Vol. 25 No. 04 Oct-Dec 2016

Hira Salam1                    BDS
Farheen Danish2          MBBS, MPhil
Lubna Avesi3                MBBS, FCPS
Talat Mirza4                  MBBS, PhD

INTRODUCTION

Despite small incidence (3.8 per million in men and 2.8 per million in women) osteosarcoma isthe second most frequently documented primary skeletal malignancy after multiple myeloma1 . The tumor usually manifests in long bones of extremities with rare (6- 9% of all osteosarcomas) occurrence in jaws2 . Incidence of osteosarcomas in jaw bones has been reported to be 0.7 per million with slight male predominance (male to female ratio 1.5:1)2,3). Osteosarcomas occurring in jaws are relatively less aggressive than those that take birth in long bones, recurrence after excision, however, is a significant  complication4 . We present a case of recurrent mandibular osteosarcoma in a 28-year-old male patient that was diagnosed and reported by histopathology department at Dow International Medical and Dental College, Ojha campus, Karachi. Three variants are described on the basis of histological picture; osteoblastic, chondroblastic, and fibroblastic osteosarcomas. The osteoblastic variant, constituting 60% of jaw lesions, is typified by disorderly arranged malignant osteoblasts with significant pleomorphism and large deep staining nuclei that deposits variable amount of osteoid matrix. Fibroblastic variant is markedly cellular, exhibiting fibroblastic proliferation along with atypical spindle cells and osteoid deposition5 . The chondroblastic variant, on the other hand, is characterized by atypical chondroid areas comprising of binucleate cells exhibiting hyperchromatism with prominent nucleoli as was observed in our case6 .

CASE REPORT

The patient was a 28-year-old male who reported with primary complaint of pain and swelling in lower border of left side mandible associated with feeding difficulty. Detailed history was acquired and revealed the patient was diagnosed and treated for osteosarcoma one year back. An orthopantomogram (OPG) (Fig. 1) was obtained and revealed mixed radiopaque and radiolucent lesion with hallmark sunburst appearance characteristic of osteosarcoma involving the body of left side mandible up to the angle but not crossing the midline. Widening of the periodontal (PDL) space and loss of lamina dura was also appreciated around left mandibular canine and premolars.

The hematological and biochemical tests were all within normal limits. Serological tests performed for hepatitis B and C were non-reactive. Hemimandibulectomy was done and specimen was received by histopathology department at Dow International Medical and Dental College, Ojha campus, Karachi for evaluation.

Histopathologic examination displayed malignant neoplasm composed of spindle shaped cells displaying moderate to marked nuclear pleomorphism and hyperchromasia. Malignant osteoid formation was noted in some places. Presence of chondroid areas was also appreciated, hence the diagnosis of chondroblastic variant of osteosarcoma was made after consideration of chondrosarcoma as differential diagnosis (Picture 1 and 2).

DISCUSSION

Osteosarcomas or osteogenic sarcomas are most commonly reported primary bone tumors in adolescents/ young adults characterized through deposition of osteoid or bone formation by neoplastic cells. Overall incidence is small encompassing merely ~ 0.2% of all malignancies7.

Occurrence in jaws is even rarer (6-9% of all osteosarcomas) with relatively greater prevalence reported in older age group (34-36 years). A broad spectrum of clinical, histological, and radiographic presentations is recognized and hamper the process of reaching a conclusive diagnosis3,8. Hallmark clinical manifestation includes swelling and pain, both observed in our case along with difficulty in feeding(3). Owing to overlapping radiographic andhistological features, it is difficult to differentiate between chondrosarcomas and osteosarcomas with chondroid differentiation. In our case,presence of large chondroid areas also posed same problem, but the characteristic sunburst radiographic picture as well as osteoid deposition histopathologically aided to rule out chondrosarcoma.

Radiography is an indispensable tool in diagnosis of mandibular osteosarcoma whereby widening of the PDL space around teeth in involved area, sunburst appearance, and widening of mandibular canal are pathognomonic9. Yen et al. 10, recognized that origin from metaphysis, boneforming matrix, marked periosteal reaction and patient from younger age group favor diagnosis of chondroblastic variant of osteosarcoma over chondrosarcoma. Fibrous dysplasia, osteomyelitis, osteoma, myositis ossificans, and cemento-osseous dysplasias are commonly occurring lesions in mandible that may resemble osteosarcoma onradiological picture11. Histopathology
remains gold standard to differentiate osteosarcoma from these differentials.

Multiple incidences of recurrence have been reported in association with osteosarcoma of jaws12-14. Two such cases13,14 were observed with chondroblastic variant of osteosarcoma, similar to our case. Wide variation exists with respect to time span observed before appearance of recurrence from3 years12 to as short as 1 month13,14. In reported case, recurrence was recorded within one year, intermediate between reported durations observed before appearance of recurrent tumor. Wide excision with clear surgical margins are mainstay of eradication and prevention of recurrence. Excision without disease-free margins renders radiation therapy inadequate12. The recurrence in presented case was recorded within one year of excision with adjuvant chemotherapy. Without resection disease progression can be fatal with 5-year survival rate of 62%.

CONCLUSION

Osteosarcomas are challenging neoplasms for both, histopathologists from a diagnostic point of view, and surgeon because of the risk for recurrence. We have reported a case of fairly rapid recurrence (within a span of one year) to highlight the significance of wide clear surgical margins in management of osteosarcomas of jaws.

CONFLICT OF INTEREST

Declared none.

ACKNOWLEDGMENTS

Declared none.

REFERENCES

1. Berner K, Johannesen TB, Berner A, Haugland HK, Bjerkehagen B, Bøhler PJ, et al. Time-trends on incidence and survival in a nationwide and unselected cohort of patients with skeletal osteosarcoma. Acta Oncologica. 2015;54: 25-33.
2. Halder GC, Patsa S, Jadav RB, Ray JG. Osteosarcoma of mandible: A case report. Int J Case Rep Imag 2015;6:280.
3. Wang S, Shi H, Yu Q. Osteosarcoma of the jaws: demographic and CT imaging features. Dentomaxillofacial Radiol. 2012; 41(1): 37-42.
4. Liu J, Cranmer L, Larsen BT, Kuo PH, Kopp LM. Recurrent Osteosarcoma Presenting as an Isolated Bone Marrow Relapse. J Pediatric Hematol Oncol. 2016.
5. Desai D, Pandith S, Jeergal PA, Arathi K, Saini R. Fibroblastic Variant of Osteosarcoma: A Challenge in Diagnosis & Management. Open Dent J. 2010; 4: 211-7.
6. Kedar S, Nagle S, Agarwal S, Bage S, Kothekar A, Kashide R, et al. Giant Chondroblastic Osteosarcoma Mandible-A Rare Case Report. Otolaryngol. 2013;3(2).
7. Raymond A, Ayala A, Knuutila S. Conventional osteosarcoma. World Health Classification of Tumours Pathology and Genetics of Tumours of Soft Tissue and Bone. 2002: 264-70.
8. Nthumba PM. Osteosarcoma of the jaws: a review of literature and a case report on synchronous multicentric osteosarcomas. World J Surg oncol. 2012; 10: 240.
9. Athar M, Chauhan S, Tripathi R, Kala S, Avasthi S, Jangra V, et al. Malignant mandibular tumors: two case reports of rare mandibular tumors in a single institution. Arch Med Biomed Res. 2014; 1: 22-9.
10. Yen CH, Chang CY, Teng MM, Wu HT, Chen PC, Chiou HJ, et al. Different and identical features of chondroblastic osteosarcoma and chondrosarcoma:highlights on radiography and magnetic resonance imaging. J Chinese Med Assoc 2009;72:76-82.
11. Camargo AJ, Cheade M, Martinelli C, Watanabe PCA. Undifferentiated osteosarcoma of the mandible oblique line: a case report. Braz Dent Sci. 2015; 18: 22-7.
12. Sivakumar T, Jeevadhas P, Raghavanpillai VB, Sivaraj S, P Joseph A, Denny SE. Recurrent osteosarcoma of maxilla presenting as fungating mass: A case report. Int J Case Rep Imag. 2016; 7: 149-53.
13. Kanala S, Gudipalli S, Ramavath MN, Venkata SK. Recurrent High Grade Chondroblastic Osteosarcoma of the Maxilla: A Rare Case Report. J Appl Dent Med Sci. 2016; 2: 2.
14. Adwani D, Bhattacharya A, Adwani N, Adwani R, Sharma VW. Massive recurrent chondroblastic osteosarcoma of maxilla: a rare case report. J Clin Diagn Res. 2014; 8: 288.

