The study of self-regulating α-TCP based composite by micro/nano scaled silk fibroin and α-CSH on physicochemical and biological properties of bone cement

Abstract

A novel self-hardening α-tricalcium phosphate (α-TCP) bone cement complexed with different content of α-calcium sulfate hemihydrate (α-CSH) and micrometer hydroxyapatite mineralized silk fibroin (HA-SF) using micro/SF as curing liquid has been investigated in this work, which was capable of tunable setting time, degradation, mechanical property and ability to anti-washout. After addition 0 ∼ 25% α-CSH to the α-TCP cement with SFFs as curing liquid, it shortened the setting time of the modified composite to 10 ∼ 30 min. Furthermore, the addition of SFFs improved the compressive strength of the composite from 5.41 MPa to 9.44 MPa. The composites with both Na₂HPO₄ and SFFs as curing liquid showed good anti-collapse performance. The weight loss ratio of bone cement was −0.18 ∼ 12.08% in 4 weeks when the content of α-CSH in α-TCP/α-CSH was between 0 ∼ 25 wt%. During the degradation of α-CSH, the amorphous α-TCP were deposited as hydroxyapatite to formed a plate-like products on the surface of composite. Compared to the composite with Na₂HPO₄ solution as the curing liquid, alkaline phosphatase (ALP) activity of the composites using SFFs as curing liquid were maintained at high levels on the 14th day especially when the Ca/P ratio was 1.7. This study provides a theoretical basis for the regeneration of bone defects guided by bone cement materials.

Keywords

Bone cement α-tricalcium phosphate α-calcium sulphate hemihydrate micro/nano scaled silk fibroin apatite deposition

Introduction

Traffic or production accidents, trauma, osteomyelitis, rheumatism, etc. usually result in bone defects. The critical size of bone defect without self-repairing capability needs to be clinically treated.¹ Calcium phosphate cement (CPC) with the injectability and in situ setting properties invented by Brown and Chow in 1978, which applied to bio-active materials for bone repair, has attracted extensive attention.² As the similarity in inorganic components of bone tissue, CPC-based materials have been widely applied to bone tissue regeneration.¹

Compared to autogenous bone, CPC has a wider range of sources in biomedical engineering and clinical application. It possesses better biocompatibility and hydration products similar to inorganic mineralized components of bone tissue.^3–6 The injectable and operable CPC could be used for minimally invasive surgery, relieving the pain of patients, and reducing medical costs.^7,8 CPC based on α-tricalcium phosphate (α-TCP) could be formed separately or combined with other components, and it also could be used between the biological scaffold and the damaged bone with the irregular shape. CPC could self-harden with water to form calcium-deficient hydroxyapatite (CDHA), thus had attracted extensive attention.⁵ Nevertheless, long setting time, low compressive strength, and especially slow degradation rate limit its application.⁹ Wiltfang et al. showed that 40% of degradation of α-TCP in vivo was achieved in 20 weeks, and a certain amount of the residue remained at the bone defect site in 86 weeks.¹⁰ Therefore, the rapidly increasing demand for artificial bone substitutes has spurred research to improve the properties of α-TCP cement bone repair materials.

It reported that α-calcium sulfate hemihydrate (α-CSH), as an accelerator, could shorten the setting time of α-TCP cement. The pores produced by the degradation of calcium sulfate dihydrate (CSD) after α-CSH reacted with water can improve the degradation rate and bone binding ability of the material.^11,12 The principle is that the crystal structure of α-CSH is a hexagonal prism. From the c-axis, Ca²⁺ and SO₄²⁻ tetrahedron are connected to form the “−Ca−SO₄−Ca−SO₄−” chain. These chains form a hexagon channel where water molecules could distribute among the crystals.¹³ The α-CSH dissolves in water, forming small amounts of Ca²⁺ and SO₄²⁻, and the oxygen atom in SO₄²⁻ forms a hydrogen bond with original -OH and water molecules. Then α-CSH forms CSD with crystal defects at the crystal nucleation site.^14,15 CSH reacts with water to form CSD, and rapidly reduces the plasticity of the materials, which lead to the setting time of the materials is shortened. Then CSD will be collapsed and peeled off when it vibrates in the PBS buffer, and the newly formed pores will also increase the contact area between the composite and PBS, and speed up the degradation rate of α-TCP.¹⁵

