Osteogenesis-synchronized degradation of Mg²⁺-doped calcium sulfate whisker bone grafts for critical-sized bone defect repair

Abstract

Background

Bone defects remain a major clinical challenge in orthopedic and reconstructive surgery. However, conventional inorganic ceramic biomaterials are limited by many problems, such as poor plasticity, limited osteogenic potential, and uncontrolled degradation kinetics, resulting in failing to keep pace with new bones’ healing. Harmonizing biomaterial degradation with osteogenesis thus remains a crucial problem to be addressed in bone tissue engineering.

Methods

In this study, Mg²⁺-doped calcium sulfate whiskers (MCSW) are synthesized by adopting a high-temperature autoclave method and are fabricated into bone grafting via die-casting molding. Their physicochemical properties, including surface morphology, solution diffusion behavior, mechanical performance, elemental distribution, and Mg²⁺ release kinetics, are systematically characterized. Biocompatibility and in vitro osteoinductive potential are evaluated through assaying cell viability and analyzing osteogenic differentiation. Furthermore, the bone grafting is implanted into critical-sized calvarial defect models in Sprague–Dawley rats, and their bones’ regenerative efficacy is assessed by micro-computed tomography (micro-CT) and histological staining.

Results

MCSW exhibits many characteristics, including a rough surface, solution-dependent degradation behavior, appropriate mechanical strength, as well as sustained and controlled Mg²⁺ release, presenting favorable physicochemical properties with tunable degradation, which shows it is suitable for applying to bone tissue engineering. In vitro studies demonstrate excellent biocompatibility, supporting the survival, proliferation, and osteogenic differentiation of BMSCs, as proved by enhanced alkaline phosphatase (ALP) activity and calcium mineral deposition. More importantly, the in vivo degradation rate of MCSW is well synchronized with osteogenesis, resulting in effective bone regeneration and satisfactory repair of critical-sized defects.

Conclusion

This Mg²⁺-doping strategy for calcium sulfate whiskers provides a novel and effective approach to enhance osteogenic activity and achieve tunable degradation in bone tissue engineering biomaterials, thereby showing strong potential for future clinical application.

Background

Bone loss can occur due to various factors, including trauma, infection, tumor, and degenerative diseases [1–3]. Current treatment strategies for managing bone defects vary greatly, with autograft, allograft, and synthetic bone substitute being the most common approaches [2, 3]. However, there are inherent limitations in these methods. Autograft, being considered the gold standard, imposes donor site morbidity and limited availability, whereas allograft carries the risk of immunogenicity and pathogen transmission [2, 4]. Synthetic calcium phosphate ceramics (e.g., hydroxyapatite and β-TCP) address these challenges partially, but they often show the drawbacks of brittle mechanical behaviors, slow degradation rates mismatched with bone regeneration timelines, and inadequate pore interconnectivity for cell colonization [2, 5–7]. Furthermore, most synthetic substitutes lack bioactive components to actively orchestrate osteogenesis and angiogenesis. These shortcomings underscore the urgent needs for advanced biomaterials that emulate both the structural and biological complexity of native bones. Among these alternatives, calcium sulfate has gained attention due to its advantages of biocompatibility, biodegradability, and ability to facilitate bone regeneration [8, 9].

Despite significant advances in synthetic bone graft materials, conventional calcium sulfate (CS)-based ceramics still have limitations, such as uncontrollable degradation rate, insufficient mechanical strength, and inadequate bioactive osteogenic stimuli [9–11]. To address these problems, this study proposes an Mg²⁺-doped calcium sulfate whiskers (MCSW) designed through a facile high-temperature autoclave reaction synthesis route. Mg²⁺, an essential trace element in bone metabolism, is strategically incorporated into the CS crystal lattice to synergistically enhance both physicochemical and biological functionalities [6, 11, 12].

The preparation process of MCSW is straightforward. Although its inherent mechanical strengths are limited, the fabrication of novel osteogenic bio-filling blocks through die-casting molding significantly enhances their mechanical properties, meeting the mechanical requirements for bone tissue engineering (BTE) applications. Owing to their identical composition, their biological activities show no significant alteration compared to conventional calcium sulfate [11]. By precisely tuning Mg²⁺ incorporation and microstructures, these materials can simultaneously achieve adjustable mechanical strengths, controllable degradation kinetics, and inherent bioactivity to stimulate cellular responses [13–16]. MCSW has demonstrated exceptional mechanical stability, while their Mg²⁺-mediated osteogenic effects outperform conventional ceramic materials.

