Gabriele Ceccarelli

A comparative analysis of the in vitro effects of pulsed electromagnetic field treatment on osteogenic differentiation of two different mesenchymal cell lineages.

Abstract Human mesenchymal stem cells (MSCs) are a promising candidate cell type for regenerative medicine and tissue engineering applications. Exposure of MSCs to physical stimuli favors early and rapid activation of the tissue repair process. In this study we investigated the in vitro effects of pulsed electromagnetic field (PEMF) treatment on the proliferation and osteogenic differentiation of bone marrow MSCs (BM-MSCs) and adipose-tissue MSCs (ASCs), to assess if both types of MSCs could be indifferently used in combination with PEMF exposure for bone tissue healing. We compared the cell viability, cell matrix distribution, and calcified matrix production in unstimulated and PEMF-stimulated (magnetic field: 2 mT, amplitude: 5 mV) mesenchymal cell lineages. After PEMF exposure, in comparison with ASCs, BM-MSCs showed an increase in cell proliferation (p<0.05) and an enhanced deposition of extracellular matrix components such as decorin, fibronectin, osteocalcin, osteonectin, osteopontin, and type-I and -III collagens (p<0.05). Calcium deposition was 1.5-fold greater in BM-MSC–derived osteoblasts (p<0.05). The immunofluorescence related to the deposition of bone matrix proteins and calcium showed their colocalization to the cell-rich areas for both types of MSC-derived osteoblast. Alkaline phosphatase activity increased nearly 2-fold (p<0.001) and its protein content was 1.2-fold higher in osteoblasts derived from BM-MSCs. The quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis revealed up-regulated transcription specific for bone sialoprotein, osteopontin, osteonectin, and Runx2, but at a higher level for cells differentiated from BM-MSCs. All together these results suggest that PEMF promotion of bone extracellular matrix deposition is more efficient in osteoblasts differentiated from BM-MSCs.

The differentiation of human adipose-derived stem cells (hASCs) into osteoblasts is promoted by low amplitude, high frequency vibration treatment

Several studies have demonstrated that tissue culture conditions influence the differentiation of human adipose-derived stem cells (hASCs). Recently, studies performed on SAOS-2 and bone marrow stromal cells (BMSCs) have shown the effectiveness of high frequency vibration treatment on cell differentiation to osteoblasts. The aim of this study was to evaluate the effects of low amplitude, high frequency vibrations on the differentiation of hASCs toward bone tissue. In view of this goal, hASCs were cultured in proliferative or osteogenic media and stimulated daily at 30Hz for 45min for 28days. The state of calcification of the extracellular matrix was determined using the alizarin assay, while the expression of extracellular matrix and associated mRNA was determined by ELISA assays and quantitative RT-PCR (qRT-PCR). The results showed the osteogenic effect of high frequency vibration treatment in the early stages of hASC differentiation (after 14 and 21days). On the contrary, no additional significant differences were observed after 28days cell culture. Transmission Electron Microscopy (TEM) images performed on 21day samples showed evidence of structured collagen fibers in the treated samples. All together, these results demonstrate the effectiveness of high frequency vibration treatment on hASC differentiation toward osteoblasts.

Investigation of low-level laser therapy potentiality on proliferation and differentiation of human osteoblast-like cells in the absence/presence of osteogenic factors

Abstract. Several studies have shown that low-level laser irradiation (LLLI) has beneficial effects on bone regeneration. The objective of this study was to examine the in vitro effects of LLLI on proliferation and differentiation of a human osteoblast-like cell line (Saos-2 cell line). Cultured cells were exposed to different doses of LLLI with a semiconductor diode laser (659 nm; 10 mW power output). The effects of laser on proliferation were assessed daily up to seven days of culture in cells irradiated once or for three consecutive days with laser doses of 1 or 3  J/cm2. The obtained results showed that laser stimulation enhances the proliferation potential of Saos-2 cells without changing their telomerase pattern or morphological characteristics. The effects on cell differentiation were assessed after three consecutive laser irradiation treatments in the presence or absence of osteo-inductive factors on day 14. Enhanced secretion of proteins specific for differentiation toward bone as well as calcium deposition and alkaline phosphatase activity were observed in irradiated cells cultured in a medium not supplemented with osteogenic factors. Taken together these findings indicate that laser treatment enhances the in vitro proliferation of Saos-2 cells, and also influences their osteogenic maturation, which suggest it is a helpful application for bone tissue regeneration.

