Veronica Papa

MMP Activity in the Hybrid Layer Detected with in situ Zymography

Dentinal proteases are believed to play an important role in the degradation of hybrid layers (HL). This study investigated the HL gelatinolytic activity by in situ zymography and functional enzyme activity assay. The hypotheses were that HLs created by an etch-and-rinse adhesive exhibit active gelatinolytic activity, and MMP-2 and -9 activities in dentin increase during adhesive procedures. Etched-dentin specimens were bonded with Adper Scotchbond 1XT and restored with composite. Adhesive/dentin interface slices were placed on microscope slides, covered with fluorescein-conjugated gelatin, and observed with a multi-photon confocal microscope after 24 hrs. Human dentin powder aliquots were prepared and assigned to the following treatments: A, untreated; B, etched with 10% phosphoric acid; or C, etched with 10% phosphoric acid and mixed with Scotchbond 1XT. The MMP-2 and -9 activities of extracts of dentin powder were measured with functional enzyme assays. Intense and continuous enzyme activity was detected at the bottom of the HL, while that activity was more irregular in the upper HL. Both acid-etching and subsequent adhesive application significantly increased MMP-2 and -9 activities (p < 0.05). The results demonstrate, for the first time, intrinsic MMP activity in the HL, and intense activation of matrix-bound MMP activity with both etching and adhesive application.

Immunohistochemical and biochemical assay of MMP-3 in human dentine

OBJECTIVE The function of endogenous MMP-3 and its distribution within the human dentine is unclear. Thus, the aim of the present study was to assay the presence and distribution of MMP-3 within human sound dentine by means of biochemical and immunohistochemical assays. METHODS Powdered dentine from extracted human teeth was prepared and (1) partially demineralised with 1% H(3)PO(4) for 10min or (2) untreated (control). The presence of MMP-3 was measured using a colorimetric assay system (QuantiSir™, Epigentek, USA). Additional cryo-fractured dentine fragments were processed for immunohistochemical identification of MMP-3 under FEI-SEM. Casein-zymography was used to investigate MMP-3 activity. RESULTS MMP-3 detected level was 2.732ng/μL in partially demineralised dentine powder, whilst it increased to 3.280ng/μL in mineralised dentine. The FEI-SEM analysis revealed positive immunolabelling patterns for MMP-3, predominantly localized on the intertubular collagen fibrillar network showing MMP-3 directly or indirectly bound to the collagen fibrils. Casein-zymograms showed positive proteolytic activity for MMP-3 in demineralised dentine powder. CONCLUSION The results of the study clearly revealed the presence and distribution of MMP3 in human sound dentine. Whilst the presence was verified, its role is still unclear. Future studies are needed to investigate the possible involvement of MMP-3 in physiological and pathological condition of the dentine-pulp complex.

MMP-2 assay within the hybrid layer created by a two-step etch-and-rinse adhesive: Biochemical and immunohistochemical analysis

OBJECTIVE Degradation of hybrid layers (HLs) within resin-infiltrated dentine results from multiple degradation factors, including collagenolytic activity of specific matrix metalloproteinases (MMPs). Inhibition of host-derived MMPs may, therefore, slow the degradation of HL. The null hypothesis tested is that the presence of MMP-2 is similar regardless of chlorhexidine (CHX) pre-treatment or the use of an adhesive. METHODS Powdered dentine prepared from extracted human teeth was divided into 4 groups: (G1) mineralised powder (control group); (G2) dentine powder treated with 1% phosphoric acid for 1 min; (G3) 1% phosphoric acid-etched dentine treated with Adper Scotchbond 1 XT (SB1XT; 3M ESPE); (G4) 1% phosphoric acid-etched dentine treated with 0.2% CHX followed by SB1XT. The concentration of detectable pro-MMP-2 and MMP-2 was assayed using a colorimetric assay system (QuantiSir). In addition, the presence of MMP-2 in the HL was assessed in 1 year-aged adhesive-dentine interfaces using an immunohistochemical approach under FEI-SEM/TEM. RESULTS In dentine powder treated with 1% phosphoric acid (G2), MMP-2 level decreased compared to controls (G1); the application of SB1XT (G3) resulted in an increase of MMP-2, whilst 0.2% CHX before SB1XT application (G4), reduced MMP-2. The FEI-SEM/TEM analysis revealed MMP-2 distribution within the HL of aged interfaces showing increase MMP-2 patterns in the control group and minor labelling in the CHX-pretreated specimens. CONCLUSION The results of this study support the use of non-toxic MMPs inhibitors, such as CHX, as an appropriate additional step in bonding procedures in order to increase the longevity of the adhesive restorations.

