Biopolymer-Based Nanocomposite Scaffolds: Methyl Cellulose and Hydroxyethyl Cellulose Matrix Enhanced with Osteotropic Metal Carbonate Nanoparticles (Ca, Zn, Mg, Cu, Mn) for Potential Bone Regeneration

Abstract

Bone fractures are a serious health problem worldwide, and up to 10% of emergency department visits are related to such injuries. The development of effective materials for bone repair remains an urgent need of modern medicine. The aim of this study was to develop new scaffolds based on biopolymers (methyl cellulose and hydroxyethyl cellulose) modified with carbonate nanoparticles (CaCO₃, MgCO₃, ZnCO₃, MnCO₃, CuCO₃) for potential applications in bone tissue engineering. FTIR spectroscopy confirmed the successful formation of stable composite structures: characteristic absorption bands of the functional groups of the molecules that make up the scaffold, as well as specific fluctuations in metal-oxygen bonds (Ca–O, Zn–O, Cu–O), were revealed. Stability tests revealed the most stable samples when changing the pH and the ionic strength of the solution. The developed scaffold matrices had a high porosity in the range from 93.3% to 98.0%, and their moisture absorption capacity ranged from 858% to 1402%. Specific gravity measurements ranged from 0.050 g/cm³ to 0.067 g/cm³, indicating optimal material density for potential biomedical applications. Biological evaluation demonstrated different cytotoxic effects depending on the type of nanoparticles. Thus, matrices with minimal toxicity and promising biocompatibility (modified CaCO₃), as well as with significant toxic effects (modified ZnCO₃ and CuCO₃) were found. As a result, it was found that CaCO₃-modified scaffolds have the most favorable combination of structural, physical, and biological properties for potential applications in bone tissue engineering. The developed innovative materials are porous scaffolds in which nanoparticles of carbonates of osteotropic elements are embedded, which presumably contribute to the acceleration of bone tissue regeneration. However, this study provides encouraging preliminary data, and further in-depth biological and functional studies are needed to fully confirm the osteogenic potential and regenerative efficacy of the scaffolds.

1. Introduction

Bone fractures are one of the most common traumatic injuries worldwide, accounting for approximately 7–10% of all emergency department visits per year [1]. This condition is a significant socio-economic burden, while in severe cases the level of long-term disability reaches 30% [2,3]. This problem is particularly acute for two demographic groups: the pediatric and geriatric populations, which are most vulnerable due to the unique features of their bone tissue [4,5]. It is noteworthy that the statistics of the winter season show a significantly higher incidence of fractures compared to the summer months [6,7], which once again underlines the urgent need for effective treatment methods. The main goal of fracture treatment is to ensure complete, correct and rapid consolidation of bone fragments while restoring lost functions [8]. Despite the wide range of available treatment methods, including both conservative and surgical approaches, up to 40% of fracture cases lead to unsatisfactory results [9,10]. These statistics highlight the urgent need for innovative solutions to combat fracturing.

Modern treatment approaches have evolved significantly, and osteosynthesis has become a widespread method that ensures reliable fixation and minimizes the risk of secondary displacement, thereby reducing the healing time of fractures [11,12]. Intramedullary or internal osteosynthesis has also become a modern treatment method involving the insertion of pins directly into the bone marrow cavity of long bones, which helps minimize tissue injury and blood loss [13]. It is worth noting that surgical methods remain the main method of fracture treatment [14]. However, combined treatment strategies are increasingly being used, including both surgical interventions and pharmacological drugs that strengthen bone tissue. Preparations based on glycosaminoglycans, which are important components of the bone extracellular matrix and can accelerate fracture healing, are particularly promising [15,16].

Recent studies have demonstrated the high efficiency of composite scaffolds in bone tissue regeneration [17,18,19,20]. Studies have shown that composite matrices with mesenchymal stem cells can regenerate bone tissue, while increasing bone mineralization [21,22]. To improve the properties of hydroxyapatite-based materials, they are often combined with natural and synthetic polymers to create scaffolds that support the restoration and adhesion of bone tissue [23].

The elemental composition of bone tissue is quite diverse. The bone mainly consists of a collagen matrix impregnated with Ca₃(PO₄)₂ nanoparticles (NPs) in the form of hydroxyapatite [24,25]. In addition, bone tissue contains various elements such as Na, K, Mg, Zn, Si, Fe, Sr, Ni, Al, Cr, Ba, Ti, Cu, Co, Mn, Sn, V, Pb, and Sr [26]. It is noteworthy that some of these elements have antioxidant and antimicrobial properties that are crucial for tissue regeneration processes [27]. Recent advances in nanotechnology have revolutionized bone regeneration strategies. Nanomaterials, especially those with a high surface-to-volume ratio, exhibit enhanced biological interactions and promote excellent cellular responses [28]. Among them, nanocomposite frameworks containing carbonate NPs of osteotropic elements (Zn, Mn, Mg, and Ca) have demonstrated significant potential [29]. Notably, osteotropic elements specifically target bone tissue and actively participate in bone metabolism. These elements possess unique properties that make them particularly suitable for bone regeneration applications. They can enhance bone formation by promoting osteoblast proliferation and differentiation, improving mineralization, and modulating bone remodeling processes. Their ability to interact with bone cells and matrix components, combined with their bioactive properties, creates a favorable environment for bone tissue regeneration [30,31].

