A Novel Approach to Monitoring Graft Neovascularization in the Human Gingiva

Summary

This study introduces a protocol for measuring microcirculation in human oral mucosa by laser speckle contrast imaging. The monitoring of wound healing after vestibuloplasty combined with a xenogenic collagen graft is presented on a clinical case.

Abstract

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids direct contact with the measured area, it is suitable for monitoring blood flow changes during wound healing in human patients. Vestibuloplasty is periodontal surgery to the oral vestibule, aiming to restore vestibular depth with simultaneous enlargement of the keratinized gingiva. In this special clinical case, a split thickness flap was elevated at the first upper premolar and a xenogenic collagen matrix was adapted to the resulting recipient bed. LSCI was used to monitor the re- and neovascularization of the graft and the surrounding mucosa for one year. A protocol is introduced for the correct adjustment of microcirculation measurement in the oral mucosa, highlighting difficulties and possible failures.

The clinical case study presented demonstrated that — following the appropriate protocol — LSCI is a suitable and reliable method for following up microcirculation in a healing wound in the human oral mucosa and gives useful information on graft integration.

Introduction

Monitoring long-term changes of human gingival microcirculation in a clinical situation is a hot topic in oral and periodontal surgery. However, reliable assessment of perfusion can be difficult. There are only a few methods that do not invasively measure changes in the blood circulation of the human mucosa. Two of these employ a laser beam^1,2,3,4, but in a different way. Laser doppler flowmetry (LDF) makes use of the Doppler shift in a laser beam^5,6, while the laser speckle contrast imaging (LSCI) method relies on the speckle pattern of the backscattered laser light to measure the velocity of red blood cells⁷.

LDF measures only in a single point, and reproducible standardization of the sensors' position is a desirable yet difficult task. Another problem is that the probe of the LDF is small in diameter (1 mm²). Measuring at predetermined points before surgery is too specific, and may be blind to postoperative circulatory changes, while edema, tissue removal, tissue movement or the implanted graft cause significant changes in the postoperative geometry of the affected soft tissue. The measuring distance of LDF is less than 1 mm which prohibits use of a dental splint with a predetermined hole for the probe in case of volumetric change of the tissue. LSCI does not require any special tools for localization and can measure in areas of several cm². As a result, wound healing can be followed throughout the surgical site. In addition, LSCI can display blood perfusion in color-coded images at a fraction of a second, with a resolution of up to 20 μm.

The LSCI device presented in this paper is used mostly for animal research applications where high resolution in small measurement areas is desired. However, since the structure and histology of the human oral mucosa are different from area to area (attached gingiva, marginal gingiva, vestibular mucosa), blood circulation is also heterogeneous⁸. Therefore, high-resolution LSCI has a great advantage over normal-resolution LSCI which is usually used in human testing.

The LSCI instrument employs an invisible laser (wavelength 785 nm). The beam is diverged to illuminate the measurement area, creating a speckle pattern. A CCD camera images the speckle pattern in the illuminated area. The CCD camera used in this system has an active imaging area of 1386 x 1034 pixels and its resolution is between 20–60 µm/pixel depending on the size of the measurement area and on the setting of the software (low, medium, high). It can take images at a speed of 16 frames per second, or even more, up to 100 frames per second, if the image size is reduced. Blood perfusion is calculated by the built-in software. It analyzes variations in the speckle pattern and quantifies the contrast. The resulting flux is color coded to produce a perfusion image. According to our previous results, LSCI assesses the blood perfusion of the gingiva with good repeatability and reproducibility⁹. This implies that it is a reliable tool for monitoring changes in the microcirculation of the oral mucosa not only in short-term experiments, but also during long-term studies to track disease progression or wound healing¹⁰.

In this paper, we present a clinical case report to demonstrate that the high spatial resolution of LSCI makes it possible to reveal the neovascularization pattern of a xenogenic collagen graft. Furthermore, this case indicates that LSCI, owing to its high reliability, could sensitively detect individual variation. This is important as significant local anatomical variation and a different systemic background between the cases makes it difficult to standardize the surgical intervention in clinical trials of periodontal surgery.

Protocol

The reported method was employed in a clinical trial which was granted ethical approval from the Hungarian Committee of the Health Registration and Training Center (approval number: 034310/2014/OTIG). 1. LSCI Setup Switch on the computer and any peripherals. Switch on the LSCI instrument to be used with the switch on the rear panel. Allow the instrument to warm up for at least 5 min. The instrument is ready for measurement when both LEDs on the rear panel have s…

Representative Results

Vestibuloplasty is periodontal surgery to the oral vestibule, aiming to increase vestibular depth, the zone of keratinized gingiva and soft tissue thickness for enhanced aesthetics and function. The apically repositioned split thickness flap combined with a collagen matrix is a frequently used vestibuloplasty procedure. Xenogenic collagen matrix is a viable alternative to autogenous gingival graft for increasing the amount of keratinized gingiva11,<sup c…

Discussion

The aim of this study was to introduce a novel technique for monitoring the neovascularization of a graft in the human gingiva. According to our previous results, LSCI assesses the blood perfusion of the gingiva with good repeatability and reproducibility⁹, when strict implementation of each step of the planned protocol as a critical requirement is met. LSCI is regarded as a semi-quantitative technique that requires calibration periodically to ensure accuracy and stability. During verification, th…

Materials

PeriCam PSI-HR	Perimed AB, Stockholm, Sweden		The PeriCam PSI System is an imaging system based on LASCA technology (LAser Speckle Contrast Analysis). The system measures superficial blood perfusion over large areas at fast capture rates. This makes it ideal for investigations of both the spatial and temporal dynamics of microcirculation in almost any tissue.
PIMSoft	Perimed AB, Stockholm, Sweden		PIMSoft is a data acquisition and analysis software, intended for use together with the PeriCam PSI System and the PeriScan PIM 3 System, for measurement and imaging of superficial blood perfusion.
Geistlich Mucograft	Geistlich, Switzerland		It's a unique 3D collagne matrix designed specifically for soft tissue regeneration. It's indicated for the gain of keratinized tissue and recession coverage.
Omron M4	Omron Healthcare Inc., Kyoto, Japan		Blood pressure monitor, which gives accurate readings.
Nikon D5200	Nikon Corportation, Tokyo, Japan		Taking intra oral photos
MS Excel	Microsoft Corporation, Redmond, Washington, USA		The software used for data management
IBM SPSS Statistics 25	IBM Corp., Armonk, NY, USA		The software used for statistical analysis