An Alternative Therapy For Bone Defects Biology Essay

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There is a growing need to search for an alternative therapy for bone defects due to various problems encountered with the current surgical techniques. Therefore, much attention has been drawn to cell-based osteoregeneration, the success of which relies on obtaining a suitable source of cells that can form bone matrix-synthesizing osteoblasts. Embryonic stem (ES) cells and primary bone-derived cells may be useful for bone tissue engineering applications as they can potentially differentiate into osteoblasts. In vitro, the differentiation process must be strictly controlled to exclude the formation of undesired cell types.

This study sought to investigate whether a combination of dexamethasone and simvastatin can stimulate osteoblastic differentiation of mouse ES and primary bone-derived cells and, to further study the effect on osteogenesis of these cells in the presence or absence of ascorbic acid (AA) and β-glycerophosphate (β-GP) supplements. The present results showed that a combination of dexamethasone and simvastatin may impede osteogenic differentiation of mouse primary bone-derived cells, whilst addition of AA and β-GP enhanced osteogenesis. However, these findings need to be further investigated. Future work examining the osteogenesis at the final phase of osteoblastic differentiation and at the osteoblast-specific gene expression levels may be undertaken to further validate the data.

2. Introduction

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The choice of cell types for bone tissue engineering is largely dependent on the ability of these cells to form bone matrix-synthesizing osteoblasts. Identification of mesenchymal stem cells (MSCs) as precursors of osteoblasts in bone development has opened up the opportunity to implicate MSCs in bone tissue engineering application. The use of MSCs is, however, limited by laborious harvesting procedure and shortage of MSCs, thus prompting a search for alternative cell sources for use in this field. Embryonic stem (ES) and primary bone-derived cells can potentially be grown into osteoblasts and they may hold a promise in bone tissue engineering. This paper will start with a brief overview of ES and primary bone-derived cells. The potential use of ES and primary bone-derived cells in bone tissue engineering, their relative merits and limitations will also be briefly discussed. With respect to osteoblastic differentiation, specific growth or differentiation factors are necessary for directing the differentiation to the desired osteoblastic lineage in vitro. The osteogenic differentiation factors of interest will be highlighted in this paper.

2.1 Background

2.1.1 Embryonic Stem (ES) Cells

ES cells are derived from the inner cell mass of the pre-implantation blastocyst. The inner cell mass is obtained by separating it from the outer trophectoderm, which forms placenta at later blastocyst developmental stage (1). ES cells have the potential to differentiate into all 3 embryonic germ layers, namely mesoderm, endoderm and ectoderm, as well as reproductive germ cells that generate sperm and ova (2, 3). Mesoderm gives rise to haematopoietic, cardiac, bone, vascular, kidney tubule cells; endoderm derivatives are composed of pancreatic, liver, thyroid and alveolar cells; whereas ectoderm commits to neural cells, keratinocytes and pigment cells (Fig. 1) (4). Because of their capacity to form any tissues (except placenta), ES cells have been described as being pluripotent (4).

ES cells were first isolated from mice, followed by non-human primates and more recently, derivation of ES cells from humans has advanced the study of early human developmental events beginning from the embryonic stage (3, 5). They could remain undifferentiated in vitro permanently after being harvested from mice, primates or humans (6). Current strategies to maintain undifferentiated ES cells in vitro include co-culture with embryonic fibroblast feeder layer in the presence or absence of specific cytokines and non-cellular coating made up of proteins. Mouse ES cells (mES) can be arrested and maintained at undifferentiated state either on mouse embryonic fibroblast feeder layer, or by leukemia inhibitory factor (LIF) alone (2, 7). Human ES (hES) cells, in contrast, are unresponsive to LIF and require basic fibroblast growth factor (bFGF) in combination with embryonic fibroblasts instead (2, 6). In terms of protein-coated methods, the embryonic fibroblast feeder layer in mES cell culture is replaced by purified bovine gelatin (1), while hES cells are maintained on matrigel- or laminin-coated surface containing mouse embryonic fibroblast-conditioned medium (2, 6).

Extracellular matrix proteins

FIG. 1. The multilineage potential of embryonic stem (ES) cells and current strategies used to initiate the differentiation process. The inner cell mass is isolated from the blastocyst and remains undifferentiated on feeder layer in the presence or absence of cytokines (leukemia inhibitory factor or basic fibroblast growth factor). ES differentiation can be initiated via embryoid body formation, co-culture with stromal cells or with a monolayer of extracellular matrix proteins, leading to formation of meso-, endo- and ectodermal derivatives. Adapted from (4).

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ES cells have been shown in vivo to spontaneously form teratomas, benign tumours comprised of all 3 embryonic germ layers, following their injection into severe combined immunodeficient (SCID) mice (5, 8), while in vitro studies demonstrate that removal of differentiation-restricted factors, i.e. embryonic fibroblast- or protein-coated feeder layer and LIF or bFGF cytokines, leads to ES cell differentiation and subsequent generation of differentiating mesodermal, endodermal and ectodermal cells (6). These indicate that ES cells must undergo differentiation process before their commitment to a specific terminal lineage. In vitro, there are 3 techniques currently used to initiate ES cell differentiation: 1) embryoid body (EB) formation, 2) co-culture with stromal cells and, 3) culture on extracellular matrix proteins, all of which are equally effective (Fig. 1) (6). The generation of EB, a 3-dimensional cell aggregate composed of all 3 differentiating embryonic germ layers, resembles gastrulation, whereby meso-, endo- and ectoderm develop during early embryogenesis, thus mimicking the sequence of human developmental process more closely (1).

