PMMA bone cement is widely used in orthopaedic surgery as an injectable artificial bone repair material due to its advantages such as desirable mechanical properties, suitable curing time and low toxicity^[1]. However, the bioinert polymer may lead to the aseptic loosening of the prostheses after long-term implantation, and the high polymerization temperature reaching 70-110 ℃ causes thermal damage to surrounding cells and tissues as well^[2-3]. The addition of bioactive materials serves as an effective way to improve the biological activity of PMMA bone cement, which can also reduce the polymerization temperature by delaying the polymerization curing of methyl methacrylate (MMA) with the second phase^[4⇓-6].

Among various bioactive materials, bioactive glasses exhibit superior biocompatibility, bioactivity and biodegradability, because they can react with body fluid to generate hydroxyapatite to form a firm bond with bone and soft tissue, and their released ions can also stimulate angiogenesis and osteogenesis^[7]. Previous studies indicated that the mesoporous borosilicate bioglass microspheres hold better bioactivity and biodegradability than do the traditional silicate bioglass because the increased specific surface area of porous bioglass leads to better surface reactivity and degradability^[8⇓-10]. Cui et al. ^[11] prepared strontium-doped borosilicate bioactive glass by melting method to improve the bioactivity and bone integration ability of PMMA bone cement.

However, the mechanical properties of composite bone cement decrease due to the lack of strong chemical bonding between hydrophilic inorganic particles and hydrophobic organic matrix^[12]. The desirable mechanical properties of bone cement are essential for the long-term stability because they undertake the function of transferring load and providing mechanical support when used to repair load-bearing bone defects. By surface modification of bioactive glass through silane coupling agent, chemical bonding between bioactive glass and PMMA matrix can be established to improve the mechanical properties of the composite bone cement^[13]. In addition, silane coupling agents do not have intrinsic toxicity^[14]. However, implementing surface modification to MBGS with silane coupling agents could change the microstructure, resulting in the loss of bioactivity. Nevertheless, the microstructure and bioactivity of MBGS effected by the silanization and the properties of PMMA based composite bone cement modified by MBGSSI have not been reported.

In this research, we prepared MBGS by template method and modified them by silane coupling agent γ-MPS to obtain MBGSSI. The constituents and microstructures of MBGS and MBGSSI were determined. MBGS and MBGSSI were combined with PMMA bone cement to prepare MBGS/PMMA and MBGSSI/PMMA composite bone cement, respectively, and the setting properties, mechanical properties and in vitro bioactivity were studied.

Tributyl borate (TBB, CP), tetraethyl orthosilicate (TEOS, AR), calcium nitrate tetrahydrate (CN, AR), triethylphosphate (TEP, CP), cetyltrimethyl ammonium bromide (CTAB, AR), ammonia solution (25%, AR), γ-methylacryloxy propyltrimethoxysilane (γ-MPS, KH 570, ≥98.0%) and benzoyl peroxide (BPO, CP) were purchased from Sinopharm Chemical Reagent Co., LTd (China). Acetate (AR, 99.5%), methyl methacrylate (MMA, >99.5%) and N, N-dimethyl-p-toluidine (DMPT, >98.0%) were purchased from Shanghai Aladdin Bio-Chem Technology Co., LTD (China). Poly(methyl methacrylate) (PMMA) was purchased from Makevale Group (UK).

According to the previous study, MBGS were prepared by template method using TBB, TEOS, CN, and TEP as raw materials to obtain the composition of 20B₂O₃-40SiO₂-36CaO-4P₂O₅^[15]. CTAB was used as template and NH₃·H₂O was used as catalyst. The reaction was carried out at 40 ℃ with a stirring rate of 400 r/min. 0.15 g CTAB was dissolved in a mixture containing 80 mL deionized water and 40 mL absolute ethanol under stirring for 30 min. Then 1.629 mL TEOS was added into the solution. After stirred for 10 min, 1 mL ammonia solution was added as a catalyst and stirred for 10 min. Subsequently 1.946 mL TBB and 0.248 mL TEP were added and stirred for 30 min. Thereafter, 1.546 g CN was added into the solution and stirred for 3 h. The micro-spheres were separated by centrifugation, washed by absolute ethanol, deionized water and absolute ethanol successively, and dried at 60 ℃ in the oven. Finally, MBGS were obtained after heat treatment at 450 ℃for 5 h at a rate of 1 ℃/min.

