Human Skin Collagen Research Articles

Native human collagen type I provides a viable physiologically relevant alternative to xenogeneic sources for tissue engineering applications: A comparative in vitro and in vivo study.

Xenogeneic sources of collagen type I remain a common choice for regenerative medicine applications due to ease of availability. Human and animal sources have some similarities, but small variations in amino acid composition can influence the physical properties of collagen, cellular response, and tissue remodeling. The goal of this work is to compare human collagen type I-based hydrogels versus animal-derived collagen type I-based hydrogels, generated from commercially available products, for their physico-chemical properties and for tissue engineering and regenerative medicine applications. Specifically, we evaluated whether the native human skin type I collagen could be used in the three most common research applications of this protein: as a substrate for attachment and proliferation of conventional 2D cell culture; as a source of matrix for a 3D cell culture; and as a source of matrix for tissue engineering. Results showed that species and tissue specific variations of collagen sources significantly impact the physical, chemical, and biological properties of collagen hydrogels including gelation kinetics, swelling ratio, collagen fiber morphology, compressive modulus, stability, and metabolic activity of hMSCs. Tumor constructs formulated with human skin collagen showed a differential response to chemotherapy agents compared to rat tail collagen. Human skin collagen performed comparably to rat tail collagen and enabled assembly of perfused human vessels in vivo. Despite differences in collagen manufacturing methods and supplied forms, the results suggest that commercially available human collagen can be used in lieu of xenogeneic sources to create functional scaffolds, but not all sources of human collagen behave similarly. These factors must be considered in the development of 3D tissues for drug screening and regenerative medicine applications.

A Preliminary Study for the Effect of Coix-seed Reactive Derivatives (CRD) on Human Skin Fibroblast Proliferation and Collagen Production

A Preliminary Study for the Effect of Coix-seed Reactive Derivatives (CRD) on Human Skin Fibroblast Proliferation and Collagen Production

Age-Related Downregulation of CCN2 Is Regulated by Cell Size in a YAP/TAZ-Dependent Manner in Human Dermal Fibroblasts: Impact on Dermal Aging

CCN2, a member of the CCN family of matricellular proteins, is a key mediator and biomarker of tissue fibrosis. We previously reported that CCN2 is significantly reduced in aged human dermis, which contributes to dermal aging through the downregulation of collagen production, the major structural protein in the skin. In this study, we investigated the underlying mechanisms of the age-related downregulation of CCN2 in human skin dermal fibroblasts. Dermal fibroblasts isolation and laser-capture microdissection‒coupled RT-PCR from human skin confirmed that age-related reduction of CCN2 expression is regulated by epigenetics. Mechanistic investigation revealed that age-related reduction of CCN2 is regulated by impaired dermal fibroblast spreading/cell size, which is a prominent feature of aged dermal fibroblasts in vivo. Gain-of-function and loss-of-function analysis confirmed that age-related downregulation of CCN2 is regulated by YAP/TAZ in response to reduced cell size. We further confirmed that restoration of dermal fibroblast size rapidly reversed the downregulation of CCN2 in a YAP/TAZ-dependent manner. Finally, we confirmed that reduced YAP/TAZ nuclear staining is accompanied by loss of CCN2 in aged human skin in vivo. Our data reveal a mechanism by which age-related reduction in fibroblast spreading/size drives YAP/TAZ-dependent downregulation of CCN2 expression, which in turn contributes to loss of collagen in aged human skin.

Low-level red plus near infrared lights combination induces expressions of collagen and elastin in human skin in vitro.

