Thermal and spectrophotometric studies of new crosslinking method for collagen matrix with glutaraldehyde acetals.

Despite the many existing crosslinking procedures, glutaraldehyde (GA) is still the method of choice used in the manufacture of bioprosthesis. The major problems with GA are: (a) uncontrolled reactivity due to the chemical complexity or GA solutions; (b) toxicity due to the release of GA from polymeric crosslinks; and (c) tissue impermeabilization due to polymeric and heterogeneous crosslinks formation, partially responsible for the undesirable calcification of the bioprosthesis. A new method of crosslinking glutaraldehyde acetals has been developed with GA in acid ethanolic solution, and after the distribution inside de matrix, GA is released to crosslinking. Concentrations of hydrochloride acid in ethanolic solutions between 0.1 and 0.001 mol/L with GA concentration between 0.1 and 1.0% were measured in an ultraviolet spectrophotometer to verify the presence of free aldehyde groups and polymeric compounds of GA. After these measurements, the solutions were used to crosslink bovine pericardium. The spectrophotometric results showed that GA was better protected in acetal forms for acid ethanolic solution with HCl at 0.003 mol/L and GA 1.0%(v/v). The shrinkage temperature results of bovine pericardium crosslinked with acetal solutions showed values near 85 degrees C after the exposure to triethylamine vapors.

Cell adhesion and cytotoxicity studies over polyanionic collagen surfaces with variable negative charge and wettability.

This work describes the cytotoxicity, and the cell adhesion behavior of K562 cell line from human erythroleukemia transfected with the DNA for the alpha(2)beta(1) integrin over type-I collagen matrices with variable degree of carboxyl group and wettability. The results showed that type-I collagen materials with variable degree of carboxyl group prepared by selective hydrolysis of carboxyamide side chains of Asn and Gln residues present in the protein, independently from the extent of side chain hydrolysis, was characterized by preserved triple helix structure for materials with a carboxyl group content up to 87 +/- 17. Imbibition and wettability increased linearly with increasing carboxyl group content from 46 +/- 12 to 87 +/- 17, and no signs of cytotoxicity were detected. Nevertheless, in comparison to native collagen, K562 cell adhesion to PACMs was significantly improved by factors ranging from 1.60 to 1.47x, with the reduction in cell adhesion observed with increasing carboxyl content attributed to a balance between the inhibition of increasing negative charge and the stimulation by increased wettability. On the other hand, the overall improvement of K562 cell adhesion to polyanionic collagen was attributed to the introduction of new distinct motifs described as the minimal active recognition sequence for alpha(2)beta(1) integrins binding with type-I collagen produced as a result of Asn-Gly Glu-Ala alpha2(I)294-297, and Gly Gln-Arg-Gly Val-Val carboxyamide side chains hydrolysis.

Subgingival utilization of a 1% chlorhexidine collagen gel for the treatment of periodontal pockets. A clinical and microbiological study.

This study evaluates the effect of subgingival irrigation with a 1% chlorhexidine collagen gel in periodontal pockets as an adjunct procedure to scaling and root planing (SRP). Thirty-seven sites with probing depth (PD) of 5-7 mm and BANA positive in 6 patients with chronic periodontal disease were selected. Sites were assigned to different treatment groups consisting of SRP only (group 1), SRP + irrigation with collagen gel (group 2), or SRP + irrigation with collagen gel containing 1% chlorhexidine (group 3). Subgingival irrigation was performed after initial SRP and at 7, 14 and 21 days. Clinical measurements including PD, plaque index (PI), gingival index (GI), gingival recession (GI), bleeding on probing (BOP) and clinical attachment level (CAL) were performed at the selected sites at baseline, 60 and 90 days and the BANA test was performed on plaque samples from the same sites at baseline and 90 days. There was an improvement in clinical parameters in all groups with a significantly greater decrease in GI and bleeding in the chlorhexidine group. There was a greater reduction of BANA positive sites in groups 2 and 3. The authors concluded that 1% chlorhexidine collagen gel is a promising adjunct to SRP in the treatment of adult periodontitis.

Hydroxylapatite implants with or without collagen in the zygomatic arch of rats. Histological study.

