Effects of Different Concentrations of Injectable Collagenase Enzyme on Capsular Tissue Around Silicone Implants: A Preliminary Experimental Study for the Development of a New Treatment Strategy.

OBJECTIVES: In recent studies, collagen organization was blamed for the formation of capsular contracture which is still a challenging problem after silicone implant-based breast operations. In this study, effects of different concentrations of collagenase enzyme derived from Clostridium histolyticum on the capsular tissue formation around the silicone implants were investigated. The injectable form of collagenase has a routine clinical use in the treatment of both Dupuytren's and Peyronie's diseases. MATERIALS AND METHODS: Thirty-two Wistar albino rats were randomized into four groups. A 2 × 1 × 0.3-cm-sized silicone block was inserted inside a dorsal subcutaneous pocket in all groups. After 2 months of insertion, capsule thicknesses around the implants were detected under ultrasonography. This was followed by injection of isotonic saline, 150, 300, and 600 IU in Gr-1, 2, 3, and 4, respectively. All the animals were sacrificed at the end of the first week for histologic sampling to determine fibroblast proliferation, vessel density of the tissue, necrosis, edema, inflammation, and capsule thickness. All the data were statistically analyzed using Kruskal-Wallis and Mann-Whitney U tests and compared for significance of the results. RESULTS: There was no significant difference in terms of capsule thinning between the 300 and 600 IU groups but in both groups thinning was significantly higher than the sham group. In the 150 IU group there was no significant thinning as compared to the sham group (p > 0.05). However, complications such as skin necrosis, infection, and seroma formation were seen only in the 600 IU injection group. The optimal safe and effective dose of the enzyme was accepted as 300 IU. The 300 IU injection provided up to 89 % thinning in the capsule tissue. There was thinning of the collagen bundles parallel to capsule thickness. In the 600 IU group, micro-pores were encountered at the thinnest points. CONCLUSION: However, the late results and recurrence rates of capsular contracture were not included in this study; collagenase seemed effective for the reduction of capsular tissue around the implants. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Isolation and characterization of human mesenchymal stem cells from gingival connective tissue.

BACKGROUND AND OBJECTIVE: The main purpose of this study was to isolate and characterize gingival connective tissue-derived mesenchymal stem cells (GMSCs). The secondary purpose was to present a modified isolation method for the GMSCs. MATERIAL AND METHODS: Collected healthy gingival tissue samples were de-epithelialized and minced into small fragments. The tissues were digested by dispase and collagenase IV for 30 min. The first digested cell suspension was discarded, and then additional digestion was performed to the remaining cells in the same solution for 90 min. The isolated cells from gingiva was incubated in 37°C humidified condition and observed by inverted microscope. Cytoskeletal morphology was evaluated by phalloidin immunofluorescence. Potency of the cells was tested by colony-forming unit fibroblast assay. GMSCs were characterized by osteogenic, adipogenic and chondrogenic differentiation, and flow cytometric, immunofluorescence analysis. RESULTS: GMSCs showed spindle-shaped, fibroblast-like morphology, colony-forming abilities, adherence to plastic and multilineage differentiation (osteogenic, adipogenic, chondrogenic) potency. GMSCs expressed CD44, CD73, CD90 and CD105, but did not express CD14, CD45, CD34 and CD19 in flow cytometry. Expression of stem cell markers (SSEA-4, STRO-1, CD146, CD166 and CD271) and a mesenchymal marker (vimentin) were observed by immunofluorescence. CONCLUSIONS: In conclusion, we isolated and characterized stem cells from human gingival connective tissue with modified protocol. GMSCs showed multipotency with high proliferation and characteristics of mesenchymal stem cells. GMSCs are promising sources for tissue engineering and may be obtained during routine procedures under local anesthesia. Further research is needed to evaluate the potential of GSMCs' proliferation and cryopreservation.

Collagenase loaded chitosan nanoparticles for digestion of the collagenous scar in liver fibrosis: The effect of chitosan intrinsic collagen binding on the success of targeting.

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

Effects of toothbrushing force on the mineral content and demineralized organic matrix of eroded dentine.

