Strontium-doped organic-inorganic hybrids towards three-dimensional scaffolds for osteogenic cells

Biomimetic organic–inorganic hybrid bioscaffolds are developed to complement or replace damaged fragments in bone tissue surgery. The aim of this work was to develop a simple and fast method to prepare composite material for bone engineering, avoiding time consuming and complex methodologies. The resulting materials (also called in this work as hybrid composites or hybrid scaffolds) have a three-dimensional macroporouspolymer-like network derived fromtriethoxyvinylsilane (TEVS) and 2hydroxyethylmethacrylate (HEMA) monomers, with incorporated calcium, strontium, and phosphate ions. The materials were fully characterized using FT-IR,biomineralization studies, scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy, scratch tests, Young‟s modulus and compressive strength tests, and gas physisorption. We report a comprehensive study on the in vitro effect of novel strontium doped materials on human bone cells.In vitro investigations were conducted using a normal human osteoblast cell line that mimics the cellular events of the in vivo intramembranous bone formation process. The materials do not have a negative impact on the survival of the normal human osteoblasts; moreover, materials doped with strontium show that not only are cells able to survive, but they also attach to and grow on a bioscaffolds surface. For this reason, they may be used in future in vivo experiments.


Introduction
Biomimetic materials with structures that have hierarchical interconnected pores and a network-like nature, have been intensively studied in recent years, because they form excellent scaffolds for biomedical applications, including controlled drug release, sophisticated substitutes for tissue engineering, bioresorbable structures, etc. Such scaffolds are used as tissue templates because they can simultaneously form an ideal structure that is necessary to ensure cell growth and viability [1]. Inside the body, such materials induce a complex series of physicochemical reactions that lead to the formation of an interfacial bone-like apatite layer [2,3]. The dissolution of biomaterial devoted to bone replacement induces ion exchanges with biological fluids (or their mimics) enabling the formation of a mineral phase(s), direct interfacial binding between bioactive material and the bone; it should be emphasized that a number of review papers described this issue [1,2]. Here, both textural properties and chemical composition must be chosen in such a way that the material might be used as an efficient implant, creating a strong interfacial bond with host tissues, and which is extremely important, stimulating bone cell adhesiveness, differentiation, and proliferation [4].
Strontium has been used as a medicinal additive for more than a hundred years and its use as a dietary supplement is reported as the way to maintain strong bones [5][6][7].
Since the development of strontium ranelate, which is a drug that reduces the incidence of fractures in patients with osteoporosis, there has been an increasing awareness of the biological role of strontiumespecially its role in bone and in interactions with calcium ions [8].Furthermore, in the literature there are several examples of various biomaterials, including scaffolds, containing Sr 2+ ions [9][10][11]. Among the trace metals present in human bone, strontium is mainly, but not solely, correlated with bone compression strength [12]. As an alkaline earth that is closely chemically related to calcium, strontium affects bone formation and resorption through direct and indirect effects on bone cells or bone minerals. It also has an anabolic activity that is very important from a bone balance point of view, as can be seen by comparing healthy and osteopenic skeletons [5,13].
There has been a greater interest in the biological role of strontium has arisen since the discovery of its beneficial effect on bone, involving the reduction of incidence of fractures in osteoporotic patients [14,15]. Osteoporosis is a disease that causes thinning of the bones with a reduction in bone mass due to depletion of calcium and bone protein. Hence, the amount of bone tissue in a certain volume of bone dramatically decreases. It has been shown that strontium, as a bone-seeking agent, inhibits bone resorption, decreases osteoclast activity, and inhibits bone-resorbing activity. In addition, strontium salts can increase bone mass without causing detectable bone mineralization defects [16][17][18][19][20]. The mechanism of the strontium action at the bone surface is not fully known, but some mechanisms are postulated. For instance, calcium receptors in some types of cell can be activated via the presence of Sr 2+ cations, resulting in the activation of secondary messenger molecules used in signal transduction in biological cells [16].
In this study, we investigated the in vitro effect of strontium-doped organic-inorganic scaffolds manufactured using the sol-gel process on biological responses associated with osteoblast viability. In this manner, we examined human bone cell behavior on three-dimensional polymer-like networks based on HEMA and TEVS, with incorporated Ca 2+ and PO 4 3− ions and, additionally, doped with strontium. Selected samples were also covered with synthetic bone-like hydroxyapatite in order to compare their in vitro behavior to new systems reported in this paper. To prepare mentioned above materials we used well-known monomers, e.g. HEMA which is safe for human and is already used in medicine [21,22]. We report on a simple and fast method to obtain interconnected porous scaffolds for bone engineering with mechanical properties similar to those in human bones. In addition, a desirable feature of the obtained formulations based on creating a strong interfacial bond with host cells and tissues was studied on a normal human osteoblast cell line (NHOst), which mimics the cellular events of the in vivo intramembranous bone formation process. According to our best knowledge, such formulations possessing this sort of matrix with mentioned above dopants have not been studied so far, both taking into consideration their chemical and mechanical properties, and also in vitro studies.

