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ObjectivesTo investigate the role of endogenous TSG-6 in human osteoarthritis (OA) and assess the disease-modifying potential of a TSG-6-based biological treatment in cell, explant and animal models of OA.MethodsKnee articular cartilages from OA patients were analysed for TSG-6 protein and mRNA expression using immunohistochemistry and RNAscope, respectively. The inhibitory activities of TSG-6 and its isolated Link module domain (Link_TSG6) on cytokine-induced glycosaminoglycan loss in OA cartilage explants were compared. Mesenchymal stem/stromal cell (MSC)-derived chondrocyte pellet cultures were used to determine the effects of Link_TSG6 and full-length TSG-6 on IL-1α-, IL-1β- or TNF-stimulated ADAMTS4, ADAMTS5 and MMP13 mRNA expression. Link_TSG6 was administered i.a. to the rat ACLTpMMx model and cartilage damage and tactile allodynia were assayed.ResultsTSG-6 is predominantly associated with chondrocytes in regions of cartilage damage and its expression is negatively correlated with MMP13, the major collagenase implicated in OA progression. Link_TSG6 is more potent than full-length TSG-6 at dose-dependently inhibiting cytokine-mediated matrix breakdown in human OA cartilage explants; about 50% of donor cartilages, from 59 tested, were responsive to Link_TSG6 treatment. Similarly, Link_TSG6 displayed more potent effects in 3D pellet cultures, suppressing aggrecanase and collagenase gene expression. Link_TSG6 treatment reduced touch-evoked pain and dose-dependently inhibited cartilage damage in a rodent model of surgically-induced OA.ConclusionsNative TSG-6 is associated with a low catabolic chondrocyte phenotype in OA cartilage. Link_TSG6, which has enhanced chondroprotective activity compared to the full-length TSG-6 protein, demonstrates potential as a disease modifying OA drug (DMOAD) and warrants further investigation and development.KEY MESSAGESWhat is already known about this subject?TSG-6, a protein with anti-inflammatory and protective effects in other tissues, is expressed in joints affected by osteoarthritis – a condition for which there are no disease-modifying drugs.What does this study add?A novel protective mechanism has been identified, whereby TSG-6 inhibits inflammatory cytokine-induced catabolic pathways in cartilage.The Link module of TSG-6 (Link_TSG6) has been shown to have greater potency than TSG-6 as an inhibitor of cartilage damage and is efficacious in a rat model of osteoarthritis.Data from cartilage explants indicate that OA patients can be stratified for Link_TSG6 responsiveness.How might this impact on clinical practice or future developments?Link_TSG6 has been identified as potential disease-modifying OA drug that mimics an intrinsic protective process.

To produce viable eggs from single primary follicles in vitro, primary follicles containing oocytes (average 39.0 ± 0.2 µm in diameter) were isolated from the ovaries of 1-week-old mice, and cultured in combination with culture membranes for the first 8 days up to the secondary follicle stage, followed by the next 12 days to the later stages. After culture with a combination of first and second culture membranes using high and low adhesion characteristics, the average oocyte diameters of the surviving follicles increased by almost two-fold in all four groups. Further, the oocyte maturation rate was the highest (74.1%) in the culture group with low adhesion with collagenase and high adhesion. In this culture group, when the O2 concentration was changed from 20% in the first culture to 5% in the second culture, the cleavage rate increased to 47.5%, which was comparable to the level of the in vivo control (34.6%). Finally, 39 embryos at the 2- to 8-cell stages were transferred into the oviducts of three pseudopregnant females, and eight live pups (20.5%) were obtained. Of the eight pups, six survived for at least six months and were fertile. The present study shows successive in vitro cultures of single isolated primary follicles for the production of viable eggs. We believe that this culture system, with a combination of culture membranes under controlled O2 conditions, is applicable to other mammalian species, including humans.

OBJECTIVES: To evaluate the effects of different polyphenols and solvents on dentin collagen’s crosslinking interactions and biostabilization against MMPs and collagenase degradation. METHODS: Two polyphenols [proanthocyanidin (PA) and quercetin (QC)] with different water solubility were prepared as treatment solutions using ethanol (EtOH) or dimethyl sulfoxide (DMSO) as solvents. 6-um-thick dentin films were microtomed from dentin slabs of third molars. Following demineralization, films or slabs were subject to 60-s treatment (PA or QC) or no treatment (control) with subsequent extended-rinse with original solvent (EtOH or DMSO) or distilled water (DW). Collagen crosslinking interactions were assessed by FTIR. Biostability was assessed through endogenous MMPs activity via confocal laser scanning microscopy, and exogenous collagenase degradation via weight loss, hydroxyproline release and SEM. Finally, direct collagenase inactivation was also evaluated. Data were analyzed by three-way ANOVA and post-hoc tests (α=0.05%). RESULTS: Distinct effects of two polyphenols and solvents on collagen crosslinking and biostabilization were observed. Higher crosslinking and biostability efficacy occurred with PA than QC (p < 0.001) that demonstrated negligible collagen interactions. With DMSO solvent, efficacy results were significantly reduced with both polyphenols (p < 0.05). DMSO-rinse further weakened interactions of PA with collagen, diminishing biostability (p < 0.05). Low biostability was detected with QC and DW-rinse, suggesting direct enzymatic inhibition due to physical presence in collagen. SIGNIFICANCE: Collagen crosslinking interactions and biostability depend on polyphenol chemical characteristics. Treatment-solution solvents may affect interactions between polyphenols and collagen, specifically, DMSO showed detrimental effects on collagen crosslinking and biostability and should be used with caution.

Chronic exposure to ultraviolet (UV) radiation leads to photoaging. There is a tremendous rise in products having a dual activity of photoprotection and antiaging. In vitro analysis in dermal fibroblasts and their biological mechanisms involved are critical to determine antiaging potential. The study aimed to investigate the antiaging potential of sunscreen formulated from nanostructured lipid carrier and tocotrienol-rich fraction (NLC-TRF sunscreen). The antioxidant activity of the NLC-TRF sunscreen was evaluated by radical scavenging and hydrogen peroxide inhibition properties. Also, collagenase, elastase and matrix metalloproteinase-1 (MMP-1) inhibition activities, and type I collagen and elastin protein expression were studied. Quantitative real-time polymerase chain reaction (qPCR) was used to evaluate the mRNA expression of fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), transforming growth factor-β1 (TGF-β1), type I collagen (COL1A1), elastin (ELN), MMP-1, MMP-2, and tissue inhibitor matrix metalloproteinase-1 (TIMP-1). The results suggested that NLC-TRF sunscreen is effective in radical, anti-hydrogen peroxide, and collagenase, elastase and MMP-1 inhibition activities. Besides, a significant increase for type I collagen (3.47-fold) and elastin (2.16-fold) protein and fibroblast regeneration genes (FGF (2.12-fold), VEGF (1.91-fold), TGF-β1 (2.84-fold), TIMP-1 (1.42-fold), ELN (2.13-fold)) were observed after sample treatment. These findings support the therapeutic potential of NLC-TRF sunscreen in antiaging.

This study was conducted to compare the correlations between free radical antioxidant activity and anti-wrinkling activity. Areca nut was extracted using various concentrations of ethanol (0, 40, 80, and 100%) and methanol (0, 40, 80, and 100%) at 100 °C for 90 min. The ethanolic and methanolic extracts of Areca nut were evaluated for total phenolic content (TPC), total flavonoid content (TFC), antioxidant activity, including total antioxidant capacity (TAC), FRAP, DPPH, ABTS, and superoxide anion radical scavenging activity (SRSA), and anti-wrinkling activity (collagenase inhibition and elastase inhibition). In addition, the antioxidant activities were correlated with collagenase inhibition. The antioxidant activities of the extracts exhibited little correlation (R=0.008-0.0760) with TPC, TFC, and TAC; however, significant correlation (R=0.825-0.891) was shown with collagenase inhibition. The 40% and 80% EtOH extracts showed the highest levels of antioxidant activities, and also had the highest phenolic and flavonoid contents. The antioxidant activities of the 80% EtOH extracts was greater than that of other extracts. These results indicate that the collagenase inhibition effects of Areca nut extracts may be due to free radical antioxidant activities, and the optimal extraction condition was 80% EtOH.

