Combined effects of melatonin and FGF-2 on mouse preosteoblast behavior within interconnected porous hydroxyapatite ceramics - in vitro analysis

ABSTRACT Objective Biocompatible materials such as interconnected porous hydroxyapatite ceramics (IP-CHA) loaded with osteogenic cells and bioactive agents are part of an evolving concept for overcoming craniofacial defects by use of artificial bone tissue regeneration. Amongst the bioactive agents, melatonin (MEL) and basic fibroblast growth factor (FGF-2) have been independently reported to induce osteoblastic activity. The present in vitro study was undertaken to examine the relationship between these two bioactive agents and their combinatory effects on osteoblastic activity and mineralization in vitro. Material and Methods Mouse preosteoblast cells (MC3T3-E1) were seeded and cultured within cylindrical type of IP-CHA block (ø 4x7 mm) by vacuum-assisted method. The IP-CHA/MC3T3 composites were subjected to FGF-2 and/or MEL. The proliferation assay, alkaline phosphatase enzyme activity (ALP), mRNA expressions of late bone markers, namely Osteocalcin (OCN) and Osteopontin (OPN), and Alizarin Red staining were examined over a period of 7 days. Results FGF-2 mainly enhanced the proliferation of MC3T3-E1 cells within the IP-CHA constructs. MEL mainly induced the mRNA expression of late bone markers (OCN and OPN) and showed increased ALP activity of MC3T3 cells cultured within IP-CHA construct. Moreover, the combination of FGF-2 and MEL showed increased osteogenic activity within the IP-CHA construct in terms of cell proliferation, upregulated expressions of OCN and OPN, increased ALP activity and mineralization with Alizarin Red. The synergy of the proliferative potential of FGF-2 and the differentiation potential of MEL showed increased osteogenic activity in MC3T3-E1 cells cultured within IP-CHA constructs. Conclusion These findings indicate that the combination of FGF-2 and MEL may be utilized with biocompatible materials to attain augmented osteogenic activity and mineralization.


INTRODUCTION
Bone grafting plays an essential role in craniofacial surgery performed for both reconstructive and aesthetic purposes, which has led to discovery of different biomaterials, including hydroxyapatite (HAp), a member of the non-resorbable calcium phosphate group of biomaterials 23 . HAp has been formed into a variety of shapes and dimensions, and shown both biocompatibility and osteoconductivity since the discovery of its similarities with natural bone 23 . Porous type HAp ceramics are expected to facilitate bone formation and become integrated with host bone tissue. However, the pores of HAp are not fully replaced with new bone for a substantial period of time due to limited interconnection, which eventually leads to undesirable results 1 . The interconnection of pores is considered to be an essential factor for osteoconduction inside an HAp ceramic appliance for cell viability and function. To overcome this problem, interconnected porous HAp ceramics (IP-CHA) components with fully interconnected and symmetrical pores have been developed 21 . This unique structure provides extensive incorporation with host cells more rapidly than other types of porous calcium HAp ceramic, while its porous architecture also provides optimum compressive strength of up to 12 MPa, similar to cancellous bone 21 . It has been suggested that IP-CHA may have an additional osteoinductive advantage if the porous architecture could be utilized to transplant osteoinductive agents or osteogenic cells or both. member of the 23-polypeptide growth factor family 13 . It has been found to participate in a variety of biological processes, such as angiogenesis, hematopoiesis, cell growth and bone development 7,13,16 been reported to stimulate osteoblast proliferation rather than differentiation in immature cells 19 . In proliferation and growth of osteoblastic MC3T3-E1 cells 5 , suggesting that in cell growth of osteoblasts.
Melatonin (MEL) is a pineal hormone that is also synthesized from other human cells and organs, such as the retina, bone marrow, and gastrointestinal tract 2,10,18 . Roth, et al. 17 (1999) demonstrated the direct effects of MEL on differentiation of rat preosteoblast cells, while it has also been reported to inhibit RANKL-induced bone resorption and thereby promote bone formation 14 . These observations imply proliferation of osteoblasts and MEL is thought to promote bone formation. The combination increased activity of osteoblast. We have previously in osseointegration around titanium implants in animal models 20 . However, their combined effects on osteoblast cell growth and bone formation have not been fully elucidated in vitro. Also, their combination within useful biomaterials, such as HAp, remains undocumented. Hence, we investigated the preosteoblast cells when cultured within an IP-CHA construct.

