In silico design of fusion keratinocyte growth factor containing collagen-binding domain for tissue engineering application.

Keratinocyte growth factor (KGF) is a potential therapeutic factor in wound healing. However, its applications have been restricted due to its low stability, short half-life, and limited target specificity. We aimed to immobilize KGF on collagen-based biomaterials for long-lasting and targeted therapy by designing fusion forms of KGF with collagen-binding domains (CBD) from natural origins. Twelve fusion proteins were designed consisting of KGF and CBDs with different lengths and amino acid compositions. Three-dimensional (3D) structures of the fusions were predicted by homology modeling. Physiochemical properties and secondary structure of the fusions were evaluated by bioinformatics tools. Moreover, the effect of the CBDs on the 3D structure and dynamic behavior of the fusions was investigated by molecular dynamics (MD) simulation. The binding affinity of the fusions to collagen, KGF receptor, and heparin was assessed using docking tools. Our results demonstrated that fusions with small CBDs like CBD of mammalian collagenase and decapeptide CBD of von Willebrand factor (VWF) were more stable and properly folded than those with larger CBDs. On the other hand, the insertion of bulky CBDs, including Fibronectin CBD and CBD of Clostridium histolyticum collagenase, into KGF resulted in stronger binding to collagen. Therefore, very small or large CBDs are inappropriate for constructing KGF fusions because they suffer from low collagen affinity or poor stability. By comparing the results of MD simulation and docking, this study proposed that CBDs belonging to Vibrio mimicus metalloprotease and A3 domain of VWF would be good candidates to produce stable fusions with proper affinities toward collagen and KGF receptors. Moreover, the secondary structure analysis showed that the overall structure of KGF and CBDs was better preserved when CBDs were inserted at the C-terminal of KGF. This computational information about novel KGF fusions may help find the best constructs for experimental studies.

The efficacies and limitations of fasciectomy and collagenase clostridium histolyticum in Dupuytren's contracture management: A meta-analysis.

BACKGROUND: We intend to assess the efficacies and limitations of collagenase clostridium histolyticum (CCH) and fasciectomy in treating Dupuytren's contracture, and the associated complications and rate of recurrences aiming to present a treatment algorithm. METHODS: A literature search within the PubMed, Web of Sciences, Cochrane Library, and EMBASE databases was performed using the combined key words 'Dupuytren, palmar aponeurosis contracture, collagenase clostridium histolyticum and fasciectomy', including all possible studies with a set of predefined inclusion and exclusion criteria. RESULTS: Thirty studies were assessed for eligibility from 215 identified records. Seventeen publications satisfied the inclusion criteria including 2142 joints in 1784 patients. The mean follow-up time was 18.0 months (3-60). CONCLUSION: Acceptable contractures release was obtained in both techniques. Severe complications associated with fasciectomy outrank those of CCH, whereas the low rate of recurrence favors the fasciectomy technique.

A state-of-art review on collagenase Clostridium Histolyticum and Peyronie's disease: drug profile, clinical evidence and safety outcomes.

Introduction: Collagenase clostridium histolyticum (CCH) is the first and only licensed medical treatment for men with Peyronie's disease (PD). Published literature shows intralesional CCH injection as an effective and safe minimally invasive treatment in a specific subgroup of PD patients.Areas covered: The authors discuss pharmacodynamics and pharmacokinetics as well as clinical outcomes and safety profile from major CCH studies in PD. All relevant CCH studies published in PubMed and EMBASE databases up to June 2019 were included.Expert opinion: Given the variability in treatment schedule and drug access coupled with the potential need for further treatment, strict patient selection and the use of adjunctive strategies are key determinants to maximize clinical efficacy of intralesional CCH. Furthermore, longer-term follow-up data on the clinical outcomes, safety and durability of CCH in larger multi-center studies and post-marketing surveillance data are necessary to provide a comparison to other standard PD treatment options.

