Optimization and mechanisms of proteolytic enzyme immobilization onto large-pore mesoporous silica nanoparticles: Enhanced tumor penetration.

Immobilization of proteolytic enzymes onto nanocarriers is effective to improve drug diffusion in tumors through degrading the dense extracellular matrix (ECM). Herein, immobilization and release behaviors of hyaluronidase, bromelain, and collagenase (Coll) on mesoporous silica nanoparticles (MSNs) were explored. A series of cationic MSNs (CMSNs) with large and adjustable pore sizes were synthesized, and investigated together with two anionic MSNs of different pore sizes. CMSNs4.0 exhibited the highest enzyme loading capacity for hyaluronidase and bromelain, and CMSNs4.5 was the best for Coll. High electrostatic interaction, matched pore size, and large pore volume and surface area favor the immobilization. Changes of the enzyme conformations and surface charges with pH, existence of a space around the immobilized enzymes, and the depth of the pore structures, affect the release ratio and tunability. The optimal CMSNs-enzyme complexes exhibited deep and homogeneous penetration into pancreatic tumors, a tumor model with the densest ECM, with CMSNs4.5-Coll as the best. Upon loading with doxorubicin (DOX), the CMSNs-enzyme complexes induced high anti-tumor efficiencies. Conceivably, the DOX/CMSNs4.5-NH2-Coll nanodrug exhibited the most effective tumor therapy, with a tumor growth inhibition ratio of 86.1 %. The study provides excellent nanocarrier-enzyme complexes, and offers instructive theories for enhanced tumor penetration and therapy.

The potential of Paeonia lactiflora pall seeds oil as a pure natural cosmetics raw material: In Vitro findings.

BACKGROUND: As a traditional Chinese herbal medicine, Paeonia lactiflora Pall is rich in various active ingredients such as polysaccharides and total flavonoids while having ornamental value. It has potential application value in the development of food and cosmetics. OBJECTIVE: To study the in vitro efficacy of Paeonia lactiflora Pall seeds oil. METHODS: Firstly, the levels of linolenic acid and linoleic acid in Paeonia lactiflora Pall seeds oil were quantified using gas chromatography. The impact of Paeonia lactiflora Pall seeds oil on the proliferation rate of B16F10 cells was assessed through the CCK-8 method, while the melanin content of B16F10 cells was determined using the sodium hydroxide lysis method. The inhibitory effects of Paeonia lactiflora Pall seeds oil on elastase, collagenase and hyaluronidase were evaluated by biochemical techniques in vitro. Lastly, the hen's egg chorioallantoic membrane test (HET-CAM) was conducted to confirm the absence of eye irritation caused by Paeonia lactiflora Pall seeds oil. RESULTS: Paeonia lactiflora Pall seeds oil within a certain volume concentration range (0.5%-4%) had no effect on the proliferation of B16F10 cells. Paeonia lactiflora Pall seeds oil showed significant inhibition of elastase, collagenase and hyaluronidase. Notably, the highest concentration tested, 4% Paeonia lactiflora Pall seed oil, yielded the most pronounced outcomes without causing any irritation. CONCLUSION: A certain concentration of Paeonia lactiflora Pall seeds oil has a significant effect on decreasing the melanin content in B16F10 cells and inhibiting the activities of elastase, collagenase, and hyaluronidase, which can provide a reference for the development of pure natural cosmetics raw materials.

Heparan sulfate selectively inhibits the collagenase activity of cathepsin K.

