In vitro osteogenesis of mesenchymal stem cells promoted by the release of La³⁺ and SiO₄⁴⁻ from sheet-shaped lanthanum dropped hexagonal mesoporous silicon

Abstract

In this study, in vitro osteogenesis was successfully promoted in mouse mesenchymal stem cells (BMSCs) by the release of La³⁺ and SiO₄⁴⁻ from a sheet-shaped lanthanum dropped hexagonal mesoporous silica (La-HMS). In comparison with previous nano-sphere shaped La-HMS, the sheet-shaped morphology was similar to the inorganic component of bone, which could provide better toughness during bone repair and regeneration. The successful incorporation of La³⁺ into the HMS was confirmed by the results of XRD and ICP-AES. In vitro proliferation was verified by CCK-8 and osteogenesis was verified by an alkaline phosphatase (ALP) activity assay, ALP staining, a calcium secretion assay and collagen secretion analysis. Considering the renewable source and highly proliferative nature of BMSCs, the achievement of engineered BMSC osteogenesis with this La-HMS could allow new applications in major bone repair and regeneration.

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1. Introduction

Lanthanides are a group of rare earth metals known for their potential medical applications¹ and diverse biological effects.^2,3 Lanthanide compounds have been widely used as diagnostic contrast media and have therapeutic applications.^4–6 Lanthanum (La, the first element of the lanthanide series) has been recognized as a “bone-seeking” element because a lot of its physicochemical characteristics are similar to calcium.^7–9 To affect bone minerals, the accumulated La³⁺ in bone may enter the crystal lattice of the hydroxyapatite or be deposited on the crystal surface. Since bone is a metabolically active tissue undergoing continuous remodeling consisting of osteoblastic bone formation and osteoclastic resorption, lanthanum could affect bone formation through interfering with cellular activities. To date, in vivo and in vitro studies on the effect of lanthanum on bone formation have been reported.^10–13 But there are still few reports mentioning osteogenesis of lanthanum and none regarding its use towards engineering bone formation. Owing to the possible osteogenic properties of lanthanum, if we could manage to engineer osteogenesis using it then this would be a great contribution to bone-related regenerative medicine.

In order to achieve efficient osteogenesis of BMSCs by La³⁺ treatment, a superior controlled release system for optimal La³⁺ loading and delivery is urgently required. To achieve the ideal release of La³⁺, the carrier must be able to ensure the retention of La³⁺ after loading and simultaneously avoid side effects such as cytotoxicity caused by a high La³⁺ dosage.¹⁴ It could be a good idea to incorporate La³⁺ into inorganic biomaterials, and release La³⁺ with these biomaterials’ degradation. Candidate materials could be calcium phosphate cements, hydroxyapatite, bioactive glass, mesoporous silica (MS), and so on.^15–18 Among them, silicon (Si) is a ubiquitous environmental element which is known to play an important role in connective tissue metabolism, especially in bone.^19–22 MS have been investigated as drug supports since they are nontoxic, and have been widely used as drug-delivery systems such as carriers for drug and gene release.^23–26 Hexagonal mesoporous silica (HMS), a typical mesoporous molecular sieve, shows great physical properties and a low degradation rate.^27,28 It was reported that bioactive glasses or MS showed significantly faster degradation and better bioactivity with the addition of some cations.^29,30 It seems reasonable to think that doping HMS with controlled amounts of La³⁺ could help to obtain an adjustable degradation performance, and has the advantages of achieving nano particles with biocompatible and osteogenic properties, with a possible capacity to deliver SiO₄⁴⁻ and La³⁺. Based on this evidence, La-HMS releasing SiO₄⁴⁻ and La³⁺ was proposed as a potential therapeutic measure for bone defects.

In this study, we fabricated and characterized a sheet-shaped La-HMS. A previously synthesized nano sphere-shaped La-containing hexagonal mesoporous solid was reported by Wusheng Guo.³¹ The inorganic part of a human bone, with a major portion of bone containing Ca-deficient hydroxyapatite in the form of a plate-shape, was 2–3 nm in thickness and tens of nanometers in length and width.^32,33 Compared with the sphere-shaped morphology, the newly synthesized sheet-shaped morphology was similar to the inorganic part of bone, which could provide better toughness during bone repair and regeneration. There has not yet been a study documenting the osteogenic effects of mouse BMSCs towards La-HMS. The physicochemical properties of La-HMS were characterized by different techniques such as transmission electron microscopy (TEM) and X-ray diffraction (XRD). Furthermore, we investigated the cytotoxicity and ALP activity, as well as carrying out ALP staining, a calcium secretion assay and collagen secretion analysis in vitro for the La-HMS.