CONTEXT Disorders associated with diabetes and the beneficial effects of pomegranate peel extract (PPE) were widely reported. However effect of diabetes and PPE on extracellular matrix (ECM) remodelling needs further investigation. OBJECTIVES The focus of this study was to investigate the involvement of diabetes in cardiac ECM and the beneficial effects of PPE. METHODS Diabetes was induced by alloxan. PPE group was injected with 100 mg/kg of PPE. The phenolic profile of PPE was analyzed by HPLC. ECM was detected by ELISA. MMP-1, -8, -13 were determined by a colorimetric assay. RESULTS Compared to control fibronectin and laminin plasma content was higher respectively by 69% and 42% (p

Spermatogonial stem cells (SSCs) are the only adult stem cells capable of passing genetic information to offspring through their ability to both self-renew and differentiate into mature spermatozoa. SSCs can be transplanted to establish donor-derived spermatogenesis in recipient animals, thus offering a novel reproductive tool for multiplication of elite individual animals to benefit livestock production. An optimal SSC culture in vitro can benefit various SSC-based studies and applications, such as mechanistic study of SSC biology, SSC transplantation process, and SSC-based transgenesis technique. However, except some model rodent animals, SSC culture remains an inefficient and unstable process. We here studied a workflow to isolate, purify, and in vitro culture porcine SSCs from neonatal pig testes. Pig testicular cells were dissociated by two step enzymatic digestion with collagenase type IV and trypsin. We enriched the spermatogonia from the testicular cell mix by differential plating for at least 3 times to remove firmly attached non-SSCs. We then tested the optimal culture medium formula by supplementation of different growth factors to the basic medium (DMEM/F12 + 1% FBS), and found that a combination of 20 ng/mL GDNF, 10 ng/mL LIF, 20 ng/mL FGF2, and 20 ng/mL IGF1 had the best effect on SSC growth in our defined experimental system. In the presence of 4 growth factors without specific feeders, the purified SSCs can be cultured in poly-L-lysine- and laminin-coated dishes for 28 days, and remain preserving a continuous proliferation without losing the undifferentiated spermatogonial phenotype.

Objective The classic chondrocyte isolation protocol is a 1-step enzymatic digestion protocol in which cartilage samples are digested in collagenase solution for a single, long period. However, this method usually results in incomplete cartilage dissociation and low chondrocyte quality. In this study, we aimed to develop a rapid, high-efficiency, and flexible chondrocyte isolation protocol for cartilage tissue engineering. Design Cartilage tissues harvested from rabbit ear, rib, septum, and articulation were minced and subjected to enzymatic digestion using the classic protocol or the newly developed sequential protocol. In the classic protocol, cartilage fragments were subjected to one 12-hour digestion. In the sequential protocol, cartilage fragments were sequentially subjected to 2-hour first digestion, followed by two 3-hour digestions. The collected cells were then subjected to analyses of cell-yield efficiency, viability, proliferation, phenotype, and cartilage matrix synthesis capacity Results Overall, the sequential protocol exhibited higher cell-yield efficiency than the classic protocol for the 4 cartilage types. The cells harvested from the second and third digestions demonstrated higher cell viability, more proliferative activity, a better chondrocyte phenotype, and a higher cartilage-specific matrix synthesis ability than those harvested from the first digestion and after the classic 1-step protocol. Conclusions The sequential protocol is a rapid, flexible, high-efficiency chondrocyte isolation protocol for different cartilage tissues. We recommend using this protocol for chondrocyte isolation, and in particular, the cells obtained after the subsequent 3-hour sequential digestions should be used for chondrocyte-based therapy.

Aims: We describe the development of a novel porcine eschar model and compare the debridement efficacy of various concentrations of a novel bromelain-based enzymatic agent with collagenase. Methods: Full thickness excisional wounds were created on pigs and injected intradermally with various doses of doxorubicin. Wounds were monitored for a period of 46 days for the development of eschar and wound closure. After determining the optimal concentration and dose of doxorubicin resulting in non-healing eschars, these conditions were used to create additional wounds on another set of animals. The resulting eschars were treated with various concentrations of a novel bromelain-based enzymatic agent (EscharEx-02) or collagenase. The primary endpoint was greater than 95% removal of the central eschar. Results: Consistent eschars composed of two distinct areas (a central area of exudate and slough representing the hard-to-heal wound bed, and a peripheral area of full-thickness mummified necrosis) were seen after injection of doxorubicin (0.5 ml/cm2 of stock solution 0.75mg/ml) at one and six days after wound creation. Complete removal of the central eschar was achieved in all wounds after five and eight treatments with 5% and 2% EscharEx-02 respectively. Complete removal of the central eschar with collagenase was achieved in 0% and 82% of the wounds after 10 and 16 treatments respectively. Conclusions: We describe a porcine model for creating eschars similar to hard-to-heal wounds in humans. A novel bromelain-based enzymatic debridement agent was more effective than a commercially available collagenase in removing eschars in this wound model.

Kidney diseases are becoming an emerging public health problem. In order to further explore the etiology of various kidney diseases, we improved the methods of isolation of primary cultures of mouse renal tubular epithelial cells. At the first stage, the kidneys were perfused with collagenase solution. To this end, the superior mesenteric artery, celiac artery and thoracic aorta were ligated and perfusion was performed through the abdominal aorta. Then, the cells were isolated ex vivo and their integrity, purity, viability, and concentration were evaluated. The proposed cost-effective and simple method provides high purity and high concentration of primary renal epithelial cells for molecular biology studies of the kidneys.

Background: Although Semaphorin 3A (Sema3A)/ Neuropilin-1(NRP1)/Plexin A1 is one of the important targets in bone metabolism, few studies are performed on this target in the glucocorticoids- induced osteoporosis. Luteolin is a chemical component of Honeysuckle and it has various bioactivities. The effect of Luteolin on the glucocorticoids-induced osteoporosis (primary osteoblasts model) remain unknown. Objective: The aim of this study was to investigate the action of Sema3A/ NRP1/Plexin A1 in Luteolin- induced osteoprotection against high-dose Dexamethasone. Methods: The effect of Luteolin on the proliferation, late differentiation, and apoptosis of primary osteoblasts model of glucocorticoids-induced osteoporosis in vitro as well as the expression of Sema3A/ NRP1/Plexin A1 are investigated by using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-Htetrazolium bromide, Cell Counting Kit-8, reverse transcription-quantitative real-time polymerase chain reaction, western bolting and so on. Results: Suckling SD rats’ calvarial osteoblasts were isolated and identified. The glucocorticoidsinduced primary osteoporosis cell model was established by 100 μM Dexamethasone in 48 h (P Conclusion: The thesis concludes that Luteolin promoted the proliferation of osteoblast and inhibited its apoptosis and late differentiation in this glucocorticoids-induced primary osteoporosis cell model. This function may be related to the expression of up-regulated Sema3A/NRP1/Plexin A1. Therefore, Luteolin may be a potential medicine for the glucocorticoids-induced osteoporosis.

The composition and location of charged and hydrophobic amino-acid residues of the collagenase molecule from Clostridium histolyticum were investigated. The percentage ratio of the various amino-acid types on the enzyme surface was determined. It was established that the charged and hydrophobic amino-acid residues are unevenly distributed over the surface of the molecule, forming local clusters. It was suggested that the most promising supports for immobilization of bacterial collagenase are hydrophobic or non-polar polymers that are likely to interact with a region on the surface of the enzyme molecules that is remote from the active center. It was established that gels of chitosan and chitosan succinate are more promising supports for immobilization of collagenase than the cation-exchange fiber VION KN-1 and the anion-exchange fiber VION AN-1.

