Aging impairs osteoblast differentiation of mesenchymal stem cells grown on titanium by favoring adipogenesis

ABSTRACT Aging negatively affects bone/titanium implant interactions. Our hypothesis is that the unbalance between osteogenesis and adipogenesis induced by aging may be involved in this phenomenon. Objective We investigated the osteoblast and adipocyte differentiation of mesenchymal stem cells (MSCs) from young and aged rats cultured on Ti. Material and Methods Bone marrow MSCs derived from 1-month and 21-month rats were cultured on Ti discs under osteogenic conditions for periods of up to 21 days and osteoblast and adipocyte markers were evaluated. Results Cell proliferation, alkaline phosphatase (ALP) activity, extracellular matrix mineralization and gene expression of RUNX2, osterix, ALP, bone sialoprotein, osteopontin, and osteocalcin were reduced in cultures of 21-month rats compared with 1-month rats grown on Ti. Gene expression of PPAR-γ , adipocyte protein 2, and resistin and lipid accumulation were increased in cultures of 21-month rats compared with 1-month rats grown on the same conditions. Conclusions These results indicate that the lower osteogenic potential of MSCs derived from aged rats compared with young rats goes along with the higher adipogenic potential in cultures grown on Ti surface. This unbalance between osteoblast and adipocyte differentiation should be considered in dental implant therapy to the elderly population.


INTRODUCTION
Titanium (Ti) implants have been largely used in dentistry thanks to their physical properties and superior ability to osseointegrate 2 . In addition to the implant characteristics, the clinical success of this treatment depends on several aspects such as surgical procedure, patient health conditions, and local bone quality and quantity 4,10,21 .
The implantology research has been focused result in unique topography and chemical features that may regulate cell adhesion, proliferation and differentiation, and ultimately the interfacial tissue formation 5,6,9,17,18,20 . Regarding the quality and amount of bone tissue, they are affected by various factors such as heart/vascular disease, estrogen deficiency, diabetes, and aging 12,15,19 . Aging also increases the risk of bone fracture and impairs the healing process in humans and animal models 8,13,22 . It has been shown that the delay in fracture repair and the reduced bone volume in the healing site in aged mice is associated with a decreased proliferation of stem cells and a disrupted osteoblast differentiation 13,22 . The effects of aging on bone repair indicate that the process of Ti implant osseointegration could also be regulated by the age of the patient. Indeed, the number of mineralized nodules and the calcium content were reduced in osteoblasts derived from old patients compared with young patients when cultured on Ti surface 23 . Corroborating this osteocalcin (OC) expression were downregulated in osteoblasts derived from aged rats compared with young ones when they were grown on Ti surface with different surface topographies 16 . Furthermore, implant contact was observed in one of the Ti surfaces placed into mouse femurs 16 .
Despite the clear evidences of the negative effects of aging on osteoblast and bone tissue interactions with Ti implants, the mechanisms behind this phenomenon have not been fully understood yet. Then, we hypothesized that the unbalance between osteogenesis and adipogenesis induced by aging may be one of the factors involved in the reduced osteoblast response to Ti surfaces. Thus, in the present study, we investigated the osteoblast and adipocyte differentiation of mesenchymal stem cells (MSCs) derived from bone marrow of young and aged rats cultured on Ti surface.

Preparation of Ti discs
Discs of commercially pure grade 2 Ti (Realum, São Paulo, SP, Brazil) with 12 mm in diameter and 1.5 mm thick were polished using 320 and 600 grit silicon carbide, cleaned by sonication and rinsed with toluene and deionized H 2 O several times, autoclaved and air-dried as previously described 18 .

Cell culture
The Committee of Ethics in Animal Research approved all animal procedures performed during the experiments (Approval number: 11.1.890.53.0). Bone marrow MSCs were obtained from the tibiae of 1-month and 21-month female Wistar rats and cultured in growth medium Island, NY, USA) supplemented with 15% fetal bovine serum (Gibco), 50 μg/mL gentamycin (Gibco), 50 μg/mL vancomycin (Acros Organics, Geel, Belgium), and 0.3 μg/mL fungizone (Gibco) cultured in osteogenic medium containing growth medium plus 5 μg/ml ascorbic acid (Gibco), 7 mM MO, USA), and 10 -7 M dexamethasone (Sigma) on Ti discs for periods of up to 21 days. During the culture period, cells were incubated at 37°C in a 2 , and the medium was changed every three days.