In vitro evaluations have shown that MCSW is conducive for promoting osteoblast proliferation and stimulating alkaline phosphatase (ALP) activity as well as upregulating osteogenic markers ALP in mesenchymal stem cells. In vivo, MCSW presents good biocompatibility and osteoinductive properties, enabling it to effectively facilitate the regeneration and repair of bone defects. These findings collectively show that MCSW represents a multifunctional biomaterial platform combining structural stability, tunable resorption kinetics, and inherent osteoinductivity—key attributes for next-generation BTE biomaterials for orthopedic defect-filling [17, 18].

This study fabricates an Mg²⁺-doped calcium sulfate whisker-based bone grafts via a combined process of high-temperature autoclave reaction and die-casting. The materials’ physicochemical properties and degradation rate can be tuned by modulating the content of Mg²⁺. Its good biocompatibility facilitates cell proliferation and development, and promotes the osteogenic differentiation of BMSCs. Furthermore, in vivo, its degradation rate is well-synchronized with the rate of bone regeneration (Fig. 1). The crucial feature is that its degradation rate aligns with the rate of bone regeneration. It is promising that in the future, the appropriate content of Mg²⁺ can be selected based on the dimensions of the bone defect, enabling a personalized approach with synchronized osteogenesis and degradation.

Fig. 1.

Graphical representation: preparations of Mg²⁺-doped calcium sulfate whisker bone graft blocks and schematic diagrams illustrating its promotion of bone regeneration with controllable degradation rate

Results

Fabrication and characterization of Mg²⁺-doped calcium sulfate whisker bone grafts

Pure calcium sulfate whiskers (PCSW) and Mg²⁺-doped calcium sulfate whiskers (5MCSW, 10MCSW, 15MCSW) are successfully fabricated via high-temperature autoclave reaction and die-casting, as illustrated in Fig. 2a–d. All four formulations exhibit excellent moldability, thus effectively circumventing the inherent moldability constraints of calcium phosphate ceramics in BTE applications. Scanning electron microscopy (SEM) reveals distinct morphological evolution processes: PCSW displays slender and elongated whisker structures, while increasing Mg²⁺ doping (5–15%) progressively transitions the morphology from whisker-like to granular configurations with improved particle size uniformity (Fig. 2a1-d4). Elemental mapping confirms a homogeneous distribution of constituent elements, particularly Mg²⁺, within the MCSW matrix (Fig. 2e-e5).

Fig. 2.

Characterization of osteogenic bio-filling blocks. a–d: Representing the gross appearance of PCSW, 5MCSW, 10MCSW, and 15MCSW, respectively; (a1-a4, b1-b4, c1-c4, d1-d4): representing SEM images of PCSW, 5MCSW, 10MCSW, and 15MCSW at different magnifications, respectively; (e, e1-e5): SEM and elemental mapping images of 10MCSW

Elemental composition, mechanical properties and osteogenic relevance

Further elemental analysis confirms the gradient content of Mg²⁺ across PCSW, 5MCSW, 10MCSW, and 15MCSW, ranging from undetectable Mg²⁺ in PCSW to progressively elevated levels in MCSW variants (Fig. 3a–d). EDS analysis indicates an increasing trend in the Mg/Ca ratio (Fig. 3e). This controlled Mg²⁺ gradient establishes a systematic platform for elucidating dose-dependent effects of Mg²⁺ on bone regeneration. And SEM analysis confirms that all four graft variants have surface roughness that is conducive for BMSCs adhesion (Fig. 2a2-d3). Mechanical testing reveals a progressive decrease in compressive resistance (PCSW: 127.6 ± 7.25 MPa → 15MCSW: 49.03 ± 9.57 MPa), compressive strength (PCSW: 6.41 ± 0.4 MPa → 15MCSW: 3.36 ± 0.36 MPa), yield strength (PCSW: 5.83 ± 1.04 MPa → 15MCSW: 3.1 ± 0.25 MPa), and elasticity modulus (PCSW: 190.34 ± 10.82 MPa → 15MCSW: 112.78 ± 5.29 MPa) with increasing Mg²⁺ doping (Fig. 3f–i).

Fig. 3.

