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Edited by Shu Chien, University of California, San Diego, La Jolla, CA, and approved January 15, 2014 (received for review November 20, 2013)
Significance
Engineered replacements for musculoskeletal tissues generally require extensive ex vivo manipulation of stem cells to achieve controlled differentiation and phenotypic stability. The ability to control cell differentiation using cell-instructive scaffolds that have biomechanical properties approximating those of native tissue would represent a transformative advance in functional tissue engineering. The goal of this study was to develop a bioactive scaffold capable of mediating cell differentiation and formation of an extracellular matrix with the biochemical composition and mechanical features that mimic native tissue properties. By combining innovative gene delivery strategies with advanced biomaterial design, we demonstrate the feasibility of generating constructs capable of restoring biological and mechanical function.
Abstract
The ability to develop tissue constructs with matrix composition and biomechanical properties that promote rapid tissue repair or regeneration remains an enduring challenge in musculoskeletal engineering. Current approaches require extensive cell manipulation ex vivo, using exogenous growth factors to drive tissue-specific differentiation, matrix accumulation, and mechanical properties, thus limiting their potential clinical utility. The ability to induce and maintain differentiation of stem cells in situ could bypass these steps and enhance the success of engineering approaches for tissue regeneration. The goal of this study was to generate a self-contained bioactive scaffold capable of mediating stem cell differentiation and formation of a cartilaginous extracellular matrix (ECM) using a lentivirus-based method. We first showed that poly-l-lysine could immobilize lentivirus to poly(ε-caprolactone) films and facilitate human mesenchymal stem cell (hMSC) transduction. We then demonstrated that scaffold-mediated gene delivery of transforming growth factor β3 (TGF-β3), using a 3D woven poly(ε-caprolactone) scaffold, induced robust cartilaginous ECM formation by hMSCs. Chondrogenesis induced by scaffold-mediated gene delivery was as effective as traditional differentiation protocols involving medium supplementation with TGF-β3, as assessed by gene expression, biochemical, and biomechanical analyses. Using lentiviral vectors immobilized on a biomechanically functional scaffold, we have developed a system to achieve sustained transgene expression and ECM formation by hMSCs. This method opens new avenues in the development of bioactive implants that circumvent the need for ex vivo tissue generation by enabling the long-term goal of in situ tissue engineering.
Footnotes
↵1C.A.G. and F.G. contributed equally to this work.
- ↵2To whom correspondence may be addressed. E-mail: charles.gersbach{at}duke.edu or guilak{at}duke.edu.
Author contributions: J.M.B., C.A.G., and F.G. designed research; J.M.B., N.P.T.H., C.M.G., P.P.-P., F.T.M., and J.S.-A. performed research; J.M.B. and N.P.T.H. analyzed data; and J.M.B., C.A.G., and F.G. wrote the paper.
Conflict of interest statement: F.T.M. and F.G. are paid employees of Cytex Therapeutics.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1321744111/-/DCSupplemental.
Scaffold-mediated transduction of MSCs
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