BACKGROUND:The restoration of collagen and elastin in human dermal fibroblasts plays a crucial role in anti-aging and skin rejuvenation therapies. Numerous studies have examined the effects of various antioxidants on skin health, but there is limited research comparing their combined effects on collagen and elastin synthesis in human dermal fibroblasts. The objective of this study was to evaluate the individual and combined effects of N-acetylcysteine (NAC), Coenzyme Q10 (CoQ10), Niacinamide (NIAC), Gamma Cyclodextrin (GAMMA), Retinol (RET), Epigallocatechin Gallate (EGCG), and Ellagic Acid (ELA) on collagen type I and elastin synthesis in human dermal fibroblasts (HDFs). Human dermal fibroblasts were treated with individual and combined antioxidants. The expression of collagen type I and elastin was measured using mRNA analysis, immunofluorescence staining, and matrix protein assays. The study focused on the effects of EGCG in combination with other antioxidants like RET, CoQ10, and NAC to identify synergistic effects. The combination of EGCG + RET and EGCG + CoQ10 showed the most significant increase in both elastin and collagen type I synthesis, surpassing the effects of individual antioxidants. EGCG demonstrated the highest fold change in elastin mRNA expression, while the combination treatments notably enhanced the extracellular matrix restoration in HDFs. The combination of EGCG with CoQ10, Retinol, or NAC presents a promising strategy for enhancing skin elasticity and firmness by promoting both elastin and collagen synthesis. These findings suggest that antioxidant combinations can be developed for effective anti-aging skincare formulations.

Collagen is extensively used in tissue engineering for various organ tissue regeneration due to the main component of human organ extracellular matrix (ECM) and their inherent nature bioactivity. Collagen various types naturally exist in different organ ECMs. Collagen fabricated with natural ECM mimics architecture, composition and mechanical properties for various organ tissue regeneration. Collagen fabrication with organ-specific biofunctionality facilitated organ tissue engineering as compared to unmodified collagen biomaterials. Collagen biofunctionality improved by subjecting collagen to synthesis, fibers and surface modifications, and blending with other components. Furthermore, collagen is loaded with bioactive molecules, growth factors, drugs and cells also enhancing the biofunctionality of collagen biomaterials. In this review, we will explore the recent advancements in biofunctional collagen biomaterials fabrication with organ-specific biofunctionality in tissue engineering to resolve various organ tissue engineering issues and regeneration challenges. Biofunctional collagen biomaterials stimulate microenvironments inside and around the implants to excellently regulate cellular activities, differentiate cells into organ native cells, enhanced ECM production and remodeling to regenerate organ tissues with native structure, function and maturation. This review critically explored biofunctional collagen biomaterials fabrication in resolving various organ tissue engineering issues and regeneration challenges, and opening new directions of biofunctional collagen biomaterials fabrication, design and applications.

Collagen plays a crucial role in platelet activation and thrombosis, yet the underlying mechanisms involving reactive oxygen species (ROS) remain incompletely understood. This study investigated how collagen modulates ROS generation and platelet aggregation both in vitro and in vivo, as well as evaluating the protective effects of antioxidants. In vitro, collagen induced dose-dependent platelet aggregation and increased ROS generation, evidenced by the enhanced EMPO adduct formation detected via electron spin resonance (ESR). In vivo experiments demonstrated that collagen administration significantly accelerated CAT-1 decay, indicating elevated oxidative stress with a transient peak around 1 minute post-treatment. Furthermore, escalating collagen doses correlated with increased ROS generation and reduced survival rates in mice, underscoring collagen's impact on oxidative stress and thrombosis severity. Importantly, treatment with enzymatic antioxidants (superoxide dismutase, catalase) and non-enzymatic antioxidants (DMTU, Tiron, mannitol) significantly attenuated collagen-induced oxidative stress and improved animal survival. Collectively, these findings elucidate the pivotal role of ROS in collagen-induced platelet activation and thrombosis and highlight antioxidants as promising therapeutic candidates for preventing thrombotic disorders and managing cardiovascular risk.

Human skin fibroblasts are an excellent in vitro model for tracking the processes occurring in human skin and studying the potential impact of various biologically active substances on these processes. Two plant hormones, which are included in the cytokinins group-kinetin (K) and N-6-benzyladenine (BA)-have a positive effect on human skin. Therefore, an attempt was made to examine the effect they have on key skin functions, cell proliferation, and migration, as well as collagen synthesis in them. The effect of phytohormones was studied at selected concentrations for kinetin-10 μM and 1 μM-and for N-6-benzyladenine-1 μM and 0.1 μM. A wound-healing assay was used in order to analyze cell migration and proliferation. The content of total protein and collagen in cells and culture medium was determined. The obtained results confirm that the studied compounds induce cell migration and proliferation, as well as collagen biosynthesis. The positive effect of kinetin and N-6-benzyladenine on fibroblast metabolism that we have demonstrated allows us to indicate them as compounds with potentially therapeutic properties. Therefore, we conclude that they should be subjected to further molecular and in vivo studies focusing on pathologies connected with skin diseases and aging.

Form-function relationships often have tradeoffs: if a material is tough, it is often inflexible, and vice versa. This is particularly relevant for the elephant trunk, where the skin should be protective yet elastic. To investigate how this is achieved, we used classical histochemical staining and second harmonic generation microscopy to describe the morphology and composition of elephant trunk skin. We report structure at the macro and micro scales, from the thickness of the dermis to the interaction of 10 μm thick collagen fibers. We analyzed several sites along the length of the trunk to compare and contrast the dorsal-ventral and proximal-distal skin morphologies and compositions. We find the dorsal skin of the elephant trunk can have keratin armor layers over 2 mm thick, which is nearly 100 times the thickness of the equivalent layer in human skin. We also found that the structural support layer (the dermis) of the elephant trunk contains a distribution of collagen-I (COL1) fibers in both perpendicular and parallel arrangement. The bimodal distribution of collagen is seen across all portions of the trunk, and is dissimilar from that of human skin where one orientation dominates within a body site. We hypothesize that this distribution of COL1 in the elephant trunk allows both flexibility and load-bearing capabilities. Additionally, when viewing individual fiber interactions of 10 μm thick collagen, we find the fiber crossings per unit volume are five times more common than in human skin, suggesting that the fibers are entangled. We surmise that these intriguing structures permit both flexibility and strength in the elephant trunk. The complex nature of the elephant skin may inspire the design of materials that can combine strength and flexibility.

DuraGen is a widely used type I collagen matrix derived from bovine Achilles tendon that promotes fibroblast ingrowth and neovascularization. However, it remains unknown the time required for dura regeneration and reabsorption of the graft. We evaluate the histopathological characteristics of implanted DuraGen in humans across multiple time points. Patients who underwent a decompressive craniectomy and duraplasty with DuraGen at our institution between January 2020 and September 2021 were prospectively enrolled. At the time of the subsequent surgery, including cranioplasty, DuraGen and associated tissues removed from patients were sent for histopathological analysis. For each patient, time from index surgery to subsequent surgery was categorized into three groups: early (0-20 days), intermediate (21-30 days), and late (> 30 days). Baseline characteristics, primary disease, operative time, complication rate, and histopathological findings were compared between groups. A total of 28 patients were enrolled in the study. Seven specimens (25.0%) were collected within 20 days after craniectomy, 9 specimens (35.7%) between 21 and 30 days, and 12 specimens (39.3%) over 31 days. Histopathologically, implanted collagen matrix, erythrocyte infiltration, and fibrin layer decreased over time, whereas fibroblasts and endogenous collagen increased. Receiver operating characteristic analysis showed that the cut-off time post-implantation for presence of endogenous collagen was 34 days after DuraGen implantation (area under the curve = 0.810). We found that DuraGen was replaced by fibroblast-derived endogenous collagen as early as 34 days post-implantation. Although certain findings remain to be further validated, the present study substantiates DuraGen as a reliable substitute, with findings derived from clinical outcomes and histopathological changes.

