ABSTRACT
Recombinant spider silk has the potential to provide a new generation of biomaterial scaffolds as a result of its degree of biocompatibility and lack of immunogenicity. These recombinant biomaterials are, however, reported to exhibit poor cellular adhesion which limits their potential for use in applications such as tissue engineering and regenerative medicine. In this study, a simple chemical functionalization approach is described that specifically addresses this issue and significantly improves the adhesion of human mesenchymal stem cells (CiMSCs) to a recombinant spider silk biomaterial. This utilizes copper-catalyzed or strain-promoted azide-alkyne cycloaddition (CuAAC/SPAAC) "click" chemistry to covalently attach cyclo(RGDfK) peptides to the azide group of l-azidohomoalanine, a methionine analogue previously site specifically incorporated into the primary sequence of a thioredoxin (TRX)-tagged silk fusion protein, TRX-4RepCT, to give TRX3Aha -4RepCT3Aha . This method is used to produce cyclo(RGDfK) functionalized films and macroscopic fibers. Over 24 h, cyclo(RGDfK) functionalized TRX3Aha -4RepCT3Aha films and 4RepCT3Aha fibers display significantly improved performance in CiMSC culture, yielding far greater cell numbers than the controls. This approach circumvents the previously observed lack of cell adhesion, thus allowing spider silk derived biomaterials to be used where such adhesion is critical, in tissue engineering, regenerative medicine and wound healing.
Subject(s)
Arthropod Proteins/chemistry , Biocompatible Materials/pharmacology , Silk/pharmacology , Wound Healing/drug effects , Alkynes/chemistry , Arthropod Proteins/chemical synthesis , Azides/chemistry , Biocompatible Materials/chemistry , Click Chemistry , Copper/chemistry , Cycloaddition Reaction/methods , Fibroins/chemistry , Fibroins/genetics , Fibroins/therapeutic use , Humans , Mesenchymal Stem Cells/drug effects , Recombinant Proteins/chemistry , Recombinant Proteins/pharmacology , Silk/chemistryABSTRACT
Polymer microparticles are widely used as acellular drug delivery platforms in regenerative medicine, and have emerging potential as cellular scaffolds for therapeutic cell delivery. In the clinic, PLGA microparticles are typically administered intramuscularly or subcutaneously, with the clinician and clinical application site determining the precise needle gauge used for delivery. Here, we explored the role of needle diameter in microparticle delivery yield, and develop a modified viscosity formulation to improve microparticle delivery across a range of clinically relevant needle diameters. We have identified an optimal biocompatible formulation containing 0.25% pluronic F127 and 0.25% carboxymethyl cellulose, which can increase delivery payload to 520% across needle gauges 21-30G, and note that needle diameter impacts delivery efficacy. We use this formulation to increase the delivery yield of PLGA microparticles, and separately, PLGA-cell scaffolds supporting viable mesenchymal stem cells (MSCs), demonstrating the first in vitro delivery of this cell scaffold system. Together, these results highlight an optimal formulation for the delivery of microparticle and microparticle-cell scaffolds, and illustrate how careful choice of delivery formulation and needle size can dramatically impact delivery payload.
