Elsevier

Cytotherapy

Volume 20, Issue 8, August 2018, Pages 1046-1060
Cytotherapy

Pericytes reduce inflammation and collagen deposition in acute wounds

https://doi.org/10.1016/j.jcyt.2018.06.011Get rights and content

Abstract

Background

Pericytes have been shown to have mesenchymal stromal cell–like properties and play a role in tissue regeneration. The goal of this study was to determine whether the addition of a pericyte sheet to a full-thickness dermal wound would enhance the healing of an acute wound.

Methods

Human muscle-derived pericytes and human dermal fibroblasts were formed into cell sheets, then applied to full-thickness excisional wounds on the dorsum of nu/nu mice. Histology was performed to evaluate epidermal and dermal reformation, inflammation and fibrosis. In addition, real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was used to determine cytokine response.

Results

Pericytes were detected in the wounds until day 16 but not fibroblasts. Decrease in wound size was noted in pericyte sheet-treated wounds. Enhanced neo-vascularization and healthy granulation tissue formation were noted in the pericyte-treated wounds. Expression of type I collagen messenger RNA (mRNA) was significantly higher in the fibroblast-treated group, whereas Type III collagen mRNA showed significant increase in the pericyte group at days 3, 6 and 9 compared with the fibroblast and no-cell groups. Trichrome staining revealed thick unorganized collagen fibrils in the fibroblast-treated wounds, whereas pericyte-treated wounds contained thinner and more alligned collagen fibrils. Tumor necrosis factor (TNF)-α mRNA levels were increased in the fibroblast-treated wounds compared with pericyte-treated wounds.

Discussion

The addition of pericytes may confer beneficial effects to wound healing resulting in reduced recruitment of inflammatory cells and collagen I deposition, potential to enhance wound closure and better collagen alignment promoting stronger tissue.

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

References (79)

  • M Ritsu et al.

    Critical role of tumor necrosis factor-alpha in the early process of wound healing in skin

    J Dermatol Dermatol Surg

    (2017)
  • SI Motegi et al.

    Mesenchymal stem cells: The roles and functions in cutaneous wound healing and tumor growth

    J Dermatol Sci

    (2017)
  • AI Caplan

    All MSCs are pericytes?

    Cell Stem Cell

    (2008)
  • MA Konig et al.

    direct transplantation of native pericytes from adipose tissue: A new perspective to stimulate healing in critical size bone defects

    Cytotherapy

    (2016)
  • YC Lin et al.

    Evaluation of a multi-layer adipose-derived stem cell sheet in a full-thickness wound healing model

    Acta Biomater

    (2013)
  • SJ Dalton et al.

    Mechanisms of chronic skin ulceration linking lactate, transforming growth factor-beta, vascular endothelial growth factor, collagen remodeling, collagen stability, and defective angiogenesis

    J Invest Dermatol

    (2007)
  • V Falanga

    Wound healing and its impairment in diabetic foot

    Lancet

    (2005)
  • T Yamamoto et al.

    Effect of interleukin-10 on the gene expression of type I collagen, fibronectin, and decorin in human skin fibroblasts: differential regulation by transforming growth factor-beta and monocyte chemoattractant protein-1

    Biochem Biophys Res Commun

    (2001)
  • L Zimmerlin et al.

    Human adipose stromal vascular cell delivery in a fibrin spray

    Cytotherapy

    (2013)
  • D-S Chae et al.

    Stromal vascular fraction shows robust wound healing through high chemotactic and epithelialization property

    Cytotherapy

    (2017)
  • DM Minteer et al.

    Adipose stem cells: biology, safety, regulation, and regenerative potential

    Clin Plast Surg

    (2015)
  • BD Humphreys et al.

    Fate tracing reveals the pericyte and not epithelial origin of myofibroblasts in kidney fibrosis

    Am J Pathol

    (2010)
  • RG Rosique et al.

    Curbing Inflammation in Skin Wound Healing: A Review

    Int J Inflam

    (2015)
  • S1 Guo et al.

    Factors affecting wound healing

    J Dent Res

    (2010)
  • FR Helmo et al.

    Fetal wound healing biomarkers

    Dis Markers

    (2013)
  • TA Järvinen et al.

    Targeted Antiscarring Therapy for Tissue Injuries

    Adv Wound Care (New Rochelle)

    (2013)
  • RJ Bodnar et al.

    Pericytes: A newly recognized player in wound healing

    Wound Repair Regen

    (2016)
  • SJ Mills et al.

    Pericytes, mesenchymal stem cells and the wound healing process

    Cells

    (2013)
  • CN Hall et al.

    Capillary pericytes regulate cerebral blood flow in health and disease

    Nature

    (2014)
  • D Proebstl et al.

    Pericytes support neutrophil subendothelial cell crawling and breaching of venular walls in vivo

    J Exp Med

    (2012)
  • V Nehls et al.

    The versatility of microvascular pericytes: from mesenchyme to smooth muscle?

    Histochemistry

    (1993)
  • A Dar et al.

    Multipotent vasculogenic pericytes from human pluripotent stem cells promote recovery of murine ischemic limb

    Circulation

    (2012)
  • CW Chen et al.

    Human pericytes for ischemic heart repair

    Stem Cells

    (2013)
  • TS Park et al.

    Placental perivascular cells for human muscle regeneration

    Stem Cells Dev

    (2011)
  • M Crisan et al.

    Perivascular cells for regenerative medicine

    J Cell Mol Med

    (2012)
  • VL Bautch

    Stem cells and the vasculature

    Nat Med

    (2011)
  • CS Lin et al.

    Defining vascular stem cells

    Stem Cells Dev

    (2013)
  • A Blocki et al.

    Not all MSCs can act as pericytes: functional in vitro assays to distinguish pericytes from other mesenchymal stem cells in angiogenesis

    Stem Cells Dev

    (2013)
  • S Paquet-Fifield et al.

    A role for pericytes as microenvironmental regulators of human skin tissue regeneration

    J Clin Invest

    (2009)
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