Matrix metalloproteinase-13 (MMP-13) is a uniquely important collagenase that promotes the irreversible destruction of cartilage collagen in osteoarthritis (OA). Collagenase activation is a key control point for cartilage breakdown to occur, yet our understanding of the proteinases involved in this process is limited. Neutrophil elastase (NE) is a well-described proteoglycan-degrading enzyme which is historically associated with inflammatory arthritis, but more recent evidence suggests a potential role in OA. In this study, we investigated the effect of neutrophil elastase on OA cartilage collagen destruction and collagenase activation. Neutrophil elastase induced significant collagen destruction from human OA cartilage ex vivo, in an MMP-dependent manner. In vitro, neutrophil elastase directly and robustly activated pro-MMP-13, and N-terminal sequencing identified cleavage close to the cysteine switch at 72 MKKPR, ultimately resulting in the fully active form with the neo-N terminus of 85 YNVFP. Mole-per-mole, activation was more potent than by MMP-3, a classical collagenase activator. Elastase was detectable in human OA synovial fluid and OA synovia which displayed histologically graded evidence of synovitis. Bioinformatic analyses demonstrated that, compared with other tissues, control cartilage exhibited remarkably high transcript levels of the major elastase inhibitor, (AAT) alpha-1 antitrypsin (gene name SERPINA1), but these were reduced in OA. AAT was located predominantly in superficial cartilage zones, and staining enhanced in regions of cartilage damage. Finally, active MMP-13 specifically inactivated AAT by removal of the serine proteinase cleavage/inhibition site. Taken together, this study identifies elastase as a novel activator of pro-MMP-13 that has relevance for cartilage collagen destruction in OA patients with synovitis.

The present study reports the feasibility and successful production of rabbit cG-CAOMECS, designed to reconstruct corneal epithelium of patients with bilateral limbal stem cell deficiency. To produce a safe, chemically defined and FDA compliant cG-CAOMECS, oral mucosal epithelial cells were isolated from a biopsy of rabbit buccal tissue and seeded on a cGMP-certified cell culture surface coated with GMP-grade extracellular matrix. A newly designed clinical-grade medium (KaFa™ medium) was utilized to carry out cell expansion. Detachment and harvesting of the produced cell sheet was accomplished using collagenase treatment. Live cell imaging and morphological analysis techniques were used to examine cell growth. Cells attached onto the surface and self-assembled into colony-forming units (CFUs). Microscopic examination showed that CFUs formed during the first 5 days, and basal monolayer cell sheet formed in less than 10 days. Cells expanded to form a multilayered epithelial cell sheet that was harvested after 17–19 days in culture. Immunostaining and Western blot analyses showed that deltaNp63 was expressed in the basal cells and K3/K12 was expressed in the apical cells, indicating the presence of corneal epithelial-like cells in the produced cell sheet. Adhesion molecules, E-cadherin, beta-catenin, and Cnx43 were also expressed and exhibited the epithelial integrity of the cell sheet. The expression of integrin-beta1 and beta4 confirmed that the collagenase treatment used for detaching and harvesting the cell sheet did not have adverse effects. Our results showed that the utilization of clinical-grade and FDA-approved reagents successfully supported the production of cG-CAMECS.

The incidence of labor induction with various indications was to increase. The key to successful labor induction is a ripe cervix. Research shows that the degree of cervical rippening as assessed by the Bishop score is influenced by the level of neutrophils contained in the cervical stroma which can be detected by performing a vaginal swab. Neutrophils will produce collagenase in the form of matrix metalloproteinase - 8 (MMPs - 8) which will degrade cervical collagen fibers, so that the cervix becomes soft and ripe. Misoprostol is the drug most widely used in labor induction today. Purpose: This study was to determine the difference in Bishop score increase between vaginal swab neutrophils ≤ 5 and > 5 in pregnancy ≥ 41 weeks induced by misoprostol.Keywords: Bishop score, neutrophil, vaginal swab, misoprostol

The collagenase enzyme proteolytic complex used in a gel form will be an alternative to invasive methods and electrophoresis, requiring medical personnel and special equipment. Taking into account the instability of proteolytic enzymes in the aquatic phase, the use of organogel as the collagenase basis makes it possible to create a stable and convenient dosage form for the scar treatment. This study’s aim was the composition of an anti-scar organogel based on Paralithodes camtschatica collagenase development. Aerosil proposed as a thickener. Various technological studies (external signs assessment, distribution on the skin, smearing, pH, proteolytic activity, stability, rheological properties) of gels samples based on lipophilic solvents: dimethicone, cyclomethicone, liquid paraffin, propylene glycol, capril/caprilic triglycerides, were carried out to select the optimal gel-forming agents. The proteolytic activity of the gel with collagenase manifested up to 24 hours, so, it may be used for stabilization. The composition of the components of the dispersion medium, which has acceptable sensory properties for the continuity of treatment, selected. It characterized by thermo- and colloidal stability, acceptable rheological properties. The oleogel on the combination of capril/caprilic triglycerides and dimethicone as a dispersion and aerosil as a thickener was justified, which may be recommended as an anti-scar agent for further research.

To investigate the use of collagenase type II for generating a rabbit model of keratoconus and to evaluate the impact of violet light (VL) irradiation on the disease model. Six Japanese White rabbits were used. After epithelial debridement, the collagenase group was treated with a collagenase type II solution for 30 min; the control group was treated with a solution without collagenase. Three rabbits also underwent VL irradiation (375 nm, irradiance 310 μW/cm2) for 3 h daily for 7 days after topical collagenase application. Slit-lamp microscopy, steep keratometry (Ks), corneal astigmatism, central corneal thickness, and axial length were examined before and after the procedure. The corneas were obtained on day 7 for biomechanical evaluation. A significant increase in Ks and corneal astigmatism was observed in the collagenase and VL irradiation groups compared with the control group at day 7. No significant difference was found in the change in corneal thickness between the groups. The elastic modulus at 10% strain but not at 3% and 5% strain in the collagenase group was significantly lower than that in the control group. There was no significant difference in the elastic modulus at each level of strain between the collagenase and VL irradiation groups. The average axial length at day 7 in the collagenase group was significantly longer than that in the control group. Collagenase type II treatment can mimic keratoconus with increased corneal keratometry and astigmatism. There was no significant difference in the observed elastic behaviour of normal and ectatic corneas under physiologically relevant stress levels. VL irradiation did not cause regression of corneal steepening in this model with short-term observation.

Background: Keloid is an abnormal scar in previously traumatic skin after going through the wound healing process. One hundred million cases have been found in developing countries with the main complaint of scar appearances. To overcome this problem, 24 literatures from various journals and textbooks are reviewed. Reviews: Keloid formation is based on high melanin amount which inhibits the collagenase enzyme. Moreover, the high melanin amount would block interleukin (IL)-1B work resulting in collagen synthesis and collagenase reduction. Depigmentation effort with 4% hydroquinone is implemented to reduce the amount of melanin presented in the skin. With melanin reduction, IL-1B can work optimally by inhibiting fibroblast growth in keloid tissue without affecting on normal skin. It also induces Matrix Metalloproteinase (MMP)-1 which is an interstitial collagenase. IL-1B has an opposing effect compared to Transforming Growth Factor (TGF)-B, thus TGF-B antibody is needed to potentiate IL-1B therapeutic effect. TGF-B antibody can neutralize TGF-B ligand and avB6 integrin resulting in blocking of COL1A1 gene expression which is responsible for MMP-1 production and type-I collagen synthesis. These three components are combined in cream with liposome as a drug carrier. This combination is applicated for adjuvant therapy after scar excision. Liposomes are chosen because of their high biocompatibility, low toxicity, and low biodegradability. Liposomes also can release slowly in the extravascular area such as skin. This advantage may carry drug components effectively to the target location. Summary: The combination of depigmentation, IL-1B, and TGF-B antibodies has a potency to be developed as a future adjuvant therapy of keloid.

