Emerging Insights On Mesenchymal Stem Cells And Wound Healing

Research has suggested mesenchymal stem cells can effectively enhance wound healing, helping repair and regenerate damaged tissue. These authors examine how these stem cells can positively affect the wound environment and review the research on potential delivery systems.

In the United States, chronic wounds of the lower extremity affect roughly 5.7 million people with an estimated financial burden on healthcare systems approaching $20 billion annually.¹ In addition to the significant cost, the impacts on patients include physical and emotional stress, limited mobility and decreased productivity.² More concerning, a chronic lower extremity wound precedes up to 85 percent of all lower extremity amputations, suggesting that failure to heal these wounds in a timely manner can result in dire consequences.³

Normal wound healing requires a precise series of coordinated events that is often impaired in the chronic wound. In recent years, research has expanded our understanding of the biochemical deficiencies involved in the chronic wound with strategies targeting these shortcomings in the form of advanced wound care products. These therapies include growth factor supplementation, scaffolding with extracellular matrices and stem cell therapy.

Research has shown stem cells are successful in repairing damaged cardiac, lung, liver, brain and kidney tissue, and research is showing equally promising results in wound care.⁴ Therapeutic benefits of mesenchymal stem cells in other organs include repair and regeneration of tissue damaged by injury or disease. There is wide variability in the mechanism of repair of other organs, showing the expanse of stem cell application. Currently, there is no stem cell-only biologic graft in use for wounds. However, there are advanced wound care products that contain a combination of growth factors, stem cells or have stem cell-like properties by recruiting growth factors and cytokines to the wound bed and stimulating healing. In both in vitro and in vivo studies, these advanced wound care products have exhibited safe and effective means for treating chronic wounds.^5,6

How Mesenchymal Stem Cells Can Kick-Start The Healing Process

Stem cells can differentiate into multiple cell lineages and have the capacity of self-renewal, which make them appealing for the healing process. We can categorize stem cells into embryonic stem cells and adult stem cells based on their origin of lineage. Due to the ethics and legality related to embryonic stem cells, much of the research regarding stem cells in the realm of wound care involve adult stem cells, specifically mesenchymal stem cells.⁷  

Mesenchymal stem cells, also known as multipotent stromal progenitor cells, possess the unique ability to differentiate into several different cell types, including osteoblasts, adipocytes and chondrocytes. These cells can be isolated from different tissues, including bone marrow, adipose tissue and the umbilical cord. The most commonly studied mesenchymal stem cells derive from bone marrow and adipose tissue due to the relative ease of obtaining these cells. Recent studies have demonstrated the ability of mesenchymal stem cells to reduce and repair tissue damage after injury to organs such as the heart, lung, kidney, liver, brain and skin in the setting of chronic wounds. Additionally, human and animal models have demonstrated the safety of use for both allogeneic and autologous mesenchymal stem cells.⁷  

Current studies on mesenchymal stem cells in wound healing, both human and animal studies, are focusing on the mechanisms and level of action in healing wounds.⁸ Differentiation, immune modulation and paracrine signaling are some proposed and studied mechanisms of action of mesenchymal stem cells in the wound healing process. Researchers have identified mesenchymal stem cell activity in each phase of the wound healing process through paracrine signaling, which is the release of biologically active molecules that affect the migration, proliferation and survival of cells, and the surrounding environment.

In addition to modulation of the wound environment through paracrine signaling, mesenchymal stem cells may also decrease inflammatory cytokines while increasing anti-inflammatory cytokines in the wound, thus helping to stimulate stalled chronic wounds beyond the inflammatory phase. In a study evaluating wound healing in mice injected with bone marrow-derived mesenchymal stem cells, there was increased secretion of cytokines in comparison with normal dermal fibroblasts, resulting in enhanced wound healing.⁵

Mesenchymal stem cells also act as chemoattractants during proliferation for macrophages, endothelial cells, epidermal keratinocytes and dermal fibroblasts, contributing to accelerated wound closure.⁸ With further secretion of growth factors, the remodeling phase accelerates by increasing collagen deposition and decreasing scar formation.⁹ In a study of diabetic mice, engrafted stem cells improved healing with increased expression of vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and Wnt3a.¹⁰ Wnt3a is a stem cell proliferation and transmigration factor, and has been associated with increased proliferation and migratory capacity of stems cells in vivo. This study also found increased host stem cell recruitment to the wound, which could be related to the Wnt3a expression. This elevation of growth factors persisted even after the engrafted mesenchymal stem cells were no longer present in the wound, suggesting the wound healing effects may persist in the absence of engrafted mesenchymal stem cells.

