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Tissue engineering represents a novel approach that aims to exploit the use of biomaterials composed mainly of scaffolds, cells (or grafts), and growth factors capable of restoring a specific tissue. Biomaterials represent the future of dental and oral regeneration due to their biocompatibility and affinity with the receiving site. The aim of this review was to collect results and considerations about a new type of biomaterial based on the use of micrografts in combination with different scaffolds. Micrografts are tissue particles enriched with progenitor cells (PCs), which are defined as descendants of stem cells that can differentiate into specialized cells belonging to the same tissue. PCs in the oral cavity might be extracted from various tissues such as dental pulp, periosteum, or periodontal ligament. Moreover, these cells are easy to isolate through a mechanical process that allows for the filtration of cells with a diameter of 80 μm, in contrast with enzymatic procedures where reagents are used and various culture periods are needed. The aim of this review was to collect data regarding the use of micrografts, developed by a Rigenera® chair-side machine, in oral regeneration evaluating the clinical, histological, and radiographical outcomes. There have been encouraging results for the application of micrografts in bone and periodontal regeneration, but further randomized clinical trials are needed to validate this promising outcome.

Today exist several techniques and processing, which permit one to obtain skin grafts, micrografts, skin substitutes offering a wide choice to clinicians in relation to needs of the patients. The techniques to obtain skin grafts are well established and still are still constantly developed to improve the healing of wounds or trauma in different areas of body. In the western world, the first techniques of skin grafting date back to 1804 and at the end of this century were better described as the methods of full-thickness grafting. In the early twentieth century, different authors described the skin expansion methods until being able to culture human keratinocytes. Dermal and epidermal grafts are constituted by a variable amount of both dermis and full-thickness skin providing a major resurfacing and stability to the wound. These grafts can be obtained through different tools, including the Meek-Waal dermatome, the flypaper technique, skin expansion with meshers, Xpansion® system, fractional skin harvesting, suction blister epidermal grafting, providing good results in terms of wound healing and re-epithelialization. In the last years, the marketing of innovative tools, such as CelluTome™ Epidermal Harvesting Device, Recell® and Rigenera® technology, offered to clinicians the advantage to reduce the execution time for skin grafting, improving the wound healing in terms of time and quality of life for the patients. Finally, the tissue engineering certainly contributed to ameliorate the field of regenerative medicine, creating engineered skin substitutes adaptable to every clinical need.

The aim of this chapter is to provide to the readers clinical evidences on the application of autologous micrografts, intended as a cell suspension obtained by mechanical disaggregation of different human tissues using the Rigenera® protocol. The human tissues mostly used to obtain micrografts are dental pulp, periosteum, or skin, all tissues easily accessible during the clinical routine of several clinicians, including, for example, plastic and general surgeons or dentists. In particular, the authors provide evidences based on their personal experience or based on the collaboration with other clinicians who wanted to test the effects of autologous micrografts obtained by Rigenera® protocol in their clinical practices. For this reason, in the different sections of this chapter are described the clinical applications of micrografts in the dentistry, wound care, and aesthetic medicine field, to show the wide versatility in the use of micrografts that can be applied alone or in combination with the most common scaffolds such as collagen and polylactic-co-glycolic acid (PLGA).

Human population is facing a revolutionary change in the demographic structure with an increasing number of elderly people requiring an unmet need to ensure a smooth aging process and dental care is certainly an important aspect that has to be considered. To date, dentistry has been conservative and the need of transferring the scientific models of regenerative dentistry into clinical practice is becoming a necessity. The aim of this study was to characterize the differentiation commitment (in vitro) and the clinical grafting ability (in vivo) of a population of progenitor stem cells obtained after mechanical digestion of dental pulp with an innovative system recently developed. This approach was successfully used in previous studies to obtain a clinical-grade ready to use dental pulp fragments that could be grafted in autologous tissues to obtain bone. We are thus showing that micro grafts resulting from mechanical digestion contain stem cells with a mesenchymal phenotype, able to differentiate toward different cell types and to generate new bone in patients. We are providing data for the establishment of standardized and routinely oral surgery approaches, having outlined the cellular properties of human stem cells obtained from the dental pulp. This method can represent a valid tool for both regenerative medicine and tissue engineering purposes not only applicable to the cranio-maxillofacial region but, likely, to different bone pathologies for a fastening and healing recovering of patients. J. Cell. Physiol. 232: 548-555, 2017. © 2016 Wiley Periodicals, Inc.

The aim of this study was to investigate the effect of the use of autologous micrografts obtained by the Rigenera&reg, Micrografting Technology and xenograft on critical size defects created in the calvaria of rats. Forty-eight rats were randomly divided into four groups for each of the two evaluation times (15 and 30 days) (n = 6). After general anesthesia, a 5-mm diameter bone defect was created in the calvaria of each animal. Each defect was filled with the following materials: blood clot, autologous bone graft, xenograft, and xenograft associated with autologous micrografts. Histomorphometric and histological analysis showed that the group that have received the Rigenera&reg, processed autologous micrografts combined with the xenograft and the group that received autologous bone graft resulted in greater bone formation in both time points when compared with the use of the xenograft alone and blood clot.

Human tissue regeneration, especially the bone, today is one of the most important chal- lenges for medicine and the need for this is particularly evident in the maxillo-facial area where it can be estimated that 1,500,000 patients in Europe undergo craniofacial reconstruction each year. Autolo- gous bone is considered as the gold standard of bone graft materials, however, this approach is very limited. Recent research of non-embryonic stem cells provides new possibilities for no invasively ob- taining new autologous bone from stem cells provided by various tissues from the same patient. Furthermore, in the litera- ture, there are limited long-term data available mainly on the safe and efficacy of the prolonged use of stem cells. In this review, we will summarize the studies conducted on the regeneration, repair and rebuilding of craniofacial tissues using stem cells both in the presence or the absence of implantable biocompatible materials and the use of new micro-graft technologies, obtained through the Rigeneracons® medical device.