Alkaline phosphatase (ALP) activity
Alkaline phosphatase activity was determined at days 4, 7, and 10 by measuring the release of thymolphthalein from thymolphthalein monophosphate using a commercial kit (Labtest with 0.5 mL of 0.3 M diethanolamine buffer, pH 10.1, and kept for 2 min at 37°C before the addition thermal shock (-20°C for 20 min and 37°C for 15 min). Then, after 10 min at 37°C, the reaction was stopped by adding 2 mL of Na 2 CO 3 (0.09 mmol/ mL) and NaOH (0.25 mmol/mL) solution, and the optical density was measured at 590 nm (μQuant, Bio-Tek). Data were obtained in quintuplicate (n=5) and expressed as ALP activity normalized by total protein content, which was determined by the Lowry method 14 .

Extracellular matrix mineralization
Extracellular matrix mineralization was detected at day 21 by alizarin red staining (Sigma-Aldrich).
temperature, dehydrated and stained with 2% alizarin red pH 4.2 (Sigma-Aldrich) for 10 min. For qualitative analysis, culture images were captured with a high-resolution digital camera (Canon EOS Digital Rebel Camera, Canon, Lake Success, NY, USA). Then, calcium content was evaluated using acetic acid was added to each well stained with alizarin red, and the plate was incubated at room temperature for 30 min under shaking. The slurry Aldrich), heated to 85°C for 10 min and transferred to ice for 5 min. The slurry was then centrifuged at 10% ammonium hydroxide to neutralize the acid. The optical density was read at 405 nm (μQuant, Bio-Tek), and data were obtained in quintuplicate (n=5) and expressed as absorbance.

Gene expression
Quantitative real-time polymerase chain reaction (PCR) was performed on day 10 to evaluate the gene expression of runt-related transcription factor 2 (RUNX2), osterix (OSX), ALP, bone sialoprotein (BSP), OC, osteopontin (OPN), peroxisome Aging impairs osteoblast differentiation of mesenchymal stem cells grown on titanium by favoring adipogenesis protein 2 (aP2), and resistin (RTN). Total RNA was extracted with Trizol reagent (Life-Technologies, Grand Island, NY, USA), and the concentration was determined by reading the optical density at the following different wavelengths: 260, 280, 230, and 320 nm (GE Healthcare, Milwaukee, WI, USA). Complementary DNA (cDNA) was synthesized using 1 μg of RNA through a reverse transcription reaction (Life Technologies-Applied Biosystems, Warrington, UK) according to the manufacturer's instructions. Real-time PCR was performed in a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Philadelphia, PA, USA) using TaqMan (Applied Biosystems) probes for the target genes. The standard PCR conditions were 50°C (2 min), 95°C (10 min), 40 cycles of 95°C (15 s), and 60°C (1 min). The relative gene expression was calculated in relation to glyceraldehyde-3phosphate dehydrogenase (GAPDH) expression and its respective control using the cycle threshold (Ct) method. This assay was performed in quadruplicate (n=4).

Lipid accumulation
Lipid accumulation was detected at day 21 by formalin for 2 h at room temperature, washed with isopropanol 60% (Merck-Germany) and stained with 0.3% oil red O (Sigma-Aldrich) for 10 min. For qualitative analysis, culture images were captured with a high-resolution digital camera (Canon EOS Digital Rebel Camera, Canon) and the lipid accumulation was measured using a colorimetric assay. The incorporated oil red O (Sigma-Aldrich) was extracted by incubation with 100% isopropanol for 10 min under shaking at room temperature. After appropriate dilution, this solution was read the data were obtained in quintuplicate (n=5) and expressed as absorbance.

Statistical analyses
The results are expressed as the mean±standard deviation and data obtained in three time-points were analyzed using two-way ANOVA followed by Student Newman Keuls post hoc test. Data obtained in one time-point were analyzed using Student's

Cell proliferation
Cell proliferation was higher in cultures from 1-month rats compared with 21-month rats at all evaluated time-points (p=0.001 for all periods; Figure 1A). Moreover, the cell number increased

ALP activity
Cells from 1-month rats showed higher ALP activity compared with cells from 21-month rats at 7 and 10 days (p=0.001 for both time-points), while no significant difference (p=0.561) was observed at 4 days ( Figure 1B). In addition, the ALP activity increased (p=0.001) over time in cultures (p>0.151) was observed between 4, 7, and 10 days in cultures from 21-month rats ( Figure 1B).

