Equisetum arvense standardized dried extract hinders age-related osteosarcopenia.

Age-associated osteosarcopenia is an unresolved syndrome characterized by the concomitant loss of bone (osteopenia) and skeletal muscle (sarcopenia) tissues increasing falls, immobility, morbidity, and mortality. Unbalanced resorption of bone in the remodeling process and excessive protein breakdown, especially fast type II myosin heavy chain (MyHC-II) isoform and myofiber metabolic shift, are the leading causes of bone and muscle deterioration in the elderly, respectively. Equisetum arvense (EQ) is a plant traditionally recommended for many pathological conditions due to its anti-inflammatory properties. Thus, considering that a chronic low-grade inflammatory state predisposes to both osteoporosis and sarcopenia, we tested a standardized hydroalcoholic extract of EQ in in vitro models of muscle atrophy [C2C12 myotubes treated with proinflammatory cytokines (TNFα/IFNγ), excess glucocorticoids (dexamethasone), or the osteokine, receptor activator of nuclear factor kappa-B ligand (RANKL)] and osteoclastogenesis (RAW 264.7 cells treated with RANKL). We found that EQ counteracted myotube atrophy, blunting the activity of several pathways depending on the applied stimulus, and reduced osteoclast formation and activity. By in silico target fishing, IKKB-dependent nuclear factor kappa-B (NF-κB) inhibition emerges as a potential common mechanism underlying EQ's anti-atrophic effects. Consumption of EQ (500 mg/kg/day) by pre-geriatric C57BL/6 mice for 3 months translated into: i) maintenance of muscle mass and performance; ii) restrained myofiber oxidative shift; iii) slowed down age-related modifications in osteoporotic bone, significantly preserving trabecular connectivity density; iv) reduced muscle- and spleen-related inflammation. EQ can preserve muscle functionality and bone remodeling during aging, potentially valuable as a natural treatment for osteosarcopenia.

Rhamnolipid 89 Biosurfactant Is Effective against Streptococcus oralis Biofilm and Preserves Osteoblast Behavior: Perspectives in Dental Implantology.

Biofilm-related peri-implant diseases represent the major complication for osteointegrated dental implants, requiring complex treatments or implant removal. Microbial biosurfactants emerged as new antibiofilm coating agents for implantable devices thanks to their high biocompatibility. This study aimed to assess the efficacy of the rhamnolipid 89 biosurfactant (R89BS) in limiting Streptococcus oralis biofilm formation and dislodging sessile cells from medical grade titanium, but preserving adhesion and proliferation of human osteoblasts. The inhibitory activity of a R89BS coating on S. oralis biofilm formation was assayed by quantifying biofilm biomass and microbial cells on titanium discs incubated up to 72 h. R89BS dispersal activity was addressed by measuring residual biomass of pre-formed biofilms after rhamnolipid treatment up to 24 h. Adhesion and proliferation of human primary osteoblasts on R89BS-coated titanium were evaluated by cell count and adenosine-triphosphate quantification, while cell differentiation was studied by measuring alkaline phosphatase activity and observing mineral deposition. Results showed that R89BS coating inhibited S. oralis biofilm formation by 80% at 72 h and dislodged 63-86% of pre-formed biofilms in 24 h according to concentration. No change in the adhesion of human osteoblasts was observed, whereas proliferation was reduced accompanied by an increase in cell differentiation. R89BS effectively counteracts S. oralis biofilm formation on titanium and preserves overall osteoblasts behavior representing a promising preventive strategy against biofilm-related peri-implant diseases.

MMP-2 Silencing through siRNA Loaded Positively-Charged Nanoparticles (AcPEI-NPs) Counteracts Chondrocyte De-Differentiation.