Eroded dentine has a complex histological structure, and its organic fraction becomes increasingly exposed as a result of the continual action of acids. The present study sought to investigate the effects of brushing forces up to 4 N on mineralized and organic dentine fractions. The study was a cyclic demineralization and remineralization experiment (carried out over 9 d). Erosion was performed with HCl (6 x 2 min d-1), pH 1.6. Samples exposed to erosion alone served as controls; test samples were eroded and brushed with a powered toothbrush (2 x 15 s d-1), applying forces of 2, 3, or 4 N. Samples were analysed (using profilometry and longitudinal microradiography) before and after the removal of superficial organic material with collagenase. Randomly selected samples were subjected to scanning electron microscopy. Demineralized organic material was present on all samples regardless of brushing force. Loss values (determined using profilometry) after erosion only, and after brushing with forces of 2, 3, and 4 N, were 11.7 +/- 5.1, 13.6 +/- 11.2, 30.7 +/- 19.0, and 25.5 +/- 20.3, respectively, before treatment with collagenase, and 111.7 +/- 11.6, 122.0 +/- 11.8, 121.9 +/- 15.7, and 123.0 +/- 12.0, respectively, after treatment with collagenase. Microradiography confirmed the results. Significant effects of brushing force were only found on the demineralized organic fraction, and mineral loss was unaffected. The notion that eroded dentine is particularly prone to abrasion should be reconsidered.

Therapy for patients with burns - an integrating review.

OBJECTIVE: to identify, through an integrative review, national studies published over the last ten years highlighting products and therapies used in burns. METHODS: integrative research with studies published in the last ten years. Including clinical studies describing the use of the already established or innovative therapies in burns and the results obtained, published in national journals in the last ten years. Excluding articles published before 2007 and those that did not present results regarding the use of products in burns. RESULTS: ten articles that met the inclusion criteria were selected. Collagenase, 1% silver sulfadiazine, and porous cellulose membrane were some of the therapies cited. CONCLUSION: the casuistry was low; however, the good results obtained with porous cellulose membrane and silver nanocrystalline dressing are highlighted, since they were used in a larger number of patients in the studies evaluated.

Localized co-delivery of collagenase and trastuzumab by thermosensitive hydrogels for enhanced antitumor efficacy in human breast xenograft.

Modulation of the collagen-rich extracellular matrix (ECM) in solid tumors by the treatment with collagenase has been proved effective in enhancement of the interstitial transport and antitumor efficacy of antibodies. We, therefore, developed a PLGA-PEG-PLGA polymer-based thermosensitive hydrogel, which incorporated a HER2-targeted monoclonal antibody trastuzumab and collagenase (Col/Tra/Gel) for peritumoral administration. HER2-positvie BT474 tumor-bearing mice were selected as a model. The Col/Tra/Gel showed the continuous and biphasic release of protein drugs for 9 days in vitro. NIR imaging studies demonstrated a long-term retention of Col/Tra/Gel hydrogel in the peritumoral area for over 20 days. Treatment with Col/Tra/Gel reduced the collagen density and enhanced apoptotic cell death in tumor tissue, resulting in superior treatments with increased efficacy and reduced toxicity compared with other control groups. Moreover, a quarter-dose of Col/Tra/Gel exhibited a better antitumor efficacy than that of intravenous injection of clinical trastuzumab formulation. This localized co-delivery system offers a potential strategy for the modulation of dense ECM and enhancement of antibody efficacy.

Proteolytic Nanoparticles Replace a Surgical Blade by Controllably Remodeling the Oral Connective Tissue.

Surgical blades are common medical tools. However, blades cannot distinguish between healthy and diseased tissue, thereby creating unnecessary damage, lengthening recovery, and increasing pain. We propose that surgical procedures can rely on natural tissue remodeling tools-enzymes, which are the same tools our body uses to repair itself. Through a combination of nanotechnology and a controllably activated proteolytic enzyme, we performed a targeted surgical task in the oral cavity. More specifically, we engineered nanoparticles that contain collagenase in a deactivated form. Once placed at the surgical site, collagenase was released at a therapeutic concentration and activated by calcium, its biological cofactor that is naturally present in the tissue. Enhanced periodontal remodeling was recorded due to enzymatic cleavage of the supracrestal collagen fibers that connect the teeth to the underlying bone. When positioned in their new orientation, natural tissue repair mechanisms supported soft and hard tissue recovery and reduced tooth relapse. Through the combination of nanotechnology and proteolytic enzymes, localized surgical procedures can now be less invasive.

