Tissue engineering therapy deals with the development of biological substitutes using a combination of cells and materials to repair or replace damaged tissues. The production of bioactive and biodegradable scaffolds, which provide a temporary 3D micro-environment for cells and guide cellular processes, is one of the most critical steps in tissue engineering.¹ A variety of natural and synthetic polymers and their composites have been synthesized by various techniques, including solvent casting and melt molding combined with particulate leaching, freeze drying, and gas foaming, to obtain highly porous, cytocompatible structures with suitable mechanical properties and biodegradability.^2,3 One of the most important drawbacks of these techniques is the presence of residual organic solvents.

Supercritical fluid technology has recently become an alternative to the traditional techniques for producing tissue scaffolds. The main advantages of this technology include the complete removal of organic solvents and avoidance of high-temperature treatments. The most commonly used supercritical fluid is carbon dioxide (CO₂) due to its mild critical parameters and non-toxic and non-corrosive properties.⁴

Among the natural polymers, chitosan (CS), which is a linear polysaccharide derived from chitin, is considered an excellent material for biomedical applications due to its biocompatible, biodegradable, and anti-allergic properties.⁵ However, the biological and mechanical properties of this natural biopolymer need to be improved in order to make CS scaffolds suitable for hard tissue replacements. Bioceramics such as hydroxyapatite are used to improve the properties of CS scaffolds and in a way to mimic the extracellular matrix of natural bone.⁶

We used a supercritical CO₂ (SC-CO₂)-assisted process to prepare CS-nanohydroxyapatite composites for potential use in bone tissue engineering applications.⁷ We synthesized nanosized hydroxyapatite particles (nHAp) by precipitation in the presence of calcium phosphor tris (CaPTris) solution.⁸ We added various fractions of nHAp to CS to prepare composite scaffolds. We performed characterization studies to determine the microstructure and physicochemical and mechanical properties. Cytotoxicity tests were done to investigate the possible use of CS-nHAp composite scaffolds in tissue engineering applications.⁹

The scanning electron microscopy (SEM) images in Figure 1 reveal the complete porous morphology of CS and CS-nHAp composite scaffolds. Based on examination by mercury intrusion porosimetry, the pore size of CS scaffolds fabricated using 2% weight/volume CS solution was ~30–150μm. The pore size distributions for CS-nHAp composite scaffolds were ~40–100μm, ~40–125μm, and ~50–125μm for 0.25%, 0.50% and 1.00% nHAp-containing scaffolds, respectively. The morphological characteristics of the scaffolds obtained from SEM analysis and pore size distribution tests pointed out a favorable microstructure for bone tissue engineering applications.

To show the successful incorporation of nHAp in CS scaffolds, we performed x-ray diffraction analysis. In the CS-nHAp scaffold, the diffraction at 20° from pure CS was weakened, indicating that the crystalline structure of CS changed in the composite scaffold. The peaks of nHAp at 25.9°, 31.9°, and 39.7° contributed to the spectrum of CS-nHAp showing the molecular interactions between CS and nHAp in the composite scaffold (see Figure 2).

The mechanical properties of scaffolds showed an increasing trend in the compression moduli of the CS scaffolds with incorporation of nHAp. The compression modulus of CS was around 11.17 kilopascals (kPa), while 0.25% CS-nHAp, 0.50 % CS-nHAp, and 1.00% CS-nHAp had compression moduli of 13.92kPa, 15.08kPa, and 16.77kPa, respectively, indicating increased mechanical strength.

We performed cytotoxicity tests for scaffolds according to International Standards Organization/European Standard 109935 guidelines. We used L929 mouse fibroblasts in cytotoxicity analysis and tested scaffolds using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test and morphological analysis. We performed the MTT assay for three days and measured the proliferation of L929 fibroblasts exposed to various dilutions of extract (25%, 50%, and 100%) obtained from incubating scaffolds in culture medium. All the cells continued to proliferate until they reached confluency at the end of the third day. We determined the morphology of cells exposed to extracts at the end of the incubation period by crystal violet staining. We did not observe cell death or morphological disorder. We concluded that the extracts of the CS-nHAp composite scaffolds did not induce any in vitro cytotoxic effect on cell viability.

In summary, we successfully prepared microporous CS and CS-nHAp composite scaffolds by using a supercritical CO₂-assisted process. Characterization studies revealed that the incorporation of nHAp in the composite structure is essential for increased osteoconductive properties in bone tissue engineering applications. The composite scaffolds showed a greater compression modulus. No cytotoxicity was observed towards fibroblasts in vitro. The results obtained from these studies demonstrated that the CS-nHAp composite scaffold prepared by using SC-CO₂ provides a suitable microenvironment for in vitro tissue development. We are currently working on cultivation of osteoblastic cells to evaluate the potential use of these composites in bone tissue engineering applications.