Basic Fibroblast Growth Factor Fused with Tandem Collagen-Binding Domains from Clostridium histolyticum Collagenase ColG Increases Bone Formation.

Basic fibroblast growth factor 2 (bFGF) accelerates bone formation during fracture healing. Because the efficacy of bFGF decreases rapidly following its diffusion from fracture sites, however, repeated dosing is required to ensure a sustained therapeutic effect. We previously developed a fusion protein comprising bFGF, a polycystic kidney disease domain (PKD; s2b), and collagen-binding domain (CBD; s3) sourced from the Clostridium histolyticum class II collagenase, ColH, and reported that the combination of this fusion protein with a collagen-like peptide, poly(Pro-Hyp-Gly)10, induced mesenchymal cell proliferation and callus formation at fracture sites. In addition, C. histolyticum produces class I collagenase (ColG) with tandem CBDs (s3a and s3b) at the C-terminus. We therefore hypothesized that a bFGF fusion protein containing ColG-derived tandem CBDs (s3a and s3b) would show enhanced collagen-binding activity, leading to improved bone formation. Here, we examined the binding affinity of four collagen anchors derived from the two clostridial collagenases to H-Gly-Pro-Arg-Gly-(Pro-Hyp-Gly)12-NH2, a collagenous peptide, by surface plasmon resonance and found that tandem CBDs (s3a-s3b) have the highest affinity for the collagenous peptide. We also constructed four fusion proteins consisting of bFGF and s3 (bFGF-s3), s2b-s3b (bFGF-s2b-s3), s3b (bFGF-s3b), and s3a-s3b (bFGF-s3a-s3b) and compared their biological activities to those of a previous fusion construct (bFGF-s2b-s3) using a cell proliferation assay in vitro and a mouse femoral fracture model in vivo. Among these CB-bFGFs, bFGF-s3a-s3b showed the highest capacity to induce mesenchymal cell proliferation and callus formation in the mice fracture model. The poly(Pro-Hyp-Gly)10/bFGF-s3a-s3b construct may therefore have the potential to promote bone formation in clinical settings.

Collagenase treatment enhances proteomic coverage of low-abundance proteins in decellularized matrix bioscaffolds.

There is great interest in the application of advanced proteomic techniques to characterize decellularized tissues in order to develop a deeper understanding of the effects of the complex extracellular matrix (ECM) composition on the cellular response to these pro-regenerative bioscaffolds. However, the identification of proteins in ECM-derived bioscaffolds is hindered by the high abundance of collagen in the samples, which can interfere with the detection of lower-abundance constituents that may be important regulators of cell function. To address this limitation, we developed a novel multi-enzyme digestion approach using treatment with a highly-purified collagenase derived from Clostridium Histolyticum to selectively deplete collagen from ECM-derived protein extracts, reducing its relative abundance from up to 90% to below 10%. Moreover, we applied this new method to characterize the proteome of human decellularized adipose tissue (DAT), human decellularized cancellous bone (DCB), and commercially-available bovine tendon collagen (BTC). We successfully demonstrated with all three sources that collagenase treatment increased the depth of detection and enabled the identification of a variety of signaling proteins that were masked by collagen in standard digestion protocols with trypsin/LysC, increasing the number of proteins identified in the DAT by ∼2.2 fold, the DCB by ∼1.3 fold, and the BTC by ∼1.6 fold. In addition, quantitative proteomics using label-free quantification demonstrated that the DAT and DCB extracts were compositionally distinct, and identified a number of adipogenic and osteogenic proteins that were consistently more highly expressed in the DAT and DCB respectively. Overall, we have developed a new processing method that may be applied in advanced mass spectrometry studies to improve the high-throughput proteomic characterization of bioscaffolds derived from mammalian tissues. Further, our study provides new insight into the complex ECM composition of two human decellularized tissues of interest as cell-instructive platforms for regenerative medicine.

