Perspectivas para el Futuro

Summary

Electromagnetic field is an effective application in bone healing treatment that promotes osteoblast growth. However, evidence of EL-EMF stimulation as therapeutic medicine is still limited. The purpose of this study is to observe bone tissue formation in vivo and cells growth when exposed to different EL-EMF intensities. Bone scaffold was developed from a mixture of 40% alginate and 60% nano-cockle shell powder. Bone scaffold was prepared in 10x3x5 mm3 _{cylindrical implant and seeded with osteoblast prior to subcutaneous} implantation on the right and left dorsum of Wistar rats. A total of 18 Wistar were randomly divided into three groups consisting of control (NC) that was not exposed to EL-EMF and treatment groups exposed to EL-EMF at 0.5 mT (T1) and 1.0 mT (T2) for an hour daily for duration of two weeks. After treatment, the scaffolds were harvested from the rats for microscopic observation, biochemical analysis and osteoblast counts. H&E staining showed osteoblast infiltration and blood vessels formation were found to be higher T2. Masson’s Trichrome staining showed the presence of collagen in NC, T1 and T2. Von Kossa staining revealed the accumulation of calcium that was found to be higher in T2. ALP analysis was found significantly higher in T1 (p<0.05) as compared to NC and T2. Osteoblast counts revealed a significant increase (p<0.001) in T2 as compare to NC and T1. In conclusion, findings from this study indicate the possible use of EL-EMF as a therapeutic alternative that could accelerate bone healing process.

Keywords: Extremely Low Electromagnetic Field (EL-EMF); Bone Scaffold; Osteoblast Cell Introduction

Electromagnetic field (EMF) was used as a physical stimulation tools in bone healing treatment (Basset et al., 1974). Ironically, low EMF exposures facilitate the production of bone matrix quality, growth factor secretion, osteoblast proliferation and differentiation (Ciombor & Aaron 2005; Lohmann et al., 2000). According to Fitzsimmons et al. (1992) and Ryaby et al. (1994), the secretion of growth factors such as IGF-2 and BMP-2 are stimulated when treated with low EMF. Growth factors serve as intermediate molecules that bind to osteoblasts receptor for cell proliferation and differentiation. Stimulation of EMF interfere the permeability of plasma membrane and increase the entry of Ca2+ _{influx to activate} calmodulin activity for DNA production (Brighton et al., 2001; Crocker et al., 1988).

In addition, the techniques from bone tissue engineering were introduced to speed up the healing process through applications of life sciences and engineering. Bone scaffold has been designed for regenerating new cells and tissues (Williams, 2004). Application of bone tissue engineering can reduce the complications such as infection, bleeding, nerves damage and defects during surgical procedure (Mountziaris & Mikos, 2008). Natural and synthetic polymers have been selected to create an ideal scaffold based on suitability and cell types (Polo-Corrales et al., 2014).

Alginate and cockle shell have been chosen for bone scaffold formation that produces a good morphological structure to facilitate osteoblast growth and attachment (Hemabarathy et al., 2014). Alginate is extracted from brown algae and consists of two

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basic monomers which are β-D guluronic acid and α-L mannuronic acid (Smidsrod & Skjak- Bræk, 1990). Alginate is an inexpensive material that often used in medical treatment such as wound healing due to low toxicity (Lee & Mooney 2012). Cockle shell from Anadara Granulosa sp. contained 98-99% of calcium carbonate; inorganic materials which are essential for cells growth and readily exist in three different form namely calcite, vaterite and aroganite. Aroganite is a suitable compound to synthesis hydroxyapatite (HA) crystals by calcination process (Bakar et al., 2011; Hemabarathy et al., 2014).

However, the evidence of EMF effectiveness for the treatment of large bone defects is still limited. Thus, the objective of this study is to observe the cells growth and bone tissue formation in vivo when exposed at different EL-EMF intensities. Hence, the combination of EL-EMF and bone tissue engineering are applied to compare and observe the cells growth and bone tissue formation, ALP activity and calcium deposition on nanobiocomposite bone scaffold.

