Leo Massari

Translocation of Akt/PKB to the nucleus of osteoblast‐like MC3T3‐E1 cells exposed to proliferative growth factors

An active phosphatidylinositol 3‐kinase (PI3K) has been shown in nuclei of different cell types. The products of this enzyme, i.e. inositides phosphorylated in the D3 position of the inositol ring, may act as second messengers themselves. Nuclear PI3K translocation has been demonstrated to be related to an analogous translocation of a PtdIns(3,4,5)P3 activated PKC, the ζ isozyme. We have examined the issue of whether or not in the osteoblast‐like clonal cell line MC3T3‐E1 there may be observed an insulin‐like growth factor‐I‐ (IGF‐I) and platelet‐derived growth factor‐ (PDGF) dependent nuclear translocation of an active Akt/PKB. Western blot analysis showed a maximal nuclear translocation after 20 min of IGF‐I stimulation or after 30 min of PDGF treatment. Both growth factors increased rapidly and transiently the enzyme activity of immunoprecipitable nuclear Akt/PKB on a similar time scale and after 60 min the values were slightly higher than the basal levels. Enzyme translocation was blocked by the specific PI3K inhibitor, LY294002, as well as cell entry into S‐phase. Confocal microscopy showed an evident increase in immunostaining intensity in the nuclear interior after growth factor treatment but no changes in the subcellular distribution of Akt/PKB when a LY294002 pre‐treatment was administered to the cells. These findings strongly suggest that the intranuclear translocation of Akt/PKB is an important step in signalling pathways that mediate cell proliferation.

Effects of electromagnetic fields on proteoglycan metabolism of bovine articular cartilage explants.

Electromagnetic field (EMF) exposure has been proposed for the treatment of osteoarthritis. In this study, we investigated the effects of EMF (75 Hz, 2,3 mT) on proteoglycan (PG) metabolism of bovine articular cartilage explants cultured in vitro, both under basal conditions and in the presence of interleukin-1 g (IL-1 g ) in the culture medium. Proteoglycan synthesis and the residual PG tissue content resulted significantly higher in EMF-exposed explants than in controls, whereas no effect was observed on PG release and nitric oxide (NO) production. IL-1 g induced both a reduction in PG synthesis and an increase in PG release, related to a strong stimulation of NO production, which resulted in a net loss of tissue PG content. In IL-1 g -treated explants, EMF increased PG synthesis, whereas in spite of a slight stimulation of NO production EMF did not modify PG release. This resulted in the residual PG tissue content being maintained at the control level. In both experimental conditions, the effects of EMF were associated with an increase in lactate production. The results of our study show that EMFs are able to promote anabolic activities and PG synthesis in bovine articular cartilage explants. This effect also is maintained in the presence of IL-1 g , thus counteracting the catabolic activity of the cytokine. Altogether, these data suggest that EMF exposure exerts a chondroprotective effect on articular cartilage in vitro.

Biophysical stimulation with pulsed electromagnetic fields in osteonecrosis of the femoral head

BACKGROUND Osteonecrosis of the femoral head is the end point of a disease process that results in bone necrosis, joint edema, and cartilage damage. It leads to joint arthritis that necessitates total hip arthroplasty in many patients. Because of its positive effects on osteogenesis and its chondroprotective effect of articular cartilage, pulsed electromagnetic field stimulation has been proposed as a method to prevent or delay the progression of osteonecrosis. METHODS A retrospective analysis of the results of treatment with pulsed electromagnetic field stimulation of seventy-six hips in sixty-six patients with osteonecrosis of the femoral head was performed. Patients with Ficat stage I, II, or III osteonecrosis of the femoral head were treated with pulsed electromagnetic field stimulation for eight hours per day for an average of five months. Clinical and diagnostic imaging information was collected at the start of the treatment and at the time of follow-up. The primary end point analyzed was the avoidance of hip surgery, and the secondary end point was limiting the radiographic progression (according to Ficat stage) of osteonecrosis of the femoral head. RESULTS Fifteen hips required a total hip arthroplasty; twelve of these hips were in patients with Ficat stage-III disease. The need for total hip arthroplasty was significantly higher in patients with Ficat stage-III disease than in patients with Ficat stage-I (p < 0.0001) or II (p < 0.01) disease at the beginning of treatment. Pulsed electromagnetic fields preserved 94% of Ficat stage-I or II hips. Furthermore, radiographic progression (according to Ficat stage) occurred in twenty hips (26%). Pain, present in all patients at the start of the treatment, disappeared after sixty days of stimulation in thirty-five patients (53%) and was of moderate intensity in seventeen patients (26%). CONCLUSIONS The results of this study confirm that pulsed electromagnetic field treatment may be indicated in the early stages of osteonecrosis of the femoral head (Ficat stages I and II). Pulsed electromagnetic field stimulation may be able to either preserve the hip or delay the time until surgery. The authors hypothesize that the short-term effect of pulsed electromagnetic field stimulation may be to protect the articular cartilage from the catabolic effect of inflammation and subchondral bone-marrow edema. The long-term effect of pulsed electromagnetic field stimulation may be to promote osteogenic activity at the necrotic area and prevent trabecular fracture and subchondral bone collapse. LEVEL OF EVIDENCE Therapeutic Level IV. See Instructions to Authors on jbjs.org for a complete description of levels of evidence.

