Pattern of adipokine expression in osteoblasts from osteoporotic and osteoarthritic bone| JOURNAL OF GERONTOLOGY AND GERIATRICS

Abstract

Background and aims. Osteoarthritis (OA) and osteoporosis (OP) are the two most common osteo-articular
diseases in elderly population, whose etiopathogenesis is complex and multifactorial. An inverse relationship
between OA and OP has been observed and both diseases are characterized by apparently opposite changes
in bone quantity and quality. In vitro studies revealed that osteoblasts from OA and OP bone present different
phenotypes. Adipokines are involved in many physiological and pathological processes, including bone
homeostasis and osteo-articular diseases. The aim of this study is to evaluate the expression of various adipokines in osteoblasts deriving from healthy subjects and patients suffering from OA or OP.
Methods. Primary human osteoblast cultures were obtained from healthy, OA, and OP subjects. In each cell
population the synthesis and gene expression of leptin, resistin and adiponectin were evaluated.
Results. Adipokines showed an opposite patterns of adipokine expression in OA and OP osteoblasts. Leptin
and resistin synthesis and expression were higher in OA osteoblasts compared to healthy and OP osteoblasts,
while adiponectin synthesis and expression were significantly lower. Conversely, in OP osteoblasts a reduced
synthesis and expression of leptin and resistin were observed, concurrently with increased adiponectin expression.
Conclusions. These data confirm that osteoblasts deriving from OA and OP are characterized by different
metabolic changes, consisting in opposite expression pattern of adipokines, and suggest that adipokines
could be involved in the pathogenesis of bone alterations in both diseases.

INTRODUCTION

Osteoarthritis (OA) and osteoporosis (OP) are the two most common osteo-articular diseases, and represent a major cause of morbidity and disability in elderly population. Whereas OP is characterized by the reduction of bone mass with a deterioration of bone microarchitecture, in OA a generalized increase of bone density is observed, together with typical changes in subchondral and periarticular bone, that are intimately related to the development and progression of cartilage alterations. Osteoblasts are crucial for the modulation of bone remodeling processes, that are determinant in the pathogenesis of bone loss and architectural alterations observed in OP and in bone changes typical of OA. It has been shown that osteoblasts derived from subchondral bone of OA joints are phenotypically different from osteoblasts deriving from healthy and osteoporotic subjects ^{1 2}. Until recently, attention was focused on a supposed inverse relationship between OA and OP, but recent studies clarified that beyond age, these disorders share several risk factors, such as low grade inflammation and alteration of body composition ³.

The relationship between adipose tissue and both OA and OP has been established a long time ago, particularly the direct correlation between Body Mass Index (BMI) and OA, and the inverse relationship between BMI and OP, considered as a consequence of mechanical load effect on bone and joints. Nevertheless, obesity is a risk factor for OA also in non load-bearing joints and in obese subjects, even with high BMD values, an increased incidence of osteoporotic fractures has been reported; these observations could be explained by structural bone changes induced by systemic metabolic alterations ⁴. Obesity can promote OA and OP through metabolic effects mediated by pro-inflammatory cytokines and adipokines produced by adipose tissue. Adipokines are a group of mediators produced mainly by adipocytes and are involved in many physiological processes, including bone homeostasis ⁵.

Increasing evidences indicate that adipokines are released not only by adipose tissue and can play a key role in the regulation of bone metabolism ^{6 7}; recent data showed that adipokines can be produced by bone cells, particularly osteoblasts ^{8 9}, but little is know about their possible local role in the pathogenesis of degenerative osteo-articular diseases. The aim of this study is to assess the production of different adipokines in primary human osteoblastic cell cultures derived from OA and OP subjects.

PATIENTS AND METHODS

NORMAL, OSTEOPOROTIC, AND OSTEOARTHRITIC HUMAN OSTEOBLASTS

OA human osteoblasts (Ob) were obtained from subchondral bone samples deriving from 12 patients (4 men and 8 women) aged 66.91 ± 6.37 years (range 56-78) undergoing total hip joint replacement who were classified as having hip OA based on clinical history, physical examination and radiographs. The bone specimens were selected depending on the integrity of overlying articular cartilage, identifying the areas in which macroscopic damage was visible. In the selected areas the cartilage surface appeared rough anderoded, yellowish, softened, with fibrillation phenomena or evencompletely absent.

None of the recruited OA subjects were affected by other metabolicbone diseases and none received medication, including corticosteroids, for 6 months prior to bone biopsy.

