Gilberto Goissis

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.

Biocompatibility of anionic collagen matrix as scaffold for bone healing.

The basic approach to the treatment of bone defects involves the use of scaffolds to favor tissue growth. Although several bioscaffolds have been proposed for this purpose, the search for new and enhanced materials continues in an attempt to address the drawbacks of the present ones. Modifying current materials can be a fast and cheap way to develop new ones. Among them, type I collagen allows its structure to be modified using relatively simple techniques. By means of an alkaline treatment, anionic collagen with enhanced piezoelectric properties can be obtained through hydrolysis of carboxyamides groups of asparagine and glutamine residues from collagen in carboxylic. The process applied to a raw source of collagen, bovine pericardium, provided a sponge-like structure, with heterogeneous pore size, and, moreover, the complete removal of interstitial cells. For the evaluation of the biocompatibility of such matrices, they were implanted in surgically created bone defects in rat tibias. Empty defects served as controls. This experimental model allowed a preliminary evaluation of the osteoconductiveness of the matrices. The histological results presented a low inflammatory response and bone formation within a short period of time, similar to that of controls. The low cost of production associated to the biocompatibility and osteoconductivity performance make the anionic collagen matrices promising alternatives for bone defects treatment.

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.

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 fibro‐blasts. 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.

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.

Tissue response to polyanionic collagen: elastin matrices implanted in rat calvaria

The tissue response to polyanionic collagen matrices, prepared from bovine pericardium and implanted subperiosteally in rat calvaria, was studied. The materials were implanted in 72 male rats (Rattus norvegicus, albinus, Holtzman), randomly divided into four groups: GI-MBP hydrolyzed for 24 h; GII-MBP hydrolyzed for 36 h; GIII-MBP hydrolyzed for 48 h; GIV-native MBP. The materials were explanted after 15, 30 and 60 days and analyzed by routine histological procedures. Except for group IV (native bovine pericardium), polyanionic collagen from groups GI, GII and GIII showed low inflammatory reaction associated with bone formation, partially or completely integrated to the cranial bone; group GIV was characterized by an intense inflammatory reaction with occasional dystrophic mineralization and with occasional bone formation at 60 days when there was a decrease in the inflammatory reaction. Thus, the MBP from groups I, II and III were biologically compatible, enhancing bone formation with a slight delay at 60 days in GII.

Hidrólise seletiva de carboxiamidas de resíduos de asparagina e glutamina em colágeno: preparação e caracterização de matrizes aniônicas para uso como biomateriais

This work describes the selective hydrolysis of carboxyamide groups of asparagine and glutamine of collagen matrices for the preparation of negatively charged collagen biomaterials. The reaction was performed in the presence of chloride and sulfate salts of alkaline and alkaline earth metals in aqueous dimethylsulfoxide solution and, selectively hydrolysis of carboxyamide groups of collagen matrices was promoted without cleavage of the peptide bond. The result is a new collagen material with controlled increase in negative charge content. Although triple helix secondary structure of tropocollagen was preserved, significative changes in thermal stabilities were observed in association with a new pattern of tropocollagen macromolecular association, particularly in respect microfibril assembly, thus providing at physiological pH a new type of collagen structure for biomaterial preparation, characterized by different charge and structural contents .

Preparation and characterization of an acellular bovine pericardium intended for manufacture of valve bioprostheses.

Major problems with biological heart valves post-implantation are associated with progressive structural deterioration and calcification attributed to glutaraldehyde processing, dead cells, and cell fragments present in the native tissue. In spite of these problems, glutaraldehyde still is the reagent of choice. The results with acellular matrix xenograft usually prepared by detergent treatment in association with enzymes are rather conflicting because while preserving mechanical properties, tissue morphology and collagen structure are process dependent. This work describes a chemical approach for the preparation of an acellular bovine pericardium matrix intended for the manufacture of heart valve bioprostheses. Cell removal was performed by an alkaline extraction in the presence of calcium salts for periods ranging from 6 to 48 h. The results showed that cell removal was achieved after 12 h, with swelling and negative charge increasing with processing time. Nevertheless, collagen fibril structure, ability to form fibrils, and stability to collagenase were progressive after 24-h processing. There was no denaturation of the collagen matrix. A process is described for the preparation of acellular bovine pericardium matrices with preserved fibril structure and morphology for the manufacture of cardiac valve bioprostheses and may be used in other applications for tissue reconstruction.

Modifications on Collagen Structures Promoted by 1,4-Dioxane Improve Thermal and Biological Properties of Bovine Pericardium as a Biomaterial

Collagen is a widely used raw material for biomaterial manufacture, which generally depends on chemical modifications of this fibrillar protein with cross-linking agents to improve biocompatibility and mechanical properties. However, cross-linking reduces the natural properties of collagen, such as low immune response, low toxicity as well as the ability to promote cellular growth and attachment. In this work, the modifications promoted by 1,4-dioxane solvent on the collagen present in native bovine pericardium (NBP) matrix routinely used in bioprosthesis manufacture, with or without subsequent cross-linking by glutaraldehyde, has been studied. The structural changes of NBP evaluated by scanning electron microscopy show that 1,4-dioxane induces a more homogeneous material by increasing aggregation of collagen fibers, while transmission scanning electron microscopy shows that natural collagen fibril arrangement, integrity, and the D-periodicity pattern are maintained by solvent treatments. Measurements of thermal stability and resistance to collagenase enzymatic digestion of NBP matrices treated with 1,4-dioxane show an increase in melting temperature and decrease in biodegradability, as compared to native pericardium. Cross-linking with glutaraldehyde improves all the analyzed NBP properties, which are not impaired by previous treatment with 1,4-dioxane. Histological evaluation of NBP submitted to 1,4-dioxane treatment shows lower lipid and cell contents and improvement in other morphologic characteristics compared to native pericardium. Altogether, these results suggest the use of 1,4-dioxane organic solvent as an alternative non-cross-linking treatment for direct utilization on rich collagen matrices, resulting in materials with improved biocompatibility and physicochemical properties suitable for tissue engineering.

Compósitos colágeno aniônico: fosfato de cálcio. Preparação e caracterização

Anionic collagen: calcium phosphate composite was obtained by controlled mixing of collagen and calcium phosphate until the consistence of a past. Material was characterized by a Ca/P ratio of 1.55, with a X-ray diffraction pattern similar to that for hydroxyapatite. Differential Scanning Calorimetry showed that the protein is not denatured under the processing conditions. Scanning Electronic Microscopy showed that the mineral phase are regularly covered with collagen fibers, indicating that anionic collagen is efficient in the preparation of stable form of calcium phosphate ceramic paste.