Alberto Smargiassi

Up-regulation of the chemo-attractive receptor ChemR23 and occurrence of apoptosis in human chondrocytes isolated from fractured calcaneal osteochondral fragments

To study the expression level of a panel of pro/anti-apoptotic factors and inflammation-related receptors in chondral fragments from patients undergoing surgical treatment for intra-articular calcaneal fractures, cartilage fragments were retrieved from calcaneal fractures of 20 patients subjected to surgical treatment. Primary cultures were performed using chondral fragments from fractured and control patients. Chondrocyte cultures from each patient of the fractured and control groups were subjected to immunofluorescence staining and quantitatively analyzed under confocal microscopy. Proteins extracted from the cultured chondrocytes taken from the fractured and control groups were processed for Western blot experiments and densitometric analysis. The percentage of apoptotic cells was determined using the cleaved PARP-1 antibody. The proportion of labelled cells was 35% for fractured specimens, compared with 7% for control samples. Quantification of caspase-3 active and Bcl-2 proteins in chondrocyte cultures showed a significant increase of the apoptotic process in fractured specimens compared with control ones. Fractured chondrocytes were positively stained for ChemR23 with statistically significant differences with respect to control samples. Densitometric evaluation of the immunoreactive bands confirmed these observations. Human articular chondrocytes obtained from patients with intra-articular calcaneal fractures express higher levels of pivotal pro-apoptotic factors, and of the chemo-attractive receptor ChemR23, compared with control cultures. On the basis of these observations, the authors hypothesize that consistent prolonged chondrocyte death, associated with the persistence of high levels of pro-inflammatory factors, could enhance the deterioration of cartilage tissue with consequent development of post-traumatic arthritis following intra-articular bone fracture.

Morphological and quantitative analysis of BCL6 expression in human colorectal carcinogenesis

The aim of the present study was to determine whether BCL6 is expressed during malignant transformation of the large bowel and to assess whether, and to what extent, immunoreactivity is related to the different stages of neoplastic progression. Samples of normal colorectal mucosa (n=22), microadenomas (n=22) and colorectal cancer (n=22), were analyzed by immunohistochemistry, immunofluorescence coupled with confocal microscopy and western blotting. Our results clearly outlined the marked increase occurring in both intensity and density of BCL6 protein expression in the normal mucosa-microadenoma-carcinoma sequence. Immunohistochemistry and immunofluorescence analyses showed that BCL6 is expressed at low levels in normal mucosa and increases in microadenoma and in cancer with statistical significance. These results were confirmed by western blotting data. The increasing expression of BCL6 in human colorectal cancer development suggests the involvement of BCL6 in tumor progression, from the earliest stages of carcinogenesis with significant increase in cancer. The enhanced understanding of the biological role of BCL6, previously shown to exert a key role in lymphomagenesis, may lead to a re-evaluation of this protein and may highlight the importance of performing further studies in order to identify novel therapeutic targets for colorectal cancer.

PTH(1-34) effects on repairing experimentally drilled holes in rat femur: novel aspects - qualitative vs. quantitative improvement of osteogenesis.

The timetable of effects on bone repair of the active fraction‐parathyroid hormone, PTH(1‐34), was analytically investigated from the morphometric viewpoint in 3‐month‐old male Sprague‐Dawley rats, whose femurs were drilled at mid‐diaphyseal level (transcortical holes). The animals were divided into groups with/without PTH(1‐34) administration, and sacrificed at different times (10, 28, 45 days after surgery). The observations reported here need to be framed in the context of our previous investigations regarding bone histogenesis (Ferretti et al. Anat Embryol. 2002; 206: 21–29) in which we demonstrated the occurrence of two successive bone‐forming processes during both skeletal organogenesis and bone repair, i.e. static and dynamic osteogenesis: the former (due to stationary osteoblasts, haphazardly grouped in cords) producing preliminary bad quality trabecular bone, the latter (due to typical polarized osteoblasts organized in ordered movable laminae) producing mechanically valid bone tissue. The primary function of static osteogenesis is to provide a rigid scaffold containing osteocytes (i.e. mechano‐sensors) for osteoblast laminae acting in dynamic osteogenesis. In the present work, histomorphometric analysis revealed that, already 10 days after drilling, despite the holes being temporarily filled by the same amount of newly formed trabecular bone by static osteogenesis independently of the treatment, the extent of the surface of movable osteoblast‐laminae (covering the trabecular surface) was statistically higher in animals submitted to PTH(1‐34) administration than in control ones; this datum strongly suggests the effect of PTH(1‐34) alone in anticipating the occurrence of dynamic osteogenesis involved in the production of good quality bone (with more ordered collagen texture) more suitable for loading. This study could be crucial in further translational clinical research in humans for defining the best therapeutic strategies to be applied in recovering severe skeletal lesions, particularly as regards the time of PTH(1‐34) administration.

