Osteogenic differentiation of stem cells on mesoporous silica nanofibers

Abstract

Electrospun nanofibrous scaffolds have gained momentum in regenerative medicine research due to their ECM-like architecture. The present study reports the fabrication of mesoporous silica nanofibers (MSF) and explores their potential to trigger osteogenic differentiation of human bone marrow derived mesenchymal stem cells (BM-MSCs) in the presence and absence of biochemical (induction) factors. BM-MSCs were seeded on MSF and allowed to differentiate into osteogenic lineage. Osteogenic differentiation of BM-MSCs was confirmed by mineralization staining, reduction in the expression of the stem cell marker CD105 and increase in the osteogenic marker osteocalcin. Cells cultured in MSF in the presence of induction media exhibited better adhesion, proliferation and differentiation. The phenotypic markers of osteoblasts such as mineralization and alkaline phosphatase (ALP) activity were higher on MSF in the presence of induction media when compared to MSF in the presence of normal media (p < 0.05). Upregulation of osteoblast specific genes (osteonectin, osteocalcin & alkaline phosphatase) suggests the potential of MSF to support osteogenic differentiation even in the absence of induction media (p < 0.05). In vitro results indicate that the nanotopography of MSF provided a favorable milieu for adhesion and proliferation of BM-MSCs. Further, the combination of the biomimetic nature of MSF, dissolution of silica ions (chemical cues) and biochemical cues presents a stable microenvironment for the differentiation of BM-MSCs into osteogenic lineage. In conclusion, the synergy of adhesion and proliferation cues in MSF along with suitable biochemical cues could be a promising design strategy to develop scaffolds for orchestrated bone healing.

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1. Introduction

Bone is an important hard tissue which constantly undergoes remodeling to manage mechanical stress, to retain healthy functions and to repair damage.¹ Though, autologous grafting is a ‘gold standard’ to treat bone loss, it has several limitations like limited availability, donor site morbidity, difficulty in matching the shape to the defect sites, etc. To overcome these issues, tissue engineering strategies have been employed to treat bone defects.¹ Bone tissue engineering involves the synergy of scaffolds, cells/stem cells and suitable growth factors. Scaffolds currently available for bone tissue engineering lack adequate vascularization at the implant site limiting their applications.² Further, the currently employed bone grafting techniques do not meet clinical demand.^3,4 The clinical success of bone substitutes relies on its ability to integrate with the host tissue, which is directed by the formation of a mineralized layer at the host bone-graft junction.⁵ Current strategies employed to design tissue engineered scaffolds for bone are aimed to have rich vascularization and also to support biomimetic regeneration.² Recent approaches for improving the success of bone scaffolds include incorporation of ligands and ECM molecules to target stem cells in regeneration, improving porous nature to augment vascularization.^6,7 In addition to this, scaffolds with nanotopography are fabricated to promote adhesion and proliferation of cells that participate in tissue regeneration.²

Scaffolds with nanotopography, cell adhesion molecules provide suitable biochemical and mechanical cues that could be ideal for tissue engineering.⁸ Various reports have indicated that nanofibers can enhance the adhesion, proliferation and differentiation of stem cells⁹ Among the various fabrication techniques, electrospinning has been widely used as a versatile and efficient method to tailor tissue-engineered scaffolds that mimic natural ECM.¹⁰ Large surface area-to-volume ratios, ECM-like geometry, high porosity are some of the desirable properties of electrospun nanofibrous scaffolds that favor tissue regeneration.⁴ Electrospun nanofibers can be fabricated as multilayered membrane in order to mimic the periosteum of bone. Such biomimetic nanofibrous scaffolds can be implanted in bone defects of different shape and size to promote regeneration.¹¹ Furthermore, electrospun nanofibers mimic the native ECM of bone (mineralized collagen nanofibers) and facilitate researchers to have a bottom-up approach to fabricate novel scaffolds for bone tissue engineering.^11,12

Biomaterials intended for bone tissue engineering applications should have the osteoinductive and osteoconductive properties which are essential to guide the neighboring cells for ectopic bone formation.^13–15 Mesoporous silica and other ceramics like alumina ceramic have been reported to have osteoinductive and osteoconductive properties.¹⁴ However, there is only scanty reports available on the use of pure ceramic nanofibers like MSF for engineering hard tissues.¹⁶ Mesoporous materials having pore sizes in range of 2–50 nm have been investigated for a myriad of biomedical applications.¹⁷ Silica based mesoporous materials are gaining increasing attention in tissue engineering due to bio-inert and biocompatible nature.¹⁸ Surface reactive functional groups in mesoporous silica have been reported to aid in biomineralization and bone tissue engineering.^18,19 Our group had earlier reported for the first time the fabrication and in vitro compatibility of MSF for bone tissue engineering.^17,18

