Osteoblast and osteoclast activity on collagen-based 3D printed scaffolds enriched with strontium-doped bioactive glasses and hydroxyapatite nanorods for bone tissue engineering.

Bone tissue engineering (BTE) aims to promote bone regeneration by means of the synergistic effect of biomaterials, cells, and other factors, as potential alternative to conventional treatments for bone fractures. To this aim, a composite material was developed, based on collagen type I, strontium-enriched mesoporous bioactive glasses, and hydroxyapatite nanorods as bioactive and biomimetic components. Nanostructured scaffolds were 3D printed and subsequently chemically crosslinked with genipin to improve mechanical properties and stability. The developed nanostructured system was maintained in culture until 3 weeks with a co-culture of human bone cells to provide anex vivomodel of bone microenvironment and examine the cellular crosstalk and signaling pathways through paracrine cell activities. Human osteoblasts (OBs), derived from trabecular bone, and human osteoclast precursors (OCs), isolated from buffy coat samples were involved, with OBs seeded on the scaffold and OC precursors seeded in a transwell device. When compared to the material without inorganic components, the bioactive and biomimetic scaffold positively influenced cell proliferation and cell metabolic activity, boosting alkaline phosphatase activity of OBs, and reducing OC differentiation. Thus, the bioactive and biomimetic system promoted an enhanced cellular response, highlighting its potential application in BTE.

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.

Enhancing osteoconduction of PLLA-based nanocomposite scaffolds for bone regeneration using different biomimetic signals to MSCs.

In bone engineering, the adhesion, proliferation and differentiation of mesenchymal stromal cells rely on signaling from chemico-physical structure of the substrate, therefore prompting the design of mimetic "extracellular matrix"-like scaffolds. In this study, three-dimensional porous poly-L-lactic acid (PLLA)-based scaffolds have been mixed with different components, including single walled carbon nanotubes (CNT), micro-hydroxyapatite particles (HA), and BMP2, and treated with plasma (PT), to obtain four different nanocomposites: PLLA + CNT, PLLA + CNTHA, PLLA + CNT + HA + BMP2 and PLLA + CNT + HA + PT. Adult bone marrow mesenchymal stromal cells (MSCs) were derived from the femur of orthopaedic patients, seeded on the scaffolds and cultured under osteogenic induction up to differentiation and mineralization. The release of specific metabolites and temporal gene expression profiles of marrow-derived osteoprogenitors were analyzed at definite time points, relevant to in vitro culture as well as in vivo differentiation. As a result, the role of the different biomimetic components added to the PLLA matrix was deciphered, with BMP2-added scaffolds showing the highest biomimetic activity on cells differentiating to mature osteoblasts. The modification of a polymeric scaffold with reinforcing components which also work as biomimetic cues for cells can effectively direct osteoprogenitor cells differentiation, so as to shorten the time required for mineralization.

Early efficacy evaluation of mesenchymal stromal cells (MSC) combined to biomaterials to treat long bone non-unions.

