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Background: To evaluate the safety of using fluoroquinolones in pediatric population in Taiwan., Methods: Patients aged 0~18 years old with fluoroquinolones prescriptions ≥5 consecutive days during year 2000 to 2013 were selected from the National Health Insurance Research Database, 4-time case number were selected as controls. We evaluated the patient's outcome after the use of fluoroquinolones by reviewing a newly diagnosis of the following collagen-associated adverse events by International Classification of Diseases, Ninth Revision, Clinical Modification codes, covering tendons rupture, retinal detachments, gastrointestinal tract perforation, aortic aneurysm or dissection., Results: Of the enrolled patients (n = 167,105), collagen-associated adverse effects developed in 85 cases (0.051%) in 6-month tracking, including 0.051% in the fluoroquinolones study cohort (17 in 33,421) and 0.051% (68 in 133,684) in the fluoroquinolones free comparison cohort. The crude hazard ratio for collagen-associated adverse events in the fluoroquinolones group was 0.997 (0.586-1.696; p = 0.990). After adjusting for age, sex, catastrophic illness, low-income household, seasons, levels of urbanization, and healthcare, the corrected hazard ratio in 6-month tracking with FQs was 1.330 (95% CI; 0.778-2.276; p = 0.255)., Conclusions: There is no significant difference of collagen-associated adverse effects between fluoroquinolones group and fluoroquinolones free group from our data. We propose that fluoroquinolones for pediatric population in clinical practice may be not so harmful as previous references reported.

Bone and teeth are derived from intrafibrillarly mineralized collagen fibrils as the second level of hierarchy. According to polymer-induced liquid-precursor process, using amorphous calcium phosphate precursor (ACP) is able to achieve intrafibrillar mineralization in the case of bone biomineral in vitro. Therefore, ACP precursors might be blended with any osteoconductive scaffold as a promising bone formation supplement for in-situ remineralization of collagens in bone. In this study, mesoporous silica nanoparticles with carboxyl-functionalized groups and ultra large-pores have been synthesized and used for the delivery of liquid like biomimetic precursors (ACP). The precursor delivery capacity of the nanoparticles was verified by the precursor release profile and successful mineralization of 2D and 3D collagen models. The nanoparticles could be completely degraded in 60 days and exhibited good biocompatibility as well. The successful translational strategy for biomineralization precursors showed that biomineralization precursor laden ultra large pore mesoporous silica possessed the potential as a versatile supplement in demineralized bone formation through the induction of intrafibrillar collagen mineralization.

Objective: to evaluate the effects of arginine on abdominal wall healing in rats., Methods: we submitted 20 Wistar rats to laparotomy and divided them into two groups, arginine and control, which then received, respectively, daily intraperitoneal treatment with arginine (300mg/kg/day) and weight-equivalent phosphate buffered solution, during five days. On the seventh postoperative day, we collected blood and scar wall samples from both groups. We evaluated serum nitrate and nitrite levels, wound evolution by tissue hydroxyproline dosages, granulation tissue formation, percentage of mature and immature collagen, myofibroblast density and angiogenesis. We used the ANOVA and the Student's t tests with p=0.05 for comparisons between groups., Results: there were no significant differences between the groups studied for nitrate and nitrite (p=0.9903), tissue hydroxyproline (p=0.1315) and myofibroblast density (p=0.0511). The arginine group presented higher microvascular density (p=0.0008), higher percentage of type I collagen (p=0.0064) and improved granulation tissue formation, with better angiofibroblastic proliferation rates (p=0.0007) and wound edge reepithelization (p=0.0074)., Conclusion: in the abdominal wall healing evaluation of Wistar rats under arginine treatment, there was no change in serum nitrate and nitrite levels, total collagen deposition and myofibroblast density. There was an increase in type I collagen maturation, microvascular density and improvement in scar granulation tissue formation by better edge reepithelization and angiofibroblastic proliferation.

It is currently challenging to eradicate cancer. In the case of solid tumors, the dense and aberrant extracellular matrix (ECM) is a major contributor to the heterogeneous distribution of small molecule drugs and nano-formulations, which makes certain areas of the tumor difficult to treat. As such, much research is devoted to characterizing this matrix and devising strategies to modify its properties as a means to facilitate the improved penetration of drugs and their nano-formulations. This contribution presents the current state of knowledge on the composition of normal ECM and changes to ECM that occur during the pathological progression of cancer. It also includes discussion of strategies designed to modify the composition/properties of the ECM as a means to enhance the penetration and transport of drugs and nano-formulations within solid tumors. Moreover, a discussion of approaches to image the ECM, as well as ways to monitor changes in the ECM as a function of time are presented, as these are important for the implementation of ECM-modifying strategies within therapeutic interventions. Overall, considering the complexity of the ECM, its variability within different tissues, and the multiple pathways by which homeostasis is maintained (both in normal and malignant tissues), the available literature - while promising - suggests that improved monitoring of ECM remodeling in vivo is needed to harness the described strategies to their full potential, and match them with an appropriate chemotherapy regimen., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Bacterial biofilms on wounds impair the healing process and often lead to chronic wounds. Chitosan is a well-known biopolymer with antimicrobial and anti-biofilm effects. S-nitrosoglutathione (GSNO) has been identified as a promising nitric oxide (NO) donor to defend against pathogenic biofilms and enhance wound healing activities. In this study, we prepared NO-releasing chitosan film (CS/NO film) and evaluated its anti-biofilm activity and in vivo wound healing efficacy against methicillin-resistant Staphylococcus aureus (MRSA) biofilm-infected wounds in diabetic mice. The in vitro release study showed sustained release of NO over 3 days in simulated wound fluid. The CS/NO film significantly enhanced antibacterial activity against MRSA by > 3 logs reduction in bacterial viability. Moreover, CS/NO film exhibited a 3-fold higher anti-biofilm activity than the control and CS film. In in vivo MRSA biofilm-infected wounds, the CS/NO film-treated group showed faster biofilm dispersal, wound size reduction, epithelialization rates, and collagen deposition than the untreated and CS film-treated groups. Therefore, the CS/NO film investigated in this study could be a promising approach for the treatment of MRSA biofilm-infected wounds., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Wound healing is a multicellular process that involves the coordinated efforts of several cell types, including keratinocytes, fibroblasts, and endothelial cells. This process is also regulated by an equally complex signaling network involving numerous growth factors, cytokines, and chemokines. The mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of cell growth, proliferation, and differentiation. A recent study showed that mTORC1 activation in epithelial cells dramatically enhanced epithelial cell proliferation, migration, and cutaneous wound healing; however, the roles of mTORC1 in fibroblasts during wound healing remain unknown. Here, we generated genetically mutated mice with activated mTORC1 in fibroblasts by conditionally deleting the mTORC1 inhibitor, TSC1. Activation of mTORC1 in fibroblasts significantly increased fibroblastic cell proliferation and contractile α-smooth muscle actin expression, thus promoting wound closure. Elevated mTORC1 activity also adversely induced excessive collagen production, leading to excessive scaring and fibrosis. Importantly, both accelerated wound healing and fibrotic phenotypes were largely reversed by the mTORC1 inhibitor, rapamycin. These observations were also replicated in primary human dermal fibroblasts. These results collectively demonstrated that mTORC1 activity in skin fibroblasts was a critical orchestrator in cutaneous wound healing and scarring., (© 2019 by the Wound Healing Society.)

Collagen is the most abundant secreted protein in vertebrates and persists throughout life without renewal. The permanency of collagen networks contrasts with both the continued synthesis of collagen throughout adulthood and the conventional transcriptional/translational homeostatic mechanisms that replace damaged proteins with new copies. Here, we show circadian clock regulation of endoplasmic reticulum-to-plasma membrane procollagen transport by the sequential rhythmic expression of SEC61, TANGO1, PDE4D and VPS33B. The result is nocturnal procollagen synthesis and daytime collagen fibril assembly in mice. Rhythmic collagen degradation by CTSK maintains collagen homeostasis. This circadian cycle of collagen synthesis and degradation affects a pool of newly synthesized collagen, while maintaining the persistent collagen network. Disabling the circadian clock causes abnormal collagen fibrils and collagen accumulation, which are reduced in vitro by the NR1D1 and CRY1/2 agonists SR9009 and KL001, respectively. In conclusion, our study has identified a circadian clock mechanism of protein homeostasis wherein a sacrificial pool of collagen maintains tissue function.

