The combination of bone grafting materials with guided bone regeneration (GBR) membranes seems to provide promising results to restore bone defects in dental clinical practice. In the first part of this work, a novel protocol for decellularization and delipidation of bovine bone, based on multiple steps of thermal shock, washes with detergent and dehydration with alcohol, is described. This protocol is more effective in removal of cellular materials, and shows superior biocompatibility compared to other three methods tested in this study. Furthermore, histological and morphological analyses confirm the maintenance of an intact bone extracellular matrix (ECM). In vitro and in vivo experiments evidence osteoinductive and osteoconductive properties of the produced scaffold, respectively. In the second part of this study, two methods of bovine pericardium decellularization are compared. The osmotic shock-based protocol gives better results in terms of removal of cell components, biocompatibility, maintenance of native ECM structure, and host tissue reaction, in respect to the freeze/thaw method. Overall, the results of this study demonstrate the characterization of a novel protocol for the decellularization of bovine bone to be used as bone graft, and the acquisition of a method to produce a pericardium membrane suitable for GBR applications.
Endometriosis is a degenerative process due to a chronic inflammatory damage leading to extracellular matrix components deposition and glandular fibrosis. It is known that mesenchymal stem cells secrete a wide range of bioactive molecules, some of them modulating the immune inflammatory response, and others providing regeneration and remodeling of injured tissue. We have performed in vitro experiments in order to analyze the capability of allogenic equine adipose-derived stem cells (ADSCs) to infiltrate mares' endometrial tissues and to stimulate the expression of cytokines and metallopeptidases. Differences in the biologic response to the exposure to ADSCs between pathological and healthy endometrial tissue have been identified. These results could challenge researchers to progress forward with future studies for the development of a biological therapy with a possible application in translational medicine.
Dental pulp tissue supports the vitality of the tooth, but it is particularly vulnerable to external insults, such as mechanical trauma, chemical irritation or microbial invasion, which can lead to tissue necrosis. In the present work, we present an endodontic regeneration method based on the use of a tridimensional (3D) hyaluronan scaffold and human dental pulp stem cells (DPSCs) to produce a functional dental pulp-like tissue in vitro. An enriched population of DPSCs was seeded onto hyaluronan-based non-woven meshes in the presence of differentiation factors to induce the commitment of stem cells to neuronal, glial, endothelial and osteogenic phenotypes. In vitro experiments, among which were gene expression profiling and immunofluorescence (IF) staining, proved the commitment of DPSCs to the main components of dental pulp tissue. In particular, the hyaluronan-DPSCs construct showed a dental pulp-like morphology consisting of several specialized cells growing inside the hyaluronan fibers. Furthermore, these constructs were implanted into rat calvarial critical-size defects. Histological analyses and gene expression profiling performed on hyaluronan-DPSCs grafts showed the regeneration of osteodentin-like tissue. Altogether, these data suggest the regenerative potential of the hyaluronan-DPSC engineered tissue.
hyaluronan; stem cells; human dental pulp stem cells (DPSCs); dental pulp; tissue engineering; regenerative endodontics
Background: Lipomas are benign solid tumours that develop in soft tissues with origin in mesenchymal progenitors. Macroscopically, they appear as soft-elastic nodules, varying in volume from a few millimiters to several centimetres and can enlarge progressively. Although they are usually asymptomatic, they can cause symptoms due to nerve or vessel compression. Microscopically they appear as fibrous connective tissue stroma with embedded adipocytes, and absence of inflammation. Up to now no characterisation of stem cell population present in this tissue has been performed.
Methods: Cytofluorimetric, biological and molecular biology analyses have been performed in order to test superficial cell markers and gene expression profile related to stemness and apoptotic activity of cells present in lipoma tissues compared to those of adipose tissue's cells.
Results: Our results confirmed that CD34+ cells in lipoma were present around small adipocytes, showing several altered biological activity such as proliferation, apoptotis and stemness.
Conclusions: The data emerging from the comparison of the lipoma cells and normal adipose tissue, suggests the presence of cell precursors involved in the development of the lipoma. This hypothesis requires further investigation and may indicate new thresholds in the study of benign tumour pathogenesis.
CD34; lipoma; cancer stem cells.
Structural fat grafting utilizes the centrifugation of liposuction aspirates to create a graded density of adipose tissue. This study was performed to qualitatively investigate the effects of centrifugation on stem cells present in adipose tissue. Liposuction aspirates were obtained from healthy donors and either not centrifuged or centrifuged at 1,800 rpm for 3 minutes. The obtained fat volumes were divided into three layers and then analyzed. The results demonstrate that centrifugation induces a different distribution of stem cells in the three layers. The high-density layer displays the highest expression of mesenchymal stem cell and endothelial markers. The low-density layer exhibits an enrichment of multipotent stem cells. We conclude that appropriate centrifugation concentrates stem cells. This finding may influence the clinical practice of liposuction aspirate centrifugation and enhance graft uptake.