BACKGROUND AND STUDY AIM: Advanced therapy medicinal products (ATMP) frequently lack of clinical data on efficacy to substantiate a future clinical use. This study aims to evaluate the efficacy to heal long bone delayed unions and non-unions, as secondary objective of the EudraCT 2011-005441-13 clinical trial, through clinical and radiological bone consolidation at 3, 6 and 12 months of follow-up, with subgroup analysis of affected bone, gender, tobacco use, and time since the original fracture. PATIENTS AND METHODS: Twenty-eight patients were recruited and surgically treated with autologous bone marrow derived mesenchymal stromal cells expanded under Good Manufacturing Practices, combined to bioceramics in the surgical room before implantation. Mean age was 39 ± 13 years, 57% were males, and mean Body Mass Index 27 ± 7. Thirteen (46%) were active smokers. There were 11 femoral, 4 humeral, and 13 tibial non-unions. Initial fracture occurred at a mean ± SD of 27.9 ± 31.2 months before recruitment. Efficacy results were expressed by clinical consolidation (no or mild pain if values under 30 in VAS scale), and by radiological consolidation with a REBORNE score over 11/16 points (value of or above 0.6875). Means were statistically compared and mixed models for repeated measurements estimated the mean and confidence intervals (95%) of the REBORNE Bone Healing scale. Clinical and radiological consolidation were analyzed in the subgroups with Spearman correlation tests (adjusted by Bonferroni). RESULTS: Clinical consolidation was earlier confirmed, while radiological consolidation at 3 months was 25.0% (7/28 cases), at 6 months 67.8% (19/28 cases), and at 12 months, 92.8% (26/28 cases including the drop-out extrapolation of two failures). Bone biopsies confirmed bone formation surrounding the bioceramic granules. All locations showed similar consolidation, although this was delayed in tibial non-unions. No significant gender difference was found in 12-month consolidation (95% confidence). Higher consolidation scale values were seen in non-smoking patients at 6 (p = 0.012, t-test) and 12 months (p = 0.011, t-test). Longer time elapsed after the initial fracture did not preclude the occurrence of consolidation. CONCLUSION: Bone consolidation was efficaciously obtained with the studied expanded hBM-MSCs combined to biomaterials, by clinical and radiological evaluation, and confirmed by bone biopsies, with lower consolidation scores in smokers.

Biomimetic calcium-silicate cements aged in simulated body solutions. Osteoblast response and analyses of apatite coating.

PURPOSE: Calcium-silicate cements have been recently proposed for application in dentistry as root-end filling and root-perforation repair materials. The aim of this study was to investigate the effect of ageing of experimental calcium-silicate cements on the chemistry, morphology and in vitro bioactivity of the surface, as well as on osteoblast viability and proliferation. METHODS: Two experimental cements (wTC-Bi, containing bismuth oxide and wTC), mainly based on dicalcium-silicate and tricalcium-silicate, were prepared and tested for their bioactivity after soaking in Dulbecco's phosphate buffered saline (DPBS), used as simulated body fluid. Human marrow stromal cells (HMSC) were seeded on the cements maintained in DPBS for 5 hr (non-aged group), 14 and 28 days (aged group). Cell viability was assessed by the Alamar blueTM test and morphology by scanning electron microscopy (SEM) at different time endpoints. The surface of the soaked cements was analyzed by environmental scanning electron microscopy or SEM coupled with energy dispersive X-ray microanalysis (ESEM/EDX or SEM/EDX respectively) and the micro-Raman technique. RESULTS: The ESEM/EDX results showed a uniform surface composed of CSH hydrogel (mainly derived from the hydration of belite and alite) on both non-aged cements. Micro-Raman spectroscopy revealed the presence of calcium carbonate, anhydrite, ettringite, alite and belite. The SEM/EDX data showed an irregular calcium-phosphate multi-layered biocoating with many sharp and protruding crystals on both the aged cements. Micro-Raman spectroscopy revealed crystalline apatite and calcite. The osteoblast response results showed that both the experimental cements exerted no acute toxicity in the cell assay systems. The non-aged samples promoted greater cell growth. SEM showed cells well spread and adherent to the non-aged materials. A reduced number of attached cells was noticed on the aged cements. Bismuth oxide-containing cement allowed a reduced cell viability suggesting some cytotoxic effects. However, the thick biocoating formed on the 28-day aged samples lowered the deleterious effect of bismuth oxide on cell growth. Actually, micro-Raman spectroscopy revealed progressive bismuth oxide depletion on the wTC-Bi surface, due to the increased thickness of the apatite deposit. CONCLUSIONS: The study demonstrated that (1) these materials support osteogenic cells growth and may induce early bone formation, (2) the ageing in DPBS reduced the growth of HMSC, but eliminated the deleterious effect of the bismuth oxide on cell growth. In conclusion, the experimental cements have adequate biological properties to be used as root-end/root repair filling materials or pulp capping materials.

