Pharmaceutical Preparations, Dental

The contributing studies were (1) Atherosclerosis Risk in Communities (ARIC); (2) The Center for Oral Health in Appalachia cohort 1 (COHRA1), which is part of the GENEVA caries consortium; (3) the Dental Registry and DNA Repository of the University of Pittsburgh School of Dental Medicine, (DRDR), also part of the GENEVA caries consortium; (4) the Hispanic Community Health Study/Study of Latinos (HCHS/SOL); (5) the Malmö Diet and Cancer Study (MDC); 6) the Northern Finland Birth Cohort 1966 (NFBC 1966); (7) the Study of Health in Pomerania (SHIP), (8) the Study of Health in Pomerania Trend (SHIP Trend); (9) TWINGENE, which is a genotyped epidemiological study recruited from the Swedish Twin Registry (TWINGENE); (10) the Women’s Genome Health Study (WGHS); (11) Biobank Japan (BBJ) and (12) Tokyo Medical and Dental University Aggressive Periodontitis Study (TMDUAGP). A detailed description of each study is included in (Supplementary Data 1 and 2).
In nine studies, analysis was conducted in individuals of European ancestry (ARIC, COHRA1, DRDR, MDC, NFBC1966, SHIP, SHIP-TREND, TWINGENE and WGHS). In one study (HCHS/SOL), participants were recruited from Hispanic and Latino communities in the USA, who self-reported ancestry from six broad groups (Cuban, Dominican, Mexican, Puerto Rican, Central American and South American). To undertake analyses within this highly admixed population, a bespoke modelling approach was undertaken. Multi-dimensional clustering was used to generate genetic analysis groups containing participants of similar ancestry. These group allocations were then used as covariates in a linear mixed model (partitioned to only fit the proportion of genetic structure due to familial relatedness rather than ancestry) alongside the first five genetic principal components, study center and log-transformed sampling weights^{53 (link)}. Subsequently, the results from HCHS/SOL were treated as a study of European ancestry and included in the primary meta-analysis.
For periodontitis only, there were two studies with participants of East Asian ancestry (BBJ, TMDUAGP), totalling 17,287 participants. For periodontitis, separate meta-analyses were performed for studies of European ancestry and studies of East Asian ancestry.

Myc, flag, GFP and GST tagged EBNA3C vectors have been described previously [14] (link), [18] (link). pcDNA3-HA-Ub was kindly provided by George Mosialos (Aristotle University of Thessaloniki, Thessaloniki, Greece). Vectors pcDNA3-Cyclin D1, pcDNA3-1x flag-Cyclin D2 and pcDNA3-1x flag-Cyclin D3 were provided by Alan Diehl (University of Pennsylvania School of Medicine, Philadelphia) and used to generate pA3F-Cyclin D by cloning PCR amplified DNA into pA3F vector [4] (link). GST Cyclin D1 vectors were cloned by inserting PCR amplified DNA into pGEX-2TK vector (GE Healthcare Biosciences, Pittsburgh, PA). pGEX-Cyclin D1 (286A) was generated by PCR using pA3F-Cyclin D1 as template. Sh-RNA vector, pGIPZ (Open Biosystems, Inc. Huntsville, AL) and lentiviral packaging vectors were described [38] (link). CDK6 cDNA cloned into pA3F vector was derived from HEK 293 cell RNA that was purified with TRIzol reagent and reverse transcribed with Superscript II (Invitrogen, Inc., Carlsbad, CA). Mouse antibodies to Cyclin D1 (DSC-6) and Sp1 (1C6), and rabbit antibody to Ub (FL-76) were from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). Rabbit antibodies to Cyclin D2 and D3 were kindly provided by Alan Diehl (University of Pennsylvania School of Medicine, Philadelphia). Mouse antibodies to flag-epitope (M2) was from Sigma-Aldrich Corp. (St. Louis, MO) and to GAPDH was from US-Biological Corp. (Swampscott, MA). Antibodies to HA-epitope (12CA5) or Myc-epitope (9E10) were prepared from cell culture supernatants as described [14] (link), [18] (link). Mouse (A10) or rabbit antibody to EBNA3C were described [14] (link), [18] (link).
HEK 293, 293T and Saos-2 (p53^-/- pRb^-/-) cells were obtained from Jon Aster (Brigham and Women's Hospital, Boston, MA, USA). Saos-2 and U2OS are human osteosarcoma cell line [39] (link). HEK 293, HEK 293T, U2OS, and Saos-2 cells were grown in Dulbecco's modified Eagle's medium (DMEM; HyClone, Logan, UT) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT), 50 U/ml penicillin (HyClone, Logan, UT), 50 µg/ml streptomycin (HyClone, Logan, UT) and 2 mM L-glutamine (HyClone, Logan, UT). BL lines BJAB, Ramos, BL41 and B95.8 infected BL41 (BL41/B95.8) were kindly provided by Elliott Kieff (Harvard Medical School, Boston, MA). MutuI, MutuIII were provided by Yan Yuan (School of Dental Medicine, University of Pennsylvania, Philadelphia, PA). These BL lines and LCL1 and LCL2 were maintained in RPMI 1640 (HyClone, Logan, UT) supplemented as described above. EBNA3C expressing BJAB lines were described [14] (link), [18] (link). Unless otherwise stated all cultures were incubated at 37°C in a humidified environment supplemented with 5% CO₂.
Adherent cells were transfected by electroporation with a Bio-Rad Gene Pulser II electroporator as described [14] (link), [18] (link).