OBJECTIVE To assess the effects of PTH (1-34) on bone and cartilage metabolism in a collagenase-induced mouse model of osteoarthritis (OA) and examine whether PTH (1-34) affects the expression of JAK2/STAT3 and WNT5A/ROR2 in this process. METHODS Eighteen 12-week-old male C57Bl/6 mice were randomly assigned into three groups as follows: sham group (Group A), the collagenase + saline injection group (Group B), and the collagenase + PTH (1-34) treatment group (Group C). Collagenase was injected (intra-articular) into the knee joint of Group B and C. The PTH (1-34)-treatment was started at 6 weeks after the operation and lasted for 6 weeks. Cartilage pathology was evaluated by gross visual, histological, and immunohistochemical assessments. Subchondral bone was evaluated by microcomputed tomography (micro-CT) and immunohistochemical analyses. RESULTS The OARSI macroscopic and microscopic scores of Group B were higher than those of Group A (P = 0.026; P = 0.002, respectively). Group C showed statistically significant differences in macroscopic and microscopic scores from Group B (P = 0.041; P = 0.008, respectively). The results showed that the Col-II and AGG expression levels in the cartilage tissue were significantly lower in Group B than Group A (P

Background: The purpose of this study was to investigate whether Sibseonsan (SSS) is an effective anti-inflammatory, anti-wrinkling, and whitening agent. Methods: To determine whether SSS had an anti-inflammatory effect, a murine macrophage cell line was used (RAW 264.7) and production of DPPH, NO, TNF-α, and PGE2 were measured. To ascertain potential anti-wrinkle effects of SSS in these cells, collagenase and elastase production were measured. To verify whether SSS had a whitening effect, tyrosinase activity and DOPA staining were performed using a melanoma cell line (B16/F10). Results: There was no significant reduction in survival of SSS-treated RAW 264.7 cells, up to 400 μg/mL. Free radical scavenging (23.96 ± 1.85%) was observed in RAW 264.7 cells treated with SSS at a concentration of 400 μg/mL. The SSS treatment group (400 μg/mL) significantly inhibited NO production compared with the LPS stimulated treatment group. The SSS treatment of macrophage cells appeared to reduce production of TNF-α in a concentration dependent manner. There was a significant reduction in the concentration of PGE2 by about 25% in the SSS treatment (400 μg/mL) group (p = 0.05). Compared with the control, the production of collagenase and elastase in B16/F10 cells treated with SSS (400 μg/mL) was greater by 26.37% and 45.71%, respectively. The SSS treatment (400 μg/mL) group showed a significant reduction by about 17% in tyrosinase production in B16/F10 cells. The SSS treatment group showed little change in DOPA staining. Conclusion: SSS extract may be useful for the treatment and prevention of inflammatory diseases and may have anti-wrinkle and whitening effects. These results may support the use of SSS in clinical practice.

Chronic salpingo-oophoritis (CSO) is one of the most common diseases among the entire gynecological pathology, the frequency of which reaches 65-68%. Despite the progress made in the treatment of CSO, there is a serious problem due to the steady growth and complications leading to infertility, ectopic pregnancy and the development of chronic pelvic pain syndrome, which significantly reduces the quality of life of patients. In this regard, the development of promising pharmaco-physiotherapeutic methods with pronounced analgesic, anti-inflammatory, immunomodulatory and bacteriological effects to increase the therapeutic effectiveness and improve the quality of life of patients with salpingo-oophoritis is an important task of modern gynecology and physiotherapy. Objective: To study in a comparative aspect the effect of the combined use of transvaginal electrophoresis of the collagenase complex and pulsed magnetic therapy and their mono-effects on the quality of life of patients with chronic salpingo-oophoritis. Materials and research methods. The study included 77 patients aged 18 to 42 years (median age was 26,7±2,8 years) with a diagnosis of chronic salpingo-oophoritis of non-specific etiology. More than half of the patients (59,7%) were women under 25 years of age. All patients were divided into three groups that were comparable according to the main clinical and anamnestic data, somatic and obstetric-gynecological status: group 1 — the main group — included 28 patients who received a course of combined application of transvaginal electrophoresis of the collagenase complex and pulsed magnetic therapy; group 2 — comparison group 1 — included 25 patients who received acourse of transvaginal electrophoresis of the collagenase complex; group 3 — comparison group 2 — included 24 patients who received a course of pulsed magnetic therapy. The quality of life was assessed by the indicators of the Quality of Life Index (QOL), which takes into account the three most important and informative aspects of patients’ lives: physical condition and its dynamics, mental (psychological) health and daily functioning. The results of the study. Based on the obtained results, it was proved that the most pronounced results were achieved in patients of the main group who received a course of transvaginal electrophoresis of the collagenase complex in combination with pulsed magnetic therapy, where the quality of life improved by 89,3% according to the QOL index, compared to the monotherapy with transvaginal electrophoresis and pulsed magnetic therapy — 75,7% and 73,1%, respectively.

The suppression of abdominal aortic aneurysm (AAA) growth by nonsurgical therapy is currently not an option, and AAA is considered an irreversible destructive disease. The formation and development of AAA is associated with the progressive deterioration of the aortic wall. Infiltrated macrophages and resident vascular smooth muscle cells oversecrete matrix metalloproteinases (MMPs), which cause the loss of crucial aortic extracellular matrix (ECM) components, thus weakening the aortic wall. Stabilization of the aortic ECM could enable the development of novel therapeutic options for preventing and reducing AAA progression. In the present work, we studied the biochemical and biomechanical interactions of pentagalloyl glucose (PGG) on mouse C2C12 myoblast cells. PGG is a naturally occurring ECM-stabilizing polyphenolic compound that has been studied in various applications, including vascular health, with promising results. With its known limitations of systemic administration, we also studied the administration of PGG when encapsulated within poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs). Treatment with collagenase and elastase enzymes was used to mimic a pathway of degenerative effects seen in the pathogenesis of human AAA. PGG and PLGA(PGG) NPs were added to enzyme-treated cells in either a suppressive or preventative scenario. Biomolecular interactions were analyzed through cell viability, cell adhesion, reactive oxygen species (ROS) production, and MMP-2 and MMP-9 secretion. Biomechanical properties were studied through atomic force microscopy and quartz crystal microbalance with dissipation. Our results suggest that PGG or PLGA(PGG) NPs caused minor to no cytotoxic effects on the C2C12 cells. Both PGG and PLGA(PGG) NPs showed reduction in ROS and MMP-2 secretion if administered after enzymatic ECM degradation. A quantitative comparison of Young's moduli showed a significant recovery in the elastic properties of the cells treated with PGG or PLGA(PGG) NPs after enzymatic ECM degradation. This work provides preliminary support for the use of a pharmacological therapy for AAA treatment.

Background and Purpose: The classic presentation of chronic (stage III) hemorrhagic stroke lesions is a fluid-filled cavity. In one of the most commonly used animal models of intracerebral hemorrhage (ICH), we noticed additional solid material within the chronic lesion. We examined the composition of those chronic ICH lesions and compared them with human autopsy cases. Methods: ICH was induced in rats by the injection of collagenase in the striatum. Tissue sections after hematoma resolution corresponding to 3 different chronic time points—28, 42, and 73 to 85 days post-ICH—were selected. Human autopsy reports at the University Hospital of Zurich were searched between 1990 and 2019 for ICH, and 3 chronic cases were found. The rat and human sections were stained with a variety of histopathologic markers. Results: Extensive collagenous material was observed in the chronic lesion after hematoma resolution in both the rat model and human autopsy cases. Additional immunostaining revealed that the material consisted primarily of a loose network of collagen 3 intermingled with occasional GFAP (glial fibrillary acidic protein)-positive processes and collagen 4. Conclusions: A key feature of the chronic ICH lesion is a loose network of collagen 3. The collagenase rat model reproduces the morphology and composition of the chronic human ICH lesion. While identifying new features of ICH lesion pathology, these results are important for treatment and recovery strategies.