Mesenchymal stem cell (MSC) therapy holds promise for treating diseases and tissue repair. Regeneration of skeletal muscle tissue that is lost during pathological muscle degeneration or after injuries is sustained by the production of new myofibers. Human Adipose stem cells (ASCs) have been reported to regenerate muscle fibers and reconstitute the pericytic cell pool after myogenic differentiation in vitro. Our aim was to evaluate the differentiation potential of constructs made from a new cross-linked hyaluronic acid (XHA) scaffold on which different sorted subpopulations of ASCs were loaded. Thirty days after engraftment in mice, we found that NG2(+) ASCs underwent a complete myogenic differentiation, fabricating a human skeletal muscle tissue, while NG2(-) ASCs merely formed a human adipose tissue. Myogenic differentiation was confirmed by the expression of MyoD, MF20, laminin, and lamin A/C by immunofluorescence and/or RT-PCR. In contrast, adipose differentiation was confirmed by the expression of adiponectin, Glut-4, and PPAR-γ. Both tissues formed expressed Class I HLA, confirming their human origin and excluding any contamination by murine cells. In conclusion, our study provides novel evidence that NG2(+) ASCs loaded on XHA scaffolds are able to fabricate a human skeletal muscle tissue in vivo without the need of a myogenic pre-differentiation step in vitro. We emphasize the translational significance of our findings for human skeletal muscle regeneration.

Dental pulp stem cells (DPSCs) are multipotent stem cells derived from neural crest and mesenchyme and have the capacity to differentiate into multiple cell lineages. It has already been demonstrated that DPSCs differentiate into melanocyte-like cells but only when cultivated in a specific melanocyte differentiating medium. In this study we have shown, for the first time, that DPSCs are capable of spontaneously differentiating into mature melanocytes, which display molecular and ultrastructural features of full development, including the expression of melanocyte specific markers and the presence of melanosomes up to the terminal stage of maturation. We have also compared the differentiating features of DPSCs grown in different culture conditions, following the timing of differentiation at molecular and cytochemical levels and found that in all culture conditions full development of these cells was obtained, although at different times. The spontaneous differentiating potential of these cells strongly suggests their possible applications in regenerative medicine.

Bone Tissue Engineering (BTE) and Dental Implantology (DI) require the integration of implanted structures, with well characterized surfaces, in bone. In this work we have challenged acid-etched titanium (AET) and Laser Sintered Titanium (LST) surfaces with either human osteoblasts or stem cells from human dental pulps (DPSCs), to understand their osteointegration and clinical use capability of derived implants. DPSCs and human osteoblasts were challenged with the two titanium surfaces, either in plane cultures or in a roller apparatus within a culture chamber, for hours up to a month. During the cultures cells on the titanium surfaces were examined for histology, protein secretion and gene expression. Results show that a complete osteointegration using human DPSCs has been obtained: these cells were capable to quickly differentiate into osteoblasts and endotheliocytes and, then, able to produce bone tissue along the implant surfaces. Osteoblast differentiation of DPSCs and bone morphogenetic protein production was obtained in a better and quicker way, when challenging stem cells with the LST surfaces. This successful BTE in a comparatively short time gives interesting data suggesting that LST is a promising alternative for clinical use in DI. © 2010 Elsevier Ltd. All rights reserved.

In this study we used a biocomplex constructed from dental pulp stem/progenitor cells (DPCs) and a collagen sponge scaffold for oro-maxillo-facial (OMF) bone tissue repair in patients requiring extraction of their third molars. The experiments were carried out according to our Internal Ethical Committee Guidelines and written informed consent was obtained from the patients. The patients presented with bilateral bone reabsorption of the alveolar ridge distal to the second molar secondary to impaction of the third molar on the cortical alveolar lamina, producing a defect without walls, of at least 1.5 cm in height. This clinical condition does not permit spontaneous bone repair after extraction of the third molar, and eventually leads to loss also of the adjacent second molar. Maxillary third molars were extracted first for DPC isolation and expansion. The cells were then seeded onto a collagen sponge scaffold and the obtained biocomplex was used to fill in the injury site left by extraction of the mandibular third molars. Three months after autologous DPC grafting, alveolar bone of patients had optimal vertical repair and complete restoration of periodontal tissue back to the second molars, as assessed by clinical probing and X-rays. Histological observations clearly demonstrated the complete regeneration of bone at the injury site. Optimal bone regeneration was evident one year after grafting. This clinical study demonstrates that a DPC/collagen sponge biocomplex can completely restore human mandible bone defects and indicates that this cell population could be used for the repair and/or regeneration of tissues and organs.

Harvesting bone for autologous grafting is a daily problem encountered by craniofacial and oral surgeons. Stem cells derived from human dental pulp are able to differentiate in osteoblasts and are a potential source of autologous bone produced in vitro. However, as stem cells are characterized by self-renewing and commitment in several cellular subtypes (ie, pluripotential differentiation), some concerns may arise as regards their potential uncontrolled proliferation. To screen the behavior of osteoblasts derived from human pulpar stem cells (ODHPSCs), we used microarray techniques to identify genes that are differently regulated in ODHPSC in comparison to normal osteoblasts (NOs). Osteoblasts derived from human pulpar stem cells were obtained from human dental pulp, and cells were selected using a cytometer. The cell profile was c-kit+/CD34+/STRO-1+/CD45−. These cells were capable of differentiation of osteoblasts in vitro. By using DNA microarrays containing 19,200 genes, we identified in ODHPSC some genes whose expression was significantly up- and downregulated compared to NO. The differentially expressed genes have different functional activities: (a) cell differentiation, (b) developmental maturation, (c) cell adhesion, and (d) production of cytoskeleton elements. Thus, some molecular differences exist between NO and ODHPSC, although the previously considered histologic parameters show a normal phenotype.

