The effects of ageing on dental pulp stem cells, the tooth longevity elixir.

Stem cells are essential for tissue homeostasis and regeneration throughout the lifespan of multicellular organisms. The decline in stem cell function during advanced age is associated with a reduced regenerative potential of tissues that leads to an increased frequency of diseases. Age-related changes also occur in the dental pulp that represents a reliable model tissue, with high regenerative capability, for studying senescence mechanisms. However, little information is available concerning the effects of ageing on dental stem-cell function. In this mini-review, recent data on how the molecular and functional alterations that accumulate in stem cell populations during ageing result in modifications of dental pulp physiology are discussed. Changes that accumulate during ageing such as how reduction of pulp chamber volume, decreased vascular supply and modifications to the stem cell niches affect stem cell functions and, therefore, dental pulp regenerative potential in response to various stressful agents. Dental pulp cells from aged individuals are still metabolically active and secrete pro-inflammatory and matrix-degrading molecules. Furthermore, miRNAs and exosomes derived from dental pulp stem cells constitute an attractive source of nanovesicles for the treatment of age-related dental pathologies. Further investigation of the epigenetic alterations in dental pulp stem cells, accumulating during ageing, might reveal crucial information for potential stem cell-based therapeutic approaches in the elderly.

Indirect co-cultures of healthy mesenchymal stem cells restore the physiological phenotypical profile of psoriatic mesenchymal stem cells.

Psoriasis microenvironment, characterized by an imbalance between T helper type 1 (Th1)/Th17 and Th2 cytokines and also influences the mesenchymal stem cells (MSCs) phenotypical profile. MSCs from healthy donors (H-MSCs) can exert a strong paracrine effect by secreting active soluble factors, able to modulate the inflammation in the microenvironment. To evaluate the influence of H-MSCs on MSCs from psoriatic patients (PsO-MSCs), H-MSCs and PsO-MSCs were isolated and characterized. Indirect co-culture of H-MSCs with PsO-MSCs was performed; effects on proliferation and expression of cytokines linked to Th1/Th17 and Th2 pathways were assayed before and after co-culture. The results show that before co-culture, proliferation of PsO-MSCs was significantly higher than H-MSCs (P < 0·05) and the levels of secreted cytokines confirmed the imbalance of Th1/Th17 versus the Th2 axis. After co-culture of H-MSCs with PsO-MSCs, healthy MSCs seem to exert a 'positive' influence on PsO-MSCs, driving the inflammatory phenotypical profile of PsO-MSCs towards a physiological pattern. The proliferation rate decreased towards values nearer to those observed in H-MSCs and the secretion of the cytokines that mostly identified the inflammatory microenvironment that characterized psoriasis, such as interleukin (IL)-6, IL-12, IL-13, IL-17A, tumour necrosis factor (TNF)-α and granulocyte-macrophage colony-stimulating factor (G-CSF), is significantly lower in co-cultured PsO-MSCs than in individually cultured PSO-MSCs (P at least < 0·05). In conclusion, our preliminary results seem to provide an intriguing molecular explanation for the ever-increasing evidence of therapeutic efficacy of allogeneic MSCs infusion in psoriatic patients.

EFFECT OF PLATELET RICH PLASMA CONCENTRATION ON SKELETAL MUSCLE REGENERATION: AN EXPERIMENTAL STUDY.

Skeletal muscle injuries are common causes of severe long-term pain and physical disability, accounting for up to 55% of all sports injuries. The phases of the healing processes after direct or indirect muscle injury are complex but clearly defined and include well-coordinated steps: degeneration, inflammation, regeneration, and fibrosis. Despite this frequent occurrence and the presence of a body of data on the pathophysiology of muscle injuries, none of the current treatment strategies have shown to be really effective in strictly controlled trials. Platelet-rich plasma (PRP) is a promising alternative approach based on the ability of autologous growth factors (GFs) to accelerate tissue healing, improve muscular regeneration, increase neovascularization and reduce fibrosis. The present study is focused on the use of different concentrations of PRP as a source of GFs. Unilateral muscle lesions were created on the longissimus dorsi muscle of Wistar rats. Twenty-four h after surgical trauma, the lesion was filled with an intramuscular injection of PRP at 2 different concentrations. A group of rats were left untreated (controls). Animals were sacrificed at 3, 15 and 60 days from surgery. Histological, immunohistochemical and histomorphometric analyses were performed to evaluate muscle regeneration, neovascularization, fibrosis and inflammation. The PRP-treated muscles showed better muscle regeneration, more neovascularization and a slight reduction of fibrosis compared with the control muscles in a dose dependent manner. However, further studies also assessing pain and functional recovery are scheduled.