The use of specially designed wound dressings could be an important alternative to facilitate the healing process of wounds in the hyperglycemic state. Biocompatible dressings combining chitosan and alginate can speed up wound healing by modulating the inflammatory phase, stimulating fibroblast proliferation, and aiding in remodeling phases. However, this biomaterial has not yet been explored in chronic and acute lesions of diabetic patients. The aim of this study was to evaluate the effect of topical treatment with a chitosan-alginate membrane on acute skin wounds of hyperglycemic mice. Diabetes mellitus was induced by streptozotocin (60 mg · kg-1 · day-1 for 5 days, intraperitoneally) and the cutaneous wound was performed by removing the epidermis using a surgical punch. The results showed that after 10 days of treatment the chitosan and alginate membrane (CAM) group exhibited better organization of collagen fibers. High concentrations of interleukin (IL)-1α, IL-1β, granulocyte colony-stimulating factor (G-CSF), and tumor necrosis factor-alpha (TNF-α) were detected in the first and second days of treatment. G-CSF and TNF-α level decreased after 5 days, as well as the concentrations of TNF-α and IL-10 compared with the control group (CG). In this study, the inflammatory phase of cutaneous lesions of hyperglycemic mice was modulated by the use of CAM, mostly regarding the cytokines IL-1α, IL-1β, TNF-α, G-CSF, and IL-10, resulting in better collagen III deposition. However, further studies are needed to better understand the healing stages associated with CAM use.

Background: Manipulating intestinal microbiota with probiotics might stimulate skin response. Understanding all stages of the healing process, as well as the gut-skin-healing response can improve the skin healing process., Aim: To evaluate the effect of perioperative oral administration of probiotics on the healing of skin wounds in rats., Methods: Seventy-two Wistar male adult rats were weighed and divided into two groups with 36 each, one control group (supplemented with oral maltodextrin 250 mg/day) and one probiotic group (supplemented with Lactobacillus paracasei LPC-37, Bifidobacterium lactis HN0019, Lactobacillus rhamnosus HN001, Lactobacillus acidophilus NCFM® at a dose of 250 mg/day), both given orally daily for 15 days. The two groups were subsequently divided into three subgroups according to the moment of euthanasia: in the 3rd, 7th and 10th postoperative days., Results: There were no significant changes in weight in both groups. Wound contraction was faster in probiotic group when compared to the controls, resulting in smaller wound area in the 7th postoperative day. As for histological aspects, the overall H&E score was lower in the probiotic group. The probiotic group showed increased fibrosis from 3rd to the 7th postoperative day. The type I collagen production was higher in the probiotic group at the 10th postoperative day, and the type III collagen increased in the 7th., Conclusion: The perioperative use of orally administrated probiotic was associated with a faster reduction of the wound area in rats probably by reducing the inflammatory phase, accelerating the fibrosis process and the deposition of collagen.

Background: There are many collagen-stimulating fillers, including calcium hydroxyapatite, polycaprolactone (PCL), and poly-L-lactic acid (PLLA), and other materials have been tested. Polydioxanone (PDO) has recently been used as absorbable thread-lifting material due to its collagen-forming effects. PDO in powdered form is expected to be a good material for collagen-producing fillers., Objectives: To evaluate the collagen-producing effects of powdered PDO injection compared with PLLA injection in a murine model., Materials and Methods: Powdered PDO mixed with sodium carboxymethyl cellulose, PLLA, and phosphate-buffered saline was injected on dorsal skin of 8-week-old rat. Tissue samples were obtained 1, 2, and 12 weeks after the procedures for histopathologic review and for real-time PCR to quantify collagen and tissue growth factors., Results: Both PLLA and powdered PDO injections induced granulomatous reactions. Collagen type 1, collagen type 3, TGF-β1, TGF-β2, and TGF-β3 showed increases 2 weeks after injection but decreased 12 weeks after injection for both powdered PDO and PLLA., Conclusion: Our results suggested that powdered PDO injection induces collagen formation more effectively than PLLA injection. Therefore, PDO can be a good option for forming collagen., (© 2019 Wiley Periodicals, Inc.)

Immunological factors play important roles in the occurrence of hypertrophic scars. Imiquimod can be used as an immunosuppressive agent to regulate the function of T-helper (Th) cell subsets Th1 and Th2. In this article, we explored the impact of imiquimod on scar hyperplasia through Th cells. A rabbit ear hypertrophic scar model was built. Four round wounds were cut in each rabbit's ears ventrally with a diameter of 1 cm and bilateral symmetry. All the right ear wounds were treated with 5% imiquimod cream. The blank control group contained all the left ear wounds, which were treated with Vaseline ointment at the same time. Haematoxylin and eosin and Masson staining showed that imiquimod collagen deposition was significantly reduced compared with the control group, scar index (SEI) showed that the proliferative degree reached its peak on the 28th day after operation in blank group, and the degree of hyperplasia was significantly higher than that of the imiquimod group (P < .05). Real-time Polymerase chain reaction results showed that the imiquimod induced the expression of Th2 cell-related chemokines CCL2, CCL3, CCL5, CCL7, and CCL13 at each time point, which were significantly lower than that of the blank control group, and the expressions of Th1 cell-associated chemokines CXCL10 and CXCL12 at each time point was significantly higher than the blank control group (P < .05). Imiquimod can be used to regulate the expression of Th1 and Th2 cell-associated chemokines to control scar hyperplasia., (© 2019 Medicalhelplines.com Inc and John Wiley & Sons Ltd.)

Purpose: To evaluate the effects of tadalafil (TD) in preventing histological alterations of the corpus cavernosum caused by isolated lesions of cavernous nerve (ILCN) and artery (ILCA) in rats., Methods: Fifty male Wistar rats were randomly assigned in five groups: G1: control; G2: bilateral ILCN; G3: bilateral ILCA; G4: ILCN+TD; G5: ILCA+TD. The cavernous bodies were submitted to histomorphometry, immunohistochemistry and biochemical analysis., Results: Nerve density was significantly higher in G2 and G4 compared to control (22.62±2.84 and 19.53±3.47 vs. 15.72±1.82; respectively, p<0.05). Smooth muscle density was significantly lower in G2 and G3 in comparison to G1 (12.87±1.90 and 18.93±1.51 vs. 21.78±1.81, respectively; p<0.05). A significant decrease in the sinusoidal lumen area was observed in G2 compared to controls (5.01±1.62 vs. 9.88±3.66, respectively; p<0.05) and the blood vessel density was increased in G2 and G3 (29.32±4.13 e 20.80±2.47 vs. 10.13±2.71, p<0.05). Collagen density was higher in G3 compared to G1 (93.76±15.81 vs. 64.59±19.25; p<0.05)., Conclusions: Histomorphometric alterations caused by ILCN were more intense than those produced by vascular injury, but the collagen analyses showed more fibrosis in animals with ILCA. TD was effective in preventing the majority of the alterations induced by the periprostatic bundle injury.

Objective: To assess the preclinical efficacy and mechanism of action of an anti-CX 3 CL1 monoclonal antibody (mAb) in systemic sclerosis (SSc)., Methods: Cultured human dermal fibroblasts were used to evaluate the direct effect of anti-CX 3 CL1 mAb on fibroblasts. In addition, bleomycin-induced and growth factor-induced models of SSc were used to investigate the effect of anti-CX 3 CL1 mAb on leukocyte infiltration, collagen deposition, and vascular damage in the skin., Results: Anti-CX 3 CL1 mAb treatment significantly inhibited Smad3 phosphorylation (P < 0.05) and expression of type I collagen and fibronectin 1 (P < 0.01) in dermal fibroblasts stimulated with transforming growth factor β1 (TGFβ1). In the bleomycin model, daily subcutaneous bleomycin injection increased serum CX 3 CL1 levels (P < 0.05) and augmented lesional CX 3 CL1 expression. Simultaneous administration of anti-CX 3 CL1 mAb or CX 3 CR1 deficiency significantly suppressed the dermal thickness, collagen content, and capillary loss caused by bleomycin (P < 0.05). Injection of bleomycin induced expression of pSmad3 and TGFβ1 in the skin, which was inhibited by anti-CX 3 CL1 mAb. Further, the dermal infiltration of CX 3 CR1+ cells, macrophages (inflammatory and alternatively activated [M2-like] subsets), and CD3+ cells significantly decreased following anti-CX 3 CL1 mAb therapy (P < 0.05), as did the enhanced skin expression of fibrogenic molecules, such as thymic stromal lymphopoietin and secreted phosphoprotein 1 (P < 0.05). However, the treatment did not significantly reduce established skin fibrosis. In the second model, simultaneous anti-mCX 3 CL1 mAb therapy significantly diminished the skin fibrosis induced by serial subcutaneous injection of TGFβ and connective tissue growth factor (P < 0.01)., Conclusion: Anti-CX 3 CL1 mAb therapy may be a novel approach for treating early skin fibrosis in inflammation-driven fibrotic skin disorders such as SSc., (© 2019, American College of Rheumatology.)

