**5. Application of cell-based therapies in veterinary dermatology**

Slow wound healing or persistent wounds are a challenge for clinicians, in both veterinary and human medicines. These wounds can result in inadequate tissue reorganization, culminating in a long period of incapacity and unsatisfactory outcomes [24]. These conditions can be related to various pathological conditions, such as autoimmune diseases, diabetes, and venous stasis, for which no definitive therapies are currently available [25]. Due to this situation, the use of MSCs in veterinary medicine has been increasing in the recent years in different fields. Regarding their application in dermatology, these cells can be used for skin tissue regeneration and for the control of dermatological pathologies, in which the immune system intervenes [14]. Due to their capacity for regeneration, differentiation, revascularization, as well as, their anti-inflammatory and immunomodulatory properties, MSCs have been used as promoters of regeneration on tissues that suffered some damage [1]. However, there are still some questions regarding the use of MSCs, such as their immunomodulation mechanism that is not fully understood. In addition, there are different routes of administration that can result in different risks for the patient. For instance, systemic administration can lead to the entrapment of MSCs in the lung or microvasculature that can cause side effects, such as pulmonary emboli. In addition, almost 90% of the cells are lost, once administered, because of hypoxia, inflammation, physical stress, or immunogenic rejection. For this reason, to reach a therapeutic efficacy, a huge number of cells may be needed, increasing the risk of teratoma formation. Therefore, new studies are needed to achieve more cost-effective treatments to overcome these obstacles [3]. Despite these, this review demonstrates the positive effects of using MSCs therapies and the need to standardize protocols.

There are several skin diseases that can produce chronic and recurring wounds, such as canine atopic dermatitis (CAD), pemphigus foliaceus (PF), and perianal fistula. The conventional treatment for the skin diseases consists in glucocorticoids, cyclosporine, and oclacitinib, which are used due to their immunomodulatory effect. However, their repeated use has several side effects, such as polydipsia, polyuria, polyphagia, vomiting, and diarrhea. It can also require increasing doses of medication over time due to drug habituation [3, 26].

#### **5.1 Application of MSCs in tissue repair and chronic non-healing wounds**

A wound is a disruption of the functional integrity and anatomic structure of the skin, so wound healing is a highly ordered process. The skin tissue repair steps need to occur in consecutive order and timing, to be successful; otherwise, the healing process will fail and cause complications, such as chronic non-healing wounds [1].

There are various types of non-healing wounds, such as bed sores, diabetic foot, and trophic ulcers of many etiologies. For instance, ulcer treatment requires necrotic tissue debridement, wound cleansing, amelioration of damaging factors (infection), improvement of arterial blood circulation, and medical management to aid with the comorbidities. Nonetheless, chronic wounds take a long time to heal and often recur after healing with extensive and intensive treatment. Thus, a great possibility in the treatment of chronic wounds is associated with cell-based therapies [27].

The use of MSCs in wound healing is due to their ability to remove necrotic and dead cells, improve vascularization and re-epithelization, and diminish scar formation and wound contraction. Transplanted MSCs release several growth factors that help coordinate different repairing activities. Fibroblasts, endothelial cells, and local stem cells are triggered to aid in tissue repair, by increasing angiogenesis, restraining leukocyte transmigration, and stimulating the proliferation, migration, and differentiation of keratinocytes and fibroblasts. MSCs also release immunosuppressive factors, helping to suppress the proliferation of immune cells, reducing inflammation, and, consequently, reducing scar formation [1].

Kuperman *et al.* demonstrated that the local application of mouse oral mucosa stem cells (mOMSCs) increased the wound healing rate and re-epithelialization and led to a larger area of granulation tissue in diabetic mice compared to the control group [28].

Gorecka *et al.* applied human-induced pluripotent stem cell-derived smooth muscle cells (hiPSC-SMCs) embedded into 3D collagen scaffolds in diabetic mice, promoting angiogenesis and accelerating diabetic wound healing [29].

A study in rabbits showed that wounds treated with a combination of plasma rich in growth factors and adipose-derived mesenchymal stem cells (PRGF+ADSCs) have higher wound healing and epithelization rates, less inflammation and scar tissue, greater collagen deposits, and better angiogenesis compared to the control group. Furthermore, the group treated with the combination PRGF+ADSCs showed a faster recovery of the damaged tissue [30].

