The effect of adipose tissue derived MSCs delivered by a chemically defined carrier on full-thickness cutaneous wound healing
Introduction
Worldwide 200 million difficult-to-treat chronic wounds in elderly individuals suffering from diabetic foot ulcers [1], pressure ulcers [2], and chronic venous leg ulcers [3] among others present an increasing socioeconomic burden and a currently unmet challenge for societies – Demographic developments suggest this will increase. In contrast to acute wounds, chronic wounds fail to progress through the normal pattern of wound repair which involves inflammation, granulation tissue formation and remodelling [4], but instead remain in a chronic inflammatory state with little signs of healing [3], [5], [6]. Common effector molecules produced from highly activated M1 macrophages, such as enhanced concentrations of tumour necrosis factor-alpha (TNF-α), play a critical in chronic wounds [7]. Scarring is another problem with poor wound healing and skin scars can range from barely visible fine white lines to major scars and keloids and also cause physical morbidity and psychological suffering and affect the quality of life of these patients [8].
There is an increasing interest in exploiting the beneficial anti-inflammatory and trophic effects of multipotent mesenchymal stem cells (MSCs) [9] to repair and regenerate non-haematopoietic tissues [10], [11], [12]. Bone marrow derived MSCs were first discovered in the 1970s [13]. Since then, MSCs have been isolated from a variety of other tissues (for a review see [14]) among them adipose tissue [15]. MSCs of different origins are endowed with the potential to differentiate into a wide variety of histogenetically distinct cell types and thus contribute to tissue repair [16]. MSCs in addition constitute the stromal niche which supports haematopoietic stem cells maintenance/differentiation and vascularisation [17], [18].
In recent studies on rodents, MSCs have been well documented to enhance cutaneous wound healing with reduced scar formation, enhanced wound closure and restoration of the skin tensile-strength [16], [19], [20], [21], [22]. In a proof of principle study on 3 patients by Falanga and coworkers [23] bone marrow aspirates were topically applied to chronic wounds which had not healed for years and resulted in complete healing. While their anti-inflammatory and regenerating properties make MSCs a highly attractive cell source for the treatment of chronic human wounds, there is a need to develop an efficient, safe and painless delivery method for applying these cells to the wound site in vivo suitable for routine clinical use. Most studies have used systemic intravenous or local subcutaneous or intraperitoneal injections, procedures which are invasive, painful and simply not convenient for any routine clinical service. Falanga and colleagues delivered bone marrow MSCs in a fibrin polymer spray, which requires a double-barrelled syringe containing fibrinogen and thrombin to deliver cells in a polymerized gel [24]. Unfortunately, fibrin spray contains blood products, which potentially induce allergic reactions in recipient patients, requires complex preparation and has a short working life [24].
The problem of delivering cultured cells from the laboratory to the clinic was solved for keratinocytes [25], [26] using a chemically defined carrier consisting of medical-grade silicone coated by plasma polymerisation with a very thin layer acrylic acid (ppAAc). This was used to deliver autologous keratinocytes to patients suffering from burns injuries [27] and chronic ulcers [28], [29]. More recently this synthetic carrier approach was extended to co-cultures of melanocytes and keratinocytes [30]. Using an in vitro human wound bed model of de-epidermized acellular demis (DED), almost all cells grown on the carrier were shown to transfer to DEDs within 48 h [31]. Very recently the in vitro delivery of bone marrow derived MSCs from ppAAc carriers to DED was reported [32]. In addition, the issue of cell transport on these carriers from culture laboratories to patients who may be at geographically distant locations was addressed as the functional properties of keratinocyte-melanocyte co-cultures were fully maintained for at least 72 h on these carriers [33]. These data indicate that the ppAAc carrier may be particularly suited for a non-invasive, painless and efficient delivery of MSCs to wounds.
Accordingly the aims of this study were to evaluate the use of a ppAAc carrier for the delivery of MSCs for wound healing in vivo and to investigate the mechanism of MSCs induced wound healing as important steps in developing MSC-based therapies for difficult-to-treat wounds. Thus we set out to evaluate the efficiency and efficacy of the ppAAc carrier to deliver human adipose tissue derived MSCs (AT-MSCs) to acute cutaneous wounds in vivo employing a full-thickness excisional murine wound model.
Section snippets
Plasma-polymerized acrylic acid (ppAAc) carriers
A thin layer of acrylic acid was deposited on the sheets of medical-grade silicone by plasma polymerization and the carrier surfaces were analysed by X-ray photoelectron spectroscopy and sterilised with gamma irradiation as detailed previously [32].
Adipose tissue derived mesenchymal stem cells (AT-MSCs)
Human AT-MSCs at passage 2 were purchased from PromoCell (Heidelberg, Germany). AT-MSCs were seeded at a density of 3000 cells/cm2 in complete MSC growth medium with SupplementMix (PromoCell), and cultured at 37 °C under 5% CO2. AT-MSCs were
Characterisation of AT-MSCs cultured on carriers
For the intended in vivo wound healing experiments, AT-MSCs were cultured on the carrier for 1 day to allow attachment and thereafter to deliver AT-MSCs grown on the carrier to the wounds for 3 days. Therefore, AT-MSCs cultured on carriers or tissue culture plastic plates for 4 days were analysed. Compared to cells cultured on tissue culture plastic plates, AT-MSCs cultured on ppAAc carriers did not show any changes with respect to gross morphology (Fig. 1A), proliferation (Fig. 1B), cell death
Discussion
In this study, we define a clinically relevant technique for a safe, efficient and painless delivery of AT-MSCs for wound healing from a previously developed chemically defined carrier. The study also characterises the biological actions of AT-MSCs in acute wounds of a murine full-thickness wound model in vivo leading to accelerated wound healing. This appears to be due to suppression of pro-inflammatory TNF-α released from M1 macrophages and enhanced TGF-β1-dependent induction of
Conclusions
In summary, this study using a synthetic chemically defined ppAAc carrier presents a method to efficiently and safely deliver MSCs to murine full-thickness wounds in vivo. Because of its ease of use, biocompatibility, xenobiotic free composition and its reliable delivery of AT-MSCs to wounds with robust suppression of TNF-α-dependent inflammation, enhancement of TGF-β1-induced granulation tissue formation, myofibroblast driven wound contraction and robust acceleration of overall wound healing,
Acknowledgements
This study is supported by contract research ‘Adulte Stammzellen II’ of the Baden-Württemberg Stiftung P-BWS-ASII/15, and EU Framework VII CASCADE grant. Nathan G. Walker is also supported by a PhD studentship from the Engineering and Physical Sciences Research Council, UK. The authors have no potential conflicts of interest.
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These authors contributed equally to this work.