Cathepsin K (CtsK) is a cysteine protease with potent collagenase activity. CtsK is highly expressed by bone-resorbing osteoclasts and plays an essential role in resorption of bone matrix. Although CtsK is known to bind heparan sulfate (HS), the structural details of the interaction, and how HS regulates the biological functions of CtsK, remains largely unknown. In this report, we discovered that HS is a multifaceted regulator of the structure and function of CtsK. Structurally, HS forms a highly stable complex with CtsK and induces its dimerization. Co-crystal structures of CtsK with bound HS oligosaccharides reveal the location of the HS binding site and suggest how HS may support dimerization. Functionally, HS plays a dual role in regulating the enzymatic activity of CtsK. While it preserves the peptidase activity of CtsK by stabilizing its active conformation, it inhibits the collagenase activity of CtsK in a sulfation level-dependent manner. These opposing effects can be explained by our finding that the HS binding site is remote from the active site, which allows HS to specifically inhibit the collagenase activity without affecting the peptidase activity. At last, we show that structurally defined HS oligosaccharides effectively block osteoclast resorption of bone in vitro without inhibiting osteoclast differentiation, which suggests that HS-based oligosaccharide might be explored as a new class of selective CtsK inhibitor for many diseases involving exaggerated bone resorption.

Enhancing the tumor penetration of multiarm polymers by collagenase modification.

Tumor penetration is a critical determinant of the therapy efficacy of nanomedicines. However, the dense extracellular matrix (ECM) in tumors significantly hampers the deep penetration of nanomedicines, resulting in large drug-untouchable areas and unsatisfactory therapy efficacy. Herein, we synthesized a third-generation PAMAM-cored multiarm copolymer and modified the polymer with collagenase to enhance its tumor penetration. Each arm of the copolymer was a diblock copolymer of poly(glutamic acid)-b-poly(carboxybetaine), in which the polyglutamic acid block with abundant side groups was used to link the anticancer agent doxorubicin through the pH-sensitive acylhydrazone linkage, and the zwitterionic poly(carboxybetaine) block provided desired water solubility and anti-biofouling capability. The collagenase was conjugated to the ends of the arms via the thiol-maleimide reaction. We demonstrated that the polymer-bound collagenase could effectively catalyze the degradation of the collagen in the tumor ECM, and consequently augmented the tumor penetration and antitumor efficacy of the drug-loaded polymers.

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

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

Chemical Profiling and Enzyme Inhibitory Activities of Essential Oil Isolated from Pistacia khinjuk Leaves: Insights On GC-MS Analysis and Skin Aging-Relevant Enzymes.

Pistacia khinjuk is a species of flowering plants belonging to family Anacardiaceae, with promising pharmacological activities like antioxidants, anti-inflammatory, antiviral, and antimicrobial. This study aimed to investigate the GC-MS chemical composition of essential oil isolated from Pistacia khinjuk leaves and its inhibitory properties against aging-relevant enzymes such a collagenase and elastase. The isolated oil showed predominance of ß-cadinene (15.34 %), γ-amorphene (8.50 %), α-cadinol (8.14 %), τ-cadinol (7.57 %), (E)-ß-caryophyllene (5.77 %), α-pinene (4.70 %), phytol (4.57 %), α-muurolene (3.30 %), (+)-epi-bicyclosesquiphellandrene (3.21 %), and cubenene (3.16 %). Further, it showed remarkable inhibitory activities against collagenase and elastase with IC50 values of 15.61±0.69 and 41.12±2.09 µg/mL, respectively compared to epigallocatechin gallate (IC50=29.52±1.3 µg/mL and 26.86±1.37 µg/mL). as a conclusion, the leaf oil is recommended for topical cosmetic preparations to retard skin aging symptoms such as wrinkles. However, the bioavailability assessment and toxicological profile should be considered in the future studies.

Anti-collagenase, Anti-elastase, Anti-urease, and Anti-cancer Potentials of Isokaempferide as Natural Compound: In vitro and in silico Study.