Several studies in rodent models have demonstrated that MSCs applied subcutaneously, topically, or intravenously can improve wound healing [12, 31–33].

In a study focusing on skin wounds in dolphins, the animals were treated with autologous ADSCs in a blinded clinical study and the group treated with ADSCs showed improved wound healing [12].

The use of secretome is an alternative treatment when dealing with chronic skin wounds. Sue *et al.* used a rat skin excisional wound healing model to demonstrate that the subcutaneous injection of ADSCs secretome around the wound could accelerate cutaneous wound healing [34].

Park *et al.* investigated if ADSCs secretome could accelerate wound healing using nude mice. In this study, a full-thickness excisional skin wound was created bilaterally on the dorsal surface of the animal. Then, the secretome was used topically in the wounds and covered with a transparent dressing. The evaluation of the lesions demonstrated that the treatment using secretome was able to stimulate angiogenesis, skin thickening, and the recruitment of immune cells, therefore enhancing the wound healing process [35].

#### **5.2 Application of MSCs in immune-mediated diseases**

CAD is a complicated disease that results from environmental factors (allergens) and a genetic predisposition (filaggrin mutation) that alter the immune response and

#### *Application of Cell-Based Therapies in Veterinary Dermatology DOI: http://dx.doi.org/10.5772/intechopen.111553*

culminate in a skin barrier dysfunction [36]. It is a common multifactorial inflammatory and pruritic skin disease in dogs, associated with the production of IgE antibodies [37]. Its prevalence is around 10–15%, and the management of these patients is a real challenge for tutors and veterinarians [38]. During the acute phase of CAD, there is an activation of Th2 cells caused by the immune dysregulation, which culminates in the production of numerous pro-inflammatory cytokines. Over time, the chronicity of the pathology is maintained by a broader roster of T helper variations. Pharmacological treatments consist in corticosteroids (less specific) and cyclosporine A (more specific), and some new approaches used to achieve the most target agents, such as lokivetmab and oclacitinib. Despite the newest therapies, approximately 25–40% of dogs with CAD endure clinical signs and do not reach a full resolution of the pathology [39]. For that reason, the use of MSCs would be a good alternative therapy, since their use in an inflammatory environment, can alter the cytokine profile of T cells and dendritic cells, which can lead to an anti-inflammatory environment [12].

In 2018, a study gathered 26 animals diagnosed with CAD refractory to conventional treatments to which an intravenous dose of 1.5 × 106 ADSCs/kg bodyweight was administered. Pruritus was evaluated using the Canine Atopic Dermatitis Extent and Severity Index, version 4 (CADESI-4), and a decrease in pruritus within a time span of 1 week to 1 month was observed, with the animals being controlled for a period of 6 months. Owners reported improvement of the animals with a satisfactory global assessment of the treatment without the occurrence of adverse events [40].

In 2019, a group of 12 canine patients diagnosed with CAD were intramuscularly inoculated with 0.5 × 106 of cryopreserved ADSCs. Injections were repeated weekly for 6 weeks. During this period, the effectiveness of the treatment was evaluated by the pruritus index and by the CAD Lesion Index (CADLI) test, and a notable reduction in both was observed. The animals were monitored at all times, and no systemic side effects or changes at the injection site were observed [41].

In 2020, a group of 16 animals diagnosed with CAD was evaluated with CADESI-4 and divided into three groups, namely mild, moderate, and severe according to the severity of their injuries. For 82 days, 2x106 MSCs were inoculated intravenously every 21 days to all animals. At the end of the 82 days, skin biopsy histopathology analysis was performed, observing a significant reduction in epidermal thickness in the moderate and severe groups. The results demonstrate that MSCs attenuated the clinical signs of CAD resulting in a safe therapy and causing no adverse effects [42].