Current adhesives bond to dentin via a micro-interlocking mechanism within the hybrid layer. Besides such mechanical retention, bonding to dentin would benefit from additional chemical interaction between collagen and resin. This study aims to synthesize a novel light-curable collagen crosslinker methacrylate (MA) functionalized grapeseed extract (GSE) and to assess MAGSE’s ability to crosslink dentin collagen in a clinically relevant setting as well as its role in light-cure as a resin. MA functionalization was accomplished by reacting GSE with methacryloyl chloride to obtain MAGSE, which was characterized by 1H-NMR and Fourier transformed infrared spectroscopy (FTIR). The 6-µm-thick dentin films were microtomed from dentin slabs of third molars. Following demineralization, they were treated for 30 s by 1% MAGSE. Collagen crosslinking and resistance to digestion of MAGSE were evaluated by FTIR, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) assay of films, and scanning electron microscopy (SEM)/transmission electron microscopy (TEM) on slabs. Meanwhile, 1% MAGSE or GSE was added to an experimental adhesive formulated with 2-hydroxyethyl methacrylate and a tricomponent photoinitiator system. Polymerization kinetics were monitored continuously in real time for 10 min using FTIR–attenuated total reflection. The results indicated that MAGSE could bind to dentin collagen and protect it from collagenase degradation as strong as GSE. Dentin collagen treated by 1% MAGSE for 30 s was scarcely digested (1.6 ± 1.6%) after 1 h in 0.1% collagenase, while untreated collagen was completely digested (100.9 ± 20.2%). SEM/TEM images indicated MAGSE efficiently crosslinked dentin collagen in 30 s and rendered it almost inert to digestion under clinically relevant settings. Unlike GSE that hindered light-curing of HEMA, MAGSE accelerated the rate of polymerization and exhibited typical traits of a resin monomer with multiple polymerizable units. In conclusion, a novel collagen crosslinking resin MAGSE is synthesized, which inherits collagen crosslinking ability from GSE and polymerization function from MA. Inclusion of this light-curable collagen crosslinker into adhesives might be a revolutionary way to improve durability of dentin bonding in composite restorations.

The objective of this study was to compare the effectiveness of ultrasound-guided injection of collagenase Clostridium histolyticum (CCH) in patients with Dupuytren's contracture (DC), with the standard injection. We hypothesised that the ultrasound-guided Injection of CCH is more effective than the standard injection. A prospective cohorts study in patients with DC was done. We treated consecutively 47 fingers with the standard injection and 43 with the ultrasound-guided. Patients in both groups had the same inclusion criteria. The degrees of contracture of the metacarpophalangeal (MCP) and proximal interphalangeal (PIP) joints were measured before treatment and after three months. We compared the effectiveness of each type of injection in respect to obtaining a complete finger extension and to the percentage of improvement in each finger and in each joint. With ultrasound-guided injection, complete finger extension was obtained in 54% of cases and an 81% mean percentage of correction of the finger contracture; with standard injection 49% and 77%, respectively. In the MCP joint, the mean percentage of correction was 92.5 % in the ultrasound-guided Injection group and 84% in the standard injection group. In the PIP joint, it was 75.1% in the ultrasound-guided injection group and 65.3% in the standard injection group. These results showed no statistical significance. Hand surgeons must balance the possible benefits of the ultrasound-guided injection with the complexity and resources needed to perform the technique.

Enzymes are biocatalysts that function as catalysts in biological functions. Collagenase enzymes have activities that can support product production. Enzyme activity for protein hydrolysis is influenced by several factors such as protein concentration, pH, temperature, substrate, inhibitor and activator. This study was aimed to determine the effect of differences in pH on the crude extract activity of the collagenase enzyme from the internal organs of malong fish. The study used a non-factorial completely randomized design, the treatments used were different pH (6.5, 7.5, and 8.5). This research consisted of several stages, namely sample preparation, extraction of internal organs to produce crude extract of the collagenase enzyme and test the activity of crude extract of the collagenase enzyme based on different pH. The analysis parameters was consisted of the proportion of fish yield, and the enzyme crude extract activity test at different pH. The results showed that malong fish has a proportion of meat 7,2 parts, bone 3,8, internal organs 1, skin 2,1. The yields of crude extract of collagenase enzyme pH 6.5, 7.5, and 8.5 were 60.83%, 64.09%, and 83.15%, respectively. The crude extract activity of collagenase enzyme from the internal organs of malong fish at pH 6.5, 7.5 and 8.5 was resulted in 0.293 U/ml, 0.5877 U/ml, and 0.7767 U/mL. The highest collagenase enzyme crude extract activity was produced at pH 8.5.

Background Joint replacement surgery provides articular cartilage samples for chondrocyte isolation. To our knowledge, the effect of the collagenase type on releasing of chondrocytes from the extracellular matrix of cartilage is not reported. Objectives To determine whether cartilage digested with collagenase IA yielded more chondrocytes than that digested with collagenase II and determine whether chondrocytes isolated with collagenase IA could be cultured in vitro. Methods Cartilage slices collected from 18 elderly patients who received joint replacement surgery (16 hips, 2 knees) were digested sequentially with 0.4% pronase E and 0.02% collagenase IA, or with 0.15% collagenase II alone, or sequentially with 0.4% pronase E and 0.02% collagenase II. We compared cell yield from each method. Cell viability by the most effective method was calculated and plotted. The morphology of cultured monolayer chondrocytes was recorded with a light microscope. Results Sequential digestion with pronase E and collagenase IA yielded 2566 ± 873 chondrocytes per mg wet cartilage, which was more effective than the other isolation methods (P = 0.018). The average chondrocyte viability could reach 84% ± 8% (n = 11). Light microscopic images showed typical chondrocyte morphology in monolayer cultures. Conclusion Sequential digestion of human articular cartilage with pronase E and collagenase IA was more effective than collagenase II alone or collagenase II combined with pronase E for releasing chondrocytes from extracellular matrix of cartilage. Chondrocytes isolated with this method could be maintained in monolayer cultures for at least 2 passages with unaltered morphology.

The intracerebral hemorrhage is associated with severe complications and high mortality. Currently there are no effective methods of treatment of this disease while the standard collagenase model of intracerebral hemorrhage is not described sufficiently. Objective. To analyze morphological characteristics of the collagenase model of intracerebral hemorrhage, and develop the regression formula predicting the hemorrhage volume based on the results of behavioral tests. Materials and methods. The experiments were carried out on 7 white male rats weighing 250-400 g aged 11-13 months. All animals underwent surgery to simulate intracerebral hemorrhage. Rats were anesthetized and then, stereotactically, using a needle with a diameter of 0.47 mm, 0.2 units of collagenase type IV were slowly injected into the left striatum. The day after the intracerebral hemorrhage, functional disabilities developed in rats were studied using beam walking test, neurological score test and adhesive removal test. Immediately after performing behavioral tests, the rats were sacrificed by decapitation. After the brain formalin fixation, serial sections on a vibromicrotome of 200 µm thick each in the anteroposterior direction with following morphological examination were made. Results. It was revealed that the collagenase model of intracerebral hemorrhage is associated with a large variability of the hemorrhage volume. It also had an irregular rugged shape and marks of repeated diapedetic hemorrhages of about 0.6 mm depth. The center of intracerebral hemorrhage along the anteroposterior axis was in average 0.5 mm posterior of the actual site of collagenase injection.The combined use of the neurological score test, the beam walking test and the adhesive removal test in the collagenase model can help estimate the probable intracerebral hemorrhage volume on the 1st day using the regression formula. Conclusions. Technical details identified in our study can help researchers in planning and conduction of correct experiments related to intracerebral hemorrhage.

