Key Extracellular Matrix Components Research Articles

Single-cell Trajectories and Pseudo-time Analysis Reveal Additional Insights into Rheumatoid Arthritis Disease Progression

As the chicken embryo grows, its increasing metabolic demand drives the development of the cardiovascular system, enabling efficient nutrient delivery through complex molecular and structural formation. Key extracellular matrix (ECM) components, such as hyaluronic acid (HA) and collagen type I alpha 1 (COL1A1), play a crucial role in shaping the biomechanical environment of the heart, particularly the sinoatrial node (SAN). These ECM elements regulate cell adhesion, migration, and the maturation of cardiac progenitor cells (CPCs), essential for the heart's electrical conductivity and rhythmicity. Based on the hypothesis that ECM mechanical properties influence the spatial patterning of conduction-related genes, we analyzed spatial transcriptomics data from chicken hearts at different developmental stages. Collagen-related genes associated with a sturdy ECM showed an increasing trend over time, while soft ECM-related genes responsible for producing proteoglycans and breaking down collagen decreased. Although conduction-related genes did not fully align with ECM trends, intriguing patterns were observed, pointing to potential interactions between ECM remodeling and the construction of the cardiac conduction system. Our findings suggest significant ECM r7emodeling between days 4 and 7, which may influence the heart's structural and functional development.

CNSC-55. UNCOVERING THE ROLE OF COLLAGEN EXTRACELLULAR MATRIX IN GLIOBLASTOMA BRAIN MICROENVIRONMENT TRANSFORMATION

Abstract Glioblastoma (GBM) is a highly malignant brain tumor characterized by diffuse infiltration and significant heterogeneity, without effective treatment. Mesenchymal-like (MES) glioma cells drive tumor aggression by promoting brain invasion and tumor microenvironment remodeling. Previous spatial transcriptomic analyses identified Collagen, particularly COL1A, as a key extracellular matrix (ECM) component, enriched in MES areas termed oncostreams. Depletion of COL1A1 disrupted oncostreams, influencing the tumor microenvironment to support cell survival, and migration. Reactive astrocytes and macrophages, essential components of the brain microenvironment, impact glioma progression. However, the interplay between glioma-secreted collagen and the brain microenvironment remains poorly understood. To elucidate this, we generated orthotopic mouse glioma models with high-collagen (NPD) and low-collagen (NPDshCol1A1). Our data revealed that downregulation of COL1A1 in glioma cells alters the abundance and spatial distribution of reactive astrocytes and macrophages/microglia. Immunofluorescence and flow cytometry analysis showed that tumors with low-collagen had decreased GFAP+ reactive astrocyte cells at the invasive edge and perivascular niche compared to high-collagen gliomas. Moreover, our results shows that high collagen levels may activate astrocytes through distant communication, as evidenced by the higher presence of reactive astrocytes in the contralateral cortex of NPD tumors. However, low-collagen tumors exhibited an increase in astrocytes (GFAP+) in the tumor core. Furthermore, high-collagen gliomas demonstrated increased macrophage infiltration, promoting an immunosuppressive environment. Using multiplex immunohistochemistry assays, we observed that reactive astrocytes along the invasive tumor edge were associated with glioma cells expressing elevated levels of COL1A1 and TGF-β ligand. In summary, our results show that glioma cell-expressed COL1A1 promotes the activation of peritumoral and perivascular brain-resident astrocytes and macrophages. The interactions of reactive astrocyte with glioma cells enriched in collagen form a feedback loop that enhances tumor invasion and vascular proliferation, suggesting that targeting the ECM-astrocyte interactions could disrupt these processes and offer a therapeutic avenue for GBM.

Optimizing prolyl hydroxylation for functional recombinant collagen in Escherichia coli

