Microporous Bio-orthogonally Annealed Particle Hydrogels for Tissue Engineering and Regenerative Medicine.

Abstract

Microporous annealed particle (MAP) hydrogels are an emerging class of biomaterials with the potential to improve outcomes in tissue repair and regeneration. Here, a new MAP hydrogel platform comprising poly(ethylene) glycol (PEG) hydrogel microparticles that are annealed in situ using bio-orthogonal tetrazine click chemistry is reported (i.e., TzMAP hydrogels). Briefly, clickable PEG-peptide hydrogel microparticles with extracellular matrix mimetic peptides to permit cell adhesion and enzymatic degradation were fabricated via submerged electrospraying and stoichiometrically controlled thiol-norbornene click chemistry. Subsequently, unreacted norbornene groups in the microparticles were leveraged for functionalization with bioactive proteins as well as annealing into TzMAP hydrogels via the tetrazine-norbornene click reaction, which is highly selective and proceeds spontaneously without requiring an initiator or catalyst. The results demonstrate that the clickable particles can be easily applied to a tissue-like defect and then annealed into an inherently microporous structure in situ. In addition, the ability to produce TzMAP hydrogels with heterogeneous properties by incorporating multiple types of hydrogel microspheres is demonstrated, first with fluorophore-functionalized hydrogel microparticles and then with protein-functionalized hydrogel microparticles. For the latter, tetrazine-modified alkaline phosphatase was conjugated to PEG hydrogel microparticles, which were mixed with nonfunctionalized microparticles and used to produce TzMAP hydrogels. A biomimetic mineralized/nonmineralized interface was then produced upon incubation in calcium glycerophosphate. Finally, platelet-derived growth factor-BB (PDGF-BB) and human periodontal ligament stem cells (PDLSC) were incorporated into the TzMAP hydrogels during the annealing step to demonstrate their potential for delivering regenerative therapeutics, specifically for periodontal tissue regeneration. In vitro characterization revealed excellent PDGF-BB retention as well as PDLSC growth and spreading. Moreover, PDGF-BB loading increased PDLSC proliferation within hydrogels by 90% and more than doubled the average volume per cell. Overall, these results demonstrate that TzMAP hydrogels are a versatile new platform for the delivery of stem cells and regenerative factors.