The abnormal matrix remodeling process, as well as inflammation, angiogenesis, and tumor metastasis, are related to an increase in the synthesis and secretion of matrix metalloproteinases (MMPs), the zinc-dependent proteolytic endopeptidases. Recent studies have evidenced MMPs' role in osteoarthritis (OA) development, during which chondrocytes undergo hypertrophic differentiation and exhibit enhanced catabolism. The trait of OA is extracellular matrix (ECM) progressive degradation regulated by many factors, in which MMPs play an important role, which indicates them as potential therapeutic targets. Herein, a small interfering RNA (siRNA) delivery system able to suppress MMPs' activity was synthetized. Results demonstrated that positively charged nanoparticles (AcPEI-NPs) complexed with MMP-2 siRNA are efficiently internalized by cells with endosomal escape. Moreover, avoiding lysosome degradation, MMP2/AcPEI nanocomplex increases nucleic acid delivery efficiency. Gel zymography, RT-PCR, and ELISA analyses confirmed MMP2/AcPEI nanocomplex activity even when embedded within collagen matrix resembling the natural extracellular matrix. Further, the inhibition of in vitro collagen degradation exerts a protective effect on chondrocyte dedifferentiation. The suppression of MMP-2 activity, preventing matrix degradation, protects chondrocytes against degeneration and supporting ECM homeostasis in articular cartilage. These encouraging results promote further investigation to validate the utilization of MMP-2 siRNA as ''molecular switch'' able to counteract osteoarthritis.

KYMASIN UP Natural Product Inhibits Osteoclastogenesis and Improves Osteoblast Activity by Modulating Src and p38 MAPK.

The imbalance in osteoblast (OB)-dependent bone formation in favor of osteoclast (OC)-dependent bone resorption is the main cause of loss of tissue mineral mass during bone remodeling leading to osteoporosis conditions. Thus, the suppression of OC activity together with the improvement in the OB activity has been proposed as an effective therapy for maintaining bone mass during aging. We tested the new dietary product, KYMASIN UP containing standardized Withania somnifera, Silybum marianum and Trigonella foenum-graecum herbal extracts or the single extracts in in vitro models mimicking osteoclastogenesis (i.e., RAW 264.7 cells treated with RANKL, receptor activator of nuclear factor kappa-Β ligand) and OB differentiation (i.e., C2C12 myoblasts treated with BMP2, bone morphogenetic protein 2). We found that the dietary product reduces RANKL-dependent TRAP (tartrate-resistant acid phosphatase)-positive cells (i.e., OCs) formation and TRAP activity, and down-regulates osteoclastogenic markers by reducing Src (non-receptor tyrosine kinase) and p38 MAPK (mitogen-activated protein kinase) activation. Withania somnifera appears as the main extract responsible for the anti-osteoclastogenic effect of the product. Moreover, KYMASIN UP maintains a physiological release of the soluble decoy receptor for RANKL, OPG (osteoprotegerin), in osteoporotic conditions and increases calcium mineralization in C2C12-derived OBs. Interestingly, KYMASIN UP induces differentiation in human primary OB-like cells derived from osteoporotic subjects. Based on our results, KYMASIN UP or Withania somnifera-based dietary supplements might be suggested to reverse the age-related functional decline of bone tissue by re-balancing the activity of OBs and OCs, thus improving the quality of life in the elderly and reducing social and health-care costs.

Thermo-Responsive Gel Containing Hydroxytyrosol-Chitosan Nanoparticles (Hyt@tgel) Counteracts the Increase of Osteoarthritis Biomarkers in Human Chondrocytes.

Although osteoarthritis (OA) is a chronic inflammatory degenerative disease affecting millions of people worldwide, the current therapies are limited to palliative care and do not eliminate the necessity of surgical intervention in the most severe cases. Several dietary and nutraceutical factors, such as hydroxytyrosol (Hyt), have demonstrated beneficial effects in the prevention or treatment of OA both in vitro and in animal models. However, the therapeutic application of Hyt is limited due to its poor bioavailability following oral administration. In the present study, a localized drug delivery platform containing a combination of Hyt-loading chitosan nanoparticles (Hyt-NPs) and in situ forming hydrogel have been developed to obtain the benefits of both hydrogels and nanoparticles. This thermosensitive formulation, based on Pluronic F-127 (F-127), hyaluronic acid (HA) and Hyt-NPs (called Hyt@tgel) presents the unique ability to be injected in a minimally invasive way into a target region as a freely flowing solution at room temperature forming a gel at body temperature. The Hyt@tgel system showed reduced oxidative and inflammatory effects in the chondrocyte cellular model as well as a reduction in senescent cells after induction with H2O2. In addition, Hyt@tgel influenced chondrocytes gene expression under pathological state maintaining their metabolic activity and limiting the expression of critical OA-related genes in human chondrocytes treated with stressors promoting OA-like features. Hence, it can be concluded that the formulated hydrogel injection could be proposed for the efficient and sustained Hyt delivery for OA treatment. The next step would be the extraction of "added-value" bioactive polyphenols from by-products of the olive industry, in order to develop a green delivery system able not only to enhance the human wellbeing but also to promote a sustainable environment.