The authors studied the behavior of calcium phosphate materials used as inlay implants into bone cavities prepared in the zygomatic arch of rats. Fifty male albino rats were divided into four groups as follows: group I-preparation of bone cavities which did not receive any implant material as controls; group II-implants of Interpore 200; group III-implants of experimental hydroxylapatite; group IV-implants of experimental hydroxylapatite combined with collagen. The animals were sacrificed after 5, 15, 30, 60 and 120 days and the specimens were submitted to histological analysis. Results showed that the experimental hydroxylapatite used in group III presented better osteogenic properties compared to the other materials. All tested materials were biocompatible, although group IV presented a more intense inflammatory response.

Characterization of polyanionic collagen prepared by selective hydrolysis of asparagine and glutamine carboxyamide side chains.

Acellular polyanionic collagen materials intended for biomaterial and tissue engineering uses were prepared by the selective and controlled hydrolysis of carboxyamides from asparagine and glutamine residues of type I collagen present in pericardium, tendon, and intestinal submucosa, all from bovine origin. The increase in carboxyl groups was from 26 +/- 14 (12 h of hydrolysis) to 134 +/- 12 (144 h of hydrolysis). Although collagen triple helix structure of polyanionic materials was preserved in all cases, a decrease in thermal stability and a gradual loss in the ability of collagen molecules to form fibrils were detected with increasing carboxyl content, probably as a result of changes in the pattern of electrostatic interaction. The resulting materials were basically acellular polyanionic collagen matrixes associated with an elastin content dependent on the time of hydrolysis. The results showed that the procedure described in this work may be a useful process for preparation of collagen biomaterials with variable physicochemical properties and macromolecular arrangement with respect to fibril formation and with potential use in tissue engineering.

Treatment of gingival recession: comparative study between subepithelial connective tissue graft and guided tissue regeneration.

BACKGROUND: Various procedures have been proposed to treat gingival recession, but few studies compare these procedures to each other. The purpose of this study was to evaluate a clinical comparison of subepithelial connective tissue graft (SCTG) and guided tissue regeneration (GTR) with a collagen membrane in the treatment of gingival recessions in humans. METHODS: Twenty-four defects were treated in 12 patients who presented canine or pre-molar Miller Class I and/or II bilateral gingival recessions. Both treatments were performed in all patients, and clinical measurements were obtained at baseline and 18 months after surgery. These clinical measurements included gingival recession height (GR), root coverage (RC), probing depth (PD), keratinized tissue width (KT), and final esthetic result. RESULTS: Both SCTG and GTR with a bioabsorbable membrane and bone graft demonstrated significant clinical and esthetic improvement for gingival recession coverage. The SCTG group was statistically significantly better than GTR for height of GR (SCTG = 0.2 mm, GTR = 1.12 mm, P= 0.02) and KT (SCTG = 4.58 mm, GTR = 2.5 mm, P<0.0001). However, PD was statistically significantly better for GTR than SCTG treatment (GTR = 1.66 mm, SCTG = 1.00, P= 0.01). The 2 procedures were statistically similar in root coverage (SCTG = 95.6%, GTR = 84.2%, P= 0.073). The esthetic condition after both treatments was satisfactory (P= 0.024). CONCLUSIONS: It was concluded that the gingival recessions treated with the SCTG group were superior for GR, RC, and KT clinical parameters, while GTR demonstrated better PD reduction. The final esthetic results were similar using both techniques.

Preparation and characterization of collagen-elastin matrices from blood vessels intended as small diameter vascular grafts.

This study describes the selective removal of cell and cell residues from small and large diameter blood vessels for the preparation of tubular collagen:elastin matrices intended for small diameter vascular prosthesis. The results showed that total or partially devitalized collagen:elastin matrices may be conveniently prepared without denaturation of the collagen:elastin matrix with a high degree of preservation of the proteins. The efficiency of cell removal and the extent collagen and elastic fiber preservation were dependent on the segment and the type of blood vessel under study, with arteries characterized by a higher susceptibility of cell removal and better preservation of the collagen-elastin matrix.

Nonstoichiometric hydroxyapatite-anionic collagen composite as support for the double sustained release of gentamicin and norfloxacin/ciprofloxacin.