Collagen, a key extracellular matrix component, is renowned for its biocompatibility, biodegradability, and bioactivity, finding wide applications in food, medicine, cosmetics, and industry. Recombinant collagen expression in Escherichia coli offers advantages such as shorter production cycles and lower costs compared to extraction from animal tissues, though it is known to lack essential post-translational modifications, such as proline hydroxylation, which are crucial for its stability and biological function. Studies have shown that certain prolyl hydroxylases, including BaP4H, DsP4H, and L593, exhibit relatively high modification efficiency in the E. coli expression system. However, structures and functions of recombinant human type III collagen after modification by three prolyl hydroxylases remain uncertain. In this study, we investigated the percentage of proline hydroxylation, hydroxylation sites, circular dichroism spectra, and biological functions of recombinant human type III collagen modified by various prolyl hydroxylases. The results indicated that the L593 exhibited the highest percentage of proline hydroxylation, and the percentage of proline hydroxylation was closely associated with the formation of the collagen triple helix, while the hydroxylation ratio of prolines is not positively correlated with the stability of the collagen triple helix structure. The biological function results showed that the cell adhesion of recombinant collagen 3-3(BaP4H) and 3-3(L593) was significantly enhanced, which was closely related to the triple helix structure of recombinant human type III collagen. Our study provides valuable insights into the industrial production and biological applications of collagen, enhancing its functional research and scalability.

Photo-crosslinked hyaluronic acid hydrogels designed for simultaneous delivery of mesenchymal stem cells and tannic acid: Advancing towards scarless wound healing

The quest for scarless wound healing is imperative in healthcare, aiming to diminish the challenges of conventional wound treatment. Hyaluronic acid (HA), a key component of the skin's extracellular matrix, plays a pivotal role in wound healing and skin rejuvenation. Leveraging the advantages of HA hydrogels, this research focuses first on tuning the physicochemical and mechanical properties of photo-crosslinkable methacrylated HA (MAHA) by varying the methacrylation degree, polymer concentration, photo-crosslinker concentration, and UV exposure time. The optimized hydrogel, featuring suitable porosity, swelling ratio, degradability, and mechanical properties, was then used for the combined delivery of tannic acid (TA), known for its hemostatic, antibacterial, and antioxidant properties, and Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) cultured on the MAHA-TA hydrogel to enhance skin regeneration. The composite MAHA-TA-MSC hydrogel demonstrated favorable pores and biocompatibility, evidenced by cell viability, and promoted cell proliferation. When applied to dorsal wounds in rats, this composite hydrogel accelerated wound healing and reduced scarring. Additionally, molecular and histopathological analyses revealed increased expression of IL-10, the TGF-β3/TGF-β1 ratio, and the Collagen III/Collagen I ratio. These findings suggest that the MAHA-TA-MSC hydrogel is a promising candidate for scarless acute wound healing.

Oral low-dose rapamycin treatment prevents aortic aneurysms in marfan mice

Abstract Background/Introduction Marfan syndrome (MFS) is a hereditary connective tissue disorder caused by fibrillin-1 (FBN1) gene mutations. Life-threatening complications are cardiovascular, e.g. thoracic aortic aneurysms and dissections. The pathophysiological cause is the increased release of transforming growth factor β-1 (TGF-β1) from the FBN1-deficient extracellular matrix (ECM) and structural disintegrity. Overactivation of the mechanistic target of rapamycin (mTOR) pathway has been demonstrated in the murine mgR/mgR model of MFS. Short-term administration of rapamycin significantly reduced aortic aneurysm formation and increased the life span in these mice. Purpose To assess the effect of an early continuous oral low-dose rapamycin treatment on the aortic aneurysm formation in the FBN1 hypomorphic mgR/mgR mouse model. Methods Male and female 3-4 week-old mgR/mgR mice were orally administered vehicle or rapamycin through diet (8mg/kg/KG) and sacrificed after 11 weeks. Rapamycin concentration was measured by liquid chromatography-mass spectrometry in whole blood. Aortic diameter was studied using echocardiography. Histopathological and immunofluorescence analyses were performed using aortic tissue sections and techniques. Results Blood rapamycin concentration following oral administration remained below detection level. Rapamycin normalized the aortic wall thickness and the diameter in female mgR/mgR mice to levels of the littermate control mice but not in male mgR/mgR mice. The circumferentially distributed elastin fibres remained significantly intact in sections of the ascending aorta of female mice. Echocardiography revealed that rapamycin-treated female mice stayed below a cut-off value for aortic aneurysms of 2 mm inner aortic diameter eight weeks after starting therapy. In contrast, vehicle-treated female animals already exceeded this value after five weeks. In female but not in male mice, the abundance of the mTOR downstream target phosphorylated ribosomal protein S6 was effectively suppressed by low-dose rapamycin up to levels detected in control animals. At the same time, extracellular matrix proteins like the proteoglycan aggrecan (ACAN) and the related chondroitin sulfate were significantly decreased in female mice. The abundance of ADAMTS5 transglutaminase-2 and xylosyltransferase-1, key enzymes involved in the turnover of proteoglycans and biosynthesis of glycosaminoglycan chains, was improved by the rapamycin treatment in female mice. No decrease in mTOR activity could be detected in male mgR/mgR animals. Here, the ACAN and chondroitin sulphate levels also remained at the level of the vehicle-treated animals. Conclusions Chronic low-dose rapamycin treatment reduced aneurysm formation only in female mgR/mgR mice, since the dose was too low for male mgR/mgR mice, by affecting the composition and organization of key ECM components essential for maintaining aortic homeostasis and mechanical properties.

