To date, the reconstructive approach addressing chronic non-healing wounds, deep tissue damage, and severe wound defects relies upon avascular dermal grafts and autologous flap techniques. Such flaps are limited by donor site availability and morbidity, while current dermal grafts rely upon host cellular invasion for neovascularization and incorporation. These products fail to include an inherent vascular network and the supporting cells necessary to ensure adequate incorporation and graft survival beyond the most optimal wound beds. Herein, we fabricate a pre-vascularized full-thickness cellularized skin equivalent containing a three-dimensional vascularized network of interconnected macro and microchannels lined with vascular cells, within a collagen neodermis populated with fibroblasts, and an epidermis comprised of human keratinocytes capable of providing whole tissue perfusion. Previously, our lab has employed a sacrificial microfiber technique to develop tissue-engineered scaffolds with an inherent hierarchical network of microvessels, which recapitulates the organization of an arteriole, venule, and capillary bed. Utilizing a type-I collagen hydrogel matrix, vascular cells were seeded within pre-fabricated channels and allowed to proliferate to generate an endothelialized microvasculature. These collagen scaffolds were subsequently anastomosed into rat models to demonstrate the clinical feasibility of such approach. The present study aims to more closely recapitulate the in vivo structure of human skin via the incorporation of vital epidermal and dermal components of native skin into a biocompatible construct containing a complex hierarchical vasculature, which may be anastomosed using standard microsurgical techniques and immediately perfused. Pluronic F127 was used as the sacrificial material: 1.5 mm diameter “U” shaped macrofibers and 100-500 µm-interwoven microfibers were heat extruded and then embedded within type-I collagen into which Cyan Fluorescent Protein (CFP)-tagged human placental pericytes and human foreskin fibroblasts (HFF1) had been encapsulated. Following pluronic sacrifice, resultant channels were intraluminally seeded with Red Fluorescent Protein (RFP)-tagged human aortic smooth muscle cells, Green Fluorescent Protein (GFP)-tagged human umbilical vein endothelial cells, and topically seeded with human epidermal keratinocytes (HEK). Construct microstructure was analyzed using multiphoton microscopy (MPM) after 7, 14 and 28 days of culture. Additionally, after 14 and 28 days of culture, endothelial cells were extracted from the construct using collagenase digestion and Real Time (RT)-qPCR performed to analyze expression of markers of angiogenesis and maturation of the vascular network. MPM demonstrated a hierarchical vascular network containing macro and microvessels lined by endothelial and smooth muscle cells, supported by perivascular pericytes, all in appropriate microanatomic arrangement. Neodermal HFF1 proliferated throughout the observation period and the HEK neoepidermis developed into a stratified epidermis along the superior aspect of the construct. Angiogenic sprouting from the nascent vascular network into neovessel like structures was noted. RT- qPCR revealed relative expression of Jagged1, Dll4, Ve-Cadherin, and CD31. We have successfully fabricated a novel tissue-engineered pre-vascularized full thickness skin flap, which recapitulates the inherent hierarchical vasculature found within human skin and is suitable for in vivo perfusion. We provide the platform for an on- demand, geometrically tunable tissue engineered skin equivalent with an anastomosable vascular network. This tissue-engineered skin flap holds the potential to transform reconstructive surgical practice by eliminating the consequences of donor site morbidity, and enabling rationally designed, patient-specific flaps for each unique wound environment and anatomic location. / 2017-06-16T00:00:00Z

Cutaneous arterial blood supply to the temporal region was evaluated in 8 dogs and 8 cats. Dissection of 4 dog and 4 cat cadavers revealed the location of cutaneous branches of the superficial telnporaJ artery supplying the frontalis tnuscle and skin of the temporal region. The frontalis 1l1uscle is a thin muscle dorsal to the temporaJis muscle that extends cranially and rostral1y from the rostral border of the scutulU111 to the forehead and upper eyelid. Microangiography and subtraction radiography of the external carotid and superficial telnporal arteries were used in 4 dogs and 4 cats to determine arterial blood supply to the temporal region and frontalis muscle. A superficial temporal artery (ST A) flap was developed in 9 dogs [ group A (n=5), group B (n=4)]. Ligation of the superficial tetnporal artery in the control dogs (n=5), rendered flaps dependent on the subdermal plexus. Dogs in group A (n=5) and the control group (n=5) had flap lengths that extended to the contralateral eye, while group B (n=4) flaps extended to the contralateral zygomatic arch. A11 flap widths were equivalent to the width of the zygomatic arch in the individual dog. Mean length of surviving tissue (mean survival length) (+/- SD) of control flaps was 7.0 (0.6) ern, compared with experimental flaps, group A 9.) (0.8) em and group B 10.4 0.1) cm. Mean survival percentage area of control flaps was 73.5 (7.4) %, compared with experimental flaps, group A 93.1 (7.5) 0/0 and group B 69.1 (4.5) 0/0. The mean survival length of control and experimental flaps was significantly different (P < 0.05). There was no significant difference between survival lengths of the experimental groups. / Master of Science

Objective. After skin flap transplants, perfusion strength monitoring is essential for the early detection of tissue perfusion disorders and thus to ensure the survival of skin flaps. Camera-based photoplethysmography (cbPPG) is a non-contact measurement method, using video cameras and ambient light, which provides spatially resolved information about tissue perfusion. It has not been researched yet whether the measurement depth of cbPPG, which is limited by the penetration depth of ambient light, is sufficient to reach pulsatile vessels and thus to measure the perfusion strength in regions that are relevant for skin flap transplants. Approach. We applied constant negative pressure (compared to ambient pressure) to the anterior thighs of 40 healthy subjects. Seven measurements (two before and five up to 90 min after the intervention) were acquired using an RGB video camera and photospectrometry simultaneously. We investigated the performance of different algorithmic approaches for perfusion strength assessment, including the signal-to-noise ratio (SNR), its logarithmic components logS and logN, amplitude maps, and the amplitude height of alternating and direct signal components. Main results. We found strong correlations of up to r = 0.694 (p < 0.001) between photospectrometric measurements and all cbPPG parameters except SNR when using the green color channel. The transfer of cbPPG signals to POS, CHROM, and O3C did not lead to systematic improvements. However, for direct signal components, the transformation to O3C led to correlations of up to r = 0.744 (p < 0.001) with photospectrometric measurements. Significance. Our results indicate that a camera-based perfusion strength assessment in tissue with deep-seated pulsatile vessels is possible.