The influence of silane coupling agents on the properties of α-TCP-based ceramic bone substitutes for orthopaedic applications

Biomaterials based on α-TCP are highly recommended for medical applications due to their ability to bond chemically with bone tissue. However, in order to improve their physicochemical properties, modifications are needed. In this work, novel, hybrid α-TCP-based bone cements were developed and examinated. The influence of two different silane coupling agents (SCAs) – tetraethoxysilane (TEOS) and 3-glycidoxypropyl trimethoxysilane (GPTMS) on the properties of the final materials was investigated. Application of modifiers allowed us to obtain hybrid materials due to the presence of different bonds in their structure, for example between calcium phosphates and SCA molecules. The use of SCAs increased the compressive strength of the bone cements from 7.24 ± 0.35 MPa to 12.17 ± 0.48 MPa. Moreover, modification impacted the final setting time of the cements, reducing it from 11.0 to 6.5 minutes. The developed materials displayed bioactive potential in simulated body fluid. Presented findings demonstrate the beneficial influence of silane coupling agents on the properties of calcium phosphate-based bone substitutes and pave the way for their further in vitro and in vivo studies.


Introduction
2][3] Due to its chemical similarity to the inorganic part of bone tissue and selfsetting properties a-TCP is commonly used as a main component of calcium phosphate-based cements (CPCs). 4CPCs possess excellent biocompatibility, osteoconductivity, and ability to bond with natural bone tissue. 5,6However, despite their numerous advantages, they still have some drawbacks, including their relatively low mechanical strength and fast resorption rates. 7,8To address these limitations, various modi-cations of powder and liquid phase of CPCs have been proposed.
2][13][14] Recently, it has been reported that calcium phosphates can be modied with silane coupling agents (SCAs), which are organosilicon compounds.Silane coupling agents can bond chemically to both the inorganic and organic surfaces, forming strong covalent bonds and improving the interfacial adhesion between different materials. 16,17][20][21][22] Suppakarn et al. 23 modied hydroxyapatite (HA) powders with different silane coupling agents, including g-aminopropyl triethoxysilane (APES), methyl trimethoxysilane (MTMS), and gglycidoxypropyl trimethoxysilane (GPMS) to obtain HA/ polypropylene composites.The results demonstrated that the silane treatment enhanced the interaction between the HA and PP, resulting in an increase in the stiffness of the composite material.The g-aminopropyl triethoxysilane (APES) was found to be the most effective silane coupling agent for this application.Ji et al. 24 developed hydroxyapatite-based scaffolds by modifying the surface of HA with different silane coupling agents: 3-methacryloxypropyltrimethoxysilane, 3-aminopropyltrimethoxysilane and carboxyethylsilanetriol sodium salt.The using of SCAs modiers enhanced mechanical strength up to 30 MPa, which was explained by the presence of electrostatic bonds between components.Furthermore, the functionalized silane-coated HAp scaffolds displayed excellent biocompatibility.Ghorbani et al. 25 incorporated 3-glycidoxypropyl trimethoxysilane (GPTMS) as a bioactive inorganic crosslinker in chitosan-polyvinyl alcohol scaffolds which led to the improvement of mechanical strength, water uptake, and biodegradation properties.It has been demonstrated that an increase in GPTMS content enhanced the compressive strength of samples, while reducing water uptake and biodegradation of materials.What is more obtained composites possessed excellent biological properties conrmed by different assays.Another common SCA is tetraethoxysilane (TEOS) which is used in the development of biomaterials due to its ability to form biocompatible silica coatings, enhancing their biocompatibility and stability within the cellular environment.Tallia et al. 26 developed promising SiO 2 -CaO/PTHF/PCL-diCOOH hybrid material with improved mechanical properties, enhanced dissolution behaviour, and cell attachment capability.Fuh et al. 27 described novel TEOS treatment method that enhances micro-porosity, reduces sintering shrinkage, and improves biodegradation in hydroxyapatite (HA) scaffolds, making it a promising technique for HA manufacturing.
Despite the potential advantages of using silane coupling agents to modify calcium phosphates powders, to the best of our knowledge, no modication of a-TCP has been carried out yet.The simultaneous hydrolysis of a-TCP and silane coupling agent may potentially lead to the formation of a novel a-TCPbased hybrid materials with improved properties due to presence of additional bonds between the components.The SCAs during hydrolysis can react with the hydroxyl groups on the surface of calcium phosphates, forming covalent bonds and modifying the properties of material.Such interactions, have been described previously in other biomaterials. 28,29In this work, two different SCAs were examined as potential moderators of interactions between materials components.We believe that the use of SCAs can lead to favourable properties of hybrid, a-TCP-based bone cements due to the formation of new bonds in the materials' structure.
The aim of this study was to develop bone cements on the basis of a-tricalcium phosphate and investigate the inuence of tetraethoxysilane (TEOS) and 3-glycidoxypropyl trimethoxysilane (GPTMS) on their physicochemical and biological properties.