2. Materials and methods

2.1 Materials

All reagents used in the La-HMS synthesis process were purchased from Tianjin DaMao Chemical reagents plant (China). Cell culture media and fetal bovine serum (FBS) were purchased from GIBCO (Carlsbad, CA, USA). CCK-8 was obtained from Dojindo (Kumamoto, Japan).

2.2 Preparation of HMS and La-HMS particles

HMS was synthesized based on the traditional process.³⁴ La-HMS was prepared as follows: dodecylamine (DDA) was dissolved in an ethyl alcohol (EtOH)–deionized water solution (pH = 9). Subsequently, tetraethoxysilane (TEOS) with different amounts of La(NO₃)₃·6H₂O (0.5 g, 1.0 g, 1.5 g and 2.0 g) was added as a lanthanum and silica source and the mixture was stirred at 350 rpm. The reaction mixture conformed to the following molar composition: TEOS: 1.0, DDA: 0.27, EtOH: 9.09, H₂O: 29.6. The mixture was stirred for 1 h, and then aged for 18 h at room temperature. The product was dried at 90 °C for 2 h, and then the DDA template was removed by EtOH extraction and calcination. The La-HMS products with 0.5 g, 1.0 g, 1.5 g and 2.0 g of added lanthanum sources were marked as La-HMS-1, La-HMS-2, La-HMS-3 and La-HMS-4, respectively.

2.3 Characterization

X-ray diffraction was carried out using a Rigaku D/max-2200 (Japan, Cu Kα, γ = 1.54056 nm) with a voltage of 40 kV and a current of 30 mA. The diffraction pattern was collected over the 2θ range of 1–10° with a scanning speed of 1° min⁻¹ and a step size of 0.02°. The morphology was characterized using scanning electron microscopy (SEM, 30XLFEG, Philips, The Netherlands) and HRTEM (JEM2011, Japan) with an accelerating voltage of 50–200 kV, a point resolution of 0.23 nm and a lattice resolution of 0.14 nm. The La³⁺ and SiO₄⁴⁻ release from the samples were measured using inductively coupled plasma-atomic emission spectrometry (ICP-AES, IRIS 1000, Thermo Elemental, USA). N₂ adsorption–desorption isotherms were obtained on a Micromeritics Tristar 3000 pore analyzer under continuous adsorption conditions. The Brunauer–Emmett–Teller (BET) and Barrett–Joyner–Halenda (BJH) methods were used to determine the surface area, the pore size distribution and the pore volume.

2.4 Cell culture

Mouse mesenchymal stem cells (BMSCs) were purchased from the American Type Culture Collection (ATCC, Manassas, VA). BMSCs were enzymatically lifted from culture dishes with trypsin/EDTA (0.125% and 0.53 mM, respectively), and centrifuged for 5 min at 250 rpm. The cells were resuspended in fresh culture medium, and then seeded on HMS and La-HMS-1–4.

2.5 Cytotoxicity assay

Cells were seeded in 96-well plates (Costa, Corning Incorporated) at a density of 2 × 10³ cells per well for cell proliferation studies. A CCK-8 assay was used to measure the cell proliferation at days 1, 3 and 7 after the BMSCs were seeded on HMS and La-HMS-1–4. At specified time intervals, the medium was removed and the wells were washed with PBS twice. 90 μl of fresh culture medium and 10 μl of CCK-8 solution were added to each well and the plate was incubated for 2 h. The absorbance was determined at 450 nm by a microplate reader (Thermo3001, USA).

2.6 Quantification of alkaline phosphatase (ALP) activity and ALP staining

Cells were seeded in 48-well plates (Costa, Corning Incorporated) at a density of 4 × 10⁴ cells per well and cultured in osteogenic media (OGM) for studies on cell osteogenic differentiation. Alkaline phosphatase (ALP) activity was determined using a pNPP assay (p-nitrophenyl phosphate liquid substrate, Sigma Diagnostics). Briefly, cells cultured in 48-well plates (Costa, Corning Incorporated) at each time point were washed gently with cold PBS, followed by lysing with 1 × lysis buffer with 0.1 M glycine, 1 mM MgCl₂ and 0.1% Triton X-100. To extract cell layers, cells were sonicated twice for 30 s and centrifuged at 12 300 rpm for 2 min at 4 °C. The supernatants were assayed for ALP activity as described previously.³⁵ ALP values (U μg⁻¹) were normalized to protein content, which was measured with a Pierce Micro BCA Protein Assay Kit (Pierce).