Elemental composition and mechanical analysis. Elemental EDS patterns of a PCSW, b 5MCSW, c 10MCSW, and d 15MCSW; e: Mg/Ca ratio; mechanical properties: f: compressive strength, g: compression strength, h: yield strength, i: elasticity modulus. “*” denotes statistically significant differences (P < 0.05); “#” indicates no statistically significant differences (P > 0.05)

XRD and ion release kinetics

XRD analysis has verified the successful incorporation of Mg²⁺ into the calcium sulfate lattice across all doped whiskers. PCSW presents characteristic peaks of bassanite (CaSO₄·0.5H₂O, PDF #41-0224). Mg²⁺-doped samples maintain the bassanite structure but show a progressive peak shifting toward higher 2θ angles, indicating lattice contraction due to smaller Mg²⁺ (0.72 Å) substituting Ca²⁺ (1.00 Å) (Fig. 4a). No secondary Mg²⁺-containing phases are detected, suggesting Mg²⁺ substitutional solid solution formation. Peak broadening increases with the content of Mg²⁺, implying reduced crystallite size and increased lattice strain from doping.

Fig. 4.

The characterization of XRD and ion release kinetics. a XRD patterns showing peak shifts with Mg²⁺ doping; b, c, and d represent the release kinetics profiles of Ca²⁺, SO₄^2-, and Mg²⁺ ions, respectively

Ion release kinetics demonstrate sustained and steady release profiles for Ca²⁺, SO₄^2-, and Mg²⁺ ions across all tested materials in simulated physiological conditions. However, 15MCSW exhibits markedly accelerated ion release rates compared with other groups. Specifically, the cumulative release of all ions (Ca²⁺, SO₄^2-, and Mg²⁺) from 15MCSW reaches near-equilibrium, which is faster than PCSW, 5MCSW, and 10MCSW over a 14-day period (Fig. 4b–d).

Degradation behavior and cellular compatibility evaluation

The dissolution kinetics of bone grafts in aqueous environments serves as a critical determinant of its degradation profile and clinical applicability. PCSW and MCSW variants are immersed in water, PBS, and α-MEM medium, showing the Mg²⁺ concentration-dependent acceleration of dissolution rates across all media (Fig. 5a). Dissolution spread measurements in cell-compatible media show reduced radial degradation distances, indicating optimized containment for defect filling (Fig. 5b). Hydrophobicity analysis reveals increasing water contact angles (PCSW: 12° ± 1.5° → 15MCSW: 21.33° ± 1.61°) (Fig. 5c).

Fig. 5.

Disintegration behavior, hydrophilicity, and cytocompatibility analysis. a: Dissolution–diffusion mapping; b: disintegration front distance; c: water contact angle; d: live/dead staining; e: CCK-8 assay. “*” denotes statistically significant differences (P < 0.05); “#” indicates no statistically significant differences (P > 0.05)

When cultivated with BMSCs in α-MEM for 24 h, all grafts support initial cell adhesion (Fig. 5d), though progressive surface dissolution’s correlating with reduced cellular retention. CCK-8 assays confirm osteoblast proliferation across all groups, with 10MCSW showing superior biocompatibility, resulting in balanced Mg²⁺ release content and surface nano topography (Fig. 5e).

Osteogenic differentiation and cellular response

Co-cultivation of BMSCs with all four graft’ variants validates the results of CCK-8, confirming that MCSW grafts support BMSCs proliferation without cytotoxicity from dissolution debris (Fig. 6a). Single-cell tracking assays demonstrate Mg²⁺-dependent enhancement of cytoskeletal development, with 10MCSW exhibiting optimal proliferation rates and filopodia-rich morphology (Fig. 6b–d).

Fig. 6.

Analysis of cell proliferation, development, and osteogenic differentiation. a: Co-cultivated system; b: staining of newly proliferated cells; c: cytoskeletal development (F-actin/DAPI); d: single-cell proliferation rate; e: qualitative ALP staining; f: quantitative alkaline phosphatase analysis; g: qualitative mineralization staining (Alizarin Red S). “*” denotes statistically significant differences (P < 0.05); “#” indicates no statistically significant differences (P > 0.05)

ALP activity, a critical early-stage osteogenic marker, is qualitatively elevated in Mg²⁺-doped groups via BCIP/NBT staining, corroborated by quantitative pNPP assays (Fig. 6e–f). Subsequent Alizarin Red S staining revealed Mg²⁺-enhanced calcium nodule formation, with 10MCSW achieving higher mineralization than other groups (Fig. 6g). This synergistic alignment of ALP expression and calcium deposition confirms 10MCSW’s superior capacity to drive osteogenic differentiation by Mg²⁺-mediated.