Materials and methods Electromagnetic Field

A solenoid was used to generate extremely low electromagnetic field. The solenoid consists of rigid PVC tube (8 inch in diameter, 15 mm thickness, Malayan Industrial Plastics Sdn. Bhd) and silicon insulated copper wire (Model: 60245 IEC 03 (YG) sheath thickness 0.78mm, diameter 4.0). The coil was attached with adjustable wire supply and EL-EMF generated was directly monitored with a Teslameter (TM-191, China) throughout the experiment. Teslameter was used to maintain the consistency between the centre and the ends of the solenoid. The rats from T1 and T2 were exposed to EL-EMF at 5.0 mT and 1.0 mT respectively for one hour dialy for a fortnight. At the end of the experiment, the rats were sacrificed and the scaffolds were harvested from left and right dorsum of Wistar rats.

Animals Study

Eighteen male Wistar rats (8 weeks old) were used in this study and had an average weight between 200-220g. The rats were received from animal house, Faculty of Medicine, Universiti Kebangsaan Malaysia. Procedures & practices of the animal was approved with approval number FSK/2016/HEMABARATHY/23 NOV./802-JAN.-2017-AUG.-2017.

Osteoblast Cell Culture

Osteoblast were differentiated from sheep mesenchymal stem cells (MSC) and received from the Centre of Tissue Engineering, Universiti Kebangsaan Malaysia Medical Centre (PPUKM). Osteoblast from single vial were culture in T-75 flask contained Dulbecco’s Modified Eagle Medium (DMEM: E15-810 PAA), 10% fetus bovine serum (FBS: Sigma F4135), 1% penisilin/streptomycin (PAA P11-010) and incubated at 37°C with 5% carbon dioxide. The culture medium was changed every 2-3 days and the cells were subcultured at 70-80% confluency.

Bone Scaffod Development

Bone scaffold was developed based on studies by Hemabarathy et al. (2014). The bone scaffold was made up of 40% alginate solution and 60% nanocockle shell powder and

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dissolved in deionized water using a magnetic stirrer. The mixture was poured into customized mold and freezed at -20°C for 24 hours. The bone scaffold was then freeze- dryed using freeze dryer (Lyolab 3000) at -50°C for 24 hours and removed from customized mold. Bone scaffold was immersed in 1% calcium chloride (CaCl2) for 20 minutes and rinsed in deionized water before stored in freezer at -20°C for 24 hours. Finally, bone scaffold was freeze-dryed at -50°C for 24 hours and prepared in 10x3x5 mm3 _{cylindrical implant.}

Bone Scaffold Implantation

Osteoblast cells were seeded on nanobiocomposite bone scaffold at 100000 cel/100 µL density and kept overnight at 37°C with 5% carbon dioxide. Animals were injected with general anesthesia of 0.2 ml/kg of 50 mg/ml ketamine, 20 mg/ml xylazil and 50 mg/ml zoletil prior to having the dorsal furs shaved and cleaned for surgical procedure. The cell seeded scaffolds were then inserted through a small incision to the left and right dorsum of the animals. Post implantation, the opened skins were sutured and treated with antiseptic. After 2 weeks treatment, bone scaffolds were harvested for histology, biochemical analysis and osteoblast counts.

Biochemical Analysis

Harvested bone scaffolds were placed in 1 mL phosphate buffer solution (PBS) and crushed using homogenizer. Lysate cell produced was centrifuged at 12000 rpm, 4 °C for 10 minutes and kept in -20 °C. ALP activity level was perfomed based on study by Tampieri et al. (2005). 100 µl lysate cells were incubated in 0.5 ml alkaline buffer solution and 0.5 ml p- nitrophenol phosphate (40 mg pNPP and 10 ml distilled water) at 37°C for 1 hour. After incubation, the level of p-nitrophenol was determined using microplate reader at 405 nm. ALP activity level was determined using equation formed by the standard graph in mg/ml.