Biomimetic Citrate-Coated Luminescent Apatite Nanoplatforms for Diclofenac Delivery in Inflammatory Environments.

Luminescent nanoparticles are innovative tools for medicine, allowing the imaging of cells and tissues, and, at the same time, carrying and releasing different types of molecules. We explored and compared the loading/release ability of diclofenac (COX-2 antagonist), in both undoped- and luminescent Terbium3+ (Tb3+)-doped citrate-coated carbonated apatite nanoparticles at different temperatures (25, 37, 40 °C) and pHs (7.4, 5.2). The cytocompatibility was evaluated on two osteosarcoma cell lines and primary human osteoblasts. Biological effects of diclofenac-loaded-nanoparticles were monitored in an in vitro osteoblast's cytokine-induced inflammation model by evaluating COX-2 mRNA expression and production of PGE2. Adsorption isotherms fitted the multilayer Langmuir-Freundlich model. The maximum adsorbed amounts at 37 °C were higher than at 25 °C, and particularly when using the Tb3+ -doped particles. Diclofenac-release efficiencies were higher at pH 5.2, a condition simulating a local inflammation. The luminescence properties of diclofenac-loaded Tb3+ -doped particles were affected by pH, being the relative luminescence intensity higher at pH 5.2 and the luminescence lifetime higher at pH 7.4, but not influenced either by the temperature or by the diclofenac-loaded amount. Both undoped and Tb3+-doped nanoparticles were cytocompatible. In addition, diclofenac release increased COX-2 mRNA expression and decreased PGE2 production in an in vitro inflammation model. These findings evidence the potential of these nanoparticles for osteo-localized delivery of anti-inflammatory drugs and the possibility to localize the inflammation, characterized by a decrease in pH, by changes in luminescence.

Osteosynthesis devices in absorbable Magnesium alloy in comparison to standard ones: a Systematic Review on effectiveness and safety.

BACKGROUND AND AIM OF THE WORK: Magnesium (Mg) is a metal physiologically present in bone tissue and essential for bone health. Mg-based-alloys exhibit mechanical properties, namely density and strength, similar to human cortical bone. These features have been exploited for the development of osteosynthesis devices in biodegradable Mg-based-alloys. Accordingly, the aim of this study was to rank the effectiveness and safety of Mg-based alloys applied in bone surgery in comparison to other suitable metals, focusing in particular on Mg superior biocompatibility and biodegradability. METHODS: a systematic-review of the literature was conducted including only primary research studies dealing with patients suffering from fractured or osteotomized bones fixed using Mg-based osteosynthesis-devices. RESULTS: literature revision suggested Mg-alloys holding comparable properties and side effects in comparison with titanium (Ti) screws, thus showing similar efficacy and safety. In particular, the gas formation in the carpal bones was identified as the main side effect of the Mg-alloys, during the corrosion/degradation phase of Mg. CONCLUSIONS: according to the considered literature, the main advantages exploiting Mg-alloys for bone implants are related to their biocompatibility, bio-absorbability/-degradability, the lack of surgical removal, osteoconductivity and antibacterial activity. On the opposite, the main limitation of Mg-alloys is due to the poor mechanical resistance of small devices for internal fixation of bone fragments that lack of sufficient strength to withstand high forces. Therefore, an important future prospect could rely in the development of innovative hybrid systems aimed at fixing high load-bearing fractures, as well as in regenerative-medicine by developing new Mg-based engineered scaffolds.

Plasma rich in growth factors (PRGF) for the promotion of bone cell proliferation and tissue regeneration.

