Collagenase Solution Research Articles

In Vitro Degradation of Collagen-Based Membranes for Guided Bone Regeneration After Zn-Ions or Doxycycline Functionalization

Collagen-based membrane is the most commonly used biomaterial for guided bone and tissue regeneration; however, its barrier function can be threatened by its rapid degradation pattern, affecting the success of the regeneration process. Differences in the origin and functionalization of the membrane to obtain better properties can alter the degradation rate. The objective of this study was to examine the biodegradation pattern of two commercially available collagen membranes (Jason® and Collprotect®) manufactured using porcine pericardium or dermis, doped or not with zinc-ions or doxycycline, in a period up to 21 days. The membrane specimens were subjected to hydrolytic and bacterial degradation tests. The different immersion times were carried out from 12 h up to 21 days. At each time point, quantitative measurements of thickness and weight were made using a digital caliper and an analytic microbalance, respectively. ANOVA and Student–Newman–Keuls tests were carried out for comparison purposes (p < 0.05). The differences between time-points within the same membranes and solutions were assessed by pairwise comparisons (p < 0.001). Unfunctionalized Jason membrane made of porcine pericardium attained the highest resistance to both degradation tests. The functionalization of the membranes did not alter the biodegradation patterns. All the membranes completely degraded before 48 h in the bacterial collagenase solution, which was the most aggressive test.

Performance of a multiphase bioactive socket plug with a barrier function for alveolar ridge preservation

The natural healing process of extraction socket and traditional socket plug material could not prevent buccal bone wall resorption and down growth of epithelium from the socket orifice. A multiphase bioactive socket plug (BP) is designed to overcome the natural healing process by maintaining the three-dimensional (3D) volume of extraction sockets, particularly in sockets with wall defects, and later provide sufficient alveolar bone volume for implant placement. The study aimed to fabricate and evaluate the physical, chemical, and biological performance of BP in vitro. The BP was fabricated through freeze-drying and layer-by-layer assembly, comprised of a base serving as a scaffold, a central portion for promoting bone regeneration, an upper buccal portion for maintaining alveolar socket dimension with a covering collagen membrane (Memb) on the top and upper buccal surface to prevent soft tissue infiltration. The BP as the experimental group and a pure collagen plug (CP) as the control group were investigated and compared. Radiograph, scanning electron microscopy, and energy-dispersive spectroscopy mapping confirmed that the four-part BP was successfully assembled and fabricated. Swelling rate analysis indicated that BP, CP, and Memb reached swelling equilibrium within 1 hour. BP exhibited a high remaining weight percentage in collagenase solution (68.81 ± 2.21% on day 90) and sustained calcium ion release, reaching the maximum 0.13 ± 0.04 mmol l−1 on day 14. In biological assays, BP exhibited excellent cell proliferation (The OD value increased from 0.02 on day 1 to 0.23 on day 21.). The BP group exhibited higher alkaline phosphatase activity and osteocalcin content than the CP group within 21 days. Memb and BP exhibited outstanding barrier function, as evidenced by Hematoxylin and eosin staining. In summary, the multiphase bioactive socket plug represents a promising scaffold for alveolar ridge preservation application.

Evaluation of Monomer-containing Isocyanate Groups on Stabilizing Demineralized Dentin Matrix Against Bond Interface Degradation.

To evaluate the effect of urethane methacrylate precursor (UMP) on the enzymatic resistance of demineralized dentin (DD) matrices. Experimental treatments containing 0 (control), 1, and 5 mmol/L UMP dissolved in an acetone (Ace) solution were formulated. Dentin matrix specimens were demineralized in vitro and immersed in the experimental treatments for 1 h. The treated specimens were then stored in 0.1 mg/mL collagenase solution for 24 h, after which their dry mass loss and hydroxyproline (HYP) release were assessed. The swelling ratios of specimens in each group were also evaluated. The interaction between UMP and the dentin matrix was observed using field-emission scanning electron microscopy (FE-SEM). Endogenous enzyme activity in dentin was evaluated using confocal laser scanning microscopy (CLSM). Compared with the other treatment groups, treatment with 1 mM and 5 mM UMP-Ace significantly decreased the dry mass loss, HYP release and swelling ratio of the DD matrix (p < 0.05). FE-SEM and CLSM observations showed that treatment with UMP-Ace protected the structure of the dentin matrix and decreased porosity within the dentin-collagen network. Treatment with 1 mM and 5 mM UMP-Ace protects DD matrix against collagenase degradation and may be clinically useful for improving the durability of the hybrid layer.