Graphene is a flat monolayer of carbon atoms, arranged in a two-dimensional hexagonal structure, with extraordinary electrical, thermal, and physical properties. Moreover, the molecular structure of graphene can be chemically modified with molecules of interest to promote the development of high-performance devices. Although carbon derivatives have been extensively employed in industry and electronics, their use in regenerative medicine is still in an early phase. Study prove that graphene is highly biocompatible, has low toxicity and a large dosage loading capacity. This review describes the ability of graphene and its related materials to induce stem cells differentiation into osteogenic, neuronal, and adipogenic lineages.
Graphene; Tissue engineering; Stem cells; Oosteogenic differentiation; Neuronal differentiation; Adipogenic differentiation
Titanium (Ti) is one of the most widely used biomaterials for manufacturing dental implants. The implant surface properties strongly influence osseointegration. The aim of the present study was to in vitro investigate the characteristics of Ti dental implants in terms of mutagenicity, hemocompatibility, biocompatibility, osteoinductivity and biological safety. The Ames test was used to test the mutagenicity of the Ti dental implants, and the hemolysis assay for evaluating their hemocompatibility. Human adipose - derived stem cells (ADSCs) were then seeded onto these implants in order to evaluate their cytotoxicity. Gene expression analyzing with real-time PCR was carried out to investigate the osteoinductivity of the biomaterials. Finally, the genetic stability of the cells cultured onto dental implants was determined by karyotyping. Our results demonstrated that Ti dental implants are not mutagenic, do not cause hemolysis, and are biocompatible. The MTT assay revealed that ADSCs, seeded on Ti dental implants, proliferate up to 30 days in culture. Moreover, ADSCs loaded on Ti dental implants show a substantial expression of some osteoblast specific markers, such as COL1A1, OPN, ALPL, and RUNX2, as well as chromosomal stability after 30 days of culture in a medium without osteogenic factors. In conclusion, the grit-blasted and acid-etched treatment seems to favor the adhesion and proliferation of ADSCs and improve the osteoinductivity of Ti dental implant surfaces.
Titanium dental implants; surface properties; adipose- derived stem cells; biocompatibility; osteogenic differentiation
Nanotechnology has gone through a period of rapid growth, thus leading to the constant increase in the application of engineered nanomaterials in daily life. Several different types of nanoparticles have been engineered to be employed in a wide array of applications due to their high surface to volume ratio that leads to unique physical and chemical properties. So far, silver nanoparticles (AgNps) have been used in many more different medical devices than any other nanomaterial, mainly due to their antimicrobial properties. Despite the promising advantages posed by using AgNps in medical applications, the possible health effects associated with the inevitable human exposure to AgNps have raised concerns as to their use since a clear understanding of their specific interaction with biological systems has not been attained yet. In light of such consideration, aim of the present work is the morphological analysis of the intracellular behavior of AgNps with a diameter of 10 nm, with a special attention to their interaction with mitochondria.
In this preliminary study, the silver nanoparticle (Ag NP)-based dressing, Acticoat™ Flex 3, has been applied to a 3D fibroblast cell culture in vitro and to a real partial thickness burn patient. The in vitro results show that Ag NPs greatly reduce mitochondrial activity, while cellular staining techniques show that nuclear integrity is maintained, with no signs of cell death. For the first time, transmission electron microscopy (TEM) and inductively coupled plasma mass spectrometry (ICP-MS) analyses were carried out on skin biopsies taken from a single patient during treatment. The results show that Ag NPs are released as aggregates and are localized in the cytoplasm of fibroblasts. No signs of cell death were observed, and the nanoparticles had different distributions within the cells of the upper and lower dermis. Depth profiles of the Ag concentrations were determined along the skin biopsies. In the healed sample, most of the silver remained in the surface layers, whereas in the unhealed sample, the silver penetrated more deeply. The Ag concentrations in the cell cultures were also determined. Clinical observations and experimental data collected here are consistent with previously published articles and support the safety of Ag NP-based dressing in wound treatment.
silver; nanoparticles; ICP-MS; SEM; TEM; Acticoat™ Flex 3; in vivo; in vitro; cytotoxicity; mitochondrial toxicity
The structural and functional fusion of the surface of the dental implant with the surrounding bone (osseointegration) is crucial for the short and long term outcome of the device. In recent years, the enhancement of bone formation at the bone-implant interface has been achieved through the modulation of osteoblasts adhesion and spreading, induced by structural modifications of the implant surface, particularly at the nanoscale level. In this context, traditional chemical and physical processes find new applications to achieve the best dental implant technology. This review provides an overview of the most common manufacture techniques and the related cells-surface interactions and modulation. A Medline and a hand search were conducted to identify studies concerning nanostructuration of implant surface and their related biological interaction. In this paper, we stressed the importance of the modifications on dental implant surfaces at the nanometric level. Nowadays, there is still little evidence of the long-term benefits of nanofeatures, as the promising results achieved in vitro and in animals have still to be confirmed in humans. However, the increasing interest in nanotechnology is undoubted and more research is going to be published in the coming years.