Aim: We investigated the effects of Ba-Wei-Die-Huang-Wan (BWDHW) on ketamine-induced cystitis (KIC) in a rat model., Methods: Female Sprague-Dawley rats were distributed into three groups: control (saline), ketamine (25 mg/kg/day for 28 days), or ketamine (25 mg/kg/day for 28 days) plus BWDHW (90 mg/kg/day, started from day 14). Functional magnetic resonance imaging (fMRI), metabolic cage study, and cystometry were evaluated. Bladder histology was evaluated. Western blots of the bladder proteins were carried out., Results: Compared with controls, ketamine-treated rats showed stronger fMRI intensity in the periaqueductal gray area and bladder overactivity in the bladder functional study, but the ketamine/BWDHW-treated rats did not. Furthermore, ketamine breached the uroplakin III membrane at the apical surface of the urothelium, enhanced substance P spread over the urothelium, induced suburothelial hemorrhage and monocyte/macrophage infiltration, and caused interstitial fibrosis deposition. By contrast, the BWDHW-treated rats exhibited less substance P spread, lower suburothelial monocyte/macrophage infiltration, and lower interstitial fibrosis deposition. The ketamine group showed significant overexpression of neuroreceptors in the bladder mucosa (the transient receptor potential vanilloid 1 and M 2 - and M 3 -muscarinic receptors) and detrusor (M 2 - and M 3 -muscarinic receptors); inflammatory mediators in the detrusor (interleukin-1β [IL-1β], IL-6, tumor necrosis factor-α, nuclear factor-κB, cyclooxygenase-2, and intercellular adhesion molecule-1); and fibrogenesis molecules in the detrusor (transforming growth factor-β1, collagen I, collagen III, and fibronectin). However, no significant changes were noted between the ketamine/BWDHW and control groups., Conclusion: BWDHW could exert therapeutic effects by inhibiting the upregulation of neuroreceptors, modulating inflammatory mediators, suppressing fibrogenesis, and ameliorating bladder overactivity in rats with KIC., (© 2019 Wiley Periodicals, Inc.)

Third-degree or full-thickness burns refer to lesions that extend to the epidermis, dermis, and subcutaneous tissue. The pathophysiology of burn wounds is characterized by tissue inflammation, edema, and hypertrophic scarring. Farnesol is a natural 15-carbon organic compound that possesses many biological effects. We have previously demonstrated that farnesol gel exerts restorative actions on ultraviolet B (UVB)-caused sunburn in vivo. The in vitro results revealed that liposomal farnesol from 0.04mM to 0.8mM significantly enhanced collagen production by murine skin fibroblasts, whereas liposomal farnesol at high (0.8mM) and low concentration (0.04mM) did not show any suppressions on skin fibroblast proliferation. We treated third-degree burns on a rat model with a formulated gel composed of various ratios of 2% hydroxypropyl methylcellulose (HPMC) and 4mM liposomal farnesol for 7 and 14 days. On days 7 and 14 post wounding, histopathological observations revealed that the HPMC:farnesol gel ratios of 1:2 and 2:1 exerted the greatest tissue-repairing effects on the skin after third-degree burns compared with skin untreated or treated with a commercial burn gel and HPMC alone. These findings were consistent with the in vivo quantitative collagen-producing assay, wound healing scoring, and IL-6 Western blot results. These findings demonstrated that the fabricated liposomal farnesol gel is potentially able to promote wound healing after third-degree burns., (Copyright © 2019 Elsevier Ltd and ISBI. All rights reserved.)

The effect of teriparatide treatment on microdamage accumulation has yet to be examined in animal studies. The purpose of this study was to investigate the effect of once-weekly teriparatide treatment on bone microdamage accumulation and the relationship between microdamage parameters and bone mass, architecture, turnover, and collagen cross-linking in the lumbar vertebral trabecular bone of ovariectomized (OVX) cynomolgus monkeys. Female monkeys were divided into four groups (n = 18-20 per group): (1) SHAM group, (2) OVX group, (3) OVX with 1.2 µg/kg once-weekly teriparatide group (LOW group), (4) OVX with 6.0 µg/kg once-weekly teriparatide group (HIGH group). After 18 months, all animals were double-labeled with calcein for histomorphometry. L3 and L7 lumbar vertebrae were harvested and analyzed for differences in histomorphometry, microdamage, and collagen cross-linking. The iliac crest was also analyzed for differences in bone turnover. In the OVX group, cancellous bone mass was reduced and microdamage accumulation was increased as compared with the SHAM control. Once-weekly teriparatide at both doses prevented the decrease in bone mass and increase in microdamage accumulation, and improved the distribution of collagen cross-linkage types. Regression analyses indicated that decreased microdamage accumulation was associated with reduced non-enzymatic cross-link pentosidine rather than increased cancellous bone mass or enzymatic cross-links. These findings suggest that once-weekly teriparatide treatment decreases microdamage accumulation by recovering the balance in collagen cross-links.

Purpose: To evaluate the long-term results of corneal collagen cross-linking (CXL) with epithelium removal in patients with progressive keratoconus., Methods: This retrospective study included 27 eyes of 18 patients who underwent CXL surgery for progressive keratoconus between April 2009 and March 2012. Best-corrected visual acuity (BCVA), manifest refraction spherical equivalent (SE), maximum keratometry reading (K max), mean of the minimum and maximum keratometry readings (mean-K), central corneal thickness (CCT), and anterior and posterior elevation at the apex preoperatively and year 1, 3 and 6 were evaluated and compared. P values<0.05 were considered to be statistically significant., Results: Mean BCVA was 0.35±0.28 logMAR preoperatively and 0.23±0.20 logMAR 6 years after the procedure (P=0.01). Mean SE decreased from -4.3±2.45 diopters (D) to -3.91±2.12 D (P=0.03). Mean K max decreased from 49.6±3.2 D to 48.6±2.8 D (P=0.04), and mean-K decreased from 47.6±2.5D to 46.9±2.6 D (P=0.04). CCT decreased insignificantly from 466.5±32.1μm to 465.4±26.6μm (P=0.65). Mean anterior elevation at the apex decreased from 12.8±7.9 to 12±8.3μm (P=0.04), and posterior elevation decreased from 27.1±17.4μm to 26.8±18.5μm (P=0.27). Mean-K, max-K, BCVA and CCT showed no change over the last 5 years. After the first year, no significant change was observed in BCVA, SE, max-K, mean-K and CCT, which were therefore considered stable. On the other hand, anterior and posterior elevation readings continued to decrease up to 6 years after CXL., Conclusion: Based on our 6-year results, CXL can halt progression of keratoconus and reduce the need for keratoplasty., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Background: Long noncoding RNAs (lncRNAs) are emerging as important mediators of cardiac pathophysiology. The aim of the present study is to investigate the effects of lncR-30245, an lncRNA, on cardiac fibrogenesis and the underlying mechanism., Methods: Myocardial infarction (MI) and transforming growth factor (TGF)-β1 were used to induce fibrotic phenotypes. Cardiac fibrosis was detected by Masson's trichrome staining. Cardiac function was evaluated by echocardiography. Western blot, quantitative reverse transcription-polymerase chain reaction, and pharmacological approaches were used to investigate the role of lncR-30245 in cardiac fibrogenesis., Results: Expression of lncR-30245 was significantly increased in MI hearts and TGF-β1-treated cardiac fibroblasts (CFs). LncR-30245 was mainly located in the cytoplasm. Overexpression of lncR-30245 promoted collagen production and CF proliferation. Knockdown of lncR-30245 significantly inhibited TGF-β1-induced collagen production and CF proliferation. LncR-30245 overexpression inhibited the antifibrotic role of peroxisome proliferator-activated receptor (PPAR)-γ and increased connective tissue growth factor (CTGF) expression, whereas lncR-30245 knockdown exerted the opposite effects. Rosiglitazone, a PPAR-γ agonist, significantly inhibited lncR-30245-induced CTGF upregulation and collagen production in CFs. In contrast, T0070907, a PPAR-γ antagonist, attenuated the inhibitory effects of lncR-30245 small interfering RNA (siRNA) on TGF-β1-induced CTGF expression and collagen production. LncR-30245 knockdown significantly enhanced ejection fraction and fractional shortening and attenuated cardiac fibrosis in MI mice., Conclusion: Our study indicates that the lncR-30245/PPAR-γ/CTGF pathway mediates MI-induced cardiac fibrosis and might be a therapeutic target for various cardiac diseases associated with fibrosis., (Copyright © 2019 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved.)