Effect of Freeze-Dried Allograft Bone With Human Basic Fibroblast Growth Factor Containing a Collagen-Binding Domain From Clostridium histolyticum Collagenase on Bone Formation After Lumbar Posterolateral Fusion Surgery in Rats.

STUDY DESIGN: An experimental study. OBJECTIVE: To evaluate the effectiveness of freeze-dried bone allograft (FDBA) with basic fibroblast growth factor (bFGF) fused with the polycystic kidney disease domain (PKD) and the collagen-binding domain (CBD) of Clostridium histolyticum collagenase, for the acceleration of lumbar posterolateral fusion in rats. SUMMARY OF BACKGROUND DATA: Reports indicate bFGF is an effective growth factor with osteogenic potential for promoting bone regeneration, although its efficiency decreases rapidly following its diffusion in body fluid from the host site. We developed a bFGF fusion protein containing the PKD and the CBD of C histolyticum collagenase (bFGF-PKD-CBD), which markedly enhanced bone formation at a relatively low concentration when applied to the surface of rat femurs in a previous study. The potential of this novel protein to accelerate bone fusion in a rat model of lumbar posterolateral fusion has yet to be investigated. METHODS: Bilateral L4-L5 posterolateral fusions were performed, using 150 mg of FDBA powder per side. A total of 20 male Sprague-Dawley rats weighing 200 to 250 g/each were divided into two groups of 10 rats: FDBA was incubated with either phosphate-buffered saline (control group) or 0.58 nmol bFGF-PKD-CBD (bFGF-PKD-CBD group) before fusion surgery. The effect of bFGF-PKD-CBD was estimated using radiographs, microcomputed tomography, and histology (hematoxylin-eosin and von Kossa staining). RESULTS: Both grafted bone volume in the posterolateral lesion and the volume of new bone formation on the surface of laminae and spinal processes were significantly higher in the bFGF-PKD-CBD group than in the control group. Histologically, new bone formation and surrounding chondrocytes and fibroblasts were prominent in the bFGF-PKD-CBD group. CONCLUSION: FDBA infused with bFGF-PKD-CBD may be a promising material for accelerating spinal fusion, and the FDBA-based delivery system for localizing bFGF-PKD-CBD may offer novel therapeutic approaches to augment spinal fusion. LEVEL OF EVIDENCE: N/A.

Role of RNase Y in Clostridium perfringens mRNA Decay and Processing.

RNase Y is a major endoribonuclease that plays a crucial role in mRNA degradation and processing. We study the role of RNase Y in the Gram-positive anaerobic pathogen Clostridium perfringens, which until now has not been well understood. Our study implies an important role for RNase Y-mediated RNA degradation and processing in virulence gene expression and the physiological development of the organism. We began by constructing an RNase Y conditional knockdown strain in order to observe the importance of RNase Y on growth and virulence. Our resulting transcriptome analysis shows that RNase Y affects the expression of many genes, including toxin-producing genes. We provide data to show that RNase Y depletion repressed several toxin genes in C. perfringens and involved the virR-virS two-component system. We also observe evidence that RNase Y is indispensable for processing and stabilizing the transcripts of colA (encoding a major toxin collagenase) and pilA2 (encoding a major pilin component of the type IV pili). Posttranscriptional regulation of colA is known to be mediated by cleavage in the 5' untranslated region (5'UTR), and we observe that RNase Y depletion diminishes colA 5'UTR processing. We show that RNase Y is also involved in the posttranscriptional stabilization of pilA2 mRNA, which is thought to be important for host cell adherence and biofilm formation. IMPORTANCE: RNases have important roles in RNA degradation and turnover in all organisms. C. perfringens is a Gram-positive anaerobic spore-forming bacterial pathogen that produces numerous extracellular enzymes and toxins, and it is linked to digestive disorders and disease. A highly conserved endoribonuclease, RNase Y, affects the expression of hundreds of genes, including toxin genes, and studying these effects is useful for understanding C. perfringens specifically and RNases generally. Moreover, RNase Y is involved in processing specific transcripts, and we observed that this processing in C. perfringens results in the stabilization of mRNAs encoding a toxin and bacterial extracellular apparatus pili. Our study shows that RNase activity is associated with gene expression, helping to determine the growth, proliferation, and virulence of C. perfringens.

