and gelatinases, MMP-2 and MMP-9, in periodontal diseases. In fact, MMP-8 (or collagenase 2) is currently one of the most promising biomarkers for periodontitis in oral fluids. Among them, salivary MMP-9 has been shown to be a more sensitive marker for periodontal inflammation during orthodontic treatment, which opens new perspectives in reducing periodontal hazards during such treatments. Both MMP-8 and MMP-9 are extremely valuable diagnostic tools in treating periodontitis, and future studies and healthcare policies should focus on implementing more accessible methods of chairside testing in order to reduce the prevalence of this disease.

Extracellular Matrix Derived From Dental Pulp Stem Cells Promotes Mineralization.

Background: Extracellular matrix (ECM) plays a pivotal role in many physiological processes. ECM macromolecules and associated factors differ according to tissues, impact cell differentiation, and tissue homeostasis. Dental pulp ECM may differ from other oral tissues and impact mineralization. Thus, the present study aimed to identify the matrisome of ECM proteins derived from human dental pulp stem cells (DPSCs) and its ability to regulate mineralization even in cells which do not respond to assaults by mineralization, the human gingival fibroblasts (GF). Methods: ECM were extracted from DPSCs cultured in normal growth medium supplemented with L-ascorbic acid (N-ECM) or in osteogenic induction medium (OM-ECM). ECM decellularization (dECM) was performed using 0.5% triton X-100 in 20 mM ammonium hydroxide after 21 days. Mass spectrometry and proteomic analysis identified and quantified matrisome proteins. Results: The dECM contained ECM proteins but lacked cellular components and mineralization. Interestingly, collagens (COL6A1, COL6A2, and COL6A3) and elastic fibers (FBN1, FBLN2, FN1, and HSPG2) were significantly represented in N-ECM, while annexins (ANXA1, ANXA4, ANXA5, ANXA6, ANXA7, and ANXA11) were significantly overdetected in OM-ECM. GF were reseeded on N-dECM and OM-dECM and cultured in normal or osteogenic medium. GF were able to attach and proliferate on N-dECM and OM-dECM. Both dECM enhanced mineralization of GF at day 14 compared to tissue culture plate (TCP). In addition, OM-dECM promoted higher mineralization of GF than N-dECM although cultured in growth medium. Conclusions: ECM derived from DPSCs proved to be osteoinductive, and this knowledge supported cell-derived ECM can be further utilized for tissue engineering of mineralized tissues.

The texture of collagen in the microenvironments of Merkel cell carcinoma.

Solid tumors typically contain high levels of fibrillar collagen. The increased stromal collagen deposition usually promotes cancer progression since biochemical and biophysical cues from tumor-associated collagen fibers stimulate neoplastic cells. Few studies have investigated the relationship between Merkel cell carcinoma (MCC) and the extracellular matrix (ECM), but there are no works evaluating collagen.This is an observational, analytical, retrospective study including 11 patients with MCC. Primary tumor-stained sections were evaluated by second harmonic generation microscopy and texture analysis.Peritumoral texture features (area fraction, mean gray value, entropy, and contrast) showed much lower values than normal skin (P < .0001) revealing extensively altered structure of peritumoral collagen fibers. These differences were not significant between tumors with unfavorable and favorable known prognostic factors.Profound changes in collagen fibers present in the stroma accompanying primary MCC may contribute to the aggressive behavior of this tumor. Our results indicate that whatever MCC histological subtype, size or anatomical location, MCC promotes the same type of ECM for its development. As an outlook, therapies using ECM macromolecules or fibroblasts (the architects of ECM remodeling) as target could be useful in the treatment of MCC.

Engineered Bacteria Enhance Immunotherapy and Targeted Therapy through Stromal Remodeling of Tumors.