Effect of calcium phosphate heparinization on the in vitro inflammatory response and osteoclastogenesis of human blood precursor cells.

The immobilization of natural molecules on synthetic bone grafts stands as a strategy to enhance their biological interactions. During the early stages of healing, immune cells and osteoclasts (OC) modulate the inflammatory response and resorb the biomaterial, respectively. In this study, heparin, a naturally occurring molecule in the bone extracellular matrix, was covalently immobilized on biomimetic calcium-deficient hydroxyapatite (CDHA). The effect of heparin-functionalized CDHA on inflammation and osteoclastogenesis was investigated using primary human cells and compared with pristine CDHA and beta-tricalcium phosphate (ß-TCP). Biomimetic substrates led to lower oxidative stresses by neutrophils and monocytes than sintered ß-TCP, even though no further reduction was induced by the presence of heparin. In contrast, heparinized CDHA fostered osteoclastogenesis. Optical images of stained TRAP positive cells showed an earlier and higher presence of multinucleated cells, compatible with OC at 14 days, while pristine CDHA and ß-TCP present OC at 21-28 days. Although no statistically significant differences were found in the OC activity, microscopy images evidenced early stages of degradation on heparinized CDHA, compatible with osteoclastic resorption. Overall, the results suggest that the functionalization with heparin fostered the formation and activity of OC, thus offering a promising strategy to integrate biomaterials in the bone remodelling cycle by increasing their OC-mediated resorption.

Feasibility and safety of treating non-unions in tibia, femur and humerus with autologous, expanded, bone marrow-derived mesenchymal stromal cells associated with biphasic calcium phosphate biomaterials in a multicentric, non-comparative trial.

BACKGROUND: ORTHO-1 is a European, multicentric, first in human clinical trial to prove safety and feasibility after surgical implantation of commercially available biphasic calcium phosphate bioceramic granules associated during surgery with autologous mesenchymal stromal cells expanded from bone marrow (BM-hMSC) under good manufacturing practices, in patients with long bone pseudarthrosis. METHODS: Twenty-eight patients with femur, tibia or humerus diaphyseal or metaphyso-diaphyseal non-unions were recruited and surgically treated in France, Germany, Italy and Spain with 100 or 200 million BM-hMSC/mL associated with 5-10 cc of bioceramic granules. Patients were followed up during one year. The investigational advanced therapy medicinal product (ATMP) was expanded under the same protocol in all four countries, and approved by each National Competent Authority. FINDINGS: With safety as primary end-point, no severe adverse event was reported as related to the BM-hMSC. With feasibility as secondary end-point, the participating production centres manufactured the BM-hMSC as planned. The ATMP combined to the bioceramic was surgically delivered to the non-unions, and 26/28 treated patients were found radiologically healed at one year (3 out of 4 cortices with bone bridging). INTERPRETATION: Safety and feasibility were clinically proven for surgical implantation of expanded autologous BM-hMSC with bioceramic. FUNDING: EU-FP7-HEALTH-2009, REBORNE Project (GA: 241876).

Polylactic acid fibre-reinforced polycaprolactone scaffolds for bone tissue engineering.