The unique properties of nanomaterials are due to their ability to interact at the cellular and molecular levels. NPs promote osteoconductivity by providing a favorable material for the deposition of minerals in bone tissue and enhancing cellular adhesion [32,33]. They also have osteoinductive properties, stimulating the differentiation of mesenchymal stem cells into osteoblasts [34]. In addition, the anti-inflammatory effects of these nanomaterials help to modulate the inflammatory response, thereby promoting tissue healing [35]. Antimicrobial activity is another important advantage of some nanomaterial compositions, which significantly reduce the risk of infection, a common complication in fracture healing [36,37,38]. The mechanical reinforcement provided by nanocomposites increases the structural integrity of the scaffolds, ensuring their functionality in vivo [32,39]. Experimental models have demonstrated that nanocomposite scaffolds can improve bone tissue integration by enhancing angiogenesis and accelerated mineralization [40]. These materials also reduce inflammatory reactions and increase the mechanical stability of the repaired bone, contributing to more effective fracture healing.

The combination of biopolymer matrices with specially selected NPs compositions deserves special attention. For instance, the incorporation of polycaprolactone, hydroxyapatite, and carbon nanotubes has been shown to increase material strength while reducing inflammatory reactions [41]. Similarly, the stabilization of CuCO₃ NPs with biological macromolecules has shown promising results in terms of biocompatibility and controlled release properties [42]. It is worth noting that materials based on cellulose derivatives with the inclusion of metal NPs, metal oxides and various inorganic compounds are promising research objects. Notably, such materials have increased antibacterial activity, the ability to regenerate bone and tissue, and cell proliferation [43,44,45,46]. While current bone tissue regeneration materials predominantly consist of calcium hydroxyapatite, emerging research on carbonate NPs of various trace elements (Ca, Mg, Mn, Cu, Zn) shows promise for bone restoration [47,48,49,50]. Despite these advancements, there remains an urgent need for innovative materials that can effectively combine mechanical stability with biological activity. Current solutions often fail to provide a balanced combination of essential properties required for successful bone regeneration, including optimal mechanical strength, controlled degradation, and biocompatibility.

Therefore, this study directly addresses this research gap and aims to develop novel biopolymer-based scaffolds modified with NPs of osteotropic elements (carbonates of Ca, Mg, Mn, Zn, Cu), offering a promising solution for improving fracture healing outcomes. To achieve this aim, the research objectives were focused on comprehensive material development and evaluation, including synthesis, characterization, and biological assessment of novel scaffolds. The proposed approach integrates several critical advantages: controlled release of bioactive factors, enhanced cell proliferation and differentiation, improved mechanical properties, tailorable porosity for tissue ingrowth, and optimal biocompatibility and biodegradability. These features make nanocomposite scaffolds a highly promising platform for next-generation bone tissue engineering applications.

2. Materials and Methods

2.1. Materials and Chemicals

For the synthesis, analytical grade reagents and class «A» glassware were utilized. The purity of the experimental setup was ensured by using distilled water with conductivity below 1 μS/cm. The core precursors for NPs synthesis included metal acetates: Zn(CH₃COO)₂, Ca(CH₃COO)₂, Mn(CH₃COO)₂, Cu(CH₃COO)₂, and Mg(CH₃COO)₂, supplied by LenReaktiv (Saint Petersburg, Russia). Ammonium carbonate ((NH₄)₂CO₃), also provided by LenReaktiv (Saint Petersburg, Russia) served as the precipitating agent in the synthesis process. To stabilize the formed NPs, 736.7 g/mol hydroxyethyl cellulose (HEC) and 658.7 g/mol methyl cellulose (MC) were employed as stabilizing agents, obtained from StavReakhim (Stavropol, Russia). Additionally, the following reagents were purchased from DIA-M (Moscow, Russia) for the experimental procedures: phosphoric acid (H₃PO₄) for pH adjustment and buffer preparation, acetic acid (CH₃COOH) as a component of the acid mixture, boric acid (H₃BO₃) for buffer solutions, sodium hydroxide (NaOH) for pH regulation, sodium chloride (NaCl) for ionic strength studies. The biological evaluation component of the study utilized Dulbecco’s Modified Eagle Medium (DMEM-1640), 10% fetal bovine serum, antibiotics (penicillin and streptomycin), MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), trypsin–versene solution and dimethyl sulfoxide (DMSO), obtained from Sigma-Aldrich (St. Louis, MO, USA).

Daphnia magna Straus was obtained from Evropolytest (Moscow, Russia) to conduct acute toxicity assessment. For cytotoxicity assessment, cells of immortalized fibroblasts WI-26 (human embryo pulmonary fibroblasts), cancer cells HeLa-123 (human cervical epithelioid carcinoma cell line, subclone M) and non-cancerous Vero-102 cells (African green monkey renal epithelial cells) were purchased from Algimed (Moscow, Russia).

2.2. Synthesis of NPs

The synthesis procedure for individual carbonate compounds followed a protocol schematically presented in Figure 1. In a 500 mL beaker, a 100 mL solution of 0.8 M metal precursor was prepared. A 1% (w/w) aqueous stabilizer solution was added and stirred at 500 rpm for 10–15 min until complete dissolution. Subsequently, a separate 100 mL solution of 0.8 M precipitant was prepared and added dropwise to the precursor-stabilizer mixture. The synthesis was conducted at room temperature. After precipitant addition, the mixture underwent centrifugation in a MicroCL 17R centrifuge (Thermo FS, Waltham, MA, USA) for 5 min at 3000 rpm. The supernatant was decanted while preserving the precipitate. The centrifuged solution was refilled with distilled water and stirred with a glass rod until complete dissolution, followed by another 5 min centrifugation at 3000 rpm. This centrifugation process was repeated 5 times. The resulting precipitate was dried in a VT 6025 drying oven (Thermo FS, Waltham, MA, USA) at 110 °C for 8 h. Throughout the synthesis process, strict adherence to procedural parameters ensured the production of high-quality NPs with consistent characteristics. The combination of controlled precipitation, thorough purification via centrifugation, and precise drying conditions contributed to the development of well-defined nanomaterial structures suitable for further application in biomedical scaffolds.

