Periodontal and biochemical bone metabolism assessment on a chronic oral anticoagulation population treated with dicoumarins.

BACKGROUND: The aim is to evaluate periodontal alteration and biochemical markers associated with bone turnover in chronic oral with dicoumarins anticoagulant treatment patients. MATERIAL AND METHODS: 80 patients treated with oral anticoagulants were divided into 2 cohort: Group A (n=36) 6 month to 1 year with anticoagulant treatment and Group B (n=44) > 2 years with anticoagulant treatment. Clinical evaluation included: Clinical attachment level (CAL), plaque index (PI) and gingival index (GI). Analytically biochemical parameters of bone remodeling (calcium and phosphorus), formation (total acid phosphatase, alkaline phosphatase and osteocalcin) and resorption (tartrate-resistant acid phosphatase and beta-crosslaps) were evaluated. RESULTS: High values of PI (67-100%) especially in men and in Group B were observed. Men with anticoagulation treatment length showed an increased GI (49.167 vs 78.083) while Group B women showed a decreased GI in comparison with Group A (59.389 vs 42.120). Women presented a greater average CAL than men as well as Group B vs Group A but without statistical significance. All biochemical markers were decreased respect to values of general population. Osteocalcin in GroupB women showed a statistically significant outcome vs GroupA (p=0.004). Acid phosphatase (total and tartrate-resistant) has a slight increase in Group B women versus Group A, and Beta-crosslap showed lower values in Group A men than Group B and slightly lower in Group A women versus Group B, without statistical significance. CONCLUSIONS: Patients showed a slight to moderate degree of periodontal affectation, especially gingivitis related to bacterial plaque. Periodontal disorders tended to be more severe in Group B. While bone remodeling showed an overall decrease with greater affectation of bone neoformation phenomena, bone destruction tended to recover and normalize in time.

Sequential keratinocytic differentiation and maturation in a three-dimensional model of human artificial oral mucosa.

BACKGROUND AND OBJECTIVE: Oral mucosa shortage may limit or condition some clinical approaches in maxillofacial, periodontal and implant treatment. The availability of a human oral mucosa model generated by tissue engineering could help clinicians to address the lack of oral mucosa. In this work, we carried out a sequential maturation and differentiation study of the epithelial cell layer of an artificial human oral mucosa substitute based on fibrin-agarose biomaterials with fibroblasts and keratinocytes. MATERIAL AND METHODS: Histological, immunohistochemical and gene expression analyses were carried out in artificial human oral mucosa models developed and cultured for 1, 2 and 3 wk. RESULTS: Artificial oral mucosa models showed expression of tight junction proteins and cytokeratins from the first week of in vitro development. Mature samples of 3 wk of development subjected to air-liquid conditions showed signs of epithelial differentiation and expressed specific RNAs and proteins corresponding to adherent and gap junctions and basement lamina. Moreover, these mature samples overexpressed some desmosomal and tight junction transcripts, with gap junction components being downregulated. CONCLUSION: These results suggest that bioengineered human oral mucosa substitutes form a well-developed epithelial layer that was very similar to human native tissues. In consequence, the epithelial layer could be fully functional in these oral mucosa substitutes, thus implying that these tissues may have clinical usefulness.

In vitro and in vivo cytokeratin patterns of expression in bioengineered human periodontal mucosa.

BACKGROUND AND OBJECTIVE: Development of human oral mucosa substitutes by tissue engineering may provide new therapeutic tools for the management of periodontal diseases. In this study we evaluated a fibrin-agarose human oral mucosa substitute both in vitro and in vivo. MATERIAL AND METHODS: In vitro bioengineered oral mucosa substitutes were developed from irrelevant biopsy samples of human oral gingiva. In vivo evaluation of the constructed tissues was performed by implantation into athymic nude mice. The expression of several epithelial markers was assessed by microarray analysis and immunohistochemistry. RESULTS: Bioengineered oral mucosa samples kept in vitro developed a multilayered epithelium that expressed several cytokeratins, including some markers of simple epithelia (cytokeratins 7, 8 and 18), along with markers of stratified epithelia (cytokeratins 5 and 13) and of cell proliferation (proliferating cell nuclear antigen). Bioengineered tissues grafted in vivo onto nude mice exhibited very good biointegration with the host, showing a cytokeratin expression pattern that was very similar to that of normal native oral mucosa controls. Histological analysis of the artificial tissues demonstrated that oral mucosa substitutes evaluated in vivo were structurally mature, showing some typical structures of human native oral mucosa such as rete ridges and chorial papillae, along with numerous blood vessels at the fibrin-agarose stromal substitute. These structures were absent in samples evaluated in vitro. CONCLUSION: The results indicate that this model of human oral mucosa, constructed using fibrin-agarose scaffolds, shows similarities to native oral mucosa controls and imply that bioengineered oral mucosa substitutes could eventually be used clinically.

