Early delivery of human umbilical cord mesenchymal stem cells improves healing in a rat model of Achilles tendinopathy.

Objective: This study aimed to explore the efficacy and optimal delivery time of human umbilical cord mesenchymal stem cells (hUC-MSCs) in treating collagenase-induced Achilles tendinopathy. Methods: Achilles tendinopathy in rats at early or advanced stages was induced by injecting collagenase I into bilateral Achilles tendons. A total of 28 injured rats were injected with a hUC-MSC solution or normal saline into bilateral tendons twice and sampled after 4 weeks for histological staining, gene expression analysis, transmission electron microscope assay and biomechanical testing analysis. Results: The results revealed better histological performance and a larger collagen fiber diameter in the MSC group. mRNA expression of TNF-α, IL-1ß and MMP-3 was lower after MSC transplantation. Early MSC delivery promoted collagen I and TIMP-3 synthesis, and strengthened tendon toughness. Conclusion: hUC-MSCs demonstrated a therapeutic effect in treating collagenase-induced Achilles tendinopathy, particularly in the early stage of tendinopathy.

Characteristic Inflammatory Biomarkers in an Equine Model of Persistent Synovitis Induced By the Intra-Articular Administration of Monoiodoacetic Acid.

Persistent synovitis damages the articular cartilage in horses. To evaluate the effectiveness of treatment for synovitis using a model induced by intra-articular administration of monoiodoacetic acid (MIA), it is necessary to identify inflammatory biomarkers characteristic of the MIA model. Synovitis was induced by administering MIA into the unilateral antebrachiocarpal joints of five horses, and saline was injected into the contralateral joints as a control on day 0. Clinical and ultrasonographic examinations and synovial fluid collection were performed on days 0, 1, 2, 7, 14, 21, 28, and 35. Leukocyte, lactate dehydrogenase (LDH), tumor necrosis factor-α (TNF-α), interleukin-1 receptor antagonist (IL-1Ra), interleukin-6 (IL-6), and transforming growth factor-ß1 (TGF-ß1) concentrations in the synovial fluid were measured. Synovium was obtained after euthanasia on day 42 and histologically examined before quantification of the gene expression of inflammatory biomarkers by real-time PCR. Acute inflammatory symptoms persisted for approximately 2 weeks before returning to control levels. However, some indicators of chronic inflammation remained elevated until day 35. On day 42, synovitis continued histologically, with osteoclasts. The expressions of matrix metalloproteinase 13 (MMP13), a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4), receptor activator of nuclear factor kappa-Β ligand (RANKL), and collagen type I α2 chain (Col1a2) were significantly higher in the MIA model than in the control. In the MIA model, representative inflammatory biomarkers in the chronic inflammatory stage were persistently expressed in both synovial fluid and tissue, suggesting that they may be useful for the assessment of the anti-inflammatory effect of drugs.

Ambient particulate matter exposure plus chronic ethanol ingestion exacerbates hepatic fibrosis by triggering the mitochondrial ROS-ferroptosis signaling pathway in mice.