2.3. Synthesis of the Biopolymer Scaffold Matrices Modified with NPs

As shown in Figure 1, the synthesis of NPs was followed by scaffold preparation. The synthesis of the biopolymer scaffold matrices modified with NPs of ostreotropic elements was carried out in several stages. Initially, suspensions were prepared by dispersing 0.75 g of metal carbonate NPs (Ca, Zn, Mg, Cu and Mn) in 100 mL of deionized water. Subsequently, a 3% aqueous solution was prepared by mixing MC and HEC in a 1:1 ratio. The prepared NPs suspension was then incorporated into this biopolymer mixture. The resulting composite solution was poured into sterile metal molds and subjected to cryogenic treatment. Freezing was performed at −50 °C for 20 h, ensuring uniform solidification of the matrix. Following the freezing stage, the samples underwent freeze-drying in a vacuum chamber for 20 h. This process was critical for complete removal of residual moisture while preserving the structural integrity of the scaffold matrix. During this process, ice crystals formed within the frozen matrix created a temporary structure that defined the primary pore architecture. As sublimation occurred, these ice crystals transformed into interconnected pores of varying sizes, creating a hierarchical porosity. The temperature gradient across the sample influenced the rate and direction of ice crystal formation, which in turn affected pore morphology and distribution. The duration of the freeze-drying process determined the extent of pore development and the overall porosity of the final scaffold [40]. The obtained scaffold samples were put in vials and sterilized by autoclaving at 120 °C for 20 min in an SPVA-75-NN steam sterilizer (Trans-Signal, Nizhniy Novgorod, Russia).

Following the synthesis of biopolymer scaffold matrices modified with NPs, the prepared samples were stored in a refrigerator at 0–4 °C until their subsequent use in experimental procedures. As illustrated in Figure 1, the experimental workflow encompassed a comprehensive range of methodologies, including material characterization techniques and toxicity assessment protocols.

2.4. Characterization Methods

The microstructure of the obtained samples was studied by scanning electron microscopy (SEM) on MIRA3-LMH device (Tescan, Brno, Czech Republic). Samples were carefully mounted on conductive stubs using double-sided carbon tape, ensuring stable adhesion and electrical conductivity. A crucial preparatory step was the application of a conductive coating to prevent charging artifacts during imaging. Samples received a uniform layer of approximately 10 nm using a QR 150 sputtering system (Tescan, Brno, Czech Republic), optimizing both conductivity and preservation of sample integrity. Prior to imaging, the microscope system was purged with nitrogen gas to create an optimal environment for observation. The imaging parameters were carefully selected to capture both macro- and micro-level details. The SEM analysis operated under the following optimized conditions: accelerating voltage set at 10 kV for optimal resolution, working distance maintained at 4.9 mm, in-beam secondary electron detector employed for detailed surface imaging. Scanning was performed at magnification 250× [40].

The elemental composition of the synthesized samples was analyzed using a scanning electron microscope MI-RA3-LMH (Tescan, Brno, Czech Republic) equipped with an energy-dispersive X-ray spectroscopy system AZtecEnergy Standard/X-max 20 (standard configuration).

The stability assessment of scaffolds was conducted to evaluate its chemical resistance under various environmental conditions. The primary focus of the investigation was to study the impact of solution pH on the material’s chemical stability. The experimental protocol involved incorporating solutions with varying pH levels into 0.5 g samples of the biopolymer matrix. Initially, a mixed acid solution was created by combining H₃PO₄, CH₃COOH, and H₃BO₃, each at a concentration of 0.04 M. The preparation process began with a 2 L volumetric flask serving as the primary vessel. Precise measurements of 5.49 mL H₃PO₄, 4.58 mL CH₃COOH, and 4.95 g H₃BO₃ were added sequentially. The mixture was then diluted to the final volume of 2 L using distilled water to ensure uniform concentration. A separate 0.2 M NaOH solution was prepared with a volume of 700 mL. To achieve the desired pH levels, a calculated volume of NaOH solution was gradually added to 100 mL of the prepared acid mixture. The pH adjustment process was carefully monitored to maintain accurate solution composition. The relationship between the volume of added NaOH and the resulting pH was systematically documented, allowing for the creation of a series of buffer solutions with defined pH levels. The prepared buffer solutions were then used to investigate the material’s response to varying pH levels. The values of the NaOH volumes with the corresponding pH values are shown in

Table 1. NaOH volumes with the corresponding pH.

NaOH Volume, mL	pH
0	1.81
10	2.21
20	3.29
30	4.56
40	5.72
50	6.8
60	7.96
70	9.15
80	10.38
90	11.58
100	11.98

.

To further investigate the stability of the biopolymer scaffold matrix under varying ionic conditions, a series of NaCl solutions was prepared with concentrations ranging from 0.1 M to 1.0 M. For the 0.1 M solution, 0.029 g of NaCl was dissolved in distilled water. To prepare the 0.25 M solution, the amount of NaCl was increased to 0.073 g. The 0.5 M solution required 0.146 g of NaCl, while the 0.75 M solution contained 0.219 g. The most concentrated solution (1.0 M) was prepared using 0.29 g of NaCl. Each prepared solution was thoroughly mixed to ensure complete dissolution of the salt. The resulting solutions were then used in stability testing procedures, where 0.5 g of the scaffold sample was incubated in 25 mL of each solution. This experimental setup allowed for the systematic evaluation of how varying ionic strengths affected the structural integrity and chemical stability of the modified scaffold matrix.

Scaffold were characterized by the method of Bartoš et al. (2018) [51]. In brief, the scaffold sample sized (6.0 ± 1) × (0.5 ± 0.1) cm weighing 0.04 ± 0.001 g was placed in a 10 mL-graduated glass cylinder; ethanol was used as a filling liquid at a temperature of 30 °C. The scaffold was placed in the cylinder, which was filled two third full of ethanol and kept for 5 min with occasional stirring. Then, the cylinder was filled to the mark with ethanol and weighed. The moisture capacity of scaffolds was determined according to the method of Kutlusoy et al. (2017) [52].