The employment of composite scaffolds with a well-organized architecture and multi-scale porosity certainly represents a valuable approach for achieving a tissue engineered construct to reproduce the middle and long-term behaviour of hierarchically complex tissues such as spongy bone. In this paper, fibre-reinforced composites scaffold for bone tissue engineering applications is described. These are composed of poly-L-lactide acid (PLLA) fibres embedded in a porous poly(epsilon-caprolactone) matrix, and were obtained by synergistic use of phase inversion/particulate leaching technique and filament winding technology. Porosity degree as high as 79.7% was achieved, the bimodal pore size distribution showing peaks at ca 10 and 200 microm diameter, respectively, accounting for 53.7% and 46.3% of the total porosity. In vitro degradation was carried out in PBS and SBF without significant degradation of the scaffold after 35 days, while in NaOH solution, a linear increase of weight lost was observed with preferential degradation of PLLA component. Subsequently, marrow stromal cells (MSC) and human osteoblasts (HOB) reached a plateau at 3 weeks, while at 5 weeks the number of cells was almost the same. Human marrow stromal cell and trabecular osteoblasts rapidly proliferate on the scaffold up to 3 weeks, promoting an oriented migration of bone cells along the fibre arrangement. Moreover, the role of seeded HOB and MSC on composite degradation mechanism was assessed by demonstrating a more relevant contribution to PLLA degradation of MSC when compared to HOB. The novel PCL/PLLA composite scaffolds thus showed promise whenever tuneable porosity, controlled degradability and guided cell-material interaction are simultaneously requested.

New Portland cement-based materials for endodontics mixed with articaine solution: a study of cellular response.

The biocompatibility of innovative tetrasilicate cements proposed for root-end filling restorations was tested. White ProRoot-MTA and AH Plus were used as control. The new cements were mixed with a local anesthetic solution (4% articaine) to form a paste. Human osteoblast-like cells Saos-2 were challenged in short-term cultures (72 hours) with solid materials and with material extracts prepared in culture medium. Cell growth and viability, cellular attachment, and morphologic features were assessed to verify cell/material interactions. No acute toxicity was exerted by the experimental cements in the assay systems. On solid samples Saos-2 adhered and proliferated on all the experimental cements and on MTA. The ultrastructural findings revealed that Saos-2 were able to adhere and to spread. The maintenance of the osteoblastic phenotype on the innovative cements was confirmed by the alkaline phosphatase assay. All experimental cements prepared with articaine supported the growth of bone-like cells, showing suitable properties to be used as canal sealers and root-end filling materials.

Expression of cell adhesion receptors in human osteoblasts cultured on biofunctionalized poly-(epsilon-caprolactone) surfaces.

This study was aimed to investigate whether the activation of poly-(epsilon-caprolactone) (PCL) surface by low-energy irradiation and/or the biofunctionalization by absorption of arginine-glycine-aspartic sequences (RGD), can modify the expression of integrins closely related to the osteoblast activity. For this purpose, we analysed the physicochemical changes induced by irradiation and RGD immobilization, the consequences on cell adhesion and spreading, and the effects on integrin expression. PCL irradiated with 5 x 10(15)He(+)/cm(2) (10 keV energy) (irr-PCL) showed an altered surface layer with a partial loss of carboxyl species and the formation of carbonyl groups. Moreover, irr-PCL showed a small smoothening effect and a less polar character in comparison to the pristine ones. The RGD immobilization was observed only on irr-PCL (surface coverage: 7.0 pmol/cm(2)). Human osteoblasts (hOB) were cultured on untreated PCL (ut-PCL), ut-PCL+RGD, irr-PCL, and irr-PCL+RGD. After 24h, ut-PCL hindered the cell adhesion, while a discrete layer of hOB with a good cytoskeleton organization was detected on irr-PCL and irr-PCL+RGD. Before seeding, the single hOB suspension expressed alpha1, alpha2, alpha3, alpha5, beta1, and alphaVbeta3; after 24h, cells cultured on tissue-plastic expressed high levels of beta1 and alphaVbeta3, while alpha1 showed a low intensity and alpha2, alpha3, and alpha5 were negative. beta1 and alphaVbeta3 were selected to evaluate the interaction between cells and PCL samples. The beta1 expression was higher in hOB cultured on irr-PCL than on the other samples. A significant increase in alphaVbeta3 expression was observed only in irr-PCL+RGD, and confirmed by the gene expression analysis. In conclusion, ion irradiation and RGD adsorption on PCL surfaces modulate the expression of integrin involved in hOB growth and function, indicating the effectiveness of biomimetic surfaces in promoting cell adhesion. Ultimately, the study of integrin expression may suggest proper changes to the surface structure in order to improve the osteoconductivity of selected materials.

