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1.  Selective Augmentation of Stem Cell Populations in Structural Fat Grafts for Maxillofacial Surgery 
PLoS ONE  2014;9(11):e110796.
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.
doi:10.1371/journal.pone.0110796
PMCID: PMC4222876  PMID: 25375632
2.  Graphene based scaffolds effects on stem cells commitment 
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.
doi:10.1186/s12967-014-0296-9
PMCID: PMC4219126  PMID: 25344443
Graphene; Tissue engineering; Stem cells; Oosteogenic differentiation; Neuronal differentiation; Adipogenic differentiation
3.  Adult Stem Cells Properties in Terms of Commitment, Aging and Biological Safety of Grit-Blasted and Acid-Etched Ti Dental Implants Surfaces 
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.
PMCID: PMC4293610  PMID: 25635249
Titanium dental implants; surface properties; adipose- derived stem cells; biocompatibility; osteogenic differentiation
4.  Nanostructured Guidance for Peripheral Nerve Injuries: A Review with a Perspective in the Oral and Maxillofacial Area 
Injury to peripheral nerves can occur as a result of various surgical procedures, including oral and maxillofacial surgery. In the case of nerve transaction, the gold standard treatment is the end-to-end reconnection of the two nerve stumps. When it cannot be performed, the actual strategies consist of the positioning of a nerve graft between the two stumps. Guided nerve regeneration using nano-structured scaffolds is a promising strategy to promote axon regeneration. Biodegradable electrospun conduits composed of aligned nanofibers is a new class of devices used to improve neurite extension and axon outgrowth. Self assembled peptide nanofibrous scaffolds (SAPNSs) demonstrated promising results in animal models for central nervous system injuries, and, more recently, for peripheral nerve injury. Aims of this work are (1) to review electrospun and self-assembled nanofibrous scaffolds use in vitro and in vivo for peripheral nerve regeneration; and (2) its application in peripheral nerve injuries treatment. The review focused on nanofibrous scaffolds with a diameter of less than approximately 250 nm. The conjugation in a nano scale of a natural bioactive factor with a resorbable synthetic or natural material may represent the best compromise providing both biological and mechanical cues for guided nerve regeneration. Injured peripheral nerves, such as trigeminal and facial, may benefit from these treatments.
doi:10.3390/ijms15023088
PMCID: PMC3958900  PMID: 24562333
nanofiber; scaffold; nerve regeneration; electrospinning; self-assembly; SAPNS
5.  Silver Nanoparticles and Mitochondrial Interaction 
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.
doi:10.1155/2013/312747
PMCID: PMC3786470  PMID: 24101927
6.  Active Silver Nanoparticles for Wound Healing 
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.
doi:10.3390/ijms14034817
PMCID: PMC3634485  PMID: 23455461
silver; nanoparticles; ICP-MS; SEM; TEM; Acticoat™ Flex 3; in vivo; in vitro; cytotoxicity; mitochondrial toxicity
7.  Hyaluronic Acid Induces Activation of the κ-Opioid Receptor 
PLoS ONE  2013;8(1):e55510.
Introduction
Nociceptive pain is one of the most common types of pain that originates from an injury involving nociceptors. Approximately 60% of the knee joint innervations are classified as nociceptive. The specific biological mechanism underlying the regulation of nociceptors is relevant for the treatment of symptoms affecting the knee joint. Intra-articular administration of exogenous hyaluronic acid (HA) in patients with osteoarthritis (OA) appears to be particularly effective in reducing pain and improving patient function.
Methods
We performed an in vitro study conducted in CHO cells that expressed a panel of opioid receptors and in primary rat dorsal root ganglion (DRG) neurons to determine if HA induces the activation of opioid peptide receptors (OPr) using both aequorin and the fluorescent dye Fura-2/AM.
Results
Selective agonists and antagonists for each OPr expressed on CHO cells were used to test the efficacy of our in vitro model followed by stimulation with HA. The results showed that HA induces stimulatory effects on the κ receptor (KOP). These effects of HA were also confirmed in rat DRG neurons, which express endogenously the OPr.