Calculations of indicators was carried out according to Equations (1)–(7):

% Porosity: P = V_p/(V_p + V_s) = [(W₂ − W₃ − W_s)/(W₁ − W₃)] × 100%

(1)

Volume of the scaffold pore: V_p = (W₂ − W₃ − W_s)/p_e, cm³

(2)

Volume of the scaffold skeleton: V_s = (W₁ − W₂ + W_s)/p_e, cm³

(3)

Density of scaffold skeleton: P_ss = W_s/V_s, g/cm³

(4)

Scaffold density: P_s = W_s/(V_p + V_s), g/cm³

(5)

Volume–mass index: V = V_p/W_s, cm³/g

(6)

% Swelling: S = [(W − W_s)/W_s] × 100%

(7)

where P is the %porosity, V_p is the volume of the scaffold pore in cm³, V_s is the volume of the scaffold skeleton in cm³, P_ss is the density of scaffold skeleton in g/cm³, P_s is the scaffold density in g/cm³, V is the volume-mass index in cm³/g, W is the scaffold mass with absorbed ethanol in g, W₁ is the mass of ethanol in a cylinder filled to the mark in g, W₂ is the mass of ethanol with scaffold in the cylinder in g, W₃ is the mass of ethanol in the cylinder after removing the ethanol-saturated scaffold in g, W_s is the mass of scaffold in g, p_e is the ethanol density in g/cm³, and S is % swelling.

2.5. Acute Toxicity Assessment

The assessment of acute toxicity of synthesized NPs was performed using a standardized biological testing method based on the mortality rate of Daphnia magna Straus [53]. The experimental protocol involved preparing aqueous dispersions of metal carbonate NPs at specific concentrations: 100 mg/L, 10 mg/L, 1 mg/L, 0.1 mg/L, 0.01 mg/L, and 0.001 mg/L. Test organisms were divided into experimental groups exposed to these concentrations and a control group maintained in NPs-free medium. The research utilized a specialized cultivator KVM-08 (Evropolytest, Moscow, Russia), which provided precise control over experimental conditions including temperature, light intensity, aeration, mixing, and convection flow simulation to replicate natural aquatic environments. This system ensured uniform distribution of NPs and consistent exposure conditions for all test organisms. The acute toxic effect was evaluated by monitoring the mortality rate of Daphnia magna over a 22 h exposure period. Mortality assessment was conducted using microscopic examination by Axio Vert A1 (Carl Zeiss Microscopy, Oberkochen, Germany) at 200× magnification to accurately determine the number of surviving organisms. The observation period was selected to capture immediate toxic responses while minimizing long-term adaptation effects [54].

2.6. Cytotoxicity Assessment of NPs in Scaffold Matrices

The comprehensive toxicity evaluation involved two sequential stages: assessment of NPs cytotoxicity followed by evaluation of modified scaffold matrices. The study employed a standardized approach to determine the biocompatibility of synthesized materials [55]. The initial stage focused on evaluating the cytotoxic potential of individual NPs. Immortalized WI-26 fibroblast cells were seeded into a 96-well plate at a density of 5000 cells per well using DMEM-1640 medium supplemented with 10% fetal bovine serum. After a 24 h incubation period, the medium was replaced with a solution containing metal carbonate NPs. The cells were incubated with NPs for 2 h, followed by medium replacement and further incubation for 48 h. Cytotoxicity assessment was performed using the MTT assay, where 10 μL of MTT reagent was added to each well. After a 2 h incubation period, the medium was removed, and formazan crystals were dissolved in 100 μL of DMSO. Optical density was measured using the SuPerMax 3100 Multi-Mode Microplate Reader (Flash Spectrum, Shanghai, China), with untreated cells serving as the 100% viability control. All measurements were normalized to the negative control (100% viability) and corrected for inter-well variability, plate-to-plate differences, day-to-day variations.

The second stage involved evaluating the cytotoxic effects of modified scaffold matrices. Matrix extracts were prepared under sterile conditions using DMEM culture medium supplemented with 10% fetal bovine serum, 0.17 M penicillin, and 0.058 M streptomycin [56]. Matrix samples (0.5 cm × 0.5 cm) were autoclaved in SPVA-75-1NN steam sterilizer (Trans-Signal, Nizhny Novgorod, Russia) for 10 min at 110 °C and incubated with 1 mL of culture medium at 37 °C for 24 h. The resulting matrix extracts are presented in Figure S1. Two cell lines were used for testing: HeLa (cervical carcinoma) and Vero-102 (African green monkey kidney cells). Cells were cultured in 25 cm² culture vials put into MCO-19AIC incubator (Sanyo, Tokyo, Japan) under standard conditions (37 °C, 5% CO₂) until reaching 80–90% confluence. After detachment using trypsin-versene solution, cells were seeded into 96-well plates (SPL Lifesciences, Gyeonggi-do, Republic of Korea) at a concentration of 4 × 10⁴ cells per well, calculated by Countess 3 FL (Thermo Fisher Scientific, Waltham, MA, USA). Following a 24 h incubation period, the culture medium was replaced with matrix extracts (100 μL per well), and cells were incubated for another 24 h. Cytotoxicity was assessed using the MTT assay. After incubation with extracts, cells were washed with DPBS buffer to remove matrix particles. MTT reagent (0.012 M in PBS, 10 μL per well) was added to wells containing complete culture medium (100 μL), followed by 2 h incubation. The medium was then removed, and 100 μL of DMSO was added to each well. The plate was placed in a PST-60HL thermoshaker (Biosan, Riga, Latvia) at 37 °C and 300 rpm for 15 min to dissolve formazan crystals. Optical density was measured using the Varioskan LUX microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at 570 nm.