Additionally, it is difficult to employ α-TCP-based bone cement in a load-bearing bone repair field because of its low compressive strength. Therefore, improving its compressive strength has always been the crux part. In the meantime, to make the material simulate human bone tissue, the researchers have added some biodegradable polymers with high mechanical strength. Silk fibroin (SF) can be a biodegradable polymer because of its good biocompatibility, toughness, bactericidal properties, and similarity to the organic components of human bone. The results showed that the mechanical strength of SF was higher than collagen, chitosan, and other biodegradable materials, which could be degraded into harmless by-products of protein hydrolysis.^16,17 Nano silk fibroin fiber solution (SFFs) has been also used as a curing liquid for bone cement materials¹⁸ to induce apatite deposition and regulate the mineralization process.¹⁹ The interface adhesion between incompatible phases is the key factor in determining the mechanical properties of inorganic-polymer hybrid composites. Cao proposed that SF when combined with hydroxyapatite (HA), could act as an interface coupling agent and provide nucleation sites for new apatite deposition to promote bone regeneration.¹⁷ This composite was called the hydroxyapatite mineralized silk fibroin protein (HA-SF). It was formed by the interaction of the hydroxyl, carboxyl, carbonyl, and amidogen groups from SF with free Ca²⁺,^17,20 which made HA self-assemble along the c-axis in the SF matrix to form a three-dimensional skeleton (Figure 1). Shao et al. have suggested that HA/SF composite scaffold has good mechanical and biological properties, and can provide a good cell survival microenvironment for bone repair.²¹ Wang also proposed that HA-SF could be combined with bone marrow mesenchymal stem cells to support bone healing in a rabbit model of a segmental bone defect.²⁰ The study conjecture HA-SF would strengthen the adhesion between α-TCP and α-CSH to improve the compaction of the material and enhance the compressive performance of the materials.

Therefore, based on the shortcomings of calcium phosphate bone cement, such as long setting time, low compressive strength, and slow degradation rate, this work mainly introduces an bone cement material formed by α-TCP combined with HA-SF and different content of α-CSH, curing by nano SF solution compared to Na₂HPO₄ solution. It aims at develop a novel bone cement composite with shorten the setting time, improved mechanical, degradation and anti-collapse properties. The effect of ingredients on the physicochemical and in vitro biological properties of composites were also studied.

Materials and methods

Preparation of materials

α-TCP was prepared by solid-state reaction method as follows. First, Ca(H₂PO₄)₂ solution was prepared by reacting 1000 ml 0.26 mol/L H₃PO₄ solution with 0.13 mol CaCO₃ for 1.5 h. Second, Ca(H₂PO₄)₂ solution was slowly added to 1000 ml 0.13 mol/L Ca(OH)₂ suspension for 4 h to get CaHPO₄ suspension. Third, 0.13 mol calcium carbonate was added and reacted with Ca(H₂PO₄)₂ suspension overnight to get Ca₃(PO₄)₂ precursor through aging, centrifugation, and ball milling. Finally, the α-TCP powder was prepared by vacuum drying, sintering, grinding, and sieving.

α-CSH was prepared through the hydrothermal method.^22,23 The CSD was placed in a vertical pressure steam sterilizer (LDZF-30KB-II, Shanghai Shen’an Medical Device Factory) at 127°C for 9 hours, and then placed in an oven for 2 hours to obtain α-CSH.

The preparation method of silk fibroin solution (SF) is: natural silks of 2 ∼ 3 cm in length were boiled in 0.02 mol/L Na₂CO₃ solution for 30 min. Then the remaining silk glue was removed with deionization water three times until completely degummed. After the degummed silk was dissolved in ternary solution at 80°C for 30 min, the solution got was dialysis and centrifuged to remove impurities. (The ternary solution consists of CaCl₂, C₂H₅OH and H₂O, and the mass ratio is 1:2:8.) Finally, the concentration of 4 wt% SF was obtained.

The preparation method of silk fibroin solution (SF) is: the SF above was concentrated to 12 wt% in an oven (60°C) and then to about 20 wt% in a fume hood. After diluting it with deionized water to 2 wt%, nano silk fibroin solution fiber solution (SFFs) was prepared by incubating 2 wt% in an oven at 60°C.

The preparation of HA-SF refers to Wang Li et al.²⁰ In short, 85 wt% of H₃PO₄ solution was dropped into a mixed solution of 18 wt% SFs and 1.1 mol/L Ca(OH)₂ solution, and then maintain the pH of solution at 9.0. HA-SF complex was obtained through mixing at 800 r/min for 3 h and then drying and grounding.