2.1.2 Primary Bone-derived Cells - The Osteoblast Lineage

Primary bone-derived cells contain a mixture of cell populations including mesenchymal stem cells (MSCs), osteoprogenitors, preosteoblasts, mature osteoblasts and terminally differentiated osteoblasts termed osteocytes (Fig. 2) (9). Interestingly, they are not restricted solely to osteoblastic cells (osteoprogenitors, preosteoblasts, osteoblasts and osteocytes), as evidenced by the fact that adipocyte precursors and adipocyte-osteoblast bipotential progenitors can be found in fetal rat calvaria-derived primary cultures, human trabecular bone-derived cells and human bone marrow stroma (10, 11).

Primary bone-derived cells have been studied extensively to provide a detailed insight of osteoblast development, which involves a series of cellular transitions originating from MSCs via intermediate osteoprogenitors and preosteoblast to differentiated osteoblasts (10). The phases of osteoblast formation are characterized as distinct sequential proliferation, differentiation and maturation of MSCs and/or osteoprogenitors (12), and the proliferative capacity of these osteoblast precursors diminishes as they are progressively transformed into terminally differentiated osteocytes, with concomitant increasing differentiation capacity along the osteoblast lineage (Fig. 2) (4).

FIG. 2. Primary bone-derived cells contain mixed cell populations. Cells along the osteoblastic lineage have reduced proliferative potential with concomitant increasing differentiation capacity as they proceed from less mature to more mature osteoblasts. Taken from (11).

MSCs are located primarily in bone marrow stroma and as such they are also termed bone marrow stromal stem cells, and their presence in other sites include adipose tissue, synovium, umbilical cord and placenta (13, 14). MSCs are multipotent, i.e. capable of differentiating into mesoderm-derived osteoblasts, chondrocytes and adipocytes that constitute bone, cartilage and fat respectively, as well as ectoderm-type astrocytes and endoderm-type hepatocytes (15, 16). Their occurrence in bone marrow is scarce, constituting about 0.003 - 0.015% of bone marrow cells (17).

Osteoprogenitors undergo cell division and differentiation into bone-forming osteoblasts. Similar to MSCs, osteoprogenitors are rare and present at relatively low frequency (< 1%) in rat calvaria culture, rat and mouse bone marrow stroma (11). They can be subdivided into 2 populations: 1) immature osteoprogenitors, which are only differentiation-inducible by osteoinductive factors e.g. dexamethasone, progesterone, bone morphogenetic proteins and, 2) mature osteoprogenitors that can constitutively differentiate into osteoblasts in the presence of standard osteogenic factors including fetal calf serum, ascorbic acid and β-glycerophosphate (11).

Osteoblasts are responsible for bone matrix production by secreting various bone extracellular matrix proteins e.g. type I collagen, osteonectin, osteopontin and osteocalcin, and they regulate bone matrix mineralization (10, 18). In addition, they express high levels of membrane-bound alkaline phosphatase (ALP) enzyme (10). Both secreted proteins and elevated ALP activity have been used as markers of osteoblastic differentiation. In regard to bone physiology, the bone-matrix synthesizing function of osteoblasts is crucial for bone remodelling, a bone replacement process by which repetitive cycles of bone formation by osteoblasts and bone resorption by osteoclasts occur to maintain calcium and phosphorus homeostasis and to repair micro-cracks induced by mechanical constraints (19, 20).

2.2 Bone Tissue Engineering

Bone defects arise as a result of disruption in bone remodeling and that disruption could be caused by trauma or diseases such as osteoporosis, diabetes and tumour (20). Conventional treatments for bone defects include artificial implants and allogenic (obtained from other individuals) or autologous (from patients themselves) bone grafting (14, 20). Issues arising from these strategies, such as prosthetic implant malfunction, limited biocompatible graft supply and immunogenic host response associated with allogenic grafts (14), have led to emergence of bone tissue engineering.

Tissue engineering, by definition, is an interdisciplinary field that combines the biomedical and engineering expertise to generate biological substitutes that can repair or replace damaged or diseased tissue with the hope to restore normal tissue function (21). In principal, there are three components involved in bone tissue engineering: 1) cells with osteogenic potential, 2) growth and differentiation factors for cell proliferation and differentiation and; 3) scaffolds (22). Each component can be used either alone or in combination (1).

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Recent interest has been drawn to the development of cell-based approaches as, in addition to overcoming the above mentioned limitations, establishment of successful cell-based regenerative therapies for cartilage and skin replacement (23) and a general belief of tissue restoration by cells is superior to any other artificial device or chemically-synthesized therapeutic compounds (24) have pointed that this relatively new approach may offer a more promising treatment for bone defects in the future. The efficacy of growth factor- and scaffold-based therapies depends partly on the availability of host endogenous osteoprogenitors, which are recruited from bone marrow or periosteum to site of application for effective bone repair (25). In this regard, cell-based therapy may be more beneficial to patients with compromised or diminished bed of bone tissues since it is independent of host osteoprogenitor infiltration as opposed to growth factor- and scaffold-based approaches (25).

Cell-based bone repair therapy requires in vitro expansion and/or differentiation of suitable cells with osteogenic potential before they are transplanted to host body. Cell transplants may be applied directly to bone defect site, or used in combination with a scaffold carrier for implantation (14). Clearly, isolation and selection of appropriate cell types are critical to success of cell-based approaches. While implication of mesenchymal stem cells (MSCs) in bone repair has widely been documented (20), ES and primary bone-derived cells may represent possible candidates of cell source for bone tissue engineering.

As indicated earlier, MSCs have the ability to form derivatives of mesenchymal lineage including bone, cartilage, tendon, muscle and adipocyte (25). In vitro, they can be expanded and remain undifferentiated under appropriate culture conditions, producing a reservoir of MSCs for transplantation (17). After implantation, MSCs still retain the capacity to differentiate into bone tissue in vivo (14). Ex vivo expanded MSCs have been proven to effect bone repair in both animal and pilot clinical studies (20, 25). Also, MSCs can be directed in vitro towards the osteoblastic lineage forming differentiated osteoblasts, which may be transplanted to recipients to effectively accelerate bone formation (25).