One of the main goals of medicinal chemistry in recent years has been the development of new enzyme inhibitors and anti-cancer medicines. The isokaempferide' ability to inhibit the enzymes urease, elastase, and collagenase were also studied. The results showed that isokaempferide was the most effective compound against the assigned enzymes, with IC 50 values of 23.05 µM for elastase, 12.83 µM for urease, and 33.62 µM for collagenase respectively. It should be emphasized that natural compound was more effective at inhibiting some enzymes. Additionally, the compound was tested for their anti-cancer properties using colon, lung, breast cancer cell lines. The chemical activities of isokaempferide against urease, collagenase, and elastase were investigated utilizing the molecular docking study. The anti-cancer activities of the compound were evaluated against lung cancer cells such as SPC-A-1, SK-LU-1, 95D, breast cancer cells like MCF7, Hs 578Bst, Hs 319.T, and UACC-3133 cell lines, and colon cancer cell lines like CL40, SW1417, LS1034, and SW480. The chemical activities of isokaempferide against some of the expressed surface receptor proteins (EGFR, estrogen receptor, CD47, progesterone receptor, folate receptor, CD44, HER2, CD155, CXCR4, CD97, and endothelin receptor) in the mentioned cell lines were assessed using the molecular docking calculations. The results showed the probable interactions and their characteristics at an atomic level. The docking scores revealed that isokaempferide has a strong binding affinity to the enzymes and proteins. In addition, the compound formed powerful contact with the enzymes and receptors. Thus, isokaempferide could be potential inhibitor for enzymes and cancer cells.

Collagenase-based wound debridement agent induces extracellular matrix supporting phenotype in macrophages.

Macrophages assume diverse phenotypes and functions in response to cues from the microenvironment. Earlier we reported an anti-inflammatory effect of Collagenase Santyl® Ointment (CSO) and the active constituent of CSO (CS-API) on wound macrophages in resolving wound inflammation indicating roles beyond debridement in wound healing. Building upon our prior finding, this study aimed to understand the phenotypes and subsets of macrophages following treatment with CS-API. scRNA-sequencing was performed on human blood monocyte-derived macrophages (MDM) following treatment with CS-API for 24 h. Unbiased data analysis resulted in the identification of discrete macrophage subsets based on their gene expression profiles. Following CS-API treatment, clusters 3 and 4 displayed enrichment of macrophages with high expression of genes supporting extracellular matrix (ECM) function. IPA analysis identified the TGFß-1 pathway as a key hub for the CS-API-mediated ECM-supportive phenotype of macrophages. Earlier we reported the physiological conversion of wound-site macrophages to fibroblasts in granulation tissue and impairment of such response in diabetic wounds, leading to compromised ECM and tensile strength. The findings that CSO can augment the physiological conversion of macrophages to fibroblast-like cells carry significant clinical implications. This existing clinical intervention, already employed for wound care, can be readily repurposed to improve the ECM response in chronic wounds.

Anti-inflammatory effects of sericin and swimming exercise in treating experimental Achilles tendinopathy in rat.

The aim of this study was to assess the effectiveness of combining sericin with swimming exercise as a treatment for type-I collagenase-induced Achilles tendinopathy (AT) in rats, with a focus on inflammatory cytokines. An experimental AT model was established using type-I collagenase in male Sprague-Dawley rats, categorized into five groups: Group 1 (Control + Saline), Group 2 (AT), Group 3 (AT + exercise), Group 4 (AT + sericin), and Group 5 (AT + sericin + exercise). Intratendinous sericin administration (0.8 g/kg/mL) took place from days 3 to 6, coupled with 30 min daily swimming exercise sessions (5 days/week, 4 weeks). Serum samples were analyzed using ELISA for tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), interleukin-10 (IL-10), and total antioxidant-oxidant status (TAS-TOS), alongside histopathological and immunohistochemical assessments of Achilles tendon samples. Elevated TNF-α and IL-1ß and decreased IL-10 levels were evident in Group 2; Of these, TNF-α and IL-1ß were effectively reduced and IL-10 increased across all treatment groups, particularly groups 4 and 5. Serum TAS was notably lower in Group 2 and significantly increased in Group 5 compared to Group 2. Histopathologically, Group 2 displayed severe degeneration, irregular fibers, and round cell nuclei, while Group 5 exhibited decreased degeneration and spindle-shaped fibers. The Bonar score increased in Group 2 and decreased in groups 4 and 5. Collagen type-I alpha-1 (Col1A1) expression was notably lower in Group 2 (P = 0.001) and significantly increased in groups 4 and 5 compared to Group 2 (P = 0.011 and 0.028, respectively). This study underscores the potential of sericin and swimming exercises in mitigating inflammation and oxidative stress linked to AT pathogenesis, presenting a promising combined therapeutic strategy.

