Abstract
The aim of this research is to evaluate in vivo the characteristics of microcirculation after taking a biopsy sample from the oral mucosa. 20 patients were recruited to the study and all underwent an oral mucosa biopsy for the excision of benign neoformations. The modifications in the oral microcirculation were evaluated in vivo in correspondence to the surgical site through videocapillaroscopy at three different times: 30 min before the biopsy; 48 h after the biopsy; and 7 days after the biopsy. The statistical significance was checked with the Mann–Whitney U -test ( P < 0.05). The analysis of videocapillaroscopic patterns showed statistically significant variations relative to the capillary loop density; the diameter of the outgoing loop; and the length of the capillary loop. In conclusion, the study describes a simple and reproducible model for the study of wound healing from a microcirculatory point of view.
Microcirculation plays a key role in the healing process, providing the necessary cellular and molecular components for re-establishing the integrity of the injured tissue. The complex reparative process of the surgical wound can be simplified in four phases: hemostasis, inflammation, proliferation and remodelling ( Fig. 1 ).
The contribution of angiogenesis and the revascularization of surgical wounds during every phase of the healing process lies in transporting oxygen, growth factors and immunological support to the tissue being regenerated. Thus postoperative revascularization and the regeneration of microcirculation are a fundamental prerequisite for proper wound healing and for the reconstitution of the metabolic integrity of the tissue.
Numerous systemic factors influence the healing process of oral surgical wounds: age, sex, alcohol, smoke, nutritional state, medicines, diabetes, stress, and obesity. The formation of new blood vessels, angiogenesis, is essential to the healing of surgical wounds. It is a dynamic process, regulated by signals coming from the serum and from the highly specialized surrounding tissue that constitutes the extracellular matrix. Angiogenesis is generally considered to be a process that occurs during the proliferative phase of wound healing, even if many of the signals that regulate the phenomenon are produced in the inflammatory phase.
Endothelial cells have a key role in the healing process; they are involved in the formation of new blood vessels, allow the migration of leucocytes, transport oxygen and nourishment to the level of the wound and biologically secrete active substances.
Among the growth factors that regulate the neoangiogenesis process are vascular endothelial growth factor (VEGF), angiopoietin, fibroblast growth factor (FGF), transforming growth factor beta (TGF-β) and the immune system.
The migration of endothelial cells and the development of new capillaries does not only depend on the cells and on the cytokines present, but also on the production and the organization of components of the extracellular matrix (ECM), among which are fibronectin, collagen, vitronectin and laminin.
The extracellular matrix has an important role in the normal growth of vessels and their maintenance, by providing a support framework through which the endothelial cells can migrate and by acting as a supplier and modulator for growth factors such as FGF2 and TGF-β in the regulation of intercellular signals.
The aim of this research is to evaluate in vivo the characteristics of districtual microcirculation linked to the tissue repair mechanisms that occur after the taking of a biopsy sample at the oral mucosa level.
Materials and methods
20 patients (10 men, 10 women; aged 20–70 years, mean ± SD = 46.4 ± 10.71 years; median age 38.5 years) were examined in the authors’ laboratory. They all gave informed consent for the processing and use of their personal medical data in scientific papers, in accordance with Italian law. The research was approved by ethical committee report number 02/2010. The authors have read the Helsinki Declaration and have followed the guidelines in this investigation. All the patients were submitted to a punch biopsy (6 mm) for benign neoformations (fibromas and papillomas) situated in correspondence to mucosa from the genian region, the lower lip and tongue. Silk suture thread of 000 diameter was used for suturing in all patients.
Microcirculation in correspondence to the surgical site was observed at three different times: t 0 , 30 min before the biopsy; t 1 , 48 h after the biopsy; and t 2 , 7 days after the biopsy.
Oral microcirculation imaging was performed using computerized videocapillaroscopic techniques and related software (Videocap 200, distributed by DS Medica S.r.l., Milan, Italy). The videomicroscope optical probe used is composed of a main unit (with an optical probe with a video-optical terminal connected to it) and a high-resolution colour monitor to view the examined area. The main unit consists of a cold halogen light source emitted by a 100 W lamp with an electronic device controlling its light intensity, and a processing unit, for the high-definition video signal (420,000 pixels), equipped with a colour-calibration device. The probe is equipped with a video-optical terminal containing a high-definition video sensor on which different variable magnification optics from 10× to 1000× can be applied.
In this study, the authors used a 200× optic, because of its good magnification and focusing properties. The video-optical terminal allows the operator to focus directly on the handpiece. Image digitization enables analysis of the fundamental parameters of microcirculation (diameter and vessel length) and calculation of the number of capillaries per square millimetre of the mucosa examined. The images were acquired and evaluated with the Videocap 3.0 proprietary software.
The capillaroscopic investigation was carried out with the patients in a sitting position on a dental chair with an angle of inclination of 90°, with the same light source, at the same room temperature (23 ± 1 °C), in the morning, and by the same operator (GAS). The investigation was repeated at least twice for each examined site.
The oral mucosa was carefully manipulated since stretching can alter microcirculation properties. Two independent observers (A and B) examined all the images. The observers carried out double-blind evaluations. The intraobserver and interobserver variability was assessed with the two observers evaluating the same selected images twice.
The following parameters were taken into account: capillary loop density (the number of visible loops per square millimetre); total loop diameter; and incoming and outgoing loop diameter (the maximum diameter of each of the two heads of the loop).
All the incoming and outgoing capillary loop vessels were measured. The images were randomly selected. The images acquired were digitalized using the Videocap 3.0 proprietary software and saved in bitmap form. A measurement for one image took about 4 min. An average measurement of 4 sites in one area was made to give a more reliable regional quantification.
It is important to point out that the parametric data originate from the software connected to the videocapillaroscope by use of the appropriate measuring tool. The system is properly calibrated, with every optical magnification corresponding to an exact metric pixel value in the digitized image.
Statistical analysis
All collected data were grouped and a statistical analysis was performed, using P.A.S.T. software (version 1.92 updated in May 2009, Øyvind Hammer, D.A.T. Harper and P.D. Ryan). The statistical significance of the differences was checked with the Mann–Whitney U -test. The level of significance was set to P < 0.05. Differences with a P -value less than 0.05 were considered statistically significant.
Results
Healing occurred without clinical signs of infection in every patient. The average density of the capillaries per mm 2 in t 0 was 13.5 ± 4.6 (mean ± SD); t 1 was 32.4 ± 3.8 (mean ± SD); t 2 was 28.3 ± 4.5 (average ± SD), thus showing a statistically significant increase between t 0 and t 1 and between t 0 and t 2 ( P < 0.05) ( Figs 2–4 ). Significant values were also found in the analysis of the capillary loop length and in the analysis of the diameter of the efferent loops ( Table 1 ). The intraobserver and interobserver variability was not statistically significant.
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