Nevertheless, MSCs are present at relatively low amounts in bone marrow and their frequency decreases with increasing age (14). This may result in low accessibility of these cells or in other words, it could be difficult to isolate sufficient number of homogenous MSC population for both in vitro expansion and bone tissue engineering application. In addition, their proliferation and differentiation capacity are reduced as the age advances (15), further contributing to the issue associated with shortage of MSCs. And, they have limited self-renewal capacity as demonstrated by finite population doublings of 24-40 before they enter senescence (17). Thus, MSCs may have to be constantly harvested from the donor for in vitro expansion.

Taken together, use of ES cells may overcome the issues associated with MSCs. As are with MSCs, ES cells have high multi-lineage potential, remain undifferentiated in vitro and can be induced, under specific signals, to cells with osteoblast phenotype (15, 20). They have been shown in vitro to propagate indefinitely without losing their pluripotency (i.e. unlimited self-renewal capacity), thus providing inexhaustible source of ES cells or more committed ES cell-derived progeny (2, 15). The later property may additionally confer ES cells an ideal cell source for bone tissue engineering application.

ES cells are still far from being used clinically, yet pre-clinical animal models of cardiac, neuronal and Parkinson's disease have demonstrated that mouse ES cell-derived progeny and terminally differentiated derivatives can, respectively, recapitulate the specific cell population and restore the normal tissue function (26). Based on these approaches, ES cell-derived osteogenic progeny or differentiated functional osteoblasts can theoretically be used for cell-based bone replacement. The harvested ES cells can be propagated and differentiated in vitro towards the osteoblastic lineage, producing osteoprogenitors or mature osteoblasts. The less differentiated osteoprogenitors may be further directed towards mature osteoblasts under the influence of in vivo signals or alternatively, transplantation of fully functional osteoblasts to hasten the bone healing process.

The success of cartilage replacement using autologous chondrocytes (27) may indicate the potential use of any mature cells including primary bone cells for re-implantation into the recipient. Furthermore, bone-derived cells have more restricted lineage potential and therefore they may generate a more homogeneous population of osteoblasts compared to multipotent MSCs or pluripotent ES cells, for which the resulting osteoblast-enriched cultures of MSCs and ES cells may contain cells of diverse phenotypes.

There are some limitations associated with the use of primary bone-derived cells. As invasive surgical intervention is needed for primary cell isolation, undesirable side effects e.g. pain and discomfort at the surgical site may arise (28). Primary bone-derived cells consist of mainly differentiating or differentiated cells with low proliferative potential, this may limit their expansion in vitro to sufficiently produce a large amount of cells available for transplantation (28). Hence, it is important to take these limitations into consideration before primary bone-derived cells are implicated in bone tissue engineering.

2.3 Osteoblastic Differentiation

There is a need to strictly control the differentiation to the desired osteoblastic lineage before the usage of ES or primary bone-derived cells for cell-based bone regeneration therapy. Osteoblastic differentiation is regulated by cytokines, growth factors and hormones. In vitro, this process can be stimulated by dexamethasone, bone morphogenetic protein-2 (BMP-2), statins, 1,25-dihydroxyvitamin D, insulin-like growth factor-1 (29), melatonin (30) and IL-6 related cytokines (31), and the list may not be exhaustive. This review will focus on the former three factors that can promote osteoblastic differentiation.

2.3.1 Dexamethasone

Dexamethasone is a common osteoinducer and has been shown to stimulate osteoblastic differentiation of various cell types including fetal calvarial cells, bone marrow stromal cells and ES cells (15, 32). It is often used in combination with ascorbic acid and β-glycerophosphate to induce osteogenesis.

2.3.2 Bone Morphogenetic Protein-2 (BMP-2)

BMP-2 is a growth factor that belongs to transforming growth factor-β super family and is present in the bone extracellular matrix (33, 34). It stimulates ectopic bone formation in vivo although the mechanism of action remains unknown (34). Its osteoinductive activity has been studied on various cell types, recombinant BMP-2 stimulates the formation of osteoblast-like cells in bone marrow mesenchymal precursor and pre-osteoblastic cell culture system (35). Also, deletion of BMP-2 gene from mouse osteoprogenitors has led to a significant reduction in proliferation and differentiation of these osteoprogenitors indicating that there is a requirement for BMP-2 gene expression in osteoblastic differentiation (33, 34).

2.3.3 Statins

Statins inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase leading to reduced production of downstream isoprenoids, therefore the subsequent cholesterol synthesis is inhibited (Fig. 3). Their pleiotropic effects such as anti-proliferation, anti-inflammation, immunosuppression, vasodilation, anti-thrombosis, anti-oxidant and increase in bone formation have been reported in various studies (36).

FIG. 3. The pleiotropic effects of statins. Statins promote osteoblastic differentiation via upregulation of bone morphogenetic protein-2 (BMP-2) gene expression and increased production of vascular endothelial growth factor (VEGF). Taken from (37).

The anabolic effect of statins on bone was first identified by Mundy et al., treatment with simvastatin, mevastatin and fluvastatin led to increased BMP-2 mRNA expression in human MG63 osteoblast-like osteosarcoma and mouse 2T3 osteoblastic cells as well as enhanced bone formation both in vitro and in vivo (38). Subsequently, the osteogenic effect of simvastatin and compactin (mevastatin) was further investigated on cultured MC3T3-E1 (mouse-derived non-transformed pre-osteoblastic cell line) (39) and mouse ES cells (35) respectively; these studies confirm that simvastatin and compactin promote osteoblastic differentiation via upregulation of BMP-2 gene expression. The second mechanism by which statins stimulate osteoblastic differentiation is mediated via production of vascular endothelial growth factor (VEGF), an anabolic factor of bone formation (37). The enhanced expression of BMP-2 and VEGF is a result of decreased isoprenylation of Ras, Rho and Rac proteins, a reduction that is caused by inhibition of upstream isoprenoid intermediates (Fig. 3) (37).

