Francesca Farinella

Biological effect of resorbable plates on normal osteoblasts and osteoblasts derived from Pfeiffer syndrome.

Biodegradable fixation devices made of the polymers polylactide, polyglycolide and their copolymers are used routinely during maxillofacial, craniofacial, and orthopedic reconstructive surgical procedures, thanks to their property of biodegradation that avoid the need for implant removal. In particular, they are used in the treatment of craniosynostosis in pediatric patients affected by Pfeiffer syndrome, where the resorption time of 1 year or less does not interfere with the normal growth of the skull. To study the mechanism how polylactide-polyglycolide (PLPG) acid plates can induce osteoblast differentiation and proliferation in normal osteoblasts and in osteoblasts derived from a patient with Pfeiffer syndrome, the expression levels of bone-related genes were analyzed using real-time reverse transcription-polymerase chain reaction. Osteoblasts grown on the PLPG acid plates resulted in significant upregulation of mRNA expression of many genes related to osteodifferentiation during the treatment, indicating that polylactide, polyglycolide biopolymers enhance proliferation, differentiation, and deposition of matrix in osteoblasts. This study also revealed some differences in gene expression between normal osteoblasts and osteoblasts derived from patients with Pfeiffer syndrome, cultivated on PLPG acid plates.

Trabecular Titanium Induces Osteoblastic Bone Marrow Stem Cells Differentiation

Pure titanium and titanium alloys are materials widely used in orthopedics and dental surgery because of their mechanical properties, chemical stability and biocompatibility. Although excellent clinical results have been shown, traditional porous metals have several inherent limitations (low volumetric porosity, relatively high modulus of elasticity and availability as a coating only). With the aim of going over these limits, improving the potentiality of osteointegration, a new highly porous titanium biomaterial (Trabecular Titaniumâ„¢, TT) has been developed. Because the molecular events due to TT and able to alter osteoblast activity to promote bone formation are poorly understood, expression of osteoblastic related genes in mesenchymal stem cells exposed to TT was investigated. The expression levels of bone related genes like RUNX2, SPP1, COL1A1, COL3A1, BGLAP, ALPL, and FOSL1) and mesenchymal stem cells marker (CD105) were analyzed, using real time Reverse Transcription-Polymerase Chain Reaction.TT causes induction of bone related genes osteopontin (SPP1), osteocalcin (BGLAP) alkaline phosphatase (ALPL) and indicating the differentiation effect of this biomaterial on mesenchymal stem cells.The obtained results can be relevant to better understand the molecular mechanism of bone regeneration and as a model for comparing other materials with similar clinical effects.

MIF Expression in Induced Peripheral Blood Mononuclear Cells by Vitamin D3 and its Potential Correlation with Resting Metabolic Rate in Obesity

Macrophage migration inhibitory factor (MIF) plays a pivotal role in systemic and local inflammatory and immune responses. The aim of this study is to assess MIF gene expression in PBMCs harvested from obese individuals and to compare it with that of lean subjects to analyze any potential relationship between resting metabolic rate as well as several different parameters and MIF expression in PBMC. We also aim to assess anti-inflammatory characteristics of vitamin D3 on MIF expression in vitro. Participants were 30 obese and 18 non-obese subjects who were assessed following an overnight fasting for RMR by means of indirect calorimetry. Body composition was measured using a Bodystat device. The PBMCs were separated from whole blood by the Ficoll-hypaque technique. The mRNA was extracted and the cDNA was synthesized. This process was followed by real-time PCR using primer pairs specific for MIF mRNA and beta actin as internal control. Our findings clearly demonstrate that there were significant differences in terms of BMI, BMR predict, fat proportion, fat mass, free fat mass, TBW, visceral fat, fasting serum glucose, TG, HDL, Hs-CRP and RMR between the two groups. Moreover, the level of MIF expression in the obese group was approximately 2.5 times higher compared to the lean group. An increased level of MIF expression in the obese group and a decreased expression of that non-obese was observed after inducing PBMCs with vitamin D3. One of the intriguing results of this study was the observed reverse correlation between MIF expression and fat mass as well as fat proportion after PBMCs were cultured in the presence of vitamin D3. Therefore, it could be concluded that MIF expression, which is in turn influenced by vitamin D3, has a role in the hyperactive immune profile and the pro-inflammatory state observed in obese individuals which is suggested to have a causal relationship with obesity.

Early Effects of P-15 on Human Bone Marrow Stem Cells

ABSTRACT Objectives Peptide-15 (P-15) is an analogue of the cell binding domain of collagen. P-15 has been shown to facilitate physiological to process in a way similar to collagen, to serve as anchorage for cells, and to promote the binding, migration and differentiation of cells. However, how P-15 alters osteoblast activity to promote bone formation is poorly understood. To study the osteoinductive properties of peptide P-15, we analyzed the expression levels of bone related genes in human mesenchymal stem cells treated with this biomaterial. Material and Methods Using real time Reverse Transcription-Polymerase Chain Reaction the quantitative expression of specific genes, like transcriptional factors (RUNX2 and SP7), bone related genes (SPP1, COL1A1, COL3A1, BGLAP, ALPL, and FOSL1) and mesenchymal stem cells marker (ENG) were examined. Results P-15 causes a considerable induction of osteoblast transcriptional factor like osterix (SP7) and of the bone related genes osteopontin (SPP1) and osteocalcin (BGLAP). In contrast the expression of endoglin (ENG) was markedly decreased in stem cells treated with P-15 respect to untreated cells, indicating the differentiation effect of this biomaterial on stem cells. Conclusions The present study shows the effect of P-15 on mesenchymal stem cells in the early differentiation stages: P-15 is an inducer of osteogenesis on human stem cells as indicated by the activation of bone related markers SP7, SPP1 and BGLAP.The results may allow a better understanding of the molecular mechanism of bone regeneration and as a model for comparing other materials with similar clinical effects.