Mesodermal iPSC–derived progenitor cells functionally regenerate cardiac and skeletal muscle

Conditions such as muscular dystrophies (MDs) that affect both cardiac and skeletal muscles would benefit from therapeutic strategies that enable regeneration of both of these striated muscle types. Protocols have been developed to promote induced pluripotent stem cells (iPSCs) to differentiate toward cardiac or skeletal muscle; however, there are currently no strategies to simultaneously target both muscle types. Tissues exhibit specific epigenetic alterations; therefore, source-related lineage biases have the potential to improve iPSC-driven multilineage differentiation. Here, we determined that differential myogenic propensity influences the commitment of isogenic iPSCs and a specifically isolated pool of mesodermal iPSC-derived progenitors (MiPs) toward the striated muscle lineages. Differential myogenic propensity did not influence pluripotency, but did selectively enhance chimerism of MiP-derived tissue in both fetal and adult skeletal muscle. When injected into dystrophic mice, MiPs engrafted and repaired both skeletal and cardiac muscle, reducing functional defects. Similarly, engraftment into dystrophic mice of canine MiPs from dystrophic dogs that had undergone TALEN-mediated correction of the MD-associated mutation also resulted in functional striatal muscle regeneration. Moreover, human MiPs exhibited the same capacity for the dual differentiation observed in murine and canine MiPs. The findings of this study suggest that MiPs should be further explored for combined therapy of cardiac and skeletal muscles.

In vitro osteoblastic differentiation of human mesenchymal stem cells and human dental pulp stem cells on poly-L-lysine-treated titanium-6-aluminium-4-vanadium

Three-dimensional (3D) titanium-6-aluminium-4-vanadium (Ti6Al4V) is a widely used biomaterial for orthopedic prosthesis and dental implants; thanks to its very high-mechanical strength and resistance to corrosion. Human mesenchymal stem cells (hMSCs) and dental pulp stem cells (hDPSCs) are responsible for bone regeneration following colonization of prosthesis or dental implants. Both hMSCs and hDPSCs have lower ability to colonize this biomaterial in comparison with tissue culture-treated plastic. Both hMSCs and hDPSCs show lack of focal adhesion kinase (FAK) activation when grown on Ti6Al4V. This signal is restored in the presence of poly-L-lysine (poly-L-lys). Poly-L-lys has been used as part of organoapatite or together with zinc and calcium ions. Our results suggest that poly-L-lys alone induces FAK activation through β1-INTEGRIN, because the presence of β1-INTEGRIN blocking antibody avoided FAK autophosphorylation. Presence of poly-L-lys also increases expression of osteoblastic differentiation marker genes in hMSCs and hDPSCs grown on Ti6Al4V.

Low-amplitude high frequency vibration down-regulates myostatin and atrogin-1 expression, two components of the atrophy pathway in muscle cells.

Whole body vibration (WBV) is a very widespread mechanical stimulus used in physical therapy, rehabilitation and fitness centres. It has been demonstrated that vibration induces improvements in muscular strength and performance and increases bone density. We investigated the effects of low‐amplitude, high frequency vibration (HFV) at the cellular and tissue levels in muscle. We developed a system to produce vibrations adapted to test several parameters in vitro and in vivo. For in vivo experiments, we used newborn CD1 wild‐type mice, for in vitro experiments, we isolated satellite cells from 6‐day‐old CD1 mice, while for proliferation studies, we used murine cell lines. Animals and cells were treated with high frequency vibration at 30 Hz. We analyzed the effects of mechanical stimulation on muscle hypertrophy/atrophy pathways, fusion enhancement of myoblast cells and modifications in the proliferation rate of cells. Results demonstrated that mechanical vibration strongly down‐regulates atrophy genes both in vivo and in vitro. The in vitro experiments indicated that mechanical stimulation promotes fusion of satellite cells treated directly in culture compared to controls. Finally, proliferation experiments indicated that stimulated cells had a decreased growth rate compared to controls. We concluded that vibration treatment at 30 Hz is effective in suppressing the atrophy pathway both in vivo and in vitro and enhances fusion of satellite muscle cells. Copyright

Emerging Perspectives in Scaffold for Tissue Engineering in Oral Surgery

Bone regeneration is currently one of the most important and challenging tissue engineering approaches in regenerative medicine. Bone regeneration is a promising approach in dentistry and is considered an ideal clinical strategy in treating diseases, injuries, and defects of the maxillofacial region. Advances in tissue engineering have resulted in the development of innovative scaffold designs, complemented by the progress made in cell-based therapies. In vitro bone regeneration can be achieved by the combination of stem cells, scaffolds, and bioactive factors. The biomimetic approach to create an ideal bone substitute provides strategies for developing combined scaffolds composed of adult stem cells with mesenchymal phenotype and different organic biomaterials (such as collagen and hyaluronic acid derivatives) or inorganic biomaterials such as manufactured polymers (polyglycolic acid (PGA), polylactic acid (PLA), and polycaprolactone). This review focuses on different biomaterials currently used in dentistry as scaffolds for bone regeneration in treating bone defects or in surgical techniques, such as sinus lift, horizontal and vertical bone grafts, or socket preservation. Our review would be of particular interest to medical and surgical researchers at the interface of cell biology, materials science, and tissue engineering, as well as industry-related manufacturers and researchers in healthcare, prosthetics, and 3D printing, too.