Throughout the wound healing process, mesenchymal stem cells theoretically alter the host immune cell by releasing immunosuppressive factors that prevent the proliferation of T cells, B cells and other cells that propagate inflammation in the chronic wound. Through the inhibition of these immune cells, mesenchymal stem cells create an environment with a decreased immune response, allowing the stalled chronic wounds to progress beyond the inflammatory phase.⁹ One such inflammatory cytokine, interleukin-6 (IL-6), is in excess in many chronic disease states, including chronic wounds.⁵ Treatment of wounds with bone marrow-derived stem cells resulted in a marked decrease of IL-6 levels in comparison to levels of dermal fibroblasts.⁵

Recognizing The Revascularization Potential Of Mesenchymal Stem Cells

Revascularization of the wound bed is an important element of the wound healing process and mesenchymal stem cells contribute by differentiating into endothelial cells for creation of new vessels and secretion of factors that promote and regulate angiogenesis. The larger role of mesenchymal stem cells in angiogenesis in theory is due to paracrine effects, producing several pro-angiogenic factors such as VEGF, angiopoietin-1 (Ang-1) and hepatocyte growth factor.¹¹ Specifically, VEGF is associated with stimulating endothelial cell proliferation, migration and organization into tubules, and increasing endothelial progenitor cells while Ang-1 is associated with new vessel maturation into more complex and large vascular structures.¹²

In a study comparing diabetic rat models, those treated with implanted bone marrow-derived mesenchymal stem cells had increased capillary density.¹² This study also investigated the paracrine effects of the stem cells and found higher levels of VEGF and angiopoietin-1 in the tissues treated with stem cells. This suggests that bone marrow-derived stem cells release pro-angiogenic factors, which could be contributing to the enhanced angiogenesis in these wounds.

Another study in ischemic murine skin found that with the injection of mesenchymal stem cells, there was decreased arteriolar vascular resistance and an increase in functional capillary density in the tissue with critical ischemia.¹³ The authors attributed these results to the paracrine expression of pro-angiogenic growth factors rather than the mesenchymal stem cells being taken up by the tissue or embedded into the wound.  

In a subsequent human study, patients with diabetes, critical limb ischemia and a foot ulcer received intramuscular injections of mesenchymal stem cells, injections of mononuclear cells or injections of normal saline.¹⁴ Authors were investigating and comparing the efficacy and safety of the application of autologous bone marrow-derived mesenchymal stem cells. The patients injected with the mesenchymal stem cells had higher levels of VEGF and basic fibroblast growth factor (bFGF) than patients treated with mononuclear cells. Researchers also found the patients treated with mesenchymal stem cells had faster time to walking without pain and decreased time to healing of the ulcers, suggesting improvement in the limb ischemia. The results associated with improved limb perfusion persisted in those patients treated with mesenchymal stem cells as they had an improved ankle brachial index, transcutaneous oxygen pressure and magnetic resonance angiography in comparison to those who received mononuclear cells and normal saline.¹⁴ These studies show that mesenchymal stem cells contribute to the process of angiogenesis, lending support to the use of mesenchymal stem cells in wound healing.

How Mesenchymal Stem Cells Can Assist With Remodeling And Scarring

In addition to paracrine signaling and angiogenesis, mesenchymal stem cell application can assist with remodeling and scarring. In normal wound healing, keratinocytes, fibroblasts and endothelial cells migrate within the wound to ensure appropriate deposition into the extracellular matrix with the proper proliferation of cells to achieve full healing.⁵ Growth factors and chemokines are crucial in recruiting these cells, and ensuring their proliferation and survival within the wounds.  

In the scarring process of chronic wounds, the normal sequence of events and the altered wound environment lead to extra deposition of the extracellular matrix, resulting in increased fibrosis and scarring. Studies have shown that the immunomodulatory effects of mesenchymal stem cells can limit fibrosis and therefore decrease scarring.¹⁵ In mice models, the application of mesenchymal stem cells decreased inflammation within the wound, therefore decreasing the fibrosis associated with scarring. This study also found decreased transforming growth factor beta (TGF-b), a fibrogenic cytokine and increased matrix metalloproteinase together decreasing the fibrosis process in the healing of wounds.

Another study investigated an umbilical cord-derived mesenchymal stem cell conditioned medium and its effects on adult dermal fibroblasts in vitro and in vivo.¹⁶ The results found reduced transforming growth factor beta in an mesenchymal stem cell-cultured medium, resulting in decreased myofibroblasts at wound sites. This suggests that mesenchymal stem cells have antifibrotic properties leading to decreased scar contractures. This study also found increased collagen type III and decreased type I collagen in adult fibroblasts treated with mesenchymal stem cells, showing that mesenchymal stem cells can help regulate the ratios of collagen types within a healing wound.

Studies, in vitro and in vivo, have demonstrated mesenchymal stem cells can differentiate into keratinocytes, fibroblasts and adipocytes that localize and fill the wound bed.¹² Research has also shown that mesenchymal stem cells increase the thickness of the regenerated epidermis along with increased dermal ridges and number of cells in the new skin with a more regular alignment of fibers, giving greater strength to the healed skin.¹⁷ These results suggest that wounds healed with mesenchymal stem cells may have better composition, increased strength and decreased chances of recurrence.

