Naturally Occurring Extracellular Matrix Scaffolds for Dermal Regeneration: Do They Really Need Cells?

The pronounced effect of extracellular matrix (ECM) scaffolds in supporting tissue regeneration is related mainly to their maintained 3D structure and their bioactive components. These decellularized matrix scaffolds could be revitalized before grafting via adding stem cells, fibroblasts, or keratinocytes to promote wound healing. We reviewed the online published literature in the last five years for the studies that performed ECM revitalization and discussed the results of these studies and the related literature. Eighteen articles met the search criteria. Twelve studies included adding cells to acellular dermal matrix (ADM), 3 studies were on small intestinal mucosa (SIS), one study was on urinary bladder matrix (UBM), one study was on amniotic membrane, and one study included both SIS and ADM loaded constructs. We believe that, in chronic and difficult-to-heal wounds, revitalizing the ECM scaffolds would be beneficial to overcome the defective host tissue interaction. This belief still has to be verified by high quality randomised clinical trials, which are still lacking in literature.


Introduction
The extracellular matrix (ECM) is a complex mixture of structural and functional proteins, glycoproteins, and proteoglycans arranged in a unique, tissue specific three-dimensional (3D) ultrastructure. The pronounced effect of ECM scaffolds in supporting tissue regeneration is related mainly to two major characteristics: the maintained 3D structure and the bioactive components. Their natural 3D structure provides structural support and tensile strength, attachment sites for cell surface receptors, and a reservoir for signaling factors that modulate angiogenesis, cell migration, cell proliferation, and orientation in wound healing [1]. The bioactive components include but are not limited to collagen, laminin, fibronectin, glycosaminoglycans, and a various group of growth factors (VEGF: vascular endothelial growth factor, bFGF: basic fibroblast growth factor, EGF: epidermal growth factor, TGFbeta: transforming growth factor-beta, KGF: keratinocyte growth factor, HGF: hepatocyte growth factor, and PDGF: platelet derived growth factor). The presence of such bioactive molecules, together with their native inhibitors, in their preserved natural 3D spatial structure provides a very convenient platform for cells to regenerate [1,2].
The decellularized dermis of the skin, submucosa of the small intestine and urinary bladder (Figure 1), and the amniotic membrane are of the commonest sources for ECM scaffolds used for tissue regeneration. Various market products were developed from naturally occurring ECM scaffolds and were approved as wound dressing for skin wounds and burns. Alloderm is one of the first approved acellular matrix materials and was extensively investigated in literature. It is processed directly from fresh cadaver skin that is treated with high salt to remove the cellular components. It is then freeze dried, leaving an immunologically inert acellular dermal matrix with intact basement membrane complex. Approved by the FDA, it has been used to treat burns since 1992. Oasis is a product derived from porcine small intestinal submucosa (SIS). It has been studied at Purdue University in West Lafayette, USA, and is now commercially available as wound dressing [3]. Graft Jacket is a cryogenically stored acellular dermal matrix (ADM) originating from cadaveric skin that is already approved for wound care purposes [4]. Epiflex is a human acellular dermal matrix transplant manufactured from screened consenting donors [5]. Endoform is an approved extracellular matrix created from the submucosa of the sheep fore-stomach, a tissue whose structure is similar to the dermis [6]. MatriStem MicroMatrix (ACell, Columbia, MD, USA) is a recently approved UBM scaffold for wound regeneration [7]. Although proved beneficial for acute and simple wounds the literature lacks high quality clinical evidences that these scaffolds can provide the desirable effects when applied to chronic, difficult-to-heal wounds. The pathophysiology of chronic wounds and ulcers is usually too complex to be reversed by adding a single factor or cellular component. Chronic ischemic or diabetic wounds as an example are thought to result from the combined comorbidities of neuropathy, vascular deficits, impaired immunity, infection, and repeated tissue trauma, all overlapping to produce a vicious cycle that is very difficult to break [8]. Standard surgical care of such chronic complicated wounds usually fails to match patient's satisfaction and restore the quality of life, and sometimes very complex surgical procedures are required to treat such wounds [9].
