Biocompatibility study of tobacco mosaic virus nanoparticles on human alveolar bone cells

Abstract

One of the most important factors in a dental implant's success is an adequate quantity of supporting bone. However, there are still some limitations for the bone substitution material. Previous studies found that tobacco mosaic virus (TMV) had the potential for bone formation induction. The aim of this study was to evaluate the biocompatibility of TMV with primary human alveolar bone cells. Primary human alveolar bone cells were cultured on TMV coated substrates. Cell viability, alkaline phosphatase activity, calcium matrix mineralization forming ability, immunofluorescence staining for osteocalcin synthesis and cell morphology were assessed. The results showed that primary human alveolar bone cells cultured on the TMV coated substrates had a higher metabolic rate than the non-TMV coated control group at days 1, 3, 7 and 14. Moreover, the calcium deposition was positive and the alkaline phosphatase activity assay was found significantly greater than the control group at day 14 (p < 0.05). The osteocalcin protein synthesis was found in both the TMV coated substrates and the control group. The immunofluorescence study revealed that in the TMV coated substrates group, the cell morphology changed into a polygonal shape and aggregated more quickly than the control group. The present findings conclude that TMV is biocompatible with primary human alveolar bone cells and also shows osteoinduction potential.

Graphical abstract

1. Introduction

Nowadays, dental implants are popular among patients who need dental prostheses. The factor that needs to be concerned, for providing implant stability, is an adequate amount of supporting bone. However, there are some limitations to the materials used for bone substitution. Previous studies found that plant virus nanoparticles such as tobacco mosaic virus (TMV), potato virus X (PVX), turnip yellow mosaic virus (TYMV) and turnip vein clearing virus (TVCV) coated substrates were able to accelerate and enhance osteogenesis of bone marrow mesenchymal stem cells (BMSCs). The reason was due to its topographical features providing surface roughness and its ability to mimic the protein matrix between cells (extracellular matrix protein) that affect cell binding, migration, proliferation and stem cell differentiation.^{1, 2, 3}

Among all the plant viruses, the TMV is of particular interest. Not only is it the first plant virus discovered, it is also one of the simplest viruses currently known. The TMV is 300 nm in length and 18 nm in diameter. The viral capsid of the TMV consists of 2,130 protein subunits that assembled into a rod-like helical surrounding a single strand of RNA. Since the TMV is a plant virus, it cannot multiply itself or cause any diseases in mammals.^4,5 The preparation of the TMV was cost-effective and the process for its genetic modifications was known and used as a biomaterial in the field of tissue engineering application.^{6, 7, 8, 9}

Previous studies in a two-dimensional (2D) model have shown that the TMV coated substrates promoted bone formation, osteoinduction, by accelerating the expression of bone morphogenic protein-2 (BMP-2). This resulted in early osteoblastic differentiation¹⁰ as well as acceleration of the expression of osteocalcin, an osteogenic marker involved in the mineralization process of rat bone marrow mesenchymal stem cells (rBMSCs).¹¹ Subsequently, the TMV was studied in combination with biological scaffold in the three-dimensional (3D) model such as the study of porous alginate hydrogel scaffolds (PAH) that contained arginine-glycine-aspartic acid-inserted tobacco mosaic virus (TMV-RGD). The study found that the TMV accelerated bone differentiation and mineralization of rBMSCs as shown by the upregulation of alkaline phosphatase (ALP) activity and osteocalcin expression as compared to the virus-free control group.¹² The combination of cysteine-inserted tobacco mosaic virus (TMV1cys) with methacrylate hyaluronic acid (MeHA) hydrogel scaffold was found to promote chondrogenesis in rBMSCs by stimulating the production of BMP-2 and collagen type II.¹³ Besides, the study from Liu T et al.¹⁴ clearly stated the effects of TMV- RGD1 on the transformation process of human bone marrow mesenchymal stem cells (hBMSCs). They found there was greater promotion of osteocalcin and BMP-2 gene expression on TMV-RGD1 coated substrates when compared with TMV coated substrates and the control group.

However, there are limited numbers of studies focusing on the roles of TMV on human alveolar bone cells. Hence, to obtain and apply the benefits of TMV on osteoinduction for regenerative therapy in Dentistry, this study aims to evaluate the biocompatibility of tobacco mosaic virus with primary human alveolar bone cells.

