Publications by authors named "Antonio Graziano"

39 Publications

The structures of E. coli NfsA bound to the antibiotic nitrofurantoin; to 1,4-benzoquinone and to FMN.

Biochem J 2021 Jul;478(13):2601-2617

School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K.

NfsA is a dimeric flavoprotein that catalyses the reduction in nitroaromatics and quinones by NADPH. This reduction is required for the activity of nitrofuran antibiotics. The crystal structure of free Escherichia coli NfsA and several homologues have been determined previously, but there is no structure of the enzyme with ligands. We present here crystal structures of oxidised E. coli NfsA in the presence of several ligands, including the antibiotic nitrofurantoin. Nitrofurantoin binds with the furan ring, rather than the nitro group that is reduced, near the N5 of the FMN. Molecular dynamics simulations show that this orientation is only favourable in the oxidised enzyme, while potentiometry suggests that little semiquinone is formed in the free protein. This suggests that the reduction occurs by direct hydride transfer from FMNH- to nitrofurantoin bound in the reverse orientation to that in the crystal structure. We present a model of nitrofurantoin bound to reduced NfsA in a viable hydride transfer orientation. The substrate 1,4-benzoquinone and the product hydroquinone are positioned close to the FMN N5 in the respective crystal structures with NfsA, suitable for reaction, but are mobile within the active site. The structure with a second FMN, bound as a ligand, shows that a mobile loop in the free protein forms a phosphate-binding pocket. NfsA is specific for NADPH and a similar conformational change, forming a phosphate-binding pocket, is likely to also occur with the natural cofactor.
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http://dx.doi.org/10.1042/BCJ20210160DOI Listing
July 2021

Cartilage Micrografts as a Novel Non-Invasive and Non-Arthroscopic Autograft Procedure for Knee Chondropathy: Three-Year Follow-Up Study.

J Clin Med 2021 Jan 17;10(2). Epub 2021 Jan 17.

Human Brain Wave, Corso Galileo Ferraris, 63, 10128 Turin, Italy.

(1) Background: Focal chondral defects of the knee can significantly impair patient quality of life. Although different options are available, they are still not conclusive and have several limitations. The aim of this study was to evaluate the role of autologous cartilage micrografts in the treatment of knee chondropathy. (2) Methods: Eight patients affected by knee chondropathy were evaluated before and after 6 months and 3 years following autologous cartilage micrografts by magnetic resonance imaging (MRI) for cartilage measurement and clinical assessment. (3) Results: All patients recovered daily activities, reporting pain reduction without the need for analgesic therapy; Oxford Knee Score (OKS) was 28.4 ± 6 and 40.8 ± 6.2 and visual analogue scale (VAS) was 5.5 ± 1.6 and 1.8 ± 0.7 before and after 6 months following treatment, respectively. Both scores remained stable after 3 years. Lastly, a significant improvement of the cartilage thickness was observed using MRI after 3 years. (4) Conclusions: Autologous cartilage micrografts can promote the formation of new cartilage, and could be a valid approach for the treatment of knee chondropathy.
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http://dx.doi.org/10.3390/jcm10020322DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7830188PMC
January 2021

Progenitor-cell-enriched micrografts as a novel option for the management of androgenetic alopecia.

J Cell Physiol 2020 05 23;235(5):4587-4593. Epub 2019 Oct 23.

Department of R&D, Human Brain Wave, Turin, Italy.

Regenerative medicine is a multidisciplinary field that combines engineering and life science principles to promote regeneration, potentially restoring the physiological condition in diseased tissues. Specifically, the developments of complex grafts enhance the intrinsic regenerative capacity of the host by altering its environment. Autologous micrografts obtained through Rigenera® micrografting technology are able to promote derma and bone regeneration. Androgenetic alopecia (AGA) leads to a progressive thinning of scalp hair affecting 60-70% of the adult population worldwide. Pharmacological treatment offers moderate results and hair transplantation represents the only permanent treatment option. The aim of this study was to demonstrate the role of dermis micrografting in the treatment of AGA by clinical and histological evaluations after 4, 6, and 12 months. Hair growth and density were improved at all indicated times. Those outcomes were also confirmed by the TrichoScan® analysis, reporting an increase of total hair count and density with an increase and reduction of anagen and telogen phases, respectively. Scalp dermoscopic analysis showed an improvement of hair density and histological analysis indicated a clear amelioration of the scalp, development of hair follicles, and a beginning of cuticle formation. Collectively, those results suggest a possible use of the micrografts as a novel therapeutic option in the management of AGA.
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http://dx.doi.org/10.1002/jcp.29335DOI Listing
May 2020

The Role of Autologous Dermal Micrografts in Regenerative Surgery: A Clinical Experimental Study.