In 2021, a double-blind study divided patients with CAD into three groups, a control group that received PBS solution, one group that received low-dose ADSCs (5 × 105 cells/kg), and the third group received a higher dose (5 × 106 cells/kg) of MSCs. Three subcutaneous treatments were performed at 4-week intervals. Pruritus was assessed by tutors using the pruritus visual analog scales (PVAS) and by veterinarians using CADESI-4. Both observed a decrease in pruritus during the 30 days following injections in the group receiving higher dose of MSCs. The animals were monitored throughout the study and did not manifest adverse side effects [43].

Recently, in 2022, a study evaluated the immunomodulatory effect of cADSCs and extracellular vesicles derived from cADSCs (cADSC-EVs) demonstrating that these cells have a beneficial effect in atopic animals. The cASCs and cADSC-EVs affect the expression levels of epidermal differentiation proteins, such as keratin1, filaggrin, loricrin, and involucrin promoting the recovery of the deficient skin barrier in these atopic animals. With the recovery of the skin barrier, it was possible to reduce the loss of water and prevent the entry of allergens *via* transepidermal route. cADSCs and cASC-EVs regulate the immune and inflammatory response

by regulating mast cell infiltration, decreasing serum levels of IgE, inflammatory cytokines and epidermal chemokines (IL-4, IL-13, and IL-31) that intervene in Th2. By decreasing IL-31, a decrease in pruritus was also observed by inhibiting the activation of JAK-STATs signaling [38].

The use of UC-MSCs secretome has been associated with the improvement of atopic dermatitis, due to the secretion of epidermal growth factor, which regulates the inflammatory response of mast cells, Th2 cells, and keratinocytes [44].

#### **5.3 Application of MSCs in autoimmune skin diseases**

Autoimmune skin diseases occur when the body itself causes self-destruction of its cells and tissues [45].

Pemphigus describes a group of cutaneous autoimmune diseases. Pemphigus vulgaris consists in the appearance of mucocutaneous blisters that are characterized by the presence of autoantibodies against desmogleins (1 and 3). It can be life threatening, and the treatment with systemic corticosteroids has improved the mortality rates [46].

PF is a common autoimmune skin disease characterized by acantholysis. This pathology is associated with the production of autoantibodies that target protein in the desmosomes of keratinocytes [47]. The desmoglein-1 and the desmocollin-1, which are epidermal adhesion proteins, are the main antigens implicated in PF [48]. It is characterized by the production of self-antibodies by B cells that attack desmoglein-1, causing cell apoptosis and disruption of the skin layers, resulting in vesicles, intra-epidermal pustules, and crust lesions [49]. The cause is usually unknown, but some situations are a sequel to a chronic inflammatory skin disease or probably drug-induced [47]. The treatment and control of PF is difficult and often requires lifelong therapy with immunosuppressive drugs (corticosteroids or cyclosporine), having severe side effects such as polyuria/polydipsia, diarrhea, weigh gain, and predisposition to recurrent infection [50]. Furthermore, it has been described that only 53% of treated cases of PF survive more than 1 year after the treatment initiation [47]. Due to their immunomodulatory, anti-inflammatory, and antiapoptotic abilities, MSCs are a promising therapeutic option, not showing the side effects of the conventional treatment.

In 2015, a 10-year-old neutered Shih-Tzu dog diagnosed with PF refractory to corticosteroid treatment was treated with cytotoxic T-lymphocyte antigen 4 (CTLA4)-overexpressing ADSCs. CTLA4-ADSCs and/or naive ADSCs were administered 21 times over a 20-month period (every 2 to 8 weeks) with a positive result. The prednisolone dose was progressively lowered with no relapse of the lesions. At the end of treatment, the lesions had improved considerably, and the disease was under control with a low dose of corticosteroids for 12 months [47].

Canine anal furunculosis is a chronic inflammatory disease that leads to perianal fistulas and that shares a large part of etiology and clinical manifestation with Crohn's disease in humans [51].

In 2015, six dogs with perianal fistulas were treated with human embryonic stem cell-derived mesenchymal stem cells (hESC-MSCs). One month after the injection, reduced serum levels of IL-2 and IL-6 and two inflammatory cytokines associated with Crohn's disease were observed, and after 3 months all animals were controlled and remained free of fistulas for 6 months [51].