Light therapy has attracted medical interests as a safe, alternative treatment for photo-ageing and photo-damaged skin. Recent research suggested the therapeutic activity of red and infrared (IR) lights may be effective at much lower energy levels than those used clinically. This study was to evaluate the efficacy of low-level red plus near IR light emitting diode (LED) combination on collagen and elastin and ATP production. Human dermal fibroblasts or skin tissues were irradiated daily by red (640nm) plus near IR (830nm) LED lights combination at 0.5mW/cm2 for 10minutes (0.3J/cm2 ). qPCR, ELISAs or histology were used to determine the gene and protein expressions. Fluorescent measurement was used to assess crosslinks of collagen and elastic fibres. ATP production was evaluated by ATP assay. Treatment of human fibroblast cell cultures with low-level red plus near IR lights combination was found to significantly increase LOXL1, ELN and COL1A1 and COL3A1 gene expressions as well as the synthesis of the procollagen type I and elastin proteins. Treating human skin explants with low-level red plus near IR lights combination similarly induced significant increases in the same gene expressions, type III collagen and elastic fibre formation and crosslinks. ATP production was increased in human dermal fibroblasts after red plus near IR lights combination treatment. Low-level red plus near IR lights combination stimulated the production of collagen and elastin production associated with anti-ageing benefits. These findings suggest that low-level red plus near IR LED light combination may provide an effective treatment opportunity for people with photo-aged skin.

Protective effects of halophyte complex extract against UVB-induced damage in human keratinocytes and the skin of hairless mice.

Limonium tetragonum, Triglochin maritimum, Artemisia scoparia and red ginseng have been used as folk remedies for treating a variety of diseases. In the current study, the protective effects of halophyte and red ginseng against ultraviolet (UV)-induced skin damage were investigated. Halophyte red ginseng complex extract (HRCE) was prepared and its effects on UV-B irradiated human keratinocytes and mouse skin were studied through ELISA, Western blotting immunofluorescence and histological staining. HRCE inhibited peroxide-induced damage in human keratinocytes. HRCE also inhibited UVB-induced collagen and elastin degradation in human keratinocytes and mouse skin. In addition, HRCE inhibited mast cell infiltration in the skin of mice irradiated with UVB light. This effect was likely due to HRCE inhibiting the activation of MAPK and NF-κB. By protecting the skin from UVB-induced skin damage, HRCE has the potential to be used in the treatment and prevention of UV-induced skin damage and photoaging.

In vivo confocal Raman spectroscopic imaging of the human skin extracellular matrix degradation due to accumulated intrinsic and extrinsic aging.

Skin aging is a dynamic process that affects the entire body, marked by molecular and structural changes. Type I collagen is the most abundant structural component and accounts 80% of total collagen in human skin. The amount of proline and hydroxyproline reflect the quantity and quality of the collagen fiber in the extracellular matrix of skin, which is alerted due to accumulated effects of intrinsic and extrinsic aging. Extrinsic aging is driven by ultraviolet radiation-induced reactive oxygen species production that activates the matrix metalloproteinase and disrupts the extracellular matrix of skin dermis, while intrinsic aging is the non-enzymatic process resulting in advanced glycation end products (AGEs). In the presence of pentosidine-AGEs, aging process is accelerated. In vivo Raman spectra of human dermis were collected from forearms of 30 volunteers and were divided into three groups: 10 young adult 25±5years, 10 old adult 65±10years and 10 diabetic old adult 65±10years old male participants. Density functional theory was performed to compute the vibration modes of AGEs, pentosidine, and glucosepane. In vivo confocal Raman spectroscopy detects the specific changes in the proline and hydroxyproline conformation, collagen fiber degradation of type I collagen and AGE protein contribution to specific Raman bands in the aged dermis because of Intrinsic and Extrinsic aging. Statistical t test marked significant differences (P<.01) in Raman peaks of proline and hydroxyproline among young adult, old adult, and diabetic old adult participants at wavenumbers 855, 875, 922, and 938cm-1 . In vivo confocal Raman spectroscopy is a useful tool to detect the AGE markers in the old adult and diabetic old adult male participants, which interacts with the ultraviolet radiations and accelerates the aging process resulting in the extracellular matrix degradation.