Stem Cells Int 2019 8;2019:9843407. Epub 2019 Sep 8.

Plastic and Reconstructive Surgery, Department of Clinical Surgical, Diagnostic and Pediatric Sciences, University of Pavia, Viale Brambilla, 74 Pavia, Italy.

The aim of the study was the objective assessment of the effectiveness of a microfragmented dermal extract obtained with Rigenera™ technology in promoting the wound healing process in an homogeneous experimental human acute surgical wound model. The study included 20 patients with 24 acute postsurgical soft tissue loss and a planned sequential two-stage repair with a dermal substitute and an autologous split-thickness skin graft. Each acute postsurgical soft tissue loss was randomized to be treated either with an Integra® dermal substitute enriched with the autologous dermal micrografts obtained with Rigenera™ technology (group A-Rigenera™ protocol) or with an Integra® dermal substitute only (group B-control). The reepithelialization rate in the wounds was assessed in both groups at 4 weeks through digital photography with the software "ImageJ." The dermal cell suspension enrichment with the Rigenera™ technology was considered effective if the reepithelialized area was higher than 25% of the total wound surface as this threshold was considered far beyond the expected spontaneous reepithelialization rate. In the Rigenera™ protocol group, the statistical analysis failed to demonstrate any significant difference vs. the controls. The old age of the patients likely influenced the outcome as the stem cell regenerative potential is reduced in the elderly. A further explanation for the unsatisfying results of our trial might be the inadequate amount of dermal stem cells used to enrich the dermal substitutes. In our study, we used a 1 : 200 donor/recipient site ratio to minimize donor site morbidity. The gross dimensional disparity between the donor and recipient sites and the low concentration of dermal mesenchymal stromal stem cells might explain the poor epithelial proliferative boost observed in our study. A potential option in the future might be preconditioning of the dermal stem cell harvest with senolytic active principles that would fully enhance their regenerative potential. This trial is registered with trial protocol number NCT03912675.
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http://dx.doi.org/10.1155/2019/9843407DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6754962PMC
September 2019

Autologous Periosteum-Derived Micrografts and PLGA/HA Enhance the Bone Formation in Sinus Lift Augmentation.

Front Cell Dev Biol 2017 27;5:87. Epub 2017 Sep 27.

Department of Clinical Surgical, Diagnostic and Pediatric Sciences, University of Pavia, Pavia, Italy.

Sinus lift augmentation is a procedure required for the placement of a dental implant, whose success can be limited by the quantity or quality of available bone. To this purpose, the first aim of the current study was to evaluate the ability of autologous periosteum-derived micrografts and Poly(lactic-co-glycolic acid) (PLGA) supplemented with hydroxyl apatite (HA) to induce bone augmentation in the sinus lift procedure. Secondly, we compared the micrograft's behavior with respect to biomaterial alone, including Bio-Oss® and PLGA/HA, commercially named Alos. Sinus lift procedure was performed on 24 patients who required dental implants and who, according to the study design and procedure performed, were divided into three groups: group A (Alos + periosteum-derived micrografts); group B (Alos alone); and group C (Bio-Oss® alone). Briefly, in group A, a small piece of periosteum was collected from each patient and mechanically disaggregated by Rigenera® protocol using the Rigeneracons medical device. This protocol allowed for the obtainment of autologous micrografts, which in turn were used to soak the Alos scaffold. At 6 months after the sinus lift procedure and before the installation of dental implants, histological and radiographic evaluations in all three groups were performed. In group A, where sinus lift augmentation was performed using periosteum-derived micrografts and Alos, the bone regeneration was much faster than in the control groups where it was performed with Alos or Bio-Oss® alone (groups B and C, respectively). In addition, the radiographic evaluation in the patients of group A showed a radio-opacity after 4 months, while after 6 months, the prosthetic rehabilitation was improved and was maintained after 2 years post-surgery. In summary, we report on the efficacy of periosteum-derived micrografts and Alos to augment sinus lift in patients requiring dental implants. This efficacy is supported by an increased percentage of vital mineralized tisssue in the group treated with both periosteum-derived micrografts and Alos, with respect to the control group of Alos or Bio-Oss® alone, as confirmed by histological analysis and radiographic evaluations at 6 months from treatment.
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http://dx.doi.org/10.3389/fcell.2017.00087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5623661PMC
September 2017

Erratum to: A Regenerative Approach with Dermal Micrografts in the Treatment of Chronic Ulcers.