Pancreatic islets are commonly isolated for research and transplantation without taking into consideration that they undergo mechanical or chemical stress during this process. In order to counteract both types of injuries, the compound AEOL10150, a novel MnSOD mimic, was added during isolation of islet at concentrations ranging from 18 to 100 µM. Mechanical or chemical stress-related pro-apoptotic signals were then studied. We demonstrate that this MnSOD mimic diminishes the negative effects of mechanical stress by blocking insulin impairment, production of non-specific islet β-cell proteins, transcription of iNOS and FAS, activation of caspase-3 and -9 and, ultimately, apoptosis. Moreover, the effects of the MnSOD mimic on isolated islets were greatly influenced by dosage: the best dose able to fully counteract mechanical stress was found to be 100 µM; doses ≥150 µM were themselves highly toxic for islet cells. On the other hand, rIL-1β-induced chemical stress is rather complex, and there was no protection in this scenario. Therefore, contrarily to what has been previously reported, MnSOD mimic administration is only capable of counteracting mechanical stress, and not cytokine-induced cytotoxicity, and that this drug acts within a limited concentration range. J. Cell. Physiol. 212: 432–438, 2007. © 2007 Wiley-Liss, Inc.

Human population is facing a revolutionary change in the demographic structure with an increasing number of elderly people requiring an unmet need to ensure a smooth aging process and dental care is certainly an important aspect that has to be considered. To date, dentistry has been conservative and the need of transferring the scientific models of regenerative dentistry into clinical practice is becoming a necessity. The aim of this study was to characterize the differentiation commitment (in vitro) and the clinical grafting ability (in vivo) of a population of progenitor stem cells obtained after mechanical digestion of dental pulp with an innovative system recently developed. This approach was successfully used in previous studies to obtain a clinical-grade ready to use dental pulp fragments that could be grafted in autologous tissues to obtain bone. We are thus showing that micro grafts resulting from mechanical digestion contain stem cells with a mesenchymal phenotype, able to differentiate toward different cell types and to generate new bone in patients. We are providing data for the establishment of standardized and routinely oral surgery approaches, having outlined the cellular properties of human stem cells obtained from the dental pulp. This method can represent a valid tool for both regenerative medicine and tissue engineering purposes not only applicable to the cranio-maxillofacial region but, likely, to different bone pathologies for a fastening and healing recovering of patients. J. Cell. Physiol. 232: 548-555, 2017. © 2016 Wiley Periodicals, Inc.

The Bio Breeding (BB) rat is a useful animal model of type 1 autoimmune diabetes. The aim of this study was to observe and follow the cytokine and antigenic expressions within the islets of Langerhans in young non-diabetic, in pre-diabetic hyperglycemic, and in overtly diabetic animals. BB rats were therefore checked at day 21 up to day 90 of life for blood glucose, insulin levels, degree of islet infiltration, expression of proinflammatory and protective cytokines and antibodies including CD4, CD8, CD25, LFA-1, and ICAM-1. Animals were treated with insulin as they became diabetic. We found that islets of non-diabetic BB rats became positive to both IL-1beta and IL-4 very early on, confirming a local but intense production of both cytokines within the islets during the initial non-diabetic period. In addition, we observed that the production of these interleukins together with the expression levels of CD4 and CD25 are events predictive for type 1 diabetes onset in non-diabetic BB rats, as for non-obese diabetic (NOD) mice. In particular, the production of IL-1beta and IL-4 during the non-diabetic period together with the lack of enhancement of CD4 and CD25, indicating selective recruitment of activated T cells, may explain the failure of anti-diabetic treatments in this animal model of type 1 diabetes.

Stem cells, derived from human adult dental pulp of healthy subjects 30-45 years of age, were cultured, and cells were selected using a FACSorter. A new c-kit+/CD34+/CD45− cell population of stromal bone producing cells (SBP/DPSCs) was selected, expanded, and cultured. These SBP/DPSCs are highly clonogenic and, in culture, differentiate into osteoblast precursors (CD44+/RUNX-2+), still capable of self-renewing, and then in osteoblasts, producing, in vitro, a living autologous fibrous bone (LAB) tissue, which is markedly positive for several bone antibodies. This tissue constitute an ideal source of osteoblasts and mineralized tissue for bone regeneration. In fact, after in vivo transplantation into immunocompromised rats, LAB formed lamellar bone-containing osteocytes. Introduction: Recently it has been reported that human dental pulp stem cells (DPSCs) are detectable, in humans, only up to the age of 30 years and that they are able to produce in vitro only sporadic calcified nodules and to form, after transplantation in vivo, a mineralized tissue. Materials and Methods: Stem cells, derived from human adult dental pulp of healthy subjects 30-45 years of age, were cultured, and cells were selected using a FACSorter. Light microscope, histochemistry, immunofluorescence, and RT-PCR analyses were performed to study both stem and differentiating cells. Results and Conclusions: A new c-kit+/CD34+/CD45− cell population of stromal bone producing cells (SBP/DPSCs) has been selected by FACSorting, expanded, and cultured. These SBP/DPSCs are highly clonogenic and, in culture, differentiate into osteoblast precursors (CD44+/RUNX-2+), still capable of self-renewing, and in osteoblasts, producing, in vitro, a living autologous fibrous bone (LAB) tissue. This new-formed tissue is markedly positive for several antibodies for bone, including osteonectin, bone sialoprotein, osteocalcin, fibronectin, collagen III, and bone alkaline phosphatase (BALP). Cells producing LAB can be stored at −80°C for a long period of time and are an extraordinary source of osteoblasts and mineralized fibrous bone tissue. In this study, we also showed that, in aged humans, stem cells can be detected from their pulps. The produced LAB is a fibrous bone tissue resembling the human bone during mineralization, with an external layer formed by osteoblasts markedly positive for osteocalcin. This newly formed tissue constitute an ideal source of osteoblasts and mineralized tissue for bone regeneration. In fact, after in vivo transplantation into immunocompromised rats, LAB formed lamellar bone containing osteocytes.

la mostra ed il catalogo relativo raccolgono gli esiti della call for proposals "luoghi dell'archeologia e usi contemporanei" organizzata nel 2009 dall'area di ricerca architettura e archeologia dello IUAV di Venezia in occasione dell'omonimo convegno internazionale. I 16 progetti selezionati tra le 130 proposte inviate da architetti italiani ed internazionali, tra i quali un progetto dell'autore Luigi Franciosini, offrono un contributo alla definizione di strategie per la valorizzazione di siti archeologici.