Based on references, we developed silanation process to modify MBGS^[16⇓-18]. The silanization of MBGS by γ-MPS was carried out at 65 ℃ with a stirring rate of 400 r/min. 2 mL γ-MPS was added into a mixture containing 20 mL deionized water and 80 mL absolute ethanol, with pH being regulated at about 4.5 by acetate. After stirring for 30 min, 2 g MBGS was added into the solution, maitaining the reaction for 2 h. The microspheres were separated by centrifugation, washed by absolute ethanol and dried at 60 ℃. Finally, the products were treated at 100 ℃ for 2 h at a rate of 1 ℃/min to obtain MBGSSI.

The solid phase of MBGS/PMMA composite bone cement is composed of 2 g PMMA powder, 0.4 g MBGS and 0.5% BPO, while the liquid phase contains 1 mL MMA and 0.6% DMPT. Their solid or liquid phase were mixed and stirred at 23 ℃ and cured in the mold to obtain MBGS/PMMA according to ISO 5833:2002^[19]. MBGSSI/PMMA was prepared by the same steps.

MBGS and MBGSSI were characterized by X-ray Diffraction (XRD, D/max2550, Japan) at a scanning rate of 5 (°)/min in the range of 10°-80°, Fourier Transform Infrared Spectroscopy (FT-IR, EQUINOXSS, Germany), Thermogravimetric Analyzer (TA, Q600, USA) from 30 ℃ to 800 ℃ at a heating rate of 10 ℃/min, high resolution Transmission Electron Microscope (TEM, H-800, Japan), specific surface area analyzer (BET, CHA-SA, China), and X-ray Photoelectron Spectrometer (XPS, ESCALAB 250XI, USA).

Setting properties and mechanical properties of PMMA, MBGS/PMMA and MBGSSI/PMMA bone cements were tested according to ISO 5833:2002 by Paperless Recorder (STR3101, China) and Electronic Universal Testing Machine (CTM2500, China).

Bone cements were observed by Field Emission Scanning Electron Microscope (SEM, Tuanta 200 FEG, USA) and analyzed by Energy Dispersive Spectroscope (EDS, Tuanta 200 FEG, USA) and X-ray Diffraction (XRD, D/max2550, Japan).

Phase and chemical composition of MBGS and MBGSSI were explored with XRD, FT-IR and TG measurement (Fig. 1 (a-c)). There is no sharp diffraction peak in the XRD patterns of MBGS and MBGSSI, indicating that the silanization does not change the amorphous phase of the bioglass. The FT-IR spectrum of MBGSSI exhibits peaks belong to both MBGS and γ-MPS, demonstrating the presence of γ-MPS in MBGSSI^[20⇓-22]. The MBGSSI are composed of about 95% inorganic composition and 5% organic composition (Fig. 1 (c)). Major decomposition of γ-MPS takes place between 100 and 200 ℃, while weight loss of MBGSSI is accelerated at 300 ℃ due to the strong chemical bond being formed between γ-MPS and bioglass^[17]. Microstructures of MBGS and MBGSSI (Fig. 1 (d, e)) consolidate a spherical structure with a diameter of about 450 nm and numerous pores, while MBGSSI show denser microstructures because γ-MPS enters the pore and binds with MBGS^[23].

According to the result of N₂ adsorption-desorption isotherm and corresponding pore size distribution, the pore structure of MBGS and MBGSSI was analyzed by BET method (Fig. 2(a, b)). Hysteresis loop back indicates their mesoporous structure (Fig. 2(a)). Pore diameter of MBGS is mainly distributed between 13- 50 nm, while pore diameter of MBGSSI presents a large differentiation, with a large number of distributions between 3-5 nm and 13-50 nm (Fig. 2(b)). The distribution of pore diameter proves that modifying MBGS with γ-MPS results in decrease of large pores and increase of small pores, leading to drastic increase of specific surface area. Besides, there exists a large number of larger pores (>50 nm) due to microspheres accumulation, evidenced by obvious N₂ adsorption in the high pressure area (Fig. 2(a)). Specific surface area, average pore diameter and total pore volume of MBGS and MBGSSI were compared in Table 1. As the schematic diagram of surface modification shown, γ-MPS binds to Si-OH on the surface of MBGS (Fig. 2(c)), causing a significant increase of specific surface area and decreases of average pore diameter and total pore volume because γ-MPS occupies a certain volume^[24].