In vivo bone regeneration assessment

The osteogenic efficacy of bone graft materials is tested through cranial defect repair in rats. PCSW, 5MCSW, 10MCSW, and 15MCSW are implanted into standardized critical defects (Fig. 7a). At the 2-month postoperative mark, only a thin layer of fibrous tissue is observed bridging the defect in the control groups, while all implantation groups show new bone regeneration. The degree of material residue varies from PCSW to 10MCSW, with 15MCSW showing the least residual material (Fig. 7b). Micro-CT analysis reveals progressive bone regeneration in all implantation groups compared with the control groups (Fig. 7c).

Fig. 7.

Analysis of osteogenic efficacy. a: Intraoperative view; b: explanted bone specimen at 2 months postoperatively; c: micro-CT reconstruction at 2 months postoperatively; d: explanted bone specimen at 3 months postoperatively; e: micro-CT reconstruction at 3 months postoperatively; f–l: quantitative micro-CT parameters of newly formed bone. “*” denotes statistically significant differences (P < 0.05); “#” indicates no statistically significant differences (P > 0.05)

By a 3-month post-surgery, the control groups display only minimal new bone formation at the defect margins. In contrast, all implantation groups demonstrate superior repair outcomes, approaching anatomical integrity with no significant material residue or evident inflammatory response (Fig. 7d). Compared with the control groups, micro-CT confirms varying degrees of new bone regeneration into the defect area across the implantation groups (Fig. 7e). Gross observation indicates that the MCSW groups achieve better repair efficacy, which correlated with the content of Mg²⁺.

Quantitative analysis of new bone volume (BV), new bone volume fraction (BV/TV), and surface area (BS) demonstrates significant bone regeneration in all grafted groups, with the 10MCSW group exhibiting the highest BV (Fig. 7f-h). Trabecular thickness (Tb.Th) markedly increases (Fig. 7i), while trabecular separation (Tb.Sp) significantly reduces in the newly formed bone (Fig. 7j). Both bone mineral density (BMD) and bone mineral content (BMC) of the regenerated bone are substantially higher in the grafted groups compared with the control groups, as shown in Fig. 7k-l.

Histopathological evaluation of bone regeneration

While micro-CT provides precise analysis of mature bone, histopathological assessment remains essential for evaluating early-stage regeneration and immature osseous tissues. The post-operation at 2 months, control groups display minimal new bone formation at defect margins with central thin fibrous tissue layers (Fig. 8a, b). In contrast, grafted groups exhibit robust tissue infilling, featuring extensive woven bone formation in defect centers, undegraded calcium sulfate whiskers dispersed within the neotissue, and moderate neovascularization.

Fig. 8.

Histological analysis of bone defects repair in SD rats treated with MCSW. a: H&E staining of the margin (dashed-line box) and center (dotted-line box) regions of the defect area in each group at 2 months’ post-surgery; b: Masson’s trichrome staining of the margin (dashed-line box) and center (dotted-line box) regions of the defect area in each group at 2 months’ post-surgery; c: H&E staining of the margin (dashed-line box) and center (dotted-line box) regions of the defect area in each group post-surgery at 3 months; d: Masson’s trichrome staining of the margin (dashed-line box) and center (dotted-line box) regions of the defect area in each group at 3 months’ post-surgery. HB host bone; NB new bone; O osteoid; F fibrous tissue; M residual material; arrows indicate neovascularization

The control groups exhibit minimal changes post-operation at 3 months compared with the timepoint at 2 months, with only slight thickening of fibrous tissue at the defect margins. In contrast, the grafted groups demonstrate vigorous expansion of new bone from the periphery into the defect zone. Central regions display maturation of osteogenic islands, characterized by the transition of woven bone into organized lamellar bone structures, as shown in Fig. 8c, d. Neovascularization is significantly enhanced, providing essential nutritional support for nascent bone regeneration.

Discussion

This study successfully fabricates an Mg²⁺-doped calcium sulfate whisker bone graft for bone defects via a controlled high-temperature autoclave reaction followed by die-casting. This process satisfies the need for improving the plasticity of ceramic biomaterials [7, 19]. The surface microstructure presents a rough and hierarchical topography, which is conducive to cell attachment [20]. The substitution of Ca²⁺ by Mg²⁺ results in alterations to the whisker length and dimensions. Concurrently, Mg²⁺ is uniformly distributed within the whiskers. This uniform distribution is a key factor governing both the ion release kinetics and the degradation rate, and it also underpins the sustained osteogenic capability.