Inhibition of extracellular matrix deposition and increased activity of matrix metalloproteinases (MMPs) with concomitant decreased activity of MMP inhibitors were suggested as mechanisms for delayed wound healing in chronic wounds. Regarding the cellular factors; fibroblasts are usually senescent, keratinocytes show impaired migration, and leukocytes exhibit impaired intracellular killing functions. Recently, an impaired function of the gap junctions has immerged as an additional pathological mechanism leading to impaired wound healing. Associated neuropathy leads to a decreased level of neuropeptides that normally contribute to healing. Neuropathy reduces capillary blood flow and vice versa [10][11][12]. These complex factors and mechanisms suggest that providing the wound with a new viable "tissue" and "milieu" is mandatory to achieve a significant response.
The ECMs are characterized by early degradation so that a major part of their role depends on the active interaction with the recipient cells and tissue. In difficult-to-heal wounds this interaction is usually defective due to a lack of reaction by recipient cells.
In an attempt to overcome this, a process of introducing cells into the biostatic graft, known as "revitalization, " could help these scaffolds perform their function, at least for the early stage after implantation. The grafted cells are usually the recipient's autologous cells (differentiated or stem cells) that are seeded either directly onto the scaffold or after retrieval and propagation in culture [13]. Revitalization of ECM scaffolds with keratinocytes, fibroblasts, or stem cells were shown to improve vascularization, scaffold integration, and cellular proliferation [14][15][16]. We reviewed the online published literature in the last five years for the studies that performed ECM revitalization and discussed the result of these studies and the related literature.

Materials and Methods
A PubMed search was performed for the articles published in English language within the previous 5 years. All the articles related to adding keratinocytes, fibroblasts, or stem cells to naturally occurring ECM scaffolds were included. The following string was used for the online search:

Results
The search string yielded 121 articles. The articles were filtered according to title, abstract, and full text resulting in 18 articles that met the search criteria. Twelve studies included adding cells to ADM, 3 studies were on SIS, one study was on UBM, one study was on amniotic membrane, and one study included both SIS and ADM loaded constructs. All in vivo studies were experimental and no single clinical study was found. The type of the study and the most relevant results and remarks are summarized in Table 1.

Discussion
Although there are no guidelines that clearly recommend the use of ECM scaffolds for wound healing, their benefit in acute wounds and burns has been demonstrated in several clinical studies. The complex mixture of structural and functional proteins, glycoproteins, and proteoglycans retained in its original 3D structure provides the key benefit of using these scaffolds for wound healing. This structure provides a temporary support into which cells can migrate and proliferate in a well-organized and controlled fashion leading to improved wound healing. The suggested mechanisms of wound improvement when applying the ECM scaffolds alone are related to providing a structural support, stimulating angiogenesis, chemotaxis for endothelial cells, and release of growth factors [17,18].
In case of chronic and difficult-to-heal wounds the challenge is much bigger. The suggested role of ECM scaffolds in improving such wounds is not fully understood. It has been suggested that they would act as a biological cover that modulates the wound environment by reducing the inflammatory activity to promote wound healing [19]. There is currently limited published data that reaches a sufficient level of evidence about the role of ECM scaffolds alone in chronic and difficult-to-heal wounds [3,[20][21][22][23][24][25][26][27].
The positive role of combining ECM scaffolds with stem cells, fibroblasts, or keratinocytes was clearly demonstrated in in vitro and experimental in vivo studies. It is believed that native stem cells play an important role in wound regeneration or healing. GFP-labelled MSCs were found in the skin of non-GFP mice after peripheral injection. This indicates that wounding stimulates MSCs to migrate via chemotaxis to the injury site and differentiate to functional skin cells [28]. Some studies have indicated that wound healing is enhanced through ADSCs that promote human dermal fibroblast proliferation by direct cell-to-cell contact and via a paracrine effect [29].