The instrument for measuring attitude towards e-professionalism among medical and dental students is part of the larger questionnaire “Exploring the impact of social networks on the professional behaviour of healthcare professionals” conducted for research on project Dangers and benefits of social networks: e-professionalism of healthcare professionals (SMePROF project [26 ]). As a part of the project a quantitative cross-sectional study on the use of SM, attitudes and ethical values of students was carried out in the School of Medicine University of Zagreb, Croatia (UZSM) and the School of Dental Medicine University of Zagreb, Croatia (UZSDM) in the academic year 2018/2019.
Students were informed about the possibility of completing the questionnaire (UZSM 2nd and 5th year, and at the UZSDM students of all 6 years of study) during regular classes. Identification information or IP addresses were not collected to ensure anonymity. All participants were informed in guidelines statement that their participation is voluntary and anonymous. The first question of the questionnaire was informed consent about participation, allowing students that do not want to participate to opt out. Students that gave electronic informed consent on that question entered the sample. Both research and questionnaires were approved by the Ethical board of University of Zagreb School of Medicine, issued on March 22nd 2018 (641–01/18–02/01) and Ethical board of University of Zagreb School of Dental Medicine, issued on February 20th 2018 (05-PA-24-2/2018). All methods were carried out in accordance with relevant guidelines and regulations.
The questionnaire was composed of seven instruments that measured as follows: (1) sociodemographic characteristics and habits of SM usage; (2) knowledge of SM; (3) reasons of SM usage; (4) impression management on SM; (5) security on SM; (6) attitudes towards professionalism; and (7) attitudes towards e-professionalism. This paper covers validation of the seventh instrument that measures students’ attitudes towards e-professionalism.
The instrument was initially composed of 32 items. All items were taken from previous research conducted on similar topics and translated to Croatian language.
Bosslet et al. measured patient-doctor relationship on online social networks with segment dedicated to ethical questions of patient-doctor communication [27 (link)]. Therefore, items 1 thru 5 (as shown in Table 1) were taken from their research.
Table 1

Average questions scoring, answers range from 1 = completely disagree to 5 = completely agree

