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The periosteum is a specialized connective tissue containing multipotent stem cells capable of bone formation. In this study, we aimed at demonstrating that human oral periosteal cells derived from three different oral sites (upper vestibule, lower vestibule, and hard palate) represent an innovative cell source for maxillo-facial tissue engineering applications in terms of accessibility and self-commitment towards osteogenic lineage. Periosteal cells (PCs) were isolated from patients with different ages (20-30 yy, 40-50 yy, 50-60 yy); we then analyzed the in vitro proliferation capacity and the bone self-commitment of cell clones culturing them without any osteogenic supplement to support their differentiation. We found that oral PCs, independently of their origin and age of patients, are mesenchymal stem cells with stem cell characteristics (clonogenical and proliferative activity) and that, even in absence of any osteogenic induction, they undertake the osteoblast lineage after 45 days of culture. These results suggest that oral periosteal cells could replace mesenchymal cells from bone marrow in oral tissue-engineering applications.

The bone regeneration is one of the most important challenges for regenerative medicine. In maxillo-facial area, bone resorption of the alveolar crest occurs after tooth extraction and leads to several risks for rehabilitation treatments, including dental implants procedures. Goal of our study was to demonstrate the efficacy of an innovative clinical protocol of bone tissue engineering called Rigenera protocol, aimed to create and optimize bio-complexes constituted by collagen biomaterial and human autologous periosteum-derived micro-grafts. We assessed the capacity of these bio-complexes to prevent the bone resorption in the alveolar crest with respect to simple collagen performing histological evaluations of neo-formed osseous tissue. We demonstrated that autologous bio-complexes significantly reduced the bone resorption of both horizontal and vertical dimension of alveolar crest when compared to collagen alone. We also showed that these bio-complexes accelerate the ossification process triggering the formation of new osseous tissue after 45 days from treatment and increasing the calcified matrix after 60 days and until to 120 days with respect to collagen alone. Taken together, these data showed the efficacy of bio-complexes composed by periosteum-derived micro-grafts and collagen in the alveolar ridge preservation through a reduction of bone resorption and an enhancement of new osseous tissue formation.

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.

article i nfo 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 30 Hz for 45 min for 28 days. 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 21 days). On the contrary, no additional significant differences were observed after 28 days cell culture. Transmission Electron Microscopy (TEM) images performed on 21 day 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.

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. ispartof: Journal of Clinical Investigation vol:125 issue:12 pages:4463-4482 ispartof: location:United States status: published

Engineered gold nanoparticles targeted with a specific antibody (GNP-HCe) to primary lung mesenchymal cells (MC) isolated from BOS patients and loaded with everolimus were able to specifically inhibit MC proliferation and increase apoptosis without stimulating the inflammatory response (Cova, 2014). The aim of the present work was to prove that GNP-HCe can be safely administered to mice by inhalation. Nanoparticles (NPs) distribution and toxicity in vivo were assayed by aerosolization of 50 µg of labeled GNP-HCe or GNP-HC (gold nanoparticles with the antibody without everolimus) for 30 min a day for 2 weeks. Control mice were treated with saline solution. NPs localization was evaluated in lungs, kidney, spleen and liver by near-infrared (NIR) light imaging technology. Electron microscopy analysis (TEM) was performed to assess NP lung distribution. Toxic effects on tissue were evaluated by hystology. Inflammation burst in lung was tested by IL-8-homologue, CXCL1/KC, in BAL by ELISA NIR showed that fluorescence was significantly higher (p Our data showed that NPs were not taken by epithelial cells and were not toxic for other organs. Moreover, their presence did not stimulate the local inflammatory response. This work confirms the feasibility of GNP-HCe to be used as novel therapeutic approach for patients affected by CLAD.

