Composite Scaffolds for Bone Tissue Regeneration Based on PCL and Mg-Containing Bioactive Glasses.

Polycaprolactone (PCL) is widely used in additive manufacturing for the construction of scaffolds for tissue engineering because of its good bioresorbability, biocompatibility, and processability. Nevertheless, its use is limited by its inadequate mechanical support, slow degradation rate and the lack of bioactivity and ability to induce cell adhesion and, thus, bone tissue regeneration. In this study, we fabricated 3D PCL scaffolds reinforced with a novel Mg-doped bioactive glass (Mg-BG) characterized by good mechanical properties and biological reactivity. An optimization of the printing parameters and scaffold fabrication was performed; furthermore, an extensive microtopography characterization by scanning electron microscopy and atomic force microscopy was carried out. Nano-indentation tests accounted for the mechanical properties of the scaffolds, whereas SBF tests and cytotoxicity tests using human bone-marrow-derived mesenchymal stem cells (BM-MSCs) were performed to evaluate the bioactivity and in vitro viability. Our results showed that a 50/50 wt% of the polymer-to-glass ratio provides scaffolds with a dense and homogeneous distribution of Mg-BG particles at the surface and roughness twice that of pure PCL scaffolds. Compared to pure PCL (hardness H = 35 ± 2 MPa and Young's elastic modulus E = 0.80 ± 0.05 GPa), the 50/50 wt% formulation showed H = 52 ± 11 MPa and E = 2.0 ± 0.2 GPa, hence, it was close to those of trabecular bone. The high level of biocompatibility, bioactivity, and cell adhesion encourages the use of the composite PCL/Mg-BG scaffolds in promoting cell viability and supporting mechanical loading in the host trabecular bone.

Nanostructure and biomimetics orchestrate mesenchymal stromal cell differentiation: An in vitro bioactivity study on new coatings for orthopedic applications.

The choice of the appropriate material having suitable compositional and morphological surface characteristics, is a crucial step in the development of orthopedic implants. The purpose of this paper is to elucidate, on this regard, the influence of two important hits, i.e., biogenic apatite with bone-like composition and nanostructured morphology, providing the evidence of the efficacy of nanostructured biogenic apatite coatings in favoring adhesion, growth, proliferation, and in vitro osteogenic differentiation of human mesenchymal stromal cells (hMSCs) isolated from the bone marrow. The specific features of this coating in terms of topographical and biochemical cues, obtained by Ionized Jet Deposition, are perceived by hMSCs, as suggested by changes in different morphologic parameters as Aspect Ratio or Elongation index, suggesting the impact exerted by the nanostructure on early adhesion events, cytoskeleton organization, and cells fate. In addition, the nanostructured CaP coating sustained the metabolic activity of the cells and facilitated the osteogenic differentiation of MSC by supporting the osteogenesis-related gene expression. These findings support the use of a combined approach between technological advancement and instructive surfaces, both from the topographical and the biochemical point of view, in order to manufacture smart biomaterials able to respond to different needs of the orthopedic practice.

Lamin A and Prelamin A Counteract Migration of Osteosarcoma Cells.

A type lamins are fundamental components of the nuclear lamina. Changes in lamin A expression correlate with malignant transformation in several cancers. However, the role of lamin A has not been explored in osteosarcoma (OS). Here, we wanted to investigate the role of lamin A in normal osteoblasts (OBs) and OS cells. Thus, we studied the expression of lamin A/C in OS cells compared to OBs and evaluated the effects of lamin A overexpression in OS cell lines. We show that, while lamin A expression increases during osteoblast differentiation, all examined OS cell lines express lower lamin A levels relative to differentiated OBs. The condition of low LMNA expression confers to OS cells a significant increase in migration potential, while overexpression of lamin A reduces migration ability of OS cells. Moreover, overexpression of unprocessable prelamin A also reduces cell migration. In agreement with the latter finding, OS cells which accumulate the highest prelamin A levels upon inhibition of lamin A maturation by statins, had significantly reduced migration ability. Importantly, OS cells subjected to statin treatment underwent apoptotic cell death in a RAS-independent, lamin A-dependent manner. Our results show that pro-apoptotic effects of statins and statin inhibitory effect on OS cell migration are comparable to those obtained by prelamin A accumulation and further suggest that modulation of lamin A expression and post-translational processing can be a tool to decrease migration potential in OS cells.

