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Purpose: Symptomatic vitreous opacifications, so-called floaters, are difficult to objectively assess majorly limiting the possibility of in vitro studies. Forward light scattering was found previously to be increased in eyes with symptomatic floaters. Using an objective setup to measure forward light scattering, we studied the effects of enzymatically digesting the components of the vitreous body on straylight to develop an in vitro model of vitreous opacifications., Methods: Fifty-seven porcine vitreous bodies were digested using hyaluronidase, collagenase, trypsin, and bromelain, as well as using a combination of hyaluronidase + collagenase and hyaluronidase + bromelain. A modified C-Quant setup was used to objectively assess forward light scattering., Results: Depletion of hyaluronic acid majorly increased vitreous straylight (mean increase 34.4 deg2/sr; P = 0.01), whereas primarily digesting the vitreous gel with collagenase or trypsin did not significantly affect straylight. When collagenase or bromelain is applied in hyaluronic acid depleted vitreous gels, the increase in forward light scattering is reversed partially., Conclusions: The age-related loss of hyaluronic acid primarily drives the increase in vitreous gel straylight induced by conglomerates of collagen. This process can be reversed partially by digesting collagen. This in vitro model allows the objective quantification and statistical comparison of straylight burden caused by vitreous opacities and, thus, can serve as a first testing ground for pharmacological therapies, as demonstrated with bromelain.

Patellar tendinopathy is a common clinical problem, but its underlying pathophysiology remains poorly understood, primarily due to the absence of a representative experimental model. The most widely used method to generate such a model is collagenase injection, although this method possesses limitations. We developed an optimized rat model of patellar tendinopathy via the ultrasound-guided injection of collagenase mixed with a thermo-responsive Pluronic hydrogel into the patellar tendon of sixty male Wistar rats. All analyses were carried out at 3, 7, 14, 30, and 60 days post-injury. We confirmed that our rat model reproduced the pathophysiology observed in human patients through analyses of ultrasonography, histology, immunofluorescence, and biomechanical parameters. Tendons that were injured by the injection of the collagenase-Pluronic mixture exhibited a significant increase in the cross-sectional area ( p < 0.01), a high degree of tissue disorganization and hypercellularity, significantly strong neovascularization ( p < 0.01), important changes in the levels of types I and III collagen expression, and the organization and presence of intra-tendinous calcifications. Decreases in the maximum rupture force and stiffness were also observed. These results demonstrate that our model replicates the key features observed in human patellar tendinopathy. Collagenase is evenly distributed, as the Pluronic hydrogel prevents its leakage and thus, damage to surrounding tissues. Therefore, this model is valuable for testing new treatments for patellar tendinopathy.

Objective: To investigate the cross-linking and protective effect of artemisinin (ART), dihydroartemisinin (DHA), and artesunate (AST) on collagen fibers of demineralized dentin surface., Methods: Molecular docking was used to predict potential interactions of ART, DHA, and AST with dentin type I collagen. Human third molars without caries were completely demineralized and treated with different solutions for 1 min. The molecular interactions and cross-linking degree of ART and its derivatives with dentin collagen were evaluated by FTIR spectroscopy, total extractable protein content, and a ninhydrin assay. Scanning electron microscopy, hydroxyproline release, and ultimate microtensile strength tests (μUTS) were employed to confirm the mechanical properties and anti-collagenase degradation properties of dentin collagen fibers., Results: ART, DHA, and AST combined with dentin type I collagen mainly through hydrogen bonding and hydrophobic interactions, and the cross-linking reaction sites were mainly C=O and CN functional groups. Compared to the control group, ART and its derivatives significantly increased the degree of cross-linking. Additionally, significant increases were observed in resistance to enzymatic digestion and mechanical properties of the artemisinin and its derivatives group., Conclusion: ART, DHA, and AST could cross-link with demineralized dentin collagen, through improving the mechanical properties and anti-collagenase degradation properties., Clinical Significance: The study endorses the potential use of ART and its derivatives as a prospective collagen cross-linking agent for degradation-resistant and long-period dentin bonding in composite resin restorations., Competing Interests: Declaration of Competing Interest The authors in this study declare that they have no conflict of financial interest or personal connections, and all authors give informed consent for the publication of this manuscript., (Copyright © 2023. Published by Elsevier Ltd.)

Patients with temporomandibular dysfunction (TMD) usually suffer from pathology or malpositioning of the temporomandibular joint (TMJ) disk, leading to the degenerative lesion of condyles. Kartogenin can promote the repair of damaged cartilage. This study aimed to explore whether intra-articular injection of kartogenin could alleviate the TMJ injury induced by type II collagenase. We measured the head withdrawal threshold and found that kartogenin alleviated the pain around TMD induced by type II collagenase. We observed the morphology of the condylar surface and found that kartogenin protected the integration of the condylar surface. We analyzed the density of the subchondral bone and found that kartogenin minimized the damage of TMJ injury to the subchondral bone. We next explored the histological changes and found that kartogenin increased the thickness of the proliferative layer and more collagen formation in the superficial layer. Then, to further ensure whether kartogenin promotes cell proliferation in the condyle, we performed immunohistochemistry of proliferating cell nuclear antigen (PCNA). The ratio of PCNA-positive cells was significantly increased in the kartogenin group. Next, immunofluorescence of TGF-β1 and SMAD3 was performed to reveal that kartogenin activated the TGF-β/SMAD pathway in the proliferative layer. In conclusion, kartogenin may have a therapeutic effect on TMJ injury by promoting cell proliferation in cartilage and subchondral bone. Kartogenin may be promising as an intra-articular injection agent to treat TMD., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: Common respiratory illnesses, such as emphysema and chronic obstructive pulmonary disease, are characterized by connective tissue damage and remodeling. Two major fibers govern the mechanics of airway tissue: elastin enables stretch and permits airway recoil, while collagen prevents overextension with stiffer properties. Collagenase and elastase degradation treatments are common avenues for contrasting the role of collagen and elastin in healthy and diseased states; while previous lung studies of collagen and elastin have analyzed parenchymal strips in animal and human specimens, none have focused on the airways to date., Methods: Specimens were extracted from the proximal and distal airways, namely the trachea, large bronchi, and small bronchi to facilitate evaluations of material heterogeneity, and subjected to biaxial planar loading in the circumferential and axial directions to assess airway anisotropy. Next, samples were subjected to collagenase and elastase enzymatic treatment and tensile tests were repeated. Airway tissue mechanical properties pre- and post-treatment were comprehensively characterized via measures of initial and ultimate moduli, strain transitions, maximum stress, hysteresis, energy loss, and viscoelasticity to gain insights regarding the specialized role of individual connective tissue fibers and network interactions., Results: Enzymatic treatment demonstrated an increase in airway tissue compliance throughout loading and resulted in at least a 50% decrease in maximum stress overall. Strain transition values led to significant anisotropic manifestation post-treatment, where circumferential tissues transitioned at higher strains compared to axial counterparts. Hysteresis values and energy loss decreased after enzymatic treatment, where hysteresis reduced by almost half of the untreated value. Anisotropic ratios exhibited axially led stiffness at low strains which transitioned to circumferentially led stiffness when subjected to higher strains. Viscoelastic stress relaxation was found to be greater in the circumferential direction for bronchial airway regions compared to axial counterparts., Conclusion: Targeted fiber treatment resulted in mechanical alterations across the loading range and interactions between elastin and collagen connective tissue networks was observed. Providing novel mechanical characterization of elastase and collagenase treated airways aids our understanding of individual and interconnected fiber roles, ultimately helping to establish a foundation for constructing constitutive models to represent various states and progressions of pulmonary disease., (© 2023. The Author(s).)