A paradigm shift that has emerged in regenerative medicine is the usage of novel cell transplantation methods in order to improve the function of the tissue, restore the lost tissue or direct the desired differentiation of stem cells to promote structural and functional regeneration.⁹ Scaffolds loaded with stem cells could offer structural support to the damaged tissue and also accelerate regeneration.²⁰ Bone marrow-derived mesenchymal stem cells (BM-MSCs) have become the major stem cell source for various tissue engineering applications due to their immuno-suppressive effect, lesser ethical issues and ease of harvest & expansion.^20,21 BM-MSCs have been shown to differentiate into various lineages like osteoblasts, hepatocytes, neurons, etc.²¹ Osteoblast cells mediate bone mineralization process and the failure to which would result in poor therapeutic outcome. Mineralization matrix (osteoid) is laid down by the mature osteoblasts.⁵ It has been well documented that BM-MSCs are the progenitor cells that generate osteoblasts.⁵ Differentiation of BM-MSCs into mature osteoblasts involves a complex interplay of multiple factors. Designing a nanofibrous scaffold, which could direct the osteogenic differentiation of BM-MSCs and maturation of osteoblasts will be a promising therapeutic strategy to improve the clinical success of bone grafts.^4,22

Two hypotheses proposed for osteoconductivity and osteoinductivity in scaffolds are (i) surface features to home and present osteoinductive factors to the cells, (ii) release of factors to trigger the differentiation of stem cells.^2,14 Based on these hypotheses, the main motivation of this study is that MSF can absorb factors and present to the cells to improve regeneration. MSF can also release factors (silicic acid along with the absorbed factors) and promote the osteogenic differentiation of BM-MSCs. The aim of the present work is to investigate the efficacy of electrospun mesoporous silica nanofibers (MSF) in supporting the adhesion, proliferation and osteogenic differentiation of BM-MSCs. The potential of MSF to favor osteogenic differentiation in presence and in absence of biochemical cues (induction factors) was also studied. To the best of our knowledge this is the first report on the osteogenic differentiation of BM-MSCs on MSF for bone tissue engineering applications.

2. Materials and methods

2.1. Materials

Tetraethyl orthosilicate (TEOS) (Fluka, USA), Pluronics P-123 (Aldrich, USA), poly(vinyl pyrrolidone) (PVP, MW 1.3 × 10⁶) (Aldrich, USA), ethanol (Jiangsu Huaxi International Trade Co. Ltd, China) were used for the fabrication of mesoporous silica nanofibers. Human bone marrow derived mesenchymal stem cells (BM-MSCs) were procured from Tran-Scell Biologics Pvt. Ltd, Hyderabad, India. Cells were cultured in minimum essential medium (Hyclone, USA) supplemented with 10% fetal bovine serum (Invitrogen, USA) and 1% penicillin–streptomycin (P/S) (Gibco, USA). Dulbecco's phosphate buffered saline (D-PBS), live/dead assay kit, Alexa Fluor 488 and Hoechst were purchased from Invitrogen, USA. Anti-CD105 was procured from Abcam, USA, anti-osteocalcin and its secondary antibody were purchased from R&D Systems, USA. Alkaline phosphatase (ALP) substrate kit and cell proliferation assay kit were purchased from Bio Rad, USA and Promega, USA, respectively. Dexamethasone, L-ascorbate, beta-glycerophosphate, alizarin red dye and pyrogallol were procured from Sigma, USA.

2.2. Fabrication of mesoporous silica nanofibrous scaffold

Mesoporous silica nanofibers were fabricated according to our earlier reports, which involve two steps.^17,18 Briefly, P123 (Pluronics 123) was used as structure directing agent and the precursor tetraethyl orthosilicate (TEOS) was dissolved in ethanol–poly(vinyl pyrrolidone) (PVP). In the first step, 1.5 g of TEOS and 0.5 g of P-123 were dissolved in ethanol and pre-hydrolyzed by the addition of water and 2 M HCl. This mixture was continuously stirred for 30 minutes to form silica sol. Then, PVP solution (prepared in ethanol) was mixed to the silica sol and the mixture was subjected to electrospinning after refluxing at 70–80 °C. Silica-sol polymer mixture was collected in a glass syringe fitted with a 24 G blunt needle. The solution was ejected at a flow rate of 0.003 mL min⁻¹ (Kent Scientific 200, USA) and a high voltage of 15 kV was applied to the needle tip (Zeonics, Bangalore, India). Nanofibers were collected on a grounded static collector and the collected fibers were subjected to calcination at 550 °C to ensure removal all organic moieties.

