# Mineralized chitin nanocrystals enhance osteoinductive ability of chitosan 3D porous biohybrid scaffolds for bone tissue regeneration

Sheila Olza, Nabil Hadj Bouzidi, Laurent Rubatat, Virginie Pellerin, Unai

Montejo, Ana Alonso-Varona, Susana C.M. Fernandes

► To cite this version:

Sheila Olza, Nabil Hadj Bouzidi, Laurent Rubatat, Virginie Pellerin, Unai Montejo, et al. Mineralized chitin nanocrystals enhance osteoinductive ability of chitosan 3D porous biohybrid scaffolds for bone tissue regeneration. Carbohydrate Polymers, 2025, 366, pp.123911. 10.1016/j.carbpol.2025.123911. hal-05136786

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# Mineralized chitin nanocrystals enhance osteoinductive ability of chitosan 3D porous biohybrid scaffolds for bone tissue regeneration

Sheila Olza<sup>a,b</sup>, Nabil M. Hadj Bouzidi<sup>a</sup>, Laurent Rubatat<sup>a</sup>, Virginie Pellerin<sup>a</sup>, Unai Montejo<sup>b</sup>, Ana Alonso-Varona<sup>b,*</sup>, Susana C.M. Fernandes<sup>a,*</sup>

<figure><img src="image_3.png" /></figure>

## ARTICLE INFO

Keywords:

Biomimetic mineralization

Hydroxyapatite

Tissue engineering

Alveolar bone

Osteogenic properties

Osteospheroids

## ABSTRACT

The loss of alveolar bone due to periodontitis has high worldwide prevalence, and Tissue Engineering (TE) emerges as an alternative to replace autologous bone grafting. Herein, TE fundamentals were applied to mimic bone extracellular matrix (ECM) and cells microenvironment. First, biomimetic hydroxyapatite-based mineralized chitin nanocrystals (MCHNC) from both alpha or beta isoforms were synthesized, showing excellent *in vitro* biocompatibility and osteoinductive properties over human adipose-derived mesenchymal stem cells (hASCs) osteogenic differentiation. Then, biohybrid 3D porous biomaterials were obtained by combining MCHNC as inorganic nanoparticles, chitosan (CS) as organic matrix and hASCs spheroids. The biohybrids showed an improvement of the mechanical properties over non-mineralized scaffolds. Furthermore, they presented high *in vitro* biocompatibility and bioactivity, providing an adequate microenvironment for cells. In particular, CS/ MβCHNC biomaterial presented the greatest mechanical properties (191 ± 10 kPa). Additionally, the incorporation of MβCHNC provided the biomaterial with osteoinductive character, since hASCs showed increased ALP activity (3.5-fold) and higher matrix mineralization (26.3-fold), even when cultured in standard cell culture medium. Finally, the osteospheroids showed very good integration into the CS/MβCHNC biomaterial, with great migration and colonization of viable cells. These findings demonstrate the potential of CS/MβCHNC biomaterial for TE as alveolar bone regeneration.

## 1. Introduction

The loss of alveolar bone, part of the periodontium located in the jaws which supports the teeth, is a common worldwide clinical problem (19 % of people over 15-year-old) with increasing prevalence rates observed in the last decades (World Health Organization, 2022). From a clinical point of view, it represents a significant challenge since current therapies such as dental prostheses or bone grafts are not effective (Liang et al., 2020). Bioinspired by the extracellular matrix (ECM) of bone tissue - composed of collagen fibrils mineralized with hydroxyapatite nanocrystals (HAp, Ca₁₀(PO₄)₆(OH)₂) - Tissue Engineering (TE) strategies focus mainly on the development of hybrid 3-dimensional (3D) scaffolds containing an organic phase (matrix), an inorganic phase at the nanoscale level (minerals) and living cells (Deepthi et al., 2016; Liu et al., 2021).

Concerning the inorganic phase of the organic-inorganic bio-nanocomposites, the most widely used compounds are HAp

nanoparticles (nHAp) from synthetic or biogenic sources (Balu et al., 2021; Munir et al., 2022; Unal et al., 2019). Due to its chemical composition, nHAp can confer osteoinductive (induction of osteogenic differentiation of stem cells), or osteoconductive (stimulation of bone cells migration and bone ECM formation) properties to the organic matrix *in vitro* and *in vivo* (Hoveidaei et al., 2024; Zafeiris et al., 2021). In addition, nHAp present a great surface area and roughness as a consequence of its nanometric dimensions, which favors cellular activities (Gurucharan et al., 2023; Kumar et al., 2020; Zakrzewski et al., 2020).

However, the use of inorganic components is usually not sufficient to overcome the structural limitations presented by natural organic matrices. This is the case of chitosan (CS), the main derivative of chitin, a polysaccharide formed by D-glucosamine and N-acetyl-D-glucosamine units linked by β- (1 → 4) glycosidic bonds. CS has been extensively used for biomedical applications as presents several advantages since: (i) its chemical structure is similar to glycosaminoglycans, components of the bone ECM favoring regeneration (Sukpaita et al., 2021); (ii) presents

<footnote><sup>a</sup> Université de Pau et des Pays de l' Adour, IPREM, E2S UPPA, CNRS, 64 000 Pau, France</footnote>

<footnote><sup>b</sup> Department of Cellular Biology and Histology, Faculty of Medicine and Nursing, University of the Basque Country (UPV/EHU), B Sarriena, s/n, 48940 Leioa, Spain</footnote>

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail address: susana.fernandes@univ-pau.fr (S.C.M. Fernandes).</footnote>
film and 3D material forming ability (Fernandes et al., 2009; Fernández-Marín et al., 2022; Salaberria, Diaz, et al., 2015a; Spitzer et al., 2024; Zubillaga et al., 2018, 2020); (iii) is biocompatible *in vitro* and *in vivo* (Guillén-Carvajal et al., 2023), and (iv) presents bioactivity such as antibacterial, mucoadhesive, etc. (Lauritano et al., 2020; Scalera et al., 2023). Nonetheless, CS as a single polymeric scaffold lacks adequate osteoinductivity, degradation rate and mechanical properties, and needs therefore be conjugated with other components (Claverie et al., 2020; Samalens et al., 2022; Zhao et al., 2022).

In the last three decades, chitin nanocrystals (CHNC) have been widely used as reinforcing nanofillers in the TE field (Olza et al., 2023; Zubillaga et al., 2018; Zubillaga et al., 2020). CHNC are nanoforms obtained by degradation of the amorphous region of chitin fibers and exhibit a rod-like morphology (with sizes from 6 to 60 nm in width and 100 to 800 nm in length; (Falgayrac et al., 2024; Fernández-Marín et al., 2021; Salaberria, Diaz, et al., 2015a; Salaberria, Labidi, et al., 2015)), with properties dependent on the crystalline allomorph of chitin from which are obtained. The alpha isoform is highly crystalline and insoluble due to its strong intrachain hydrogen bonds (antiparallel chains) so it's part of support structures of marine organisms such as arthropods. Contrarily, beta isoform is less crystalline, more soluble and reactive as consequence of the weaker hydrogen bonds (parallel chains) and is present on flexible structures in cephalopods (Cabrera-Barjas et al., 2021; Olza et al., 2023). Thus, studies with alpha CHNC are predominant.

CHNC has a similar morphology of native nHAp (needle- or thin plate-shaped nanocrystals; (Du et al., 2019)) and different works have successfully mineralized materials based on chitin or chitin nanofibers (Duan et al., 2017; Kawata et al., 2015; Lin et al., 2021; Yao et al., 2020). Indeed, chitin acts as a template in mineralization processes of marine organisms where it occurs naturally (Falini & Fermani, 2004). In this context, we hypothesize that CHNC can act as a nanoscale organic carrier for mineral nucleation and growth in a biomimetic mineralization process, giving rise to hydroxyapatite-based mineralized chitin nanocrystals that would acquire a novel function as inorganic-organic nanoparticles with osteoinductive properties. And, thus, to obtain CHNC with dual function: as a reinforcing nanofiller and an inorganic osteoinductive agent.