Desmoplastic solid tumors are characterized by the rapid build-up of extracellular matrix (ECM) macromolecules, such as hyaluronic acid (HA). The resulting physiological barrier prevents the infiltration of immune cells and also impedes the delivery of anticancer agents. The development of a hypervesiculating Escherichia coli Nissle (ΔECHy) based tumor targeting bacterial system capable of distributing a fusion peptide, cytolysin A (ClyA)-hyaluronidase (Hy) via outer membrane vesicles (OMVs) is reported. The capability of targeting hypoxic tumors, manufacturing recombinant proteins in situ and the added advantage of an on-site OMV based distribution system makes the engineered bacterial vector a unique candidate for peptide delivery. The HA degrading potential of Hy for stromal modulation is combined with the cytolytic activity of ClyA followed by testing it within syngeneic cancer models. ΔECHy is combined with immune checkpoint antibodies and tyrosine kinase inhibitors (TKIs) to demonstrate that remodeling the tumor stroma results in the improvement of immunotherapy outcomes and enhancing the efficacy of biological signaling inhibitors. The biocompatibility of ΔECHy is also investigated to show that the engineered bacteria are effectively cleared, elicit minimal inflammatory and immune responses, and therefore could be a reliable candidate as a live biotherapeutic.

Tailoring the biofunctionality of collagen biomaterials via tropoelastin incorporation and EDC-crosslinking

Recreating the cell niche of virtually all tissues requires composite materials fabricated from multiple extracellular matrix (ECM) macromolecules. Due to their wide tissue distribution, physical attributes and purity, collagen, and more recently, tropoelastin, represent two appealing ECM components for biomaterials development. Here we blend tropoelastin and collagen, harnessing the cell-modulatory properties of each biomolecule. Tropoelastin was stably co-blended into collagen biomaterials and was retained after EDC-crosslinking. We found that human dermal fibroblasts (HDF), rat glial cells (Rugli) and HT1080 fibrosarcoma cells ligate to tropoelastin via EDTA-sensitive and EDTA-insensitive receptors or do not ligate with tropoelastin, respectively. These differing elastin-binding properties allowed us to probe the cellular response to the tropoelastin-collagen composites assigning specific bioactivity to the collagen and tropoelastin component of the composite material. Tropoelastin addition to collagen increased total Rugli cell adhesion, spreading and proliferation. This persisted with EDC-crosslinking of the tropoelastin-collagen composite. Tropoelastin addition did not affect total HDF and HT1080 cell adhesion; however, it increased the contribution of cation-independent adhesion, without affecting the cell morphology or, for HT1080 cells, proliferation. Instead, EDC-crosslinking dictated the HDF and HT1080 cellular response. These data show that a tropoelastin component dominates the response of cells that possess non-integrin based tropoelastin receptors. EDC modification of the collagen component directs cell function when non-integrin tropoelastin receptors are not crucial for cell activity. Using this approach, we have assigned the biological contribution of each component of tropoelastin-collagen composites, allowing informed biomaterial design for directed cell function via more physiologically relevant mechanisms. Statement of significanceBiomaterials fabricated from multiple extracellular matrix (ECM) macromolecules are required to fully recreate the native tissue niche where each ECM macromolecule engages with a specific repertoire of cell-surface receptors. Here we investigate combining tropoelastin with collagen as they interact with cells via different receptors. We identified specific cell lines, which associate with tropoelastin via distinct classes of cell-surface receptor. These showed that tropoelastin, when combined with collagen, altered the cell behaviour in a receptor-usage dependent manner. Integrin-mediated tropoelastin interactions influenced cell proliferation and non-integrin receptors influenced cell spreading and proliferation. These data shed light on the interplay between biomaterial macromolecular composition, cell surface receptors and cell behaviour, advancing bespoke materials design and providing functionality to specific cell populations.

Diagnostic value of MMP-1 in chronic liver diseases

Background: Chronic liver disorders in children constitute an increasing concern with major public health impacts, the MMP is degrading diverse extracellular matrix macromolecules and involves tissue reshaping and repair in physiological processes. The purpose of this research was to assess the diagnostic usefulness of MMP-1 in children with chronic liver disorders. Methods: 60 children undertook this research; Group I (hepatic group): 60 children with chronic liver disease, Group II (control group): 20 seemingly healthy youngsters, The entire history of taking, comprehensive clinical examination, abdominal US and laboratory testing like a complete blood count and liver function tests have been performed for all children (Alt, AST, Albumin, PT, PTT, INR, Total and direct bilirubin), Liver biopsy was conducted in all patients, with human enzyme-connected immunosorbent testing measuring Serum MMP-1 levels (ELISA). Results: the mean MMP-1 in hepatic group was 11.2±2.1 ng/ml and in control group 32.7±17.4 ng/ml. In the hepatic group controlled, p F1), AUC was 0.872 (95 percent confidence interval: 0.709-0.487), p F3); AUC p < 0.001. The sensitivity was 90% at cutoff point < 4.5 ng/ml and the specificity was 80%. Conclusion: Fibrosis identification in children with chronic liver disorders might benefit from MMP-1.