Modication of a-TCP
The a-TCP powder was modied with 1, 2 and 5 wt% TEOS (T) ($99.5%,Sigma-Aldrich, St. Louis, MO, USA) or GPTMS (G) ($98.0%,Sigma-Aldrich, St. Louis, MO, USA) (Table 1).To modify the initial powders, solutions of the appropriate silane coupling agents were prepared using anhydrous ethanol as a solvent (99.8%,POCH, Gliwice, Poland).The alcoholic solvent was used to avoid hydrolysis of the calcium phosphate powders and SCAs.Aer adding the powders to the SCA solutions, they were stirred on a magnetic stirrer for 4 hours.The resulting precipitates were subjected to aging (1 h) and subsequently silanised by thermal treatment at 115 °C (4 h).Prior to preparing the samples, the powders were sieved through a sieve below 63 mm.

Bone cements preparation
Seven types of materials were prepared by mixing the solid phase, i.e., non-modied and SCA-modied a-TCP with the liquid phase.The initial composition of the prepared cements, as well as a liquid to powder ratio (L/P), are presented in Table 2.
2.4.Methods 2.4.1.Specic surface area.The specic surface area (SSA) of the initial a-TCP and SCAs-modied a-TCP powders was determined by the BET (Brunauer-Emmett-Teller) method using accelerated surface area and porosimetry system ASAP 2010 (Micromeritics, Norcross, GA, USA).
2.4.2.Powders size distribution and zeta potential.The size distribution of the initial a-TCP and SCAs-modied a-TCP powders as well as zeta potential was based on Brownian motion and the Dynamic Light Scattering (DLS) technique and combination of electrophoresis and the Laser Doppler Velocimetry technique using system Zetasizer Nano-ZS (Malvern Panalytical, Malvern, UK).
2.4.3.Chemical and phase composition.The X-ray uorescence method (XRF) was applied to check the chemical composition of the initial powders (WDXRF Axios Max, PANalytical, Malvern, UK).The X-ray diffraction (XRD) analysis was performed using Cu Ka radiation (1.54 Å) at 30 kV and 10 mA.The analysis was conducted in the 2q range of 10-60°at 0.04 intervals with a scanning speed of 2.5°min −1 using D2 Phaser diffractometer (Bruker, Ballerica, MA, USA).The obtained diffractograms were compared with the International Centre for Diffraction Data a-TCP (00-009-0348) and hydroxyapatite (HA; a-TCP powder modied with 1 wt% of TEOS a-TCP/T2 a-TCP powder modied with 2 wt% of TEOS a-TCP/T5 a-TCP powder modied with 5 wt% of TEOS a-TCP/G1 a-TCP powder modied with 1 wt% of GPTMS a-TCP/G2 a-TCP powder modied with 2 wt% of GPTMS a-TCP/G5 a-TCP powder modied with 5 wt% of GPTMS 01-076-0694) to identify the crystalline phases.TOPAS soware (Bruker, Ballerica, MA, USA) was used for phase quantication based on Rietveld renement.All measurements were performed in triplicate.The mean ± standard deviation (SD) was used to present the results.FT-IR and Raman spectroscopy methods were used for the characterization of obtained non-modied and modied powders, as well as the hardened bone cements.FTIR investigations of materials were conducted on a BioRad FTS 6000 spectrometer (Bruker, Ballerica, MA, USA) with the Raman attachment (NdYAG laser excitation line of 1064 nm) in the wavenumber range of 3800-200 cm −1 .The transmission technique was used and the samples were prepared as standard KBr pellets.This methodology was used to obtain an accurate and detailed understanding of the chemical and structural properties of the materials, which is crucial for determining their suitability for specic biomedical applications.
2.4.4.Setting times.The setting times were determined in accordance with the ASTM C266-20 standard, using Gilmore Needles (Humbold MFG Co., Norridge, IL, USA). 31The Gillmore Apparatus, consisting of two steel-weighted needles, was used for this purpose.The initial setting needle weighed 113 g and had a diameter of 2.12 mm, while the nal setting needle weighed 453.6 g and had a diameter of 1.06 mm.To determine the setting times, the cementitious pastes were placed in a special form measuring 8 mm × 10 mm × 5 mm, and the needle of the apparatus was lightly applied to its surface.The setting time was measured as the point at which the needle penetrated the surface without leaving a complete circular mark.All experiments were carried out at room temperature (22 ± 1 °C), and the results were presented as the average of three measurements, along with their corresponding standard deviations (SD).
2.4.5.Mechanical strength.To determine the compressive strength of the materials, cylindrical samples with a height of 12 mm and diameter of 6 mm were tested using a universal material testing machine (Instron 3345, Norwood, MA, USA) at a crosshead displacement rate of 1 mm min −1 .The compressive strength results were expressed as the mean value ± the standard deviation (SD) of twenty-fold determinations.To determine if there were any statistically signicant differences between the materials, a one-way ANOVA followed by post hoc Tukey's Honest Signicant Difference (HSD) test was applied with a signicance level of p < 0.01.
2.4.6.Microstructure.For microstructure observations of the fractured samples, scanning electron microscopy (SEM) was employed using a PhenomPure instrument (Thermo Fisher Scientic, Waltham, MA, USA).To evaluate the bioactive potential of the materials, their surfaces were assessed aer 7 days of incubation in simulated body uid (SBF) at 37 °C.Before examination, the samples were coated with a thin layer of gold lm using a low deposition rate to prevent any charge build-up and to enhance the imaging resolution.
2.4.7.In vitro bioactivity and chemical stability.To evaluate the chemical stability and bioactivity of the bone cements, they were incubated in distilled water or simulated body uid (SBF) prepared according to Kokubo's protocol. 32Cylindrical samples were placed in containers with 20 mL SBF or water and stored at 37 °C.The chemical stability of the materials was determined by measuring the pH and ionic conductivity of the solutions around incubated samples at various time intervals.Measurements were taken using a Seven Compact Duo pH/ conductometer (Mettler Toledo, Columbus, OH, USA).Results were expressed as the average value of three measurements ± standard deviation (SD).Aer immersion, the samples were removed from solution, rinsed with distilled water, and dried at temperatures below 40 °C.To conrm the bioactive potential, the surfaces of the samples were observed using SEM.