Collagenase Type I and Probucol-Loaded Nanoparticles Penetrate the Extracellular Matrix to Target Hepatic Stellate Cells for Hepatic Fibrosis Therapy.

Hepatic fibrosis is a common pathological process in chronic liver diseases, characterized by excessive reactive oxygen species (ROS), activated hepatic stellate cells (HSCs), and massive synthesis of extracellular matrix (ECM), which are important factors in the development of liver cirrhosis, liver failure, and liver cancer. During the development of hepatic fibrosis, ECM collagen produced by activated HSCs significantly hinders medication delivery to targeted cells and reduces the efficiency of pharmacological therapy. In this study, we designed a multifunctional hyaluronic acid polymeric nanoparticle (HA@PRB/COL NPs) based on autophagy inhibitor probucol (PRB) and collagenase type I (COL) modification, which could enhance ECM degradation and accurately target HSCs through specificity binding CD44 receptor in hepatic fibrosis therapy. Upon encountering excessive collagen I-deposition formed barrier, HA@PRB/COL NPs performed the nanodrill-like function to effectively degrade pericellular collagen I, leading to greater ECM penetration and prominent HSCs internalization capacity of delivered PRB. In mouse hepatic fibrosis model, HA@PRB/COL NPs were efficiently delivered to HSCs through binding CD44 receptor to achieve efficient accumulation in fibrotic liver. Further, we showed that HA@PRB/COL NPs executed the optimal anti-fibrotic activity by inhibiting autophagy and activation of HSCs. In conclusion, our novel dual-functional co-delivery system with degrading fibrotic ECM collagen and targeting activated HSCs exhibits great potentials in the treatment of hepatic fibrosis in clinic. STATEMENT OF SIGNIFICANCE: The excess release of extracellular matrix (ECM) such as collagen in hepatic fibrosis hinders medication delivery and decreases the efficiency of pharmacological drugs. We aimed to develop a nano-delivery carrier system with protein hydrolyzed surfaces and further encapsulated an autophagy inhibitor (PRB) to enhance fibrosis-related ECM degradation-penetration and hepatic stellate cells (HSCs) targeting in hepatic fibrosis niche (HA@PRB/COL NPs). The COL of HA@PRB/COL NPs successfully worked as a scavenger to promote the digestion of the ECM collagen I barrier for deeper penetration into fibroid liver tissue. It also accurately targeted HSCs through specifically binding to the CD44 receptor and subsequently released PRB to inhibit autolysosome and ROS generation, thus preventing HSCs activation. Our HA@PRB/COL NPs system provided a promising therapeutic strategy for hepatic fibrosis in a clinic setting.

The extracellular matrix dictates regional competence for tumour initiation.

The skin epidermis is constantly renewed throughout life1,2. Disruption of the balance between renewal and differentiation can lead to uncontrolled growth and tumour initiation3. However, the ways in which oncogenic mutations affect the balance between renewal and differentiation and lead to clonal expansion, cell competition, tissue colonization and tumour development are unknown. Here, through multidisciplinary approaches that combine in vivo clonal analysis using intravital microscopy, single-cell analysis and functional analysis, we show how SmoM2-a constitutively active oncogenic mutant version of Smoothened (SMO) that induces the development of basal cell carcinoma-affects clonal competition and tumour initiation in real time. We found that expressing SmoM2 in the ear epidermis of mice induced clonal expansion together with tumour initiation and invasion. By contrast, expressing SmoM2 in the back-skin epidermis led to a clonal expansion that induced lateral cell competition without dermal invasion and tumour formation. Single-cell analysis showed that oncogene expression was associated with a cellular reprogramming of adult interfollicular cells into an embryonic hair follicle progenitor (EHFP) state in the ear but not in the back skin. Comparisons between the ear and the back skin revealed that the dermis has a very different composition in these two skin types, with increased stiffness and a denser collagen I network in the back skin. Decreasing the expression of collagen I in the back skin through treatment with collagenase, chronic UV exposure or natural ageing overcame the natural resistance of back-skin basal cells to undergoing EHFP reprogramming and tumour initiation after SmoM2 expression. Altogether, our study shows that the composition of the extracellular matrix regulates how susceptible different regions of the body are to tumour initiation and invasion.