BACKGROUND: Chronic ethanol ingestion causes persistent oxidative stresses in the liver, leading to hepatic injury and fibrosis, but the underlying mechanisms remain unclear. Recently, ambient particulate matter (PM) has been confirmed to aggravate high-fat diet-induced liver fibrosis by enhancing oxidative stress. Thus, we hypothesized that oxidative stress induced by ambient PM exposure increases the severity of liver fibrosis caused by ethanol ingestion. METHODS AND RESULTS: C57BL/6 mice were subjected to ambient PM inhalation, ethanol ingestion or ambient PM-plus-ethanol ingestion for 12 weeks. Oxidative stress, mitochondrial reactive oxygen species (MtROS), liver fibrosis and ferroptosis indicators in the liver were evaluated. In vitro, oxidative stress, MtROS, ferroptosis indicators, profibrotic molecules and fibrosis markers in hepatic stellate (LX-2) cells were also determined. We found that ethanol ingestion markedly elevated hepatic oxidative stress and MtROS levels, triggered hepatic ferroptosis, and induced liver fibrosis, along with upregulation of the profibrotic molecule TGF-ß1 and fibrosis marker collagen-I, in mice. Moreover, the combination of ambient PM and ethanol accelerated these adverse effects. Importantly, the combination of PM exposure and ethanol ingestion had a synergistic effect on these changes. In vitro, LX-2 cells activated with PM2.5 alone or combined with ethanol showed upregulation of TGF-ß1 and collagen-I. In addition, the levels of MtROS, the oxidative stress marker 4-hydroxynonenal (4-HNE) and ferroptosis-related proteins and the GSH/GSSG ratio were significantly increased in PM2.5 plus ethanol-treated LX-2 cells. After pretreatment with a MtROS scavenger (Mito-TEMPO), we found that Mito-TEMPO treatment inhibited ferroptosis and oxidative stress in PM2.5 plus ethanol-treated LX-2 cells. Furthermore, a specific ferroptosis inhibitor (Fer-1) decreased the levels of ferroptosis-related proteins and profibrotic molecules in activated LX-2 cells co-exposed to PM2.5 and ethanol. CONCLUSION: In this study, we revealed that ambient PM exposure induced profibrotic effects and that combined exposure to ambient PM and chronic ethanol ingestion exacerbated hepatic fibrosis, which may trigger ferroptosis by increasing MtROS, thereby activating hepatic stellate cells.

Ligustrazine Attenuates Liver Fibrosis by Targeting miR-145 Mediated Transforming Growth Factor-ß/Smad Signaling in an Animal Model of Biliary Atresia.

To investigate therapeutic target for ligustrazine during liver fibrosis in an ethanol-induced biliary atresia rat model and transforming growth factor-ß (TGF-ß) induced hepatic stellate cell activation cell model, and the underlying mechanism, a total of 30 rats were randomly assigned into five groups (n = 6 per group): control, sham, ethanol-induced biliary atresia model, model plus pirfenidone, and model plus ligustrazine groups. The liver changes were assessed using H&E and Masson staining and transmission electron microscopy. Expression of miR-145 and mRNA and protein levels of TGF-ß/smads pathway-related proteins were detected. HSC-T6 cells were infected with LV-miR or rLV-miR-145 in the presence or absence of SMAD3 inhibitor SIS3 and treated with 2.5 ng/ml TGF-ß1 and then with ligustrazine. Collected cells were subjected to detect the expression of miR-145 and mRNA and protein expression levels of TGF-ß/smads pathway-related proteins. Ligustrazine rescued liver fibrogenesis and pathology for ethanol-caused bile duct injury, revealed by decreased α-smooth muscle actin and collagen I expression and liver tissue and cell morphology integrity. Further experiments showed that ligustrazine inhibited intrinsic and phosphorylated Smad2/3 protein expression and modification. Similar results were obtained in cells. In addition, ligustrazine altered miR-145 expression in both animal and cell models. Lentivirus mediated miR-145 overexpression and knockdown recombinant virus showed that miR-145 enhanced the TGF-ß/Smad pathway, which led to hepatic stellate cell activation, and ligustrazine blocked this activation. This work validated that ligustrazine-regulated miR-145 mediated TGF-ß/Smad signaling to inhibit the progression of liver fibrosis in a biliary atresia rat model and provided a new therapeutic strategy for liver fibrosis. SIGNIFICANCE STATEMENT: With an ethanol-induced biliary atresia rat model, ligustrazine was found to rescue liver fibrogenesis and pathology for ethanol caused bile duct injury, revealed by decreased α-smooth muscle actin and collagen I expression and liver tissue and cell morphology integrity. Furthermore, we found ligustrazine upregulated miR-145 expression and inhibited TGF-ß/SMAD signaling pathway both in vivo and in vitro. In addition, overexpression and knockdown of miR-145 confirmed that miR-145 is involved in the ligustrazine inhibition of liver fibrosis through the TGF-ß/SMAD signaling pathway.