Fabrication and characterization of biomimetic hydroxyapatite thin films for bone implants by direct ablation of a biogenic source.

Biomimetic bone apatite coatings were realized for the first time by the novel Ionized Jet Deposition technique. Bone coatings were deposited on titanium alloy substrates by pulsed electron ablation of deproteinized bovine bone shafts in order to resemble bone apatite as closely as possible. The composition, morphology and mechanical properties of the coatings were characterized by GI-XRD, FT-IR, SEM-EDS, AFM, contact angle measurements, micro-scratch and screw-insertion tests. Different post-treatment annealing conditions (from 350 °C to 425 °C) were investigated. Bone apatite coatings exhibited a nanostructured surface morphology and a composition closely resembling that of the deposition target (i.e. natural bone apatite), also regarding the presence of magnesium and sodium ions. Crystallinity and composition of the coatings were strongly influenced by annealing temperature and duration; in particular, upon annealing at 400 °C and above, a crystallinity similar to that of bone was achieved. Finally, adhesion to the titanium substrate and hydrophilicity were significantly enhanced upon annealing, all characteristics being known to have a strong positive impact on promoting host cells attachment, proliferation and differentiation.

Magnetic forces and magnetized biomaterials provide dynamic flux information during bone regeneration.

The fascinating prospect to direct tissue regeneration by magnetic activation has been recently explored. In this study we investigate the possibility to boost bone regeneration in an experimental defect in rabbit femoral condyle by combining static magnetic fields and magnetic biomaterials. NdFeB permanent magnets are implanted close to biomimetic collagen/hydroxyapatite resorbable scaffolds magnetized according to two different protocols . Permanent magnet only or non-magnetic scaffolds are used as controls. Bone tissue regeneration is evaluated at 12 weeks from surgery from a histological, histomorphometric and biomechanical point of view. The reorganization of the magnetized collagen fibers under the effect of the static magnetic field generated by the permanent magnet produces a highly-peculiar bone pattern, with highly-interconnected trabeculae orthogonally oriented with respect to the magnetic field lines. In contrast, only partial defect healing is achieved within the control groups. We ascribe the peculiar bone regeneration to the transfer of micro-environmental information, mediated by collagen fibrils magnetized by magnetic nanoparticles, under the effect of the static magnetic field. These results open new perspectives on the possibility to improve implant fixation and control the morphology and maturity of regenerated bone providing "in site" forces by synergically combining static magnetic fields and biomaterials.

MRMT-1 rat breast carcinoma cells and models of bone metastases: improvement of an in vitro system to mimic the in vivo condition.

Breast cancer frequently metastasizes to the skeleton thus interrupting the normal bone remodeling process and causing bone degradation. Having suitable in vitro and in vivo models is important for understanding the pathogenesis and developing treatment strategies for bone metastasis in humans. In order to improve and characterize an in vitro model of bone metastasis from breast cancer an MRMT-1 rat breast carcinoma cell line or their conditioned medium were directly co-cultured with rat monocytes. To confirm the in vitro results, an in vivo model, in which MRMT-1 cells were inoculated into the proximal surface of the tibia, was also adopted. Osteoclast viability, activity and differentiation showed a significant increase (p<0.05, p<0.0005, p<0.0005, respectively) between co-culture with MRMT-1 cells and the other culture conditions. Moreover, scanning electron microscopy analysis, phalloidin staining and 4'-6-diamidino-2-phenylindole (DAPI) nuclear acid staining confirmed that co-culture with MRMT-1 cells also induced a greater differentiation in osteoclast structure and morphology. Finally, the in vivo outcome at 3 weeks showed the presence of a severe osteolytic lesion, thus confirming the effectiveness of the present in vitro results. These results demonstrated an improvement of an in vitro model of bone metastases from breast cancer in which co-culture with MRMT-1 cells was shown to be a dynamic system that closely mimics the in vivo situation. The present study may help improve our understanding of the complex "vicious cycle" between osteoblasts, osteoclasts and tumor cells.

Pulsed plasma deposition of zirconia thin films on UHMWPE: proof of concept of a novel approach for joint prosthetic implants.