Umbelliferone alleviates impaired wound healing and skin barrier dysfunction in high glucose-exposed dermal fibroblasts and diabetic skins.

Skin wound healing is a complex process involving various cellular and molecular events. However, chronic wounds, particularly in individuals with diabetes, often experience delayed wound healing, potentially leading to diabetic skin complications. In this study, we examined the effects of umbelliferone on skin wound healing using dermal fibroblasts and skin tissues from a type 2 diabetic mouse model. Our results demonstrate that umbelliferone enhances several crucial aspects of wound healing. It increases the synthesis of key extracellular matrix components such as collagen I and fibronectin, as well as proteins involved in cell migration like EVL and Fascin-1. Additionally, umbelliferone boosts the secretion of angiogenesis factors VEGF and HIF-1α, enhances the expression of cell adhesion proteins including E-cadherin, ZO-1, and Occludin, and elevates levels of skin hydration-related proteins like HAS2 and AQP3. Notably, umbelliferone reduces the expression of HYAL, thereby potentially decreasing tissue permeability. As a result, it promotes extracellular matrix deposition, activates cell migration and proliferation, and stimulates pro-angiogenic factors while maintaining skin barrier functions. In summary, these findings underscore the therapeutic potential of umbelliferone in diabetic wound care, suggesting its promise as a treatment for diabetic skin complications. KEY MESSAGES: Umbelliferone suppressed the breakdown of extracellular matrix components in the skin dermis while promoting their synthesis. Umbelliferone augmented the migratory and proliferative capacities of fibroblasts. Umbelliferone activated the release of angiogenic factors in diabetic wounds, leading to accelerated wound healing. Umbelliferone bolstered intercellular adhesion and reinforced the skin barrier by preventing moisture loss and preserving skin hydration.

Bioinspired Hyaluronic Acid‐Based Hydrogel Fuels Bi‐Directional Lung Organoid Maturation via PIEZO1 and ITGB1 Mediated Mechanosensation

AbstractLung diseases are one of the leading causes of global mortality. Advances in induced pluripotent stem cell (iPSC) differentiation have enabled the creation of bronchiolar and alveolar lung organoids, advancing research on lung conditions. Traditional Matrigel encapsulation, reliant on the spontaneous assembly and propagation of cells with limited external intervention, often results in variability and low reproducibility. The absence of hyaluronic acid (HA) in Matrigel, a key lung extracellular matrix component, limits bronchiolar and alveolar cell differentiation, reducing the efficacy and reproducibility of iPSC‐derived organoid generation. To address this, a novel hybrid hydrogel combining HA and 23% Matrigel, inspired by the natural lung environment, is developed. This hydrogel offers improved biochemical support and viscoelastic properties, significantly accelerating organoid development. Within eight days, the hydrogel produces uniformly sized organoids containing both bronchiolar and alveolar epithelial cells. Increased levels of active mechanosensors and transducers, including PIEZO1, Integrin, and Myosin, suggest that the hydrogel's altered viscoelasticity triggers a mechanotransduction cascade. This bioinspired hydrogel provides a robust, fast model for biomedical research, facilitating rapid drug screening, respiratory disease treatment studies, and surfactant trafficking investigations. Furthermore, it enables the exploration of underlying biomechanical mechanisms to enhance the controllability of organoid generation and maturation.