And the compaction process increases calcium sulfate content per unit volume while reducing porosity, thereby mitigating the mismatch between its accelerated degradation kinetics and the osteogenesis rate [21]. Regulating ceramic grain growth through Mg²⁺ doping content represents a crucial method to modulate the mechanical properties of calcium phosphate (CaP) ceramics, while simultaneously striking a balance between degradation kinetics and osteogenic bioactivity [10, 19]. This structural refinement enhances the controllability and stability of MCSW in biological applications.

The increased Mg²⁺ doping content induces grain refinement while accelerating degradation kinetics and excessive Mg²⁺ release [19, 22], die-compaction molding and the uniform Mg²⁺ dispersion may effectively mitigate these adverse effects of undesirable degradation acceleration and establish a foundation for sustained ion release for predictable osteogenic modulation in vivo. Determining the optimal concentration of osteoactive elements constitutes a critical objective in BTE [22]. The Mg²⁺/Ca²⁺ ratios of the four implant groups are lower than the theoretical values but align with the predetermined gradient. The precise controllability of elemental composition in these grafts, through doping modulation, represents an important technological advancement, ensuring their suitability for tailored bone repair applications.

Mechanical strength constitutes a critical parameter for bone defect grafts, where appropriate rigidity not only supports adjacent soft tissues but also delivers essential mechanical stimulation to the defect microenvironment while providing structural scaffolding for BMSCs migration [17, 23]. The compressive strength of PCSW is doubled compared to conventional calcium phosphates (3 MPa) [10], demonstrating that die-casting molding serves as an effective method to enhance the mechanical robustness of osteogenic bio-filling blocks. However, this inversely proportional relationship indicates that Mg²⁺ doping concentration serves as a primary modulator of the whiskers’ mechanical properties. Strategic balancing of Mg²⁺ content and biomechanical performance is therefore imperative during clinical graft selection.

As proved by prior SEM observations, Mg²⁺ doping content serves as a critical modulator of crystallite dimensions in calcium sulfate whiskers, and by extension, governs their degradation kinetics. Crucially, Mg²⁺ concentration and crystallite size construct a dual regulatory mechanism essential for optimizing osteogenic performance in clinical applications [24, 25]. This accelerated elution profile indicates enhanced degradation kinetics in the 15MCSW. The faster dissolution likely stems from increased lattice strain and reduced crystallite size (as evidenced by XRD peak broadening) induced by high Mg²⁺ substitution, which disrupts the sulfate crystal stability. While all materials meet the baseline requirement for sustained ion delivery in bone regeneration, the rapid degradation of 15MCSW may compromise long-term structural support but can benefit scenarios demanding early-stage bioactive ion mobilization.

The application of magnesium in BTE has been previously constrained by its excessively rapid degradation and hydrogen gas creation [26, 27]. The fabrication into composite materials represents a highly effective strategy for enhancing their functionality [24, 25]. However, incorporating magnesium into osteogenic biomaterials not only enhances osteogenic activity but also effectively modulates its degradation kinetics, thus providing viable solutions for its practical implementation in bone regeneration [26, 28, 29]. Die-casting of MCSW enables controllable release kinetics of osteoinductive factors and maintains localized therapeutic concentrations, serving as the fundamental basis for sustained and effective osteogenic differentiation of BMSCs [19, 30, 31].

In contrast to the disintegration results, the water contact angle gradually increases, but all samples maintain good hydrophilicity. Hydrophilicity is an essential prerequisite for histophilicity [32, 33]. BMSCs viability results show that as the Mg²⁺ content increases, cell detachment increases along with the material’s disintegration. This discrepancy is attributed to the difference between the solution environment and the in vivo environment. The assays of CCK-8 confirm that MCSW supports the proliferative activity of BMSCs. Microscopic images from the co-cultivation and observations of individual cell proliferation provide further evidence of its excellent cytocompatibility.

Encouragingly, from PCSW to 10MCSW, the induction of cytoskeleton development in BMSCs becomes progressively stronger. The cytoskeletal protein F-actin is a key driver of BMSCs migration, which is conducive to the recruitment of BMSCs to the bone defect site [34, 35]. Results from in vitro osteogenic induction demonstrate favorable ALP expression levels, while calcium salt deposition also varies from the Mg²⁺ content. However, possibly due to factors such as excessive disintegration and degradation caused by its high Mg²⁺ content, the osteogenic capacity of 15MCSW at the cellular level does not surpass that of 10MCSW. Overall, 10MCSW proves superior in promoting the proliferation, development, and osteogenic differentiation of BMSCs, suggesting it as a promising candidate for bone defect filler.