ALP activity on the cell layers was measured using ALP staining. Briefly, cultured cells were rinsed with PBS and fixed in 10% neutral formalin solution for 30 min. The cells were stained using BCIP/NBT as a dye for 30 min at 37 °C, followed by washing with distilled water.

2.7 Alizarin red staining

Osteogenesis mineralization was assessed by alizarin red staining at day 14 after the initial osteogenic induction. Briefly, cultured cells were rinsed three times with PBS and fixed with 10% neutral formalin solution for 30 min, carefully washed five times with distilled H₂O and then stained with ARS (40 mM) for 30 min at room temperature. After several washes with distilled H₂O to remove excess dye, scaffolds were examined under an optical microscope.

2.8 Collagen secretion

Collagen secretion by the cells on the samples was assessed by Sirius Red staining at day 21.³⁶ After washing with PBS and fixing, the constructs were stained using 0.1% Sirius Red (Sigma) to reveal the collagen. The unbound stain was removed with 0.1 M acetic acid before the images were taken.

2.9 Statistical analysis

Six completely independent experiments were performed for every assay and the results were expressed as means ± standard deviations. Statistical significance was calculated using one way analysis of variance (one-way ANOVA). Comparison between the two means was performed using Tukey’s test and the data were determined to be statistically significant at p < 0.05.

3 Results

3.1 Characteristics of La-HMS

3.1.1 Morphology determination. The morphology and structure of the HMS and La-HMS were investigated by SEM and HRTEM. The representative SEM images in Fig. 1 demonstrate the surface structure. The HMS and La-HMS had an irregular sheet-shaped surface morphology. The representative HRTEM images of HMS and La-HMS given in Fig. 2 show their hive or sponge-like porous structure. Highly ordered mesoporous structure can be clearly seen in the HMS (Fig. 2A). Similar behavior was observed in the La-HMS-2 (Fig. 2B), further confirming that the partial substitution of La for Si in HMS does not destroy the mesoporous structure.

Fig. 1 SEM images of HMS (A) and La-HMS-2 (B) (bar = 3 μm).

Fig. 2 HRTEM images of HMS (A) and La-HMS-2 (B) (bar = 50 nm).

3.1.2 XRD. To further confirm the incorporation of La³⁺ into the HMS, XRD was used to characterize the crystalline structure of the HMS and La-HMS. As shown in Fig. 3, the XRD patterns of the HMS and all the La-HMS samples exhibit a single d(100) reflection as typical HMS system mesoporous materials. The peak of the d(100) reflection for the La-HMS was higher when less of the lanthanum source was added.

Fig. 3 The XRD patterns of HMS and La-HMS-1–4.

3.1.3 N₂ adsorption–desorption. N₂ adsorption–desorption isotherms and the corresponding pore size distributions of HMS and La-HMS-1–4 are shown in Fig. 4. The data for the surface area, pore volume and peak pore size are listed in Table 1. It can be seen that the substitution of La for Si in HMS has not changed the mesoporous structure, which is consistent with the result of the HRTEM analysis. The type IV isotherms indicate the p6mm mesoporous structure of the HMS and La–HMS-1–4 (Fig. 4A). The HMS had a surface area of 1299.4 m² g⁻¹, while the surface areas of La-HMS-1, La-HMS-2, La-HMS-3 and La-HMS-4 were 849.2, 806.9, 727.5 and 533.1 m² g⁻¹, respectively. The total adsorption volume at P/P₀ = 0.97 was 0.668 cm³ g⁻¹ for HMS, 0.652 cm³ g⁻¹ for La-HMS-1, 0.659 cm³ g⁻¹ for La-HMS-2, 0.583 cm³ g⁻¹ for La-HMS-3 and 0.508 cm³ g⁻¹ for La-HMS-4. The pore size distribution curves in Fig. 4B were calculated from the adsorption branches using the BJH model. The peak pore sizes of all samples were between 2 and 3 nm.

Fig. 4 N₂ adsorption–desorption isotherms (A) and the corresponding pore size distributions (B) of HMS and La-HMS-1–4.