The biliary atresia susceptibility gene, EFEMP1, regulates extrahepatic bile duct elastic fiber formation and mechanics

IntroductionEGF-Containing Fibulin Extracellular Matrix Protein 1 (EFEMP1, also called fibulin-3) is an extracellular matrix protein linked in a genome-wide association study to biliary atresia, a fibrotic disease of the neonatal extrahepatic bile duct. Fibulin-3 is deposited in most tissues and Efemp1 null mice have decreased elastic fibers in visceral fascia; however, fibulin-3 does not have a role in the development of large elastic fibers and its overall function in the extrahepatic bile ducts remains unclear. MethodsWe used staining and histology to define the amount and organization of key extracellular matrix components in the extrahepatic bile ducts. We also repurposed pressure myography, a technique heretofore applied to the vasculature, to determine the contribution of elastin and fibulin-3 to extrahepatic bile duct mechanics. We examined extrahepatic bile duct structure and mechanics in three models: neonatal vs. adult rat ducts (n=6 each), elastase-treated adult rat ducts (n=6-7 each), and Efemp1+/- vs. wild type mouse ducts (n=6 each). ResultsWe demonstrated that fibulin-3 is expressed in the submucosa of both neonatal and adult mouse, rat and human extrahepatic bile ducts and that, in adult Efemp1+/- mouse ducts, elastin organization into fibers is decreased by approximately half. Pressure myography showed that Efemp1+/- ducts have altered mechanics compared to control ducts, with Efemp1+/- ducts displaying significant stretch compared to controls (p=0.0376); these changes in stretch are similar to those observed in elastase-treated compared to normal ducts (p<0.0001) and in neonatal ducts compared to adult ducts (p< 0.0001). ConclusionFibulin-3 has an important role in the formation of elastic fibers and the mechanical properties of the extrahepatic bile duct. This provides functional relevance for the biliary atresia susceptibility gene EFEMP1. Impact and ImplicationsThe gene EFEMP1 was found via a genome wide association study to be a susceptibility gene for the neonatal disease biliary atresia. EFEMP1 encodes the protein fibulin-3, which regulates elastic fiber organization in the extrahepatic bile duct (EHBD), the major site of disease in biliary atresia. We showed that neonatal EHBDs as well as mice heterozygous for Efemp1 have decreased numbers of elastic fibers, and that this alters EHBD mechanics. This work is important for understanding the mechanism of biliary atresia, in particular susceptibility to obstruction.

Recent Advancements of Hyaluronic Acid Nanoscaffolds in Arthritis Management

AbstractArthritis is a significant health concern affecting over 528 million people globally, reducing quality of life. The chronic nature of arthritis, coupled with insufficient therapeutic effectiveness, recurrence, and adverse side effects, complicates its management. Hyaluronic acid (HA) has emerged as a promising solution due to its biocompatibility, non‐toxicity, and targeted drug delivery capabilities, being a key component of the cartilage extracellular matrix widely used in managing arthritis. HA's inherent viscoelastic property and ability to target CD44 have spurred interest in developing HA‐based systems for delivering drugs to manage arthritis. Hyaluronic acid nanoscaffolds (HA‐NSCfs) show improved outcomes over treatments, including enhanced drug uptake through receptor‐mediated targeting, prolonged retention in the synovium, reduced proinflammatory mediator expression, enhanced cartilage regeneration, reduced drug toxicity due to sustained release, and more cost‐effective treatments. This review highlights the rationale for using HA‐NSCfs for the management and advanced drug delivery systems in arthritis, emphasizing preclinical and clinical results. It discusses various solid‐lipid nanoparticles, hydroxyapatite nanoparticles, polyelectrolyte complexes, liposomes, bilosomes, drug‐polymer conjugates, hydrogels, inorganic nanoparticles, and polymer nanoparticles, etc reported for arthritis management. Additionally, the review identifies key challenges and offers suggestions to address them, ultimately highlighting the potential of HA‐NSCfs as a topical treatment for arthritis.