Le pubblicazioni di Luigi Franciosini dal titolo "Mercati di Traiano in Roma: l'asse di strutturazione orizzontale e il recupero della via Biberatica" e "La riqualificazione urbana della zona dell'antico Lavatoio a Sutri" sono raccolte nel "Trattato di restauro architettonico", volume secondo, Il progetto di restauro e il cantiere, curato da Giovanni Carbonara per i tipi UTET. I due interventi, descritti da grafici di progetto e da immagini del realizzato, vengono proposti dal curatore, quali modelli esemplari di rilettura di contesti archeologici e storico-monumentali nell'ambito di adeguamenti architettonici indirizzati alla migliore fruizioni e valorizzazione dei siti. Il restauro e la sistemazione museale dell'area archeologica dei Mercati di Traiano e del giardino delle Milizie ha avuto come obiettivo principale quello di conciliare i caratteri di un monumento storico e artistico con i requisiti di un centro archeologico museale dotato di servizi tecnologici, didattici, di ricezione, di consultazione ed accessibile ad un'utenza ampliata. In questo quadro il progetto di architettura assume il ruolo di strumento diretto a mediare le istanze di conservazione con quelle di ammodernamento e di adeguamento alle nuove esigenze.

none 11 si Abstract Mesenchymal stem cells deriving from dental pulp differentiate into osteoblasts capable of producing bone. In previous studies, we extensively demonstrated that, when seeded on collagen I scaffolds, these cells can be conveniently used for the repair of human mandible defects. Here, we assess the stability and quality of the regenerated bone and vessel network 3 years after the grafting intervention, with conventional procedures and in-line holotomography, an advanced phase-imaging method using synchrotron radiation that offers improved sensitivity toward low-absorbing structures. We found that the regenerated tissue from the graft sites was composed of a fully compact bone with a higher matrix density than control human alveolar spongy bone from the same patient. Thus, the regenerated bone, being entirely compact, is completely different from normal alveolar bone. Although the bone regenerated at the graft sites is not of the proper type found in the mandible, it does seem to have a positive clinical impact. In fact, it creates steadier mandibles, may well increase implant stability, and, additionally, may improve resistance to mechanical, physical, chemical, and pharmacological agents. Giuliani A; Manescu A; Langer M; Rustichelli F; Desiderio V; Paino F; De Rosa A; Laino L; d'Aquino R; Tirino V; Papaccio G. Giuliani A; Manescu A; Langer M; Rustichelli F; Desiderio V; Paino F; De Rosa A; Laino L; d'Aquino R; Tirino V; Papaccio G.

Mesenchymal stem cell (MSC) therapy holds promise for treating diseases and tissue repair. Regeneration of skeletal muscle tissue that is lost during pathological muscle degeneration or after injuries is sustained by the production of new myofibers. Human Adipose stem cells (ASCs) have been reported to regenerate muscle fibers and reconstitute the pericytic cell pool after myogenic differentiation in vitro. Our aim was to evaluate the differentiation potential of constructs made from a new cross-linked hyaluronic acid (XHA) scaffold on which different sorted subpopulations of ASCs were loaded. Thirty days after engraftment in mice, we found that NG2(+) ASCs underwent a complete myogenic differentiation, fabricating a human skeletal muscle tissue, while NG2(-) ASCs merely formed a human adipose tissue. Myogenic differentiation was confirmed by the expression of MyoD, MF20, laminin, and lamin A/C by immunofluorescence and/or RT-PCR. In contrast, adipose differentiation was confirmed by the expression of adiponectin, Glut-4, and PPAR-γ. Both tissues formed expressed Class I HLA, confirming their human origin and excluding any contamination by murine cells. In conclusion, our study provides novel evidence that NG2(+) ASCs loaded on XHA scaffolds are able to fabricate a human skeletal muscle tissue in vivo without the need of a myogenic pre-differentiation step in vitro. We emphasize the translational significance of our findings for human skeletal muscle regeneration.

Stem cell migration is a critical step during the repair of damaged tissues. In order to achieve appropriate cell-based therapies for tooth and periodontal ligament repair it is necessary first to understand the dynamics of tissue-specific stem cell populations such as dental pulp stem cells (DPSC) and dental follicle stem cells (DFSC). Using time-lapse imaging, we analysed migratory and proliferative capabilities of these two human stem cell lines in vitro. When cultured alone, both DPSC and DFSC exhibited low and irregular migration profiles. In co-cultures, DFSC, but not DPSC, spectacularly increased their migration activity and velocity. DFSC rapidly surrounded the DPSC, thus resembling the in vivo developmental process, where follicle cells encircle both dental epithelium and pulp. Cell morphology was dependent on the culture conditions (mono-culture or co-culture) and changed over time. Regulatory genes involved in dental cell migration and differentiation such as TWIST1, MSX1, RUNX2, SFRP1 and ADAM28, were also evaluated in co-cultures. MSX1 up-regulation indicates that DPSC and DFSC retain their odontogenic potential. However, DPSC lose their capacity to differentiate into odontoblasts in the presence of DFSC, as suggested by RUNX2 up-regulation and TWIST1 down-regulation. In contrast, the unchanged levels of SFRP1 expression suggest that DFSC retain their potential to form periodontal tissues even in the presence of DPSC. These findings demonstrate that stem cells behave differently according to their environment, retain their genetic memory, and compete with each other to acquire the appropriate territory. Understanding the mechanisms involved in stem cell migration may lead to new therapeutic approaches for tooth repair.

Purpose To investigate and compare the influence of surgical difficulty on postoperative pain after treatment of impacted mandibular third molars by rotatory osteotomy or Piezoelectric surgery. Materials and methods A prospective, randomized, split-mouth study was performed of 52 patients with bilateral and symmetrically oriented impacted mandibular third molars, who were surgically treated using a burr (Group A) on one random side of the lower jaw and a Piezoelectric device (Group B) on the contralateral side. Surgical difficulty was evaluated using a modified version of the Parant scale to categorize “simple extractions” and “complex extractions”. Primary outcome parameters were the comparison of the postoperative pain evaluation rated on the Visual Analogue Scale from day 0 to day 6 postsurgery, and the assessment of differences in surgery time between the groups. Bone biopsies were taken during surgery to assess differences in bone tissue damage levels between the two different techniques. Results In “complex extractions” lower pain evaluation and significantly shorter surgery times were recorded when rotatory instruments were used. In “simple extractions”, similar surgery times were observed for both techniques, but pain was greatest on the day of surgery when the burr was used. Bone heat osteonecrosis was observed only in the rotatory group and a high level of alkaline phosphatase was noted only in the Piezoelectric group. Conclusion Pain after extraction of a mandibular third molar increases with increased surgical difficulty and especially in longer interventions. The integrity of the bony structure observed after the ultrasonic technique may favour the bone healing process.

