Golding A et al. (2016), A Tunable Silk Hydrogel Device for Studying Lim...

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PLoS One 2016 Jan 01;116:e0155618. doi: 10.1371/journal.pone.0155618.

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A Tunable Silk Hydrogel Device for Studying Limb Regeneration in Adult Xenopus Laevis.

Golding A , Guay JA , Herrera-Rincon C , Levin M , Kaplan DL .

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In certain amphibian models limb regeneration can be promoted or inhibited by the local wound bed environment. This research introduces a device that can be utilized as an experimental tool to characterize the conditions that promotes limb regeneration in the adult frog (Xenopus laevis) model. In particular, this device was designed to manipulate the local wound environment via a hydrogel insert. Initial characterization of the hydrogel insert revealed that this interaction had a significant influence on mechanical forces to the animal, due to the contraction of the hydrogel. The material and mechanical properties of the hydrogel insert were a factor in the device design in relation to the comfort of the animal and the ability to effectively manipulate the amputation site. The tunable features of the hydrogel were important in determining the pro-regenerative effects in limb regeneration, which was measured by cartilage spike formation and quantified by micro-computed tomography. The hydrogel insert was a factor in the observed morphological outcomes following amputation. Future work will focus on characterizing and optimizing the device's observed capability to manipulate biological pathways that are essential for limb regeneration. However, the present work provides a framework for the role of a hydrogel in the device and a path forward for more systematic studies.

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Species referenced: Xenopus laevis
Genes referenced: tuba4b tubb4b

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	Fig 4. Histology, Immunohistology, and Micro-CT Images of Spike Formation from Animals. (A) and (B) Histology and Micro-CT. Red Arrows: Porous calcified tissue growth or old bone growth remodeling. Signs of osteoblast and osteoclast reconstruction can be observed in the tissues dense structure that is filled with marrow pockets. Orange Arrow: Bony callus formation, this is typically connected to the old bone around the amputation site and is hollow. Black Arrows: Formation of hyaline cartilage behind the old bone. Green Arrow: Connection site of old marrow cavity to new tissue growth. Brown Arrow: Signs that the old marrow cavity did not connect with the new tissue growth. Purple Arrow: Invasion of tissue into the cartilage spike core. (C) and (D) Immunohistological Stains. Green: Acetyl- N-Alpha Tubulin (Î±-Tubulin) (Nerve) Red: Smooth Muscle Actin (SMA) (Vasculature) Yellow: Collagen type II (Col2) (cartillage type II only found in abundance in hyaline cartilage) Purple: Phosphorylated Mothers Against Decapentaplegic Homolog (pSMAD 5/9) (TGF-Î² activator, signs of proliferation and differentiation/ not cartilage behavior) Orange Arrow: Location where protein would be typically found in spike. White Arrow: Location where protein would not be typically found in spike. http://dx.doi.org/10.1371/journal.pone.0155618.g004
	Fig 5. Histology and Micro-CT of Animals with Variations in Technical Design. (A) Variations Observe in Different Outer Sleeve Design. Red Arrow: Signs of Spike core isolation Brown Arrow: Connection of center cartilage tissue with without outside tissue. Orange Arrow: Ectopic Bone Growth. Purple Arrow: Bone Marrow Pocket. Green Arrow: Cartilage tissue behind the old bone. (B) Variations Observed in Different Modes of Attachment. Red Arrow: Ectopic (beyond plane of amputation) calcified tissue formation Green Arrow: Calcified tissue behind site of amputation White Arrow: Calcified Tissue at the site of amputation. http://dx.doi.org/10.1371/journal.pone.0155618.g005
	Fig 1. Design and Fabrication Process for the Device.(A) Image of the device attached to the animal using a silicone wrap for attachment. (B) Final version of the soft Dragonskin. The purple arrow points out a device with an outer sleeve with no inner hydrogel insert, while the black arrow points out an experimental device with a hydrogel. (C) Schematic of the outer silicone and silk hydrogel insert. (D) Diagram of the hydrogel insert fabrication process.
	Fig 2. Mechanical and Material Properties of the Device.(A) Change in Stiffness of Hydrogel insert Pre and Post (24 hr) Device Attachment with varying gelation parameters (B) Contraction rate of hydrogel inserts with varying gelation conditions (C) Loss of mass of hydrogel over different time periods of attachment (D) Schematic of the silk hydrogelâs dynamic nature during device attachment. (E) Images of two HRP silk hydrogels. One of the hydrogels was cast with frog water (FW) in its gelation solution, while the other hydrogel had only distilled water (diH2O) in its gelation solution. Images are taken at time of gelation, and one hour after gelation. (Statistical significance: * for p < 0.05, for p < 0.01, and * for p < 0.001).
	Fig 3. Measurements and Micro-CT Analysis of Animals.(A) Resultant spike with animals with and without device attachment (B) Non-destructive measurements of animals with and without device. (C) Micro-CT bone volume and surface area of animals that had a device attached versus those who did not. (Statistical significance: * for p < 0.05, ** for p < 0.01, and * for p < 0.001).
	Fig 6. Morphological Differences in Device Animals with (Hydrogel) and without (No Hydrogel) Hydrogel inserts.(A) Micro-CT of the change in calcified tissue in two animals in the current soft Dragonskin generation of the device. (B) Comparison of resultant morphological outcomes with a stiff outer sleeve device, with and without the hydrogel insert. The hydrogel animal has an additional Micro-CT image in order to confirm that the observed changes in the histological stain are not due to tissue processing error. The dotted line represents the point of amputation.

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Agha, A review of the role of mechanical forces in cutaneous wound healing. 2011, Pubmed