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Mesenchymal Stem Cell Therapy for Chronic Wound Healing in Dogs.

Introduction

Non healing chronic wounds constitute a major problem for Veterinary practitioner as 50% of these wounds do not respond to current treatments (Mustoe et al., 2006). The existence of mesenchymal stem cells (MSCs) in normal skin (Sellheyer and Krahl, 2010) and their definite role in wound healing. Paquet et al. (2009) suggests their application potential for management of non-healing wounds. MSCs have been well characterized to be multipotent cells that can differentiate into multiple tissue forming cell lineages (Krampera et al., 2006). They can provide not only desired cells for healing by trans-differentiation but also stimulate local healing process by providing different cytokines and growth factors. Thus application of exogenous MSCs in wound healing process for non healing wounds resulting from trauma, diabetes, vascular insufficiency and numerous other conditions, seems to be logical. The present study was undertaken to evaluate the clinical efficacy of bone marrow derived mesenchymal stem cells (MSCs) in management of chronic non-healing wounds in dogs.

Material and Methods

Dogs of different sex and breed with traumatic or post-operative wounds located at different body parts presented were subjected to routine treatment protocols including wound debridement, antiseptic dressing, systemic antibiotic etc. The six dogs which did not respond to routine treatment protocols were enrolled for the study. The physiological parameters were within normal range in all animals, on presentation and remained so during the treatment period. After 3-4 weeks, the wounds were subjected to allogenic bone marrow derived mesenchymal stem cell therapy. The wounds were debrided and stitched using quill suturing technique, when possible. MSC were obtained from IVRI stem cell laboratory, where canine (BMSC's) are routinely isolated and propagated for research application. MSC were injected locally around the edges of wound at concentration of 2.5X[10.sup.6] BMSC's in 1 ml PBS. The first administration was counted as day 0 and second administration was made after a gap of 7 days. In addition, the wounds were dressed with diluted Povidone (1%) daily, 2-3 times, sparing the day of administration of MSC The condition of wound was accessed on third, seventh, tenth and fourteenth days. The degree of exudation at site of repair was graded on 1-4 scale (Table 1). The degree of swelling at site of repair was graded on 1-4 scales (Table 1). Pain on palpation was graded on scale 1-4 (Table 2). Percentage contraction with respect to original wound size was calculated as per following formula:

Percentage contracture = 100 - [Total wound area [day.sub.n]/Original wound area [day.sub.o] X100

Data for wound contraction at different intervals were compared with 0 day values using paired 't' test in SPSS. Data for degree of exudation, degree of swelling, pain on palpation at different intervals were compared with 0 day values using Wilcoxon test in SPSS. P<0.05 was considered significant.

Results and Discussion

Normal skin depends on pool of adult stem cells, such as epidermal stem cells for its renewal and maintenance (Fuchs and Segre, 2000). After skin injury, an elaborate series of events progress, involving haemostasis, inflammation, cellular proliferation, angiogenesis and extracellular matrix production (Wu et al., 2007; Branski et al., 2009). Cytokines, chemokines and growth factors, produced from inflammatory cells, skin progenitor cells, fibroblasts and BM derived progenitor cells, are crucial in this process (Nakagawa et al., 2005). This underlines the utility of MSCs for enhancing wound healing in chronic non-healing wounds.

Six wounds like maggot wounds, wounds on tension sites like knee joint, electro-surgical tumor excision wounds and suture dehiscing laparotomy wounds were observed unresponsive to routine wound treatment protocol. These types of wounds may take long time to heal and may require special efforts. There are reports that in pathological conditions, such as diabetes mellitus, immune suppression, wounds at tension sites, following large injuries or burns and electro-surgical incisions, wound healing is delayed or not completed (Kim et al., 2003). The use of electro-cautery for tissue incision decreases capacity of wound healing due to tissue damage by thermal effect (Rappaport et al., 1990) which could also be the reason for delayed healing in present study. Impairment in production of cytokines and reduced angiogenesis may also contribute to non healing of chronic wounds (Wu et al., 2007; Kim et al., 2009). In patients with BM depletion or extensive skin tissue destruction, host adult stem cell pool cannot function effectively, resulting in delayed healing or non- healing skin wounds (Nakagawa et al., 2005). The Mean[+ or -] SD values of score for extent of wound contraction, exudation, degree of swelling and pain on palpation on 0, 3rd, 7th, 12th are given in Table 3.