Satellite cells are unipotent stem cells involved in muscle regeneration. However, the skeletal muscle microenvironment exerts a dominant influence over stem cell function. The cell intrinsic complexity of the skeletal muscle niche located within the connective tissue between fibres includes motor neurons, tendons, blood vessels, immune response mediators and interstitial cells. All these cell types modulate the trafficking of stimuli responsible of muscle fibre regeneration. In addition, several types of stem cell have been discovered in skeletal muscle tissue, mainly located in the interstitium. The majority of these stem cells appear to directly contribute to myogenic differentiation, although some are mainly implicated in paracrine effects. This review focuses on one of these classes of stem cells known as adult stem cells, which following their identification in the last decade have been used for therapeutic purposes, mainly in animal models of chronic muscle degeneration. Emerging literature identifies other myogenic progenitors generated from pluripotent stem cells as potential candidates for the treatment of skeletal muscle degeneration. However, adult stem cells still represent the gold standard for future comparative studies. ispartof: Current Gene Therapy vol:4 issue:15 pages:348-363 ispartof: location:United Arab Emirates status: published

Most recent advances in tissue engineering in the fields of oral surgery and dentistry have aimed to restore hard and soft tissues. Further improvement of these therapies may involve more biological approaches and the use of dental tissue stem cells in combination with inorganic/organic scaffolds. In this study, we analyzed the osteoconductivity of two different inorganic scaffolds based on poly (lactic-co-glycolic) acid alone (PLGA-Fisiograft) or in combination with hydroxyapatite (PLGA/HA-Alos) in comparison with an organic material based on equine collagen (PARASORB Sombrero) both in vitro and in vivo. We developed a simple in vitro model in which periosteum-derived stem cells were grown in contact with chips of these scaffolds to mimic bone mineralization. The viability of cells and material osteoconductivity were evaluated by osteogenic gene expression and histological analyses at different time points. In addition, the capacity of scaffolds to improve bone healing in sinus lift was examined. Our results demonstrated that the osteoconductivity of PLGA/HA-Alos and the efficacy of scaffolds in promoting bone healing in the sinus lift were increased. Thus, new clinical approaches in sinus lift follow-up should be considered to elucidate the clinical potential of these two PLGA-based materials in dentistry.

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.

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.

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.

Skeletal muscle is one of the most plastic tissues of vertebrates since it may able upon exercises to double in size due to a physiological hypertrophy. Despite the fact that it is mainly a syncytial tissue, it contains a relevant number of mononucleated cells that can be involved in its homeostasis and repair. Although the mononuclear cell types with the highest myogenic potential are the satellite cells located underneath the basal lamina of muscle fibres, other interstitial cells have been shown to contribute to muscle regeneration. Adding complexity to this scenario is the fact that several authors revealed myogenic potential in pluripotent stem cells, which can be generated from patient somatic cells and eventually manipulated to correct the genetic defect. Notwithstanding the copiousness of myogenic cell types, their use in ex vivo cell therapies for muscular degenerative diseases is still questionable. However, new discovers on their biological properties have advanced our comprehension in handling myogenic stem cells significantly. In this review, we will focus on the myogenic potential of multi- and pluri-potent stem cells and their use in preclinical and clinical studies. New insights from direct reprogramming and epigenetic signalling to generate myogenic stem cells are also considered.

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.

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.

In the last few years? stem cell research contributed to gain a fundamental understanding of how organisms grow and develop and how tissues are maintained throughout adult life. Mesenchymal stem cells (MSC) are self renewing, multipotent cells that are present in many adult tissues, such as bone marrow, adipose tissue, trabecular bone and muscle. More recently they have been found also in skin, liver and other tissues. Dermal skin-derived fibroblasts exhibit mesenchymal surface antigen immunophenotype and differentiation capabilities versus the three main mesenchymal tissues (bone, fat and cartilage). Hereditary Hemorrhagic Telangiectasia affects 1 in 5000 people and leads to abnormal blood vessel formation in skin and mucous membranes. We isolated human dermal fibroblasts from patients with Hereditary Hemorrhagic Telangiectasia (HHT) and healthy controls. In order to evaluate future applications of these cells in tissue engineering we compared mesenchymal properties (self-renewal, differentiation potential) of human gingival fibroblasts isolated from healthy and HHT-affected subjects using a combination of phenotypic (flow cytometry), morphologic (senescence), and functional (in vitro differentiation, colony forming unit assay and proliferation assay) criteria. Our results suggest that HHT cells were ideal candidates for tissue engineering.