At present, there is a significant population of individuals experiencing bone deficiencies caused by injuries, ailments affecting the bones, congenital abnormalities, and cancer. The management of substantial bone defects a significant global orthopedic challenge due to the intricacies involved in promoting and restoring the growth of fresh osseous tissue. Autografts are widely regarded as the "gold standard" for repairing bone defects because of their superior tissue acceptance and ability to control osteogenesis. However, patients undergoing autografts may encounter various challenges, including but not limited to hernia, bleeding, nerve impairment, tissue death. Therefore, researchers in regenerative medicine are striving to find alternatives. Collagen is the most abundant protein in the human body, and its triple helix structure gives it unique characteristics that contribute to its strength and functionality in various tissues. Collagen is commonly processed into various forms such as scaffolds, sponges, membranes, hydrogels, and composite materials, due to its unique compatibility with the human body, affinity for water, minimal potential for immune reactions, adaptability, and ability to transport nutrients or drugs. As an alternative material in the field of bone regeneration, collagen is becoming increasingly important. The objective of this review is to provide a comprehensive analysis of the primary types and sources of collagen, their processes of synthesis and degradation, as well as the advancements made in bone regeneration research and its potential applications. A comprehensive investigation into the role of collagen in bone regeneration is undertaken, providing valuable points of reference for a more profound comprehension of collagen applications in this field. The concluding section provides a comprehensive overview of the prospective avenues for collagen research, underscoring their promising future and highlighting their significant potential in the field of bone regeneration. The Translational Potential of this Article. The comprehensive exploration into the diverse functions and translational potential of collagen in bone regeneration, as demonstrated in this review, these findings underscore their promising potential as a treatment option with significant clinical implications, thus paving the way for innovative and efficacious therapeutic strategies in this domain.

PURPOSE:To evaluate whether the joint use of autologous platelet-rich plasma (aPRP) and rosuvastatin (RSV) in biopsies of dermal wounds induced in rabbits results in an additive effect on the immunoexpression of collagens type I and III, optimizing the healing process and increasing collagen production during the proliferative phase of healing to improve the quality of tissue repair. METHODS:Thirty-two biopsy samples from eight clinically healthy adult male New Zealand rabbits were used. They were treated with aPRP, RSV, or aPRP + RSV and analyzed zero, three, seven, ten, and 14 days post wound induction. RESULTS:Type I collagen immunoexpression was significantly higher in wounds treated with aPRP when compared to the control group. This study demonstrated that type III collagen is predominant during the proliferation phase of the healing process, highlighting its critical role in tissue repair and regeneration. CONCLUSION:The association of aPRP and RSV in wound treatment may have an additive effect in the immunoexpression of type III collagen and can thus be used as an alternative in tissue repair and collagen formation, optimizing the healing process.

As skin ages, its structure and function undergo significant transformations driven by complex cellular and molecular processes. In this study, we explore these changes using the axolotl, an amphibian model known for its transparent skin, allowing detailed observation of both epidermal and dermal layers. We found that axolotl skin, composed of an epidermis and a collagen-rich dermis with three distinct layers (stratum baladachinum, spongiosum, and compactum), shows clear age-related alterations. These changes include reduced fibroblast numbers, altered lattice-patterned cell morphology, disruption of the lattice patterned collagen fiber pattern, thickening the stratum spongiosum, and thinning of the stratum compactum. Notably, fibroblasts, which play a crucial role in collagen braiding, displayed diminished functionality in older axolotls. This study highlights how aging affects both the structural integrity of dermal collagen and cellular dynamics. Given the similarity between axolotl and mammalian skin, these findings may provide valuable insights into the mechanisms of skin aging and potential avenues for anti-aging therapies. This research offers a foundation for future studies aimed at understanding skin aging and regeneration.

AIMS:Fibrosis is a multifactorial process characterized by the excessive accumulation of extracellular matrix (ECM), increased tissue stiffness, and decreased elasticity. This study examined how individual cytokines and a cytokine combination alter collagen production and biomechanics in a 3D in vitro model of the human ECM. METHODS:Cultured human fibroblasts were seeded into a circular agarose trough molded in 24 well plates. The fibroblasts aggregated and formed a 3D ring-shaped tissue that synthesized de novo a collagen-rich human ECM complete with collagen fibrils. Unlike existing models, no macromolecular crowders were added, nor artificial scaffolds or exogenous ECM proteins. Rings were treated with TGF-β1, IL-13 or the combination of TGF-β1 and IL-13 for up to 3 weeks. Morphology, histology, collagen, DNA, fibril formation, gene expression and tensile properties of the rings were measured. RESULTS:As the rings compacted, cellularity and total DNA decreased, whereas total collagen accumulated. TGF-β1 stimulated collagen accumulation and increased ring biomechanics at day 7, but these increases stalled and declined by day 21. When treated with IL-13, a cytokine exclusive to the immune system, there were no significant differences from control. However, when TGF-β1 was combined with IL-13, collagen levels and ring biomechanics increased over the entire three weeks to levels higher than TGF-β1 alone. Gene expression was differentially regulated by cytokine treatment over the entire three weeks suggesting that increased collagen accumulation was not due to upregulation of collagen gene expression. CONCLUSIONS:These results suggest that TGF-β1 requires a second signal, such as IL-13, to sustain the long-term pathological increases in collagen accumulation and biomechanics that can compromise the function of fibrotic tissues.

Given the exponential growth of the recombinant human collagen market, it is paramount to devise a robust and straightforward design strategy aimed at preserving the remarkable biological activity of recombinant human collagen while endowing it with tailored mechanical properties and stable morphologies. This innovative approach stands to broaden its applicability in hard tissue repair endeavors. Our study employed a synergistic approach of alkali hydrolysis and Schiff's base chemistry to graft Type I recombinant human collagen (rhCol-I) onto poly (L-lactic acid) (PLLA) membranes, yielding PLLA-rhCol composites. In vitro evaluations substantiated that this reengineered material not only retained the biological efficacy of rhCol-I but also imparted mechanical robustness and processability ideal for bone implant applications. Notably, it exhibited superior tissue engineering attributes, fostering proliferation, adhesion, osteogenic differentiation, mineralization of bone marrow mesenchymal stem cells (BMSCs), and encouraging vascularization. In a rat model of critical-sized bone defects, PLLA-rhCol exhibited markedly enhanced bone repair efficiency over conventional PLLA bone implants, achieving a bone volume fraction (BV/TV) of up to 32.57 ± 3.77 %, while promoting angiogenesis and effectively mitigating inflammatory cell infiltration. This pioneering method of modifying recombinant human collagen onto the side chains of polymeric macromolecules portends broad applicability in enhancing various biocompatible, yet mechanically robust and processable polymers, thereby expanding the horizons of recombinant human collagen utilization in tissue engineering and catering to the ever-evolving market demands.

Background:The extracellular matrix (ECM) provides essential physical support and biochemical cues for diverse biological activities, including tissue remodelling and regeneration, and thus is commonly applied in the construction of artificial peripheral nerve grafts. Nevertheless, the specific functions of essential peripheral nerve ECM components have not been fully determined. Our research aimed to differentially represent the neural activities of main components of ECM on peripheral nerve regeneration. Methods:Schwann cells from sciatic nerves and neurons from dorsal root ganglia were isolated and cultured . The cells were seeded onto noncoated dishes, Matrigel-coated dishes, and dishes coated with the four major ECM components fibronectin, laminin, collagen I, and collagen IV. The effects of these ECM components on Schwann cell proliferation were determined via methylthiazolyldiphenyl-tetrazolium bromide (MTT), Cell Counting Kit-8, and 5-ethynyl-2'-deoxyuridine (EdU) assays, whereas their effects on cell migration were determined via wound healing and live-cell imaging. Neurite growth in neurons cultured on different ECM components was observed. Furthermore, the two types of collagen were incorporated into chitosan artificial nerves and used to repair sciatic nerve defects in rats. Immunofluorescence analysis and a behavioural assessment, including gait, electrophysiology, and target muscle analysis, were conducted. Results:ECM components, especially collagen I, stimulated the DNA synthesis and movement of Schwann cells. Direct measurement of the neurite lengths of neurons cultured on ECM components further revealed the beneficial effects of ECM components on neurite outgrowth. Injection of collagen I into chitosan and poly(lactic-co-glycolic acid) artificial nerves demonstrated that collagen I facilitated axon regeneration and functional recovery after nerve defect repair by stimulating the migration of Schwann cells and the formation of new blood vessels. In contrast, collagen IV recruited excess fibroblasts and inflammatory macrophages and thus had disadvantageous effects on nerve regeneration. Conclusions:These findings reveal the modulatory effects of specific ECM components on cell populations of peripheral nerves, reveal the contributing roles of collagen I in microenvironment construction and axon regeneration, and highlight the use of collagen I for the healing of injured peripheral nerves.

