Introduction:Wooden Breast (WB) is a myopathy affecting the skeletal breast muscle (Pectoralis major) in broiler chickens and is characterized by muscle fiber damage and varying degrees of fibrosis, ECM remodeling and inflammation. Several key factors such as pro-inflammatory cytokines like TGF-β1 and IL-1β, drive fibrosis in WB myopathy. We have previously shown that the expression of syndecan-4 (SDC4), a transmembrane proteoglycan, was increased in WB poultry skeletal muscle tissue. Furthermore, the ectodomain shedding of SDC4 by matrix metalloproteinases (MMPs) differed in the skeletal muscle satellite cells from isolated affected chickens compared with normal. While SDC4 has been previously implicated as a key driver for regulating myofibroblast activity in mechanically induced fibrosis in cardiac tissue, its specific role and shedding activity in chicken fibroblasts in relation to WB myopathy remain poorly understood. Methods:In vitro the overexpression system was used to mimic the previously detected increased SDC4 levels in WB and to further investigate fibrotic markers and syndecans at the gene and protein levels. Furthermore, we used blocking peptides derived from the SDC4 ectodomain and investigated their effect on SDC4 shedding and fibrotic markers. Additionally, TGF-β1 treatment, the main trigger of myofibroblasts, fibrosis, and cytokines, was used to investigate the connection between SDC4 shedding and fibrosis. Results and discussion:Overexpression of SDC4 in chicken fibroblasts reduced the gene and protein expression of fibrotic markers such as collagen I, collagen III and MMP-2. At the same time, we observed an increase in the gene expression of TGFB1 and IL1B. SDC4 overexpression also modulated intracellular proteins connected to fibrosis-relevant signaling pathways, with increased phosphorylation of p38 MAPK and decreased phosphorylation of Akt. Moreover, we could observe a decreased production of ribosomal protein S6 and β-catenin. SDC4 overexpression induced shedding of a 15 kDa SDC4 fragment, while a 20 kDa fragment was produced at similar level regardless of overexpression. Modulation of ectodomain shedding using blocking peptides targeting various ectodomain regions (amino acids 76-106 and 100-130) significantly reduced the production of the 20 kDa endogenous fragment. However, it did not affect the expression level of the 15 kDa fragment. This contrasts with what we have seen with the same blocking peptides in chicken skeletal muscle satellite cells where the expression level of the 15 kDa fragment was reduced. When we stimulated the cells with TGF-β1, an increase in gene expression of fibrotic marker was observed as expected. No change in SDC4 gene expression was observed, while SDC4 fragment of 20 kDa was increased. Taken together, our results reveal a complex role of SDC4 and shedding in modulating fibrotic responses in chicken fibroblasts, suggesting a potential dual function where the full-length SDC4, produced by overexpression, may act as an anti-fibrotic regulator, while the TGF-β1 induced shed ectodomain fragments could promote pro-fibrotic and inflammatory processes.

IntroductionWooden breast disease is a myopathy of the skeletal muscle in chickens of commercial breeding. Although the underlying pathophysiology remains unknown, we and others have previously shown that affected broilers display varying degrees of fibrosis, extracellular matrix (ECM) remodeling, inflammation, and alterations in various molecular signaling pathways. Other myopathy conditions, such as Duchenne muscular dystrophy, also affect the cardiac muscle and are associated with fibrosis and reduced cardiac function. To determine potential cardiac implications of wooden breast disease and identify whether molecular and fibrotic changes were similar to what we have previously found in the breast, we have investigated the hearts of commercial Ross 308 broilers.MethodsHearts from male Ross 308 broiler chickens from mildly and severely wooden breast-affected chickens categorized in previous studies were analyzed. Ventricles from the hearts were analyzed by immunoblotting, real-time qPCR, near-infrared spectroscopy, Raman spectroscopy, and Masson`s trichrome histology. RNA sequencing was also conducted to identify the molecular footprint of the mildly and severely wooden breast-affected chickens.ResultsCompared to mildly affected chickens, the severely wooden breast-affected chickens did not show an increase in heart weight, water-binding capacity, or macronutrient composition. The hearts did also not display any differences in fibrosis development, extracellular matrix gene expression, or typical cardiac and inflammatory markers. The severely affected chickens did, however, show a reduction in protein levels of biglycan and fibromodulin, as well as alterations in matrix metalloproteinase 2, Wnt ligands, mTOR signaling, heat shock protein 70, and muscle LIM protein. Functional enrichment analysis of RNA sequencing also suggested a different molecular footprint of biological processes and pathways between the two groups.ConclusionHearts from wooden breast-affected chickens did not display the same fibrotic alterations as those previously found in the breast. Despite few alterations detected in the markers and signaling molecules tested, RNA sequencing indicated a different molecular footprint in the hearts of severely compared to mildly wooden breast-affected chickens.

