Significant Cell Populations for Regenerative Skin Wound Therapies

Significant Cell Populations for Regenerative Skin Wound Therapies: History

Please note this is an old version of this entry, which may differ significantly from the current revision.

Subjects: Medicine, Research & Experimental

Contributor:

Andrew J. Dalley

Considerable progresses have been accomplished in cell biology fields, and the existing evidence has revealed the effectiveness of cell therapy for pathologic wounds. Transplantation of keratinocytes, fibroblasts, platelets, and more recently, stem cells (SCs) can promote wound healing through de-novo synthesis, secretion, and release of a wide range of cell signaling molecules such as growth factors (GFs) and cytokines.

biofabrication
biomaterial
cell delivery
cell therapy
keratinocyte
matrix
regeneration
skin
stem cell

1. Keratinocytes

The cultivation of human keratinocytes was first introduced by Rheinwald and Green in 1975 [12]. Using this original technique, complete sheets of keratinocytes can be cultured and enzymatically released from their supporting substrate as cultured epithelial autograft (CEA) and used to surface optimally prepared partial-thickness cutaneous wounds. Six years after the pioneering work of Rheinwald and Green, O’Connor et al. reported, for the first time, the transplant of cultured autologous epithelium onto two patients with full-thickness burn wounds [13]. The cultured epithelia developed an epidermal structure similar to the split-thickness skin grafts and survived for about eight months. Importantly, given sufficient time, large numbers of CEA can be manufactured from a comparatively small initial biopsy of a patient’s skin, making CEA engraftment suitable for large wounds [13]. Decades later, cultured autologous keratinocytes have been applied for the treatment of numerous different skin wound types, and their ability to promote wound healing has been widely acknowledged [14].

Some studies have even explored the application of allogeneic keratinocytes in the treatment of deep partial-thickness burns and other wounds [15,16]. The beneficial effects of keratinocytes on wound healing are related mainly to their physical presence in re-epithelializing the wound. In addition, keratinocytes secrete and deposit numerous GFs, such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), basic fibroblast GF, transforming growth factor (TGF)-α, TGF-β, and cytokines such as interleukin (IL)-1, -6, -8, and -10 directly into the wound site [17,18]. Keratinocytes alone, however, are unable to produce the large, structural, and voluminous components of the ECM that comprise the dermis [19]; therefore, in isolation they are not appropriate for the re-epithelialization of full-thickness wounds. The biggest disadvantage of therapeutic autologous keratinocytes culture for the re-epithelialization of extensive cutaneous wounds is the time taken to grow sufficient CEA sheets. Using the techniques of Rheinwald and Green and starting with an initial skin biopsy of 7 cm²–10 cm², it takes a minimum of three weeks to produce sufficient sheets to cover three quarters (75%) of an adult human’s body surface area. Other reports have suggested disadvantages including the inconsistent graft take rates, poor long-term durability, infections, mechanical shear, scarring, and high production cost [20]; however, such criticisms often reflect deficiencies in how the CEA sheets have been used clinically, rather than the fundamental properties of CEA. When used as part of a considered wound management plan in appropriate surgical settings on optimally prepared wound beds where effective post engraftment wound management practices are implemented, CEA provides a highly reliable, therapeutic adjunct for timely epithelial closure of partial-thickness wounds.

2. Fibroblasts

Autologous cultured fibroblasts are another cell therapy candidate that has been utilized for burn wounds [10,11], gingival tissue repair [21,22], and the prevention of acne scarring [23]. When transplanted effectively, fibroblasts can release numerous GFs and cytokines into the wound bed that contribute to endogenous cell proliferation, stimulate angiogenesis, and modulate the immune responses [24,25]. Of note, fibroblasts can secrete various ECM proteins such as collagens and proteoglycans and improve the wound healing rate [26]. It has been suggested that the injection of fibroblasts into the wound site could heal chronic wounds rapidly and with no adverse clinical or immunopathologic effects [27,28]. Allogenic fibroblasts can be prepared and stored in advance, but are known to be highly immunogenic and may be the source of cross-infections. Autologous fibroblasts, on the other hand, present little additional risk of infection or immune response, but require a long cultivation period which is a major practical challenge that limits their clinical applications. To date, most of the studies that have utilized fibroblasts or their derivatives to promote wound healing have used allogenic cryopreserved products that comprise poor therapeutic benefits. Some of these reports have cited the harsh recipient tissue microenvironment, which is deficient in oxygen and nutriments, as the underlying cause of poor engraftment efficiency when introducing cryopreserved fibroblasts into wounds. In addition, it has been suggested that cryopreservation causes functional impairments in fibroblasts, leading to decreased viability, impaired protein synthesis, and reduced angiogenic factor secretion [29,30,31].