Rapid and Efficient Enrichment of Mouse Spinal Cord Microglia.

The vertebral column defines a vertebrate and shapes the spinal canal, a cavity that encloses and safeguards the spinal cord. Proper development and function of the mammalian central nervous system rely significantly on the activity of resident macrophages known as microglia. Microglia display heterogeneity and multifunctionality, enabling distinct gene expression and behavior within the spinal cord and brain. Numerous studies have explored cerebral microglia function, detailing purification methods extensively. However, the purification of microglia from the spinal cord in mice lacks a comprehensive description. In contrast, the utilization of a highly purified collagenase, as opposed to an unrefined extract, lacks reporting within central nervous system tissues. In this study, the vertebral column and spinal cord were excised from 8-10 week-old C57BL/6 mice. Subsequent digestion employed a highly purified collagenase, and microglia purification utilized a density gradient. Cells underwent staining for flow cytometry, assessing viability and purity through CD11b and CD45 staining. Results yielded an average viability of 80% and a mean purity of 95%. In conclusion, manipulation of mouse microglia involved digestion with a highly purified collagenase, followed by a density gradient. This approach effectively produced substantial spinal cord microglia populations.

Insufficiency of collagenases in establishment of primary chondrocyte culture from cartilage of elderly patients receiving total joint replacement.

Background Collagenases are frequently used in chondrocyte isolation from articular cartilage. However, the sufficiency of this enzyme in establishing primary human chondrocyte culture remains unknown. Methods Cartilage slices shaved from femoral head or tibial plateau of patients receiving total joint replacement surgery (16 hips, 8 knees) were subjected to 0.02% collagenase IA digestion for 16 h with (N = 19) or without (N = 5) the pre-treatment of 0.4% pronase E for 1.5 h. Chondrocyte yield and viability were compared between two groups. Chondrocyte phenotype was determined by the expression ratio of collagen type II to I. The morphology of cultured chondrocytes was monitored with a light microscope.Results Cartilage with pronase E pre-treatment yielded significantly higher chondrocytes than that without the pre-treatment (3,399 ± 1,637 cells/mg wet cartilage vs. 1,895 ± 688 cells/mg wet cartilage; P = 0.0067). Cell viability in the former group was also significantly higher than that in the latter (94% ± 2% vs. 86% ± 6%; P = 0.03). When cultured in monolayers, cells from cartilage with pronase E pre-treatment grew in a single plane showing rounded shape while cells from the other group grew in multi-planes and exhibited irregular shape. The mRNA expression ratio of collagen type II to I was 13.2 ± 7.5 in cells isolated from cartilage pre-treated with pronase E, indicating a typical chondrocyte phenotype. Conclusions Collagenase IA was not sufficient in establishing primary human chondrocyte culture. Cartilage must be treated with pronase E prior to collagenase IA application.

Anti-cancer, Anti-collagenase and Anti-elastase Potentials of Some Natural Derivatives: In vitro and in silico Studies.