Background: Polypropylene meshes cause severe complications in some patients. Materials that are biomechanically compatible and can better integrate into host tissues are urgently needed., Objective: To design an oestradiol-releasing electrospun poly-l-lactic acid (PLA) mesh and evaluate its ability to stimulate new extracellular matrix and new blood vessel formation., Design, Setting, and Participants: Human adipose derived mesenchymal cells (ADMSC) were isolated from fat. PLA meshes with micro- to nano-sized fibres containing 1%, 5%, and 10% oestradiol were constructed and used for in vitro and in vivo experiments., Intervention: The angiogenic potential of the fibrous meshes was evaluated using an in vivo chorioallantoic membrane and an in vitro chick aortic arch assays., Outcome Measurements and Statistical Analysis: Oestradiol release was measured fluorometrically. The effect of fibrous meshes on proliferation and extracellular matrix (ECM) production of ADMSC was assessed using immunohistology. Mechanical properties were tested using a tensiometer., Results and Limitations: The ultrastructure of the mesh was not affected by the inclusion of oestradiol and mechanical properties were only slightly modified. Oestradiol was released from PLA meshes over a 5-mo period. ADMSCs cultured on oestradiol-releasing PLA meshes produced more ECM involving collagen I, collagen III, and elastin. Oestradiol-releasing meshes doubled new blood vessel formation in the chorioallantoic membrane assay (p=0.001) and outgrowth of pro-angiogenic cells in the aortic arch assay (p=0.001). Further studies in longer-term animal models are required to confirm these results., Conclusions: Oestradiol-releasing PLA meshes increase ECM production and stimulate angiogenesis. As such, they are promising candidate materials to be used in pelvic floor repair and to improve the initial healing phase of a repair material following implantation., Patient Summary: In this study, we designed a tissue engineered material to be used to support weakened pelvic floor tissues in women to avoid the complications associated with current surgical mesh. Our results showed that this material can stimulate new blood vessel formation in simple chick assays and tissue production in vitro. Both properties should help with the integration of this material into patients' tissues and merit further study in physiologically relevant animal models., (Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.)

Purpose: To report our initial experience in the treatment of Acanthamoeba keratitis with accelerated corneal collagen cross-linking., Methods: Retrospective chart review of patients diagnosed with Acanthamoeba keratitis with progressive corneal melting who were treated with accelerated collagen cross-linking., Results: A total of 6 eyes (5 patients) were reviewed. All the patients received adjuvant therapy with moxifloxacin and chlorhexidine. In 4 cases, the ulcer healed with a mean interval to epithelialization of 108.8 days (range 59-217). In 2 eyes, there was a persistent neurotrophic ulcer. The melting was not progressive in any case, nor did any eye required emergency penetrating keratoplasy., Conclusion: This study suggests a beneficial effect of accelerated collagen cross-linking in cases of Acanthamoeba keratitis with corneal melting. Thus, collagen cross-linking may be considered as adjuvant treatment for Acanthamoeba keratitis.

Fibrosis is characterized by the excessive deposition of extracellular matrix and crosslinked proteins, in particular collagen and elastin, leading to tissue stiffening and disrupted organ function. Lysyl oxidases are key players during this process, as they initiate collagen crosslinking through the oxidation of the ε-amino group of lysine or hydroxylysine on collagen side-chains, which subsequently dimerize to form immature, or trimerize to form mature, collagen crosslinks. The role of LOXL2 in fibrosis and cancer is well documented, however the specific enzymatic function of LOXL2 and LOXL3 during disease is less clear. Herein, we describe the development of PXS-5153A, a novel mechanism based, fast-acting, dual LOXL2/LOXL3 inhibitor, which was used to interrogate the role of these enzymes in models of collagen crosslinking and fibrosis. PXS-5153A dose-dependently reduced LOXL2-mediated collagen oxidation and collagen crosslinking in vitro. In two liver fibrosis models, carbon tetrachloride or streptozotocin/high fat diet-induced, PXS-5153A reduced disease severity and improved liver function by diminishing collagen content and collagen crosslinks. In myocardial infarction, PXS-5153A improved cardiac output. Taken together these results demonstrate that, due to their crucial role in collagen crosslinking, inhibition of the enzymatic activities of LOXL2/LOXL3 represents an innovative therapeutic approach for the treatment of fibrosis., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Aim: To establish a method using Fourier Transform Infra-Red spectroscopy (FTIR) to characterize the nature and depth of changes in dentinal collagen following exposure to sodium hypochlorite (NaOCl) during root canal irrigation in an ex vivo model., Methodology: Fourier Transform Infra-Red spectroscopy was used to assess the changes in dentinal collagen when the root canal was exposed to NaOCl. The changes in dentinal collagen caused by NaOCl irrigation of root canals in transverse sections of roots, at 0.5 mm from the canal wall and 0.5 mm from the external root surface, were assessed by FTIR. The data were analysed using paired t-test with 5% significance level., Results: Fourier Transform Infra-Red spectroscopy confirmed that NaOCl exposure caused alterations in the chemistry and structure of collagen in dentine. FTIR spectra obtained from dentine surfaces and dentine adjacent to root canals exposed to NaOCl, all consistently showed degradation and conformational change of the collagen structure. FTIR data from the ex vivo model showed that the depth of effect of NaOCl extended to at least 0.5 mm from the canal wall., Conclusion: In extracted human teeth, NaOCl caused changes in dentinal collagen that were measurable by FTIR. In an ex vivo model, the depth of effect into dentine extended at least 0.5 mm from the canal wall., (© 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

This aim of this study was to assess the molecular mechanism of osteoporosis in schizophrenia patients with risperidone use. Here, we investigated the effects of risperidone on cellular proliferation and apoptosis of a preosteoblast cell line, MC3T3-E1. Cell viability and apoptotic rate of MC3T3-E1 were detected by cell counting kit-8 and flow cytometry at a serial dose of risperidone and at different time points, respectively. Bone transformation relevant gene serum osteocalcin (BGP), collagen 1, tumor necrosis factor-α (TNF-α), osteoprotegerin (OPG), and receptor activator of nuclear factor-κB ligand (RANKL) mRNA levels were determined by real-time PCR (qPCR). Their protein expression patterns were evaluated using western blot. The results revealed that risperidone dramatically inhibited MC3T3-E1 cell proliferation in a dose-dependent manner. It also significantly induced MC3T3-E1 cell apoptosis. TNF-α gene and protein levels were greatly enhanced after risperidone treatment. In contrast, BGP, collagen 1, OPG, and RANKL gene and protein levels were markedly downregulated. Our study indicated that risperidone suppressed MC3T3-E1 cell proliferation and induced apoptosis. It also regulated BGP gene and protein expression.

Microarchitectural features of collagen-rich extracellular matrices provide the mechanical foundation for tissue function and exhibit topographical cues that influence cellular behavior including proliferation, migration and protein expression. Preservation of tissue microarchitecture is required for accurate evaluation of tissue characteristics and pathology. It is unclear whether common tissue preservation methods possess equal ability to preserve microarchitecture. We investigated collagen microarchitecture in samples that had been flash frozen, fixed in formalin or preserved in RNAlater®, and which contained both collagen-rich and collagen-sparse regions. Fibrillar collagen organization was characterized using picrosirius red staining and second harmonic generation (SHG) microscopy. Maintenance of collagen fiber characteristics compared to the gold standard of flash freezing depended on both the method of preservation and the local collagen content of the tissue. Both formalin fixation and RNAlater® preserved collagen fiber characteristics similar to flash freezing in collagen-rich areas of the tissue, but not in collagen-sparse regions. Analysis using picrosirius red staining indicated preservation-dependent changes in overall tissue architecture and suprafibrillar organization. Together with considerations of cost, ease of use, storage conditions and ability to use the preserved tissue for RNA or protein analysis, our quantitative characterization of the effects of preservation method on collagen microarchitecture may help investigators select the most appropriate preservation approach for their needs.

Tendon rupture is a very frequent accident involving average people and high-performance athletes. Clinical studies describe tendon recovery as a painful and slow process involving different biochemical and histological events. Ascorbic acid (AA) is a potent antioxidant as well as an important cofactor for collagen synthesis. In the current study, we evaluated if local treatment with AA is able to promote tendon repair in tenotomized rats. Animals were submitted to Achilles tendon rupture followed by surgical suture. Control and AA groups received in loco injection of saline solution (0.9% NaCl) and 30 mM AA, respectively. Histological and functional recovery of Achilles tendon tissue was evaluated at 7, 14, and 21 days post-surgery. Hematoxylin/eosin staining and collagen fluorescence analysis showed intense disarrangement of tendon tissue in the saline group. Tenotomized animals also showed hypercellularity in tendon tissue compared with non-tenotomized animals. The Achilles functional index (AFI) showed a significant decrease of tendon functionality in tenotomized animals at 7, 14, and 21 days post-surgery. AA accelerated tissue organization and the recovery of function of the Achilles tendons. The beneficial effect of AA treatment was also observed in the organization of the collagen network. Data presented in the current work showed that in loco treatment with AA accelerated the recovery of injured Achilles tendon post-surgery.