The Molisch test
In a standard procedure, 0.5 mL of the filtrate was mixed with 5 mL of cold 75% H 2 SO 4 .
Three drops of a 3% α-naphthol solution in ethanol were added to the acid mixture. A yellow color is produced by the addition of the naphthol. After this, the mixture was warmed up on a water bath at 80 °C. Depending on the amount of carbohydrates, a red to blue-violet color appears throughout the whole mixture. In the absence of carbohydrates, the examined solution remains yellow.

Biomineralization assay
The biomineralization process was performed by soaking the hybrid samples in Dulbecco"s modified Eagle"s medium (DMEM), maintaining the solution at 37 °C. The process was conducted using a static protocol; this means that the DMEM was introduced once, without exchanging the biological fluid in the container. Samples after exposition in DMEM were washed out using distilled water (three washes with 5 mL), to remove any water-soluble inorganic salts, and then using acetone (three washes with 5 mL) and dried under compressed air. The newly formed inorganic layer was characterized using FT-IR spectroscopy, X-ray powder diffraction, energy dispersive Xray spectroscopy, and elemental analysis. The infrared spectra and elemental analyses were measured on approximately 1 mg of material directly scraped from the samples" surfaces. The X-ray powder diffraction and energy dispersive X-ray spectroscopy measurements were carried out on the surfaces of the samples. The ion concentration in the biological medium was measured for different soaking times (0.5, 1, 6, and 12 h, and 1, 2, 3, 4, and 7 days), using inductively coupled plasma analysis. Additionally, X-ray powder diffraction for all samples was performed after 7-9 weeks of soaking in DMEM solution, to confirm the nature of the mineral phase that crystallized or precipitated on the materials" surfaces. Parafilm, to reduce the diffusion of ammonia, and placed in a sealed chamber (volume, 1 L). After 48 h, the scaffolds were removed from the solution, washed out using distilled water (three washes of 15 mL) and dried under vacuum at 60 °C. The composition of the synthetic hydroxyapatite was confirmed using FT-IR spectroscopy and X-ray powder diffraction.

Young's modulus and compressive strength
The Young"s modulus and compressive strength were determined for monoliths and scaffolds for both 1HCP and 1HCPS systems. These preliminary studies were carried out to determine the materials" utility as potential implants in bone tissue engineering.
The collected data were compared with results obtained for cortical [23][24][25][26] and cancellous bone [27][28][29][30] in the case of monoliths and scaffolds, respectively. The mechanical tests were performed with ElectroForce 5100 BioDynamic Test Instrument from BOSE company and using a TA-XT texture analyzer (TA Plus, Lloyd Instruments) equipped with mechanical grips. Test instruments provide accurate characterization of biomaterials and biological specimens. They can be used for the evaluation of a variety of specimens, including biomaterials, cellular and cell-seeded scaffolds, native tissue samples and tissue-engineered constructs. The obtained data were average value of measured factor ± standard deviation. All samples were measured at least 7 times.