Immune-mediated inflammatory disease (IMID) is a major public health issue which can affect a number of organs and tissues with a profound impact on quality of life and often present with comorbidities. Psoriasis is a chronic IMID affecting the skin which presents with both local and systemic inflammation as part of its pathophysiology. An oil rich in polar lipids extracted from the roe sacks of herring has been shown to have immunomodulatory functions and to improve the clinical symptoms and impact inflammatory cytokine pathways in psoriasis in a clinical trial. The lipidic nature of herring roe oil and its high content of marine polyunsaturated fatty acids could suggest involvement of lipid mediator pathways for the observed alleviation of psoriatic inflammation. Of particular interest is the super-family of lipid mediators termed specialized pro-resolving mediators (SPMs), due to their known involvement in the resolution of inflammation and return to homeostasis. We have therefore explored the influence of herring roe oil and phospholipid esters from herring roe on lipid mediator and SPM biosynthesis in IFN-γ and LPS-stimulated human monocyte-derived macrophages and an IL-17A-stimulated skin cell co-culture with keratinocytes and fibroblasts. Lipid mediators including SPMs were quantified from resulting cell supernatants using a validated LC-MS/MS protocol. In these experiments we observed broad SPM biosynthesis with dominant upregulation of RvE2 and RvE3 in both cell systems and upregulation of DHA-derived SPMs such as RvD2 and PDX. Observations of PCTR2 in the macrophage cell supernatants also indicate activation of reparative pathways upon treatment with herring roe oil. In conclusion, we observed a promotion of SPM biosynthesis associated with a shift towards a protective and possibly reparative macrophage phenotype as well as promotion of biosynthesis of pro-resolving lipid mediators in a skin cell co-culture, thus demonstrating a possible mechanism for resolution of inflammation in the skin niche using herring roe oil. ### Competing Interest Statement Thomas A. Ringheim-Bakka, Maftuna Busygina, and Runhild Gammelsæter are employed by Arctic Bioscience AS. The authors declare that all experiments performed were partially funded by Arctic Bioscience AS. Jesmond Dalli is an inventor on patents related to the composition of matter and/or use of pro-resolving mediators some of which are licensed by Brigham and Women's Hospital or Queen Mary University of London for clinical development.

