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Evaluation of persistence and distribution of intra-dermally administered PKH26 labelled goat bone marrow derived mesenchymal stem cells in cutaneous wound healing model

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Abstract

The current study was designed to study the persistence and distribution of caprine bone marrow derived mesenchymal stem cells (cBM-MSCs) when administered intra-dermally in experimentally induced cutaneous wounds in rabbits. MSC’s from goat bone marrow were isolated and their differentiation potential towards adipogenic and osteogenic lineages were assayed in vitro. The isolated cells were phenotypically analysed using flow cytometry for the expression of MSC specific matrix receptors (CD73, CD105 and Stro-1) and absence of hematopoietic lineage markers. Further, these in vitro expanded MSCs were stained with PKH26 lipophilic cell membrane red fluorescent dye and prepared for transplantation into cutaneous wounds created on rabbits. Five, 2 cm linear full thickness skin incisions were created on either side of dorsal midline of New Zealand white rabbits (n = 4). Four wounds in each animal were implanted intra-dermally with PKH26 labelled cBM-MSCs suspended in 500 µl of Phosphate Buffer Saline (PBS). Fifth wound was injected with PBS alone and treated as negative control. The skin samples were collected from respective wounds on 3, 7, 10 and 14 days after the wound creation, and cryosections of 6 µM were made from it. Fluorescent microscopy of these cryosections showed that the PKH26 labelled transplanted cells and their daughter cells demonstrated a diffuse pattern of distribution initially and were later concentrated towards the wound edges and finally appeared to be engrafted with the newly developed skin tissues. The labelled cells were found retained in the wound bed throughout the period of 14 days of experimental study with a gradual decline in their intensity of red fluorescence probably due to the dye dilution as a result of multiple cell division. The retention of transplanted MSCs within the wound bed even after the complete wound healing suggests that in addition to their paracrine actions as already been reported, they may have direct involvement in various stages of intricate wound healing process which needs to be explored further.

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Acknowledgements

Authors are thankful to Director, ICAR-IVRI for providing necessary financial assistance.

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Authors and Affiliations

  1. Division of Physiology and Climatology, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, UP, India

    M. D. Pratheesh, Nitin E. Gade, Amar Nath, Pawan K. Dubey, T. R. Sreekumar & G. Taru Sharma

  2. Division of Surgery, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, UP, India

    T. B. Sivanarayanan, D. N. Madhu &  Amarpal

  3. Division of Pathology, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, UP, India

    G. Saikumar

  4. Kerala Veterinary and Animal Sciences University, Pookode, India

    M. D. Pratheesh & T. R. Sreekumar

  5. College of Veterinary and Animal Sciences, Durg, India

    Nitin E. Gade

  6. Central Drug Research Institute, Lucknow, India

    Amar Nath

  7. Centre for Genetic Disorders, Institute of Science, Banaras Hindu University, Varanasi, India

    Pawan K. Dubey

Authors
  1. M. D. Pratheesh
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  2. Nitin E. Gade
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  3. Amar Nath
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  4. Pawan K. Dubey
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  5. T. B. Sivanarayanan
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  6. D. N. Madhu
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  7. T. R. Sreekumar
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  8. Amarpal
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  9. G. Saikumar
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  10. G. Taru Sharma
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Correspondence to G. Taru Sharma.

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10616_2017_97_MOESM1_ESM.jpg

Supplementary material (Figure)-Negative control wound cryo-sections administered with PBS alone when observed under bright field and fluorescent microscopy did not observe any red fluorescent signals at 3rd and 14th day post surgery (JPEG 172 kb)

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Pratheesh, M.D., Gade, N.E., Nath, A. et al. Evaluation of persistence and distribution of intra-dermally administered PKH26 labelled goat bone marrow derived mesenchymal stem cells in cutaneous wound healing model. Cytotechnology 69, 841–849 (2017). https://doi.org/10.1007/s10616-017-0097-0

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  • DOI: https://doi.org/10.1007/s10616-017-0097-0

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