In this work, CHNC from both alloforms – alpha and beta chitin – were first *in situ* mineralized by the formation of an inorganic layer of HAp on the surface of the CHNC mimicking the mineral formation process that occurs in bone (Zhao et al., 2022). Then, the ensuing mineralized CHNC were incorporated in the CS matrices and the 3D porous hybrid scaffolds were characterized in terms of physicochemical and biological properties. Finally, the best 3D construct was combined with osteospheroids from human adipose-derived mesenchymal stem cells (hASCs) to obtain TE building blocks with potential for alveolar bone regeneration.

## 2. Materials and methods

### 2.1. Raw materials and chemicals

Chitosan powder (CS, from squid pens) with 98 % of degree of deacetylation (DDA, determined by ¹H NMR spectroscopy in D₂O containing a 1 % of CD₃COOD) and an average molecular weight of 350,000 g·mol⁻¹ (determined by viscometry according to (Rinaudo et al., 1993), β-chitin (from squid pens, >98 %) with a degree of acetylation (DA) of 95 %, and α-chitin (from shrimp shells, >98 %) with a DA of 88 % were purchased from Mahtani Chitosan Pvt. Ltd., India. Chitosan serves as scaffold matrix and β-chitin and α-chitin as (bio) active nanofillers. Hydrochloric acid (HCl, 37 %), calcium chloride dihydrate (CaCl₂·2H₂O, 99 %), sodium phosphate dibasic (Na₂HPO₄) and genipin were purchased from Sigma-Aldrich, Europe. Acetic acid (99–100 %) was purchased from VWR Chemicals, USA. All reagents were used as received without further purification.

### 2.2. Chitin nanocrystals (CHNC) mineralization

Rod-like αCHNC (115 nm average length and 27 nm width) and sphere-like βCHNC (38 nm average diameter) (Fig. S1) were first *in house* isolated using acid hydrolysis (Salaberria, Diaz, et al., 2015a). The physicochemical properties are displayed in (Fig. S2): αCHNC presented a degree of acetylation (DA) of 96 % and crystallinity index (CI) of 90 %, whereas βCHNC showed a DA of 90 % and CI of 86 %.

Mineralized CHNC (MCHNC) were prepared using a wet chemical precipitation reaction between CaCl₂·2H₂O and Na₂HPO₄ aqueous solutions. Briefly, 10 % wt. suspensions of both CHNC in ultrapure water were homogenized using a homogenizer (X 1000D Unidrive, CAT, Germany) at 16000 rpm for 15 min, and placed in a shaker (NB-T205L, N-BIOTEK,Inc, Korea) with orbital stirring at 37 °C at 150 rpm before subsequent addition of CaCl₂ and Na₂HPO₄ solutions. Three different concentrations of minerals precursors solutions were tested to find the best HAp-like minerals forming conditions in which the ratio CaCl₂: Na₂HPO₄ (Ca/P) was 1.67 (Unal et al., 2019): 100:60 mM, 200:120 mM and 400:240 mM, identified as 1 ×, 2 × and 4 ×, respectively. All the reactions were held for 2, 4 or 7 days. Control samples were prepared using the same protocol but with only CHNC suspension. After mineralization, the suspensions were washed and kept at 4 °C or freeze-dried for further characterization (Table 1).

### 2.3. Synthesis of chitosan 3D porous hybrid scaffolds

The chitosan (CS) 3D porous hybrid scaffolds (CS/MCHNC) were synthesized by incorporating MαCHNC or MβCHNC as nanofillers (Zubillaga et al., 2018). Briefly, 1 % m/v CS solution was prepared by dissolving CS powder in aqueous 1 % v/v acetic acid solution and stirred at room temperature (RT) for 48 h until complete dissolution. Then, 0.5 % w/w of genipin (related to CS dry weight) and never-dried nanochitin (weight ratio CS:nanofillers of 1:0.5) were added and dispersed by homogenizer (20,000 rpm for 15 min twice). The resulting suspension was poured into 24-well cell culture plates (Sarstedt, Germany) and left at 40 °C for 24 h in a ventilated oven (Memmert GmbH + Co.KG, Germany) for genipin-crosslinking to occur. Finally, the obtained hydrogels, were freeze-dried to obtain 3D porous hybrid scaffolds (CS/MαCHNC or CS/MβCHNC). CS 3D porous scaffolds containing αCHNC or βCHNC were prepared as controls.

### 2.4. General characterization of MCHNC and CS/MCHNC

#### 2.4.1. Physico-chemical characterization

##### 2.4.1.1. Attenuated Total Reflectance-Fourier Transform Infrared spectroscopy (ATR-FTIR).
The chemical composition of the samples was carried out by using a Nicolet iS50 FTIR spectrometer (Thermo Fisher Scientific, USA) equipped with a diamond crystal and a DLaTGS diamond detector. The spectra were recorded in transmittance mode in the wavenumber range of 400–4000 cm⁻¹, with a resolution of 4 cm⁻¹ and 128 scan accumulations.

##### 2.4.1.2. ¹³C Nuclear Magnetic Resonance (¹³C NMR).
Solid-state Cross-Polarized Magic Angle Spinning (CP-MAS) ¹³C NMR was recorded

**Table 1**

Summary of the conditions employed for the mineralization process. Ca/P solutions of 100:60, 200:120 and 400:240 mM CaCl₂:Na₂HPO₄ referred as 1 ×, 2 × and 4 ×, respectively.

<table><thead><tr><td>Sample Identification</td><td>CHNC suspension</td><td>Ca/P solution</td></tr></thead><tbody><tr><td>αCHNC</td><td rowspan="2">αCHNC</td><td rowspan="2">—</td></tr><tr><td>MαCHNC</td></tr><tr><td>βCHNC</td><td rowspan="2">βCHNC</td><td>1 × or 2 × or 4 ×</td></tr><tr><td>MβCHNC</td><td>1 × or 2 × or 4 ×</td></tr></tbody></table>
using a Bruker 400WB Plus (Bruker, USA) spectrometer with a 4 mm CP-MAS probe, to chitin and isolated CHNC spectra. The DA was estimated according to Kasaai (Kasaai, 2010) using Eq. (1):

$$DA = (I_{N-CH3}) / (1/6 \sum I_{\text{main chain carbon}}) \times 100 \quad (1)$$

where $I_{N-CH3}$ is the integral of methyl carbon and $I_{\text{main chain carbon}}$ is the integral of carbon atoms of the D-glucopyranosyl ring.

### 2.4.2. Thermogravimetric analysis (TGA)

The thermal properties of the MCHNC and the scaffolds were obtained using a TGA 2 Mettler Toledo thermogravimeter (Mettler Toledo, USA). Dried samples of around 4 mg were placed in 70 or in 90 µL alumina pans and were heated to 900 °C at a constant rate of 10 °C. min⁻¹ under air atmosphere with a flow rate of 50 mL.min⁻¹.

### 2.4.3. Morphological analysis

The morphology of the isolated α- and βCHNC was evaluated by high-resolution microscopy. First, Scanning Electron Microscopy (SEM) using a Hitachi S-4800 microscope (Hitachi, Japan) with an accelerating voltage of 5 kV and were analyzed in an air-dried drop from a 1000 times diluted suspension. All samples had a 15 nm thick gold layer sputtered with an Emitech K550X sputter coater (Emitech, France) before imaging. Then, Scanning Transmission Electron Microscopy (STEM) was employed to corroborate the morphology of the nanoforms using a STEM detector in the SEM-FEG Apreo 2S (Thermo Fisher Scientific, USA) with an accelerating voltage of 30 kV in DF mode. Samples were visualized by air-drying a drop of a 10 % wt. CHNC suspension in a TEM FCF400-Cu (Formvar/Carbon film, 400 mesh, copper) grid. Finally, the size of the CHNC was determined by Atomic Force Microscopy (AFM) using a MultiMode8 microscope (Bruker, USA). The images were taken in PeakForce Quantitative NanoMechanics (QNM) mode under ambient conditions using ScanAsyst-air tips, 2 kHz Z-piezo and 150 nm amplitude. A 1:100 times dilution was prepared, and homogenized using ultrasound treatment for 10 min before air dried onto a silica wafer piece. Ten measurements of each isoform were taken and average length and width values were calculated with NanoScope Analysis 2.0 software.

