Towards Complex Tissues Replication: Multilayer Scaffold Integrating Biomimetic Nanohydroxyapatite/Chitosan Composites

Abstract

This study explores an approach to design and prepare a multilayer scaffold mimicking interstratified natural tissue. This multilayer construct, composed of chitosan matrices with graded nanohydroxyapatite concentrations, was achieved through an in situ biomineralization process applied to individual layers. Three distinct precursor concentrations were considered, resulting in 10, 20, and 30 wt% nanohydroxyapatite content in each layer. The resulting chitosan/nanohydroxyapatite (Cs/n-HAp) scaffolds, created via freeze-drying, exhibited nanohydroxyapatite nucleation, homogeneous distribution, improved mechanical properties, and good cytocompatibility. The cytocompatibility analysis revealed that the Cs/n-HAp layers presented cell proliferation similar to the control in pure Cs for the samples with 10% n-HAp, indicating good cytocompatibility at this concentration, while no induction of apoptotic death pathways was demonstrated up to a 20 wt% n-Hap concentration. Successful multilayer assembly of Cs and Cs/n-HAp layers highlighted that the proposed approach represents a promising strategy for mimicking multifaceted tissues, such as osteochondral ones.

Keywords: biomineralization; chitosan; hydroxyapatite; multilayered scaffolds.

Figure A1

SEM micrographs of transverse (a,c,e,g) and longitudinal (b,d,f,h) sections of pure chitosan scaffold stabilized through arginine solution (0.2 M) (a,b) and composite chitosan/nano-hydroxyapatite scaffolds: Cs/n-HAp 10% (c,d), Cs/n-HAp 20% (e,f), and Cs/n-HAp 30% (g,h), obtained through biomineralization in an arginine solution (0.2 M). Magnification 60×, scale bar 500 μm. White arrows represent the areas in which the porous structure is most altered.

Figure 1

(A) Scheme of Cs/n-HAp composite scaffold preparation. (B) XRD pattern of Cs/n-HAp 30% compared with reference HA powder (JCPDS 9-432). Asterisk (*) refers to Cs diffraction maxima. Since Cs is a semi-crystalline material, a broad diffraction peak at about 2θ ≈ 20° was detected. (C) XRD patterns of Cs/n-HAp 30% obtained from Cs/Ca-P precursor 30 at three different soaking times (5 min, 6 h, and 24 h). The Miller indices of two diffraction peaks characteristic of HA are shown (002), (211). Asterisk (*) refers to Cs diffraction maxima.

Figure 2

(A) SEM micrographs of pure Cs and Cs/n-Hap composite scaffolds’ transverse sections [left side: mag. 60×, scale bar 200 µm; right side: mag. 300×, scale bar 100 µm). (B) Pore size distribution analysis of pure Cs and Cs/n-Hap composite scaffolds. (C) Calcium (Ca) and phosphorus (P) mapping by EDS analysis of pure Cs and Cs/n-Hap composite scaffolds’ transverse sections (Mag. 150×; scale bar 100 μm).

Figure 3

(A) FTIR spectra of L-arginine powder, pure Cs membrane, and Cs membrane interacted with L-arginine. (B) Representation of the chemical interaction between chitosan and L-arginine. (C) Swelling ratio (expressed as percentage) of pure Cs scaffold and composites Cs/n-HAp 10%, Cs/n-HAp 20%, and Cs/n-HAp 30%. (D) Stress–strain compression curves of pure Cs scaffold and composites Cs/n-HAp 10%, Cs/n-HAp 20%, and Cs/n-HAp 30%.

Figure 4

(A) MTT proliferation assay of NIH-3T3 cells cultured on pure Cs scaffold and composite scaffolds (Cs/n-HAp 10%, Cs/n-HAp 20%, and Cs/n-HAp 30%) after 48 h of cell seeding. Data are expressed as mean percentage ± SEM (n = 3). The cell proliferation on the pure Cs scaffold has been normalized to 100% (control). Statistical analysis was conducted with the Student’s t-test (** p <0.01), and the difference between the other groups is not significant. (B) Transcriptional regulation of Casp-3 gene in NIH-3T3 cells grown for 72 h on Cs and composite scaffolds. Quantitative expression analysis by real-time RT-PCR. Expression of the identified target mRNA is represented as the 2^−ΔCt function value, obtained from amplification data (Ct, threshold cycle), normalized by 28S RNA values (internal control for calibration). The 2^−ΔCt values are relative to the control mean value (Cs = 1) and are expressed as fold-change on the y-axis in the graphic. Mean values (±S.E.M.) are derived from n = 3 independent assays.

Figure 5

Scheme for the Cs and Cs/precursor assembly mode and SEM image of the obtained multilayer graded structure. Arrows highlight the border line between layers with increasing n-HAp loading.