Comparative ultrasonographic evaluation of the Achilles paratenon in symptomatic and asymptomatic subjects: an imaging study.

Achilles tendon analysis represents one of the most frequently requested ultrasonographic evaluations, due to the high incidence of tendinopathy. Various authors have described inflammatory features of the paratenon recruited 22 subjects complaining of pain in the mid-portion of the Achilles tendon and 22 healthy subjects. Both groups underwent ultrasonographic examination and Victorian Institute of Sport Assessment-Achilles questionnaire administration. It was found statistically significant inter-group differences of the paratenon (p = 0.0001) as well as tendon thickness (p < 0.0001). Our results show that Achilles symptoms could also be associated with an increase in the paratenon thickness. We suggest that clinicians should carefully analyze paratenon thickness when evaluating patients with Achillodynia using ultrasound. It may be that the paratenon, when thickened, may explain some of the painful symptoms reported by patients and it is associated with a tendinopathy process, hence we suggest careful analysis in patients with Achillodynia.

An innovative, easily fabricated, silver nanoparticle-based titanium implant coating: development and analytical characterization.

Microbial colonization and biofilm formation on implanted devices represent an important complication in orthopaedic and dental surgery and may result in implant failure. Controlled release of antibacterial agents directly at the implant site may represent an effective approach to treat these chronic complications. Resistance to conventional antibiotics by pathogenic bacteria has emerged in recent years as a major problem of public health. In order to overcome this problem, non-conventional antimicrobial agents have been under investigation. In this study, polyacrylate-based hydrogel thin coatings have been electrosynthesised on titanium substrates starting from poly(ethylene glycol diacrylate)-co-acrylic acid. Silver nanoparticles (AgNPs) with a narrow size distribution have been synthesized using a "green" procedure and immobilized on Ti implant surfaces exploiting hydrogel coatings' swelling capabilities. The coatings have been characterized by XPS and SEM/EDX, while their silver release performances have been monitored by ICP-MS. The antibacterial activity of these AgNP-modified hydrogel coatings was tested evaluating in vitro inhibition growth of Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli, among the most common pathogens in orthopaedic infections. Moreover, a preliminary investigation of the biocompatibility of silver-loaded coatings versus MG63 human osteoblast-like cells has been performed. An important point of strength of this paper, in fact, is the concern about the effect of silver species on the surrounding cell system in implanted medical devices. Silver ion release has been properly tuned in order to assure antibacterial activity while preserving osteoblasts' response at the implant interface.

In vitro evaluation of mesenchymal stem cell isolation possibility from different intra-oral tissues.

Mesenchymal stem cells (MSCs) are of great interest for the regeneration of tissues and organs. Bone marrow is the first sources of MSCs, but in the recent years there has been interest in other tissues for the isolation of these pluripotent cells. In this study, we investigated the features of MSCs isolated from different oral regions in order to evaluate their potential application in the regeneration of damaged maxillofacial tissues. Sampling from human periodontal ligament, dental pulp, maxillary periosteum as well as bone marrow were collected in order to obtain different stem cell populations. Cells were morphologically and immunophenotipically characterized. Their proliferation potential and their ability to differentiate in osteoblasts were also assessed. All tested cell population showed a similar fibroblast-like morphology and superimposable immunophenotype. Slight differences were observed in proliferation and differentiation potential. Cells isolated from human periodontal ligament, dental pulp, maxillary periosteum had the characteristics of stem cells. Considering their peculiar feature they may alternatively represent interesting cell sources in stem cell-based bone/periodontal tissue regeneration approaches.

Antibiotic-modified hydrogel coatings on titanium dental implants.