OP human Ob were obtained from bone samples deriving from 11 patients (2 man and 9 women) aged 68.63 ± 3.9 years (range 66-75), undergoing total hip joint replacement for fragility fractures of the femoral neck. In all OP patients, the cartilage surface corresponding to the bone fragments did not present signs of degeneration.

As control, specimens obtained from 8 healthy subjects (3 men, 5 women) aged 61.1 ± 5.05 years (mean ± SD, range 54-68) who were undergoing surgery for reduction of traumatic proximal femur fractures were used. Subjects affected by metabolic bone diseases and whose received medication which could interfere with bone metabolism, including corticosteroids, for 6 months prior to bone biopsy were excluded.

Appropriate informed consent was obtained from each patient and the study was approved by Institutional Ethics Committee.

PRIMARY SUBCHONDRAL BONE OSTEOBLAST CELL CULTURE

Human osteoblasts were isolated from subchondral bone specimens as previous described ¹⁰. Each fragment was washed using sterile polysaline buffer solution 1X (PBS 1X) to eliminate bone marrow cells and digested with 0.5 mg/ml type II collagenase (Millipore) with Dulbecco’s modified Eagle’s medium (DMEM) without serum and supplemented with antibiotics (penicillin 100 IU/ml and streptomycin 100 mg/ml) for 1 h at 37°C to remove all fibroblasts and residual blood cells. After digestion, bone fragments were washed in DMEM supplemented with antibiotics and fungizone and containing 20% foetal calf serum (FCS) to inactivate and remove collagenase and then cultured in sterile flasks in the same medium at 37°C in a water-saturated atmosphere containing 5) CO_2. When osteoblasts began to grow out from the bone specimens in the flasks (approximately 1 week), the culture medium was changed every 3 days with a fresh medium containing 10) FCS until the confluence within 3-4 weeks.

OSTEOCALCIN SYNTHESIS AND ALKALINE PHOSPHATASE ACTIVITY

To confirm the osteoblast lineage of cultured cells, osteocalcin synthesis and basal alkaline phosphatase (ALP) synthesis were evaluated, by ELISA assay (Human Osteocalcin and Human Alkaline Phosphatase ELISA Kit, MyBioSource) in cell culture supernatants. In both tests, results were normalized to the respective total protein concentration, measured using the Bradford Method in cell lysate (Amresco, Solon, Ohio, USA), and were expressed as pg/mg intracellular proteins. The intracellular protein content of each flasks was determined by the Bradford method (Bio-Rad protein assay, Bio-Rad Laboratories, Richmond, CA).

MEASUREMENT OF ADIPOKINES

The synthesis of adiponectin, leptin and resistin was evaluated in culture medium using an enzyme-linked immunoassorbent assay kit (ELISA Kit, Novex by Life Technologies, USA). For each adipokine, according to the manufacturer’s instructions, cell medium samples and standards were added to the pre-coated plates with the specific antibodies and incubated for 2 h. The no-bound proteins were removed and a biotinylated antibody was added. In the second step, the plates were washed and streptavidin-horseradish peroxidase (HRP) was added before incubation. After the last washing, 3.3′,5.5′-tetramethylbenzidine (TMB) was added as substrate of HRP. The acidic stopping solution was subsequently added and absorbance of the resulting products was measured spectrophotometrically at 450 nm using a microplate ELISA reader. The results obtained were normalized to the respective total protein concentration, measured using the Bradford Method in cell lysate (Amresco, Solon, Ohio, USA), and were expressed as pg/mg intracellular protein (leptin) or ng/mg intracellular protein (adiponectin, resistin). All measurements were performed in triplicate for each sample.

RNA ISOLATION AND REAL-TIME PCR ANALYSIS

Total RNA was collected in a QIazol Lysing Reagent and extracted using an RNeasy Mini Kit (Qiagen, Hilden), following the manufacturer’s protocol for cultured cells. RNA concentration and purity were measured by the 260 and 280 nm absorbance ratio. 1 μg of total RNA was reverse transcribed with a SuperScript IV VILO Master Mix (Invitrogen) to generate cDNA template for the following real-time PCR. The reverse transcription was performed on a Mastercycler Epgradient thermocycler (Eppendorf). Real-time PCR (qPCR) was carried out with Applied Biosystems 7300 Real-Time PCR System. 20-μl reactions were prepared with TaqMan fast Advanced Master Mix (Applied Biosystem) and the expression of adiponectin, resistin and leptin (using primers Hs00605917_m1, Hs00220767_m1, Hs00174877_m1 respectively; Applied Biosystem) was normalized to endogenous gene GAPDH (Hs02786624_g1, Applied Biosystem). The relative quantitation of target gene expression was determined using the comparative method Ct (cycle-threshold fluorescence values), by normalization to the endogenous gene GAPDH. Results were expressed fold change over control (healthy cells).

