Vincenzo Sollazzo

Chitosan, hyaluronan and chondroitin sulfate in tissue engineering for cartilage regeneration: A review

Injection of hyaluronan into osteoarthritic joints restores the viscoelasticity, augments the flow of joint fluid, normalizes endogenous hyaluronan synthesis, and improves joint function. Chitosan easily forms polyelectrolyte complexes with hyaluronan and chondroitin sulfate. Synergy of chitosan with hyaluronan develops enhanced performances in regenerating hyaline cartilage, typical results being structural integrity of the hyaline-like neocartilage, and reconstitution of the subchondral bone, with positive cartilage staining for collagen-II and GAG in the treated sites. Chitosan qualifies for the preparation of scaffolds intended for the regeneration of cartilage: it yields mesoporous cryogels; it provides a friendly environment for chondrocytes to propagate, produce typical ECM, and assume the convenient phenotype; it is a good carrier for growth factors; it inactivates metalloproteinases thus preventing collagen degradation; it is suitable for the induction of the chondrogenic differentiation of mesenchymal stem cells; it is a potent means for hemostasis and platelet delivery.

Effects of Pulsed Electromagnetic Fields on Human Articular Chondrocyte Proliferation

Low-energy, low-frequency pulsed electromagnetic fields (PEMFs) can induce cell proliferation in several cell culture models. In this work we analysed the proliferative response of human articular chondrocytes, cultured in medium containing 10% FBS, following prolonged exposure to PEMFs (75 Hz, 2.3 mT), currently used in the treatment of some orthopaedic pathologies. In particular, we investigated the dependence of the proliferative effects on the cell density, the availability of growth factors and the exposure lengths. We observed that PEMFs can induce cell proliferation of low density chondrocyte cultures for a long time (6 days), when fresh serum is added again in the culture medium. In the same conditions, in high density cultures, the PEMF-induced increase in cell proliferation was observed only in the first three days of exposure. The data presented in this study show that the availability of growth factors and the environmental constrictions strongly condition the cellular proliferative response to PEMFs.

Effects of pulsed electromagnetic fields on human chondrocytes: an in vitro study.

Abstract.3H-thymidine incorporation was studied in cultured human nasal and articular chondrocytes exposed to low-energy, low-frequency pulsed electromagnetic fields (PEMFs) (75 Hz, 2.3 mT). The reverse transcriptase polymerase chain reaction (RT-PCR) analysis shows that human secondary chondrocytes derived from both nasal and articular cartilage express collagen type II mRNA, which is a specific marker of the chondrocyte phenotype. In a preliminary series of experiments, cells were exposed to PEMF for different time periods ranging from 6 to 30 hours (time-course), in medium supplemented with 10% or 0.5% fetal calf serum (FCS) and in serum-free medium. The ratios between the 3H-thymidine incorporation in PEMFs and control cultures show an increase of the cell proliferation in cultures exposed to PEMFs when serum is present in the culture medium, whereas no effect was observed in serum-free conditions. The increase in DNA synthesis, induced by PEMFs, was then evaluated only at the times of maximum induction and the results were analyzed by the three-factor analysis of variance (ANOVA).The data presented in this study show that even if 3H-thymidine incorporation is higher in nasal than in articular chondrocytes, PEMF induce an increase in the proliferation of both cell types. Moreover, the concentration of FCS in the culture medium greatly influences the proliferative response of human chondrocytes to the PEMF exposure. Though normal human osteoblast cells increase their proliferation when exposed to PEMFs if only 10% FCS is present in the medium, human chondrocytes are able to increase their cell proliferation when exposed to PEMFs in the presence of both 0.5% and 10% of FCS in the medium. The results obtained may help to explain the basic mechanisms of PEMF stimulation of fracture healing.

Responses of Human MG-63 Osteosarcoma Cell Line and Human Osteoblast-Like Cells to Pulsed Electromagnetic Fields

We have studied the effects of low-energy, low-frequency pulsed electromagnetic fields (PEMF) on cell proliferation, in both human osteoblast-like cells obtained from bone specimens and in human MG-63 osteosarcoma cell line. Assessment of osteoblastic phenotype was performed both by immunolabeling with antiosteonectin antibody and by verifying the presence of parathyroid hormone receptors. The cells were placed in multiwell plates and set in a tissue culture incubator between a pair of Helmholtz coils powered by a pulse generator (1.3 ms, 75 Hz) for different periods of time. [3H]Thymidine incorporation was used to evaluate cell proliferation. Since it had previously been observed that the osteoblast proliferative response to PEMF exposure may also be conditioned by the presence of serum in the medium, experiments were carried out at different serum concentrations. [3H]Thymidine incorporation increases in osteoblast-like cells, when they are exposed to PEMF in the presence of 10% fetal calf serum (FCS). The greatest effect is observed after 24 hours of PEMF exposure. No effects on cell proliferation are observed when osteoblast-like cells are exposed to PEMF in the presence of 0.5% FCS or in a serum-free medium. On the other hand, PEMF-exposed MG-63 cells show increased cell proliferation either at 10% FCS, 0.5% FCS and in serum-free medium. Nevertheless, the maximum effect of PEMF exposure on MG-63 cell proliferation depends on the percentage of FCS in the medium. The higher the FCS concentration, the faster the proliferative response to PEMF exposure. Our results show that, although MG-63 cells display some similarity with human bone cells, their responses to PEMFs exposure are quite different from that observed in normal human bone cells.