Stem Cell Rev Rep 2017 Feb;13(1):149

Multidisciplinary Department of Medical-Surgical and Dental Specialties, Second University of Naples, Miraglia Square, 80122, Naples, Italy.

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http://dx.doi.org/10.1007/s12015-016-9698-9DOI Listing
February 2017

A Regenerative Approach with Dermal Micrografts in the Treatment of Chronic Ulcers.

Stem Cell Rev Rep 2017 Feb;13(1):139-148

Multidisciplinary Department of Medical-Surgical and Dental Specialties, Second University of Naples, Miraglia Square, 80122, Naples, Italy.

Background: The etiology of non-healing ulcers depends on both systemic and local factors. The introduction of advanced dressing, negative wound therapy and compression therapy have undoubtedly improved clinical outcomes. The principal aim of study was to demonstrate the efficacy of dermal micrografts in the treatment of ulcers with different etiologies. The second aim was to investigate in vitro the action of micrografts in the regenerative process.

Methods: The dermal micro-grafts were obtained from mechanical disaggregation of small pieces of skin tissue through a medical device called Rigeneracons.

Results: We observed in vivo the ability of dermal autologous micrografts to improve the healing of venous, diabetic, pressure and post-traumatic ulcers after few week of treatment accomplished in general with a better quality of life for the patients. In vitro results showed that these micrografts express mesenchymal stem cells (MSCS) marker such as CD34, CD73, CD90 and CD105, and are able to form a viable and proliferative biocomplex with collagen sponge. Finally, the site of ulcers displayed a different expression of epidermal growth factors, insulin-like growth factors, platelet-derived growth factors and their receptors and tumor necrosis factor-β with respect to healthy skin samples.

Conclusion: We reported a good outcome for the treatment of chronic ulcers using dermal autologous micrografts. Finally, we suggest that the positivity to MSCs markers and the ability to interact with a scaffold can play a key role in their regenerative properties.
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http://dx.doi.org/10.1007/s12015-016-9692-2DOI Listing
February 2017

In Vitro and In Vivo Differentiation of Progenitor Stem Cells Obtained After Mechanical Digestion of Human Dental Pulp.

J Cell Physiol 2017 Mar 21;232(3):548-555. Epub 2016 Jun 21.

Department of Clinical-Surgical Diagnostic and Pediatric Sciences, School of Dentistry, University of Pavia, Pavia, Italy.

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.
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http://dx.doi.org/10.1002/jcp.25452DOI Listing
March 2017

Rigenera protocol in the treatment of surgical wound dehiscence.

Int Wound J 2017 Feb 29;14(1):277-281. Epub 2016 Apr 29.

SHRO Center of Biotechnology, Temple University, Philadelphia, PA, USA.

The effective management of post-operative wounds is important to prevent potential complications such as surgical-site infections and wound dehiscence. The purpose of this study was to treat wound dehiscence in elderly patients who were subjected to orthopaedic surgical interventions. The dehisced wounds were treated with autologous micro-grafts obtained using a promising CE-certified medical device called Rigeneracons. This instrument is a biological disruptor of human tissues able to specifically select progenitor cells that, as already reported in previous studies, maintain high cell viability but mainly have a high regenerative potential, allowing the repair of damaged tissues. Autologous micro-grafts obtained by Rigeneracons are ready to use and can be applied alone or in combination with biological scaffolds directly on the injured area. We observed in our patients a complete remission of dehisced wounds, on average, after 30 days from micro-grafts application and a total wound re-epithelialisation after 1 year from the surgical intervention. In conclusion, although we reported only three patients, autologous micro-grafts can be considered a promising approach for the treatment of dehisced wounds, improving the wound-healing process and in general the patient's quality of life without using other dressings.
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http://dx.doi.org/10.1111/iwj.12601DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7949493PMC
February 2017

Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation.

J Vis Exp 2016 Mar 4(109):e53579. Epub 2016 Mar 4.