Contact angle and surface free energy measurements using the Owens-Wendt method
The surface free energy (ɣ) and its dispersive (ɣ d ) and polar (ɣ p ) components were determined for the obtained materials using contact angle measurements and the Owens-Wendt method [31,32]. The aqueous contact angle analysis was carried out using sessile drop method and a DataPhysics OCA20 instrument to determine the wettability of the resulting coatings. The averages of at least five readings taken at different locations on each sample surface were calculated.

Scratch tests
Scratch tests were performed on a glass surface covered by a thin film of resulting materials. A pencil hardness test was carried out according to Chinese standard GB/T6739-2006.

Human osteoblast cell culture
Cells from NHOst were cultured in osteoblast growth basal medium supplemented with 10% fetal bovine serum, 0.1% ascorbic acid, and 0.1% gentamycin. The cell line and supplements were obtained from Lonza, Belgium. The adherent cells were cultured in 75 cm 2 flasks (Becton Dickinson, USA). The cells were maintained at 37 °C in a fully humidified atmosphere at 5% CO 2 in air (Hera Cell Heraeus, Germany). The cell line populations used in this experiments were taken between passages 5 and 6.

Cytotoxicity evaluation of hybrid biocomposites on the NHOst cell line
In this studies both monoliths and foam-like samples were considered. Cylindrical specimens from each type of material with a "diameter x height" dimensions: 6 mm x 2 mm and 12 mm x 10 mm for monoliths and scaffolds, respectively were investigated. In the SRB assay 1 sample (monolith or scaffold) per well of plate was added.Cells in the exponential growth phase were seeded at 0.2 × 10 6 cells/well on 24-well plates (Becton Dickinson, USA), and then incubated for 24 h. On the second day, after fixing the cells, the culture medium was refreshed and the following biocomposite samples were placed on the cells: 1HCP, 1HCP_H, 1HCPP, 1HCPS, 1HCPS_H, and 1HCPSP. The plates were incubated for 72 h. After this time, a sulforhodamine B assay (SRB assay) (Sigma, USA) was performed [33], to measure cell survival, after prior fixation of cells with trichloroacetic acid (Sigma, USA) [34]. Absorbance at a wavelength of 562 nm was measured on a plate reader (Tecan Infinite M200 Pro, Austria). Three independent repetitions of each test were performed.

Preparation of NHOst cell line for SEM
Prior to seeding the cells on the hybrid biocomposites, samples of 1HCPS were incubated for 24 h in osteoblast growth basal medium (Lonza, Belgium). The next day, cells at the exponential growth phase were seeded at 10 5 cells per sample and placed in a 12-well plate (Becton Dickinson, USA). This seeding procedure was repeated twice more, on days 9 and 16 of incubation. Throughout the whole experiment, the medium was changed every 2-3 days. After 20 days, the cells were fixed for SEM.

Statistical analysis
All results were obtained by performing three independent replicates of each experiment. The results are expressed as means ± standard deviations. Statistical analysis was carried out using Dunnett"s multiple comparison test. Values of P < 0.05 were considered statistically significant.

Synthesis of organic-inorganic hybrid materials
Organic-inorganic hybrid materials were synthesized using the sol-gel process. The Moreover, silanols are able to promote the exchange of ions between the composite and the outer environment. As a consequence, the formation of various inorganic salts, such as phosphates and carbonates, is possible [35]. It is worth noting that the crystallization of such salts initiates bone growth and enables the treated bone fragment to recover. This also has a positive impact on cellular behavior and activity [37].
During syntheses, inorganic salts such as calcium and strontium chlorides and triethylphosphate were added. These salts constitute a source of calcium Ca 2+ , strontium Sr 2+ , and phosphate PO 4 3-