Blockade of checkpoint ILT3/LILRB4/gp49B binding to fibronectin ameliorates autoimmune disease in BXSB/Yaa mice.

The extracellular matrix (ECM) is the basis for virtually all cellular processes and is also related to tumor metastasis. Fibronectin (FN), a major ECM macromolecule expressed by different cell types and also present in plasma, consists of multiple functional modules that bind to ECM-associated, plasma, and cell-surface proteins such as integrins and FN itself, thus ensuring its cell-adhesive and modulatory role. Here we show that FN constitutes an immune checkpoint. Thus, FN was identified as a physiological ligand for a tumor/leukemia/lymphoma- as well as autoimmune-associated checkpoint, ILT3/LILRB4 (B4, CD85k). Human B4 and the murine ortholog, gp49B, bound FN with sub-micromolar affinities as assessed by bio-layer interferometry. The major B4-binding site in FN was located at the N-terminal 30-kDa module (FN30), which is apart from the major integrin-binding site present at the middle of the molecule. Blockade of B4-FN binding such as with B4 antibodies or a recombinant FN30-Fc fusion protein paradoxically ameliorated autoimmune disease in lupus-prone BXSB/Yaa mice. The unexpected nature of the B4-FN checkpoint in autoimmunity is discussed, referring to its potential role in tumor immunity.

A guide to the composition and functions of the extracellular matrix.

Extracellular matrix (ECM) is a dynamic 3-dimensional network of macromolecules that provides structural support for the cells and tissues. Accumulated knowledge clearly demonstrated over the last decade that ECM plays key regulatory roles since it orchestrates cell signaling, functions, properties and morphology. Extracellularly secreted as well as cell-bound factors are among the major members of the ECM family. Proteins/glycoproteins, such as collagens, elastin, laminins and tenascins, proteoglycans and glycosaminoglycans, hyaluronan, and their cell receptors such as CD44 and integrins, responsible for cell adhesion, comprise a well-organized functional network with significant roles in health and disease. On the other hand, enzymes such as matrix metalloproteinases and specific glycosidases including heparanase and hyaluronidases contribute to matrix remodeling and affect human health. Several cell processes and functions, among them cell proliferation and survival, migration, differentiation, autophagy, angiogenesis, and immunity regulation are affected by certain matrix components. Structural alterations have been also well associated with disease progression. This guide on the composition and functions of the ECM gives a broad overview of the matrisome, the major ECM macromolecules, and their interaction networks within the ECM and with the cell surface, summarizes their main structural features and their roles in tissue organization and cell functions, and emphasizes the importance of specific ECM constituents in disease development and progression as well as the advances in molecular targeting of ECM to design new therapeutic strategies.

The action of hyaluronan in functional properties, morphology and expression of matrix effectors in mammary cancer cells depends on its molecular size.

Breast cancer constitutes a heterogeneous disease. The expression profiles of estrogen receptors (ERs), as well as the expression patterns of extracellular matrix (ECM) macromolecules, determine its development and progression. Hyaluronan (HA) is an ECM molecule that regulates breast cancer cells' properties in a molecular size-dependent way. Previous studies have shown that 200-kDa HA fragments modulate the functional properties, morphology, and expression of several matrix mediators of the highly metastatic ERα- /ERβ+ MDA-MB-231 cells. In order to evaluate the effects of HA fragments (<10, 30 and 200-kDa) in ERβ-suppressed breast cancer cells, the shERβ MDA-MB-231 cells were used. These cells are less aggressive when compared with MDA-MB-231 cells. To this end, the functional properties, the morphology, and the expression of the molecules associated with breast cancer cells metastatic potential were studied. Notably, both cell proliferation and invasion were significantly reduced after treatment with 200-kDa HA. Moreover, as assessed by scanning electron microscopy, 200-kDa HA affected cellular morphology, and as assessed by qPCR, upregulated the epithelial marker Ε-cadherin. The expression profiles of ECM mediators, such as HAS2, CD44, and MMP7, were also altered. On the other hand, cellular migration and the expression levels of syndecan-4 (SDC-4) were not significantly affected in contrast to our observations regarding MDA-MB-231 cells. These novel data demonstrate that the molecular size of the HA determines its effects on ERβ-suppressed breast cancer cells and that 200-kDa HA exhibits antiproliferative effects on these cells. A deeper understanding of this mechanism may contribute to the development of therapeutic strategies against breast cancer.

