Pericytes reduce inflammation and collagen deposition in acute wounds
Introduction
Skin protects the body against environmental influences and prevents water loss. The loss of this barrier through burns, trauma and surgical excision requires the elementary processes of tissue repair for any organism to survive. Wound healing is a natural adaptive response to tissue injury. However, it remains a big challenge to enhance the healing of chronic ulcers and to prevent scarring in acute traumatic wounds [1]. In skin wounds of healthy adults, the barrier function of skin is efficiently restored; however, repair of deep dermal structures culminates in scar formation with loss of the original tissue structure and function. Wounds that exhibit impaired healing frequently enter a state of pathological inflammation due to a delayed, incomplete or uncoordinated healing process [2]. Although no two wounds are the same, there is a commonality in the dysfunction of specific factors promoting the formation of chronic wounds and scars. Studies of fetal skin wounds and oral mucosa that heal exceptionally quickly and with low/no scar formation show a reduction in inflammation and minimal fibrosis [3].
The ability to encourage cutaneous injuries to heal with limited to no scarring would significantly enhance healing of lacerations, incisions and burns, reducing morbidity and mortality rates. Current treatments only slightly improve tissue regeneration and integrity, resulting in a need for the development of new therapies to enhance wound healing [4], [5]. One promising approach to enhance tissue regeneration and reduce fibrosis is the use of pericyte-based therapies [6], [7].
Pericytes (mural cells or Rouget cells) are cells that reside on the outer surface of the microvasculature. The cells tightly encircle capillaries, arterioles and venules, regulating microvascular physiology, blood flow and inflammatory cell trafficking [8], [9]. In addition to their ability to promote vessel stabilization and maturation, detachment from the vessel promotes dedifferentiation into a stem-like progenitor cell [10]. Pericytes are thought to be of mesodermal origin and display differentiation potential. They have been found to exhibit multi-lineage potential, having the ability to differentiate into adipocytes, chondrocytes and myoblasts [11], [12]. However, the heterogeneity of pericytes has led to questions regarding the origins of these cells from different organs. Pericytes purified from muscle, adipose, placenta and other organs have been shown to repair and regenerate damaged or defective tissues [13], [14], [15], [16]. Thus, pericytes share similarities to mesenchymal stromal cells (MSC) and have been hypothesized to be the in vivo counterpart of MSCs [16], [17], [18]. A study by Blocki et al. showed that pericytes represented a subpopulation of MSCs and that pericyte behavior is not intrinsic to all MSCs [19]. Additionally, a study by Chen et al. (2013) showed that pericyte homing to perivascular sites may support the long-term survival of the pericytes and thus enhance overall outcome [13].
In the central nervous system (CNS), microvascular pericytes are thought to have macrophage-like properties and possess the ability to perform at least some immune function [20], [21]. Pericytes are thought to play a role in regulating cell activation including T cells and neutrophils. Their interactions with macrophages and fibroblasts may foster vascular expansion and granulation tissue formation [22]. Also, pericytes have been shown to signal to keratinocytes to promote re-epithelialization [23]. In excessive fibrosis, it has been suggested that a population of pericytes may migrate into the perivascular space and develop into collagen-synthesizing fibroblasts [24].
Pericytes represent a potent cell population in the skin that can regulate the skin microenvironment, promoting wound healing, vascularization and cellular activation [6]. Therapeutic use of pericytes has shown the ability to enhance muscle regeneration and improve the repair of lung, kidney, cardiac and CNS tissues [25], [26]. These studies demonstrate a novel use for pericytes in tissue repair. Pericyte interaction with inflammatory, dermal and epidermal cells makes them an enticing therapeutic candidate for various type of acute wounds and non-healing chronic wounds. The contributions of pericytes to skin tissue repair and regeneration are not well understood. To determine the direct influence of transplanted pericytes on the wound environment, we developed a cell sheet free of biomaterials that contains a homogenous cell type composed of either fibroblasts or pericytes with an extracellular matrix protein secreted from the cells. This exogenous scaffold-free cell system can be directly transplanted to the wound site without additional factors. Cell sheets derived from cell populations other than pericytes have been applied to a variety of tissues including the skin, cornea, myocardium, blood vessels and muscle but have not had significant or consistent positive outcomes [27]. This study provides new evidence that a pericyte cell sheet enhances the acute wound-healing process. Herein we show a one-time application of a pericyte sheet to full-thickness wounds significantly changes the wound bed by altering the inflammatory, cytokine and collagen profile, enhancing the wound-healing process and reducing fibrosis.