Osteoclast differentiation from human blood precursors on biomimetic calcium-phosphate substrates.

The design of synthetic bone grafts to foster bone formation is a challenge in regenerative medicine. Understanding the interaction of bone substitutes with osteoclasts is essential, since osteoclasts not only drive a timely resorption of the biomaterial, but also trigger osteoblast activity. In this study, the adhesion and differentiation of human blood-derived osteoclast precursors (OCP) on two different micro-nanostructured biomimetic hydroxyapatite materials consisting in coarse (HA-C) and fine HA (HA-F) crystals, in comparison with sintered stoichiometric HA (sin-HA, reference material), were investigated. Osteoclasts were induced to differentiate by RANKL-containing supernatant using cell/substrate direct and indirect contact systems, and calcium (Ca++) and phosphorus (P5+) in culture medium were measured. We observed that OCP adhered to the experimental surfaces, and that osteoclast-like cells formed at a rate influenced by the micro- and nano-structure of HA, which also modulate extracellular Ca++. Qualitative differences were found between OCP on biomimetic HA-C and HA-F and their counterparts on plastic and sin-HA. On HA-C and HA-F cells shared typical features of mature osteoclasts, i.e. podosomes, multinuclearity, tartrate acid phosphatase (TRAP)-positive staining, and TRAP5b-enzyme release. However, cells were less in number compared to those on plastic or on sin-HA, and they did not express some specific osteoclast markers. In conclusion, blood-derived OCP are able to attach to biomimetic and sintered HA substrates, but their subsequent fusion and resorptive activity are hampered by surface micro-nano-structure. Indirect cultures suggest that fusion of OCP is sensitive to topography and to extracellular calcium. STATEMENT OF SIGNIFICANCE: The novelty of the paper is the differentiation of human blood-derived osteoclast precursors, instead of mouse-derived macrophages as used in most studies, directly on biomimetic micro-nano structured HA-based surfaces, as triggered by osteoblast-produced factors (RANKL/OPG), and influenced by chemistry and topography of the substrate(s). Biomimetic HA-surfaces, like those obtained in calcium phosphate cements, are very different from the conventional calcium phosphate ceramics, both in terms of topography and ion exchange. The role of these factors in modulating precursors' differentiation and activity is analysed. The system is closely reproducing the physiological process of attachment of host cells and further maturation to osteoclasts toward resorption of the substrate, which occurs in vivo after filling bone defects with the calcium phosphate grafts.

The effect of irradiation modification and RGD sequence adsorption on the response of human osteoblasts to polycaprolactone.

Using techniques of tissue engineering, synthetic substitutes can be applied for the repair and regeneration of damaged bone. It has been found that material surface properties are crucial for cell adhesion and spreading, i.e. cell activities that are related directly to the ability of osteoblasts to proliferate. This fact has promoted the strategy of creating an ECM-like layer onto materials, so as to influence the cell response. In this study human bone-derived osteoblasts have been used to test the effects of surface modification by low energy ion beams of a poly epsilon-caprolactone (PCL) substrate and subsequent RGD adsorption. Osteoblasts were seeded and grown onto untreated and irradiated poly epsilon-caprolactone films, with or without RGD-adsorption step, and viability, morphology, and spreading of the osteoblasts were studied at different time endpoints. Differences were observed in the organization of cytoskeleton within cells: stress fibers were more evident in irradiated samples vs. untreated and total cell adhesion was higher. Surface characterization by X-ray Photoelectron Spectroscopy, Atomic Force Microscopy, and surface free energy measurements showed that the polar character of PCL, i.e., the acid-base term, was increased following irradiation treatment. Moreover the irradiated PCL had a nano-sized topography, which also could improve osteoblasts adhesion. We found that the treatment of the surface with ion beam is per se improving osteoblasts adhesion and spreading onto PCL. Furthermore, also if a significant RGD adsorption was obtained for irradiated PCL surfaces, it was found that in the investigated conditions it seems to have only a minor effect on the cell response. This study suggests that new strategies involving irradiation-based treatments can be adopted to promote the initial steps of bone deposition onto synthetic surfaces, exploiting the surface-induced reorganization of the ECM matrix.