ANALYSIS OF EFFICACY OF COLLAGEN DRESSING VERSUS CONVENTIONAL DRESSING IN CHRONIC WOUND

A breach in continuity of the covering epithelium either of skin or of mucus membrane is defined as ulcer. Chronic or non-healing ulcers are defined as ulcers that are unresponsive to initial treatment or that persist for more than 3 weeks after appropriate care and management. Chronic nonhealing ulcers not only have a medical challenge but have social, economical and occupational effects on the patient. Ulcers have been treated conventionally by cleansing, debridement and removal of necrotic and infected tissue which was followed by dressing with topical antiseptic like Povidone iodine, antibiotic ointment, silver ointment, etc. Collagen is a group of naturally occurring proteins found in all phases of wound healing. Collagen is a key component of extracellular matrix which plays critical roles in the regulation of the phases of wound healing in its native fibrillar conformation or as a soluble component.Collagen helps wound healing through deposition and organisation of freshly formed fibres and granulation tissue in the wound bed which creates a good environment for wound healing.Collagen sheets when applied to a wound promote angiogenesis and also enhance bodyÂ’s repair mechanisms Our Aim is To compare the efficacy of the conventional dressing and collagen dressing in chronic wound in terms of Average time required for appearance of Granulation tissue, Requirement of Skin Grafting and mean Hospital Stay. Based on the type of dressing used, patients were divided into 2 groups, Group A(49) patients were dressed with conventional dressing, Group B patients (51) were dressed with collagen making a total of 100 patients. Appearance of healthy granulation tissue was observed to be significantly earlier among patients of collagen dressing group as compared to other group .The observed difference between number of days of hospital stay of patients with collagen dressing was statistically significant as compared to other group Thus Collagen based dressing were observed to be superior from conventional dressings in terms of aspects of healing viz. appearance of healthy granulation tissue.

Comprehensive evaluation of the efficacy and safety of a new multi-component anti-aging topical eye cream.

The delicate periorbital region is susceptible to skin dehydration, wrinkles, and loss of elasticity. Thus, targeted and effective anti-aging interventions are necessary for the periorbital area. To evaluate the efficacy and safety of a new anti-aging eye cream formulated with the active complex (Yeast/rice fermentation filtrate, N-acetylneuraminic acid, palmityl tripeptide-1, and palmitoyl tetrapeptide-7). The cell viability and expressions of key extracellular matrix (ECM) components of the active complex were evaluated using a human skin fibroblast model. In the 12-week clinical trial, skin hydration, elasticity, facial photographs, and collagen density following eye cream application were assessed using Corneometer, Cutometer, VISIA, and ultrasound device, respectively. Dermatologists and participants evaluated clinical efficacy and safety at baseline, and after 4, 8, and 12weeks. PCR and immunofluorescent analyses revealed that the active complex significantly stimulated fibroblast proliferation (p<0.05) and markedly promote the synthesis of collagen and elastin. Clinical findings exhibited a substantial enhancement in skin hydration (28.12%), elasticity (18.81%), and collagen production (54.99%) following 12weeks of eye cream application. Dermatological evaluations and participants' assessments reported a significant improvement in skin moisture, roughness, elasticity, as well as fine lines and wrinkles by week 8. The new anti-aging eye cream, enriched with the active complex, demonstrates comprehensive rejuvenating effects, effectively addressing aging concerns in the periorbital area, coupled with a high safety profile.

NFS-20. DECIPHERING CELLULAR CROSSTALK IN PERIPHERAL NERVOUS SYSTEM TUMORS ASSOCIATED WITH NEUROFIBROMATOSIS TYPE 1 THROUGH SINGLE-CELL RNA SEQUENCING

Abstract BACKGROUND Neurofibromatosis Type 1 (NF1) is a genetic disorder characterized by mutations and dysfunctional expression of neurofibromin 1 gene, a critical tumor-suppressor gene. NF1’s complex pathophysiology involves dysregulation of the RAS-MAPK signaling pathway, resulting in sustained MAPK signaling. Among others, the spectrum of clinical features includes low-grade tumors (e.g. plexiform neurofibromas (pNFs)) and malignant peripheral nerve sheath tumors (MPNST). While surgical removal of peripheral nerve tumors is common, total resection is often unattainable. Additionally, approved therapies comprising MEK inhibitors fail to prevent malignization, highlighting the urgent need for novel targets for effective therapies. METHODS To explore novel potential targets across a broad spectrum, we analyzed published scRNA-seq datasets (Kershner et al. 2021, Wu et al. 2022) from human patients, including pNFs and MPNSTs. We further investigated key pathways in vitro with patient-derived cell culture models. RESULTS ScRNA-seq provides insight into complex networks of cell-to-cell interactions that drive the malignant phenotype of cancerous tissues. Due to the rich presence of ECM in NF1 tumors we aimed to investigate cell types contributing to key extracellular matrix (ECM) components and associated signaling pathways. Analysis of 19 NF1 tumor samples revealed consistent presence of Schwann cells (SCs), fibroblasts, endothelial cells, and immune cells. Proliferating cells were found in MPNSTs, while pericytes were exclusive to pNFs. We compared secreted and ECM signaling between the cell types of both tumors. Our analysis discovered, among other findings, collagen signaling, highlighting fibroblasts as emitters and SCs as receivers. We further confirmed these findings through culturing of NF1-derived cells in vitro with and without collagen, observing increased cell proliferation in the presence of collagen. Further experiments and analyses are ongoing. CONCLUSIONS The emphasis on ECM and signaling adds depth to our understanding of NF1 pathogenesis and suggests potential therapeutic targets.