OBJECTIVES: Over the past few years, studies about growth factors have been increasingly developed and the knowledge of their role in stimulating cell proliferation and differentiation used for therapeutic purposes. This study aims to compare a platelets concentrate, the plasma rich in growth factors (PRGF) to a control, consisting of cellulose membranes, to evaluate in vitro the cellular adhesion and migration of human osteoblasts (hOb) and understand if the use of platelets concentrates could be an advantage in view of bone tissue regeneration. STUDY DESIGN: Twenty-seven human donors provided 27 blood samples used to make 54 samples: 27 for PRGF and 27 for the control group. PRGFs and controls were incubated for 48 h in sterility in 1 ml of culture with 105 hOb and hOb in the scaffolds were then quantified. RESULTS: In PRGF samples, hObs were more numerous than in controls. (T = 6.6964, p < 0.0001). CONCLUSIONS: Human osteoblasts are driven to colonize PRGFs with a greater efficacy than negative controls, probably due to the presence of chemokines and growth factors in PRGFs.

ICOS-Ligand Triggering Impairs Osteoclast Differentiation and Function In Vitro and In Vivo.

Osteoblasts, osteocytes, and osteoclasts (OCs) are involved in the bone production and resorption, which are crucial in bone homeostasis. OC hyperactivation plays a role in the exaggerated bone resorption of diseases such as osteoporosis, rheumatoid arthritis, and osteolytic tumor metastases. This work stems from the finding that OCs can express B7h (ICOS-Ligand), which is the ligand of the ICOS T cell costimulatory molecule. Because recent reports have shown that, in endothelial, dendritic, and tumor cells, B7h triggering modulates several activities of these cells, we analyzed the effect of B7h triggering by recombinant ICOS-Fc on OC differentiation and function. The results showed that ICOS-Fc inhibits RANKL-mediated differentiation of human monocyte-derived OC-like cells (MDOCs) by inhibiting the acquirement of the OC morphology, the CD14- cathepsin K+ phenotype, and the expression of tartrate-resistant acid phosphatase, OSCAR, NFATc1, and DC-STAMP. Moreover, ICOS-Fc induces a reversible decrease in the sizes of cells and nuclei and cathepsin K expression in mature MDOCs. Finally, ICOS-Fc inhibits the osteolytic activities of MDOCs in vitro and the development of bone loss in ovariectomized or soluble RANKL-treated mice. These findings open a novel field in the pharmacological use of agonists and antagonists of the ICOS-B7h system.

Postoperative blood salvage versus allogeneic blood transfusion in total knee and hip arthroplasty: a literature review.

BACKGROUND AND AIM OF THE WORK: We aimed to compare Postoperative Blood Salvage (PBS) with Allogeneic Blood Transfusion (ABT) in patients undergoing Total Hip and Knee Arthroplasty (THA, TKA). METHODS: A bibliographic research was carried out in order to review the literature dedicated to postoperative blood salvage in major orthopaedic surgery, excluding papers dealing exclusively with preoperative autologous donation, intraoperative blood salvage and ABT. PBS and ABT were compared according to complications, costs and duration of hospitalization. PBS effectiveness in reducing ABT was also assessed. RESULTS: PBS system is useful for reducing the complication rate and the length of hospital stay if compared to ABT. Costs for the reinfusion of unwashed shed blood, washed blood, and allogeneic transfusion are controversial among the different authors. Several papers demonstrate that PBS significantly reduces the need of postoperative ABT in both THA and TKA, while there is low evidence that PBS does not affect the risk of surgical wound complications. To reduce potential risks related to PBS, including non-hemolytic febrile reaction, the reinfusion of saved blood should begin within 4-6 hours after the start of collection through the wound drainage. CONCLUSIONS: According to literature, PBS appears to be a valid alternative to ABT, which is the standard treatment for postoperative anemia in THA and TKA. Contraindications to PBS must be ruled out before recommending it to patients undergoing major orthopaedic procedures.

Human Lipoaspirate as Autologous Injectable Active Scaffold for One-Step Repair of Cartilage Defects.