1 Department of Oral Pathology, Dow International Medical and Dental College, Ojha Campus, Karachi, Pakistan
2 Assistant Professor, Department of Pathology, Dow International Medical and Dental College, Ojha Campus, Karachi, Pakistan
3 Associate Professor, Department of Pathology, Dow International Medical and Dental College, Ojha Campus, Karachi, Pakistan
4 Meritorious Professor, Director IBMS, Dow International Medical and Dental College, Ojha Campus, Karachi, Pakistan
Corresponding author: “Dr. Hira Salam” < hirasalam@gmail.com >

Hira Salam1                    BDS
Farheen Danish2          MBBS, MPhil
Lubna Avesi3                MBBS, FCPS
Talat Mirza4                  MBBS, PhD

Zohaib Khurshid1                         BDS, MRes
Jameela Abdul Haq2                   BDS
Rabia Sannam Khan3                 BDS, MSc
Muhammad Sohail Zafar4        BDS, MSc, PhD
Maria Altaf 5                                   BDS, MPhil
Shariq Najeeb6                              BDS, MSc

INTRODUCTION

The Saliva – a clinically informative biological fluid, has gained a lot of interest because of its physiologic diagnostic medium.1 Its major components include a broad spectrum of proteins and peptides, nucleic acids, electrolytes, and hormones.2 The major salivary gland contributes to secrete 93% of the saliva, whereas the remaining 7% is secreted by the minor glands. The salivary composition contains 99% water and 1 % solvent molecules (organic and inorganic) with a paramount value of quantity as well as quality.3 The salivary glands are composed of acinar and ductal cells. A large serous secretion is produced by the acinar cells of the parotid glands. The production of calcium by acinar gland is lesser as compared to submandibular gland; however, it does synthesize most of the alpha-amylase. The submandibular glands are responsible for producing mucins along with the sublingual glands. It also produces proline and histatin-rich proteins in conjunction with the parotid.4 .In Table 1, detailed discussion on how composition of saliva helps in different function for the maintenance of oral health.2

The daily secretion rate of minor salivary gland essentially secretes mucous up to 500ml to 700ml whereas, the average volume in the mouth is 1.1 ml/per minute. The saliva production is controlled by the nervous system and the secretion ranges from 0.25 ml/min to 0.35 ml/min at the basal metabolic rate. Certain sensory, electrical, or mechanical stimulation can raise the secretion rate to 1.5ml/min.6

The fact that the highest volume of saliva is produced around meals has been proved by various research studies. The maximum peak is reached at around 12 a.m., with a significant fall while sleeping.7 The salivary function includes maintaining the integrity of oral structures in personal relationships, digestion of food, and regulating the cause of oral infections.7
Saliva also plays a major role in protecting the teeth from caries.8 These can be subdivided into four categories: (i) diluting and eliminating sugars and other substances (ii) buffer capacity (iii) balancing demineralisation and (iv) anti-microbial action.

1. Physiology of Saliva

The three major salivary glands are Parotid, submandibular, and sublingual glands, 800 to 1000 minor salivary glands are present throughout the oral submucosa. Approximately 600 ml of serous and mucinous saliva, which includes 99% water and 1% other important compounds like minerals, electrolytes, buffers and enzymes, are produced by the human salivary glands each day.9 Each type of gland is responsible for producing a special type of secretion. The parotid glands produce serous fluids, the submandibular glands produce sero-mucous secretion, and the sublingual glands secrete mucous saliva. Secretions from individual salivary glands are also evaluated for detecting glandspecific diseases, such as infection and obstruction. However, the whole saliva is most frequently studied when the salivary analysis is used for the evaluation of systemic disorders. A collection of saliva can take place with as well as without stimulation.10 Stimulated saliva is collected by masticatory action (i.e. from a subject chewing on paraffin) or by gustatory stimulation (i.e. application of citric acid on the subject’s tongue). Unstimulated saliva is collected without exogenous gustatory, masticatory, or mechanical stimulation. The whole saliva can be collected by the draining method, in which saliva is allowed to drip off the lower lip, and the splitting method, in which the subjects expectorates saliva into a test tube.11

2. Transfer of Biomolecules from Blood to Saliva

Ultrafiltration through the tight junctions between the cells of secretary units is the most common route. Ultrafiltration also occurs through the spaces between the acinus and ductal cells. The molecules must be relatively small in order to follow this type of system.12 A serum molecule reaching saliva by diffusion must cross 5 stages: the capillary wall, the interstitial space, the basal cell membrane of the acinus cell or duct cell, and the cytoplasm of the acinus or duct cell and the luminal cell membrane. The molecule’s size and the electric charge it carries are partly the decisive factors of the ability of a molecule to diffuse passively through cell membranes. It will be difficult
for a polar molecule, or a charged ion in a solution to pass through the phospholipid membrane. In addition, molecules are transported into saliva through transudation of plasma compounds into the oral cavity. This could be from gingival crevicular fluid or directly by the oral route.13

THE DIAGNOSTIC ROLE OF SALIVA

It has been analysed that human saliva can be obtained by various non-invasive techniques and can demonstrate various biomarkers for the detection and monitoring of body diseases.14 Therefore, there is an increased interest of investigators to screen, diagnose and monitor the normal functioning of the human body using saliva. The screening of steroid and peptide hormones, antibodies and the therapeutic drug abuse can be monitored via salivary functions. The steroid hormone analysis can be measured at home by multiple specimens; this may be helpful in measuring the reproductive cycles, stress anxiety and menopausal variations. The drug abuse can be monitored without using any blood drawing procedures.15,16 The onset of infectious diseases can be monitored via measuring the antibody level in human body.17 Therefore, saliva has been observed as a diagnostic tool for various diseases. Saliva, besides serving as an effective and inexpensive diagnostic fluid has also served as a rapid medium for evaluation of various analytes.18A number of studies have been conducted which have analysed multiple scenarios associated with the blood and saliva as a diagnostic tool.8,16,19Some of them have favoured the salivary diagnosis whereas; some of them formulated difficult setups.

1. Advantages of Saliva as a diagnostic tool

As the saliva can be collected by a non-invasive method, it has been proved to be beneficent to avoid the complications associated with needle infections. This method has been proved to be valuable for the elderly patients who have a decreased blood count, or subjects with physical or mental challenges.20 This prevents the crosscontamination, which could eventually occur due to improperly collected blood specimens. For steroid hormone evaluation, saliva depicts an active level free hormone concentration rather than the bound form as observed with the serum level. Therefore, saliva serves as a more efficient fluid for the steroid hormone monitoring for measuring th menopausal fluctuations whereas, steroids in the blood are bound to the globulin molecules.21Due to the diurnal variations in the reproductive cycle, steroid hormones are needed to be collected late night or early morning, or daily at the same time of day to observe the variations in the cycle. These multiple specimens when collected via blood servestoo expensive for the patient.

2. Saliva provides potential diagnostics

The methodology for saliva assay has been improving with time due to the ease in its collection. The human salivary proteins serve diagnostic value for several systemic diseases,

2.1. Cardiovascular disease

The cardiovascular disease is being the major cause of mortality worldwide. The biomarkers in saliva such as; Creactive proteins, myoglobin, brain natriuretic peptide (NTproBNP) and cardiac troponin (cTnl) may be helpful for postoperative cardiovascular patients.22 This includes the total salivaryamylase; the estimation of salivary amylase is being evaluated for every six hours after the surgery. Decreased salivary amylase level has been found for the patient with a ruptured aortic aneurysm. The salivary aamylase provides a direct and end point for catecholamine activity for the patients with altered heart rate in a stressed condition.23,24

2.2. Endocrine Function

The plasma steroid levels are monitored for the clinical assessment of endocrine function, the steroid hormone in plasma shows the active level of these hormones. Currently, the saliva is being more commonly used to evaluate the steroid hormone levels. These include the cortisol, dehydroepiandrosterone, estradiol, estriol, testosterone and progesterone. 21,25This may be helpful to evaluate the cognitive-emotional behaviour and to predict the individual’s sexual activity. Additionally, the ovarian function and the risk of preterm birth can be further evaluated by assaying the salivary steroid levels.

2.3. Infectious Diseases (Viral and Bacterial)

The potential use of salivary estimation is beneficial to evaluate human immunodeficiency virus (HIV). The enzyme-linked fluorescence technique combined with Western blot assays has been used for saliva testing to determine the sensitivity and specificity. The efficacy of saliva as a diagnostic tool has alleviated its association along with the traditional diagnostic methodology. HIV-infected patient can now be screened for HIV-1 and HIV-2 with help of salivary-based enzyme enzyme-linked immunosorbent assay (ELISA) accompanied by a western blot for further confirmation.26 Additionally, a significant progress has been observed with the identification of various other infections which includes viral Hepatitis, Dengue, Zika virus and Malaria.27–29

3. The Technological Discovery of Salivary Biomarkers

The diagnostic role of saliva is still facing the technological barriers due to the presence of complex constituents with lower quantity. However, with the emergence of advanced technology during the last decade, the analyses of proteins and nucleic acids have provided a broader horizon to overcome these challenges.30 The current research on salivary proteins has shown that albeit a lower concentration of proteins are present in saliva, but these proteins can still play an important role in thediagnosis of various acute and chronic diseases. The principle analysis of salivary proteomes briefs the complex spectrum of oral and general health consequences and unveils the disease progression at anearly stage. The protein expression is generally analysed by PAGE (polyacrylamide gel electrophoresis).31,32 PAGE discriminates between the similar types different complex compounds, these even provide help to identify different isoforms of the same polypeptide. The mass spectrometry introduces more specific separation along with PAGE, these constituents can further be categorized by using electro spray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI).33

4. Barriers and Challenges Associated with Salivary Collection

As the salivary specimens are collected by direct spitting into the tube or the method of absorption by cotton balls, these specimens are non-sterile and serve as amedium for bacterial progression. However, many different companies patented theirsaliva collection devices such as Oasis diagnostics, DNA Genotek, and Salimetrics. 34,35Other barriers are clinician and insurance companies have to accept this test as authenticate so the investment can be promising for the development ofstandardisation of sampling.

