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Combined Local Delivery Of Tacrolimus And Stem Cells In Fibrin Gel Is A Viable Potential Treatment For Enhancing Peripheral Nerve Regeneration
Tiam M. Saffari, MD1, Katelyn Chan, B.Eng BioSci2, Kevin J. Zuo, MD2, Gregory H. Borschel, Prof, MD2, Alexander Y. Shin, Prof, MD1.
1Mayo Clinic, Rochester, MN, USA, 2SickKids Hospital, Toronto, ON, Canada.

PURPOSE: The outcome of scaffold-based stem cell transplantation in peripheral nerve injury remains unsatisfactory due to poor survival of transplanted cells, most likely associated with the immune rejection. Systemic immunosuppression could overcome rejection, but causes various undesired side effects. Recently, the neuroregenerative potential and bimodal dose effects of tacrolimus (an FDA approved immunosuppressant) have been explored in addition to its immunosuppressive role. The aim of this study was to determine the feasibility and effectiveness of combining local, sustained delivery of tacrolimus with stem cells.
METHODS: First, the drug release profile from the local tacrolimus delivery system was validated. Tacrolimus was incorporated into fibrin gel in poly(lactic-co-glycolic) acid (PLGA) microspheres in concentrations of 0.01 ng/ml and 100 ng/ml, as previously described by Tajdaran et al. (2015). Microspheres were prepared and characterized using the validated protocol by Tajdaran et al. (2015). Drug release from the tacrolimus-delivery system was analyzed by incubating the gels in phosphate-buffered saline (PBS) at physiological temperature, replenishing and collecting release media for every 3 days up to 35 days. Release media was characterized using liquid chromatography mass spectrometry to plot drug release profiles. Secondly, adipose derived mesenchymal stem cells (MSCs) were cultured in the fibrin gel to evaluate their interaction with tacrolimus and the gel. 103 MSCs were seeded in (i) gel only, (ii) PLGA empty microspheres+gel, (iii) 0.01, and (iv) 100 ng/ml of tacrolimus encapsulated in PLGA microspheres+gel. MSCs were cultured in drug release media collected at days 7, 15 and 28 to mimic systemic exposure representing released concentrations at these days from both concentrations microspheres. After 72h, cytotoxicity assays and immunofluorescence staining against MSC surface marker CD90 were conducted to confirm stem cell viability and characterization, respectively. Live/dead staining was performed at 24h, 48h, 72h, and seven days.
RESULTS: From tacrolimus microspheres containing 100 ng/ml, a sustained release of up to 35 days was detected, with a total of 200 g tacrolimus released for the duration of the study. Microspheres containing 0.01 ng/ml showed depletion of tacrolimus by 13 days. Drug release profiles were consistent with those of Tajdaran et al. (2015), in which an inflection point is observed around 15 days due to almost complete erosion of fibrin gel, which results in an increase in the rate of tacrolimus release. For MSCs cultured in the gel groups, and in the tacrolimus drug release media from 7, 15 and 28 days release, cell viability was approximately 80% and 100% respectively, and insignificantly different across groups, when compared to MSCs cultured in untreated media (negative control). CD90 staining confirmed stem cell characterization and live/dead staining confirmed viability up to seven days.
CONCLUSION: Encapsulation of 100 ng/ml tacrolimus in PLGA microspheres and MSCs in fibrin gel is feasible and has strong potential to enhance survival of transplanted cells, which may ultimately lead to improved nerve regeneration.

Figure 1. Viability of MSCs(A) and CD90 staining(B).


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