Spontaneous and LPS-induced cytokine production in supernatants of cell cultures isolated from a skin biopsy in a mouse model of acute psoriasis-like dermatitis

The purpose of the work performed was to obtain data on spontaneous and LPS-stimulated cytokine production in cell culture supernatants isolated from isolated from skin bioptates of C57BL/6 mice in an imiquimod-induced psoriasis model. Methods. Skin cells were isolated by spontaneous migration and enzymatic dissociation in a collagenase solution. Cells isolated from the skin were cultured for 24 hours in RPMI-1640 (spontaneous cytokine production) or RPMI-1640 medium with the addition of a solution (10 micrograms/ml) of E. coli LPS (induced). Cytokine levels were determined by flow laser cytofluorometry using the Mouse Th1/Th2/Th17 multiplex test system (Antigenix America, USA) on an FC-500 cytometer. Results. In the focus of imiquimod-induced inflammation, an increase in the spontaneous production of proinflammatory cytokines IL-1, IL-5, IL-17 was observed by 2.4-3.8 times. At the same time, a 1.9-fold inhibition of IFNy synthesis was detected when cultivating skin cells in the presence of bacterial LPS.

Fabrication and characterization of a bioactive composite scaffold based on polymeric collagen/gelatin/nano β-TCP for alveolar bone regeneration

One strategy to correct alveolar bone defects is use of bioactive bone substitutes to maintain the structure of defect site and facilitate cells and vessels' ingrowth. This study aimed to fabricate and characterize the freeze-dried bone regeneration scaffolds composed of polymeric Type I collagen, nano Beta-tricalcium phosphate (β-TCP), and gelatin. The stable structures of scaffolds were obtained by thermal crosslinking and EDC/NHS ((1-ethyl-3-(3-dimethylaminopropyl) carbodiimide)/(N-hydroxysuccinimide)) chemical crosslinking processes. Subsequently, the physicochemical and biological properties of the scaffolds were characterized and assessed. The results indicated the bioactive composite scaffolds containing 10% and 20% (w/v) nano β-TCP exhibited suitable porosity (84.45 ± 25.43 nm, and 94.51 ± 14.69 nm respectively), a rapid swelling property (reaching the maximum swelling rate at 1 h), excellent degradation resistance (residual mass percentage of scaffolds higher than 80% on day 90 in PBS and Type I collagenase solution respectively), and sustained calcium release capabilities. Moreover, they displayed outstanding biological properties, including superior cell viability, cell adhesion, and cell proliferation. Additionally, the scaffolds containing 10% and 20% (w/v) nano β-TCP could promote the osteogenic differentiation of MC3T3-E1. Therefore, the bioactive composite scaffolds containing 10% and 20% (w/v) nano β-TCP could be further studied for being used to treat alveolar bone defects in vivo.

Epigenetic modification of histone acetylation in the sensorimotor cortex after intracerebral hemorrhage.

Epigenetic regulation is involved in post-stroke neuroplasticity. We investigated the effects of intracerebral hemorrhage (ICH) on histone acetylation and gene expression related to neuronal plasticity in the bilateral sensorimotor cortices, which may affect post-stroke sensorimotor function. Wistar rats were randomly divided into the SHAM and ICH groups. We performed ICH surgery stereotaxically based on the microinjection of a collagenase solution in the ICH group. Foot fault and cylinder tests were performed to evaluate motor functions at 4-time points, including pre-ICH surgery. The amount of acetyl histones and the mRNA expression of neurotrophic factors crucial to neuroplasticity in the bilateral sensorimotor cortices were analyzed approximately 2 weeks after ICH surgery. Sensorimotor functions of the ICH group were inferior to those of the SHAM group during 2 weeks post-ICH. ICH increased the acetylation of histone H3 and H4 over the sham level in the ipsilateral and contralateral cortices. ICH increased the mRNA expression of IGF-1, but decreased the expression of BDNF compared with the sham level in the ipsilateral cortex. The present study suggests that histone acetylation levels are enhanced in bilateral sensorimotor cortices after ICH, presenting an altered epigenetic platform for gene expressions related to neuronal plasticity.