STATISTICAL ANALYSIS

Results were expressed as means ± SD. The differences in adipokine levels between normal, osteoarthritic and osteoporotic osteoblasts were assessed with the non-parametric Kruskal-Wallis test, using the Dunn’s test for multiple comparisons. p values < 0.05 were considered as significant.

RESULTS

There were no significant differences in demographic factors (age, sex) between the subjects included in the study. BMI values were slightly higher in OA subjects (25.57 ± 3.44) compared to OP (23.96 ± 1.44) and healthy (24.05 ± 1.3) subjects; age of osteoporotic subjects was slightly higher compared to healthy subjects, but the differences did not reach a statistically significant level. All cultured cells produced osteocalcin and alkaline phosphatase, confirming the osteoblast lineage of cultured cells (data not shown).

The results of this study show that adiponectin, leptin and resisitin are synthetized by primary cultured human osteoblasts both in normal than in pathological conditions.

Compared to osteoblasts deriving from healthy donors, leptin and resistin synthesis was significantly increased in OA osteoblasts (38.42 pg/ml ± 21.83 and 0.043 ng/ml ± 0.024 versus 12.98 pg/ml ± 3.2 and 0.032 ng/ml ± 0.01 respectively), whereas it was significantly reduced in OP osteoblasts (6.4 pg/ml ± 1.74 and 0.024 ng/ml ± 0.007). Conversely, OP osteoblasts produced a significantly higher amount of adiponectin compared to healthy osteoblasts (4.775 ng/ml ± 0.3 vs 2.38 ng/ml ± 0.77), whereas in OA osteoblasts the adiponectin production was reduced (1.70 ng/ml ± 0.65) (Figs. 1A, 1B, 1C).

MRNA EXPRESSION AND PROTEIN LEVELS OF ADIPOKINES IN CULTURED OSTEOBLASTS

The analysis of mRNA expression confirms that adiponectin, resistin and leptin are expressed in primary cultured human osteoblasts derived from healthy subjects, osteoarthritic and osteoporotic subjects, with an opposite patterns of adipokine expression in OA and OP osteoblasts. OA osteoblasts showed a significantly higher mRNA expression of leptin and resistin compared to osteoblasts derived from healthy donors; conversely, in OP osteoblasts a significantly lower mRNA expression of both leptin and resistin was observed. On the other hand, compared to normal cells, adiponectin mRNA expression was significantly increased in OP osteoblasts whereas was reduced in OA osteoblasts (Fig. 2).

DISCUSSION

In the latest years many studies revealed the crucial role of adipokines in various physiological processes, including inflammation, immune function, glucose and lipid metabolism, and bone homeostasis ⁵. Several data suggest that adipokines can affect the bone remodelling processes and are involved in the pathogenesis of degenerative osteoarticular diseases.

OA and OP are the two most common osteo-articular disorders, which occurs mainly in the elderly, and are associated with substantial morbidity and disability. These diseases have complex etiopathogenesis and different pathologic findings, both affecting bone tissue.

While OP is characterized by a reduction of bone mass combined to qualitative changes of bone microarchitecture which lead to an increased fracture risk, OA is associated to high bone mass and to local typical bone changes, represented by subchondral bone thickening, bone sclerosis and osteophytes development that are strictly related to the concomitant cartilage degenerative alterations ^{11 12}. The possibility of an inverse relationship between OA and OP was proposed several decades ago ¹³; subsequently, this hypothesis has been confirmed by many clinical, epidemiolological and experimental observations ^{14 15}.