Histology

Ten percent formalin was used to preserve the harvested scaffold sample structure. The samples were processed using automated tissue processors and followed by a standard tissue processing protocol. The slides obtained from each sample were stained with Hematoxylin dan Eosin (H&E), Von Kossa (VK) and Masson’s Trichrome (MT). In addition, the scaffold samples for scanning electron microscopy (SEM) analysis were dehydrated in 70%, 80%, 85%, 90% and 100% for 10 minutes respectively prior to be soaked in hexamethyldisilazine (Sigma-Aldrich, USA) solution for 30 minutes and observed under scanning electron microscope (SU5000, HITACHI).

Osteoblast Counts

Cells counts were performed through histological slides observation which were stained with H&E. Histological slides were observed under light microscope (Zeiss, Germany) at x40 magnification. Five slides from each samples were taken (a, …,e) and five areas on each slides were randomly chosen (a1, …,a5).Osteoblast counts were then performed based on the equation below:

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a= (a1+a2+a3+a4+a5)/5 (1)

A=(a+b+c+d+e)/5 (2)

Average osteoblast count =(A+B+C+D+E)/5 (3)

(a: average number of cells in five calculated area per slide, A: average number of cells from each sample)

Statistical analysis

One way ANOVA has been used to analyze data and results expressed as mean + standard error of mean. Post-hoc test is performed using Turkey’s multiple comparison test and significant value at p<0.05.

Results

Comparison and observation of cells growth and bone tissue formation on nanobiocomposite bone scaffold

Figure 1 shows osteoblast number on nanobiocomposite bone scaffold. The results found that osteoblast counts in EL-EMF treated groups (T1 and T2) were significantly higher (p<0.001) by 50% as compared to NC (11 + 0.84). Meanwhile, osteoblast counts in T2 (41 + 1.42) was significantly higher (p<0.001) by 50% as compared to T1 (20 + 1.64). Microscopic examination revealed a significant difference between control and EL-EMF treated groups as shown in Figures 2, 3 and 6. Figure 2 shows H&E staining of nanobiocomposite bone scaffold at x40 magnification. The results showed presence of blood vessels and osteoblast infiltrations in T1 scaffolds that were observed to be higher than NC but lower compared to scaffolds from T2. In addition, EL-EMF treated group in T2 showed neutrophils and osteoblasts infiltration on nanobiocomposite bone scaffold and more intact blood vessel structure than NC and T1. Figure 3 shows Masson’s Trichrome staining on nanobiocomposite bone scaffold at x40 magnification. The results showed no deposition of bone matrix and little formation of collagen fibers in NC.

Meanwhile, EL-EMF treated group in T1 showed little deposition of bone matrix and collagen fibers as compared to NC. While scaffolds from T2 group showed more deposition of bone matrix and collagen fibers as compared to NC and T1. Figure 4 (A1-A3) shows the presence of cells and structures that involved in bone tissue formation under scanning electron microscopy. Collagen was observed as long rod fibers while osteoblast was noted as irregular shaped cells. Osteocyte was also observed in image A1 as single flatten cell with long finger like projections.

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Figure 1: Osteoblast counts, ap < 0.001vscontrol, bp < 0.001vs 0.5 mT EMF and cp < 0.00 1 vs control. Data are presented as mean + standard error of mean.

Control (NC) 0.5 mT EMF (T1) 1.0 mT EMF (T2)

Figure 2: Histological image of H&E stained nanobiocomposite scaffolds at x40 magnification. BV: blood vessel, N: neutrophil, OS: osteoblast cell, RBC: erythrocyte

0 5 10 15 20 25 30 35 40 45

Control 0.5 mT EMF 1.0 mT EMF

O steo b las ts Co u n ts (cm 2) Groups Control 0.5 mT EMF 1.0 mT EMF a b,c BV BV BV BV BV BV RBC N OS _OS OS