Background Several systemic treatment options are available for moderate to severe psoriasis, while low-risk oral treatments for milder forms are limited. Herring roe oil (HRO) has previously shown beneficial effects on the clinical presentation of psoriasis, however, cell-specific effects remained unknown. Objective This study aimed to elucidate cellular effects of HRO and whether signaling pathways in skin and immune cells relevant to the pathophysiology of psoriasis are affected by HRO. Methods We used cell-culture models of human primary macrophages and T-cells, the keratinocyte cell line HaCat and human primary fibroblasts to represent important site-specific pathological aspects of psoriasis in the skin and immune system. The cells were supplemented with HRO emulsions and cellular signaling was analyzed on protein and mRNA level. Skin cell proliferation was analyzed in real-time by a Live-Cell Analysis System. Conclusion We here present results which suggest that HRO exerts effects on different cell types on major psoriasis-driving aspects, reducing the secretion of IL-23 and IL-17 from macrophages and T-cells, respectively, as well as interfering with IL-17-induced signaling and proliferation of keratinocytes in co-culture with fibroblasts. [Clinicaltrials.gov][1] (or equivalent) listing (if applicable): not applicable Key Points ### Competing Interest Statement F. Petrucelli and R. Gammelsaeter were employed by Arctic Bioscience AS. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. * ALA : Alpha linoleic acid AMPs : Antimicrobial peptides CXCL10 : C-X-C motif chemokine ligand 10 DC : Dendritic cell DHA : Docosahexaenoic acid ELISA : Enzyme-linked immunosorbent assay EPA : Eicosapentaenoic acid HRO : Herring roe oil IFN : Interferon IκBζ : NF-kB inhibitor zeta (protein) IL : Interleukin IMID : Immune-mediated inflammatory disease IRF : Interferon regulatory factor LPS : Lipopolysaccharide A M-CSF : Macrophage colony-stimulating factor MDM : Monocyte-derived macrophages N3-PUFA : Omega-3 polyunsaturated fatty acid NF-kB : Nuclear factor kappa B NFKBIZ : NF-kappa-B inhibitor zeta PBMC : Peripheral blood mononuclear cells PGE2 : Prostaglandin E2 RvD1 : Resolvin D1 S100A7 : S100 calcium-binding protein A7 (psoriasin) SEAP : Secreted embryonic alkaline phosphatase SPM : Specialized pro-resolving mediator TNF-α : Tumor necrosis factor alpha TW : Transwell Qrt-PCR : Quantitative real-time PCR The Research Council of Norway, , 327953, 35147633 Norwegian Fund for Research Fees for Agricultural Products (FFL), , 354160 [1]: http://Clinicaltrials.gov

Cellular Agriculture (CellAg) represents a groundbreaking approach to food production that can potentially transform various aspects of our agricultural practices significantly. This encompasses agricultural output, land utilization, ownership dynamics, policy-making, dietary patterns, and, notably, ethical inquiries. This chapter delves into two primary categories of CellAg food production systems, focusing on their technological intricacies and the ethical considerations they prompt. The emergence of CellAg heralds a paradigm shift in food production, holding the capacity to reshape fundamental elements of agriculture, encompassing production levels, land management, ownership structures, policy formulation, eating behaviors, and particularly, ethical deliberations. Nonetheless, the field of Cellular Agriculture is still in its nascent stages. A substantial portion of pioneering innovations and advancements remains veiled within the confines of private enterprises. Nevertheless, through government funding and an escalating commitment to public research, the trajectory of CellAg is poised to progress toward heightened transparency, trustworthiness, and openness. This concerted effort will unveil challenges and facilitate exploring strategies to address them.

IntroductionSkeletal muscle satellite cells (MuSCs or stem cells) play a crucial role in muscle development, maintenance, and regeneration, supporting both hypertrophy and regenerative myogenesis. Syndecans (SDCs) act as communication bridges within the muscle microenvironment, regulating interactions with extracellular matrix components and contributing significantly to tissue repair and inflammation. Specifically, syndecan-4 (SDC4) is involved in muscle regeneration at multiple stages.MethodsThis study delves into the emerging challenge of wooden breast (WB) myopathy and its connection with SDC4. Our hypothesis proposes that disruptions in MuSC dynamics through SDC4 contribute to the increased incidence of breast myopathies observed in growing broilers. To test our hypothesis, non-affected and affected broilers were systematically selected, and the characteristics of WB myopathy were studied both in vitro and in vivo. SDC4 overexpression in MuSCs and blocking peptides (BPs) corresponding to the SDC4 ectodomain were used for investigating the role of SDC4 in muscle development and its shedding levels.Results and discussionIn vivo examination of affected muscles revealed smaller fibers and changes in metabolic pathways. In vitro studies unveiled disrupted proliferation of MuSCs in WB myopathy, accompanied by the downregulation of several muscle markers. Investigation of the potential role of SDC4 in the pathogenesis of WB myopathy revealed a decreased tendency in SDC4 gene expression and increased shedding of its ectodomain. Moreover, we showed that SDC4 overexpression is linked to reduced proliferation in MuSCs and affected myogenesis. We detected an impaired proliferation of WB-affected MuSCs, revealing critical insights into the dysfunctional state of these cells in myopathy. Additionally, by treating MuSCs with blocking peptides derived from the SDC4 ectodomain, we identified altered proliferation. Taken together, this work contributes with valuable knowledge on the molecular mechanisms underlying WB myopathy and the role of SDC4 in this chicken myopathy.