A Closer Look At Methods Of Effective Delivery Of Stem Cells

With all the benefits stem cells have shown in wound healing, there is still much debate related to the ideal application of stem cells in the wound setting to ensure adequate dosing and delivery of mesenchymal stem cells.

Direct topical application, sprays, scaffolds, patches and injections are all proposed vehicles for the application of the mesenchymal stem cells to the wound or patient. Topical application of mesenchymal stem cells has been associated with improved wound healing in diabetic mice with increased reepithelialization, granulation tissue formation and neovascularization.¹⁸ In a canine model, topically applied  mesenchymal stem cells to full-thickness wounds decreased inflammation with decreased pro-inflammatory cytokines and increased healing through increased collagen synthesis, cellular proliferation and angiogenesis.¹⁹

An extracellular matrix patch made from small intestine mucosa embedded with adipose-derived mesenchymal stem cells had increased survival and localization in an excisional wound model in mice.²⁰ The healed wounds treated with the stem cell patch also had less scarring upon healing. In comparison to the injection of mesenchymal stem cells, stem cells delivered in a hydrogel scaffold to wounds demonstrated accelerated wound healing and increased survival in wounds in a dermal wound microenvironment.²¹ Also, the wounds treated with the mesenchymal stem cell hydrogel scaffold had increased angiogenesis, increased levels of vascular endothelial growth factor and other cytokines. Hydrogels appeared to augment the function of mesenchymal stem cells and enhance the wound healing process.²¹

A fibrin spray system to deliver mesenchymal stem cells to the wound was safe in one study in human and animal wounds.²² Through this application, mesenchymal stem cells were able to migrate and establish themselves into the wound bed. The wounds treated with the mesenchymal stem cell fibrin spray also had accelerated wound healing in the mouse model.

Dash and coworkers found that injection of mesenchymal stem cells at the edges of the wounds in patients with diabetes or vasculitis accelerates wound healing.²³ Injected stem cells into femoral veins in rats promoted wound healing with increased granulation tissue formation, angiogenesis, cellular proliferation and high levels of growth factor secretion. Shi and colleagues also found that the stem cells can migrate to the site of the ulcer, most likely signaled by the inflammation and injury at the ulcer site.²⁴

Although researchers have not identified any one delivery system as superior, all systems have been safe and effective means for facilitating the role of mesenchymal stem cells in wound healing. These delivery models can contribute to identifying a successful stem cell biologic in the future.

Where Further Investigation On Mesenchymal Stem Cells Should Focus

Mesenchymal stem cells have promising results related to wound healing but there are still aspects of their creation, use and application that need further investigation. Mesenchymal stem cells have a high variability of viability within the wound. Studies have shown that mesenchymal stem cells do not survive in the wound for an extended period of time and, on some occasions, were not engrafted by the wound at all.^6,11,20 This may not be a large barrier to mesenchymal stem cell success due to the role of paracrine signaling associated with mesenchymal stem cells but is a perceived benefit to cellular persistence over time.

We need to investigate strategies to increase survival of the mesenchymal stem cells in the wound or further expand the paracrine effects of mesenchymal stem cells. The wound environment may also hinder the success of mesenchymal stem cells. We know wound tissue is hypoxic because of the decreased blood supply and uptake of oxygen by local cells. Research has shown that mesenchymal stem cells stimulate an increased release of growth factors and cytokines in hypoxic conditions. Increased cell proliferation, neovascularization and recruitment of macrophages are present in hypoxic conditions as well.⁹ For mesenchymal stem cells to gain their full potential in wounds, researchers would need to identify the environment in which they thrive most.   

The source of mesenchymal stem cells is also a limitation. Although mesenchymal stem cells from bone marrow, adipose tissue and other sources have promising results, many differences in the means of obtaining and cell proliferation are still present. Stem cells can derive from both autogenic and allogenic sources. Research has shown that a host response typically does not occur in the presence of allogenic mesenchymal stem cells due to their low immunogenicity and immunosuppressive features, which is beneficial in an immunocompromised patient or one with chronic disease.¹¹

A more unified protocol would need to arise for stem cell harvest and proliferation. No guidelines have been established for the retrieval, expansion, use or development of stem cells for therapeutic purposes. More research needs to be completed to further establish the mechanism of action and function of mesenchymal stem cells within wounds at different stages of the healing process. Current research is aiming to address many of these concerns with mesenchymal stem cells and once further understanding is achieved, mesenchymal stem cells may become more common in the practice of chronic wound healing in podiatry.

Dr. Iosue is a Clinical Fellow in Surgery at Harvard Medical School and a second-year resident at the Beth Israel Deaconess Medical Center in Boston.

Dr. Dinh is an Assistant Professor of Surgery at Harvard Medical School. She is the Program Director of the Podiatry Surgical Residency Program at the Beth Israel Deaconess Medical Center in Boston. Dr. Dinh is a Fellow and member of the Board of Directors for the American College of Foot and Ankle Surgeons.

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