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Ultrasonication of Human Tendon Hydrogel Improves Cellular Integration into a Nano Matrix Scaffold
Anais Legrand, MD; Michael Galvez, MD; Arhana Chattopadhyay, BA; Daniel Cohen, PhD; Lydia-Marie Joubert, PhD; Hung Pham, BS; James Chang, MD
Stanford University, Stanford, CA

Background: Hydrogels are an important class of biomaterial used in drug delivery and tissue engineering. They can act a delivery vehicle for cells and as a scaffold for three-dimensional guided tissue regeneration, with the potential to provide an ideal healing environment for improved repair strength after tendon injury or chronic tendinopathies. The aim of this study is to optimize human tendon hydrogel with ultrasonication to further decrease collagen fibril size, enhance cellular integration and create a modulable collagen network for delivery.
Methods: Ultrasonication at several settings were applied to human tendon hydrogel and compared to control hydrogel. Cell proliferation and viability were assessed by MTS and Live/Dead cells assays after one week. Hydrogel dimensions, porosity and cell-to-matrix interactions were characterized using a Scanning Electron Microscope and a Confocal Laser Microscope. Fiber measurements were quantified utilizing ImageJ Software. In vivo injection of control hydrogel and ultrasonicated hydrogel were injected into the rat dorsum.
Results: Increased cell proliferation was seen at day 7 with ultrasonicated hydrogel compared to control (p=0.0002) and Live/Dead assay confirmed viability. Scanning Electron Microscope demonstrated the creation of a porous network with an average pore diameter of 1.380.73 M. The average collagen fiber diameter was 0.170.06 M, which confirmed nano-hydrogel properties. Scanning Electron Microscope and Confocal Laser Microscope demonstrated the presence of cell clusters at day 1 post-reseeding in ultrasonicated gel with preserved hydrogel properties and therefore easier cell-hydrogel injection delivery compared to controls.
Conclusion: Human tendon hydrogels are a viable therapeutic delivery agent to enhance tissue healing. The application of ultrasonication further enhances the collagen network into nano fibers resulting in improved reseeding with retained hydrogel properties and therefore facile clinical application for combined cell-hydrogel delivery.

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