Time-course study of histological and genetic patterns of differentiation in human engineered oral mucosa.

The lack of sufficient oral mucosa available for intra-oral grafting is a major surgical problem, and new sources of oral tissues for clinical use are needed. In this regard, some models of engineered oral mucosa have been reported to date, but little is known about the structural and genetic mechanisms that occur during the process of development and maturation of these tissue substitutes. We have carried out a time-course study of the genes and morphological patterns of cell and tissue differentiation that develop in oral mucosa constructs after 3, 7, 11 and 21 days of development. Our electron microscopy and microarray analyses demonstrated that the oral mucosa constructs generated by tissue engineering undergo a progressive process of cell differentiation with the sequential formation and maturation of several layers of epithelium (with expression of stratifin, sciellin, involucrin, trichohyalin and kallikrein 7), intercellular junctions (with expression of plakophilin, desmocollin, desmoglein and cadherins), cytokeratins, a basement membrane (laminins, collagen IV) and the extracellular matrix (biglycan, matrix metalloproteinases). In conclusion, although the level and type of keratinization developed in vitro could be different, the oral mucosa substitutes were very similar to the native tissues.

Histological and histochemical evaluation of human oral mucosa constructs developed by tissue engineering.

Reconstruction of large oral mucosa defects is often challenging, since the shortage of healthy oral mucosa to replace the excised tissues is very common. In this context, tissue engineering techniques may provide a source of autologous tissues available for transplant in these patients. In this work, we developed a new model of artificial oral mucosa generated by tissue engineering using a fibrin-agarose scaffold. For that purpose, we generated primary cultures of human oral mucosa fibroblasts and keratinocytes from small biopsies of normal oral mucosa using enzymatic treatments. Then we determined the viability of the cultured cells by electron probe quantitative X-ray microanalysis, and we demonstrated that most of the cells in the primary cultures were alive and had high K/Na ratios. Once cell viability was determined, we used the cultured fibroblasts and keratinocytes to develop an artificial oral mucosa construct by using a fibrin-agarose extracellular matrix and a sequential culture technique using porous culture inserts. Histological analysis of the artificial tissues showed high similarities with normal oral mucosa controls. The epithelium of the oral substitutes had several layers, with desmosomes and apical microvilli and microplicae. Both the controls and the oral mucosa substitutes showed high suprabasal expression of cytokeratin 13 and low expression of cytokeratin 10. All these results suggest that our model of oral mucosa using fibrin-agarose scaffolds show several similarities with native human oral mucosa.

Regional differences in cell surface patterns in normal human sulcular epithelium.

Studies with scanning electron microscopy in the normal human sulcular epithelium are scarce, and no precise information exists about cell surface patterns along the epithelium, the frequencies of these patterns, or possible regional differences within the mouth. In five periodontal biopsy specimens each from the anterior and posterior region of the mouth, we observed three cell patterns on the basis of the overall appearance of morphological surface markers in the coronal and apical zones of sulcular epithelium: microvilli; microplicae; and pits. The percentage of keratinocytes showing the microvillous pattern in the surface of apical sulcular epithelium of the posterior region of the mouth was significantly higher than in the anterior region. We posit that the presence, in the bottom of the normal sulcular epithelium in the posterior region of the mouth, of mainly microvillous keratinocytes (the most undifferentiated and least desquamative type of keratinocyte, and thus the most vulnerable to bacterial colonization) can be associated with observations of longitudinal clinical studies of periodontal disease, which suggest that more severe clinical findings are found in the region of the molars.

Electron microprobe analysis in periodontal guided tissue regeneration.

Electron microprobe analysis was used to determine the evolution of Ca, P and S in regenerated tissue surrounding incisors roots after periodontal treatment with guided tissue regeneration. Our results, which showed increased Ca and P, and decreased S are discussed in relation to the process of mineralization electron probe microanalysis with potentially provided an accurate means of assessing the degree of mineralization in extremely small tissue samples.