Molecular basis of osteoclastogenesis induced by osteoblasts exposed to wear particles.

In this study, we investigate the molecular mechanisms by which human osteoblasts (HOB) challenged with wear debris promote the differentiation of osteoclast precursors. HOB were obtained from trabecular bone and exposed to alumina (Al(2)O(3)) or 'ultra-high molecular weight polyethylene' (UHMWPE) particles for 24h. The supernatant (HOB-CM) was used for the immunoenzymatic detection of receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG), as well as for inducing the osteoclast differentiation from peripheral blood mononuclear cells (PBMC). The OPG-to-RANKL ratio was significantly decreased in the conditioned medium of UHMWPE-challenged HOB. Morphological and cytochemical analysis showed that HOB-CM induced by itself the osteoclast formation, but a large amount of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive giant cells were obtained when PBMCs were cultured with 1 microg/mL UHMWPE HOB-CM. The expression of genes involved in osteoclast differentiation and activation was evaluated, i.e. c-fms, RANK, c-src, c-fos, cathepsin-K (CATK), TRAP, and calcitonin R (CTR). The UHMWPE HOB-CM increases c-src expression, suggesting that polyethylene debris favour the paracrine activity of HOB in inducing the pathway involved in osteoclast polarization and adhesion. On the contrary, Al(2)O(3) HOB-CM downregulates c-fos expression, suggesting that the passage from macrophages into the osteoclast lineage is deviated. These results show that Al(2)O(3) wear debris is less active than UHMWPE in inducing osteoclast differentiation. Moreover, they provide new insight into the molecular basis of particle-induced osteoclastogenesis, that is the starting point for planning mode-specific targeting of periprosthetic osteolysis.

Effect of four acrylic bone cements on transforming growth factor-beta1 expression by osteoblast-like cells MG63.

Based on the hypothesis that bone cements cause changes in the production of transforming growth factor-beta 1 (TGF-beta1) by bone cells, the effects of four acrylic bone cements (Sulfix-60, CMW 1, CMW 2 and CMW 3) were examined using the osteoblast-like cell line MG63. The extracts in MEM of the cements were tested, following 1 h- and 7 day-curing. MG63 cells seldom expressed mRNA specific for TGF-beta1 in basal conditions. The cultures expressed mRNA constantly after incubation with the extract of CMW 1 cured for 1 h. TGF-beta1 specific mRNA was seldom expressed after incubation with the other cement extracts. The release of TGF-beta1 into the conditioned medium was increased significantly by CMW 1 extract at 1 h-curing, but was not changed significantly by CMW 1 extract at 7 day-curing and by the extracts of the other cements, at both curing times. The stimulating effect of CMW 1 on the secretion of TGF-beta1, even with all the restrictions of an in vitro study of continuous cell lines, if confirmed in vivo, might favor the development of the synovial-like membrane around the implant, and therefore impair the chance of success of the prosthesis.

Thrombomodulin expression in endothelial cells after contact with bone cement.

The expression of thrombomodulin after contact with CMW 1 bone cement extracts was studied in human umbilical vein endothelial cells. Cement extracts after 1 h and 7-day curing induced no significant variations in thrombomodulin antigen levels and in mRNA expression. Significant increase of thrombomodulin was observed when endothelial cells were treated with all-trans retinoic acid (ATRA). ATRA induced the increase of thrombomodulin also in cells incubated with cement extracts. These results suggest that CMW 1 bone cement does not impair the expression of thrombomodulin in endothelial cells.