Functional results of surgical treatment of macular holes fully preserving the internal limiting membrane as compared with the traditional method

Purpose: to assess the functional results of surgical treatment of macular holes (MH) using the authors’ developed technology preserving the internal limiting membrane (ILM) and compare them with the results of traditional surgery technology involving ILM peeling.Material and methods: 150 MH patients aged 50 to 82 were divided into 2 groups: the main group who underwent surgical MH treatment with ILM preservation and the control group, whose traditional surgical treatment involved ILM peeling. After vitrectomy, the main group patients’ macular areas were treated with a bacterial collagenase solution (1 kU) for three minutes, whereupon it was washed out, the vitreal cavity was dried and treated with platelet-rich plasma. In the control group, ILM was stained and removed, whereupon platelet-rich plasma was applied in the air medium. The anatomical result was assessed by optical coherence tomography data. To assess the functional results, the patients were tested for the best corrected visual acuity (BCVA) and subjected to microperimetry. The observation period was 24 months.Results: in all cases, a significant increase in BCVA was achieved after surgical treatment. After 6 months, the control group showed a tendency to a BCVA decrease, while in the main group BCVA remained stable. In the main group, not a single case showed a decrease in the total retinal photosensitivity below the threshold level of the norm of 24 dB, whilst in the control group, 35 cases (47 %) revealed a retinal sensitivity decrease below 24 dB. The total retinal sensitivity turned out to be significantly lower in the control group as compared to the main group over the entire observation period (p &lt; 0.05). Postoperative scotomas were significantly more frequent after ILM peeling. Absolute scotomas were detected in 7 control group cases, whilst in the main group they were not noted in any case.Conclusion. The developed technology makes it possible to practically eliminate the formation of absolute scotomas and to significantly reduce the formation of relative scotomas in the postoperative period, as well as improve functional results against the traditional method of surgical treatment of MH with ILM peeling.

Dentin remineralization using a stimuli-responsive engineered small molecule GSK3 antagonists-functionalized adhesive

ObjectivesTideglusib has shown great performance in terms of dentin regenerative properties. This study aims to evaluate bonding ability, of demineralized dentin infiltrated with polymeric nanoparticles (NPs) doped with tideglusib (TG) (TG-NPs). MethodsDentin conditioned surfaces were infiltrated with NPs and TG-NPs. Bonded interfaces were created and stored for 24 h and then submitted to mechanical, chemical and thermal challenging. The resin-dentin interface was evaluated through a doubled dye fluorescent technique and a calcium chelator fluorophore under a confocal laser scanning microscopy, and by field emission scanning electron microscopy. ResultsDentin surfaces treated with TG-NPs and load cycled produced higher bond strength than the rest of the groups. Immersion of dentin specimens treated with undoped-NPs in collagenase solution attained the lowest microtensile bond strength (MTBS) values. Both porosity and nanoleakage decreased when dentin was infiltrated with TG-NPs, that revealed strong signals of xylenol orange stain at both hybrid layer and dentinal tubules. The presence of NPs, in general, inducted the presence of mineralized interfaces after mechanical loading and thermocycling. ConclusionsNanoparticles doped with tideglusib promoted the highest dentin bonding efficacy among groups, as they facilitated the maximum bond strength values with creation of mineral deposits at the hybrid layer and dentinal walls. Tideglusib enabled scarce porosity, nanoleakage and advanced sealing among dentin groups. SignificanceDoping hydrophilic polymeric NPs with tideglusib, infiltrated in etched dentin represents a reproducible technique to create reparative dentin at the resin-dentin interface, by inducing therapeutic bioactivity.

Repairable ex vivo model of functional and degenerative mitral regurgitation.