Implant-associated infections represent an occasional but serious problem in dental and/or orthopaedic surgery. A possible solution to prevent the initial bacterial adhesion may be the coating of the implant surface with a thin layer of antibiotic-loaded biocompatible polymer. Hydrogels are one of the promising and versatile materials as antibiotic controlled release systems. In this work, antibiotic-modified poly(ethylene-glycol diacrylate) hydrogel coatings on titanium substrates were prepared by electrochemical polymerization and tested against methicillin resistant Staphylococcus aureus (ATCC 33591). Two different methods to load vancomycin and ceftriaxone were used. We show that the proposed titanium coatings displayed an interesting antibacterial activity, however, further studies on their effective cytotoxicity will furnish evidence of their real clinical efficacy.

Platelet rich fibrin matrix effects on skeletal muscle lesions: an experimental study.

Even though muscle injuries are very common, few scientific data on their effective treatment exist. Growth Factors (GFs) may have a role in accelerating muscle repair processes and a currently available strategy for their delivery into the lesion site is the use of autologous platelet-rich plasma (PRP). The present study is focused on the use of Platelet Rich Fibrin Matrix (PRFM), as a source of GFs. Bilateral muscular lesions were created on the longissimus dorsi muscle of Wistar rats. One side of the lesion was filled with a PRFM while the contralateral was left untreated (controls). Animals were sacrificed at 5, 10, 40 and 60 days from surgery. Histological, immunohistochemical and histomorphometric analyses were performed to evaluate muscle regeneration, neovascularization, fibrosis and inflammation. The presence of metaplasia zones, calcifications and heterotopic ossification were also assessed. PRFM treated muscles exhibited an improved muscular regeneration, an increase in neovascularization, and a slight reduction of fibrosis compared with controls. No differences were detected for inflammation. Metaplasia, ossification and heterotopic calcification were not detected. This preliminary morphological experimental study shows that PRFM use can improve muscle regeneration and long-term vascularization. Since autologous blood products are safe, PRFM may be a useful and handy product in clinical treatment of muscle injuries.

Synovial mast cells from knee and hip osteoarthritis: histological study and clinical correlations.

PURPOSE: The aim of this study was to investigate the presence of synovial mast cells (MCs) in hip and knee tissue from osteoarthritis (OA) patients and to correlate them with clinical and radiological data. METHODS: Synovial tissue was obtained during arthroplasty from 60 patients, 30 with knee OA and 30 with hip OA. Control synovial tissue was obtained from 30 patients without OA, 15 undergoing above-knee amputation and 15 receiving a hip replacement for fracture. Before surgery, the radiographic findings were graded according to the Kellgren-Lawrence system and clinical data including pain (VAS) and functional information (KOOS and HOOS) was collected. The tissue was stained with hematoxylin-eosin and toluidine blue for histochemistry and incubated with CD117 and CD31 antibodies for immunohistochemistry. MC and vessel number and synovitis score were determined in all samples. RESULTS: Mean MC number, synovitis score and vessel number were significantly higher in the OA samples (p < 0.05) than in control tissue. MC number correlated with the synovitis score and disease severity in both patient groups. CONCLUSIONS: The prevalence of MCs in synovium from OA patients and their association with synovial inflammation and pain suggest a role for them in OA pathophysiology.

Carboxymethyl cellulose-based hydrogel film combined with berberine as an innovative tool for chronic wound management.

Polysaccharide-based hydrogels are achieving remarkable performances in chronic wounds treatment. In this work, a carboxymethyl cellulose-based hydrogel film was developed to support skin repair. The hydrogel was loaded with berberine, a polyphenolic molecule endowing antioxidant and cytoprotective features. The film was physico-chemically characterized and in vitro tested on keratinocytes and fibroblasts subjected to oxidative stress. The biocomposite showed high thermal stability (onset decomposition temperature 245 °C) and significant fluid uptake performances, both in free conditions (up to 6510%) and under external pressure (up to 3400%). Moreover, it was able to control oxidative stress and inflammation markers involved in wound chronicity. Keratinocytes hyperproliferation, features that normally hamper injury restoration, was reduced of 25%. Our results showed that the combination of berberine and hydrogel provides a synergic improvement of the material properties. The biocomposite represents a promising candidate for dermatological applications against oxidative stress at the chronic wound site, promoting the healing process.