Collagen I Induces Preeclampsia-Like Symptoms by Suppressing Proliferation and Invasion of Trophoblasts.

Preeclampsia is a common obstetric disorder affecting 2-8% of pregnancy worldwide. Fibrosis is an important histological change occurring in preeclamptic placenta, and might depend on the excess deposition of collagen I. However, the role of fibrotic placenta and collagen I in the pathogenesis of preeclampsia remains unclear. Therefore, we analyzed the collagen deposition and the expression of Collagen I in human placenta by Masson staining, Sirius red staining and western blotting. Further, the role of collagen I in preeclampsia pathogenesis was studied in C57BL/6 mice. HTR-8/SVneo cells were used to investigate the mechanisms underlying the effects of collagen I in trophoblasts by transcriptome sequencing and pharmacological agonists. Human preeclamptic placenta exhibited a significantly higher degree of fibrosis in stem villi and terminal villi than normal placenta, and was characterized by collagen I deposition. In vivo, a single injection of collagen I on gestational day 0.5 led to an increase in systolic pressure of pregnant mice from gestational days 4.5-17.5, to a decrease in weight and number of embryos, and to enhanced placental collagen I expression and degree of fibrosis compared with control mice. In vitro, collagen I attenuated the proliferation and invasion of HTR-8SV/neo cells. This effect could be reversed by treatment with agonists of ERK and ß-catenin. Moreover, transcriptome sequencing demonstrated that signaling pathways related to cell proliferation and invasion were significantly downregulated in HTR-8SV/neo cells. Thus, we propose that collagen I induced preeclampsia-like symptoms by suppressing the proliferation and invasion of trophoblasts through inhibition of the ERK phosphorylation and WNT/ß-catenin signaling pathways. Our findings could pave the way to the discovery of small-molecule inhibitors for preeclampsia treatment and future studies with larger sample size are required.

Cell-free cartilage repair in large defects of the knee: increased failure rate 5 years after implantation of a collagen type I scaffold.

INTRODUCTION: Cartilage defects of the knee remain a challenging problem in orthopedic surgery despite the ongoing improvements in regenerative procedures such as the autologous chondrocyte transplantation. Due to the lack of donor-site morbidity and the single-stage procedure cell-free scaffolds are an interesting alternative to cell-based procedures. But as currently mid- and long-term data are lacking, the aim of the present study was to present mid-term clinical, radiological and histological results of a cell-free collagen type I scaffolds for cartilage repair. MATERIALS AND METHODS: Twenty-eight patients were followed prospectively. Clinical evaluation using patient-reported outcome measures (KOOS, IKDC; VAS for pain, Tegner score for activity) as well as radiologic evaluation of the repair tissue (MOCART) was performed at 1 year, 2 years and 5 years. Histologic evaluation of the repair tissue was done in case of revision surgery using the ICRS II score for human cartilage repair. RESULTS: In these large cartilage defects with a mean defect size of 3.7 ± 1.9 cm2, clinical failure necessitating revision surgery was seen in 5 of 28 patients (18%). While the remaining patients showed good-to-excellent clinical results (KOOS, IKDC, VAS, Tegner), the radiologic appearance of the repair tissue showed a reduction of the MOCART score between the 2- and 5-year follow-up. Histologic evaluation of the repair tissue showed a cartilage-like appearance with no signs of inflammation or cell death but an overall medium tissue quality according to the ICRS II Score. CONCLUSION: The use of this cell-free collagen type I scaffold for large defects showed increased wear of the repair tissue and clinical failure in 18% of cases at 5-year follow-up.

Oral Intake of Low-Molecular-Weight Collagen Peptide Improves Hydration, Elasticity, and Wrinkling in Human Skin: A Randomized, Double-Blind, Placebo-Controlled Study.