Biocompatibility Analyses of Al2O3-Treated Titanium Plates Tested with Osteocyte and Fibroblast Cell Lines

Osseointegration of a titanium implant is still an issue in dental/orthopedic implants durable over time. The good integration of these implants is mainly due to their surface and topography. We obtained an innovative titanium surface by shooting different-in-size particles of Al2O3 against the titanium scaffolds which seems to be ideal for bone integration. To corroborate that, we used two different cell lines: MLO-Y4 (murine osteocytes) and 293 (human fibroblasts) and tested the titanium scaffolds untreated and treated (i.e., Al2O3 shot-peened titanium surfaces). Distribution, density, and expression of adhesion molecules (fibronectin and vitronectin) were evaluated under scanning electron microscope (SEM) and confocal microscope (CM). DAPI and fluorochrome-conjugated antibodies were used to highlight nuclei, fibronectin, and vitronectin, under CM; cell distribution was analyzed after gold-palladium sputtering of samples by SEM. The engineered biomaterial surfaces showed under SEM irregular morphology displaying variously-shaped spicules. Both SEM and CM observations showed better outcome in terms of cell adhesion and distribution in treated titanium surfaces with respect to the untreated ones. The results obtained clearly showed that this kind of surface-treated titanium, used to manufacture devices for dental implantology: (i) is very suitable for cell colonization, essential prerequisite for the best osseointegration, and (ii) represents an excellent solution for the development of further engineered implants with the target to obtain recovery of stable dental function over time.

Morphological study: Ultrastructural aspects of articular cartilage and sub-chondral bone in patients affected by post-traumatic shoulder instability.

Post‐traumatic shoulder instability is a frequent condition in active population, representing one of most disabling pathologies, due to altered balance involving joints. No data are so far available on early ultrastructural osteo‐chondral damages, associated with the onset of invalidating pathologies, like osteoarthritis‐OA. Biopsies of glenoid articular cartilage and sub‐chondral bone were taken from 10 adult patients underwent arthroscopic stabilization. Observations were performed under Transmission Electron Microscopy‐TEM in tangential, arcuate and radial layers of the articular cartilage and in the sub‐chondral bone. In tangential and arcuate layers chondrocytes display normal and very well preserved ultrastructure, probably due to the synovial liquid supply; otherwise, throughout the radial layer (un‐calcified and calcified) chondrocytes show various degrees of degeneration; occasionally, in the radial layer evidences of apoptosis/autophagy were also observed. Concerning sub‐chondral bone, osteocytes next to the calcified cartilage also show signs of degeneration, while osteocytes farther from the osteo‐chondral border display normal ultrastructure, probably due to the bone vascular supply. The ultrastructural features of the osteo‐chondral complex are not age‐dependent. This study represents the first complete ultrastructural investigation of the articular osteo‐chondral complex in shoulder instability, evaluating the state of preservation/viability of both chondrocytes and osteocytes throughout the successive layers of articular cartilage and sub‐chondral bone. Preliminary observations here collected represent the morphological basis for further deepening of pathogenesis related to shoulder instability, enhancing the relationship between cell shape and microenvironment; in particular, they could be useful in understanding if the early surgical treatment in shoulder instability could avoid the onset of OA. Anat Rec, 300:1208–1218, 2017.