Specic surface area
The specic surface area is an important parameter for characterizing the performance of catalysts, adsorbents, and other porous materials, as it can affect the reactivity, selectivity, and stability. 33,34The specic surface area of obtained powders is shown in Fig. 1.
The specic surface area of the obtained calcium phosphate powders was in the range of 1.81 ± 0.04 to 3.67 ± 0.04 and decreased with increasing amounts of the silane coupling agent.The highest value of specic surface area was noticed for non-modied a-TCP powder.From the results obtained, it can be seen that the specic surface area of the powders decreases with use and increasing amount of silane coupling agent.Generally, the specic surface area of a-TCP ranged from 2.0 to 11.0 g m −2 and depends on many factors, such as synthesis, processing routes and presence of modiers. 35,36The decreasing value of the specic surface area of silane coupling agent-modied powders is due to the connement of micropores in the powder grains with the silane coupling agents.The silane coupling agents form a thin layer on the surface of the grains, which reduce the overall surface area of calcium phosphate powders.Similar results were obtained by Sonn et al. 37 and Chuang et al., 38 who modied the surface of silica nanoparticles with various coupling agents.They have also shown that the specic surface area of nanoparticles decreased with increasing concentration of the silane coupling agents due to reduction of micropores in the powders.

Powders size distribution and zeta potential
The powder size distribution and zeta potential are crucial parameters in powder technology, especially for calcium phosphates, as they can affect the properties and performance of the powders.Optimal values of this parameters depend on specic requirements.The powder size distribution (Fig. 2) and zeta potential (Table 3) of obtained powders are shown below.
It was observed that the surface modication of the a-TCP powders with both silane coupling agents changed a powders size distribution.For non-modied a-TCP pore size were in the range of approximately 1.0 to 100.0 mm.However, the SCA modication caused change of powders size distribution to the range between ∼1.0 to 10.0 mm.When silane coupling agents are used to modify the surface of the powder particles, they can form a monolayer or multilayer made of organic molecules, which can reduce the surface energy and enhance the dispersibility of the particles in the surrounding medium. 391][42] The differences in zeta potential measurements also were observed.The non-modied powder possessed the least electro-negative zeta potential (i.e.−4.65 ± 0.3 mV).However, the surface modication caused changes in zeta potential values.As in the case of powders size distribution the changes in zeta potential values were caused by the presence of layer of SCA molecules, which can reduce the electrostatic repulsion between CaPs particles and decrease the zeta potential 43 (up to −24.4 ± 0.9 mV for a-TCP/G5).