Cell culture & IP-CHA
MC3T3-E1 mouse preosteoblast cells were (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) (Biowest, Miami, FL, USA), 1% penicillin-streptomycin, and 2 at 37°C. We used a cylindrical type of porous IP-CHA block (NEOBONE ® , MMT, Osaka, Japan) that was 4 mm in diameter, 7 mm in height, with 75% porosity. The mean pore diameter was 150 μm and the pores interconnections were 40 μm. Prior to cell seeding, IP-CHA blocks were pre-coated with cell-free medium to enhance cell adhesion in the interior of the scaffold. Medium was trickled onto the block and then it was subjected to vacuum, which moved the air out of the porous IP-CHA and drew medium in. determined by trypan blue staining. Next, 1x10 5 viable cells were resuspended in 130 μl of expansion medium and concentrated cell suspensions were pipetted onto the IP-CHA in a 24-well plate. To ensure cell penetration within the IP-CHA construct, each one was subjected to vacuum of 100 mmHg for 100 milliseconds 4 . The samples were then placed in an incubator for 1 hour to allow the cells to adhere to the interior of the construct. An additional 1.5 ml of expansion medium was added later to the IP-CHA/MC3T3 composite to aid proliferation within and/or MEL.

FGF-2 and MEL treatment
We determined the optimum concentration previous reports 15,24 (Sigma-Aldrich) was used to evaluate the optimum therefore the samples were subjected to different 24 . MEL (Sigma-Aldrich) was used to evaluate the optimum concentration of MEL needed for cell differentiation, therefore the samples were subjected to different concentrations of MEL (50, 200, 1000 nM) 15 .

Proliferation assay
treatment was evaluated using an MTS Assay (Aqueous One Cell Proliferation Assay, Promega, Madison, WI, USA) after 1, 3, and 5 days. The principle behind the MTS assay is the formation of formazan crystals by dehydrogenase enzyme in functionally active cell mitochondria. The amount of purple formazan formed is directly proportional to the number of viable cells. The method was performed according to the manufacturer's protocol.
rinsed in PBS and transferred to a new 24-well in vitro analysis plate. MTS solution (100 μl per 1 ml of expansion medium) was added to the composite and subjected to vacuum at 100 mmHg for 100 milliseconds to ensure that the MTS solution entered the core. Next, the composite was allowed to incubate for 2 hours, after which medium in the wells was gently aspirated and discarded. Finally, 750 μl of dimethyl sulfoxide was added for dissolving the formazan crystals formed by the cells in the composite and 250 ml of this solution was transferred to a 96-well plate, and absorbance at 490 nm was measured using a microplate reader (Bio-Rad, Hercules, CA, USA). Results are expressed as the mean±SD of 3 independent experiments.

Quantitative RT-PCR analysis
RNA was extracted using an RNAeasy micro kit into cDNA was performed using SuperScript III First Strand Synthesis Supermix (Invitrogen, was carried out using Eppendorf Master Cycler and The reaction mixture consisted of 1.0 μg of cDNA, the reference mRNA control. The PCR protocol was as follows: initial melting at 95°C for 10 minutes, followed by 40 cycles at 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 40 seconds. Reverse transcribed Human Total Reference RNA (Stratagene, Cheshire, UK) was used to plot a standard curve. Results are expressed as the mean±SD of 3 independent experiments.

ALP enzyme activity
ALP enzyme activity was determined using an ALP assay measurement kit (TRACP & ALP Assay MC3T3 composites after 3, 5, and 7 days of culture PBS, then homogenized in the provided extraction solution and sonicated for 3 minutes. Cell lysates were then collected by centrifugation at 16,000 g for phosphate) provided in the assay kit. The solution was then incubated at 37°C for 1 hour before measuring absorbance at 405 nm (Bio-Rad). Next, we calculated the ratio of absorbance of each sample in relation to the control sample at day 3. Results are expressed as the mean±SD of 3 independent experiments.
Extracellular calcium deposits were examined by was freshly prepared by dissolving 2 g of Alizarin Red (Sigma-Aldrich) in 100 ml of deionized distilled water, then pH was incrementally adjusted to 4.1-4.3 using 0.1% NH 4 OH solution. Both monolayer cultures and treated IP-CHA/MC3T3 composites in enough 10% neutral buffered formalin (Sigma-Aldrich) to submerge the cells or composite. After 30 minutes, formalin was gently aspirated and the cells were washed with deionized distilled water. Finally, prepared Alizarin Red solution was added to cover the cells and incubated at room temperature in the dark for 45 minutes, after which the monolayer cells were examined under a microscope. Later, both the monolayers and treated IP-CHA/MC3T3 composites IP-CHA/MC3T3 composites were submerged in 20% methanol and 10% acetic acid solution in water. After substantial vortexing, readings were obtained using a spectrophotometer at 450 nm of absorbance. Results are expressed as the mean±SD of 3 independent experiments.