The anti-cancer activities of the compounds were evaluated against KYSE-150, KYSE-30, and KYSE-270 cell lines and also on investigated esophageal line HET 1 A as a standard. Modified inhibitory impact on enzymes of collagenase and elastase were used Thring and Moon methods, respectively. Among both compounds, both of them recorded impact on cancer cells being neutral against the control, both had IC50 lower than 100 µM and acted as a potential anticancer drug. The chemical activities of Skullcapflavone I and Skullcapflavone II against elastase and collagenase were investigated utilizing the molecular modeling study. IC50 values of Skullcapflavone I and Skullcapflavone II on collagenase enzyme were obtained 106.74 and 92.04 µM and for elastase enzyme were 186.70 and 123.52 µM, respectively. Anticancer effects of these compounds on KYSE 150, KYSE 30, and KYSE 270 esophageal cancer cell lines studied in this work. For Skullcapflavone I, IC50 values for these cell lines were obtained 14.25, 19.03, 25.10 µM, respectively. Also, for Skullcapflavone II were recorded 20.42, 34.17, 22.40 µM, respectively. The chemical activities of Skullcapflavone I and Skullcapflavone II against some of the expressed surface receptor proteins (CD44, EGFR, and PPARγ) in the mentioned cell lines were assessed using the molecular docking calculations. The calculations showed the possible interactions and their characteristics at an atomic level.

Linear and nonlinear rheology of liberase-treated breast cancer tumors.

Extracellular matrix (ECM) rigidity has been shown to increase the invasive properties of breast cancer cells, promoting transformation and metastasis through mechanotransduction. Reducing ECM stiffness via enzymatic digestion could be a promising approach to slowing breast cancer development by de-differentiation of breast cancer cells to less aggressive phenotypes and enhancing the effectiveness of existing chemotherapeutics via improved drug penetrance throughout the tumor. In this study, we examine the effects of injectable liberase (a blend of collagenase and thermolysin enzymes) treatments on the linear and nonlinear rheology of allograft 4T1 mouse mammary tumors. We perform two sets of in vivo mouse studies, in which either one or multiple treatment injections occur before the tumors are harvested for rheological analysis. The treatment groups in each study consist of a buffer control, free liberase enzyme in buffer, a thermoresponsive copolymer called LiquoGel (LQG) in buffer, and a combined, localized injection of LQG and liberase. All tumor samples exhibit gel-like linear rheological behavior with the elastic modulus significantly larger than the viscous modulus and both independent of frequency. Tumors that receive a single injection of localized liberase have significantly lower tumor volumes and lower tissue moduli at both the center and edge compared to buffer- and free liberase-injected control tumors, while tissue viscoelasticity remains relatively unaffected. Tumors injected multiple times with LQG and liberase also have lower tissue volumes but possess higher tissue moduli and lower viscoelasticities compared to the other treatment groups. We propose that a mechanotransductive mechanism could cause the formation of smaller but stiffer tumors after repeated, localized liberase injections. Large amplitude oscillatory shear (LAOS) experiments are also performed on tissues from the multiple injection study and the results are analyzed using MITlaos. LAOS analysis reveals that all 4T1 tumors from the multiple injection study exhibit nonlinear rheological behavior at high strains and strain rates. Examination of the Lissajous-Bowditch curves, Chebyshev coefficient ratios, elastic moduli, and dynamic viscosities demonstrate that the onset and type of nonlinear behavior is independent of treatment type and elastic modulus, suggesting that multiple liberase injections do not affect the nonlinear viscoelasticity of 4T1 tumors.

Intraventricular hemorrhage induces inflammatory brain damage with blood-brain barrier dysfunction in immature rats.