CONCLUSIONS

The molecular diagnostics of saliva have proved a valuable early stage detection of various infectious and systemic disease. Saliva, as biological fluid is rich in diagnostic biomarkers for both the oral and systemic disorders. The interest in saliva as a diagnostic tool is due to the fact that the collection of thesample is easy and simple with non-invasive interventions, therefore, avoiding the risk of infection spread. A new technology known as Point of Care (PoC) technology will soon be available in dental practices that can be utilizedfor an early screening or detection of various systematic diseases on spot. However, further studies are warranted in this area.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

ACKNOWLEDGMENTS

We acknowledge thePakistan Human Saliva Research Group (PakSRG) for supporting us for the write-up of this manuscript and providing the technical data as well.

AUTHORS’ CONTRIBUTION

ZK and JH: bring this idea and start compiling papers from different database such as Pubmed and scopus.
RSK and MA: help in write up and designing of tables.
MSZ and SN: did their expert help for the improvement and final proof read.

REFERENCES

1. Wong DT. Salivary diagnostics powered by nanotechnologies, proteomics and genomics. J Am Dent Assoc; 2006; 137: 313–21. 2.
2. Khurshid Z, Naseem M, Sheikh Z, Najeeb S, Shahab S, Zafar MS. Oral antimicrobial peptides: Types and role in the oral cavity. Saudi Pharm J. 2016 30; 24: 515-24.
3. Streckfus C, Bigler L. Saliva as a diagnostic fluid.Oral Diseases. 2002;8:69–76.
4. Khurshid Z, Najeeb S, Mali M, Moin SF, Raza SQ, Zohaib S, Sefat F, Zafar MS. Histatin peptides: Pharmacological functions and their applications indentistry. Saudi Pharm J. 2016 May 4.
5. Dawes C, Pedersen AML, Villa A, Ekström J, Proctor GB, Vissink A, et al. The functions of human saliva: A review sponsored by the World Workshop on Oral Medicine VI. Arch Oral Biol. 2015;60:863–74.
6. Henson BS, Wong DT. Collection, Storage, and Processing of Saliva Samples for Downstream Molecular Applications. In: Methods in molecular biology (Clifton, NJ). 2010 p. 21–30.
7. Zhang Y, Sun J, Lin C-C, Abemayor E, Wang MB, Wong DTW. The emerging landscape of salivary diagnostics. Periodontol 2000. 2016;70:38–52.
8. Sun X, Huang X, Tan X, Si Y, Wang X, Chen F, et al. Salivary peptidome profiling for diagnosis of severe early childhood caries. J Transl Med. 2016; 14: 240.
9. Proctor GB. The physiology of salivary secretion. Periodontol 2000. 2016; 70: 11–25.
10. Khurshid Z, Zohaib S, Najeeb S, Zafar M, Slowey P, Almas K. Human Saliva Collection Devices for Proteomics: An Update. Int J Mol Sci. 2016; 17: 846.
11. Khurshid Z, Najeeb S, Khan RS, Zafar MS Salivaomics: An Emerging Approach in Dentistry. J Pak Dent Assoc. 2016; 25: 1–3.
12. Lee YH, Wong DT. Saliva: An emerging biofluid for early detection of diseases. Am J Dent. 2009; 22: 241–8.
13. Chiappin S, Antonelli G, Gatti R, De Palo EF. Saliva specimen: A new laboratory tool for diagnostic and basic investigation. Clin Chim Acta. 2007; 383: 30–40.
14. Rudney JD, Staikov RK, Johnson JD. Potential biomarkers of human salivary function: A modified proteomic approach. Arch Oral Biol. 2009; 54: 91–100.
15. Johnson GF, Dechtiaruk WA, Solomon HM. Gaschromatographic determination of theophylline in human serum and saliva. Clinical chemistry. 1975 1; 21:144-7.
16. Kargul B, Tanboga I, Ergeneli S, Karakoc F, Dagli E. Inhaler medicament effects on saliva and plaque pH in asthmatic children. J Clin Pediatr Dent. 1998; 22:137–40.
17. Speicher DJ, Wanzala P, D’Lima M, Johnson KE, Johnson NW. Detecting DNA viruses in oral fluids:evaluation of collection and storage methods. Diagn Microbiol Infect Dis. 2015; 82:120–7.
18. Hu S, Loo JA, Wong DT. Human body fluid proteome analysis. Proteomics. 2006;6:6326-53.
19. Streckfus CF, Bigler LR. Saliva as a diagnostic fluid. Oral Dis. 2002;8:69–76.
20. Punyadeera C, Slowey PD. Saliva as an emerging biofluid for clinical diagnosis and applications of MEMS/NEMS in salivary diagnostics. InNanobiomaterials in Clinical Dentistry 2012 Dec 14 (pp. 453-473). William Andrew [Elsevier].
21. Gröschl M. Current status of salivary hormone analysis. Clin Chem. 2008;54:1759-69.
22. Yin X, Subramanian S, Hwang SJ, O’Donnell CJ, Fox CS, Courchesne P, Muntendam P, Gordon N, Adourian A, Juhasz P, Larson MG, Levy D. Protein Biomarkers of New-Onset Cardiovascular DiseaseSignificance. Arterioscler Thromb Vasc Biol. 2014; 34:939-945.
23. Ajwani S, Mattila KJ, Narhi TO, Tilvis RS, Ainamo A. Oral health status, C-reactive protein and mortality – a 10 year follow-up study. Gerodontology . 2003 ; 20: 32–40.
24. Punyadeera C, Dimeski G, Kostner K, Beyerlein P, Cooper-White J. One-step homogeneous C-reactive protein assay for saliva. J Immunol Methods. 2011; 373:19–25.
25. Kirschbaum C, Hellhammer DH. Salivary cortisol in psychoneuroendocrine research: Recent developments and applications. Psychoneuroendocrinology. 1994; 19: 313–33.
26. Poloni TR, Oliveira AS, Alfonso HL, Galvão LR, Amarilla AA, Poloni DF, Figueiredo LT, Aquino VH. Detection of dengue virus in saliva and urine by real time RT-PCR. Virol J. 2010;7:22.
27. Musso D, Roche C, Nhan TX, Robin E, Teissier A, Cao-Lormeau VM. Detection of Zika virus in saliva. J Clin Virol. 2015;68:53-5.
28. Ho DD, Byington RE, Schooley RT, Flynn T, Rota TR, Hirsch MS. Infrequency of isolation of HTLV-III virus from saliva in AIDS [Letter]. New Eng J Med. 1985;313:1606.
29. Balcarek KB, Warren W, Smith RJ, Lyon MD, Pass RF. Neonatal screening for congenital cytomegalovirus infection by detection of virus in saliva. J Infect Dis 1993;167:1433–6.
30. Wong DT. Towards a simple, saliva-based test for the detection of oral cancer “oral fluid (saliva), which is the mirror of the body, is a perfect medium to be explored for health and disease surveillance”. Expert Rev Mol Diagn [Internet]. 2006;6:267–72.
31. Latterich M, Abramovitz M, Leyland-Jones B. Proteomics: New technologies and clinical applications. Eur J Cancer 2008;44:2737–41.
32. Rabilloud T, Lelong C. Two-dimensional gel electrophoresis in proteomics: A tutorial. J Proteomics 2011;74:1829–41.
33. Xiao H, Wong DTW. Proteomic analysis of microvesicles in human saliva by gel electrophoresis with liquid chromatography-mass spectrometry. Anal Chim Acta 2012;723:61–7.
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35. Michishige F, Kanno K, Yoshinaga S, Hinode D, Takehisa Y, Yasuoka S. Effect of saliva collection method on the concentration of protein components in saliva. J Med Invest. 2006;53:140–6.