IntroductionAvian eggshell membrane (ESM) is a complex extracellular matrix comprising collagens, glycoproteins, proteoglycans, and hyaluronic acid. We have previously demonstrated that ESM possesses anti-inflammatory properties in vitro and regulates wound healing processes in vivo. The present study aimed to investigate if oral intake of micronized ESM could attenuate skeletal muscle aging associated with beneficial alterations in gut microbiota profile and reduced inflammation.MethodsElderly male C57BL/6 mice were fed an AIN93G diet supplemented with 0, 0.1, 1, or 8% ESM. Young mice were used as reference. The digestibility of ESM was investigated using the static in vitro digestion model INFOGEST for older people and adults, and the gut microbiota profile was analyzed in mice. In addition, we performed a small-scale pre-clinical human study with healthy home-dwelling elderly (>70 years) who received capsules with a placebo or 500 mg ESM every day for 4 weeks and studied the effect on circulating inflammatory markers.Results and discussionIntake of ESM in elderly mice impacted and attenuated several well-known hallmarks of aging, such as a reduction in the number of skeletal muscle fibers, the appearance of centronucleated fibers, a decrease in type IIa/IIx fiber type proportion, reduced gene expression of satellite cell markers Sdc3 and Pax7 and increased gene expression of the muscle atrophy marker Fbxo32. Similarly, a transition toward the phenotypic characteristics of young mice was observed for several proteins involved in cellular processes and metabolism. The digestibility of ESM was poor, especially for the elderly condition. Furthermore, our experiments showed that mice fed with 8% ESM had increased gut microbiota diversity and altered microbiota composition compared with the other groups. ESM in the diet also lowered the expression of the inflammation marker TNFA in mice and in vitro in THP-1 macrophages. In the human study, intake of ESM capsules significantly reduced the inflammatory marker CRP. Altogether, our results suggest that ESM, a natural extracellular biomaterial, may be attractive as a nutraceutical candidate with a possible effect on skeletal muscle aging possibly through its immunomodulating effect or gut microbiota.

IntroductionSuccessful long-term expansion of skeletal muscle satellite cells (MuSCs) on a large scale is fundamental for cultivating animal cells for protein production. Prerequisites for efficient cell expansion include maintaining essential native cell activities such as cell adhesion, migration, proliferation, and differentiation while ensuring consistent reproducibility.MethodThis study investigated the growth of bovine MuSC culture using low-volume spinner flasks and a benchtop stirred-tank bioreactor (STR).Results and discussionOur results showed for the first time the expansion of primary MuSCs for 38 days in a bench-top STR run with low initial seeding density and FBS reduction, supported by increased expression of the satellite cell marker PAX7 and reduced expression of differentiation-inducing genes like MYOG, even without adding p38-MAPK inhibitors. Moreover, the cells retained their ability to proliferate, migrate, and differentiate after enzymatic dissociation from the microcarriers. We also showed reproducible results in a separate biological benchtop STR run.

Introduction: The skeletal muscle deformity of commercial chickens (Gallus gallus), known as the wooden breast (WB), is associated with fibrotic myopathy of unknown etiology. For future breeding strategies and genetic improvements, it is essential to identify the molecular mechanisms underlying the phenotype. The pathophysiological hallmarks of WB include severe skeletal muscle fibrosis, inflammation, myofiber necrosis, and multifocal degeneration of muscle tissue. The transmembrane proteoglycans syndecans have a wide spectrum of biological functions and are master regulators of tissue homeostasis. They are upregulated and shed (cleaved) as a regulatory mechanism during tissue repair and regeneration. During the last decades, it has become clear that the syndecan family also has critical functions in skeletal muscle growth, however, their potential involvement in WB pathogenesis is unknown.Methods: In this study, we have categorized four groups of WB myopathy in broiler chickens and performed a comprehensive characterization of the molecular and histological profiles of two of them, with a special focus on the role of the syndecans and remodeling of the extracellular matrix (ECM).Results and discussion: Our findings reveal differential expression and shedding of the four syndecan family members and increased matrix metalloproteinase activity. Additionally, we identified alterations in key signaling pathways such as MAPK, AKT, and Wnt. Our work provides novel insights into a deeper understanding of WB pathogenesis and suggests potential therapeutic targets for this condition.