Decellularization of native blood vessels is a promising technology to generate 3D biological scaffolds for vascular grafting. Blood vessel decellularization has been performed in previous studies under various experimental conditions, that complicates comparison and optimization of suitable protocols. The goal of this work was to systematically compare the decellularization and recellularization efficacy of 5 different protocols utilizing the detergents sodium dodecyl sulfate (SDS), sodium deoxycholate (SDC), CHAPS and TritonX-100 together with DNA-removing enzymes on porcine vena cava in a perfusion bioreactor setup. Additionally, we tested the effect of DNase on the extracellular matrix (ECM) properties. We found that all protocols could efficiently decellularize blood vessels. Mechanical strength, collagen preservation and ECM integrity were similar among all tested detergents, yet TritonX protocols required long-term DNase application for complete decellularization. However, TritonX-based protocols showed the greatest recellularization efficacy with HUVECs in vitro. Furthermore, we developed a novel protocol for TritonX which improved recellularization and reduced total process time and ECM stiffness compared to previous protocols. SDS, SDC and CHAPS based protocols had a lower recellularization potential. In conclusion, decellularization of blood vessels can be achieved with all tested reagents, but TritonX treated ECM can be most efficiently recellularized with endothelial cells., Competing Interests: The authors have read the journal’s policy and the authors of this manuscript have the following competing interests: RS is collaborating with the company VERIGRAFT AB, which seeks to commercialize tissue engineering products, including products that underwent decellularization processes. There are no patents, products in development or marketed products associated with this research to declare. VERIGRAFT AB did not offer funding in form of salaries or consumables, but provided lab space and equipment. The study was performed independently without funding by Verigraft, and Verigraft did not affect the study design, collection and evaluation of data, drawing of conclusion or reporting of results. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

In recent years, the use of collagen cross-linking with ultraviolet A and riboflavin is being developed universally due to its significance in the improvement of the mechanical strength of collagen fibers in clinical and basic research. It has been used in treating some corneal diseases including keratoconus, keratectasia and keratohelcosis. Thus, it might become a new treatment to prevent progressive myopia by strengthening scleral tissue. This article focuses on the effectiveness and safety of ultraviolet A-riboflavin cross-linking, and discusses the application prospect, focal points and problems related to cross-linking therapy. (Chin J Ophthalmol, 2018, 54:948 - 953) .

Objective: Matrix metalloproteinases (MMPs) and cysteine cathepsins (CCs) are two distinct enzymatic pathways responsible for the degradation of collagen fibrils in demineralized dentin. NaF and KF have been shown to inhibit salivary MMP-2, -9 and CCs. This study investigated the inhibitory effect of calcium fluoride (CaF 2 ) on the dentin matrix-bound MMPs and CCs., Design: Phosphoric acid (10%)-demineralized dentin beams (1 × 2×6 mm) were incubated at 37 °C in an 1 ml of artificial saliva (AS, control), or AS with 6, 12, 24, 48, 120. 179 and 238 mM F containing CaF 2 (n = 10/group) for 1, 7 and 21 days. All groups were further incubated in AS only for 6 months. Total MMP activity, dry mass loss, CTX and hydroxyproline (HYP) analyses were performed after each incubation. The beams were examined under scanning electron microscopy (SEM). MMP-2 and MMP-9 activities were screened with gelatin zymography. Data were analyzed by using ANOVA and Tukey HSD tests (p = .05)., Results: The total MMP activity was similar for all groups after 21 days and 6 months. After 21 days, the cumulative mass loss and CTX levels were lower compared to control for the CaF 2 ≥48 and CaF 2 ≥120 mM, respectively (p < .05). After 6 months, no significant difference was detected in the dry mass loss and CTX compared to the control (p > .05), whereas HYP level was higher with F 24 and 238 mM groups. CaF 2 -like minerals were observed on the beams under SEM. There was no gelatinase inhibition in zymography., Conclusion: CaF 2 does not prevent the degradation of demineralized dentin matrices due to the catalytic activity of MMPs and CCs., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Tumor initiation and growth is associated with significant changes in the surrounding tissue. During carcinoma progression, a global stiffening of the extracellular matrix is observed and is interpreted as a signature of aggressive invasive tumors. However, it is still unknown whether this increase in matrix rigidity promotes invasion and whether this effect is constant along the course of invasion. Here we have developed a biomimetic in vitro assay that enabled us to address the question of the importance of tissue rigidity in the chronology of tumor invasion. Using low concentrations of the sugar threose, we can effectively stiffen reconstituted collagen I matrices and control the stiffening in time with no direct effect on residing cells. Our findings demonstrate that, depending on the timing of its stiffening, the extracellular matrix could either inhibit or promote cancer cell invasion and subsequent metastasis: while matrix stiffening after the onset of invasion promotes cancer cell migration and tumor spreading, stiff matrices encapsulate the tumor at an early stage and prevent cancer cell invasion. Our study suggests that adding a temporal dimension in in vitro models to analyze biological processes in four dimensions is necessary to fully capture their complexity.

The aim of this study was to investigate the effects of pilocarpine-induced ciliary body constant contraction for a long time period on the elastic modulus and collagen in corneal and scleral tissues in the early developmental stage. Twelve one-month-old New Zealand white rabbits were randomly monocularly treated with pilocarpine to cause ciliary body constant contraction. After 1- and 2-months, the sclera tissues and the eyeballs were obtained to assess the cornea and three regions of the sclera-anterior, equatorial and posterior. The corneal tissues and the three regions of the scleral tissues were cut into strips for elastic modulus measurement using an Instron 5544, the size distribution of collagen fibrils was examined using electron microscopy, the samples were homogenized, and the concentration of hydroxyproline was measured to determine the collagen content. For corneal tissues, long-term pilocarpine-induced ciliary body constant contraction did not affect the elastic modulus and collagen. For scleral tissues, long-term pilocarpine-induced ciliary body constant contraction affected the elastic modulus, diameter of collagen fibrils and collagen content in the equatorial and posterior sclera, however, the anterior sclera were unaffected. These results suggested that pilocarpine-induced ciliary body contraction could affect the scleral structure and lead to deformation of the sclera and eyeball, thereby affecting visual functions function in the process of ocular emmetropization., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Objectives/hypothesis: Our laboratory recently described NR4A1 as an endogenous inhibitor of TGF-β-induced vocal fold (VF) fibrosis. Our prior report described the temporal expression of NR4A1 during VF healing in vivo and the effects of NR4A1 knockdown on fibroplastic cell activities in vitro. Based on these findings, we hypothesized that cytosporone-B (Csn-B), an NR4A1 agonist, may hold significant therapeutic potential., Study Design: In vitro., Methods: Human VF fibroblasts were exposed to TGF-β1+/-Csn-B. Expression of genes related to fibrosis were quantified. In addition, contraction was assayed as a surrogate for the fibrotic phenotype in our cell line., Results: TGF-B1 stimulated COL1A1 and ACTA2, as expected. Csn-B significantly downregulated TGF-β1-mediated upregulation of these genes (P = .009, P = .03, respectively). Csn-B had no effect on genes related to TGF-β/Smad signaling. Csn-B also decreased the TGF-β1-mediated contractile phenotype in our cells (P = .004)., Conclusions: NR4A1 is an endogenous inhibitor of fibrosis in the vocal folds and Csn-B, as an NR4A1 agonist, may evolve as an ideal, therapeutic candidate for this challenging condition., Level of Evidence: NA Laryngoscope, 128:E425-E428, 2018., (© 2018 The American Laryngological, Rhinological and Otological Society, Inc.)

