Impact of Hydrogel Cross-Linking Chemistry on the in Vitro and in Vivo Bioactivity of Recombinant Human Bone Morphogenetic Protein-2

Abstract

Designing strategies to deliver functional proteins at physiologically relevant concentrations using chemically cross-linked biocompatible hydrogels is a major field of research. However, the impact of cross-linking chemistry on the encapsulated protein bioactivity is rarely studied. Here we examine the two well-known cross-linking reactions namely; hydrazone cross-linking chemistry and thiol-Michael addition reaction to form hyaluronic acid (HA) hydrogels. As a therapeutic protein, we employed recombinant human bone morphogenetic protein-2 (rhBMP-2) for this study. Incubation of rhBMP-2 with HA functionalized with a thiol diminished phosphorylation of Smad 1/5/8, a signal transducer for osteogenic differntiation, whereas an aldehyde functionalized HA had no effect. This indicates that thiol functionalized polymers indeed has an impact on protein function. To validate this result in an in vivo setting we performed BMP-2 induced bone formation in a rat ectopic model. These experiments revealed that the hydrazone-cross-linked HA-hydrogel induced significantly higher bone formation (18.90 ± 4.25 mm3) as compared to the HA-thiol-Michael hydrogels (1.25 ± 0.52 mm3) after 8 weeks as determined by micro-computed tomography. The histological examination of the neo-bone indicated that hydrazone-hydrogels promoted a better quality of bone formation with improved mineralization and collagen formation as compared to the thiol-Michael hydrogels. We believe such a direct comparison of two cross-linking chemistries will provide new insight for developing biomaterials for protein delivery for in vivo applications.

Influence of Charge and Heat on the Mechanical Properties of Scaffolds from Ionic Complexation of Chitosan and Carboxymethyl Cellulose

“…Thus, the properties of scaffolds have been improved by chemical cross-linking, but this procedure often requires chemical modification or prior chemical treatment with reactive functional groups. 30 − 33 This can be associated with cytotoxicity and require extensive purification. Therefore, an alternative process, namely, dehydrothermal (DHT) treatment in the dry state after PEC can be considered.…”

“…Scaffolds based on CMC and CS have been prepared via polyelectrolyte charge complexation (PEC) followed by freeze-drying and reported for tissue engineering applications. , Even though they can easily be fabricated via PEC, most products lack dimensional stability or load-bearing capacity in biological environments under physiological conditions (37 °C, pH 7.4). Thus, the properties of scaffolds have been improved by chemical cross-linking, but this procedure often requires chemical modification or prior chemical treatment with reactive functional groups. − This can be associated with cytotoxicity and require extensive purification. Therefore, an alternative process, namely, dehydrothermal (DHT) treatment in the dry state after PEC can be considered.…”

Stability of proteins encapsulated in Michael‐type addition polyethylene glycol hydrogels

“…We have previously shown that proteins can be encapsulated in similar PEG hydrogels without causing structural changes or aggregation (Zustiak & Leach, 2010, 2011). Contrarily, others have shown that proteins might interact with functionalized PEG during gelation via nucleophilic reactions with α,β‐unsaturated carbonyl compounds, disruption of disulfide bridges from thiol‐functionalized polymers, or hydrophobic interactions (Hammer et al, 2015; Paidikondala et al, 2019). Note that lysozyme has four disulfide bonds and a relatively hydrophobic surface and may be susceptible to such interactions.…”

Smart Hydrogels for Advanced Drug Delivery Systems

“…Studies in the field of bone tissue engineering have recently showed that the incorporation of bone morphogenetic protein (BMP) as a growth factor into the hydrogels can promote in vivo bone formation on the implants’ surface [ 69 ]. Li et al constructed a porous Ti alloy (Ti-6Al-4V) and injected BMP-laden chitosan TRHs into pores of the Ti6Al4V scaffolds [ 70 ].…”