Wear of ultra-high molecular weight polyethylene (UHMWPE) has been recognized as the main cause for long-term revision in joint arthroplasty. A new approach to overcome this detrimental issue is here presented: zirconia (ZrO2) thin films were directly deposited onto the surface of UHMWPE by Pulsed Plasma Deposition (PPD) technique. The obtained films were structurally, morphologically and mechanically characterized by X-ray diffraction, scanning electron microscopy and nanoindentation tests, respectively. The critical fracture load was estimated by the analysis of the indenter footprints, while the adhesion degree was evaluated by a cross-cut tape test. Zirconia films exhibited a fully cubic structure, with densely packed grains, whereas mechanical tests showed that hard, tough and well-adherent films were deposited. These preliminary results suggested the feasibility of pursuing this alternative route to improve UHMPWE performances while preserving its well-established mechanical properties.

Ultraviolet B (UVB) irradiation-induced apoptosis in various cell lineages in vitro.

Ultraviolet B (UVB) radiation acts as a strong apoptotic trigger in many cell types, in tumor and normal cells. Several studies have demonstrated that UVB-induced cell death occurs through the generation of reactive oxygen species. The consequent oxidative stress includes the impairment of cellular antioxidants, the induction of DNA damage and the occurrence of apoptosis. In this review, we investigated UVB apoptotic action in various cell models by using ultrastructural, molecular and cytofluorimetric techniques. Myeloid leukemia HL-60, T-lymphoblastoid Molt-4 and myelomonocytic U937 human cells, generally affected by apoptotic stimuli, were studied. Human chondrocytes and C2C12 skeletal muscle cells, known to be more resistant to damage, were also considered. All of them, when exposed to UVB radiation, revealed a number of characteristic apoptotic markers. Membrane blebbing, cytoplasm shrinkage and chromatin condensation were detected by means of electron microscopy. DNA cleavage, investigated by using agarose gel electrophoresis and TUNEL reaction, was observed in suspended cells. Differently, in chondrocytes and in skeletal muscle cells, oligonucleosomic DNA fragmentation did not appear, even if a certain TUNEL positivity was detected. These findings demonstrate that UVB radiation appears to be an ideal tool to study the apoptotic behavior.

New PMMA-based composites for preparing spacer devices in prosthetic infections.

Even though the systemic antibiotic therapy is usually applied after prosthetic infections surgical treatments, it is unable to reach the infection site in sufficient concentrations to eradicate bacteria. Delivering antibiotics locally with the use of custom made device (spacer or nail coating) might eradicate or reduce the infection and the risk of recolonization, providing a very high concentration of antibiotic. PMMA-based (Mendec Spine) composites with BaSO(4) were enriched with ß-tricalcium phosphate (Porosectan-TCP) or only a slightly higher BaSO(4) concentration (Porosectan-BaSO(4)) to obtain higher porosity. The aim of the study was to evaluate: (i) drug absorption capability and drug release kinetics in vitro soaking them with a combined solution of gentamicin and vancomycin, (ii) their in vitro and in vivo biocompatibility, and finally, (iii) they were tested preliminarily in an experimental model of bone infection. The simultaneous presence of ß-TCP and BaSO(4) resulted in the formation of a texture of interconnecting channels with different diameters, from a few microns to several hundred microns, which totally filled the material. The porosity, determined by microcomputed tomography, was significantly higher in both tested plain composites (Porosectan-TCP: +17.3%; Porosectan-BaSO(4): +7.5%) in comparison to control composite material (Mendec Spine). The kinetics of antibiotic release from composites was rapid and complete, producing high drug concentrations for a short period of time. Both composites showed a good level of biocompatibility. The osteomyelitic model confirmed that both composites, soaked in antibiotic solution, were able to cure bone infection. These composites could be useful for preparing devices for prosthetic joint infections treatment also allowing the use of antibiotics solution at required concentrations.

Lights and shadows concerning platelet products for musculoskeletal regeneration.

Various types of platelet (PLT) products, such as Platelet Rich Plasma (PRP) and Platelet Gel (PG), derived from autologous peripheral blood, have been used for tissue repair. The good clinical outcomes, due mainly to their safety and Growth Factor (GF) content, have led to a wide use of PLT products in many fields of medicine. However, until now the existing literature adds controversies to the use of PLT concentrates. When talking about PLTs and their products, a great number of variables have to be considered. These variables are mainly related to PRP preparation methods, the type of activators, intra- and inter-species variability, types of pathology to be treated, the ways and times of administration and the association of PRP or PG with other treatments. This review considers and discusses these causes of variability with particular attention to orthopaedic implications. The possibility of improving the knowledge on variables affecting therapeutic efficacy will surely help in addressing the best combination of factors implied in the different steps of PLT concentrate preparation and use.