1 Department of Prosthodontics and Implantology, College of Dentistry, King Faisal University, Al-Ahsa, Saudi Arabia
2 College of Dentistry, University of Sharjah, UAE
3 Department of Oral Pathology, College of Dentistry, Baqai University, Karachi, Pakistan
4 Department of Dental Materials, College of Dentistry, Al-Taibah University, Saudi Arabia
5 Department of Epidemiology and Public health. Dadabhoy Institute of Higher Education, Karachi, Pakistan;
6 Restorative Dental Sciences, Al-Farabi Colleges, Riyadh, Saudi Arabia
Corresponding author: “Dr. Zohaib Khurshid” < drzohaibkhurshid@gmail.com >

Zohaib Khurshid1                         BDS, MRes
Jameela Abdul Haq2                   BDS
Rabia Sannam Khan3                 BDS, MSc
Muhammad Sohail Zafar4        BDS, MSc, PhD
Maria Altaf 5                                   BDS, MPhil
Shariq Najeeb6                              BDS, MSc

Salman Aziz                                 FCPS, FICD, MFDS RCPSG, MFGDP, BDS

INTRODUCTION

Regenerative therapy is no doubt a reality! In this era of Hi-tech dentistry it occupies one of the greatly explored aspects of both clinical and laboratory dental medicine. The last few decades have shown an interesting paradigm shift towards procedures directed into conservation, rejuvenation and/or replacement of missing/ lost or endangered oro-dental tissues. It has been quite an intriguing, thought provoking and interesting journey to have moved progressively based upon evidence from xenografts, allografts, alloplasts and autografts alone, to the era of growth factors (GF), proteins and stem cells.

We can broadly categorize the use of ongoing regenerative techniques into three main specialties of dentistry namely periodontics, endodontics/conservative dentistry and oral and maxillofacial surgery. Detailed insight into all specialties would be out of scope for this review however the following discussion will focus on the most practically happening periodontal regenerative techniques at the current time concentrating on the biological mediators of regenerative therapy and stem cells and provide some directives towards future of regeneration in periodontology.

All efforts these days are directed towards either the possibility of re-growing the entire periodontium related to an individual tooth or group of teeth or repairing each individual constituent (periodontal ligaments, cementum, bone or gingiva) as close as possible to the lost natural counterpart. Both require the utility of biomaterials, scaffolds, stem cells, and/or growth factors. Biomaterials and scaffolds have been talked about in many publications however the utilization of the latter two techniques are scarce in literature. The following discussion therefore will be divided into two main parts:

• Advances in Growth factors, signaling molecules and cytokine therapies.
• Advances in Stem cell techniques.

GROWTH FACTORS, SIGNALING MOLECULES, CYTOKINES AND GENE THERAPY

1. Autologous Platelet Products

These products are biological factors extracted from and utilized in the same patients. The main aim behind these techniques is that platelets being a natural source of growth factors are utilized as tools for promotion of regenerative process. These techniques/products could be used solely or in combination with scaffolds like collagen membranes, bone substitutes etc. and include the following:

1.1. Platelet Rich Plasma (PRP)

This is an autologous product derived from patient’s own blood and contains high concentration of platelets suspended in a small amount of plasma. In contrast to Normal platelet count in blood which ranges from 150,000 to 300,000/ µl, PRP by definition contains 1000,000 platelets/µl in 5 ml volume of plasma2. The main idea behind a solution with concentrated platelets is that alpha granules in the platelets contain growth factors which are released by platelet activation initiated by thrombin, calcium chloride or collagen3. The released growth factors include platelet derived growth factor (PDGF), transforming growth factorb1 (TGF-b1), insulin-like growth (IGF), vascular endothelial growth factor (VEGF),fibroblast growth factor and epidermal growth factor (EGF), which promote tissue healing and repair of damaged periodontal tissues, modulate inflammatory process and promote angiogenesis4,5.
The formation of PRP includes collection of patient’s blood in a vacutainer/ collection tube containing anticoagulant6. Blood is mixed with anticoagulant and the vacutainer tube is then subjected to two step centrifugation process. In the first centrifugation RBC’s are separated from plasma and in the second centrifugation the platelets and leukocytes are separated from a cellular plasma also known as platelet poor plasma7. The advantage PRP holds is the ready provision of growth factors (GF) to the site of defect. However as the release is immediate and in high quantities, there is a concern that the GF can be lost without much effect8. Therefore a combination of bone substitutes and PRP is more advantageous in sustaining and retaining GF levels to the site of interest9,10 .

1.2. Plasma Rich in Growth Factors (PRGF)

This technique has been projected as one step ahead of PRP in respect to duration and sustenance of release of GF11,12. Also the concentration of White Blood Cells (WBCs) is nonexistent in PRGF as compared to PRP13,14.
The main difference in the preparation includes onetime centrifugation for PRGF and addition of calcium chloride for activation leading to formation of a gel11. The advantage of this gelatinous substance is that it increases the duration of growth factor release. PRGF can be used in a variety of ways i.e. as supernatant, liquid coating of dental implant surfaces, as scaffold gel or as elastic hemostatic fibrin11.

1.3. Platelet Rich Fibrin (PRF)

This is a fibrin membrane consisting of a collection of immune and platelet concentrate15. The PRF membrane has three fold benefits in that it provides a platform for development of angiogenesis, wound coverage and immune support at the site of interest16-18. The technique for PRF generation is much easier as compared to the previously mentioned methods. It has the advantage of exclusion of addition of any sort of activator or anticoagulant and therefore is less technique sensitive. There is a handsome constituency of leukocytes in PRF which release cytokines, and therefore PRF acts as a double edge sword by regulating immune process on one hand and promoting bone and tissue regeneration on the other19. The biggest advantage PRF has is that the duration of release of growth factors is enhanced up to several days as compared to PRP and PRGF20. In addition to this the PRF membrane has better mechanical properties than PRGF membrane21.

2. Enamel Matrix Derivatives

These proteins are available in the market by the name Emdogain since the past nineteen years. It is mainly used for periodontal regeneration. The main constituents of Emdogain include enamel matrix derivatives (mainly amelogenins isolated from developing porcine teeth), water and propylene glycol alginate (carrier)22,23. Its use and effectiveness is backed by considerable evidence24 however it faces some religious issues in countries which do not use porcine products for health care purposes. Moreover in a fairly recent Cochrane review it was summarized that, although there are less post-operative complications, post one year application Emdogain has neither been able to save more compromised teeth as compared to the other regenerative methods nor It has been able to demonstrate Patient’s perceived esthetic improvement one year post procedure25.
The techniques of using Emdogain is simple. After open flap debridement and root surface conditioning, the gel is applied directly to the denuded root and flap is repositioned26,27.

3. Recombinant / Synthetic Products Produced by Virtue of Gene Therapy

As opposed to their autologous counterparts there are a few commercially available products manufactured using recombinant (genetic) technologies. One of the uses of gene therapy is to introduce vectors into the target cells which are then programmed to yield the required protein or growth factor28. These provide a source of one growth factor/protein in high potency to fulfill specific regenerative needs.

3.1. Recombinant Human Platelet Derived Growth Factor (rhPDGF)

PGDF is a potent cytokine with the ability to mediate wound healing and regeneration29. This is also released by platelets as mentioned above, however the concentration available varies depending upon the platelet product and its generation technique used. Commercially a genetically engineered Food and drug Administration (FDA) approved product available in the market is GEM-21S (growth-factor enhanced matrix) which contains 0.5 ml (at a concentration of 0.3mg/ml i.e. 0.15mg) of PDGF along with beta tri calcium phosphate (β-TCP) 0.5 cc and is indicated for use in intra bony defects, furcation defects and gingival recession associated with periodontal defects30

3.2. Recombinant Human Bone Morphogenetic Protein 2 (rhBMP 2)

This is one of the most happening and researched upon product these days in my humble opinion. It belongs to the transforming growth factor β (TGF β) family31. It regulates the differentiation of stem cells located in the bone tissues. The main reason for its popularity is its osteoinductive nature32. Recombinant human BMP-2 (rhBMP-2) is available in the market by the name of Infuse an FDA approved product from Medtronic. The Infuse kit is available in various sizes from 0.7 cc to 8.0 cc and consists of sterile water vial/vials, absorbable collagen sponge, and vial/vials of lyophilized rhBMP-2. The sterile saline is mixed with lyophilized rhBMP-2 and the absorbable collagen sponge is dipped in the resulting solution. The BMP dipped collagen sponge is then placed in the defect. This product has provided with more than satisfactory results to be considered a promising solution for bone regeneration33.

3.3. Recombinant Human Fibroblast Growth Factor 2 (rhFGF 2)

Recombinant human Fibroblast growth factor (rhFGF-2) has been reported to exert potent angiogenic and mitogenic effects on mesenchymal cells34. To date very few randomized control trials have been carried out utilizing rhFGF-2. A phase II trial previously conducted did not reveal any significant findings35. However a recently published phase III study provides some more promising results, however post marketing stage of this product in the near future will enlighten us more about rhFGF-2’s efficacy and capability in periodontal regeneration36.

3.4. Recombinant Human Growth Differentiation Factor 5 (rhGDF 5) or BMP 14, rhBMP 7 and rhBMP

These are other potential products undergoing animal trials and would be commercially available in the near future for oral and maxillofacial purposes37.

ADVANCES IN STEM CELL TECHNIQUES

In the quest for approaches to restore oral structures to their normal function and form, and possibly to engineer the entire tooth and periodontium, novel technologies have come up in the form of stem cell tissue engineering techniques.

1. Stem Cells Types and Utility

These are unspecialized and immature cells with selfrenewal programmability potential and the ability to differentiate into a variety of cell lines38,39. Due to their unique traits, stem cells have gained popularity in tissue engineering to regenerate or replace missing or damaged tissues and/or organs.