2.3. SEM and TEM analysis

Morphology of as-electrospun MSF and calcined MSF were analyzed using field emission scanning electron microscope (FE-SEM, JSM 6701F, JEOL, Japan). A thin layer of gold was sputtered on the sample placed on brass stubs. The sputter-coated samples were imaged at an accelerating voltage of 3 kV. Internal structure of calcined MSF was investigated using field emission transmission electron microscopy (FE-TEM, JEM 2100F, JEOL, Japan). Silica nanofibers were spun on the TEM sample grid and calcined before imaging using FE-TEM.

2.4. Cell culture & seeding

BM-MSCs were cultured in minimum essential medium supplemented with 15% FBS and 1% P/S. Culture flasks were incubated in CO₂ incubator maintained at 37 °C (NuAire NU-8700E, USA) with 5% CO₂. Culture media was replaced every 48 hours until the cells reached 80% confluency. Confluent cells were subcultured and cells at passage 2 were used for the study. Mesoporous silica fibrous scaffolds (0.2–0.3 mm thick & 10 mm diameter) were sterilized by exposure to UV light for 1 hour on each side and placed in 48-well culture plates. The scaffolds were washed with D-PBS thrice and pre-incubated with culture media overnight. Ten thousand cells were seeded on the scaffolds for evaluating cell adhesion, proliferation, live/dead assay, ALP assay while 25 000 cells were seeded to carry out real-time PCR and other staining experiments. Osteogenic induction media was prepared in MEM growth media supplemented with 100 nM dexamethasone, 10 mM β-glycerophosphate, and 0.05 mM ascorbic acid-2-phosphate.²³ Tissue culture polystyrene (TCPS) served as positive control, and the medium was replenished every 48 hours. Comparison was made between the cells cultured in normal and induction media.

2.5. Cell adhesion & proliferation

The adhesion of BM-MSCs on MSF was qualitatively evaluated using FE-SEM. After 1, 3 and 7 days of culture, cells were fixed using 4% buffered glutaraldehyde at 4 °C overnight. The scaffolds were washed with D-PBS and allowed to air dry. The samples were sputter coated with gold and imaged using FE-SEM.²⁰

After 3 & 5 days of culture the viability of BM-MSCs on MSF scaffold was evaluated using live/dead cell viability kit following standard protocol. The samples were observed using a laser scanning confocal microscope (FV1000, Olympus, Japan)²⁴ where live cells were stained green and dead cells appear red.

The proliferation of cells was determined after 1, 3 & 10 days using MTS assay (CellTiter⁹⁶ AQueous one solution, Promega, USA).²⁵ At the end of each time point, the samples were washed with D-PBS solution to remove the non-adherent cells. Serum-free media (1 mL) and 200 μL of MTS reagent were added to each well and incubated at 37 °C for 2 hours. The reaction was stopped by the addition of 100 μL of sodium dodecyl sulfate (10% SDS) solution and the absorbance was read at 490 nm using a multimode reader (Infinite 200M, Tecan, USA).

2.6. Alkaline phosphatase assay

Alkaline phosphatase (ALP) (a phenotypic marker of bone formation) was measured after 7 & 14 days of culture using an alkaline phosphatase substrate kit (Bio Rad, USA).^26,27 At the end of each time point, the media was removed, the scaffolds were washed with D-PBS and cells were lysed using 1% Triton X 100. Hundred microliter of the cell lysate was added to 400 μL of the substrate and incubated at 37 °C for 30 minutes. The reaction was stopped by the addition of 0.4 M sodium hydroxide solution and the absorbance was measured at 410 nm using a multimode reader (Infinite 200M, Tecan, USA).

2.7. Real-time RT-PCR analysis

The expression levels of bone specific markers namely alkaline phosphatase, collagen I, RUNX2, osteonectin, osteocalcin and bone morphogenetic protein were evaluated using a real-time RT-PCR (Eppendorf AG22331, Germany). Real-time PCR analysis was carried out using the protocol described earlier.^28,29 Quantitative values were determined by the delta–delta method and normalized with the house-keeping gene, hypoxanthine phosphoribosyltransferase 1 (HPRT1). HPRT1 was used as a housekeeping gene to calculate the change in the target gene expression. BM-MSCs cultured in TCPS using induction media served as control. The primer sequences used in this study are listed in Table 1.³⁰

