Elisa Leonardi

Growth factors in bone repair

The role of growth factors (GF) in bone repair is widely recognised, particularly for bone morphogenetic proteins (BMPs), fibroblast growth factor (FGF), insulin-like growth factors (IGFs), platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β) and vascular endothelial growth factor (VEGF). GF are usually stored in the extracellular matrix (ECM), but after injury are actively released by ECM, cells and platelets. In this paper, the use of different recombinant GF for bone repair stimulation is summarised in experimental research and clinical applications. Drug delivery systems, including carriers, cell or gene therapy, are needed to ensure a sustained local release of the factors, but efficacy and potential side effects of such systems require additional research prior to clinical applications. Current sources for delivery of a GF mixture into the site of bone repair are platelet gel and demineralised bone matrix. Nevertheless, the levels of GF in such preparations are affected by variability among donors and differences in preparation. Autogenous GF, produced by the patient himself during the bone repair process, potentially interfere with prosthetic devices or even have a role in implant loosening due to the periprosthetic tissue reaction. In conclusion, GF are key components of functional bone regeneration: screening of basic research results and controlled clinical trials are accelerating the development of GF in orthopaedic surgery.

Response of human bone marrow stromal cells to a resorbable P2O5–SiO2–CaO–MgO–Na2O–K2O phosphate glass ceramic for tissue engineering applications

This work focuses on the synthesis and characterization of a novel bioresorbable glass ceramic phosphate-based material (GC-ICEL). More specifically, its solubility in different aqueous media (water, Tris-HCl and acellular simulated body fluid) and the response of human stromal cells cultured on it were investigated. X-ray diffraction analysis showed the presence of two crystalline phases identified as Na(2)Mg(PO(4))(3) and Ca(2)P(2)O(7) and dissolution tests highlighted a preferential dissolution of the Na(2)Mg(PO(4))(3) phase and of the residual amorphous phase in all the chosen media. Soaking tests in simulated body fluid showed precipitation of a hydroxyapatite layer, demonstrating the bioactivity of GC-ICEL, which is partially due to the reported bioactivity of Ca(2)P(2)O(7). The effect of GC-ICEL on adhesion, proliferation and osteoblastic gene expression of human bone marrow-derived stromal cells was also studied. Combining molecular and biochemical analyses, it was found that bone marrow cell differentiation was stimulated over proliferation on GC-ICEL. Moreover, the expression of bone-related genes in cells cultured on GC-ICEL confirmed the bioactivity of this phosphate-based glass ceramic, which might have a stimulatory effect on osteogenesis.

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.

Galectin-3 expression in pituitary adenomas as a marker of aggressive behavior

The purpose of this retrospective study was to investigate the role of galectin-3 (LGALS3) expression in predicting the recurrence and the progression potential of prolactin (PRL) and adrenocorticotropic hormone (ACTH)-producing pituitary adenomas and its correlation with the RUNX1 and RUNX2 transcription factors involved in the regulation mechanism of LGALS3 expression. Clinical, neuroradiologic, and follow-up data from 92 pituitary adenomas, including 59 PRL cell adenomas and 33 ACTH-functioning pituitary adenomas, were collected. The LGALS3 expression was analyzed by both immunohistochemistry and quantitative real time-polymerase chain reaction, whereas RUNX1 and RUNX2 were analyzed by quantitative real time-polymerase chain reaction only. The data obtained indicated that invasive growth with suprasellar extension, Ki-67 labeling index, and LGALS3 immunohistochemical and/or LGALS3 messenger RNA levels are the most important histologic features for assessing a high risk of progression or recurrence of PRL- and ACTH-functioning pituitary adenomas. Multivariate Cox regression analysis assessed LGALS3 immunohistochemical positivity in at least 30% of neoplastic cells and/or LGALS3 messenger RNA positivity (P < .001) as strong predictive factors of recurrence/tumor progression followed by a Ki-67 labeling index greater than 3% (P = .019) in the 81 cases in which follow-up data were available. In addition, a significant correlation between LGALS3 and RUNX1 expression levels (P = .0435) was found. This retrospective immunohistochemical and molecular study demonstrated that LGALS3 expression appeared to be a predictive factor of the aggressive behavior of PRL- and ACTH-functioning pituitary adenomas, and its expression was correlated with RUNX1 expression levels.

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.

Effects of osteogenic differentiation inducers on in vitro expanded adult mesenchymal stromal cells

Purpose For bone regeneration therapy using stem cells, well-defined ex vivo protocols to expand mesenchymal stromal cells (MSC), as well as assays to show their potential differentiation into the osteogenic lineage, are needed. Aim of this study was to analyze the role of the biochemical osteogenic inducers, i.e. ascorbic acid, dexamethasone, and β-glycerophosphate, employed in the current protocols for osteogenic differentiation of MSC in vitro, to address the requirements for reliable differentiation systems. Methods MSC were isolated from the bone marrow of donors (46–73 years of age) undergoing total hip replacement, and expanded in vitro. At confluence, MSC were cultured under four different conditions: α-MEM plus serum (basal medium or C1), basal medium plus ascorbate (C2), basal medium plus ascorbate and dexamethasone (C3), or basal medium plus ascorbate, dexamethasone and β-glycerophosphate (C4). Morphology, proliferation, mineralization, alkaline phosphatase, collagen and expression of bone-related genes of MSC under the different media were analyzed at fixed time points. Results MSC proliferation and the number of colony forming units were increased by ascorbic acid, whereas dexamethasone enhanced the proportion of ALP-positive CFU and was critical for mineral deposition. Runx-2 and type I collagen gene expression decreased along with additive-induced MSC differentiation, i.e. from C1 to C4, while ALP and osteocalcin were differently regulated. Conclusion Our findings support the role of different inducers on the sequential stages of MSC expansion and osteogenic differentiation in vitro, suggesting the addition of DEX following proliferation to ensure mineralization, as an index of in vivo osteogenic potency of human mesenchymal cells.