The two main sources of stem cells in dentistry are adult stem cells and pluripotent stem cells. The latter includes embryonic stem cells and induced pluripotent stem cells (iPS) Pluripotent variant are the most promising, having the ability to develop into all cell types from all germ layers in contrast to adult stem cells which can only differentiate into limited cell types40.

1.1. Adult Stem Cells
There are many variants of these somatic or postnatal stem cells depending upon the specific ability of differentiation and include:

• Mesenchymal stem cells or mesenchymal stromal cells (MSCs)
• Bone marrow derived MSC (BMSCs)
  From iliac crest
  From orofacial bones
• Dental tissues derived stem cells
• Epithelial stem cells

MSC-like cells: These are called MSC-like because they have similar phenotypic characteristics of BMSCs and include:

• • Dental Pulp Stem Cells (DPSC)
  • Stem cells from human exfoliated deciduous teeth (SHED)
  • Periodontal ligament stem cells (PDLSCs)
  • Dental Follicle Stem Cells (DFSCs)
  • Tooth Germ Progenitor Cells (TGPCs)
  • Stem cells from apical papilla (SCAP)
    Oral Mucosa derived stem cells/ Gingiva derivedMesenchymal Stem Cells (OMSCs/GMSCs)
  • Periosteum derived stem/progenitor cells
  • Salivary gland derived stem cells
  • Adipose tissue derived stem cells (ASCs)

A detailed account of each of the above would require another review as it is not possible to accommodate the details they deserve, in current manuscript. Moreover the above mentioned adult stem cell techniques are in their infancy and it will take considerable amount of time for all to reach the clinical settings. Currently only periodontal .

ligament stem cells (PDLSCs) have been introduced in the clinics and are discussed below.
1.2. Pluripotent Stem Cells
1.2.1. Embryonic Stem Cells (ES Cells

ES are extracted from undifferentiated inner mass of blastocyst, which is an early stage of embryonic development post fertilization41. This is the main reason for major ethical and moral concerns whenever the topic of ES cell extraction from human beings comes up. Therefore ES cell use in regenerative dentistry is still a grey area with unpredictable future.
1.2.2. Induced Pluripotent SC (iPS Cells

When human or mouse somatic cells undergo nuclear reprogramming by introduction of certain genetic transcription factors, they can be transformed to embryonic state thus unlocking pluripotency. These induced cells then act similarly to ES cells42. Ever since their discovery, iPS cells have been the research focus of regenerative medicine and dentistry. To the dentists advantage luckily the oral cavity is a unique and rich source of stem cells43. iPS have been successfully generated from SCAP44, DPSCs45, gingival fibroblasts and Periodontal ligament fibroblasts46.

Induced pluripotent stem cells have great potential in regenerative dentistry in the upcoming future however their clinical utility currently is limited to animal models46.

CURRENT PRACTICAL SUITABILITY OF STEM CELLS IN CLINICAL DENTISTRY

Apart from the ability of differentiation into specific target tissues, and ease of collection and preparation, in order to ensure patient safety in regenerative dentistry, complete control of cellular fate is a mandatory requirement for all stem cells as well. Based upon this only adult MSCs currently seem to have a convincing clinical potential47,48. Initial case reports and randomized control trials (RCTs) utilizing stem cells for periodontal regeneration are coming up48,49.

The autologous periodontal ligament stem cell technique reported in very recent literature involves prior extraction of third molars from subjects scheduled to receive stem cell treatment for periodontal defects. Post Extraction, production of single cell suspensions is carried out by cell isolation process, which involves separation of Periodontal ligament (PDL) cells from the roots followed by digestion, straining and centrifugation processes. The cells then undergo culture and cell characterization procedures, which are followed by creation of cell sheets. Once the PDLSC sheets are ready for use, freshly prepared Bone particles e.g. Bio-Oss® etc. (in a concentration of 0.25g/sheet) are sprinkled over each sheet to be used. The periodontal ligament stem cell sheets are then rolled up to pack the bone particles and introduce into the defect49.

Although the results of Chen and co-worker’s RCT49 showed no significant difference between the control group and stem cell group for pocket depth fill, the emergence of such clinical trials are affirming and encouraging for us to start preparing for the future of stem cell use in clinics.

We must therefore not hesitate to incorporate the above mentioned techniques in our daily practices as soon as they are available, but after achieving adequate skills through continuing education programs. The utility of regenerative techniques might, not only, serve to provide the patients with comparatively non-invasive treatment plans but also bring more and more promising results as the use becomes wide spread. We are therefore looking towards a future that would be the beginning of the end of material based therapies and start of stem cell based regenerative therapies.

CONFLICT OF INTEREST

Declared none.

ACKNOWLEDGEMENTS

Declared none

REFERENCES

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Consultant in Restorative Dentistry, Institute for Advanced Dental Sciences and Research and Ministry of Higher Education,

Al Jouf University, Saudi Arabia < drsalmanaziz@gmail.com >

Salman Aziz                                 FCPS, FICD, MFDS RCPSG, MFGDP, BDS

Faisal Rehan1                                 BDS
Rabia Sannam Khan2                  BDS, MSc
Zohaib Khurshid3                         BDS, MRes
Mohammed Sohail Memon4     BDS, MSc, PhD
Sadia Naqvi5                                    BDS
Muhammad Sohail Zafar6         BDS, MSc, PhD

INTRODUCTION

Saliva is a clear mucoserous fluid, secreted by major and minor salivary glands of oral cavity. Appropriate ecological balance of oral health is maintained by salivary functions such as lubrication, protection, buffering action and pH balance, tooth integrity maintenance, taste sensation digestion1-4 and antibacterial activity through antimicrobial peptides5-7. The source of saliva, location and anatomy of salivary glands has an impact on salivary flow rate in relation to localised and systemic disease8. It is widely used in diagnosis of various oral and systemic conditions as it is easily accessible, reliable and non-invasive diagnostic medium9,10. Salivary contents are supposed to be altered by drugs (anti cholinergic, anti-hypertensive, antihistamines, diuretics and psychoactive substances) and conditions such as post-surgery, metabolic, nutritional, psychological and neurological diseases9. Hence, alterations in any property of saliva, whether pH or flow rate could be associated with oral and dental diseases, pharyngeal, esophageal, neoplastic changes, autoimmune diseases, inflammatory changes and systemic diseases1,11.
The use of tobacco is well-known to affect the oral health12,13. The active ingredient of tobacco is nicotine which stimulates cholinergic receptors in brain and other organs which results in neural activation leading to altered salivary secretion.14Widely consumed chewable form of tobacco is the areca nut; approximately 600 million people around the globe use it. It is the 4th most commonly used psychoactive agent. Adverse effects of areca nut includes attrition, staining, caries, periodontal diseases, lichenoid reactions, burning sensation in oral mucosa, oral sub-mucous fibrosis, oral leukoplakia and oral squamous cell carcinoma.15Saliva is the first fluid that gets exposed to tobacco whether smoked or smokeless form. 16The aim of current study was to analyze and compare the long term effect smoked and smokeless tobacco on SFR and pH of saliva. The aim of this study was to assess the severity of adverse effects of tobacco consumption using the saliva flow rate and pH as diagnostic parameters. In addition, the effects of the chewing tobacco and smoking tobacco were also compared.

METHODOLOGY

The research protocol was reviewed and approved by the research ethics committee at the College of Dentistry, Baqai Medical University, Karachi, Pakistan. A total of 210 patients attending the outpatient Department of Oral Medicine and Periodontology of Baqai Dental College were recruited for this study. Each patient was explained about the study protocol and an informed consent was obtained to participate in the research. Patients were divided in three groups (70 subjects in each group; Group A; include tobacco chewers, Group B included smokers and Group C included non-consumers of tobacco) and all required data were collected using the questionnaire within three months.

The questionnaire was used to collect demographic information, and subjects reported of smoking and chewing habits.

Inclusion Criteria
1) Subjects in the age range of 20 to50 years.
2) Patients who consumed tobacco either in smokeless form or in smoke form.
3) Apparent healthy patients; no systematic disease.

Exclusion Criteria
1) Subjects who had history of trauma to head and neck.
2) Subjects who wore dentures.
3) Subjects who had undergone radiotherapy.
4) Subjects who had salivary gland diseases.

Saliva Collection

After obtaining the informed consent, saliva of each subject was collected under resting condition using the simple drooling method for 5 minutes. The salivary flow rate expressed in ml/min. Salivary pH was determined using specific pH strips. Saliva was collected between 10 am to 1pm. Each subject was requested not to eat, drink, perform any oral hygiene, chew or smoke before and during the entire procedure. Saliva was collected in graduated container every 1 min for 5 minutes. During saliva collection subject was instructed not to speak or swallow. After collection, SFR was measured and expressed in ml/min for 5 minutes. pH Measurement Salivary pH was measured immediately after measuring SFR. PH was assessed in accordance with the colour change on the indicator paper strips (SIMPLEXTM), which gets either lighter or darker in colour when it comes in contact with saliva. Subsequent increase in lightness of shade suggests increase in acidity and increase in darkness suggests increase in basicity.