Specimens from the defect area reveal that the degradation rate of MCSW matches the new bone regeneration rate, indicating its suitability for critical-sized bone defect repair [36]. Among the groups, 10MCSW presents the optimal osteogenic performance. Mg²⁺ doping synergistically enhances osteogenesis while accelerating calcium sulfate whisker degradation, necessitating further synchronization of degradation–osteogenesis rates [37–39]. Die-casting molding enhances material density and prolongs both the degradation period and osteointegration window, thereby illuminating the application prospects of magnesium phosphate in bone defect repair.

As a dynamically complex hierarchical structure, bone presents challenges where calcium sulfate’s osteogenic activity and biocompatibility are overshadowed by its degradation limitations in biomimetic porous designs [40, 41]. A paradigm shift toward compacted bio-filling graft design warrants exploration. This study demonstrates that compaction simulating cortical bone density (1.8–2.1 g/cm³) and composition achieves remarkable osteogenic outcomes while mitigating historical constraints of calcium sulfate applications. Concurrently, it exhibits potent angiogenic capacity, fulfilling critical requirements for vascularized tissue ingrowth [40, 42]. This synergistic osteogenic outcome stems from the integrated optimization of composition, mechanical properties, and processing refinement in Mg²⁺-doped calcium sulfate osteogenic bio-filling grafts [13, 43].

Bone regeneration is a multifaceted process influenced by numerous biochemical and mechanical factors [23]. Our study shows that MCSW significantly enhances osteogenic activity compared with standard calcium sulfate and control conditions. The gradual improvement in outcomes with Mg²⁺ content increasing suggests a dose-dependent effect on bone healing. The mechanisms underlying these findings may involve Mg²⁺’s role in improving osteoblast proliferation and differentiation, which has been supported by previous studies [30, 44]. Moreover, magnesium has been reported to improve the mechanical properties of bone, further aiding in overall structural integrity during the healing phase [36]. In this study, die-casting molding of MCSW significantly enhances mechanical robustness for BTE applications.

Conclusion

This study provides compelling evidence that Mg²⁺ doping enhances the bioactivity of calcium sulfate whisker in bone regeneration. The results demonstrate that 10MCSW processes via die-compaction molding achieve an optimal equilibrium among mechanical strengths, degradation kinetics, and osteogenesis. This balanced profile endows the materials with superior clinical potential for bone defect repair. However, this study focuses on the repair of non-load-bearing bone defects, defining its scope and application. Further experiments into its long-term performance and underlying mechanisms are essential for translating these findings into effective therapeutic applications.

Materials and methods

Preparation and characterization of Mg²⁺-doped calcium sulfate whiskers bone graft

Preparation of Mg²⁺-doped calcium sulfate whiskers bone graft

Magnesium sulfate and calcium sulfate are uniformly mixed according to Mg/Ca molar ratios of 0%, 5%, 10%, and 15%. Then, aluminum sulfate octadecahydrate is incorporated as a catalyst at a mass fraction of 0.12%, and the blend mixed thoroughly. Subsequently, the mixture is placed into a high-pressure reactor and the reaction conditions are set to 134 °C and 0.14 MPa for 8 h. Finally, they are compacted into sheets and dried in an 80 °C oven for 12 h. Thus, four types of bone grafts, pure calcium sulfate whisker (PCSW), 5% Mg²⁺ doped calcium sulfate whiskers (5MCSW), 10% Mg²⁺ doped calcium sulfate whiskers (10MCSW), and 15% Mg²⁺ doped calcium sulfate whiskers (15MCSW) are successfully prepared.

Scanning electron microscopy analysis

The surface microstructures of PCSW and MCSW scaffolds are characterized using field-emission scanning electron microscopy (FE-SEM). Prior to imaging, the die-cast samples are sputter-coated with a 5-nm gold–palladium layer to enhance conductivity. Micrographs are acquired at accelerating voltages of 5–15 kV with working distances of 8–12 mm, employing both secondary electron (SE) and backscattered electron (BSE) detection modes.

EDS elemental mapping and quantitative analysis

The elemental distribution and composition of both PCSW and MCSW die-cast grafts are systematically analyzed by using energy-dispersive X-ray spectroscopy (EDS) coupled with scanning electron microscopy (SEM).

Mechanical property characterization

Cylindrical grafts (Ø15 × 6 mm, n = 3 per group) are prepared by die-casting. The mechanical properties of both PCSW and MCSW die-cast scaffolds are systematically evaluated under compressive loading through using a universal testing machine equipped with a 5 kN load cell.

