Nanohydroxyapatite-reinforced chitosan composite hydrogel for bone tissue repair in vitro and in vivo

Abstract

BackgroundBone loss during trauma, surgeries, and tumor resection often results in critical-sized bone defects that need to be filled with substitutionary materials. Complications associated with conventional grafting techniques have led to the development of bioactive tissue-engineered bone scaffolds. The potential application of hydrogels as three-dimensional (3D) matrices in tissue engineering has gained attention in recent years because of the superior sensitivity, injectability, and minimal invasive properties of hydrogels. Improvements in the bioactivity and mechanical strength of hydrogels can be achieved with the addition of ceramics. Based on the features required for bone regeneration, an injectable thermosensitive hydrogel containing zinc-doped chitosan/nanohydroxyapatite/beta-glycerophosphate (Zn-CS/nHAp/β-GP) was prepared and characterized, and the effect of nHAp on the hydrogel was examined.MethodsHydrogels (Zn-CS/β-GP, Zn-CS/nHAp/β-GP) were prepared using the sol–gel method. Characterization was carried out by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDX), Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD) as well as swelling, protein adsorption, and exogenous biomineralization studies. Expression of osteoblast marker genes was determined by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and western blot analyses. In vivo bone formation was studied using a rat bone defect model system.ResultsThe hydrogels exhibited sol–gel transition at 37°C. The presence of nHAp in the Zn-CS/nHAp/β-GP hydrogel enhanced swelling, protein adsorption, and exogenous biomineralization. The hydrogel was found to be non-toxic to mesenchymal stem cells. The addition of nHAp to the hydrogel also enhanced osteoblast differentiation under osteogenic conditions in vitro and accelerated bone formation in vivo as seen from the depositions of apatite and collagen.ConclusionsThe synthesized injectable hydrogel (Zn-CS/nHAp/β-GP) showed its potential toward bone formation at molecular and cellular levels in vitro and in vivo. The current findings demonstrate the importance of adding nHAp to the hydrogel, thereby accelerating potential clinical application toward bone regeneration.

Alginate-nanohydroxyapatite hydrogel system: Optimizing the formulation for enhanced bone regeneration

“…No mineral layer was observed over Alg hydrogels, but the presence of nanoHA induced significantly the mineral layer growth with apatite structures developing on Alg-nanoHA hydrogels, which increased with increasing nanoHA content. This increase of apatite on Alg-nanoHA surfaces is probably due to the availability of negatively charged hydroxyl groups on nanoHA that acted as a nucleation site for crystal deposition [48,49]. However, the observed Ca/P molar ratios were lower than that of stoichiometric hydroxyapatite (HAP), i.e.…”

“…Additionally, the bioactivity response of the composites was characterized regarding the apatite deposition on the materials' surface, upon immersion in SBF. The formation of bone-like mineral on the surfaces is correlated with an increase in the implant's calcium phosphate solubility [48] and is an essential property to indicate the bonding with living bone [48,49]. No mineral layer was observed over Alg hydrogels, but the presence of nanoHA induced significantly the mineral layer growth with apatite structures developing on Alg-nanoHA hydrogels, which increased with increasing nanoHA content.…”

“…The Ca 2+ ions could affect the remineralization of demineralized areas, since Ca 2+ concentration leads to a growth in the saturation of mineralized tissues with nanoHA, favoring the deposition of apatite in the lesions and eventually promoting remineralization [52]. Moreover, the amount of Ca 2+ ions released from biomaterials can play a vital role in regulating of the chemotaxis, proliferation and osteogenic differentiation of osteoblast cells [49].…”

Chitosan‐based hydrogels: recent design concepts to tailor properties and functions

“…In addition to Zn, which also possesses excellent antimicrobial properties, Dhivya et al . added HAp NPs, a mineral component of natural bone, to reinforce CS hydrogels for bone tissue repair applications . Zn and HAp NPs were added before the formation of the CS hydrogels by addition of β ‐GP.…”

Chitosan in Surface Modification for Bone Tissue Engineering Applications

“…Chitosan (CS) is a linear cationic heteropolymer composed of randomly distributed N ‐acetylglucosamine and β‐1,4‐linked glucosamine residues . It has structural and compositional similarity to glycosaminoglycans and therefore produces a minimal immune response . CS has properties such as biocompatibility and biodegradability .…”