Table 1 Structural parameters of HMS and La-HMS-1–4

Sample	SBET (m^2 g^−1)	Vp (cm^3 g^−1)	Dp (nm)
HMS	1299.4	0.668	2.06
La-HMS-1	849.2	0.652	3.07
La-HMS-2	806.9	0.659	3.26
La-HMS-3	727.5	0.583	3.2
La-HMS-4	533.1	0.508	3.81

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3.2 Degradation behavior

3.2.1 ICP-AES. The degradation behavior was observed over 3 weeks to allow the release of SiO₄⁴⁻ and La³⁺. The ion release from 10 mg ml⁻¹ HMS and La-HMS-1–4 was investigated in Tris–HCl solution after immersion for various time periods. La-HMS-1–4 immersed in Tris–HCl solution showed a gradual increase in La³⁺ and SiO₄⁴⁻ concentration, and a higher amount of La³⁺ incorporated into the La-HMS always gave rise to higher La³⁺ and SiO₄⁴⁻ concentrations during the examined time interval (Fig. 5). Compared with La³⁺, the SiO₄⁴⁻ concentration increased slightly at a slow rate with increasing immersion time up to 3 weeks. For example, with La-HMS-2, the La³⁺ and SiO₄⁴⁻ concentrations changed from 4.88 ± 0.53 to 11.93 ± 0.45 ppb, and 61.48 ± 0.41 to 66.22 ± 0.25 ppm, respectively.

Fig. 5 Release of La³⁺ (A) and SiO₄⁴⁻ (B) from HMS and La-HMS-1–4 into the Tris–HCl solution versus soaking time.

3.2.2 pH value. The pH change in the Tris–HCl solution (pH = 7.4) incubated with 50 μg ml⁻¹ HMS and La-HMS-1–4 was also evaluated. During the incubation of HMS, the pH value in the Tris–HCl solution decreased to about 7.38 after 3 weeks (Fig. 6). At the same concentration, when more of the lanthanum source was added, the pH value in the Tris–HCl solution incubated with La-HMS increased.

Fig. 6 Change in the pH value of the Tris–HCl solution during the degradation of HMS and La-HMS-1–4.

3.3 In vitro cell investigation

3.3.1 Cytotoxicity properties of La-HMS. The CCK-8 assay was used to measure the cytotoxicity of HMS and La-HMS-1–4. According to the CCK-8 measurements, the cell viabilities after treatment with the prepared La-HMS with varying amounts of La³⁺ showed similar trends after 1 day, 3 days and 7 days of exposure (Fig. 7). As shown, compared with the untreated control (cells cultured on TCPS (tissue culture polystyrene plates) without materials), after treatment with a high concentration of La-HMS-1–4 (50 μg ml⁻¹), the cell viability of BMSCs significantly decreased, in particular after 3 days and 7 days of exposure (Fig. 7B and C). The inhibition efficiency was particularly enhanced when the amount of incorporated La³⁺ was increased. But the viabilities of cells treated with 5 μg ml⁻¹ and 0.5 μg ml⁻¹ of La-HMS-1–4 were close to or higher than the untreated control in all cases, which suggested that they had good cytocompatibility. And compared with the 5 μg ml⁻¹ group, the 0.5 μg ml⁻¹ group seem to have better cytocompatibility.

Fig. 7 In vitro assessment of the cytotoxicity of HMS and La-HMS-1–4 to BMSCs. The cells were seeded on HMS and La-HMS-1–4 at concentrations ranging from 0.5 to 50 μg ml⁻¹ for 1 (A), 3 (B) and 7 (C) days. The cytotoxicity was evaluated by the CCK8 assay. (*) indicates statistical significance when compared with the untreated control (cells cultured on TCPS without materials) (p < 0.05).

3.3.2 Osteogenic properties of La-HMS. In this study, BMSCs were made to undergo osteogenic differentiation. After confirming the better cytocompatibility of 0.5 μg ml⁻¹ La-HMS-1–4, the osteogenic properties of 0.5 μg ml⁻¹ HMS and La-HMS-1–4 were also evaluated. The secretion of ALP, a standard marker of osteogenic differentiation, was determined by the pNPP assay (Fig. 8). Positive ALP production was detected at 7 and 10 days exposure, followed by significant downregulation at 14 days. BMSCs cultured on La-HMS-2 always gave rise to the highest ALP activity during the examined time interval, and displayed significantly higher levels of ALP secretion compared to the control group at 10 days. When the amount of La³⁺ incorporated in the La-HMS was decreased or increased compared to La-HMS-2, the ALP activity decreased. Consistent with the quantitative analysis, direct staining of intracellular ALP on the cell layers was carried out on day 10 and the staining on the La-HMS-2 was significantly denser than that on the other groups (Fig. 9), which is in agreement with the quantification of ALP activity.

Fig. 8 Alkaline phosphatase (ALP) activity for BMSCs in the presence or absence of 0.5 μg ml⁻¹ HMS and La-HMS-1–4 for 3, 7 and 10 days. (*) and (#) indicate statistical significance when compared with the control (cells cultured on TCPS without materials and only treated by OGM) and HMS, respectively (p < 0.05).