Table 1 Primers used to amplify bone specific genes

Gene	Primer sequence	Length (bp)
HPRT1	5′-CCTGGCGTCGTGATTAGTG-3′	125
	5′-TCAGTCCTGTCCATAATTAGTCC-3′
ALP	5′-GCACCTGCCTTACTAACTC-3′	162
	5′-AGACACCCATCCCATCTC-3′
Collagen type I	5′-TGGAGCAAGAGGCGAGAG-3′	122
	5′-CACCAGCATCACCCTTAGC-3′
RUNX2	5′-GCCTTCAAGGTGGTAGCCC-3′	67
	5′-CGTTACCCGCCATGACAGTA-3′
Osteonectin	5′-AGGTATCTGTGGGAGCTAATC-3′	224
	5′-ATTGCTGCACACCTTCTC-3′
Osteocalcin	5′-GCAAAGGTGCAGCCTTTGTG-3′	80
	5′-GGCTCCCAGCCATTGATACAG-3′
BMP2	5′-TGCGGTCTCCTAAAGGTC-3′	186
	5′-AACTCGAACTCGCTCAGG-3′

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2.8. Immunostaining of stem cell and osteogenic markers

The osteogenic differentiation of BM-MSCs was confirmed using immunofluorescent staining of stem cell specific marker CD105 and osteoblast specific marker osteocalcin.¹ After 1 day of culture, cells were fixed in 3.7% paraformaldehyde for 15 minutes and washed with D-PBS. Scaffolds were incubated with blocking buffer to block the non-specific sites. Primary antibody for CD-105 (1 : 100) (Abcam, USA) was added and the secondary antibody conjugated with Alexa Fluor 488 (Invitrogen, USA) (1 : 200) (Green) was added at room temperature. Osteocalcin was immunostained to determine osteogenic differentiation after 21 days of culture. Cells were fixed in 3.7% paraformaldehyde and the non-specific sites were blocked. Primary antibody for osteocalcin (1 : 100) (Sigma, USA) was added for 90 minutes at room temperature and the cells were incubated with the secondary antibody conjugated with Alexa Fluor 594 (Invitrogen, USA) (1 : 200) (red). The scaffolds were washed with D-PBS to remove excess stains. The scaffolds were incubated with Hoechst in the dilution of 1 : 5000 at room temperature, washed with D-PBS and imaged using laser scanning confocal microscope (Olympus FV 1000), Japan.

2.9. Alizarin red staining

After 14 and 21 days of culture, the spent media was removed. The cells were washed with D-PBS and fixed in 10% formalin for 15 minutes at room temperature. The formalin was removed gently and washed twice with double distilled water. Following formalin fixation, the scaffolds were incubated in alizarin red dye (40 mM, pH 4.3) at room temperature.³¹ The dye was removed and washed for 3–4 times with double distilled water. The stained scaffolds were transferred to a fresh culture dish and imaged using phase contrast inverted microscope (Carl Zeiss-Axiovert, Germany).

2.10. von Kossa staining

After 14 and 21 days of culture, cells were fixed with 10% formalin and washed twice with double distilled water. The cells were stained with 5% silver nitrate solution and washed twice with double distilled water. The cells were fixed with 5% sodium thiosulfate and the scaffolds were washed for 3–4 times with double distilled water.²³ The stained scaffolds were transferred to a fresh culture dish and imaged using phase contrast inverted microscope (Carl Zeiss-Axiovert, Germany).

2.11. Statistical analysis

Analysis of variance (two-way ANOVA) was used to evaluate the significance between the incubation days for cell proliferation, alkaline phosphatase activity and gene expression. Statistical significance was evaluated at p < 0.05.

3. Results

3.1. MSF surface analysis

Defect-free MSF was fabricated using the optimized electrospinning parameters reported previously by our group.^17,18 We have also reported the surface properties, FTIR spectrum, thermogravimetric and surface area analysis results of as-spun and calcined MSF.^17,18 The average fiber diameter of as-spun MSF was 755 ± 45 nm, which decreased to 490 ± 40 nm after calcination (Fig. 1A & B). The MSF retained intact nanofibrous morphology post-calcination. Fig. 1C shows the cross-section of the calcined MSF and Fig. 1D shows the presence of mesopores on the surface.

Fig. 1 Morphology of mesoporous silica nanofibers (MSF). FE-SEM images of [A] as-spun MSF; [B] calcined MSF; [C] FE-SEM cross section image and [D] FE-TEM image calcined MSF.