Structure of hybrid scaffolds
Cancellous bone is a highly porous structure with a wide distribution of pore sizes and wall thicknesses [27][28][29][30]. The specific three-dimensional scaffold with a highly interconnected macroporous network and tunable mechanical properties is highly desirable in various medical applications [38]. The porosity and pore size of bioscaffolds play a crucial role in bone formation [39]. For pore sizes larger than 50 µm, tissue mineralization is observed; for bigger pores (>100 µm) this process extends to a depth of 1000 µm and there is a good chance that hard tissue can be firmly rebuilt. Moreover, the roughness of the scaffold"s surface is indispensable for efficient cells" attachment, both in vitro and in vivo. It strongly affects cellular morphology, proliferation, and phenotype expression [40].
In this study, the resulting organic-inorganic hybrid materials constitute a perfect platform in the formation of biomimicking scaffolds for hard tissue engineering. Porous structures were created using sugar templates. This easily-leachable and biocompatible porogen enables the formation of macropores of 150-350 µm in size that guarantee natural tissue ingrowth, mass transport, osteoinductive agent attachment, and ion exchange [41] (Fig. 3).
The total porous fraction for pure 1HCPP and 1HCPSP was approximately 90%, corresponding to an apparent density of ~0.25 ± 0.01 g cm −3 . In turn, the skeleton densities of the scaffolds were 2.488 ± 0.009 g cm −3 and 2.479 ± 0.008 g cm −3 for 1HCPP and 1HCPSP, respectively.
In addition to macroporosity, mesoporosity studies for both systems were also performed. Here, the Brunauer−Emmett−Teller surface area, average and modal pore diameters, and total pore volume were calculated. Surface areas were measured using the Brunauer-Emmett-Teller models for relative pressures of 0.05 < p/p 0 < 0.30. The surface areas were of the order of 21 m 2 ·g−1 for both 1HCPP and 1HCPSP. By comparison with, e.g., macroporous bioactive glasses [42], these surface areas are smaller, probably arising from the deeper packing inside the network. Surface area also affects nanopore sizes. Analysis of nanopore size distribution was based on the Horvath−Kawazoe model. Nanopores were present in a wide range of sizes from 1.6 to 2.1 nm for both materials. It should be noted, as a general comment, that in vitro lower porosity effectively stimulates osteogenesis by suppressing cell proliferation and forcing cell aggregation. Conversely, such a trend results in diminished mechanical properties, thereby setting an upper functional limit for pore size and porosity [40].
The roughness of the scaffolds" surface was achieved via the addition of ammonia carbonate to sugar porogen. During decomposition of carbonate salt at about 60 °C, gaseous carbon dioxide and ammonia are spontaneously liberated. Such vapor products play a dual role. While CO 2 serves as an agent that additionally opens noninterconnected macropores, the released NH 3 provides roughness of the surface. This is clearly shown in Fig. 4.

Biomineralization
The resulting organic-inorganic hybrids were soaked in biological medium ( In this study, biological medium was used because it additionally contains amino acids and vitamins and provides better biomimetic conditions. However, owing to the presence of various proteins in this solution, lower rates for the material"s dissolution and a subsequent delay in surface layer formation are expected [43]. The dissolution experiments revealed that Ca 2+ cations were released from the material in a different way for each of the 1HCPP and 1HCPSP systems (Fig. 5). In the During biomineralization, changes in pH were also observed (Fig. 6). In general, pH for 1HCPP is slightly higher than for 1HCPSP. This closely corresponds with the amount of PO 4 3− anions released into the solution. In the case of 1HCPP, the PO 4 3− concentration is higher. Phosphates undergo hydrolysis and this leads to a decrease in H + concentration and increasing pH values. This may also confirm that, in the case of 1HCPSP, the surface is more favorable to phosphate salts than carbonates. This phenomenon is also confirmed by X-ray powder diffraction analysis of the precipitate scraped away from the scaffolds after biomineralization, e.g. diffraction peaks assigned to a bone-like apatite are observed at about 31.9 and 46.6°.