General method for classification of fiber families in fiber-reinforced materials: application to in-vivo human skin images

Fiber structures play a major role for the function of fiber-reinforced materials such as biological tissue. An objective classification of the fiber orientations into fiber families is crucial to understand its mechanical properties. We introduce the Fiber Image Network Evaluation Algorithm (FINE algorithm) to classify and quantify the number of fiber families in scientific images. Each fiber family is characterized by an amplitude, a mean orientation, and a dispersion. A new alignment index giving the averaged fraction of aligned fibers is defined. The FINE algorithm is validated by realistic grayscale Monte-Carlo fiber images. We apply the algorithm to an in-vivo depth scan of second harmonic generation images of dermal collagen in human skin. The derived alignment index exhibits a crossover at a critical depth where two fiber families with a perpendicular orientation around the main tension line arise. This strongly suggests the presence of a transition from the papillary to the reticular dermis. Hence, the FINE algorithm provides a valuable tool for a reliable classification and a meaningful interpretation of in-vivo collagen fiber networks and general fiber reinforced materials.

Effects of carboxytherapy on skin laxity.

Carboxytherapy is capable of inducing tissue repair which results in an increase in elastic and collagen fibers. To evaluate the effects of carboxytherapy upon human skin collagen and elastic fiber synthesis. Case study of nine volunteers who received a single application of carboxytherapy in the left infraumbilical region, with infusion rate of 100mL/min and 0.6mL/kg weight over an area of 25cm2 . After 60days on average, the skin was collected for histological analysis and stained with picrosirius red for collagen and Verhoeff for elastic fibers. The percentage of fibers found was marked by the Image J® program and recorded in a Microsoft Excel spreadsheet. Statistical analysis was performed using SPSS (version 20), with a significance level of 95%. An increase in the collagen and elastic fibers sample was observed in the treated group. Morphometrically, a significant increase in the percentage of collagen in the Carboxytherapy group (41.44±4.50%) was observed compared with the control group (37.44±3.87%) with P=.04; for elastic fibers, the percentage showed no significant difference between the control group (10.55±4.33%) and the carboxytherapy group (10.44±3.71%). Carboxytherapy with the parameters used in this study was able to stimulate collagen and elastic fiber synthesis, with significant differences in the morphometry for collagen fibers.

Jak Inhibition Prevents Bleomycin-Induced Fibrosis in Mice and Is Effective in Patients with Morphea

Jak Inhibition Prevents Bleomycin-Induced Fibrosis in Mice and Is Effective in Patients with Morphea

Prostaglandins Contribute to the Effects of Solar Ultraviolet Irradiation on Type I Collagen and Matrix Metalloproteinase-1 Expression in Human Skin

Prostaglandins Contribute to the Effects of Solar Ultraviolet Irradiation on Type I Collagen and Matrix Metalloproteinase-1 Expression in Human Skin

The pecking order of skin Advanced Glycation Endproducts (AGEs) as long-term markers of glycemic damage and risk factors for micro- and subclinical macrovascular disease progression in Type 1 diabetes.

To date more than 20 glycation products were identified, of which ~15 in the insoluble human skin collagen fraction. The goal of this review is to streamline 30years of research and ask a set of important questions: in Type 1 diabetes which glycation products correlate best with 1) past mean glycemia 2) reversibility with improved glycemic control, 2) cross-sectional severity of retinopathy, nephropathy and neuropathy and 3) the future long-term risk of progression of micro- and subclinical macrovascular disease. The trio of glycemia related glycation markers furosine (FUR)/fructose-lysine (FL), glucosepane and methylglyoxal hydroimidazolone (MG-H1) emerges as extraordinarily strong predictors of existing and future microvascular disease progression risk despite adjustment for both past and prospective A1c levels. X(2) values are up to 25.1, p values generally less than 0.0001, and significance remains after adjustment for various factors such as A1c, former treatment group, log albumin excretion rate, abnormal autonomic nerve function and LDL levels at baseline. In contrast, subclinical cardiovascular progression is more weakly correlated with AGEs/glycemia with X(2) values < 5.0 and p values generally < 0.05 after all adjustments. Except for future carotid intima-media thickness, which correlates with total AGE burden (MG-H1, pentosidine, fluorophore LW-1 and decreased collagen solubility), adjusted FUR and Collagen Fluorescence (CLF) are the strongest markers for future coronary artery calcium deposition, while cardiac hypertrophy is associated with LW-1 and CLF adjusted for A1c. We conclude that a robust clinical skin biopsy AGE risk panel for microvascular disease should include at least FUR/FL, glucosepane and MG-H1, while a macrovascular disease risk panel should include at least FL/FUR, MG-H1, LW-1 and CLF.