The composite bone cement with a liquid-solid ratio of 0.4 ml/g was prepared by mixing solid powders of α-TCP, α-CSH, and HA-SF with Na₂HPO₄ solution or SFFs as curing liquid, respectively. The proportion of powders (HA-SF and α-CSH) is shown in Table 1. The concentration of Na₂HPO₄ solution and SFFs was 2.5 wt% and 0.4 wt% separately.

Table 1.

The proportion of powders.

Total weight of powders (g) = 3 g
Weight percentage of HA-SF in powders (wt%)	The weight ratio of α-CSH in the remaining powders (wt%)
3	0
	5
	10
	15
	20
	25

Characterization of the composite

Setting time determination

The preparation of cylindrical mold (Փ 10 mm × 15 mm) (ISO 13779-1): the total mixed materials (3 g) were put into the mold with curing liquid (the liquid to solid ratio is 0.4 ml/g), then made both ends of the materials level with the mold. Then the setting time was measured by Vicat apparatus, with five repeated samples in every group of different α-TCP materials, and was recorded as means ± SD

Injectability test and the anti-washout test

Injectability of the bone cement was measured using a syringe (Փ = 12.5 mm) which was fitted with a round needle (Փ = 2 mm). 1 g bone cement powder was mixed with curing liquid, and the liquid to solid ratio is 0.4 ml/g. Then it was transferred into the syringe and pushed by a propulsion pump at a speed of 1 mm/min for 2 min. The injectability of the cement was calculated as the mass percentage of the paste extruded from the syringe by equation (1).⁴

I n j (%) = (W_{2} - W_{1}) / (W_{2} - W_{0}) \times 100 %

(1)

Where Inj is the percentage of injectability, W₀ is the weight of the empty syringe, W₂ is the weight of the syringe and the composites, and W₁ is the weight of the syringe and the composites after extrusion. Each test was repeated three times.

The anti-washout property of the bone cement was measured by a thermostatic oscillator (DF-101SZ, gongyi kori instrument Co., LTD). The bone cement powder was mixed with a curing liquid at 0.4 ml/g. After being placed in the thermostatic oscillator, it was incubated at 37°C for 24 h, and at the end of the incubation, the bone cement was immersed in a Petri dish with PBS solution. The anti-washout property of the bone cement was observed after shaking for 30 min at 80 rpm. The appearance for different groups of the cement was photographed along with time change.

Mechanical test

The bone cement column (Փ 10 mm × 15 mm) was prepared in the air (The preparation method was consistent with the Setting Time Determination). The mechanical test was carried out on samples after drying for about 1 week using the universal material testing machine (INSTRON5544, British strang company). Different materials of α-TCP in each group with four parallel samples were compressed at the loading rate of 1 mm/min.

Scanning electron microscopy

The microstructure and micromorphology of bone cement particles (Փ 5 mm × 3 mm), which were dried for about 1 week, were analyzed by a scanning electron microscope (SEM, JSM-7100F, Japan Electronics Co., Ltd).

X-ray diffraction and FT-IR

The phase components of composite materials, which were dried after degradation for about 2 weeks, were identified by X-ray diffraction (XRD, DX-2700BH, DANDONG HAOYUAN Instrument Co., Ltd The condition is: 2θ_min = 5°, 2θ_max = 60°, step size = 0.05).

The structure of the composite materials were analyzed with Fourier transform infrared spectroscopy (FT-IR, ALPHA-Ⅱ, Bruker Optics), performed over a range of 400–4000 cm⁻¹. The method was used as FTIR measurements is KBr pellet.

Degradation performance test

The vitro degradation of different α-TCP parallel samples (Փ 5 mm × 3 mm) was evaluated via soaking in 5 ml of phosphate buffer solution (PBS) for 4 weeks by using a thermostatic oscillator (DF-101SZ, gongyi kori instrument Co., LTD) on the constant temperature oscillation (90 r/min). PBS was used as a body fluid simulator and was refreshed every 3 days. After drying the samples soaked in PBS for 2 and 4 weeks, the weight loss ratio of the material was calculated by weight loss as a percentage of original weight by equation (2).

T h e w e i g h t l o s s r a t i o = (W_{3} - W_{4}) / W_{3} \times 100 %

(2)

Where W₃ is the original weight of the composites, W₄ is the weight after degradation of the composites. Each test has four parallel samples.