EDITORIAL article Front. Cell Dev. Biol., 13 March 2023Sec. Cell Growth and Division Volume 11 - 2023 | https://doi.org/10.3389/fcell.2023.1139044

This study was conducted to evaluate the effect of adding eggshell membrane (ESM), a by-product of the chicken egg processing industry, to emulsified meat models with different NaCl concentrations. The aim was to see if ESM could help reduce the amount of NaCl that is usually added to this type of product. The effects of ESM were investigated in terms of cooking loss, water distribution, color, and texture properties using a simplified meat emulsion model with no other additives than ESM. Emulsified meat samples were made with three different NaCl concentration (0.5, 0.1, 1.5%), without and with three levels of ESM (0.5, 1.0, 1.5%). In general addition of ESM reduced cooking loss (CL), improved texture, and increased redness. Effects of ESM were explained by structural changes in the protein matrix, as shown by two different histological methods. Addition of ESM explained 86% of the variation in LF-NMR T2 relaxation times in uncooked samples, while 97% of the variation in cooked samples was explained by the NaCl concentration. FTIR micro-spectroscopic measurements revealed that samples supplemented with ESM had a higher proportion of α-helical structures and reduced amount of protein ß-sheet aggregation in samples with 1.0 and 1.5% NaCl. It was shown that ESM increased the pH of the emulsified meat, and it was therefore suggested that increased negative repulsion effects had a positive heat stabilizing effect on the protein network. The fact that the cooked samples were redder was probably related to the antioxidant effect of ESM which was measured as MDA (malondialdehyde) equivalents after in vitro digestion of the samples. ESM can thus help reduce the salt content in sausages while ESM also has a positive antioxidant effect improving color.

A major challenge for successful cultured meat production is the requirement for large quantities of skeletal muscle satellite cells (MuSCs). Commercial microcarriers (MCs), such as Cytodex®1, enable extensive cell expansion by offering a large surface-to-volume ratio. However, the cell-dissociation step post cell expansion makes the cell expansion less efficient. A solution is using food-grade MCs made of sustainable raw materials that do not require a dissociation step and can be included in the final meat product. This study aimed to produce food-grade MCs from food industry by-products (i.e., turkey collagen and eggshell membrane) and testing their ability to expand bovine MuSCs in spinner flask systems for eight days. The MCs' physical properties were characterized, followed by analyzing the cell adhesion, growth, and metabolic activity. All MCs had an interconnected porous structure. Hybrid MCs composed of eggshell membrane and collagen increased the mechanical hardness and stabilized the buoyancy compared to pure collagen MCs. The MuSCs successively attached and covered the entire surface of all MCs while expressing high cell proliferation, metabolic activity, and low cell cytotoxicity. Cytodex®1 MCs were included in the study. Relative gene expression of skeletal muscle markers showed reduced PAX7 and increased MYF5, which together with augmented proliferation marker MKI67 indicated activated and proliferating MuSCs on all MCs. Furthermore, the expression pattern of cell adhesion receptors (ITGb5 and SDC4) and focal adhesion marker VCL varied between the distinct MCs, indicating different specific cell receptor interactions with the various biomaterials. Altogether, our results demonstrate that these biomaterials are promising prospects to produce custom-fabricated food-grade MCs intended to expand MuSCs.