Osteoinduction properties of different growth factors on cells from non-union patients: in vitro study for clinical application.

This report compares the effect of rhBMPs and PRG on cells derived from human non-union sites. Treatment of non-union continues to be a challenging task for the trauma surgeon often resulting in unsatisfactory results and long-term morbidity. Over the past two decades, the possibility to use growth factors in bone regeneration has been investigated. In this study we compared the in vitro capability of two recombinant human bone morphogenetic proteins (rhBMP-2 and rhBMP-7) and activated platelet-rich plasma (PRG) to stimulate proliferation and/or differentiation of cells derived from non-union patients. Cells derived from the lesion sites, osteoblasts and mesenchymal stem cells (MSCs) derived from other bone sites of the same patients were used. Treatment with rhBMP-7 or rhBMP-2 showed an improvement in the expression of osteoblastic markers (osteonectin and osteocalcin) in cells derived from human non-union sites. This enhancement was more marked in MSCs, while no significant changes were observed in osteoblast cultures. The PRG treatment produced in all analysed samples a considerable increase in cell proliferation without affecting cell differentiation. On the basis of our results, for an effective biological treatment of non-unions, small amounts of autologous bone marrow (MSCs) are necessary in the lesion site in order to provide both growth factors and a sufficient number of responsive cells. Finally, our results prove that sequential timing administration of PRG and rhBMPs may be used in new therapeutic strategy.

Skeletal muscle repair in a rat muscle injury model: the role of growth hormone (GH) injection.

OBJECTIVE: Muscle injury tends to heal with incomplete functional recovery. Among the growth factors released in the physio-pathological response of muscle lesion, the Insulin-like Growth-Factor-1 (IGF-1) results in an engine factor of the reparation program. The therapeutic use of growth factors has been exploited to improve healing. As IGF-1 is a primary mediator of the effects of growth hormone (GH), we exploited its systemic administration to muscle recovery in a rat model of muscle injury. MATERIALS AND METHODS: Monolateral lesion of the longissimus dorsi muscle of rats was performed. Animals were divided into 5 groups: four groups for histological studies and serum hormone dosage, whilst the fifth group represented the uninjured control. Rat GH was intraperitoneally administered after 24h from the surgical lesion at three different concentrations (0.1, 0.2, 0.4 mg/kg). At 3 days from surgery, immunohistochemical and histological analyses evaluated the expression of MyoD and Myogenin, and the presence of neovascularization and inflammation, respectively. After 2 months, we analyzed the presence of muscle regeneration and fibrosis. RESULTS: The treatment with GH resulted in a significant increase in neovascularization and Myogenin expression at 24h from injury in comparison with the control. This suggested speed up biological recovery times. After two-months, a dose-dependent increase of the connective component was observed. CONCLUSIONS: The potential effect of GH on muscle repair and regeneration, through the activation of satellite cells already demonstrated in vitro, was confirmed in this in vivo experimental approach. This study sheds light on the role of growth factors in damage repair mechanisms to find an appropriate biological treatment for muscle injury.

Vitamin K and D association stimulates in vitro osteoblast differentiation of fracture site derived human mesenchymal stem cells.

There is growing interest in osteoinductive agents for fracture healing especially in patients with non-union or delayed-union fractures. The aim of the present study is the assessment of the association of Vitamins D3 and K1 on proliferation and differentiation of human mesenchymal stem cells (hMSCs) derived from fracture sites in view of a possible clinical use. The synergic effect of Vitamin D3 and Vitamin K2 in preventing osteoporosis has been documented in clinical practice; however no reports investigating this association for fracture healing are present. Our data show a different outcome on cell proliferation linked to the different timing of drug administration as well as a synergic effect of the two vitamins on cell differentiation. The high level of osteocalcin and carboxylated osteocalcin detected in hMSCs treated with the association of the two vitamins in comparison with controls and with single vitamin administration underline the differentiation of these cells into osteoblastic phenotype. Our results indicate for the first time that vitamin D3 and K1 association is able to modulate in vitro the differentiation towards osteoblastic phenotype of hMSCs derived from fracture sites, thus offering clinicians a promising and low-cost strategy for reparative osteogenesis.