Dental pulp stem cells (DPSCs), originating from neural crests, can be found within dental pulp. Up to now, it has been demonstrated that these cells are capable of producing bone tissue, both in vitro and in vivo and differentiate into adipocytes, endotheliocytes, melanocytes, neurons, glial cells, and can be easily cryopreserved and stored. Moreover, recent attention has been focused on tissue engineering and on the properties of these cells. In addition, adult bone tissue with good vascularisation has been obtained in grafts. The latest use in clinical trials for bone repair enforces the notion that DPSCs can be used successfully in patients. Therefore, their isolation, selection, differentiation and banking is of great importance. The isolation and detection techniques used in most laboratories are based on the use of antibodies revealed by flow-cytometers with cell sorter termed FACS (fluorescent activated cell sorter). In this report, we focus our attention on the main procedures used in the selection of DPSCs by flow cytometry, cell culture, freezing/thawing, cell cycle evaluation, histochemistry/immunofluorescence and differentiation of DPSCs. In addition, new methods/protocols to select and isolate stem cells without staining by fluorescent markers for implementation in biomedical/clinical laboratories are discuss. We emphasize that the new methods must address simplicity and short times of preparation and use of samples, complete sterility of cells, the potential disposable, low cost and complete maintenance of the viability and integrity of the cells with real-time response for subsequent applications in the biomedical/clinical/surgical fields.

Mesenchymal stem cells are present in many tissues of the human body, including amniotic fluid (AF) and dental pulp (DP). Stem cells of both AF and DP give rise to a variety of differentiated cells. In our experience, DP stem cells (DPSCs) display a high capacity to produce bone. Therefore, our aim was to investigate if AF-derived stem cells (AFSCs) were able to undergo bone differentiation in the presence of DPSCs. AFSCs were seeded under three different conditions: (i) cocultured with DPSCs previously differentiated into osteoblasts; (ii) cultured in the conditioned medium of osteoblast-differentiated DPSCs; (iii) cultured in the osteogenic medium supplemented with vascular endothelial growth factor and bone morphogenetic protein-2 (BMP-2). Results showed that AFSCs were positive for mesenchymal markers, and expressed high levels of Tra1-60, Tra1-80, BMPR1, BMPR2, and BMP-2. In contrast, AFSCs were negative for epithelial and hematopoietic/endothelial markers. When AFSCs were cocultured with DPSCs-derived osteoblasts, they differentiated into osteoblasts. A similar effect was observed when AFSCs were cultured in the presence of a conditioned medium originated from DPSCs. We found that osteoblasts derived from DPSCs released large amounts of BMP-2 and vascular endothelial growth factor into the culture medium and that those morphogens significantly upregulate RUNX-2 gene, stimulating osteogenesis. This study highlights the mechanisms of osteogenesis and strongly suggests that the combination of AFSCs with DPSCs may provide a rich source of soluble proteins useful for bone engineering purposes. Mesenchymal stem cells are present in many tissues of the human body, including amniotic fluid (AF) and dental pulp (DP). Stem cells of both AF and DP give rise to a variety of differentiated cells. In our experience, DP stem cells (DPSCs) display a high capacity to produce bone. Therefore, our aim was to investigate if AF-derived stem cells (AFSCs) were able to undergo bone differentiation in the presence of DPSCs. AFSCs were seeded under three different conditions: (i) cocultured with DPSCs previously differentiated into osteoblasts; (ii) cultured in the conditioned medium of osteoblast- differentiated DPSCs; (iii) cultured in the osteogenic medium supplemented with vascular endothelial growth factor and bone morphogenetic protein-2 (BMP-2). Results showed that AFSCs were positive for mesenchymal markers, and expressed high levels of Tra1-60, Tra1-80, BMPR1, BMPR2, and BMP-2. In contrast, AFSCs were negative for epithelial and hematopoietic/endothelial markers. When AFSCs were cocultured with DPSCs-derived osteoblasts, they differentiated into osteoblasts. A similar effect was observed when AFSCs were cultured in the presence of a conditioned medium originated from DPSCs. We found that osteoblasts derived from DPSCs released large amounts of BMP-2 and vascular endothelial growth factor into the culture medium and that those morphogens significantly upregulate RUNX-2 gene, stimulating osteogenesis. This study highlights the mechanisms of osteogenesis and strongly suggests that the combination of AFSCs with DPSCs may provide a rich source of soluble proteins useful for bone engineering purposes. © Mary Ann Liebert, Inc. 2011.

The aim of this study was to evaluate the behavior of human Dental Pulp Stem Cells (DPSCs), as well as human osteoblasts, when challenged on a Biocoral scaffold, which is a porous natural hydroxyapatite. For this purpose, human DPSCs were seeded onto a three-dimensional (3D) Biocoral scaffold or on flask surface (control). Either normal or rotative (3D) cultures were performed. Scanning electron microscopic analyses, at 8, 24 and 48 h of culture showed that cells did not adhere on the external surface, but moved into the cavities inside the Biocoral structure. After 7, 15 and 30 days of culture, morphological and molecular analyses suggested that the Biocoral scaffold leads DPSCs to hook into the cavities where these cells quickly start to secrete the extra cellular matrix (ECM) and differentiate into osteoblasts. Control human osteoblasts also moved into the internal cavities where they secreted the ECM. Histological sections revealed a diffuse bone formation inside the Biocoral samples seeded with DPSCs or human osteoblasts, where the original scaffold and the new secreted biomaterial were completely integrated and cells were found within the remaining cavities. In addition, RT-PCR analyses showed a significant increase of osteoblast-related gene expression and, above all, of those genes highly expressed in mineralized tissues, including osteocalcin, OPN and BSP. Furthermore, the effects on the interaction between osteogenesis and angiogenesis were observed and substantiated by ELISA assays. Taken together, our results provide clear evidence that DPSCs differentiated into osteoblasts, forming a biocomplex made of Biocoral, ECM and differentiated cells.

