Forward Light Scattering of the Vitreous Gel After Enzymatic Aging: An In Vitro Model to Study Vitreous Opacification.

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.

A Novel Tendon Injury Model, Induced by Collagenase Administration Combined with a Thermo-Responsive Hydrogel in Rats, Reproduces the Pathogenesis of Human Degenerative Tendinopathy.

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.

The cross-linking and protective effect of artemisinin and its derivatives on collagen fibers of demineralized dentin surface.

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.

Effects of tissue degradation by collagenase and elastase on the biaxial mechanics of porcine airways.

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.

Therapeutic effects of kartogenin on temporomandibular joint injury by activating the TGF-ß/SMAD pathway in rats.

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-ß1 and SMAD3 was performed to reveal that kartogenin activated the TGF-ß/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.

Effect of silver nanoparticles associated with fluoride on the progression of root dentin caries in vitro.

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.

Development on potential skin anti-aging agents of Cosmos caudatus Kunth via inhibition of collagenase, MMP-1 and MMP-3 activities.

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, 1H and 13C 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.

A Comparison of Topical Agents for Eschar Removal in a Porcine Model: Bromelain-enriched vs Traditional Collagenase Agents.

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.

Benzene, 1,2,4-Trimethoxy-5-(2-Methyl-1-Propen-1-yl), a New Neuroprotective Agent, Treats Intracerebral Hemorrhage by Inhibiting Apoptosis, Inflammation, and Oxidative Stress.

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 GABAA 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.

Sequential Nano-Penetrators of Capillarized Liver Sinusoids and Extracellular Matrix Barriers for Liver Fibrosis Therapy.

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.

A Photochemical Crosslinking Approach to Enhance Resistance to Mechanical Wear and Biochemical Degradation of Articular Cartilage.

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.

Clostridium Collagenase Impact on Zone of Stasis Stabilization and Transition to Healthy Tissue in Burns.

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.

Isolation of Adult Mouse Cardiomyocytes Using Langendorff Perfusion Apparatus.

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 CaCl2. The isolated cardiac myocytes can be utilized in a broad range of experiments including screening for drugs.

Novel strategy of cartilage repairing via application of P. atlantica with stem cells and collagen.

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.

Glibenclamide does not improve outcome following severe collagenase-induced intracerebral hemorrhage in rats.

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.

Mouse IgG3 binding to macrophage-like cells is prevented by deglycosylation of the antibody or by Accutase treatment of the cells.

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.

Defining optimal enzyme and matrix combination for replating of human induced pluripotent stem cell-derived cardiomyocytes at different levels of maturity.

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.

Co-encapsulation of collagenase type I and silibinin in chondroitin sulfate coated multilayered nanoparticles for targeted treatment of liver fibrosis.

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.

Study on patterned photodynamic cross-linking for keratoconus.

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.

Comparison of Islet Characterization from Use of Standard Crude Collagenase to GMP-Grade Collagenase Enzyme Blends in Preweaned Porcine Islet Isolations.

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.