Electromagnetic fields (EMFs) and adenosine receptors modulate prostaglandin E(2) and cytokine release in human osteoarthritic synovial fibroblasts.

Synovial fibroblasts (SFs) contribute to the development of osteoarthritis (OA) by the secretion of a wide range of pro‐inflammatory mediators, including cytokines and lipid mediators of inflammation. Previous studies suggest that electromagnetic fields (EMFs) may represent a potential therapeutic approach to limit cartilage degradation and control inflammation associated to OA, and that they may act through the adenosine pathway. Therefore, we investigated whether EMFs might modulate inflammatory activities of human SFs from OA patients (OASFs) treated with interleukin‐1β (IL‐1β), and the possible involvement of adenosine receptors (ARs) in mediating EMF effects. EMF exposure induced a selective increase in A2A and A3 ARs. These increases were associated to changes in cAMP levels, indicating that ARs were functionally active also in EMF‐exposed cells. Functional data obtained in the presence of selective A2A and A3 adenosine agonists and antagonists showed that EMFs inhibit the release of prostaglandin E2 (PGE2) and the proinflammatory cytokines interleukin‐6 (IL‐6) and interleukin‐8 (IL‐8), while stimulating the release of interleukin‐10 (IL‐10), an antinflammatory cytokine. These effects seem to be mediated by the EMF‐induced upregulation of A2A and A3 ARs. No effects of EMFs or ARs have been observed on matrix degrading enzyme production. In conclusion, this study shows that EMFs display anti‐inflammatory effects in human OASFs, and that these EMF‐induced effects are in part mediated by the adenosine pathway, specifically by the A2A and A3 AR activation. Taken together, these results open new clinical perspectives to the control of inflammation associated to joint diseases. J. Cell. Physiol. 227: 2461–2469, 2012.

Chondroprotective effects of pulsed electromagnetic fields on human cartilage explants

This study investigated the effects of pulsed electromagnetic fields (PEMFs) on proteoglycan (PG) metabolism of human articular cartilage explants from patients with osteoarthritis (OA). Human cartilage explants, recovered from lateral and medial femoral condyles, were classified according to the International Cartilage Repair Society (ICRS) and graded based on Outerbridge scores. Explants cultured in the absence and presence of IL-1β were treated with PEMF (1.5  mT, 75  Hz) or IGF-I alone or in combination for 1 and 7 days. PG synthesis and release were determined. Results showed that explants derived from lateral and medial condyles scored OA grades I and III, respectively. In OA grade I explants, after 7 days exposure, PEMF and IGF-I significantly increased (35) S-sulfate incorporation 49% and 53%, respectively, compared to control, and counteracted the inhibitory effect of IL 1β (0.01 ng/ml). The combined exposure to PEMF and IGF-I was additive in all conditions. Similar results were obtained in OA grade III cartilage explants. In conclusion, PEMF and IGF-I augment cartilage explant anabolic activities, increase PG synthesis, and counteract the catabolic activity of IL-1β in OA grades I and III. We hypothesize that both IGF-I and PEMF have chondroprotective effects on human articular cartilage, particularly in early stages of OA.

The electrical stimulation of tibial osteotomies. Double-blind study.

The effect of electromagnetic field stimulation was investigated in a group of 40 consecutive patients treated with valgus tibial osteotomy for degenerative arthrosis of the knee. All patients were operated on by the same author and followed the same postoperative program. After surgery, patients were randomly assigned to a control group (dummy stimulators) or to a stimulated one (active stimulators). Four orthopedic surgeons, unaware of the experimental conditions, were asked to evaluate the roentgenograms taken 60 days postoperatively and to rate the osteotomy healing according to four categories (the fourth category being the most advanced stage of healing). In the control group, 73.6% of the patients were included in the first and second category. In the stimulated group, 72.2% of the patients were included in the third and fourth category. On a homogeneous group of patients, electromagnetic field stimulation had positive effects on the healing of tibial osteotomies.

Expression and functional role of adenosine receptors in regulating inflammatory responses in human synoviocytes

Background and purpose:  Adenosine is an endogenous modulator, interacting with four G‐protein coupled receptors (A1, A2A, A2B and A3) and acts as a potent inhibitor of inflammatory processes in several tissues. So far, the functional effects modulated by adenosine receptors on human synoviocytes have not been investigated in detail. We evaluated mRNA, the protein levels, the functional role of adenosine receptors and their pharmacological modulation in human synoviocytes.