3. Platelets

Platelets contribute to normal wound healing and have been used experimentally to deliver GFs into the wound sites [32,33] such as PDGFs (PDGF-AA, PDGF-AB, and PDGF-BB), TGF-β (TGF-β1 and TGF-β2), VEGF, and epidermal growth factor (EGF). It has been suggested that platelet-derived GFs could effectively restore impaired GF activity in the wounds that are deficient in leading cells [34]. Studies have reported positive results of wound healing upon treatment with autologous platelets. The majority of patients whose platelet treatment continued for more than three months showed complete healing and reported fewer adverse events [35,36]. The major disadvantage of using autologous platelets therapeutically centers around the large-volume blood withdrawals required to isolate the platelets which may give rise to severe adverse effects, such as hemodynamic instability in patients with chronic wounds, bleeding disorders, or anemic conditions [37].

4. Stem/Progenitor Cell Therapies

Differentiated cells have limited self-renewal capacity; therefore, the use of stem/progenitor cells, especially mesenchymal stem cells (MSCs), has received a great deal of interest for wound healing (Table 1). MSCs present considerable immunomodulatory potential [38], and after transplantation into the wound sites, MSCs secrete ECM molecules that activate re-epithelialization, improve wound closure, and induce angiogenesis [39]. MSCs also contribute to the recruitment of several immune cell types via the release of cytokines [40,41], and can induce the differentiation of multiple progenitor cells and prompt the release of bioactive factors that support wound healing [42,43].

Table 1. Clinical studies on stem/progenitor cell-based therapy for wound healing. Abbreviations: AVLU: leg ulcers of arterial-venous, CLI: critical limb ischemia, DFU: diabetic foot ulcers, MSCs: mesenchymal stem cells, PAD: peripheral arterial disease, VLU: leg ulcers of venous.

Stem/Progenitor Cells	Treatment Group(s)	Wound Type	Remarks	Reference
Adipose-derived MSCs	Adipose tissue derived MSCs	CLI	- 66.7% of patients showed ulcer healing - The treatment showed the formation of numerous vascular collateral networks	[44] *
	1: Autologous adipose-derived stem and regenerative cells plus traditional methods and advanced dressings 2: Only traditional methods and nonadherent dressings	Chronic ulcer of lower limbs	- There was a reduction in both the diameter and depth of the ulcer - In 6 of 10 cases, there was complete healing of the ulcer	[46]
	Autologous cultured adipose-derived stroma/SCs	Non-revascularizable critical limb ischemia	- Ulcer evolution and wound healing showed improvement	[47] **
	Non-culture-expanded autologous, adipose-derived stromal vascular fraction cells	CLI	- 6 of the 10 patients with non-healing ulcers had a complete closure - There was evidence of neovascularization in 5 patients	[48] *
	Adipose-derived SCs	Hypertensive leg ulcers	- Wound surfaces constantly and significantly decreased (wound closure rate of 73.2% at month 3 and 93.1% at month 6) - Percentages of fibrin and necrosis decreased, whereas granulation tissue increased significantly - There was no recurrence	[49] *
	1: Autologous stromal vascular fraction cells plus a wound dressing 2: A standard dressing	Chronic VLU and AVLU	- All VLU patients and 4 of 9 AVLU patients showed complete epithelialization of the ulcers within 71–174 days - In 3 patients with large ulcerations on both legs, ulcerations on the non-treated, contralateral leg also epithelialized (paracrine effects seemed to stimulate the regenerative changes even at a large distance)	[50]
Bone marrow derived MSCs	1: Allogeneic bone marrow-derived MSCs 2: PlasmaLyte A	CLI	- The use of allogeneic BM-MSCs was safe in patients with CLI - All ulcers at two-year follow-up healed in group 2, whereas one patient in group 1 continued to have ulcers but with reduced size	[51]
	1: Bone marrow-derived cells 2: Autologous peripheral blood plus regular wound care treatments	Chronic lower limb wounds due to diabetes mellitus	- The average decrease in wound area at 2 (17.4% vs. 4.84%) and 12 (36.4% vs. 27.32%) weeks was higher in group 1 compared to in group 2	[52]
	1: Bone marrow MSCs 2: Bone marrow-derived mononuclear cells 3: normal saline	Diabetic critical limb ischemia	- The ulcer healing rate was significantly higher in group 1 - They reached 100% four weeks earlier than group 2 - Ulcer healing rate in group 2 was significantly higher than in group 3, which appeared at 12 weeks	[53]
	Autologous bone marrow nuclear cells	Pressure ulcers	- Pressure ulcers had fully healed after a mean time of 21 days in 86.36% of the patients - During a mean follow-up of 19 months, none of the resolved ulcers recurred	[54] *
	1: Autologous bone marrow aspirate 2: Saline dressings	Chronic wounds	- Group 1 achieved a significant reduction in the wound surface area	[55]
Progenitor cells	CD34+ cells isolated from bone marrow	Sacral pressure sore	- The treatment positively affected granulation tissue formation and wound contraction, which showed about a 50% reduction in the pressure sore volume on the treated side versus a 40% reduction on the control side	[45] **
	Genetically modified epidermal stem cells	Junctional epidermolysis bullosa	- Complete engraftment was achieved following 8 days - Transduced stem cells enabled the regeneration of epidermis	[56] **
	Genetically modified epidermal stem cells	Junctional epidermolysis bullosa	- The human epidermis is supported not by equipotent progenitors, but by long-lived stem cells with an extensive self-renewal ability so that they could generate progenitors to renew terminally differentiated keratinocytes	[57] **
Bone marrow-derived mononuclear cells	Mononuclear bone marrow cells	Chronic venous and neuro-ischemic wounds	- The treatment led to a wound size reduction, a markedly increased vascularization, and infiltration of mononuclear cells	[58] **
Placental MSCs	1: Cryopreserved human placental tissue in a human viable wound matrix plus standard compression therapy 2: Standard compression therapy	VLU	- Complete healing in 53% of the cases in group 1 - Reduction in wound size by half (80% in group 1 vs. 25% in group 2)	[59]
Placental MSCs	Human placenta-derived mesenchymal stromal-like cells (cenplacel)	DFUs with PAD	- There was preliminary evidence of ulcer healing in seven patients (five complete; two partial) within 3 months of cenplacel treatment - Circulating endothelial cell levels (a biomarker of vascular injury in PAD) were decreased within 1 month - Cenplacel was generally safe and well-tolerated in patients with chronic DFUs and PAD	[60] *
Umbilical cord MSCs	1: Human umbilical cord MSCs plus a percutaneous angioplasty treatment 2: A percutaneous angioplasty treatment	Ulcer wounds	- 3 months after treatment, there was a significant increase in neovessels accompanied by complete or gradual ulcer healing in group 1	[61]