Dual growth factor delivery from degradable oligo(poly(ethylene glycol) fumarate) hydrogel scaffolds for cartilage tissue engineering.

This work describes the development of a non-invasive means of simultaneously delivering insulin-like growth factor-1 (IGF-1) and transforming growth factor-beta1 (TGF-beta1) to injured cartilage tissue in a controlled manner. This novel delivery technology employs the water-soluble polymer, oligo(poly(ethylene glycol) fumarate) (OPF), in the fabrication of biodegradable hydrogels which encapsulate gelatin microparticles. Release studies first examined the effect of gelatin isoelectric point (IEP) and crosslinking extent on IGF-1 release from these microparticles. In the presence of collagenase, highly crosslinked, acidic gelatin (IEP=5.0) provided sustained release of IGF-1, 95.2+/-2.9% cumulative release at day 28, while less crosslinked microparticles and microparticles of alternate IEP exhibited similar release values after only 6 days. Encapsulation of these highly crosslinked microparticles in a network of OPF provided a means to further control release, reducing final cumulative release to 70.2+/-4.7% in collagenase-containing PBS. Final release values from OPF-gelatin microparticle composites could be altered by incorporating less crosslinked, non-loaded microparticles within these constructs. Finally, this technology was extended to the dual delivery of IGF-1 and TGF-beta1 by loading these growth factors into either the OPF hydrogel phase or gelatin microparticle phase of composites. Release profiles were successfully manipulated by altering the phase of growth factor loading and microparticle crosslinking extent. For instance, by loading TGF-beta1 into the gelatin microparticle phase, a burst release of 10.8+/-0.7% was achieved, while loading this growth factor into the OPF hydrogel phase resulted in a burst release of 25.2+/-1.5%. With either system, simultaneous, slow release of IGF-1 over a 4-week period was accomplished by selectively loading this protein into highly crosslinked, encapsulated microparticles. These results demonstrate the utility of these systems in future studies to assess the interplay and time course of multiple growth factors in cartilage repair.

Transurethral enzyme injection--future management of benign prostatic hyperplasia.

Although benign prostatic hyperplasia, a common condition among elderly men, has been effectively treated with transurethral resection of the prostate, this surgical procedure is associated with many well-recognized risks and complications. Because of this potential morbidity and mortality, various alternative treatment strategies for benign prostatic hyperplasia have been proposed. The use of enzyme solubilization and ablation of prostatic tissue to alleviate urinary outlet obstruction has proved effective in dogs and warrants investigation in human trials. Transurethral enzyme injection of the prostate has the potential for being a treatment modality with minimal invasiveness, limited requirements for anesthesia, and minimal associated toxicity for the management of benign prostatic hyperplasia.

Transurethral enzymatic ablation of the prostate: canine model.

OBJECTIVES: The purpose of this study was to investigate the toxicity and potential usefulness of transurethral prostatic injection of a collagenase-based solution in treating benign prostatic hyperplasia in dogs. METHODS: The injected solution contained collagenase, hyaluronidase, Triton X-100, and gentamicin. Twenty-one dogs were randomly divided into three groups for transurethral prostatic needle injection: two treatment groups were observed for 6 and 12 weeks and a control group was observed for 12 weeks. Laboratory studies, clinical monitoring, and complete postmortem examination were performed in all animals. RESULTS: Gross hematuria was noted in all dogs for a mean of 4 days after injection. No significant postoperative morbidity was noted. There were no significant differences in the values of laboratory tests among the three groups except for a mean increase in serum level of aspartate transaminase for treatment groups on postoperative day 1; this resolved by postoperative day 7. Histologically, all treated prostates had stromal atrophy and cystic acinar dilation involving about 30% of the gland without extraprostatic extension of these changes. The urethra, bladder, rectum, testicles, kidney, liver, and lungs were normal and intact in all animals. CONCLUSIONS: Transurethral injection of this enzyme solution creates a predictable, favorable histologic response in the canine prostate. The procedure appears safe and warrants further investigation for treatment of human benign prostatic hyperplasia.