X-ray diffraction (XRD) analysis

The phase composition and crystallinity of four material groups—PCSW, 5MCSW, 10MCSW, and 15MCSW—are characterized using an X-ray diffractometer (Rigaku Ultima IV, Japanese). Powders are scanned from 10° to 70° (2θ) with a step size of 0.02° and a dwell time of 0.5 s/step. Cu-Kα radiation (λ = 1.5406 Å) is operated at 40 kV and 40 mA. Phase identification is performed by matching peaks to ICDD PDF standards for calcium sulfate hydrates and Mg²⁺-containing phases. Crystallite size and lattice strain are calculated from peak broadening by utilizing the Scherrer equation and Williamson–Hall analysis.

Ion release kinetics

Ion release profiles (Ca²⁺, SO₄^2-, Mg²⁺) are quantified in simulated physiological conditions. According to ISO 10993-12, specimens (*n* = 3 per group) are immersed in 20 mL phosphate-buffered saline (PBS, pH 7.4) at 37 °C under gentle agitation (60 rpm). At predetermined intervals (12 h, 24 h, 72 h, 168 h, 336 h), 1 mL supernatant is extracted (replaced with fresh PBS) and filtered (0.22 μm). Ca²⁺ and Mg²⁺ concentrations are measured via inductively coupled plasma optical emission spectrometry (ICP-OES). SO₄^2- concentration is determined by ion chromatography (IC).

Dissolution behavior and hydrophilicity analysis

The dissolution–diffusion characteristics of PCSW and MCSW are evaluated by immersing cylindrical samples (Ø15 × 6 mm) in three liquid media: water, phosphate-buffered saline (PBS, pH 7.4), and α-MEM cultivation medium at 37 °C. After 30 min of incubation, the dissolution fronts are visually tracked, with diffusion distances being measured from the graft  periphery using calibrated ImageJ software.

Complementary wettability analysis is performed via static water contact angle measurements (Dataphysics OCA20) at room temperature. Graft’s surfaces are polished to Ra < 0.5 μm prior to testing. A 5 μL ultrapure water droplet is dispensed onto the surface, with contact angles being recorded at 5-s intervals over 60 s using Young–Laplace fitting. Three measurements per sample ensure statistical reliability.

In vitro biological activity evaluation

Cell isolation

Rat bone marrow-derived mesenchymal stem cells (rBMSCs) are extracted from femurs of 6-week-old Sprague–Dawley (SD) rats and expanded to passage 2–5.

Cell viability assay

PCSW, 5MCSW, 10MCSW, and 15MCSW (Ø15 × 3 mm) are sterilized, then pre-wetted in complete cultivation medium (α-MEM + 10% FBS + 1% penicillin/streptomycin). Fillers are placed in 12-well plates, and rBMSCs are seeded at a density of 5 × 10⁴ cells/graft in 50 µL medium for 5 min (to enhance attachment) before adding 1 mL medium per well. Cultivated for 3 days, grafts are gently rinsed with PBS and incubated in 2 µM Calcein-AM (live, green fluorescence) and 4 µM propidium iodide (PI, dead, red fluorescence) for 30 min at 37 °C. Upright fluorescent microscopes are utilized for imaging cellular samples.

BMSCs proliferation assay

Cultivated to 1, 3, and 5 days, CCK-8 reagent (10% v/v in medium, 500 µL/well) is added and incubated for 2 h at 37 °C. Supernatant (100 µL) is transferred to a 96-well plate, and absorbance is measured at 450 nm by using a microplate reader. About BMSCs proliferation rate, BMSCs are cultivated with bone filler substrates for 72 h and pulse-labeled with 10 μM EdU for 2 h. Cells are fixed (4% PFA, 15 min, RT), permeabilized (0.5% Triton X-100, 20 min), and stained with Alexa Fluor 647-azide (Click-iT^™ kit, 30 min, dark) for EdU detection, followed by DAPI (1 μg/mL, 5 min) nuclear counterstaining. Fluorescent images are acquired on an upright fluorescent microscope. In ImageJ, 8-bit thresholded images are analyzed with the “Analyze Particles” tool to quantify EdU + cells. Proliferation rate is calculated as (EdU + cells/DAPI + cells) × 100%.

