Does chronic raise of metal ion levels induce oxidative DNA damage and hypoxia-like response in patients with metal-on-metal hip resurfacing?

Metal-on-metal hip resurfacing (MOM-HR) represents a viable alternative to traditional arthroplasty. Nevertheless, in MOM coupling both metal nanoparticles and ions are released, whose toxicity remains a matter of concern. We investigated whether 'endogenous' chronic exposure to cobalt and chromium induced a state of oxidative stress, DNA damage and a hypoxia-like response in patients with well-functioning MOM-HR. Twenty-two patients with unilateral MOM-HR were recruited at long-term. Twenty-one osteoarthritic subjects were enrolled for comparison. Serum ion levels were measured and correlated with 8-hydroxydeoxyguanosine and circulating-free-DNA, as markers of oxidative DNA damage. Moreover, the hypoxia-inducible factor-1α, marker of hypoxic state, was evaluated. Ion concentrations were found to be 5-to-15 times higher in MOM-HR patients than in presurgery subjects (p < 0.001); circulating-free-DNA, 8-hydroxydeoxyguanosine, and hypoxia-inducible factor-1α levels were not significantly different between groups and did not correlate with ion levels. Analyzing the results according to gender, MOM-HR males had higher 8-hydroxydeoxyguanosine levels (p = 0.01) compared with MOM-HR females. Similarly, circulating-free-DNA values were higher in males than females, even if this difference did not reach statistical significance. This research is the first that attempted to investigate the long-term effects of ion dissemination in subjects with well-fixed MOM implants. A significant correlation between biomarkers increase and ion levels was not demonstrated. Nevertheless, both circulating-free-DNA and 8-hydroxydeoxyguanosine showed a tendency to increase in MOM-HR males. Further studies with a larger sample size should be performed to detect the clinical relevance of biomarkers increase especially in younger subjects, where a chronic moderately elevated exposure has to be faced. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 460-466, 2017.

Effects of hypoxia on osteogenic differentiation of mesenchymal stromal cells used as a cell therapy for avascular necrosis of the femoral head.

BACKGROUND AIMS: Avascular necrosis of the femoral head (AVN) occurs as common result of various conditions or develops as a primary entity, with a high freqency in young adults. Because of its tendency toward osteoarthritis requiring total hip arthroplasty, alternative treatments are being advocated, including cell therapy with mesenchymal stromal cells (MSCs). Because osteonecrotic bone is a severely hypoxic tissue, with a 1-3% oxygen tension, the survival and function of multipotent cells is questionable. METHODS: In this study, the proliferative, immunophenotypic and osteogenic properties of bone marrow (BM)-derived MSCs from a clinical series of patients with AVN were evaluated under in vitro conditions mimicking the hypoxic milieu of AVN to verify the rationale for cell therapy. MSCs retrieved from the iliac crest (BM-MSC) were isolated, expanded and induced to osteogenic differentiation under a 2% pO2 atmosphere (hypoxia) in comparison with the standard 21% pO2 (normoxia) that is routinely used in cell culture assays. RESULTS: Both proliferation and colony-forming ability were significantly enhanced in hypoxia-exposed BM-MSCs compared with BM-MSCs under normoxia. The expression of bone-related genes, including alkaline phosphatase, Type I collagen, and osteocalcin was significantly increased under hypoxia. Moreover, mineral deposition after osteogenic induction was not hampered, but in some cases even enhanced under low oxygen tension. CONCLUSIONS: These findings support autologous cell therapy as an effective treatment to stimulate bone healing in the hypoxic microenvironment of AVN.

Acridine Orange is an Effective Anti-Cancer Drug that Affects Mitochondrial Function in Osteosarcoma Cells.