2.4 Objectives

As discussed above, ES and primary bone-derived cells can be used as a cell source for bone tissue engineering and each cell type has its own merits and limitations in terms of lineage potential and proliferative capacity. ES cells are pluripotent and highly proliferative, whereas primary bone-derived cells have the advantage of generating more restricted lineage, specifically osteoblastic lineage, but with low proliferative capacity. To accurately and efficiently direct the differentiation along the osteoblastic lineage in vitro, considerable research is focused on designing and optimizing the protocols for osteoblastic differentiation in vitro with the hope of generating cultures enriched with pure osteoblastic cells. The initial objective of the experiment was therefore to compare the growth and osteoblastic differentiation of mouse ES and primary bone-derived cells in response to dexamethasone at varying concentrations. However, mouse ES cells did not grow as they should during the culture period and so the experimental design was changed slightly in such a way that simvastatin was included and that much focus has been drawn to osteogenesis of primary bone-derived cells.

The current objectives were:

1) To examine the effect of dexamethasone and simvastatin combination on proliferation of mouse ES and mouse primary bone-derived cells;

2) To investigate whether the combination of dexamethasone and simvastatin will stimulate the osteoblastic differentiation of mouse primary bone-derived cells;

3) To further study the effect on osteogenesis of primary bone-derived cells in the presence or absence of ascorbic acid-2-phosphate (AA) and β-glycerophosphate (β-GP) supplements.

3. Materials and Methods

3.1 Cell Culture

3.1.1 Mouse ES cells

Mouse ES cells were maintained on mitomycin C-inactivated mouse SNL fibroblast feeder layer in a standard culture medium containing Dulbecco's Modified Eagles Medium (DMEM), 10% fetal calf serum (FCS), 1% penicillin/streptomycin (Pen/Strep), 1% L-glutamine and 100 µM 2-mercaptoethanol under stationary conditions (37°C, humidified atmosphere with 5% CO2). Leukemia inhibitory factor (LIF) was added to maintain mouse ES cell pluripotency or undifferentiated state.

To initiate mouse ES cell differentiation, embryoid bodies (EBs) were allowed to form in mass suspension culture as previously described (40). Briefly, mouse ES cell colonies were lightly trypsinized with trypsin-ethylenediaminetetraacetic acid (EDTA), cetrifuged, resupended in standard culture medium and plated onto bacteriological grade petri dishes for 5 days. Removal of mouse SNL fibroblasts and differentiation-restricted LIF resulted in formation of EBs. The EBs were then dissociated with 0.25% trypsin-EDTA, resuspended in standard culture medium and cultured where appropriate in gelatinised 12- or 96-well plates at a seeding density of 5 x 104 cells/mL in each well.

3.1.2 Mouse primary bone-derived cells

Mouse primary bone-derived cells were isolated from neonatal mouse calvariae by sequential enzymatic digestion of calvariae. Cells were seeded at a density of 5 x 104 cells/mL and maintained in a standard culture medium containing α-Minimal Essential Medium (α-MEM), 10% FCS, 1% Pen/Strep and 1% L-glutamine at 37°C in a 5% CO2 humidified incubator.

3.1.3 Cell Density

As mentioned above, cells were seeded at a cell density of 5 x 104 cells/mL per well (unless otherwise specified) for each indicated assay prior to osteogenic stimulation.

3.1.3 Osteodifferentiation

Both mouse ES and primary bone-derived cells were cultured in control and differentiation media, both media either supplemented with (+) or without (-) 50 µg/mL ascorbic acid-2-phosphate (AA) and 50 mM β-glycerophosphate (β-GP) supplements. The control media were standard culture media, whereas the differentiation media contained additional dexamethasone and simvastatin in various combinations - dexamethasone (0, 0.1, 1 and 100 µM) + 5 µM simvastatin and simvastatin (0, 0.5, 5 and 50 µM) + 1 µM dexamethasone. The cultures were incubated at 37°C in 5% CO2 humidified atmosphere. The medium in each well was replenished every 2-3 days until the indicated assays were performed.

3.2 Cell Proliferation Assay

Mouse primary bone-derived cells plated in 96-well plate at an increasing cell density (0, 1 x 104, 2.5 x 104, 5 x 104 and 1 x 105 cells/mL per well) were cultured in control media in the presence or absence of AA and β-GP supplements. This plate was used to generate a standard cell density curve. Mouse ES and primary bone-derived cells seeded in each respective 96-well plate were treated with control or differentiation media in the presence or absence of supplements. The volume of medium added to each well was 100 µL.

After 2 days of treatment, cell proliferation was determined using CellTiter® 96 AQueous Non-Radioactive Cell Proliferation assay (Promega, UK). The combined MTS ([3-(4,5-dimehtylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]/PMS (phenazine methosulfate) solution (20 µL) was added to each well containing 100µL medium. The 96-well plates were then incubated at 37°C in 5% CO2 humidified atmosphere for 2 hours, after which the plates were read at 490nm absorbance using ELISA (enzyme-linked immunosorbent assay) plate reader. Data were expressed as corrected 490nm absorbance, from which the background absorbance was subtracted to eliminate any physical or chemical interference that may contribute to increased 490nm absorbance readings.

3.3 Cytotoxicity Assay

CytoTox-ONE™ Homogeneous Membrane Integrity assay (Promega, UK) was performed to assess the cytotoxic effect of dexamethasone-simvastatin combination. Mouse primary bone-derived cells seeded in 96-well plate was treated with control or differentiation media in the presence or absence of supplements. The volume of medium added to each well was 100 µL.