	Items	Mean	SD
1	It is ethically acceptable for a physician to communicate with a patient through social media as part of his/her care for patients and the patient healthcare process.a	3.30	1.00
2	It is ethically acceptable for a physician to communicate (e.g. share personal messages) with a patient through personal social media account for easier social interaction.a	3.25	1.057
3	Social media have the potential to improve communication between a physician and a patient.a	3.52	1.00
4	Communication with a patient through social media can be achieved without compromising physician-patient confidentiality.a	3.44	1.036
5	It is ethically acceptable for a physician to visit patient social media profile.a	3.18	.972
6	It is possible that your potential employer will not hire you or invite you for an interview due to information about you found online.	3.95	.901
7	There is a possibility that your online behaviour might have an impact on perception of others in your profession.	4.09	.800
8	People can make wrong assumptions about you based solely on the content of your post.	4.19	.795
9	You may lose a position you already hold (as an employee or student) due to information about you found online.	3.64	1.006
10	Sharing privileged patient information on social media without their consent is deemed to be inadmissible.	4.68	.747
11	Healthcare professionals should be banned from using social networking software due to too much of a risk.a	1.68	1.043
12	Healthcare professionals should be restricted from using social networking software due to too much of a risk.a	2.13	1.118
13	I should be able to do whatever I want online.a	3.41	1.250
14	The School has no right to interfere in my online activities.a	3.86	1.103
15	I believe that my online activities do not affect me as a professional.a	3.39	1.228
16	I strongly agree with expectations for professional behaviour and make a conscious effort to comply with them in every aspect of my life.a	3.94	.976
17	I know well what constitutes professional behaviour and what is expected of me as a current/future professional.a	4.09	.850
18	High-level professional behaviour should also be expected of students from the very beginning of their studies.a	3.52	1.196
19	Guiding patients to online information is a new responsibility of physicians in the digital age.a	2.81	1.283
20	As a medicine / dental medicine graduate, it is my obligation to keep abreast with the current trends in the use of social media.a	3.79	1.100
21	One of the responsibilities of a teacher is to counsel students on the appropriate use of social media.a	2.98	1.315
22	Professionals cannot actually fully relax.a	3.42	1.182
23	Social media have removed protection of professionals against the public.a	3.34	1.143
24	It is not always possible to maintain professionalism in online activities.a	3.68	1.027

Items marked with asterisk (^a) were measured without neutral answer

In similar research, White et al. measured attitudes to guidelines relating Facebook use [28 (link)]. Total of 21 items were taken from their research, out of which 18 entered the final scale (6 thru 18 and 22 thru 24), and 5 items that didn’t (25, 26, 29, 30, 31 as shown in Table 2).
Table 2

Items excluded after validation

	Items	Reason for exclusion
25	People have the opportunity to post photos and document aspects of their professional life which would otherwise remain private.	Low saturation on all factors
26	A little leniency should be shown if unprofessional behaviour occurs in the first years of professional education.	Not contributing to one factor; low inter-item correlation;
27	The risks of social networking software greatly overweigh the benefits.	Equally low contribution to all factors
28	Patients use social media to get medical / dental information.	Ambiguous direction of item, not contributing to one factor
29	Professionalism in online activities is as important as in traditional (offline) environments.	Low contribution to multiple factors
30	I believe discussion on online professionalism to be more important for my profession than for any other.	These two items form specific dimension that stays uncorrelated with rest of instrument, suggesting they do not measure a part of e-professionalism
31	I believe discussion on online professionalism to be more important for my healthcare profession (physicians of different specialties) in respect to any other profession.
32	The benefits of social media overweight the risks of their use.	Equally low contribution to multiple factors