High-speed atomic force microscopy reveals strongly polarized movement of clostridial collagenase along collagen fibrils.

Bacterial collagenases involved in donor infection are widely applied in many fields due to their high activity and specificity; however, little is known regarding the mechanisms by which bacterial collagenases degrade insoluble collagen in host tissues. Using high-speed atomic force microscopy, we simultaneously visualized the hierarchical structure of collagen fibrils and the movement of a representative bacterial collagenase, Clostridium histolyticum type I collagenase (ColG), to determine the relationship between collagen structure and collagenase movement. Notably, ColG moved ~14.5 nm toward the collagen N terminus in ~3.8 s in a manner dependent on a catalytic zinc ion. While ColG was engaged, collagen molecules were not only degraded but also occasionally rearranged to thicken neighboring collagen fibrils. Importantly, we found a similarity of relationship between the enzyme-substrate interface structure and enzyme migration in collagen-collagenase and DNA-nuclease systems, which share a helical substrate structure, suggesting a common strategy in enzyme evolution.

Identification and Characterization of Novel Matrix-Derived Bioactive Peptides: A Role for Collagenase from Santyl® Ointment in Post-Debridement Wound Healing?

Debridement, the removal of diseased, nonviable tissue, is critical for clinicians to readily assess wound status and prepare the wound bed for advanced therapeutics or downstream active healing. Removing necrotic slough and eschar through surgical or mechanical methods is less specific and may be painful for patients. Enzymatic debridement agents, such as Clostridial collagenase, selectively and painlessly degrade devitalized tissue. In addition to its debriding activities, highly-purified Clostridial collagenase actively promotes healing, and our past studies reveal that extracellular matrices digested with this enzyme yield peptides that activate cellular migratory, proliferative and angiogenic responses to injury in vitro, and promote wound closure in vivo. Intriguingly, while collagenase Santyl® ointment, a sterile preparation containing Clostridial collagenases and other non-specific proteases, is a well-accepted enzymatic debridement agent, its role as an active healing entity has never been established. Based on our previous studies of pure Clostridial collagenase, we now ask whether the mixture of enzymes contained within Santyl® produces matrix-derived peptides that promote cellular injury responses in vitro and stimulate wound closure in vivo. Here, we identify novel collagen fragments, along with collagen-associated peptides derived from thrombospondin-1, multimerin-1, fibronectin, TGFß-induced protein ig-h3 and tenascin-C, generated from Santyl® collagenase-digested human dermal capillary endothelial and fibroblastic matrices, which increase cell proliferation and angiogenic remodeling in vitro by 50-100% over controls. Using an established model of impaired healing, we further demonstrate a specific dose of collagenase from Santyl® ointment, as well as the newly-identified and chemically-synthesized ECM-derived peptides significantly increase wound re-epithelialization by 60-100% over saline-treated controls. These results not only confirm and extend our earlier studies using purified collagenase- and matrix-derived peptides to stimulate healing in vitro and in vivo, but these Santyl®-generated, matrix-derived peptides may also represent exciting new opportunities for creating advanced wound healing therapies that are enabled by enzymatic debridement and potentially go beyond debridement.

Inhibition of Collagenase by Mycosporine-like Amino Acids from Marine Sources.