Chemical and phase composition
The XRD analysis revealed that the initial a-tricalcium phosphate and silane coupling agents modied a-TCP powders composed mainly of a-TCP (97-98 wt%) and a small amount of hydroxyapatite phase (2-3 wt%) (Fig. 3A).As expected, the modication of the powders via SCAs in anhydrous medium did not result in hydrolysis of a-TCP to non-stoichiometric hydroxyapatite.Additionally, by using silane coupling agents to modify the surface of the powders, silicon ions were introduced.The applied method of modication allowed for introduction to a-tricalcium phosphate, and no other crystalline phases containing silicon were observed.The X-ray uorescence method conrmed the presence of silicon in all modied  Paper RSC Advances powders and in amount of 0.035 ± 0.001 wt% for non-modied powder.As expected, the silicon content increased with the increasing amount of SCAs used and was 0.218 ± 0.004 wt% and 0.280 ± 0.002 wt% for powders modied with 5 wt% of TEOS and GPTMS respectively.Presence of silicon may improve the biological properties of the nal material, such as osteoconductivity. 44The modication allowed the powder to be enriched with silicon ions aer a-TCP synthesis.Overwhelming majority of scientic publications describe the introduction of silicon ions into calcium phosphate-based materials at the synthesis stage. 45he diffractograms of bone cements aer setting and hardening in air for 7 days (Fig. 3B and Table 4), as well as cements incubated in SBF for 7 days (Fig. 3C and Table 3), revealed two crystalline phases, i.e., a-TCP and hydroxyapatite, regardless the amount of the silane coupling agents.It has been demonstrated that in the simulated body uid, the a-TCP phase is thermodynamically metastable and almost completely hydrolyses to nonstoichiometric hydroxyapatite.This transformation is accompanied by a change in the chemical composition and crystallographic structure of the material, which can affect its properties and behaviour in biological and biomedical applications. 46nfrared spectra of initial powders (Fig. 4A), cements aer setting and hardening (Fig. 4B) as well as cements aer 7 days of incubation in simulated body uid (Fig. 4C) revealed the presence of several characteristic bands, which provided valuable insights into the composition and structure of the materials.Similar results were obtained for all SCAs concentrations, and no signicant differences were observed for the various amount of silane coupling agents.Specically, bands at around 565 and 605 cm −1 were observed and assigned to triply degenerate y4 P-O-P bending modes.Additionally, a strong doublet near 600 and 670 cm −1 was connected to SO 4 bending vibrations (y4).Interestingly, the coincidence of SO 4 and PO 4 bending vibration bands was observed at around 603 cm −1 .The other important bands were observed at 1060 cm −1 and around 1040 cm −1 , which were assigned to y3 non-symmetric stretching modes of P-O and are characteristic of calcium phosphates.Furthermore, a band at 962 cm −1 was observed, which was associated with the y1 symmetric stretching vibration of P-O bands.In addition, a wide band around 3500 cm −1 indicates the presence of residual water in the studied samples.Low concentration of silane coupling agents in the cements, as well as an overlapping of bands, may explain the lack of visible peaks of Si-O-Si, Si-OH, Si-C and C-H bands assigned to SCAs. 47In addition bone cements aer setting and hardening in air, as well as aer incubation in SBF possessed the absorption bands arising from HPO 4 2− .The weak band at around 870 cm −1 assigned as P-OH stretch of HPO 4 2− to calcium decient materials.Thus, obtained data conrmed the presence of calcium-decient hydroxyapatite (CDHA) in material at the same time shows its bioactive potential due to hydrolysis of a-TCP to nonstoichiometric hydroxyapatite.Raman spectra of non-modied powder and cements (Fig. 5A), TEOS-modied powder and cements (Fig. 5B) as well as TEOS-modied powder and cements (Fig. 5C) revealed the presence of bands characteristic for