Collagen-peptide supplementation could be an effective remedy to improve hydration, elasticity, and wrinkling in human skin. The aim of this study was to conduct a double-blind, randomized, placebo-controlled trial to clinically evaluate the effect on human skin hydration, wrinkling, and elasticity of Low-molecular-weight Collagen peptide (LMWCP) with a tripetide (Gly-X-Y) content >15% including 3% Gly-Pro-Hyp. Individuals (n = 64) were randomly assigned to receive either placebo or 1000 mg of LMWCP once daily for 12 weeks. Parameters of skin hydration, wrinkling, and elasticity were assessed at baseline and after 6 weeks and 12 weeks. Compared with the placebo group, skin-hydration values were significantly higher in the LMWCP group after 6 weeks and 12 weeks. After 12 weeks in the LMWCP group, visual assessment score and three parameters of skin wrinkling were significantly improved compared with the placebo group. In case of skin elasticity, one parameter out of three was significantly improved in the LMWCP group from the baseline after 12 weeks, while, compared with the placebo group, two parameters out of three in the LMWCP group were higher with significance after 12 weeks. In terms of the safety of LMWCP, none of the subjects presented adverse symptoms related to the test material during the study period. These results suggest that LMWCP can be used as a health functional food ingredient to improve human skin hydration, elasticity, and wrinkling.

Prevention of postcardiopulmonary bypass pericardial adhesions by a new resorbable collagen membrane.

Reduction in mediastinal adhesions is an issue in cardiac surgery. To evaluate a porcine-bioengineered collagen membrane (Cova™ CARD) intended to promote tissue regeneration, 18 sheep underwent a sternotomy and a 30 min period of cardiopulmonary bypass. They were divided into three equal groups: pericardium left open, placement of an e-polytetrafluoroethylene membrane (Preclude(®)) taken as a non-absorbable substitute comparator and placement of the absorbable Cova™ CARD membrane. Four months thereafter, the study animals underwent repeat sternotomy and were macroscopically assessed for the degree of material resorption and the intensity of adhesions. Explanted hearts were evaluated blindly for the magnitude of the inflammatory response, fibrosis and epicardial re-mesothelialization. The bioengineered membrane was absorbed by 4 months and replaced by a loosely adherent tissue leading to the best adhesion score. There was no inflammatory reaction (except for a minimal one in an animal). Fibrosis was minimal (P = 0.041 vs Preclude(®)). The highest degree of epicardial re-mesothelialization, albeit limited, was achieved by the bioengineered group in which five of six sheep demonstrated a new lining of mesothelial cells in contrast to two animals in each of the other groups. This collagen membrane might thus represent an attractive pericardial substitute for preventing post-operative adhesions.

[The curative effect and safety of collagen sponge with different pH and content of protein implanted into orthopedic bone defect].

This paper presents a 3mm fracture model of radial bone of rabbits using operation method. The bone defect was treated with collagen sponge with different pH and different content. After the operation, the body temperature, blood routine measurement, serum antibody, X-ray examination and histological observation in each group were examined to evaluate and study the curative effect and safety of collagen sponge. Collagen sponge had a good result of safety, but there was slightly change in blood routine, serum antibody, and histological observation, etc, with the pH changing and different content of collagen. The results showed that there was no obvious influence of safety to tissues after treatment of the collagen sponge at different pH implanted into bone defect. Collagen sponge at lower pH could promote the healing of bone defect partly, while the safety of collagen sponge with lower content was better.

Differences in chemical composition and internal structure influence systemic host response to implants of biomaterials.

In reconstructive surgery, implantable devices are used to supply a missing function. In tissue engineering, biomaterials serve to guide and eventually deliver cells and/or molecules where a tissue regenerative response is needed. The host organism always reacts to implants of any biomaterial, in some instances even triggering a local cascade of events called the foreign body response (FBR), whose mechanisms are well defined. What has yet to be completely unraveled are the biomarkers systemically mirroring the FBR and the regeneration processes, which would be helpful for assessing the therapeutic efficacy of the bioscaffold. Our goal was to identify a biomarker fingerprint of the systemic reaction of host response to bioscaffold implants. Different biomaterials chosen for their osteoconductive properties, including collagen, hydroxyapatite, in foam or granules, and poly-ε-caprolactone, were implanted in immunocompetent mice. We analyzed serum concentrations of cells and cytokines involved in the inflammatory/immune response, and the histological features of grafts. Within two weeks after implantation, a wave of proinflammatory cytokines was flowing in the blood stream and the concentration of blood cells changed, revealing specific patterns depending on the chemistry and structure of the implanted biomaterials. Cells secreting pro-inflammatory, chemoactractant, and pro-angiogenic cytokines required for the early events in tissue repair were locally recruited because of the presence of a bioscaffold.