Matrix metalloproteinases play an important role in extracellular matrix remodeling. Excessive activity of these enzymes can be induced by UV light and leads to skin damage, a process known as photoaging. In this study, we investigated the collagenase inhibition potential of mycosporine-like amino acids, compounds that have been isolated from marine organisms and are known photoprotectants against UV-A and UV-B. For this purpose, the commonly used collagenase assay was optimized and for the first time validated in terms of relationships between enzyme-substrate concentrations, temperature, incubation time, and enzyme stability. Three compounds were isolated from the marine red algae Porphyra sp. and Palmaria palmata, and evaluated for their inhibitory properties against Chlostridium histolyticum collagenase. A dose-dependent, but very moderate, inhibition was observed for all substances and IC50 values of 104.0 µM for shinorine, 105.9 µM for porphyra, and 158.9 µM for palythine were determined. Additionally, computer-aided docking models suggested that the mycosporine-like amino acids binding to the active site of the enzyme is a competitive inhibition.

Synergistic Effect of Neutral Protease and Clostripain on Rat Pancreatic Islet Isolation.

BACKGROUND: Islet isolation currently requires collagenase, neutral protease and other components. Thermolysin (TL) from Bacillus thermoproteolyticus is the gold standard neutral protease. However, we speculated that neutral protease derived from Clostridium histolyticum (Ch; ChNP) would be biologically superior for islet isolation. Tryptic-like activity has also been reported to be important. Therefore, we focused on clostripain (CP), since it is one of the main proteases in Clostridium histolyticum which possesses tryptic-like activity. We then examined the synergistic effects of highly purified ChNP and CP on rat islet isolation. METHODS: The same amount of collagenase was used in all four groups (TL, ChNP, TL+CP and ChNP+CP; n = 12/group). The efficiency was evaluated by the islet yield and function. An immunohistochemical analysis, in vitro digestion assay for each enzyme component and evaluation of the activation of endogenous exocrine proteases during islet isolation were also performed. RESULTS: The islet yield of the TL group was significantly higher than that of the ChNP group (P < 0.01). The islet yield was dose dependently increased in the ChNP+CP group, but was decreased in the TL + CP group. The islet yield in the ChNP + CP group was significantly higher than that in the TL group, but their islet function was similar. Different specificities for laminin, especially laminin-511, were observed in the TL, ChNP, and CP groups. CONCLUSIONS: Clostripain had a strong synergistic effect with ChNP, but not with TL. Therefore, ChNP and CP, in combination with collagenase derived from the same bacteria, may effectively increase the isolation efficiency without affecting the quality of islets.

Aeromonas piscicola AH-3 expresses an extracellular collagenase with cytotoxic properties.

UNLABELLED: The aim of this study was to investigate the presence and the phenotypic expression of a gene coding for a putative collagenase. This gene (AHA_0517) was identified in Aeromonas hydrophila ATCC 7966 genome and named colAh. We constructed and characterized an Aeromonas piscicola AH-3::colAh knockout mutant. Collagenolytic activity of the wild-type and mutant strains was determined, demonstrating that colAh encodes for a collagenase. ColAh-collagen interaction was assayed by Far-Western blot, and cytopathic effects were investigated in Vero cells. We demonstrated that ColAh is a gluzincin metallopeptidase (approx. 100 kDa), able to cleave and physically interact with collagen, that contributes for Aeromonas collagenolytic activity and cytotoxicity. ColAh possess the consensus HEXXH sequence and a glutamic acid as the third zinc binding positioned downstream the HEXXH motif, but has low sequence similarity and distinct domain architecture to the well-known clostridial collagenases. In addition, these results highlight the importance of exploring new microbial collagenases that may have significant relevance for the health and biotechnological industries. SIGNIFICANCE AND IMPACT OF THE STUDY: Collagenases play a central role in processes where collagen digestion is needed, for example host invasion by pathogenic micro-organisms. We identified a new collagenase from Aeromonas using an integrated in silico/in vitro strategy. This enzyme is able to bind and cleave collagen, contributes for AH-3 cytotoxicity and shares low similarity with known bacterial collagenases. This is the first report of an enzyme belonging to the gluzincin subfamily of the M9 family of peptidases in Aeromonas. This study increases the current knowledge on collagenolytic enzymes bringing new perspectives for biotechnology/medical purposes.