Cell experiment

MC3T3 culture

Mouse osteoblasts (MC3T3) were cultured in Durbeco’s modified Eagle medium at 37°C and 5% CO₂ humidity, which was consisted of high glucose original medium (DMEM), 15% fetal bovine serum, 2% 100 μg/ml penicillin and 100 μg/ml streptomycin mixed solution.²²

Cell counting kit-8 assay

To evaluate cell proliferation, CCK-8 assay was performed according to the manufacturer’s instructions (Japan Dongren). Four parallel samples (Փ 5 mm × 3 mm) were set for each group of materials with different α-TCP ratios (the material was pre-sterilized by Co-γ), and a total of three groups were placed in three 96-well plates. The 200 μl cell suspension was seeded onto the sterilized composite bone cement materials at a density of 2.5 × 10⁵ cells/ml. After 2, 4, and 6 days of culture, CCK-8 10 μl was added to each sample and kept for 4 h. Finally, the absorbance of supernatant fluid, which was transferred into 96-well plates and oscillated under an enzyme standard instrument, was measured at 450 nm using a Micro-plate Reader (Bio-Rad, iMark).

ALP assay

To evaluate the effect of the addition of SFFs on the apatite deposition of composite bone cement, alkaline phosphatase (ALP) was performed according to the manufacturer’s instructions (Biyuntian). The principle is that ALP can promote the formation of mineralized matrix proteins by hydrolyzing phosphate, increasing the concentration of calcium and phosphorus, thus affecting the mineralization of the extracellular matrix of osteoblasts.²⁴ Three parallel samples (Փ 5 mm × 3 mm) were set for each group of materials with different α-TCP ratios (pre-sterilized by Co-γ), and a total of three groups were placed in three 96-well plates. The 200 μl cell suspension was seeded onto the sterilized composite bone cement at the density of 2.5 × 10⁴ cells/ml. After 7, 14 days of culture, the materials were washed twice with PBS buffer, added 200 μl lysate to each well, and kept on ice for 3 ∼ 4 min. Then the supernatant was transferred into a 2 ml centrifuge tube and stored at a low temperature. In the final detection, 50 μl of each sample was taken and 50 μl chromogenic substrate was added. After shaking 2 min (400 r/min) on the shaker, and incubating in the incubator for 30 min, 100 μl reaction termination solution was vibrated for 2 min (400 r/min) again. The absorbance was measured at 415 nm to evaluate the activity of ALP.

Results

Characterization of materials

The morphology of α-TCP powder observed by SEM is shown in Figure 2(a), it has an irregular shape from ball milling.⁹ α-CSH is a rod-shaped crystal, and a channel can be observed in the cross-section of the crystal in Figure 2(b), the channel allows α-CSH to react rapidly with water. After being treated, the SFFs shows fibrous distribution under SEM in Figure 2(c). The particle size of HA-SF powder observed by SEM is larger than α-TCP and the surface is covered with fibrous SF materials in Figure 2(d).

The XRD patterns of α-TCP and α-CSH in Figure 2(e) show the diffraction peak of α-TCP is mainly at 2θ = 27.76°, 31.02° and 34.37°, the diffraction peak of α-CSH is mainly at 2θ = 14.85°, 25.50°, 29.80°, and 49.45°. Figure 2(g) shows the XRD patterns of the HA and HA–SF complex. The HA is verified by PDF#09-0432. The diffraction peak of HA is mainly at 2θ = 25.87°, 31.73°, 39.82°, and 49.46°. And the diffraction peaks of HA-SF are widened and partially overlapped compared with HA, such as at 2θ = 26.10°, 32.15°, and 39.96°. It indicates that the crystallinity of HA in HA-SF is not high.¹⁷

As can be seen from the FTIR absorption spectrum in Figure 2(f). The absorption bands at 1639, 1540, and 1246 cm⁻¹ of SF are the amides I, II, and III, respectively, and at 3275 cm⁻¹ is the –OH. The bands at 1011 and 545 cm⁻¹ of α-TCP are derived from phosphate ions, showing that PO₄³⁻ sites. However, shifting of peaks occurs because of the molecular interactions between the Ca²⁺ in the HA and the negatively charged functional groups such as carboxyl and amine groups of the SF.²⁰ For example, the band of amide I shifts from 1639 cm⁻¹ to 1633 cm⁻¹, the band of amide II shifts from 1540 cm⁻¹ to 1514 cm⁻¹, while the amide III band shifts from 1246 cm⁻¹ to 1225 cm⁻¹. At the same time, a new band at 1417 cm⁻¹ appears because of the absorption of amide I in the β-sheet conformation of the SF. It showed that there was a chemical interaction between HA and SF, which may be the interaction between Ca²⁺ and SF in HA.¹⁷