The aim of this study was to report hand function, disability and satisfaction and patients perception of functionally troublesome contractures 5 years after injection with collagenase Clostridium histolyticum and hand therapy for Dupuytrens disease. Data from 79 patients were collected before and at 3, 12 and 60 months after treatment. Hand function was significantly improved, and 70% achieved a functional range of motion in the treated hand. QuickDASH scores and range of motion were best at 3 months follow-up. At 60 months, mean total extension deficit was 48 degrees, which was 57% of the deficit before treatment. Thirty-seven patients (47%) had developed recurrent contractures in treated finger(s) meeting the criteria for new treatment. The threshold for functionally troublesome contractures was found to be 30 degrees-60 degrees in the finger joints. Treatment was experienced as painful, but few hand function problems occurred. Most patients would choose this treatment method again. Funding Agencies|Orthopaedic Clinic at Kalmar County Hospital; Department of Occupational Therapy at Kalmar County Hospital; Department of Hand Surgery, Plastic Surgery and Burns, Linkoping University Hospital

BACKGROUND Fibrous connective tissue (referred to as fiber) in lipoaspirates would be discarded before lipotransfer in case of cannula blockage. However, the fiber contains extracellular matrix which provide structure support and is rich in stromal vascular fractions (SVFs). Removal of the fiber might theoretically affect the survival quality and mechanical properties of fat grafts. But there is few evidence in vivo and vitro about how the fiber affects the fat grafts. OBJECTIVE To assess the effect of fibers on the survival quality and mechanical property of fat grafts. METHODS The SVFs in both fat and fiber were obtained by collagenase digestion for cells counting and comparison. Three groups were designed according to the different proportions of fat and fiber: the fat group (100% fat), the mixed group (50% fat, 50% fiber in volume ratio), and the fiber group (100% fiber). Three groups of grafts were transplanted in vivo to evaluate the differences in volume retention rate, histological characteristics and mechanical properties. RESULTS The amount of SVF cells in fibers (3.47 ± 1.49 × 104 cells/mL) was significantly lower than that in fat (12.3 ± 4.95 × 104 cells/mL) (P

For birds, the uterus is an important part for eggshell mineralization, and the establishment of the endometrial epithelial cell (EEC) model was beneficial to the study of uterine function. This study was conducted to establish a culture model of primary EECs of laying hens and explore the effects of zinc on primary EEC proliferation, zinc transport, and calcium deposition in vitro. The EECs were isolated and cultured via type I collagenase digestion, and in the logarithmic phase during 2–5 days, and then reached the plateau phase on the 7th day of inoculation. Results showed that the proliferation of EECs treated by 50 μM ZnSO4 or zinc-methionine (Zn-Met) were markedly promoted at 24-h or 48-h treating time (P

Ficus carica produces, in addition to the cysteine protease ficin, a serine protease. Earlier study on a serine protease from F. carica cultivar Brown Turkey showed that it specifically degraded collagen. In this study, we characterized the collagenolytic activity of a serine protease in the latex of F. carica cultivar Masui Dauphine. The serine protease degraded denatured, but not undenatured, acid-solubilized type I collagen. It also degraded bovine serum albumin, while the collagenase from Clostridium histolyticum did not. These results indicated that the serine protease in Masui Dauphine is not collagen-specific. The protease was purified to homogeneity by two-dimensional gel electrophoresis, and its partial amino acid sequence was determined by liquid chromatography-tandem mass spectrometry. BLAST searches against the Viridiplantae (green plants) genome database revealed that the serine protease was a subtilisin-like protease. Our results contrast with the results of the earlier study stating that the serine protease from F. carica is collagen-specific.

Matrix metalloproteinases (MMPs) are a large group of proteolytic enzymes involved in extracellular matrix turnover in the ovary. Under physiological conditions, the activity of MMPs is controlled by specific tissue inhibitors of MMPs (TIMPs). Information concerning the role and regulation of MMPs in the chicken ovary is scarce. This study was undertaken to examine the expression of selected MMPs and their TIMPs in the chicken ovary during a pause in egg laying induced by feed deprivation. The activities of MMP-2 and MMP-9 were investigated as well. Real-time polymerase chain reaction and Western blot analyses showed changes in the expression of gelatinases (MMP-2, MMP-9), stromelysin (MMP-10), collagenase (MMP-13), TIMP-2, and TIMP-3 on mRNA and/or protein levels in the prehierarchical white (WFs) and yellowish (YFs) follicles, as well as in the largest yellow preovulatory (F3-F1) follicles. In feed-deprived hens, the occurrence of ovarian regression was accompanied by (1) a pronounced decrease in mRNA expression of the examined MMPs and TIMP-3 in all tissues except the YFs where the expression of MMP-13 was higher than in the control hen ovary; (2) an increase in the transcript abundance of TIMP-2 in the yellow atretic follicles; (3) a decrease or no changes in MMP-2 and MMP-9 protein expression in all tissues; (4) an increase in the total activity of gelatinases in the YFs and theca layer of F3; and (5) a decrease in the activity of MMP-2 in F3-F1 follicles and MMP-9 in the theca of F3. In summary, the results of the current study suggest that the selected MMPs and TIMPs may not be involved in the regulation of the advanced stages of atresia of the largest yellow preovulatory follicles in the chicken ovary. This event may require different cell signaling pathways.

Raman spectroscopy is promising in vivo tool in various biomedical applications; moreover, in recent years, its use for characterizing articular cartilage degeneration has been developing. It has also shown potential for scoring the severity of cartilage lesions, which could be useful in determining the optimal treatment strategy during cartilage repair surgery. However, the effect of different cartilage injury types on Raman spectra is unknown. This study aims to investigate the potential of Raman spectroscopy for detecting changes in cartilage due to different injury types. Artificial injuries were induced in cartilage samples using established mechanical and enzymatic approaches to mimic trauma‐induced and natural degeneration. Mechanical damage was induced using surface abrasion (ABR, n = 12) or impact loading (IMP, n = 12), while enzymatic damage was induced using three different treatments: 30 min trypsin digestion (T30, n = 12), 90 min collagenase digestion (C90, n = 12), and 24 h collagenase digestion (C24, n = 12). Raman spectra were obtained from all specimens, and partial least squares discriminant analysis (PLS‐DA) was used to distinguish cartilage injury types from their respective controls. PLS‐DA cross‐validation accuracies were higher for C24 (88%) and IMP (79%) than for C90 (67%), T30 (63%), and ABR (58%) groups. This study indicates that Raman spectroscopy, combined with multivariate analysis, can discern different cartilage injury types. This knowledge could be useful in clinical decision‐making, for example, selecting the optimal treatment remedy during cartilage repair surgery.

