Chronic no...

Aim: The study was conducted to reveal the efficacy of Bone marrow derived mesenchymal stem cells (BM-MSCs) based therapy in healing of chronic non-healing and ulcerative wound in bovine species. Materials and Methods: One 2 years old Jersey heifer affected with chronic ulcerative wound involving full thickness skin and under lying muscle at dorsal side of lumbar region since four months at the time of presentation. Bone marrow was collected from tibia, cultured and grown and after achievement of optimum confluence it was applied at the site. Different parameters of clinical, physiological, haematological, biochemical, histochemical, histological, tensile strength and photographic evaluations were done during the study period. Results: The estimated values of above mentioned parameters on zero day and after healing (18 days) showed significant difference (P<0.05) in relation to collagen content, tensile strength and physical characteristics of wound like extent of wound, size of wound, type of exudates and photography. But clinical, haematological and biochemical data showed no significant difference. Conclusion: The BM-MSCs were the main pioneers to bring the chronic ulcerative wound towards healing. The procedure is simple, safe and effective in bringing out healing without showing any adverse effect on host.

Introduction autologous bone marrow derived stem cell (BM-Chronic non-healing or slow healing ulcers MSCs) therapy and to know the progress and duration represent a major health burden and continue to pose a of healing with this application.challenge not only to the patient owners but to the

Materials and Methods
treating physician and medical procedures as a whole.The healing of wound occurs by primary intention and The owner was fully informed and written secondary intention [1, 2,3].The therapeutic application consent was obtained about the clinical trial, risk and of autologous BM derived MSCs (Bone marrow benefits of the proposed cell based therapy.It was also derived mesenchymal stem cells) have revolutionized approved by the Institutional Animal Ethics the field of regenerative medicine.The BM-MSCs Committee of the institution since it was a clinical case were been used for the treatment of chronic wound in and related to Agricultural Production Research.diabetic patients [4], for non-healing ulcers of lower Measurement of the wound dimension was taken extremities in human being [5], cutaneous radiation along with swab from wound bed for bacteriological syndrome in minipig model [6] and in dogs [7] proved culture and sensitive test (Fig. 1).Biopsy was done successful in their findings.A clinical case of 2 years from wound bed for histopathological and old Jersey heifer was presented with one large size histochemical study.Wound was washed, cleaned and chronic non-healing wound at lumbar region since 4 dressed with fly repellent spray.Under peroneal nerve months.It was treated with different standard therapeutic block and local infiltration with 2% lignocaine regimens since then but showing no tendency towards hydrochloride, the proposed site i.e antero-medial aspect of tibia was prepared aseptically for bone Quantitative estimation of collagen content of tissue TM marrow collection.A 0.5 cm long incision was made was also done with Sircol method by help of and tibia was drilled with 2 mm drill bit. 10 ml of bone spectrophotometer at 555 nm wave length [8,9].The marrow was aspirated in a sterile syringe primed with study of tensile strength and strain of tissue was EDTA (Himedia) @ 1mg/ml.The EDTA mixed bone carried out after 60 days of healing and compared with marrow was despatched keeping inside ice packed normal skin by help of Universal testing machine thermo cool to the stem cell laboratory for culture and (Instron 3382, USA) with running rate 50 mm/minute 0 growth.
at 27 C temperature and 65% humidity.Conditioned media was prepared for culture

with commercially available basic ingredients as per Table-1. The medium was changed reguraly and cell
During the study period the physiological, morphology was examined under a Nikon phase haematological and biochemical parameters through contrast microscope.After complete colony formation varied but remained within the normal physiological the cultured MSCs were taken for therapeutic range.On the basis of culture and sensitivity test application (Fig. 2).As the stem cell therapy requires antibiotic inj.Ciprofloxacine @ 5 mg/kg body weight complete sterile medium, the ulcerated wound site was was administered parentrally for 5 days.Regarding prepared aseptically.The prepared BM-MSCs was collection of bone marrow it was collected from femur diluted with normal saline solution (NSS) at 2: 1 ratio and tibia of rats [10], mouse [11], mongrel dogs [12], and implanted intra-dermally and topically on wound iliac crest of femur [7], humerus in minipigs [6].In this bed (Fig. 3) and then bandaged with paraffin wet study the bone marrow was collected from proximal bandage.Outwardly fly repellent spray was sprinkled.antero-medial aspect of tibia owing to its superficial in 0 The clinical parameters like rectal temperature ( C), position, very thin layer of tissue over the bone and pulse rate (beats/min), respiration rate (breaths/min) easy for collection.Histopathology after therapy showed and status of visible mucous membrane were recorded neovascularization with appearance of fibroblasts, on the day of presentation, BM collection, BM-MSCs sebaceous glands and epithelialisation which supports th the progression of healing process (Fig. 4).implantation and 18 days after healing.The Histo-chemical study showed formation of more photographic evaluation along with dimension of the collagen content after stem cell therapy.The collagen ulcerative wound was measured during healing TM th period.Haematological and biochemical parameters content with Sircol method on zero day, 14 day and were estimated during the study (  elongation at break of normal skin and BM-MSCs wound healing effects of skin is being mediated by the treated skin were 20.23 MPa, 13.95 MPa and regenerative effects of transforming growth factor 104.85%, 35.79% respectively.While comparing the (TGF), fibroblast growth factor (FGF), fibronectintensile stress and strain between normal skin and like peptide, keratinocytes growth factor, epidermal healed skin after BM-MSCs therapy, though normal growth factor and growth hormone releasing factor skin shows higher value for breaking strength, the [21] and these factors might have been accelerated due healed tissue was advancing towards proper to MSCs application.This result indicates that it may stretchability and gaining the normal tensile strength.emerge as an effective therapeutic approach for The wound assessment was studied for gross chronic non-healing and ulcerative cutaneous wound observation of wound viz types of exudates, extent of in future.Therefore large scale randomised studies and exudates, swelling and wound contraction.JD and IN designed the study as this is a part of JD's In this case it was studied on 0-3 scale and found PhD thesis and IN was his major advisor.PR helped in significant difference (P<0.05) while comparing processing of BM.RKD analyzed the study during healing with that of zero day.The photographs of the post therapy period.SSB helped during collection of wound were evaluated by three surgeons and showed BM and assisting throughout the study.All authors th complete healing on 18 day (Fig. 5).The result read and approved the final manuscript.supports the findings of comparison of photographs by

Fig- 1 .Fig- 2 .Fig- 3 .Fig- 4 .Fig- 5 .
Fig-1.Measurement of wound dimension (length) Fig-2.Photomicrograph of cultured BM cells on day-5.40X Fig-3.Application of BM-MSCs by intradermal injection Fig-4.Formation of granulation tissue and abundant neovascularisation after BM-MSCs application Fig-5.Photograph showing healing after 18 days of BM-MSCs application Table-2).Histo-18th day were 11.98 µg/mg, 26.54 µg/mg and 29.24 pathology with H and E stain and Masson-Trichrome µg/mg which supports the findings of Ghani et al. [13] stain for study of collagen content were carried out.regarding wound healing.The tensile strength and The wound clinical trials are needed to prove this promising option.healingwasassessed byBose et al. [14]in bovines on

Table - 1
Autologous stem cell therapy to treat chronic ulcer in heifer-A case study .Constituents of the Conditioned media.

Table - 2
. Biochemical parameters measured before and after implantation.