Wounds started to show decrease in size from 3rd day onwards. The wound contraction was significant (P< 0.05) on day 3rd, 7th and 12th day. By 12th day on an average, there was more than 99% wound contraction, where 5 out of 6 wounds healed completely, the sixth wound healed completely by 14th day. Wound contraction plays vital role in healing of skin wounds. The wound undergoes physical contraction throughout the entire wound healing process, which is believed to be mediated by myofibroblasts (Gosain and Dipietro, 2004; Campos et al., 2008). The significant wound contraction from 3rd day onwards might be due to wound contraction due to myofibroblasts as well as enhanced epithelisation by stem cell administered locally. MSCs improve tissue repair by trans differentiation as well as by paracrine signalling. Mansilla et al. (2005) have proposed the three basic mechanisms to explain how MSCs could repair tissue injury. (i) Creation of milieu that enhances regeneration of endogenous cells (ii) Trans-differentiation of MSCs in to dermal fibroblast like cells (iii) fusion and integration with host cells. MSC paracrine signalling is the primary mechanism accounting for beneficial effects of MSCs on responses to injury such as inflammation, angiogenesis and fibro-proliferation (Lau et al., 2009). The results of present study also confirmed to the findings of earlier studies on bone marrow derived mononuclear cells (Borena et al., 2009) and bone marrow derived mesenchymal stem cells (Nanjappa et al., 2014).

There was gradual reduction in wound exudation which was on 7th day as compared to 0 day and by 12th day. it was totally abolished. The change was significant (P[less than or equal to]0.05) with respect to baseline on 7th and 12th day. The decrease in exudation could be attributed to decrease in inflammatory process and subsequent enhanced healing process by stem cells. However, moist wound environment was maintained during the healing process which is important and allows optimal healing by hastening debridement, promoting granulation tissue formation, differentiation of administered stem cells into epithelial cells and epithelialisation (Fossum et al., 2007; Kataoka et al., 2003). Slight to moderate swelling was recorded in all groups on 0 day. The degree of swelling decreased significantly (P<0.05) on 3rd day after application of stem cells to wounds. It decreased further and by 7th day no appreciable swelling was observed in any of the wounds. Moderated pain on palpation at site of wound was observed on day of first therapy which reduced significantly (p<0.05) to mild pain by 7th day and by 12th day the pain was abolished completely from all the wounds. Slight to moderate swelling and pain in earlier stages of wound healing could be attributed to inflammatory mechanism (Kumar, 1996) and is reduced significantly (p<0.05) due to decrease in inflammatory process and subsequent enhanced healing process by stem cells.

It was concluded that mesenchymal stem cell therapy hastens the healing in chronic non-healing wounds following electro surgery, wounds at tension sites etc. in dogs and can be advocated in such clinical cases with positive outcome.

Acknowledgments

Authors are highly thankful to the Director of Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India for providing necessary facilities.

References

Borena, B.M., Pawde, A.M., Amarpal, Aithal, H.P., Kinjavdekar, P., Singh, R. and Kumar, D. (2009). Autologous bone marrow-derived cells for healing excisional dermal wounds of rabbits. Vet. Rec. 165: 563-68.

Branski, L.K., Gauglitz, G.G. and Herndon, D.N. (2009). A review of gene and stem cell therapy in skin wound healing. Burns 35: 171-80.

Campos, A.C., Groth, A.K. and Branco, A.B. (2008). Assessment and nutritional aspects of wound healing. Curr. Opin. Clin. Nutri. Met. Care. 11: 281-88.

Fossum, T.W., Hedlund, C.S., Johnson, A.L., Schulz, K.S., Seim, H.B., Willard, M.D., Baer, A. And Carroll, G.L. (2007). Surgery of integumentary system. In: Manual of Small Animal Surgery. Mosby. P. 159-75.

Fuchs, E. and Segre, J.A. (2000). Stem cells: a new lease on life. Cell 100: 143-55.

Gosain, A. and Dipietro, L.A. (2004). Aging and wound healing. World J. Surg. 28: 321-26.