Cell surface patterns in normal human oral gingival epithelium. A quantitative scanning electron microscopy approach.

We used scanning electron microscopy to study the morphological surface patterns of cells that cover the attached gingiva and intervestibular papilla of the human oral gingival epithelium. Five patterns are described on the basis of the overall appearance of morphological surface markers: microvilli, parallel, fingerprint, reticular and pitted. Statistical analyses detected significant differences in the frequency of each pattern in both regions of the oral gingival epithelium, and showed the reticular and fingerprint types to predominate. We propose that our description of the different morphological surface types may be of use as a standard for subsequent cytological studies and characterizations of morphological alterations in diseased gingiva.

Evidence that dopamine is acting at alpha-adrenoceptors of the rat vas deferens.

In the rat vas deferens, a vast number of experiments have shown that the alpha-adrenoceptors present are of two types: alpha 1 and alpha 2. This series of experiments with the isolated rat vas deferens was designed to probe by pharmacological means, the nature of the responses elicited by neurogenic transmural stimulation and also those responses evoked by exogenous NE and DA. The methodology required the production of chemical denervation, neurotransmitter depletion, and the use of specific adrenoceptor blockers. The results obtained with the blocking agents, yohimbine or prazosin versus NE and DA, were pA2 values that were virtually interchangeable. The effects of chemical alteration with 6-OH-DA or reserpine point to a certain similarity and interdependence of the mechanism of action for the two neurotransmitters. Therefore, it is suggested that these two transmitters act at the same receptor site or share a common receptive microenvironment in the rat vas deferens.

Effects of haloperidol on neurotransmitter activity in the rat vas deferens.

1. The effects of haloperidol on the responses of the isolated rat vasa deferentia to catecholamines and ACh were studied. 2. Haloperidol produced a competitive antagonism to responses elicited by NA and DA in the vas deferens. 3. The M1 and M2 muscarinic responses to ACh of the vas deferens were potentiated by this neuroleptic. 4. The AChE activity of the vas deferens was significantly depressed by pretreatment with haloperidol. 5. The ability of haloperidol to lower AChE activity was compared with that of neostigmine and it may be due to a similar molecular mechanism. 6. The present results suggest that haloperidol has anti-AChE properties that may be responsible for the potentiation of the responses to ACh. 7. The study indicates that haloperidol has a wider range of pharmacological actions than previously reported.

Castration and tolerance induces changes in the levels of the activity of acetylcholinesterase in the isolated vas deferens of the rat.

Changes in the activity of acetylcholinesterase (AChE) of the isolated vas deferens from normal, castrated, morphine and ethanol-tolerant rats were studied. Three days after the termination of treatment with morphine and on the last day of treatment with ethanol, a significant inhibition of the activity of AChE was detected. This reduction in the enzymatic activity persisted in morphine-tolerant rats for 15 days, but not for 30 days, at which time the levels of AChE were determined to be normal. However, in ethanol-tolerant rats, there were no significant changes found at days 15 or 30. The activity of AChE was decreased significantly in castrated rats, but this effect was reversed by treatment with testosterone. During withdrawal from morphine or ethanol, the levels of AChE were significantly increased. The results indicate that morphine and ethanol may be inducing changes in the feedback mechanism which regulates the levels of AChE at post-synaptic sites, and these changes could play an important role in the development of tolerance to morphine and to ethanol.

Mixed actions of hemicholinium at autonomic receptor sites of the rat vas deferens.

1. The effects of hemicholinium (HC-3) on several autonomic agents in the isolated rat vas deferens were investigated. 2. HC-3 reduced slightly the effects of NE and DA. 3. The responses by cholinergic agents on the M1-ACh receptors were not modified, however HC-3 reduced, significantly, the responses on the M2-ACh receptors. 4. These results suggest that HC-3 besides its anticholinergic properties possesses ability to interact with adrenoceptors in the isolated rat vas deferens.

Ontogenesis of autonomic receptors and AChE activity in the rat vas deferens.

The study was undertaken to obtain some information about the development of the autonomic receptors and the AChE activity in the rat vas deferens. The results suggest that the adrenoceptors were fully developed at birth. The M1-ACh receptors were developed before the M2-ACh receptors. The AChE activity developed before the ACh muscarinic receptors of the rat vas deferens.