Effects of bone cement extracts on the cell-mediated immune response.

The aim of the study was to evaluate some aspects of the immunocompatibility of 10 acrylic bone cements. Mononuclear cells harvested from healthy individuals were cultured with cement extracts which were tested to assess their effect on the viability of lymphocytes, unstimulated and phytohaemoagglutinin (PHA)-stimulated, activating resting lymphocytes, and changing the reactivity of PHA-stimulated lymphocytes. After 24 h the extracts did not increase the percentage of dead cells in unstimulated or PHA-stimulated lymphocytes. The early apoptotic events of culture were evaluated after 4 and 24 h in PHA-stimulated lymphocytes: at 4 h three cements, namely Zimmer-dough type, Palacos R and CMW-1, increased significantly the percentage of apoptotic cells, while at 24 h no differences were found. Cement extracts did not activate the resting lymphocytes, whereas the response of the PHA-stimulated cells was significantly modified. All cements decreased the expression of the interleukin 2 receptor (CD25) and the lymphocyte proliferation, whereas only two materials (Zimmer-dough type, CMW 1) affected the expression of early activation antigen (CD69). These findings show that the products released from bone cement are not able, by themselves, to elicit a specific immune response; on the contrary they hamper the function of lymphocytes activated by an exogenous stimulus.

Bone cement extracts modulate the osteoprotegerin/osteoprotegerin-ligand expression in MG63 osteoblast-like cells.

The osteoprotegerin-ligand (OPG-L) has been identified as the essential factor required for osteoclastogenesis, and its effects are prevented by the osteoprotegerin (OPG). The OPG-L/OPG balance plays a crucial role in coordinating the sequence of osteoclast and osteoblast differentiation during the bone remodeling. The aim of the study was to investigate if polymethylmethacrylate-based cements are able to modulate the expression of OPG-L/OPG in MG63 cells, which are known to have high levels of OPG and inducible expression of OPG-L. Four radio-opaque cements. namely Sulfix-60, CMW1, CMW2 and CMW3, were polymerized for either 1 h or 7 d. MG63 were incubated for 24 h with culture medium only, cement extracts and 2 microg/ml of human recombinant IL-1beta as positive control. An RT-PCR was performed to detect the OPG and OPG-L expression, and the house-keeping gene, GAPDH, was used as a reference for the semi-quantitative analysis. An increase in the OPG-L band density was observed for all cements, and consequently, the OPG-L/OPG ratio also increased. The ability of bone cements to induce the expression of OPG-L could be a co-factor in the development of osteolysis at the bone-cement interface.

Immunological changes in patients with primary osteoarthritis of the hip after total joint replacement.

We aimed to assess whether the immunological abnormalities which have been observed in patients with loose total hip replacements (THRs) are present in patients with a well-fixed prosthesis. We examined blood samples from 39 healthy donors, 22 patients before THR and 41 with well-fixed THRs of different types (15 metal-on-metal, 13 metal-on-polyethylene, 13 ceramic-on-ceramic). Before THR, the patients showed a decrease in leukocytes and myeloid cells in comparison with healthy donors, and a prevalence of type-1 T lymphocytes, which was confirmed by the increase in ratio of interferon-gamma to interleukin 4. Moreover, patients with metal-on-metal or metal-on-polyethylene implants showed a significant decrease in the number of T lymphocytes and a significant increase in the serum level of chromium and cobalt, although no significant correlation was observed with the immunological changes. In the ceramic-on-ceramic group, leukocytes and lymphocyte subsets were not significantly changed, but a significant increase in type-2 cytokines restored the ratio of interferon-gamma to interleukin 4 to normal values. We conclude that abnormalities of the cell-mediated immune response may be present in patients with a well-fixed THR, and that the immunological changes are more evident in those who have at least one metal component in the articular coupling.