Kataoka, K., Medina, R.J., Kageyama, T., Miyazaki, M., Yoshino, T., Makino, T. and Huh, N. (2003). Participation of adult mouse bone marrow cells in reconstitution of skin. Am J Pathol. 163: 1227-31.

Kim, B.C., Kim, H.T. and Park, S.H. (2003). Fibroblasts from chronic wounds show altered TGF-b-signaling and decreased TGF-B Type II receptor expression. J Cell Physiol. 195: 331-36.

Kim, S.S., Song, C.K. and Shon, S.K. (2009). Effects of human amniotic membrane grafts combined with marrow mesenchymal stem cells on healing of full-thickness skin defects in rabbits. Cell Tissue Res. 336: 59-66.

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Kumar, A. (1996). Veterinary Surgical Techniques. Vikas Publishing House, New Delhi. p. 151-65.

Lau, K., Paus, R., Tiede, S., Day, P. and Bayat, A. (2009). Exploring the role of stem cells in cutaneous wound healing. Exp. Dermatol. 18: 921-33.

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Mustoe, T.A., O'Shaughnessy, K. and Kloeters, O. (2006). Chronic wound pathogenesis and current treatment strategies: A unifying hypothesis. Plast. Reconstr. Surg. 117: 35- 41.

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Nanjappa, M.D., Ahmad, R.A., Amarpal, Gugjoo, M.B., Pawde, A.M., Kinjavedkar, P., Aithal, H.P., Ansari, M., Chandra, V. and Sharma, G.T. (2014). Caudal superficial epigastric axial pattern flap and stem cell therapy for the management of large wound on medial aspect of thigh in a dog. Adv. Anim. Vet. Sci. 2: 188-91.

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Malik Abu Rafee (1), Amarpal (2), Vikash Chandra (3), G. Taru Sharma (3) and P. Kinjavdekar (4)

Division of Veterinary Surgery

Indian Veterinary Research Institute (IVRI)

Izatnagar

Bareilly - 243122 (Uttar Pradesh)

(1.) Veterinary Assistant Surgeon, Government of J & K

(2.) Principal Scientist cum HOD and Corresponding

author. E-mail: amarpal@ivri.res.in

(3.) Principal Scientists, Veterinary Physiology Division

(4.) Principal Scientist
Table 1: Score for degree of exudation and swelling at repair site

Score  Degree of exudation      Degree of
                                swelling

1      None (apparently dry     No swelling
       wound)
2      Slight exudates          Mild swelling
       (wound is moist, non
       oozing on pressing
       the wound)
3      Moderate exudates        Moderate
       (wound is moist, slight  swelling
       oozing on pressing the
       wound)

4      Extreme exudates         Severe
       (exudate is visible and  swelling
       pressure lead to
       extensive exudation)

Table 2: Score for pain on palpation

S.N.  Degree  Parameters

1.      0     None
2.      1     Mild (discomfort)
3.      2     Moderate (struggles)
4.      3     Severe pain (tries to
              bite, tries to escape)

Table 3: Mean [+ or -] SD values of score for extent of wound
contraction exudation, degree of swelling and pain on palpation

Days     Wound                   Exudation
         Contraction (%)

0      0[+ or -]0             2.67[+ or -]0.52
3     43.85[+ or -]20.49 (*)  2.17[+ or -]0.41
7     90.11[+ or -]5.74 (*)   1.33[+ or -]0.52 (*)
12    99.27[+ or -]0.62 (*)   1.00[+ or -]0.00 (*)

Days     Degree of             Pain on
         Swelling              palpation

0     2.00[+ or -]0.63      2.00[+ or -]0.00
3     1.33[+ or -]0.52 (*)  1.67[+ or -]0.52
7     1.00[+ or -]0.00 (*)  0.67[+ or -]0.82 (*)
12    1.00[+ or -]0.00 (*)  0.00[+ or -]0.00 (*)

Values with (*) superscripts are significantly different (P[less than
or equal to]0.05) with respect to zero day
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Title Annotation:Research Article
Author:Rafee, Malik Abu; Amarpal; Chandra, Vikash; Sharma, G. Taru; Kinjavdekar, P.
Publication:Intas Polivet
Article Type:Report
Date:Jan 1, 2017
Words:2148
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