Collagen is extensively used in fabrication of hydrogels for biomedical applications but needs improvement after its isolation from tissues due to slow gelation and poor mechanical properties. Crosslinking could tailor such properties. Collagen has previously been crosslinked by chemical or photochemical methods. Chemical crosslinkers are often toxic, and the crosslinking reaction is difficult to control. Photochemical crosslinkers are usually biocompatible compounds that are activated upon irradiation. Riboflavin (vitamin B2), a photochemical crosslinker of collagen, photodegrades to lumichrome upon irradiation. Cyclodextrins have previously been used to increase the aqueous solubility of lumichrome and regulate collagen self-assembly. In this study, lumichrome dissolved by cyclodextrin complexation was used as a photochemical crosslinker of collagen. Lumichrome photocrosslinking reduced the gelation time to 10 s, compared to 90 min for physical crosslinking. The formed hydrogels exhibited increased elasticity, water absorption properties and water holding capacity compared to physically crosslinked collagen hydrogels and riboflavin photocrosslinked collagen hydrogels. Fibroblasts achieved a myofibroblastic phenotype when cultivated in 2D on lumichrome photocrosslinked gels as observed from histology. These biocompatible photocrosslinked hydrogels could have potential applications in biomedical applications, such as wound healing.

Recently, two chicken breast fillet abnormalities, termed Wooden Breast (WB) and Spaghetti Meat (SM), have become a challenge for the chicken meat industry. The two abnormalities share some overlapping morphological features, including myofiber necrosis, intramuscular fat deposition, and collagen fibrosis, but display very different textural properties. WB has a hard, rigid surface, while the SM has a soft and stringy surface. Connective tissue is affected in both WB and SM, and accordingly, this study’s objective was to investigate the major component of connective tissue, collagen. The collagen structure was compared with normal (NO) fillets using histological methods and Fourier transform infrared (FTIR) microspectroscopy and imaging. The histology analysis demonstrated an increase in the amount of connective tissue in the chicken abnormalities, particularly in the perimysium. The WB displayed a mixture of thin and thick collagen fibers, whereas the collagen fibers in SM were thinner, fewer, and shorter. For both, the collagen fibers were oriented in multiple directions. The FTIR data showed that WB contained more β-sheets than the NO and the SM fillets, whereas SM fillets expressed the lowest mature collagen fibers. This insight into the molecular changes can help to explain the underlying causes of the abnormalities.

The most significant cost driver for efficient bio-production of edible animal proteins is the cell culture media, where growth factors account for up to 96% of the total cost. The culture media must be serum-free, affordable, contain only food-grade ingredients, be efficient to promote cell growth and available in massive quantities. The commercially available serum substitutes are expensive and not necessarily food-grade. Identifying inexpensive food-safe alternatives to serum is crucial. By-products from food production are available in massive quantities, contain potential factors that can promote growth and are promising ingredients for serum replacement. The main goal of this study was to explore if food-grade by-product materials can be used as growth promoting agents in skeletal muscle cell culture to develop a tailor-made serum free media. Different by-products, including chicken carcass, cod backbone, eggshell membrane, egg white powder and pork plasma were enzymatically or chemically hydrolyzed. The hydrolysates in addition to lyophilized pork plasma and yeast extract were further characterized by size-exclusion chromatography, elemental combustion analysis and degree of hydrolysis. The materials were used as supplement to or replacement of commercial serum and further evaluated for their effect on metabolic activity, cell proliferation and cell cytotoxicity in muscle cells cultured in vitro. Our results indicate that none of the materials were cytotoxic to the skeletal muscle cells. Hydrolysates rich in peptides with approximately 2-15 amino acids in length were shown to improve cell growth and metabolic activity. Of all the materials tested pork plasma hydrolysates and yeast extract were the most promising. Pork plasma hydrolysates increased metabolic activity by 110% and cell proliferation with 48% when cultured in serum-free conditions for 3 days compared with control cells cultured with full serum conditions. Most interestingly, this response was dependent on both material and choice of enzyme used. We suggest that these materials have the potential to replace serum during cultivation and as such be included in a tailor-made serum-free media.