Adult mesenchymal stem cells for bone and cartilage engineering: effect of scaffold materials.

Bone marrow is a useful cell source for skeletal tissue engineering approaches. In vitro differentiation of marrow mesenchymal stem cells (MSCs) to chondrocytes or osteoblasts can be induced by the addition of specific growth factors to the medium. The present study evaluated the behaviour of human MSCs cultured on various scaffolds to determine whether their differentiation can be induced by cell-matrix interactions. MSCs from bone marrow collected from the acetabulum during hip arthroplasty procedures were isolated by cell sorting, expanded and characterised by a flow cytometry system. Cells were grown on three different scaffolds (type I collagen, type I + II collagen and type I collagen + hydroxyapatite membranes) and analysed by histochemistry, immunohistochemistry and spectrophotometry (cell proliferation, alkaline phosphatase activity) at 15 and 30 days. Widely variable cell adhesion and proliferation was observed on the three scaffolds. MSCs grown on type I+II collagen differentiated to cells expressing chondrocyte markers, while those grown on type I collagen + hydroxyapatite differentiated into osteoblast-like cells. The study highlighted that human MSCs grown on different scaffold matrices may display different behaviours in terms of cell proliferation and phenotype expression without growth factor supplementation.

Pressure-activated microsyringe (PAM) fabrication of bioactive glass-poly(lactic-co-glycolic acid) composite scaffolds for bone tissue regeneration.

The aim of this work was the fabrication and characterization of bioactive glass-poly(lactic-co-glycolic acid) (PLGA) composite scaffolds mimicking the topological features of cancellous bone. Porous multilayer PLGA-CEL2 composite scaffolds were innovatively produced by a pressure-activated microsyringe (PAM) method, a CAD/CAM processing technique originally developed at the University of Pisa. In order to select the optimal formulations to be extruded by PAM, CEL2-PLGA composite films (CEL2 is an experimental bioactive SiO2 -P2 O5 -CaO-MgO-Na2 O-K2 O glass developed at Politecnico di Torino) were produced and mechanically tested. The elastic modulus of the films increased from 30 to > 400 MPa, increasing the CEL2 amount (10-50 wt%) in the composite. The mixture containing 20 wt% CEL2 was used to fabricate 2D and 3D bone-like scaffolds composed by layers with different topologies (square, hexagonal and octagonal pores). It was observed that the increase of complexity of 2D topological structures led to an increment of the elastic modulus from 3 to 9 MPa in the composite porous monolayer. The elastic modulus of 3D multilayer scaffolds was intermediate (about 6.5 MPa) between the values of the monolayers with square and octagonal pores (corresponding to the lowest and highest complexity, respectively). MG63 osteoblast-like cells and periosteal-derived precursor cells (PDPCs) were used to assess the biocompatibility of the 3D bone-like scaffolds. A significant increase in cell proliferation between 48 h and 7 days of culture was observed for both cell phenotypes. Moreover, qRT-PCR analysis evidenced an induction of early genes of osteogenesis in PDPCs. Copyright © 2015 John Wiley & Sons, Ltd.

Resin-based dentin restorative materials under accelerated ageing: bio-functional behavior.

OBJECT: The aim of the present study was the evaluation of the effect of different polishing and finishing procedures on Filtek Z250 FZ ESPE restorative material. Particularly, the consequence of artificial aging (UV-irradiation) on this resin-based dental material was investigated determining also its outcome on cell behavior. METHODS: 96 specimens of restorative material were prepared using a light emitting diode curing unit and randomly divided into four finishing and polishing groups: (I) No treatment (FZ); (II) Identoflex rubbers (ID); (III) Enhance System (EN) and (IV) Sof-Lex Pop-on XT discs (SF). The surface morphology of native and artificially aged materials was assessed with Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM). FTIR and biological (biocompatibility and bacterial adhesion) analyses were also performed. RESULTS: Among all, the ID procedure represented an acceptable compromise for efficiency of polymerization and biocompatibility both before and after artificial ageing. SF and EN techniques showed better interactions with the biological environment. CONCLUSION: UV artificial ageing of the tested specimens has shown an acceleration of the surface degrading processes, favoring a possible decrease in the mechanical properties and the release of toxic free radicals. Finishing and polishing procedure seemed to affect the photodegrading pathways, even though no differences among the techniques were observed. As the cytotoxicity of materials undergoing accelerated aging is relevant, further improvement of dental restorative materials are required to limit the long-term biological damage.