Preliminary observations on scleral ossicles in performing functionalized 3D vascularized scaffolds for “critical-size” bone defect healing

The problem of “critical-size” bone defects occurs when a severe lesion is difficult to be self-recovered. Many strategies of regenerative medicine were used in the last decade, with translational approaches, to mimic both structure and function of the native bone tissue, making use of synthetic materials, nanotechnologies, bio/synthetic constructs or some of their combination. The main obstacle to engineering strategies is mostly due to the lack of a proper vascularization of the construct used. In this feasibility study, our attention is directed towards the main tissue engineering items: scaffolds, cells and conditioning factors. We propose the use of scleral ossicles of lower vertebrates (1), as natural scaffolds which will be functionalized to allow the best adhesion of endothelial cells along a geometrically controlled pattern on the bony surface of the construct; successively, on the functionalized scaffold, osteogenic cell lines will be cultured. In the preliminary phases of the study, the ossicles were scratched to remove soft tissue residues, variously flattened with different methods to reach a regular morphology on both sides, and finally autoclaved to eliminate cellular remnants and to annul antigenic properties. Ossicles were observed under SEM and subjected to micro-assay, to establish the best scaffold preparation and to characterize morphological properties more suitable for engineering phases. Functionalization will be made by immobilizing on the engineered ossicles specific growth factors for endothelial cells; later, mouse primary lung endothelial cells (ECs) and immortalized osteogenic cells (IDG-SW3) will be used. As expected results ECs should adhere to the ossicle surface and organize to form lumenized microvascular-like structures; later, supported by the vascular-like network, the osteogenic lineage should produce bone matrix on the construct. The production of newly-formed bone around vascular-like buds will be verify. 3D tissue constructs generated in vitro will be used in successive in vivo study for the healing of “critical-size” bone defects experimentally induced in mice.

Expression and functional proteomic analyses of osteocytes from Xenopus laevis tested under mechanical stress conditions: preliminary observations on an appropriate new animal model

Hitherto, the role of the osteocyte as transducer of mechanical stimuli into biological signals is far from settled. In this study, we used an appropriate model represented by the cortex of Xenopus laevis long bone diaphysis lacking (unlike the mammalian one) of vascular structures and containing only osteocytes inside the bone matrix. These structural features allow any change of protein profile that might be observed upon different experimental conditions, such as bone adaptation to stress/mechanical loading, to be ascribed specifically to osteocytes. The study was conducted by combining ultrastructural observations and two‐dimensional electrophoresis for proteomic analysis. The osteocyte population was extracted from long bones of lower limbs of amphibian skeletons after different protocols (free and forced swimming). The experiments were performed on 210 frogs subdivided into five trials, each including free swimming frogs (controls) and frogs submitted to forced swimming (stressed). The stressed groups were obliged to swim (on movable spheres covering the bottom of a pool on a vibrating plate) continuously for 8 h, and killed 24 h later along with the control groups. Long bones free of soft tissues (periosteum, endosteum and bone marrow), as well as muscles of posterior limbs, were processed and analyzed for proteins differentially expressed or phosphorylated between the two sample groups. The comparative analysis showed that protein phosphorylation profiles differ between control and stressed groups. In particular, we found in long bones of stressed samples that both Erk1/2 and Akt are hyperphosphorylated; moreover, the different phosphorylation of putative Akt substrates (recognized by specific Akt phosphosubstrates‐antibody) in stressed vs. control samples clearly demonstrated that Akt signaling is boosted by forced swimming (leading to an increase of mechanical stress) of amphibian long bones. In parallel, we found in posterior limb muscles that the expression of heat shock protein HSP27 and HSP70 stress markers increased upon the forced swimming condition. Because the cortexes of frog long bones are characterized by the presence of only osteocytes, all our results establish the suitability of the X. laevis animal model to study the bone response to stress conditions mediated by this cell type and pave the way for further analysis of the signaling pathways involved in these signal transduction mechanisms.

Osteocytes signaling events induced by intermittent vs continuous Teriparatide treatment affect in vitro osteoblast differentiation and mineralization

