MAPLE Deposition of Resorbable Calcium Phosphates on Electrospun Nylon Nanofibres for Bone Tissue Engineering

1. Introduction

Bone defects resulting from trauma, disease, or surgical resection represent a significant clinical challenge [1], with more than two million bone graft procedures performed annually worldwide [2]. While autologous and allogeneic bone grafts remain the gold standard, they are constrained by donor site morbidity, limited tissue availability, immunogenic responses, and disease transmission risk [3]. Consequently, engineered biomaterials designed to recapitulate not only the hierarchical structure but also the dynamic bioactivity of native bone are urgently needed to address this unmet clinical demand [4]. The extracellular matrix (ECM) of bone is fundamentally a composite structure combining a collagenous organic phase with an inorganic mineral phase composed predominantly of calcium phosphate apatite [5]. This hierarchical organization, spanning scales from nanometers to millimeters, provides mechanical resilience, load transfer, and cell-instructive biochemical cues [6]. Scaffolds designed for bone tissue engineering must therefore reproduce this multiscale architecture, maintain interconnected porosity [7] for nutrient diffusion and vascular ingrowth [8], and present osteoconductive surfaces [9] capable of supporting progenitor cell recruitment and differentiation [10].

Electrospun nanofibres have emerged as a powerful platform for recreating the nano- to micro-structure of the ECM [11]. They offer precise control over fibre diameter [12] in the submicron regime, very high surface area-to-volume ratio for protein adsorption and cell interaction [6], tunable pore size and orientation through adjustment of process parameters and collector design [13], and compatibility with a wide range of natural and synthetic polymers that can be blended, functionalized, or loaded with bioactive agents [14].

Natural polymers such as collagen [15] and gelatine provide inherent cell-recognition motifs [16] but often display limited mechanical strength and rapid, difficult-to-control degradation [17]. In contrast, synthetic polymers afford superior mechanical tunability and degradation control, yet they are typically bioinert and require additional strategies to introduce osteogenic signalling [14]. Nylon (polyamide) represents a compelling compromise [18]. Its repeating amide bonds structurally resemble peptide linkages found in proteins [19], which supports good biocompatibility and relatively low immunogenicity, while its mechanical robustness and thermal stability enable the reproducible fabrication of defect-free nanofibres across broad electrospinning windows [20]. Nylon has already been used in sutures, catheters [21] and preliminary bone tissue engineering constructs [12], but systematic studies addressing how different bioactive mineral coatings modulate its performance as a bone scaffold are still scarce [12].

Calcium phosphates (CaP) are the most physiologically relevant inorganic phases for bone regeneration [22], as their composition closely mirrors the mineral content of native bone [23]. Among them, monetite (CaHPO₄, anhydrous dicalcium phosphate), brushite (CaHPO₄·2H₂O, hydrated dicalcium phosphate) and related orthophosphate (Ca₃(PO₄)₂) and pyrophosphate (Ca₂P₂O₇) phases exhibit distinct solubility, resorption and ion-release profiles, which translate into different osteogenic responses [24]. Monetite possesses higher solubility than hydroxyapatite, enabling sustained calcium and phosphate release and relatively fast but controlled resorption [25]. Brushite resorbs even more rapidly, but it can transform in vivo into less soluble apatite, altering its long-term behaviour [26]. Rare-earth-doped CaP, such as cerium-substituted phosphate [11], retain the osteoconductivity of CaP while introducing additional antioxidant [27] and antimicrobial functionalities arising from the redox activity of cerium ions [28]. These compositional levers make CaP phases attractive candidates for tailoring the biological performance of otherwise bioinert nanofibrous scaffolds [11].