In vitro evaluation of bio-functional performances of Ghimas titanium implants.

Titanium is the most widely used material for dental implants. The natural formation, in presence of oxygen, of different oxide films (passivation films) is correlated to titanium implant biocompatibility, resistance to corrosion and is responsible for implant bacteriostatic action. Surface roughness is another surface property of Ti-implants that, affecting implant-to-bone contact, improves integration. In the present study data concerning composition, surface roughness and biocompatibility of Ghimas implants and mini-implants undergoing sandblasting with Calcium Magnesium Carbonate (CaMg(CO3)2) are reported. AFM, SEM/EDX, XRD analyses and morpho-functional tests (MTT and ALP) were performed. Cell actin cytoskeletal modification (fluorescence phalloidin staining) was also observed with confocal laser microscopy (CLSM). Data related to surface geometry and chemical properties, associated with evidence of high purity of all the tested materials (XRD and EDX), highlighted the elevated biocompatibility of tested implants and mini-implants. CLSM investigation confirmed osteoblast features of an active cell behavior able to fit cell to chemico-mechanical stimuli present at the bone/implant interface and suggests an effective implant/alveolar bone integration in vivo.

Data on glycerol/tartaric acid-based copolymer containing ciprofloxacin for wound healing applications.

This data article is related to our recently published research paper "Exploiting a new glycerol-based copolymer as a route to wound healing: synthesis, characterization and biocompatibility assessment", De Giglio et al. (Colloids and Surfaces B: Biointerfaces 136 (2015) 600-611) [1]. The latter described a new copolymer derived from glycerol and tartaric acid (PGT). Herein, an investigation about the PGT-ciprofloxacin (CIP) interactions by means of Fourier Transform Infrared Spectroscopy (FT-IR) acquired in Attenuated Total Reflectance (ATR) mode and Differential Scanning Calorimetry (DSC) was reported. Moreover, CIP release experiments on CIP-PGT patches were performed by High Performance Liquid Chromatography (HPLC) at different pH values.

HA/alginate hybrid composites prepared through bio-inspired nucleation.

Poorly crystalline apatite has been directly nucleated on self-assembling alginate chains by neutralization synthesis to obtain a biomimetic artificial bone-like composite. It has been observed that in preparing HA/alginate composites, Ca2+ ions present on the apatitic surface cross-link the alginate chains to produce a material with different morphology and thermal stability, both functions of the HA/alginate weight ratio. In vitro tests were performed on different samples in terms of both the HA/alginate ratio and synthesis temperature. All the samples were cultured for seven days with MG63 osteoblast-like cells and then underwent morphological and biochemical analyses (MTT and ALP tests). Scaffolds showed a different solubility into the culture media, which was related to the temperature of synthesis and to the HA/alginate ratio. All our data confirm the ability of the tested materials to favour cell growth and to maintain their osteoblastic functionality, at least during the examined period.

Cell dynamics in the correct control of bone metabolism using natural treatments.

The present study was undertaken in order to assess the efficacy of a commercial product containing calcium and silicon (Osteosil-Calcium) on cell metabolism. MG-63 osteblast-like cells were cultured in the presence of three different drug concentrations (10, 5 and 2.5 microg/mL). Either serum-free culture and standard culture with serum were investigated. Morpho-functional tests (MTT and ALP), scanning electron microscopy (SEM), microanalysis (EDAX) and time-lapse video microscopy were performed. Cell actin cytoskeletal modification with fluorescence phalloidin staining was also tested. Our data show the in vitro functional efficacy of Osteosil-Calcium on MG63 cell viability and ALP production. This study demonstrates its positive effect on the metabolism of the single cell and suggests wider uses of this drug in health protection and or in Regenerative Medicine therapies which are currently applied to the elderly.