Extracellular Matrix Mimicking Wound Microenvironment Responsive Amyloid-Heparin@TAAgNP Co-Assembled Hydrogel: An Effective Conductive Antibacterial Wound Healing Material.

Bioengineered composite hydrogel platforms made of a supramolecular coassembly have recently garnered significant attention as promising biomaterial-based healthcare therapeutics. The mechanical durability of amyloids, in conjunction with the structured charged framework rendered by biologically abundant key ECM component glycosaminoglycan, enables us to design minimalistic customized biomaterial suited for stimuli responsive therapy. In this study, by harnessing the heparin sulfate-binding aptitude of amyloid fibrils, we have constructed a pH-responsive extracellular matrix (ECM) mimicking hydrogel matrix. This effective biocompatible platform comprising heparin sulfate-amyloid coassembled hydrogel embedded with polyphenol functionalized silver nanoparticles not only provide a native skin ECM-like conductive environment but also provide wound-microenvironment responsive on-demand superior antibacterial efficacy for effective diabetic wound healing. Interestingly, both the cytocompatibility and antibacterial properties of this bioinspired matrix can be fine-tuned by controlling the mutual ratio of heparin sulfate-amyloid and incubated silver nanoparticle components, respectively. The designed biomaterial platform exhibits notable effectiveness in the treatment of chronic hyperglycemic wounds infected with multidrug-resistant bacteria, because of the integration of pH-responsive release characteristics of the incubated functionalized AgNP and the antibacterial amyloid fibrils. In addition to this, the aforementioned assemblage shows exceptional hemocompatibility with significant antibiofilm and antioxidant characteristics. Histological evidence of the incised skin tissue sections indicates that the fabricated composite hydrogel is also effective in controlling pro-inflammatory cytokines such as IL6 and TNFα expressions at the wound vicinity with significant upregulation of angiogenesis markers like CD31 and α-SMA.

Essential growth factor receptors for fibroblast homeostasis and activation: Fibroblast Growth Factor Receptor (FGFR), Platelet Derived Growth Factor Receptor (PDGFR), and Transforming Growth Factor β Receptor (TGFβR).

Fibroblasts are cells of mesenchymal origin that are found throughout the body. While these cells have several functions, their integral roles include maintaining tissue architecture through the production of key extracellular matrix components, and participation in wound healing after injury. Fibroblasts are also key mediators in disease progression during fibrosis, cancer, and other inflammatory diseases. Under these perturbed states, fibroblasts can activate into inflammatory fibroblasts or contractile myofibroblasts. Fibroblasts require various growth factors and mitogenic molecules for survival, proliferation, and differentiation. While the activity of mitogenic growth factors on fibroblasts in vitro was characterized as early as the 1970s, the proliferation and differentiation effects of growth factors on these cells in vivo are unclear. Recent work exploring the heterogeneity of fibroblasts raises questions as to whether all fibroblast cell states exhibit the same growth factor requirements. Here, we will examine and review existing studies on the influence of fibroblast growth factor receptors (FGFRs), platelet-derived growth factor receptors (PDGFRs), and transforming growth factor β receptor (TGFβR) on fibroblast cell states.

O10 Tuneable Caf1 protein polymers as an innovative and scalable solution for regenerative wound care