Comparative histological controlled study of human skin collagen fibers after cell therapy with fibroblasts

Comparative histological controlled study of human skin collagen fibers after cell therapy with fibroblasts

Oxidant exposure induces cysteine-rich protein 61 (CCN1) via c-Jun/AP-1 to reduce collagen expression in human dermal fibroblasts.

Human skin is a primary target of oxidative stress from reactive oxygen species (ROS) generated from both extrinsic and intrinsic sources. Oxidative stress inhibits the production of collagen, the most abundant protein in skin, and thus contributes to connective tissue aging. Here we report that cysteine-rich protein 61 (CCN1), a negative regulator of collagen production, is markedly induced by ROS and mediates loss of type I collagen in human dermal fibroblasts. Conversely, antioxidant N-acetyl-L-cysteine significantly reduced CCN1 expression and prevented ROS-induced loss of type I collagen in both human dermal fibroblasts and human skin in vivo. ROS increased c-Jun, a critical member of transcription factor AP-1 complex, and increased c-Jun binding to the AP-1 site of the CCN1 promoter. Functional blocking of c-Jun significantly reduced CCN1 promoter and gene expression and thus prevented ROS-induced loss of type I collagen. Targeting the c-Jun/CCN1 axis may provide clinical benefit for connective tissue aging in human skin.

Colorful protein-based fluorescent probes for collagen imaging.

Real-time visualization of collagen is important in studies on tissue formation and remodeling in the research fields of developmental biology and tissue engineering. Our group has previously reported on a fluorescent probe for the specific imaging of collagen in live tissue in situ, consisting of the native collagen binding protein CNA35 labeled with fluorescent dye Oregon Green 488 (CNA35-OG488). The CNA35-OG488 probe has become widely used for collagen imaging. To allow for the use of CNA35-based probes in a broader range of applications, we here present a toolbox of six genetically-encoded collagen probes which are fusions of CNA35 to fluorescent proteins that span the visible spectrum: mTurquoise2, EGFP, mAmetrine, LSSmOrange, tdTomato and mCherry. While CNA35-OG488 requires a chemical conjugation step for labeling with the fluorescent dye, these protein-based probes can be easily produced in high yields by expression in E. coli and purified in one step using Ni2+-affinity chromatography. The probes all bind specifically to collagen, both in vitro and in porcine pericardial tissue. Some first applications of the probes are shown in multicolor imaging of engineered tissue and two-photon imaging of collagen in human skin. The fully-genetic encoding of the new probes makes them easily accessible to all scientists interested in collagen formation and remodeling.

Tumor necrosis factor-α-accelerated degradation of type I collagen in human skin is associated with elevated matrix metalloproteinase (MMP)-1 and MMP-3 ex vivo

Tumor necrosis factor (TNF)-α induces matrix metalloproteinases (MMPs) that may disrupt skin integrity. We have investigated the effects and mechanisms of exogenous TNF-α on collagen degradation by incubating human skin explants in defined serum-free media with or without TNF-α (10ng/ml) in the absence or presence of the nonselective MMP inhibitor GM6001 for 8 days. The basal culture conditions promoted type I collagen catabolism that was accelerated by TNF-α (p<0.005) and accomplished by MMPs (p<0.005). Levels of the collagenases MMP-8 and MMP-13 were insignificant and neither MMP-2 nor MMP-14 were associated with increased collagen degradation. TNF-α increased secretion of MMP-1 (p<0.01) but had no impact on MMP-1 quantities in the tissue. Immunohistochemical analysis confirmed similar tissue MMP-1 expression with or without TNF-α with epidermis being the major source of MMP-1. Increased tissue-derived collagenolytic activity with TNF-α exposure was blocked by neutralizing MMP-1 monoclonal antibody and was not due to down-regulation of tissue inhibitor of metalloproteinase-1. TNF-α increased production (p<0.01), tissue levels (p<0.005) and catalytic activity of the endogenous MMP-1 activator MMP-3. Type I collagen degradation correlated with MMP-3 tissue levels (rs=0.68, p<0.05) and was attenuated with selective MMP-3 inhibitor. Type I collagen formation was down-regulated in cultured compared with native skin explants but was not reduced further by TNF-α. TNF-α had no significant effect on epidermal apoptosis. Our data indicate that TNF-α augments collagenolytic activity of MMP-1, possibly through up-regulation of MMP-3 leading to gradual loss of type I collagen in human skin.