Surgical excision and grafting of deep partial-thickness (DPT) and full-thickness (FT) burns is a cornerstone of wound care. The use of commercially available topical enzymatic agents has been limited due to slower and less complete eschar removal than surgical excision. Using a porcine model of DPT and FT burns, we compared the eschar removal efficacy of a bromelain-enriched enzymatic agent derived from the stems of pineapple plants and a commercially available collagenase. We created 40 DPT and 40 FT burns on four anesthetized Yorkshire pigs. Eschar removal was initiated 24 hours later. Two pigs each were randomly assigned to collagenase or the bromelain-enriched agent. The bromelain-enriched agent was applied topically once for 4 hours followed by a 2-hour soaking. The collagenase was applied topically daily until complete removal of eschar or for up to 14 days. All bromelain-enriched treated FT burns underwent complete removal of the eschar after a single application while none of the collagenase-treated FT burns underwent complete removal of the eschar even after 14 days of treatment. All bromelain-enriched treated DPT burns had complete eschar removal after the single application. None of the collagenase-treated DPT burns experienced complete removal of eschar after 10 days; by day 14, 35% had complete eschar removal, 30% had >50% eschar removed, and 35% had <50% eschar removed. We conclude that eschar removal is quicker and more complete with the bromelain-enriched compared with collagenase debriding agent., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Burn Association. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Skin aging is associated with degradation of collagen by matrix metalloproteinases (MMPs), which leads to loss of skin elasticity and formation of wrinkles. Cosmos caudatus Kunth (CC) has been traditionally claimed as an anti-aging agent in Malaysia. Despite its well-known antioxidant activity, the anti-aging properties of CC was not validated., Purpose: This study aimed to investigate the anti-aging potential of CC extracts and fractions, particularly their inhibition of collagenase, MMP-1 and MMP-3 activities in human dermal fibroblasts CCD-966SK, followed by isolation, identification and analysis of their bioactive constituents., Study Design and Methods: DPPH assay was firstly used to evaluate the antioxidant activity throughout the bioactivity-guided fractionation. Cell viability was determined using MTS assay. Collagenase activity was examined, while MMP-1 and MMP-3 expression were measured using qRT-PCR and western blotting. Then, chemical identification of pure compounds isolated from CC fractions was done by using ESIMS, 1 H and 13 C NMR spectroscopies. HPLC analyses were carried out for bioactive fractions to quantify the major components., Results: Throughout the antioxidant activity-guided fractionation, fractions CC-E2 and CC-E3 with antioxidant activity and no toxicity towards CCD-966SK cells were obtained from CC 75% ethanol partitioned layer (CC-E). Both fractions inhibited collagenase activity, MMP-1 and MMP-3 mRNA and protein expression, as well as NF-κB activation induced by TNF-α in CCD-966SK cells. 14 compounds, which mainly consists of flavonoids and their glycosides, were isolated. Quercitrin (14.79% w/w) and quercetin (11.20% w/w) were major compounds in CC-E2 and CC-E3, respectively, as quantified by HPLC. Interestingly, both fractions also inhibited the MMP-3 protein expression synergistically, compared with treatment alone., Conclusion: The quantified CC fractions rich in flavonoid glycosides exhibited skin anti-aging effects via the inhibition of collagenase, MMP-1 and MMP-3 activities, probably through NF-κB pathway. This is the first study reported on MMP-1 and MMP-3 inhibitory activity of CC with its chemical profile, which revealed its potential to be developed as anti-aging products in the future., Competing Interests: Declaration of Competing Interest The authors have no competing interests regarding the publication of this study., (Copyright © 2023. Published by Elsevier GmbH.)

Objectives: To assess the anti-proteolytic effect and potential to inhibit dentin root caries progression of a silver nanoparticle and fluoride solution (CNanoF) in comparison to silver diamine fluoride (SDF)., Methods: 48 specimens of root dentin artificial caries lesion were treated with 38% SDF, CNanoF, CNano or F (n = 6 per group). Ph cycling with demineralization and remineralization solutions simulated caries lesion progression. In addition, specimens were incubated with or without bacterial collagenase in the remineralization solution to induce dentin proteolytic degradation. Dentin degradation was assessed by weight loss rate and hydroxyproline (Hyp) release. Changes in cross-sectional microhardness, and lesion permeability and collagen integrity as determined by confocal laser scanning microscopy indicated potential for further demineralization inhibition. The effect of the solutions on the activity of metalloproteinases (MMP) -2 and -9 was also investigated. Statistical analysis consisted of ANOVA, Kruskal-Wallis, and linear mixed models with post-hoc pairwise Tukey, Dunn, and t-tests (α = 0.05)., Results: Treatment with SDF resulted in lower weight loss rate than did other solutions, but all groups showed similar Hyp release (p = 0.183). SDF resulted in greater microhardness at superficial layers of the caries lesions (p<0.05), while there were no differences among CNanoF, CNano, and F. Lesion permeability was similar among all groups after pH cycling (p>0.05), with or without the use of collagenase (p = 0.58). No statistically significant difference was noted among solutions regarding collagen integrity after pH cycling; however, SDF-treated dentin had a significant decrease in collagen integrity when collagenase was used (p = 0.003). Interestingly, only SDF was able to completely inactivate MMP-2 and -9., Conclusions: CNanoF and SDF both potentially prevent dentin degradation during caries lesion progression in vitro; however, SDF was more effective at inhibiting further tissue demineralization., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Sandrine Bittencourt Berger is inventor of patent #NBR102019024385-6., (Copyright: © 2023 Dias et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The highest disability rates and mortality among neurodegenerative diseases were caused by intracerebral hemorrhage (ICH). We previously proved that Benzene, 1,2,4-trimethoxy-5-(2-methyl-1-propen-1-yl) (BTY) has an inhibitory effect on sodium ion channel and an activation effect on GABA A receptor, which were related to the brain injury. Based on this, we aimed to investigate BTY's neuroprotection on intracerebral hemorrhage and its underlying mechanism. In the in vivo study, a stereotactic injection of collagenase VII in Sprague Dawley rats (0.5 U) induced ICH and the BTY was intraperitoneally injected at 2 h after ICH. The neurological deficit scores, blood-brain barrier (BBB) permeability, and other indicators were assessed 24 h after ICH. The results showed that the BTY reduced brain edema and hematoma volume, improved neurological function and BBB permeability, and inhibited inflammatory factors and neuron apoptosis. The cell experiments proved that the BTY suppressed oxidative stress, cell apoptosis, intracellular calcium influx, and stabilized mitochondrial membrane potential by reducing glutamate's excitotoxicity. This study for the first time exhibited desirable neuroprotection of BTY, indicating it may be a promising neuroprotective agent for ICH therapy., (Copyright © 2022 IBRO. Published by Elsevier Ltd. All rights reserved.)

During liver fibrogenesis, liver sinusoidal capillarization and extracellular matrix (ECM) deposition construct dual pathological barriers to drug delivery. Upon capillarization, the vanished fenestrae in liver sinusoidal endothelial cells (LSECs) significantly hinder substance exchange between blood and liver cells, while excessive ECM further hinders the delivery of nanocarriers to activated hepatic stellate cells (HSCs). Herein, an efficient nanodrug delivery system was constructed to sequentially break through the capillarized LSEC barrier and the deposited ECM barrier. For the first barrier, LSEC-targeting and fenestrae-repairing nanoparticles (named HA-NPs/SMV) were designed on the basis of the modification with hyaluronic acid and the loading of simvastatin (SMV). For the second barrier, collagenase I and vitamin A codecorated nanoparticles with collagen-ablating and HSC-targeting functions (named CV-NPs/siCol1α1) were prepared to deliver siCol1α1 with the goal of inhibiting collagen generation and HSC activation. Our in vivo results showed that upon encountering the capillarized LSEC barrier, HA-NPs/SMV rapidly released SMV and exerted a fenestrae-repairing function, which allowed more CV-NPs/siCol1α1 to enter the space of Disse to degrade deposited collagen and finally to achieve higher accumulation in activated HSCs. Scanning electronic microscopy images showed the recovery of liver sinusoids, and analysis of liver tissue sections demonstrated that HA-NPs/SMV and CV-NPs/siCol1α1 had a synergetic effect. Our pathological barrier-normalization strategy provides an antifibrotic therapeutic regimen.

Objective: The objective of this study was to evaluate photochemical crosslinking using Al(III) phthalocyanine chloride tetrasulfonic acid (CASPc) and light with a wavelength of 670 nm as a potential therapy to strengthen articular cartilage and prevent tissue degradation., Design: Changes in viscoelastic properties with indentation were used to identify 2 crosslinking protocols for further testing. Crosslinked cartilage was subjected to an in vitro , accelerated wear test. The ability of the crosslinked tissue to resist biochemical degradation via collagenase was also measured. To better understand how photochemical crosslinking with CASPc varies through the depth of the tissue, the distribution of photo-initiator and penetration of light through the tissue depth was characterized. Finally, the effect of CASPc on chondrocyte viability and of co-treatment with an antioxidant was evaluated., Results: The equilibrium modulus was the most sensitive viscoelastic measure of crosslinking. Crosslinking decreased both mechanical wear and collagenase digestion compared with control cartilage. These beneficial effects were realized despite the fact that crosslinking appeared to be localized to a region near the articular surface. In addition, chondrocyte viability was maintained in crosslinked tissue treated with antioxidants., Conclusion: These results suggest that photochemical crosslinking with CASPc and 670 nm light holds promise as a potential therapy to prevent cartilage degeneration by protecting cartilage from mechanical wear and biochemical degradation. Limitations were also evident, however, as an antioxidant treatment was necessary to maintain chondrocyte viability in crosslinked tissue.

Osteoarthritis (OA) is an inflammatory joint condition, still lacking effective treatments. Some factors consider as the main causes of OA, including biochemical, mechanical, and genetic factors. The growth of studies confirmed that modern medicine in combination with folk medicine regarding the arrival of reliable, efficient, and safe therapeutic products against OA. In the present study, the effects of various single and combinatorial treatments of knee articular cartilage, including stem cells, collagen, and P. atlantica hydroalcoholic leaves extract were investigated in a rat-induced OA model. On week 12 after OA confirmation, histopathology and radiography assessments were evaluated and the serum and synovial fluid levels of TAC, TNF-α, PEG2, MPO, MMP3, MMP13, and MDA were also measured. Combination therapy of OA-induced rats with hydroalcoholic extract of P. atlantic leaves, stem cells, and collagen considerably increased the efficacy of treatment as evidenced by increasing the TAC and lowering TNF-α, MPO, MMP3, and MMP13 compared to control group and even groups received single therapy. This is in agreement with a high amount of total phenolic compounds and antioxidant capacities of the hydroalcoholic extract of P. atlantic leaves. It is concluded that multifunctional agents targeting the pathophysiology of OA has exhibited significant therapeutic effects against OA., (© 2021 International Center for Artificial Organs and Transplantation and Wiley Periodicals LLC.)