Angiogenesis, the process of new blood vessel formation, involves endothelial cell proliferation and migration, accompanied by the remodeling of the extracellular matrix (ECM). Type IV collagen, a major ECM component, plays a critical role in vascular basement membrane regeneration, influencing cell polarity, migration, and survival. This study examines the regulatory role of Notch signaling, mediated by Notch3, in type IV collagen expression using TIG-1 fibroblasts and a co-culture angiogenesis model with human umbilical vein endothelial cells (HUVECs). Using small interfering RNA (siRNA) to suppress Notch3 expression, we observed a significant reduction in COL4A1 gene expression, which encodes the α1 chain of type IV collagen. Conversely, transient expression of the Notch3 intracellular domain (NICD3) activated Notch signaling, resulting in increased COL4A1 expression. In the co-culture model, pre-treatment of TIG-1 cells with Notch signaling inhibitors, including siNotch3 and DAPT, decreased the number of α1(IV)-positive TIG-1 fibroblasts adjacent to HUVECs. This reduction highlights the essential role of Notch3-mediated signaling in promoting type IV collagen expression during angiogenesis. Our findings suggest that Notch signaling regulates type IV collagen expression levels, supporting basement membrane formation and vascular maturation. These results provide insight into the molecular mechanisms of angiogenesis, potentially contributing to therapeutic strategies targeting vascular-related pathologies.

Type III collagen, a fundamental constituent of the extracellular matrix (ECM), is extensively utilized across the biomedicine, tissue engineering, and cosmetic industries because of its exceptional biocompatibility and biodegradability. Despite its widespread application, traditional methods of collagen production are often hindered by the limit yield, potential immunogenicity, and batch-to-batch variability. In the present study, we describe a synthetic biological approach for expressing full-length recombinant type III collagen (RCIII) in the Komagataella phaffii system. Furthermore, the protein expression level was effectively increased by co-expression of the MPR1 gene, which improved the intrinsic antioxidant defenses of yeast cells, thereby reducing oxidative stress damage. The resulting RCIII maintains its native secondary structure and exhibits robust biological activities, including the promotion of platelet coagulation, enhancement of cell migration, and anti-inflammatory efficacy. Our findings suggest a viable pathway for large-scale, industrial production of type III collagen, offering a promising biomaterial for advanced biomedical applications, and contributing to the circular economy of the biomedical sector.

Collagen (Col) types I and III are integral components in wound healing and tissue regeneration, influencing tissue development, homeostasis, and related pathologies. Col I and Col III expression changes during different stages of wound healing and understanding the regulation of collagen phenotype determination is crucial for unraveling the complexities of these processes. Transcription factors and microRNAs, directly and indirectly, play a critical role in regulating collagen expression, however, a comprehensive understanding of the factors regulating Col I and III phenotypes remains elusive. This critically analyzed published reports with focuses on various factors regulating the expression of Col I and Col III at the transcriptional and translational levels. We performed bioinformatics analysis with an input of proinflammatory mediators, growth factors, elastases, and matrix metalloproteinases and predicted transcription factors and microRNAs involved in the regulation of collagen expression. Network analysis revealed an interaction between genes, transcription factors, and microRNAs and provided a holistic view of the regulatory landscape governing collagen expression and unveils intricate interconnections. This analysis lays a founda-tional framework for guiding future research and therapeutic interventions to promote extracellular matrix remodeling, wound healing, and tissue regeneration after an injury by modulating collagen expression. In essence, this scientific groundwork offers a comprehensive exploration of the regulatory dynamics in collagen synthesis, serving as a valuable resource for advancing both basic research and clinical interventions in tissue repair.

The structure of many native tissues consists of aligned collagen (Col) fibrils, some of which are further composited with dispersed hydroxyapatite (HAp) nanocrystals. Accurately mimicking this inherent structure is a promising approach to enhance scaffold biocompatibility in tissue engineering. In this study, biomimetic sheets composed of highly aligned Col fibrils were fabricated using a plastic compression and tension method, followed by the deposition of HAp nanocrystals on the surface via an alternate soaking method. The fabricated Col sheets exhibited high solid density, retained the native periodicity (D-band) of Col fibrils, and displayed plate-like HAp nanocrystals dispersed on their surface. In vitro experiments demonstrated that these sheets could regulate fibroblasts adhesion, inducing more elongated nuclei and oriented actin bundles on the aligned Col sheets. Analysis of focal adhesion assembly revealed greater cell focal adhesions on the aligned composite sheets compared to those with random Col fibril structures. Fibroblasts cultured on aligned Col with partly HAp-mineralized sheets exhibited the highest cell-extracellular matrix (ECM) protein secretion, highlighting that HAp incorporation and fibroblast alignment synergistically promote early ECM formation and wound healing. These results suggest that highly aligned Col fibrils with dispersed HAp nanocrystals, closely mimicking the microarchitecture of natural tissues, have significant potential to control cell adhesion and protein secretion for tissue engineering applications.

Collagen peptides have been reported to display various bioactivities and high bioavailability. Recently, increasing evidence has revealed the excellent wound-healing activity of collagen peptides, but their molecular mechanisms remain incompletely elucidated. This review systematically evaluates the therapeutic efficacy of collagen peptides from diverse sources based on various wound models. Furthermore, the structure-activity relationships of collagen peptides and wound-healing mechanisms are discussed and summarized. Characterized by their low molecular weight and abundant imino acids, collagen peptides facilitate efficient absorption by the body to deliver nutrition throughout the wound-healing process. The specific mechanism of collagen peptide for wound healing is mainly through up-regulation of related cytokines and participation in the activation of relevant signaling pathways, such as TGF-β/Smad and PI3K/Akt/mTOR, which can promote cell proliferation, angiogenesis, collagen synthesis and deposition, re-epithelialization, and ECM remodeling, ultimately achieving the effect of wound healing. Collagen peptides can offer a potential therapeutic approach for treating incision and excision wounds, mucosal injuries, burn wounds, and pressure ulcers, improving the efficiency of wound healing by about 10%-30%. The present review contributes to understanding of the wound-healing potential of collagen peptides and the underlying molecular mechanisms.

Type I collagen, a crucial component maintaining the structural integrity and physiological function of various tissues, is widely regarded as one of the most suitable biomaterials for healthcare applications. In this study, shad scales, used for treating ulcers, scalds, and burns in traditional Chinese medicine, were exploited for type I collagen extraction. After self-assembly into hydrogels, the extracted collagen was subsequently freeze-dried to form collagen sponges. The collagen sponge promoted rapid hemostasis, neovascularization, and immune regulation. Additionally, it accelerated the formation of granulation tissue, re-epithelialization, and collagen remodeling at the wound site in full-thickness skin wound rat models. Consequently, the shad scale collagen sponge holds great promise for the treatment of chronic wounds and skin regeneration. Notably, the shad was sourced from sustainably recirculating aquaculture systems (RAS) farms that adhere to the Traceable Management of Animal Products Safety, ensuring that the derived collagen possesses potential in the medical apparatus market.

Advanced Glycation End Products (AGEs) are the end result of the irreversible, non-enzymatic glycation of proteins by reducing sugars. These chemical modifications accumulate with age and have been associated with various age-related and diabetic complications. AGEs predominantly accumulate on proteins with slow turnover rates, of which collagen is a prime example. Glycation has been associated with tissue stiffening and reduced collagen fibril remodelling. In this study, we investigate the effects of glycation on the stability of type I collagen, its molecular-level mechanics and its ability to perform its physiological role of self-assembly. Collagen AGEing is induced in vitro by incubation with ribose. We confirm and assess glycation using fluorescence measurements and changes in collagen's electrophoretic mobility. Susceptibility to trypsin digestion and circular dichroism (CD) spectroscopy are used to probe changes in collagen's triple helical stability, revealing decreased stability due to glycation. Atomic Force Microscopy (AFM) imaging is used to quantify how AGEing affects collagen flexibility, where we find molecular-scale stiffening. Finally we use microscopy to show that glycated collagen molecules are unable to self-assemble into fibrils. These findings shed light on the molecular mechanisms underlying AGE-induced tissue changes, offering insight into how glycation modifies protein structure and stability.