Fig. 9 ALP staining of BMSCs after 10 days of incubation. BMSCs treated by OGM without materials (A), OGM with 0.5 μg ml⁻¹ HMS (B) and OGM with 0.5 μg ml⁻¹ La-HMS-1–4 (C–F) (bar = 200 μm).

An important function of osteogenic cells is their participation in biological mineralization. Calcium deposition by osteogenic cells was assessed by alizarin staining at day 14 (Fig. 10). After 14 days of culture, a higher level of calcium secretion was observed from cells cultured with HMS and La-HMS-1–4 in OGM when compared with the control group. BMSCs cultured on La-HMS-2 gave rise to the most calcium secretion.

Fig. 10 Mineralization and calcium secretion by BMSCs after 14 days of incubation. BMSCs treated by OGM without materials (A), OGM with 0.5 μg ml⁻¹ HMS (B) and OGM with 0.5 μg ml⁻¹ La-HMS-1–4 (C–F) (bar = 200 μm).

Collagen secretion assayed by Sirius Red staining is shown in Fig. 11. The optical images show that the BMSCs secrete abundant collagen on all groups after 3 weeks. More collagen is secreted on the La-HMS-2 compared to the others. Incorporation of a suitable amount of La³⁺ into HMS increases collagen secretion, which is in agreement with the quantification of ALP activity, ALP staining and alizarin red staining.

Fig. 11 Collagen secreted by BMSCs after 21 days of incubation. BMSCs treated by OGM without materials (A), OGM with 0.5 μg ml⁻¹ HMS (B) and OGM with 0.5 μg ml⁻¹ La-HMS-1–4 (C–F) (bar = 100 μm).

4. Discussion

Over the past decades, given the frequency and relevance of pathological situations of bone, attention to the regeneration and healing of this tissue has grown. Significant progress has been made in the development of surgical techniques for skeletal reconstruction. As the first element of the lanthanide series, lanthanum has a preferential uptake in bone due to its marked bioinorganic similarity to Ca²⁺ in ionic radii and coordination properties.^7–9,37 Low doses of La³⁺ could improve the osteogenesis of cells through interfering with cellular activities, Xi Wang et al. found that La³⁺ at a concentration of 1 × 10⁻⁶ mol l⁻¹ could promote osteoblast activity through the phosphorylation of FAK and reorganization of the cytoskeleton,¹⁰ and promote osteoblast proliferation and differentiation in vitro via a pertussis toxin-sensitive Gi protein and the ERK signaling pathway.¹² Dawei Zhang et al. reported that 1 × 10⁻⁵ mol l⁻¹ La³⁺ could significantly increase the ALP activity of rat calvarial osteoblast-like cells.³⁸

However, high doses of La³⁺ may cause toxicity to cells or retard bone maturation, which has reduced its application for bone repair. In the previous studies, the authors mainly investigated the effect of lanthanum chloride or lanthanum nitrate solutions on cell proliferation or osteoblastic differentiation, and there were few reports on the effects of lanthanum doped materials on cells. Data on the biocompatibility of La³⁺ were limited, sometimes with controversial findings.^11,39,40 So, in this study, we evaluated the cytotoxicity of La-HMS with five different amounts of incorporated La³⁺ and at three different concentrations. La-HMS-1–4 at 50 μg ml⁻¹ exhibited toxicity to the BMSCs and the toxicity was enhanced when the amounts of incorporated La³⁺ were increased. This may be due to the higher La³⁺ concentration released from higher concentrations of La-HMS (Fig. 5A), which will cause some cytotoxicity to BMSCs. All of the ionic products (SiO₄⁴⁻ and La³⁺) from 0.5 μg ml⁻¹ La-HMS-1–4 were non-cytotoxic to surrounding tissues after immersion in our study. These results are consistent with others' findings, in that the viability of osteoblasts was suppressed after incubation with La³⁺ at high concentrations.¹² We may surmise that when the La-HMS concentration is decreased to a low value, it will have good cytocompatibility.