Plastic and Reconstructive Surgery, AOU "Ospedali Riuniti"

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one step treatments for acute and chronic skin lesions. Moreover, the management of chronic but also acute wounds resulting from trauma, diabetes, infections and other causes, remains challenging. In this study we describe a new method to create autologous micro-grafts from cutaneous tissue of a single patient and their clinical application. Moreover, in vitro biological characterization of cutaneous tissue derived from skin, de-epidermized dermis (Ded) and dermis of multi-organ and/or multi-tissue donors was also performed. All tissues were disaggregated by this new protocol, allowing us to obtain viable micro-grafts. In particular, we reported that this innovative protocol is able to create bio-complexes composed by autologous micro-grafts and collagen sponges ready to be applied on skin lesions. The clinical application of autologous bio-complexes on a leg lesion was also reported, showing an improvement of both re-epitalization process and softness of the lesion. Additionally, our in vitro model showed that cell viability after mechanical disaggregation with this system is maintained over time for up to seven (7) days of culture. We also observed, by flow cytometry analysis, that the pool of cells obtained from disaggregation is composed of several cell types, including mesenchymal stem cells, that exert a key role in the processes of tissue regeneration and repair, for their high regenerative potential. Finally, we demonstrated in vitro that this procedure maintains the sterility of micro-grafts when cultured in Agar dishes. In summary, we conclude that this new regenerative approach can be a promising tool for clinicians to obtain in one step viable, sterile and ready to use micro-grafts that can be applied alone or in combination with most common biological scaffolds.
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http://dx.doi.org/10.3791/53579DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4828224PMC
March 2016

An innovative regenerative treatment of scars with dermal micrografts.

J Cosmet Dermatol 2016 Sep 30;15(3):245-53. Epub 2016 Jan 30.

Multidisciplinary Department of Medical-Surgical and Dental Specialties, Second University of Naples, Naples, Italy.

Background: Pathological scars occur following injuries and are often considered esthetically unattractive. Several strategies have been attempted to improve these types of scars using both surgical and nonsurgical methods. The most common treatments include cryotherapy, intralesional corticosteroid injections, 5-fluorouracil, bleomycin, interferon, and verapamil.

Aims: In this study, we aim to investigate the effectiveness of dermal autologous micrografts in the treatment of pathological scars resulting from burns, trauma, or any iatrogenic source.

Methods: We used a new clinical practice called Rigenera Protocol to obtain autologous micrografts which were in turn injectable in the patients.

Results: A significant improvement was observed in appearance and texture of the exaggerated scars in all cases following already 4 months of autologous micrograft treatment We have also shown that these micrografts are composed of mesenchymal stem cells and in addition, histological evaluation verified restoration of the structural layers immediately below the epidermis and a horizontal realignment of collagen fibers in the papillary dermis.

Conclusion: Our results clearly demonstrate the optimal outcomes obtained following treatment with dermal micrografts on exaggerated scars with different etiologies. However, further studies are required to confirm the efficacy of this new technique.
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http://dx.doi.org/10.1111/jocd.12212DOI Listing
September 2016

Dermoscopy of verrucous epidermal nevus: large brown circles as a novel feature for diagnosis.

Int J Dermatol 2016 Jun 16;55(6):653-6. Epub 2015 Oct 16.

Plastic and Reconstructive Surgery Unit, Campus Bio-Medico University of Rome, Rome, Italy.

Background: The epidermal nevus is a hamartomatous proliferation of the epithelium that can involve keratinocytes, sebaceous glands, the pilosebaceous unit, and eccrine or apocrine glands. It occurs in one in 1000 live births and most commonly presents as the verrucous type. Dermoscopy is a non-invasive technique commonly used to differentiate between melanocytic and non-melanocytic lesions.

Objectives: This study was performed to analyze dermoscopic aspects of verrucous epidermal nevi, which have not previously been described.

Methods: Dermoscopic analyses of eight different verrucous epidermal nevi were conducted. Each lesion was excised, and its diagnosis was histopathologically confirmed.

Results: In non-melanocytic lesions, a new dermoscopic feature of large brown circles was observed. This characteristic is useful in the diagnosis of verrucous epidermal nevi.

Conclusions: Large brown circles represent a specific dermoscopic feature for the diagnosis of verrucous epidermal nevus.
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http://dx.doi.org/10.1111/ijd.12948DOI Listing
June 2016

Osteogenic Potential of Human Oral-Periosteal Cells (PCs) Isolated From Different Oral Origin: An In Vitro Study.

J Cell Physiol 2016 Mar;231(3):607-12

Department of Public Health, Experimental Medicine and Forensics, University of Pavia, Pavia, Italy.

The periosteum is a specialized connective tissue containing multipotent stem cells capable of bone formation. In this study, we aimed at demonstrating that human oral periosteal cells derived from three different oral sites (upper vestibule, lower vestibule, and hard palate) represent an innovative cell source for maxillo-facial tissue engineering applications in terms of accessibility and self-commitment towards osteogenic lineage. Periosteal cells (PCs) were isolated from patients with different ages (20-30 yy, 40-50 yy, 50-60 yy); we then analyzed the in vitro proliferation capacity and the bone self-commitment of cell clones culturing them without any osteogenic supplement to support their differentiation. We found that oral PCs, independently of their origin and age of patients, are mesenchymal stem cells with stem cell characteristics (clonogenical and proliferative activity) and that, even in absence of any osteogenic induction, they undertake the osteoblast lineage after 45 days of culture. These results suggest that oral periosteal cells could replace mesenchymal cells from bone marrow in oral tissue-engineering applications.
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http://dx.doi.org/10.1002/jcp.25104DOI Listing
March 2016

Use of dermoscopy in the diagnosis of sebaceoma.