Acceleration of periosteal bone formation by human basic fibroblast growth factor containing a collagen-binding domain from Clostridium histolyticum collagenase.

Basic fibroblast growth factor 2 (bFGF) is a potent mitogen for mesenchymal cells, and the local application of recombinant bFGF accelerates bone union and defect repair. However, repeated dosing is required for sustained therapeutic effect as the efficacy of bFGF decreases rapidly following its diffusion from bone defect sites. Here, we attempted to develop a collagen-based bone formation system using a fusion protein (collagen binding-bFGF, CB-bFGF) consisting of bFGF and the collagen-binding domain (CBD) of Clostridium histolyticum collagenase. The addition of the CBD to bFGF did not modify its native biological activity, as shown by the capacity of the fusion protein to promote the in vitro proliferation of periosteal mesenchymal cells. The affinity of the fusion protein towards collagen and demineralized bone matrix (DBM) was also confirmed by collagen-binding assays. Moreover, in vivo periosteal bone formation assays showed that the combination of CB-bFGF with a collagen sheet induced periosteal bone formation at protein concentrations lower than those required for bFGF alone. In addition, grafts of DBM loaded with CB-bFGF accelerated new bone formation in rat femurs compared to the same concentration of bFGF administered alone. Taken together, these properties suggest that the CB-bFGF/collagen composite is a promising material for bone repair in the clinical setting.

Acceleration of bone formation during fracture healing by injectable collagen powder and human basic fibroblast growth factor containing a collagen-binding domain from Clostridium histolyticum collagenase.

Growth factor delivered with implantable biomaterials has been used to both accelerate and ensure healing of open fractures in human patients. However, a major limitation of implantable biomaterials is the requirement for open surgical placement. Here, we developed an injectable collagen material-based bone formation system consisting of injectable collagen powder with fibril morphology and collagen triple helix conformation, and basic fibroblast growth factor (bFGF) fused to the collagen-binding domain (CBD) of Clostridium histolyticum collagenase. The affinity of the CBD towards collagen was confirmed by the results of collagen-binding assays. Moreover, the combination of the collagen binding-bFGF fusion protein (CB-bFGF) with injectable collagen powder induced bone formation at protein concentrations lower than those required for bFGF alone in mice fracture models. Taken together, these properties suggest that the CB-bFGF/collagen powder composite is a promising injectable material for bone repair in the clinical setting.

Structural basis for activity regulation and substrate preference of clostridial collagenases G, H, and T.

Clostridial collagenases are among the most efficient enzymes to degrade by far the most predominant protein in the biosphere. Here we present crystal structures of the peptidases of three clostridial collagenase isoforms (ColG, ColH, and ColT). The comparison of unliganded and liganded structures reveals a quaternary subdomain dynamics. In the unliganded ColH structure, this globular dynamics is modulated by an aspartate switch motion that binds to the catalytic zinc. We further identified a calcium binding site in proximity to the catalytic zinc. Both ions are required for full activity, explaining why calcium critically affects the enzymatic activity of clostridial collagenases. Our studies further reveal that loops close to the active site thus serve as characteristic substrate selectivity filter. These elements explain the distinct peptidolytic and collagenolytic activities of these enzymes and provide a rational framework to engineer collagenases with customized substrate specificity as well as for inhibitor design.

Enhancement of the structural stability of full-length clostridial collagenase by calcium ions.