To exploit these phases effectively, deposition techniques must integrate minerals onto delicate polymer fibres without damaging their morphology [29]. Matrix-assisted pulsed laser evaporation (MAPLE) [30] has recently gained attention as a gentle, highly adaptable method for this purpose [31]. Unlike conventional pulsed laser deposition, which directly ablates a solid target and can induce significant thermal and mechanical stress on heat-sensitive substrates, MAPLE employs a frozen target in which the solute of interest is dispersed in a volatile solvent [32]. Upon laser irradiation, the solvent predominantly absorbs the energy [33], undergoes rapid sublimation and entrains the solute species toward the substrate in a plume, where they condense as a thin coating [34]. This indirect transfer mechanism minimizes substrate heating, preserves polymer integrity, and allows deposition on three-dimensional architectures such as electrospun mats, including their internal fibre junctions and shadowed regions [28]. Furthermore, MAPLE readily accommodates multicomponent targets , enabling deposition of doped CaP phases or composite organic–inorganic layers with controlled thickness and composition [35].

In this context, the present study systematically evaluates how the controlled deposition of monetite, brushite and cerium-doped calcium phosphate phases onto electrospun nylon nanofibres via MAPLE affects scaffold architecture, compositional uniformity and early osteogenic performance. By combining structurally robust, ECM-mimetic nylon nanofibres with phase-specific CaP coatings produced by a gentle laser-based method, this work aims to establish a tunable platform for designing next-generation fibrous bone grafts tailored to different clinical requirements.

2. Materials and Methods

2.1. Materials

Nylon 66 (Mw ≈ 15,000–30,000 Da) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Formic acid (99.5 %) and the inorganic precursors calcium nitrate tetrahydrate (Ca(NO₃)₂·4H₂O), diammonium hydrogen phosphate ((NH₄)₂HPO₄), calcium carbonate (CaCO₃), phosphoric acid (H₃PO₄, 85 %) and cerium ammonium nitrate ((NH₄)₂Ce(NO₃)₆) were obtained from Merck or Sigma-Aldrich at analytical grade. Dimethyl sulfoxide (DMSO, (CH₃)₂SO) was used for MAPLE target preparation, and all media components and solvents for cell culture (α-MEM, fetal bovine serum, penicillin–streptomycin, PBS, trypsin–EDTA) were supplied by standard commercial vendors and handled according to the manufacturers’ instructions.

2.2. Synthesis of Calcium Phosphates

Brushite (CaHPO₄·2H₂O), monetite (CaHPO₄) and cerium-doped phosphate powders used in this study were synthesized and fully characterized in our previous works [11,23], where their phase purity, crystallinity and morphology were confirmed by XRD, FTIR and SEM/EDS. Briefly, monetite and brushite were obtained by aqueous coprecipitation routes optimized to yield phase-pure dicalcium phosphates with controlled particle size, while cerium-doped phosphate was prepared by co-precipitation in basic medium followed by calcination at 500 °C or 800 °C to give the CP-Ce-500 and CP-Ce-800 powders [11], respectively. In the present work, these previously validated CaP phases were used as targets for MAPLE deposition onto electrospun nylon scaffolds without further modification.

2.3. Nylon Nanofibre Synthesis via Electrospinning

Nylon solutions (15 wt% and 25 wt%) were prepared by dissolving nylon 66 in formic acid at room temperature under magnetic stirring for 48 h. For the 15 % solution, complete dissolution was achieved; whereas the 25 % solution required extended stirring and mild warming to reach homogeneity.

Electrospinning was performed using a custom-built apparatus with the following standard configuration: a high-voltage power supply (0–30 kV), a syringe pump delivering polymer solution at controlled flow rates, and a grounded aluminium foil collector.

The 15 % nylon solution was electrospun using a 20-gauge needle with applied voltage 20–30 kV, a needle–collector distance of 19–31 cm and a feed rate of 0.1–0.5 mL/h; under these conditions, droplet formation and beading defects were frequently observed on the collector, indicating suboptimal jet stability. In contrast, the 25 % solution electrospun at 30 kV, 0.1 mL/h feed rate and 20 cm working distance produced uniform, bead-free nanofibres, and was therefore selected as the base formulation for subsequent CaP MAPLE coatings.

2.4. MAPLE Deposition of Calcium Phosphate Coatings

MAPLE targets were prepared by freezing the CaP suspensions in dedicated copper holders using liquid-nitrogen (77 K), as shown in Figure 1. For each composition, 0.06 g of ultradispersed CaP powder was suspended in 6 mL DMSO, corresponding to a concentration of 10 g/L, and rapidly frozen to obtain a solid target.