The de novo design of self-assembling peptides has garnered significant attention in scientific research. While alpha-helical assemblies have been extensively studied, exploration of polyproline type II helices, such as those found in collagen, remains relatively limited. In this study, we focus on understanding the sequence-structure relationship in hierarchical assemblies of collagen-like peptides, using defense collagen Surfactant Protein A as a model. By dissecting the sequence derived from Surfactant Protein A and synthesizing short collagen-like peptides, we successfully construct a discrete bundle of hollow triple helices. Amino acid substitution studies pinpoint hydrophobic and charged residues that are critical for oligomer formation. These insights guide the de novo design of collagen-like peptides, resulting in the formation of diverse quaternary structures, including discrete and heterogenous bundled oligomers, two-dimensional nanosheets, and pH-responsive nanoribbons. Our study represents a significant advancement in the understanding and harnessing of collagen higher-order assemblies beyond the triple helix.

OBJECTIVE:This study aimed to investigate the collagen fiber structure of the subcutaneous fascia, a connective tissue layer between the skin and epimysium. METHODS:Fascia samples with varying extensibility were examined using biochemical and microscopic methods. RESULTS:Loose fascia, the more extensible type, displayed sparsely distributed collagen fibers, while dense fascia showed tightly packed collagen fiber bundles. Elastase treatment, after urea pretreatment, caused the loosening of collagen fiber bundles and increased collagen fiber generation as the treatment time increased. This suggests that elastic fibers contribute to collagen fiber bundle formation. Additionally, elastase treatment stretched the fascia, indicating the presence of twodimensional tensile stress generated by elastic fibers. Either enzymes capable of cleaving elastic fibers may be activated or the stretching of elastic fibers accompanying tissue deformation may increase the enzyme sensitivity to elastic fibers, leading to the formation of localized collagen fibers in vivo. Tissue staining confirmed that loose and dense fascia corresponded to areas with sparse and dense collagen fibers, respectively. Some dense collagen fibers appeared to migrate and disperse into loose areas. CONCLUSION:These findings provide insights into the structural organization and functional significance of collagen fibers within the subcutaneous fascia. They particularly highlight the role of elastic fibers in maintaining tissue integrity and facilitating dynamic remodeling.

Macrophages displaying a pro-repair and anti-inflammatory polarization have been implicated in resolution of acute liver injury. Macrophage receptor with collagenous structure (MARCO) expression marks tolerogenic hepatic macrophages and is expressed by pro-resolution macrophages in the injured liver. We tested the hypothesis that MARCO promotes repair of the acetaminophen (APAP)-injured liver. Robust and sustained induction of MARCO mRNA and protein expression was evident in livers of mice challenged with a hepatotoxic dose of APAP (i.e. 300 mg/kg), whereas hepatic MARCO induction failed in mice with APAP-induced liver failure (i.e. 600 mg/kg). Serum proteomics identified a significant increase in serum MARCO levels in surviving acute liver failure (ALF) patients, but not in ALF patients who died. MARCO expression was high in F480+ liver macrophages, and MARCO deficiency reduced macrophage expression of pro-resolution markers such as Gpnmb and Mertk during the repair phase (i.e. 48 h). The results suggested a delay in necrosis resolution along with a trend toward increased mortality in APAP-challenged MARCO-/- mice. Notably, a robust increase in peak hepatic injury (i.e. 6- to 24-h post-APAP challenge) was evident in MARCO-/- mice, which could not be ascribed to differences in NAPQI/APAP-adduct generation nor changes in hepatic neutrophil/macrophage numbers. Interestingly, a reduction in hepatic CD11c+ cells, shown previously to limit APAP-induced liver injury, was evident 24 h after APAP challenge in MARCO-/- mice. The results indicate that MARCO deficiency worsens APAP-induced acute liver injury in mice and provide experimental and initial translational evidence linking MARCO induction to positive outcomes in acute liver injury.

Collagen-I fibrillogenesis is crucial to health and development, where dysregulation is a hallmark of fibroproliferative diseases. Here, we show that collagen-I fibril assembly required a functional endocytic system that recycles collagen-I to assemble new fibrils. Endogenous collagen production was not required for fibrillogenesis if exogenous collagen was available, but the circadian-regulated vacuolar protein sorting (VPS) 33b and collagen-binding integrin α11 subunit were crucial to fibrillogenesis. Cells lacking VPS33B secrete soluble collagen-I protomers but were deficient in fibril formation, thus secretion and assembly are separately controlled. Overexpression of VPS33B led to loss of fibril rhythmicity and overabundance of fibrils, which was mediated through integrin α11β1. Endocytic recycling of collagen-I was enhanced in human fibroblasts isolated from idiopathic pulmonary fibrosis, where VPS33B and integrin α11 subunit were overexpressed at the fibrogenic front; this correlation between VPS33B, integrin α11 subunit, and abnormal collagen deposition was also observed in samples from patients with chronic skin wounds. In conclusion, our study showed that circadian-regulated endocytic recycling is central to homeostatic assembly of collagen fibrils and is disrupted in diseases.

Collagenolytic degradation is a process fundamental to tissue remodeling. The microarchitecture of collagen fibril networks changes during development, aging, and disease. Such changes to microarchitecture are often accompanied by changes in matrix degradability. In a matrix, the pore size and fibril characteristics such as length, diameter, number, orientation, and curvature are the major variables that define the microarchitecture. , collagen matrices of the same concentration but different microarchitectures also vary in degradation rate. How do different microarchitectures affect matrix degradation? To answer this question, we developed a computational model of collagen degradation. We first developed a lattice model that describes collagen degradation at the scale of a single fibril. We then extended this model to investigate the role of microarchitecture using Brownian dynamics simulation of enzymes in a multi-fibril three dimensional matrix to predict its degradability. Our simulations predict that the distribution of enzymes around the fibrils is non-uniform and depends on the microarchitecture of the matrix. This non-uniformity in enzyme distribution can lead to different extents of degradability for matrices of different microarchitectures. Our simulations predict that for the same enzyme concentration and collagen concentration, a matrix with thicker fibrils degrades more than that with thinner fibrils. Our model predictions were tested using experiments with synthetic collagen gels of different microarchitectures. Experiments showed that indeed degradation of collagen depends on the matrix architecture and fibril thickness. In summary, our study shows that the microarchitecture of the collagen matrix is an important determinant of its degradability.

The use of collagen in cell cultures promotes cell proliferation and differentiation, and it has been commercialized. In this study, we separated and purified collagen from adipose tissue discarded during liposuction and prepared collagen-coated dishes. After collagen was identified from human adipose tissue, type identification and quantification were performed using SDS-PAGE and FPLC. Collagen type I was used to coat culture dishes. Human skin fibroblasts and human adipose tissue-derived stem cells were seeded at a density of 2.5 × 105 cells/ml on prepared dishes at a collagen concentration of 3 mg/ml and cultured for 7 days. Cell viability was then measured and analyzed. The WST-1 assay was used to evaluate the results. The amount of collagen in 300 g of adipose tissue was 25.5 mg for type I, 41.4 mg for type III, 10.6 mg for type IV, 6.5 mg for type V and 15 mg for type VI. The highest rates were observed for adipose stem cells cultured on human adipose tissue-derived collagen-coated dishes. In cell cultures, cell affinity was higher when cells and the substrate used were of the same origin, and affinity was stronger when the tissue of origin was the same.