The clostridial collagenases G and H are multidomain proteins. For collagen digestion, the domain arrangement is likely to play an important role in collagen binding and hydrolysis. In this study, the full-length collagenase H protein from Clostridium histolyticum was expressed in Escherichia coli and purified. The N-terminal amino acid of the purified protein was Ala31. The expressed protein showed enzymatic activity against azocoll as a substrate. To investigate the role of Ca(2+) in providing structural stability to the full-length collagenase H, biophysical measurements were conducted using the recombinant protein. Size exclusion chromatography revealed that the Ca(2+) chelation by EGTA induced interdomain conformational changes. Dynamic light scattering measurements showed an increase in the percent polydispersity as the Ca(2+) was chelated, suggesting an increase in protein flexibility. In addition to these conformational changes, differential scanning fluorimetry measurements revealed that the thermostability was decreased by Ca(2+) chelation, in comparison with the thermal melting point (T(m)). The melting point changed from 54 to 49°C by the Ca(2+) chelation, and it was restored to 54°C by the addition of excess Ca(2+). These results indicated that the interdomain flexibility and the domain arrangement of full-length collagenase H are reversibly regulated by Ca(2+).

Cranberry proanthocyanidins act in synergy with licochalcone A to reduce Porphyromonas gingivalis growth and virulence properties, and to suppress cytokine secretion by macrophages.

AIMS: Periodontitis is an inflammatory disease of polymicrobial origin that affects the tooth-supporting tissues. With the spread of antibiotic resistance among pathogenic bacteria, alternative strategies are required to better control infectious diseases such as periodontitis. The aim of our study was to investigate whether two natural compounds, A-type cranberry proanthocyanidins (AC-PACs) and licochalcone A, act in synergy against Porphyromonas gingivalis and the host inflammatory response of a macrophage model. METHODS AND RESULTS: Using a checkerboard microtitre test, AC-PACs and licochalcone A were found to act in synergy to inhibit P. gingivalis growth and biofilm formation. Fluorescein isothiocyanate-labelled P. gingivalis adhesion to oral epithelial cells was also inhibited by a combination of the two natural compounds in a synergistic manner. Fluorometric assays showed that although AC-PACs and licochalcone A reduced both MMP-9 and P. gingivalis collagenase activities, no synergy was obtained with a combination of the compounds. Lastly, AC-PACs and licochalcone A also acted in synergy to reduce the lipopolysaccharide (LPS)-induced secretion of the pro-inflammatory mediators IL-1ß, TNF-α, IL-6 and IL-8 in a macrophage model. CONCLUSIONS: A-type cranberry proanthocyanidins and licochalcone A, natural compounds from cranberry and licorice, respectively, act in synergy on both P. gingivalis and the host immune response, the two principal etiological factors of periodontitis. SIGNIFICANCE AND IMPACT OF THE STUDY: The combined use of AC-PACs and licochalcone A may be a potential novel therapeutic strategy for the treatment and prevention of periodontal disease.

In vitro study of novel collagenase (XIAFLEX®) on Dupuytren's disease fibroblasts displays unique drug related properties.

Dupuytren's disease (DD) is a benign, fibroproliferative disease of the palmar fascia, with excessive extracellular matrix (ECM) deposition and over-production of cytokines and growth factors, resulting in digital fixed flexion contractures limiting hand function and patient quality of life. Surgical fasciectomy is the gold standard treatment but is invasive and has associated morbidity without limiting disease recurrence. Injectable Collagenase Clostridium histolyticum (CCH)--Xiaflex®--is a novel, nonsurgical option with clinically proven in vivo reduction of DD contractures but with limited in vitro data demonstrating its cellular and molecular effects. The aim of this study was to delineate the effects of CCH on primary fibroblasts isolated from DD and non-DD anatomical sites (using RTCA, LDH, WST-1, FACS, qRT-PCR, ELISA and In-Cell Quantitative Western Blotting) to compare the efficacy of varying concentrations of Xiaflex® against a reagent grade Collagenase, Collagenase A. Results demonstrated that DD nodule and cord fibroblasts had greater proliferation than those from fat and skin. Xiaflex® exposure resulted in dose- and time-dependent inhibition of cellular spreading, attachment and proliferation, with cellular recovery after enzyme removal. Unlike Collagenase A, Xiaflex® did not cause apoptosis. Collagen expression patterns were significantly (p<0.05) different in DD fibroblasts across anatomical sites - the highest levels of collagen I and III were detected in DD nodule, with DD cord and fat fibroblasts demonstrating a smaller increase in both collagen expression relative to DD skin. Xiaflex® significantly (p<0.05) down-regulated ECM components, cytokines and growth factors in a dose-dependent manner. An in vitro scratch wound assay model demonstrated that, at low concentrations, Xiaflex® enabled a faster fibroblast reparatory migration into the wound, whereas, at high concentrations, this process was significantly (p<0.05) inhibited. This is the first report elucidating potential mechanisms of action of Xiaflex® on Dupuytren fibroblasts, offering a greater insight and a better understanding of its effect in DD.