The setting time and compressive strength

As shown in Figure 3(a), when Na₂HPO₄ solution was the curing liquid, the setting time of α-TCP/α-CSH/HA-SF cements was shortened obviously to 7 ∼ 12 min with the addition of α-CSH. This indicates that the addition of α-CSH can effectively shorten the setting time of the composite. When SFFs was curing liquid, the setting time of the materials was 10 ∼ 30 min. As the material is single α-TCP bone cement, SFFs shorten the setting time. But when the materials are composite with α-CSH, the setting time became longer in different degree comparing to the materials with Na₂HPO₄ solution as the curing liquid. In this paper, it is suggested that there exists a kind of interaction between α-CSH and the SFFs when the SFFs was in a hydrogel state. SFFs is hydrophilic, and there is a reversible conversion of SFF from solution to hydrogel in other study reports because SF remains a changeable aggregation state with various sizes in aqueous solutions, peptides in hydrogel state could assemble into ECM-like nanofibers.^25,26 So it is more difficult for α-CSH to capture water from SFFs than directly from Na₂HPO₄ solutions, which results in the slower reduction of moisture in the materials.

As shown in Figure 3(b), when Na₂HPO₄ solution was curing liquid, the maximum compressive strength of the materials reached to 5.41 ± 0.58 MPa from 0.2 ± 0.11 MPa with the content of α-CSH in α-CSH/α-TCP powders increasing from 0 to 20%. When SFFs was curing liquid, the maximum compressive strength of the materials cement reached 9.44 ± 1.86 MPa with 15 wt% of α-CSH in α-CSH/α-TCP powders. This indicates that compared with Na₂HPO₄ solution as curing liquid, SFFs can significantly improve the compressive strength of the bone cement.

Injectability and the anti-washout test

As can be seen in Figure 3(c), the injectability of the materials decreases from 20.99% to 9.29% with the increase of α-CSH content as Na₂HPO₄ solution is the curing liquid, and increases from 6.50% to 18.20% as SFFs is the curing liquid.

Because Na₂HPO₄ solution is an inorganic aqueous solution, in which the H₂O molecules can react with the solid phase of bone cement directly when it is the curing liquid. With the addition of α-CSH, the water molecules are absorbed and generated CSD, which increases the viscosity of the material and gradually reduces the injectability. However, the SFFs is in the gel state before solidification, with the addition of α-CSH, the water around SFFs may be captured by α-CSH for transition to CSD gradually, the hydrogel state may be changed to sol state and the materials became progressively injectable. The result is consistent that SFFs can prolong the setting time because of the high water content as a hydrogel.²⁵

Figure 3(d) is the result of the anti-washout test. After demoulding and curing, all composite materials with Na₂HPO₄ or SFF as curing liquid did not collapse obviously at 37°C under oscillation of 80 rpm within 30 min.

The degradation rate and surface morphology

Figure 4 shows the different degradation behavior of bone cement solidified by different curing liquids. When Na₂HPO₄ was curing liquid (Figure 4(a)) and as the degradation time increased, the mass of composite cement increased with the content of α-CSH from 0 to 15 wt% in α-CSH/α-TCP powders, but decreased with the content of α-CSH in α-CSH/α-TCP powders at 20 wt% and 25 wt%. Compared with single-phase α-TCP bone cement, the α-CSH additive improved the degradation rate of the materials. The composites mediated by α-CSH displayed a plate-like structure with the prolongation of degradation time (Figure 4(c)).¹⁵ In contrast, rod-like materials appeared in the undegraded materials with the content of α-CSH at 20 wt%, larger flake-like crystals were distributed in a higher ratio of α-CSH composites. When SFFs was curing liquid (Figure 4(b)), the weight loss ratio of composite bone cement decreased with α-CSH at 0 ∼ 15 wt% and increased at 20 wt% and 25 wt%. In particular, the degradation rate of composite bone cement was higher than that with Na₂HPO₄ as a curing liquid. When the content of α-CSH was less than 15 wt%, SFFs also affected the degradation of composite bone cement. It can also be observed that flake-like crystals in the SEM diagram (Figure 4(d)) deposited on the surface of the composites with the increase of α-CSH content and the prolongation of degradation time. And after 2 weeks of degradation, the surface of the materials were no longer dense but had pores, which is because the connected channel-like pores can be formed after the degradation of SFFs in the bone cement.²⁷ Figure 4(c) and (d) also show the EDS diagram of the composite materials degraded 4 w. It proved that the ratio of Ca/P of the composite materials with Na₂HPO₄ as curing liquid gradually increased from 1.52. The ratio of Ca/P of the composite materials with SFFs as curing liquid is around 1.67, which is very close to that of hydroxyapatite.