calcium phosphates, as well as band unique for TEOS and GPTMS.Specically, CPCs based on a-TCP are characterised by bands around 450 and 560 cm −1 attributed to the symmetric stretching and bending modes of the Ca-O bonds.This bands may shi slightly depending on the conditions.Additionally, phosphate groups at around 960, 1005, and 1080 cm −1 are attributed to the symmetric stretching, bending, and asymmetric stretching modes, respectively.The small amounts of carbonate ions, which are conrmed by bands at around 1060 and 1415 cm −1 .These bands are attributed to the symmetric stretching and bending modes of the carbonate  ion, especially for cements aer its incubation in SBF.The presence of remaining water in CPCs was also conrmed by the presence of characteristic bands at around 3300 and 3500 cm −1 .
In addition to the bands characteristic for calcium phosphates, bands characteristic for silane coupling agents also were observed.TEOS exhibits a Si-O-Si band at around 1100 cm −1 , which is attributed to the symmetric stretching mode of the Si-O-Si bond.It also exhibits band at around 2940 cm −1 , attributed to the stretching mode of the Si-CH 3 bond and band around 1250 cm −1 is assigned to the stretching mode of the Si-O-CH 3 bond in TEOS.The presence of Si-OH groups in TEOS were conrmed by bands around 3600-3700 cm −1 , which is attributed to their stretching mode.On the other hand, GPTMS presence in obtained materials was conrmed by the presence of Raman band at around 910 cm −1 , attributed to the stretching mode of the CH 2 -O bond.This characteristic bond of epoxide groups is more visible for incubated materials.Similarly, to TEOS, GPTMS also exhibits bands assigned to a Si-O-Si (∼1100 cm −1 ), Si-CH 3 (∼2940 cm −1 ) and Si-O-CH 2 (∼1250 cm −1 ).
During the simultaneous hydrolysis of a-TCP and silane coupling agents several potential new bonds can be formed between the coupling agents and the surface of the a-TCP particles.This includes the Si-O bond, which can be formed when the silanol group of the TEOS or GPTMS molecule reacts with the groups on the surface of CaPs grains.The stretching vibration of the Si-O bond typically appears between 1000-1200 cm −1 , while the stretching vibration of the -OH appears around 3500 cm −1 .The formation of the Si-O bond results in a shi of the Si-O stretching vibration.Ribeiro et al. 48demonstrated interaction between ultra-ne calcium carbonate and natural rubber by applying a supercial treatment to the calcium carbonate which provides materials with improved properties.They observed the similar bonds between coupling agents and calcium carbonate groups.Bi et al. 49 investigated the effects of trimethoxysilanes on the properties of CaCO 3 -based composites.They conrmed the presence of chemical interaction between components, which allow to improve the tensile strength and abrasion resistance of the materials.Moreover, Bi et al. stated that the silanol groups of two coupling agents react with each other and form a covalent bond Si-O-Si, creating the hybrid material.Purcar et al. 50synthesized hybrid nanomaterials based on zinc oxide via the sol-gel method, using different silane coupling agents.They conrmed the presence of Si-O-Si bands in the materials and showed their positive inuence on the materials' properties.Furthermore, the Fig. 4 FT-IR spectra of obtained materials: powders (A), bone cements after setting and hardening in air for 7 days (B) and bone cements incubated in SBF for 7 days (C).presence of Si-O-P bond at 1000 and 1200 cm −1 can conrm the interactions between the silanol group on the TEOS or GPTMS molecule and the phosphate groups, forming a covalent bond between the silicon atom of the coupling agent and the oxygen atom of the phosphate group.These bonds can enhance the further properties of the materials, such as mechanical strength and bioactivity.In addition, the introduction of coupling agents to cements composition may allow for its further modication using polymers with different functional groups that can also react with SCAs.