Research on mesenchymal stem cells from adipose tissue shows promising results for cell-based therapy in cartilage lesions. In these studies, cells have been isolated, expanded, and differentiated in vitro before transplantation into the damaged cartilage or onto materials used as scaffolds to deliver cells to the impaired area. The present study employed in vitro assays to investigate the potential of intra-articular injection of micro-fragmented lipoaspirate as a one-step repair strategy; it aimed to determine whether adipose tissue can act as a scaffold for cells naturally present at their anatomical site. Cultured clusters of lipoaspirate showed a spontaneous outgrowth of cells with a mesenchymal phenotype and with multilineage differentiation potential. Transduction of lipoaspirate clusters by lentiviral vectors expressing GFP evidenced the propensity of the outgrown cells to repopulate fragments of damaged cartilage. On the basis of the results, which showed an induction of proliferation and ECM production of human primary chondrocytes, it was hypothesized that lipoaspirate may play a paracrine role. Moreover, the structure of a floating culture of lipoaspirate, treated for 3 weeks with chondrogenic growth factors, changed: tissue with a high fat component was replaced by a tissue with a lower fat component and connective tissue rich in GAG and in collagen type I, increasing the mechanical strength of the tissue. From these promising in vitro results, it may be speculated that an injectable autologous biologically active scaffold (lipoaspirate), employed intra-articularly, may 1) become a fibrous tissue that provides mechanical support for the load on the damaged cartilage; 2) induce host chondrocytes to proliferate and produce ECM; and 3) provide cells at the site of injury, which could regenerate or repair the damaged or missing cartilage.

Effects of erythropoietin on adipose tissue: a possible strategy in refilling.

BACKGROUND: The increased resorption and the difficulty of the fat graft take following autologous fat transplantation procedure are associated with reduced fat tissue revascularization and increased apoptosis of adipose cells. We suppose that the lipofilling procedure induces an inflammatory environment within the fat graft mass, whose evolution influences the efficacy of autologous fat graft survival. Erythropoietin (EPO) is a glycoprotein hormone known to exert angiogenetic and anti-inflammatory effects; therefore, our purpose was to investigate its reaction with adipose tissue used in lipofilling. METHODS: Fat masses were harvested using manual suction lipectomy and then seeded on dishes in appropriate culture and treated for 3 weeks with 3 doses of EPO. CD31 and CD68 immunohistochemistry was used to identify microvessels and several infiltrating leukocyte cells. RESULTS: Following EPO administration, we have detected an increase in the number of CD31-positive microvessel endothelium cells and CD31-positive small leukocytes and a reduction of CD68-positive cells. These effects were more conspicuous following higher EPO dose. CONCLUSIONS: Our findings evidence EPO treatment as a useful strategy to sustain the revascularization of grafted tissue and to reduce its inflammatory state.

Enhanced gene silencing through human serum albumin-mediated delivery of polyethylenimine-siRNA polyplexes.

Small interfering RNA (siRNA) targeted therapeutics (STT) offers a compelling alternative to tradition medications for treatment of genetic diseases by providing a means to silence the expression of specific aberrant proteins, through interference at the expression level. The perceived advantage of siRNA therapy is its ability to target, through synthetic antisense oligonucleotides, any part of the genome. Although STT provides a high level of specificity, it is also hindered by poor intracellular uptake, limited blood stability, high degradability and non-specific immune stimulation. Since serum proteins has been considered as useful vehicles for targeting tumors, in this study we investigated the effect of incorporation of human serum albumin (HSA) in branched polyethylenimine (bPEI)-siRNA polyplexes in their internalization in epithelial and endothelial cells. We observed that introduction of HSA preserves the capacity of bPEI to complex with siRNA and protect it against extracellular endonucleases, while affording significantly improved internalization and silencing efficiency, compared to bPEI-siRNA polyplexes in endothelial and metastatic breast cancer epithelial cells. Furthermore, the uptake of the HSA-bPEI-siRNA ternary polyplexes occurred primarily through a caveolae-mediated endocytosis, thus providing evidence for a clear role for HSA in polyplex internalization. These results provide further impetus to explore the role of serum proteins in delivery of siRNA.

Adhesion and differentiation of neuronal cells on Zn-doped bioactive glasses.

To verify the compatibility of rigid supports with neuronal cells for biomechanical application, we have evaluated the biocompatibility of Zn-doped bioglasses versus neuronal cell line SKNBE. Undifferentiated and retinoic acid-differentiated cells were used. We have observed that bioglasses doped with low concentration of Zn favored cell adhesion and proliferation of undifferentiated SKNBE neuronal cells, while the high Zn concentration strongly interfered with cell proliferation. Instead the high Zn concentration lightly stimulates the adhesive and strongly stimulates the phenotype characterization of differentiated SKNBE cells. Focal contact sites were observed in cells performing spread adhesive morphology, while they were down-regulated in cells performing differentiation behavior. GAP-43 and neurofilament were expressed in differentiated cells. However, GAP-43 was also found to be expressed in undifferentiated cells, where its expression seems related to proliferative behavior of cells. This work evidenced the importance of the biomaterial chemical structure in influencing proliferation or differentiation pathways of neuronal cells.