Genetic Effects of Trabecular Titanium™ on Human Osteoblast-Like Cells (MG-63): An In Vitro Study

Pure titanium and titanium alloys are materials widely used in orthopaedics and dental surgery because of their mechanical properties, chemical stability and biocompatibility. Although excellent clinical results have been shown, traditional porous metals have several inherent limitations (low volumetric porosity, relatively high modulus of elasticity and low frictional characteristics, availability as a coating only). With the aim of moving beyond these limits, improving the potentiality of osteointegration and consequently the stability of prosthetic implants, a new highly porous titanium biomaterial (Trabecular Titanium™, TT) has been developed. In this in vitro study, we are testing the effects of TT on osteoblast-like cells (MG63) cultured on disks of this biomaterial by using DNA microarrays containing 20,000 genes. We identified several genes covering a broad range of functional activities whose expression was significantly up- or down-regulated. Findings from this study can give a detailed description of the genetic effects TT has on human osteoblast-like cells in vitro. We demonstrated that TT stimulates osteoblast proliferation and differentiation, and reduces osteoblast apoptosis. Moreover, TT limits osteoclastogenesis, inflammation and reduces the immunologic events against the material, modulating genes related to the immune system. Therefore, all these effects contribute to improve osseointegration and consequently survival of the prosthetic implants. These reported data encourage the clinical application of Trabecular Titanium™ to prosthetic devices.

Idiopathic pulmonary fibrosis and polymorphisms of the folate pathway genes

OBJECTIVES This study aims to determine the possible association between folate pathway gene polymorphisms and idiopathic pulmonary fibrosis. This represents the first study carried out on folate pathway gene polymorphisms as possible risk factors in this kind of pathology. The premise is that several polymorphisms mapping on genes responsible for folate uptake are associated with the risk of numerous diseases occurring between pregnancy and old age, and that too little is currently known about idiopathic pulmonary fibrosis. DESIGN AND METHODS We genotyped 9 single nucleotide polymorphisms and 1 polymorphic insertion in 7 essential genes belonging to the folate pathway in 32 Italian idiopathic pulmonary fibrosis patients and 81 control subjects. This was done by PCR and restriction analysis. RESULTS Allelic and genotypic association tests indicated that for all the analysed polymorphisms there were no significant differences between patients and controls. Nevertheless, the haplotype association analysis revealed a significant association between idiopathic pulmonary fibrosis and transcobalamin II gene polymorphisms: specifically the haplotype 776G (rs1801198)-c.1026-394G (rs7286680)-444C (rs10418) (OR=2.84; 95% C.I. 1.36-5.93, P value=0.004). CONCLUSIONS This small-scale preliminary study would suggest the importance of further research focusing on the role of folate in the onset of idiopathic pulmonary fibrosis.

No evidence of HAND2 involvement in nonsyndromic cleft lip with or without cleft palate.

Craniofacial morphogenesis is determined by multistep processes involving signalling molecules and transcription factors, which are organised into highly coordinated pathways. Derailment from this intricate network can lead to congenital malformations. Cells migrate from neural crests to populate different structures, such as branchial arches, involved in embryonal orofacial development. The EDN1 pathway is involved in branchial arch development. Gene knockout and knockdown experiments on EDN1 or its downstream effector dHAND resulted in mice that were characterised by craniofacial defects and cleft palate. Our aim was to evaluate whether the transcription factor HAND2 could be implicated in non-syndromic cleft lip with or without cleft palate (CL/P) aetiology. A sample study composed of 39 multiplex Italian pedigrees was enrolled to test linkage between two microsatellite flanking HAND2 locus and CL/P. No evidence of linkage between HAND2 and CL/P was obtained. Indeed, formal levels of exclusion were obtained with different inheritance models. Investigation results did not support a role of HAND2 in CL/P aetiology. Nevertheless a minor contribute of the gene in clefting could not be ruled out.

Engipore acts on human bone marrow stem cells.

OBJECTIVES Porous HA scaffolds are promising materials for tissue engineering because they offer a tridimensional support and serve as template for cell proliferation and at last tissue formation. Engipore provide a natural 3D scaffold with organic fibrous material in bone. However, how this material alters osteoblast activity to promote bone formation is poorly understood. MATERIALS AND METHODS To study how Engipore can induce osteoblast differentiation in mesenchymal stem cells, the expression levels of bone related genes and mesenchymal stem cells marker were analyzed. RESULTS Engipore causes a significant induction of osteoblast transcriptional factors like SP7 and RUNX2 and of the bone-related gene osteocalcin (BGLAP). The expression of CD105 was not significantly changed in stem cells treated with Engipore with respect to untreated cells, while SSP1 (osteopontin) was significantly down expressed thus reducing osteoclast activity. CONCLUSIONS The obtained results can be relevant to better understand the molecular mechanism of bone regeneration.