During human embryonic development, odontogenic tissues, deriving from the neural crest, remain undifferentiated until the adult age. This study was aimed at characterising the cells of the follicle enveloping the dental germ, due to its direct origin from neural crests. Sixty dental follicles were collected from patients aged 18 to 45 years. This research has clarified that dental follicles, if extracted in a very early stage, when dental roots did not start to be formed, contain a lineage of cells, characterised by a high degree of plasticity in comparison with other adult stem cell populations. In particular, we found that these cells share the following features with ES: (i) high levels of embryonic stem cell markers (CD90, TRA1-60, TRA1- 81, OCT-4, CD133, and SSEA-4); (ii) mRNA transcripts for Nanog and Rex-1; (iii) broader potency, being able to differentiate in cell types of all three germ layer, including smooth and skeletal muscle, osteoblasts, neurons, glial cells, and adipocytes; (iv) high levels of telomerase activity; (v) ability to form embryoid bodies; (vi) ability, after injection in murine blastocysts, to be localised within the inner cell mass; (vii) no teratoma formation after injection; (viii) in vivo tissue formation after transplantation. Our results demonstrate that these cells represent a very easy accessible and extraordinary source of pluripotent cells and point out the fact that they own the cardinal feature of embryonic stem cells. During human embryonic development, odontogenic tissues, deriving from the neural crest, remain undifferentiated until the adult age. This study was aimed at characterising the cells of the follicle enveloping the dental germ, due to its direct origin from neural crests. Sixty dental follicles were collected from patients aged 18 to 45 years. This research has clarified that dental follicles, if extracted in a very early stage, when dental roots did not start to be formed, contain a lineage of cells, characterised by a high degree of plasticity in comparison with other adult stem cell populations. In particular, we found that these cells share the following features with ES: (i) high levels of embryonic stem cell markers (CD90, TRA1-60, TRA1-81, OCT-4, CD133, and SSEA-4); (ii) mRNA transcripts for Nanog and Rex-1; (iii) broader potency, being able to differentiate in cell types of all three germ layer, including smooth and skeletal muscle, osteoblasts, neurons, glial cells, and adipocytes; (iv) high levels of telomerase activity; (v) ability to form embryoid bodies; (vi) ability, after injection in murine blastocysts, to be localised within the inner cell mass; (vii) no teratoma formation after injection; (viii) in vivo tissue formation after transplantation. Our results demonstrate that these cells represent a very easy accessible and extraordinary source of pluripotent cells and point out the fact that they own the cardinal feature of embryonic stem cells.

Dental pulp stem cells (DPSCs) can be found within the "cell rich zone" of dental pulp. Their embryonic origin, from neural crests, explains their multipotency. Up to now, two groups have studied these cells extensively, albeit with different results. One group claims that these cells produce a "dentin-like tissue", whereas the other research group has demonstrated that these cells are capable of producing bone, both in vitro and in vivo. In addition, it has been reported that these cells can be easily cryopreserved and stored for long periods of time and still retain their multipotency and bone-producing capacity. Moreover, recent attention has been focused on tissue engineering and on the properties of these cells: several scaffolds have been used to promote 3-D tissue formation and studies have demonstrated that DPSCs show good adherence and bone tissue formation on microconcavity surface textures. In addition, adult bone tissue with good vascularization has been obtained in grafts. These results enforce the notion that DPSCs can be used successfully for tissue engineering.

Drs. Antonio Graziano and Antonio Giordano were not included in the author byline. Antonio Graziano should be listed as the sixth author and is affiliated with Dipartimento di Scienze Odontostomatologiche, Ortodontiche e Chirurgiche, Secondo Ateneo di Napoli, Napoli, Italy. His contributions were as follows: Performed the experiments. Antonio Giordano should be listed as the 11th author and is affiliated with Sbarro Institute for Cancer Research and Molecular Medicine, Center of Biotechnology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania, United States of America, and Department of Human Pathology and Oncology, University of Siena, Siena, Italy. His contributions were as follows: Conceived and designed the experiments, contributed reagents/materials/analysis tools.

Human tissues are different in term of regenerative properties. Stem cells are a promising tool for tissue regeneration, thanks to their particular characteristics of proliferation, differentiation and plasticity. Several “loci” or “niches” within the adult human body are colonized by a significant number of stem cells. However, access to these potential collection sites often is a limiting point. The interaction with biomaterials is a further point that needs to be considered for the therapeutic use of stem cells. Dental pulp stem cells (DPSCs) have been demonstrated to answer all of these issues: access to the collection site of these cells is easy and produces very low morbidity; extraction of stem cells from pulp tissue is highly efficiency; they have an extensive differentiation ability; and the demonstrated interactivity with biomaterials makes them ideal for tissue reconstruction. SBP-DPSCs are a multipotent stem cell subpopulation of DPSCs which are able to differentiate into osteoblasts, synthesizing 3D woven bone tissue chips in vitro and that are capable to synergically differentiate into osteoblasts and endotheliocytes. Several studied have been performed on DPSCs and they mainly found that these cells are multipotent stromal cells that can be safety cryopreserved, used with several scaffolds, that can extensively proliferate, have a long lifespan and build in vivo an adult bone with Havers channels and an appropriate vascularization. A definitive proof of their ability to produce dentin has not been yet done. Interestingly, they seem to possess immunoprivileges as they can be grafted into allogenic tissues and seem to exert anti-inflammatory abilities, like many other mesenchymal stem cells. The easy management of dental pulp stem cells make them feasible for use in clinical trials on human patients.