Evaluation of antioxidant properties and biofunctions of polar, nonpolar, and water-soluble fractions extracted from gonad and body wall of the sea urchin Tripneustes gratilla

This study was performed to compare the various bioactivities of water-soluble, polar, and nonpolar extracts from the edible part (gonad) and waste (body wall) of the sea urchin Tripneustes gratilla. During antioxidant activity screening, these three extracts from both gonad and body wall showed various antioxidant activities in different tests. During human skin fibroblast (CCD966SK) viability and collagen screening, only water-soluble and polar extracts from gonad and body wall were observed to promote cell viability, while the water-soluble gonad extract promoted the collagen-generating activity of CCD966SK cells. The water-soluble and polar extracts from gonad and body wall showed proliferative activity towards different cell types. In contrast, the nonpolar extracts of gonad and body wall exerted antiproliferative effects on most tumor cells. These results indicate that the bioactivities of sea urchin extracts depend on the part of the urchin that the extract was taken from as well as the extraction method used.

Comparative study of various growth factors and cytokines on type I collagen and hyaluronan production in human dermal fibroblasts

Dermal fibroblast is a primary cell type responsible for synthesis and remodeling of extracellular matrix in human skin. Type I collagen and hyaluronan are main components that have roles in skin fibrosis, wound healing, tissue remodeling as well as skin aging. Several studies have reported cytokine-dependent changes in collagen expression or hyaluronan production; however, the cytokines' effect was controversial in human dermal fibroblasts. To clarify the role of various growth factors, cytokines or chemokines on the production of interstitial type I collagen and hyaluronan in dermal fibroblasts. We confirmed the presence of various corresponding receptors and assessed the effects of 33 human recombinants on the production of type I collagen and hyaluronan using the assay system in dermal fibroblasts. Platelet-derived growth factor (PDGF)-AA, PDGF-BB, epidermal growth factor (EGF), transforming growth factor (TGF)-β1, MCP-1, IP-10, interleukin (IL)-1α, IL-1β, and IL-15 were effective on both type I collagen and hyaluronan production, as compared with no stimulated control. On the other hand, IL-10 and IFN- α caused a significant decrease in type I collagen production, and IL-8 and GM-CSF caused a decrease in hyaluronan production compared with no cytokine-treated control. Interestingly, some chemokines, such as MCP-1 (CCL2), RANTES (CCL5), eotaxin-2 (CCL24), IP-10 (CXCL10), or fractalkine (CX3CL1) significantly induced the type I collagen or hyaluronan production. Various growth factors and cytokines on the regulation of type I collagen and hyaluronan in human dermal skin probably function as key factors in skin remodeling and skin aging. Our profile may help to apply to cosmeceutical area maintaining as young skin through the increase in extracellular matrix.

Cutaneous wound healing after treatment with plant-derived human recombinant collagen flowable gel.