Four distinct targets were prepared in this way: monetite, brushite, and cerium-doped phosphate, calcined at 500 °C, and 800 °C [11]. Deposition was carried out in a stainless steel reaction chamber using a KrF excimer laser (λ = 248 nm, τ_FWHM ≈ 20 ns), operated at a fluence of 300 mJ/cm² and a repetition rate of 20 Hz. The laser beam was focused on the rotating frozen target and 100,000 pulses were applied for each deposition, with all parameters kept constant to allow direct comparison between CaP phases. To ensure a uniform energy distribution across the laser spot, a laser beam homogeniser was used. During the MAPLE experiments, the target (maintained frozen by the cooling system) and the electrospun nylon substrates were placed 5 cm apart, in parallel configuration, inside a high-vacuum chamber evacuated to 5×10⁻⁵ mbar. Under these conditions, the ejected CaP species condensed uniformly onto the fibres, forming thin, conformal coatings. The obtained samples are described in Table 1.

2.5. Characterization Methods

2.5.1. Scanning Electron Microscopy and Energy Dispersive X-Ray Spectroscopy

The morphology of the electrospun and MAPLE-coated scaffolds, as well as of the CaP powders, was examined using a FEI Quanta Inspect F50 scanning electron microscope (Thermo Fisher Scientific, Waltham, MA, USA). Samples were mounted on aluminium stubs with carbon adhesive and sputter-coated with a 5–10 nm gold layer to reduce charging under the electron beam. Images were acquired at accelerating voltages of 10–20 kV over a wide range of magnifications to evaluate fibre continuity, coating morphology and pore architecture.

Elemental composition was assessed by energy-dispersive X-ray spectroscopy (EDS) using the detector integrated in the same equipment. Semi-quantitative analysis of peak intensities was used to compare relative CaP loading between coatings.

2.5.2. Fourier-Transform Infrared Spectroscopy

Chemical structure of the scaffolds was analysed by Fourier-transform infrared (FTIR) spectroscopy using a Thermo Scientific Nicolet iS50 spectrometer equipped with an attenuated total reflectance (ATR) module. Spectra were collected at room temperature in the 4000–400 cm⁻¹ range, averaging 32 scans at 4 cm⁻¹ resolution. The data were used to identify nylon amide bands and phosphate- and hydroxyl-related vibrations specific to monetite, brushite and cerium-doped phosphate.

2.5.3. Fibre Diameter Measurements

Fibre diameters were determined from representative scanning electron microscopy (SEM) images using Fiji (ImageJ) software [36]. For each scaffold composition, at least 50 individual fibres were randomly selected and measured. The resulting values were used to calculate mean diameter, standard deviation and diameter histograms to describe size distribution.

2.5.4. Fibre Orientation Analysis

Fibre orientation was quantified on low-magnification SEM micrographs using the OrientationJ plugin [37] implemented in Fiji. Images were converted to 8-bit grayscale and minimally filtered to enhance contrast without altering fibre geometry. OrientationJ’s structure tensor analysis [38] was applied to compute the local orientation of fibres over the whole image and to generate orientation histograms expressed as frequency versus angle (−90 ° to 90 °). The mean preferred direction angle and standard deviation were extracted to compare anisotropy between uncoated and MAPLE-coated scaffolds. Experimental data was subsequently smoothed using a Savitzky–Golay filter (second-order polynomial) to reduce high-frequency noise while preserving peak shape. Curves were plotted in Origin 8.5.