Section snippets
Preparation of cell sheets
Hs68 fibroblasts were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco, ThermoFisher Scientific Inc.) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific Inc.). Human muscle-derived pericytes [11] were a kind gift from Dr. Bruno Peaúlt (University of California, Los Angeles, Los Angeles, California, USA) and were grown in pericyte growth medium (ZenBio, Inc.). Pericytes used in all experiments were between passage five and seven. The protocol for cell sheet
Histology
The entire wound/scar bed with a border of normal skin was excised from the animal at each designated time point (post wound days 3, 6, 9 and 16) and one half of the wound was fixed in 10% neutral buffered formalin for 3 days. The fixed tissue was embedded in paraffin for subsequent histological evaluation. Five-micron paraffin sections were stained with hematoxylin and eosin (H-E) and Masson's trichrome to determine inflammation and collagen content. The presence of blood vessels was confirmed
Wound area measurements
Digital images were captured on post-wound days 3, 6, 9 and 16 using a Nikon CoolPix AW120 camera. All images were taken with the camera placed at the same distance and the same angle (90° perpendicular) relative to the wound. A metric ruler was positioned in the plane of the wound during image capture for later processing and calibration of the images to determine the extent of wound contraction. Wound area measurements of treated and untreated groups were calculated using NIS-Elements AR3.1
Inflammatory cell scoring
A semi-quantitative inflammatory cell scoring system was developed and performed by a board-certified veterinary pathologist (L.H.R.) to compare inflammatory infiltrates within H-E–stained sections. The pathologist was blinded to the experimental group. Each cell type was individually analyzed, and a subjective score was given, taking into consideration all cells within the histological section. Only viable cells were included in score analysis. Evaluations were made by scoring a minimum of ten
RNA extraction
Wound biopsies collected and stored in RNA Later (Ambion) on days 3, 6, 9 and 16 were subjected to RNA extraction. Samples were homogenized, and total RNA was isolated using the RNeasy Mini Kit (Qiagen Inc.) following manufacturer's instructions. The concentration of the extracted total RNA was measured using the Infinite 200 PRO spectrophotometer (Tecan). The quality of RNA extracted was determined using capillary electrophoresis using an Agilent 2100 BioAnalyzer and a Nano6000 RNA chip. All
Quantitative real-time RT-PCR
The protocols for reverse transcription and real-time RT-PCR were performed as previously described [29], [30]. In brief, reverse transcription was performed using 225 ng of total RNA with Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV)-reverse transcriptase (Invitrogen) with random primers at a concentration of 150 ng/μL. Real-time RT-PCR amplification and detection of template were carried out using Applied Biosystems PRISM 7900HT system using Applied Biosystems Taqman
Statistical analysis
Statistical analysis was performed using Student t test to determine the statistical significance between the groups. P < 0.05 was considered statistically significant. Two groups were compared at a time to determine the significance.
Transplanted pericyte cell sheets persist in the wounded skin
To understand if pericytes play a role in modulating the wound environment to enhance wound closure and healing, we used human muscle-derived pericyte cell sheets as a cell graft to enhance deep dermal wound healing. To verify the purity of the pericyte cell culture, cells were stained for CD146, a well-established pericyte cell marker. As seen, all the pericytes stain positive for CD146, indicating a homogenous pericyte (CD146+) population (Figure 1). In addition, pericytes were also stained
Discussion
Managing wound healing represents one of the most relevant clinical burdens in the world. The management of scarring and fibrosis remains a major issue in wound healing, representing a staggering $12 billion annual market [42], thus optimizing current therapies is critical. Various cell-based therapies such as cultured skin grafts using keratinocytes, epithelial cells and fibroblasts have been developed but with limited success [43], [44]. A current strategy has been to use MSCs for tissue
Acknowledgments
We extend our thanks to the Department of Plastic Surgery, University of Pittsburgh, for their funding support to complete this study. We extend our thanks to Kacey Marra PhD, Department of Plastic Surgery, University of Pittsburgh, for her technical expertise in preparing the cell sheets. Our thanks to the research histology core facility at the University of Pittsburgh for their service in completing the histological stains. Our thanks to Shriners Hospitals for Children-Cincinnati for their
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