Setting times
Setting time is critical parameter for calcium phosphate cements because it affects their handling, injectability, and mechanical properties. 51The setting times of the obtained bone cements depended on the amount of used silane coupling agent and varied between 3.5 and 6.5 min (initial setting time) and 6.5 and 11.5 min (nal setting time) (Table 5).
The results showed that the addition of silane coupling agent used for calcium phosphate modication decreased both, the initial and nal setting time.The process responsible for setting and hardening of calcium phosphate cements involves a-TCP hydrolysis.When a-TCP reacts with water, it forms calcium-decient apatite, as described by the following eqn (1): 52,53 3Ca 3 (PO 4 ) 2 + H 2 O / Ca 9 (PO 4 ) 5 (HPO 4 )OH Several factors can affect the setting reaction of bone cements, such as the amount of highly reactive a-TCP phase in the material, such as: the presence of setting accelerators in the liquid phase, the type and amount of polymeric additive in the liquid or powder phase, and a-TCP powder modications. 54esults of our studies revealed that the presence of silane coupling agents also inuence the setting time values.It seems that SCAs accelerate the setting process of a-TCP-based bone cements.Previous studies have demonstrated that the incorporation of silicon into calcium phosphate cement alters its properties in comparison to the non-modied cements.For example, Czechowska et al. 45 developed calcium phosphatebased bone llers based on silicon doped a-TCP with hybrid, gold-modied granules.Their study showed a shortening of both initial and nal setting times for silicon-containing materials.The study by Wei et al. 55 conrmed a higher solubility of silicon-modied a-TCP, additionally Mestres et al. 56 found a faster hydrolysis of silicon-modied a-TCP in comparison with non-modied powders.All this nding corresponds with the results obtained during experiments.Although the measured setting times were reduced due to the use of a silane coupling agent, their values remained in the range suitable for clinical application (4-8 min for initial setting, up to 15 min for nal setting time 46 ).Fig. 7 Morphology of initial a-TCP powders (magnification: 100 00×).