The cytotoxicity of the compounds was assessed 2 days after the treatment according to manufacturer's protocol. The 96-well plate was equilibrated to 22°C for about 20-30 minutes. Lysis Solution (2 µL) was added to 3 control wells to get a maximum lactate dehydrogenase (LDH) release. CytoTox-ONE™ Reagent (100 µL) was added to each remaining well and again incubated at 22°C. After 10 minutes, Stop Solution (50 µL) was added to each well and the plate was shaked gently to allow thorough mixing. The fluorescence was recorded at excitation wavelength of 560nm and emission wavelength of 590nm. Data were expressed as mean percentage cytotoxicity, which was calculated as follows:

3.4 Alkaline Phosphatase (ALP) Assay

Mouse primary bone-derived cells were seeded in 12-well plates and ALP activity was measured on Day 9 of culture using SensoLyte® p-Nitrophenyl Phosphate (pNPP) Assay Kit (Anaspect). Cells in each well were washed twice with 1X lysis buffer, lysed with Triton X-100, followed by scrapping off adherent cells to collect the cell suspension, which was incubated and centrifuged sequentially at 4°C for 10 minutes. The supernatant (50 µL/well) of each sample was then added with 50 µL/well of pNPP substrate and incubated for 30 minutes at 37°C in 5% CO2 humidified atmosphere. After incubation, stop solution was immediately added and the absorbance was recorded at a wavelength of 405 nm. Results were expressed as ALP activity in µg/mL. ALP activity was then normalized to the parallel total protein content, which was determined by bicinchoninic acid (BCA) assay.

3.5 Immunostaining Assay

Immunostaining assay was performed on Day 14 of culture to detect the markers of osteoblastic differentiation. Culture media were disposed off and cells were fixed in phosphate buffered saline (PBS) containing 4% paraformaldehyde for 20 minutes, washed in PBS and immunostained using avidin-biotin-peroxidase complex method, whereby the cell slides were immersed for 30 minutes in 0.03% hydrogen peroxide in methanol to inhibit endogenous peroxidase. After 3 times 10-minute washes with PBS, non-specific binding was blocked by incubation with normal goat serum for 20 minutes, blot dried and incubated overnight at 4°C with rabbit primary antibodies against core binding factor α-1 (cbfa-1), cadherin-11 and osteocalcin [1:1000 dilution in PBS:bovine serum albumin (BSA; 0.05 %):sodium azide (0.01%) for each marker]. Next, after washing in PBS, cells were first incubated with biotinylated anti-serum against rabbit IgG (1:100 dilution in PBS:BSA) for 30 minutes, followed by another incubation with avidin-biotin-peroxidase complex (Vectastain, Vector Laboratories) for 60 minutes. Diaminobenzidine-hydrogen peroxide method was used to detect the peroxidase activity. Images were captured at a magnification of 20.

3.6 Morphological Examination

Mouse primary bone-derived cells were morphologically examined on Day 8 of culture using phase contrast microscope. Images were taken at a magnification of 20.

3.7 Statistical Analysis

Results of cell proliferation, cytotoxicity and ALP activity were expressed as mean ± standard error of mean (SEM) of triplicate. Unpaired Student's two-tailed t-test was performed to assess whether the differences observed between different data pairs were statistically significant. A value of P < 0.05 was considered statistically significant.

4. Results

4.1 Cell Proliferation

Viable cells contain dehydrogenase, which reduces MTS [3-(4,5-dimehtylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium], an active component of CellTiter® 96 AQueous Assay reagent, into aqueous formazan. Amount of formazan formed as quantified by the amount of 490 nm absorbance increases exponentially with viable cell number (Fig. 5). This means that cell proliferation, if occurring, will result in a greater number of viable cells and thus amount of 490nm absorbance.

FIG. 4. Standard cell density curve for mouse primary bone-derived cells seeded at an increasing cell density in α-Minimal Essential Medium (α-MEM) supplemented with or without ascorbic acid-2-phosphate (AA) and 50 β-glycerophosphate (β-GP) supplements.

Dexamethasone + 5 µM Simvastatin

(a)

(b)

FIG. 5. Effect of dexamethasone-5 µM simvastatin combination, supplemented with or without AA and β-GP supplements, on proliferation of mouse ES cells (a) and mouse primary bone-derived cells (b).

Data are means of 490nm absorbance ± SEM.

* P < 0.05 statistically different from control (+ supplements).

# P < 0.05 statistically different from control (- supplements).

- P < 0.05 for comparison between with and without supplements.

For both mouse ES and primary bone-derived cells, a combination of dexamethasone (0.1-100 µM, with or without supplements) and 5 µM simvastatin showed a significant dose-dependent decrease in 490 nm absorbance amount compared to control (*, #P < 0.05). The optimal amount of 490 nm absorbance was observed in 0.1 µM dexamethasone, whilst 100 µM dexamethasone had the smallest amount of absorbance among the combinations in the presence or absence of supplements. Also, there was a significant reduction in proliferation of both mouse ES and primary bone-derived cells when simvastatin (5 µM) was added singly. A higher 490nm absorbance was observed in the untreated mouse ES cell cultures compared to that in the cultures of primary bone-derived cells. Treatment with AA and β-GP supplements resulted in a comparatively higher 490 nm absorbance than that without supplements (Fig. 6a and 6b).

Simvastatin + 1 µM Dexamethasone

(a)

(b)

FIG. 6. Effect of simvastatin-1 µM dexamethasone combination, supplemented with or without AA and β-GP supplements, on proliferation of mouse ES cells (a) and mouse primary bone-derived cells (b).

Data are means of 490nm absorbance ± SEM.

* P < 0.05 statistically different from control (+ supplements).

# P < 0.05 statistically different from control (- supplements).

- P < 0.05 for comparison between with and without supplements.