This work studied the sustained release of ciprofloxacin or norfloxacin and gentamicin from nonstoichiometric hydroxyapatite (nHA) and anionic collagen composite. Within the first 24 and 48 h, the total antibiotic supply was significantly higher than the minimal inhibitory concentration required for the majority of the gram-negative bacteria. Although gentamicin was completely released from the matrix after 48 h by a normal diffusion mechanism, ciprofloxacin or norfloxacin release was characterized by a 2-phase release mechanism due to binding to nHA by complexation with calcium ion. Under the conditions studied, most of the norfloxacin or ciprofloxacin only will be disposable due to bioresorption or dissociation of the complexes. In conclusion, due to its biocompatibility nHA-anionic collagen composite may be a convenient support for the double sustained release of the antibiotics gentamicin and ciprofloxacin/norfloxacin for the control of bone infection while promoting bone tissue growth.

Biocompatibility studies of anionic collagen membranes with different degree of glutaraldehyde cross-linking.

The work describes the biocompatibility and biodegradation studies of anionic collagen membranes casted form collagen gels collagen, that were selective hydrolyzed at the carboxyamide groups, as a function of the degree of cross-links induced by glutaraldehyde. Independently from the degree of cross-links, all membranes studied were characterized by a similar inflammatory response, inversely dependent on glutaraldehyde reaction time, that decreased from the time of the implant. Cell alterations, mineralization or contact necrosis were not observed in any of the membranes studied. Rates for membrane tissue biodegradation were directly related to glutaraldehyde reaction time, and ranged from 30 to periods longer than 60 days, associated with good biocompatibility. Although other properties must be considered, their use in the treatment of periodontal diseases, the biological behavior observed with the 8 h GA cross-linked membrane suggests that, anionic collagen membrane described in this work may be of potential use, not only in association with guided tissue regeneration technique for periodontal tissue reconstruction, but also in other collagen biomaterial applications where controlled biodegradability is required.

Anionic collagen: polymer composites with improved dielectric and rheological properties.

This work describes the preparation and characterization of anionic collagen composites with rhamsan and vinylidene fluoride-trifluorethylene with improved rheological and dielectric properties without loss of collagen secondary structure with an interaction occurring between both macromolecules of the composites. On a comparative basis, the force needed for the extrusion of anionic collagen:rhamsan composites was in the range from 0.088 to 0.080 J compared to that for collagen of 0.189 J. Anionic collagen:vinylidene fluoride-trifluorethylene composites were characterized, in the case of the 1:1 composite, by a pyroelectric coefficient of 1.89 x 10(-4) cm(-2) K(-1), which was significantly higher than those determined under the same conditions for native anionic collagen and vinylidene fluoride-trifluorethylene.

The chemical protecting group concept applied in crosslinking of natural tissues with glutaraldehyde acetals.

This work describes the results of the controlled crosslinking of collagen matrices by glutaraldehyde based on a double protection strategy, glutaraldehyde acetals and lysine protonation due to the acidic conditions of acetal formation. Materials crosslinked by this approach were characterized by thermal stability comparable to those obtained by conventional procedures with mechanical properties expected for bioprosthesis manufacture and with a higher stability toward collagenase hydrolysis. The integrity of the microfibrillar structure was confirmed by optical and scanning electronic microscopy. The results indicate that the glutaraldehyde acetals procedure may be of potential use for the crosslinking of bovine pericardium used in the manufacture of bioprosthetic devices. Advantages may be related to the production of materials with homogeneous crosslinking distributions, associated with better definition in the nature of the chemical link that they introduce, due to a better distribution of glutaraldehyde within the tissue matrix before the crosslinking reaction is allowed to occur. As a result, materials with improved biological and mechanical properties are expected.

The controlled release of antibiotic by hydroxyapatite: anionic collagen composites.

Major problems with the treatment of osteomyelitis are associated with poor antibiotic distribution at the site of infection due to limited blood circulation to the skeletal tissue. Improved treatment procedures have been used in drug delivery systems that include bioceramics and natural and synthetic polymers. This work reports the development of anionic collagen:hydroxyapatite composite paste for sustained antibiotic release. Antibiotic release by the composite was characterized by two steps. In the first, 15.0+/-4.9% was released in the first 5 h (n = 53) by a normal Fick diffusion mechanism. In the second step, only 16.8+/-2.2% was released after 7 days. In conclusion, hydroxyapatite:anionic collagen composite can be an efficient support for sustained antibiotic release in the treatment of osteomyelitis because most of the antibiotic release may be associated with composite bioresorption, thus permitting antibiotic release throughout the healing process. Hydroxyapatite:anionic collagen paste showed good biocompatibility associated with bone tissue growth with material still being observed after 60 days from the time of implants.