The experimental design included both negative and positive controls to validate the results. The negative control consisted of cells cultivated on the same medium, while positive control included cells cultivated on the same medium with addition of MC:HEC extract without NPs. Cell viability was calculated relative to control cells cultured in complete medium. Results were presented as average cell survival percentages based on 5 replicates for each sample. Morphological assessment of cell cultures was performed using an Axio Vert A1 inverted microscope (Carl Zeiss Microscopy, Oberkochen, Germany) at 400× magnification after 24 h of incubation with matrix extracts. Images were obtained using ZEN 3 Pro software (Carl Zeiss Microscopy, Oberkochen, Germany), allowing for detailed evaluation of cell morphology and viability.

2.7. Statistical Data Processing

Biological and analytical experiments were repeated three to five times to assess the reproducibility of the measurements. Results are expressed as the mean ± standard error of the mean. Statistical analyses were performed with GraphPad Prism v. 6.01 (GraphPad Software, Boston, MA, USA). Data were subjected to one-way variance analysis (ANOVA). The normality and uniformity of the variance were checked using Pearson’s chi-squared criterion. Differences were considered significant at p ≤ 0.05.

3. Results and Discussion

3.1. Characterization of Samples

The microstructural characteristics of the developed scaffolds were thoroughly investigated using SEM, providing valuable insights into the morphology and composition of the synthesized materials. The SEM analysis revealed distinct microstructural features for each type of carbonate NPs-modified scaffold (Figure 2). The CaCO₃ NPs modified scaffold exhibited a unique morphology characterized by spherical primary particles ranging from 50 to 500 nm in size. These NPs aggregated into larger clusters measuring 10–30 µm in diameter. The material demonstrated an interconnected porous network with well-defined through pores throughout the structure. The MgCO₃ NPs modified scaffold displayed a different microstructural arrangement. The NPs formed spherical particles with a size distribution between 100 and 500 nm. The material exhibited high NPs dispersion, resulting in a uniform structure with consistent particle distribution across the entire polymer matrix. The microstructure showed minimal agglomeration and a homogeneous distribution of pores. The MnCO₃ NPs modified scaffold presented a distinctive morphology with spherical particles measuring 60–300 nm in diameter. These NPs formed larger agglomerates ranging from 10 to 20 µm, creating porous architecture characterized by parallel-oriented layers. The CuCO₃ NPs modified scaffold showed spherical NPs ranging from 40 to 300 nm in size. These particles aggregated into larger structures measuring 10–20 µm, resulting in a porous microstructure with a uniform distribution of pores throughout the material. The ZnCO₃ NPs modified scaffold displayed spherical primary particles measuring 80–300 nm, which formed smaller agglomerates ranging from 3 to 10 µm. This formulation exhibited a well-developed porous structure with a distinct distribution of NPs within the polymer matrix. These microstructural differences contribute to the unique properties of each scaffold formulation, influencing their potential biological responses and mechanical characteristics. The observed variations in NPs size, distribution, and pore morphology suggest that each material may exhibit distinct behavior in biological environments [57,58,59].

The analysis of elemental composition distribution maps demonstrated a homogeneous distribution of carbon (C) and oxygen (O) elements across the matrix surface, along with the presence of various metal elements. Quantitative evaluation of the elemental composition revealed that the samples contained carbon with a mass fraction ranging from 58.84% to 66.67%, oxygen with a mass fraction varying between 32.06% and 39.20%, and metal elements (Ca, Mg, Zn, Cu, Mn) present in concentrations from 1.27% to 2.97%. These findings confirm the uniform distribution of major elements and provide quantitative confirmation of the presence of trace metal elements within the specified concentration ranges throughout the sample matrix.

Next step evaluated the chemical stability and structural characteristics of biopolymer scaffold matrices modified with metal carbonate NPs. This comprehensive analysis was essential for understanding the potential applications of these materials in biomedical fields [60,61]. Visual analysis of the obtained samples, presented in Figures S2–S6, provided valuable insights into the materials’ behavior under different environmental conditions. The results revealed significant differences in stability among the modified samples. Notably, the scaffold matrix incorporating ZnCO₃ NPs demonstrated resistance to both pH and solution ionic strength. After 60 min of exposure, this sample maintained its original form without any signs of dissolution. Samples modified with CuCO₃ and MnCO₃ NPs exhibited intermediate stability, retaining their shape only in acidic environments. In contrast, matrices containing MgCO₃ and CaCO₃ NPs displayed the lowest stability across all tested conditions.

Following the stability assessment, a comprehensive microstructural analysis was performed on all obtained scaffold matrices. This analysis provided detailed insights into the materials’ structural characteristics, with results summarized in

Table 2. Structural and physical characteristics of scaffold matrices.

NPs	Specific Gravity, g/cm3	Porosity, %	Swelling, %	Volume-Mass Index, cm3/g	Thickness, mm	Density, g/cm3
MgCO3	0.058 ± 0.003	93.3 ± 2.4	1083.0 ± 18	16.3 ± 0.49	7.7 ± 0.23	0.119 ± 0.006
CaCO3	0.056 ± 0.003	98.0 ± 2.5	1089.0 ± 19	17.0 ± 0.51	1.43 ± 0.04	0.073 ± 0.003
ZnCO3	0.050 ± 0.0025	95.6 ± 2.4	1402.0 ± 24	19.9 ± 0.6	1.67 ± 0.05	0.079 ± 0.004
MnCO3	0.067 ± 0.0033	93.9 ± 2.4	858.0 ± 15	15.3 ± 0.46	1.66 ± 0.05	0.092 ± 0.005
CuCO3	0.060 ± 0.003	95.8 ± 2.4	1192.0 ± 20	16.2 ± 0.49	2.38 ± 0.07	0.085 ± 0.004
control	0.066 ± 0.0033	95.3 ± 2.4	1359.0 ± 23	17.7 ± 0.53	2.5 ± 0.08	0.087 ± 0.004

.