15 pags, 5 figs, 3 tabs. -- Supplementary data to this article can be found online at https://doi.org/10.1016/j.csbj.2021.11.027., Many microorganisms feed on the tissue and recalcitrant bone materials from dead animals, however little is known about the collaborative effort and characteristics of their enzymes. In this study, microbial metagenomes from symbionts of the marine bone-dwelling worm Osedax mucofloris, and from microbial biofilms growing on experimentally deployed bone surfaces were screened for specialized bone-degrading enzymes. A total of 2,043 taxonomically (closest match within 40 phyla) and functionally (1 proteolytic and 9 glycohydrolytic activities) diverse and non-redundant sequences (median pairwise identity of 23.6%) encoding such enzymes were retrieved. The taxonomic assignation and the median identity of 72.2% to homologous proteins reflect microbial and functional novelty associated to a specialized bone-degrading marine community. Binning suggests that only one generalist hosting all ten targeted activities, working in synergy with multiple specialists hosting a few or individual activities. Collagenases were the most abundant enzyme class, representing 48% of the total hits. A total of 47 diverse enzymes, representing 8 hydrolytic activities, were produced in Escherichia coli, whereof 13 were soluble and active. The biochemical analyses revealed a wide range of optimal pH (4.0-7.0), optimal temperature (5-65 °C), and of accepted substrates, specific to each microbial enzyme. This versatility may contribute to a high environmental plasticity of bone-degrading marine consortia that can be confronted to diverse habitats and bone materials. Through bone-meal degradation tests, we further demonstrated that some of these enzymes, particularly those from Flavobacteriaceae and Marinifilaceae, may be an asset for development of new value chains in the biorefinery industry., We acknowledge financial support of ERA-NET Marine Biotechnology (GA no.: 604814) funded under the FP7 ERA-NET scheme and nationally managed from the German Federal Ministry of Education and Research and Norwegian Research Council, the grants PCIN-2017-078 (within the Marine Biotechnology ERA-NET), BIO2017-85522-R, PID2020-112758RB-I00 and PDC2021-121534-I00 from the Ministerio de Economía, Industria y Competitividad, Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación (AEI) (Digital Object Identifier 10.13039/501100011033), Fondo Europeo de Desarrollo Regional (FEDER) and the European Union (“NextGenerationEU/PRTR”), and the grant 2020AEP061 from the Agencia Estatal CSIC. F.J.P and J.S-A. acknowledges grants PID2019-105838RB-C31 and PID2019-105838RB-C33 from the Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación (AEI), Fondo Europeo de Desarrollo Regional (FEDER) and the European Union (EU). Additional funding was received from the Norwegian Biodiversity Information Centre (PA 809116 knr. 47-14) and NORCE Norwegian Research Centre. J.W. acknowledge the use of de.NBI cloud and the support by the High Performance and Cloud Computing Group at the Zentrum für Datenverarbeitung of the Eberhard Karls University of Tübingen and the Federal Ministry of Education and Research (BMBF) through grant no 031 A535A. K.S.M. is supported by intramural funds of the US Department of Health and Human Services (to the National Library of Medicine).

There are several sources of collagen, one of which is the yellowfin tuna bones. Collagen is a bioactive peptide with specific biological characteristics so that it can be used widely. However, hydrolysis is carried out to produce collagen hydrolysate to increase the biological effectiveness of collagen and its application. This study aimed to produce collagen hydrolysate from yellowfin tuna bones using collagenase from Bacillus sp. 6-2 and evaluate its ability as an antibacterial agent. The Hydrolysis process was optimized by varying the concentration of substrate, enzyme and hydrolysis time. To determine the effectiveness of the hydrolysis process in inhibiting bacterial growth, the hydrolysis degree and antibacterial activity of collagen hydrolyzate have been investigated. Results showed that hydrolysis at a substrate concentration of 10 %, enzyme of 10 % and hydrolysis time of 4 h was the optimum conditions to obtain the highest degree of hydrolysis (22.48 %). All collagen hydrolysates obtained have antibacterial activity against Escherichia coli and Staphylococcus aureus with the highest inhibition zones of 11.50 mm and 12.60 mm respectively. Collagen hydrolysate from yellowfin tuna bones is bactericidal so it is a very good candidate to be developed as an antibacterial agent.

An investigation into the methanol extracts obtained from the stems of Dodonaea viscosa led to the isolation of one nor-clerodane diterpene (1) and two labdane diterpenes (2, 3), as well as 17 known compounds (4-20). The structures of these compounds were elucidated based on chemical and spectral evidence. The stereochemical structure of the nor-clerodane diterpene was confirmed via its circular dichroism spectrum and calculated electronic circular dichroism spectrum. Isolated compounds were evaluated for their inhibitory effects on collagenase and tyrosinase. Since 5,7,4'-trihydroxy-3'-(4-hydroxy-3-methylbutyl)-5'-(3-methylbut-2-enyl)-3,6-dimethoxyflavone (9) showed collagenase inhibitory activity and scopoletin (12) had significant tyrosinase inhibitory activity, they were considered to be good candidates for cosmetic agents.

The dense extracellular matrix (ECM) in tumor tissue severely hinders the penetration and enrichment of antitumor nanomedicines, which could significantly affect their efficiency. In this study, we used pH-sensitive nanocarriers loaded with collagenase (Col) to remold the tumor microenvironment (TME). Furthermore, we combined the collagenase delivery system with a nanomedicine to improve its penetration and enrichment in the tumor, thereby improving efficacy. We synthesized acetalated dextran (Ace-DEX) with an ideal pH-sensitivity as the carrier material of collagenase. Under mild preparation conditions, collagenase was loaded into Ace-DEX nanoparticles (NPs) with a high loading capacity (>4%) and remained highly active (>90%). Col-carrying NPs (Col-NPs) significantly reduced the tumor collagen content by 15.1%. Pretreatment with Col-NPs increased the accumulation of doxorubicin (DOX)-loaded liposome (DOX-Lipo) in the tumor by 2.8-fold. There were no safety concerns as the Col-NP showed no significant toxicity and reduced Col-induced damage to healthy tissues. Additionally, the number of circulating tumor cells remained unchanged after Col-NP treatment, suggesting no increased risk of tumor metastasis. Because the Col-NP acts essentially independent of the subsequent treatment, it has considerable potential for enhancing many existing delivery systems and drugs for cancer treatment. It may also be used for treating other collagen-related diseases.

A face serum composed of a combination of biologically active compounds was evaluated for safety and efficacy in vitro, in a repeat insult patch test and in a human clinical efficacy trial. The serum inhibited tyrosinase activity modestly, decreased collagenase activity and exhibited notable free radical scavenging activity in vitro. It is gentle to the skin, as the serum did not irritate the skin or produce symptoms of allergic contact dermatitis in the 55 healthy adults that participated in the repeat insult patch test. In the efficacy trial, daily application of the face serum for 30 days significantly increased skin hydration, with all 35 volunteers experiencing improvement. Substantial improvements in skin elasticity, roughness (fine lines and wrinkles), and brightness also occurred during the trial. Dermatological examination also revealed a trend for reduced comedone count with use of the serum. Self-assessment responses revealed that all volunteers experienced improvements in multiple skin quality parameters and that participant perceptions are consistent with the results of the instrumental analyses. These findings indicated that the measured improvements in skin quality are not only statistically significant but are also clinically relevant as they were great enough for users of the face serum to feel and recognize.

Matrix metalloproteinases (MMPs) are one of the most important endopeptidases in periodontal disease that generally degrade extracellular matrix (ECM) components of periodontal supporting tissues, leading to tooth loss. Among the MMP family, MMP-1, -8 and -13, which are also known as the collagenase group, play a vital role in the degradation of ECM collagen and non-collagen substances. Elevated levels of MMP -1, -8 and -13 are markedly significant within tissue, gingival crevicular fluid (GCF), and saliva of patients with periodontitis, which help to explain the progression pattern of the disease. This review provides an overview of MMP -1, -8, and -13 on their structures, functions and their critical role in periodontitis.