Over-expression of stromal periostin correlates with poor prognosis of cutaneous squamous cell carcinomas.

Periostin, an extracellular matrix macromolecule implicated in tumorigenesis, serves as a prognostic marker for many cancer types. However, there are no data on periostin expression in cutaneous squamous cell carcinoma (cSCC). This study examined periostin expression in patients with cSCC and explored its clincopathological relationship and prognosis. Using immunohistochemistry and ImageJ analysis, we compared periostin expression in 95 cSCCs across a spectrum of cSCC aggressiveness: cSCC in situ (SCCIS) (n=25), low-risk cSCC (LR-cSCC) (n=26), high-risk cSCC (HR-cSCC) (n=38), and cSCC in recessive dystrophic epidermolysis bullosa patients (RDEB cSCC) (n=6). Immunohistochemistry demonstrated periostin expression within the intra-tumoral stroma but not within tumor cells. Periostin levels significantly (P<0.001) increased from SCCIS, LR-cSCC, HR-cSCC to RDEB SCC. The stroma of most of the cSCCs we evaluated contained cancer-associated fibroblasts with a myofibroblastic (α -SMA-positive) phenotype. Co-localization of periostin with α-SMA, evidence of fibroblast periostin expression, and absence of keratinocyte or tumor cell periostin expression suggest that, in cSCC, periostin is a product of the peritumoral microenvironment and not the tumor cells themselves. Our data indicate that fibroblast periostin expression is highly correlated with the aggressiveness of cSCC, and may thereby provide a molecular marker that will be useful for subtyping and diagnosing cSCCs according to their biological nature.

MicroRNAs and Regulation of Autophagy in Chondrocytes.

Chondrocytes are the main cells responsible for the maintenance of cartilage homeostasis and integrity. During development, extracellular matrix (ECM) macromolecules are produced and deposited by chondrocyte precursors. Autophagy, a highly dynamic process aimed at degradation of dysfunctional or pathogenic proteins, organelles, and intracellular microbes that can damage tissues, is one of the key processes required for sustained cartilage homeostasis. In different cell types it has been shown that, among others, autophagy is regulated by epigenetic mechanisms such as small noncoding RNAs (miRNAs, ~22 base pairs). Increasing evidence suggests that miRNAs are also involved in the regulation of autophagy in chondrocytes. Based on our previous research of gene and miRNA expression in articular cartilage, in this chapter we provide a summary of the tools models to direct in vitro and in vivo studies aimed at gaining a better understanding of the regulatory roles of miRNAs in chondrocyte autophagy.

The Human Epidermal Basement Membrane: A Shaped and Cell Instructive Platform That Aging Slowly Alters.

One of the most important functions of skin is to act as a protective barrier. To fulfill this role, the structural integrity of the skin depends on the dermal-epidermal junction—a complex network of extracellular matrix macromolecules that connect the outer epidermal layer to the underlying dermis. This junction provides both a structural support to keratinocytes and a specific niche that mediates signals influencing their behavior. It displays a distinctive microarchitecture characterized by an undulating pattern, strengthening dermal-epidermal connectivity and crosstalk. The optimal stiffness arising from the overall molecular organization, together with characteristic anchoring complexes, keeps the dermis and epidermis layers extremely well connected and capable of proper epidermal renewal and regeneration. Due to intrinsic and extrinsic factors, a large number of structural and biological changes accompany skin aging. These changes progressively weaken the dermal–epidermal junction substructure and affect its functions, contributing to the gradual decline in overall skin physiology. Most changes involve reduced turnover or altered enzymatic or non-enzymatic post-translational modifications, compromising the mechanical properties of matrix components and cells. This review combines recent and older data on organization of the dermal-epidermal junction, its mechanical properties and role in mechanotransduction, its involvement in regeneration, and its fate during the aging process.