The morphology of the MCHNC in the solid freeze-dried form, the internal structure and the pores size of the freeze-dried materials were assessed by SEM using the mentioned configuration. To determine the dimensions of the pores, 40 random pores were measured and quantified with ImageJ computer-based image analysis software (NIH-National Institute of Health, USA).

### 2.4.4. X-ray crystallography

The chitin and CHNC crystallinity was assessed collecting X-Ray Diffractometry (XRD) patterns by using a X'PERT PRO (Malvern Panalytical, UK) automatic diffractometer operating at 40 kV and 40 mA, in theta-theta configuration, using a secondary monochromator with Cu-Kα radiation (λ = 1.5418 Å) and a PiXcel solid state detector (active length in 2θ 3.3477°). Data were collected from 5 to 70° 2θ, step size = 0.039° and time per step of 350 s (scan speed 0.029°/s). Fixed soller and divergence slit with constant volume of sample illumination were used.

The crystal structure of the MCHNC was evaluated by Wide Angle X-ray Scattering (WAXS) analysis. The freeze-dried powder samples were pressed to make pellets using a hydraulic press (15 T, Specac Ltd., UK). Data was collected on the SWING Beamline synchrotron (SOLEIL, France) using an X-ray wavelength, λ, of 1.03317 Å, with a sample-to-detector distance of 0.128 m. The scattering data were collected as function of the scattering vector (q) defined by Eq. (2) and, then, curves were plotted as a function of the scattering angle (2θ) using the Cu Kα wavelength, λ, of 1.5418 Å.

$$q = \frac{4\pi}{\lambda} \times \sin\theta \quad (2)$$

### 2.4.5. Mechanical properties

Uniaxial compressive tests were conducted in a Physica MCR 302 Rheometer (Anton Paar Co., Austria) with a 25 mm plan-plan geometry parallel plate apparatus, and data collected with Anton Paar Rheo-Compass™ v1.30 software. Five cylindrical (15 ± 1 mm diameter, 12 ± 3 mm height) dry freeze-dried samples per condition were compressed until 1 mm height from down plate or until the normal force reached 40 N, with a compression velocity of 0.1 mm·s⁻¹ (ISO 844:2021 for cylindrical specimens) at RT. The stress-strain curves were then plotted and the compressive modulus was calculated at 80 % strain (σ₈₀) and the toughness with the area under the curves.

### 2.4.6. Swelling

Dry cubic samples (n = 3) of 0.5 cm were weighted (W₉) and sterilized in 70 % ethanol (Panreac AppliChem, Spain) for 2 h, followed by three washes in sterile phosphate-buffered saline (PBS, pH 7.4, Sigma-Aldrich, USA). Then, each sample was immersed in sterile PBS and incubated in cell culture incubator (Sanyo MCO-18AIC(UV), Marshall Scientific, USA) at 37 °C in a humidified atmosphere containing 5 % CO₂ for 24 h under static conditions. After removing the excess PBS on the surface of the swollen samples with filter paper, they were immediately weighted (W₅) and the swelling calculated using Eq. (3).

$$\text{Swelling (\%)} = (W_s - W_d)/W_d \times 100 \quad (3)$$

where W₅ is the weight of the swollen sample and W₉ is the weight of the dry sample.

### 2.4.7. In vitro degradability

The evaluation of the hydrolytic and enzymatic degradation of the CS 3D porous materials was determined by immersing the samples in sterile PBS or in 1.5 mg·mL⁻¹ lysozyme/PBS solution (Sigma-Aldrich, USA), respectively. First, cubic samples (Section 2.4.6) were pre-wetted in PBS for 24 h, and the initial wet weight (Wᵢ) was obtained by removing excess PBS. Then, PBS was replaced with fresh PBS or lysozyme/PBS solution and incubated for ten weeks. After 4 days, and every 7 days, the weight of the 3D porous materials (Wₜ) was measured. The solutions were replaced twice a week and four samples of each material were used for measurements. The remaining mass of the materials was calculated by Eq. (4):

$$\text{Remaining mass (\%)} = W_t/W_i \times 100 \quad (4)$$

where Wₜ is the weight of the swollen sample at time t and Wᵢ is the initial weight of the swollen sample.

## 2.5. In vitro biological activity of MCHNC

### 2.5.1. Determination of the non-cytotoxic concentration

To determine the maximum CHNC and MCHNC suspension concentration with no cytotoxic cell response, sterilized suspensions of low (20 µg·mL⁻¹), medium (200 µg·mL⁻¹) and high concentration (2 mg·mL⁻¹) were prepared in complete cell culture medium containing Minimum Essential Medium (MEM) supplemented with 1 mM sodium pyruvate, 1 % v/v non-essential amino acids, 1 % v/v penicillin-streptomycin (all from Gibco®, UK), and 10 % v/v fetal bovine serum (FBS, Gibco®, UK). The suspensions were maintained for 24 h at 37 °C in a cell culture incubator. Cell viability of murine L929 fibroblasts (ATCC, American Type Culture Collection, USA) was determined in 96-well cell culture plates (Sarstedt, Germany) at a density of 4 × 10³ cells/well after incubation throughout 72 h with the CHNC and MCHNC suspensions. Complete cell culture media was used as negative control, and 10 % v/v dimethyl sulfoxide (DMSO, Sigma-Aldrich, USA) in complete cell culture media as positive control. PrestoBlue® reagent (10 % v/v, Invitrogen, USA) was used to quantify the viability of the cells, by measuring the absorbance at 570 nm and 600 nm using a microplate reader (Synergy HT spectrophotometer, Biotek, USA). The viability with respect to the
negative control was calculated with Eq. (5):

$$Viability (\%) = A_{test}/A_{control} \times 100 \qquad (5)$$

where Atest is the absorbance of the sample cells and Acontrol is the absorbance of the negative control.

## 2.5.2. Osteoinductive ability

### 2.5.2.1. Human adipose-derived mesenchymal stem cells (hASCs) culture and osteogenic differentiation.
hASCs kindly provided by Histocell S.L. (Zamudio's Scientific Park, Spain) were expanded in control culture medium consisting on low glucose DMEM (Dulbecco's Modified Eagle Medium, Gibco®, UK) supplemented with 1 % v/v Glutamax™ (100×, Gibco®, UK), 1 % v/v Penicillin-Streptomycin, 0.5 % v/v Gentamicin (50 mg·ml⁻¹, Gibco®, UK) and 10 % v/v FBS; and used up to passage six. The osteoinductive ability of MCHNC was assessed by evaluating the osteogenic differentiation of hASCs seeded in 24-well cell culture plates at a density of 2 × 10⁴ hASCs/well, in direct contact with 200 µg·mL⁻¹ MCHNC or CHNC suspension (Section 2.5.1) for 21 days at 37 °C in a cell culture incubator. The culture medium was replaced twice a week, and cells maintained in control culture media was used as negative control.