During the process of photoaging in the skin, Succinylated type I collagen has a significant effect on reversing the damage caused by UVB radiation, with the regulation of cellular ferroptosis being one of its important pathophysiological mechanisms. Specifically, Succinylated type I collagen reduces the expression of key cell cycle regulators P16, P21, and P53, as well as the ferroptosis-related factor Acyl-CoA Synthetase Long Chain Family Member 4 (ACSL4), induced by UVB radiation in cells and tissues. Meanwhile, it increases the expression of key factors Glutathione Peroxidase 4 (GPX4) and Solute Carrier Family 7 Member 11 (SLC7A11), which inhibit ferroptosis. Additionally, our study also reveals the impact of Succinylated type I collagen on the levels of malondialdehyde (MDA), glutathione (GSH), and reactive oxygen species (ROS) in cells and tissues, directly affecting the cells' ability to cope with oxidative stress. This further suggests that Succinylated type I collagen may improve skin photoaging through various pathways, including regulating ferroptosis, antioxidation, promoting collagen synthesis, protecting the skin barrier, reducing pigmentation, and inhibiting inflammatory responses, contributing to maintaining healthy and youthful skin.

Collagen proteins contain a characteristic structural motif called a triple helix. During the self-assembly of this motif, three polypeptides form a folding nucleus at the C-termini and then propagate towards the N-termini like a zip-chain. While polypeptides from human collagens contain up to a 1000 amino acids, those found in bacteria can contain up to 6000 amino acids. Additionally, the collagen polypeptides are also frequently interrupted by non-helical sequences that disrupt folding and reduce stability. Given the length of polypeptides and the disruptive interruptions, compensating mechanisms that stabilize against local unfolding during propagation and offset the entropic cost of folding are not fully understood. Here, we show that the information for the correct folding of collagen triple helices is encoded in their sequence as interchain electrostatic interactions, which likely act as molecular clamps that prevent local unfolding. In the case of humans, disrupting these electrostatic interactions is associated with severe to lethal diseases.

Collagen comprises approximately 30% of the body's protein content and is essential for maintaining the structural integrity, support, and strength of the skin, muscles, bones, and connective tissues. Recent research has further elucidated its role in various aspects of tumor biology, including tumorigenesis, invasion, migration, drug resistance, and recurrence. Furthermore, collagen is involved in prognostic assessments, the evaluation of therapeutic efficacy, immunoregulation, and the identification of potential treatment targets in oncology. This review examines a range of tumor types, including lung, gastric, breast, melanoma, and colorectal cancers, among others. Our objective is to differentiate these tumors based on the specific types of collagen present and to analyze the roles of various collagen types in tumor development, progression, prognosis, and their potential as therapeutic targets.

Collagen-based skincare products can replenish collagen in the skin; however, collagen cannot easily penetrate the dermis, limiting its effectiveness. Therefore, nanomaterials that can enable collagen to effectively penetrate the dermis are urgently needed. This study aimed to determine the potential role of the supramolecular collagen nanoparticles, namely, lactoferrin, recombinant human collagen, and palmitoyl tripeptide-5, in improving the effectiveness of skincare products. Lactoferrin and recombinant collagen served as carriers encapsulating palmitoyl tripeptide-5, with an encapsulation rate of 94.18 %. The supramolecular collagen nanoparticles demonstrated good stability after 1 month. Transdermal efficiency was improved by 69.90 %, allowing the nanoparticles to penetrate deeply into the dermis. Within 28 days of use, the moisture content of the stratum corneum increased by 10.51 %, facial elasticity improved by 8.15 %, skin firmness increased by 12.53 %, facial melanin index decreased by 1.84 %, and individual type angle increased by 19.10 %. Within 14 days, there was a 24.69 % reduction in eye bag wrinkles and a 37.61 % reduction in nasolabial wrinkles. Wrinkle lengths decreased by 10.22 % and 21.57 %, and areas decreased by 34.41 % and 27.92 %, respectively. The supramolecular collagen nanoparticles displayed multiple skincare benefits, including moisturizing, whitening, wrinkle reduction, and firming. In conclusion, the supramolecular collagen nanoparticles are promising candidates for cosmetic products.

Materials-mediated piezoelectric signals have been widely applied in bone regeneration. Collagen is the most abundant protein in the human body, and native collagen with complete tertiary structure shows efficient piezoelectricity. However, the traditional collagen scaffolds are lack of piezoelectricity due to the destruction of the complete tertiary structure. Here, natural collagen scaffolds with the complete tertiary structure were prepared. Alkali treatment made the collagen scaffold lose piezoelectricity. The collagen with/without piezoelectricity (PiezoCol/NCol) scaffolds both possessed good cytocompatibility and promoted cell adhesion. After being implanted subcutaneously, the NCol scaffold almost did not affect bone regeneration with/without ultrasound treatment. However, under ultrasound treatment, the PiezoCol scaffold promoted the new bone formation with enhanced osteogenic differentiation, angiogenesis, and neural differentiation, meaning that piezoelectricity endows collagen with satisfactory promotion for bone regeneration. Meanwhile, the PiezoCol scaffold can also accelerate bone formation without ultrasound treatment, which should be attributed to the daily exercise-caused weak piezoelectric stimulation. Further, the proteomic analysis revealed the mechanism by which the PiezoCol scaffold promoted bone tissue formation via mainly upregulating the PI3K-Akt signaling pathway. This study provides a new strategy to enhance the osteoinduction of collagen scaffold for bone regeneration by maintaining intrinsic piezoelectricity.

Biomaterials have been the subject of extensive research, and their applications in medicine and pharmacy are expanding rapidly. Collagen and its derivatives stand out as valuable biomaterials due to their high biocompatibility, biodegradability, and lack of toxicity and immunogenicity. This review comprehensively examines collagen from various sources, its extraction and processing methods, and its structural and functional properties. Preserving the native state of collagen is crucial for maintaining its beneficial characteristics. The challenges associated with chemically modifying collagen to tailor its properties for specific clinical needs are also addressed. The review discusses various collagen-based biomaterials, including solutions, hydrogels, powders, sponges, scaffolds, and thin films. These materials have broad applications in regenerative medicine, tissue engineering, drug delivery, and wound healing. Additionally, the review highlights current research trends related to collagen and its derivatives. These trends may significantly influence future developments, such as using collagen-based bioinks for 3D bioprinting or exploring new collagen nanoparticle preparation methods and drug delivery systems.

The hierarchic assembly of fibrillar collagen into an extensive and ordered supramolecular protein fibril is critical for extracellular matrix function and tissue mechanics. Despite decades of study, we still know very little about the complex process of fibrillogenesis, particularly at the earliest stages where observation of rapidly forming, nanoscale intermediates challenges the spatial and temporal resolution of most existing microscopy methods. Using video rate scanning atomic force microscopy (VRS-AFM), we can observe details of the first few minutes of collagen fibril formation and growth on a mica surface in solution. A defining feature of fibrillar collagens is a 67-nm periodic banding along the fibril driven by the organized assembly of individual monomers over multiple length scales. VRS-AFM videos show the concurrent growth and maturation of small fibrils from an initial uniform height to structures that display the canonical banding within seconds. Fibrils grow in a primarily unidirectional manner, with frayed ends of the growing tip latching onto adjacent fibrils. We find that, even at extremely early time points, remodeling of growing fibrils proceeds through bird-caging intermediates and propose that these dynamics may provide a pathway to mature hierarchic assembly. VRS-AFM provides a unique glimpse into the early emergence of banding and pathways for remodeling of the supramolecular assembly of collagen during the inception of fibrillogenesis.

Collagen is the most abundant protein in human and mammalian structures and is a component of the mammalian extracellular matrix (ECM). Recombinant collagen is a suitable alternative to native collagen extracted from animal tissue for various biomaterials. However, due to the limitations of the expression system, most recombinant collagens are collagen fragments and lack triple helix structures. In this study, Chinese hamster ovary (CHO) cells were used to express the full-length human type I collagen α1 chain (rhCol1α1). Moreover, Endo180 affinity chromatography and pepsin were used to purify pepsin-soluble rhCol1α1 (PSC1). The amino acid composition of PSC1 was closer to that of native human type I collagen, and PSC1 contained 9.1 % hydroxyproline. Analysis of the CD spectra and molecular weight distribution results revealed that PSC1 forms a stable triple helix structure that is resistant to pepsin hydrolysis and has some tolerance to MMP1, MMP2 and MMP8 hydrolysis. Atomic force microscopy (AFM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) revealed that PSC1 can self-assemble into fibers at a concentration of 1 mg/ml; moreover, PSC1 can promote the proliferation and migration of NIH 3T3 cells. In conclusion, our data suggest that PSC1 is a highly similar type of recombinant collagen that may have applications in biomaterials and other medical fields.