XPS survey spectra of MBGS and MBGSSI prove the existence of γ-MPS on the surface of MBGSSI (Fig. 3(a)). It can be further observed (Fig. 3(b, c)) that atomic percentage of C1s C=O on MBGSSI decreases from 1.11% to 0.32% as etching depth increases from 0 to 100 nm, demonstrating that the binding between γ-MPS and MBGS mainly occurs on the near surface of mesoporous microspheres. Atomic concentrations of MBGS, MBGSSI and MBGSSI (etching depth at 100 nm) were compared in Table 2.

The setting properties and mechanical properties of PMMA, MBGS/PMMA and MBGSSI/PMMA bone cements were summarized in Table 3. Compared with PMMA, MBGS/PMMA and MBGSSI/PMMA show shorter dough time and longer setting time that are beneficial for clinical operation due to the rise of solid/liquid and decrease of organic concentration in the composite. In addition, polymerization curing is prolonged by addition of the second phase, resulting in lower peak temperature which also benefits clinical application.

However, after adding MBGS in PMMA, the flexural strength of bone cement significantly decreases to (44.53±2.59) MPa, which is lower than the standard strength of ISO 5833:2002 (≥50 MPa)^[19]. This is because physical mixture between hydrophilic bioglass and hydrophobic PMMA results in agglomeration of inorganic phase in the organic matrix, and brittle fracture was caused by stress concentration in the test of bending resistance^[25-26]. While allyl oxy group in γ-MPS reacts with C=C in MMA monomer, whose strong chemical bond makes the inorganic phase dispersed evenly in the organic matrix, leading to better flexural strength of MBGSSI/PMMA^[27]. In addition, the composite bone cements show increased compressive modulus and flexural modulus because the inorganic rigid mesoporous microspheres improve the ability of bone cements to resist the deformation^[26].

Fig. 4 shows the dispersity of MBGS and MBGSSI in MMA monomer. MBGS settle obviously in 5 min, while MBGSSI disperse in the MMA for a longer period of time. Moreover, SEM images of PMMA, MBGS/ PMMA and MBGSSI/PMMA bone cements (Fig. 5) show that MBGS on the surface of composite bone cement agglomerate, while MBGSSI disperse evenly in the organic matrix, which matching the mechanical properties of composite bone cements.

After immersing in SBF for 42 d, morphologies of PMMA, MBGS/PMMA and MBGSSI/PMMA bone cements were characterized by SEM, EDS and XRD measurements. As can be observed that dense lamellar structure appears on the surface of MBGS/PMMA and MBGSSI/PMMA, while the surface of pure PMMA still keeps smooth (Fig. 6(a, c, e)). The diffraction peaks corresponding to (002), (210) and (211) of HA (PDF#09-0432) can be observed in the XRD patterns of MBGS/PMMA and MBGSSI/PMMA (Fig. 6(g)), indicating HA being formed on the surfaces of composite bone cements. This result proves the excellent in vitro bioactivity of MBGS/PMMA and MBGSSI/PMMA. Moreover, the lower element content ratio of Ca and P of MBGSSI/PMMA studied by EDS indicates that formation of calcium-deficient hydroxyapatite, which is caused by the limited ion dissolution of MBGSSI due to the silane modification.

MBGS with high specific surface area were successfully prepared by template method and then modified by γ-MPS to obtain MBGSSI. BET and XPS results indicates that the binding between γ-MPS and MBGS mainly occurs on the near surface of mesoporous microspheres, resulting in the increase of specific surface area and decrease of average pore diameter and total pore volume. Moreover, the silanization improves the bonding between hydrophilic bioglass and hydrophobic organic PMMA matrix. Hence, MBGSSI/PMMA composite bone cement presents suitable setting properties and improved mechanical properties compared with MBGS/PMMA. In addition, MBGSSI/PMMA shows excellent in vitro bioactivity, which can be a promising bone repair material.