ALP is an important marker for the osteogenesis of MSCs.⁴¹ It specifically degrades the organic phosphoesters in bone and cartilage, which promotes calcium deposition in bone and inhibits cartilage mineralization.⁴² Expression of ALP increases before mineralization, and subsequently decreases after the initiation of mineralization. In our study, the ALP activity decreases at day 14, which may be because the mineralization period has begun, which is in agreement with others' studies.^43–45 The deposition of a calcified mineralized matrix is universally recognized as an optimal indicator of the maturation of the osteoblast phenotype derived from the differentiation of BMSCs. Like most MSC cultures, the cells showed mineralization after culture in OGM for 14 days. The initial stage of collagen biosynthesis is critical to the development of mature bone tissue. The strongest ALP staining, alizarin red staining and Sirius Red staining in the cells treated by La-HMS-2 supports its increased osteogenesis promoting properties (Fig. 8–11), indicating that only a suitable amount of La³⁺ incorporated in La-HMS could effectively promote the osteogenic properties of BMSCs. The osteogenic outcome from La-HMS-2 could also be mainly attributed to its chemical composition, the ionic products of La-HMS degradation (i.e. La³⁺ and SiO₄⁴⁻) are known for their beneficial role in increasing the ALP activity, calcium secretion and collagen secretion involved in bone growth.^10,46 In our study, from the ICP-AES data, the ALP activity, calcium secretion and collagen secretion of BMSCs treated by HMS was higher than the control group but lower than the La-HMS-2 group at each time point, suggesting that the promotion of osteogenic commitment by BMSCs might also come from SiO₄⁴⁻. Si was reported to have mitogenic effects on osteoblasts and to stimulate collagen I synthesis,^47,48 which play a supporting role here. The osteogenic markers of BMSCs treated with La-HMS-2 and OGM indicate successful osteogenic induction and may provide a foundation for developing a brand new osteogenic medium recipe based on La-HMS-2 and OGM for BMSCs.

Because of the calcination process for the removal of the surfactant, La-HMS exhibits a low degradation rate.⁴⁹ The degradation of pure sintered MS is quite slow due to its network structure. Shula Radin et al. reported that the degradation rate of silica-based materials was related to the composition.⁵⁰ In this work, we adjusted the amount of La³⁺ doped in HMS to obtain an adjustable degradation performance. Incorporating more La³⁺ into the HMS reduced the order of La-HMS (Fig. 3), reduced the surface area and pore volume, increased the pore size (Fig. 4), and released more ions (Fig. 5). Comparing La-HMS-4 with HMS, the intensity of the d(100) reflection of the HMS was almost more than three times that of La-HMS-4, which suggested that the degree of order of the HMS was significantly higher than the La-HMS-4. The surface area of the La-HMS-4 exhibited a relatively large decrease compared to the HMS. The La³⁺ released from La-HMS-4 was significantly higher than the other three groups at all time points. When more of the lanthanum source was added, more La was substituted for Si, more Si–O–La bonds were formed and the T–O–T bond angle was changed, causing a large number of defects in the mesoporous framework due to the difference in atom diameter, which made the degree of order decrease in the La-HMS and resulting in enhanced degradation and ion release from the La-HMS, which is in agreement with other studies.^15,28,51 The right concentration of La-HMS-2 had the capacity to deliver suitable doses of La³⁺ and SiO₄⁴⁻, which had good biocompatibility and could effectively promote the osteogenic commitment of BMSCs.

Besides releasing suitable amounts of La³⁺ from La-HMS to promote the osteogenesis of BMSCs, the sheet-shaped La-HMS also takes on another active role. Bone is an organic/inorganic composite with the inorganic apatite phase having a plate-shaped morphology. Compared with the previously reported sphere-shaped morphology, this newly synthesized sheet-shaped morphology is similar to the inorganic apatite phase of bone, which is more suitable for bone repair. Furthermore, with more of the lanthanum source added, the pH value in the Tris–HCl solution increased. We may surmise that increasing the amount of La³⁺ incorporated in the La-HMS will increase the materials' alkalinity. In fact, cells including bone marrow mesenchymal stem cells are sensitive to changes in extracellular pH, and this may cause apoptosis or necrosis as the potential across the cell membrane can be altered.⁵² After 3 weeks of soaking, the pH showed little change during the in vitro test, and the incorporation of La³⁺ into the HMS may make the materials weakly alkaline, which is good for the cells.⁵³

MS have also been proven to be a good drug carrier for bone regeneration applications.⁵⁴ In this study, La-HMS-2 had large surface areas of 807 m² g⁻¹ and mesoporous channels of 3.3 nm, which mean it will still be a good drug carrier. Besides its potential use as a drug carrier, La-HMS-2 could also be used to fabricate polymer-based composite scaffolds through the technique developed by Borden et al.⁵⁵

5. Conclusion

In this study, the osteogenic differentiation of BMSCs on a sheet-shaped La-HMS was promoted successfully in OGM. The sheet-shaped La-HMS exhibited the release of suitable amounts of La³⁺ and SiO₄⁴⁻. 0.5 μg ml⁻¹ La-HMS promoted the proliferation of BMSCs. Strong osteogenic markers, such as ALP, collagen secretion and mineralization were highly expressed when BMSCs were cultured on La-HMS-2 in OGM. To sum up, La-HMS is a drug carrier with wide ranging applications, and this low La³⁺ dose-releasing system could be utilized to fabricate polymer-based composite scaffolds for engineered osteogenesis.