Transcatheter mitral valve repair is an emerging alternative to the surgical repair. This technology requires preclinical studies to assess efficacy in mitigating mitral regurgitation (MR). However, ex vivo MR models are not established. We developed 2 novel repairable models, functional and degenerative, which can quantitatively assess regurgitation and effect of intervention. We used porcine mitral valves and a pulsatile flow circulation system. In the functional MR model, the annulus was immersed in 0.1% collagenase solution and dilated using 3D-printed dilators. To control the regurgitation grade, the sizes of the dilator and silicone sheet in which the valve was sutured to were adjusted. Chordae of P2 were severed in the degenerative model, and the number of severed chordae was adjusted to control the regurgitation grade. Models were repaired using the edge-to-edge or artificial chordae technique. The mean regurgitant fraction of the moderate-severe functional and degenerative models were 47.9% [standard deviation (SD): 2.2%] and 58.5% (SD: 8.0%), which were significantly reduced to 28.7% (SD: 4.4%) (P < 0.001) and 26.0% (SD: 4.4%) (P < 0.001) after the valve repair procedures. Severe functional model had a mean regurgitant fraction of 59.4% (SD: 6.0%). Both functional and degenerative models could produce sufficient MR levels that meet the interventional indication criteria. The repairable models are valuable in evaluating the efficacy of valve repair procedures and devices. The ability to control the amount of regurgitation enhances the versatility and reliability of these models. These reproducible models could expedite the development of novel devices.

The Use of Stromal Vascular Fraction in Long Bone Defect Healing in Sheep.

The objectives of the present study were to evaluate (a) the feasibility of using stromal vascular fraction (SVF) and nanocrystalline hydroxyapatite (nHA) paste in combination for the treatment of segmental bone defect, (b) the quality of the callus produced, (c) the potential improvement of the autograft technique, and (d) the direct comparison of the biomaterial to the use of autogenous cancellous bone. Unilateral, segmental mid-diaphyseal bone defect was created on the right metatarsus of skeletally mature sheep animals (n = 24) under anesthesia (D0). Residual segments were stabilized by stainless-steel plates and appropriate screws. Defects were managed as follows: group A: use of nHA paste to filling, group B: use of autogenous bone graft mixed with nHA bone paste, placed in defect, group C: use of SVF mixed with nHA bone paste injected into defect, group D: use of bone graft and SVF with nHA paste before apposition in bone defect. SVF had been previously isolated from adipose tissue of the animals intra-operatively after digestion with collagenase solution and neutralization. Animals were evaluated clinically and by X-raying and ultrasonographic examination of the defect, at regular intervals, until D90. Ultrasonographic assessment performed along the length of the defect included calculation of the length of the bone defect and assessment of vascularization. SVF was successfully isolated from group C and D animals, with the average yield being 1.77 × 106 cells. The comparison of clinical scores (based on the 'Kaler scale') on each post-operative day indicated significant differences between the four groups on D1 to D30 (p < 0.01); the median clinical score within group A was 2.5 for D1-D30 and 1 for the entire period; respective scores for other groups were 1.5 (p = 0.07) and 0 (p = 0.033). Differences in radiographic assessment scores were significant for scores obtained on D60 (p = 0.049) and D90 (p = 0.006). There was a significant difference between the four groups in the length of the bone defect, as assessed ultrasonographically, for the entire length of the study; median values were 8, 8.5, 6, and 8 mm for groups A, B, C, and D, respectively (p = 0.008). There was a significance in the differences between median scores obtained during the histopathological examination: 2, 11, 13.5, and 12 for group A, B, C, and D (p = 0.022). There was an inverse correlation between the overall scores of histopathological evaluations and the length of the bone defect (observed on D90) (p < 0.0001) and a correlation between the overall scores and the radiographic assessment scores (obtained on D90) (p < 0.0001). This is the first study in which the efficacy of fresh autologous Stromal Vascular Fraction (SVF) from adipose tissue in enhancing bone healing in a long, weight-bearing, diaphyseal bone was evaluated. It is concluded that the lumbosacral region was an attractive site for harvesting adipose tissue, the use of SVF contributed to faster rehabilitation post-operatively, and SVF significantly enhanced bone formation; in general, the results indicated an osteogenic potential of SVF comparable to the gold standard autologous bone graft.

