Elsevier

Matrix Biology

Volume 29, Issue 8, October 2010, Pages 690-700
Matrix Biology

Extracellular matrix-derived products modulate endothelial and progenitor cell migration and proliferation in vitro and stimulate regenerative healing in vivo

https://doi.org/10.1016/j.matbio.2010.08.007Get rights and content

Abstract

Most adult mammals heal without restorative replacement of lost tissue and instead form scar tissue at an injury site. One exception is the adult MRL/MpJ mouse that can regenerate ear and cardiac tissue after wounding with little evidence of scar tissue formation. Following production of a MRL mouse ear hole, 2 mm in diameter, a structure rapidly forms at the injury site that resembles the amphibian blastema at a limb amputation site during limb regeneration. We have isolated MRL blastemal cells (MRL-B) from this structure and adapted them to culture. We demonstrate by RT-PCR that even after continuous culturing of these cells they maintain expression of several progenitor cell markers, including DLK (Pref-1), and Msx-1. We have isolated the underlying extracellular matrix (ECM) produced by these MRL-B cells using a new non-proteolytic method and studied the biological activities of this cell-free ECM. Multiplex microELISA analysis of MRL-B cell-free ECM vs. cells revealed selective enrichment of growth factors such as bFGF, HGF and KGF in the matrix compartment. The cell-free ECM, degraded by mild enzyme treatment, was active in promoting migration and proliferation of progenitor cells in vitro and accelerating wound closure in a mouse full thickness cutaneous wound assay in vivo. In vivo, a single application of MRL-B cell matrix-derived products to full thickness cutaneous wounds in non-regenerative mice, B6, induced re-growth of pigmented hair, dermis and epidermis at the wound site whereas scar tissue replaced these tissues at wound sites in mice treated with vehicle alone. These studies suggest that matrix-derived products can stimulate regenerative healing and avert scar tissue formation in adult mammals.

Introduction

The MRL/MpJ wild type strain of mice (MRL) was originally generated by interbreeding C57BL/6 J (0.3%), C3H/HeDi (12.1%), AKR/J (12.6%) and LG/J (75%) strains (Theofilopoulos and Dixon, 1985, Clark et al., 1998). MRL mice at particular sites of the body exhibit increased wound healing capacity as compared to other inbred strains (Heber-Katz, 1999, Samulewicz et al., 2002). For example, ~ 4 weeks after receiving a 2 mm ear hole injury, MRL mice showed ~ 80% reduction in ear hole size whereas C57BL/6 mice only exhibited a 36% closure (Samulewicz et al., 2002). Histological examination revealed that the process of healing in MRL mice was faster and more complete than that of the C57BL/6 mice and exhibited parallels to amphibian regeneration, including the formation of a blastemal structure, a dynamic extracellular matrix, re-appearance of cartilage and hair follicles, healing without scarring, and up-regulation of tenascin, Pref-1 and down-regulation of MSX-2 in cells present within the blastema (Clark et al., 1998, Gourevitch et al., 2003). MRL mice also have the ability to heal burn-induced infarct of the ventricle with restoration of normal structures and function of the heart and reduced cardiac scarring, a finding that does not occur in other adult mice such as C57BL/6 mice (Leferovich et al., 2001, Leferovich and Heber-Katz, 2002). During ear hole healing in MRL mice, a blastemal-like structure forms as early as day 4 post-injury in the form of a swelling along the circumference of the wound (Heber-Katz, 1999). We have isolated cells from this MRL blastemal structure (MRL-B) on day 18 post injury, adapted the cells to culture and characterized the cells. The present work focused on the isolated cell-free matrix of these cells and its ability to reduce scar tissue formation and stimulate regenerative healing.

An intact ECM is present in all tissues and organs and it is secreted by the tissue cells to provide a physical bioscaffold for cellular support. The ECM is a complex mixture of molecules comprised of collagen, elastic fibers, heparin, proteoglycans, as well as associated non-matrix molecules such as growth factors (Brown et al., 2006, Badylak, 2007, Paige et al., 1991). The ECM is known to serve as a natural reservoir of these growth factors and other signaling molecules. Thus, the ECM functions as a physical support for cells; but it is also in a state of continuous remodeling catalyzed by the rate limiting degradation enzymes, the matrix metalloproteinases (MMPs). Remodeling of the ECM is involved in survival, migration and proliferation of many cell types and has been implicated in the wound healing response. Recently the ECM has been shown to have important roles in the normal and abnormal response of cells and tissues to injury (Chen et al., 1999, Armour et al., 2006). ECMs from such decellularized tissues and organs as the small intestine (Badylak et al., 1995, Kropp et al., 1995), liver (Brown et al., 2006), urinary bladder (Chen et al., 1999), arterial vasculature, heart valves (Cebotari et al., 2006), heart, and dermis (Armour et al., 2006) have been used as biological scaffolds and have also been shown to promote reconstruction of injured tissue and facilitate tissue remodeling and healing processes (Sclamberg et al., 2004, Badylak et al., 2005, Badylak, 2007). It has been noticed that scaffold degradation is necessary for the realization of these accelerated processes (Badylak, 2007). The heparin found within ECMs has been shown to bind to and protect growth factors from degradation caused by heat, trypsin or chymotrypsin digestion, as well as highly basic or acid pH changes in the wound environment (Onda et al., 1990). Thus, during matrix remodeling, numerous stored matrix-associated molecules are mobilized. Previous studies have shown that matrix-derived molecules produced in the course of acid, heat and enzymatic treatments of ECM retain bioactivity and can go on to affect angiogenesis (Li et al., 2004), cell migration (Li et al., 2004, Badylak et al., 2001, Reing et al., 2009) and proliferation (Reing et al., 2009) and possess antimicrobial activity (Brennan et al., 2006, Sarikaya et al., 2002). The present study focused on products derived from an ECM produced by cells isolated from the MRL blastema and their ability to evoke tissue regeneration.

Section snippets

Materials and methods

All animal study protocols were in compliance with the University of Massachusetts guidelines and the National Research Council's criteria for the humane care of animals and were conducted using approved IACUC procedures.

Ear hole closure and isolation of blastemal cells from MRL wounds

It has previously been reported that MRL mice will close a 2.0 mm ear hole punch over a 30–40 day period (Clark et al., 1998, Samulewicz et al., 2002). During the course of ear hole closure, we noted the formation of an avascular structure that others have described as a blastema-like structure. We produced ear holes in MRL (panel A–D) vs. B6 (Panel E–H) mice and monitored the formation of a blastemal like structure (*) and ear hole closure over a 64 day time course (Fig. 1). We noted the

Discussion

MRL mice have the ability to replace numerous tissues and organs, including ear and heart tissue, without scarring (Clark et al., 1998, Heber-Katz, 1999, Leferovich et al., 2001, Colwell et al., 2006). In the course of the present study, we observed an MRL blastemal like structure of the ear as a distinct region apparently devoid of new blood vessels during the regenerative process. This blastema like structure is pronounced in regenerating but not seen in non-regenerating strains of mice at

Acknowledgements

The work described was funded by an award from the Defense Advanced Research Program Agency, award # W911NF-06-1-0067.

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