In this study, we have observed dental pulp stem cells (SBP-DPSCs) performances on different scaffolds, such as PLGA 85:15, hydroxyapatite chips (HA) and titanium. Stem cells were challenged with each engineered surface, either in plane cultures or in a rotating apparatus, for a month. Gingival fibroblasts were used as controls. Results showed that stem cells exerted a different response, depending on the different type of textured surface: in fact, microconcavities significantly affected SBP-DPSC differentiation into osteoblasts, both temporally and quantitatively, with respect to the other textured surfaces. Actually, stem cells challenged with concave surfaces differentiated quicker and showed nuclear polarity, an index of secretion, cellular activity and matrix formation. Moreover, bone-specific proteins were significantly expressed and the obtained bone tissue was of significant thickness. Thus, cells cultured on the concave textured surface had better cell-scaffold interactions and were induced to secrete factors that, due to their autocrine effects, quickly lead to osteodifferentiation, bone tissue formation, and vascularization. The worst cell performance was obtained using convex surfaces, due to the scarce cell proliferation on to the scaffold and the poor matrix secretion. In conclusion, this study stresses that for a suitable and successful bone tissue reconstruction the surface texture is of paramount importance. J. Cell. Physiol. 214:166–172, 2008. © 2007 Wiley-Liss, Inc.

Cancer stem cells (CSCs) are tumoral cells which have stem features such as self-renewal, high migration capacity, drug resistance, high proliferation abilities. In the last 10 years the pathological meaning and the existence of CSCs have been matter of discussion and a large number of articles have been published about the role that these cells play in the development and maintenance of the tumors. Head and neck squamous-cell carcinoma (HNSCC) is the sixth most common cancer worldwide: early diagnosis of high-risk premalignant lesions are high priorities for reducing deaths due to head and neck cancer. In the last years the CSCs hypothesis has been faced also for head and neck cancer, with the aim of a better comprehension of the tumor biology and an early diagnosis. The evidence that the development of a tumor comes from a small number of cells with stem-like characteristic, could bring too to the identification of therapies against these cellular target, fundamental for maintenance and progression of the lesion. Here, a literature review has been reported about the detection of supposed CSCs in head and neck cancer.

BACKGROUND Scaffold surface features are thought to be important regulators of stem cell performance and endurance in tissue engineering applications, but details about these fundamental aspects of stem cell biology remain largely unclear. METHODOLOGY AND FINDINGS In the present study, smooth clinical-grade lactide-coglyolic acid 85:15 (PLGA) scaffolds were carved as membranes and treated with NMP (N-metil-pyrrolidone) to create controlled subtractive pits or microcavities. Scanning electron and confocal microscopy revealed that the NMP-treated membranes contained: (i) large microcavities of 80-120 microm in diameter and 40-100 microm in depth, which we termed primary; and (ii) smaller microcavities of 10-20 microm in diameter and 3-10 microm in depth located within the primary cavities, which we termed secondary. We asked whether a microcavity-rich scaffold had distinct bone-forming capabilities compared to a smooth one. To do so, mesenchymal stem cells derived from human dental pulp were seeded onto the two types of scaffold and monitored over time for cytoarchitectural characteristics, differentiation status and production of important factors, including bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF). We found that the microcavity-rich scaffold enhanced cell adhesion: the cells created intimate contact with secondary microcavities and were polarized. These cytological responses were not seen with the smooth-surface scaffold. Moreover, cells on the microcavity-rich scaffold released larger amounts of BMP-2 and VEGF into the culture medium and expressed higher alkaline phosphatase activity. When this type of scaffold was transplanted into rats, superior bone formation was elicited compared to cells seeded on the smooth scaffold. CONCLUSION In conclusion, surface microcavities appear to support a more vigorous osteogenic response of stem cells and should be used in the design of therapeutic substrates to improve bone repair and bioengineering applications in the future.

Non-hypercalcemic analogs of vitamin D3 modulate the immune response through antigen-presenting cells (APCs) and activated T-cells. A large population-base case-control showed that vitamin D3 intake significantly decreases the risk of type 1 diabetes development. The aim of this study was, therefore, to observe the in vivo effects of a vitamin D3 analog administered to Bio Breeding (BB) rats. 1,25-Dihydroxy-16,23Z-diene-26,27-hexafluoro-19-nor vitamin D3 (BXL-219, formerly Ro 26-2198) (BioXell, Milan, Italy) was administered in vivo to BB rats from days 42 to 110 of life at 0.2 µg/Kg BW. Control animals received only vehicle (olive oil, 4.8 µl/100 g BW). The animals of these two groups were subjected to insulin treatment as they became diabetic. Insulin (Humulin, 28.6 UI/day) was administered irrespective of diabetes occurrence to another group of rats for comparison. Blood glucose, insulin levels, glycosuria, degree of islet infiltration, and the expression of some antigens were observed. Results showed that the vitamin D3 analog reduced diabetes incidence, although limitedly, in BB rats while administration of oral insulin increased diabetes incidence. In addition, the vitamin D3 analog did not stimulate an enhancement in the expression of CD4 and CD25 in BB rats as it does in NOD mice, which may explain the failure of this as well as other antidiabetic treatments in the BB animal model of type 1 diabetes. J. Cell. Biochem. 100: 808–814, 2007. © 2006 Wiley-Liss, Inc.

[EN] This article illustrates the methodology, criteria and design options considered in the intervention to visually and materially restore the original flooring of the basilica of Santo Stefano Rotondo. The author examines the choices and different effects in a carefully considered intervention with an extremely detailed design and execution. The second part of the text includes a similar reflection on the challenge of planning lighting and designing appropriate lamps for each situation according on the desired atmosphere for the space and the distinctive connotations of the types and shapes of lamps proposed. Although the age and the religious involved great complexity, the character of the basilica intervention dealt sensitively with pre-existing elements., [ES] El artículo ilustra la metodología, los criterios y las opciones proyectuales barajadas en la intervención de reintegración visual y material de los antiguos pavimentos de la histórica basílica de Santo Stefano Rotondo. El autor razona sobre las alternativas barajadas y su diversa implicación, en una intervención caracterizada por su esmero y su exquisito detalle en el concepto y la ejecución. En una segunda parte del texto, el autor realiza una reflexión similar y paralela en la tarea de proyectar la iluminación del conjunto y diseñar las luminarias adecuadas en cada caso en base a la atmósfera que se deseaba para el espacio y las connotaciones propias de los tipos y las formas de luminarias proyectadas. No obstante la antigüedad y carácter venerable dificultaba en gran medida cualquier tipo de actuación, esta intervención ha obtenido un resultado especialmente delicado con las preexistencias.