Osteogenic properties of late adherent subpopulations of human bone marrow stromal cells

The nonadherent (NA) population of bone-marrow-derived mononuclear cells (MNC) has been demonstrated to be a source of osteogenic precursors in addition to the plastic-adherent mesenchymal stromal cells (MSC). In the current study, two subpopulations of late adherent (LA) osteoprogenitors were obtained by subsequent replating of NA cells, and their phenotypic, functional, and molecular properties were compared with those of early adherent (EA) MSC. Approximately 35% of MNC were LA cells, and they acquired a homogeneous expression of MSC antigens later than EA cells. In EA-MSC, the alkaline phosphatase (ALP) activity increased significantly from time of seeding to the first confluence, whereas in LA cells it raised later, after the addition of mineralization medium. All subpopulations were able to produce type I collagen and to deposit extracellular matrix with organized collagen fibrils. The proportion of large colonies with more than 50% of ALP positive cells as well as the calcium content was higher in LA than in EA cells. Molecular analysis highlighted the upregulation of bone-related genes in LA-MSC, especially after the addition of mineralization medium. Our results confirm that bone marrow contains LA osteoprogenitors which exhibit a delay in the differentiation process, despite an osteogenic potential similar to or better than EA-MSC. LA cells represent a reservoir of osteoprogenitors to be recruited to gain an adequate bone tissue repair and regeneration when a depletion of the most differentiated component occurs. Bone tissue engineering and cell therapy strategies could take advantage of LA cells, since an adequate amount of osteogenic MSCs may be obtained while avoiding bone marrow manipulation and cell culture expansion.

Genetic clonal mapping of in situ and invasive ductal carcinoma indicates the field cancerization phenomenon in the breast

Nearly 80% of well-differentiated in situ duct carcinomas (g1 DCIS) have been shown to be multicentric (multilobar) lesions, while most in situ poorly differentiated duct carcinomas (g3 DCIS) were unifocal (unilobar) lesions. Here we present a clonality study of 15 cases of DCIS, all showing multiple foci. Twelve of these cases were associated with an invasive duct carcinoma. Fifteen cases of female breast cancer patients all showing multiple DCIS foci (5 g1 DCIS, 5 g2 DCIS, 5 g3 DCIS) were randomly selected and histologically studied using large histological sections. Care was taken to laser-microdissect DCIS foci that were most distantly located from one another in the same large section, and pertinent cells were genetically studied. Invasive duct carcinoma and ipsilateral lymph node metastases and/or contralateral lesions, whenever present, were additionally microdissected. DNA of neoplastic cells was purified, and the mtDNA D-loop region was sequenced. Genetic distance of different foci from the same case was visualized by phylogenetic analyses using the neighbor-joining method. Patients ranged in age from 36 to 87 years (mean 65.1). All 9 cases of widely spread DCIS were not clonal. Four of 6 cases that showed multiple adjacent foci were clonally related on mtDNA analysis. In the present series, 11/15 DCIS appeared as multiple synchronous primary breast tumors, genetically not related to one another. The present data enhance the view that breast can also show the field cancerization phenomenon, paralleling what has already been proposed in other organs.

Podoplanin and E-cadherin Expression in Preoperative Incisional Biopsies of Oral Squamous Cell Carcinoma Is Related to Lymph Node Metastases

Metastases to cervical lymph nodes (LNs) are an important independent adverse indicator in the prognosis of oral squamous cell carcinoma (OSCC). An accurate evaluation of molecular patterns favoring the metastatic process can be helpful in predicting cases of OSCC with elevated probability of early or late metastases and, moreover, in planning the proper therapeutic procedures before surgery. To this end, immunohistochemical expressions of both E-cadherin and podoplanin were evaluated on preoperative incisional biopsies of OSCC from 102 patients. The probability to have or develop metastases was very low when high E-cadherin expression was found in a preoperative sample or when a low podoplanin expression was found. Therefore, because of the strong association with LN metastases, high E-cadherin/low podoplanin immunohistochemical expression should also be assessed on preoperative incisional biopsies as a useful tool for evaluating the probability of early or late LN metastases of OSCCs.

Immunohistochemical expression of p16INK4A protein in oral lichen planus

The expression of p16(INK4A) has been investigated in oral leukoplakias (OLK), but no data are available about oral lichen planus (OLP). In this study, p16(INK4A) immunohistochemical expression was evaluated in 56 OLP and 36 OLK (12 without inflammation [NI-OLK] and 24 with chronic inflammation [I-OLK]) and compared with 23 reactive nonspecific inflammations (INF) and 14 normal control samples. The p16(INK4A) immunostaining was considered to be positive when >5% of keratinocytes were stained. All normal control samples were negative. Positive p16(INK4A) was detected in OLP, IOLK, and INF. Significant differences in p16(INK4A) positivity were found between OLP (64%) and OLK (28%) (χ(2) = 17.7; P < .01), and between I-OLK and NI-OLK (χ(2) = 4.5; P < .05). No significant difference was found between OLP and INF (43%). In conclusion, positive p16(INK4A) in OLP patients seems to be related to reactive inflammatory processes rather than to a risk of progression to oral squamous cell carcinoma.