Pharmacological inhibition of histone deacetylases ameliorates cognitive impairment after intracerebral hemorrhage with epigenetic alteration in the hippocampus

ObjectivesPost-stroke cognitive impairment (PSCI) interferes with neurorehabilitation in patients with stroke. Epigenetic regulation of the hippocampus has been targeted to ameliorate cognitive function. In particular, the acetylation level of histones is modulated by exercise, a potent therapy for patients with stroke. Materials and MethodsWe examined the effects of exercise and pharmacological inhibition of histone deacetylase (HDAC) using sodium butyrate (NaB) on cognitive function and epigenetic factors in the hippocampus after intracerebral hemorrhage (ICH) to seek beneficial neuronal conditioning against PSCI. Forty rats were randomly assigned to five groups: sham, control, NaB, exercise, and NaB plus exercise groups (n=8 in each group). Except for those in the sham group, all rats underwent stereotaxic ICH surgery with a microinjection of collagenase solution. Intraperitoneal administration of NaB (300 mg/kg) and treadmill exercise (11 m/min for 30 min) were conducted for approximately 4 weeks starting 3 days post-surgery. ResultsICH reduced cognitive function, as detected by the object location test, accompanied by enhanced activity of HDACs. Although exercise did not modulate HDAC activity or cognitive function, repetitive NaB administration increased HDAC activity and ameliorated cognitive impairment induced by ICH. ConclusionsThis study suggests that pharmacological treatment with an HDAC inhibitor could potentially present an enriched epigenetic platform in the hippocampus and ameliorate PSCI for neurorehabilitation following ICH.

2D Collagen Membranes from Marine Demosponge Chondrosia reniformis (Nardo, 1847) for Skin-Regenerative Medicine Applications: An In Vitro Evaluation.

Research in tissue engineering and regenerative medicine has an ever-increasing need for innovative biomaterials suitable for the production of wound-dressing devices and artificial skin-like substitutes. Marine collagen is one of the most promising biomaterials for the production of such devices. In this study, for the first time, 2D collagen membranes (2D-CMs) created from the extracellular matrix extract of the marine demosponge Chondrosia reniformis have been evaluated in vitro as possible tools for wound healing. Fibrillar collagen was extracted from a pool of fresh animals and used for the creation of 2D-CMs, in which permeability to water, proteins, and bacteria, and cellular response in the L929 fibroblast cell line were evaluated. The biodegradability of the 2D-CMs was also assessed by following their degradation in PBS and collagenase solutions for up to 21 days. Results showed that C. reniformis-derived membranes avoided liquid and protein loss in the regeneration region and also functioned as a strong barrier against bacteria infiltration into a wound. Gene expression analyses on fibroblasts stated that their interaction with 2D-CMs is able to improve fibronectin production without interfering with the regular extracellular matrix remodeling processes. These findings, combined with the high extraction yield of fibrillar collagen obtained from C. reniformis with a solvent-free approach, underline how important further studies on the aquaculture of this sponge could be for the sustainable production and biotechnological exploitation of this potentially promising and peculiar biopolymer of marine origin.

Construction of enzyme digested holes on hydrogel surface inspired by cell migration processes

The construction of in vitro capillary network models for drug testing and toxicity evaluation has become a major challenge in the field of tissue engineering. Previously, we discovered a novel phenomenon of hole formation by endothelial cell migration on the surface of fibrin gels. Interestingly, the hole characteristics, such as depth and number, were strongly influenced by the gel stiffness, but the details of hole formation are not to be clarified.In this study, we tried to understand the effect of hydrogel stiffness on the hole formation by dropping collagenase solution onto the surface of the hydrogels because the endothelial cell migration was made possible by the metalloproteinases' digestion. We found that smaller hole structures were formed on stiffer fibrin gels, but larger ones were formed on softer fibrin gels after the hydrogel digestion of the collagenase. This is consistent with our previous results in experiments on hole structures formed by endothelial cells. Furthermore, deep and small hole structures were successfully obtained by optimizing the volume of collagenase solution and incubation time. This unique approach inspired by endothelial cell hole formation may provide new methods of fabricating hydrogels with opening hole structures.