3.2. BM-MSCs adhesion and viability on MSF

The potential of MSF to promote the adhesion and viability of BM-MSCs was evaluated using FE-SEM and laser scanning confocal microscopy. Fig. 2 shows the adhesion of BM-MSCs on MSF after 1, 3 and 7 days of culture. BM-MSCs exhibit better adhesion on MSF within initial days of culture and the cell number was found to be higher with well preserved morphology after 7 days of culture. Live/dead staining was performed after 3 and 5 days of culture to assess the cytocompatibility of BM-MSCs and the 3-D reconstruction from the z-stacks are shown in Fig. 2D & E. Z-section video of the BM-MSCs cultured on MSF was captured (ESI data: video 1†). There was an increase in cell number as a function of culture time and no dead cells were found at later culture time point.

Fig. 2 BM-MSCs adhesion on MSF. FE-SEM images showing cells adhered to MSF after [A] 1 day; [B] 3 days; and [C] 7 days of culture. Confocal 3-D reconstruction images of BM-MSCs stained using live/dead stain after [D] 3 days and [E] 5 days of culture on MSF.

3.3. BM-MSCs viability on MSF

Viability of BM-MSCs in presence and absence of induction media was evaluated using MTS assay. In order to assess the effect of nanotopography, biochemical cues and their combination on the viability of BM-MSCs, four different groups were studied viz.; (i) MSF in presence of induction media (MSF-I), (ii) TCPS with induction media (TCPS-I); (iii) MSF in presence of normal media (MSF-N) and (iv) TCPS with normal media (TCPS-N).

After 10 days of culture, the cell viability on MSF-I was found to be significantly higher when compared to other groups (p < 0.05) (Fig. 3A). Even in the absence of induction media, the MSF-N exhibited significantly higher cell viability than its TCPS counterpart. After 10 days of culture, the viability of cells cultured in MSF-N was comparable to those cultured in TCPS in presence of induction media thereby demonstrating the superiority of MSF-N for tissue engineering applications. Cell proliferation in TCPS-I was comparable with MSF-N and TCPS-N showed less proliferation among the groups after 10 days of culture.

Fig. 3 [A] MTS assay results showing BM-MSCs proliferation on MSF after 1, 3 and 10 days of culture and [B] ALP expression of BM-MSCs indicating osteogenic differentiation after 7 and 14 days of culture (*p < 0.05).

3.4. Alkaline phosphatase expression

Alkaline phosphatase (ALP) is an early phenotypic marker expressed by osteoblast cells. Evaluating the levels of ALP is therefore imperative to confirm the commitment of BM-MSCs towards osteogenic lineage. After 7 and 14 days of culture, the ALP expression of BM-MSCs on MSF-I and MSF-N was significantly higher than TCPS-I and TCPS-N (Fig. 3B) (p < 0.05). As expected, ALP activity in cells cultured in TCPS-I was significantly higher than TCPS-N (p < 0.05). However, the ALP activity in cells cultured in normal conditions on mesoporous silica nanofibers was significantly higher than those cultured in the presence of induction medium on MSF.

3.5. Gene expression profile

Real time RT-PCR of the genes expressed by BM-MSCs such as type I collagen (TIC), bone morphogenetic protein (BMP2), alkaline phosphatase (ALP), osteocalcin (OCN), RUNX2, and osteonectin (ON) were evaluated after 7 and 14 days of culture (Fig. 4). TIC expression was up-regulated in all the groups but was significantly higher in MSF-I after 14 days of culture (Fig. 4A) (p < 0.05). ALP expression was significantly up regulated in MSF-I after 7 days of culture while it was down regulated in other groups (Fig. 4B). After 14 days of culture, the expression levels were significantly reduced in both MSF-I and MSF-N though they were slightly higher than the TCPS-N group. BMP & OCN levels were up-regulated and comparable on MSF-I and MSF-N after 7 and 14 days of culture but down-regulated on TCPS-I (Fig. 4C & E). RUNX2 expression was down regulated after 7 days in all the groups and up-regulated in cells cultured on MSF-I and MSF-N after 14 days of culture (Fig. 4D). ON expression levels were up-regulated in cells cultured on MSF-I after 14 days of culture (p < 0.05) (Fig. 4F).

Fig. 4 Bone specific gene expression of BM-MSCs on MSF. Gene expression profile of BM-MSCs differentiating towards osteogenic lineage. [A] Collagen I; [B] alkaline phosphatase; [C] bone morphogenetic protein; [D] RUNX2; [E] osteocalcin and [F] osteonectin.