Statistical methods
Data obtained were analyzed using one-way analysis of variance (ANOVA) and the results are presented as the mean±standard deviation. At least 3 independent IP-CHA blocks were used for each experiment in statistical analysis.

Optimum concentration of FGF-2 and MEL
To determine the optimum concentration of were cultured and treated accordingly. Firstly, we for the growth of MC3T3-E1 cells by MTT assay. The high proliferative potential of MC3T3-E1 cells on monolayer culture was observed in the presence study ( Figure 1).
Secondly, we determined the optimum concentration of MEL on monolayer cultures. Osteopontin (OPN) and osteocalcin (OCN) are considered to be late osteogenic markers, and have roles in the onset of the mineralization phase of osteoblast lineage 3 . Therefore, we examined OPN and OCN mRNA expression to determine the induced OPN (Figure 2a) and OCN mRNA ( Figure  2b) at 200 nM compared to the controls and 50 nM treatments. Therefore, 200 nM could be considered the lowest concentration of MEL that can elicit an osteoblastic response for our study.

SEM analysis of IP-CHA/MC3T3-E1 cells composites
CHA, IP-CHA/MC3T3-E1 composites were examined by SEM after 3 days of culture. We found the seeded MC3T3-E1 cells in the interior walls of porous IP-CHA, 3.5 mm deep from the surface (Figures 3a  and 3b). expression of these markers from day 5, though 5). On the other hand, combined treatment had an even greater effect on increased OCN and OPN expression ( Figure 5). Figure 6 shows the relative ALP enzyme activities of MC3T3-E1 cells cultured within IP-CHA constructs enzyme activity is known to be closely associated independently induced ALP activity from day 3, though induction by the latter was more prominent greater ALP enzyme activity compared to the individual treatments ( Figure 6).

DISCUSSION
HAp is a biocompatible material and possesses the advantage of protein adhesion whereby it can facilitate osteoblastic cell binding, proliferation, and differentiation, leading to matrix organization 12 . Fully interconnected porous HAp is thought to be a suitable candidate for transplantation of both osteoinductive agents and osteoblastic cells. In this study, we found that mouse preosteoblastic cells can penetrate and grow inside the construct. In addition, attachment of MC3T3-E1 cells to porous hydroxyapatite was well documented in a study presented by Smith, et al. 19 (2006). These results suggest that IP-CHA blocks are suitable scaffold for osteoblastic cells.
Bone formation is a cascade of events that occur in the initial proliferation phase, followed by the mineralization phase marked by OPN and OCN expressions 14 eminent growth factor which is more favorable to cell proliferation than differentiation 11 , which was also shown in our study using IP-CHA constructs.
OCN highlight its role in the proliferative phase of osteoblast activity in contrast to differentiation 11 . This phenomenon is of particular interest, because population within the IP-CHA constructs in our study, ensuring that more cells were available for entering the maturation phase of bone formation.
MEL has been reported to positively stimulate bone formation by suppressing RANKL-mediated osteoclast formation and resultant bone resorption in the bone remodeling cycle of MC3T3-E1 cells 6 . In addition, Roth, et al. 17 (1999) have reported that MEL can induce differentiation of MC3T3-E1 cells and mineralization of matrix in culture. These role in bone formation. Although MEL had no study, as anticipated, its differentiation potential was largely highlighted by the mRNA expressions of the late osteogenic markers OPN and OCN. Furthermore, we found that MEL induced mineralization, as shown differentiation and mineralization.
MEL may induce increased cellular activity and differentiation because of its activity as an inherent free radical scavenger 22 . MC3T3 cells expel various free radicals during proliferation and growth 8 , while buildup of a large amount of free radicals hampers the natural activity of MC3T3 cells, leading to inhibition of mineralization 9 . Therefore, we speculate that MEL assists MC3T3 cellular activity and mineralization by neutralizing free radicals. In the present study, the OCN, and also increased mineralization compared to treatment with each one alone. We concluded that number of cells for MEL to induce to differentiation into mature osteoblasts and therefore positively regulate mineralization.
other growth factors, such as bone morphogenic proteins (BMPs), and shown to significantly stimulate cell proliferation, while BMPs alone significantly stimulated differentiation and in 3 . study may operate in a similar manner, in which help to consider delayed administration of MEL in a future in vivo model. We found that MEL induced differentiation of preosteoblasts into mature bone 20 (2008), in which superior osseointegration was achieved by use of titanium screws in rat tibias after systemic CONCLUSIONS of MC3T3-E1 cells within IP-CHA constructs by targeting different phases of the osteoblast lineage.