Collagenase Solution for Isolation of Oocytes

Collagenase Solution for Isolation of Oocytes

Effect of Collagen and GelMA on Preservation of the Costal Chondrocytes' Phenotype in a Scaffold in vivo.

Chondrocytes were isolated from the costal cartilage of newborn rats using 0.15% collagenase solution in DMEM. The cells was characterized by glycosaminoglycan staining with alcian blue. Chondrocyte scaffolds were obtained from 4% type I porcine atelocollagen and 10% GelMA by micromolding and then implanted subcutaneously into the withers of two groups of Wistar rats. Histological and immunohistochemical studies were performed on days 12 and 26 after implantation. Tissue samples were stained with hematoxylin and eosin, alcian blue; type I and type II collagens were identified by the corresponding antibodies. The implanted scaffolds induced a moderate inflammatory response in both groups when implanted in animals. By day 26 after implantation, both collagen and GelMA had almost completely resorbed. Cartilage tissue formation was observed in both animal groups. The newly formed tissue was stained intensively with alcian blue, and the cells were positive for both types of collagen. Cartilage tissue was formed among muscle fibers. The ability of collagen type I and GelMA hydrogels to form hyaline cartilage in animals after subcutaneous implantation of scaffolds was studied. Both collagen and GelMA contributed to formation of hyaline-like cartilage tissue type in animals, but the chondrocyte phenotype is characterized as mixed. Additional detailed studies of possible mechanisms of chondrogenesis under the influence of each of the hydrogels are needed.

Human umbilical-cord-derived mesenchymal stem cells in combination with rapamycin reduce cartilage degradation via inhibition of the AKT/mTOR signaling pathway

Background and aims Mesenchymal stem cell (MSC) therapy is a promising strategy for treating osteoarthritis (OA). However, the inflammatory microenvironment, apoptosis of transplanted cells, and shear forces during direct injection limit the therapeutic efficacy. This study aimed to explore the role of rapamycin combined with human umbilical-cord-derived mesenchymal stem cells (hUMSCs) in OA rabbits in vivo. Methods OA rabbits received an intra-articular injection of a collagenase solution. Gross observations, X-ray examinations, and histological examinations were performed to detect cartilage degradation levels. The fluorescent membrane dye DiR was used to label hUMSCs. In the combination therapy group, rapamycin was injected into the rabbit knee joint one day post the intra-articular injection of hUMSCs. Bioinformatics and transcriptome profiling of the knee meniscus were used to evaluate the potential molecular mechanisms of the combination therapy. Results Our study shows that rapamycin combined with hUMSCs significantly ameliorated OA severity in vivo, enhancing matrix synthesis and promoting cartilage repair. The combination therapy was more efficient than rapamycin or hUMSC treatment alone. Moreover, bioinformatics and transcriptomic analyses revealed that combination therapy might enhance autophagy in chondrocytes, partially by inhibiting the mTOR pathway. Conclusions Our study indicates that the combination therapy of rapamycin and hUMSCs may promote cartilage repair in OA rabbits through the mTOR pathway and offers a novel approach for OA therapy. The translational potential of this article Our study provides new evidence to support the use of hUMSCs in combination with rapamycin as a potential candidate for OA treatment.

Comparison of motor function recovery and brain changes in intracerebral hemorrhagic and ischemic rats with similar brain damage.

