DOI: 10.14457/tu.the.2022.1472
title: A study on effects of hydroxyapatite nanoparticles and calcium carbonate microcapsules on properties and biocompatibility of a thermosensitive chitosan/collagen hydrogel

description: To develop thermosensitive chitosan/collagen hydrogels that are osteoconductive andinjectable for alveolar bone regeneration. The current study aims to improve the propertiesof hydrogel by adjusting ratio of calcium carbonate (CaCO3) microcapsules andnanohydroxyapatite particles (nHA) in the beta glycerophosphate chitosan/collagen hydrogel.Material & method: There were 2 parts of study. Part I: Hydrogel characterizations, thereare two subsets for the nHA and CaCO3 microcapsule sets (Groups I-IV) as followed, nHA set:nHA 2% and nHA 5% (w/v), and CaCO3 set: Group I: CaCO3 0%, Group II: CaCO3 1%, Group III:CaCO3 2%, and Group IV: CaCO3 5% (w/v). Including microstructures, mechanical properties,physical properties, and cytotoxicity test. Part II: Subcutaneous implants in mice forhistology analysis to evaluate biocompatibility and new bone formation, there were fourgroups of study (Groups A-D), Group A: Calcium carbonate microcapsules, Group B:thermosensitive CaCO3-nHA-chitosan/collagen hydrogel, Group C: thermosensitive CaCO3-nHAchitosan/collagen hydrogel with BMP-2, and Group D: sham operation. The CaCO3 wereprepared by precipitating sodium carbonate that with calcium chloride in the carboxymethylcellulose solution, then morphology of the microcapsules was examined using scanningelectron microscope (SEM). After that the thermosensitive 0%, 1%, 2%, and 5% (w/v) CaCO3-52% and 5% (w/v) nHA-chitosan/collagen hydrogels were prepared and the 10% (w/v) betaglycerophosphatewas used as an accelerator for the sol-gel transition under the temperaturechange at 37ºC. After that, the effects of the 0% - 5% CaCO3 and 2% and 5% nHA on themicrostructures, mechanical, and physical strengths of the hydrogels were investigated. Cellviability assay was performed on the human osteoblast cell line (hFOB1.19, ATTC, USA) thatwere cultured with the culture mediums pre-incubated with the hydrogels. The 2% CaCO3-2% nHA-chitosan/collagen hydrogel with and without BMP-2 were implanted in subcutaneouspockets of mouse for 4 weeks. Then histological examination of the hematoxylin eosin andMasson trichrome staining was performed to determine levels of foreign body reaction andectopic bone formation. The significant differences were set at p&lt;0.05 (n=3-5, Mean ± SD).Results: It was found that CaCO3 microcapsules were 2-5 μm round shaped particles. The 5%nHA was strong particulate gel that supports distribution of the microcapsule in a threedimensionalmatrix of the hydrogels. The CaCO3 microcapsules increased mechanical andphysical strength, while decreased porosity and swelling and degradation rates of thehydrogels. The CaCO3-nHA hydrogels were non-cytotoxic demonstrated by high levels of cellviability of cells incubated in the culture medium pre-incubated with the hydrogels. Themicrocapsules and hydrogels were biocompatible by stimulating mild foreign body tissuereaction and the inflammatory responses in the sample groups were comparable to the shamgroup. Moreover, in a group of the hydrogel with BMP-2, the patches of the newly formedbone or mineralized tissue were identified. The patches exhibited light pink stain ofhaematoxylin and eosin staining and blue staining of Masson trichrome staining with multiplelacunae with nuclei on the matrixes of the hydrogel with BMP-2 group. Conclusion: Propertiesof the hydrogel could be modified by adjusting ratios of the CaCO3 microcapsules and nHA.The 2% CaCO3 with 2% nHA-chitosan/collagen hydrogels exhibited mechanical and physicalstrength superior and suitable than other groups for promoting bone regeneration and couldfunction as a delivery vehicle for BMP-2. The hydrogels were noncytotoxic and bioinert.