### 2.5.2.2. Alkaline phosphatase (ALP) activity.
At 7, 14 and 21 days of osteogenic differentiation, cells were washed with PBS, incubated with 0.25 % v/v Trypsin-EDTA (Gibco®, UK), and collected in Eppendorf tubes. After centrifugation, cells were resuspended in 0.5 % v/v Triton™ X-100 (Sigma-Aldrich, USA) containing 1 % v/v protease inhibitor cocktail (Sigma-Aldrich, USA) and incubated for 1 h at 4 °C with stirring. Permeabilized samples were then lysed by ultrasonication, and finally centrifuged to keep the supernatants. ALP activity of cell lysates was determined by the enzymatic assay of p-nitrophenylphosphate (pNPP, Sigma-Aldrich, USA). The DNA content of cell lysates was also quantified using a DNA quantification Kit (Sigma-Aldrich, USA) in order to normalize the ALP activity level. Each experimental group consisted of triplicates.

### 2.5.2.3. Matrix mineralization.
Calcium deposits of differentiated hASCs were evaluated by selective staining with Alizarin red S (ARS, Sigma-Aldrich, USA). After 7, 14 and 21 days of osteogenic differentiation, samples were washed with PBS, fixed in 4 % v/v paraformaldehyde (Panreac AppliChem, Spain) at 4 °C for 20 min, washed again and, then, stained with 2 % w/v ARS in PBS for 30 min in the dark with stirring at RT. Then, samples were washed with distilled water to remove unbound ARS and images were taken with a phase-contrast microscope (DMi1, Leica Microsystems, Germany). Finally, aqueous 10 % v/v acetic acid solution was added and stirred in the dark for 30 min at RT, to dissolve ARS-calcium crystals, and absorbance at 405 nm was measured in a microplate reader. Triplicates of each sample were conducted.

## 2.6. In vitro biological evaluation of CS 3D porous (hybrid) scaffolds

### 2.6.1. In vitro biocompatibility

#### 2.6.1.1. Cytotoxicity.
The ISO 10993 part 5:2009 and part 12:2021 was used to evaluate the cytotoxicity (indirect method of the extracted media) of the samples. The extracted media of the materials (CS/αCHNC, CS/βCHNC, CS/MαCHNC and CS/MβCHNC) were obtained by incubating previously sterilized materials in complete L929 cell culture media for 24 h at 37 °C. Negative control consisting of complete cell culture media, and positive control corresponding to 10 % v/v DMSO in complete cell culture media were prepared in the same conditions. See cell seeding and viability measurement procedure in Section 2.5.1.

#### 2.6.1.2. Live/dead assay.
Previously sterilized materials (0.5 cm side),

were placed in 24-well ultralow attachment cell culture plates (Corning, USA) and pre-wetted in complete L929 culture medium for 24 h at 37 °C in cell culture incubator. Then, a total of 5·10⁴ L929 cells in a drop of complete culture medium were seeded in each material. At day 3 post-seeding, samples were washed twice with PBS, and double stained with 3 µM calcein-AM (Sigma-Aldrich, USA) and 5 µM propidium iodide (Invitrogen, USA) for 20 min at RT in the dark, to visualize adherent viable cells in green fluorescence (λex⁻λem: 495–515 nm) and dead cells in red fluorescence (λex⁻λem: 535–617 nm), respectively. Samples were observed in a Zeiss LSM800 confocal microscope (Carl Zeiss, USA) and images treated with Image J analysis software.

#### 2.6.1.3. Cell adhesion.
Samples were prepared as described for Live/Dead assay and after 24 and 72 h of post-seeding were processed to visualize under SEM microscope. Briefly, all samples were washed with PBS and fixed using 2 % v/v glutaraldehyde in 1× Sorensen buffer (both from Panreac AppliChem, Spain) for 30 min at 4 °C. Then, samples were washed three times with Sorensen buffer, dehydrated using a series of increasing concentrations of ethanol solutions (from 30 % v/v to absolute ethanol), desiccated to the critical point with hexamethyldisilazane (Electron Microscopy Science, USA) and allowed to air-dry. Finally, samples were sputtered with a gold layer and observed with an accelerating voltage of 5 kV on a Hitachi S-4800 microscope.

### 2.6.2. Osteoinductive ability

#### 2.6.2.1. Bioactivity assay.
The ability of the materials to induce bone-like apatite formation using Simulated Body Fluid (SBF) was evaluated in vitro following the procedure described in ISO 23317:2014. Briefly, cylindrical samples (15 ± 1 mm diameter, 2 ± 0.3 mm height) were placed in 50 mL Falcon tubes (Sarstedt, Germany) and soaked in 2 mL of SBF at 37 °C for 1 or 4 weeks. After, the samples were washed with distilled water and dried in a desiccator without heating. The formation of apatite on the surface was observed by SEM images at both high (× 10,000) and low (× 1000) magnifications (Section 2.4.3). Additionally, XRD patterns, using a X'PERT PRO (Malvern Panalytical, UK) automatic diffractometer operating at 40 kV and 40 mA, in theta-theta configuration, using a secondary monochromator with Cu-Kα radiation (λ = 1.5418 Å) and a PIXcel solid state detector (active length in 2θ 3.3477°), were collected from 3 to 50° 2θ (step size = 0.026°) using a variable divergence slit with constant 7 mm area illumination.

#### 2.6.2.2. Osteoinduction ability.
A total of 2·10⁴ hASCs in a drop of control culture medium were seeded in the selected hybrid material, previously sterilized and pre-wetted as described in Section 2.6.1. After 24 h, osteogenic differentiation was induced using osteogenic differentiation (OD) media consisting of StemPro® Osteogenesis Differentiation Kit (Gibco®, UK) supplemented with 0.01 % v/v Gentamicin. As control group, seeded materials were incubated with complete hASCs culture media. The culture medium of the samples was replaced twice a week for 21 days and, non-mineralized material was used as control. Osteogenic differentiation of hASCs was determined at 7, 14 and 21 days by evaluating ALP enzymatic activity and matrix mineralization as described in Section 2.5.2. In this case, for ARS quantification, materials were removed from the wells before absorbance measurement and, as blank, samples without hASCs seeded were used.

### 2.6.3. Colonization and integration of osteospheroids

#### 2.6.3.1. Preparation and characterization of osteospheroids.
Osteospheroids from hASCs were obtained following hanging drop method and later induced osteogenic differentiation. Briefly, 25 µL drops of control culture medium containing 10 × 10³ cells were seeded on the lid of a plastic Petri dish (Sarstedt, USA) with PBS on the base, and allowed to aggregate upside down in a cell culture incubator for 48 h. Then, two
spheroids per well were seeded in 24-well ultralow attachment cell culture plates, and OD medium or control culture medium was added to obtain osteospheroids or control group spheroids, respectively. The culture medium was replaced twice a week until analysis.

The viability, volume and osteogenic differentiation capacity of the osteospheroids was assessed at 7, 14 and 21 days of culture, before their incorporation into the materials. Cell viability on spheroids was assessed by Live/Dead assay as described in Section 2.6.1. To determine the size and volume, images were taken using an image capture program (IC Capture 2.3, The Imaging Source, Germany) under a phase-contrast microscope. The average diameter of the spheroids was measured using a computer-based image analysis software (Image J), by making two distance measurements. Then, the volume (V) was calculated considering the spheroids as perfect spheres following Eq. (6). Ten spheroids were measured per each time and culture condition. Finally, the osteogenic differentiation evaluation was done by determination of ALP enzymatic activity and matrix mineralization quantification as described in Section 2.5.2.

$$ Volume (mm^3) = \frac{4}{3} \times \pi \times r^3 \qquad (6) $$

where r is the average radius of the spheroid in mm.

### 2.6.3.2. Seeding of osteospheroids into materials.
An evaluation of the integration of hASCs spheroids in the selected material was done. First, dry cubic samples with a side of 0.5 cm were sterilized and pre-wetted as for Live/Dead assay (Section 2.6.1). Then, a total of ten previously formed hASCs spheroids in a drop of control culture medium were seeded in the material, and were led to adhere for 2 h before osteogenic differentiation was induced by adding the OD medium. As control group, seeded materials were incubated with control culture media. The culture media was replaced twice a week until analysis. On day 7, 14 and 21 of differentiation, spheroid adhesion, cell colonization and morphology of adherent spheroids on the material were evaluated by SEM (Section 2.6.1.3); and viability of adhered spheroids was evaluated by Live/Dead assay.

