Decellularized amnion scaffold with activated PRP: a new paradigm dressing material for burn wound healing

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Decellularized amnion scaffold with activated PRP: a new paradigm dressing material for burn wound healing Jeevitaa Kshersagar . Ravi Kshirsagar . Shashikant Desai . Raghvendra Bohara . Meghnad Joshi

Received: 22 May 2017 / Accepted: 20 February 2018 Ó Springer Science+Business Media B.V., part of Springer Nature 2018

Abstract Direct application of amnion has greater risk of immunological rejection and infection. Decellularization is an effective method to lower the risk of immune complications and infections. The bioreactor assembly with multiple cassettes was designed for decellurization of multiple amnions with different cell types simultaneously in single run. A detergent-based protocol was modified to remove all cellular components from amnion and diminish the DNA content to render it non-immunogenic. Amnion (n = 10) were treated with 2% sodium dodecyl sulphate (SDS), 5% dimethyl sulfoxide (DMSO) and 2% sodium deoxycholeate (SD). Decellularized amnion samples were analyzed by haematoxylin–eosin staining (HE), Alcian blue pH 1 (AB-pH-1), 4,6-diamnionidino-2phenylindol (DAPI), Massion’s trichrome stain, DNA quantification, mechanical testing and scanning electron microscopy (SEM). Histological analysis showed complete removal of cellular components and the histoarchitecture of scaffold remained intact. Amnion scaffold activated with platelet rich plasma (PRP) and calcium chloride composition supported better

adherence to the wound than amnion alone. Only single application showed good healing. In vivo assessment of activated amnion revealed stable dressing. It has good promising outcome. At day 7, histologically the wounds treated with activated amnion were almost closed without scarring and showed well differentiated epidermis, proliferation of keratinocytes, hair follicles and basement membrane as compared to controls and silver nitrate gel dressings in a mouse (Mus musculus). Cryopreservation had no adverse effect on the mechanical properties of the amnion scaffold. Cryopreservation of decellularized amnion by Dulbecco’s modified eagle medium (DMEM) was expected to prepare off-the-shelf skin substitutes and preserve them to be immediately available upon request of patients’ needs. Keywords Burn wound  Bioreactor  Scaffold  Activated PRP  Cryopreservation

Introduction J. Kshersagar  R. Kshirsagar  R. Bohara  M. Joshi (&) Department of Stem Cells and Regenerative Medicine, D.Y.Patil University, D. Y. Patil Vidyanagar, Kasba Bawda, Kolhapur, MS 416006, India e-mail: [email protected] S. Desai Stem Plus Biotech, Block #11, Shiv Meridian, M G Road, Sangli, MS 416416, India

Amnion is the thin innermost semitransparent membrane separated from the chorion, devoid of blood vessels, lymphatic, nerves and biological viable which is ideal for creating scaffold (Augustine et al. 2014; Niknejad et al. 2008). It can produce growth factors (Zhang et al. 2013) and has anti-inflammatory (Kesting et al. 2014), anti-microbial, anti-fibrosis, anti-

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scarring (Niknejad et al. 2008) and an