BACKGROUND: We aimed to characterize a preclinical model of intraventricular hemorrhage-induced brain damage (IVH-BD) in extremely low birth weight newborns (ELBWN), to identify potential therapeutic targets based on its pathophysiology. METHODS: IVH was induced in 1-day-old (P1) Wistar rats by left periventricular injection of clostridium collagenase (PVCC). At P6, P14, and P45 IVH-BD (area of damage, motor and cognitive deficits, Lactate/N-acetylaspartate ratio), white matter injury (WMI: ipsilateral hemisphere and corpus callosum atrophy, oligodendroglial population and myelin basic protein signal reduction), blood-brain barrier (BBB) dysfunction (occludin and Mfsd2a expression, Gadolinium leakage) and inflammation (TNFα, TLR4, NFkB, and MMP9 expression; immune cell infiltration), excitotoxicity (Glutamate/N-acetylaspartate), and oxidative stress (protein nitrosylation) were assessed. Sham animals were similarly studied. RESULTS: IVH-BD leads to long-term WMI, resulting in motor and cognitive impairment, thus reproducing IVH-BD features in ELBWN. BBB dysfunction with increased permeability was observed at P6 and P14, coincident with an increased inflammatory response with TLR4 overexpression, increased TNFα production, and increased immune cell infiltration, as well as increased excitotoxicity and oxidative stress. CONCLUSIONS: This model reproduced some key hallmarks of IVH-BD in ELBWN. Inflammation associated with BBB dysfunction appears as relevant therapeutic target to prevent IVH-BD-induced WMI. IMPACT: Paraventricular injection of clostridium collagenase (PVCC) to 1-day-old Wistar rats uniquely reproduced the neuroimaging, histologic and functional characteristics of intraventricular hemorrhage-induced brain damage (IVH-BD) in extremely low birth weight newborns (ELBWN). PVCC-induced IVH triggered a prolonged inflammatory response associated with blood-brain barrier increased permeability, which in turn facilitates the infiltration of inflammatory cells. Thus, PVCC led to white matter injury (WMI) resulting in long-term motor and cognitive impairment. This model offers a valuable tool to obtain further insight into the mechanisms of IVH-BD in ELBWN and proposes some key therapeutic targets.

NDGA enhances the physicochemical and anti-biodegradation performance of dentin collagen.

OBJECTIVES: Collagen fibrils from carious dentin matrix are prone to enzymatic degradation. This study investigates the feasibility and mechanism of nordihydroguaiaretic acid (NDGA), as a collagen crosslinker, to bio-modify the demineralized dentin matrix. METHODS: The physicochemical properties of the crosslinked dentin matrix were characterized by swelling ratio, ninhydrin assay, Fourier Transform Infrared spectroscopy, and atomic force microscopy. The collagenase degradation resistance was evaluated by measuring loss of dry mass, hydroproline release, loss of elasticity, and micro-nano structure integrity. The cytotoxicity of NDGA-crosslinked dentin collagen was evaluated by flow cytometry. RESULTS: NDGA crosslinked dentin matrix without destroying the integrity of collagen. Mechanistically, NDGA formed bisquinone bond between two adjacent o-quinone groups, resulting in NDGA polymeric matrix in which collagen fibrils were embedded. NDGA modification could significantly enhance the stiffness of dentin matrix at macro-nano scale. The NDGA-crosslinked dentin matrix exhibited remarkably low collagen degradation and sustained bulk elasticity after collagenase challenge, which were attributed to decreased water content, physical masking of collagenase bind sites on collagen, and improved stiffness of collagen fibrils. Notably, NDGA-crosslinked dentin matrix exhibited excellent biocompatibility. CONCLUSION: NDGA, as a biocompatible collagen crosslinker, improves the mechanical properties and biodegradation resistance of demineralized dentin matrix.

A combination of omega-3 and exercise reduces experimental Achilles tendinopathy induced with a type-1 collagenase in rats.

This study aimed to evaluate the effectiveness of omega-3 supplementation with exercise in a collagenase-induced Achilles tendinopathy (AT) rat model. Experimental groups (healthy control (HC), AT, exercise (Ex), omega-3 (W), and Ex+W) were randomly allocated. After a week of adaptation, oral omega-3 was initiated for 8 weeks (5 days/week). The exercise groups performed treadmill running for 30 min/day (5 days/week, 20 m/min, 8 weeks) following one week of adaptation (10 m/min, 15 min/day). Matrix metalloproteinase-13 (MMP-13), interleukin-1 beta (IL-1ß), tumor necrosis factor-alpha (TNF-α), and total antioxidant-oxidant status (TAS) levels were determined in serum samples. Tendon samples were obtained for biomechanical, histopathological, and immunohistochemical assessments. Ultimate tensile force, yield force, stiffness values, collagen type-I alpha 1 expression, and serum TAS significantly decreased (P < 0.05) in AT vs. HC. These values and expression significantly increased in the Ex+W group vs. AT. Serum MMP-13, IL-1ß, and TNF-α levels decreased in all treatment groups vs. AT. The most significant decrease was found in the Ex+W group (P < 0.01). Histopathologically, the improvement in degeneration was statistically significant in the Ex+W group (P < 0.05). Immunohistochemically, MMP-13, IL-1ß, TNF-α, and nitric oxide synthase-2 expression was decreased in all treatment groups vs. AT. In conclusion, omega-3 and exercise might be recommended in AT patients.