2.5.5. MC3T3-E1 Cell Culture

MC3T3-E1 preosteoblasts (ATCC, USA) [40,41] were cultured in Alpha Minimum Essential Medium (α-MEM, Gibco, Thermo Fisher, USA) supplemented with 10 % foetal bovine serum, 1% L-glutamine and 1 % penicillin–streptomycin under standard conditions (37 °C, 5 % CO₂, 90 % humidity). The scaffolds were thermally sterilized at 120 °C for 1 h before cell seeding. Cells were seeded at a density of 40,000 cells/100 µL/sample in 12-well plates and incubated for 1 h to allow initial adhesion, after which 1 mL of fresh culture medium was added to each well. Constructs were then incubated for 2 days under standard conditions, after which cell viability assays were performed and selected samples were prepared for morphological investigations using SEM.

2.5.6. MTT Viability Assay

Cell viability was assessed after 2 days of direct contact using the MTT tetrazolium salt assay [42]. Following the incubation period, the culture medium was removed and replaced with an MTT solution prepared by diluting a 5 mg/mL MTT stock in PBS to 10 % (v/v) in complete α-MEM; samples were then incubated for 2 h under standard culture conditions. In this assay, metabolically active cells reduce MTT to insoluble formazan crystals, and the amount of formazan formed is proportional to cell viability [42]. After incubation, the MTT-containing medium was discarded and the formazan crystals were solubilized in DMSO.

The resulting solutions were analysed spectrophotometrically at 570 nm, and cell viability for each scaffold was calculated relative to the negative control, consisting of cells cultured on tissue-culture plastic in complete medium, which was set to 100 %. Data were expressed as mean ± standard error of the mean (SEM), and statistical significance was evaluated using the Student t-test, with *p < 0.05, **p < 0.01 and ***p < 0.001 considered significant.

2.5.7. Cell Fixation for SEM

For SEM, the culture medium was removed, samples were gently rinsed with PBS and fixed in 2.5 % glutaraldehyde for 1 h at room temperature. After fixation, specimens were washed with PBS and dehydrated in graded ethanol solutions (70–100 %, 30 min at each step), followed by incubation in increasing hexamethyldisilazane (HDMS):ethanol mixtures (50:50, 75:25) and finally 100 % HMDS. Dried samples were subsequently mounted and sputter-coated prior to electron microscopy imaging.

3. Results

3.1. Nylon Nanofibre and MAPLE Deposition Characterization

3.1.1. Scanning Electron Microscopy and X-Ray Energy Dispersive Spectroscopy

Electrospinning of 25 % nylon solutions yielded uniform, non-woven fibre mats. SEM analysis of the 15 % formulation revealed fibres with diameters ranging from 39 nm to 98 nm (mean ± SD: 68 ± 18 nm, n = 50). The 25 % solution produced fibres with slightly larger diameters (mean ± SD: 75 ± 22 nm, n = 50) due to higher polymer concentration. Both solutions exhibited smooth fibre surfaces, with random fibre orientation characteristic of conventional electrospinning geometry.

SEM analysis of the uncoated nylon scaffold (Ny_25%) in Figure 2. revealed a highly entangled mat of smooth nanofibres with a mean diameter of $62.1\pm 23.8$ nm (range 31–147 nm), with only occasional thicker strands and no evidence of beading, confirming stable electrospinning conditions and preservation of high porosity. At higher magnification, individual fibres displayed well-defined contours and a consistent circular cross-section within this nanometric range, indicative of a mechanically robust network suitable as a base substrate for subsequent mineral functionalization. The corresponding EDS spectrum showed only the signals associated with the polymer and mounting system (C, N and O from nylon, Al from the sample holder and Au from the sputter-coating layer), with no detectable Ca or P, confirming that Ny_25% is a purely organic reference scaffold without intrinsic mineral content.

In the case of the Ny_monetite sample, the nanofibrous architecture remained clearly visible, but SEM micrographs (Figure 3) revealed that the nylon fibres were covered by a granular coating consisting of sub-micrometric particles tightly attached to the surface. These particles formed a fine, almost conformal layer along the fibre length without generating large bridges between adjacent fibres, so that the open pore structure of the electrospun mat was largely preserved while surface roughness was markedly increased. EDS spectra exhibited pronounced Ca and P peaks superimposed on the characteristic C, N, O, Al and Au signals, demonstrating successful deposition of a CaP phase over the whole analysed area and confirming that MAPLE deposition efficiently mineralized the nylon scaffold with monetite.