Statistical Analysis

Data was analyzed using IBM SPSS (v 23.0, Statistical Package for Social Service; IBM, USA) computer software. The frequency and percentage was computed for qualitative variables.One-way ANOVA test was applied to compare mean or median of the outcome variable.

RESULTS

The current study compared the effects of tobacco on salivary flow rate and its pH. The data were analysed using the frequency and percentage of participants. In group A, 27.1% was the highest frequency noted among the subjects which had 0.20 ml/min mean resting mouth SFR.Where as in the group B, 25.7% was the highest percentage that had 0.30 ml/min mean SFR (Table ). In group C, 25.7% was the highest frequency which had mean SFR of 0.20 ml/min, while second highest frequency of subjects i.e. 24.3% had 0.5 ml/min SFR. The subjects presented in our study were in the age group from 30-40 years. The mean age (±SD) in the group A, was 36.85 (±0.77), group B‑ 35.55 (±0.56) and group C 34.55 (±0.85).

There was a statistically insignificant difference between groups as determined by one-way ANOVA (F = 7.053, p = .281& .274). There was no statistically significant difference between and within the chewers, smokers and control groups (p = .281 & .274).

Each group consisted of 70 subjects in total with 30 females and 40 males. Subjects in group A consisted of tobacco chewers, subjects consumed tobacco for more than 10 years, with the Mean (±SD) duration, consumption and frequency of habit was 12.05 (±1.16), 9.9 (±0.8 pieces/ day) and 8.75 (±0.89) in Group A. Analysis was done by the calculated frequency.
In group A, 27.1% was the highest frequency noted among the subjects which had 0.20 ml/min mean resting mouth SFR. In group A, 75.7% subjects had pH of 6. Subjects in the group B smoked 14.8±8.30 cigarettes per day (minimum=2, maximum=40 cigarettes). 25.7% was the highest percentage in group B who had 0.30 ml/min mean SFR. In group B, 68.6% subjects had pH of 6 while 8.6% showed pH level of 5 (Table ).

Subjects who smoked and chewed tobacco per day had the habit of intake for longer periods having the greater risk for developing xerostomia. In group C which was the control group 25.7% was the highest frequency which had mean SFR of 0.20 ml/min, while second highest frequency of subjects i.e. 24.3% had 0.5 ml/min SFR. In group C, 91.4% subjects had pH level of 7, while 8.6% had pH levels of 8 (Table 2 ).

DISCUSSION

Salivary flow is under control of higher salivary centres present within medulla oblongata of CNS and depends on afferent stimulations. Sympathetic as well as Parasympathetic nervous system determines the quality of saliva. Para-sympathetic system involves vasodilation of blood vessels within salivary glands and thus increased mobility of liquid within saliva, thus producing serous saliva. In contrast Sympathetic system has an influential role in producing thick concentrated saliva. 1,17In absence of any external stimuli, salivary flow rate (SFR) is termed as resting mouth SFR which in normal healthy individuals is found to be 0.3- 0.5 ml/minutes1,18.In contrast stimulated salivary flow rate is found to be as high as 10 ml/minutes18,19. Although It has been noted that salivary flow rate even in same individual does not remain constant and can vary when noted at different times which can be due to age, gender, circadian rhythms and other factors. 20,21 Carbonic acid/bicarbonate system, phosphates system, protein system plays a key role in maintenance of Salivary pH17 which in numerous studies have been found to be 5.5-7.9 in resting mouth19,22.

Use of tobacco has always been associated with poor oral hygiene, halitosis, local red and white lesions etc. A symptom which remains common with these conditions is Xerostomia. Xerostomia, the subjective sensation of dry mouth, is a frequent complaint and the most common symptom of salivary gland hypo-function (SGH)23-25 which reflects an objective, measurable decrease in salivary flow (hypo-salivation). Symptoms of dry mouth may range from mild oral discomfort to significant oral disease that can compromise patients health, dietary intake and quality of life23,26-29 . Signs and symptoms of hypo-salivation includes Increased incidence of tooth decay, demineralization, attrition, erosion, plaque accumulation, mucositis, fungal candidiasis and can occur in response to oral, pharyngeal esophageal, neoplastic, metabolic nutritional, inflammatory, genetic, auto-immune and nervous system disorders and require early diagnosis and intervention1,18,19

The main ingredient of tobacco is nicotine which itself is Para-sympathomimetic and acts on cholinergic receptors and induce Para-sympathetic response and thus it has been seen that initially with use of tobacco SFR increase, in short term. 30,31 Whereas in long term, some studies suggest that SRF remains unaffected30. However, there are also studies which suggest that salivary flow reduces with long term consumption of tobacco in any form. 11,30 Kanwar et al.13 compared long term effect of tobacco among tobacco chewers, smokers and non-tobacco consumers. They found significant difference with most decreased SFR for smokers, while chewers also had decreased SFR in comparison to non-consumers. Although they did not find any significant difference for pH among these three groups but salivary pH for tobacco consumers was found to be lower in comparison to non-consumers13. Rad et al. 31 although did not include tobacco chewers, but with pool of 100 patients in both groups i.e. tobacco smokers and non-smokers they found significant difference with lower SFR values in smokers group. They also found increased occurrence of calculus, gingivitis, periodontitis, mobility, halitosis and cervical caries in smokers then non-smokers.

Rooban et al. conducted their study regarding SFR and pH analysis of patient consuming different types of tobacco in comparison to non-consumers. They found the mean SFR for chewers to be 3.35 ±1.7 and for non-chewers 3.55 ± 1.39. The difference was not statistically significant (p=0.5). The pH of chewers was 6.57 ± 0.52 and for non-chewers it was 6.77 ± 0.41. The difference for pH was statistically significant11. Khan et al. proposed that long term habit of smoking leads to development of tolerance to salivary effects in some individuals, which may be the reason why some studies suggest that long term effects of tobacco consumption remains unclear, they also compared pH values between smokers and non-smokers and found lower pH in smokers. 30 In contrast Reddy et al. found non-significant difference in salivary pH between chewers and non-chewers 32.There is a profound relationship seen between SFR and pH. With increase in SFR pH increases and vice versa. It is believed that increase in SFR increases bicarbonates in saliva which increases pH33,34. There are multiple factors and complex oral environment that may affect the outcome. Further long term studies are required to analyse their interactions.

CONCLUSIONS

Based on calculated frequencies, it can be concluded that, the mean resting mouth SFR does not get affected by consumption of tobacco, but pH levels certainly decreases with tobacco consumption, more by smoked tobacco. The altered levels of salivary pH for tobacco consumers suggests that tobacco usage can impair the salivary defence mechanism and may eventually result in multiple mucosal and dental diseases.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

ACKNOWLEDGEMENTS

Authors have not received any financial support for this research.We acknowledge the contribution of Dr. Syed Fareed Mohsin (Associate Professor, Baqai Medical University) in conceptualization of the study.

AUTHORS’ CONTRIBUTION

FR: gives the main research proposal and intervention to this research.
RSK: contributed in the collection and interpretation of data.
ZK: developed methodology and wrote part of the manuscript.
MSM: wrote discussion and conclusion.
SN: helped in data collection and writing of the manuscript.
MSZ: critically reviewed for intellectual contents, revised the manuscript and correspondence.