PTH(1-34), also known as Teriparatide, is an active anabolic drug used in the treatment of some forms of osteoporosis and occasionally exploited to speed fracture healing. The effect of such therapies are dependent on the type of administration, in fact it has been largely demonstrated that a short administration of Teriparatide (also called intermittent) increases the bone mass, meanwhile a long administration of the same agent (known as continuous) leads to an increased resorption. The molecular reason why the type of administration is so critical for the fate of the bone remodeling is still largely unknown but it is probably due to the fact that it affects several signaling pathways and alters the biological activity of a cohort of cells: osteoblasts, lining cells, osteoclasts, and osteocytes. In the present work, we firstly focused the attention on molecular events induced by intermittent vs continuous Teriparatide treatment in a well-known osteocytes in vitro model, the MLO-Y4 cells. By the use of a gene array platform, we found many molecules upregulated or downregulated depending on the the temporal administration modes, suggesting that the drug affects in diverse manner the osteocytes related signaling pathways. In particular, we paid attention to Wisp-2, a protein of the Wnt pathway that has been demonstrated to be able to interact and influence the differentiation of osteoblasts into osteocytes and their mineralization. Secondly, through the mineralization assay, we analyzed the functional effects, involving the differentiation of osteoblast IDG-SW3 cell line, upon the conditioning culture with MLO-Y4 medium, that were pre-treated with short and long time administration of Teriparatide. These findings, consistent with the crucial role performed by osteocytes on osteoblast differentiation, clarify the molecular events downstream the short treatment with Teriparatide, suggesting that the perturbation of certain signaling patwhays, such as the Wnt pathway, is crucial for the positive regulation of bone formation.

Proposal of a Novel Natural Biomaterial, the Scleral Ossicle, for the Development of Vascularized Bone Tissue In Vitro

Recovering of significant skeletal defects could be partially abortive due to the perturbations that affect the regenerative process when defects reach a critical size, thus resulting in a non-healed bone. The current standard treatments include allografting, autografting, and other bone implant techniques. However, although they are commonly used in orthopedic surgery, these treatments have some limitations concerning their costs and their side effects such as potential infections or malunions. On this account, the need for suitable constructs to fill the gap in wide fractures is still urgent. As an innovative solution, scleral ossicles (SOs) can be put forward as natural scaffolds for bone repair. SOs are peculiar bony plates forming a ring at the scleral-corneal border of the eyeball of lower vertebrates. In the preliminary phases of the study, these ossicles were structurally and functionally characterized. The morphological characterization was performed by SEM analysis, MicroCT analysis and optical profilometry. Then, UV sterilization was carried out to obtain a clean support, without neither contaminations nor modifications of the bone architecture. Subsequently, the SO biocompatibility was tested in culture with different cell lines, focusing the attention to the differentiation capability of endothelial and osteoblastic cells on the SO surface. The results obtained by the above mentioned analysis strongly suggest that SOs can be used as bio-scaffolds for functionalization processes, useful in regenerative medicine.

Effects of PTH(1-34) during fracture healing after experimental bone drilling in rat femur: novel aspects

The study concerns the role of PTH(1-34) during bone lesion repair. 3-month-old male Sprague-Dawley rats, in which trans-cortical holes were drilled at femur mid-diaphysis, were divided in groups with/without Teriparatide administration (40𝜇g/ Kg/day), and sacrificed at different times (10, 28, 45 days). In 2002 (1) we demonstrated the occurrence of two successive bone forming processes during both skeletal organogenesis and bone repair, i.e. static (SO) and dynamic (DO) osteogenesis: the former (due to stationary osteoblasts, haphazardly grouped in cords) producing pre- liminary bad quality trabecular bone, the latter (due to typical polarized osteoblasts organized in ordered movable laminae) producing mechanically valid bone tissue. In brief, the primary function of SO is to provide a rigid scaffold, containing osteocytes (i.e. mechano-sensors), to DO-osteoblastic laminae; therefore, in DO mechanical factors can play a crucial role in transduction of mechanical stresses into biological signals. In the present work, histomorphometric analysis showed that, already after 10 days from drilling, notwithstanding the holes are temporarily filled by the same amount of newly-formed trabecular bone (produced by SO) independently from the treatment, the number of movable osteoblast laminae (typical of DO), covering the trabecular surface, is statistically higher in animals submitted to PTH(1-34) administration than in the control ones; this suggests that the mere effect of Teriparatide is to anticipate the occurrence of dynamic osteogenesis involved in the production of good quality bone more suitable to loading. These findings are also supported by the higher values of microhardness as well as the more ordered-fibered texture (observed by polarized light) in treated animals with respect to control ones that strongly indi- cates the qualitative (instead of quantitative) effect of PTH (1-34) in improving bone healing. The present investigation could be of crucial importance in further translational clinical research in humans to define the best therapeutic strategies in recovering skeletal lesions, particularly in terms of time of administration of PTH(1-34).