Enhanced adjuvantic property of polymerized liposome as compared to a phospholipid liposome.

Liposome, although intensively researched as vaccine or drug delivery vehicle, has been of limited use due to the low and unpredictable long-term stability. In order to overcome such problems, polymerized liposome (PL) that could initiate polymerization under very mild reaction condition was examined and compared to a conventional liposome. The polymerizable lipid, 1,2-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphorylcholine (DLL), was synthesized according to the literature, and 1,2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC) was used as the conventional lipid counterpart. Polymerization of liposome was as easy and convenient as just shaking in pH 7.4 buffer. The protein encapsulation efficiency of DLL was higher than that of DSPC, and its protein release rate was lower. Immunoglobulin G (IgG) activity examined after intraperitoneal injection of antigen encapsulated by either DLL or DSPC showed that ca. 2 times as much antibody was formed by DLL-encapsulated lysozyme compared with DSPC-encapsulated form. The reasons for the superior adjuvantic properties of DLL and its future application as a drug delivery system are briefly discussed.

Changes in urinary bone resorption markers (pyridinoline, deoxypyridinoline) resulting from experimentally-induced osteoarthritis in the temporomandibular joint of rats.

The purpose of this study was to quantify the urinary bone resorption markers, pyridinoline (Pyr) and deoxypyridinoline (Dpyr), excreted from experimentally-induced osteoarthritis (OA) in the temporomandibular joint (TMJ) of rats. Osteoarthritic lesions were induced by intra-articular injection of collagenase into the right TMJs of 16-week-old male rats. The whole day's urine was collected from each animal one day before the injection and 5, 7, 11 and 14 days after the injection. Urine samples were analyzed by high-perfomance liquid chromatography and fluorescence spectroscopy. Histological changes in condyle were examined by using paraffin sections with toluidine blue staining. Degenerative changes were observed in the articular cartilage of the experimental group on day 7 and day 14 after the injection of collagenase. The concentration of Pyr was remarkably high in the experimental group, and consequently the Pyr to Dpyr ratio was significantly higher (p<0.05) in the experimental group than in the control group from 7-14 days after the injection. These findings suggest that a urinary Pyr/Dpyr ratio would be available for the detection of degenerative changes in condyle relevant to temporomandibular joint osteoarthritis (TMJ OA).

Therapy for patients with burns - an integrating review

SUMMARY OBJECTIVE: to identify, through an integrative review, national studies published over the last ten years highlighting products and therapies used in burns. METHODS: integrative research with studies published in the last ten years. Including clinical studies describing the use of the already established or innovative therapies in burns and the results obtained, published in national journals in the last ten years. Excluding articles published before 2007 and those that did not present results regarding the use of products in burns. RESULTS: ten articles that met the inclusion criteria were selected. Collagenase, 1% silver sulfadiazine, and porous cellulose membrane were some of the therapies cited. CONCLUSION: the casuistry was low; however, the good results obtained with porous cellulose membrane and silver nanocrystalline dressing are highlighted, since they were used in a larger number of patients in the studies evaluated.

RESUMO OBJETIVO: Identificar, por meio de revisão integrativa, estudos nacionais publicados nos últimos dez anos que destaquem produtos e terapêuticas utilizados nas queimaduras. MÉTODOS: Pesquisa integrativa com estudos publicados nos últimos dez anos. Incluídos os estudos clínicos que descreveram a utilização de terapias já consagradas ou inovadoras em queimaduras e os resultados obtidos e publicados em periódicos nacionais nos últimos dez anos. Excluídos os artigos publicados antes de 2007 e os que não apresentaram resultados quanto ao uso de produtos nas queimaduras. RESULTADOS: Selecionados dez artigos que atenderam aos critérios de inclusão, sendo colagenase, sulfadiazina de prata 1% e membrana celulósica porosa algumas das terapias descritas. CONCLUSÕES: A casuística foi baixa, porém, ressaltam-se os bons resultados obtidos com a membrana celulósica porosa e o curativo com prata nanocristalina, em virtude de terem sido utilizados em um maior número de pacientes nos estudos avaliados.