When compared to control, simvastatin (0.5-50 µM, with or without supplements) dose dependently decreased 490nm absorbance amount in both cell types, with 0.5 µM simvastatin giving the greatest absorbance amount among the combinations. Again, 490nm absorbance was relatively higher in control cultures of mouse ES cells compared to mouse primary bone-derived cells, as well as in both control and simvastatin-1 µM dexamethasone combination supplemented with AA and β-GP supplements compared to that without supplements.

4.2 Cytotoxicity

Cytoplasmic lactace dehydrogenase (LDH) is released into surroundings upon cell lysis. LDH works synergistically with diaphorase to reduce CytoTox-ONE™ Reagent component resazurin into fluorescent resorufin. The measured amount of fluorescence is proportional to the number of lysed cells, thus the cytotoxicity of the tested compounds can be indirectly determined.

FIG. 7. Mean percentage cytotoxicity for mouse primary bone-derived cells treated with control or differentiation (dexamethasone-simvastatin combination) media in the presence or absence of AA and β-GP supplements.

Data are means of percentage cytotoxicity ± SEM.

* P < 0.05 statistically different from control (+ supplements).

# P < 0.05 statistically different from control (- supplements).

- P < 0.05 for comparison between with and without supplements.

Dexamethasone + 5 µM Simvastatin

Dexamethasone-simvastatin combination (0.1-100 µM, with or without supplements) showed a significant lower mean percentage (%) cytotoxicity compared to control (*, #P < 0.05). Upon addition of supplements, there was a reduction in mean % cytotoxicity for both control and dexamethasone-5 µM simvastatin combination (Fig. 8).

Simvastatin + 1 µM Dexamethasone

Mean % cytotoxicity increased with increasing simvastatin concentrations, but it was significantly lower in 0-5 µM simvastatin-1 µM dexamethasone than that in control (*, #P < 0.05). The decreased mean % cytotoxicity was observed too in the presence of supplements for both control and simvastatin-1 µM dexamethasone combination (Fig. 8).

4.3 Alkaline Phosphatase (ALP) Activity

ALP is expressed by osteoblasts and it is an early phenotypic marker of osteoblastic differentiation.

Dexamethasone + 5 µM Simvastatin

(a)

(b)

FIG. 8. Effect of dexamethasone-5 µM simvastatin combination on ALP activity of mouse primary bone-derived cells. ALP activity was assessed on Day 9 of culture and normalized to total protein content. (a) Comparison of ALP activity of mouse primary bone-derived cells between controls and treatment groups. (b) ALP activity of mouse primary bone-derived cells for dexamethasone-5 µM simvastatin.

Data are means of ALP activity ± SEM.

* P < 0.05 statistically different from control (+ supplements).

# P < 0.05 statistically different from control (- supplements).

- P < 0.05 for comparison between with and without supplements.

ALP activity for dexamethasone-5 µM simvastatin-treated groups (0.1-100 µM, with or without supplements) was significantly lower compared to controls (*, #P < 0.05), and a significant higher ALP activity was seen upon addition of supplements (-P < 0.05). Dexamethasone-5 µM simvastatin showed an optimal ALP activity at concentration of 0.1 µM (Fig. 9b).

Simvastatin + 1 µM Dexamethasone

(a)

FIG. 9. Effect of simvastatin-1 µM dexamethasone combination on ALP activity of mouse primary bone-derived cells. ALP activity was assessed on Day 9 of culture and normalized to total protein content. (a) Comparison of ALP activity of mouse primary bone-derived cells between controls and treatment groups. (b) ALP activity of mouse primary bone-derived cells for simvastatin-1 µM dexamethasone.

Data are means of ALP activity ± SEM.

* P < 0.05 statistically different from control (+ supplements).

# P < 0.05 statistically different from control (- supplements).

- P < 0.05 for comparison between with and without supplements.

Simvastatin-1 µM dexamethasone combination (0.5-50 µM, with and without supplements) had a significantly lower ALP activity compared to control (*, #P < 0.05), with 0.5 µM simvastatin showing greater ALP activity among the combinations (Fig. 10a and 10b). ALP activity was enhanced in the presence of supplements for both control and simvastatin-1 µM dexamethasone combination (Fig. 10a).

4.4 Immunostaining

Osteoblastic differentiation can be examined by immunostaining the markers of osteoblastic differentiation. Core binding factor α-1 (cbfa-1) is a transcription factor regulating osteoblastic differentiation, cadherin-11 is a cell adhesion molecule expressed on osteoblasts and osteocalcin is a marker of late osteoblastic differentiation as it is expressed only in the mature osteoblasts.

b

a

c

FIG. 10. Immunostaining of mouse primary bone-derived cells for markers of osteoblastic differentiation: cbfa-1 (a), cadherin-11 (b), osteocalcin (c). Red arrows indicate presence of the respective stained markers. Osteoblastic differentiation was confirmed by the localization of these markers in mouse primary bone-derived cells. Original magnification x 20.

4.5 Morphological Examination

Control (+)

Dex 100 µM (+)

Dex 0.1 µM (+)

Simva 50 µM (+) (+)

Simva 0.5 µM (+) (+)

FIG. 11. Microscopic examination of mouse primary bone-derived cells in control or differentiation culture media (with AA and β-GP supplements) on Day 8 of culture. Spindle-like cell structures indicate that the cells are differentiating, as seen in 0.1 µM dexamethasone-5 µM simvastatin and 0.5 µM simvastatin-1 µM dexamethasone culture, although cell death represented as small clusters of rounded cells are observed. At high concentrations (100 µM dexamethasone and 50 µM simvastatin), cell debris is observed. Dex = dexamethasone; simva = simvastatin. Original magnification x 20.