## 2.7. Statistical analysis

According to the design of each experiment, when required, one-way or two-way analysis of variance (ANOVA) followed by Bonferroni post hoc test was used to perform the statistical analysis using Prism® statistical software (GraphPad, USA). All data is expressed as mean ± SD and values of *p* < 0.05 were considered to be statistically significant.

# 3. Results and discussions

## 3.1. Mineralized CHNC characterization

As mentioned before, nHAp was mimicked by mineralization of CHNC through an approach inspired by biological process. The formation of nHAp on both CHNC isoforms was induced by the coprecipitation of inorganic phosphate ions (PO₄³⁻) and calcium ions (Ca²⁺) on their surface using the wet chemical method (Hu et al., 2020). Consequently, the resulting mineralized CHNC (MCHNC) were expected to acquire an osteoinductive character, in addition to their well-known reinforcing character (Olza et al., 2023). To optimise the mineralization, the effect of Ca/P solution concentration and mineralization time of the different samples were assessed (Section 2.2 and Table 1).

The presence of both chitin and mineral structure after the biomimetic mineralization process was confirmed by ATR-FTIR spectra and TGA curves (Fig. 1). The ATR-FTIR spectra corresponding to mineralized CHNC (MαCHNC and MβCHNC) display the bands corresponding to the chitin amides including: amide I double band (1655 and 1622 cm⁻¹) for alpha and a single band (1633 cm⁻¹) for beta structure; amide II at

~1557 cm⁻¹; and amide III at ~1306 cm⁻¹ (Fernández-Marín et al., 2021; McReynolds et al., 2022) (Fig. 1A). In the CO/COC region (1200 and 950 cm⁻¹), corresponding to chitin glycoside ring, of the MCHNC samples, a broad and strong peak around 1022 cm⁻¹ is visible, which corresponds to the asymmetric phosphate (PO₄³⁻) group peak v₃ vibration (in grey). The peaks corresponding to the PO₄³⁻ vibration v₁ (960 cm⁻¹) and v₄ (doublet at 601 and 559 cm⁻¹) are also visible. At the highest concentration (4 × ), a peak appeared at 880 cm⁻¹ (in purple), which can be attributed to the HPO₄²⁻ group. All of these absorption wavelengths are characteristic of synthetic HAp (Coelho et al., 2019; Moradi et al., 2022). The phosphate bands become more intense and sharper with increasing concentration, showing a greater effect on mineral formation than the mineralization time.

TGA was performed to quantify the mineral residue remaining after degradation of the organic component (Fig. 1B and Fig. S3). MαCHNC and MβCHNC showed a degradation profile similar to that of CHNC with two or three characteristic weight losses assigned to water evaporation, maximum degradation temperature (Tmax) of the organic component, and a last loss corresponding to combustion reaction of the carbonised organic part (Falgayrac et al., 2024; Fernández-Marín et al., 2021). Interestingly, the degraded weight percentage varied according to the mineralization conditions. Suggesting that CHNC are covered by the inorganic part and, therefore, more or less accessible to heat suffering degradation at higher temperatures. There was no clear trend that suggested a direct correlation between the mineralization conditions used and the Tmax values. However, there was a clear relationship between the amount of residue and the mineralization conditions, since the residue depends on the concentration and for MβCHNC was also time dependent. Table S1 summarizes the Tmax and residue (%) values for each condition.

Since the presence of phosphate groups and mineral residues was identified in MCHNC samples, it is suggested that the biomimetic mineralization approach carried out worked correctly to form Ca/P family minerals through coprecipitation. We hypothesize that the carboxyl, carbonyl and amino groups present on the nanochitin backbone act as a biotemplate to induce initial nucleation and later growth of minerals leading to a Ca/P coating over CHNC (Fig. 2).

To better understand the effect of the Ca/P concentration and mineralization time in the final properties of MCHNC, morphological and crystallographic analysis (Fig. 3) were carried out. Taking 4 days mineralization time as a reference (Fig. 3A), it could be observed that the morphology of MCHNC was dependent of the CHNC isoform and concentration. Interestingly, MαCHNC showed flake-like morphology and MβCHNC a needle-like shape, with a degree of compaction increasing with the concentration. The evaluation of the effect of mineralization time at 4× Ca/P concentration (Fig. 3C) showed the presence of integrated minerals between the CHNC, reaching at 4d the most homogeneous inorganic-organic network.

In Fig. 3B and D crystallographic planes attributed to the structure of alpha or beta chitin from CHNC could only be identified at 2θ 19.5° (110) and 23° (101) in some cases and at 26° (013) in all the samples (Fernández-Marín et al., 2021). The other peaks present in the patterns were in accordance with those reported in the JCPDS card no. 09-0432 attributed to pure HAp and other peaks reported in literature to HAp minerals (Mawuntu & Yusuf, 2018; Pangon et al., 2016; Zhao et al., 2018), including: 2θ 26° (002), 28.5° (102), 31.8° (211), 32.3° (300), 34° (202), 39.4° (310), 45.3° (203), 46.5° (222), 48.6° (320) and 49.5° (213). Interestingly, the intensity of the HAp peaks increased with the increase of the concentration. No clear relationship was found with the mineralization time.

It can be concluded that the biomimetic mineralization allowed the formation of flake- or needle-shaped HAp minerals covering both CHNC isoforms. As reported before for chitin macromolecule (Lin et al., 2021; Muzzarelli & Muzzarelli, 2002), herein, we demonstrated that CHNC also create an environment suitable for HAp formation. Likewise, a directly proportional relationship was observed between the
<figure><img src="image_4.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Chemical composition and thermal analysis of the MCHNC. ATR-FTIR spectra (A); and TGA curves (B) of MaCHNC and MβCHNC. For each sample the mineralization time and the effect of the different Ca/P concentration (100:60 mM (1 × ), 200:120 mM (2 × ) and 400:240 mM (4 × )) are compared.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>Fig. 2. Proposed mineralization process of CHNC by wet chemical method and final MCHNC.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>Fig. 3. Morphological and crystallographic analysis of MαCHNC and MβCHNC. Effect of Ca/P solution concentration (1 ×, 2 ×, 4 ×) on sample morphology (SEM) (A) and crystallographic structure (WAXS) (B) after 4 days of mineralization; and effect of mineralization time (2d, 4d, 7d) on sample morphology (C) and crystallographic structure (D) after immersion on 4× Ca/P solution.</figcaption></figure>

concentration of the Ca/P solutions used and the degree of mineraliza-
tion, whereas the time favoured the growth of minerals until 4d since
latter gave rise to the formation of large aggregates. Taking into account
the obtained results, the selected condition to produce MCHNC was 4×
Ca/P solution for 4 days, and further analysis was performed (Section
3.2). The general aspect of the selected MCHNC suspensions is displayed
in Fig. S4.

3.2. *In vitro* biological response of MCHNC

The high biocompatibility of CHNC (Barbalinardo et al., 2021;
Zubillaga et al., 2018) and of nHAp has been previously reported
(Cestari et al., 2021; Coelho et al., 2019). Thus, we determined the
maximum concentration of the MCHNC suspension of both isoforms that
does not cause a cytotoxic response. The concentrations of MCHNC - low
(l, 20 µg·mL⁻¹), medium (m, 200 µg·mL⁻¹) and high (h, 2 mg·mL⁻¹) - were selected based on previous studies (Jiang et al., 2017), to test the viability of mouse fibroblast L929 cell line. The results showed in Fig. 4A indicated that MCHNC were not cytotoxic since, in all cases, viability values were higher than 90 % respect to the negative control. Thus, the concentration of 200 µg·mL⁻¹ was suggested as the maximum concentration of MCHNC that does not significantly affect the viability of the cells. This agrees with a previous work, in which same concentration of marine origin nHAp applied to MC3T3-E1 mouse preosteoblasts presented high cell viability (Shi et al., 2018).