However, the relation between the efficacy of wound healing and the number of transplanted MSCs does not seem to be a linear one. Yeum et al. [30] have shown that repeated injection of additional MSCs did not increase the number of MSCs participating in wound healing beyond a certain constant maximum amount. The number of MSCs in the wound site remains constant in the range 2-3 × 10 5 from day 1 to day 10. MSCs were not detected after day 10, probably because the role of transplanted MSCs ended thereafter. Lam et al. [31] also could not detect the signals after 12 days postwounding. It was suggested that the stem cells would have been engulfed by macrophages or migrated to other body sites speculating that after the completion of the MSCs' roles, the wound site no longer needs the MSCs as it has recovered completely by 14 days.
Although the effect of stem cells is well documented in promoting wound healing, these cells usually do not survive well when directly transplanted to the wound site. Many studies have shown that a great number of cells die during transplantation and this effect would be diminished if cells were allowed to proliferate in an optimal milieu [32,33]. Attempts for aiding stem cell survival often involve The study focuses on the role of the bioactive structure of SIS rather than its scaffolding properties codelivery with slow release and survival-promoting gels such as Matrigel or collagen gel. In several in vivo and in vitro studies Matrigel was found to be superior probably due to its basement membrane component [34][35][36]. Similar studies on SIS have demonstrated that the ECM patch allowed the stem cells to remain localized to the wound area rather than migrate to other regions as evidenced by in vivo cell tracking [31].
Orbay et al. [37] concluded that ADSCs could attach to ADM and decrease its in vivo resorption suggesting that this construct may be a useful tool for soft tissue augmentation with stable long-term results. This effect was thought to be due to stimulatory effects of ADSCs on fibroblasts leading to an indirect increase in the synthesis of collagen and extracellular matrix components.
In an attempt to enhance wound epithelialization, keratinocytes were added to ECM scaffolds in various studies. Based on the in vitro behaviour of the keratinocytes, Zajicek et al. [38] suggested that the ADM promotes wound healing through supporting the growth of patient's own keratinocytes from the adnexa remnants in the wound by providing optimal conditions for their attachment, proliferation, and migration. Peramo et al. [39] proved that Alloderm could also permit the differentiation and stratification of nonkeratinized, buccal mucosa in vitro.
Regarding their effect on the dermal regeneration, Seland et al. [40] have shown that implantation of a single cell layer of keratinocytes to the ADM added nothing to the dermal thickness in the wound healing process. Interestingly keratinocytes loaded on microcarriers showed a significantly thicker epithelium and neodermis at both 16 and 21 days after grafting compared to the wounds treated with a single layer. This led to the hypothesis that these carriers could act as a facilitator for the dermal regeneration beside their role in transportation and transplantation of autologous keratinocytes.
For the recipient keratinocytes to proliferate and uniformly stratify above/within the ECM, it was traditionally known that an optimal environment would require the presence of fibroblasts [41]. This is probably due to the paracrine interaction between the two cell types [42,43]. Deshpande et al. have concluded in their in vitro study, however, that the formation of a well-organized epithelium on the acellular dermal matrix depends mainly on the presence of intact basement membrane but is largely independent of the presence of cultured fibroblasts. They have noticed that incorporating fibroblasts in the absence of a basement membrane had no significant effect on the keratinocyte behavior [44]. Other groups have demonstrated an enhanced keratinocyte migration on a sterilized dermis after removal of basement membrane antigens but in the presence of fibroblasts under conditions of normal extracellular calcium concentration [45]. These conditions probably represent the in vivo situation during normal wound healing, when the basement membrane has been traumatically disrupted and fibroblast numbers are upregulated in order to heal the wound [46]. We guess that the solution for these contradictory results is the establishment of a well-standardized in vivo study for the assessment of the definite role of fibroblasts and basement membrane factors.