Acridine orange (AO) is an antimalarial drug that accumulates into acidic cellular compartments. Lysosomes are quite acidic in cancer cells, and on this basis we have demonstrated that photoactivated AO is selectively toxic in sarcomas. However, photodynamic therapy is only locally effective, and cannot be used to eradicate systemic residual disease. In this study, we have evaluated the activity of non-photoactivated AO on sensitive and chemoresistant osteosarcoma (OS) cells to be considered for the systemic delivery. Since lysosomes are even more acidic in chemoresistant cells (MDR), we found that AO accumulation was significantly higher in the lysosomes of MDR in respect to parental cells, and in both cell types, therapeutic doses of AO significantly inhibited cell growth. However, the level of growth inhibition was inversely related to the level of lysosomal uptake of AO, suggesting that the main target of this agent is indeed extralysosomal. A significant reduction of intracellular ATP content and of the expression of mitochondrial complex III suggests a mitochondrial targeting. Notably, MDR cells showed a lower mitochondrial activity. Finally, the combined treatment of AO with the anticancer agent doxorubicin (DXR) significantly increased chemotoxicity by promoting DXR mitochondrial targeting, as revealed by the further reduction in ATP intracellular content. In conclusion, AO is able to effectively target both sensitive and resistant OS cells through mitotoxicity.

Prolonged exposure to hypoxic milieu improves the osteogenic potential of adipose derived stem cells.

Mesenchymal stem cells (MSC) have been widely used in orthopedics for several applications. Conventionally, MSC are maintained under 21% O2 which does not reflect the real O2 tension in vivo. Recently, it was reported that different O2 conditions can give different cellular responses. Here, we investigated whether prolonged exposure to hypoxia affects the osteogenic differentiation of adipose-derived stem cells (ASC). ASC from six individuals were cultured under "low" (2-3%) or "air" (21%) oxygen tensions, either without or with osteogenic stimuli. The effect of the O2 tension was evaluated on cell proliferation, surface antigens, stemness and bone-related genes expression, alkaline phosphatase activity (ALP), mineralization activity, and release of osteogenic growth factors. Without differentiating stimuli, hypoxia favored ASC proliferation, reduced the number of CD184+ and CD34+ cells, and preserved the expression of NANOG and SOX2. The combination of hypoxia and osteogenic medium induced a high proliferation rate, a rapid and more pronounced mineralization activity, a higher expression of genes related to the MSC differentiation, a higher release of mitogenic growth factors (bFGF, PDGF-BB), and the decrease in TGF-ß secretion, an inhibitor of the early stage of the osteoblast differentiation. We demonstrated that hypoxia acts dually, favoring ASC proliferation and the maintenance of the stemness in the absence of osteogenic stimuli, but inducing the differentiation in a bone-like microenvironment. In conclusion, prolonged cell culture in hypoxic microenvironment represents a proper method to modulate the stem cell function that may be used in several applications, for example, studies on bone pathophysiology or bone-tissue engineering.

Hypoxia enhances proliferation and stemness of human adipose-derived mesenchymal stem cells.

The aim of the study was to obtain the highest number of multipotent adipose-derived mesenchymal stem cells (ADMSCs) by using culture conditions which favour cell expansion without loss of mesenchymal stem cells (MSC)-like properties. Based on the assumption that stem cells reside in niches characterized by hypoxic condition, we investigated if the low oxygen tension may improve the proliferation and stemness of ADMSCs. Intact adipose tissue was resected from eight subjects, and the stromal vascular fraction was obtained by using type II collagenase. The heterogeneity of cellular lineages was confirmed by immunophenotypic analysis that showed the presence of leukocytes (CD45+), endothelial cells (CD34+), and pericytes (CD140+). The immunophenotype of confluent ADMSCs was similar to that of bone marrow-derived MSCs, except for the expression of CD34, which was variable (donor-dependent) and inversely correlated to the CD36 expression. ADMSCs showed a high clonal efficiency (94.5 ± 1 %) and were able to generate osteoblastic, chondrocytic and adipocytic lineages. ADMSCs were cultured under normoxic (21 % O2) and hypoxic (1 % O2) conditions, and we found that hypoxia significantly favoured ADMSC proliferation and preserved the expression of stemness genes, i.e. Nanog and Sox2. Since hypoxia reflects the microenvironment in which ADMSCs must proliferate and differentiate, the culture in hypoxic condition allows to better understand the biology of these cells and their regenerative potential. Low oxygen concentrations promote cell proliferation and stemness, thus enriching the pool of cells potentially able to differentiate into multi-lineages, and extending the possibility of a long-term expansion.