SEM micrographs of Ny_brushite (Figure 3) showed a distinct coating morphology compared with monetite, with the nylon fibres partially covered by thicker, plate-like mineral formations that overlapped and occasionally formed small clusters along the filaments. While the overall fibrous structure and inter-fibre spacing were retained, local regions appeared more heavily mineralized, suggesting that brushite tends to form sheet-like deposits that enhance surface roughness but may slightly narrow pore openings compared with the finer monetite particles. The EDS spectrum again revealed clear Ca and P signals in addition to the polymer- and substrate-derived elements, confirming uniform CaP deposition; the similar elemental composition but different microstructure relative to Ny_monetite indicates that the observed morphological differences arise from the intrinsic crystallization behaviour of the brushite phase rather than from differences in overall mineral loading.

For Ny_CP_Ce_500, SEM imaging (Figure 4) indicated that the cerium-doped phosphate deposited from the 500 °C calcined powder formed a finely textured coating, with discrete mineral particles scattered over the nanofibre surfaces and only limited coalescence into larger aggregates. The fibres remained individually discernible and the network kept its open, highly porous architecture, suggesting that moderate calcination temperature favors the retention of nanoscale particles that primarily modify local surface topography and chemistry without masking the underlying nylon scaffold. EDS analysis showed Ca and P peaks together with the characteristic C, O, Al and Au signals, and a low-intensity contribution assigned to the Ce dopant, confirming successful deposition of a cerium-containing CaP layer over the fibres while maintaining a relatively thin and dispersed coating.

In contrast, the Ny_CP_Ce_800 sample (Figure 4) displayed a markedly different morphology, with SEM revealing large, petal-like mineral clusters anchored to the nanofibre network and composed of densely packed particles that coalesced during calcination at 800 °C. Although individual nylon fibres could still be recognized, these compact aggregates locally spanned multiple filaments and partially obstructed the pores, indicating that higher calcination temperature promotes sintering and thickening of the cerium-doped phosphate coating, which may alter cell accessibility. The corresponding EDS spectrum again contained intense Ca and P peaks together with C, O, Al and Au, confirming that the aggregates consist of CaP; the similar elemental composition but significantly coarser microstructure compared with Ny_CP_Ce_500 highlights the strong influence of thermal history on coating morphology and suggests a trade-off between mineral continuity and preservation of fine porosity.

3.1.2. Fourier-Transform Infrared Spectroscopy

FTIR analysis (Figure 5) of the electrospun nylon scaffold (Ny_25%) displayed the characteristic polyamide-6,6 bands: N–H stretching at 3300–3500 cm⁻¹, CH₂ stretching in the range 2930–2850 cm⁻¹, amide I and II at ≈1640 and ≈1540 cm⁻¹, and amide III vibrations between 1260 and 1170 cm⁻¹, confirming preservation of the nylon backbone after processing. Upon MAPLE deposition of monetite (Ny_monetite), additional phosphate bands corresponding to [PO₄]^3‒ groups emerged in the 1200–900 cm⁻¹ region, together with a prominent [PO₄]^3‒band at ~1070 cm⁻¹ and a P–OH vibration at ~880 cm⁻¹, matching the reference monetite spectrum and indicating formation of anhydrous dicalcium phosphate (DCPA, CaHPO₄). Brushite-coated fibres (Ny_brushite) exhibited a broad O–H stretching envelope between 3600 and 3100 cm⁻¹ and a strong H–O–H bending mode near 1640 cm⁻¹, together with characteristic [PO₄]^3‒ vibrations, consistent with hydrated dicalcium phosphate dihydrate (DCPD, CaHPO₄·2H₂O), as characterized in previous work [24]. In contrast, Ny_CP_Ce_500 and Ny_CP_Ce_800 samples showed the typical apatite [PO₄]^3‒ bands at ≈1040–1090, 960 and 600/560 cm⁻¹, confirming successful deposition of cerium-doped phosphate, without detectable degradation of the nylon substrate [11].