Clostridium collagenase has provided superior clinical results in achieving digestion of immediate and accumulating devitalized collagen tissue. Recent studies suggest that debridement via Clostridium collagenase modulates a cellular response to foster an anti-inflammatory microenvironment milieu, allowing for a more coordinated healing response. In an effort to better understand its role in burn wounds, we evaluated Clostridium collagenase's ability to effectively minimize burn progression using the classic burn comb model in pigs. Following burn injury, wounds were treated with Clostridium collagenase or control vehicle daily and biopsied at various time points. Biopsies were evaluated for factors associated with progressing necrosis as well as inflammatory response associated with treatment. Data presented herein showed that Clostridium collagenase treatment prevented destruction of dermal collagen. Additionally, treatment with collagenase reduced necrosis (HMGB1) and apoptosis (CC3a) early in burn injuries, allowing for increased infiltration of cells and protecting tissue from conversion. Furthermore, early epidermal separation and epidermal loss with a clearly defined basement membrane was observed in the treated wounds. We also show that collagenase treatment provided an early and improved inflammatory response followed by faster resolution in neutrophils. In assessing the inflammatory response, collagenase-treated wounds exhibited significantly greater neutrophil influx at day 1, with macrophage recruitment throughout days 2 and 4. In further evaluation, macrophage polarization to MHC II and vascular network maintenance were significantly increased in collagenase-treated wounds, indicative of a pro-resolving macrophage environment. Taken together, these data validate the impact of clostridial collagenases in the pathophysiology of burn wounds and that they complement patient outcomes in the clinical scenario.

Components of the extracellular matrix (ECM) are overexpressed in fibrotic liver. Collagen is the main component of the liver fibrosis stroma. Here we demonstrate that chondroitin sulfate coated multilayered 50-nm nanoparticles encapsulating collagenase and silibinin (COL + SLB-MLPs) break down the dense collagen stroma, while silibinin inhibits activated hepatic stellate cells. The nanoparticles were taken up to a much greater extent by hepatic stellate cells than by normal hepatocytes, and they down-regulated production of type I collagen. In addition, chondroitin sulfate protected the collagenase from premature deactivation. COL + SLB-MLPs were delivered to the cirrhotic liver, and the collagenase and silibinin synergistically inhibited fibrosis in mice. Immunofluorescence staining of liver tissues revealed that CD44, mediated by chondroitin sulfate, delivered the nanoparticles to hepatic stellate cells. This strategy holds promise for degrading extracellular stroma and thereby facilitating drug penetration into fibrotic liver and related diseases such as liver cirrhosis and liver cancer., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) create an unlimited cell source for basic and translational research. Depending on the maturity of cardiac cultures and the intended applications, obtaining hiPSC-CMs as a single-cell, monolayer or three-dimensional clusters can be challenging. Here, we defined strategies to replate hiPSC-CMs on early days (D15-30) or later more mature (D60-150) differentiation cultures. After generation of hiPSCs and derivation of cardiomyocytes, four dissociation reagents Collagenase A/B, Collagenase II, TrypLE, EDTA and five different extracellular matrix materials Laminin, iMatrix-511, Fibronectin, Matrigel, and Geltrex were comparatively evaluated by imaging, cell viability, and contraction analysis. For early cardiac differentiation cultures mimicking mostly the embryonic stage, the highest adhesion, cell viability, and beating frequencies were achieved by treatment with the TrypLE enzyme. Video-based contraction analysis demonstrated higher beating rates after replating compared to before treatment. For later differentiation days of more mature cardiac cultures, dissociation with EDTA and replating cells on Geltrex or Laminin-derivatives yielded better recovery. Cardiac clusters at various sizes were detected in several groups treated with collagenases. Collectively, our findings revealed the selection criteria of the dissociation approach and coating matrix for replating iPSC-CMs based on the maturity and the requirements of further downstream applications., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Intracerebral hemorrhage (ICH) is a devastating insult with few effective treatments. Edema and raised intracranial pressure contribute to poor outcome after ICH. Glibenclamide blocks the sulfonylurea 1 transient receptor potential melastatin 4 (Sur1-Trpm4) channel implicated in edema formation. While glibenclamide has been found to improve outcome and reduce mortality in animal models of severe ischemic stroke, in ICH the effects are less clear. In our previous study, we found no benefit after a moderate-sized bleed, while others have reported benefit. Here we tested the hypothesis that glibenclamide may only be effective in severe ICH, where edema is an important contributor to outcome. Glibenclamide (10 μg/kg loading dose, 200 ng/h continuous infusion) was administered 2 hours post-ICH induced by collagenase injection into the striatum of adult rats. A survival period of 24 hours was maintained for experiments 1-3, and 72 hours for experiment 4. Glibenclamide did not affect hematoma volume (~81 μL) or other safety endpoints (e.g., glucose levels), suggesting the drug is safe. However, glibenclamide did not lessen striatal edema (~83% brain water content), ionic dyshomeostasis (Na+, K+), or functional impairment (e.g., neurological deficits (median = 10 out of 14), etc.) at 24 hours. It also did not affect edema at 72 h (~86% brain water content), or overall mortality rates (25% and 29.4% overall in vehicle vs. glibenclamide-treated severe strokes). Furthermore, glibenclamide appears to worsen cytotoxic edema in the peri-hematoma region (cell bodies were 46% larger at 24 h, p = 0.0017), but no effect on cell volume or density was noted elsewhere. Overall, these findings refute our hypothesis, as glibenclamide produced no favorable effects following severe ICH., Competing Interests: The authors have declared that no competing interests exist.

The binding of mouse IgG3 to Fcγ receptors (FcγR) and the existence of a mouse IgG3-specific receptor have been discussed for 40 years. Recently, integrin beta-1 (ITGB1) was proposed to be a part of an IgG3 receptor involved in the phagocytosis of IgG3-coated pathogens. We investigated the interaction of mouse IgG3 with macrophage-like J774A.1 and P388D1 cells. The existence of an IgG3-specific receptor was verified using flow cytometry and a rosetting assay, in which erythrocytes clustered around the macrophage-like cells coated with an erythrocyte-specific IgG3. Our findings confirmed that receptors binding antigen-free IgG3 are present on J774A.1 and P388D1 cells. We demonstrated for the first time that the removal of N-glycans from IgG3 completely abolished its binding to the cells. Moreover, we discovered that the cells treated with Accutase did not bind IgG3, indicating that IgG3-specific receptors are substrates of this enzyme. The results of antibody-mediated blocking of putative IgG3 receptors suggested that apart from previously proposed ITGB1, FcγRII, FcγRIII, also additional, still unknown, receptor is involved in IgG3 binding. These findings indicate that there is a complex network of glycan-dependent interactions between mouse IgG3 and the surface of effector immune cells.

Purpose: This study examined the patterned treatment of corneal collagen cross-linking (CXL) for keratoconus to reduce the complications caused by ultraviolet (UV) irradiation. By modifying the method of UV irradiation during the cross-linking process, cross-linking with a special structure is achieved, and the cross-linking effect is analyzed and compared to that of traditional cross-linking. By constructing an animal model of keratoconus, the process and effect of corneal cross-linking can be investigated more fundamentally. These studies provide valuable references for future cross-linking precision improvement and specialization., Method: By injecting exogenous collagenase into the corneal stroma of rabbits, the balance between collagenase and collagenase inhibitor in the corneal stroma was disrupted, the collagen fiber structure of the cornea was broken to simulate the pathogenesis of keratoconus, and an animal model of keratoconus was thus constructed. Two custom cross-linking patterns were designed with reference to the cable dome structure, and these two special patterns were irradiated and cross-linked by a DMD chip. The cross-linking effect was evaluated by optical coherence tomography (OCT), corneal topography and corneal biaxial tensile tests. The experimental rabbits were divided into four groups: group A, cross-linking of the bird's nest structure; group B, cross-linking of the honeycomb structure; group C, cross-linking of the traditional spot structure; and group D, normal (without modeling and cross-linking)., Result: Following collagenase treatment, the collagen fiber structure of the rabbit cornea was destroyed, the central thickness of the cornea was reduced, the mechanical properties of the cornea were weakened, and no keratitis, ulcers or haze occurred. After the three cross-linking treatments, the morphology of the cornea improved, the density of the stromal layer increased, and the mechanical properties were enhanced. For the improvement of keratoconus mechanical properties, the average relative difference (Δ) of the four outcome measures was 61.98% for bird's nest cross-linking versus keratoconus (Wilcoxon rank sum test, P = 0.024), 16.13% for honeycomb cross-linking versus keratoconus (Wilcoxon rank sum test, P = 0.025), and 21.07% for traditional spot cross-linking versus keratoconus (Wilcoxon rank sum test, P = 0.014). All these differences are statistically significant., Conclusion: All three methods of cross-linking can improve the morphology and tissue structure of keratoconus and significantly improve the biomechanical properties of the cornea. Among them, the corneal cross-linking of the bird's nest structure attains the best biomechanical properties, followed by the corneal cross-linking of the traditional spot structure and of the honeycomb structure. This suggests that similar or better cross-linking effects can be achieved by designing custom structures with less UV exposure. This provides a direction for future research on better and more accurate pattern cross-linking treatments., (Copyright © 2021. Published by Elsevier Ltd.)