The investigation of matrix porosity demonstrated a direct influence of carbonate type on the structural properties of the scaffolds. The structural and physical properties of the developed scaffolds demonstrate a clear correlation between the type of incorporated nanoparticles and the resulting material characteristics. The specific gravity of the scaffolds shows a direct relationship with the density of incorporated nanoparticles. The highest specific gravity (0.067 g/cm³) is observed in the MnCO₃-modified scaffold, which correlates with the higher density of MnCO₃. Conversely, the ZnCO₃-modified scaffold exhibits the lowest specific gravity (0.050 g/cm³), reflecting the lower density of ZnCO₃. The porosity varied significantly across different formulations, ranging from a minimum of 93.3% observed in the MnCO₃-modified matrix to a maximum of 98.0% in the CaCO₃-modified variant. Notable, the average porosity across all samples consistently exceeded 95%, indicating an optimal porous architecture conducive to cellular ingrowth and tissue integration [62,63]. The density measurements indicate that ZnCO₃-modified matrices have the lowest density (0.050 g/cm³), while MgCO₃-modified matrices exhibit the highest density (0.067 g/cm³). These variations in density correlate with differences in NPs distribution and matrix structure. The moisture absorption capacity exhibited notable differences among the tested matrices. The highest values were recorded in ZnCO₃-modified matrices (1402 ± 23.8%) and CuCO₃-modified matrices (1192.0 ± 20.2%). This elevated moisture absorption capability suggests enhanced potential for nutrient delivery and metabolic exchange within the matrix structure [64,65]. The volume-mass index reflects the relationship between the material’s structure and its mass. The ZnCO₃-modified scaffold shows the highest value (19.9 cm³/g), suggesting a more open structure with larger pores. The lower values in other samples (ranging from 15.3 to 17.7 cm³/g) indicate variations in pore size distribution and matrix density. Thickness measurements range from 1.43 mm (CaCO₃-modified scaffold) to 2.5 mm (control sample).

These physical characteristics have important implications for the potential biomedical applications of the developed scaffolds. For instance, high porosity (>93%) ensures adequate cell penetration and tissue ingrowth. Optimal specific gravity (0.050–0.067 g/cm³) indicates suitable mechanical properties for bone tissue engineering. Variations in swelling capacity reflect differences in moisture absorption and nutrient delivery capabilities. Thickness measurements provide information about structural stability and potential load-bearing capacity [40]. Based on the observed structural and functional properties, matrices modified with ZnCO₃, CaCO₃, and CuCO₃ demonstrate the most promising regenerative characteristics. The superior performance of ZnCO₃-modified matrices can be attributed to the unique combination of high porosity and high moisture absorption capacity, which collectively enhance cellular interaction and metabolic activity [66]. CaCO₃-modified matrices benefit from their biocompatibility and structural stability, closely mimicking natural bone composition [67]. CuCO₃-modified matrices exhibit an optimal balance between porosity and moisture absorption properties [68].

3.2. Acute Toxicity Assessment

Having thoroughly evaluated the structural and physical properties of the developed materials, the subsequent phase of the investigation focused on assessing biological safety of synthesized NPs (Figure 3A) through acute toxicity testing. The acute toxicity assessment was conducted using Daphnia magna Straus as a test organism (Figure 3B). The study employed a range of concentrations (100 mg/L to 0.001 mg/L) to determine dose-dependent effects. The results obtained are shown in Figure 3C.

The experimental findings revealed significant differences in toxicity among the tested NPs. Notably, CuCO₃ and ZnCO₃ NPs exhibited pronounced acute toxic effects, demonstrating the highest mortality rates among all tested materials. This heightened toxicity can be attributed to the higher solubility and bioavailability of Cu²⁺ and Zn²⁺ ions in aqueous media, which interfere with cellular metabolic processes [69]. Notably, the results obtained are in the line with data of similar tests on ZnO and CuO NPs towards Daphnia magna [70,71]. In contrast, CaCO₃ NPs showed moderate toxicity, which is likely due to their biological relevance and controlled release of Ca²⁺ ions [72,73]. Remarkably, MgCO₃ and MnCO₃ NPs demonstrated minimal toxic effects, with mortality rates not exceeding 20% at concentrations of ≤1 mg/L. Notable, both MgCO₃ and MnCO₃ NPs have relatively lower solubility in water, which limits the release of Mn²⁺ and Mg²⁺ ions into the environment. Furthermore, Mg²⁺ ions are essential for many biological processes and are naturally present in living organisms, which reduces their toxic potential [74]. Mn²⁺ ions, although less common than Mg²⁺, are also biocompatible and have a lower tendency to disrupt cellular functions [75]. The observed concentration-dependent response provides critical insights into the materials’ safety profiles. At higher concentrations (100 mg/L and 10 mg/L), significant toxic effects were consistently observed across all NPs. However, as the concentration decreased to 1 mg/L, 0.1 mg/L, and 0.01 mg/L, the mortality rate of test organisms decreased proportionally, indicating a clear dose–response relationship.

3.3. Cytotoxicity Assessment of NPs in Scaffold Matrices

The comprehensive cytotoxicity evaluation of carbonate NPs involved a multi-stage experimental design to assess material biocompatibility under different conditions. The initial phase focused on investigating individual stabilizers (MC and HEC) to determine their independent effects on NPs behavior and cellular response of WI-26 fibroblasts. This approach allowed to isolate the specific contributions of each stabilizing agent to the overall biocompatibility profile. Consequently, the cytotoxicity evaluation of MC- and HEC-based scaffolds modified with carbonate NPs revealed significant differences in cellular response depending on the type of metal ion and stabilizing agent (Figure 4A).