3.1.3. Electrospun Fibre Diameter

Uncoated Ny_25% fibres exhibited a relatively narrow diameter distribution mainly in the 40–80 nm range, with a modest tail toward larger values, as shown in Figure 6, confirming that the electrospinning conditions yield a uniform nanofibrous scaffold prior to mineralization.

The fibre diameter histograms of the MAPLE-coated scaffolds (Figure 6) show that mineral functionalization modulates but does not radically alter these nanoscale dimensions. Ny_monetite exhibits the narrowest distribution, centred around 55–60 nm, indicating that the fine granular monetite layer preserves the original nylon diameter and even slightly reduces variability. Ny_brushite and Ny_CP_Ce_500 shift the distribution toward larger values, with most fibres in the 70–100 nm range, consistent with the formation of thicker phosphate coatings on the polymer core. Ny_CP_Ce_800 displays the largest mean diameter, approaching 100 nm and extending beyond 130 nm, which correlates with the presence of coarser aggregates along the fibres.

3.1.4. Electrospun Fibre Orientation

Fibre orientation analysis (Figure 7) showed that all samples exhibited a pronounced preferential alignment rather than a fully random distribution, with major peaks centred close to 90 ° (horizontal) and a secondary component near 0 °, indicative of a cross-aligned nanofibre network. The alignment remained moderate, and MAPLE coating with different CaP phases did not significantly disrupt this anisotropy, as Ny_monetite, Ny_brushite, Ny_CP_Ce_500 and Ny_CP_Ce_800 all followed the same bimodal trend observed for Ny_25%. This preserved directional organization is expected to contribute to anisotropic mechanical behaviour and may provide contact guidance cues for adherent cells along the predominant fibre direction.

3.2. Cell Viabilty Evaluation

3.2.1. MTT Cell Viability Assay

MTT assays after 48 h revealed that all MAPLE-functionalized scaffolds maintained and enhanced MC3T3-E1 metabolic activity compared to the negative control, confirming good cytocompatibility of the coatings. Uncoated nylon (Ny_25%) showed a response similar to the negative control, with a cellular viability of about 108.4±2.4% (not significant compared to negative control). Brushite-coated fibres showed the highest viability, reaching 178.5±1.7 % of the negative control (p<0.001), which indicates a pronounced stimulatory effect on preosteoblast metabolism. Monetite and cerium-doped phosphate coatings also increased cell viability above the negative control (p<0.05), but to a lesser extent, suggesting that CaP chemistry and microstructure modulate the early osteogenic response. The lower, but still statistically significant, cellular metabolic values measured for Ny_CP_Ce_500 (119±0.6%, p=0.04) and Ny_CP_Ce_800 (134.3±1%, p=0.003) in comparison to the negative control, are consistent with their finer or more aggregated morphologies and indicate that the contribution of cerium does not translate into an acute boost of osteoblastic metabolism at 48 h.

Figure 8. Cell viability of MC3T3-E1 pre-osteoblasts after 48 h culture on uncoated nylon nanofibres (Ny_25%) and MAPLE-coated scaffolds (Ny_monetite, Ny_brushite, Ny_CP_Ce_500, Ny_CP_Ce_800), expressed as percentage relative to the tissue-culture plastic negative control (mean ± SD, n = 3; *p < 0.05, **p < 0.01, ***p < 0.001 vs. negative control).

Figure 8. Cell viability of MC3T3-E1 pre-osteoblasts after 48 h culture on uncoated nylon nanofibres (Ny_25%) and MAPLE-coated scaffolds (Ny_monetite, Ny_brushite, Ny_CP_Ce_500, Ny_CP_Ce_800), expressed as percentage relative to the tissue-culture plastic negative control (mean ± SD, n = 3; *p < 0.05, **p < 0.01, ***p < 0.001 vs. negative control).