Heart disease is one of the leading causes of death in the United States. Isolation and culture adult cardiomyocytes are important for studying cardiomyocyte contractility, heart hypertrophy, and cardiac failure. In contrast to neonatal cardiomyocyte isolation, adult mice cardiomyocytes isolation is challenging due to firm connections among cardiomyocytes through intercalated discs. The availability of newly generated genetically modified mouse lines requires to establish protocols to isolation and culture adult mouse cardiomyocyte for in vitro studies. In this manuscript, we described a straightforward method of isolating adult mouse cardiomyocytes using Langendorff perfusion apparatus. Briefly, the hearts were harvested from adult mice and the heart was mounted to Lagendorff apparatus. After perfusion with calcium depletion and collagenase digestion, the left ventricles were minced and filtered. Lastly, the separated cardiomyocytes were treated with CaCl 2 . The isolated cardiac myocytes can be utilized in a broad range of experiments including screening for drugs., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Background and Purpose: Intracerebral hemorrhage (ICH) is a known risk factor for the development of seizures, but little is known about the pathophysiology of seizures in the acute phase post-ICH and their influence on functional outcome. With the use of an animal model, the underlying pathophysiology could be further unraveled. The aim of our study was to optimize the rat collagenase stroke model for the detection of acute symptomatic seizures using video-EEG monitoring., Methods: Male Sprague-Dawley rats were implanted with scalp electrodes and a craniotomy was made for later injection of collagenase. After one week of baseline video-EEG recording, rats were injected with 0.6 U collagenase in 0.7 μL saline in left striatum, in close proximity of the piriform cortex, and immediately reconnected to the video-EEG setup for 7 days. Occurrence of clinical and electrographic seizures was assessed and functional deficits were evaluated on several time points using the cylinder test, Neurological Deficit Scale (NDS) and forelimb placing test. At day 7 post-ICH, animals were euthanized. The volume and cortical involvement of the hemorrhage were assessed by histological examination of the brain tissue, using Cresyl violet stain., Results: Collagenase injection induced ICH in all animals with a mean volume of 27 mm³ (SEM 7 mm³, range 4-92 mm³). Functional deficits were present in all animals injected with collagenase (pre-ICH vs post-ICH, p < 0.001). Epileptic seizures occurred in 5/11 animals and started between 1 and 61 h after ICH induction. Behavioral changes were observed in 13/15 seizures., Conclusions: Injecting rats with 0.6 U of collagenase is a useful model to study the occurrence of acute symptomatic seizures post-ICH as it results in ICH in all animals without mortality, 45% incidence of ICH-induced acute symptomatic seizures and measurable functional deficits., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The decellularization of long segments of tubular tissues such as blood vessels may be improved by perfusing decellularization solution into their lumen. Particularly, transmural flow that may be introduced by the perfusion, if any, is beneficial to removing immunogenic cellular components in the vessel wall. When human umbilical arteries (HUAs) were perfused at a transmural pressure, however, very little transmural flow was observed. We hypothesized that a watertight lining at the abluminal surface of HUAs hampered the transmural flow and tested the hypothesis by subjecting the abluminal surface to enzyme digestion. Specifically, a highly viscous collagenase solution was applied onto the surface, thereby restricting the digestion to the surface. The localized digestion resulted in a water-permeable vessel without damaging the vessel wall. The presence of the abluminal lining and its successful removal were also supported by evidence from SEM, TEM, and mechanical testing. The collagenase-treated HUAs were decellularized with 1% sodium dodecyl sulfate (SDS) solution under either rotary agitation, simple perfusion, or pressurized perfusion. Regardless of decellularization conditions, the decellularization of HUAs was significantly enhanced after the abluminal lining removal. Particularly, complete removal of DNA was accomplished in 24 h by pressurized perfusion of the SDS solution. We conclude that the removal of the abluminal lining can improve the perfusion-assisted decellularization.

Fibrosis is a complication of tendon injury where excessive scar tissue accumulates in and around the injured tissue, leading to painful and restricted joint motion. Unfortunately, fibrosis tends to recur after surgery, creating a need for alternative approaches to disrupt scar tissue. We posited a strategy founded on mechanobiological principles that collagen under tension generated by fibroblasts is resistant to degradation by collagenases. In this study, we tested the hypothesis that blebbistatin, a drug that inhibits cellular contractile forces, would increase the susceptibility of scar tissue to collagenase degradation. Decellularized tendon scaffolds (DTS) were treated with bacterial collagenase with or without external or cell-mediated internal tension. External tension producing strains of 2-4% significantly reduced collagen degradation compared with non-tensioned controls. Internal tension exerted by human fibroblasts seeded on DTS significantly reduced the area of the scaffolds compared to acellular controls and inhibited collagen degradation compared to free-floating DTS. Treatment of cell-seeded DTS with 50 mM blebbistatin restored susceptibility to collagenase degradation, which was significantly greater than in untreated controls (p < 0.01). These findings suggest that therapies combining collagenases with drugs that reduce cell force generation should be considered in cases of tendon fibrosis that do not respond to physiotherapy.

Dense extracellular matrix (ECM) severely impedes the spread of drugs in solid tumors and induces hypoxia, reducing chemotherapy efficiency. Different proteolytic enzymes, such as collagenase (Col) or bromelain, can directly attach to the surface of nanoparticles and improve their diffusion, but the method of ligation may also impair the enzymatic activity due to conformational changes or blockage of the active site. Herein, a "nanoenzyme capsule" was constructed by combining collagenase nanocapsules (Col-nc) with heavy-chain ferritin (HFn) nanocages encapsulating the chemotherapy drug doxorubicin (DOX) to enhance tumor penetration of the nanoparticles by hydrolyzing collagen from the ECM. Col-nc could protect the activity of the enzyme before reaching the site of action while being degraded under mildly acidic conditions in tumors, and the released proteolytic enzyme could digest collagen. In addition, HFn as a carrier could effectively load DOX and had a self-targeting ability, enabling the nanoparticles to internalize into cancer cells more effectively. From in vivo and in vitro studies, we found that collagen was effectively degraded by Col-nc/HFn(DOX) to increase the accumulation and penetration of nanoparticles in the solid tumor site and could alleviate hypoxia inside the tumor to enhance the antitumor effects of DOX. Therefore, the strategy of increasing nanoparticle penetration in this system is expected to provide a potential approach for the clinical treatment of solid tumors.

Purpose : The objective of this study was to determine the responses of normal meniscus to collagenase activity. It was hypothesized that meniscal explants exposed to collagenase would significantly increase release of pro-inflammatory cytokines and degradative enzymes, in a dose-dependent manner, compared to control. Methods : Menisci were harvested from adult dogs (n = 6) euthanized for reasons unrelated to this study. Meniscal explants were created from the central portion of lateral and medial meniscus. Explants were injected with 100 µl collagenase at a concentration of 50 µg/ml, 5 µg/ml, or 0 µg/ml of collagenase. Explants were cultured for 12 days, and media were changed and collected every 3 days for biomarker analyses. Differences among collagenase concentrations were determined by a three factor ANOVA with adjustment for multiple comparisons, with pre-adjustment statistical significance set at p < 0.05. Results : When data from all explants were compared, the 50 µg group released significantly higher IL-6 and PGE2, and the 5 µg group released significantly higher levels of MMP-3 and CTX-II compared to the 0 µg group. Explants from the medial meniscus released significantly more MMP-1, MMP-2, MMP-3, and MMP-13 in response to stimulation with 5 µg/ml of collagenase compared to explants from the lateral meniscus. Discussion : The data from this study indicate that in response to localized degradative enzyme activity, the meniscus increases the release of pro-inflammatory and degradative biomarkers in a dose-dependent manner. Further, these data indicate potential differences in metabolic responses of lateral versus medial menisci to collagenase insult.

Background: Noncultured extracted hair follicle outer root sheath cell suspension (NC-EHF-ORS-CS) is an upcoming surgical technique to treat stable vitiligo. Conventionally it employs trypsin to tap the hair follicle (HF) reservoir for autologous melanocytes and their precursors for transplantation. However, a perifollicular dermal sheath composed of type 1 collagen encases the target 'bulge' region of the HF. Adding collagenase type 1 would digest the ORS, facilitating better release of cells., Aim: To compare the repigmentation achieved using trypsin and a combination of collagenase plus trypsin, respectively, with dermabrasion alone, and to compare cell counts, viability and composition of both suspensions., Methods: This was a randomized, double-blind, comparative, therapeutic trial, conducted as a pilot study on 22 patients with stable vitiligo. Three similar patches were randomized into three parallel treatment arms [(A) trypsin plus collagenase, (B) trypsin alone and (C) dermabrasion with vehicle alone]. Each patient's HF sample was divided and digested by the two methods, and transplanted as suspensions onto dermabraded patches, while a third dermabraded patch received the vehicle only. Suspensions were sent for laboratory analysis. Repigmentation was assessed over a follow-up of 6 months., Results: There was a significant increase in cell yield and comparable viability when collagenase was added. Immunohistochemical and flow cytometry studies showed a nonsignificant increase in HMB45+ melanocytes and their precursor stem cells in group A. This trend was reflected clinically in the extent of repigmentation [group A (33.22%) > B (24.31%) > C (16.59%); P = 0.13]. Adding collagenase induced significantly higher repigmentation than dermabrasion alone (P < 0.05)., Conclusions: Incorporating collagenase type I into the conventional NC-EHF-ORS-CS technique resulted in enhanced retrieval of pigment-forming cells and subsequently improved repigmentation in vitiligo., (© 2019 British Association of Dermatologists.)