The obtained IC₅₀ values provide quantitative confirmation of the observed cytotoxic effects, with significant differences noted between various formulations. The MC:HEC control scaffold demonstrated excellent biocompatibility with IC₅₀ values exceeding 25 mg/mL, indicating a safe polymer matrix foundation. The most pronounced cytotoxic effects were observed for CaCO₃ NPs stabilized with MC (IC₅₀ = 0.5444 mg/mL) and ZnCO₃ NPs stabilized with MC (IC₅₀ = 0.6995 mg/mL). Conversely, CaCO₃ and ZnCO₃ NPs stabilized with HEC exhibited no significant cytotoxic effects, with IC₅₀ values exceeding 25 mg/mL. This suggests that the HEC stabilizer may effectively form a protective layer around the NPs, reducing their interaction with cellular membranes and mitigating cytotoxic effects, as was shown by Mansfield et al. [76] and Motelica et al. [77]. The intermediate cytotoxicity observed for other samples, ranging from 1.51 mg/mL for MnCO₃ stabilized with HEC to 12.66 mg/mL for MgCO₃ stabilized with MC, confirmed a complex relationship between type of stabilizing agent (MC vs. HEC), metal ion release kinetics, NPs-polymer interactions, and surface properties of modified scaffolds. The differences in cytotoxic potential can be attributed to variations in NPs surface properties, aggregation behavior, and cellular uptake mechanisms [78].

These findings have important implications. The low cytotoxicity of CaCO₃ and ZnCO₃ NPs stabilized with HEC, combined with the biological relevance of these elements, suggests their potential for use in regenerative materials. Ca, being a major component of bone, and Zn, playing an important role in enzyme activity, can contribute to bone regeneration processes [79,80]. MC stabilization may lead to more efficient cellular uptake and metal ion release, resulting in higher cytotoxicity. In contrast, HEC stabilization likely provides a protective barrier that minimizes NPs-cell interactions. However, MC possesses several critical advantages that justify its inclusion in the composite stabilizer system. MC contributes significantly to the structural integrity and mechanical stability of the NPs formulations. Its ability to enhance particle dispersion stability and provide optimal surface properties for biomedical applications cannot be overlooked [81]. The combination of MC with HEC in a mixed stabilizer system aims to leverage these strengths while mitigating the cytotoxic effects observed with MC alone.

Subsequent cytotoxicity testing employed a combination of MC:HEC stabilizers to evaluate the synergistic effects of the stabilizing system, which more closely mimics the final formulation intended for biomedical applications. The decision to proceed with MC:HEC mixtures for further testing on HeLa and Vero cell lines is based on the hypothesis that the synergistic effects of the combined stabilizers may result in a more favorable biocompatibility profile while preserving the essential material properties required for biomedical applications.

The results of MTT assay, presented in Figure 4B, reveals significant differences in cellular response to various carbonate matrix formulations. The study demonstrates a striking dichotomy in cytotoxic effects, with matrices containing CuCO₃ and ZnCO₃ exhibiting severe toxicity, while those with CaCO₃, MgCO₃, and MnCO₃ showing minimal to no adverse effects. The extreme cytotoxicity observed with CuCO₃ matrices is particularly noteworthy, as these formulations reduced Vero cell viability to just 2.5% and HeLa cell viability to 3% compared to the control group. Similarly, ZnCO₃ matrices demonstrated substantial toxicity, decreasing Vero cell viability to 5.5% and HeLa cell viability to 3%. This severe response can be attributed to the unique biochemical properties of Cu²⁺ and Zn²⁺ ions, which at elevated concentrations disrupt essential cellular processes [82]. Cu²⁺ ions are known to interfere with cellular metabolism and induce oxidative stress through reactive oxygen species generation [83], while Zn²⁺ ions, despite their essential biological role, can become toxic at high concentrations by disrupting cellular signaling pathways and enzyme function [84].

In stark contrast, matrices containing CaCO₃, MgCO₃, and MnCO₃, along with the control MC:HEC formulation, exhibited remarkably different behavior. The viability of Vero cells exposed to these matrices ranged from 107% to 112%, while HeLa cell viability ranged from 103% to 108%. These results suggest either a lack of significant cytotoxic effects or a mild stimulatory response that may fall within the assay’s margin of error.

Micrographs, presented in Figure 4C, confirms preservation of normal cellular morphology in cultures exposed to extracts from matrices containing CaCO₃, MgCO₃, and MnCO₃. The observed cell morphology remained identical to that of the positive control (MC:HEC) and the negative control with complete culture medium. This morphological consistency indicates the absence of any disruptive effects on cellular structure and confirms the non-cytotoxic nature of these formulations. At the same time, extracts from matrices containing CuCO₃ and ZnCO₃ NPs demonstrated profound morphological alterations in both cell lines. The most notable changes included the formation of small cell clusters and the presence of isolated cells detaching from the substrate. These morphological changes are indicative of advanced stages of cell damage leading to cell death [85], which correlates with the pronounced cytotoxic effects observed in these samples. Thus, the morphological analysis provides visual confirmation of the cytotoxic effects observed in the viability assays and offers additional insights into the mode of action of these materials.

The dual nature of CuCO₃ and ZnCO₃ NPs effects presents both opportunities and challenges for their potential therapeutic application. While their ability to selectively target cancer cells (HeLA) is promising, the concomitant effect on normal cells (Vero) requires careful consideration. Further research is needed to develop strategies that maximize their antitumor potential while minimizing cytotoxic effects on healthy tissues. Considering the primary aim of this study, general analysis of the results obtained revealed that CaCO₃ NPs are most suitable for modification of MC:HEC scaffolds for bone regenerations. At the same time, this study demonstrates that CuCO₃ and ZnCO₃ NPs integrated in MC:HEC matrix could serve as a basis for developing anticancer agents, but their current formulation requires significant optimization to achieve selective cytotoxicity and minimize side effects on normal cells.