Outcome of 47 consecutive sinus lift operations using aragonitic calcium carbonate associated with autologous platelet-rich plasma: clinical, histologic, and histomorphometrical evaluations.

BACKGROUND: The reconstruction of the maxillary bone frequently represents a real challenge for maxillofacial surgeons especially regarding the best choice of a suitable material to produce the required bone augmentation. AIM: In this study, we summarize our clinical experience on 47 sinus lifts with lateral approach using a mixture of aragonitic calcium carbonate and autologous platelet-rich plasma compared with that of a previous published study in which bovine bone (LADDEC) and autologous bone were used in 50 sinus lift operations (Br J Oral Maxillofac Surg 2005;43:309-313). MATERIALS AND METHODS: We subjected 34 patients to sinus lift operation, for a total of 47 sinus lifts, using natural coral as osteoconductive material. This material, combined with autologous platelet-rich plasma, was placed onto the maxillary sinus floor, after carefully lifting the endosteum. Cases were clinically, radiographically, and histologically analyzed. Histomorphometrical analysis, tests of microhardness, and x-ray microanalysis were conducted comparing the various sample to controls obtained from the same patients. RESULTS AND CONCLUSIONS: Histomorphometrical analysis, microhardness test, and x-ray microanalysis demonstrated that the newly formed bone showed morphologic and structural characteristics that were similar for all the grafting materials compared (bovine bone, autologous bone, and coral). Although all the grafting materials did yield good results of maturation of the newly formed bone, best results were achieved using autologous bone.

Outcome of 50 consecutive sinus lift operations.

We report 50 patients who had sinus lift operations between 1995 and 1999 using different grafting materials [autologous bone, heterologous bone, and hydroxyapatite]. The casenote were analysed clinically (intraoperative and postoperative results and complications), radiographically (amount of augmented bone 6 months postoperatively), and histologically (quality of the new bone 12 months postoperatively). Our aim was to find out if the various bone grafting materials used resulted in different outcomes after adjustment for significant predictors. We found that hydroxyapatite was associated with the lowest rate of loss of grafted material (median, 2.4 mm) followed by the autologous bone from iliac crest (2.8 mm). Bovine bone shown the highest loss (3.3 mm).

Leiomyosarcoma of the spermatic cord: a light and ultrastructural description of one case.

A case of leiomyosarcoma arising in the spermatic cord is described. A 83-year-old man required medical care for an irreducible inguinal hernia. The patient underwent herniorraphy and transinguinal radical orchiectomy. Macroscopically, the spermatic cord was enlarged by a gray-tan and ill-defined neoplasm measuring 4 x 4 x 3 cm. Histologically, this proliferation was composed of atypical spindle cells with blunted end nuclei and eosinophilic cytoplasm. Immunohistochemistry and ultrastructure confirmed the smooth muscle nature of the neoplastic cells. The diagnosis of leiomyosarcoma of the spermatic cord was made. To improve the assignment of this rare lesion to its specific anatomic location, we analyzed the immunohistochemical and ultrastructural characteristics of the smooth muscle tumoral cells and in particular those of the intracellular filament aggregates.

In vitro behaviour of bone marrow-derived mesenchymal cells cultured on fluorohydroxyapatite-coated substrata with different roughness.

Among ceramic materials, recent interest has been risen on fluorinated hydroxyapatites, which undergo slow in vivo degradation and offer a more stable interface for osseointegration and bone fixation. Apart from the information available on the chemistry, little is known of their biological properties and, more specifically, of their interaction with bone cells. The aim of the present study has been to investigate the behaviour and differentiation of human bone marrow-derived mesenchymal cells cultured on two substrata consisting of fluorohydroxyapatite (FHA)-coated titanium and characterised by different surface roughness. To this end, osteoprogenitor cells were seeded on the test materials and, once adhered, were induced to differentiate to osteoblastic cells. The cell behaviour on the different surfaces was monitored for a period of up to 2 weeks. The results obtained indicate that FHAs are cytocompatible materials, which allow the adhesion and growth of osteogenic cells. Mesenchymal cells promptly adhered, covering the surface of the test materials, and subsequently expressed some typical markers of osteoblasts. No significant difference in alkaline phosphatase specific activity was observed when comparing the two test material surfaces to plastic control. At 7 days the number of adhered cells and the presence in the medium of C-terminal propeptide of type I collagen appeared lower or slightly lower for cultures on FHA substrata than on plastic control. These differences tended to subside with time.