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5. Khurshid Z, Zohaib S, Najeeb S, Zafar MS, Rehman R, Rehman IU. Advances of proteomic sciences in dentistry. Int J Mol Sci 2016; 17: 728.
6. Khurshid Z, Najeeb S, Mali M, Moin SF, Raza SQ, Zohaib S, Sefat F, Zafar MS. Histatin peptides: Pharmacological functions and its applications in dentistry. Saudi Pharm J 2016; E-pub ahead of prints,doi:10.1016/j.jsps.2016.04.027.
7. Khurshid Z, Naseem M, Sheikh Z, Najeeb S, Shahab S, Zafar MS. Oral antimicrobial peptides: Types and role in the oral cavity. Saudi Pharm J 2015;24:515-24.
8. Edgar WM. Saliva: Its secretion, composition and functions. Br Dent J 1992;172:305-12.
9. Mandel ID. The diagnostic uses of saliva. J Oral Pathol Med 1990;19:119-25.
10. Khurshid Z, Najeeb S, Khan RS, Zafar MS. Salivaomics: An emerging approach in dentistry. J Pak Dent Assoc 2016; 25: 41.
11. Rooban T, Mishra G, Elizabeth J, Ranganathan K, Saraswathi TR. Effect of habitual arecanut chewing on resting whole mouth salivary flow rate and pH. Indian J Med Sci 2006; 60: 95-105.
12. Fahad K, Aziz A, Shahab S, Zafar M. Laboratorial and clinical impacts of tobacco on periodontal health: A systematic review. Int Dent J Student’s Res 2015; 3: 72-8.
13. Kanwar A, Sah K, Grover N, Chandra S, Singh RR. Long-term effect of tobacco on resting whole mouth salivary flow rate and pH: An institutional based comparative study. Eur J Gen Dent 2013; 2: 296.
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16. Burket LW, Greenberg MS, Glick M, and Ship JA. Burket’s oral medicine. PMPH-USA. 2008.
17. Nanci A. Ten Cate’s oral histology: development, structure, and function. St. Louis, Mo.; London: Mosby. 2008: 411.
18. Ghezzi EM, Lange LA, Ship JA. Determination of variation of stimulated salivary flow rates. J Dent Res 2000; 79: 1874-8.
19. Choo RE, Huestis MA. Oral fluid as a diagnostic tool. Clinl Chem Laborat Med 2004; 42: 1273-87.
20. Bennett J. Oral Anatomy, Histology and Embryology 2004.
21. Dawes C. Physiological factors affecting salivary flow rate, oral sugar clearance, and the sensation of dry mouth in man. J Dent Res 1987; 66 Spec No: 648-53.
22. Drobitch RK, Svensson CK. Therapeutic drug monitoring in saliva. Clin Pharmacokinet 1992; 23: 365-79.
23. Jensen SB, Pedersen AML, Vissink A, Andersen E, Brown C, Davies A, Dutilh J, Fulton J, Jankovic L, Lopes N. A systematic review of salivary gland hypofunction and xerostomia induced by cancer therapies: Management strategies and economic impact. Support Care Cancer 2010; 18: 1061-79.
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1 College of Dentistry, Baqai Medical University, Karachi, Pakistan
2 College of Dentistry, Baqai Medical University, Karachi, Pakistan
3 Department of Dental Biomaterials, College of Dentistry, King Faisal University, Al-Ahsa, Saudi Arabia
4 Department of dentistry, University of Sydney, Sydney, Australia
5 College of Dentistry, Baqai Medical University, Karachi, Pakistan
6 Department of Restorative Dentistry, College of Dentistry, Taibah University, Al Madinah, Al Munawwarah, Saudi Arabia
Corresponding author: “Dr. Muhammad Sohail Zafar” drsohail_78@hotmail.com

Faisal Rehan1                                 BDS
Rabia Sannam Khan2                  BDS, MSc
Zohaib Khurshid3                         BDS, MRes
Mohammed Sohail Memon4     BDS, MSc, PhD
Sadia Naqvi5                                    BDS
Muhammad Sohail Zafar6         BDS, MSc, PhD

Salwa A. AlSadhan1             BDS, MSc
Amel G. Darwish2                BDS, MSc, PhD
Abeer AlSwailem3                BDS
Amal AlShehri4                     BDS
AlHanouf AlMutrafi5          BDS

INTRODUCTION

Awareness of occupational hazards started as far back as the 18th century when Bernardino Ramazzini, often called “the father of occupational medicine”, founded the bases of occupational medicine in his book “Diseases of Workers”1 . Modern Dentistry has been cited as the least hazardous of all occupations, but despite the numerous advancements, many risks still challenge the status of it2 . In comparison with other high-risk medical professionals, dentists report worse and more frequent health problems3 . Dental personnel are exposed to various occupational hazards including exposure to infections, percutaneous exposure incidents, dental materials, radiation, noise, musculoskeletal disorders, dermatitis, respiratory disorders, eye insults, and psychological problems4-6. Some factors such as patients’ interaction, physical strain and financial pressure negatively related to the psychological wellbeing of the dental professionals7 .

Dentistry is unequivocally a highly demanding profession in which dentists struggle to manage long working hours, anxieties regarding cross-infection control, pain control, stressful noise pollution, communication with patients, medically compromised cases, time management8-10 and – often- handle all the above with exhausting perfectionism.(11) The difficulty of finding balance can stir-up issues of a psychological nature which metastasize to social impairments at work and home.(12,13) The physical aspect of professional hazards was acknowledged long ago, which lead to finding ergonomic approaches that provided a longer professional life and improved career satisfaction. The social and psychological hazards challenging dentists include burnout, decline in professional performance, job control, stress, depression, effects on family life, and early retirement12,14.

Job burnout is described as a prolonged response to chronic emotional and interpersonal stressors on the job, and is defined by the three dimensions of exhaustion, cynicism, and inefficacy12. Eventually, it leads to withdrawal from people, patients, colleagues or employees and from work in general15,16. Burnout has also been associated with absenteeism, intention to leave the job, and actual career change. However, for people who stay on the job, it leads to lower productivity and effectiveness at work, thus raising quality-of-care issues17-19. People who are experiencing burnout can have a negative impact on their colleagues, both by causing greater personal conflict and by disrupting job tasks. (12)

Nervous psychological state, tension, depression and other signs of psychological impairment should be taken into account when talking about job related stress in dental practice20. Dentists indicated that running behind schedule, causing pain, heavy workload and late and anxious patients as being the most intense stressors in their work21,22. Dentistry is a prestigious and a respected profession, but it is under public pressure, which may cause more stress to some practitioners23,24. This is particularly true for dentists who practice in a less than desirable environment and those who feel professionally isolated23. Like most depressed people, depressed dentists tend not to seek treatment. Some find it difficult to overcome problems if they associate them with personal failure25,26. In 2005, specialty and gender were studied as predictors of depression among dentists chosen randomly from the ADAs mailing list. The most interesting finding was that only 15%of depressed dentists were receiving treatment17. Specialty and gender were studied as predictors of depression among dentists, and results showed that gender was associated with depression, but specialty was not17.

Nowadays, a remuneration system has led dentists to long working hours, leaving little time to relax from work, and participate in family life25. Most dentists experience difficulties in balancing professional and family life, which renders them lacking time for hobbies, family leisure activities, neglecting family duties, and taking their workrelated anxieties home. These occupational hazards were mainly related to long working hours, which negatively affected all areas of family life19.
A study conducted among Australian dentists found that the most common reasons dentists expected to retire were to have more leisure time, to afford a life after retirement, and job stress or pressure27. Few studies were conducted to assess the prevalence and distribution of work-related musculoskeletal disorders among dentists in Saudi Arabia, and the factors associated with the disorders28,29. However, there is limited data concerning the social challenges caused by the dental profession among dentists in Saudi Arabia. This study was conducted to assess the prevalence of the social occupational hazards encountered by dentists working in Riyadh, Saudi Arabia and to evaluate the factors that might be related to these hazards.

METHODOLOGY

This cross-sectional descriptive study was approved by College of Dentistry Research Center (CDRC), King Saud University, Riyadh, Saudi Arabia.

Each selected dentist was asked to complete a questionnaire; which included questions concerning the different psychosocial hazards of the dental profession. The questionnaire was divided into 5 parts; the first part was concerning personal information (age, gender, nationality, marital status, number of children, age of the youngest child and region of residence in Riyadh), the second part included professional information, the third part included 8 questions inquiring about how Dentistry as a profession affected their personal life, the fourth part contained 7 questions concerning challenges and difficulties in the dental profession, and the last part was about early retirement. The questions of the last 3 parts called for a yes/no answer. A tick box layout was used for the provision of the appropriate answer.

The questionnaire was adopted and modified from forms of previous studies4,12-18,22. In addition, face and content validity of the questionnaire was evaluated by 3 independent faculty members of the College of Dentistry. A pilot study was conducted through distributing the questionnaire on 20 dentists, who were not included in the main study. Adjustments to the questions were done accordingly. Eight-hundred questionnaires were distributed in governmental and private dental health sectors in Riyadh. It was distributed to both male and female, Saudi and nonSaudi dentists from all age groups and with any qualifications. Regarding the governmental sector, 250 questionnaires were distributed in hospitals, and 150 were distributed in the College of Dentistry, King Saud University. For the private sector, 250 questionnaires were distributed in private hospitals and clinics, and 150 were distributed in the private dental colleges. The questionnaires were handed to the dentists and were collected at the end of the day in each site.

The governmental hospitals included Al Iman Hospital, King Abdulaziz and King Khalid University Hospitals, King Fahadand King Saud Medical Cites, King Faisal Specialist Hospital, Prince Sultan Military Medical City, National Guard Hospital, Security Forces Hospital and Al Yamamah Hospital. The private hospitals included Al-Hammadi Hospital, Sulaiman Alhabib Medical Group and Specialized Medical Center Hospital and the private dental colleges included AlFarabi Dental College and Riyadh College of Dentistry.

Scores were recorded and the data analysis was performed using Micro Soft Office and the Statistical Package for Social Sciences (SPSS version 20). Descriptive statistics were used to explore frequencies, minimum and maximum values, the mean, and the standard deviation. Relations between psychosocial hazards of dental profession and socio-demographic and personal characteristics of dentists working in Riyadh were tested using Chi-square test with a 95% confidence (p≤0.05). The relation of psychosocial hazards of the dental profession and the worries among dentists working in Riyadh were also investigated in this study.

RESULTS

Six hundred and twenty-three out of 800 randomly distributed questionnaires were returned, giving a response rate of 77.8%. The majority of the respondents (38.6%) fell in the age range of 23-30 years, while the least percentage (4.7%) were those of the age group of above 50 years old. The response rate tipped on the female end of the spectrum (57%). Two thirds of the participants were married (66.6%) and over half were Saudi citizens (55.8%).

