Mansoura Journal of Dentistry 2014;1(3):142-145.

Ahmed R. Zaheret al.

Introduction

ooth development starts at 5th week of human

gestation and at 10th embryonic day of mouse

development. Each tooth passes through four morphological stages: initiation, bud, cap, and bell stages,

named according to the shape of enamel organ [1]. At late

bell stage, odontoblasts secrete first layer of dentin starting

dentinogenesis, dentin formation, and amelogenesis,

enamel formation, through reciprocal induction.

Collagen is the major insoluble fibrous protein in the

extracellular matrix and in connective tissue [2]. Type I

collagen forms a fibrous three-dimensional network which

builds up the dentin matrix. Compared to bone, the collagen

matrix in dentin is more interwoven with numerous

crossings of fibrils [3]. Excessive degradation of type I collagen is associated with a variety of human diseases such

as RA, tumor metastasis, and atherosclerosis [4].

Rheumatoid arthritis (RA) is a chronic inflammatory

disease characterized by joint swelling, joint tenderness,

and destruction of synovial joints, leading to severe

disability and premature mortality [5]. RA is considered an

autoimmune disease due to presence of auto-antibodies,

such as rheumatoid factor (RF) and anticitrullinated protein antibody (ACPA) (tested as anticyclic citrullinated peptide [anti-CCP]), which can precede the clinical

manifestation of RA by many years [6].

Many women with RA are of childbearing age, which highlights the importance to study if there is any effect on

tooth development of their children. To our best

knowledge, this is the first study that evaluates the effect of

rheumatoid arthritis rat mother on tooth development of

their offspring.

Materials and methods

Animals

20 female Albino rats weighting (180-200 g) were used in

the study and divided into 2groups: control group (CG), and

rheumatoid non treated group (RhG).

Collagen preparation Arthritis was induced successfully according to the protocol

recommended by Chondrex Inc. Type II collagen (CII),

isolated and puried from bovine articular cartilage (Chondrex, Inc), dissolved overnight at C in 0.01 M acetic acid at a concentration of 2 mg/ml. The solution was

then emulsied in an equal volume of incomplete Freunds adjuvant (IFA) (Sigma) in a drop-wise fashion with

continuous stirring with electric homogenizer. The stability

of the emulsion was tested by adding one drop of emulsion

into a beaker of water. A stable emulsion remained, as a

solid clump in water without dispersing, while the

spreading onto the water surface indicated an unstable emulsion [7].

Histological and immunohistochemical analysis

Tongue specimens were fixed in 10% neutral buffered

formalin for 24 hours then were trimmed and processed by

standard paraffin-embedding methods. Sections were cut at

m, deparaffinized, and then stained with: H&E &Immunohistochemical staining using monoclonal

antibodies to collagen I (COL-1).

Results

Histological analysis

H&E stained coronal sections were examined of fetuses of

CG and RhG for developing tooth germ of the 1st molar. In

CG, at 1st day after birth tooth germ of developing 1st molar

was at late bell stage with definite layer of dentin and at 10th day dentin and enamel matrix formation were almost

completed. In RhG, at 1st day after birth tooth germ of

developing 1st molar was at early bell stage; without dentin

formation and at 10th day normal thickness of enamel

matrix and dentin were formed.(Fig. 1 A&B) and (Fig. 2

A&B).

Immunohistochemical analysis

COL-1 was more brown intense in developing dentin and

bone in CG than in bone in RhG at 1st day after birth. On

the other hand, the colour intensity of COL-1 at 10th day in

T

Ahmed R. Zaher1, Mohamad E. Helal

2, Asmaa S. Elmahdy

3

1 Professor of Oral Biology, Faculty of Dentistry, Mansoura University, Egypt. 2 Professor of Oral Biology, Faculty of Dentistry, Mansoura University, Egypt. 3 Teaching assistant, Department of Oral Biology, Faculty of Dentistry, Mansoura University, Egypt.