Comparative in vitro study of four commercial biomaterials used for bone grafting.

PURPOSE: This work aimed to study and compare the behavior of macrophages and human osteoclast-like cells cultured in contact with four commercial bone substitute materials used as graft for ridge augmentation techniques. Two materials are of natural origin and two are synthetic bone substitutes. METHODS: Macrophage activation and cytokine release were assessed using SEM analysis and a sandwich ELISA kit while the activity of human osteoclast-like cells was studied quantifying calcium released from the substrata studied together with SEM analysis of cell morphology and pit formation on the bone substitutes. RESULTS: Hydroxyapatite proved to be the greatest macrophage activator, while the bovine derived material turned out to be the only bone substitute that does not induce macrophages to release IFNγ. No material had direct induction of osteoclast precursor differentiation even if mature osteoclasts showed the highest activity on the human derived material followed by bone ceramic, while the bovine derived material was the substrate with the least osteoclast activity. CONCLUSIONS: The four materials reacted differently with the cells tested and between them, the bovine derived bone, being the lowest macrophage and osteoclast activator may be considered a good bone substitute for clinical situations requiring greater time of permanence of the material at the site of implantation, while human derived bone will be more suitable for clinical applications that require lower time of permanence.

Chondrogenic induction of human mesenchymal stem cells using combined growth factors for cartilage tissue engineering.

The objective of this study was to evaluate whether growth factors (FGF-2, FGF-4 and FGF-6) used alone or in combination with TGFß2 are able to increase the proliferation and induce the differentiation of human bone marrow mesenchymal stem cells (hMSCs) to chondrocytes, with a view to using them in cartilage tissue engineering. Cells cultured in monolayer, used to test the activity of the growth factors on cell proliferation, showed that a combination of FGFs with TGFß2 increases cell proliferation compared to cells cultured in control medium or in the presence of growth factors alone. The chondrogenic potential, evaluated in three-dimensional (3D) cell aggregates, showed that FGF-2 and FGF-6, when used in combination with TGFß2 increased the size and glycosaminoglycan content of the cell aggregates without increasing cell number. Extracellular matrix (ECM) also showed higher collagen type II immunoreactivity, which was particularly evident in an area similar to a germinative pole that was observed only in pellets cultured with FGF-2 and FGF-6 combined with TGFß2, or in pellets cultured with FGF-2 alone. Moreover, the RT-PCR assay has highlighted an increased expression of collagen type II and Sox9, used as gene markers for chondrogenesis. We can conclude that combinations of FGF-2 or FGF-6 with TGFß2 may provide a novel tool to induce the differentiation of adult human mesenchymal stem cells for applications in cartilage tissue engineering.

Controlled delivery of the heparan sulfate/FGF-2 complex by a polyelectrolyte scaffold promotes maximal hMSC proliferation and differentiation.

Growth factors and other regulatory molecules are required to direct differentiation of bone marrow-derived human mesenchymal stem cells (hMSC) along specific lineages. However, the therapeutic use of growth factors is limited by their susceptibility to degradation, and the need to maintain prolonged local release of growth factor at levels sufficient to stimulate hMSC. The aim of this study was to investigate whether a device containing heparan sulfate (HS), which is a co-factor in growth factor-mediated cell proliferation and differentiation, could potentiate and prolong the delivery of fibroblast growth factor-2 (FGF-2) and thus enhance hMSC stimulation. To this aim, we synthesized cationic polyelectrolyte polymers covalently and non-covalently anchored to HS and evaluated their effect on hMSC proliferation. Polymers non-covalently bound to HS resulted in the release of an HS/FGF-2 complex rather than FGF-2 alone. The release of this complex significantly restored hMSC proliferation, which was abolished in serum-free medium and only partially restored by the release of FGF-2 alone as occurred with polymer covalently bound to HS. We also demonstrate that exposure to HS/FGF-2 during early growth but not during post-confluence is essential for hMSC differentiation down the fibroblast lineage, which suggests that both factors are required to establish the correct stem cell commitment that is necessary to support subsequent differentiation. In conclusion, the delivery platform described here is a step towards the development of a new class of biomaterial that enables the prolonged, non-covalent binding and controlled delivery of growth factors and cofactors without altering their potency.