Presumably, the increased porosity of the developed materials can create a kind of environment for bone and vascular growth, moisture capacity can ensure the supply of essential nutrients, and durability determines the stability and integrity of the structure [40]. As osteotropic elements, Ca, Zn, Mg, Cu and Mn can promote accelerated bone tissue growth due to proliferation and differentiation of osteoblasts and antibacterial effect [86,87]. Solubility and redox properties play important roles in determining the behavior and toxicity of metal carbonates. Solubility measures how readily metal ions detach from a compound, while redox ability indicates the aggressiveness and stability of a reagent [88]. The differences in these properties explain the varying behavior of the materials studied. Notably, Ca, Zn and Mg carbonates exhibit low solubility and high stability, making them resistant to oxidation and reduction processes. This inherent stability results in minimal toxicity, which makes these compounds suitable for biomedical applications [89]. In contrast, Cu and Mn compounds demonstrate higher solubility levels. As known, Cu participates in the generation of superoxide anions and reactive radicals [90], while Mn undergoes oxidation to the trivalent state [91]. These properties contribute to the observed toxicity of materials containing these elements. This distinction in material behavior directly correlates with the cytotoxic effects observed during biological testing, where Ca-based materials exhibited minimal toxicity, while Cu- and Zn-based materials showed more pronounced toxic effects. Understanding these relationships between solubility, redox properties, and toxicity is essential selecting appropriate materials in bone tissue engineering applications.

The results of this study demonstrate that the incorporation of Ca, Mg, Mn, Zn, and Cu carbonate NPs into the structure of a biopolymer scaffold enables targeted modification of its key physicochemical parameters. The observed improvement in mechanical strength and controlled degradation is consistent with literature data on the role of mineral fillers in polymer matrices. Furthermore, the absence of a cytotoxic effect and the high viability of cells in direct contact with the material indicate its preliminary cytocompatibility. It is important to emphasize that these data form the necessary fundamental basis for subsequent experiments.

4. Conclusions

A comprehensive study has demonstrated the successful development and characterization of new biopolymer scaffolds modified with carbonate NPs. Structural analysis confirmed the formation of stable structures with preserved functional groups, as evidenced by characteristic IR spectra describing the positions and presence or absence of characteristic functional groups. Stability tests have shown high resistance of ZnCO₃-modified matrices to acidic environments, which makes it possible to maintain structural integrity in difficult conditions. This property opens up new possibilities for their potential application in specific biomedical fields. The structural and physical and mechanical characteristics of the frames were investigated, and based on the results obtained, samples with the most favorable parameters (high porosity and moisture absorption capacity) were determined. The porosity level reached 93.3–98.0%, and the moisture absorption capacity ranged from 858% to 1402%. These parameters are highly favorable for potential biomedical applications.

Biological evaluation revealed significant differences in cytotoxic effects between different types of NPS and cell lines. Evaluation of the cytotoxicity of MC and HEC scaffolds modified with carbonate NPS in relation to WI-26 fibroblasts revealed significant differences in the cellular response depending on the type of metal ion and stabilizing agent. Stabilization of MC can lead to more efficient uptake by cells and release of metal ions, which leads to increased cytotoxicity. On the contrary, HEC stabilization probably provides a protective barrier that minimizes the interaction of NPs with cells. However, MC has a number of important advantages that justify its inclusion in the integrated stabilization system. The combination of MC with HEC in a mixed stabilizing system aims to take advantage of these advantages while reducing the cytotoxic effects observed only with MC. Notably, the modified CaCO₃ matrices demonstrated minimal cytotoxicity with up to 112% HeLa and Vero cell viability and promising biocompatibility. On the contrary, ZnCO₃ and CuCO₃ preparations showed a pronounced cytotoxic effect against these cell lines, reducing cell viability to only 2.5–3%.

The developed biopolymer scaffolds represent a significant advancement in bone tissue engineering, offering a novel platform that combines several critical advantages. The unique combination of natural polymers with osteotropic metal carbonates creates a biomimetic material with optimal porosity (93.3–98.0%) and controlled degradation rates, which are essential for successful bone integration and tissue regeneration. The material’s ability to provide controlled release of bioactive ions opens new possibilities for enhancing bone regeneration processes, offering a dual therapeutic effect through mechanical support and biological activity. The favorable mechanical properties and biocompatibility of CaCO₃-modified scaffolds make them particularly promising for clinical applications, with potential use in fracture repair and bone defect filling. This innovative approach addresses several critical challenges in bone tissue engineering, including the need for biocompatible, osteoconductive, and mechanically stable materials with controlled degradation rates and bioactive properties. The successful integration of these components provides a versatile platform for developing next-generation bone graft substitutes with enhanced regenerative potential, capable of meeting the unmet clinical needs in bone tissue engineering.

However, the current study has certain limitations that should be considered. The study was mainly conducted in vitro, which may affect the live broadcast of the results in vivo. In addition, long-term biodegradation profiles and mechanical properties under dynamic conditions have not been fully studied. The potential long-term effects of metal ion leaching also require further assessment. Therefore, future research directions should focus on conducting in vivo studies to assess biocompatibility and biodegradability. Studying long-term metal ion release profiles and optimizing mechanical properties for specific clinical applications are important next steps. Studying the potential of ZnCO₃ and CuCO₃ drugs in antitumor therapy, as well as studying the synergistic effects between different combinations of carbonates, may open up new possibilities for use in biomedicine.

These preliminary results open a promising area of research into biopolymer-based osteogenic materials. Overall, the study provides compelling preliminary evidence for the potential of this approach. However, final confirmation of the bone-regenerative potential of the developed scaffolds requires additional functional studies, including an assessment of osteogenic differentiation in vitro and regeneration of bone defects in in vivo models.