A variety of hepatic insults result in the accumulation of collagen-rich new extracellular matrix in the liver, ultimately culminating in liver fibrosis and cirrhosis. For such reasons, approaches looking into digestion of the collagen-rich extracellular matrix present an interesting therapeutic approach for cases of chronic liver disease, where the fibrotic scar is well established. Portal collagenase administration has recently led to the successful reversion of cirrhosis in an experimental rabbit model. Notwithstanding, the question of how such a sensitive therapeutic macromolecule could be administered in a less invasive manner, and in a way that preserves its functionality and avoids digestion of other non-hepatic vital collagen presents itself. Chitosan is a biodegradable polymer that has been reported to interact and bind to collagen. Chitosan nanoparticles (CS NPs) have also been reported to encapsulate therapeutic proteins, maintaining their functional form and protecting them from in-vivo degradation. For such reasons, CS NPs were loaded with collagenase and evaluated in-vitro and in-vivo for their ability to target and digest collagen. CS NPs were able to encapsulate collagenase (≈ 60% encapsulation efficiency) and release its content in active form. To determine whether chitosan's collagen interaction would enable NP collagen binding or whether the modification with collagen binding peptides (CBPs) is necessary, CS NPs were modified with the CBP; CCQDSETRTFY. Since the density of targeting ligand and the length of tether play a significant role in the success of active targeting, the surface of NPs was modified with different densities of the CBP either directly or using a polyethylene glycol (PEG) spacer. PEGylated NPs showed higher levels of CBP tagging; high, intermediate and low density of CBPs corresponded to 585.8 ± 33, 252.9 ± 25.3 and 56.5 ± 8.8 µg/mL for PEGylated NPs and 425.56 ± 12.67, 107.91 ± 10.3 and 49.86 ± 3.2 µg/mL for unPEGylated NPs, respectively. In-vitro collagen binding experiments showed that unmodified CS NPs were able to bind collagen and that modification with CBPs either directly or via PEG did not enhance collagen binding. In-vivo experiments demonstrated that unmodified CS NPs were able to reverse fibrosis with a survival rate of 100% at the end of the study, indicating the ability of CS NPs to deliver functional collagenase to the fibrotic liver and making the use of CBPs unnecessary., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Intracerebral hemorrhage (ICH) is a devastating stroke often modelled in rats. Isoflurane anesthetic, commonly used in preclinical research, affects general physiology (e.g., blood pressure) and electrophysiology (e.g., burst suppression) in many ways. These physiological changes may detract from the clinical relevance of the model. Here, we revised the standard collagenase model to produce an ICH in rats without anesthetic. Guide cannulas were implanted stereotaxically under anesthetic. After 3 days of recovery, collagenase was infused through an internal cannula into the striatum of animals randomly assigned to the non-anesthetized or isoflurane group. We assessed whether isoflurane affected hematoma volume, core temperature, movement activity, pain, blood pressure, and seizure activity. With a small ICH, there was a hematoma volume increased from 8.6 (±3.3, 95% confidence interval) µL in anesthetized rats to 13.2 (±3.1) µL in non-anesthetized rats (P = 0.008), but with a larger ICH, hematoma volumes were similar. Isoflurane decreased temperature by 1.3 °C (±0.16 °C, P < 0.001) for 2 h and caused a 35.1 (±1.7) mmHg group difference in blood pressure (P < 0.007) for 12 m. Blood glucose increased twofold after isoflurane procedures (P < 0.001). Pain, as assessed with the rat grimace scale, did not differ between groups. Seizure incidence rate (62.5%) in non-anesthetized ICH rats was similar to historic amounts (61.3%). In conclusion, isoflurane appears to have some significant and injury size-dependent effects on the collagenase model. Thus, when anesthetic effects are a known concern, the use of the standardized cannula infusion approach is scientifically and ethically acceptable., (Copyright © 2019 The Author(s). Published by Elsevier B.V. All rights reserved.)

For the advancement of porcine xenotransplantation for clinical use in type 1 diabetes mellitus, the concerns of a sustainable and safe digestion enzyme blend must be overcome. Incorporating good manufacturing practices (GMP) can facilitate this through utilizing GMP-grade enzymes. In conjunction, still taking into account the cost-effectiveness, a wide concern. We evaluated how GMP-grade enzyme blends impact our piglet islets and their long-term effects.  Preweaned porcine islets (PPIs) were isolated from 8- to 10-day-old pigs. Digestion enzyme blends, collagenase type V (Type V), collagenase AF-1 GMP-grade with collagenase NB 6 GMP-grade (AF-1 and NB 6), and collagenase AF-1 GMP-grade with collagenase neutral protease AF GMP-grade (AF-1 and NP AF) were compared. Islet quality control assessments, islet yield, viability, and function, were performed on days 3 and 7, and cell content was performed on day 7.  GMP-grade AF-1 and NB 6 (17,209 ± 2,730 islet equivalent per gram of pancreatic tissue [IE/g] on day 3, 9,001 ± 1,034 IE/g on day 7) and AF-1 and NP AF (17,214 ± 3,901 IE/g on day 3, 8,833 ± 2,398 IE/g on day 7) showed a significant increase in islet yield compared to Type V (4,618 ± 1,240 IE/g on day 3, 1,923 ± 704 IE/g on day 7). Islet size, viability, and function showed comparable results in all enzyme blends. There was no significant difference in islet cellular content between enzyme blends.  This study demonstrated a comparison of GMP-grade collagenase enzyme blends and a standard crude collagenase enzyme in preweaned-aged porcine, a novel topic in this age. GMP-grade enzyme blends of AF-1 and NB 6 and AF-1 and NP AF resulted in substantially higher yields and as effective PPIs compared to Type V. In the long run, considering costs, integrity, and sustainability, GMP-grade enzyme blends are more favorable for clinical application due to high reproducibility in comparison to undefined manufacturing processes of standard enzymes.

Isolation of mesenchymal stromal cells (MSCs) from pretreated, hematologic patients is challenging. Especially after allogeneic hematopoietic cell transplantation (HCT), standard protocols using bone marrow aspirates fail to reliably recover sufficient cell numbers. Because MSCs are considered to contribute to processes that mainly affect the outcome after transplantation, such as an efficient lymphohematopoietic recovery, extent of graft-versus-host disease as well as the occurrence of leukemic relapse, it is of great clinical relevance to investigate MSC function in this context. Previous studies showed that MSCs can be isolated by collagenase digestion of large bone fragments of hematologically healthy patients undergoing hip replacement or knee surgeries. We have now further developed this procedure for the isolation of MSCs from hematologic patients after allogeneic HCT by using trephine biopsy specimens obtained during routine examinations. Comparison of aspirates and trephine biopsy specimens from patients after allogeneic HCT revealed a significantly higher frequency of clonogenic MSCs (colony-forming unit-fibroblast [CFU-F]) in trephine biopsy specimens (mean, 289.8 ± standard deviation 322.5 CFU-F colonies/1 × 10 6 total nucleated cells versus 4.2 ± 9.9; P < 0.0001). Subsequent expansion of functional MSCs isolated from trephine biopsy specimen was more robust and led to a significantly higher yield compared with control samples expanded from aspirates (median, 1.6 × 10 6 ; range, 0-2.3 × 10 7 P0 MSCs versus 5.4 × 10 4 ; range, 0-8.9 × 10 6 ; P < 0.0001). Using trephine biopsy specimens as MSC source facilitates the investigation of various clinical questions., Competing Interests: Declaration of Competing Interest The project was funded by the Deutsche Forschungsgemeinschaft (Clinician Scientist position and Seed grant within the Centre for Regenerative Therapies Dresden, www.crt-dresden.de). The authors have no conflicts of interest to disclose., (Copyright © 2019 International Society for Cell and Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Adipocytes attached to the extracellular matrix (ECM) mainly consist of collagen in adipose tissues, while the degradation of ECM by collagenase induces the apoptosis of adipocytes, leading to a decrease in local subcutaneous adipose. To achieve this goal, we are developing a mutant collagenase H (ColH) to remove local subcutaneous fat such as submental fat (SMF). Three vectors were constructed for expressing rColH(FM, mutant for fat melting, with 6xHis tag), rColH(WT, wild-type, with 6xHis tag), and rColH(E451D, E451D mutant, without 6xHis tag) in Escherichia coli. rColH(FM) & rColH(WT) were purified by Ni Sepharose on a laboratory scale, while rColH(E451D) was purified by five chromatography purification steps on a large scale. Then, the stability of rColH(FM) and rColH(WT) was tested by SDS-PAGE to investigate the influence of the E451D mutation on stability. Afterwards, the enzyme kinetics of ColH (mutant or wild-type, with or without His tag) were investigated and compared. Finally, the adipolysis of rColH(E451D) at various doses was tested in vitro and in vivo. The ultrasound results in minipigs suggested that effective adipolysis was induced by rColH(E451D) compared with the negative control, and the histological results suggest dose-dependent fibrosis, necrosis, inflammation and cholesterol cleft formation. These findings indicate the possibility of rColH(E451D) becoming a new injectable drug to safely remove subcutaneous adipose., Competing Interests: All experiments are sponsored by Rejuven Dermaceutical Co., Ltd., a biopharmaceutical company based in Hangzhou, China, of which F.C., G.D., P.B, S.S., Z.Z. are salaried employees and Y.C. and S.S. are shareholders. M.S. and H.Y. are salaried employees of PharmaLegacy Laboratories Co., Ltd., a contract research organization company based in Shanghai, China. All work performed in PharmaLegacy has been funded by Rejuven and belongs to the properties of the latter. The current arrangement does not alter our adherence to PLOS ONE policies on sharing data and materials.