Role of cellulose family in fibril organization of collagen for forming 3D cancer spheroids: In vitro and in silico approach.

Introduction: Cell aggregation of three-dimensional (3D) culture systems (the so-called spheroids) are designed as in vitro platform to represent more accurately the in vivo environment for drug discovery by using semi-solid media. The uniform multicellular tumor spheroids can be generated based on the interaction of cells with extracellular matrix (ECM) macromolecules such as collagen and integrin. This study aimed to investigate the possible interactions between the cellulose family and collagen using both in vitro and in silico approaches. Methods: The 3D microtissue of JIMT-1 cells was generated using hanging drop method to study the effects of charge and viscosity of the medium containing cellulose family. To determine the mode of interaction between cellulose derivatives (CDs) and collagen-integrin, docking analysis and molecular simulation were further performed using open source web servers and chemical simulations (GROMACS), respectively. Results: The results confirmed that the addition of CDs into the 3D medium can promote the formation of solid spheroids, where methylcellulose (MC) yielded uniform spheroids compared to carboxymethyl cellulose (CMC). Moreover, the computational analysis showed that MC interacted with both integrin and collagen, while sodium carboxymethyl cellulose (NaCMC) only interacted with collagen residues. The stated different behaviors in the 3D culture formation and collagen interaction were found in the physicochemical properties of CDs. Conclusion: Based on in vitro and in silico findings, MC is suggested as an important ECM-mimicking entity that can support the semi-solid medium and promote the formation of the uniform spheroid in the 3D culture.

A Role for HAPLN1 During Phenotypic Modulation of Human Lung Fibroblasts In Vitro.

Hyaluronan and proteoglycan link protein 1 (HAPLN1) stabilizes interactions between two important extracellular matrix (ECM) macromolecules, versican and hyaluronan, which facilitate proliferation of fibroblasts and their conversion to myofibroblasts. However, the role of HAPLN1 in these events has not been studied. Using immunocytochemistry, cellular and ECM locations of HAPLN1 were evaluated in cultured human lung fibroblasts during proliferation and conversion to myofibroblasts. HAPLN1 localized to pericellular matrices, associating with both versican and hyaluronan in the ECM and on the cell surface. Nuclear and total HAPLN1 immunostaining increased after myofibroblast induction. Confocal microscopy showed HAPLN1 predominant in the ECM under cells while versican predominated above cells. Versican and HAPLN1 were also juxtaposed in columnar inclusions in the cytoplasm and nucleus. Nuclear HAPLN1 staining in interphase cells redistributed to the cytosol during mitosis. In the absence of TGF-β1, addition of exogenous bovine HAPLN1 (together with aggrecan G1) facilitated myofibroblast formation, as seen by significant upregulation of α-smooth muscle actin (SMA) staining, while adding full-length bovine versican had no effect. Increased compaction of hyaluronan-rich ECM suggests that HAPLN1 plus G1 addition affects hyaluronan networks and myofibroblast formation. These observations demonstrate changes in both extracellular and intracellular localization of HAPLN1 during fibroblast proliferation and myofibroblast conversion suggesting a possible role in fibrotic remodeling.

Going beyond RGD: screening of a cell-adhesion peptide library in 3D cell culture

In tissue engineering, cell-adhesion peptides (CAPs) such as the ubiquitous arginine–glycine–aspartic acid (RGD) sequence have allowed the functionalization of synthetic materials to mimic macromolecules of the extracellular matrix (ECM). However, the variety of ECM macromolecules makes it challenging to reproduce all of the native tissue functions with only a limited variety of CAPs. Screening of libraries of CAPs, analogous to high-throughput drug discovery assays, can help to identify new sequences directing cell organization. However, challenges to this approach include the automation of cell seeding in three dimensions and characterization methods. Here, we report a method for robotically generating a library of 16 CAPs to identify a microenvironment capable of directing a chain-like morphology in olfactory ensheathing cells (OECs), a cell type of particular interest for guiding axon growth in spinal cord injury repair. This approach resulted in the identification of one CAP not previously reported to interact with OECs to direct their morphology into structures suitable for potential axon guidance. The same screening approach should be applicable to any range of cell types to discover new CAPs to direct cell fate or function.