J Am Acad Dermatol 2015 Jun;72(6):e143-5

Plastic and Reconstructive Surgery Unit, Campus Bio-Medico University, Rome, Italy. Electronic address:

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http://dx.doi.org/10.1016/j.jaad.2014.12.004DOI Listing
June 2015

A New Medical Device Rigeneracons Allows to Obtain Viable Micro-Grafts From Mechanical Disaggregation of Human Tissues.

J Cell Physiol 2015 Oct;230(10):2299-303

Human Brain Wave srl, Turin, Italy.

Autologous graft is considered the gold standard of graft materials; however, this approach is still limited due to both small amount of tissue that can be collected and to reduced cell viability of cells that can be obtained. The aim of this preliminary study was to demonstrate the efficacy of an innovative medical device called Rigeneracons® (CE certified Class I) to provide autologous micro-grafts immediately available to be used in the clinical practice. Moreover, Rigeneracons® is an instrument able to create micro-grafts enriched of progenitors cells which maintain their regenerative and differentiation potential. We reported preliminary data about viability cell of samples derived from different kind of human tissues, such as periosteum, cardiac atrial appendage biopsy, and lateral rectus muscle of eyeball and disaggregated by Rigeneracons®. In all cases we observed that micro-grafts obtained by Rigeneracons® displayed high cell viability. Furthermore, by cell characterization of periosteum samples, we also evidenced an high positivity to mesenchymal cell markers, suggesting an optimal regenerative potential.
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http://dx.doi.org/10.1002/jcp.24973DOI Listing
October 2015

Hormesis and vitagenes in aging and longevity: mitochondrial control and hormonal regulation.

Horm Mol Biol Clin Investig 2013 Dec;16(2):73-89

Abstract Average life span has increased because of medical and environmental factors, but maximal life span remains unchanged. Understanding the mechanisms of aging will help to reduce age-related morbidity and facilitate healthy aging. Unlike female menopause, which is accompanied by an abrupt and permanent cessation of ovarian function (both folliculogenesis and estradiol production), male aging does not result in either cessation of testosterone production or infertility. Although the circulating serum testosterone concentration does decline with aging, in most men this decrease is small, resulting in levels that are generally within the normal range. Age-related hypogonadism has been referred to as andropause or late-onset hypogonadism (LOH), with LOH considered to be the most suitable term for this condition. Hormone therapy (HT) trials have caused both apprehension and confusion about the overall risks and benefits associated with HT treatment. During aging, a gradual decline in the potency of the heat shock response occurs, and this may prevent the repair of protein damage. Thus, the interest in developing pharmacological agents capable of inducing stress responses is growing within the broad frame of hormesis, which underlie strategies for optimal patient treatment of numerous diseases. Vitagenes encode for heat shock proteins, thioredoxin, and sirtuin protein systems. Nutritional antioxidants have recently been demonstrated to be neuroprotective through the activation of hormetic pathways, including vitagenes. Here, we focus on possible signaling mechanisms involved in the activation of vitagenes resulting in enhanced defense against bioenergetic defects leading to degeneration and cell death with consequent impact on longevity processes.
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http://dx.doi.org/10.1515/hmbci-2013-0051DOI Listing
December 2013

Cellular stress response, sirtuins and UCP proteins in Alzheimer disease: role of vitagenes.

Immun Ageing 2013 Oct 17;10(1):41. Epub 2013 Oct 17.

Department of Biomedical Sciences, University of Catania, Catania, Italy.