3.6. Immunostaining of CD105 and osteocalcin

To confirm the differentiation of BM-MSCs towards osteogenic lineage, the osteogenic marker (osteocalcin) expression was immunostained and imaged after 21 days of culture using confocal microscopy (Fig. 6). Further, the BM-MSC marker (CD105) expression was studied using immunostaining and imaged after 1 day of culture (ESI data: video 2†) (Fig. 5A & B). BM-MSCs expressed their native CD105 expression after 1 day of culture and cells also exhibited spindle (fibroblast-like) morphology. After 21 days of culture in MSF-I, BM-MSCs had differentiated into osteoblasts and expressed osteocalcin (Fig. 6) (ESI data: video 3†). The BM-MSCs in MSF-N also exhibited better osteogenic differentiation than TCPS but their degree of osteogenic differentiation was lesser when compared to cells cultured in MSF-I (Fig. 6).

Fig. 5 CD105 expression of BM-MSCs after one day of culture. Confocal images showing the expression of CD105 in BM-MSCs cultures on TCPS. [A] Hoechst; [B] CD105 and [AB] merged image of [A] and [B]. [C] & [D] are the 3-D reconstruction from confocal image stacks of CD105 (green) and Hoechst (blue) in BM-MSCs on MSF.

Fig. 6 Osteocalcin expression of BM-MSCs on MSF. Osteocalcin expression of BM-MSCs on MSF after 21 days of culture in presence of induction media [A] nucleus; [B] osteocalcin [AB] merged image of [A] & [B] and in presence of normal media [C] nucleus; [D] osteocalcin [CD] merged image of [C] & [D].

3.7. Mineralization staining

Alizarin red and von Kossa staining have been used to detect the early differentiation marker, ALP (stained red) and late differentiation indicator mineralization matrix (stained green) formation. After 14 days of culture, the alizarin red staining was deeper in MSF-I when compared to MSF-N and there was no stain in TCPS-N and very trace amounts observed in TCPS-I. After 21 days, alizarin stain remained highly intense in MSF-I when compared to 14 days and also remained superior to MSF-N after 21 days (Fig. 7).

Fig. 7 Alizarin red staining to confirm the osteogenic differentiation of BM-MSCs. Cells were cultured on MSF for [A] 14 days & [C] 21 days in absence of induction media and for [B] 14 days & [D] 21 days in presence of induction media (light micrograph, magnification: ×100).

von Kossa staining (deep green) is used to detect the matrix mineralization, which is a hall mark activity in mature osteoblasts. After 14 days, the von Kossa staining was more intense in MSF-I when compared to MSF-N. After 21 days, deep blackish green von Kossa stain was observed in MSF-I and less intense stain was observed in MSF-N (Fig. 8). This could be attributed to the fact that BM-MSCs cultured in induction medium produced higher levels of alkaline phosphatase which is a key enzyme involved in in vivo bone mineralization by cleaving phosphates from organic phosphates. Induction medium contains organic sources of phosphates which are substrates for alkaline phosphatase and hence there is enhanced mineralization.³²

Fig. 8 von Kossa staining to confirm the osteogenic differentiation of BM-MSCs. Cells were cultured on MSF for [A] 14 days & [C] 21 days in absence of induction media and for [B] 14 days & [D] 21 days in presence of induction media (light micrograph, magnification: ×100).

4. Discussion

Bone defects which do not heal in a timely and orchestrated fashion are treated by surgical interventions using autografts and allografts.³³ The current clinical procedures have limitations such as donor-site morbidity, unpredictable healing and high risk of transmission of diseases.³¹ Hence, tissue engineering has emerged as a promising alternative to conventional treatment strategies for bone defects. Tissue engineering attempts to retain functional tissue-specific cells in a scaffold that provides optimal topographical, chemical and mechanical cues similar to the native environment to restore the function of the tissue.³⁴ To achieve this, scaffolds with nano-architecture, which can mimic the native extracellular matrix of bone tissue have been fabricated. Electrospinning has emerged as a popular technique to tailor biomimetic scaffolds similar to the fibrous protein network found in extracellular matrix, which could be incorporated with stem cells to promote functional tissue regeneration.¹ Bone scaffolds must provide a 3-D microenvironment for cells to aid in regeneration and hence scaffolds designed for bone tissue engineering should be osteoconductive, osteoinductive and also be able to bond to the host bone (osteointegration).³⁵ Synthetic bone-graft substitutes have been developed using a wide range of materials such as polymers, ceramics and composites.^17,18 Among these, bioactive ceramics (bioactive glass) have been reported to integrate with bone and stimulate bone healing.³⁵ Another facet that has an important role in regeneration of tissues is the topography of the scaffold. It has been reported that nanofibers can provide a suitable milieu for stem cells and also provide stability during differentiation towards specific lineage.³⁶