2. Material and methods

2.1. Human alveolar bone cells isolation and culture

Human alveolar bone cells were harvested and characterized from our research group (Areevijit K et al.)¹⁵ In brief, two primary osteoblastic cell lines OB1 and OB2 (osteoblast; OB) were harvested and characterized from healthy patients, ages between 20 and 50 years old, who underwent the third molar surgical removal at the Department of Oral Surgery and Oral Medicine, Faculty of Dentistry, Srinakharinwirot University, Bangkok, Thailand. This process was performed under informed consent from the patients and this study was approved by the Srinakharinwirot University Ethics Committee (approval number SWUEC-087/2562E).

The alveolar bone chips were cultured in Dulbecco's Modified Eagle Medium/Ham's F-12 advance (DMEM/F-12 advance) with 10% FBS, 1% of 200 mM l-glutamine and 1% of 10,000 Units/ml Penicillin/10,000 μg/ml Streptomycin/25 μg/ml Amphotericin B at 37 °C in a 5% CO₂ incubator. The media was replenished every three days. The cells from passages 3–8 were used in this study.

2.2. TMV isolation and characterization

The TMV was isolated and purified from tobacco leaves which were previously TMV infected following the protocol from Metavarayuth K et al.³ In brief, the purification of TMV started with the infected leaves being blended with three volumes of 0.1 M potassium phosphate buffer (pH 7.8 and 0.1–0.3% 2-mercaptoethanol). The mixture was filtered, and the filtrate was subjected to centrifugation to remove the bulk plant materials. The supernatant was collected and clarified by adding an equal volume of n-butyl alcohol and Chloroform (50:50). The aqueous layer was then collected followed by precipitation of virus with 8% PEG 8K and 0.2 M NaCl. The pellet was centrifuged and resuspended in the buffer before it was subjected to a low-speed centrifugation to remove PEG. The virus in the supernatant was finally pelleted by ultracentrifugation and resuspended in buffer and final pelleting by sucrose gradient, the pellet was dissolved in the buffer then centrifuged and kept supernatant. UV absorbance (260 nm, 280 nm) was used to check the concentration. MALDI-TOF mass spectrometry was used with 1 μl TMV solution: 9 μl matrix solution (Sinapic acid in 70% acetonitrile, 0.1% TFA) to confirm the modifications. The structure of the TMV was characterized by Transmission electron microscopy (TEM) analysis. Briefly, 20 μl of TMV concentration 1 mg/ml was dropped onto the grids with an addition of 1% uranyl acetate and then viewed with a JEM-2100/HR (200 kV, JEOL Japan) microscope.

2.3. Preparation of the TMV coated substrates

The 24 well tissue culture plates (TCPs) were first coated with 0.1 mg/mL Poly-d-lysine (PDL) which is a positively charged biocompatible polymer that can absorb the negative charge of TMV particles by electrostatic interaction. The TCPs were coated with 0.1 mg/mL TMV in 18.2 mΩ water and incubated overnight under sterile cells culture hood. The bottoms of each of the wells were rinsed briefly with 18.2 mΩ water before being used for cell culture. The virus coverage on the TCPs was characterized by atomic force microscopy (AFM) using Nanosurf CoreAFM (dynamic mode: Trapping mode, Contact mode with Dyn190Al).³

2.4. Cell viability assay

The 24 wells without the TMV coated substrates (the control group) and the 24 wells with TMV coated substrates (the experimental group) were seeded with 20,000 cells per well in DMEM-F12 advance. The Resazurin (Prestoblue™ Invitrogen, ThermoFisher Scientific) was used to determine cell viability by monitoring the metabolic rate of cells on days 1, 3, 7 and 14. Resazurin was added to DMEM-F12 advance with a ratio of 1:10 per well and incubated for 1 h at 37 °C in 5%CO₂ incubator. The metabolic rate of cells was determined by fluorescence intensity at 560/590 nm then analyzed by microplate reader (CLARIOSTAR, BMG Labtech, USA) and normalized fluorescence intensities against initial signal intensity on day 1.^10,16