Overexpressed extracellular matrix (ECM) in pancreatic ductal adenocarcinoma (PDAC) limits drug penetration into the tumor and is associated with poor prognosis. Here, we demonstrate that a pretreatment based on a proteolytic-enzyme nanoparticle system disassembles the dense PDAC collagen stroma and increases drug penetration into the pancreatic tumor. More specifically, the collagozome, a 100 nm liposome encapsulating collagenase, was rationally designed to protect the collagenase from premature deactivation and prolonged its release rate at the target site. Collagen is the main component of the PDAC stroma, reaching 12.8 ± 2.3% vol in diseased mice pancreases, compared to 1.4 ± 0.4% in healthy mice. Upon intravenous injection of the collagozome, ∼1% of the injected dose reached the pancreas over 8 h, reducing the level of fibrotic tissue to 5.6 ± 0.8%. The collagozome pretreatment allowed increased drug penetration into the pancreas and improved PDAC treatment. PDAC tumors, pretreated with the collagozome followed by paclitaxel micelles, were 87% smaller than tumors pretreated with empty liposomes followed by paclitaxel micelles. Interestingly, degrading the ECM did not increase the number of circulating tumor cells or metastasis. This strategy holds promise for degrading the extracellular stroma in other diseases as well, such as liver fibrosis, enhancing tissue permeability before drug administration.

The pathophysiology of osteoarthritis (OA) involves dysregulation of anabolic and catabolic processes associated with a broad panel of proteins that ultimately lead to cartilage degradation. An increased understanding about these protein interactions with systematic in vitro analyses may give new ideas regarding candidates for treatment of OA related cartilage degradation. Therefore, an ex vivo tissue model of cartilage degradation was established by culturing tissue explants with bacterial collagenase II. Responses of healthy and degrading cartilage were analyzed through protein abundance in tissue supernatant with a 26-multiplex protein profiling assay, after exposing the samples to a panel of 55 protein stimulations present in synovial joints of OA patients. Multivariate data analysis including exhaustive pairwise variable subset selection identified the most outstanding changes in measured protein secretions. MMP9 response to stimulation was outstandingly low in degrading cartilage and there were several protein pairs like IFNG and MMP9 that can be used for successful discrimination between degrading and healthy samples. The discovered changes in protein responses seem promising for accurate detection of degrading cartilage. The ex vivo model seems interesting for drug discovery projects related to cartilage degradation, for example when trying to uncover the unknown interactions between secreted proteins in healthy and degrading tissues., Competing Interests: The authors have declared that no competing interests exist.

In this study, we present a method to experimentally quantify and numerically identify the constituent-specific material behavior of soft biological tissues. This allows the clear identification of the individual contributions of major load-bearing constituents and their interactions in the constitutive law. While the overall approach is applicable for many tissues, here it will be presented for the identification of a sophisticated constituent-specific material model of viable lung parenchyma. This material model will help to better model the effects of various lung diseases that feature altered fiber content in the lungs, such as emphysema or fibrosis. To experimentally quantify the mechanical properties of collagen, elastin, collagen-elastin-fiber interactions, and ground substance, we examined 18 collagenase and elastase treated rat lung parenchymal slices. The mechanical contributions of the collagen and elastin fibers in the living tissue were inferred from uniaxial tension tests comparing the behavior before and after the selective digestion of the respective fibers. In order to also obtain the mechanical influence of the ground substance, we consecutively treated the samples with both proteases. Collagen and elastin fibers are morphologically interconnected. Thus, a mechanical interaction between these fibers appears likely, but has not yet been experimentally verified. In this paper, we propose an experimental method to quantitatively assess the mechanical behavior of these collagen-elastin-fiber interactions. Based on our experiments, we have identified individual material models within a nonlinear continuum mechanics framework for each load-bearing component via an inverse analysis. The proposed constituent-specific material law can be incorporated into computational models of the respiratory system to simulate and even predict the behavior and alteration of the individual constituents and their effect on the whole respiratory system during normal and artificial breathing, in particular in the case of diseases that alter the fibers in the tissue.

Aims: The extracellular matrix (ECM) within the tumor nest plays a key role in cancer cell proliferation and invasion. It has been proven that the increased density of ECM, especially collagen network, correlates with the poor distribution of gold-nanoparticles (GNPs) to the tumor mass. Here, for the first time, we examined the combined effect of collagenase (COL) with metformin (MET)-conjugated GNPs on mammosphere generated from JIMT-1 breast cell line in vitro., Main Methods: Mammospheres (on days 7 and 14) and monolayer culture were treated with MET, MET-GNPs, and a mixture of COL-GNPs and MET-GNPs for 5 days. To assess the impacts of the engineered nanoparticles (NPs) on the survival/apoptosis of cancer cells and cancer stem cells (CSCs), the amount/activity of collagen and the expression of pyruvate kinase M2, different methods were applied, including MTT, flow cytometry, immunofluorescence, ELISA and real-time PCR analyses. Our results confirmed the enhanced cytotoxic effects of MET-GNPs combined with COL-GNPs on mammospheres compared to the cells treated with MET alone or MET-GNPs., Key Findings: Upon treatment with the mixture of MET-GNPs and COL-GNPs, the population of the apoptotic cells was significantly increased. A marked reduction was found in the number of CD24 - /CD44 + CSCs and the amount of collagen in the group received a mixture of MET-GNPs and COL-GNPs., Significance: Based on our findings, the use of COL can improve the cellular interaction/penetration of MET-GNPs in mammospheres and its antitumor impacts on the CSCs., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Cellulose biomembranes may be used for difficult-to-treat ulcers. To assess the efficacy and safety of cellulose biomembranes compared to a collagenase dressing for the treatment of chronic venous ulcers. A randomized, controlled clinical trial was performed using two groups: one treated with collagenase dressing and the other with cellulose biomembrane over a 90-day (T90) period. Both groups received compression therapy. The primary outcome was reduction in ulcer area at T90. Secondary outcomes were healing status, decrease in devitalized tissue and exudate, vascular fraction, change in quality of life, and safety. We randomized 46 participants with 73 venous ulcers (21 with 36 ulcers in the collagenase group and 25 with 37 ulcers in the biomembrane group). A decrease in ulcer area occurred at T90 in both groups, but the difference was not significant. Complete healing before T90 occurred for seven ulcers in the collagenase group and 12 ulcers in the biomembrane group, without significant difference. However, the biomembrane promoted increased precocious healing (p = 0.02). Improved bed vitality and quality of life was observed as a function of time (p < 0.01), but this was not significant between the groups. Immunohistochemical analysis of CD34 showed an increase in vascular fraction in both groups (p < 0.01), with a higher level in the biomembrane group (p < 0.05). No adverse events were considered related to the products. Cellulose biomembranes, in combination with compressive therapy, are effective and safe for the treatment of venous ulcers and provide results similar to those with collagenase treatment.

Aberrant c-Jun N terminal kinase (JNK) activation is broadly involved in the pathogenesis of several acute and chronic neurological diseases. However, the mechanism of JNK activation leading to aggravation of injury after ICH remains unclear. In this study, we confirmed that using NIMoEsh to inhibit JNK activation effectively reduced the level of brain injury following ICH. We evaluated brain outcomes by histology, immunofluorescence, Luxol fast blue/Cresyl violet staining and other experimental methods. We found that NIMoEsh could significantly inhibit the activity of JNK and thus improve inflammation, white-matter damage and neuronal cell death after ICH in mice. Our results suggest that JNK activation plays an important role of brain damage after acute stage of ICH and that NIMoEsh may be a potential target drug for the treatment of ICH., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Whilst demonstrated extensively in vitro, the control of cell behaviour via modulation of substrate compliance in live tissues has not been accomplished to date. Here we propose that stem cells can be regulated solely through in situ modulation of tissue biomechanics. By first establishing, via high-resolution Brillouin spectro-microscopy, that the outer edge (limbus) of live human corneas has a substantially lower bulk modulus compared to their centre, we then demonstrate that this difference is associated with limbal epithelial stem cell (LESC) residence and YAP-dependent mechanotransduction. This phenotype-through-biomechanics correlation is further explored in vivo using a rabbit alkali burn model. Specifically, we show that treating the burnt surface of the cornea with collagenase effectively restores the tissue's mechanical properties and its capacity to support LESCs through mechanisms involving YAP suppression. Overall, these findings have extended implications for understanding stem cell niche biomechanics and its impact on tissue regeneration.