In this study, we compared the mechanisms of brain recovery in intracerebral hemorrhage and ischemia, focusing on synapses, glial cells, and dopamine expression, which are considered fundamental for neural recovery after stroke. Male Wistar rats were divided into intracerebral hemorrhage, ischemia, and sham surgery (SHAM) groups. The intracerebral hemorrhage group was injected with a collagenase solution, the ischemia group was injected with an endothelin-1 solution, and the SHAM group was injected with physiological saline. The motor function of these rats was evaluated using a rotarod test on days 7, 14, 21, and 28 post-surgery. On postoperative day 29, lesion volume was analyzed using Nissl staining. In addition, the protein expression levels of NeuN, GFAP, tyrosine hydroxylase, and PSD95 were analyzed in the striatum and motor cortex. There was no significant difference between the ischemia and intracerebral hemorrhage groups in terms of lesion volume in the striatum; however, the motor recovery of the intracerebral hemorrhage group occurred more rapidly than that of the ischemia group, and the intracerebral hemorrhage group exhibited higher GFAP protein expression in the motor cortex. The rapid motor recovery in intracerebral hemorrhage rats relative to that in ischemia rats may be associated with changes in astrocytes in brain regions remote from the injury site.

Polymer – flufenamic acid delivery systems for injured skin

Collagen, the main protein of the body, is extracted in different forms and used as reservoir for drug delivery. The aim of this work was to obtain a drug delivery system based on collagen-dextran matrices cross-linked with glutaraldehyde as support and flufenamic acid and/or microcapsules with flufenamic acid as drug. The flufenamic acid was encapsulated in polymeric microcapsules consisting in gelatin, alginate, and sodium carboxymethyl cellulose. The morphology of matrices was determined by water absorption and contact angle. The biodegradation was performed in collagenase solution. In vitroflufenamic acid release profiles were built and the kinetic mechanism was set according to different mathematical models. The pharmacological studies followed the effect of collagen formulations treatment on the healing process of Wistar rats which were induced experimental wounds. The studied matrices proved that flufenamic acid delivery can be controlled, and the healing can be completed using the designed spongious matrices.

Effectiveness of experimental adhesive systems containing carbodiimide and solvated in different solvents

ObjectivesTo evaluate the influence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide – EDC (C) on the degree of conversion (DC%), resin-dentin bonding stability, collagen degradation, and MMP inactivation produced by experimental adhesive systems solvated in acetone (A), ethanol (E) or tetrahydrofuran (T). MethodsEDC was added to experimental primers consisting of HEMA, PMGDM, and water, solvated in A, E, or T, performing six groups (A, AC, E, EC, T, TC). The adhesive consisted of BISGMA, HEMA, camphorquinone, and EDMAB. DC% was evaluated by FT-IR spectroscopy. The μTBS was evaluated after 24 h or 1 yr of artificial saliva storage at 37°C. The degradation of the dentin collagen was evaluated using FT-IR spectroscopy at three periods: after primer application, 15, and 30 days, and the enzymatic activity within the hybrid layer using in situ zymography. Data was analyzed using ANOVA and Tukey's HSD test (α = 0.05). ResultsNo statistical significance in DC% was found among all adhesive systems (p > 0.05). EDC-containing primers maintained μTBS stability after 1 yr of artificial saliva storage (p > 0.05). The intensity of the Amide I and II bands from the dentin collagen was preserved for AC and EC after immersion in collagenase solution. The dentin treated with EDC-containing primers presented reduced fluorescein in the hybrid layer. ConclusionsThe incorporation of EDC into primers contributed to resin-dentin bonding stability and reduced the enzymatic activity and collagen degradation.

Isolation of Hepatocytes from Liver Tissue by a Novel, Semi-Automated Perfusion Technology.

Primary hepatocytes are a major tool in biomedical research. However, obtaining high yields of variable hepatocytes is technically challenging. Most protocols rely on the two-step collagenase perfusion protocol introduced by Per Ottar Seglen in 1976. In this procedure, the liver is perfused in situ with a recirculating, constant volume of calcium-free buffer, which is maintained at 37 °C and continuously oxygenated. In a second step, the liver is removed from the carcass and perfused with a collagenase solution in order to dissociate the extracellular matrix of the liver and liberate individual cells. Finally, the dissected hepatocytes are further purified and concentrated by density-based centrifugation. However, failure in proper cannulation, incomplete enzymatic digestion or over-digestion can result in low cell yield and viability. Here we present a novel semi-automated perfusion device, which allows gentle, rapid and efficient generation of a single-cell suspension from rodent livers. In combination with prefabricated buffers, the system allows reliable and highly reproducible isolation of primary hepatocytes.