3.2.2. Cell Distribution via SEM Imaging

For the uncoated nylon scaffold (Ny_25%), SEM imaging of MC3T3-E1 (Figure 9) pre-osteoblasts after 48 h revealed a continuous but relatively sparse cellular layer, with cells displaying mainly flattened morphologies and limited spreading compared to the mineral-coated samples. At low magnification, cells were distributed across the fibrous mat, but numerous bare regions of exposed fibres remained visible, indicating that the pristine nylon surface anchored cells at multiple contact points to the nanofibres, with short filopodia bridging neighbouring strands and following the underlying fibre orientation.

On monetite-functionalized fibres (Ny_monetite), SEM images (Figure 10a) revealed dense cellular coverage after 48 h, with pre-osteoblasts forming an almost continuous layer that closely followed the underlying nanofibre topography. At higher magnification, cells exhibited extensive spreading and numerous filopodial extensions bridging adjacent fibres, suggesting strong focal adhesion and intimate contact with the monetite particles, in line with the elevated MTT metabolic activity measured for this composition. Ny_brushite scaffolds also supported robust MC3T3-E1 attachment, with SEM micrographs (Figure 10b) showing confluent cell monolayer regions interspersed with small pores corresponding to the underlying brushite-coated fibre bundles.

On Ny_CP_Ce_500, SEM micrographs (Figure 11a) revealed a continuous but somewhat more heterogeneous cellular distribution, with regions of well-spread cells interspersed with sparsely populated areas where the cerium-doped phosphate-coated fibres remained visible. At high magnification, adherent cells extended filopodia that anchored to individual fibres and appeared to probe the nanoscale phosphate particles, suggesting that this coating maintains good cytocompatibility, in agreement with the viability values. In contrast, Ny_CP_Ce_800 samples displayed more discontinuous cell coverage, with SEM images (Figure 11b) revealing clusters of pre-osteoblasts separated by regions dominated by large apatitic aggregates and exposed fibres. Higher-magnification images showed cells that were still able to attach and spread but often wrapped around or perched on top of the coarse mineral clusters, indicating that excessive aggregate size and surface roughness may hinder uniform colonization.

4. Discussion

Electrospun nylon nanofibres functionalized with bioactive CaP phases via MAPLE represent a versatile platform for bone tissue engineering. Systematic variation of phosphate chemistry (monetite, brushite, cerium-doped phosphate) and processing parameters revealed brushite-coated fibres to have the most bone cell stimulating properties, enhancing pre-osteoblasts metabolism to about 178 % of control levels. The gentle MAPLE deposition process preserved fibre diameter and three-dimensional architecture while enabling mineral deposition across the nanofibre network.

The distinct morphologies observed for brushite, monetite, and cerium-doped phosphate reflect differences in particle size, crystallinity, and interaction with the polymeric substrate. Monetite fine, uniform granular coating provides maximal surface area for cellular interaction while minimizing aggregate bridging between adjacent fibres, preserving three-dimensional porosity. Brushite sheet-like morphology, while promoting roughness, may create regions of local fibre fusion that reduce pore accessibility. Cerium-doped phosphate phase-dependent morphology [11] illustrates how thermal processing modulates material behaviour: the moderate temperature (500 °C) preserves fine particle size and dispersed distribution, while aggressive calcination (800 °C) promotes sintering and coalescence, potentially limiting cellular accessibility to mineral surfaces.

The reduced measured performances of cerium-doped phases at 48 h suggests that additional functionalities induced by Ce ions do not necessary improve the bone cells response, as compared with monetite. Given the thinness of the deposited layer and the low overall cerium content, these coatings are not expected to induce cytotoxic effects based on concentration alone, but extended culture periods, antimicrobial evaluation and in vivo implantation studies will be necessary to fully evaluate cerium-doped phosphate utility [43].

The preservation of fibre diameter and structural integrity following MAPLE deposition validates this approach as a gentle functionalization method compatible with nanofibrous substrates. Unlike conventional pulsed laser deposition [44] or thermal spraying techniques that can alter or destroy the underlying fibre architecture [29], MAPLE deposition process and room-temperature substrate exposure maintain scaffold porosity and mechanical continuity [35]. Similar behaviour has been reported for MAPLE coatings on flexible polymers and three-dimensional architectures, where mineral, polymeric or hybrid films [28] are deposited without compromising substrate morphology or mechanics [34]. In the presented system, this compatibility enables a modular design in which electrospun nylon mats are first optimized solely for structural performance [18] and subsequently functionalized with diverse bioactive CaP [45] or composite phases [14], thereby reducing process complexity and expanding the accessible material design space.