Phosphatidylinositol 3-Kinase Translocates to the Nucleus of Osteoblast-Like MC3T3-E1 Cells in Response to Insulin-Like Growth Factor I and Platelet-Derived Growth Factor But Not to the Proapoptotic Cytokine Tumor Necrosis Factor α

Changes in the metabolism of nuclear inositides phosphorylated in the D3 position of the inositol ring, which may act as second messengers, mainly have been linked to cell differentiation. To clarify a possible role of this peculiar class of inositides also during cell proliferation and/or apoptosis, we have examined the issue of whether or not in the osteoblast‐like clonal cell line MC3T3‐E1 it may be observed an insulin‐like growth factor‐I (IGF‐I)‐ and platelet‐derived growth factor (PDGF)‐dependent nuclear translocation of an active phosphatidylinositol 3‐kinase (PI 3‐K). We found that both the growth factors increased rapidly and transiently both the amount and the activity of immunoprecipitable nuclear PI 3‐K. Intranuclear PI 3‐K exhibited a massive tyrosine phosphorylation on the p85 regulatory subunit. Moreover, by means of coimmunoprecipitation experiments, we showed the presence, in isolated nuclei, of the p110β catalytic subunit of PI 3‐K. Enzyme translocation was blocked by the specific PI 3‐K inhibitor LY294002. In contrast, intranuclear translocation of PI 3‐K did not occur in response to the proapoptotic cytokine tumor necrosis factor α (TNF‐α). IGF‐I was able to counteract the apoptotic stimulus of TNF‐α and this was accompanied by the intranuclear translocation of PI 3‐K. LY294002 inhibited both intranuclear translocation of PI 3‐K and the rescuing effect of IGF‐I. These findings strongly suggest that an important step in the signaling pathways that mediate both cell proliferation and survival is represented by the intranuclear translocation of PI 3‐K.

Oxidative Stress and Bone Resorption Interplay as a Possible Trigger for Postmenopausal Osteoporosis

The underlying mechanism in postmenopausal osteoporosis (PO) is an imbalance between bone resorption and formation. This study was conducted to investigate whether oxidative stress (OxS) might have a role in this derangement of bone homeostasis. In a sample of 167 postmenopausal women, we found that increased serum levels of a lipid peroxidation marker, hydroperoxides, were negatively and independently associated with decreased bone mineral density (BMD) in total body (r = −0.192, P < 0.05), lumbar spine (r = −0.282, P < 0.01), and total hip (r = −0.282, P < 0.05), as well as with increased bone resorption rate (r = 0.233, P < 0.05), as assessed by the serum concentration of C-terminal telopeptide of type I collagen (CTX-1). On the contrary, the OxS marker failed to be correlated with the serum levels of bone-specific alkaline phosphatase (BAP), that is, elective marker of bone formation. Importantly, multiple regression analysis revealed that hydroperoxides is a determinant factor for the statistical association between lumbar spine BMD and CTX-1 levels. Taken together, our data suggest that OxS might mediate, by enhancing bone resorption, the uncoupling of bone turnover that underlies PO development.

Risk factors for development of atypical femoral fractures in patients on long-term oral bisphosphonate therapy

Bisphosphonates (BPs) are the first-line therapy for osteoporosis. In recent years, atypical femoral fractures (AFF) have been described in patients on BPs therapy. However, the relationship between BPs and AFF remains to be clarified. We evaluated clinical and hormonal characteristics of AFF patients, in order to determine AFF risk factors. We studied 11 females with AFF and 58 females with typical femoral fractures (TFF), admitted to our Department for surgical repair between January 2008 and December 2011. All AFF patients received BPs therapy for 6 to 13 yrs, whereas 36.2% (p<0.0001) of TFF patients received BPs for shorter period (TFF, 6.1±1.8 yr vs. AFF, 8.6±1.9 yr, p<0.0001). A higher prevalence of hypocalcemia was observed in AFF patients compared with TFF (p<0.02), with significantly (p<0.05) lower corrected calcium levels in AFF patients. By contrast a reduced prevalence of elevated PTH levels (p<0.05) was found in AFF patients. No significant difference in prevalence of vitamin D defect was observed between the two groups. Younger age (p<0.004), higher BMI (>30 kg/m2, p<0.03) and early menopausal age (p<0.05) were observed in AFF patients. At time of fracture, prevalence of osteopenia/osteoporosis and levels of bone turnover markers were significantly (p<0.01) lower in AFF compared with TFF patients. By multivariate analysis hypocalcemia, obesity, and younger age (<70 yr) were confirmed to be independent predictors of AFF; elevated PTH level was the predominant independent protective factor (p<0.004). In conclusion, our data indicate that clinical characteristics and metabolic factors may favor the development of AFF in BP treated patients. We identified hypocalcemia due to latent hypoparathyroidism as primary risk factor for AFF; age, obesity, early menopause, and BMD may also influence the development of AFF. An adequate clinical and metabolic assessment is suggested to prevent the development of AFF in BP treated patients.