It has been demonstrated that calcium polyphosphate (CaP) nano-particles supplied in cell culture media can induce osteogenic differentiation of mesenchymal stem cells by providing alternative inorganic source of phosphate, which is used in the synthesis of HAp through pyrophosphate release by the enzyme ALP (Chen et al., 2015; Hatt et al., 2019). Thus, we hypothesized that MCHNC could also act as bioactive molecules incorporated into the cell culture medium to induce osteogenic differentiation of stem cells.

First, ALP enzymatic activity of hASCs was analyzed after cultured in medium supplemented with MCHNC (Fig. 4B). The results showed high and increasing ALP activity over time (296-fold MαCHNC and 219-fold MβCHNC), when were added as supplements. Thus, the presence of MCHNC led to a statistically significant and time-dependent increase in ALP activity with respect to CHNC-supplemented. It is worth noting that the stimulation obtained of this early differentiation marker is greater than that reported by Hatt et al. with CaP nanoparticles on hMSCs (Hatt et al., 2019) and by Shi et al. on MC3T3-E1 mouse preosteoblasts with marine origin nHAp (Shi et al., 2018).

We also evaluated a late osteogenic differentiation marker such as the formation of calcified nodules of the ECM, that can be detected by ARS staining. The ARS concentration values respect to the negative control of the MCHNC-supplemented samples were significantly higher reaching the highest values at 14 days (2.4-fold for MαCHNC and 1.8-fold for MβCHNC, Fig. 4C). In the case of MβCHNC this value was maintained at 21 days. This high degree of mineralization induced by MCHNC was also observed in Fig. 4D, by the high presence of red-

<figure><img src="image_8.png" /><figcaption>**Fig. 4.** *In vitro* biological response of MCHNC. Cytotoxicity evaluation of low (l, 20 µg·mL⁻¹), medium (m, 200 µg·mL⁻¹) and high (h, 2 mg·mL⁻¹) concentration of CHNC- or MCHNC-suspensions on L929 cells along 72 h of incubation time (A); ALP enzymatic activity (B); and ARS quantification, dot line represents control medium value (C) and staining (D) of hASCs after 7, 14 and 21 days of culture with 200 µg·mL⁻¹ CHNC- or MCHNC-supplemented medium. Data is expressed as mean ± SD (n = 3). \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001. Scale bar represents 100 µm.</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>Fig. 4. (continued).</figcaption></figure>

colored calcium deposits.

These results suggest that the inorganic component (HAp) confers an osteoinductive character to CHNC, since MCHNC added as supplement stimulated the differentiation towards the early stages of osteoblasts with greater impact on ALP enzymatic activity than on matrix mineralization. Similar approach has been proposed by Fragal et al. using the method with SBF solution of surface-modified cellulose nanocrystals, obtaining a hybrid nanocomponent highly biocompatible with L929 cells (Fragal et al., 2016). Additionally, Ribeiro et al. demonstrated that biomimetically mineralized cellulose nanocrystals can be used to develop mineralized nanocomposite scaffolds with osteoinductive properties (Ribeiro et al., 2022). In this context, MCHNC could result in promising osteoinductive nanofillers in the development of hybrid bio-materials for alveolar bone regeneration.

### 3.3. Structure and physicochemical properties of hybrid CS-based 3D materials

Once osteoinductive character of the synthesized MCHNC was demonstrated, we evaluated whether synthesized MCHNC could be used as osteoinductive nanofillers in the development of hybrid organic-inorganic biomaterials for bone regeneration. With this purpose, MCHNC were used in combination with CS (a non-osteoinductive organic matrix) to develop 3D porous scaffolds (CS/MαCHNC and CS/MβCHNC). Their non-mineralized counterparts (CS/αCHNC and CS/βCHNC) were used as control materials.

In Fig. S5A the cylindrical 3D porous materials obtained by freeze-drying showed that mineralized materials had less intense bluish colour, which indicates that the inorganic component was able to

establish linkages with the CS and CHNC functional groups or cover the surface of the CHNC making them less accessible to genipin crosslinker (Salaberria, Diaz, et al., 2015b). The presence of the MCHNC in the hybrid organic-inorganic materials was assessed by ATR-FTIR (Fig. S5B), revealing both characteristic bands of the CS matrix (C—H bond, amide I and amide II) and those corresponding to the vibration of the PO₄³⁻ groups (asymmetric P—O stretching and symmetrical O-P-O bending) typical of HAp type minerals (Moradi et al., 2022; Salaberria, Diaz, et al., 2015a). The presence of the inorganic component in the hybrid 3D materials was also demonstrated by TGA analysis (Fig. S6), which showed a residual mass of 23–25 % (Table S2).

The presence of MCHNC and their good dispersion into the CS matrix was evidenced by the appearance of particles corresponding to inorganic elements, such as HAp, in the surface of the porous walls of the 3D materials (Fig. 5A). Besides, the cross-section images showed a structure with high porosity and irregularly-shaped interconnected pores (Fig. 5B). Both micro and macropores were simultaneously present along the 3D materials, which is advantageous to promote protein adsorption and cell adhesion, and new bone ingrowth, respectively (Iviglia et al., 2016).

An average pore size >120 µm was reached in all materials as displayed in Fig. 5C. The hybrid materials (147 ± 18 µm for MαCHNC and 121 ± 14 µm for MβCHNC) surpassed the minimum pore size established in the literature (100 µm) as required for correct cellular infiltration and nutrient exchange (Iviglia et al., 2019; Saravanan et al., 2019). Indeed, since BAMs-based biomaterials in wet state expand due to their hydrophilic nature (Wack & Bertling, 2014), it is expected that in humid conditions, such as in the presence of body fluids *in vivo*, the pore size will be larger. The swelling capacity (Fig. 5D) decreased for
<figure><img src="image_10.png" /><figcaption>**Fig. 5.** SEM images of the surface (A) and internal structure (B), average pore size (C) and swelling capacity (D) of the CS-based 3D porous materials. Chitosan materials with CHNC (CS/αCHNC, CS/βCHNC) and MCHNC (CS/MαCHNC and CS/βMCHNC). Data is expressed as mean ± SD (n = 40). \* *p* < 0.05; \*\*\* *p* < 0.001.</figcaption></figure>

CS/MCHNC materials as a consequence of the lower number of OH and NH₂ groups available in MCHNC surface for protonation in contact with the aqueous medium. A reduction in pore size and on swelling capacity due to nHAp incorporation on CS matrix has been previously reported (Akman et al., 2010; Demirtaş et al., 2017).

Fig. 6A displays the effect of MCHNC incorporation on the strain-stress curves, showing in general three differentiated sections as for CS-based 3D porous materials (Zubillaga et al., 2018): i) a linear elastic deformation at low stresses (up to 10–15 %); ii) cell collapse indicated by the section with the lowest slope, (up to 65–70 %); and iii) densification at large strains, represented by the last section of the exponential curve where the collapse of the materials occurs. The CS/MαCHNC scaffold’s curve didn’t show the same behaviour as the integration of the MαCHNC gave rise to a more compact scaffold as shown in Fig.S5A. Interestingly, toughness and compressive strength modulus (Fig. 6B and C) increased for CS/MβCHNC material (5.70 ± 0.25 J·m⁻³ and 191 ± 10 kPa) with respect to CS/βCHNC (4.23 ± 0.06 J·m⁻³ and 182 ± 8 kPa). Therefore, the presence of the inorganic component had a positive effect combined with βCHNC, making MβCHNC a more effective nano-filler to improve the mechanical properties of the final material. Other works using nHAp into CS-based materials also showed an improvement in the compressive strength (Akman et al., 2010; Zhao et al., 2022).