BACKGROUND Idiopathic pulmonary fibrosis (IPF) is a deadly disease characterized by excessive collagen in the extracellular matrix (ECM) of the lungs. Collagen is the primary protein component of the ECM. However, the exact mechanisms underlying the formation and deposition of collagen in the ECM under normal and pathological conditions remain unclear. Previous studies showed that lysyl hydroxylase (LH) plays a crucial role in the formation of collagen. Minoxidil is an FDA-approved anti-hypertensive agent that inhibits LH that reduces fibrosis. In this study, we investigated the functional roles of LHs (LH1, LH2, and LH3) in pulmonary fibrosis and the anti-fibrotic effects of minoxidil. MATERIAL AND METHODS Patient serum samples were examined for their expression of procollagen-lysine, 2-oxoglutarate 5-dioxygenases (PLOD) 1-3, the genes encoding LH 1-3. Mice with bleomycin (BLM 2.5 mg/kg)-induced pulmonary fibrosis were administered a minoxidil solution (30 mg/kg) by oral gavage. RESULTS The PLOD mRNA levels were significantly higher in the IPF patients than in the healthy control subjects. Minoxidil suppressed the BLM-induced pulmonary fibrosis in vivo. These effects were associated with blocking TGF-β₁/Smad3 signal transduction and attenuating the expression and activity of LHs, resulting in decreased collagen formation, thus reducing the pulmonary fibrosis. The anti-fibrotic effects of minoxidil may be mediated through competitive inhibition of LHs activity, resulting in decreased pyridine cross-link formation and collagen production and deposition. CONCLUSIONS The results of this study suggest that LH represents a target to prevent or treat pulmonary fibrosis, and minoxidil may provide an effective agent to inhibit LHs.

Background: Pneumatic injections of non-cross-linked hyaluronic acid are effective in skin rejuvenation, however, the associated biophysical parameters and appearance have not been evaluated., Objectives: To determine the changes in skin biophysical parameters after facial pneumatic injections of non-cross-linked hyaluronic acid., Patients and Methods: Twenty-eight healthy female volunteers received pneumatic injections of non-cross-linked hyaluronic acid into the face for consecutive 5 weeks. Skin biophysical parameter assessment and clinical evaluation were performed using the CK Multi-Probe Adapter and Visia system. Five of the volunteers also underwent retroauricular skin biopsy before and after the last treatment. The skin tissues were all stained with Masson-trichrome, Verhoeff-van Gieson stain, and hematoxylin-eosin to evaluate the changes in collagen, elastic fibers, and the epidermis, before and after the last treatment., Results: Transepidermal water loss was significantly lower in week 4 (18.46 ± 4.70 g/h/m 2 ) than at the baseline (22.03 ± 7.15 g/h/m 2 , p < 0.05). Skin texture was better in week 4 (599.29 ± 354.32) than at the baseline (668.43 ± 342.55, p < 0.05). Skin pores also improved significantly at week 4 (934.07 ± 458.78) compared to the baseline (1024.57 ± 415.31, p < 0.05). Skin wrinkles were improved at the 3-month follow-up (29.29 ± 11.11) compared to the baseline (35.83 ± 16.05, p < 0.05)., Conclusion: Pneumatic injections of non-cross-linked hyaluronic acid improved skin TEWL, texture, pores, and wrinkles.

Background Aortic valve sclerosis ( AVS c), the early asymptomatic presentation of calcific aortic valve (AV) disease, affects 25% to 30% of patients aged >65 years. In vitro and ex vivo experiments with antioxidant strategies and antagonists of osteogenic differentiation revealed that AVS c is reversible. In this study, we characterized the underlying changes in the extracellular matrix architecture and valve interstitial cell activation in AVSc and tested in vitro and in vivo the activity of a clinically approved SOD (superoxide dismutase) mimic and redox-active drug MnTnBu OE -2-PyP 5+ ( BMX -001). Methods and Results After receiving informed consent, samples from patients with AVS c, AV stenosis, and controls were collected. Uniaxial mechanical stimulation and in vitro studies on human valve interstitial cells were performed. An angiotensin II chronic infusion model was used to impose AV thickening and remodeling. We characterized extracellular matrix structures by small-angle light scattering, scanning electron microscopy, histology, and mass spectrometry. Diseased human valves showed altered collagen fiber alignment and ultrastructural changes in AVS c, accumulation of oxidized cross-linking products in AV stenosis, and reversible expression of extracellular matrix regulators ex vivo. We demonstrated that MnTnBu OE -2-PyP 5+ inhibits human valve interstitial cell activation and extracellular matrix remodeling in a murine model (C57 BL /6J) of AVS c by electron microscopy and histology. Conclusions AVS c is associated with architectural remodeling despite marginal effects on the mechanical properties in both human and mice. MnTnBu OE -2-PyP 5+ controls AV thickening in a murine model of AVS c. Because this compound has been approved recently for clinical use, this work could shift the focus for the treatment of calcific AV disease, moving from AV stenosis to an earlier presentation ( AVS c) that could be more responsive to medical therapies.

The aim of this study was to evaluate the effect of acute administration of nicotine and ethanol on tooth movement in rats. Two hundred rats were divided into eight groups: S: saline; N: nicotine; E: ethanol; NE: nicotine and ethanol; SM: saline with tooth movement; NM: nicotine with tooth movement; EM: ethanol with tooth movement; and NEM: nicotine and ethanol with tooth movement. All the solutions were applied for 32, 44, or 58 days, according to the subgroup. Orthodontic movement (25 cN) was initiated 30 days after solution administration in the groups with tooth movement. The rats were euthanized 2, 14, or 28 days after initiation of tooth movement. Tooth sections were stained using picrosirius and tartrate-resistant acid phosphatase (TRAP). The data were compared by ANOVA using Tukey's HSD and Games-Howell. On day 28 of tooth movement, the NEM group had a lower percentage of type I collagen compared to the SM group (p = 0.0448), and the S group had a higher number of osteoclasts/μm2 compared to the N group (p = 0.0405). Nicotine and ethanol did not affect the tooth movement rate, regardless of induction of orthodontic movement. Nicotine influenced the number of osteoclasts by decreasing their quantity when dental movement was not induced. When nicotine was associated with ethanol, it interfered in the maturation of collagen fibers during orthodontic movement.

Continuous contraction of 3D skin equivalents in construction and use restricts their applications in clinical and pharmaceutical practices. So far, no effective method has been developed to inhibit such contraction. Hence, low cytotoxic cross-linkers, 1-ethyl-3-3-dimethylaminopropylcarbodiimide hydrochloride (EDC) and genipin, are investigated to reduce the contraction in this study. As found, both genipin and EDC at 0.2 and 0.4 mmol/L are nontoxic to collagen-entrapped fibroblasts and upregulate the extracellular matrix expression of fibroblasts in cross-linked collagen. Particularly, collagen cross-linking by intermediate concentrations of genipin, specifically 0.4 mmol/L, greatly reduces the contraction of 3D skin equivalents from 87% to 28% (n = 9, P < 0.05), while the collagen after EDC cross-linking at 0.4 mmol/L still presented severe contraction of 64% over a 21-day follow-up period. The inhibited contraction might relate to the increased gel stiffness and slowed collagen degradation. Moreover, the genipin cross-linking does not impair the formation of epidermal layers and improves the epidermal-dermal junction of skin equivalents as well. In this regard, genipin cross-linking might facilitate the applications of 3D skin equivalents in clinical practices and pharmacology testing., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Collagen cross-linking, as a form of collagen post-translational modification, plays a crucial role in maintaining bone mechanical properties as well as in regulating cell biological functions. Shifts in cross-links profile are found apparently correlated to kinds of skeletal pathology and diseases, whereas little is known about the relationship between collagen cross-links and osteogenesis. Here, we hypothesized that the inhibition of collagen cross-links could impair skeletal microstructure and inhibit osteogenesis. A mouse model of collagen cross-linking defects has been established using subcutaneous injection of 350 mg/kg β-aminopropionitrile (BAPN) daily for 4 weeks, and same dose of phosphate buffered saline (PBS) served as control group. The analysis of bone microstructural parameters revealed a significant decrease of bone volume fraction (BV/TV) and trabecular thickness (Tb.Th), and increase of bone surface ratio (BS/BV), structure model index (SMI) as well as trabecular separation (Tb.Sp) in the experimental group (p < 0.05), whereas there was no difference observed in bone mineral density (BMD). Histological staining displayed that the BAPN treatment caused thinner trabeculae and decrease of collagen content in proximal tibiae. The analysis of osteogenesis PCR (Polymerase Chain Reaction) array reflected that BAPN remarkably influenced the expression of Alpl, Bglap, Bgn, Bmp5, Col10a1, Col1a1, Col1a2, Col5a1, Itga2b, and Serpinh1. The results of immunohistochemistry displayed a significant reduction in the mean optical densities of OCN and COL1 at the presence of BAPN. The overall results of this study suggested that BAPN alters bone microstructure and hinders the expression of osteogenic genes without affecting mineralization processes, indicating the influences of collagen cross-links on osteogenesis may be a potential pathological mechanism in skeletal diseases.

