S. A. Hilton1, L. C. Dewberry1, C. Zgheib1, M. M. Hodges1, E. J. Burtch2, J. Jacot2, P. Rozance3, S. Seal4, K. W. Liechty1 1University Of Colorado Denver,Department Of Surgery,Aurora, CO, USA 2University Of Colorado Denver,Department Of Bioengineering,Aurora, CO, USA 3University Of Colorado Denver,Department Of Pediatrics,Aurora, CO, USA 4University of Central Florida,Department of Material Science Engineering, AMPAC And NSTC Center,Orlando, FL, USA
Introduction
Spina bifida encompasses a range of caudal neural tube defects affecting 1 in every 2000 live births in the United States, the most severe being myelomeningocele where the spinal cord herniates out of the back. Amniotic fluid stem cells are an easily obtained source of autologous cells capable of differentiation into all three germ lines. Silk fibroin (SF) is a biocompatible and biodegradable polymer, which can be tailored to different nanostructures for biomedical application. We have tested multiple formulations of nanosilk including a liquid and matte formulation. We have previously shown that the nanosilk liquid improves biomechanical properties of human skin and both liquid and matte formulations and can be used for drug delivery. We hypothesized, we could isolate and differentiate ovine amniotic fluid stem cells and use these cells to populate a nanosilk matte that could be applied to a variety of wound healing applications including fetal repair of myelomeningocele.
Methods
Nanosilk (matte formulation) was created from silk fibroin isolated from the cocoons of Bombyx mori silk worms, cut into small pieces, and dissolved in solution to obtain the viscosity needed for electrospinning nanofibers. Ovine amniotic fluid mesenchymal stem cells (oAF MSCs) were isolated from mid-gestation amniotic fluid collected during fetal intervention. oAF MSCs were cultured with the nanosilk for 7 days.
Nine female 12 week old mice that are bred homozygous diabetic (Db/Db) were used. Db/Db mice were used as this is an established murine model of abnormal diabetic wound healing. A single 8mm wound was made on the dorsal neck skin of each mouse with a punch biopsy. Wounds were treated with one time administration of phosphate buffered saline control or Nanosilk only and Nanosilk + oAF MSCs at the time of wounding.
Results
oAF MSC cells were isolated from 7 ewes within 7-21 days after amniotic fluid harvest. oAF MSCs were confirmed with IHC markers (positive for CD29, CD90, CD44, and negative for CD45) and showed the ability to differentiate into multiple cell lineages including adipocytes, osteocytes, and neural cells (Figure 1). Diabetic mice treated with nanofiber had improved wound healing (day 16 compared to day 18). Nanofiber cultured with ovine amniotic stems cells had similar wound healing to the nanofiber. The nanofiber did not incorporate into the wound and acted as a biologic dressing that was shed as the wound healed (Figure 2).
Conclusions
oAF MSCs can be quickly isolated and cultured for incorporation into nanofiber silk patch that can be applied to wounds and possibly for fetal repair of myelomeningocele. The nanofiber silk patch is able to hold the MSCs stable in culture for several weeks and presents a possible delivery method for these cells. No adverse effects on wound healing are seen when the nanosilk or nanosilk + oAF MSCs are used and may have improved wound healing.
