Elsevier

Biomaterials

Volume 34, Issue 11, April 2013, Pages 2674-2682
Biomaterials

The effect of platelet rich plasma on angiogenesis in ischemic flaps in VEGFR2-luc mice

https://doi.org/10.1016/j.biomaterials.2013.01.016Get rights and content

Abstract

To improve skin flap healing, one promising strategy in reconstructive surgery might be to optimize platelet rich plasma (PRP) bioactivity and the ischemia-altered expression of genes. We studied both the effect of PRP on ischemic flaps, and whether in vivo bioluminescence imaging (BLI) is a suitable method for the longitudinal monitoring of angiogenesis in surgical wounds. Axial murine skin flaps were created in four experimental groups. In vivo measurements of VEGFR2 expression levels were made every other day until the 14th day. The local VEGF level and microvessel density were quantified on the 14th day via ELISA and immunohistochemistry, and flap survival rates were measured. We demonstrated that PRP and induced ischemia have a beneficial influence on angiogenesis and flap healing. Combining the two resulted in a significantly robust increase in angiogenesis and flap survival rate that was corroborated by bioluminescence imaging of VEGFR2 activity. This study shows that angiogenic effects of PRP may be potentialized by the stimulus of induced ischemia during free flap harvesting, and thus the two procedures appear to have a synergistic effect on flap healing. This study further demonstrates that BLI of modulated genes in reconstructive surgery is a valuable model for longitudinal in vivo evaluation of angiogenesis.

Introduction

Skin flaps are routinely used for reconstruction of large skin defects and deep wounds secondary to trauma, ablative surgery or congenital defects. Ways of improving skin flap survival and the mechanisms involved in the healing process at the recipient site are much-studied matters. The persistence of both partial and complete skin flap necrosis despite the greatest of efforts presents a challenging, multifactorial problem. Adequate vascular supply to the integument is crucial to skin flap survival, especially in comorbid patients. Elevating a skin flap results in varying degrees of hypoxia, which acts as the primary stimulus for vascular changes. The effects of hypoxia on vascular growth and angiogenesis in ischemic tissues have been reported in several studies [1], [2], [3], [4], [5]. Several preconditioning techniques are used to increase ischemic tolerance [6], [7]. Following these procedures, angiogenic agents such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) are increased, leading to new blood vessel formation [8], [9].

A skin flap actually survives as a consequence of flawless wound healing process. Angiogenesis, the process of capillary sprouting from preexisting blood vessels, is essential for wound healing [10], [11]. VEGF, a potent angiogenic, mitogenic and vascular permeability-enhancing protein, is secreted in the cutis by keratinocytes and fibroblasts [5], [12], [13]. VEGF is a pleiotropic factor that acts on a variety of cells, but especially on dermal vascular structures involved in cutaneous angiogenesis. This is because VEGF is one of the growth factors with strong effects on endothelial cells [14], [15]. Activation entails circulating VEGF binding to tyrosine kinase receptors on endothelial cell surfaces; of these, VEGFR2 is the one responsible for regulating angiogenesis [16], [17], [18]. VEGFR2 is expressed in vascular endothelial progenitors [19], and its expression is up-regulated during wound healing [20].

Inadequate angiogenesis leads to loss of tissue function and necrosis of skin flaps. Several studies have examined topical or systemic exogenous VEGF used to improve wound healing processes and reduce flap failure during flap surgery [21], [22], [23]. Platelet-rich plasma (PRP) is an available alternative to exogenous VEGF therapy as a source of growth factors to stimulate angiogenesis. At present, however, no study has used bioluminescence imaging (BLI) to evaluate the effect of PRP on VEGFR2. PRP is a rich source of growth factors, has been found effective in accelerating significant tissue repair and regeneration, and releases massive quantities of platelet growth factors [24], [25]. It is a concentration of autologous platelets in a small volume of plasma containing several growth factors including epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), keratinocyte growth factor (KGF), platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-β) and vascular endothelial growth factor (VEGF) [26], [27]. The secretion of growth factors in PRP starts with platelet activation immediately following the addition of calcium, a necessary cofactor for degranulation of the alpha granules [28]. Recently the use of PRP gel in wound healing has become popular in nearly all fields of surgery, including orthopedic, thoracic and vascular surgery, as well as in dentistry and reconstructive oral maxillofacial surgery [25], [28], [29], [30], [31], [32].