BACKGROUND It is well documented that the Blood-Brain barrier (BBB) can be damaged by matrix metalloproteases (MMPs) after intracerebral hemorrhage (ICH), but little is known about the mechanism of this effect. MATERIAL AND METHODS We established an ICH model in rats by injecting collagenase VII into the striatum. Afterwards, intraperitoneal injection of these rats with 40 mg/kg GM6001 (a MMPs inhibitor). The effects of GM6001 on ICH were investigated by neurological severity score, brain water content, Evans blue staining, hematoxylin-eosin staining, immunohistochemical staining, and Western blot assays. RESULTS We demonstrated that the neurological damage caused by ICH was relieved at 5 and 7 days following administration of GM6001. The impaired BBB induced by ICH was improved in response to GM6001 treatment at around 3 days, as evidenced by alleviated cerebral edema, decreased Evans blue extravasation, and a reduction in inflammatory cellular infiltration. Mechanism analysis revealed that ICH induced the generation of β-dystroglycan cleavage, which could be suppressed by GM6001 treatment. Furthermore, we found that recombinant MMP2 and MMP9 triggered the cleavage of β-dystroglycan in vitro, and this action could be inhibited by GM6001 administration. CONCLUSIONS Taken together, our results suggest that MMPs-mediated cleavage on β-dystroglycan may play an important role in BBB after ICH.

Aging of extracellular proteins colloidal systems is one of major synchronizing mechanism in mammal`s «biological clock». We hypothesized that induced controllable modification of connective tissue composition could reverse aging. In murine experimental models collagenase was used for selective destruction of old collagen. Oxygen consumption, urine hydroxyproline excretion, density and distribution of mature and old collagen and elastine fibers in dermal biopsies were determined. Collagenase injections significantly increased hydroxyproline excretion. We observed reduced density of mature and old collagen fibers and increased oxygen consumption in dermal biopsies after course of collagenase injections. Collagenase treatment intensified the destruction of mature and old collagen matrix and enhanced synthesis of new collagen and elastine fibers. Furthermore oxygen consumption increased. Our findings can be considered as indicator of collagenase systemic anti-aging (rejuvenation) activity.

The abundant tumor extracellular matrix (ECM) could result in insufficient tumor retention and ineffective intratumor penetration of therapeutic agents as well as an acidic and hypoxic tumor microenvironment (TME), leading to unsatisfactory therapeutic outcomes for many types of therapies. Therefore, developing strategies to modulate the TME by selectively degrading the condensed ECM may be helpful to improve existing cancer therapies. Herein, collagenase (CLG)-encapsulated nanoscale coordination polymers (NCPs) are synthesized based on Mn 2+ and an acid-sensitive benzoic-imine organic linker and then modified by polyethylene glycol (PEG). Upon intravenous (iv) injection, these CLG@NCP-PEG nanoparticles show efficient accumulation within the tumor, in which CLG would be released because of the collapse of NCP structures within the acidic TME. The released CLG enzyme could then specifically degrade collagens, the major component of ECM, leading to a loosened ECM structure, enhanced tumor perfusion, and relieved hypoxia. As a result, the second wave of nanoparticles, chlorin e6 (Ce6)-loaded liposomes (liposome@Ce6), would exhibit enhanced retention and penetration within the tumor. Such phenomena together with relieved tumor hypoxia could then lead to greatly enhanced photodynamic therapeutic effect of liposome@Ce6 for mice pretreated with CLG@NCP-PEG. Our work thus presents a unique strategy for TME modulation using pH-responsive NCPs as smart enzyme carriers.

This study aims to explore the optimized digestive method of collagenase to nucleus pulposus (NP) cells by observing the digestive effects of type I and II collagenase in different concentrations to NP in degenerated intervetebral discs.NP were collected from 18 human herniated intervertebral disc samples, and digested by type I and II collagenase, which were separated or combined in different concentrations. NP cells were counted using an inverted microscope, and the activities were determined by trypan blue staining at 4, 8, 16, and 24 hours after digestion. The growth of NP cells was also observed.The amount of NP cells with combined collagenases was greater than that separated in an identical concentration. With the combined collagenases at 4 and 8 hours, the higher concentration, the greater the amount of NP cells became. The amount of cells in extremely low concentrations of collagenase increased after 16 and 24 hours, and its activities remained at a higher level.The optimized digestion of extremely low concentrations of type I and II collagenase combined could save enzymes, was less harmful to NP cells, and was more adapted to separated and cultured NP cells.

OBJECTIVE To evaluate effects of various concentrations of collagenase and dimethyl sulfoxide (DMSO) on yield of equine adipose-derived multipotent stromal cells (ASCs) before and after cryopreservation. SAMPLE Supragluteal subcutaneous adipose tissue from 7 Thoroughbreds. PROCEDURES Tissues were incubated with digests containing 0.1%, 0.05%, or 0.025% type I collagenase. Part of each resulting stromal vascular fraction was cryopreserved in 80% fetal bovine serum (FBS), 10% DMSO, and 10% Dulbecco modified Eagle medium F-12 and in 95% FBS and 5% DMSO. Half of each fresh and cryopreserved heterogeneous cell population was not immunophenotyped (unsorted) or was immunophenotyped for CD44 + , CD105 + , and major histocompatability complex class II (MHCII; CD44 + -CD105 + -MHCII + cells and CD44 + -CD105 + -MHCII - cells). Cell proliferation (cell viability assay), plasticity (CFU frequency), and lineage-specific target gene and oncogene expression (reverse transcriptase PCR assays) were determined in passage 1 cells before and after culture in induction media. RESULTS Digestion with 0.1% collagenase yielded the highest number of nucleated cells. Cell surface marker expression and proliferation rate were not affected by collagenase concentration. Cryopreservation reduced cell expansion rate and CD44 + -CD105 + -MHCII - CFUs; it also reduced osteogenic plasticity of unsorted cells. However, effects appeared to be unrelated to DMSO concentrations. There were also variable effects on primordial gene expression among cell isolates. CONCLUSIONS AND CLINICAL RELEVANCE Results supported the use of 0.1% collagenase in an adipose tissue digest and 5% DMSO in cryopreservation medium for isolation and cryopreservation, respectively, of equine ASCs. These results may be used as guidelines for standardization of isolation and cryopreservation procedures for equine ASCs.

Earlier it was shown that number of retrieved follicles was significantly higher in Tumor Dissociation Enzyme (TDE)-treatment group compare to standard Liberase TM-group. The aim of our present investigations was to examine the effect of TDE on appearance of apoptosis and necrosis in follicles and stromal cells after digesting of cryopreserved ovarian cortex. Fresh and frozen ovarian cortex fragments (OCF) from 14 patients (29 ± 6 years old), sized 20-210 mm 3 were randomly distributed into four treatment groups and digested with 16% TDE or 0.05 mg/ml Liberase TM: Group 1 frozen OCF digested with TDE; Group 2 frozen OCF digested with LiberaseTM; Group 3 fresh OCF digested with TDE; Group 4 fresh OCF digested with Liberase TM. To differentiate the live, early apoptotic, late apoptotic and necrotic cells in digested ovarian cortex suspension, a flow cytometric apoptosis/necrosis assay with FITC Annexin V Apoptosis Detection Kit and with 7-AAD was performed. Most of fresh (not frozen) cells digested with TDE or Liberase TM (95 ± 2.4% vs. 90.4 ± 3.1%, respectively) as well as in frozen ovarian cortex digested with TDE or Liberase TM (93.1 ± 3.4% vs. 89.7 ± 4.4%, respectively) has located in Q3 quadrant and these cells both negative to 7-AAD and Annexin V were considered as viable. It was established that both types of enzymatic treatment applying to fresh as well as to frozen ovarian cortex resulted to high rate of viable cells (Group 1: 93.8 ± 3.4%; Group 2: 91.8 ± 6.0%; Group 3: 90.5 ± 6.9%; Group 4: 87.3 ± 2.3%) and are non significantly different (P > 0.1) between all treatment groups. The amount of early apoptotic (Group 1: 3.5 ± 1.6%; Group 2: 4.4 ± 1.6%; Group 3: 1.6 ± 1.1%; Group 4: 2.4 ± 1.5%), late apoptotic (Group 1: 2.7 ± 2.4%; Group 2: 44.0 ± 1.9%; Group 3: 3.1 ± 1.1%; Group 4: 2.8 ± 0.7% and necrotic (Group 1: 0.9% ± 0.1%; Group 2: 2.9 ± 0.8%; Group 3: 3.4 ± 4.5%; Group 4: 1.1 ± 0.6%) cells was low and was not significantly different in all treatment groups (P > 0.1). It was concluded that the use of Tumor Dissociation Enzyme, effectiveness of which is higher than Liberase TM, does not lead to increasing of apoptosis and necrosis in follicles and stromal cells after enzymatic digesting of cryopreserved ovarian cortex., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objective: This study aimed to compare the behavior of dental pulp stem cells (DPSCs) after isolation using solutions containing either collagenase/dispase or collagenase alone., Design: DPSCs were isolated by two digestion methods (collagenase/dispase or collagenase alone) from human third molars. Immunophenotypic features were confirmed by flow cytometry for cell markers STRO-1, cluster of differentiation (CD) 146, CD45, and collagen type-I. The proliferation potential of cells was evaluated by 5-bromo-2'-deoxyuridine (brdU) incorporation assay, and finally they were assessed for multi-lineage differentiation potential. Data were analyzed using one-way analysis of variance and independent t-tests., Results: DPSCs isolated by either method showed similar levels of STRO-1, CD45, and collagen type-I and similar incorporation of brdU (P > 0.05). However, DPSCs obtained by collagenase I/dispase treatment had significantly higher numbers of CD146 + cells and osteogenic and chondrogenic capacities compared to those obtained by treatment with collagenase I alone (P < 0.05). On the other hand, more STRO-1+/CD164-DPSCs were found in the collagenase alone group with higher adipogenic potential., Conclusions: Different enzyme solutions gave rise to different populations of DPSCs. Dispase enhanced isolation of CD146 + DPSCs probably by disrupting the basement membranes of blood vessels and releasing DPCSs embedded in the perivascular niche. Furthermore, the differentiation potential of DPSCs was influenced by the change in enzyme solution., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