Three items (19 thru 21) measuring the aspect of responsibility towards guiding patients on social networks were taken from the study of Kitsis et al., as well as two items that were not included in the final version (27, 28 as shown in Table 2) [2 (link)].
In order to assess the developed scale, it was administered to 714 students at the School Medicine and School of Dental Medicine, University of Zagreb, Croatia in the period from 10th of November 2018 to the 4th of January 2019. After screening results, 698 answers entered the analysis.
Before administering, content validity was reviewed and affirmed by the research team of experts from various scientific fields: sociologist, doctor of medicine, doctors of dental medicine, psychiatrist and communicologist. Following the example of Ichikawa et al., six researchers repeatedly checked whether items represented e-professionalism, whether item content was suitable for judging attitude towards e-professionalism of dental and medical students, and whether concept of e-professionalism was covered in the entire scale [29 (link)]. The researchers agreed that all items included in this scale reflected attitude towards e-professionalism.
Data analysis was performed using IBM SPSS statistics 25. Demographic data were summarized as descriptive statistics.
Construct validity was investigated using exploratory factor analysis, followed by scale reliability analysis using Cronbach’s alpha coefficient of the internal consistency. In an iterative process of repeating dimensionality analysis and reliability analysis after each item that was discarded instruments was improved and validated. Internal consistency was investigated for each factor and for the instrument as a whole.
As the extraction method, principal component analysis was used with oblimin as selected oblique rotation method.

Two investigators conducted individual interviews in a private exam room at the FQHC. While (SA) was interviewing the participant, (HA) took field notes for data enhancement to provide additional context for analysis. Additionally, SA kept a research journal as evidence of the interview process and enable reflection on personal roles and biases that might have influenced the analysis [21 (link)]. The journal was shared with (TC) to ensure reflexivity, a crucial component of phenomenology. The interviews were conducted in a natural conversations manner to evoke authenticity and develop rapport and establish a safe space for participants to share their personal narratives and experiences. Each overarching question was followed up with more specific questions and probes as appropriate [22 ]. Interview questions explored participants’ past history in their home country including access to oral health care and related issues, and some of the possible barriers to access oral health in the United States, oral health preventative behaviors, social and emotional behaviors. The interviews were recorded using a tape‐recorder and later uploaded to a password protected computer via a secure server at Harvard University School of Dental Medicine (HSDM) using a virtual private network. Each interview was transcribed and translated verbatim to English by the first author SA, and all identifying information was removed before this process.

This study was conducted in collaboration between Harvard School of Dental Medicine and two Federally Qualified Health Centers (FQHC) in the State of Massachusetts. The refugees list was provided by the Health Centers’ administrations. Participants were recruited by inviting them through mail and then following up with a phone call to schedule appointments, with a second call as a reminder two days before their scheduled visit. Appointments were held at both FQHC. Before data collection, participants were given enough time to read and sign a written informed consent form available in either Arabic or English. Children were given a child assent form and a consent from to their parents to sign. Participants were given a copy of the consent information.
A mixed method approach, combining both qualitative and quantitative methods, was used to provide context, via the in-depth, open-ended investigation, of matters that cannot be quantified or broken down into defined choices per se. The participants’ sample for the quantitative and qualitative study were different and exclusive of each other.

Ocy454 cells, a murine osteocyte line, were kindly provided by Prof. Paola Divieti Pajevic (Boston University, Henry M. Goldman School of Dental Medicine). MC3T3-E1 cells were purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. Ocy454 cells were differentiated in α-minimum essential medium (α-MEM, Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (Gibco) at 37 °C for 10 days. Cells were then trypsinised and subcultured in 6-well plates (2 × 10⁵ cells per well) or 96-well plates (1 × 10⁴ cells per well) for 12 h before subsequent experiments. For coculture experiments, Ocy454 cells were seeded onto 0.4-mm-thick polycarbonate inserts (Costar Corning, Life Sciences, Acton, MA, USA) in 24-well plates while MC3T3-E1 cells were plated in 24-well plates in α-MEM medium. Ocy454 and MC3T3-E1 cells were cocultured in a mixed medium (α-MEM and osteogenic differentiation medium, 1:1 ratio; Cyagen) for 28 days. The medium was replaced every 2 days, and the inserts seeded with Ocy454 cells were replaced every 4 days to prevent unwanted effects associated with excessive cell density.