To evaluate the biodegradability and degradation rate that the hybrid 3D materials would have in physiological conditions, a long-term *in vitro* study (10 weeks) of hydrolytic degradation in PBS (Fig. 7A) and enzymatic degradation in PBS/lysozyme solution (Fig. 7B) were carried out. The degradation curves showed in both cases: a major degradation within the first 28 days followed by a slowdown until stabilization of the remaining weight. The incorporation of the inorganic component

greatly reduced the hydrolytic degradation of the materials, since the residual mass at 28 days was 1.4 times higher with MCHNC (Table 2), which is the minimum time that the scaffolds must maintain their integrity for regenerative process *in vivo* (Sperber, 2018). Furthermore, in the long-term, the degradation ratio until stabilization was reduced 2.9 times for CS/MαCHNC and 3.7 times for CS/MβCHNC with respect to its non-mineralized counterparts. It has been previously reported that the incorporation of minerals contributes to reduce the degradation of the biomaterial (Liang et al., 2020). The enzymatic degradation was only significantly reduced in the case of CS/MβCHNC scaffold (1.4-fold at 28 days and 1.7-fold in the long term). This is attributed to the different accessibility of the acetamide groups of the mineralized nanoforms, leading to a more protected mineralized surface against lysozyme activity in the case of MβCHNC.

With these first analyses, it was demonstrated that the presence of mineral phase in the MCHNC decreased the pore size and swelling capacity, and improved the resistance to mechanical compression and hydrolytic degradation.

### 3.4. *In vitro* biocompatibility of hybrid materials

The short-term *in vitro* biocompatibility of the materials was assessed as a preliminary approach to evaluate their suitability for biomedical applications. None of the materials showed a cytotoxic effect on L929 murine cells, since the cell viability values were higher than 79 % of the negative control, and ISO standard 10,993–5 sets 70 % as the threshold for a material to be considered not cytotoxic (Fig. 8A). These results are in line with Zubillaga et al. work where the biocompatibility of CS/αCHNC scaffolds was demonstrated (Zubillaga et al., 2018). The high
<figure><img src="image_11.png" /><figcaption>**Fig. 6.** Compressive stress-strain curves (A), toughness (B) and compressive moduli (C) of synthesized CS-based 3D porous materials with CHNC (CS/αCHNC, CS/βCHNC) and MCHNC (CS/MαCHNC and CS/MβCHNC). Data is expressed as mean ± SD (n = 5). *** p < 0.001.</figcaption></figure>

biocompatibility of βCHNC (Barbalinardo et al., 2021; Hassanzadeh et al., 2016) and nHAp (Gurucharan et al., 2023; Zou et al., 2019) has been also reported.

Then, a Live/Dead assay was performed at 72 h to semi-qualitatively evaluate the viability of the cells adhered directly to the materials (Fig. 8B). All the materials presented a large number of green/viable cells adhered, with the presence of very few red/dead cells. A very good adaptation of the L929 cells was observed, since the cells uniformly colonized the surface and the internal structure even delimiting the perimeter of the pores. Also, SEM images showed the surface of the hybrid materials full of crystalline mineral deposits (Fig. 8C), attributed to the reaction between the culture medium used and the exposed functional groups of the MCHNC distributed along the scaffold. Interestingly, the adaptation process of L929 cells slowed down without compromising cellular activity, since after 72 h of culture, the cells were well adhered to the substrate as indicated by the multiple adhesion points established (see enlarged images). This demonstrated the high in vitro biocompatibility of the materials, highlighting that MCHNC did not negatively interfere with cellular activity and that the topography of the materials offered a friendly environment that favors cell adhesion. This

agrees with other works that showed great osteoblast affinity by HAp incorporating composite (Zafeiris et al., 2021) or HAp coated scaffolds (Duan et al., 2017; Wu et al., 2020).

### 3.5. Osteoinductive ability of hybrid scaffolds

Here, we checked whether the incorporation of MCHNC conferred biomineralizing or osteoconductivity capacity to the hybrid 3D porous CS-based biomaterials. Fig. 9A shows the surface of the biomaterials before and after 1 or 4 weeks of incubation in SBF. Progressive mineral deposition over time was evident, with different morphology, size and distribution depending on the biomaterial composition. CS/MαCHNC surface gave rise to flake-type minerals that covered the entire surface, while on CS/MβCHNC biomaterials high-ordered crystal aggregates were observed. In both, formed minerals corresponded to morphologies of more crystalline stages of CaP minerals (Gourgas et al., 2019; Shao et al., 2021).

The nature of the minerals in SBF for 4 weeks was confirmed by XRD (Fig. 9B). In addition to the crystallographic planes attributed to the structure of alpha chitin identified at 2θ 9.3° (020), 12.5° (021), 19.5°
<figure><img src="image_12.png" /><figcaption>**Fig. 7.** *In vitro* degradation evolution of the 3D porous materials. With CHNC (CS/αCHNC, CS/βCHNC) and MCHNC (CS/MαCHNC and CS/MβCHNC) up to 70 days. Incubated with PBS (hydrolytic degradation) (A); or PBS/Lysozyme solution (enzymatic degradation) (B). Data is expressed as mean ± SD (n = 4).</figcaption></figure>

<caption>**Table 2**</caption>

Remaining mass (%) at 28 days and the day that stabilizes below 50 % remaining mass of the 3D porous materials. With CHNC (CS/αCHNC, CS/βCHNC) and MCHNC (CS/MαCHNC and CS/MβCHNC), incubated with PBS or PBS/Lysozyme solution. Data is expressed as mean ± SD (n = 4).

<table><thead><tr><th rowspan="2">Sample</th><th colspan="2">Remaining mass (%) at 28 days</th><th colspan="2">Day that stabilizes below 50 % remaining mass</th></tr><tr><th>PBS</th><th>PBS/Lysozyme</th><th>PBS</th><th>PBS/Lysozyme</th></tr></thead><tbody><tr><td>CS/αCHNC</td><td>41 ± 6</td><td>38 ± 3</td><td>23 ± 4</td><td>28 ± 0</td></tr><tr><td>CS/MαCHNC</td><td>56 ± 4</td><td>43 ± 4</td><td>67 ± 7</td><td>35 ± 6</td></tr><tr><td>CS/βCHNC</td><td>35 ± 4</td><td>35 ± 2</td><td>19 ± 7</td><td>25 ± 4</td></tr><tr><td>CS/MβCHNC</td><td>50 ± 8</td><td>50 ± 3</td><td>> 70 ± 4</td><td>42 ± 0</td></tr></tbody></table>

(110, 20.7° (120), 23.2° (101) and 26° (013) (Fernández-Marín et al., 2021); and those of beta chitin at 2θ 8.2° (010), 19.6° (110) and 25.8° (013) (McReynolds et al., 2022); several peaks corresponding to the crystallographic planes of HAp were found at 26° (002), 27.4° (102), 31.7° (211), 34.3° (202), 39° (310), 45.4° (203), 46.4° (222) and 49.2° (213), according to JCPDS card no. 09-0432. The mineralized biomaterials showed more defined and sharper peaks attributed to the HAp crystalline structure, indicating improved crystallinity of the hybrid biomaterials. These results are similar to previously reported (Gurucharan et al., 2023; Scalera et al., 2023; Zhao et al., 2018) and proved the bioactivity of the hybrid CS/MCHNC biomaterials as they accelerated high crystalline particle deposition and induced more apatite minerals formation.

Based on these results together with those obtained in the physical-chemical and *in vitro* biocompatibility characterization, the CS/ MβCHNC biomaterial was selected as the one with the greatest potential for use in bone regeneration and to continue the characterization. To determine whether it had osteoinductive ability over hASCs, osteogenic differentiation was analyzed after incubating in control (C) or osteogenic differentiation (OD) culture medium (Fig. 10).