Acknowledgements

This study was supported by the National Basic Research Program of China (A00102110400), China Postdoctoral Science Foundation funded project (2013M540787), Fundamental Research Funds for the Central Universities (No. 2013ZB0005) and Postdoctoral Science Foundation of China (No. 2013M531853).

References

1. K. Wang, Y. Cheng, X. Yang and R. Li, Met. Ions Biol. Syst., 2003, 40, 707–751.
2. T. Das, A. Sharma and G. Talukder, Biol. Trace Elem. Res., 1988, 18, 201–228.
3. M. E. Switzer, Sci. Prog., 1978, 65, 19–30.
4. C. A. Barta, K. Sachs-Barrable, J. Jia, K. H. Thompson, K. M. Wasan and C. Orvig, Dalton Trans., 2007, 5019–5030,  10.1039/b705123a.
5. I. Kostova, Curr. Med. Chem.: Anti-Cancer Agents, 2005, 5, 591–602.
6. P. Caravan, J. J. Ellison, T. J. McMurry and R. B. Lauffer, Chem. Rev., 1999, 99, 2293–2352.
7. L. Jarup, Nephrol., Dial., Transplant., 2002, 17, 35–39.
8. M. J. Hardie, C. L. Raston and A. Salinas, Chem. Commun., 2001, 1850–1851,  10.1039/b105517h.
9. F. Jalilehvand, D. Spangberg, P. Lindqvist-Reis, K. Hermansson, I. Persson and M. Sandstrom, J. Am. Chem. Soc., 2001, 123, 431–441.
10. X. Wang, J. Huang, T. Zhang and K. Wang, Prog. Nat. Sci., 2009, 19, 331–335.
11. C. Ergun, H. Liu and T. J. Webster, J. Biomed. Mater. Res., Part A, 2009, 89, 727–733.
12. X. Wang, L. Yuan, J. Huang, T.-L. Zhang and K. Wang, J. Cell. Biochem., 2008, 105, 1307–1315.
13. B. Lacour, A. Lucas, D. Auchere, N. Ruellan, N. M. D. S. Patey and T. B. Drüeke, Kidney Int., 2005, 67, 1062–1069.
14. G. J. Behets, S. C. Verberckmoes, P. C. D'Haese and M. E. De Broe, Curr. Opin. Nephrol. Hypertens., 2004, 13, 403–409.
15. D. Chenglong, G. Han, L. Jingxiong, S. Jianlin, W. Jie and L. Changsheng, Biomaterials, 2011, 32, 8506–8517.
16. M. P. Ginebra, T. Traykova and J. A. Planell, J. Controlled Release, 2006, 113, 102–110.
17. M. Stigter, J. Bezemer, K. de Groot and P. Layrolle, J. Controlled Release, 2004, 99, 127–137.
18. W. Xia and J. Chang, J. Controlled Release, 2006, 110, 522–530.
19. K. Schwarz and D. B. Milne, Nature, 1972, 239, 333–334.
20. E. M. Carlisle, Science, 1970, 167, 279–280.
21. E. M. Carlisle, Science, 1972, 178, 619–621.
22. E. M. Carlisle, J. Nutr., 1980, 110, 1046–1056.
23. C. Barbe, J. Bartlett, L. Kong, K. Finnie, H. Q. Lin, M. Larkin, S. Calleja, A. Bush and G. Calleja, Adv. Mater., 2004, 16, 1959–1966.
24. Y.-F. Zhu, J.-L. Shi, Y.-S. Li, H.-R. Chen, W.-H. Shen and X.-P. Dong, J. Mater. Res., 2005, 20, 54–61.
25. Y. Zhao, J. L. Vivero-Escoto, I. I. Slowing, B. C. Trewyn and V. S. Y. Lin, Expert Opin. Drug Delivery, 2010, 7, 1013–1029.
26. I. I. Slowing, J. L. Vivero-Escoto, C.-W. Wu and V. S. Y. Lin, Adv. Drug Delivery Rev., 2008, 60, 1278–1288.
27. P. T. Tanev and T. J. Pinnavaia, Science, 1995, 267, 865–867.
28. X. Li, L. Zhang, X. Dong, J. Liang and J. Shi, Microporous Mesoporous Mater., 2007, 102, 151–158.
29. B. Bunker, J. Non-Cryst. Solids, 1994, 179, 300–308.
30. L. L. Hench, J. Am. Ceram. Soc., 1991, 74, 1487–1510.