Teaching Anatomy in the XXI Century: New Aspects and Pitfalls

Anatomy has historically been a cornerstone in medical education regardless of nation, racial background, or medical school system. By learning gross anatomy, medical students get a first “impression” about the structure of the human body which is the basis for understanding pathologic and clinical problems. Although the importance of teaching anatomy to both undergraduate and postgraduate students remains undisputed, there is currently a relevant debate concerning methods of anatomy teaching. In the past century, dissection and lectures were its sole pedagogy worldwide. Recently, the time allocated for anatomy teaching was dramatically reduced to such an extent that some suggest that it has fallen below an adequate standard. Traditional anatomy education based on topographical structural anatomy taught in lectures and gross dissection classes has been replaced by a multiple range of study modules, including problem-based learning, plastic models or computer-assisted learning, and curricula integration. “Does the anatomical theatre still have a place in medical education?” And “what is the problem with anatomic specimens?” We endeavor to answer both of these questions and to contribute to the debate on the current situation in undergraduate and graduate anatomy education. Doctors without anatomy are like moles.They work in the dark and the work of their hands are mounds. Friedrich Tiedemann The foundation of the study of the art of operating must be laid in the dissecting room. Robert Liston

3D printing: a valuable resource in human anatomy education

Anatomy is essential to the health and medical professions: by learning anatomy, medical students learn about the structure of the human body, providing them with the basic tools needed for understanding pathology and clinical problems. In the past century, dissection and lectures formed the basis of anatomy education worldwide. More recently, traditional anatomy education based on topographical structural anatomy taught in lectures and in gross dissection classes, has been replaced by a multiple range of study modules, including problem-based learning, plastic models and/or computer-assisted learning and curricula integration (Louw et al. 2009). The anatomy field is strongly confident that donated bodies can still benefit new medical students significantly, and that dissection and pro-section procedures cannot be underestimated in a modern medical curriculum (Louw et al. 2009). Nevertheless, dissection and light microscopy are not problem-free. Storing human bodies is expensive, and other issues such as preservation and reduced suitability for dissection due to illness, age or obesity could be a problem; moreover, careful dissection is time-consuming and microscopy equipment can be expensive. Aside from biological and methodological matters, dissection and prosection have also issues concerning ethical convictions and legal restrictions or simply logistical problems due to lack of space, funds, recruitment, or proper furniture and equipment. Considerable variations in the legal and ethical frameworks concerning body bequests for anatomical examination exist worldwide based on cultural and religious variations as well as different legal and constitutional backgrounds. For instance, there are different views concerning the ‘‘ownership’’ of cadavers or the acceptability of using unclaimed bodies that have not given informed consent (McHanwell et al. 2008). In addition to known methods such as plastination and Thiel method embalming, a new three-dimensional printing system (3D printing) has been developed recently—an innovative approach that could become a valuable resource in anatomy education. 3D printing (also known as additive manufacturing or rapid prototyping) has existed since the late 1980s but has seen rapid advancements more recently because of decreased cost, computer engineering, and expanding applications. Rapid prototyping involves creating a physical 3D model from a computerised mould. The technology has been used in industrial processes to create forerunners of intended final products; models can be also analysed and modified before production is planned (Gibson et al. 2010). Basically, the principle of rapid prototyping is to use 3D computer models for the reconstruction of a 3D physical model by the addition of material layers (Gibson et al. 2010). With additive fabrication, the machine reads in data from a CAD drawing and lays down successive layers of liquid, powder, or other sheet material, and in this way builds up the model from a series of cross sections. These M. Vaccarezza (&) V. Papa Department of Human, Social and Health Sciences, University of Cassino and Southern Lazio, Campus Folcara, via S. Angelo in Theodice, 03043 Cassino, FR, Italy e-mail: m.vaccarezza@uq.edu.au; m.vaccarezza@unicas.it

RFID as a new ICT tool to monitor specimen life cycle and quality control in a biobank