The XRD diffraction patterns

The XRD diffraction patterns of the composite bone cement with Na₂HPO₄ and SFFs as curing liquid respectively degraded for 2 weeks were revealed in Figure 5(a) and (b) and the HA absorption peak at 2θ = 31.80° appeared in all the degraded samples. α-TCP can be seen in the diffraction peaks at 2θ = 27.90°, 31.10°, and 34.50°, and the other peaks disappeared. And only two diffraction peaks of α-CSH moving slightly to the right at 2θ = 25.80°, 53.15° can be observed. The α-CSH can be transformed into CSD during the reaction process, whereas it also can be dissolved and collapsed because of degradation after 14 days, so its peak was rarely observed in the XRD diffraction pattern. At the same time, apatite deposition may occurred on the surface of the materials during the slow degradation of α-TCP and the hydration reaction of α-CSH. The content of α-CSH decreased gradually during the degradation process, and the Ca²⁺ dissolved from α-CSH leading to the increase of concentration for Ca²⁺, which induced the transition of α-TCP to HA. The diffraction peak intensity of HA in bone cement became stronger in the XRD diffraction pattern and the crystal size increased in the SEM image (Figure 4(c) and (d)) with the increase of α-CSH content when Na₂HPO₄ was curing liquid. The diffraction peak intensity of HA became obvious when the initial Ca/P ratio of the materials was close to 1.67, using SFFs as curing liquid. Theoretically, as the content of α-CSH in α-CSH/α-TCP powders is 15 wt%, the initial Ca/P of the composite is closest to HA at 1.70, and the diffraction peak of HA should be the strongest and the crystal size should be the largest. But the Ca/P of the composite would be less than 1.7 because of the presence of PO₄³⁻ in the buffer. So the experimental results showed that when the content of α-CSH was 20 wt%, the diffraction peak of HA was the strongest and the crystal size was the largest. Therefore, it was speculated that when α-CSH provides Ca²⁺ and buffer provides PO₄³⁻, SFFs can induce apatite deposition as Ca/P is close to 1.67. When α-CSH was 25 wt%, the high Ca/P resulted in calcium loss during PBS buffer replacement before binding with SFFs during the degradation experiment. It is further suggested that SFFs can better induce apatite deposition only when the Ca/P is close to 1.67.

The FTIR spectrum

Figure 5(c) and (d) shows the FTIR spectrum of composite bone cement using Na₂HPO₄ solution and SFFs as curing liquid. Compared with the composite bone cement with Na₂HPO₄ solution as curing liquid, the N–H deformation of amide Ⅱ can be identified in the composite bone cement with SFF as curing liquid in the range of 1580 ∼ 1500 cm⁻¹. In addition, the tensile vibration peak of C = O along the fiber coincides with the bending vibration peak of –OH at 1621 cm⁻¹, leading to the difference of absorption peak. And in this spectrum, the strongest bonds are found at the regions 1019, 608 and 554 cm⁻¹ attributed to the vibrations of phosphate groups, which are provided by α-TCP. At 1019 cm⁻¹, ν₁ bands assigned to P–O symmetric stretching vibrations and ν₃ bands assigned to triply degenerate P–O asymmetric stretching could be detected. At 608 cm⁻¹ and 554 cm⁻¹, ν₄ band was detected relevant to triply degenerate P–O asymmetric bending. Besides the characteristic bonds of α-TCP, the peaks at 675 cm⁻¹ were related to sulphate (S-O) bands confirming SO₄²⁻. With the content of α-CSH in α-CSH/α-TCP beyond 15 wt%, at 675 cm⁻¹, the absorption peak of SO₄²⁻ gradually became sharper.

Cell counting kit-8

To investigate the proliferation of osteoblast cells on bone cement, two kinds of curing liquid were evaluated in CCK-8 assay as illustrated in Figure 6(a) at the culture time was 2 days, 4 days, and 6 days. According to Figure 6(a), the absorbance of each materials show an increasing trend with the growth of culture time, indicating that the materials have cytocompatibility. There is no significant difference in absorbance between the materials with Na₂HPO₄ solution as the curing liquid after the 6 days of culture and so does the materials with SFFs as the curing liquid. Cells are observed on composite bone cement with different content of α-CSH in α-CSH/α-TCP powders at 5 wt%, 15 wt%, 25 wt% after 6 days of culture (Figure 6(b) and (c)). In particular, cells are well-attached and spread on the surface of composite bone cement using SFFs as a curing liquid (Figure 6(c)). And the morphology of the cells on the materials solidified with SFFs is fusiform, and stretching out pseudopodia.