V-ATPase as an effective therapeutic target for sarcomas.

Malignant tumors show intense glycolysis and, as a consequence, high lactate production and proton efflux activity. We investigated proton dynamics in osteosarcoma, rhabdomyosarcoma, and chondrosarcoma, and evaluated the effects of esomeprazole as a therapeutic agent interfering with tumor acidic microenvironment. All sarcomas were able to survive in an acidic microenvironment (up to 5.9­6.0 pH) and abundant acidic lysosomes were found in all sarcoma subtypes. V-ATPase, a proton pump that acidifies intracellular compartments and transports protons across the plasma membrane, was detected in all cell types with a histotype-specific expression pattern. Esomeprazole administration interfered with proton compartmentalization in acidic organelles and induced a significant dose-dependent toxicity. Among the different histotypes, rhabdomyosarcoma, expressing the highest levels of V-ATPase and whose lysosomes are most acidic, was mostly susceptible to ESOM treatment.

How do metal ion levels change over time in hip resurfacing patients? A cohort study.

Metal-on-metal hip resurfacing (MOM-HR) is offered as an alternative to traditional hip arthroplasty for young, active adults with advanced osteoarthritis. Nevertheless, concerns remain regarding wear and corrosion of the bearing surfaces and the resulting increase in metal ion levels. We evaluated three cohorts of patients with Birmingham hip resurfacing (BHR) at an average follow-up of 2, 5, and 9 years. We asked whether there would be differences in ion levels between the cohorts and inside the gender. Nineteen patients were prospectively analyzed. The correlation with clinical-radiographic data was also performed. Chromium, cobalt, nickel, and molybdenum concentrations were measured by atomic absorption spectrophotometry. Chromium and cobalt levels demonstrated a tendency to decrease over time. Such tendency was present only in females. An inverse correlation between chromium, implant size, and Harris hip score was present at short term; it disappeared over time together with the decreased ion levels. The prospective analysis showed that, although metal ion levels remained fairly constant within each patient, there was a relatively large variation between subjects, so mean data in this scenario must be interpreted with caution. The chronic high exposure should be carefully considered during implant selection, particularly in young subjects, and a stricter monitoring is mandatory.

Hyaluronan-based pericellular matrix: substrate electrostatic charges and early cell adhesion events.

Cells are surrounded by a hyaluronan-rich coat called 'pericellular matrix' (PCM), mainly constituted by hyaluronan, a long-chain linear polysaccharide which is secreted and resorbed by the cell, depending on its activity. Cell attachment to a surface is mediated by PCM before integrins and focal adhesions are involved. As hyaluronan is known to bear a negative charge at physiological pH, the relevance of its electrical properties in driving the early cell adhesion steps has been studied, exploring how PCM mediates cell adhesion to charged surfaces, such as polyelectrolyte multilayer (PEM) films. Poly(ethylene imine) (PEI) and poly(sodium 4-styrene sulphonate) (PSS), assembled as PEI/PSS and PEI/PSS/PEI layers, were used. The nanoscale morphology of such layers was analysed by atomic force microscopy, and the detailed surface structure was analysed by X-ray photoemission spectroscopy. PCM-coated and PCM-depleted MG63 osteoblast-like cells were used, and cell density, morphology and adhesive structures were analysed during early steps of cell attachment to the PEM surfaces (1-6 h). The present study demonstrates that the pericellular matrix is involved in cell adhesion to material surfaces, and its arrangement depends on the cell interaction with the surface. Moreover, the PCM/surface interaction is not simply driven by electrostatic effects, as the cell response may be affected by specific chemical groups at the material surface. In the development of biomimetic surfaces promoting cell adhesion and function, the role of this unrecognised outer cell structure has to be taken into account.