Development of the foremost light-curable calcium-silicate MTA cement as root-end in oral surgery. Chemical-physical properties, bioactivity and biological behavior.

AIM: An innovative light-curable calcium-silicate cement containing a HEMA-TEGDMA-based resin (lc-MTA) was designed to obtain a bioactive fast setting root-end filling and root repair material. METHODS: lc-MTA was tested for setting time, solubility, water absorption, calcium release, alkalinizing activity (pH of soaking water), bioactivity (apatite-forming ability) and cell growth-proliferation. The apatite-forming ability was investigated by micro-Raman, ATR-FTIR and ESEM/EDX after immersion at 37°C for 1-28 days in DPBS or DMEM+FBS. The marginal adaptation of cement in root-end cavities of extracted teeth was assessed by ESEM/EDX, and the viability of Saos-2 cell on cements was evaluated. RESULTS: lc-MTA demonstrated a rapid setting time (2min), low solubility, high calcium release (150-200ppm) and alkalinizing power (pH 10-12). lc-MTA proved the formation of bone-like apatite spherulites just after 1 day. Apatite precipitates completely filled the interface porosities and created a perfect marginal adaptation. lc-MTA allowed Saos-2 cell viability and growth and no compromising toxicity was exerted. SIGNIFICANCE: HEMA-TEGDMA creates a polymeric network able to stabilize the outer surface of the cement and a hydrophilic matrix permeable enough to allow water absorption. SiO(-)/Si-OH groups from the mineral particles induce heterogeneous nucleation of apatite by sorption of calcium and phosphate ions. Oxygen-containing groups from poly-HEMA-TEGDMA provide additional apatite nucleating sites through the formation of calcium chelates. The strong novelty was that the combination of a hydraulic calcium-silicate powder and a poly-HEMA-TEGDMA hydrophilic resin creates the conditions (calcium release and functional groups able to chelate Ca ions) for a bioactive fast setting light-curable material for clinical applications in dental and maxillofacial surgery. The first and unique/exclusive light-curable calcium-silicate MTA cement for endodontics and root-end application was created, with a potential strong impact on surgical procedures.

Resorbable glass-ceramic phosphate-based scaffolds for bone tissue engineering: synthesis, properties, and in vitro effects on human marrow stromal cells.

Highly porous bioresorbable glass-ceramic scaffolds were prepared via sponge replication method by using an open-cell polyurethane foam as a template and phosphate-based glass powders. The glass, belonging to the P2O5-SiO2-CaO-MgO-Na2O-K2O system, was synthesized by a melting-quenching route, ground, and sieved to obtain powders with a grain size of less than 30 µm. A slurry containing glass powders, polyvinyl alcohol, and water was prepared to coat the polymeric template. The removal of the polymer and the sintering of the glass powders were performed by a thermal treatment, in order to obtain an inorganic replica of the template structure. The structure and properties of the scaffold were investigated from structural, morphological, and mechanical viewpoints by means of X-ray diffraction, scanning electron microscopy, density measurements, image analysis, and compressive tests. The scaffolds exhibited a trabecular architecture that closely mimics the structure of a natural spongy bone. The solubility of the porous structures was assessed by soaking the samples in acellular simulated body fluid (SBF) and Tris-HCl for different time frames and then by assessing the scaffold weight loss. As far as the test in SBF is concerned, the nucleation of hydroxyapatite on the scaffold trabeculae demonstrates the bioactivity of the material. Biological tests were carried out using human bone marrow stromal cells to test the osteoconductivity of the material. The cells adhered to the scaffold struts and were metabolically active; it was found that cell differentiation over proliferation occurred. Therefore, the produced scaffolds, being biocompatible, bioactive, resorbable, and structurally similar to a spongy bone, can be proposed as interesting candidates for bone grafting.