Non-invasive Real-time Monitoring of Perfusion and Vascularization of an Engrafted Soft Tissue Engineered Oral Mucosa
Repair of soft tissue defects of the lips, as seen in complex maxillofacial injuries and post-ablative defects, requires pre-vascularized multi-tissue composite grafts. Protocols for fabrication of human exvivo produced oral mucosal equivalents (EVPOME) composed of epithelial cells and a dermal equivalent are now available to create prelaminated flaps for grafting in patients for lip reconstruction. However, invivo assessment of vascularization of the buried prelaminated flaps remains clinically challenging. Here, we use diffuse reflectance spectroscopy (DRS) and diffuse correlation spectroscopy (DCS) to non-invasively quantify longitudinal changes in the vascular saturation and blood-flow within EVPOME grafts, and explore the utility of these optical techniques for assessing vascularization of implanted grafts.
Materials and Methods
Twenty SCID mice were implanted with an EVPOME graft subcutaneously in their backs. DRS and DCS measurements were obtained from each animal both atop the graft site and a distance from the graft site (off-site). Measurements were obtained from each implanted graft at four consecutive weeks post-implantation.
Methods of Data Analysis
DRS measurements provided information derived from tissue reflectance, which is determined in turn by the concentration of oxy and deoxyhemoglobin. DCS monitors high-frequency fluctuations of the source laser speckle, which depends on the motion of red-blood cells in the blood vessels. DRS measurements were analyzed using a quantitative Monte Carlo based photon transport model to extract data, representing vascular saturation. The DCS data were analyzed using theoretical methods of diffuse optics to obtain the relative blood flow (RBF) index. Random-effect regression model was used to obtain differences in means at the graft site versus off-site at each week along with their 95% confidence intervals and P values.
Results
There were significant differences in the mean optical parameters (averaged across all mice) at the graft site versus the off-site measurements. For week 1 and 2 measurements, DRS instrument was significant with vascular saturation differences of 9.03 (p= 0.003; 95% CI = 2.97, 15.09) and 13.49 (p=0.001; 95% CI = 5.38, 21.60), respectively. At week 3, DCS instrument was significant with RBF index difference of 0.075 (p = 0.02; 95% CI = 0.01, 0.14). At week 4, neither DRS nor DCS instrument measurements showed statistically significant difference.
Conclusions
Both DRS and DCS measurements detected changes in vascularity occurring in (or around) the implanted EVPOME constructs. DRS data indicated increases in vascular saturation when measured atop the site of the grafts, significantly in the first two weeks post-implantation. DCS data showed that the relative blood flow was higher on-graft, statistically significant at 3 weeks post-operatively, though the measurements were reliably higher for all four weeks. Although it is difficult to control for factors such as inflammation leading to vasodilatation and transient hyper-vascularization during wound healing, DRS and DCS techniques were able to discern significantly between on and off-graft. Furthermore, these optical techniques were able to quantify perfusion and vascularization, characterizing the changes longitudinally. Therefore, our study shows the potential of these techniques for challenging clinical scenarios such as monitoring of buried flaps.
2 References
1. Holze F, Loeffelbein DJ, et. al. Free flap monitoring using simultaneous non-invasive laser Doppler flowmetry and tissue spectrophotometry. J Craniomaxillofac Surg. 2006 34, 25-33.
2. Mesquita RC, Schenkel SS, et. al. Influence of probe pressure on the diffuse correlation spectroscopy blood flow signal: extra-cerebral contributions. Biomed Opt Express. 2013. 4(7): 978-94