Chronic wounds, particularly diabetic ulcers, represent a main public health concern with significant costs. Ulcers often harbor an additional obstacle in the form of tunneled or undermined wounds, requiring treatments that can reach the entire wound tunnel, because bioengineered grafts are typically available only in a sheet form. While collagen is considered a suitable biodegradable scaffold material, it is usually extracted from animal and human cadaveric sources, and accompanied by potential allergic and infectious risks. The purpose of this study was to test the performance of a flowable gel made of human recombinant type I collagen (rhCollagen) produced in transgenic tobacco plants, indicated for the treatment of acute, chronic, and tunneled wounds. The performance of the rhCollagen flowable gel was tested in an acute full-thickness cutaneous wound-healing rat model and compared to saline treatment and two commercial flowable gel control products made of bovine collagen and cadaver human skin collagen. When compared to the three control groups, the rhCollagen-based gel accelerated wound closure and triggered a significant jumpstart to the healing process, accompanied by enhanced re-epithelialization. In a cutaneous full-thickness wound pig model, the rhCollagen-based flowable gel induced accelerated wound healing compared to a commercial product made of bovine tendon collagen. By day 21 post-treatment, 95% wound closure was observed with the rhCollagen product compared to 68% closure in wounds treated with the reference product. Moreover, rhCollagen treatment induced an early angiogenic response and induced a significantly lower inflammatory response than in the control group. In summary, rhCollagen flowable gel proved to be efficacious in animal wound models and is expected to be capable of reducing the healing time of human wounds.

In vivoobservation of age-related structural changes of dermal collagen in human facial skin using collagen-sensitive second harmonic generation microscope equipped with 1250-nm mode-locked Cr:Forsterite laser

In vivo visualization of human skin aging is demonstrated using a Cr:Forsterite (Cr:F) laser-based, collagen-sensitive second harmonic generation (SHG) microscope. The deep penetration into human skin, as well as the specific sensitivity to collagen molecules, achieved by this microscope enables us to clearly visualize age-related structural changes of collagen fiber in the reticular dermis. Here we investigated intrinsic aging and/or photoaging in the male facial skin. Young subjects show dense distributions of thin collagen fibers, whereas elderly subjects show coarse distributions of thick collagen fibers. Furthermore, a comparison of SHG images between young and elderly subjects with and without a recent life history of excessive sun exposure show that a combination of photoaging with intrinsic aging significantly accelerates skin aging. We also perform image analysis based on two-dimensional Fourier transformation of the SHG images and extracted an aging parameter for human skin. The in vivo collagen-sensitive SHG microscope will be a powerful tool in fields such as cosmeceutical sciences and anti-aging dermatology.

Gadolinium Contrast Agent Associated Stimulation of Human Fibroblast Collagen Production

Nephrogenic systemic fibrosis occurs in patients with poor renal function who receive gadolinium-based contrast agents (Gd-CAs). Several reports suggest that this is more likely to occur with the less stable forms of Gd chelates, suggesting a release of cytotoxic free Gd ions from these. There is evidence that Gd can stimulate human fibroblast proliferation but the evidence is less clear concerning the production of collagen by these cells. Our aim was to assess effects of Gd chelates on human skin cell activity and collagen production. Keratinocytes and fibroblasts were cultured with 3 Gd chelates (Gd-EDTA, Omniscan [nonionic linear Gd-CA], and Dotarem [ionic macrocyclic Gd-CA]) for up to 7 days, and cell viability and collagen production were assessed using the colorimetric assays of MTT (3-(4, 5-dimethylthiazol-2-yl) 2, 5-diphenyltetrazolium bromide) and Sirius Red, respectively. The uptake of Gd by cultured fibroblasts was also undertaken using the techniques of inductively coupled plasma mass spectrometry and relaxometry. Our data show that Gd-EDTA and Omniscan significantly stimulated both fibroblast and keratinocyte viability and fibroblast (but not keratinocyte) collagen production. In contrast, Dotarem had little, if any, effect on these cultured cells. The Omniscan-induced increase in fibroblast collagen was around 40% over 7 days-a similar increase to that seen for cell viability, suggesting that collagen production was secondary to an initial stimulatory effect on fibroblast viability. Studies of the uptake of Gd by the cultured fibroblasts showed that these took up Gd when cultured with Omniscan for 7 days, and our study also suggests that some of this Gd was in a free dissociated form. We conclude that these results support a simple nephrogenic systemic fibrosis causative role of low-stability nonionic linear Gd-CA inducing dermal collagen via the release of dissociated Gd which enters fibroblasts and stimulates their activity and associated collagen production.