Alzheimer's Disease (AD) is a neurodegenerative disorder affecting up to one third of individuals reaching the age of 80. Different integrated responses exist in the brain to detect oxidative stress which is controlled by several genes termed Vitagenes. Vitagenes encode for cytoprotective heat shock proteins (Hsp), as well as thioredoxin, sirtuins and uncouple proteins (UCPs). In the present study we evaluate stress response mechanisms in plasma and lymphocytes of AD patients, as compared to controls, in order to provide evidence of an imbalance of oxidant/antioxidant mechanisms and oxidative damage in AD patients and the possible protective role of vitagenes.We found that the levels of Sirt-1 and Sirt-2 in AD lymphocytes were significantly higher than in control subjects. Interestingly, analysis of plasma showed in AD patients increased expression of Trx, a finding associated with reduced expression of UCP1, as compared to control group.This finding can open up new neuroprotective strategies, as molecules inducing this defense mechanisms can represent a therapeutic target to minimize the deleterious consequences associated to oxidative stress, such as in brain aging and neurodegenerative disorders.
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http://dx.doi.org/10.1186/1742-4933-10-41DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3842652PMC
October 2013

Sinus lift tissue engineering using autologous pulp micro-grafts: A case report of bone density evaluation.

J Indian Soc Periodontol 2013 Sep;17(5):644-7

Dental Clinic, Don Orione Hospital, Bergamo, Italy.

Background: Although autografts are the standard procedure for bone grafting, the use of bone regeneration by means of dental pulp stem cell is an alternative that opens a new era in this field. Rigenera Protocol is a new technique able to provide the surgeon autologous pulp micro-grafts.

Materials And Methods: At the Department of Oral Surgery, Don Orione Hospital, Bergamo, Italy, one patient underwent sinus lift elevation with pulp stem micro-grafts gentle poured onto collagen sponge. A CT scan control was performed after 4 months and DICOM data were processed with medical imaging software which gives the possibility to use a virtual probe to extract the bone density. Pearson's Chi-square test was used to investigate difference in bone density (BD) between native and newly formed bone.

Results: BD in newly formed bone is about the double of native bone.

Conclusion: This report demonstrated that micro-grafts derived from dental pulp poured onto collagen sponge are a useful method for bone regeneration in atrophic maxilla.
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http://dx.doi.org/10.4103/0972-124X.119284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3808021PMC
September 2013

Stress responses, vitagenes and hormesis as critical determinants in aging and longevity: Mitochondria as a "chi".

Immun Ageing 2013 Apr 25;10(1):15. Epub 2013 Apr 25.

Department of Chemistry University of Catania, Viale Andrea Doria, 95100 Catania, Italy.

Understanding mechanisms of aging and determinants of life span will help to reduce age-related morbidity and facilitate healthy aging. Average lifespan has increased over the last centuries, as a consequence of medical and environmental factors, but maximal life span remains unchanged. Extension of maximal life span is currently possible in animal models with measures such as genetic manipulations and caloric restriction (CR). CR appears to prolong life by reducing reactive oxygen species (ROS)-mediated oxidative damage. But ROS formation, which is positively implicated in cellular stress response mechanisms, is a highly regulated process controlled by a complex network of intracellular signaling pathways. By sensing the intracellular nutrient and energy status, the functional state of mitochondria, and the concentration of ROS produced in mitochondria, the longevity network regulates life span across species by coordinating information flow along its convergent, divergent and multiply branched signaling pathways, including vitagenes which are genes involved in preserving cellular homeostasis during stressful conditions. Vitagenes encode for heat shock proteins (Hsp) Hsp32, Hsp70, the thioredoxin and the sirtuin protein systems. Dietary antioxidants, have recently been demonstrated to be neuroprotective through the activation of hormetic pathways, including vitagenes. The hormetic dose-response, challenges long-standing beliefs about the nature of the dose-response in a lowdose zone, having the potential to affect significantly the design of pre-clinical studies and clinical trials as well as strategies for optimal patient dosing in the treatment of numerous diseases. Given the broad cytoprotective properties of the heat shock response there is now strong interest in discovering and developing pharmacological agents capable of inducing stress responses. Here we focus on possible signaling mechanisms involved in the activation of vitagenes resulting in enhanced defense against energy and stress resistance homeostasis dysiruption with consequent impact on longevity processes.
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http://dx.doi.org/10.1186/1742-4933-10-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3644272PMC
April 2013

Traumatic brain injury: oxidative stress and neuroprotection.

Antioxid Redox Signal 2013 Sep 10;19(8):836-53. Epub 2013 May 10.

Department of Clinical and Experimental Medicine and Pharmacology, School of Medicine, University of Messina, Messina, Italy.

Significance: A vast amount of circumstantial evidence implicates high energy oxidants and oxidative stress as mediators of secondary damage associated with traumatic brain injury. The excessive production of reactive oxygen species due to excitotoxicity and exhaustion of the endogenous antioxidant system induces peroxidation of cellular and vascular structures, protein oxidation, cleavage of DNA, and inhibition of the mitochondrial electron transport chain.