Collagen possesses distinctive chemical properties and biological functions due to its unique triple helix structure. However, recombinant collagen expressed in without post-translational modifications such as hydroxylation lacks full function since hydroxylation is considered to be critical to the stability of the collagen triple-helix at body temperature. Here, a proline-deficient strain was constructed and employed to prepare hydroxylated recombinant collagens by incorporating proline (Pro) and hydroxyproline (Hyp) from the culture medium. By controlling the ratio of Pro to Hyp in the culture medium, collagen with different degrees of hydroxylation (0-88%) can be obtained. When the ratio of Pro and Hyp was adjusted to 12:8 mM, the proline hydroxylation rate of recombinant human collagen (rhCol, 55 kDa) ranged from 40-50%, which was also the degree of natural collagen. After proline hydroxylation, both the thermal stability and cell binding of rhCol were significantly enhanced. Notably, when the hydroxylation rate approached that of native human collagen (40-50%), the improvements were most pronounced. Moreover, the cell binding of rhCol with a hydroxylation rate of 43% increased by 29%, and the melting temperature (Tm) rose by 5 °C compared to the non-hydroxylated rhCol. The system achieved a yield of 1.186 g/L of rhCol by batch-fed in a 7 L fermenter. This innovative technology is expected to drive the development and application of collagen-related biomaterials with significant application value in the fields of tissue engineering, regenerative medicine, and biopharmaceuticals.

Collagens are the foundational component of diverse tissues, including skin, bone, cartilage, and basement membranes, and are the most abundant protein class in animals. The fibrillar collagens are large, complex, multidomain proteins, all containing the characteristic triple helix motif. The most prevalent collagens are heterotrimeric, meaning that cells express at least two distinctive procollagen polypeptides that must assemble into specific heterotrimer compositions. The molecular mechanisms ensuring correct heterotrimeric assemblies are poorly understood - even for the most common collagen, type-I. The longstanding paradigm is that assembly is controlled entirely by the ~30 kDa globular C-propeptide (C-Pro) domain. Still, this dominating model for procollagen assembly has left many questions unanswered. Here, we show that the C-Pro paradigm is incomplete. In addition to the critical role of the C-Pro domain in templating assembly, we find that the amino acid sequence near the C terminus of procollagen's triple-helical domain plays an essential role in defining procollagen assembly outcomes. These sequences near the C terminus of the triple-helical domain encode conformationally stabilizing features that ensure only desirable C-Pro-mediated trimeric templates are committed to irreversible triple-helix folding. Incorrect C-Pro trimer assemblies avoid commitment to triple-helix formation thanks to destabilizing features in the amino acid sequences of their triple helix. Incorrect C-Pro assemblies are consequently able to dissociate and search for new binding partners. These findings provide a distinctive perspective on the mechanism of procollagen assembly, revealing the molecular basis by which incorrect homotrimer assemblies are avoided and setting the stage for a deeper understanding of the biogenesis of this ubiquitous protein.

Recreating the structural and mechanical properties of native tissues presents significant challenges, particularly in mimicking the dense fibrillar network of extracellular matrixes such as skin and tendons. This study develops a reversible collagen film through cycling collagen self-assembly and disassembly, offering an innovative approach to address these challenges. We first generated an engineered collagen scaffold by applying plastic compression to the collagen hydrogel. The reversibility of the collagen assembly was explored by treating the scaffold with lactic acid, leading to its breakdown into an amorphous gel─a process termed defibrillogenesis. Subsequent immersion of this gel in phosphate buffer facilitated the reassembly of collagen into fibrils larger than those in the original scaffold yet with the D-banding pattern characteristic of collagen fibrils. Transfer learning of the mobileNetV2 convolutional neural network trained on atomic force microscope images of collagen nanoscale D-banding patterns was created with 99% training and testing accuracy. In addition, extensive external validation was performed, and the model achieved high robustness and generalization with unseen data sets. Further innovation was introduced by applying collagen hybridizing peptides, which significantly accelerated and directed the assembly of collagen fibrils, promoting a more organized and aligned fibrillar structure. This study not only demonstrates the feasibility of creating a reversible collagen film that closely mimics the density and structural properties of the native matrix but also highlights the potential of using collagen hybridizing peptides to control and enhance collagen fibrillogenesis. Our findings offer promising tissue engineering and regenerative medicine strategies by enabling precise manipulation of collagen structures .

The extracellular matrix (ECM) is present in all tissues and crucial in maintaining normal tissue homeostasis and function. Defects in ECM synthesis and remodeling can lead to various diseases, while overproduction of ECM components can cause severe conditions like organ fibrosis and influence cancer progression and therapy resistance. Collagens are the most abundant core ECM proteins in physiological and pathological conditions and are predominantly synthesized by fibroblasts. Previous efforts to target aberrant collagen synthesis in fibroblasts by inhibiting pro-fibrotic signaling cascades have been ineffective. More recently, metabolic rewiring downstream of pro-fibrotic signaling has emerged as a critical regulator of collagen synthesis in fibroblasts. Here, we propose that targeting the metabolic pathways involved in ECM biomass generation provides a novel avenue for treating conditions characterized by excessive collagen accumulation. This review summarizes the unique metabolic challenges collagen synthesis imposes on fibroblasts and discusses how underlying metabolic networks could be exploited to create therapeutic opportunities in cancer and fibrotic disease. Finally, we provide a perspective on open questions in the field and how conceptual and technical advances will help address them to unlock novel metabolic vulnerabilities of collagen synthesis in fibroblasts and beyond.

Collagen is the oldest and most abundant extracellular matrix protein and has many applications in biomedical, food, cosmetic, and other industries. Previous reviews have already introduced collagen's sources, structures, and biosynthesis. The biological and mechanical properties of collagen-based composite materials, their modification and application forms, and their interactions with host tissues are pinpointed. It is worth noting that self-assembly behavior is the main characteristic of collagen molecules. However, there is currently relatively little review on collagen-based composite materials based on self-assembly. Herein, we briefly reviewed the biosynthesis, extraction, structure, and properties of collagen, systematically presented an overview of the various factors and corresponding characterization techniques that affect the collagen self-assembly process, and summarize and discuss the preparation methods and application progress of collagen-based composite materials in different fields. By combining the self-assembly behavior of collagen with preparation methods of collagen-based composite materials, collagen-based composite materials with various functional reactions can be selectively prepared, and these experiences and outcomes can provide inspiration and practical techniques for the future development directions and challenges of collagen-based composite biomaterials in related applications fields.

Collagens have dual functions in the extracellular matrix (ECM), acting as both structural components and signaling molecules in matricellular communication. Although collagen molecules share a common triple helix motif, the supramolecular organization helps classify them into nearly 30 different types of collagens. Collagen type VIII is a non-fibrillar, short-chain, network-forming collagen that is expressed throughout the vasculature. Collagen VIII expression is aberrant in cardiovascular, lung, and renal disease, as well as in several different types of cancer. It plays active roles in angiogenesis, vessel injury repair, maintenance of arterial compliance, atherosclerotic plaque formation and stability modulation, fibrosis, and ECM remodeling. This review presents an overview of the characteristics of collagen VIII in vascular-related disorders, from clinical significance to laboratory studies, with a major focus on highlighting the signaling properties of collagen VIII in the vascular ECM. The expression patterns of collagen VIII in human diseases and experimental animal models highlight the protein's important yet underexplored functions. A deeper understanding of its mechanisms and downstream signaling pathways may pave the way for translational and tissue engineering applications of collagen VIII.

The collagen triple helix is one of the structurally simplest protein motifs that still holds a lot of secrets. The Gly-X-Y repeat is a business card of collagens, where Gly is required for the tight packing of three helices into a superhelix and X and Y residues are important for stabilizing the triple helix and communicating with the world. On its way to a functional molecule, collagen sequences undergo unique post-translational modifications inside and outside of the cell. Moreover, folding and secretion of collagens require specific proteins and mechanisms. Cracking the collagen triple helix codes opens up opportunities for curing associated diseases and developing new biomaterials. Here, we summarized my journey through some mysteries of the collagen triple helix and point out key unaddressed questions and problems for other researchers to pursue.