Regulation of osteoblast and osteoclast functions by FGF-6.

Fibroblast growth factor-6 (FGF-6) is known to be the key ligand for fibroblast growth factor receptor 4 (FGFR4) during muscle regeneration but its role in bone has yet to be verified. FGFR signaling is known to be important in the initiation and regulation of osteogenesis, so in this study the actions of FGF-6 on human osteoblasts and osteoclasts were investigated. Human primary osteoblasts (hOB) were used to study the effect of FGF-6 on proliferation (by ATP quantification), signal transduction (by ERK and AKT phosphorylation), differentiation (by alkaline phosphatase activity, APA), and mineralization (by calcein staining). To study FGF-6 activity on osteoclast differentiation, human bone marrow cells were used and tartrate-resistant acid phosphatase (TRAP) multinucleated cells together with actin filaments arrangements were quantified. Human primary mature osteoclasts were used to evaluate the effect of FGF-6 on osteoclast reabsorbing activity by reabsorbed pit measurements. FGF-6 >10(-9) M as FGF-2 10(-7) M induced hOB proliferation mediated by pERK together with a reduction in APA and reduced mineralization of the treated cells. Moreover FGF-6 increased the formation of TRAP-positive multinucleated cells in a dose-dependent manner (maximal effect at 10(-8) M). FGF-6-treated cells showed also a greater percentage of cells that formed typical osteoclast sealing zones. Mature osteoclasts cultured on dentine slice increased the area of reabsorption with a maximal effect of FGF-6 at 10(-12) M. FGF-6 may be considered a regulator of bone metabolism as shown by its activity on both osteoblasts and osteoclasts.

Different apoptosis modalities in periprosthetic membranes.

This study reports on an investigation into apoptotic and proliferation signals in leukocyte and membrane fibroblasts in periprosthetic membranes collected during revision surgery for loosened total hip joint arthroplasty. Cementless and cemented prosthesis were studied under both aseptic and septic conditions. Fluorescence colocalization immunohistochemistry and colorimetric immunohistochemistry were used to investigate cell death signals. In aseptic cementless prosthesis macrophages and membrane fibroblasts show high bax signal, implying the occurrence of toxic/oxidative cell death caused by the debris of titanium alloy metal implant. Instead in aseptic cemented prosthesis only a moderate number of apoptotic leukocytes were observed, whilst the fibroblasts were affected by a diffuse apoptotic-like cell death, the Co-Cr ions debris released from cemented stem, may be at basis of apoptotic cell death induction. Furthermore cement debris is recognized to induce macrophages to produce cytokine, that may be responsible for the cell death observed and implant failure. The septic environment seems to protect leukocytes cell death. Septic cementless prosthesis showed only a few apoptotic leukocytes, instead fibroblasts remain affected by cell death signals. Similarly in septic cemented prosthesis, scanty apoptotic leukocytes were detected, whereas membrane fibroblasts showed an increase in proliferation index (Ki-67) along with caspase-3 activation. These findings indicate some kind of caspase-3 involvement in tissue proliferation, rather than in cell death pathway. Apoptotic periprosthetic sites have been interpreted as signs of inflammation resolution and normal tissue turnover. Nevertheless apoptosis may also be a sign of cell renewal associated to tissue proliferation.

Effects of phosphatidylserine coatings on titanium on inflammatory cells and cell-induced mineralisation in vitro.

Ideally an active bone biomaterial should increase the mineralisation rate at the bone healing sites, keeping at the same time the inflammation process to levels required for tissue regeneration. Our studies suggest that in addition to improving the nucleation process for new bone formation, coating titanium with phospholipids may reduce the inflammatory response, which was shown to vary depending on the formulation employed. As phosphatidylserine reduced the inflammatory response to the greatest extent, in the second part of this study we examined its effect on osteoblast mineralisation. These studies demonstrated that phosphatidylserine improves the nucleation process for bone formation, by promoting the formation of bone-like tissue, so the high mineralisation potential of phosphatidylserine-coated titanium, together with the lower level of inflammatory response, supports the further development of this technology for coating osteointegrative devices.