Recent Advances: Different integrated responses exist in the brain to detect oxidative stress, which is controlled by several genes termed vitagens. Vitagens encode for cytoprotective heat shock proteins, and thioredoxin and sirtuins.

Critical Issues And Future Directions: This article discusses selected aspects of secondary brain injury after trauma and outlines key mechanisms associated with toxicity, oxidative stress, inflammation, and necrosis. Finally, this review discusses the role of different oxidants and presents potential clinically relevant molecular targets that could be harnessed to treat secondary injury associated with brain trauma.
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http://dx.doi.org/10.1089/ars.2012.4981DOI Listing
September 2013

In vitro osteoblastic differentiation of human mesenchymal stem cells and human dental pulp stem cells on poly-L-lysine-treated titanium-6-aluminium-4-vanadium.

J Biomed Mater Res A 2011 May 2;97(2):118-26. Epub 2011 Mar 2.

Interdepartmental Center of Tissue Engineering, University of Pavia, Italy.

Three-dimensional (3D) titanium-6-aluminium-4-vanadium (Ti6Al4V) is a widely used biomaterial for orthopedic prosthesis and dental implants; thanks to its very high-mechanical strength and resistance to corrosion. Human mesenchymal stem cells (hMSCs) and dental pulp stem cells (hDPSCs) are responsible for bone regeneration following colonization of prosthesis or dental implants. Both hMSCs and hDPSCs have lower ability to colonize this biomaterial in comparison with tissue culture-treated plastic. Both hMSCs and hDPSCs show lack of focal adhesion kinase (FAK) activation when grown on Ti6Al4V. This signal is restored in the presence of poly-L-lysine (poly-L-lys). Poly-L-lys has been used as part of organoapatite or together with zinc and calcium ions. Our results suggest that poly-L-lys alone induces FAK activation through β1-INTEGRIN, because the presence of β1-INTEGRIN blocking antibody avoided FAK autophosphorylation. Presence of poly-L-lys also increases expression of osteoblastic differentiation marker genes in hMSCs and hDPSCs grown on Ti6Al4V.
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http://dx.doi.org/10.1002/jbm.a.32996DOI Listing
May 2011

Stem cells in cancer therapy: From their role in pathogenesis to their use as therapeutic agents.

Drug News Perspect 2010 Apr;23(3):175-83

Department of Biochemistry & Microbiology and the Department of Surgery, Joan C. Edwards School of Medicine, Marshall University, Huntington, West Virginia, USA; Department of Basic and Applied Biology, University of L'Aquila, Italy.

Advances in the understanding of the intriguing properties of stem cells are prompting the development of new therapeutic approaches in oncology. Stemness is a crucial condition for the homeostasis of the human body. Nevertheless, pathways that regulate self-renewal and cell fate of normal stem cells, such as Wnt and hedgehog, are also involved in the regulation of cancer stem cells and tumor growth and progression, and may thus represent novel therapeutic targets in cancer treatment. In addition, the ability of stem cells to self-renew, migrate to tumor sites and differentiate into multiple cell types makes them perfect candidates for being used as tools for delivering therapeutic genes and proteins and as drug vectors to eliminate malignant cells.
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http://dx.doi.org/10.1358/dnp.2010.23.3.1489979DOI Listing
April 2010

Human mandible bone defect repair by the grafting of dental pulp stem/progenitor cells and collagen sponge biocomplexes.

Eur Cell Mater 2009 Nov 12;18:75-83. Epub 2009 Nov 12.

Department of Experimental Medicine, Section of Histology and Embryology, TERM Division, 2nd University of Naples, Naples, Italy.

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.
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http://dx.doi.org/10.22203/ecm.v018a07DOI Listing
November 2009

Detection and characterization of CD133+ cancer stem cells in human solid tumours.

PLoS One 2008 21;3(10):e3469. Epub 2008 Oct 21.

Dipartimento di Medicina Sperimentale, Sezione di Istologia ed Embriologia, Seconda Università degli Studi di Napoli, Napoli, Italy.

Background: Osteosarcoma is the most common primary tumour of bone. Solid tumours are made of heterogeneous cell populations, which display different goals and roles in tumour economy. A rather small cell subset can hold or acquire stem potentials, gaining aggressiveness and increasing expectancy of recurrence. The CD133 antigen is a pentaspan membrane glycoprotein, which has been proposed as a cancer stem cell marker, since it has been previously demonstrated to be capable of identifying a cancer initiating subpopulation in brain, colon, melanoma and other solid tumours. Therefore, our aim was to observe the possible presence of cells expressing the CD133 antigen within solid tumour cell lines of osteosarcoma and, then, understand their biological characteristics and performances.