Mechanical strength
The mechanical strength of calcium phosphate-based bone cements is important parameter, because it determines the ability of the cement to withstand the mechanical forces and stresses in the bone environment, ensuring successful bone repair and regeneration.The compressive strength of obtained biomaterials ranged from 7.24 ± 0.35 MPa to 12.17 ± 0.48 MPa (Fig. 6).The results of the mechanical tests revealed that the presence of SCAs modier positively affected the compressive strength of the bone cements.The highest values were obtained for material G5 (with 5 wt% of GPTMS).Based on the results of a one-way ANOVA and a subsequent Tukey HSD post-hoc analysis, it can be concluded that the observed differences between the control and SACs modied materials were statistically signicant.
The silane coupling agents as modiers of a-TCP may provide the mechanical improvement of obtained bone cements due to formation of additional bonds in materials' structure during the simultaneous hydrolysis of a-TCP and SCAs.The increase in the mechanical strength of the developed cements is possibly related to the chemical interactions between their components and formation of additional Si-O-Si, Si-O, or Si-O-P bonds what led to creation of hybrid-type materials.Similar, positive inuence of using silane coupling agents on mechanical behaviour of different biomaterials where previously described in the literature.In the study by Ma et al. 57 the trimethoxysilane was used as a modier of hydroxyapatite/ polyether ether ketone (PEEK) composites to obtain materials with improved mechanical strength.Furthermore, Ma et al. shoved promising results from in vivo studies.Vaz et al. 58 Fig. 8 Microstructure of obtained materials after seven days of setting and hardening in air (cross-sections, magnifications: 1000, 5000, 100 00×).shoved benecial application of different coupling agents to obtain starch/ethylene-vinyl alcohol copolymer/hydroxyapatite composites characterised by increased mechanical resistance due to improved adhesion between material components.Harper et al. 59 presented the benecial properties of hydroxyapatite loaded acrylic bone cements.By modication of hydroxyapatite by using 3-trimethoxysilylpropylmethacrylate authors obtained materials characterised by increased mechanical parameters.It should be noted that the compressive strength of cancellous bone ranges from approximately 4 to 12 MPa. 60Thus, the hybrid-type, SACs modied materials developed in our study possessed mechanical strength suitable for implantation in non-load or low-load bearing locations.

Microstructure
To evaluate the potential of the developed materials as a bone tissue substitutes and detect any microstructural defects, their microstructure was examined.The initial powders and obtained biomaterials were observed using scanning electron microscopy (SEM) aer setting and hardening in air, as well as aer incubation in SBF.SEM observations revealed that the morphology of powder grains of non-modied and TEOS or GPTMS modied a-TCP did not differ signicantly (Fig. 7).
The set and hardened materials possessed compact, homogenous microstructure formed by cementitious matrix with visible micropores (Fig. 8).Regardless the amount of silane coupling agent no differences in materials' microstructure were observed.Similar microstructures of a-TCP-based bone cements can be found in other studies. 61er 7 days of incubation in simulated body uid (SBF) all prepared materials were completely covered by plate-like apatitic structure conrmed their bioactivity in vitro according to Kokubo and Takadama's criteria (Fig. 9). 62Furthermore, the microstructure analysis aer incubation showed that the developed materials retained their bioactive potential despite the using silane coupling agents as a modier of a-TCP powders.