The natural compound Alizarin as an osteotropic drug for the treatment of bone tumors.

Despite significant clinical improvements, conventional therapies for bone cancer treatment are limited by significant systemic toxicity and lack of specific targeting. In this study, we considered Alizarin, a natural hydroxyanthraquinone derived from madder root with high affinity to calcium and remarkable osteotropic features, as a novel approach for bone cancer treatment. Due to its antitumor properties, as demostrated in colon cancer cells, and to its tropism to bone, Alizarin may be an ideal drug to reduce bone tumor growth. We demonstrated that low dosages of Alizarin strongly inhibited the osteosarcoma (IC(50) for Saos-2, MG-63, and U-2 OS cells, 27.5, 29.0, and 69.9 µg/ml, respectively) and breast carcinoma (IC(50) for MDA-MB-231 cells, 62.1 µg/ml) cell proliferation in vitro. Importantly, Alizarin had a significantly lower inhibitory activity on normal cells (IC(50) for MSC, 828.6 µg/ml), thereby revealing a selective activity towards malignant cells. Furthermore, we found that Alizarin acted through the inhibition of ERK phosphorylation and cell cycle arrest in the S-phase. Finally, Alizarin significantly and strongly impaired both osteosarcoma and breast cancer tumorigenesis. Our results highlight a selective and effective inhibitory activity of Alizarin towards cancerous cells, laying the basis for further studies to investigate its application in bone cancer therapy.

The effect of poly(d,l-lactide-co-glycolide)-alendronate conjugate nanoparticles on human osteoclast precursors.

Nanoparticles (NPs) formed from polymers conjugated with bisphosphonates (BPs) allow the bone targeting of loaded drugs, such as doxorubicin, for the treatment of skeletal tumours. The additional antiosteoclastic effect of the conjugated BP could contribute to the inhibition of tumour-associated bone degradation. With this aim, we have produced NPs made of poly(d,l-lactide-co-glycolide) (PLGA) conjugated with alendronate (ALE). To show if ALE retained the antiosteoclastic properties after the conjugation with PLGA and the production of NPs, we treated human osteoclasts, derived from circulating precursors, with PLGA-ALE NPs and compared the effects on actin ring generation, apoptosis and type-I collagen degradation with those of free ALE and with NPs made of pure PLGA. PLGA-ALE NPs disrupted actin ring, induced apoptosis and inhibited collagen degradation. Unexpectedly, also NPs made of pure PLGA showed similar effects. Therefore, we cannot exclude that in addition to the observed antiosteoclastic activity dependent on ALE in PLGA-ALE NPs, there was also an effect due to pure PLGA. Still, as PLGA-ALE NPs are intended for the loading with drugs for the treatment of osteolytic bone metastases, the additional antiosteoclastic effect of PLGA-ALE NPs, and even of PLGA, may contribute to the inhibition of the disease-associated bone degradation.

Idiopathic and secondary osteonecrosis of the femoral head show different thrombophilic changes and normal or higher levels of platelet growth factors.