Increased production of VEGF in ischemic tissues such as myocardial ischemia or ischemic retinopathy is well described [2], [3], [4], [15]. Since local hypoxia is an inducer of VEGF activity in wounds, and since PRP improves wound healing, it would be beneficial to study their combined use in flap survival and healing.

In vivo BLI in living transgenic mice has been employed for several years in widely ranging research fields, particularly in cancer research, where it is used to monitor the efficacy of drug treatment on the survival of implanted cancer cells or to analyze tumor metastasis [33], [34]. The technique allows non-invasive longitudinal imaging of markers of gene expression in vivo by luciferase-catalyzed reactions. However, the technique has not yet been used to study flap healing and flap survival.

For the present study, we used BLI to monitor the angiogenesis in a murine axial pattern skin flap experimental model designed to examine the effects of PRP and hypoxia in ischemic tissue during flap elevation and microsurgical anastomosis. Even though the beneficial effects of PRP and hypoxia have been described separately in several previous studies, no conclusive experimental therapeutic evidence is available regarding their combined use. Nor have VEGFR2 analyses been used to study their effects. BLI is a simple, quantifiable and non-invasive technique that enables longitudinal studies on transgenic mice. Whether BLI could be used to determine VEGFR2 expression levels during the flap healing process was unclear. In order to address these questions we performed our experiments on transgenic VEGFR2-luc mice, evaluated VEGFR2 activity and flap viability, and analyzed VEGF concentrations and newly formed blood vessels in tissue sections.

Section snippets

Surgical procedure

All animal experiments were conducted in accordance with the guidelines of the German animal protection statute and approved by the governmental review committee.

FVB/N-Tg(VEGFR2-luc)Xen homogenously gender-matched mice of 10–12 weeks of age were used in this study. The animals were housed in a special, temperature- and humidity-controlled, pathogen-free environment on a cycle of 12 h of light and 12 h of darkness, and were allowed free access to food and water.

After flap elevation, only those

Assessment of VEGFR2 gene activation during flap healing in vivo

In vivo longitudinal measurements on VEGFR2-luc mice were performed using an IVIS Lumina Imaging System 100 Series in order to study transcriptional activation of VEGFR2 and its translation into immunohistological vessel formation. To mimic neovascularisation by flap healing we performed an axial skin island flap on mice (Fig. 1). The main vascular network of the lateral thoracic artery and vein along with their abundant connections could be easily observed on the underface of this flap (Fig. 1

Discussion

Improved angiogenesis is known to facilitate flap healing and survival and thus to reduce flap loss. Local hypoxia in ischemic tissue triggers the hypoxia response element HIF-1 alpha, which results in enhanced production of VEGF [35], [36]. Autologous PRP may be capable of improving wound healing in that it releases growth factors such as VEGF, PDGF, FGF [26], [27], [37]. In this study, we investigated the clinical benefits of PRP and ischemia altered growth factors and genes, for which it has

Conclusions

In the present study our data have revealed that PRP and ischemia together produce synergistic therapeutic stimuli for improved angiogenesis by enhancing VEGFR2 and VEGF expression in a surgical wound. Thus, we can provide local application of PRP on the undersurface of a tissue flap following ischemic period can lead to a significant improvement of flap healing and flap survival levels. In conclusion, these results suggest that using PRP in microsurgical free tissue transfers might reduce the

Disclosures

The authors have no conflicts of interest to disclose.

Acknowledgments

We wish to thank Mr. Wolfgang Graulich for the drawing. Furthermore we thank S. Echterhagen, M. Nicolau, L. Shen and A. Rüben for their excellent technical assistance and Assoc. Prof. Cengizhan Acikel from FAVOR laboratories of Gülhane Military Medical Academy, Ankara. This study was supported in part by the Deutsche Forschungsgemeinschaft (DFG) (DFG No. PU 214/3-2: 4-2; 5-2) and by a grant from the Interdisciplinary Centre for Clinical Research (IZKF) within the faculty of Medicine at the RWTH

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