A series of Plectranthus spp. plant extracts (aqueous, acetonic, methanolic and ethyl acetic) obtained from eight different species, and previously isolated compounds (ranging from polyphenols, diterpenes and triterpenes), were assayed for in vitro inhibition of the skin-related enzymes tyrosinase, collagenase and elastase, and for studying their antioxidant properties. The ethyl acetic extracts of P. grandidentatus and P. ecklonii registered the highest antioxidant activity, whereas acetonic, methanolic and ethyl acetic extracts of P. ecklonii, P. grandidentatus, P. madagascariensis and P. saccatus concerning the enzymatic inhibition assays revealed high anti-tyrosinase and anti-collagenase activities. From the isolated compounds tested, abietane diterpenes and triterpenes were highly active against tyrosinase and elastase activity. Overall, the experimental results showed the powerful antioxidant and inhibitory action on skin-related enzymes tyrosinase, collagenase and elastase of Plectranthus spp. extracts and/or isolated compounds, supporting their further research as bioactive metabolites against skin sagging and hyperpigmentation in cosmetic and pharmaceutical formulations., Graphical Abstract

Purpose of the study:to investigate the impact of hydroxyethyl starch (HES) and polyethylene oxide (PEO) on the indicators of preservation of murine testis interstitial cells (IC) under cryopreservation. Materials and methods.To isolate IC the enzymes were used: 0.2 mg/ml collagenase and 0.1 mg/ml DNase. The obtained cell suspension was cryopreserved in the solutions that contained 0; 0,7; 1,4; 2,1; 2,8 M of dimethyl sulfoxide (DMSO) and/or 10%, 20% fetal cow serum, 10 mg/ml PEO or HES. The samples (1 ml) were cooled at a rate of 1 °C/min to -80 °C then stored in liquid nitrogen (-196 °C). They were warmed at 37 °C in the water bath. Cryopreservation solution was removed. The number of cells and their preservation were assessed before and after with the assistance of Goryaev’s camera. Viability of IC, Leydig cell preservation and preservation of metabolic activity were measured with trypan blue dye, histochemical staining for 3β-hydroxysteroid dehydrogenase activity. Results.It was shown that 1,4 M DMSO without supplements favored IC preservation. Addition to the cryopreservation solution 10% and 20% of fetal cow serum or 10 mg/ml HES increased total preservation of IC by more than 10% and Leydig cell cryopreservation by an average 15%. HES 10 mg/ml may decrease DMSO concentration to 0,7 M. This combination had the best indicators of total preservation of IC, preservation of viable cells and Leydig cells: 75,8 (53,3; 93,3), 55,6 (45,1; 69,4), 57,1 (40,2;70,3) %, respectively. PEO was ineffective. Conclusion.High-molecular weight synthetic polymers such as HES can substitute protective properties of blood serum under cryopreservation and allow decreasing effective concentration of permeable cryoprotective such as DMSO.

We report a multiplexed imaging mass spectrometry method which spatially localizes and selectively accesses the extracellular matrix on formalin-fixed paraffin-embedded tissue sections. The extracellular matrix (ECM) consists of (1) fibrous proteins, post-translationally modified (PTM) via N- and O-linked glycosylation, as well as hydroxylation on prolines and lysines, and (2) glycosaminoglycan-decorated proteoglycans. Accessing all these components poses a unique analytical challenge. Conventional peptide analysis via trypsin inefficiently captures ECM peptides due to their low abundance, intra- and intermolecular cross-linking, and PTMs. In previous studies, we have developed matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) techniques to capture collagen peptides via collagenase type III digestion, both alone and after N-glycan removal via PNGaseF digest. However, in fibrotic tissues, the buildup of ECM components other than collagen-type proteins, including elastin and glycosaminoglycans, limits efficacy of any single enzyme to access the complex ECM. Here, we have developed a novel serial enzyme strategy to define the extracellular matrix, including PTMs, from a single tissue section for MALDI-IMS applications. Graphical Abstract.

Anacardium occidentale (cashew) has been used in the traditional system of medicine for curing many inflammatory disorders. The present study investigates the antiarthritic effects of cashew leaves extract using the rat model of FCA-induced rheumatoid arthritis. Arthritic rats were treated with 100 and 200 mg/kg b.w. ethanolic extract of cashew leaves. Animals were sacrificed at day 23, and before sacrificing the animals, gross pathological changes were observed. Histopathology of ankle joint was evaluated with hematoxylin and eosin staining, whereas the serum levels of C-reactive protein (CRP) were evaluated by the agglutination method. Inflammatory cells and other hematological parameters were assessed by employing an automated hemocytometer and chemistry analyzer. Rheumatoid factor (Rf) and lysosomal enzymes levels were determined in blood. Results indicated that A. occidentale significantly decreased the CPR levels, macroscopic arthritic score, and rheumatoid factor as compared to the diseased group. Histopathological evaluation showed significant attenuation in bone erosion, joint inflammation, and pannus formation by plant extract. Treatment with A. occidentale significantly suppressed the levels of acid phosphatase, β-galactosidase, β-glucuronidase, N-acetylglucosaminidase, and collagenase. Moreover, A. occidentale significantly raised the HB levels and RBCs counts which were found depleted in the diseased group. The raised counts of total leukocytes, platelets, neutrophils, lymphocytes, and monocytes were also significantly decreased by treatment with plant extract. Comparative analysis showed that higher dose of A. occidentale demonstrated superior amelioration of rheumatoid arthritis as compared to low dose. In conclusion, A. occidentale possesses significant antiarthritic potential, which may be attributed to the suppression of lysosomal enzymes and collagenase levels.

Local unwinding of the collagen triple helix is a necessary step for initiating the collagen degradation cascade in extracellular matrices. A few matrix metalloproteinases (MMPs) are known to support this key process, but its energetic aspects remain unknown. Here, we captured the thermodynamics of the triple helix unwinding by monitoring interactions between a collagen peptide and MMP-1(E200A) – an active-site mutant of an archetypal vertebrate collagenase – at increasing temperatures, using isothermal titration calorimetry (ITC). Coupled binding and unwinding manifests as a curved relationship between the total enthalpy change and temperature of the reaction, producing increasingly negative heat capacity change (ΔΔCp≈ −36.3 kcal/molK2). A specially designed solid-phase binding and cleavage assay (SPBCA) reported strain in the catalytically relevant unwound state, suggesting that this state is distinct from the horizon of sampled conformations of the collagenase-susceptible site. MMP-1 appears to blend selected fit with induced fit mechanisms to catalyse collagen unwinding prior to cleavage of individual collagen chains.Graphical AbstractHighlightsThe unwinding of the collagen triple helix by matrix metalloproteinases (MMPs) is critical for collagen catabolism, but how MMPs harness bioavailable energy for this energetically expensive process is enigmaticRising temperature causes linear increases of the negative heat capacity change associated with the interaction of MMP-1 with a collagenase-susceptible triple-helical peptide, indicating enrichment of hydrophobic contacts during the triple helix unwindingThe complex of MMP-1 with locally unwound collagen holds considerable structural strain, which becomes relieved upon cleavage of the unwound collagen strand(s)MMP-1 consumes heat to drive specific (structurally distinct) collagen unwinding by increasing entropy associated with evolving hydrophobic anchor points between the enzyme and the substrateThe prototypic collagenase MMP-1 appears to blend conformational selection with induced fit, shedding light on the thermodynamic principles by which MMPs trigger collagen breakdown and supporting general mechanistic conclusions concerning conformational changes coupled to protein-protein binding

This study investigated the impacts of gelatin hydrolysate addition on the technological properties and lipid oxidation stability of cooked sausage. Gelatin hydrolysate was prepared from pork and duck skin gelatin, through stepwise hydrolysis using collagenase and pepsin. The cooked sausages were formulated without gelatin (control) or with 1% pork skin gelatin, 1% duck skin gelatin, 1% pork skin gelatin hydrolysate, and 1% duck skin gelatin hydrolysate. The pH, color characteristics, protein solubility, cooking loss, and textural properties of cooked sausages were evaluated, and the 2-thiobarbituric acid reactive substances (TBARS) value was measured weekly to determine lipid oxidation stability during 4 wk of refrigerated storage. Enzymatic hydrolysis of gelatin decreased protein content and CIE L* but increased redness and yellowness (p0.05). The addition of 1% duck skin gelatin hydrolysate increased the cohesiveness and chewiness of cooked sausages. The inclusion of 1% duck skin gelatin accelerated lipid oxidation of cooked sausages during refrigerated storage (p