Methylation analysis of the promoter F of estrogen receptor α gene: effects on the level of transcription on human osteoblastic cells

In this study, the methylation status of the distal promoter F of estrogen receptor alfa (ERalpha) gene in human osteoblastic cells was investigated. The activity of this promoter is responsible for the ERalpha gene transcription in bone tissue. The methylation status of promoter F was here evaluated, for the first time, by direct sequencing of bisulfite-treated genomic DNA, at 10 CpG specific sites localized in a region of about 800 bp. An heterogeneous methylation pattern was observed. The most notable difference was found at four particular CpGs, distant from the exon F transcription start site, showing a methylation status that correlates with the expression level, being ERalpha mRNA transcription reduced in a partially methylated cells but preserved in demethylated cells. The other CpG sites, localized around the transcription start site, were always demethylated except for MG-63 cells showing the lowest level of ERalpha expression. By quantitative RT-PCR analysis we demonstrated that ERalpha gene expression was higher in primary osteoblasts than in bone-derived cells (MG-63 and SaOS-2) and in all cases the ERalpha mRNA is represented by the isoform F. The same 10 CpG sites were investigated in non-osseous cell lines and were found fully methylated in ERalpha-negative breast cancer cells (MDA-MB-231) and completely demethylated in ERalpha-positive breast cancer cells (MCF7). The overall results suggest that methylation of the CpG sites inside ERalpha gene promoter F here analyzed may contribute to ERalpha transcriptional control, directly or indirectly, influencing the tissue specific expression of the gene.

Anatase Nanosurface Regulates Micrornas

Titanium is the criterion standard among materials used for prosthetic devices because of its good mechanical and chemical properties. When exposed to oxygen, titanium becomes an oxide that is biocompatible and able to induce osseointegration. There are 3 allotropic forms of titanium dioxide: brookite, rutile, and anatase. Anatase can be prepared as a colloidal suspension and then used to coat surfaces. Anatase coating (AC) can potentially have specific biologic effects and specifically induce bone formation. To get more information as regards the osteogenic effect of AC, we used microRNA (miRNA) microarray techniques to investigate translation regulation in osteoblasts exposed to AC. Transduction, transcription, and translation are the 3 levels of regulation of cell activity. Recently, a new type of translation regulation has been identified: RNA interference. RNA interference is a process in which miRNA (i.e., noncoding RNAs of 19-23 nucleotides) can induce sequence-specific mRNA degradation and/or translational repression. The human genome encodes a few hundred miRNAs that can posttranscriptionally repress thousands of genes. miRNA oligonucleotide microarray provides a novel method of carrying out genome-wide miRNA profiling in human samples. By using miRNA microarrays containing 329 probes designed from human miRNA sequences, we identified in osteoblast-like cell line (MG-63) cultured with AC several miRNA whose expression had been significantly modified. The data reported constitute, to our knowledge, the first study on translation regulation in osteoblasts exposed to AC. They can be relevant to a better understanding of the molecular mechanism of bone regeneration and as a model for comparing other materials with similar clinical effects.

Assessment of Pain Associated with Insertion Torque of Dental Implants. A Prospective, Randomized-Controlled Study

This study investigated pain experience following dental implant placement in relation to insertion torque using questionnaires. A total 80 implants were placed in 20 patients. Each patient received 4 implants at different times. One implant was inserted and, then, after 40 days was placed the second implant, after 80 days was placed the third implant and after 120 days was placed the fourth implant. At each time the peri-implant bone levels were evaluated on intraoral radiographs taken with the paralleling technique. The implants were placed with a dynamometric key at 35N, 50N, 65N, 85N. Patients were asked to evaluate their pain experience during surgery, 24 hours after surgery, and at 2 days, 4 days, 1, 2, and 4 weeks after surgery on special pain assessment forms. A separate form was used for each time point. Pain was assessed using a descriptive numerical rating scale of 0 to 10, with 0 indicative of no pain and 10 representing the worst pain imaginable. Patients were instructed that a score of 1 to 3 was indicative of mild pain, 4 to 6 was indicative of moderate pain, and 7 to 10 was indicative of severe pain. A significant correlation pain scores and insertion torque was found between group III and group IV vs group II and group I during surgery, at 24 hours, 2 days, 4 days, 1 week, 2 weeks p⩽0.05. No statistical difference was found between group I vs. group II during surgery, at 24 hours, 2 days, 4 days, 1 week, 2 weeks p⩾0.05. In conclusion, elevated insertion torque values produces pain and resoption of the crestal bone around the implants.

Effects of Low-Frequency Pulsed Electromagnetic Fields on Human Osteoblast-Like Cells In Wtro

We have studied the effects of low-frequency, low-energy pulsed electromagnetic fields (PEMFs) on human osteoblast-like cells in vitroin order to investigate the biochemical mechanism(s) by which electrical stimulation can effect changes in skeletal metabolism. Bony nature of the cells was assayed by immunolabeling with antiosteonectin antibody. Multidish plates were placed between a pair of Helmoltz coils powered by a pulse generator (1.3 ms, 75 Hz) in a tissue culture incubator for 24 h. 3H-thymidine was added to the culture medium to evaluate cell proliferation. The acid-precipitable 3H-thymidine incorporation significantly increases in PEMF-exposed cells as compared to the control. PEMF exposure induces a two- to fivefold increase of DNA synthesis. Osteoblasts cultured for 24 h in a serum-free medium show a very low proliferation rate, and the exposure to PEMF does not stimulate DNA synthesis.

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.