Synthetic hydroxyapatite
Owing to its chemical similarity to the mineral component of the bone [38], hydroxyapatite is considered one of the most important materials in biomedical applications. Synthetic hydroxyapatite (Ca 10 (PO 4 ) 6 (OH) 2 ) is a material that has been successfully used as a filler of small bone defects and coating material to improve the biocompatibility of various implants. The particular interest in hydroxyapatite is for use as a bone substitute in dental and orthopedic implants, but because of its poor mechanical properties it cannot be applied to highly loaded components. In such cases, hydroxyapatite can only be used as a coating to improve materials properties [46].
In this study, synthetic hydroxyapatite was used to cover the synthesized scaffolds. As shown in Fig. 7, synthetic hydroxyapatite ideally covers the scaffold"s surface. It is worth noting that the macropores are not closed or blocked, so do not preventing mass transport inside the network.

Properties of the organic-inorganic hybrid system
These studies are preliminary in nature, to collect data concerning the properties of the resulting materials. Depending on the properties to be determined, samples were examined in the form of thin films, monoliths, or scaffolds. Only pure samples were used in these studies. No samples after biomineralization or coating with synthetic hydroxyapatite were analyzed.
Compressive strength and Young"s modulus were studied for both 1HCPP and 1HCPSP. Studies were performed for two different structures, monoliths and threedimensional scaffolds. To obtain monoliths, composites were collected into polypropylene molds directly after synthesis and cured at 65 °C for 12 h. Scaffolds were obtained as described in Experimental Section. The results are shown in Table 2 and The mechanical properties of monoliths are similar to those observed for cortical bone, whereas scaffolds seems to be perfect candidates for sophisticated cancellous bone replacements [24,38,47]. The values of Young"s modulus and compressive strength are better than those observed for tricalcium phosphate and its composites with 2-hydroxyethylmethacrylate, 3-methacryloxypropyltrimethoxysilane, and methacrylate [48][49][50][51][52][53]. It should be noted the target mechanical properties which have been explicitly stated in the literature, span several different approaches which have been taken with regard to the design for mechanical properties. Some of them have stated that bone scaffold properties should match those of natural bone [54]. Moreover, such scaffold must be sufficient and not collapse during handling and patient"s normal activity [55].
The results shown in Table 3 demonstrate that the presence of Sr 2+ -dopant in the organic-inorganic hybrid composition had some influence on the wettability of the resulting coatings. The systems with Sr 2+ ions exhibited smaller contact angles than the systems without. It should be emphasized that in the case of cells and proteins, an adhesion contact angle of ~65° distinguishes between hydrophobic and hydrophilic surfaces [56]. Here, additional factors, such as roughness and composition of the resulting materials may have a dominant influence on cell behavior.
The synthesized hybrids can also be used as coatings on various bioinert materials, such as metallic or ceramic implants. With this use in mind, the scratch test of the resulting coatings was evaluated to determine their resistance to scratching [57][58][59]. We employed a calibrated set of drawing pencils ranging from 9B, as the softest grading, through B, HB, F and H, to the hardest grading, 9H. Surprisingly, the hardness of 1HCP and 1HCPS differs drastically. The surface preparation and coating process were the same in both cases. 1HCP seems much softer material than 1HCPS; their values in the pencil scratch hardness tests were "2B" and "8H", respectively. Studies were conducted with ten independent repeats and the results were recurrent. Unfortunately, at this stage of studies it is hard to irrefutably clarify the observed values or draw any appropriate conclusions.

Evaluation of cytotoxicity of hybrid biocomposites on human osteoblast cell line
The  These materials possess a sufficient shelf life (~6 months), in the liquid form before curing, which is important for an easy and wide distribution. The incorporation of Sr 2+ ions into the hybrid scaffolds was non-cytotoxic to the osteoblast cells. Moreover, obtained materials create a favourable rough topography which is friendly for cellular attachment and growth on the material surface. For this reason, they may be used in future in vivo experiments. Moreover, it should be noted that formulations possessing this sort of matrix with mentioned above dopants have not been studied so far, both taking into consideration their chemical and mechanical properties, and also in vitro studies. Figure 1. Ramified structure of the organic-inorganic hybrid network.