Background Biospecimen quality is crucial for clinical and translational research and its loss is one of the main obstacles to experimental activities. Beside the quality of samples, preanalytical variations render the results derived from specimens of different biobanks or even within the same biobank incomparable. Specimens collected along the years should be managed with a heterogeneous life cycle. Hence, we propose to collect detailed data concerning the whole life cycle of stored samples employing radio-frequency identification (RFID) technology. Methods We describe the processing chain of blood biosamples that is operative at the biobank of IRCSS San Raffaele, Rome, Italy (BioBIM). We focus on the problem of tracing the stages following automated preanalytical processing: we collected the time stamps of all events that could affect the biological quality of the specimens by means of RFID tags and readers. Results We developed a pilot study on a fragment of the life cycle, namely the storage between the end of the preanalytics and the beginning of the analytics, which is usually not traced by automated tools because it typically includes manual handling. By adopting RFID devices we identified the possible critical time delays. At 1, 3 and 6 months RFID-tagged specimens cryopreserved at -80°C were successfully read. Conclusions We were able to record detailed information about the storage phases and a fully documented specimen life cycle. This will allow us to promote and tune up the best practices in biobanking because i) it will be possible to classify sample features with a sharper resolution, which allows future utilization of stored material; ii) cost-effective policies can be adopted in processing, storing and selecting specimens; iii) after using each aliquot, we can study the life cycle of the specimen with a possible feedback on the procedures.

Immunohistochemical and biochemical identification of MMP-2 in dentin

Matrix metalloproteinases (MMPs) play an important role in many biological and pathological processes because of their ability to degrade all extracellular matrix (ECM) components. The purpose of this study was to identify MMP-2 in human dentin by immunohistochemical and biochemical methods. Dentin cryo-fractured fragments were obtained from human sound teeth, partially decalcified in 0.5 M EDTA pH 7.4 for 30min and submitted to a pre-embedding immunolabeling technique, using primary monoclonal antibodies anti-MMP-2 and exposed to a secondary antibody conjugated with gold nano-particles. Observations were performed by means of a FEI-SEM. Furthermore,the presence of MMP-2 was correlatively assayed by a colorimetric assay system (Quantisir) that allows direct measurement of MMP-2 levels. The immunohistochemical analysis revealed an intricate three-dimensional network of type I collagen and positive immunolabeling patterns for MMP-2 showing its distribution along with the collagen fibrils. The colorimetric assay resulted in higher presence of MMP-2 in mineralized dentin, compared to the partially demineralized counterpart. The role and function of dentin MMPs is not well known, but they have shown to contribute to auto-degenerative processes in dentin, such as inflammation of dental pulp, progression of caries lesions. This study demonstrated using an immunohistochemical and a biochemical approach that MMP-2 is an intrinsic component of the human dentin organic matrix, with possible roles in dentin matrix turnover and degenerative processes.

Identification of MMP-8 in human sound dentin

Objective Matrix metalloproteinases (MMPs), a class of Ca/Zn-dependent endopeptidases, are secreted by odontoblasts as proenzymes that remain trapped within the mineralized dentin matrix. Activation of these proenzymes is a critical step that leads to dentin extracellular matrix breakdown. The study aimed to assess presence, localization and distribution of MMP-8 in human sound dentin by biochemical and immunohistochemical assays. Methods The presence of MMP-8 in human sound dentin was assayed using a colorimetric assay system that allows direct measuring of MMP-8 levels (QuantisirTM, Epigentek, USA): powdered dentin prepared from extracted human teeth was prepared and 1) partially demineralized with 1% H3PO4 for 10 min or 2) untreated (control). To further detect the localization and distribution of MMP-8 within the dentin organic matrix, dentin cryo-fractured fragments were obtained from human sound teeth, partially decalcified in 0.5 M EDTA pH 7.4 for 30min and submitted to a pre-embedding immunolabeling technique, using primary monoclonal antibodies anti-MMP-8 and exposed to a secondary antibody conjugated with gold nano-particles. Observation was performed under FEI-SEM. Results In mineralized dentin the amount of MMP-8 was 3.32 ng/μL, while the MMP-8 level in the partially demineralized dentin powder significantly decreased (2.45 ng/μL) compared to levels obtained in mineralized dentin. FEI-SEM analyses confirmed that MMP-8 is an endogenous component of the human dentin organic matrix. The labelling pattern of the gold particles bound to the antibody anti-MMP-8 was clearly detectible in the dentin fragments as spherical, highly electron reflective spots localised in the peritubular dentin, revealing the three-dimensional relationship between these proteinase and the collagen fibrils. Conclusion The role and function of dentin MMPs is not well known, but they have shown to contribute to auto-degenerative processes in dentin, such as inflammation of dental pulp, progression of caries lesions and, more recently, degradation of dentin matrix exposed during dental bonding procedures. This study demonstrated that MMP-8 is an intrinsic constituent of the fibrillar network of the human dentin organic matrix, thus supporting that this enzyme may play important roles in the degradation of the dentin organic matrix.