2.5. Alkaline phosphatase (ALP) activity

Osteogenic differentiation was determined by ALP activity assay. To induce osteogenesis, after cells attachment the media was replaced with osteogenic media containing DMEM-F12 advance supplemented with 50 μg/ml Ascorbic acid, 5 mM β–glycerophosphate and 250 nM Dexamethasone and evaluated on days 7 and 14. The samples were washed with Tris-buffer saline 1x (TBS) then combined with 1.5 g/l p-nitrophenyl phosphate (pNPP) (alkaline phosphatase yellow, Sigma Aldrich®, USA) at room temperature, covered with aluminum foil and incubated for 1 h. The absorbance was read at 405 nm by a microplate reader (CLARIOSTAR, BMG Labtech, USA). The measured ALP activity from each sample was normalized to the corresponding cell viability at each time point. The enzyme activity was calculated from Beer-Lambert law as follows:

$$Enzyme(\mu moles/\min /\mu g)=\frac{V(\mu l)\times OD405nm(c{m}^{-1})}{\epsilon \times incubationtime(\min )\times enzyme(\mu g)}$$

where ϵ is the molar extinction coefficient (M⁻¹ × cm⁻¹) p-nitrophenol, ϵ = 1.78 × 10⁴ M⁻¹ × cm⁻¹, OD 405 nm (cm⁻¹) is the absorbance at 405 nm divided by the light-path length (cm), and V is the final assay volume¹² (In this study, V = 120 μl).

2.6. Immunofluorescence staining

To determine the morphology and localization of the osteogenic marker, osteocalcin (OCN), immunofluorescence was performed after the cells were cultured on the TMV coated substrates for 7 and 14 days in osteogenic media. The cells were fixed with 10% formalin for 20 min at room temperature. Each of the samples were permeabilized with 0.1% TRITON® X-100 (AMRESCO, Ohio) for 15 min and blocked with 1% bovine serum albumin (BSA) for 1 h. After blocking, the cells were incubated with 1.51 mg/ml Rabbit anti human osteocalcin (Primary antibody 1:200 dilution, Thermo scientific™, USA) at 37 °C for 1 h. After that, 4 μg/ml Goat anti rabbit antibody-AF546 (Secondary antibody, Thermo scientific™, USA) was spilled over for 45 min at room temperature. Then stained the nucleus with 1 mg/ml DAPI (4,6′-diamidino-2-phenylindole, Thermo scientific™, USA) at 1:100 PBS dilution for 30 min and used 6.6 μM FITC-Phalloidin (Thermo scientific™, USA) at 1:40 PBS dilution for 30 min to stain actin. For the result analysis, we took the images of the stained substrates and examined them under a fluorescence microscope (Olympus IX51) with 10x objective lens. Image J software was used for the data acquisition of different staining.^4,12

2.7. Alizarin red staining

In this research, we selected Alizarin red testing for the qualitative assessment of the cell color staining which reflects the calcium deposition from human alveolar bone cells. Alizarin red staining was performed after the cells were cultured on the TMV coated substrates for 7 and 14 days in osteogenic media. Briefly, the cells were fixed with 10% formalin for 20 min at room temperature then rinsed twice with ddH₂O. After that, they were stained with 40 mM Alizarin red solution (Sigma Aldrich, USA) pH 4.1–4.3, covered with aluminum foil and incubated for 30 min. Finally, the cells were rinsed with ddH₂O for 5 min 4 times and viewed by a bright-field optical microscope.

3. Results

3.1. Characterization of the virus coated substrates

Characterization of the TMV particles was confirmed by MALDI-TOF MS analysis, the peak of the coated protein expression was 17534 m/z, consistent with the previous study^9,17,18 (Fig. 1 (A)) and Transmission electron microscopy (TEM) to visualize the morphology of TMV particles by JEM-2100/HR (200 kV, JEOL Japan) microscope. The TMV particles were presented in a rod shape,15–18 nm in diameter and 280–350 nm in length (Fig. 1 (B)). The presence of the TMV coated substrates was verified by AFM. The AFM images showed that 0.1 mg/ml TMV concentration was able to cover the substrates almost completely. The average height of the virus that was collected from AFM images (n = 4) was 23.37 ± 4.87 nm which was similar in diameter to the single virus 18 nm (Fig. 1 (C)).

Fig. 1.