The present study aimed at verifying “in vivo” and “in vitro” the effects of mechanical vibrations on muscle development and on differentiation of satellite cells, the “stem cells” of muscle tissue. We realized a bioreactor composed by an eccentric motor which produces a displacement of 11 mm at frequencies between 1 and 120 Hz on a plate connected to the motor. On the plate we fixed a cage used for animals and the dishes for satellite cells and linear acceleration provoked by the motor to samples was measured. We used 30 Hz as stimulating frequency and we treated newborn mice from their birth for the next four weeks one 1h/day and satellite cells 1 and 4 days 1h/day always at 30 Hz. Every week we collected from a control and a treated mouse tibialis anterior muscles and we performed Western Blot analysis and quantitative Real-Time PCR (qRT-PCR) to investigate proteins and genes involved in hypertrophy and atrophy pathways of skeletal muscle. On satellite cells we studied some genes involved in differentiation and fusion of primary myoblasts. Results demonstrate that mechanical vibration induces muscle hypertrophy within the first week of treatment and enhances terminal differentiation of myoblasts of treated satellite cells respect to control ones.

The aim of this work is to test the effects of a specific mechanical stimulation (Low Amplitude, high Frequency Vibrations) on the bone matrix formation of hBMSCs. Previous studies demonstrated that chemical culture conditions could influence the differentiation of hBMSCs toward bone: by plating the cells in appropriate osteogenic culture medium hBMSCs differentiate into osteoblasts [1-3].

The aim of the work was to understand the consequences of low-amplitude, high-frequency vibrations on proliferation and differentiation of SAOS-2 cells (sarcoma osteogenetic), an osteoblastic and tumorigenic cell line. We realized a bioreactor composed of an eccentric motor that produces a displacement of 11 mm at frequencies between 1 and 120 Hz on a plate connected to the motor. The cultures of SAOS-2 cells were fixed on the plate, and the linear acceleration provoked by the motor to the cultures was measured. We used 30 Hz as stimulating frequency after a preliminary test on the effect of different frequencies on differentiation of cells. Afterward, SAOS-2 cells were stimulated with 30 Hz for different durations, every day for 4 days. The expression of some genes involved in the differentiation process was analyzed first with a reverse transcriptase-polymerase chain reaction and afterward with a real-time polymerase chain reaction on the most expressed genes. Moreover, the proliferation of cells was evaluated. The results suggest a strong increase in the expression of the genes involved in tissue differentiation in the treated groups with respect to the controls. On the other hand, the proliferation seems to be slowed down, so probably the acceleration perceived by the mechanosensors of the cells changes the cellular cycle by blocking the duplication to early differentiate toward bone tissue.

The present study aimed at verifying “in vivo” the effects of mechanical vibrations on muscle development. We realized a bioreactor composed by an eccentric motor which produces a displacement of 11 mm at frequencies between 1 and 120 Hz on a plate connected to the motor. On the plate we fixed a cage used for animals and the linear acceleration provoked by the motor to the cage was measured. We used 30 Hz as stimulating frequency and we treated newborn mice from their birth for the next five weeks one hour/day. Every week we collected from a control and a treated mouse tibials and quadriceps anterior muscles and we performed morfometric analysis on muscle fibers. Moreover, with PCR and Real-Time PCR (RT-PCR) we analysed for the second and the third week the expression of genes involved in muscle differentiation. Preliminary results suggest that whole-vibration treatment increases the rate of proliferation of muscle fibers with respect to the control, while from the third week the total number of fibers of treated or non-treated mice tend to balance. The results of RT-PCR of some of the genes involved in terminal differentiation demonstrate that mechanical vibration seems to increase the rate of differentiation of the treated muscles with respect to the control ones.

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/cm(2). 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.