Objectives/hypothesis: Photodynamic therapy (PDT) is a promising treatment modality for laryngeal dysplasia, early-stage carcinoma, and papilloma, and was reported to have the ability to preserve laryngeal function and voice quality without clinical fibrotic response. We aimed to investigate the mechanism behind the antifibrotic effects of PDT on primary human vocal fold fibroblasts (VFFs) in vitro., Study Design: In vitro analysis from one human donor., Methods: Cell viability of VFFs in response to varying doses of PDT was investigated by the Cell Counting Kit-8 method. Sublethal-dose PDT (SL-PDT) was used for the following experiments. Expression of genes related to vocal fold extracellular matrix formation was analyzed by real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blotting. Effects of PDT on cell migration, collagen contraction, and transforming growth factor β-1 (TGF-β1)-induced myofibroblast differentiation were also analyzed., Results: PDT affects the viability of VFFs in a dose-dependent manner. SL-PDT significantly changed the expression profile of VFFs with antifibrotic effects. It also inhibited cell migration, reduced collagen contraction, and reversed the fibroblast-myofibroblast differentiation induced by TGF-β1., Conclusions: SL-PDT induces antifibrotic alterations in VFFs. This could explain the low incidence of vocal fold scar associated with PDT. Moreover, PDT may be useful in treating existing vocal fold scars. Further studies should focus on the in vivo effect of PDT on vocal fold wound healing and scar remodeling., Level of Evidence: NA Laryngoscope, 128:E323-E331, 2018., (© 2018 The American Laryngological, Rhinological and Otological Society, Inc.)

Purpose: To investigate the use of kartogenin (KGN) in augmenting healing of the repaired enthesis after rotator cuff repair in a murine model., Methods: Seventy-two C57BL/6 wild-type mice underwent unilateral detachment and transosseous repair of the supraspinatus tendon augmented with either fibrin sealant (control group; n = 36) or fibrin sealant containing 100 μmol/L of KGN (experimental group; n = 36) applied at the repair site. Postoperatively, mice were allowed free cage activity without immobilization. Mice were humanely killed at 2 and 4 weeks postoperatively. Repair site integrity was evaluated histologically through fibrocartilage formation and collagen fiber organization and biomechanically through load-to-failure testing of the supraspinatus tendon-bone construct., Results: At 2 weeks, no differences were noted in percent area of fibrocartilage, collagen organization, or ultimate strength between groups. At 4 weeks, superior collagen fiber organization (based on collagen birefringence [17.3 ± 2.0 vs 7.0 ± 6.5 integrated density/μm 2 ; P < .01]) and higher ultimate failure loads (3.5 ± 0.6 N vs 2.3 ± 1.1 N; P = .04) were seen in the KGN group. The percent area of fibrocartilage (13.2 ± 8.4% vs 4.4 ± 5.4%; P = .04) was higher in the control group compared with the KGN group., Conclusions: Rotator cuff repair augmentation with KGN improved the collagen fiber organization and biomechanical strength of the tendon-bone interface at 4 weeks in a murine model., Clinical Relevance: These findings have implications for improving the structural integrity of the repaired enthesis and potentially reducing the retear rate after rotator cuff repair, which can ultimately lead to improvements in clinical outcomes., (Copyright © 2018 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.)

Objective: Rheumatoid arthritis (RA) is a chronic and degenerative autoimmune joint disease that leads to disability, reduced quality of life, and increased mortality. Although several synthetic and biologic disease-modifying antirheumatic drugs are available, there is still a medical need for novel drugs that control disease progression. As only 10% of experimental drug candidates for treatment of RA that enter phase I trials are eventually registered by the Food and Drug Administration, there is an immediate need for translational tools to facilitate early decision-making in drug development. In this study, we aimed to determine if the inability of fostamatinib (a small molecule inhibitor of Syk) to demonstrate sufficient efficacy in phase III of a previous clinical study could have been predicted earlier in the development process., Methods: Biomarkers of bone, cartilage, and interstitial matrix turnover (C-telopeptide of type I collagen [CTX-I], matrix metalloproteinase-derived types I, II, and III collagen neoepitopes [C1M, C2M, and C3M]) were measured in 450 serum samples from the Oral Syk Inhibition in Rheumatoid Arthritis 1 study (OSKIRA-1, a phase III clinical study of the efficacy of fostamatinib in RA) at baseline and follow-up. Additionally, the same biomarkers were subsequently measured in conditioned media from osteoclast, cartilage, and synovial membrane cultured with the active metabolite of fostamatinib, R406, to assess the level of suppression induced by the drug., Results: In OSKIRA-1 serum samples and osteoclast and cartilage cultures, fostamatinib suppressed the levels of CTX-I and C2M. In OSKIRA-1 serum samples and synovial membrane cultures, fostamatinib did not mediate any clinical or preclinical effect on either C1M or C3M, which have previously been associated with disease response and efficacy., Conclusion: These data demonstrate that translational biomarkers are a potential tool for early assessment and decision-making in drug development for RA treatment., (© 2018 The Authors Arthritis & Rheumatology published by Wiley Periodicals, Inc. on behalf of American College of Rheumatology.)

Although the presence of intraepithelial lymphocytosis with surface epithelial damage is a unifying feature of lymphocytic colitis, there are nonclassical features that create morphologic heterogeneity between cases. Limited data on the significance of these secondary histologic features are available. Cases of lymphocytic colitis diagnosed between 2002 and 2013 were identified using the Research Patient Data Registry of a tertiary referral center. Diagnostic biopsy slides were reviewed and evaluated for histologic features of lymphocytic colitis. Clinical data including type of therapy and response to treatment were collected. χ 2 Test (or Fisher exact test) and logistic regression analysis were used where appropriate. Thirty-two cases of lymphocytic colitis with complete clinical data and slides available for review were identified. The mean age was 56.4 years, and the female-to-male ratio was 3:2. Eleven patients improved with minimal intervention (group 1), 14 patients responded to steroid therapy (group 2), and 7 patients responded to mesalamine, bismuth subsalicylate, and/or cholestyramine therapy (group 3). Histologic differences in the characteristics of the subepithelial collagen table (P = .018), the severity of lamina propria inflammation (P = .042), and the presence of eosinophil clusters (P = .016) were seen between groups 2 and 3. Patients in group 1 were more likely to have mild crypt architectural distortion in their biopsies than patients in groups 2 and 3. Lymphocytic colitis is a heterogeneous disease, and the evaluation of histologic factors may help identify various subtypes and predict therapy response., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The physical strength of the collagen fibre network in the extracellular matrix is due to the covalent crosslinks between the molecules within the fibres (intra-fibrillar crosslinks). Citric acid was investigated as an agent to introduce crosslinks within marine collagen electrospun fibres. We used collagen films to understand the ideal conditions for citric acid crosslinking. This information was used to develop an optimised method for intra-fibrillar crosslinking in electrospun marine collagen fibres, which increased the stability of these fibres in aqueous environments. The optimised method included a spinning solution containing collagen and citric acid at pH3.5 at high concentrations (260:1 citric acid:collagen molar ratio) coupled with high temperature annealing (165°C), which resulted in the highest intra-fibrillar crosslinking density in electrospun fibres., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Introduction: A number of studies indicate that endothelin-1 (ET-1) may act as an inflammatory cell "gatekeeper," by regulating the influx of neutrophils following pulmonary injury. To further examine the role of ET-1 in modulating lung inflammation, hamsters were treated with an endothelin receptor antagonist (ERA), HJP272, either 1 h prior to intratracheal instillation of amiodarone (AM) or 24 h afterwards., Methods: In both cases, the extent of lung injury and repair was determined by (1) histopathological changes; (2) neutrophil content in bronchoalveolar lavage fluid (BALF); (3) lung collagen content; (4) tumor necrosis factor receptor 1 expression by BALF macrophages; (5) BALF levels of (a) transforming growth factor beta-1, (b) stromal cell-derived factor 1 (commonly referred to as CXCL12), and (c) platelet-derived growth factor BB; (6) alveolar septal cell apoptosis., Results: For each parameter, pretreatment with HJP272 resulted in a significant reduction compared to AM alone, whereas post-treatment was either ineffective or produced only a marginally significant change, suggesting that the course of lung inflammation and repair is programmed at a very early stage., Conclusions: This finding may explain why ERAs are not an effective treatment for human pulmonary fibrosis. Nevertheless, they may be useful as an adjunct to therapeutic regimens involving drugs that have fibrogenic potential.