31. K. Wei, W. Guo, C. Lai, N. Zhao and X. Li, J. Alloys Compd., 2009, 484, 841–845.
32. S. Weiner and H. D. Wagner, Annu. Rev. Mater. Sci., 1998, 28, 271–298.
33. S. J. Eppell, W. Tong, J. L. Katz, L. Kuhn and M. J. Glimcher, J. Orthop. Res., 2001, 19, 1027–1034.
34. P. T. Tanev, M. Chibwe and T. J. Pinnavaia, Nature, 1994, 368, 321–323.
35. A. Shioi, Y. Nishizawa, S. Jono, H. Koyama, M. Hosoi and H. Morii, Arterioscler., Thromb., Vasc. Biol., 1995, 15, 2003–2009.
36. L. Zhao, L. Liu, Z. Wu, Y. Zhang and P. K. Chu, Biomaterials, 2012, 33, 2629–2641.
37. K. Wang, S. Afr. J. Chem., 1997, 50, 232–239.
38. D. Zhang, J. Zhang, Y. Chen, M. Yang and X. Yao, Prog. Nat. Sci., 2007, 17, 618–623.
39. Y.-L. Shi, L.-W. Wang, J. Huang, B.-D. Gou, T.-L. Zhang and K. Wang, J. Cell. Biochem., 2009, 108, 1184–1191.
40. J. Huang, T.-L. Zhang, S.-J. Xu, R.-C. Li, K. Wang, J. Zhang and Y.-N. Xie, Calcif. Tissue Int., 2006, 78, 241–247.
41. X. Shi, Y. Wang, R. R. Varshney, L. Ren, F. Zhang and D.-A. Wang, Biomaterials, 2009, 30, 3996–4005.
42. D.-A. Wang, C. G. Williams, F. Yang, N. Cher, H. Lee and J. H. Elisseeff, Tissue Eng., 2005, 11, 201–213.
43. H. Yoshikubo, N. Suzuki, K. Takemura, M. Hoso, S. Yashima, S. Iwamuro, Y. Takagi, M. J. Tabata and A. Hattori, Life Sci., 2005, 76, 2699–2709.
44. Y. Li, S. Dånmark, U. Edlund, A. Finne-Wistrand, X. He, M. Norgård, E. Blomén, K. Hultenby, G. Andersson and U. Lindgren, Acta Biomater., 2011, 7, 751–758.
45. H. F. Al-Jallad, Y. Nakano, J. L. Chen, E. McMillan, C. Lefebvre and M. T. Kaartinen, Matrix Biol., 2006, 25, 135–148.
46. I. D. Xynos, A. J. Edgar, L. D. Buttery, L. L. Hench and J. M. Polak, Biochem. Biophys. Res. Commun., 2000, 276, 461–465.
47. P. E. Keeting, M. J. Oursler, K. E. Wiegand, S. K. Bonde, T. C. Spelsberg and B. L. Riggs, J. Bone Miner. Res., 1992, 7, 1281–1289.
48. D. M. Reffitt, N. Ogston, R. Jugdaohsingh, H. F. J. Cheung, B. A. J. Evans, R. P. H. Thompson, J. J. Powell and G. N. Hampson, Bone, 2003, 32, 127–135.
49. J. Andersson, J. Rosenholm, S. Areva and M. Lindén, Chem. Mater., 2004, 16, 4160–4167.
50. S. Radin, S. Falaize, M. H. Lee and P. Ducheyne, Biomaterials, 2002, 23, 3113–3122.
51. A. E. Porter, C. M. Botelho, M. A. Lopes, J. D. Santos, S. M. Best and W. Bonfield, J. Biomed. Mater. Res., Part A, 2004, 69, 670–679.
52. J. E. Gough, J. R. Jones and L. L. Hench, Biomaterials, 2004, 25, 2039–2046.
53. C. Vitale-Brovarone, E. Verne, L. Robiglio, P. Appendino, F. Bassi, G. Martinasso, G. Muzio and R. Canuto, Acta Biomater., 2007, 3, 199–208.
54. M. Zhu, H. Wang, J. Liu, H. He, X. Hua, Q. He, L. Zhang, X. Ye and J. Shi, Biomaterials, 2011, 32, 1986–1995.
55. M. Borden, M. Attawia, Y. Khan and C. T. Laurencin, Biomaterials, 2002, 23, 551–559.

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Footnotes

† These authors contributed equally to this manuscript.

‡ Present address: South China University of Technology, Room 333, Building B12, Panyu District, Guangzhou City, Guangdong Province, China.

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