In vitro bioactivity and chemical stability
The chemical stability of implantable biomaterials is crucial parameter in determining their potential clinical application.The pH changes of the SBF during the samples' immersion are illustrated in Fig. 10.
The pH changes of SBF around incubated samples remained close to the physiological values and ranged from 7.34 to 7.40.The addition of TEOS and GPTMS only slightly inuenced the solution's pH values.Similar pH values of incubated calcium phosphate-based bone substitutes were observed elsewhere. 15onic conductivity during the incubation in distilled water of the control material was in the range of ∼72-81 mS cm −1 .A CPCs modiers caused slightly increase in ionic conductivity to the range of ∼73-87 mS cm −1 for TEOS, and to the range of ∼84-96 for GPTMS (Fig. 11).This phenomenon can be explained by Fig. 9 Microstructure of obtained materials after 7 days incubation in SBF (magnification: 100 00×).

RSC Advances Paper
a higher degradation rate silane coupling agents and the releasing of ion during its continuous hydrolysis in aqueous solutions. 63Ionic conductivity of all the obtained materials is in the range typical for a-TCP-based bone cements.It can therefore be concluded that the modication with silane coupling agents did not signicantly affect the degradation rate of bone cements.
The bioactive potential of obtained materials was estimated through its incubation in SBF. 62The SEM observations show that the evenly distributed, bone-like apatite layer was present on the sample's surfaces and cross-sections aer seven days of incubation in SBF at 37 °C (Fig. 9).The presence of apatite forms indirectly indicates the high bioactive potential of the obtained materials.

Conclusions
The positive effect of silane coupling agents on the mechanical behaviour of various biomaterials has been previously described, but there is little information about the effect of SACs on the properties of calcium phosphate bone cements.In this study, we developed and examined the novel, hybrid-type calcium phosphate bone cements based on a-TCP powders modied with silane coupling agents (SCAs)tetraethoxysilane (TEOS) or 3-glycidoxypropyl trimethoxysilane (GPTMS).Developed materials, due to their hybrid nature, combine the excellent biological properties and improved physicochemical properties.Application of SACs allowed to increase the compressive strength of the composites from 7.24 ± 0.35 MPa to 12.17 ± 0.48 MPa.The strengthening effect may be explained by the chemical interactions between the components during the simultaneous hydrolysis of a-TCP and silane coupling agents.It has been shown that SCAs during hydrolysis can react with the hydroxyl groups on the surface of calcium phosphates, forming covalent bonds (e.g.Si-O-Si, or Si-O-P).In addition, the silanol groups have the tendency to form hydrogen bonding between each other.Furthermore, the results of our studies revealed that modication of a-TCP by SCAs inuenced the setting process of bone cements.The decrease of nal setting time from 11.0 to 6.5 minutes was observed.SEM observations showed that all of the developed bone substitutes have a homogenous microstructure.All of the studied materials possessed a bioactive potential, proven in in vitro studies in simulated body uid.By using the silane coupling agents, we developed materials containing silicon, which may positively impact their osteoconductivity.Based on the obtained results, it can be concluded that the most favourable enhancement of the physicochemical properties of the developed materials was achieved by the modication of a-TCP with 5 wt% GPTMS.
All presented ndings conrm the benecial inuence of silane coupling agents on the properties of calcium phosphatebased bone cements and pave the way to further in vitro and in vivo studies.Developed hybrid-type materials may be further modied, by introducing natural polymers, what should lead to creation of chemically coupled hybrid-type bone cements.

Fig. 1
Fig. 1 Specific surface area vs. amount of used silane coupling agent.

Fig. 2
Fig.2Powders size distribution of non-modified and modified a-TCP powders.

Fig. 3
Fig. 3 Diffractograms of obtained materials: powders (A), bone cements after setting and hardening in air for 7 days (B) and bone cements incubated in SBF for 7 days (C).

Fig. 5
Fig. 5 Raman spectra of obtained materials: non-modified powder and cements (A), TEOS-modified powder and cements (B) and GPTMSmodified powder and cements (C).

Fig. 6
Fig.6Compressive strength of obtained bone cements after 7 days of setting and hardening (*statistically significant difference, p < 0.01).

Table 1
Composition of the initial powders

Table 2
Composition of the bone cements

Table 5
Setting times of developed materials