Extracellular matrix mineralization
Extracellular calcium deposits were detected on Ti discs irrespective of cell source, with cells from 1-month rats producing the most dense and regularly distributed matrix ( Figure 1C). times higher (p=0.001) in cultures from 1-month rats compared with 21-month rats ( Figure 1C).

Gene expression
The gene expression of all evaluated bone markers, RUNX2 (Figure 2A Figure 3C) was higher in cultures from 21-month rats compared with 1-month rats.

Lipid accumulation
Lipid accumulation was detected on Ti discs irrespective of cell source, with cells from 21-month rats producing the most dense lipid droplets ( Figure  3). Additionally, the lipid content was nearly twice higher (p=0.010) in cultures from 21-month rats compared with 1-month rats (Figure 4).

DISCUSSION
The present study was designed to investigate if disruption of the balance between osteogenesis and adipogenesis induced by aging may be one of the factors involved in the reduced osteoblast response to Ti surfaces. Our results showed that MSCs derived from 21-month rats exhibit lower osteogenic and higher adipogenic potential compared with MSCs derived from 1-month rats when cultured on Ti surface under osteogenic conditions. The capacity to expand is a key feature of the osteoblastic cells in order to colonize the implant surface, and distinct results are reported regarding the effect of aging on cell proliferation 3 . Here, we observed a relevant decrease in the proliferation activity of cells from 21-month rats compared with 1-month rats cultured on Ti surface at all evaluated time-points. According to this result, a reduced cell number in osteoblast cultures derived from the calvaria of old rats compared with young ones was observed 16 . In contrast, MSCs from young and aged rats exhibited similar proliferation rate when cultured on polystyrene in a non-inducing differentiation medium 7 . Such discrepancies may be related to the use of cells in different stages of differentiation and culture conditions, since the Another relevant characteristic of osteoblastic cells to interact with implants is the ability to complete the differention process and to synthesize an extracellular matrix that will be subsenquently mineralized. In this study, all evaluated markers of osteoblast genotype and phenotype expression were higher in cultures derived from 1-month rats compared with 21-month rats, including gene expression of RUNX2, OSX, ALP, BSP, OPN and OC, ALP activity and extracellular matrix mineralization. mineralized nodules and calcium content were noticed in osteoblasts derived from old patients compared with young ones grown on Ti surface 23 . Furthermore, a low osteogenic potential induced by aging was demonstrated in vivo in mice and in vitro in cells from rats in response to different Ti surfaces 16 .
The reduced osteoblast differentiation of MSCs grown on Ti surface induced by aging was paralleled by an increased genotype and phenotype expression level of adipocyte markers, such as and more lipid accumulation, the later not reported in studies investigating the effect of aging on the interaction between osteoblasts and Ti 16,23 . In were no differences between MSCs from young and aged rats in terms of osteoblast and adipocyte differentiation; however, this study evaluated cells obtained from 3-week and 12-month rats cultured on polystyrene under osteogenic and adipogenic conditions, respectively 7 . It is worth noting that, in our study, cells were cultured only in osteogenic medium on Ti surface, indicating that even in a nonadipogenic milieu aging may disrupt the balance between osteoblast and adipocyte differentiation in favor of adipogenesis. Also, it has been shown that adipocytes inhibit the osteoblast phenotype expression by releasing tumor necrosis factor alpha to the tissue environment 1 are of relevance from the clinical perspective as the process of Ti implant osseointegration occurs in a more osteogenic environment than in an adipogenic one.
The unbalance between osteoblast and adipocyte differentiation induced by aging could be related to the extracellular signal-regulated kinase 1/2 (ERK 1/2) that acts as a switch for the reciprocal regulation of osteogenesis and adipogenesis by modulating 11 . While ERK 1/2 phosphorylation is sustained during osteoblast differentiation, it is transient and decreased during adipocyte differentiation of human MSCs, suggesting that a reduced ERK 1/2 activation favors adipogenesis 11 . However, further studies are necessary to investigate the participation of ERK 1/2 on the reduced osteoblast differentiation of MSCs cultured on Ti surface induced by aging.

CONCLUSIONS
The results of our study indicate that the reduced osteogenic potential of MSCs derived from aged rats compared with young ones goes along with the increased adipogenic potential in cultures grown on Ti surface. In this context, the unbalance between osteoblast and adipocyte differentiation should be taken into consideration in dental implant therapy to the older-adult population.

Disclosure statement
interest associated with this study and there has our results.