Obesity is a recognized risk factor of OA in bearing joint, but epidemiological data confirmed an increased risk of OA also in non bearing joint in obese patients ¹⁶ and animal studies of diet-induced obesity showed that mechanical overload cannot entirely explain the development and the progression of knee OA ¹⁷. A relationship between systemic inflammatory factors associated to obesity and OA has been suggested, as adipose tissue is involved in the production of various adipokines and inflammatory citokines ¹⁸ and can promote systemic low grade inflammation which play a key role in the cartilage and bone changes ¹⁹. Contrary to what is observed in OA, low body weight is a well-known risk factor for the development of OP; a direct correlation has been found between BMI and Bone Mineral Density (BMD) and until recently, subjects with high BMI was considered to be protected against fracture risk ^{20 21}. Nevertheless, epidemiologic evidences showed an increased fracture risk in obese subjects, suggesting the hypothesis that obesity could negatively affect bone quality through various mechanisms, including the release of inflammatory cytokines by adipose tissue ^{22 23}, which can increase bone resorption by stimulating the activation, differentiation and proliferation of osteoclasts trough the up-regulation of RANK-L ²⁴. The role of inflammation in the development of bone loss is observed also in healthy subjects in which low-grade inflammation is a significant and independent risk factor of non-traumatic fractures ²⁵. Adipokines have been identified as central mediators in the interplay between obesity, inflammation and bone alterations, but their role in OA and OP is not clearly established as the great part of published studies have been performed on mall case-series, had a cross sectional design and had a short follow-up duration ⁷.

Few cross-sectional studies have been performed on the association between serum adipokine concentrations and Bone Mineral Density (BMD) in vivo. A negative correlation was found between serum adiponectin concentrations and both lumbar and femoral neck BMD ^26-30. Serum adiponectin has be found to be significantly higher in women with fractures and was associated with a significant increased risk of fracture. Apparently, no significant association was found between serum leptin and BMD ²⁷; however, after adjustment for age, sex and BMI, serum levels of leptin showed a positive correlation with BMD ²⁷. In another study leptin seems to be positively related with BMD at total body in men postmenopausal women ³¹. Also plasma levels of resistin showed a significant correlation with femoral BMD in osteoporosis women after adjusting for age and BMI, but did not show a significant correlation with BMD at lumbar spine ²⁹.

A large cross sectional 5 years follow up study showed that serum levels of leptin and resistin significantly correlated to biochemical markers of cartilage degradation and to radiographic progression of knee OA, but this correlation disappeared for leptin after adjustment for BMI ³². High levels of serum leptin have been found in subjects with OA and were related to joint damage in term of cartilage degradation, bone marrow oedema, osteophytes, meniscal abnormalities and synovitis ³³, but these data were not confirmed ³⁴. It has been found that in patients with knee OA the synovial levels of leptin and circulating levels of leptin and adiponectin did not vary depending on the stage of disease, but the ratio between synovial and serum levels of leptin was significantly lower in advanced OA compared to early OA ³⁵. A positive relationship has been found between serum resistin levels and radiographic changes in hand OA ³⁶. A longitudinal study performed on patients with hand OA showed that higher adiponectin serum levels at baseline were associated to a decreased risk of OA progression ³⁷. In a cross-sectional study a negative association between both systemic and intra-articular adiponectin levels and the severity of knee OA was found; in this study the association between serum adiponetin levels and radiographic changes disappeared after adjusting for BMI, age and gender, while association between intra-articular adiponectin and OA severity was confirmed after the same adjustment ³⁸. These data suggest that different adipokines may have distinct effects in different diseases and in different disease stages and these effects can vary when considering local or systemic levels, supporting the hypothesis that intra-articular produced adipokines might be more relevant markers for bone changes in OA and OP than circulating adipokines. By their contribution to the control of osteoclast recruitment, differentiation and activation and being directly responsible for formation of new bone tissue, osteoblasts are relevant for the regulation of physiological bone remodelling processes and are crucial in the pathogenesis of both OA and OP ³⁹. Many studies showed and altered and often opposite osteoblast phenotype in OA and OP ^{2 40-42}, represented by distinct production of regulatory cytokines and growth factors and different response to various stimuli ^{43 44}. In this study we confirm that osteoblasts derived from subchondral bone are able to produce adipokines; interestingly we found an opposite phenotype of adipokine production between osteoblasts derived from osteoporotic and osteoarthritic bone. Particularly, leptin and resistin production is significantly higher in osteoblasts from OA subjects compared to osteoblasts from healthy subjects. whereas adiponectin production is significantly reduced. Conversely, osteoblasts derived from subchondral osteoporotic bone show a significantly lower leptin and resistin synthesis and gene expression and higher adiponectin synthesis and gene expression compared to normal cells.