Mesoporous silica possesses well-oriented pores and high surface area that can accommodate signaling molecules and help in the promotion of cell adhesion, proliferation and native gene expression. Mesoporous silica can be fabricated as nanofibers and the advantages of mesoporous geometry could be combined with nanotopography, which will further improve its in vitro activities.¹⁸ We have earlier reported the fabrication of mesoporous silica nanofibers and its in vitro potential in osteoblast (MG-63) maturation.¹⁸ In the present study it was revealed that the fabricated MSF had nanofibrous topography and also had mesopores in the surface of MSF. This is in agreement with the report published earlier by our group.¹⁷ MSF have been reported to be able to support the adhesion and proliferation of osteoblast cells but to the best of our knowledge there is no report on the osteogenic differentiation of BM-MSCs on MSF and hence the present work is the first to investigate the osteogenic properties of MSF.

The in vitro results have clearly indicated that the adhesion and proliferation of BM-MSCs were higher on MSF-I when compared to other groups. This may be attributed to the nanotopography of MSF, which resembles the extracellular matrix (ECM) and the induction factors present in the media. Initial cell adhesion is an important parameter in determining the effectiveness of a tissue engineered scaffold. The adhesion of BM-MSCs on MSF was evaluated using FE-SEM after 1, 3, 7 days of culture and proliferation was evaluated using MTS assay after 1, 3 and 10 days of culture. MSF-I showed more number of cells with intact morphology and proliferation was significantly higher after 10 days of culture. This could be due to the fact that cells can respond and adhere to nanofibers through integrin receptors which can stimulate the proliferation of cells.³⁷ It has been well established that the integrin mediated binding of cells to the ECM-like scaffolds can promote cellular fate processes (adhesion, proliferation and differentiation).³⁸ In addition, the presence of mesopores and micropores in the MSF surface will serve as a host to the bioactive factors (ions, vitamins etc.) and release them in a sustained manner, which can trigger cell proliferation.^17,18 Live/dead staining after 3 and 5 days of culture revealed no dead cells in MSF indicating its cytocompatibility. Similar observations have been reported for poly(γ-glutamic acid)/silica sol–gel hybrid scaffolds suggesting that silica is a major contributor to the cytocompatibility factor rather than the topography.³⁹

Osteoblasts secrete alkaline phosphatase, an ectoenzyme, which degrades inorganic pyrophosphate and causes an increase in phosphate levels thereby activating mineralization process.⁴⁰ Therefore, ALP activity is considered as a direct measure of the functional activity of osteoblasts.⁴⁰ Significantly higher levels of ALP activity was observed in MSF-I & MSF-N after 7 and 14 days of culture in the present study confirming that MSF can induce the osteogenic differentiation of BM-MSCs in presence and absence of induction factors. In our previous study, we had shown that MSF could help in the maturation of osteoblasts (MG-63) and thus increase in ALP levels after 7 days of culture.¹⁷

BM-MSCs cultured on MSF were analyzed for the expression of bone specific genes such as collagen I, ALP, BMP-2, RUNX2, osteonectin and osteocalcin after 14 days of culture. While collagen I is a major ECM protein and ALP is expressed only by mature osteoblasts.^41,42 Collagen I expression was up-regulated on all the groups but was significantly higher in MSF-I after 14 days of culture (Fig. 4) (p < 0.05). ALP activity was significantly higher in MSF-I after 7 days and the levels up-regulated in both MSF-I and MSF-N after 14 days of culture. Silicic acid which is a dissolution product of silica is known to promote osteoblast proliferation and maturation.¹⁸ This may be the reason for the observed enhancement in the osteogenic differentiation of BM-MSCs on MSF. Up-regulation of collagen-I and ALP in MSF could also be attributed to the stimulation by silicic acid. BMP2 is a growth factor responsible for the differentiation of osteoblasts and helps in bone formation.⁴³ RUNX2 is a transcription factor that mediates the maturation of osteoblasts.⁴⁴ OCN and ON are markers of osteoblasts that indicate mineralization or progress of osteoblasts maturation.⁴⁴ BMP & OCN levels were up-regulated and comparable on MSF-I and MSF-N after 7 and 14 days of culture but down regulated on TCPS-I. RUNX2 expression was down regulated after 7 days in all the groups and up-regulated in MSF-I and MSF-N after 14 days of culture. ON expression levels were up-regulated in MSF-I after 14 days of culture. These gene expression profiles indicate the differentiation of BM-MSCs on MSF.