Abstract Introduction and aims Wound healing is a complex process essential for tissue restoration, where the extracellular matrix (ECM) plays a crucial role by providing structural and biochemical support to cells. Laminin 332, a key ECM component, is a promising target for protein-based therapeutics in view of its pathogenic role in junctional epidermolysis bullosa (JEB). Bioengineered capsular antigen F1 protein (Caf1) has emerged as a potential candidate molecule. We have developed the technology to enable bacteria to produce Caf1 polymers with multiple tailored bioactive motifs. Additionally, the unique properties of Caf1 minimize the potential for a host immune response. In this study, we engineered Caf1 proteins to express specific synthetic basement membrane components related to laminin 332 with the aim of restoring ECM equilibrium and achieving effective wound healing. Methods To evaluate Caf1 construct effects on human epidermal keratinocytes (HEKs) (CCD 1106) and human dermal fibroblasts (HDFs), scratch assays were performed under different treatment with Caf constructs. The Incucyte® ZOOM system (Sartorius AG, Göttingen, Germany) automated scratch wound and Live-Cell Analysis System screened constructs enhancing wound healing, capturing images every 2 h. Analysis involved quantifying the area under the curve of wound confluence (%) over 72 h to assess wound closure. Results The migration of HDFs was enhanced by 10.4% ± 0.95% (P &amp;lt; 0.05 Anova, n = 9 independent experiments) when there was interaction with Caf1-CTB4 (PMQKMRGDVFSP). The migration of CCDs was enhanced by 12.7% ± 1.03% when interacting with Caf1-J3 (KNSFMALYLSKGR) and by 14.4% ± 1.11% when interacting with Caf1-J4 (TALIRATYGEYSTGYI) (P &amp;lt; 0.05 Anova; n = 3 independent experiments). Combining Caf1-J3 and Caf1-J4 synergistically boosts CCD migration by 19.1% ± 1.42% (P &amp;lt; 0.05 Anova; n = 3 independent experiments), yielding better Results compared with individual treatments. Conclusions Caf1 exhibits promise as a platform for motif delivery, and the incorporation of cell migration motifs into Caf1 holds the potential to enhance wound healing.

Biomimetic design of fibril-forming non-immunogenic collagen like proteins for tissue engineering

Collagen, a key component of extracellular matrix serves as a linchpin for maintaining structural integrity and functional resilience. Concerns over purity and immunogenicity of animal-derived collagens have spurred efforts to develop synthetic collagen-based biomaterials. Despite several collagen mimics, there remains limited exploration of non-immunogenic biomaterials with the capacity for effective self-assembly. To combat the lacuna, collagen like protein (CLP) variants were rationally designed and recombinantly expressed, incorporating human telopeptide sequences (CLP-N and CLP-NC) and bioactive binding sites (CLP-NB). Circular dichroism analyses of the variants confirmed the triple helical conformation, with variations in thermal stability and conformation attributed to the presence of telopeptides at one or both ends of CLP. The variants had propensity to form oligomers, setting the stage for fibrillogenesis. The CLP variants were biocompatible, hemocompatible and supported cell proliferation and migration, particularly CLP-NB with integrin-binding sites. Gene expression indicated a lack of significant upregulation of inflammatory markers, highlighting the non-immunogenic nature of these variants. Lyophilized CLP scaffolds maintained their triple-helical structure and offered favorable biomaterial characteristics. These results accentuate the potential of designed CLP variants in tissue engineering, regenerative medicine and industrial sectors, supporting the development of biocompatible scaffolds and implants for therapeutic and cosmetic purposes.

The Role of Hyaluronan/Receptor for Hyaluronan-Mediated Motility Interactions in the Modulation of Macrophage Polarization and Cartilage Repair

Hyaluronan (HA), a negatively charged linear glycosaminoglycan, is a key macromolecular component of the articular cartilage extracellular matrix. The differential effects of HA are determined by a spatially/temporally regulated display of HA receptors, such as CD44 and Receptor for Hyaluronan Mediated Motility (RHAMM). HA signaling through CD44 with RHAMM has been shown to stimulate inflammation and fibrotic processes. This study shows an increased expression of RHAMM in pro-inflammatory macrophages. Interfering with HA/RHAMM interactions using a 15mer RHAMM-mimetic, HA binding peptide (P15-1) together with high molecular weight (HMW)HA reduced the expression and release of inflammatory markers and increased the expression of anti-inflammatory markers in pro-inflammatory macrophages. Interfering with HA/RHAMM interactions in vivo during the regeneration of a full thickness cartilage defect after microfracture surgery in rabbits using three intra-articular injections of P15-1 together with HMWHA reduced the number of pro-inflammatory macrophages and increased the number of anti-inflammatory macrophages in the injured knee joint and greatly improved the repair of the cartilage defect compared to intra-articular injections of HMWHA alone. These findings suggest that HA/RHAMM interactions play a key role in cartilage repair/regeneration via stimulating inflammatory and fibrotic events, including increasing the ratio of pro-inflammatory to anti-inflammatory macrophages. Interfering with these interactions reduced inflammation and greatly improved cartilage repair.