Characterization of TMV particles. (A) MALDI-TOF MS spectra of the subunit proteins of TMV (17534 m/z) (B) Transmission electron microscopy (TEM) image of TMV, magnification 150,000x scale bar 200 nm. (C) Atomic force microscopy (AFM) height image (5x5 µm) showing the coverage of substrates with TMV particles.

3.2. Viability of human alveolar bone cells

The viability test of two cell lines was determined by Resazurin to assess their metabolic rate. The normalized metabolic rate data was calculated based on the metabolic rate on day 1. The metabolic rate of TMV coated substrates in both cell lines increased from day 1 to day 14 when compared with the control group. Statistically, the analysis showed significant differences (p < 0.05) on days 3, 14 (in cell line OB1) and on days 7, 14 (in cell line OB2) (Fig. 2).

Fig. 2.

The average metabolic rates base on Resazurin (Prestoblue^TM) to determine cell viability in TMV coated substrates and no TMV coated substrates (control) in 2 different time points. (A) cell line OB1. (B) cell line OB2. Normalized fluorescence intensity 560/590 nm against day 1 was expressed as mean ± SD (n = 4 * p<0.05 based on Independent Sample T-test).

3.3. Osteogenic differentiation

Osteogenic differentiation was assessed by ALP activity assay, osteocalcin protein expression and calcium deposition. ALP expression was detected in both the experimental and the control group in osteogenic media on days 7 and 14. The measured ALP activity from each sample was normalized to the corresponding cell viability at each time point as shown in Fig. 3. In OB1, ALP activity of the TMV group was significantly higher than the control group on days 7 and 14 (p < 0.05) (Fig. 3(A)) whereas in OB2, ALP activity was found statistically higher in the TMV group than the control group on day 14 (Fig. 3(B)).

Fig. 3.

Qualification ALP activity by calculated enzyme activity to determine cell differentiation in TMV coated substrates and no TMV coated substrates (control) in 2 different time points. (A) cell line OB1. (B) cell line OB2. Normalized absorbance 405 nm against the viability test was expressed as mean ± SD (n = 4 * p<0.05 based on Independent Sample T-test).

3.4. Immunofluorescence staining

After days 7 and 14 in the osteogenic conditions, both cell lines demonstrated no difference in osteocalcin protein expression between the experimental and the control groups as shown by immunostaining (Fig. 4).

Fig. 4.

Images of immunofluorescence staining of cells in TMV coated substrates and no TMV coated substrates (control) at day 7 and 14. (A) cell line OB1. (B) cell line OB2. Color representation: osteocalcin (red), Nucleus (blue) and actin (green). Scale bars are 500 µm, x10 objective lens.

Having evaluated the morphologic changes of OB1 cells during the differentiation process on day 7, the cells in the experimental group showed more cell aggregation with polygonal shapes, while those in the control group were a long narrow fibroblast-like appearance and remained spread out over the substrates. However, on day 14, the OB1 cells in the control group became aggregated and polygonal shapes like those in the experimental group (Fig. 4(A)).

On the other hand, OB2 cells demonstrated long spindle-shaped, fibroblast-like at day 7 for both experimental and control groups. The cells seemed fully spread out on the substrates and cell aggregation was not recognized. However, on day 14, the experimental group with TMV showed the transformation of cell shape to polygonal shapes and aggregation. The control group, however, did not show any change and remained spread out to cover the entire substrates (Fig. 4(B)). The findings suggested that the cell line OB1 differentiated faster than the cell line OB2.

3.5. Alizarin red staining

On day 7, the cells in TMV coated substrates and the control group showed similar results of positive red staining, indicating calcium deposition. However, on day 14, the TMV group showed intense red color-like nodules whereas the control group did not show any changes from day 7 (Fig. 5).

Fig. 5.

Alizarin red staining to determine the mineralization of cells in TMV coated substrates and no TMV coated substrates (control) at day 7 and 14.

4. Discussion

TMV, the first plant virus discovered, has a structure at the nanomolecular level. It is currently in the interest of a wide range of biomaterial applications^{6, 7, 8, 9} since it was proved that it does not cause diseases or multiply itself in mammals.^4,5 The study of the TMV effect on cells biocompatibility is very limited, so far there have been only two studies in rBMSCs¹⁹ and hBMSCs¹⁴ in which biocompatibility was found in both lines. Our present study also found that TMV was biocompatible to human alveolar bone cells as shown by metabolic rate monitoring. Interestingly, the TMV significantly stimulated cell viability after a period of time. The stimulating mechanism is still unexplained and will require further detailed studying.