Conclusions
HA activates the KOP receptor in a concentration dependent manner, with a pEC50 value of 7.57.
doi:10.1371/journal.pone.0055510
PMCID: PMC3557250  PMID: 23383210
8.  Nanostructured Surfaces of Dental Implants 
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.
doi:10.3390/ijms14011918
PMCID: PMC3565355  PMID: 23344062
adult stem cells; nanotechnologies; differentiation; osteogenesis; surfaces; dental implant
9.  Donor Age-Related Biological Properties of Human Dental Pulp Stem Cells Change in Nanostructured Scaffolds 
PLoS ONE  2012;7(11):e49146.
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.
doi:10.1371/journal.pone.0049146
PMCID: PMC3509126  PMID: 23209565
10.  Adipose Tissue Regeneration: A State of the Art 
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.
doi:10.1155/2012/462543
PMCID: PMC3488420  PMID: 23193362
12.  ALMS1-Deficient Fibroblasts Over-Express Extra-Cellular Matrix Components, Display Cell Cycle Delay and Are Resistant to Apoptosis 
PLoS ONE  2011;6(4):e19081.
Alström Syndrome (ALMS) is a rare genetic disorder (483 living cases), characterized by many clinical manifestations, including blindness, obesity, type 2 diabetes and cardiomyopathy. ALMS is caused by mutations in the ALMS1 gene, encoding for a large protein with implicated roles in ciliary function, cellular quiescence and intracellular transport. Patients with ALMS have extensive fibrosis in nearly all tissues resulting in a progressive organ failure which is often the ultimate cause of death. To focus on the role of ALMS1 mutations in the generation and maintenance of this pathological fibrosis, we performed gene expression analysis, ultrastructural characterization and functional assays in 4 dermal fibroblast cultures from ALMS patients. Using a genome-wide gene expression analysis we found alterations in genes belonging to specific categories (cell cycle, extracellular matrix (ECM) and fibrosis, cellular architecture/motility and apoptosis). ALMS fibroblasts display cytoskeleton abnormalities and migration impairment, up-regulate the expression and production of collagens and despite the increase in the cell cycle length are more resistant to apoptosis. Therefore ALMS1-deficient fibroblasts showed a constitutively activated myofibroblast phenotype even if they do not derive from a fibrotic lesion. Our results support a genetic basis for the fibrosis observed in ALMS and show that both an excessive ECM production and a failure to eliminate myofibroblasts are key mechanisms. Furthermore, our findings suggest new roles for ALMS1 in both intra- and extra-cellular events which are essential not only for the normal cellular function but also for cell-cell and ECM-cell interactions.
doi:10.1371/journal.pone.0019081
PMCID: PMC3082548  PMID: 21541333
13.  Hyaluronan and Fibrin Biomaterial as Scaffolds for Neuronal Differentiation of Adult Stem Cells Derived from Adipose Tissue and Skin 
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.
doi:10.3390/ijms12106749
PMCID: PMC3211008  PMID: 22072917
adipose derived stem cells; skin; adipose tissue; stem cells; Schwann cell; karyotypes
14.  Human Bone Marrow Mesenchymal Stem Cells Display Anti-Cancer Activity in SCID Mice Bearing Disseminated Non-Hodgkin's Lymphoma Xenografts 
PLoS ONE  2010;5(6):e11140.
Background
Although multimodality treatment can induce high rate of remission in many subtypes of non-Hodgkin's lymphoma (NHL), significant proportions of patients relapse with incurable disease. The effect of human bone marrow (BM) mesenchymal stem cells (MSC) on tumor cell growth is controversial, and no specific information is available on the effect of BM-MSC on NHL.