Psoriasis is another chronic autoimmune disease, characterized by silvery white scaly patches projecting from the inflamed skin, mainly present in humans, but that can also appear in dogs and monkeys [52]. In patients with psoriasis, the histology of damaged skin demonstrates that epidermal keratinocytes are hyperproliferative and

#### *Application of Cell-Based Therapies in Veterinary Dermatology DOI: http://dx.doi.org/10.5772/intechopen.111553*

a huge number of immune cells infiltrate into the skin layers (dermis and epidermis). The first treatment used for this pathology was crude coal tar or a combination of the last with ultraviolet irradiation, both with the purpose to restrain keratinocyte proliferation and therefore restoring normal epidermopoiesis. With time, retinoids were also proved to be an efficient treatment for psoriasis since they were able to inhibit the keratinocytes proliferation. For that reason, in the beginning keratinocytes were considered as the main inducer of psoriasis. Later, cyclosporine was discovered to be efficient in psoriasis treatment by inhibiting cytokines produced by T cells [53]. Therefore, keratinocytes can trigger psoriasis and actively participate in a complex environment coordinated by cytokines [54]. The treatments depend on the severity of the disease, from topical agents and phototherapy to the usual immunosuppressant drugs [55]. However, these therapies are expensive and are related to adverse reactions, and therefore, there is a need to discover a more effective and safer treatment. For that reason, there are some expectations in the use of MSCs for psoriasis.

A study testing a treatment with umbilical cord-derived MSCs (UC-MSCs) resulted in comparison with the conventional treatments (betamethasone cream) on imiquimod-induced psoriasis-like skin lesion in adult male albino rat model. MSCs showed efficacy in reducing the severity of the disease, demonstrating that the cells had anti-inflammatory and immunomodulatory effects by inhibiting the clinical manifestation [55].

The use of the MSCs secretome in psoriasis is also able to inhibit the maturation and activation of DC and IL17, decreasing psoriasis score in a rat model [56].

Systemic lupus erythematosus (SLE) is a multisystemic autoimmune disease, with clinical manifestations on every organ. The production of antigen-antibodies complexes that settle in the basement membrane zone of the skin can make this disease manifest as cutaneous lupus erythematosus, with the appearance of rashes. It is a multifactorial disease that can be caused by genetic factors, B cell hyperactivity, T cell defects, virally induced antigen-antibody complex formation, and even hormonal alterations [57]. The cutaneous inflammation is due to Th1 cells, neutrophils, B cells, and even complex cascades of native skin cell types (keratinocytes and endothelial cells) [58]. The conventional treatment options consist in the use of corticosteroids and cyclophosphamide.

The use of allogenic MSCs administrated intravenously has improved multiorgan dysfunction in both MRL/lpr mice and NZB/W1 F1 mice, used as SLE animal models [59].

The use of human AD-MSCs also improved the survival rate and histological, serological, and immunological function of NZB × NZW mice, models for SLE, without adverse effects [57].

#### **5.4 Application of MSCs in alopecia**

Alopecia is a frequent dermatologic disorder, affecting both veterinary species and humans. The pathophysiology of this group of disorders is still unclear, but can be associated with trauma, stress, autoimmune disorders, hormones, and even genetics [60]. Alopecia can be caused by a several disorders, such as infections, allergenic reasons, nutritional deficits (mineral deficiency), parasites, bacteria or hot spots, and fungi. A complete physical examination and history are needed to an accurate diagnosis for the reason of hair loss [61]. Hair loss is usually accompanied by a section of thinned skin at the site with hair loss. Additionally, these clinical symptoms occur because of the functional loss of follicular stem cell activity. Nowadays, there are various treatments for alopecia, such as oral medications, that only improve the situation

temporary and often with reduced effectiveness. For this reason, alternative strategies are fundamental for alopecia [62]. The use of BM-MSCs and UC-MSCs *in vitro* and in an athymic nude mouse model has been able to create dermal papilla-like structures, as well as hair follicles [12]. In a study, the use of UC-MSCs increased the regeneration of new follicles and improved onset of anagen phase [60]. To determine if UC-MSCs promote the hair cycle, these cells were injected intra-dermally at multiple sites in the dorsal skin field of depilated mouse. The control was the use of daily topical treatments with 3% minoxidil, and the hair cycle stage was determined with the measurement of skin hair regrowth. The results showed that the UC-MSCs emphasized hair follicle morphogenesis, but only without the ablation of the neonatal dermal cell, indicating that UC-MSCs facilitate hair growth and regeneration *via* a paracrine mechanism [62]. Several studies using ADSCs secretome had positive results when used to treat alopecia, as they were able to restore hair loss, due to the activation of hair regeneration pathways [63, 64].