High-Frequency Vibration Treatment of Human Bone Marrow Stromal Cells Increases Differentiation toward Bone Tissue

In order to verify whether differentiation of adult stem cells toward bone tissue is promoted by high-frequency vibration (HFV), bone marrow stromal cells (BMSCs) were mechanically stimulated with HFV (30 Hz) for 45 minutes a day for 21 or 40 days. Cells were seeded in osteogenic medium, which enhances differentiation towards bone tissue. The effects of the mechanical treatment on differentiation were measured by Alizarin Red test, (q) real-time PCR, and protein content of the extracellular matrix. In addition, we analyzed the proliferation rate and apoptosis of BMSC subjected to mechanical stimulation. A strong increase in all parameters characterizing differentiation was observed. Deposition of calcium was almost double in the treated samples; the expression of genes involved in later differentiation was significantly increased and protein content was higher for all osteogenic proteins. Lastly, proliferation results indicated that stimulated BMSCs have a decreased growth rate in comparison with controls, but both treated and untreated cells do not enter the apoptosis process. These findings could reduce the gap between research and clinical application for bone substitutes derived from patient cells by improving the differentiation protocol for autologous cells and a further implant of the bone graft into the patient.

Molecular signature of amniotic fluid derived stem cells in the fetal sheep model of myelomeningocele

Abnormal cord development results in spinal cord damage responsible for myelomeningocele (MMC). Amniotic fluid-derived stem cells (AFSCs) have emerged as a potential candidate for applications in regenerative medicine. However, their differentiation potential is largely unknown as well as the molecular signaling orchestrating the accurate spinal cord development. Fetal lambs underwent surgical creation of neural tube defect and its subsequent repair. AFSCs were isolated, cultured and characterized at the 12th (induction of MMC), 16th (repair of malformation), and 20th week of gestation (delivery). After performing open hysterectomy, AF collections on fetuses with sham procedures at the same time points as the MMC creation group have been used as controls. Cytological analyses with the colony forming unit assay, XTT and alkaline-phosphatase staining, qRT-PCR gene expression analyses (normalized with aged match controls) and NMR metabolomics profiling were performed. Here we show for the first time the metabolomics and molecular signature variation in AFSCs isolated in the sheep model of MMC, which may be used as diagnostic tools for the in utero identification of the neural tube damage. Intriguingly, PAX3 gene involved in the murine model for spina bifida is modulated in AFSCs reaching the peak of expression at 16 weeks of gestation, 4 weeks after the intervention. Our data strongly suggest that AFSCs reorganize their differentiation commitment in order to generate PAX3-expressing progenitors to counteract the MMC induced in the sheep model. The gene expression signature of AFSCs highlights the plasticity of these cells reflecting possible alterations of embryonic development.

Nanostructured TiO2 Surfaces Promote Human Bone Marrow Mesenchymal Stem Cells Differentiation to Osteoblasts

Micro- and nano-patterning/modification are emerging strategies to improve surfaces properties that may influence critically cells adherence and differentiation. Aim of this work was to study the in vitro biological reactivity of human bone marrow mesenchymal stem cells (hBMSCs) to a nanostructured titanium dioxide (TiO2) surface in comparison to a coverglass (Glass) in two different culture conditions: with (osteogenic medium (OM)) and without (proliferative medium (PM)) osteogenic factors. To evaluate cell adhesion, hBMSCs phosphorylated focal adhesion kinase (pFAK) foci were analyzed by confocal laser scanning microscopy (CLSM) at 24 h: the TiO2 surface showed a higher number of pFAK foci with respect to Glass. The hBMSCs differentiation to osteoblasts was evaluated in both PM and OM culture conditions by enzyme-linked immunosorbent assay (ELISA), CLSM and real-time quantitative reverse transcription PCR (qRT-PCR) at 28 days. In comparison with Glass, TiO2 surface in combination with OM conditions increased the content of extracellular bone proteins, calcium deposition and alkaline phosphatase activity. The qRT-PCR analysis revealed, both in PM and OM, that TiO2 surface increased at seven and 28 days the expression of osteogenic genes. All together, these results demonstrate the capability of TiO2 nanostructured surface to promote hBMSCs osteoblast differentiation and its potentiality in biomedical applications.