Discipline: Biological Sciences
Subcategory: Biomedical Engineering
Aundrya Montgomery - University of Connecticut Health Center
Co-Author(s): Dr. Cato T. Laurencin, University of Connecticut Health Center, Farmington, C; Dr. Lakshmi Nair, University of Connecticut Health Center, Farmington, CT
Although currently impossible, our lab continues to make conscious efforts in regenerating a human limb. Over time, studies have evaluated children’s ability to regenerate the digit tip and reasoning why these properties decrease with age. Humans possess the ability to regenerate digit tips dependent upon the level of amputation; moreover, successful regeneration relies on the presence of key tissues, such as the nail organ. The hard nail plate, the shiny keratinous structure on the dorsal aspect of the fingertip, regenerates continuously and is physically required to maintain the interaction between the proximal nail fold and nail matrix to promote the growth of the nail plate. The proximal nail matrix houses a population of stem cells that contribute to the continuous extension of the fingernail and play a key role in mammalian digit tip regeneration. Nail matrix removal, as a result of traumatic injury, precludes the regeneration of a fingertip and results in scar tissue formation. To better understand the mammals’ ability to regenerate a digit tip, fingernail regeneration has become an ideal model in examining the nail matrix’s participation in the process. Overall, our goal is to fabricate a biomaterial capable of supporting the delivery of nail stem cells and growth factors to ultimately regenerate the hard nail plate and assist in digit tip regeneration. The present study focuses on developing a biodegradable and biocompatible extracellular matrix mimic nanofibrous scaffold to facilitate the interaction between the proximal nail fold and nail matrix and to use a stem cell-delivery system to accelerate the regeneration process of hard nail tissue.
MATERIALS AND METHODS:
Poly(lactide-co-glycolide) (85:15) was dissolved in 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) to compose 15%-25% (w/v) polymer solutions for the electrospinning process. Constant parameters of 10 kV applied voltage, 10 cm needle tip to collector distance, and flow rate of 1.5 mL/hour was used to develop the nanofibers. The diameter and morphology of the electrospun PLGA nanofibers was characterized by scanning electron microscopy (SEM) and analyzed using ImageJ software. Adipose-derived stem cells (ADSCs) were isolated from the inguinal region of Sprague Dawley rats according to standard protocol. Nail stem cells (NSCs) were isolated from the germinal matrix region of Sprague Dawley rat claws. Flow cytometry was used to determine the purity of the stem cell populations (positive markers: CD 29 and CD 90; negative markers: CD 45, CD 11b, CD 34). Live/Dead Assay was used to evaluate the biocompatibility PLGA electrospun scaffolds as substrates for both ADSCs and NSCs in vitro.
RESULTS AND DISCUSSION:
Hard nail tissue is composed of keratin nanofibers, therefore, the aim of the present study is to fabricate a biomimetic synthetic matrix as a stem cell delivery system for hard nail tissue regeneration. Among the different PLGA (85:15) concentrations studied, 16% (w/v) led to the development of bead-free fibers with fiber diameter distribution ranging between 143-1109 nm. The bioactivity of PLGA nanofibrous matrices was increased by adsorbing epidermal growth factor (EGF) on the surface to modulate cell functions. EGF is found within the matrix of hard nail tissue and is necessary for maintaining the morphological development of the nail organ. The incorporation of EGF in the nanofiber matrix will therefore support the regeneration of the hard nail plate. Nail stem cells housed in the proximal nail matrix give rise to the hard nail plate and are credited for the nail plate’s constant regeneration. NSCs isolated from the proximal nail matrix expressed the stem cell markers (CD 29 and CD 90). ADSCs were also evaluated as potential stem cells for this application due to their availability, ability to differentiate to the keratinocyte cell lineage, as well as the translational potential of ADSCs compared to NSCs. ADSCs displayed mesenchymal stem cell markers CD 29 and CD 90 as well. Live/Dead analysis revealed that both NSCs and ADSCs seeded on PLGA nanofiber scaffolds remained viable after 48 hours in culture. PLGA nanofiber matrices, due to their biocompatibility and biomimetic structure could serve as an ideal cell carrier to promote hard nail tissue regeneration.
Our work in limb regeneration has taken many forms and for the first time we have engineered a scaffold capable of mimicking the structure and orientation of nail matrix to support hard nail tissue regeneration. Further work will center on the regeneration of a nail in vitro and in vivo.
Funder Acknowledgement(s): 1. National Institutes of Health Grant #: DP1 AR068147 2. Biomedical Research Trust Fund Grant#: 600940-10301-20
Faculty Advisor: Dr. Cato T. Laurencin, firstname.lastname@example.org
Role: Throughout efforts to regenerate hard nail tissue, I have been responsible for the isolation, culture, and characterization of both nail stem cells and adipose-derived stem cells. Also, I have conducted the scaffold fabrication as well as characterization experiments needed to reach the optimal parameters. Furthermore, I have carried out the in vitro experiments and in the future I will continue in vivo studies that determine the efficacy of a regenerative engineering approach in regenerating the nail organ.