Mineral deposition in BM-MSCs was visualized using alizarin red stain, which binds to Ca²⁺. Cells in MSF-N were stained red while those in MSF-I exhibited even more intense red stain on treatment with alizarin red, which demonstrates the intrinsic osteoinduction properties of MSF. Similar observations were reported for BM-MSCs cultured on PLGA–HA sintered scaffolds without any induction factors.³¹ The von Kossa staining has been performed to detect the extent of mineralization matrix formation using silver staining. Osteoblasts produce mineralization matrix during their maturation or late differentiation stages.⁵ After 21 days of culture, an intense green von Kossa staining was observed in MSF-N and a deeper blackish green stain in MSF-I was observed. BM-MSCs cultured on poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) (PHBVHHx) films in induction media showed deeper alizarin and von Kossa staining when compared to normal media which confirmed the osteogenic differentiation.⁴⁵ Thus, these results indicate the potential of MSF to favor osteogenic differentiation of BM-MSCs.

CD-105 is a surface maker protein expressed by mesenchymal stem cells.³⁰ CD105 expression was observed through immunostaining in BM-MSCs cultured on MSF after one day of culture. It was observed that all the cells expressed their native morphology and significant CD105 expression, which suggests the compatibility of MSF. Osteocalcin is a non-collagenous protein expressed by mature osteoblasts. It is made up of three γ-carboxyglutamic acid, which helps in calcium binding. OCN is responsible for ossification process of bone healing.⁴⁶ Immunostaining of osteocalcin was performed after 21 days of culture to confirm the osteogenic differentiation of BM-MSCs on MSF. OCN expression was higher in MSF-I when compared to MSF-N indicating the superiority of the synergistic effect of topography, chemical factors and biochemical cues. Furthermore, the dissolution of ions from MSF could have also promoted the adhesion, proliferation and differentiation of BM-MSCs.⁴¹ This may be the reason MSF-N expresses osteoblast differentiation marker even in the absence of induction factors.

Ravichandran et al., have demonstrated the osteogenic differentiation of ADSCs on poly(L-lactic acid)/poly-benzyl-L-glutamate/collagen nanofibers incorporated with hydroxyapatite, which could be used as a bone graft for better osteogenesis.¹ Osteocalcin expression and gene expression of BM-MSCs on the surface of MSF-I confirms that MSF provides a stable microenvironment for BM-MSCs differentiation towards osteogenic lineage. The results presented in the present work clearly demonstrate that MSF along with suitable biochemical cues could help in accelerated bone regeneration. This novel construct could be an implantable stem cell-seeded scaffold to improve bone healing and also offers a promising alternative for the existing therapies.

5. Conclusion

The aim of the present study was to fabricate mesoporous silica nanofibers and to investigate its potential to support the adhesion, proliferation and osteogenic differentiation of BM-MSCs. The surface morphology of MSF resembles the native ECM and provides adhesion & proliferation cues for BM-MSCs. Cell infiltration was better in MSF and the proliferation rate was significantly higher in MSF-I as compared to MSF-N, TCPS-I and TCPS-N. It was observed that MSF possess osteoinductive cues, which are evident from the alizarin red staining, von Kossa staining and immunostaining results. However, the differentiation and osteoblast maturation was greatly enhanced in presence of induction factors (biochemical cues). The gene expression results have clearly demonstrated that BM-MSCs cultured on MSF-I express the genes that are necessary for BM-MSCs differentiation towards osteogenic lineage and also the genes responsible for osteoblast maturation. Overall, in this study we have attempted to synergize the effect of stem cells, surface & topographical cues of the scaffold and biochemical cues to engineer a novel nanofibrous scaffold for orthopedic applications. We believe that this new approach may improve the design strategies that are currently employed to fabricate novel scaffolds for bone tissue engineering.

Conflict of interest

The authors declare that they have no competing interests.

Acknowledgements

We sincerely acknowledge the Nano Mission (SR/S5/NM-07/2006 & SR/NM/PG-16/2007) and FIST (SR/FST/LSI-327/2007 & SR/FST/LSI-058/2010), Department of Science & Technology, India. We also acknowledge the financial support from Prof. T. R. Rajagopalan R&D Cell of SASTRA University. The first author acknowledges the SRF support from the Council of Scientific & Industrial Research (09/1095/(0002)/2013/EMR-I). The financial support from Drugs and Pharmaceuticals Research Programme (VI-D & P/267/08-09/TDT) is also acknowledged for providing FE-TEM.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra07014g

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