BACKGROUND AND PURPOSE: Thrombophilia represents a risk factor both for idiopathic and secondary osteonecrosis (ON). We evaluated whether clotting changes in idiopathic ON were different from corticosteroid-associated ON. As platelet-rich plasma has been proposed as an adjuvant in surgery, we also assessed whether platelet and serum growth factors were similar to those in healthy subjects. METHODS: 18 patients with idiopathic ON and 18 with corticosteroid-associated ON were compared with 44 controls for acquired and inherited thrombophilia. Platelet factor 4 (PF4), transforming growth factor-ß1, platelet-derived growth factor-BB (PDGF-BB), and vascular endothelial growth factor were assayed in the supernatants of thrombin-activated platelets, in platelet lysates, and in serum from 14 ON patients and 10 controls. RESULTS: Idiopathic ON patients had higher plasminogen levels (median 118%) than controls (101%) (p = 0.02). Those with corticosteroid-associated ON had significantly higher D-dimer (333 ng/mL) and lower protein C levels (129%) than controls (164 ng/mL, p = 0.004; 160%, p = 0.02). The frequency of inherited thrombophilia was not different from the controls. No statistically significant differences were found between idiopathic and corticosteroid-associated ON. 20 of the 36 ON patients were smokers. (The controls were selected from smokers because nicotine favors hypercoagulability). ON patients had significantly higher serum PF4 levels (7,383 IU/mL) and PDGF-BB levels (3.1 ng/mL) than controls (4,697 IU/mL, p = 0.005; 2.2 ng/mL, p = 0.02). INTERPRETATION: Acquired hypercoagulability was common in both ON types, but the specific changes varied. The release of GF from platelets was not affected, providing a biological basis for platelet-rich plasma being used as an adjuvant in surgical treatment.

Platelet-rich plasma impairs osteoclast generation from human precursors of peripheral blood.

Platelet-rich plasma is used to accelerate bone repair for the release of osteogenic growth factors from activated platelets. To date, the effects on osteoclasts have been only scarcely investigated, even though these cells are crucial for bone remodeling. The aim of this research was the evaluation of the effects of thrombin-activated platelets (PRP) on osteoclastogenesis from human blood precursors. We evaluated both the ability to influence osteoclast differentiation induced by the receptor activator of nuclear factor-kappaB ligand (RANKL), and the ability to induce osteoclast differentiation without RANKL. In both assays, the incubation with PRP supernatant at 10% did not significantly affect the formation of tartrate-resistant acid phosphatase (TRACP)-positive multinucleated cells that were able to form the F-actin ring. However, when PRP at 25 and 50% was added to the medium without RANKL, the generation of TRACP-positive multinucleated cells was inhibited. PRP, even at 10%, reduced the osteoclast-mediated bone collagen degradation, suggesting inhibition of osteoclast activation. Similarly, after incubation with PRP supernatant, calcitonin receptor mRNA was lower than the untreated samples. In conclusion, PRP at 10% interfered with the complete differentiation process of human osteoclast precursors. At higher concentration it impaired osteoclast formation also at an early stage of differentiation.

Background and rationale of platelet gel in orthopaedic surgery.

Autologous platelet gel, which is usually prepared by adding thrombin and calcium to a platelet concentrate, is used to accelerate bone repair as a possible alternative to recombinant growth factors (GF), through the osteogenic GF released from alpha-granules. The advantages of platelet gel lie in its mimicking the GF effects of the physiological bone healing and regenerative processes, in addition to a relatively simple and low cost technique. Moreover, if autologous platelet gel is used, immunological reactions are avoided. In in vitro systems, platelet gel stimulated osteogenic differentiation of bone marrow stromal cells, while it inhibited complete osteoclast differentiation and activation. Moreover, platelet gel favoured endothelial cell proliferation and expression of pro-osteogenic functions. In experimental animals and in clinical application, the efficacy of platelet gel was increased by the combination with bone allografts, acting as scaffolds, and with bone marrow stromal cells.

Endothelial cells incubated with platelet-rich plasma express PDGF-B and ICAM-1 and induce bone marrow stromal cell migration.