Cytoskeleton development staining

BMSCs are cultivated on bone filler substrates for 72 h in vitro. After fixation with 4% paraformaldehyde (15 min, RT) and PBS washes, cells are permeabilized with 0.1% Triton X-100 (10 min). F-actin is labeled using Alexa Fluor^™ 488-phalloidin (1:200, 37 °C, 60 min in darkness), followed by nuclear counterstaining with DAPI (1 μg/mL, 5 min, RT). Samples are imaged by using an upright fluorescent microscope objective.

Osteogenic differentiation

BMSCs are co-cultivated with bone filler materials in 12-well plates using osteogenic induction medium. At days 7 and 14, ALP activity is assessed qualitatively via BCIP/NBT staining and semi-quantitatively using a p-nitrophenyl phosphate (pNPP) assay kit. For calcium deposition analysis, a parallel set of samples cultivated for 14 days undergoes Alizarin Red S staining: cells are fixed with 70% ethanol (10 min), incubated with 2% Alizarin Red S (20 min), and thoroughly washed to remove nonspecific dye. All stained samples are imaged under bright-field microscopy. Semi-quantitative ALP data are normalized to total cellular protein content measured by BCA assay.

In vivo osteogenic efficacy evaluation

Animal model and implantation

Thirty male SD rats are randomly assigned to 5 material-based groups. After 6 h of fasting with water deprivation, anesthesia is induced via intraperitoneal injection of 3% pentobarbital sodium (1 mL/kg body weight). Following anesthesia, the cranial hair is removed, and the surgical site is disinfected. A midline scalp incision exposes the skull surface, and bilateral 5 mm diameter bone defects are created using a cranial drill at the mid-region adjacent to the sagittal suture. In control groups, defects are closed via layer-by-layer suture post-modeling; other groups receive corresponding bone graft implants prior to closure. Postoperative analgesia (e.g., meloxicam) and antibiotics (e.g., enrofloxacin) are administered for 3 days. Animals are housed with ad libitum access to food/water until euthanasia via anesthetic overdose at 2 and 3 months post-surgery.

Micro-CT analysis

The bone defect regions are subjected to micro-CT scanning and reconstruction. Quantitative analyses of the regenerated bone include new bone volume (BV), bone surface area (BS), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), bone mineral content (BMC), and bone mineral density (BMD). These parameters are systematically evaluated to assess the osteogenic efficacy of MCSW.

Histological assessment

Following micro-CT scanning, cranial specimens from all groups are immersion-fixed in neutral buffered formalin and subsequently underwent EDTA decalcification. Decalcified samples are paraffin-embedded, sectioned into 5-μm-thick slices, and subjected to adjacent-section staining: hematoxylin–eosin (HE) for cellular morphology and Masson’s trichrome for collagen distribution. Stained slides are digitized using a whole-slide scanner at 20× magnification.

Statistical analysis

Data were analyzed using ANOVA followed by post hoc tests. The results are shown as means ± standard deviation (SD). A p-value of < 0.05 was considered statistically significant.

Abbreviations

BTE

Bone tissue engineering

MCSW

Mg²⁺-doped calcium sulfate whiskers

ALP

Alkaline phosphatase

PCSW

Pure calcium sulfate whiskers

5MCSW

5% Mg²⁺-doped calcium sulfate whiskers

10MCSW

10% Mg²⁺-doped calcium sulfate whiskers

15MCSW

15% Mg²⁺-doped calcium sulfate whiskers

SEM

Scanning electron microscopy

XRD

X-ray diffraction

WCA

Water contact angle

SEM

Scanning electron microscopy

EDS

Energy dispersive spectroscopy

PBS

Phosphate-buffered saline

ICP-OES

Inductively coupled plasma optical emission spectrometry

FBS

Fetal bovine serum

rBMSCs

Rat bone marrow-derived mesenchymal stem cells

BMSCs

Bone marrow mesenchymal stem cells

CCK-8

Cell counting Kit-8

Author contributions

CL, ZS, and HY conceived the idea and designed the experiment; CL, XL, and ZS carried out the experiments; CL and ZS analyzed the data and wrote the manuscript; YY and LZ assisted with the experiments; TY and HY contributed to scientific discussion of the paper. All authors read and approved the final manuscript.

Funding

This work was financially supported by the Scientific Research Fund Project of Yunnan Provincial Department of Education (Grant No. 2024J0021; grant recipient: Chengyong Li).

Data availability

All data generated or analyzed during this study are included in the published article. Additional research-related data are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

All animal experiments were conducted in strict accordance with protocols approved by the Animal Welfare and Ethics Committee of Yunnan University (Approval ID: YNU20230698), with the entire project duration under continuous oversight by the Committee.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.