5. Discussion

5.1 Effect of Dexamethasone-simvastatin Combination

Cell Proliferation

Publications on the proliferative effect of simvastatin are scarce and the findings have been variable. While two studies reported that simvastatin, as low as 1 µM, decreased the proliferation of human bone marrow stromal cells (41, 42), it was mitogenic at concentrations of 1 µM and 10 µM in fetal mouse calvarial osteoblast-like cells and thereafter a reduction in proliferation was seen (43). These reported data may suggest that simvastatin inhibits cell prolifetation in a dose-dependent manner. Dexamethasone at pharmacological doses (0.1 µM and 1 µM) has been shown to stimulate proliferation of fetal rat calvarial cells, rat and human bone marrow stromal cells and human alveolar bone cells (44, 45), the proliferation effect is paradoxical in mouse species such that treatment with dexamethasone leads to inhibition of cell growth in mouse calvaria and mouse bone marrow stromal cells (46, 47). Taken together, that diminished proliferation of both mouse ES cells and primary bone-derived cells observed in either dexamethasone-5 µM simvastatin or simvastatin-1 µM dexamethasone may be attributed to concentration-dependent anti-proliferative function of simvastatin and species-specific inhibition of proliferation by dexamethasone. This is evidenced by the suppression of cell proliferation upon single treatment with 5 µM simvastatin or 1 µM dexamethasone (Fig. 6 and 7).

Consistent with one study showing that dexamethasone (0.003-0.3 µM) dose dependently decreased proliferation of mouse osteoprogenitors (46), the present findings demonstrated that dexamethasone-5 µM simvastatin enhanced cell proliferation most effectively at 0.1 µM dexamethasone despite the presence of anti-proliferative simvastatin (5 µM) indicating that dexamthasone may have limited inductive effect on cell proliferation at lower concentrations, presumably at and/or less than 0.1 µM. Similarly, simvastatin-1 µM dexamethasone diminished cell proliferation in a dose-dependent manner and this may imply that simvastatin is likely to be proliferative at lower concentration and that high concentrations of simvastatin (5 µM and 50 µM) appear to be anti-proliferative as mentioned above.

When compared between mouse ES and primary bone cells in control cultures, cell proliferation of mouse ES cells appeared to be more pronounced as the 490nm absorbance amount for mouse ES cells was relatively higher than that for primary bone-derived cells (1.2-1.4 vs 0.9 absorbance units; Fig. 6 and 7). Mouse ES cells exhibit extremely high proliferative and differentiation potential, this may account for the observation of greater cell growth of mouse ES cells in untreated cultures compared to primary bone-derived cells, in which osteoprogenitors capable of proliferating and differentiating towards osteoblasts are at relatively low number and have comparatively limited self-renewal capacity. In the presence of dexamthasone-simvastatin combination, mouse ES cells, however, did not give a relatively higher cell proliferation profile than mouse primary bone-derived cells, implying that dexamethasone-simvastatin combination may restrict cell proliferation to a similar extent irrespective of the cell types.

Differentiation is coupled with down-regulation/cessation of proliferation and requires deposition of collagen in bone extracellular matrix (48, 49). Although the implication of growth arrest induced by dexamethasone-simvastatin combination remains unclear, it is unlikely that this suppression of cell proliferation may contribute to and accelerate osteodifferentiation of mouse ES or primary bone-derived cells because the ALP activity was not correspondingly increased, but rather decreased in the presence of either dexamethasone-simvastatin combination (Fig. 9a and 10a).

Cytotoxicity

Dexamethasone-5 µM simvastatin combination had relatively lower mean percentage cytotoxicity compared to control indicating that combination of dexamethasone and simvastatin may have a protective effect against any cytotoxic cellular events that could cause cell death. However, the finding remains to be elucidated due to as cell debris can be found in dexamethasone-5 µM simvastatin-treated cultures as shown in Fig. 12.

There was a reduction in mean percentage cytotoxicity in simvastatin-1 µM dexamethasone-treated groups, implying that simvastatin-1 µM dexamethasone combination at lower concentrations (0.5 µM and 5 µM) is less cytotoxic and may protect the cells against cytotoxic events. When comparing among the combinations, simvastatin-1 µM dexamethasone showed a dose-dependent increase in mean percentage cytotoxicity and this suggests that simvastatin is more cytotoxic at higher concentrations.

Osteodifferentiation

The present data demonstrated that either dexamethasone-simvastatin combination significantly decreased the ALP activity of mouse primary bone-derived cells, a finding that is consistent with that noted in the cell proliferation assay. Proliferation precedes and is required for later-stage differentiation of mesenchymal stem cells or osteoprogenitors present in the fetal mouse calvarial population (50). Suppressing cell proliferation may prevent their progression to differentiate which could in turn decrease the formation of differentiated osteoblasts expressing ALP. Therefore, that inhibition of cell proiliferation at the earlier phase may subsequently account for the reduced osteodifferentiation of mouse primary bone-derived cells.

Also, when correlated with the findings from cell proliferation assay, a parallel dose-dependent decrease in ALP activity of mouse primary bone-derived cells was observed too. Both dexamethasone-5 µM simvastatin and simvastatin-1 µM dexamethasone dose dependently decreased the ALP activity, with the maximal osteodifferentiation occurred at 0.1 µM dexamethasone and 0.5 µM simvastatin respectively. This finding may further support the above notion, i.e osteoblastic differentiation is closely linked to cell proliferation and that maximal osteodifferentiation at lower concentrations of dexamethasone-simvastatin combination is likely to result from the preceding increase in cell proliferation.