Platelet-rich plasma (PRP) is used to accelerate bone repair through the growth factors released by platelets. The purpose of this study was to evaluate if PRP induce human umbilical vein endothelial cells (HUVEC) to express mRNA for osteogenic growth factors and stimulate the migration of bone marrow stromal cell (BMSC). The effects of PRP were compared to those induced by vascular endothelial growth factor-A (VEGF-A) or, as a negative control, by platelet poor plasma (PPP). After incubation with PRP, but not with PPP, HUVEC showed an increased expression of mRNA for platelet derived growth factor-B (PDGF-B), and this effect was not inhibited by an anti-VEGF-A antibody. The migration of BMSC was more stimulated by HUVEC incubated with PRP than by HUVEC incubated with low serum medium or PPP. Besides, PRP increased the expression of intercellular adhesion molecule-1 (ICAM-1) and osteoprotegerin, but did not affect the expression either of the receptor activator for nuclear factor kappaB ligand (RANKL) or of RANK. These findings support the hypothesis that PRP contribute to bone repair by favoring the pro-osteogenic function of endothelial cells, including the recruitment of osteoblast precursors and the expression of adhesion molecules for monocyte/macrophages, while inhibiting their pro-osteolytic properties.

A novel biomaterial for osteotropic drug nanocarriers: synthesis and biocompatibility evaluation of a PLGA-ALE conjugate.

BACKGROUND & AIMS: Osteotropic drug-delivery systems have been proposed as a means to provide drugs with affinity to bone tissues. Drugs or proteins have been linked chemically to bone-seeking agents, such as bisphosphonates (BPs); alternatively, drug-loaded nanoparticles have been used to target specific tissues, such as tumor areas. In our current research, these approaches were merged by synthesizing a novel bone-seeking polymer conjugate, from which targetable nanoparticles can be produced. MATERIALS & METHODS: An amino-BP, alendronate (ALE) was bound covalently to a biodegradable polymer, poly(lactic-co-glycolide) (PLGA), containing a free end carboxylic group. Blood compatibility and cytotoxicity were assessed in vitro. RESULTS & DISCUSSION: By a classical solvent-evaporation method, nanoparticles with a mean size of 200-300 nm were prepared from the conjugate; sterilization was achieved by gamma-irradiation, confirming their potential as injectable drug nanocarriers. Owing to the presence of the BP residue, PLGA-ALE nanoparticles were adsorbed onto hydroxyapatite to a higher extent than pure PLGA nanoparticles. The PLGA-ALE conjugate did not induce either hemolysis or alterations of the plasmatic phase of coagulation, or cytotoxic effects on endothelial cells and trabecular osteoblasts. CONCLUSION: The prepared conjugate represents a novel biomaterial that is able to provide nanoparticles, which can be further loaded with drugs, such as anticancer agents, and addressed to osteolytic or other bone diseases.

Biocompatibility of poly(D,L-lactide-co-glycolide) nanoparticles conjugated with alendronate.

Nanoparticles made of a conjugate of poly(D,L-lactide-co-glycolide) with alendronate (PLGA-ALE NPs), were prepared by emulsion/solvent evaporation technique. The conjugation yield, determined by MALDI TOF analysis, was 30-35%. PLGA-ALE NPs size, evaluated by photon correlation spectroscopy, was 198.7+/-0.2 nm. Haemocompatibility studies using different concentrations of PLGA-ALE NPs did not show any significant effect on haemolysis, leukocyte number, platelet activation, APTT and complement consumption, in comparison with blood incubated with phosphate buffered saline (PBS). A significant reduction of the prothrombin activity was demonstrated after incubation with 560 microg/ml of PLGA-ALE NPs; a significant increase was observed at the highest dilutions. The viability of human umbilical vein endothelial cells and bone marrow stromal cells (BMSC), evaluated through the neutral red test, was not affected by PLGA-ALE NPs. There were no significant differences in cell-associated alkaline phosphatase between BMSC incubated with PLGA-ALE NP- and PBS-added media. These results demonstrated that PLGA-ALE NPs had an acceptable degree of blood compatibility and were not cytotoxic; therefore, they may be considered suitable for intravenous administration.