Lenaldo B. Rocha

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.

Biofilm on the apical region of roots in primary teeth with vital and necrotic pulps with or without radiographically evident apical pathosis

AIM To evaluate, by scanning electron microscopy (SEM), the presence of biofilms on the external surfaces of the apical third of roots of human primary teeth with vital or necrotic pulps with and without radiographically evident periradicular pathosis. METHODOLOGY Eighteen teeth were selected: group I - normal pulp (n = 5), group II - pulp necrosis without radiographic evidence of periapical pathosis (n = 7) and group III - pulp necrosis with well-defined radiographic periapical pathosis (n = 6). After extraction, the teeth were washed with saline and immersed in 0.03 g mL(-1) trypsin solution for 20 min. The teeth were then washed in sodium cacodilate buffer and stored in receptacles containing modified Karnovsky solution. The teeth were sectioned, dehydrated in an ethanol series, critical-point dried with CO(2), sputter coated with gold and the external root surface in the apical third examined by SEM. RESULTS In the teeth of groups I and II, the apical root surfaces were covered by collagen fibres, with no evidence of bacteria (100%). In the teeth of group III, the root apices had no collagen fibres but revealed resorptive areas containing microorganisms (cocci, bacilli, filaments and spirochetes) in all cases (100%). CONCLUSION Microorganisms organized as biofilms on the external root surface (extraradicular infection) were detected in primary teeth with pulp necrosis and radiographically visible periapical pathosis.

Shannon's entropy and fractal dimension provide an objective account of bone tissue organization during calvarial bone regeneration

The regeneration of compact bone involves the deposition of a poorly organized connective tissue template that remodels into compact lamellar bone. An objective description of this process is difficult because classical histomorphometry is unable to correctly characterize qualitative changes in tissue complexity. In this study, we demonstrated the use of two distinct methods of image texture analysis, the Shannons entropy [standard error (SE)], and the fractal dimension (FD) to characterize the formation and remodeling of newly formed compact bone within two different polyanionic collagen‐elastin matrices. The matrices were implanted in defects created into parietal bones of rats. The SE and FD were calculated for histological images of the experimental groups collected 3, 7, 15, 30, 60, and 365 days postsurgery and for the original bone only at day 365. Results showed that the SE and the FD initially increased and then diminished for all groups from day 3 to day 365 approaching the values of the original bone. These results are consistent with poor tissue organization during early osteogenesis that remodels into an organized lamellar structure, showing that these methods can be valuable tools to describe bone tissue remodeling during the regeneration process of compact bones. Microsc. Res. Tech., 2008.

In Situ Zymography and Immunolabeling in Fixed and Decalcified Craniofacial Tissues

In situ zymography is a very important technique that shows the proteolytic activity in sections and allows researchers to observe the specific sites of proteolysis in tissues or cells. It is normally performed in non-fixed frozen sections and is not routinely performed in calcified tissues. In this study, we describe a technique that maintains proteolytic activity in fixed and decalcified sections obtained after routine paraffin sectioning in conventional microtome and cryostat sections. We used adult rat hemimandibles, which presented bone, enamel, and dentine matrices; the substrate used was dye-quenched-gelatin. Gelatinolytic activity was colocalized with MMP-2 using fluorescent antibodies. Specific proteolytic activity was observed in all sections, compatible with metalloproteinase activity, particularly in dentine and bone. Furthermore, matrix metalloproteinase-2 was colocalized to the sites of green fluorescence in dentine. In conclusion, the technique presented here will allow in situ zymography reactions in fixed, decalcified, and paraffin-embedded tissues, and we showed that paraformaldehyde-lysine-periodate–fixed cryostat sections are suitable for colocalization of gelatinolytic activity and protein labeling with antibodies. (J Histochem Cytochem 57:615–622, 2009)

In vitro and ex vivo infection models help assess the molecular aspects of the interaction of Trichophyton rubrum with the host milieu

Dermatophytes are fungal pathogens that cause cutaneous infections such as onychomycosis and athletes foot in both healthy and immunocompromised patients.Trichophyton rubrum is the most prevalent dermatophyte causing human nail and skin infections worldwide, and because of its anthropophilic nature, animal infection models are limited. The purpose of this work was to compare the expression profile of T. rubrum genes encoding putative virulence factors during growth in ex vivo and in vitro infection models. The efficiency of the ex vivo skin infection model was confirmed by scanning electron microscopy (SEM), which showed that the conidia had produced hyphae that penetrated into the epidermis. Quantitative RT-PCR (qRT-PCR) analysis showed that the expression of some genes is modulated in response to the infection model used, as compared to that observed in cells grown in glucose-containing media. We concluded that ex vivo infection models help assess the molecular aspects of the interaction of T. rubrum with the host milieu, and thus provide insights into the modulation of genes during infection.

SEM Study of Apical Morphological Alterations in Primary Teeth with Vital and Necrotic Pulps

This study evaluated, by SEM, the morphology of human primary teeth roots. Twenty-four teeth were divided into 3 groups: pulp vitality (group I) and pulp necrosis without (group II) and with apical periodontitis (group III). Roots were analyzed by the presence of periodontal ligament (PDL) fibers and resorption areas. In groups I and II, presence of PDL fibers and absence of resorption were observed in all cases (100%), while all specimens (100%) of group III showed no PDL fibers and resorption areas. In conclusion, there are morphological differences in the apical region of primary teeth with different pulpal and periapical pathologies.