This article presents a damage model for collagenous tissue under monotonic loading. Given that the true stretch of collagen fibers is not uniform and is regulated by fiber waviness, we postulate that damage commences from more stretched (i.e. straighter) fibers and progresses to less stretched (i.e. wavier) ones. The complicated nonlinear response is regarded as the outcome of two competing mechanisms: the recruitment of wavy intact fibers and the loss of taut functioning fibers. The progression of damage is modeled by an evolving damage front in the waviness domain. A power law is proposed for the evolution of damage front. The model was fitted to four groups of published uniaxial and biaxial tests data of vascular tissues. Spot-on fits were observed in all groups.

Recombinantly produced collagens present a sustainable, ethical, and safe substitute for collagens derived from natural sources. However, controlling the folding of the recombinant collagens, crucial for replicating the mechanical properties of natural materials, remains a formidable task. Collagen-like proteins from willow sawfly are relatively small and contain no hydroxyprolines, presenting an attractive alternative to the large and post-translationally modified mammalian collagens. Utilizing CD spectroscopy and analytical ultracentrifugation, we demonstrate that recombinant willow sawfly collagen assembles into collagen triple helices in a concentration-dependent manner. Interestingly, we observed that the lower concentration threshold for the folding can be overcome by freezing or adding crowding agents. Microscopy data show that both freezing and the addition of crowding agents induce phase separation. We propose that the increase in local protein concentration during phase separation drives the nucleation-step of collagen folding. Finally, we show that freezing also induces the folding of recombinant human collagen fragments and accelerates the folding of natural bovine collagen, indicating the potential to apply phase separation as a universal mechanism to control the folding of recombinant collagens. We anticipate that the results provide a method to induce the nucleation of collagen folding without any requirements for genetic engineering or crosslinking.

Skin undergoes mechanical alterations due to changes in the composition and structure of the collagenous dermis with aging. Previous studies have conflicting findings, with both increased and decreased stiffness reported for aging skin. The underlying structure-function relationships that drive age-related changes are complex and difficult to study individually. One potential contributor to these variations is the accumulation of nonenzymatic crosslinks within collagen fibers, which affect dermal collagen remodeling and mechanical properties. Specifically, these crosslinks make individual fibers stiffer in their plastic loading region and lead to increased fragmentation of the collagenous network. To better understand the influence of these changes, we investigated the impact of nonenzymatic crosslink changes on the dermal microstructure using discrete fiber networks representative of the dermal microstructure. Our findings suggest that stiffening the plastic region of collagen's mechanical response has minimal effects on network-level stiffness and failure stresses. Conversely, simulating fragmentation through a loss of connectivity substantially reduces network stiffness and failure stress, while increasing stretch ratios at failure.

Collagens are ubiquitous in biology functioning as the backbone of the extracellular matrix, forming the primary structural components of key immune system complexes, and fulfilling numerous other structural roles in a variety of systems. Despite this, there is limited understanding of how triple helices, the basic collagen structural units, pack into collagenous assemblies. Here we use a peptide self-assembly system to design collagenous assemblies based on the C1q collagen-like region. Using cryo-EM we solve a structure of one assembly to 3.5 Å resolution and build an atomic model. From this, we identify a triple helix conformation with no superhelical twist, starkly in contrast to the canonical right-handed triple helix. This non-twisting region allows for unique hydroxyproline stacking between adjacent triple helices and also results in the formation of an exposed cavity with rings of hydrophobic amino acids packed symmetrically. We find no precedent for such an arrangement of collagen triple helices and have designed mutant assemblies to probe key stabilizing amino acid interactions in the complex. The mutations behave as predicted by our atomic model. Our findings, combined with the extremely limited experimental structural data on triple helix packing in the literature, suggest that collagen and collagen-like assemblies may adopt a far more varied conformational landscape than previously appreciated. We hypothesize that this is particularly likely adjacent to the termini of these helices and at discontinuities to the required Xaa-Yaa-Gly repeating primary sequence; a discontinuity found in the majority of this class of proteins and in many collagen-associated diseases.

Highly abundant in mammals, collagens define the organization of tissues and participate in cell signalling. Most of the 28 vertebrate collagens, with the exception of collagens VI, VII, XXVI and XXVIII, can be categorized into five subgroups: fibrillar collagens, network-forming collagens, fibril-associated collagens with interrupted triple helices, membrane-associated collagens with interrupted triple helices and multiple triple-helix domains with interruptions. Collagen peptides are synthesized from the ribosome and enter the rough endoplasmic reticulum, where they undergo numerous post-translational modifications. The collagen chains form triple helices that can be secreted to form a diverse array of supramolecular structures in the extracellular matrix. Collagens are ubiquitously expressed and have been linked to a broad spectrum of disorders, including genetic disorders with kidney phenotypes. They also have an important role in kidney fibrosis and mass spectrometry-based proteomic studies have improved understanding of the composition of fibrosis in kidney disease. A wide range of therapeutics are in development for collagen and kidney disorders, including genetic approaches, chaperone therapies, protein degradation strategies and anti-fibrotic therapies. Improved understanding of collagens and their role in disease is needed to facilitate the development of more specific treatments for collagen and kidney disorders.

Collagen, the most abundant protein in the human body, must withstand high mechanical loads due to its structural role in tendons, skin, bones, and other connective tissue. It was recently found that tensed collagen creates mechanoradicals by homolytic bond scission. We here employ scale-bridging simulations to determine the influence of collagen's mesoscale fibril structure on molecular breakages, combining atomistic molecular dynamics simulations with a newly developed mesoscopic ultra-coarse-grained description of a collagen fibril. Our simulations identify a conserved structural feature, a length difference of the two helices between pairs of crosslinks, to play a critical role. The release of the extra hidden length enables collagen to buffer mechanical stress. At the same time, this topology funnels ruptures such that the potentially harmful mechanoradicals are readily stabilized, buffering the arising oxidative stress. Our results suggest collagen's hidden length to exploit a sweet spot in the trade-off between breakage specificity and strength.

Collagens are ubiquitous in biology: functioning as the backbone of the extracellular matrix, forming the primary structural components of key immune system complexes, and fulfilling numerous other structural roles in a variety of systems. Despite this, there is limited understanding of how triple helices, the basic collagen structural units, pack into collagenous assemblies. Here we use a peptide self-assembly system to design collagenous assemblies based on the C1q collagen-like region. Using cryo-EM we solved a structure of one assembly to 3.5 Å resolution and built an atomic model. From this, we identify a triple helix conformation with no superhelical twist, starkly in contrast to the canonical right-handed triple helix. This nontwisting region allows for unique hydroxyproline stacking between adjacent triple helices and also results in the formation of an exposed cavity with rings of hydrophobic amino acids packed symmetrically. We find no precedent for such an arrangement of collagen triple helices and designed assemblies with substituted amino acids in various locations to probe key stabilizing amino acid interactions in the complex. The stability of these altered complexes behaves as predicted by our atomic model. Our findings, combined with the extremely limited experimental structural data on triple helix packing in the literature, suggest that collagen and collagen-like assemblies may adopt a far more varied conformational landscape than previously appreciated. We hypothesize that this is particularly likely in packed assemblies of triple helices, adjacent to the termini of these helices and at discontinuities in the required Xaa-Yaa-Gly repeating primary sequence, a discontinuity found in the majority of this class of proteins and in many collagen-associated diseases.

Type I collagen is the most abundant structural protein in the body and, with other fibrillar collagens, forms the fibrous network of the extracellular matrix. Another group of extracellular matrix polymers, the glycosaminoglycans, and glycosaminoglycan-modified proteoglycans, play important roles in regulating collagen behaviors and contribute to the compositional, structural, and mechanical complexity of the extracellular matrix. While the binding between collagen and small leucine-rich proteoglycans has been studied in detail, the interactions between collagen and the large bottlebrush proteoglycan versican are not well understood. Here, we report that versican binds collagen directly and regulates collagen structure and mechanics. Versican colocalizes with collagen fibers in vivo and binds to collagen via its C-terminal G3 domain (a non-GAG-modified domain present in all known versican isoforms) in vitro; it promotes the deposition of a highly aligned collagen-rich matrix by fibroblasts. Versican also shows an unexpected effect on the rheology of collagen gels in vitro, causing decreased stiffness and attenuated shear strain stiffening, and the cleavage of versican in the liver results in reduced tissue compression stiffening. Thus, versican is an important collagen-binding partner and plays a role in modulating collagen organization and mechanics.