It is not known whether cells derived from stem cells retain their differentiation and morpho-functional properties after long-term cryopreservation. This information is of importance to evaluate their potential for long-term storage with a view to subsequent use in therapy. Here, we describe the morpho-functional properties of dental pulp stem cells (SBP-DPSCs), and of their differentiated osteoblasts, recovered after long-term cryopreservation. After storage for 2 years, we found that stem cells are still capable of differentiation, and that their differentiated cytotypes proliferate and produce woven bone tissue. In addition, cells still express all their respective surface antigens, confirming cellular integrity. In particular, SBP-DPSCs differentiated into pre-osteoblasts, showing diffuse positivity for ALP, BAP, RUNX-2, and calcein. Recovered osteoblasts expressed bone-specific markers and were easily recognizable ultrastructurally, with no alterations observed at this level. In addition, after in vivo transplantation, woven bone converted into a 3D lamellar bone type. Therefore, dental pulp stem cells and their osteoblast-derived cells can be long-term cryopreserved and may prove to be attractive for clinical applications.

Stem cells were obtained from deciduous dental pulp of healthy subjects, aged 6-10 years. This stem cell population was cultured, expanded, and specifically selected, detecting using a FACsorter, c-kit, CD34, and STRO-1 antigen expression. Then, c-kit+/ CD34+/STRO-1+cells were replaced in the culture medium added of 20% FBS, leading to osteoblast differentiation. In fact, these cells, after a week, showed a large positivity for CD44, osteocalcin, and RUNX-2 markers. To achieve an adipocytic differentiation, cells, after sorting, were challenged with dexamethason 10-8mM in the same culture medium. To obtain myotube fusion, sorted cells were co-cultured in ATCC medium with mouse myogenic C2C12 cells and, after a week, human stem cell nuclei were found to be able to fuse, forming myotubes. Differentiated osteoblasts, as assessed by a large positivity to several specific antibodies, after 30 days of culture and already in vitro, started to secrete an extracellular mineralized matrix, which, 2 weeks later, built a considerable number of 3D woven bone samples, which showed a strong positivity to alkaline phosphatase (ALP), alizarin red, calcein, other than to specific antibodies. These bone samples, after in vivo transplantation into immunosuppressed rats, were remodeled in a lamellar bone containing entrapped osteocytes. Therefore, this study provides strong evidence that human deciduous dental pulp is an approachable "niche" of stromal stem cells, and that it is an ideal source of osteoblasts, as well as of mineralized tissue, ready for bone regeneration, transplantation, and tissue-based clinical therapies. © 2005 Wiley-Liss, Inc.

IL-1beta is an important mediator in the pathogenesis of type 1 diabetes both in vivo and in vitro and it has been shown to induce islet beta-cell apoptosis. Most of the IL-1beta effects seem to be mediated by NF-kappaB transcription factor activation, but its role in the induction of islet beta-cell apoptosis has not yet been clarified. Taking advantage of the protease inhibitor TPCK (N-tosyl-L-phenylalanine chloromethyl ketone), which specifically inhibits the nuclear transcription factor NF-kappaB activation, we studied the role of NF-kappaB in the rIL-1beta treated rat pancreatic islets. Our results show that TPCK blocked rIL-1beta-mediated early increase of MnSOD activity and beta-cell defence/repair protein expression, suggesting a protective role for NF-kappaB at the beginning of IL-1beta treatment; but, in a second phase, NF-kappaB induces a sustained decrease of specific beta-cell proteins like insulin, GLUT-2 and PDX-1 with a concomitant increase of aspecific proteins and iNOS transcription. The appearance of iNOS expression correlates with increased levels of nitrite + nitrate levels and appearance of mitochondrial damage detected either at morphological and biochemical level. After 36 h of IL-1beta treatment islet beta-cells begin to undergo apoptosis. Since IL-1beta induction of apoptosis is completely prevented by TCPK treatment, this finding underscores the central role of NF-kappaB in this process. Thus, our results clearly indicate that NF-kappaB regulates MnSOD genes expression and MnSOD activity, which protects islet beta-cells by IL-1beta damage. Furthermore, when the IL-1beta stress impairs islet beta-cell function, NF-kappaB activation regulates the entrance of islet beta-cell into the cell death program.

Interleukin (IL)-1beta-treated rat islets of Langerhans were exposed in vitro either to the imidazoline compound, Efaroxan, or to the selective inducible nitric oxide synthase (iNOS) inhibitor, 1400W, in a medium containing a high concentration of glucose (16.7 mmol/L). Our data have evidenced the following: (i) addition of Efaroxan to islet cultures inhibited IL-1beta activation of ICE (cysteine protease IL-1beta converting enzyme) while addition of 1400W did not; (ii) Efaroxan completely inhibited IL-1beta-induced suppression of insulin secretion and induction of iNOS mRNA transcripts, and, in addition, counteracted islet beta-cell protein profile alterations, Bax-cytochrome c translocation, caspase activation, and apoptosis; (iii) 1400W inhibited IL-1beta induction of iNOS, but failed to completely counteract the other cytotoxic effects; (iv) the two compounds, moreover, exerted different effects on manganese superoxide dismutase (MnSOD), in fact, while Efaroxan inhibited the early stimulatory effect of IL-1beta on MnSOD, 1400W did not. Thus, Efaroxan completely protected islet beta cells from damage caused by IL-1beta-induced toxicity, while compound 1400W only inhibited NO radical production without altering the cytokine's cytotoxicity. Our observations have evidenced that suppression of ICE activation is required to counteract IL-1beta-mediated islet beta cell toxicity, and that IL-1beta-induced apoptosis is NO-independent and involves the cytochrome c-mitochondrial pathway.