The exact role and function of the locally produced adipokines by bone cells are controversial ⁴⁵ and can vary depending on the experimental model. Several data suggest that locally produced leptin might regulate bone metabolism ⁴⁶ and it has been proposed that it is able to decrease bone resorption by the inhibition of osteoclast activity and to enhance bone formation by increasing osteoblast proliferation and differentiation ⁴⁸, thus promoting osteogenesis ^47-49. Also resistin has been reported to increase bone formation by promoting osteoblast proliferation ⁵⁰.

Some studies showed a negative effect of adiponectin on bone formation; adiponectin has been found to inhibits alkaline phosphatase and osteoprotegerin expression in osteoblastic cells ⁵¹ and to have a direct negative effect of osteoblastogenesis ⁵², exerting a negative effect on bone formation ⁵³. Further, adiponectin stimulates the production of pro-inflammatory citokines that are involved in the pathogenesis of joint damage ⁵⁴.

The results of this study showing an increased leptin and resistin synthesis and a reduced adiponectin synthesis in OA osteoblasts indicate that local adipokine production can be involved in the pathogenesis of changes of bone remodeling leading a new bone formation and support the hypothesis that locally produced leptin and resistin could promote the formation of osteophytes and subchondral bone sclerosis, acting with a pro-osteogenic effect ⁵⁵; conversely, the reduced synthesis of leptin end resistin in osteoporotic osteoblasts, associated to the enhanced adiponectin synthesis, is suggestive of a possible involvement of these adipokines in the pathogenesis of the reduced bone mass.

Nevertheless, other authors found contrasting results; it has been found that adiponectin can promote osteoblastogenesis ⁵⁶ and it is able to inhibit osteoclastogenesis and bone resorption processes, thus exerting a positive effect on bone metabolism ^57-60, whereas resistin has been reported to induce osteoclastogenesis via activation of NF-kjB signaling, thus promoting bone resorption processes ⁵⁰.

It is possible that changes in adipokine production by osteoblasts could alter the physiological balance of bone remodelling; the opposite bone structural alterations that characterize osteoporotic and osteoarthritic subchondral bone are reflected by an opposite patterns of adipokine expression by articular osteoblasts.

The main limitations of this study were the restricted number of healthy and pathological subjects, even if it should be taken into account that fewer patients are needed for in vitro studies; further, the in vitro experimental conditions might not truly represent the complete in vivo conditions, in which many other factors (such as growth factors, cytokines, and interactions with other cell types) might be involved. However, these preliminary data support the hypothesis that osteoblasts might contribute to the pathogenesis of both OA and OP trough autocrine/paracrine mechanisms that involves the local synthesis of adipokines. Additional studies are needed to investigate the role of these and other adipokines in the development and progression of OA and OP

CONCLUSIONS

The opposite adipokine expression we found between OP and OA osteoblasts could reflect differences in cell metabolism related to the specific pathology. It is possible to hypothesize that intra-articularly produced adipokines might be more relevant markers for the pathogenesis of OP and OA than systemic adipokine levels and that difference in adipokine production by subchondral osteoblasts is associated with differences in cell phenotypes that partially could contribute to explain the proposed inverse relationship between OA and OP.

Figures and tables

Figure 1(A-C).Adipokines levels in human osteoblast culture medium. Leptin, resistin and adiponectin in healthy (normal - N), osteoporotic (OP) and osteoarthritic (OA) osteoblasts detected in cell culture medium through ELISA. Leptin and resistin production is significantly greater in OA cells compared to the normal cells (*p < 0.01). OP cells sow significantly lower production of leptin and resistin (**p < 0.001; ‡p < 0.01) (A,B). Compared to normal cells, adiponectin production is significantly lower in OA osteoblasts (*p < 0.05) and significantly higher in OP ones (**p < 0.001). Results are normalized per mg/cell proteins.

Figure 2.Adipokines mRNA expression in healthy (normal), osteoporotic (OP) and osteoarthritic (OA) osteoblasts. Leptin and resistin mRNA expression is significantly greater in OA cells compared to the normal cells (*p < 0.01), whereas in OP osteoblasts, leptin and resistin expression is significantly lower (**p < 0.001). Conversely, adiponectin expression is significantly lower in OA cells (§p < 0.01), whereas adiponectin expression in OP cells is significantly higher (§§p < 0.001) compared to normal osteoblasts. Results are expressed as mean ± SD of fold change over control (normal osteoblasts - N).

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