Delivery of bioactive macromolecules from microporous polymer matrices: Release and activity profiles of lysozyme, collagenase and catalase.

Microporous polycaprolactone (PCL) matrices containing lysozyme, collagenase and catalase respectively with molecular weight covering a wide range from 14.3 to 240kDa were produced by a novel method involving rapid cooling of particle suspensions in dry ice. The enzyme loading efficiency (lysozyme (50%), collagenase (75%) and catalase (90%)) depended on the enzyme molecular weight and the non-solvent used to extract acetone from the hardened matrices. Sustained enzyme release occurred from the PCL matrices over 11 days with retained activity dependent on the particular enzyme used (collagenase 100% activity at 11 days, lysozyme 75-80% at 11 days, catalase 10-20% at 5 days). The present findings confirm the potential of microporous PCL matrices for delivering bioactive macromolecules from implantable/insertable depot-type formulations and tissue engineering scaffolds and recommend catalase as a challenging model protein for evaluating such devices.

Collagenase-aided intracortical microelectrode array insertion: effects on insertion force and recording performance.

Intracortical microelectrodes puncture the intact pia mater membrane during insertion, a process that can cause brain dimpling and trauma. To ensure that the device is able to withstand forces during implantation without buckling, the selection of acceptable implant materials and geometries is limited to rigid designs with large cross-sectional areas. Such designs likely increase insertion trauma and potentially exacerbate the chronic tissue response. In this paper, a technique that may relax the mechanical requirements of implanted microelectrodes through enzymatic (collagenase mediated) manipulation of the pia mater is quantified experimentally. Measurements of the insertion force profiles were obtained with a load cell during computer controlled (10 microm/s) insertion of microwire arrays into the cortex of rats. It was observed that collagenase application reduced the peak insertion force experienced by the microwire arrays by almost 40% on average (4.04 +/-2.03 mN versus 2.36 +/-1.17 mN; control versus treated sites). Peak insertion force magnitudes were highly dependent on implant location with anterior sites registering lower peaks than more posterior sites. Chronic neural recording performance (up to one month) did not appear to be adversely affected by the collagenase treatment, suggesting the overall safety of the technique. Our data suggest that controlled application of collagenase is a useful method in enabling implantation of thinner microelectrodes, potentially facilitating reduced insertion trauma and lower immune response. Furthermore, due to dependence of insertion force on anatomical location, the intended target region should be considered in implant design.

In vitro dentin pretreatment: surface roughness and adhesive shear bond strength.

The aim was to define the morphology and roughness of dentin from different tooth areas after various pretreatments to identify the effect of hybrid layer, resin tags, and mineralised dentin surface on shear bond strength. Thirty-eight extracted molars were used, each providing two sections of cervical (c) and lateral (l) dentin. Five pretreatments were performed: A) 0.2% EDTA; B) abrasion with Al2O3 particles, 0.2% EDTA; C) 10% H3PO4; D) 10% H3PO4 and immersion in a collagenase solution; E) control: no treatment. Z100 composite resin cylinders were bonded to the specimens with All Bond 2 bonding resin and tested for shear bond strength. Twelve other specimens from each group were analysed with an optical profilometer and an atomic force microscope, and four were further examined by scanning electron microscopy (SEM). Mean shear strength values in MPa were: Ac: 8.36 +/- 4.23; Al: 8.77 +/- 3.68; Bc: 6.05 +/- 3.62; Bl: 8.39 +/- 4.60; Cc: 6.87 +/- 3.45; Cl: 9.00 +/- 5.62; Dc: 13.30 +/- 5.45; Dl: 8.44 +/- 4.47; Ec: 4.10 +/- 1.54; El: 6.09 +/- 4.34. No statistically significant difference for cervical versus lateral dentin was found within treatments except for group D. Treatments performed on lateral dentin did not differ significantly. In cervical dentin, A differed from E; C from E; and D from A, B, C and E. An increased surface roughness was found in group D. Shear bond strength to dentin did not seem to depend on a hybrid layer formation, but on the direct contact of the adhesive with the mineralised dentinal surface and partly on the orientation of the dentinal tubules.