Alkaline phosphatase

The activity of ALP can reflect the content of mineralized matrix protein.²⁴ ALP can hydrolyze phosphate esters in the process of osteogenesis to provide necessary phosphate for HA deposition, and hydrolyze pyrophosphate to relieve its inhibitory effect on bone salt formation, which is conducive to osteogenesis. To investigate the effect of SFFs on promoting osteogenesis of composite bone cement, the ALP activity in two kinds of curing liquid was determined by the ALP kit. Among them, the higher the absorbance, the greater the activity of ALP, and the better the effect of promoting osteogenesis of the materials. As illustrated in Figure 7, the result showed that the ALP activity of the composite with SFFs as curing liquid was higher than Na₂HPO₄ solution after 14 days of culture when the content of α-CSH in α-CSH/α-TCP powders at 15 ∼ 25 wt%. This suggests that SFFs could promote osteogenesis under long-term culture. The highest ALP activity was at 15 wt% of α-CSH in α-CSH/α-TCP powders, and the effect was more significant at the ratio of 1.7 of Ca/P. ALP activity of the composite solidified with Na₂HPO₄ solution increased at 5 ∼ 10 wt% of α-CSH after 14 days of culture compared to the seventh day, and ALP had highest expression levels on the seventh day between 15 to 25 wt% of α-CSH then decreased on the 14th day. This is probably because the former Ca/P ratio of less than 1.7 increases with the degradation of α-CSH in the culture medium, but thelatter Ca/P ratio is equal to or greater than 1.7 originally. However, the bone cement with SFFs as curing liquid still has a high expression level on the 14th day.

Discussion

The bone cement operation is influenced by the setting time to a great extent.^9,25,28 In injecting the materials, if it takes too long setting time before into the human body, the tissue around the wound may be contaminated in the air²³ or directly dissolved in the tissue fluid, resulting in the operation’s failure. If the setting time is too short, bone cement clotting may occur, which makes it impossible for the materials to be injected into the body. Bone cement must be injected into damaged tissue before it sets²⁵ and not cause other harm to the human body. The addition of α-CSH shortened the solidification time of composite when Na₂HPO₄ was curing liquid. Because the formed CSD can be used as a coagulant to speed up the solidification speed.²⁵ The α-TCP in single-phase when SFFs was curing liquid was more compact probably because of the combining of C = O and N–H with Ca²⁺. It could increase the viscosity of the material, accelerate the setting reaction and shorten the setting time. However, α-CSH could not react with water quickly to form CSD when the SFFs was curing liquid because of its water-remaining effect, which slowing down the setting reaction. Finally, the setting time of composite with the different α-CSH content using SFFs as curing liquid was prolonged by 10 ∼ 30 min, which was fitter for the operation procedure.

In addition to setting time, the injectability and anti-washout properties of materials are also parameters for evaluating bone cement.^25,28 With the increase of α-CSH content, because of the procoagulant action of α-CSH, the setting time of the materials decreased, and the injectability property also gradually decreased. When the curing liquid was SFFs, because of the water retention of SFFs, the pro-coagulability of α-CSH decreased, whereas the injectability increased, which was related to the setting time of the composite materials.

Improving the maximum compressive strength of composite bone cement has always been the focus of research. When the SFFs was curing liquid, the compressive strength of the composites was increased comparing to Na₂HPO₄ as curing liquid. Furthermore, the shorter the setting time was, the higher the compressive strength was. It showed that SFF can affect the properties of α-TCP-based bone cement to a certain extent. And SFFs can induce apatite deposition on its surfaces,¹⁹ which may increase the maximum compressive strength of materials. And when the content of α-CSH in α-CSH/α-TCP powders was 15 wt%, the Ca/P was 1.7 in the degradation process, which is closest to the calcium-phosphorus ratio of the inorganic component HA in human bone, and the compressive strength of the composite bone cement material was the strongest.