The purpose of this study was to investigate the tissue regenerating and biomechanical properties of processed eggshell membrane powder (PEP) for use in 3D-scaffolds. PEP is a low-cost, natural biomaterial with beneficial bioactive properties. Most importantly, this material is available as a by-product of the chicken egg processing (breaking) industry on a large scale, and it could have potential as a low-cost ingredient for therapeutic scaffolds. Scaffolds consisting of collagen alone and collagen combined with PEP were produced and analyzed for their mechanical properties and the growth of primary fibroblasts and skeletal muscle cells. Mechanical testing revealed that a PEP/collagen-based scaffold increased the mechanical hardness of the scaffold compared with a pure collagen scaffold. Scanning electron microscopy (SEM) demonstrated an interconnected porous structure for both scaffolds, and that the PEP was evenly distributed in dense clusters within the scaffold. Fibroblast and skeletal muscle cells attached, were viable and able to proliferate for 1 and 2 weeks in both scaffolds. The cell types retained their phenotypic properties expressing phenotype markers of fibroblasts (TE7, alpha-smooth muscle actin) and skeletal muscle (CD56) visualized by immunostaining. mRNA expression of the skeletal muscle markers myoD, myogenin, and fibroblasts marker (SMA) together with extracellular matrix components supported viable phenotypes and matrix-producing cells in both types of scaffolds. In conclusion, PEP is a promising low-cost, natural biomaterial for use in combination with collagen as a scaffold for 3D-tissue engineering to improve the mechanical properties and promote cellular adhesion and growth of regenerating cells.

Background Extracellular matrix (ECM) remodeling is essential for skeletal muscle development and adaption in response to environmental cues such as exercise and injury. The cell surface proteoglycan syndecan-4 has been reported to be essential for muscle differentiation, but few molecular mechanisms are known. Syndecan-4 -/- mice are unable to regenerate damaged muscle, and display deficient satellite cell activation, proliferation, and differentiation. A reduced myofiber basal lamina has also been reported in syndecan-4 -/- muscle, indicating possible defects in ECM production. To get a better understanding of the underlying molecular mechanisms, we have here investigated the effects of syndecan-4 genetic ablation on molecules involved in ECM remodeling and muscle growth, both under steady state conditions and in response to exercise. Methods Tibialis anterior (TA) muscles from sedentary and exercised syndecan-4 -/- and WT mice were analyzed by immunohistochemistry, real-time PCR and western blotting. Results Compared to WT, we found that syndecan-4 -/- mice had reduced body weight, reduced muscle weight, muscle fibers with a smaller cross-sectional area, and reduced expression of myogenic regulatory transcription factors. Sedentary syndecan-4 -/- had also increased mRNA levels of syndecan-2, decorin, collagens, fibromodulin, biglycan, and LOX. Some of these latter ECM components were reduced at protein level, suggesting them to be more susceptible to degradation or less efficiently translated when syndecan-4 is absent. At the protein level, TRPC7 was reduced, whereas activation of the Akt/mTOR/S6K1 and Notch/HES-1 pathways were increased. Finally, although exercise induced upregulation of several of these components in WT, a further upregulation of these molecules was not observed in exercised syndecan-4 -/- mice. Conclusions Altogether our data suggest an important role of syndecan-4 in muscle development.

Natural deep eutectic solvents (NADES) have previously shown antibacterial properties alone or in combination with photosensitizers and light. In this study, we investigated the behavior of the structural protein collagen in a NADES solution. A combination of collagen and NADES adds the unique wound healing properties of collagen to the potential antibacterial effect of the NADES. The behavior of collagen in a NADES composed of citric acid and xylitol and aqueous dilutions thereof was assessed by spectroscopic, calorimetric and viscosity methods. Collagen exhibited variable unfolding properties dependent on the type of material (telo- or atelocollagen) and degree of aqueous dilution of the NADES. The results indicated that both collagen types were susceptible to unfolding in undiluted NADES. Collagen dissolved in highly diluted NADES showed similar results to collagen dissolved in acetic acid (i.e., NADES network possibly maintained). Based on the ability to dissolve collagen while maintaining its structural properties, NADES is regarded as a potential excipient in collagen-based products. This is the first study describing the solubility and structural changes of an extracellular matrix protein in NADES.