The triple helix and D-period are distinctive features of native collagen, crucial for its physicochemical properties and bioactivities. However, developing recombinant humanized collagen with D-period features remains elusive. Here, we present a strategy for preparing a novel recombinant humanized collagen using a 'charged-hydrophobic-charged amino acid' sequence with the capacity of self-assembling. The hydrophobic amino acids in the middle region are believed to be crucial for the triple helix formation while the charged amino acids at the C- and N-terminal drive the triple-helix to self-assemble into higher-order structures like fibrils, with D-period formation during this process. To prove this concept, the particular fragment of Gly1059-Ala1103 of human type III collagen, featuring arginine (R), lysine (K), aspartic acid (D), and glutamic acid (E)-rich termini and a Glycine-Proline-Alanine (G-P-A) central motif, was selected and repeated to construct a recombinant humanized collagen, designated as rhCL04. This construct successfully formed hierarchical structures, including triple helices, rod-like fibrils, and hydrogels, exhibiting a distinct 10 nm D-period across a broad pH range from 4 to 10. Additionally, cell adhesion and biocompatibility were confirmed using L929 mouse fibroblast cells, demonstrating the ability to promote cell adhesion activity and no significant cytotoxicity. Our study provides valuable insights into the self-assembling mechanisms of native collagens. Moreover, these results highlight the efficacy of this strategy in producing recombinant humanized collagen with collagen-like characteristics. The simplicity and versatility of the approach, combined with the excellent self-assembling properties and biological activity of rhCL04, underscore its potential for biomaterial production.

Optimizing cell-matrix interactions for effective bone regeneration remains a significant hurdle in tissue engineering. This study presents a novel approach by developing a human mesenchymal stem cells (hMSCs)-embedded 3D aligned collagen for enhanced bone regeneration. A one-step mechanical strain was applied to a mixture of hMSCs and collagen, producing an hMSC-embedded, aligned 3D collagen hydrogel patch that mimics the natural bone matrix. Notably, the hMSCs embedded in the aligned collagen spontaneously differentiated into osteoblasts without external inducing reagents. Immunofluorescence analysis revealed that the BMP2-smad1/5 signaling pathway, critical for osteogenic differentiation, were activated by aligned collagen. In vivo experiments using a calvarial defect model confirmed that this approach effectively promotes new bone formation, starting centrally within the defect rather than from the edges adjacent to the existing bone. Our findings suggest that this simple method of pre-straining to create aligned 3D collagen embedded with hMSCs holds promise as a novel cell therapy platform for bone regeneration. STATEMENT OF SIGNIFICANCE: This study introduces a novel method for enhancing bone regeneration by developing a 3D aligned collagen patch embedded with hMSCs. A single mechanical strain applied to the hMSC-collagen mixture produces an aligned collagen matrix that mimics natural bone tissue. Remarkably, the hMSCs spontaneously differentiate into osteoblasts in the absence of exogenous inducing reagents triggered by activation of the bone morphogenetic protein signaling pathway. In vivo studies using a calvarial defect model confirm effective bone regeneration, initiating the new bone generation from the center of the defect. This approach offers a promising and simple cell therapy platform for bone repair, with broad implications for tissue engineering and regenerative medicine.

Fibrosis is a pathological condition characterised by excessive accumulation of extracellular matrix (ECM) components, particularly collagens, leading to tissue scarring and organ dysfunction. In fibrosis, an imbalance between collagen synthesis (fibrogenesis) and degradation (fibrolysis) results in the deposition of fibrillar collagens disrupting the structural integrity of the ECM and, consequently, tissue architecture. Fibrosis is associated with a wide range of chronic diseases, including cirrhosis, kidney fibrosis, pulmonary fibrosis, and autoimmune diseases. Recently, the concept of "hot" and "cold" fibrosis has emerged, referring to the immune status within fibrotic tissues and the nature of fibrogenic signalling. Hot fibrosis is characterised by active immune cell infiltration and inflammation, while cold fibrosis is associated with auto- and paracrine myofibroblast activation, immune cell exclusion and quiescence. In this article, we explore the relationship between hot and cold fibrosis, the role of various types of collagens and their biologically active fragments in modulating the immune system, and how serological ECM biomarkers can help improve our understanding of the disease-relevant interactions between immune and mesenchymal cells in fibrotic tissues. Additionally, we draw lessons from immuno-oncology research in solid tumours to shed light on potential strategies for fibrosis treatment and highlight the advantage of having a "hot fibrotic environment" to treat fibrosis by enhancing collagen degradation through modulation of the immune system.

BACKGROUND:Collagen is currently a widely used injectable filler material. Due to the complexity and specificity of the infraorbital structure and function, it undergoes various aging changes earlier in the facial aging process. Additionally, continuous exposure to Ultraviolet (UV) leads to photoaging of the skin. In this study, we utilized type I and type III composite collagen as a filler material for injecting the infraorbital region of the human face and the dorsal region of a nude mouse skin photoaging model to assess its effectiveness and safety. OBJECTIVE:To assess the effectiveness of type I and type III composite collagen in treating age-related changes in the human infraorbital area, as well as in a nude mouse model of skin aging. METHODS:A total of 36 patients with infraorbital aging were enrolled to receive type I and type III composite collagen injections. The improvement of infraorbital aging was assessed at pre-injection, immediate post-injection, 1 week, 4 weeks, and 12 weeks. Additionally, nude mice photodamaged models were prepared and collagen injections were administered to treat photodamaged skin. The therapeutic efficacy was assessed by gross view, histological staining, gene expression analysis, and ELISA assay. RESULTS:Type I and Type III composite collagen injection can immediately fill depressed areas. However, one week after the injection, the collagen dehydrates and contracts, causing the material to be absorbed by the tissues and resulting in a slight regression of the filling effect. After one month, some of the collagen has degraded, with most of it degrading after three months. Additionally, in treating photoaging, type I and type III composite collagen has a more pronounced therapeutic effect on photoaging and demonstrates better results in collagen regeneration, inflammation reduction, and melanin control. CONCLUSION:Type I and Type III composite collagen demonstrates superior efficacy and safety in addressing infraorbital aging and photoaging in nude mice skin, making it a more favorable choice as an injection material. LEVEL OF EVIDENCE II:This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors   www.springer.com/00266 .

Endothelial dysfunction, defined as a reduction in the bioavailability of nitric oxide (NO), is a risk factor for the occurrence and progression of various vascular diseases. This study investigates the effect of endothelial dysfunction on age-related changes in aortic extracellular matrix (ECM) microstructure and the relationship between microstructural adaptation and the mechanical response. Here, we used groups of NOS3 knockout (KO), NOS3 heterozygotes (Het), and wild-type (WT) B6 mice (controls) to study changes in hemodynamic parameters, collagen fiber organization, and both active and passive aortic mechanics using biaxial pressure myography over a time course from 1.5 to 12 mo. Our results show that homeostatic levels of passive circumferential stress and stretch were preserved in KO mice by remodeling adventitial collagen fibers toward a more predominantly circumferential direction with age, rather than by increased fibrosis, in response to hypertension induced by endothelial dysfunction. However, passive aortic stiffness in KO mice was significantly increased owing to geometrical changes, including significant increases in wall thickness and decreases in inner diameter, and by ECM microstructural reorganization, during this maladaptive vascular remodeling. Furthermore, long-term NO deficiency significantly increased smooth muscle cell (SMC) contractility initially, but this effect was attenuated with age. These findings improve our understanding of microstructural and mechanical changes during the maladaptive vascular remodeling process, demonstrating a role for adventitial collagen fiber reorientation in the response to hypertension. Endothelial dysfunction facilitates the reorganization of collagen fibers toward a more predominantly circumferential orientation with age, consequently promoting homeostatic normalization of passive circumferential stress and stretch in the vessel subjected to hypertension.