adult stem cells; nanotechnologies; differentiation; osteogenesis; surfaces; dental implant
The aim of the present work is to study how biological properties, such as proliferation and commitment ability, of human adult dental pulp stem cells (DPSCs) relate to the age of the donor. Human dental pulps were extracted from molars of healthy adult subjects aged 16 to >66 years. DPSCs were isolated and cultured in the presence of osteogenic, neurogenic, or vasculogenic differentiation medium. Proliferation ability was evaluated by determining doubling time, and commitment ability was evaluated by gene expression and morphological analyses for tissue-specific markers. The results confirm a well-defined proliferative ability for each donor age group at an early in vitro passage (p2). DPSCs from younger donors (up to 35 years) maintain this ability in long-term cultures (p8). Stem cells of all age donor groups maintain their commitment ability during in vitro culture. In vivo tests on the critical size defect repair process confirmed that DPSCs of all donor ages are a potent tool for bone tissue regeneration when mixed with 3D nanostructured scaffolds.
Adipose tissue pathologies and defects have always represented a reconstructive challenge for plastic surgeons. In more recent years, several allogenic and alloplastic materials have been developed and used as fillers for soft tissue defects. However, their clinical use has been limited by further documented complications, such as foreign-body reactions potentially affecting function, degradation over time, and the risk for immunogenicity. Tissue-engineering strategies are thus being investigated to develop methods for generating adipose tissue. This paper will discuss the current state of the art in adipose tissue engineering techniques, exploring the biomaterials used, stem cells application, culture strategies, and current regulatory framework that are in use are here described and discussed.
Recently, we have described a simple protocol to obtain an enriched culture of adult stem cells organized in neurospheres from two post-natal tissues: skin and adipose tissue. Due to their possible application in neuronal tissue regeneration, here we tested two kinds of scaffold well known in tissue engineering application: hyaluronan based membranes and fibrin-glue meshes. Neurospheres from skin and adipose tissue were seeded onto two scaffold types: hyaluronan based membrane and fibrin-glue meshes. Neurospheres were then induced to acquire a glial and neuronal-like phenotype. Gene expression, morphological feature and chromosomal imbalance (kariotype) were analyzed and compared. Adipose and skin derived neurospheres are able to grow well and to differentiate into glial/neuron cells without any chromosomal imbalance in both scaffolds. Adult cells are able to express typical cell surface markers such as S100; GFAP; nestin; βIII tubulin; CNPase. In summary, we have demonstrated that neurospheres isolated from skin and adipose tissues are able to differentiate in glial/neuron-like cells, without any chromosomal imbalance in two scaffold types, useful for tissue engineering application: hyaluronan based membrane and fibrin-glue meshes.
adipose derived stem cells; skin; adipose tissue; stem cells; Schwann cell; karyotypes
Interactions between Z-disc proteins regulate muscle functions and disruption of these interactions results in muscle disorders. Mutations in Z-disc components myotilin, ZASP/Cypher, and FATZ-2 (calsarcin-1/myozenin-2) are associated with myopathies. We report here that the myotilin and the FATZ (calsarcin/myozenin) families share high homology at their final C-terminal five amino acids. This C-terminal E[ST][DE][DE]L motif is present almost exclusively in these families and is evolutionary conserved. We show by in vitro and in vivo studies that proteins from the myotilin and FATZ (calsarcin/myozenin) families interact via this novel type of class III PDZ binding motif with the PDZ domains of ZASP/Cypher and other Enigma family members: ALP, CLP-36, and RIL. We show that the interactions can be modulated by phosphorylation. Calmodulin-dependent kinase II phosphorylates the C terminus of FATZ-3 (calsarcin-3/myozenin-3) and myotilin, whereas PKA phosphorylates that of FATZ-1 (calsarcin-2/myozenin-1) and FATZ-2 (calsarcin-1/myozenin-1). This is the first report of a binding motif common to both the myotilin and the FATZ (calsarcin/myozenin) families that is specific for interactions with Enigma family members.
The Gene Ontology Project provides structured controlled vocabularies for molecular biology that can be used for the functional annotation of genes and gene products. In a collaboration between the Gene Ontology (GO) Consortium and the muscle biology community, we have made large-scale additions to the GO biological process and cellular component ontologies. The main focus of this ontology development work concerns skeletal muscle, with specific consideration given to the processes of muscle contraction, plasticity, development, and regeneration, and to the sarcomere and membrane-delimited compartments. Our aims were to update the existing structure to reflect current knowledge, and to resolve, in an accommodating manner, the ambiguity in the language used by the community.
The updated muscle terminologies have been incorporated into the GO. There are now 159 new terms covering critical research areas, and 57 existing terms have been improved and reorganized to follow their usage in muscle literature.
The revised GO structure should improve the interpretation of data from high-throughput (e.g. microarray and proteomic) experiments in the area of muscle science and muscle disease. We actively encourage community feedback on, and gene product annotation with these new terms. Please visit the Muscle Community Annotation Wiki .