Essential growth factor receptors for fibroblast homeostasis and activation

Fibroblasts are cells of mesenchymal origin that are found throughout the body. While these cells have several functions, their integral roles include maintaining tissue architecture through the production of key extracellular matrix components, and participation in wound healing after injury. Fibroblasts are also key mediators in disease progression during fibrosis, cancer, and other inflammatory diseases. Under these perturbed states, fibroblasts can activate into inflammatory fibroblasts or contractile myofibroblasts. Fibroblasts require various growth factors and mitogenic molecules for survival, proliferation, and differentiation. While the activity of mitogenic growth factors on fibroblasts in vitro was characterized as early as the 1970s, the proliferation and differentiation effects of growth factors on these cells in vivo are unclear. Moreover, recent work exploring the heterogeneity of fibroblasts raises questions as to whether all fibroblast cell states exhibit the same growth factor requirements. Here, we will examine and review existing growth factors known to influence fibroblast homeostasis to begin unpacking the potential growth factors that may influence in vivo fibroblast cell states.

The role of human extracellular matrix proteins in defining Staphylococcus aureus biofilm infections.

Twenty to forty one percent of the world's population is either transiently or permanently colonized by the Gram-positive bacterium, Staphylococcus aureus. In 2017, the CDC designated methicillin-resistant S. aureus (MRSA) as a serious threat, reporting ∼300 000 cases of MRSA-associated hospitalizations annually, resulting in over 19 000 deaths, surpassing that of HIV in the USA. S. aureus is a proficient biofilm-forming organism that rapidly acquires resistance to antibiotics, most commonly methicillin (MRSA). This review focuses on a large group of (>30) S. aureus adhesins, either surface-associated or secreted that are designed to specifically bind to 15 or more of the proteins that form key components of the human extracellular matrix (hECM). Importantly, this includes hECM proteins that are pivotal to the homeostasis of almost every tissue environment [collagen (skin), proteoglycans (lung), hemoglobin (blood), elastin, laminin, fibrinogen, fibronectin, and fibrin (multiple organs)]. These adhesins offer S. aureus the potential to establish an infection in every sterile tissue niche. These infections often endure repeated immune onslaught, developing into chronic, biofilm-associated conditions that are tolerant to ∼1000 times the clinically prescribed dose of antibiotics. Depending on the infection and the immune response, this allows S. aureus to seamlessly transition from colonizer to pathogen by subtly manipulating the host against itself while providing the time and stealth that it requires to establish and persist as a biofilm. This is a comprehensive discussion of the interaction between S. aureus biofilms and the hECM. We provide particular focus on the role of these interactions in pathogenesis and, consequently, the clinical implications for the prevention and treatment of S. aureus biofilm infections.

DEVELOPMENT OF PHOTO-CROSSLINKABLE DECELLULARIZED EXTRACELLULAR MATRIX HYDROGELS FOR CARTILAGE TISSUE ENGINEERING

Cartilage lacks the ability to self-repair when damaged, which can lead to the development of degenerative joint disease. Despite intensive research in the field of cartilage tissue engineering, there is still no regenerative treatment that consistently promotes the development of hyaline cartilage. Extracellular matrix (ECM) derived hydrogels have shown to support cell adhesion, growth and differentiation [1,2]. In this study, porcine articular cartilage was decellularized, solubilised and subsequently modified into a photo-crosslinkable methacrylated cartilage ECM hydrogel. Bone marrow derived mesenchymal stem/stromal cells (MSCs) were encapsulated into both methacrylated ECM hydrogels (ECM-MA) and gelatin methacryloyl (GelMA) as control hydrogel, and their chondrogenic potential was assessed using biochemical assays and histological analysis. We found that successful decellularization of the cartilage tissue could be achieved while preserving key ECM components, including collagen and glycosaminoglycans. A live-dead assay demonstrated good viability of MSCs withing both GelMA and ECM-MA hydrogels on day 7. Large increases in sGAG accumulation was observed after 21 days of culture in chondrogenic media in both groups. Histological analysis revealed the presence of a more fibrocartilage tissue in the GelMA group, while cells embedded within the ECM-MA showed a round and chondrocytic-like morphology. Both groups stained positively for proteoglycans and collagen, with limited evidence of calcium deposition following Alizarin Red staining. These results show that ECM-MA hydrogels support a hyaline cartilage phenotype and robust cartilaginous matrix production. Future studies will focus on the printability of ECM-MA hydrogels to enable their use as bioinks for the biofabrication of functional tissues.