A new enzyme mixture to increase the yield and transplant rate of autologous and allogeneic human islet products.

BACKGROUND: The optimal enzyme blend that maximizes human islet yield for transplantation remains to be determined. In this study, we evaluated eight different enzyme combinations (ECs) in an attempt to improve islet yield. The ECs consisted of purified, intact or truncated class 1 (C1) and class 2 (C2) collagenases from Clostridium histolyticum (Ch), and neutral protease (NP) from Bacillus thermoproteolyticus rokko (thermolysin) or Ch (ChNP). METHODS: We report the results of 249 human islet isolations, including 99 deceased donors (research n=57, clinical n=42) and 150 chronic pancreatitis pancreases. We prepared a new enzyme mixture (NEM) composed of intact C1 and C2 collagenases and ChNP in place of thermolysin. The NEM was first tested in split pancreas (n=5) experiments and then used for islet autologous (n=21) and allogeneic transplantation (n=10). Islet isolation outcomes from eight different ECs were statistically compared using multivariate analysis. RESULTS: The NEM consistently achieved higher islet yields from pancreatitis (P<0.003) and deceased donor pancreases (P<0.001) than other standard ECs. Using the NEM, islet products met release criteria for transplantation from 8 of 10 consecutive pancreases, averaging 6510 ± 2150 islet equivalent number/gram (IEQ/g) pancreas and 694,681 ± 147,356 total IEQ/transplantation. In autologous isolation, the NEM yielded more than 200,000 IEQ from 19 of 21 pancreases (averaging 422,893 ± 181,329 total IEQ and 5979 ± 1469 IEQ/kg recipient body weight) regardless of the severity of fibrosis. CONCLUSIONS: A NEM composed of ChNP with CIzyme high intact C1 collagenase recovers higher islet yield from deceased and pancreatitis pancreases while retaining islet quality and function.

Probing the 3-D structure, dynamics, and stability of bacterial collagenase collagen binding domain (apo- versus holo-) by limited proteolysis MALDI-TOF MS.

Pairing limited proteolysis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to probe clostridial collagenase collagen binding domain (CBD) reveals the solution dynamics and stability of the protein, as these factors are crucial to CBD effectiveness as a drug-delivery vehicle. MS analysis of proteolytic digests indicates initial cleavage sites, thereby specifying the less stable and highly accessible regions of CBD. Modulation of protein structure and stability upon metal binding is shown through MS analysis of calcium-bound and cobalt-bound CBD proteolytic digests. Previously determined X-ray crystal structures illustrate that calcium binding induces secondary structure transformation in the highly mobile N-terminal arm and increases protein stability. MS-based detection of exposed residues confirms protein flexibility, accentuates N-terminal dynamics, and demonstrates increased global protein stability exported by calcium binding. Additionally, apo- and calcium-bound CBD proteolysis sites correlate well with crystallographic B-factors, accessibility, and enzyme specificity. MS-observed cleavage sites with no clear correlations are explained either by crystal contacts of the X-ray crystal structures or by observed differences between Molecules A and B in the X-ray crystal structures. The study newly reveals the absence of the ßA strand and thus the very dynamic N-terminal linker, as corroborated by the solution X-ray scattering results. Cobalt binding has a regional effect on the solution phase stability of CBD, as limited proteolysis data implies the capture of an intermediate-CBD solution structure when cobalt is bound.