In-Situ Forming pH and Thermosensitive Injectable Hydrogels to Stimulate Angiogenesis: Potential Candidates for Fast Bone Regeneration Applications

Abstract

Biomaterials that promote angiogenesis are required for repair and regeneration of bone. In-situ formed injectable hydrogels functionalised with bioactive agents, facilitating angiogenesis have high demand for bone regeneration. In this study, pH and thermosensitive hydrogels based on chitosan (CS) and hydroxyapatite (HA) composite materials loaded with heparin (Hep) were investigated for their pro-angiogenic potential. Hydrogel formulations with varying Hep concentrations were prepared by sol–gel technique for these homogeneous solutions were neutralised with sodium bicarbonate (NaHCO3) at 4 °C. Solutions (CS/HA/Hep) constituted hydrogels setting at 37 °C which was initiated from surface in 5–10 minutes. Hydrogels were characterised by performing injectability, gelation, rheology, morphology, chemical and biological analyses. Hydrogel solutions facilitated manual dropwise injection from 21 Gauge which is highly used for orthopaedic and dental administrations, and the maximum injection force measured through 19 G needle (17.191 ± 2.296N) was convenient for manual injections. Angiogenesis tests were performed by an ex-ovo chick chorioallantoic membrane (CAM) assay by applying injectable solutions on CAM, which produced in situ hydrogels. Hydrogels induced microvascularity in CAM assay this was confirmed by histology analyses. Hydrogels with lower concentration of Hep showed more efficiency in pro-angiogenic response. Thereof, novel injectable hydrogels inducing angiogenesis (CS/HA/Hep) are potential candidates for bone regeneration and drug delivery applications.

Evolving applications of the egg: chorioallantoic membrane assay and ex vivo organotypic culture of materials for bone tissue engineering

“…Chitosan and hydroxyapatite hydrogels with varying concentrations of heparin were applied to the CAM; 100 however, there was a poor survival rate using this ex ovo method (described by Mangir et al 4 ) with 40%-50% surviving to implantation of materials and 70%-80% proceeding to survive until sample harvest. With this low and limited survival rate, and low numbers of eggs initially used for each condition, statistical analysis is difficult in this report and the lack of positive controls limits further comparison.…”

“…40 Older methods still in use today, typically detail defining a radius around the scaffold or implantation site and counting blood vessels. 96,100,133 The Chalkley scoring method provides an estimate of blood vessels, rather than a real counted value of vessels, as three counts of the dots aligned over blood vessels are taken and averaged to give the Chalkley count to reduce the variability in readings. 134 This method has been found to give consistent results when used for histological tissue samples in oncology research.…”

Combination Therapy of Killing Diseases by Injectable Hydrogels: From Concept to Medical Applications

“…The total survival rates of CAM experiments before and after sample implantation were between 45-50% and 70-80%, respectively. [21] For the treatment of rheumatoid arthritis (RA) as a disease with complex pathogenicity, the treatment systems should be acted through multiple mechanisms around anti-inflammation effects and osteogenesis, while traditional methods suffer from side effects and also they were limited to one or two mechanisms and ignored the other mechanisms. In an exploration by Pan et al, [127] a new platform composed of black phosphorus nanosheets (BPNs) into platelet-rich plasma (PRP)-CS thermoresponsive IH was employed for RA treatment (Figure 11A).…”

“…In another interesting example, Kocak et al. [ 21 ] developed pH and thermosensitive IHs based on CS and hydroxyapatite (HA) composite materials loaded with heparin (Hep) to improve angiogenesis toward repair and regeneration of bone ( Figure A). Hydrogel precursor solutions with various concentrations of Hep were prepared using sol–gel technique.…”

“…[ 13 ] Besides, some polymeric hydrogel systems have shear thinning and self‐recovering properties or are capable of being in situ formed within the body after injecting in a liquid form. [ 14 ] These properties provide the chance of preparing injectable hydrogels (IHs) [ 15,16 ] that are promising candidates in cancer therapy, cartilage repair, [ 17,18 ] angiogenesis and vascularization, [ 19,20,21 ] tissue engineering, [ 22 ] nervous system repair, [ 19 ] therapeutics delivery, [ 23,24 ] wound healing, [ 25,26 ] etc. The in situ forming hydrogels can be fabricated through physical interactions (ionic and hydrophobic interactions as well as supramolecular chemistry), non‐toxic chemical crosslinkers, or biological cross‐linking by enzymes.…”