#### **5.5 Application of MSCs in scar tissue**

Scar tissue is produced due to cutaneous wound healing, in which there is excessive deposition of extracellular matrix, allowing the skin to restore its integrity after a lesion. It occurs due to specific mechanisms that are modulated by local proinflammatory mediators, in the inflammatory phase of wound healing [65]. It has a different appearance, and the tissue does not function as normal skin, lacking some structures, such as hair follicles, sensory nerve receptors, and even sebaceous glands. Its tensile strength is also diminished in about 20%, which leads to re-injures, as the scar is considered a weak point [65].

The current treatments include sterile dressing and topical antibiotics to diminish infection risk and to advance into the proliferation and remodeling phases of the wound healing process, then allowing the regeneration of the tissue. It may also be necessary to debride the wound and irrigate it, to remove necrotic tissue [65].

The use of ADSCs has been reported to have the capacity to remodel scar tissue or block its formation, when used with a fat graft, in human plastic surgery [12].

A study has shown that the use of silk fibroin scaffolds containing human Wharton's jelly MSCs (Wj-MSCs-SF) in a murine model reduced the formation of fibrotic scar tissue, improving reepithelization and vascularized granulation tissue [66].

Gentile *et al* have demonstrated that the use of ADSCs contained in fat grafts has positive effects in scar signs and symptoms [67].

The use of ADSCs secretome in full skin defects demonstrated that it is capable of reducing scar formation, as well as accelerate wound closure and improve angiogenesis [68].

#### **5.6 Application of MSCs in burns**

Burns are very common lesions both in veterinary and human medicine and occur due to thermal, electrical, chemical, or radiation exposure. They are associated with the loss of normal tissue and cells, which complicates the wound healing process. Their severity is characterized by the depth and size of tissue damage, as first-, second-, third-, or fourth-degree burns. A first-degree burn is red or pink and sensitive, and can swell slightly. A second-degree burn is painful and often associated with the presence of blisters that reach the reticular dermal layer and have a high risk of chronic inflammation and keloid/hypertrophic scar formation. A third-degree burn

presents necrosis of the skin and deep tissues, and is compact and immobile. Fourthdegree burns presents as dry, very compact, and wrinkled. There are alterations in blood composition associated with a burn that occupies 10% or more of the body, which leads to metabolic complications [69, 70].

Current treatments consist in antibiotics, fluids, and detoxification. First- and second-degree burns are treated with antibiotics and anti-inflammatory drugs. Third- and fourth-degree burns require regular dressing change and sometimes surgery [70]. In several studies, the use of local injections of UC-MSCs and ADSCs in rodent models has diminished burn wound progression, as well as burn-induced inflammation. They have also accelerated wound healing in burned lesions, increased re-epithelization, vascularization, and granulation tissue formation [71–73].

The use of a hydrogel containing unsaturated arginine-based poly(ester amide) and chitosan associated with BM-MSCs also demonstrated an increased re-epithelization, granulation tissue formation, and wound healing rate [73, 74]. Also, the use of systemically delivered BM-MSCs in rodent models has increased wound healing [73].

Hackers *et al.* demonstrated the effects of secretome from peripheral blood mononuclear cells on pig models of skin burns. The results showed a decrease in the amount of mast cells in the wound area, demonstrating a lower inflammation of the burn area when using MSCs secretome [75].

In the study by Kudinov et al. using secretome from UC-MSCs combined with a chitosan hydrogel in burned rats, the results showed that this combination cleared the wound of bacteria, promoted re-epithelization and the formation of vascularized granulation tissue, and decreased inflammation [76].