A multicenter, randomized, double-blind, placebo-controlled trial of oral type I collagen treatment in patients with diffuse cutaneous systemic sclerosis: I. oral type I collagen does not improve skin in all patients, but may improve skin in late-phase disease.

OBJECTIVE: To investigate the safety and efficacy of oral bovine type I collagen (CI) treatment in patients who have had diffuse cutaneous systemic sclerosis (dcSSc; scleroderma) for

Cellular and humoral responses to collagen-polyvinylpyrrolidone administered during short and long periods in humans.

Collagen, particularly type I, and its related derivatives have been extensively employed in many areas of pharmacology. The present study was performed to determine the safety of collagen-polyvinylpyrrolidone (collagen-PVP) by in vitro and in vivo studies. Sera and peripheral blood cells from healthy donors without treatment and patients treated with collagen-PVP were evaluated. We observed that the biodrug does not stimulate lymphoproliferation or DNA damage in vitro, nor does it induce human anti-porcine type I collagen or anti-collagen-PVP antibodies in vivo. Furthermore, no hepatic or renal metabolic dysfunctions were observed when collagen-PVP was administered by intradermal or intramuscular routes in short- or long-term treatments. In conclusion, the present work shows that no cellular damage or immunological adverse effects (cellular and humoral) occurred during collagen-PVP treatment, even after more than 400 weeks of consecutive administrations.

Dr. Gary J. Becker young investigator award: comparison of small-diameter type 1 collagen stent-grafts and PTFE stent-grafts in a canine model--work in progress.

PURPOSE: To report an in-progress experiment in a canine model in which two types of small-diameter stent-grafts-one constructed of polytetrafluoroethylene (PTFE) and the other of a new, type 1 collagen material-were compared regarding vessel patency, intimal hyperplasia formation, and tissue reaction. MATERIALS AND METHODS: Six mongrel dogs weighing 30-35 kg were used. Stent-grafts of 4-mm diameter and 20-mm length were constructed with use of balloon-expandable stainless-steel stents wrapped with either PTFE or a new type 1 collagen graft. Stent-grafts were placed in deep femoral arteries bilaterally (PTFE on one side, collagen on the other). Animals were followed for 2 weeks (n = 2), 6 weeks (n = 2), or 12 weeks (n = 2). Percent stenosis based on angiographic findings as well as thickness and area of neointimal hyperplasia were compared at each time point and compared with use of the Student t test. RESULTS: All devices were patent in the immediate postimplantation period. Five of six collagen stent-grafts and five of six PTFE implants were patent at follow-up. In-stent stenosis was undetectable angiographically in all five patent collagen stent-grafts. All five patent PTFE stent-grafts showed demonstrable in-stent stenosis (10%-60%), indicating a trend toward improved patency in collagen stent-grafts versus PTFE stent-grafts (P = .07). Neointimal hyperplasia was absent at 2 weeks in the collagen stent-grafts. Neointimal thickness increased to a maximum of 360 microm at 12 weeks in the collagen stent-grafts. For PTFE stent-grafts, neointimal hyperplasia was present in all samples and reached a maximum of 770 microm at 12 weeks (P = .03). CONCLUSIONS: Even in small-diameter vessels, type 1 collagen stent-grafts demonstrate excellent patency rates and favorable histologic findings. The type 1 collagen stent-graft technology merits further developmental efforts in preclinical models.