Methodology And Principal Findings: In this study, using SAOS2, MG63 and U2OS, three human sarcoma cell lines isolated from young Caucasian subjects, we were able to identify and characterize, among them, CD133+ cells showing the following features: high proliferation rate, cell cycle detection in a G2\M phase, positivity for Ki-67, and expression of ABCG2 transporters. In addition, at the FACS, we were able to observe the CD133+ cell fraction showing side population profile and forming sphere-clusters in serum-free medium with a high clonogenic efficiency.

Conclusions: Taken together, our findings lead to the thought that we can assume that we have identified, for the first time, CD133+ cells within osteosarcoma cell lines, showing many features of cancer stem cells. This can be of rather interest in order to design new therapies against the bone cancer.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0003469PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2565108PMC
January 2009

Comparison between genetic portraits of osteoblasts derived from primary cultures and osteoblasts obtained from human pulpar stem cells.

J Craniofac Surg 2008 May;19(3):616-25; discussion 626-7

Department of DMCCC, Section of Maxillofacial Surgery, University of Ferrara, Ferrara, Italy.

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.
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http://dx.doi.org/10.1097/SCS.0b013e31816aabc8DOI Listing
May 2008

Comparison between osteoblasts derived from human dental pulp stem cells and osteosarcoma cell lines.

Cell Biol Int 2008 Jul 4;32(7):733-8. Epub 2008 Mar 4.

Centre of Molecular Genetics, CARISBO Foundation, Institute of Histology and General Embryology, School of Medicine, University of Bologna, Bologna, Italy.

Stem cells derived from human dental pulp are able to differentiate into osteoblasts and are a potential source of autologous bone. The aim of this study was to compare genes differentially expressed in osteoblastoids from human dental pulp (OHDP) to osteosarcoma cells (OCs). Human dental pulp was extracted and immersed in a digestive solution. Cells were cultured and selected using c-kit, CD34, CD45 and STRO-1 antibodies. In parallel, two OCs (i.e., SAOS2 and TE85) were cultured. RNA was extracted from different populations of cells and cDNA was used for the hybridisation of human 19.2K DNA microarrays. We identified several differences in gene expression between OHDP and OCs. Some down-regulated OHDP genes, such as RUNX1, MAP4K4 and PRDM2, are involved in bone development, cell motility and transcript regulation. Gene expression in OHDP is significantly different from that in OCs, suggesting differences in cell function and activity between these cells.
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http://dx.doi.org/10.1016/j.cellbi.2008.02.003DOI Listing
July 2008

Dental pulp stem cells: a promising tool for bone regeneration.

Stem Cell Rev 2008 ;4(1):21-6

Dipartimento di Discipline Odontostomatologiche, Ortodontiche e Chirurgiche, Secondo Ateneo di Napoli (Italy), Naples, Italy.

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.
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http://dx.doi.org/10.1007/s12015-008-9013-5DOI Listing
September 2008

Scaffold's surface geometry significantly affects human stem cell bone tissue engineering.

J Cell Physiol 2008 Jan;214(1):166-72

Dipartimento di Medicina Sperimentale, Sezione di Istologia ed Embriologia, Secondo Ateneo di Napoli, Napoli, Italy.

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.
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http://dx.doi.org/10.1002/jcp.21175DOI Listing
January 2008

Concave pit-containing scaffold surfaces improve stem cell-derived osteoblast performance and lead to significant bone tissue formation.

PLoS One 2007 Jun 6;2(6):e496. Epub 2007 Jun 6.

Dipartimento di Medicina Sperimentale, Sezione di Istologia ed Embriologia, Secondo Ateneo di Napoli, Naples, Italy.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0000496PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1876259PMC
June 2007

The tissue banking in cancer and stem cell research.

J Cell Physiol 2007 Aug;212(2):345-7

Dipartimento di Medicina Sperimentale, Sezione di Istologia ed Embriologia, Secondo Ateneo di Napoli, Napoli, Italy.

The Sapio Award was established in 1999 by the Sapio Group along with several Italian universities and research centers to recognize Italian scientists who have made a major contribution to the discovery or development of novel technologies in the fields of biotechnology, social and health services, nonotechnology and biosecurity in agricultural production and scientific distribution. The 2006 edition of the award meeting centered around the issues of tissue banks and biorepositories and translational medicine. The organizing committee divided this edition into a pre-meeting held in Milan on October 18, 2006 and a master meeting on October 19, 2006, held at the ISS in Rome. A summary of these meetings is given.
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http://dx.doi.org/10.1002/jcp.21098DOI Listing
August 2007