Methylglyoxal (MG), a highly reactive dicarbonyl, interacts with proteins to form advanced glycation end products (AGEs). AGEs include a variety of compounds which were shown to have damaging potential and to accumulate in the course of different conditions such as diabetes mellitus and aging. After confirming collagen as a main target for MG modifications in vivo within the extracellular matrix, we show here that MG-collagen disrupts fibroblast redox homeostasis and induces endoplasmic reticulum (ER) stress and apoptosis. In particular, MG-collagen-induced apoptosis is associated with the activation of the PERK-eIF2α pathway and caspase-12. MG-collagen contributes to altered redox homeostasis by directly generating hydrogen peroxide and oxygen-derived free radicals. The induction of ER stress in human fibroblasts was confirmed using collagen extracts isolated from old mice in which MG-derived AGEs were enriched. In conclusion, MG-derived AGEs represent one factor contributing to diminished fibroblast function during aging., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Aim: To evaluate the efficacy of sonic irrigation (EndoActivator ® ) using various polymer tips and power settings in a stained collagen ex vivo model., Methodology: The root canals of fifty human, straight single-rooted extracted teeth were prepared to size 40, .08 taper. The roots were split longitudinally; stained collagen applied to the canal surfaces, photographed and re-assembled. The canals were subjected to syringe without supplementary (group 1, n = 10) or with supplementary sonic (groups 2-5, n = 10) irrigation. EndoActivator ® tip sizes (size 15, .02 taper for groups 2 and 3; size 35, .04 taper for groups 4 and 5) and power settings (low for groups 2 and 4; high for groups 3 and 5) were tested. After irrigation, the canals were re-photographed and the area of residual stained collagen was quantified using the UTHSCA Image Tool program (Version 3.0). The data were analysed using Wilcoxon signed rank test and general linear mixed models., Results: Supplementary sonic irrigation using EndoActivator ® resulted in significantly (P < 0.0001) less residual collagen compared with syringe irrigation only. Agitation of irrigant using the large EndoActivator ® tip with high power resulted in significantly less (22.4% - 29.5%) residual collagen compared to other combinations (large tip/low power P = 0.001; small tip/low power P = 0.01; small tip/high power P = 0.04). There was no significant difference amongst the latter three groups (P > 0.5)., Conclusions: Supplementary sonic irrigation using the EndoActivator ® system was significantly more effective in removing stained collagen from the canal surface than syringe irrigation alone. EndoActivator ® used with large tip (size 35, .04 taper) and high power setting in size 40, .08 taper canals was more effective than other combinations., (© 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

This study aimed to investigate the effects of gallium-aluminum-arsenium (GaAlAs) (670 nm) laser therapy on neoangiogenesis and fibroplasia during tissue remodelling. Forty male Wistar rats underwent cutaneous surgery and were divided into 2 experimental groups: the Control and Laser group (9 mW, 670 nm, 0.031 W/cm 2 , 4 J/cm 2 ). After 14, 21, 28, and 35 days, the animals were euthanised. Descriptive and quantitative analyses were performed in sections stained with haematoxylin-eosin and Sirius Red, respectively. The amounts of VEGF+ and CD31+ cells were evaluated by immunohistochemistry and histomorphometric analysis, respectively. Statistical analysis was performed using the Mann-Whitney, Friedman, and Spearman correlation test, P < 0.05. The collagen expression was significantly higher in the laser group compared with the control group on days 14 and 21 after the creation of the skin wound (P = 0.008; P = 0.016) and in the control group between 14 and 28 and 14 and 35 days (P = 0.001; P = 0.007). There were more blood vessels in three periods of the study only in the (Laser) treated group, with statistical significance at day 14 (P = 0.016). There was no statistically significant difference in VEGF+ cell count in the different experimental groups throughout the study, although a positive correlation was shown with the area of collagen on days 14 and 28 (P = 0.037). Laser treatment had a positive effect in the late course of healing, particularly with regards to collagen expression and the number of newly formed vessels. VEGF+ cells were present in both experimental groups, and VEGF appeared to influence fibroplasia in the treated group., (© 2017 Medicalhelplines.com Inc and John Wiley & Sons Ltd.)

Fibrosis, tightly associated with fibroblasts collagen synthesis, is related closely with inflammatory response. Our previously study found that acute downregulation of miR-155 at wound sites leads to a reduced fibrosis, however its particular mechanism is unclear. Herein, we aimed to explore the mechanism of miR-155 in reducing fibrosis. We first found that down-regulation of miR-155 inhibited macrophages transforming growth factor-β1 (TGF-β1) and IL-1β secretion. Next, we found that co-cultured with macrophages increased the proliferation and collagen synthesis of fibroblasts, and downregulation of miR-155 in macrophages could effectively attenuate the accelerative effects. We further identified SH2 domain containing inositol-5-phosphatase 1 (SHIP1) as a direct target of miR-155 in macrophages, and the expression of SHIP1 was negatively correlated with the level of miR-155. We further confirmed that PI3K/Akt pathway was involved in this process. Last, we found that downregulation of miR-155 leads to a reduced fibrosis in sever burn rat. Taken together, these results indicate that down-regulation of miR-155 leads to a reduced fibroblasts proliferation and collagen synthesis through attenuating macrophages TGF-β1 and IL-1β secretion by targeting SHIP1 via PI3K/Akt pathway, suggesting its potential therapeutic effects on the treatment of skin fibrosis.

BACKGROUND This study investigates the effects and mechanism of sulfur dioxide (SO2) inhalation and exercise on rat myocardial collagen fiber. MATERIAL AND METHODS The rats were randomly divided into 4 groups: a control group (RG), an exercise group (EG), an SO2 pollution group (SRG), and an SO2 pollution and exercise group (SEG). Body weight, cardiac index, and left ventricular index in each group were compared. The myocardial hydroxyproline (Hyp) concentration was determined by pepsin acid hydrolysis. The interstitial myocardial collagen expression was measured by Sirius Red F3B in saturated carbazotic acid. The local myocardial angiotensin II type 1 receptor (AT1R) and connective tissue growth factor (CTGF) expression was tested by immunohistochemistry SABC method. RESULTS Compared with RG, the weight growth rate of EG, SRG, and SEG decreased significantly (P<0.01). Compared with EG, the body weight growth rate of SEG significantly decreased (P<0.01) and cardiac index and left ventricular index decreased but without a significant difference. Compared with EG, myocardial Hyp and collagen concentration, myocardial collagen volume fraction (CVF), perivascular collagen area (PVCA), and the expression of AT1R and CTGF in myocardium of SEG increased significantly (P<0.01). CONCLUSIONS SO2 inhalation and exercise will not only offset beneficial health effects of movement on the cardiovascular system, but also produce more unfavorable influences. People should pay attention to their environment when exercising, and try to avoid exercising in environments with SO2 pollution.

Objective: Management of laryngotracheal stenosis (LTS) remains primarily surgical, with a critical need to identify targets for adjuvant therapy. Laryngotracheal stenosis scar fibroblasts exhibit a profibrotic phenotype with distinct metabolic shifts, including an increased glycolysis/oxidative phosphorylation ratio. This study examines the effects of the glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) on collagen production, gene expression, proliferation, and metabolism of human LTS-derived fibroblasts in vitro., Method: Paired normal and scar-derived fibroblasts isolated from subglottic and proximal tracheal tissue in patients with iatrogenic laryngotracheal stenosis (iLTS) were cultured. Proliferation rate, gene expression, protein production, and cellular metabolism were assessed in two conditions: 1) fibroblast growth medium, and 2) fibroblast growth medium with 1 × 10 -4 M DON., Results: DON treatment reduced cellular proliferation rate (n = 7, P =  0.0150). Expression of genes collagen 1 and collagen 3 both were reduced (n = 7, P =  0.0102, 0.0143, respectively). Soluble collagen production decreased (n = 7, P =  0.0056). As measured by the rate of extracellular acidification, glycolysis and glycolytic capacity decreased (n = 7, P =  0.0082, 0.0003, respectively). adenosine triphosphate (ATP) production and basal respiration decreased (n = 7, P = 0.0045, 0.0258, respectively), determined by measuring the cellular rate of oxygen consumption., Conclusion: The glutamine antagonist DON reverses profibrotic changes by inhibiting both glycolysis and oxidative phosphorylation in iLTS scar fibroblasts. In contrast to untreated iLTS scar fibroblasts, collagen gene expression, protein production, metabolic rate, and proliferation were significantly reduced. These results suggest DON and/or its derivatives as strong candidates for adjuvant therapy in the management of iatrogenic laryngotracheal stenosis. Enzymes involved in glutamine metabolism inhibited by DON offer targets for future investigation., Level of Evidence: NA. Laryngoscope, 128:E59-E67, 2018., (© 2017 The American Laryngological, Rhinological and Otological Society, Inc.)