Novel strategy for primary epithelial cell isolation: Combination of hyaluronidase and collagenase I.

OBJECTIVE: Different strategies for epithelial cell isolation significantly affect the viability and physiological properties of primary cells. Trypsin digestion, a conventional method, causes collateral damage owing to its strong digestive potential. To better preserve the physiological properties of epithelial tissues, we aimed to develop a modified method (hyaluronidase and collagenase I combination) for primary cell isolation. METHOD: We used conventional and modified methods to compare cell viability, morphology and stemness. Additionally, we investigated the passaging stability of epithelial cells and their capacity for organoid formation. Finally, we compared the two methods for isolating urothelial, oesophageal, lingual, and epidermal epithelial cells. RESULT: Gingival epithelial cells obtained using the modified method had higher viability, better morphology and stronger stemness than those obtained using the conventional method. Additionally, primary cells obtained using the modified method were stably passaged. Regarding organoid culture, adopting the modified method led to a significant increase in the growth rate and expression of the stem cell markers cytokeratin (CK)-19 and Ki-67. Furthermore, the modified method outperformed the conventional method for isolating urothelial, epidermal, oesophageal and lingual epithelial cells. CONCLUSION: We demonstrated that the combination of hyaluronidase and collagenase I outperformed trypsin in preserving the physiological properties of primary cells and organoid formation. The modified method could be broadly applied to isolate different types of epithelial cells and facilitate studies on organoids and tissue engineering.

Standardized Pomegranate (Pomella®) and Red Maple (Maplifa®) Extracts and Their Phenolics Protect Type I Collagen by the Inhibition of Matrix Metalloproteinases, Collagenase, and Collagen Cross-Linking.

Phenolics enriched pomegranate fruit (Pomella®) and red maple leaf (Maplifa®) extracts and their major phenolic constituents have demonstrated beneficial skin effects through the protection of human skin keratinocytes from oxidative-stress-induced damage. However, their mechanisms of protection of cutaneous collagen are still unclear. Herein, the collagen protective effects of Pomella® and Maplifa®, and their major bioactive phytochemicals, namely, punicalagin (PA) and ginnalin A (GA), respectively, were evaluated using enzymatic assays including collagenase, anti-glycation and cell-based models as well as computational methods. The importance of the modulatory effects was validated at the protein level for type I collagen and matrix metalloproteinases (MMPs) using human-skin-derived keratinocytes. The synergistic collagenase inhibitory effects upon combinations of Pomella® + Maplifa® and PA + GA at a combination ratio of 1:2 and 1:1, respectively, were evaluated using their combination index (CI; a well-established assessment of synergism). Pomella® (50-400 µg/mL), Maplifa® (100-800 µg/mL), PA (50-400 µM), and GA (50-400 µM) dose-dependently inhibited collagenase activity by 26.3-86.3%, 25.7-94.0%, 26.2-94.0%, and 12.0-98.0%, respectively. The CI of the anti-collagenase activity of Pomella® and Maplifa® ranged from 0.53-0.90, while that of PA and GA (12.5/12.5 and 25/25 µM) ranged from 0.66 and 0.69, respectively, suggesting a synergistic inhibitory effect. Interestingly, in the cell-based assays by Western blotting, Pomella® and Maplifa® reduced the protein expression levels of collagen degradation enzymes (MMPs), while simultaneously increasing that of type I collagen in epidermoid carcinoma A431 cells. This is the first report to show that these extracts exert synergistic collagen protective effects. Taken together, these findings provide molecular insights into the usefulness of Pomella® and Maplifa® or their phenolics as bioactive ingredients for skin care products to slow down aging and enhance skin tone.