Considering the stem/progenitor cells’ capability to enhance wound healing, many clinical studies have been conducted to use these cells for the treatment of non-healing wounds. Different types of MSCs, including bone marrow-derived MSCs, adipose-derived MSCs, placental-derived MSCs, and umbilical cord-derived MSCs, have been considered for cell therapy of chronic wounds. For example, Lee et al. recruited 15 patients with critical limb ischemia to be treated with multiple intramuscular adipose tissue-derived MSCs injections [44]. No complications were reported during follow-ups at a mean time of 6 months. There was clinical improvement in 66.7% of the patients. Although five patients underwent minor amputation, the amputation sites indicated complete wound healing. Additionally, the cell transplantation resulted in collateral vessel development across the affected arteries [44]. In another study, three patients with sacral pressure sore were treated with CD34+ cells isolated from bone marrow (NCT00535548) [45]. The treatment improved granulation tissue formation and wound contraction, leading to around a 50% decrease in the volume of the pressure sore on the treated side, as opposed to a 40% decrease on the control side [45].

Although highly promising, therapeutic stem/progenitor cell engraftment has encountered several practical challenges. Obtaining high-quality progenitors for therapeutic engraftment is a slow and laborious process that requires a high level of capital investment and technical expertise making it a complex therapeutic option [62]. In addition, the potential for some progenitors to differentiate into divergent phenotypic lineages adds to the need for long-term post-engraftment surveillance to ensure that only beneficial effects have been delivered by the progenitors.

Compounding this situation, co-morbidities such as diabetes, vascular disease, and aging have the potential to drive progenitors mal-differentiation through pathological changes in the wound microenvironment [63]. Regarding post-engraftment surveillance, cutaneous wounds are particularly suitable for exploring the therapeutic potential of progenitors, since the skin is an easily accessible tissue from which problematic areas can be rapidly identified and readily excised. Progenitor cell therapy for cutaneous wounds is a developing field; the beneficial outcomes of which have so far been limited and inconsistent. There is a paucity of data for post-engraftment progenitor cells take rates, with reports of engraftment failure arising from bacterial colonization and unsuitable wound conditions [19].

Indeed, the injected cells have low retention rates in the transplantation sites in parts because of washout by blood flow [64,65]. In addition, ischemia and inflammation within the wound microenvironment can jeopardize the survival and proliferation of administered cells and may cause cell death in vivo [66]. Despite the technical challenges and practical setbacks that stem/progenitor therapy has encountered, it is still a very exciting field with the potential to deliver great clinical outcomes when it has been mastered.

This entry is adapted from the peer-reviewed paper 10.3390/pharmaceutics14122749

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