Evaluation of anionic collagen membranes in the treatment of class II furcation lesions: an histometric analysis in dogs.

This study aims to evaluate the effect of using anionic collagen membranes in guided tissue regeneration treatment of Class II furcation lesions in dogs. The defects were created in the buccal furcation of 16 mandibular premolars of four dogs. After 56 days without plaque control, the sites were scaled and divided into two groups according to the treatment applied: control sites, open flap debridement; and test sites, guided tissue regeneration treatment. The animals were killed after 3 months. Histological and histometrical analyses showed that the collagen membrane was better than open flap debridement in terms of newly formed cementum and epithelial migration prevention. It provided effective blockade of epithelial tissue and promoted regeneration of lost periodontal tissues, suggesting that the membrane warrants further study.

Captopril reduces collagen and mast cell and eosinophil accumulation in pig serum-induced rat liver fibrosis.

The effect of captopril on the development of hepatic septal fibrosis in a specific experimental model produced by repeated injections of whole pig serum into the peritoneal cavity of rats was studied. The results afforded four basic conclusions. First, the experimental model used seems to be a pure form of septal fibrosis, which depends on active tissue fibroplasia, without hepatocyte necrosis. The fibrotic septa, located between limiting plates of adjacent classic hepatic lobules, and delimiting the classic liver lobule, consisted of collagen fibers infiltrated by eosinophils, mast cells, fat-storing cells (Ito cells), transitional cells and interstitial fibroblasts. Second, the angiotensin-converting enzyme inhibitor captopril attenuated the hepatic fibrosis induced by pig serum administration, as proven by a decrease in hepatic hydroxyproline concentration and histological examination of the liver. Third, this attenuation of hepatic fibrosis might be related, at least in part, to diminished mast cell and eosinophil accumulation in the hepatic tissue. Finally, these data may indicate a novel action of angiotensin-converting enzyme inhibitor in general, and for captopril in particular, as drugs potentially capable of reducing eosinophils in fibrotic processes.

Effect of tyrosine ionization upon biological activities of angiotensin II and two new peptide analogues.

The role of the tyrosine side-chain in the smooth muscle contracting activity of angiotensin III was investigated by determining intrinsic activities and ED50 values of [4-(3-chlorotyrosine)]angiotensin II and [4-(3-benzyltyrosine)]angiotensin II in the isolated guinea-pig ileum and rat uterus. [4-(3-chlorotyrosine)]angiotensin II activity was compared with that of angiotensin II at different pH values, in which the ratio of their degrees of phenolic ionization varied. The results indicated that deprotonation of the phenolic group hinders binding to smooth muscle cell receptors, but not triggering of the response by the hormone-receptor complex. Steric hindrance by the benzyl substituent in [4-(3-benzyltyrosine)]angiotensin II reduced both receptor-binding and triggering of the response.

Synthesis of biologically active retroenantiomers of angiotensin peptides.

The effect of the reversal of the direction of amide bonds in the peptide chain of angiotensin was determined by the synthesis and study of retroenantiomers of the following peptides: 1 [Val5]angiotensin II (angiotensin); 2, [Suc1]angiotensin (desamino-angiotensin); 3, [Ala7]desamino-angiotensin; 4, [beta-Ala7]desamino-angiotensin. In all of these retroenantiomers, the N-terminal Phe residue was replaced by a benzylmalonyl moiety in order to maintain the topological features of angiotensin's C terminus which are important for biological activity. The separation of the diasteromeric peptides containing D- or L-benzylmalonyl residues was possible in the cases of the retroenantiomers of 1 and 2 but not in those of 3 and 4. The retroenantiomers of 1 and 2 were devoid of smooth muscle contracting activities, while those of 3 and 4 contracted the isolated guinea-pig ileum and rat uterus with activities ranging from 8 to 24%, when compared with the respective parent compounds. The results indicate that (a) the sense of the peptide bonds in angiotensin's backbone is not essential for activity, and (b) the Pro7 residue in angiotensin is important for maintaining an "active" conformation of the molecule. The compounds reported in this paper are the first retroenantiomers of linear peptide hormones that have been shown to retain the biological activities of the parent compounds.