ALP enzymatic activity (Fig. 10A) showed increasing values over time. At 14 days there was a significant difference related to the use of OD medium (1.6-fold increase, p < 0.001) compared with the control. Nonetheless, at day 21, it was the mineralized biomaterials that showed significantly higher values than their non-mineralized counterparts, even with C medium (3.5-fold increase, p < 0.001). Demonstrating that the presence of the MβCHNC seems to exert an osteoinductive effect, since the hybrid materials incubated with C medium reached higher ALP

activity than with OD medium. Increase in ALP activity is frequently achieved in the presence of nHAp (Lauritano et al., 2020; Lee et al., 2014; Palaveniene et al., 2020). On the other hand, matrix mineralization evaluation (Fig. 10B) confirmed the osteoinductive character conferred by the presence of MβCHNC in the biomaterial, since only the mineralized biomaterials had increased ARS values over time. Furthermore, the difference between ARS values in C and OD medium reduced over time (from 6.4-fold at 7 days to 1.7-fold at the end of the test). The data indicated that MβCHNC had a great effect on the production of mineralized matrix by the cells maintained 21 days in C medium (26.3-fold increase) and, a synergistic effect when combined with OD medium (35.2-fold increase). Similarly, Larrañaga et al. obtained osteogenic differentiation of hASCs in standard culture medium when using polymeric scaffold with bioactive glass particles (Larrañaga et al., 2015).

### 3.6. Osteospheroids colonization/integration in hybrid CS/MβCHNC biomaterial

Having demonstrated the osteoinductive character, we went further by combining CS/MβCHNC biomaterial with osteospheroids to assess its ability as TE building block for alveolar bone regeneration. To do so, first hASCs spheroids were produced followed by differentiation. The results displayed in Fig. S7 confirmed the obtention of high viable and well-organized spheroids, in particular, for osteospheroids presenting high cell viability with few dead cells disaggregating and greatly maintained the sphere organization along the 21 days. Also, volume quantification indicated time-dependent decrease with low compaction and cell disaggregation during the differentiation process. According to the osteogenic differentiation degree, ALP enzymatic activity showed the highest values at 7 days (4.9-fold, p < 0.01) and 14 days (3.4-fold, p < 0.05). For its part, matrix mineralization, as late marker, revealed high values at 21 days.

The establishment of the hybrid biomaterial-osteospheroid 3D model was made by evaluating their integration into the CS/MβCHNC biomaterial. As displayed in Fig. 11, both control group (spheroids) and osteospheroids adhered, colonized and proliferated into the CS/ MβCHNC biomaterial throughout 21 days of culture. In both cases, the spheroid disaggregated in a time-dependent manner and, cells migrated towards the biomaterial with adhered flattened cells forming a homogeneous layer of tissue appearance at 21 days of culture. Interestingly, some small mineral crystals appeared in the osteospheroids group, possibly due to the osteogenic induced differentiation process and the
A

<figure><img src="image_13.png" /></figure>

B

<figure><img src="image_14.png" /></figure>

C

<figure><img src="image_15.png" /><figcaption>**Fig. 8.** *In vitro* biocompatibility of the hybrid CS-based 3D porous materials with L929 cells. Cytotoxicity test (A); Live/Dead assay (B) and cell adhesion evaluation by SEM microscopy (C) of the materials with CHNC (CS/αCHNC and CS/βCHNC) and MCHNC (CS/MαCHNC and CS/MβCHNC). Data is expressed as mean ± SD (n = 3). The dotted line represents the limit of acceptance established by the ISO 10993-5:2009 (70 % of the value of the negative control); *** *p* < 0.001.</figcaption></figure>
<figure><img src="image_16.png" /><figcaption>Fig. 9. Bioactivity assessment of hybrid CS-based biomaterials. SEM micrographs of the biomaterials surface before and after immersion in SBF for 1 and 4 weeks (A); and XRD patterns at 4 weeks (B).</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>Fig. 10. Osteogenic differentiation of hASCs on hybrid CS/MβCHNC biomaterial. ALP enzymatic activity (A) and matrix mineralization (B). \* *p* < 0.05; \*\*\* *p* < 0.001.</figcaption></figure>

consequent synthesis of bone ECM. Additionally, the colonization and integration process on the biomaterial did not have a detrimental effect on the cell's viability.

These results showed better integration of the spheroids and cell migration towards the 3D hybrid scaffold than reported by Kronemberger et al., in which the ASC spheroids remained as single spheroids into a 3D printed scaffold (Kronemberger et al., 2023).

## 4. Conclusion

In conclusion, MCHNC were successfully synthesized using a biomimetic mineralization approach that enabled the formation of flake- or needle-shaped HAp minerals, demonstrating, for the first time, that CHNC of both isoforms could serve as templates for mineralization. The

degree of mineralization achieved was found to be directly proportional to the concentration of the Ca/P solution, with optimal conditions at 400:240 mM over 4 days of mineralization. *In vitro* biocompatibility test showed that a maximum concentration of 200 µg·mL⁻¹ MCHNC added as a supplement to the culture medium did not significantly affect cell viability. Likewise, this MCHNC concentration significantly increased the ALP enzymatic activity of hASCs and the mineralization of the matrix was favoured.

When MCHNC were employed as inorganic osteoinductive nanofillers in CS organic matrix, the resulted hybrid 3D porous biomaterials showed improved mechanical properties, hydrolytic degradation, and high *in vitro* biocompatibility and bioactivity, providing an adequate microenvironment for cells. Unambiguously, the hybrid CS/MβCHNC biomaterial presented the greatest potential for TE applications since
<figure><img src="image_18.png" /><figcaption>**Fig. 11.** Evaluation of the adhesion, cell colonization and viability of control group and osteospheroids at 7, 14 and 21 days of culture into hybrid CS/MβCHNC 3D porous biomaterial. Scale bar represents 100 µm.</figcaption></figure>

presented the best mechanical properties, swelling capacity and resistance to enzymatic degradation. Furthermore, the incorporation of MβCHNC provided the hybrid biomaterial with osteoinductive character, since hASCs showed increased ALP activity and higher matrix mineralization even cultured in standard cell culture medium. Finally, preliminary study with osteospheroids showed a very good integration into the CS/MβCHNC biomaterial, with great migration and colonization of viable cells. In conclusion, the developed CS/MβCHNC biomaterial combined with osteospheroids showed great potential for alveolar bone regeneration. Further studies are needed to evaluate the *in vivo* biocompatibility and osteointegration of CS/MβCHNC biomaterial for clinical application.

### CRediT authorship contribution statement

**Sheila Olza:** Writing – review & editing, Writing – original draft, Validation, Methodology, Formal analysis, Conceptualization. **Nabil M. Hadj Bouzidi:** Writing – review & editing, Methodology, Formal analysis, Conceptualization. **Laurent Rubatat:** Writing – review & editing, Validation, Methodology, Investigation, Formal analysis. **Virginia Pellerin:** Writing – review & editing, Validation, Methodology, Formal analysis. **Unai Montejo:** Writing – review & editing. **Ana Alonso-Varona:** Writing – review & editing, Validation, Supervision, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization. **Susana C.M. Fernandes:** Writing – review & editing, Validation, Supervision, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.

### Funding

This work was carried under the framework of E2S UPPA Partnership Chair MANTA (Marine Materials) funded by the ‘Investissements d’Avenir’ French program managed by ANR, grant number #ANR-16-IDEX-0002 (France). This research also received funding from the University of the Basque Country UPV/EHU (Spain), grant reference

GIU21/054 and PES21/50.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgements

S.O. would like to thank UPPA for her international joint PhD thesis fellowship supported by the “UPPA/AAP tranfrontalier UPV” cotutelle programme between UPPA and UPV/EHU. We acknowledge SOLEIL for provision of synchrotron radiation facilities and we would like to thank J. Perez for assistance in using beamline SWING. Finally, the authors express their appreciation to the POLYCAT platform at IPREM/UPPA for providing access to their facilities and thank for technical and human support provided by SGIker services (UPV/EHU).

### Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.carbpol.2025.123911.

### Data availability

No data was used for the research described in the article.

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