In chronic and difficult-to-heal wounds, vascularisation of the wound bed is a major concern. If STSG is to be implanted over the ADM, then adequate scaffold neovascularisation would be an essential prerequisite. Neovascularisation of the matrix occurs during the early stages of complete adherence of ADM to the recipient wound bed [47]. Increasing and accelerating this neovascularisation and estimating its timing are thus important for an optimal treatment plan [48]. An enhanced angiogenesis through the application of ECM scaffolds was also suggested as an important factor in decreasing wound fibrosis [31]. Sahin et al. [48] have demonstrated that adding MSCs to the ADM has a significant positive effect on the vascularisation probably due to enhanced secretion of VEGF [49]. Han et al. [50] have also demonstrated that enhancement of ADM engraftment and wound angiogenesis could be achieved by seeding of microencapsulated VEGF-expressing fibroblasts below the scaffold. Huang et al. [51] have also demonstrated that DiIlabeled cells were colocalized with staining for VEGF and vWF (Von Willebrand factor) well 14 days after seeding on ADM and implantation in full thickness wounds, suggesting that the grafted cells might improve angiogenesis via the indirect paracrine effect or contribute to newly formed vasculature. Our research group has also demonstrated an enhanced angiogenic activity with autologous keratinocyte grafting with porcine UBM, which could be attributed to a cross talk between the keratinocyte and endothelial cells and release of angiogenic factors from UBM degradation, or even from the dying keratinocytes after grafting [52].
In difficult-to-heal wounds as in chronic or irradiated wounds, it is always wise to bring new healthy "tissue" to the wound bed. Applying the same concept makes adding cells to the scaffold crucial for wound regeneration in such difficult situations where the wound regeneration capacity is subnormal. Roessner et al. [15] have demonstrated that adding fibroblasts to ADM in irradiated wounds would improve wound healing evidenced by enhanced wound tensile strength. This effect was abolished when the transplanted cells where irradiated in an adjuvant-radiotherapy setting.
In a clinical setting, these difficult-to-heal wounds were almost exclusively treated with cell-loaded non-ECM scaffolds such as Apligraf, Dermagraft, and GammaGraft [53]. From all the available ECM scaffolds, only the SIS (Oasis) and to a lesser extent Graft Jacket have been reported clinically in a considerable number of patients to improve chronic wounds without adding cells [3,21,25]. The role of SIS in promoting wound closure was extensively investigated. Shi et al. [16] have demonstrated that MMPs inhibit keratinocyte migration in vitro and that preincubating the MMP solution with SIS could significantly reduce this inhibitory effect. MMPs are important contributors to wound chronicity and are abundantly expressed in chronic ulcers and not in acute wounds [54]. MMPs inhibit keratinocyte migration and degrade fibronectin, growth factors, and other proteins vital to wound healing and thus reducing elevated levels of MMPs in chronic wounds should promote healing [55].
A high quality randomized controlled clinical study comparing the wound healing potential of cell free versus cell loaded ECM scaffolds is unfortunately still lacking. Lev-Tov et al. [56] have introduced a protocol to compare the standard surgical care either alone or with Dermagraft (bioengineered ECM containing living fibroblasts) or with UBM (Oasis). Although Dermagraft is not a naturally occurring ECM scaffold, the data coming out of such a study would be useful in understanding the relative role of ECM and added cells in a clinical context.
We think that in difficult-to-heal wounds adding cells to the ECM scaffolds would enhance their regenerative capacity. In acute and simple wounds, however, the regenerative capacity of the native tissues are usually preserved so that the high costs and time linked to adding autologous cells within good clinical practice guidelines could be avoided as the relative benefit would be negligible. These conclusions are based on our surgical and experimental experiences and still have to be verified by high quality randomised clinical trials.

List of Abbreviations
ADM: Acellular dermal matrix ADSC: Adipose derived stem cells bFGF: Basic fibroblast growth factor bMSC: Bone marrow derived mesenchymal stem cells EGF: Epidermal growth factor HGF: Hepatocyte growth factor KGF: Keratinocyte growth factor MMP: Matrix metalloproteinases PDGF: Platelet derived growth factor STSG: Split thickness skin graft TGF-beta: Transforming growth factor-beta UBM: Urinary bladder matrix VEGF: Vascular endothelial growth factor vWF: Von Willebrand factor.