Distribution of socio-demographic and personal characteristics of the study subjects is illustrated in Table 1. The highest percentage of the dentists included in the study were general practitioners (44.1%) followed by specialists (27.8%) and the least percentage (2.1%) were professors. Regarding the type of dental practice, 38.9% were from governmental hospitals, 34.9% from academic institutions and 24.9% were from private hospitals. The duration of working in the dental profession ranged from 6 month to 40 years. The range of the number of working hours per week was (0-84 hours). The range of the average number of patients seen per week was (0-80 patients).

The prevalence of psychosocial hazards among dentists working in Riyadh is presented in Fig. 1. The most prevalent social problem reported by the study sample was lack of time to spend with the family (78.6%). Being under stress was reported by 75.7% of the dentists, followed by dentists whose profession affected their commitment to social gatherings (72.9%). Plans of marriage and plans to establish a family were not affected by the dental profession in 55.7% and 52.3% of the participants, respectively. Over two third (67.5%) of the study subjects felt that their profession affected their ability to enjoy favorite sports and hobbies.

The prevalence of worries caused by the dental profession among dentists working in Riyadh is demonstrated in Fig. 2. Dealing with uncooperative or anxious patients was reported to be the highest worry encountered by the dentists participating in this study (75.4%) followed by worries of causing pain (73.5%), then running behind schedule (71.1%).

Table 2 presents the relation of psychosocial hazards of the dental profession with socio-demographic and personal characteristics of dentists working in Riyadh. There were statistically significant relations between age and marital status and postponing either plans of marriage or establishing a family (p<0.0001 and 0.002, respectively) as younger dentists stated that practicing dentistry had an effect on their plans of marriage and establishing a family. The relation of gender with reduced time to spend with the family was statistically significant (p=0.024) as females were more effected than their male counterparts. There were also statistically significant relations between type of practice and both lack of free time and neglected family needs (p=0.028 and 0.007, respectively) since dentists working in governmental hospitals indicated that they had less free time and neglected their family needs more than dentists working in other sectors. No statistically significant relations were found between the professional rank and any of the psychosocial hazards (Table 2).

Table 3 presents the relation of psychosocial hazards of dental profession and the worries among dentists working in Riyadh. Running behind schedule and meeting society’s expectations of the profession had statistically significant relations with all the psychosocial hazards (p<0.0001). There were also statistically significant relations of worries of causing pain, maintaining high quality work and heavy work load with all the social hazards (Table 3). There were no statistically significant relations between dealing with uncooperative and anxious patients and postponing marriage plans and neglected family needs (p= 0.071 and 0.075, respectively). No statistically significant relations were found between having late or broken appointments and postponing marriage plans, postponing plans to establish family and inability to enjoy favorite sports or hobbies (p= 0.088, 0.147 and 0.304, respectively).

DISCUSSION

This cross-sectional study assessed the prevalence of the psychosocial hazards encountered by dentists and evaluated the factors that might be related to these hazards .The data was collected from dentists working in governmental hospitals, private hospitals and clinics, and academic institutions in Riyadh. About 39% of the respondents fell in the age range of 23-30 years, which reflects the increased dental workforce in the late years and the general increase of the younger population. The response rate tipped on the female end of the spectrum (57%), probably because higher response rates could be yielded from respondents who are acquainted with the researchers who were females as well. Also, it might be the result of the easier access to the female faculty for the researchers.

The highest percentage of the dentists included in the study was of the general practitioners, followed by specialists and the least percentage of the participants was of professors. This may be due to the fact that most of the respondents were young dentists who had not got postgraduate degrees yet.

The psychosocial challenges with the highest prevalence among research subjects were lack of family and free time, having to skip social events and lack of leisure time for sports and hobbies. This agrees with Purine et al (2011)(13) who found that most of Lithuanian dentists had insufficient time to spend with their families and that they had insufficient time for hobbies due to work-related stressors. Being under stress was also highly prevalent among the study sample which is consistent with the findings of several other studies5,22,23.

Dentists in this study indicated that dealing with uncooperative or anxious patients, causing pain and running behind schedule, as being the highest worries in their work. These have also been reported by Moore and Brodsgaard (2001) as being the most intense stressors faced by dentists during their work15. Type of dental specialty was not investigated in this study, as there is limited evidence that the degree of stress experienced by dental practitioners may vary according to the type of dental specialty24. A statistically significant relation was found between the dentists’ available free time and the type of practice in this study. This might be because governmental hospitals have to meet all the family needs while private sectors have more flexible schedule and the academic sectors have longer vacations.

There was also statistically significant relation between gender and the effect of the dental profession on the time available to spend with family as females were affected more than males. Females usually have a complex burden of responsibilities as having to fulfill both roles of being a wife and/ or mother and a dentist. It might also be possible that females tend to report their psychological complains more than males. This however disagrees with another study that found that male and female dentists’ responses to stress were similar25. The older aged group did not report any effect of the dental profession on either marriage plans or of establishing family. This might be due to the fact that at that age, participants could not recall exactly what happened in the younger age.

There were statistically significant relations between psychosocial hazards and worries such as running behind schedule, causing pain, dealing with uncooperative or anxious patients, maintaining the quality of work, heavy load of work and meeting society’s expectations of the prestigious profession. This agrees with Moore and Brodsgaard (2001) who found that running behind schedule and causing pain were sources of stress in the dental profession15. With regards to anxious patients, dentists need to spend more time with them and they might ask for changing the treatment plan or refuse to pay their bills which put dentists under more stress. Ayers et al (2008) also reported that time pressure was a main source of stress among dental professionals22.
This study was cross-sectional which does not allow the examination of cause-and-effect relations, limiting our conclusions. Based on the findings of this study, further analytical studies are recommended to evaluate the correlation between the worries and psychosocial hazards hypothesized through this study and to try to determine with more accuracy the causes of occupational health problems affecting dentists to help reduce the prevalence and impact.

CONCLUSION

It can be concluded from this study that lack of family time, free time and leisure time for favorite sports or hobbies was the main psychosocial hazard encountered by dentists working in Riyadh. Being under stress was the second prevalent psychosocial hazard of the dental profession. The most prominent related factors were found to be; dealing with uncooperative or anxious patients, worrying of causing pain to patients, running behind schedule, enduring heavy work load, maintaining a high quality of work and meeting society’s expectations of the prestigious profession.

RECOMMENDATIONS

To overcome the psychosocial problems faced by dentists, balance between work and family should be met. Practitioners are advised to establish a schedule in accordance to their priorities, thus creating enough time for their spouses, children and themselves to satisfy their social life. Maintaining good physical health is also emphasized so that they can enjoy their professional and personal lives. Practitioners are also encouraged to avoid isolation, strengthen friendship ties and join support groups of other dentists. Lastly, they should not hesitate to reach out for professional help when work takes its toll on their social life. Stress management together with personal and professional awareness training should be included in the undergraduate curriculum so that threats to mental and social wellbeing, which might occur during the professional life, might be avoided or addressed. This could be done through providing more specialized courses and activities regarding occupational health and safety.

On a larger scale, it is recommended that dental institutions in Riyadh establish well-organized and flexible schedules more suitable to dentists. On a national level, it is suggested that legislations would be applied by health authorities endorsing an increase in the number of clinics and practitioners to meet the population’s high demands, and laws concerning working hours and working conditions would be amended, to protect health professionals from being overloaded with work and ultimately burnt out.

CONFLICT OF INTEREST

The authors declare that they have no competing interests.

ACKNOWLEDGEMENTS

We thank Mr. Nasser Al-Maflehi for sharing his expertise on statistical analysis generously. His contribution was greatly appreciated.

AUTHORS’ CONTRIBUTIONS

SA: Participated in the design of the study, revising the questionnaire, the data analysis and the final review of manuscript.
AD: Contributed in the design of the study, revising the questionnaire, data analysis and the manuscript.
AS: Contributed in the selection of the topic, development of the questionnaire, collection and analysis of the data and writing the draft of the manuscript.
AA: Contributed in the selection of the topic, development of the questionnaire, collection and analysis of the data and writing the draft of the manuscript.
AM: Contributed in the selection of the topic, development of the questionnaire, collection and analysis of the data and writing the draft of the manuscript.

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1 Head of Community Service Unit, Associate Professor, Department of Periodontics and Community Dentistry, College of Dentistry, King Saud University, Riyadh, Saudi Arabia
2 Lecturer, Department of Pediatric and Community Dentistry, Faculty of Dentistry, Alexandria University, Egypt
3 Intern, College of Dentistry, King Saud University, Riyadh, Saudi Arabia
4 Intern, College of Dentistry, King Saud University, Riyadh, Saudi Arabia
5 Intern, College of Dentistry, King Saud University, Riyadh, Saudi Arabia
Corresponding author: “Dr. Salwa A. AlSadhan” < ssadhan@ksu.edu.sa >

Salwa A. AlSadhan1             BDS, MSc
Amel G. Darwish2                BDS, MSc, PhD
Abeer AlSwailem3                BDS
Amal AlShehri4                     BDS
AlHanouf AlMutrafi5          BDS