A thermostable metallo-collagenase enzyme (150 kDa), recently identified in a newly isolated actinomycestes strain (Nocardiopsis dassonvillei NRC2aza), has been purified from natural source, characterized to have application in wound healing. A simple 3 step purification procedure gave an increase of purity by 6.23 fold with a specific activity of 387.2 U mg -1 . The enzyme activity showed stability across a range of pH (7.0-8.5) and temperature (40-55 °C) with optima at pH 8.0 and 60 °C, respectively. Activators include Mg +2 , Ca +2 , Zn +2 , Na + , K + and Ba +2 , while Mn +2 , Co +2 , Ni +2 and Ag + ions gave partial inhibition. Full inhibition was given by other tested ions and metalloproteinase inhibitors. Broad substrate specificity was demonstrated including activity against a native collagen. The K m and V max of the enzyme using azocollagen were 5.5 mg/ml and 1280 U, respectively. The purified collagenase enhanced wound closure in vitro and in vivo and the repair process was dose dependent. Topical application of the purified collagenase (either of 25 or 50 U) to cutaneous wounds significantly accelerated the rate of wound healing and the formation of granulation tissue. Hence, the purified collagenase has a great potential as a therapeutic agent in wound care and collagen related diseases., (Copyright © 2018. Published by Elsevier B.V.)

Tissue engineers utilize a battery of expensive, time-consuming and destructive techniques to assess the composition and function of engineered tissues. A nondestructive solution to monitor tissue maturation would reduce costs and accelerate product development. As a first step toward this goal, two nondestructive, label-free optical techniques, namely multispectral fluorescent lifetime imaging (FLIm) and time-resolved fluorescence spectroscopy (TRFS), were investigated for their potential in evaluating the biochemical and mechanical properties of articular cartilage. Enzymatic treatments were utilized to selectively deplete cartilage of either collagen or proteoglycan, to produce a range of matrix compositions. Samples were assessed for their optical properties using a fiber-coupled optical system combining FLIm and TRFS, their biochemical and mechanical properties and by histological staining. Single and multivariable correlations were performed to evaluate relationships among these properties. FLIm- and TRFS-derived measurements are sensitive to changes in cartilage matrix and correlate with mechanical and biochemical assays. Mean fluorescence lifetime values extracted from FLIm images (375-410 nm spectral band) showed strong, specific correlations with collagen content (R2 = 0.79, p < 0.001) and tensile properties (R2 = 0.45, p = 0.02). TRFS lifetime measurements centered at 520 nm (with a 5 nm bandwidth) possessed strong, specific correlations with proteoglycan content (R2 = 0.59, p = 0.001) and compressive properties (R2 = 0.71, p < 0.001). Nondestructive optical assessment of articular cartilage, using a combination of FLIm- and TRFS-derived parameters, provided a quantitative method for determining tissue biochemical composition and mechanical function. These tools hold great potential for research, industrial and clinical settings.

Fibrosis is a common lesion in different pathologic diseases and defined by the excessive accumulation of collagen. Different approaches have been used to treat different conditions characterized by fibrosis. The FDA and EMA approved the use of collagenase to treat palmar fibromatosis (Dupuytren's contracture). The EMA approved additionally its use in severe Peyronie's disease, but it has been used off label in other conditions [1,2]. The approved treatment includes up to three (in palmar fibromatosis) or up to eight (in penile fibromatosis) injections followed by finger extension or penile modeling procedures, typically causing severe pain. Frequent single injections are adequate to treat palmar fibromatosis [3]. The need to repeatedly inject doses of this enzyme can be due to the labile nature of collagenase, which exhibits a complete activity loss after a short period of time. This study presents a novel strategy to manage this enzyme based on the synthesis of polymeric nanocapsules that contain collagenase encapsulated within their matrix. These nanocapsules have been engineered for achieving a gradual release of the encapsulated enzyme for a longer time, which can be up to ten days. The efficacy of these nanocapsules has been tested in a murine model of local dermal fibrosis, and the results demonstrate a reduction in fibrosis greater than that with the injection of free enzyme; this type of treatment showed a significant improvement compared to conventional therapy of free collagenase., Statement of Significance: The use of proteins as therapeutic molecules has recently attracted great interest. Collagenase injection is the current treatment for fibrotic diseases. Unfortunately, proteins have a low stability and presume several repetition cycles to obtain an effective treatment. This article describes a novel treatment for these types of diseases using collagenase nanocapsules designed to exhibit a sustainable release of the encapsulated enzyme, which maintains the enzymatic activity for a long period of time. The therapeutic effect of nanocapsules was tested in a murine mouse model of local dermal fibrosis, and the results showed an important improved effect compared to the effect of the administration of free enzyme. These results indicate a high potential for this novel system to improve the current treatment for fibrotic diseases., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Collagen and hyaluronan are the most abundant components of the extracellular matrix (ECM) and their overexpression in tumors is linked to increased tumor growth and metastasis. These ECM components contribute to a protective tumor microenvironment by supporting a high interstitial fluid pressure and creating a tortuous setting for the convection and diffusion of chemotherapeutic small molecules, antibodies, and nanoparticles in the tumor interstitial space. This review focuses on the research efforts to deplete extracellular collagen with collagenases to normalize the tumor microenvironment. Although collagen synthesis inhibitors are in clinical development, the use of collagenases is contentious and clinically untested in cancer patients. Pretreatment of murine tumors with collagenases increased drug uptake and diffusion 2-10-fold. This modest improvement resulted in decreased tumor growth, but the benefits of collagenase treatment are confounded by risks of toxicity from collagen breakdown in healthy tissues. In this review, we evaluate the published in vitro and in vivo benefits and limitations of collagenase treatment to improve drug delivery.

Background: To investigate use of collagenase type II for generating a rabbit model of corneal ectasia., Methods: Ten New Zealand white rabbits were used with right eyes treated as the experimental group and left eyes treated as the control group. After epithelial debridement, a collagenase type II solution (200 μL of 5 mg/mL) was applied in the experimental group at room temperature (24 °C) for 30 min, and a 200 μL solution without collagenase was applied in the control group. Slit-lamp microscopy, the mean keratometry (Km), and central cornea thickness (CCT) were examined before and after the procedure. Corneas were obtained on day 14 for biomechanical evaluation., Results: No obvious inflammatory reaction was observed in all eyes after the procedure. A statistically significant increase in Km (1.54 ± 1.29D vs - 0.82 ± 0.44D at day7 and 0.89 ± 0.89D vs - 2.11 ± 1.02D at day14) and a statistically significant decrease in CCT (- 23.10 ± 12.17 μm vs 6.20 ± 16.51 μm at day7 and - 16.10 ± 10.46 μm vs 11.60 ± 0.88 μm at day14) were observed in the experimental group compared with the control group. The mean stresses and elastic modulus at 5%, 10%, 15%, and 20% deformities in the experimental group decreased and the differences in elastic modulus between the two groups were statistically significant at 10% and 15% deformities., Conclusions: Collagenase type II treatment results in mimic KC with increased corneal keratometry and corneal thinning and a lower elastic modulus. An animal model for corneal ectasia can be generated by treatment with collagenase type II.

Background: Three-dimensional (3D) cultured clumps of mesenchymal stem cell (MSC)/extracellular matrix (ECM) complexes (C-MSCs) consist of cells and self-produced ECM. C-MSCs can regulate cellular functions in vitro and can be grafted into a defect site without an artificial scaffold to induce bone regeneration. Long-term cryopreservation of C-MSCs, which can enable them to serve as a ready-to-use cell preparation, may be helpful in developing beneficial cell therapy for bone regeneration. Therefore, the aim of this study was to investigate the effect of cryopreservation on C-MSCs., Methods: MSCs isolated from rat femurs were cultured in growth medium supplemented with ascorbic acid. To obtain C-MSCs, confluent cells that had formed on the cellular sheet were scratched using a micropipette tip and were then torn off. The sheet was rolled to make a round clumps of cells. The C-MSCs were cryopreserved in cryomedium including 10% dimethyl sulfoxide., Results: Cryopreserved C-MSCs retained their 3D structure and did not exhibit a decrease in cell viability. In addition, stem cell marker expression levels and the osteogenic differentiation properties of C-MSCs were not reduced by cryopreservation. However, C-MSCs pretreated with collagenase before cryopreservation showed a lower level of type I collagen and could not retain their 3D structure, and their rates of cell death increased during cryopreservation. Both C-MSC and cryopreserved C-MSC transplantation into rat calvarial defects induced successful bone regeneration., Conclusion: These data indicate that cryopreservation does not reduce the biological properties of C-MSCs because of its abundant type I collagen. More specifically, cryopreserved C-MSCs could be applicable for novel bone regenerative therapies.