In Vitro Characteristics of Porcine Tendon Hydrogel for Tendon Regeneration

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Abstract

Purpose

Previous work has characterized the development of a human tendon hydrogel capable of improving mechanical strength after tendon injury. Animal tendon hydrogel has not yet been described, but would prove beneficial due to the cost and ethical concerns associated with the use of human cadaveric tendon. This study details the manufacture and assesses the biocompatibility of porcine tendon hydrogel seeded with human adipoderived stem cells (ASCs).

Materials and Methods

Porcine tendon was dissected from surrounding connective and muscle tissue and decellularized via 0.2% sodium dodecyl sulfate and 0.2% sodium dodecyl sulfate/ethylenediaminetetraacetic acid wash solutions before lyophilization. Tendon was milled and reconstituted by previously described methods. Decellularization was confirmed by hematoxylin-eosin staining, SYTO Green 11 nucleic acid dye, and DNeasy assay. The protein composition of milled tendon matrix before and after digestion was identified by mass spectrometry. Rheological properties were determined using an ARG2 rheometer. Biocompatibility was assessed by live/dead assay. The proliferation of human ASCs seeded in porcine and human hydrogel was measured by MTS assay. All experimental conditions were performed in triplicate.

Results

Decellularization of porcine tendon was successful. Mass spectrometry showed that collagen composes one third of milled porcine tendon before and after pepsin digestion. Rheology demonstrated that porcine hydrogel maintains a fluid consistency over a range of temperatures, unlike human hydrogel, which tends to solidify. Live/dead staining revealed that human ASCs survive in hydrogel 7 days after seeding and retain spindle-like morphology. MTS assay at day 3 and day 5 showed that human ASC proliferation was marginally greater in human hydrogel.

Conclusions

After reconstitution and digestion, porcine hydrogel was capable of supporting growth of human ASCs. The minimal difference in proliferative capacity suggests that porcine tendon hydrogel may be an effective and viable alternative to human hydrogel for the enhancement of tendon healing.

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