To evaluate bone differentiation, the assessment of ALP activity, osteocalcin expression and calcium deposition were performed. The ALP enzyme level is normally high during early osteoblastic differentiation and extracellular matrix synthesis. The level is usually reduced later during the mineralization process.³ This study found that the TMV coated substrates significantly stimulated ALP activity in both osteoblastic lines. This finding was in agreement with Sitasuwan P et al.¹⁰ who found ALP stimulating effect of TMV on rBMSCs. However, Liu T et al.¹⁴ showed no different ALP activity in hBMSCs after TMV stimulation.

The enhancement in bone cell differentiation was also assessed by the osteogenic marker. Osteocalcin is a protein-bound to calcium ion and hydroxyapatite. It is the most specific marker for osteogenic differentiation and mineralization.^11,14 Normally, osteocalcin is expressed during late osteoblastic differentiation and reaches the maximum level during mineralization by mature osteoblast and osteocyte.^3,20 In our present study, TMV showed no effect on osteocalcin expression in both osteoblast lines. Osteocalcin staining was detected around the nucleus under a fluorescent microscope after day 7 with no difference between TMV treated and control group. In contrast, Kaur G et al.¹¹ found TMV stimulated osteocalcin expression in rBMSCs. It appeared as darkened red-fluorescent staining in the TMV group. The color was even more intensified where the cells changed to be like sheet structure. In another study by Liu T et al.,¹⁴ a slightly higher level of osteocalcin expression by immunostaining was found in hBMSCs grown on TMV than the control group. Since immunostaining may not be a proper quantitative indication, future studies to identify the osteocalcin gene is recommend to confirm the effect of TMV on osteocalcin expression.

Observation of cell morphology using a fluorescence microscope revealed that the TMV coated substrates induced cellular aggregation in both osteoblastic lines after day 7. In addition, the aggregated cells changed the morphology from fibroblast-like structure to polygonal shape. This characteristic has resulted from the cytoskeletal change during cell differentiation.²¹ Our study was in accordance with Metavarayuth K et al.³ and Kaur G et al.¹¹ who found that TMV promoted morphology changed and cell aggregation in rBMSCs. The mechanism could possibly be explained by the stress of unfavorable surface from the TMV coated substrates which caused the reduction in focal adhesion area of cells grown on substrates. The cells then increased mobility, facilitating cell aggregation³

The Alizarin red staining for calcium deposition in the late stages of osteogenesis was performed to determine the mineralization process. We found that calcium deposition of cells grown on TMV coated substrates was stimulated after day 7 as shown by the intense red-color-like nodules. This observation was supported by previous studies which indicated rBMSCs grown on TMV substrates expressed deep red color in large nodules than the control counterpart.^3,10,11 For hBMSCs, TMV coated substrates also showed slightly more mineral nodules as compared to the control group.¹⁴ In agreement, our present study indicated a promising quality of TMV to promote osteogenic differentiation and mineralization process in primary human alveolar bone cells.

It would also be interesting to explore other types of plant viruses such as the potato virus Y or cucumber mosaic virus coated substrates. Since they can be easily obtained, they have the potential to be an important tool for medical research. Some limitations of our current in vitro experiment include the limited numbers of human alveolar bone cells in which we selected 2 of the most stable proliferation lines. Further studies are required to evaluate the expression of other osteogenic markers such as bone morphogenic protein-2 (BMP-2), osteocalcin, osteopontin, Runt-related transcription factor 2 (RunX2) and also to find the appropriate scaffold for implantation. Most importantly, careful monitoring of cells behavior is needed before future implantation application.

5. Conclusion

The TMV is biocompatible to primary human alveolar bone cells. It can stimulate the metabolic rate, ALP activity, cell aggregation and calcium deposition. This study shows the possibility of bone differentiation induction potential of the TMV to human alveolar bone cells. Thus, the TMV can function as an alternative matrix mimetic to promote bone cells in hard tissue engineering work.