The developed phosphate-coated nylon fibre scaffolds address several critical design criteria for bone regeneration [46]: micro-architecture mimicking native ECM porosity, bioactivity through CaP deposition, mechanical resilience from nylon inherent strength, and customizability through modular MAPLE deposition. The hierarchical fibre organization combined with nanoscale mineral coatings creates a multi-scale architecture conducive to osteogenic differentiation [3] and mineralization. Importantly, although calcium phosphate ceramics are intrinsically brittle, confining them to thin, conformal layers on a ductile fibrous substrate mitigates this limitation, allowing the scaffold to retain overall flexibility while still presenting a bioactive mineral interface to cells.

Future investigations should include: extended cell culture assays (14, 21 days) to evaluate proliferation and osteogenic marker expression (alkaline phosphatase, osteocalcin) [47]; incorporation of osteoinductive factors (bone morphogenetic proteins, growth factors) via dual MAPLE deposition or subsequent chemical functionalization; optimization of coating thickness and porosity through laser parameter variation; and manufacturing scale-up and regulatory pathway assessment for clinical translation.

These findings establish a rational design framework for customizable, bioactive fibrous scaffolds poised for clinical translation in bone regeneration and other hard tissue engineering applications.

5. Conclusions

This study demonstrates that combining electrospinning with matrix-assisted pulsed laser evaporation enables precise, phase-specific mineral functionalization of nylon nanofibres without compromising their nanoscale architecture. Monetite, brushite and cerium-doped phosphate could all be deposited as conformal coatings, yet their morphology and biological performance diverged markedly, underscoring the importance of calcium phosphate chemistry in scaffold design. Brushite-coated fibres were particularly effective, generating a homogeneous granular layer that preserved high porosity and produced the highest early preosteoblast metabolism, highlighting this phase as a strong candidate for promoting rapid osteogenic activation at the implant interface. Monetite coatings enhanced surface roughness and supported robust cell adhesion but showed comparatively lower metabolic stimulation, suggesting that their lower solubility and tendency to form thinner structures may moderate short-term bone cells signalling. Cerium-doped phosphate provided a route to introduce rare-earth functionality; however, the strong sintering and aggregate growth observed at 800 °C indicate that careful control of calcination is required to balance the well-known antibacterial or antioxidant benefits against potential reductions in accessible surface area.

From a processing standpoint, the results confirm that MAPLE can uniformly deposit inorganic phases into nanofibrous scaffolds while maintaining fibre diameter, orientation and mechanical continuity, which are often compromised by conventional high-energy coating methods. This compatibility opens the possibility of decoupling scaffold fabrication from surface functionalization [48]: electrospinning can be optimized solely for structural and mechanical performance, after which MAPLE can independently tune the mineral phase, loading and distribution. Such modularity is particularly attractive for clinical translation, where different calcium phosphate chemistries, dopants or drug payloads may be required for distinct indications or patient populations [49].

The phosphate-coated nylon scaffolds developed here meet several central criteria for next-generation bone graft substitutes: they mimic the fibrous architecture of the native extracellular matrix, incorporate bioactive mineral phases capable of delivering osteoconductive and potentially osteoinductive cues, and rely on a processable synthetic backbone. Together, these attributes create a multi-scale environment that can support cell attachment, guide tissue organization and accommodate physiological loading. Looking ahead, extending in vitro assays to longer time points and relevant osteogenic markers, followed by in vivo evaluation in critical-size defect models, will be essential to confirm whether the early advantages observed for monetite translate into superior bone formation and integration. Parallel optimization of MAPLE, coating thickness and dopant content, could ultimately position this platform as a flexible manufacturing route for mineralized fibrous scaffolds aimed at repairing challenging defects in craniofacial, and dental applications.