The performance of SFFs in the degradation experiment showed different from Na₂HPO₄. The degradation rate of the materials was about 12.5 wt% in single-phase α-TCP with SFFs as curing liquid, whereas the rate of the same materials with Na₂HPO₄ as curing liquid showed up as weight gain. During the process, the weight loss in PBS buffer occurred due to the degradation of SFFs leads to the formation of pores on the surface of bone cement with SFFs as curing liquid simultaneously.²⁷ The weight loss ratio of composite with the addition of α-CSH decreased gradually, probably because HA was easy to deposit on SFFs. Additionally, the collapsibility of α-CSH became obvious, when the content of α-CSH was 15 wt%, and the degradation rate accelerated again, which was the main reason for the weight loss of composites when SFFs was curing liquid. What’s more, it was concluded from the EDS that SFFs can induce apatite deposition with the ratio of Ca/P close to 1.67. Whereas this phenomenon did not occur when Na₂HPO₄ was curing liquid. The Ca/P ratio increased gradually with the addition of α-CSH so that the dissolved Ca²⁺ and PO₄³⁻ in PBS reached a saturation value quickly and were rearranged as soon as possible to form HA.

In the cell proliferation experiment, it could also be found that there was no significant difference in the proportion of α-CSH prepared by the two solidification solutions, and the cell absorbance showed an upward trend with the increase of culture days. However, when SFFs was curing liquid, the absorbance of cells cultured on the materials for 6 days was higher than that of Na₂HPO₄ as curing liquid. Cells morphology immobilized by glutaraldehyde and gradient dehydration of anhydrous ethanol was fusiform and formed pseudopodia in SEM images. More mineral deposits could be seen on the surface of composites with SFFs as curing liquid.

In the ALP experiment, it was found that when SFFs was curing liquid, the composites could effectively promote osteogenesis on the seventh day, especially when the Ca/P ratio is 1.7, and maintained high levels on the 14th day. ALP activity of the composites solidified with Na₂HPO₄ solution increased on the seventh day but decreased on the 14th day with the content of α-CSH from 5 to 25 wt%. It was more closely related to the Ca/P, the closer the Ca/P ratio is to 1.7, the higher ALP activity. On the seventh day, the degradation of the composite was accelerated with the increase of α-CSH content, resulting in higher Ca²⁺ in the cell culture medium. On the 14th day, the concentration of Ca²⁺ decreased due to the degradation of α-CSH, and mainly be provided by α-TCP at this stage. This change made the Ca/P ratio of the composites unstable. However, the ALP activity maintained a high level at 14 days when SFFs was curing liquid, showing that SFFs could induce the apatite deposition with the ratio of Ca/P close to 1.67 of the bone cement longer than Na₂HPO₄ as curing liquid.

Conclusions

In this study, α-TCP bone cement was complexed with changeable α-CSH using nano SF as curing liquid was prepared at a liquid-solid ratio of 0.4 ml/g, and its properties were studied. The results showed that the setting time of the modified composite using SFFs as curing liquid was shortened to 10 ∼ 29 min, and the compressive strength was increased from 5.41 MPa to 9.44 MPa. The composites prepared with two curing liquids had good anti-washout properties, but the injectability was lower than 20.99%. When the content of α-CSH in α-CSH/α-TCP powders was 0 ∼ 25 wt%, the weight loss ratio of bone cement degraded was −0.18% ∼ 12.08% for 4 weeks. And the degradation process of α-CSH was accompanied by the deposition and transition from amorphous α-TCP to hydroxyapatite. It was shown that α-CSH can be used as a coagulant to shorten the setting time of α-TCP cement. As the degradation of α-CSH can provide calcium ions and pore nucleation sites, which promoted the further deposition and apatite deposition of α-TCP. Nano SF can improve the compressive strength of the materials. When the ratio of Ca/P was 1.67, the existence of SFFs induces the expression of ALP. This study would be beneficial to the migration of osteoblasts and the growth of bone trabecula in further study, which would provide a theoretical basis for bone defect tissue regeneration repaired by bone cement materials.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was funded by the Key Research and Development Program of Shanxi Province, China (International Cooperation) (grant numbers 201803D421076), Fundamental Research Program of Shanxi Province, China (grant numbers 20210302123132, 20210302124405, 202203021221047 and 201901D111115), Joint Construction Agreement of Shanxi Provincial Key Laboratory for Functional Proteins (grant number 213310462-J), China Scholarship Council (grant number 201906935015), National Natural Science Foundation of China (grant number 51502192 and 12272253), Science and Technology Innovation Project of Colleges and Universities in Shanxi Provincial Education Department (grant number 2020L0053).

Data availability

All data that support the findings of this study are included within the article.

ORCID iD

Xiaojie Lian

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