Recent studies have reported that simvastatin increases ALP activity of fetal mouse calvarial osteoblasts, MC3T3-E1, mouse and human bone marrow stromal cells (39, 43, 49, 51). This positive effect, however, occurred at considerably low doses of simvastatin (≤ 1 µM) and its effect on these cells at concentrations beyond 1 µM was not examined. The present data showed that simvastatin (5 µM) significantly reduced ALP activity of mouse primary bone-derived cells when it was added on its own (Fig. 9a) and such discrepancy could be due to the difference in simvastatin concentration, i.e. a much higher concentration (5 µM) was used in this experiment. Based on the data obtained, it may be postulated that simvastatin at high concentration may inhibit osteoblast differentiation. Accordingly, that strikingly decreased osteoblastic differentiation as reflected by a significant suppression of ALP activity in dexamethasone-5 µM simvastatin-treated cell cultures may be explained, at least in part, by the concomitant presence of relatively high concentration of simvastatin.

The effect of dexamethasone on osteoblastic differentiation seems to be species-specific, while several findings show that dexamethasone induces osteoblastic differentiation of fetal rat calvarial osteoblast-like cells, rat and human bone marrow cells (46), other studies have reported that opposite effect is seen in mouse-derived bone marrow stromal cells, MC3T3-E1 cell line and fetal mouse calvarial cells (44, 46, 47). When dexamethasone was added singly, reduction of osteoblastic differentiation as shown by a significant diminished ALP activity (Fig. 10a) is consistent with the reported studies, implying that inhibition of osteoblastic differentiation in either dexamethsone-simvastatin combination could be caused by species-specific inhibitory effect of dexamethasone on osteogenesis.

5.2 Effect of Ascorbic Acid (AA) and β-Glycerophosphate (β-GP)

Addition of AA and β-GP supplements enhanced cell proliferation of both mES and primary bone-derived cells in control or dexamethasone-simvastatin combination-treated cultures. The stimulatory effect, however, was not considered to be statistically significant in most of the control and treatment groups (Fig. 6 and 7), indicating that these supplements may not have a pronounced stimulation on cell growth.

A decline in cytotoxicity was noted in the presence of AA and β-GP supplements for both control and combination of dexamethasone and simvastatin. The anti-oxidant property of ascorbic acid may protect the cultured mouse primary bone-derived cells against any toxic events that could lead to cell burst, thus reducing the number of dead cells in the cultures. Interestingly, that reduction was not statistically significant in dexamethasone-simvastatin combination (except dexamethasone-5 µM simvastatin at 1 µM and 100 µM dexamethasone) as opposed to controls. This suggests that the protective effect AA is less remarkable when it is in combination with either dexamethasone-simvstatin combination and these agents are probably interfering with each other through an undefined mechanism that limits the anti-oxidant activity of AA. Despite that, AA may still serve as a powerful anti-cytotoxicity agent to prevent cell death associated with oxidative stress as shown by a significantly lower mean percentage cytotoxicity observed in the control culture with AA and β-GP supplements.

AA and β-GP markedly enhanced osteogenesis as reflected by a significantly higher ALP activity of mouse primary bone-derived cells in supplemented than that in non-supplemented cultures. This is in good agreement with previous observations showing that requirement for AA and β-GP in in vitro osteoblastic differentiation of primary osteoblasts and mouse MC3T3-E1 cells (50). AA has been known as an important co-factor for collagen hydroxylation, which forms the basis of collagenous extracellular matrix production (50, 52). Thus, as collagen deposition in extracellular matrix is a prerequisite for osteodifferentiation, treatment of mouse primary bone-derived cells with AA may favour osteoblastic differentiation through induction of AA-mediated collagen deposition. β-GP, on the other hand, has no stimulatory effect on its own but works synergistically with AA to further increase collagen deposition and ALP activity (52). Also, it is required to accelerate mineralization of extracellular matrix (53).

5.3 Limitations and Future Directions

The present data showed that dexamethasone-simvastatin combination had a negative effect on osteodifferentiation as reflected by reduced cell proliferation and ALP activity, while treatment with AA and β-GP supplements enhanced osteodifferentiation. These findings need to be further investigated due to a number of limitations. First, the current experiment was conducted over a relatively short duration (15 days) without an examination of the effect of these agents on final phase of osteoblastic differentiation. Formation of mature osteoblasts is characterized by production of mineralized bone nodules, which can be quantified and used as an indication of successful osteoinduction. It is possible that the levels of ALP activity may not be equivalent to the amount of bone nodules formed. Second, although the osteoblast phenotype markers such as cbfa-1, cadherin-11, and osteocalcin were determined by immunohistochemical staining, the levels of gene expression were not quantitatively measured. Increased mRNA expression activity of osteoblast-specific markers usually results in increased production of these protein markers. If dexamethasone-simvastatin combination showed a relatively lower mRNA expression activity, this may confirm the negative effect of dexamethasone-simvastatin combination on osteoblastic differentiation.

Bone nodule counting and reverse transcription polymerase chain reaction (RT-PCR) may be performed if the experiment was extended in order to address the above-mentioned limitations. Bone nodule counting is a useful analysis for confirming osteoblastic differentiation. Alizarin red or von Kossa staining may be used to detect the deposition of minerals in bone extracellular matrix. As mineralization represents the final phase of osteoblastic differentiation, the inhibitory effect on osteogenesis may be confirmed if a parallel reduction in the amount of mineralized bone nodules was observed in dexamethasone-simvastatin-treated groups. Also, RT-PCR may be used to examine the osteoblastic differentiation at the molecular levels. The intensity of the gene expression of interest in the treatment groups can be compared with that in the controls, a weaker expression of the genes of interest in the treatment groups may suggest that dexamethasone-simvastatin combination inhibits osteoblastic differentiation.

6. Conclusion

The present findings showed that combination of dexamethasone and simvastatin may impede osteogenic differentiation of primary primary bone-derived cells and that addition of AA and β-GP supplements facilitated osteogenesis. With respect to the inhibitory effect of dexamethasone-simvastatin combination on osteoblastic differentiation, further investigation is needed to support the results. Future work investigating the osteoblastic differentiation at the mineralization phase and at the osteoblast-specific gene expression levels may be undertaken to further validate the present finding