Methodology/Principal Findings
The effect of BM-MSC was analyzed in two in vivo models of disseminated non-Hodgkin's lymphomas with an indolent (EBV− Burkitt-type BJAB, median survival = 46 days) and an aggressive (EBV+ B lymphoblastoid SKW6.4, median survival = 27 days) behavior in nude-SCID mice. Intra-peritoneal (i.p.) injection of MSC (4 days after i.p. injection of lymphoma cells) significantly increased the overall survival at an optimal MSC∶lymphoma ratio of 1∶10 in both xenograft models (BJAB+MSC, median survival = 58.5 days; SKW6.4+MSC, median survival = 40 days). Upon MSC injection, i.p. tumor masses developed more slowly and, at the histopathological observation, exhibited a massive stromal infiltration coupled to extensive intra-tumor necrosis. In in vitro experiments, we found that: i) MSC/lymphoma co-cultures modestly affected lymphoma cell survival and were characterized by increased release of pro-angiogenic cytokines with respect to the MSC, or lymphoma, cultures; ii) MSC induce the migration of endothelial cells in transwell assays, but promoted endothelial cell apoptosis in direct MSC/endothelial cell co-cultures.
Conclusions/Significance
Our data demonstrate that BM-MSC exhibit anti-lymphoma activity in two distinct xenograft SCID mouse models of disseminated NHL.
doi:10.1371/journal.pone.0011140
PMCID: PMC2886845  PMID: 20585401
15.  Hyaluronan Benzyl Ester as a Scaffold for Tissue Engineering 
Tissue engineering is a multidisciplinary field focused on in vitro reconstruction of mammalian tissues. In order to allow a similar three-dimensional organization of in vitro cultured cells, biocompatible scaffolds are needed. This need has provided immense momentum for research on “smart scaffolds” for use in cell culture. One of the most promising materials for tissue engineering and regenerative medicine is a hyaluronan derivative: a benzyl ester of hyaluronan (HYAFF®). HYAFF® can be processed to obtain several types of devices such as tubes, membranes, non-woven fabrics, gauzes, and sponges. All these scaffolds are highly biocompatible. In the human body they do not elicit any adverse reactions and are resorbed by the host tissues. Human hepatocytes, dermal fibroblasts and keratinocytes, chondrocytes, Schwann cells, bone marrow derived mesenchymal stem cells and adipose tissue derived mesenchymal stem cells have been successfully cultured in these meshes. The same scaffolds, in tube meshes, has been applied for vascular tissue engineering that has emerged as a promising technology for the design of an ideal, responsive, living conduit with properties similar to that of native tissue.
doi:10.3390/ijms10072972
PMCID: PMC2738906  PMID: 19742179
HYAFF; hyaluronan; tissue engineering
16.  Characteristics of repair tissue in second-look and third-look biopsies from patients treated with engineered cartilage: relationship to symptomatology and time after implantation 
Arthritis Research & Therapy  2008;10(6):R132.
Introduction
The present study established characteristics of tissue regrowth in patients suffering knee lesions treated with grafts of autologous chondrocytes grown on three-dimensional hyaluronic acid biomaterials.
Methods
This multicentred study involved a second-look arthroscopy/biopsy, 5 to 33 months post implant (n = 63). Seven patients allowed a third-look biopsy, three of which were performed 18 months post implant. Characteristics of tissues were histologically and histochemically evaluated. The remaining bone stubs were evaluated for cartilage/bone integration. For data analysis, biopsies were further divided into those obtained from postoperative symptomatic patients (n = 41) or from asymptomatic patients (n = 22).
Results
The percentage of hyaline regenerated tissues was significantly greater in biopsies obtained after, versus within, 18 months of implantation. Differences were also observed between symptomatic and asymptomatic patients: reparative tissues taken from symptomatic patients 18 months after grafting were mainly fibrocartilage or mixed (hyaline–fibrocartilage) tissue, while tissues taken from asymptomatic patients were hyaline cartilage in 83% of biopsies. In a small group of asymptomatic patients (n = 3), second-look and third-look biopsies taken 18 months after surgery confirmed maturation of the newly formed tissue over time. Cartilage maturation occurred from the inner regions of the graft, in contact with subchondral bone, towards the periphery of the implant.
Conclusions
The study indicates that, in asymptomatic patients after chondrocyte implantation, regenerated tissue undergoes a process of maturation that in the majority of cases takes longer than 18 months for completion and leads to hyaline tissue and not fibrous cartilage. Persistence of symptoms might reflect the presence of a nonhyaline cartilage repair tissue.
doi:10.1186/ar2549
PMCID: PMC2656234  PMID: 19014452

Results 1-16 (16)