Full length articleMultilayered polycaprolactone/gelatin fiber-hydrogel composite for tendon tissue engineering☆
Graphical abstract
Introduction
Tendons and ligaments are prone to injuries such as rupture and laceration due to their load-bearing nature [1], [2]. In cases of severe tendon injury, surgical intervention is employed to repair or replace the damaged tendon with autografts, allografts, xenografts, or prosthetic devices [3], [4], [5], for the natural healing process is slow and insufficient [6], [7]. To date, the clinical outcomes of tendon repair remain limited and unsatisfactory due to donor site morbidity, high failure rates, risk of injury recurrence, and limited long-term function restoration [8], [9], [10]. These limitations have spurred the development of tendon tissue engineering approaches, which apply combination of cells, scaffolds and bioactive molecules, as a promising strategy to create functional tissue replacements or to enhance the innate healing process [11], [12]. Ultimately, tendon tissue engineering aims at improving the quality of healing in order to fully restore tendon structure and function [13].
Tendon tissues are composed of densely packed aligned collagen fibrils that connect muscle to bone [7], [14]. Therefore, aligned nano- and micro-fibrous scaffolds fabricated by electrospinning have been extensively explored in attempts to recapitulate the mechanical and topographical characteristics of native tendon tissue [15], [16], [17]. Electrospun poly-ε-caprolactone (PCL) scaffolds are frequently used in tendon tissue engineering as well as applications for other soft tissues. PCL is an aliphatic linear polyester approved by the U.S. Food and Drug Administration for clinical use [18]. It is biocompatible, bioresorbable and a low-cost synthetic polymer. Of equal importance, PCL exhibits low degradation rate due to its semi-crystalline and hydrophobic nature [19], [20], making it a suitable graft material to facilitate the relatively slow healing process of injured tendons [21], [22]. However, the hydrophobic nature of PCL often results in poor wettability [23], inadequate cell attachment, and poor tissue integration [24] when used in tissue engineering. Moreover, as a synthetic polyester, its lack of bioactivity is a major challenge for PCL to direct cell behavior after seeding due to the absence of cell-binding motifs found in natural extracellular matrix (ECM) proteins [25].
Hydrogels prepared from collagen and its derivative, gelatin, represent another class of scaffolds for regenerating and repairing a wide variety of tissues and organs [26], [27]. Unlike other types of scaffolds, hydrogels retain a large volume of water and thus provide a highly hydrated environment similar to that in native tissues. Cells encapsulated within collagen/gelatin hydrogels can be easily distributed homogeneously by simple mixing during gel preparation [28], [29]. Importantly, collagen and gelatin, as constituents of natural ECM, better mimic at least in part the native tissue microenvironment, as compared to synthetic polymers [30], [31]. Nevertheless, improvement in the mechanical properties and introduction of topographical cues are needed to apply these hydrogels to tendon grafts that aim at reproducing the mechanical and structural features of native tendon tissues.
The organization of native ECM may be viewed as a cell-containing hydrogel reinforced by structural fibers. An engineered scaffold consisting of hydrogels and electrospun fibers may thus be considered as a biomimetic of the ECM. For example, a microfiber-reinforced silk hydrogel displayed a greatly improved modulus compared to a fiber-free hydrogel [32]. In addition, hydrogels composed of natural proteins could provide the bioactive motifs absent from synthetic polymeric scaffolds to enhance control of cell binding and fate determination [28], [33]. In terms of tendon tissue engineering, an ideal composite scaffold consisting of hydrogel and fibrous scaffold has yet to be developed. Consequently, little is known about the effects such a composite scaffold may have on the activities of encapsulated cells.
In this study, we have developed a novel composite scaffold as a tendon graft consisting of electrospun PCL microfibers and methacrylated gelatin (mGLT). We have optimized the retention of mGLT by photocrosslinking and its integration with the fibrous scaffold. Simultaneous cell seeding and photocrosslinking between scaffold layers were performed to create cell-impregnated multilayered constructs, and their mechanical properties and architecture and the activity of encapsulated cells were assessed. Our results show that this novel cell-scaffold construct combines the advantages of PCL nanofibrous scaffolds and gelatin hydrogels to mimic the mechanical feature, structure and cell phenotype of native tendon tissue.
Section snippets
Synthesis of methacrylated gelatin
Methacrylated gelatin (mGLT) was synthesized using an established protocol with slight modification [29], [34]. Gelatin (GLT, Sigma–Aldrich) was dissolved in deionized H2O at 37 °C (30%, w/v). Methacrylic anhydride (Sigma–Aldrich) was then added dropwise into the mixture at 37 °C under mild agitation to react with amine groups on GLT for 24 h (Supplementary Fig. S1 A). Reacted mGLT solution was dialyzed against water to completely remove low molecular-weight byproducts using 3500 NMWCO dialysis
Organization of fibers in composite scaffold
Microscopic observation of the co-electrospun scaffold showed that the fluorescently labeled fibers were interspersed within the dry scaffold (Fig. 2A, Composite Dry, red: PCL; green: mGLT). Hydration of the scaffold in an aqueous environment resulted in rapid dissolution of mGLT fibers, whereas PCL fibers remained intact (Fig. 2A, Composite Wet, red: PCL; green: mGLT). SEM revealed a fraction of fibers with distinct ribbon-like morphology, in addition to the cylindrically shaped fibers seen in
Discussion
Scaffolds are of critical importance in the context of tissue engineering, serving to provide a physical substrate that mimics the in vivo milieu of healthy tissues and thus orchestrate the activity of therapeutic cells in a tissue-specific fashion [38]. In terms of tendon tissue engineering, a number of scaffold designs have been developed to reproduce one or multiple structural/compositional characteristics of tendon tissues, among which aligned, electrospun fibrous scaffolds and the 3D
Conclusion
In this study, we have developed a novel composite scaffold consisting of fibrous PCL and methacrylated gelatin (mGLT) interspersed by dual-electrospinning. The crosslinkable nature of the composite scaffold, together with the excellent integration of the gelatin within the PCL mesh, allowed the creation of a multilayered construct as a tendon graft through photo-crosslinking of stacked scaffold sheets. Human ASCs were impregnated into the scaffold to generate a cell-laden construct and were
Disclosure statement
No competing financial interests exist.
Acknowledgement
The authors would like to thank Dr. Jian Tan for hASC characterization and Morgan Jessup (Center of Biological Imaging, University of Pittsburgh) for technical support with the confocal microscope. This work is supported in part by grants from the Commonwealth of Pennsylvania Department of Health (SAP 4100050913), NIH (5R01 AR062947), and U.S. Department of Defense (W81XWH-08-2-0032, W81XWH-14-2-0003, W81XWH-15-1-0104, and W81XWH-11-2-0143). Benjamin B. Rothrauff is a pre-doctoral trainee
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This work was done at the University of Pittsburgh, Pittsburgh, PA.
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Address: 450 Technology Drive, Room 239, Pittsburgh, PA 15219, USA.
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The abstract and some of the data in this manuscript were presented in the 2015 International Symposium on Ligaments & Tendons.