Abstract: Objectives: In this study, we evaluated the impact of maternal rheumatoid arthritis (RA) in Albino rats on tooth development in their

offspring. Methods: 30 female rats were divided into 2 groups control group (CG), and rheumatoid non-treated group (RhG). Induction of RA was performed in (RhG) by injection of collagen II, followed by induction of pregnancy in both groups. Results: Fetuses of (RhG) showed slower rate of development both histologically and immunohistochemically at first few days followed by rapid normal growth at 10th day. Conclusion: Maternal RA, temporarily, slow down the rate of tooth development in their fetuses, then rapid catch up of normal rate of growth occurs.

Keywords: Rheumatoid arthritis, collagen, tooth development.

Impact of Systemically Induced Rheumatoid

Arthritis on Tooth Development in Rats

Offsprings

Mansoura Journal of Dentistry 2014;1(3):142-145.

Ahmed R. Zaheret al.

both groups was nearly the same. (Fig. 3 A&B) and (Fig. 4 A&B).

Discussion

Systemic auto-immune diseases have higher pervelance in woman and mainly at child bearing age [8]. Many studies

observed that autoimmune disease in women during

pregnancy may be associated with an increased risk for

learning disabilities in their sons. In this study, RA is one of

systemic autoimmune disease and is strongly associated

with oral health [9]. This is to say because immunological

and pathological processes occurring in periodontitis and

RA are nearly identical. Prospective studies on pregnant

mothers suggested that maternal periodontal disease may

cause preterm birth, low birth weight and may increase

their offspring's risk of developing early and severe dental caries [10]. So this study was conducted to answer the

question, whether RA diseased albino rat mothers has any

effect on tooth development in their offsprings.

In the present study, histological examination of

developing 1st molar tooth germ at 1st day in RhG showed

stage of early bell rather than late bell stage that was found

in CG. This delay in tooth development of group B,

compared with that in group A, can be explained by

findings of Scott [11] who demonstrated that pregnant women with connective tissue diseases, such as RA, are at

risk for preterm birth and intra-uterine fetal growth

restriction. As a contradict, many clinical experience and the reports of over 500 patients in clinical studies

demonstrated that RA activity decreases for many women

during pregnancy and their baby born healthy [12].

However, this may be due to either low disease activity or

good choice of medication taken by mothers during

pregnancy.

Histolofical findings of RhG at 10th day, in this study, showed normal development as in CG. This was in

agreement with findings of a cohort study of Dutch women

with RA and was accomplished by de Steenwinkel et al.

[13] who demonstrated that active rheumatoid arthritis

(RA) during pregnancy, without medication, and the

presence of RF or anticitrullinated protein antibodies (ACPAs) are associated with lower birth weight of the child

followed by rapid postnatal catch-up in weight.

In the present study, Immunohistochemical analysis

showed that brown colour intensity of COL-1 in RhG at 1st

day was less than that of CG indicating that secreted collagen I was decreased in RhG. This result could be

explained ad the following; auto-antibodies against

collagen type I that circulating in blood of high disease

activity RA pregnant mothers were able to cross placenta

and attack collagen type I in developing bone and dentin

resulting in transient slow rate of formation and decreased

amount of collagen in these tissues [14,15]. However, this

was not the case at 10th day, where the colour intensity of

COL-1 was the same in RhG and CG which may be also

due to rapid catch up mechanism.

Conclusion

With the limitation of our study as it was the first study that

investigate the effect of one of maternal autoimmune

disease, RA, on tooth development, we can conclude that

RA, under certain circumstances as absence of medication or high disease activity, leads to slow rate of development

and decreased amount of collagen I production at first few

days after birth and compensated by rapid compensatory

mechanism and restoration of normal rate of development

and growth at the following days.

Figure 1: Photomicrograph of developing 1st molar at 1st day, CG (A) and RhG (B). (H&E stain, x 100)

A B

Mansoura Journal of Dentistry 2014;1(3):142-145.

Ahmed R. Zaheret al.

Figure 2: Photomicrograph of developing 1st molar at 10th day, CG (A) and RhG (B). (H&E stain, x 400)

Figure 3: Photomicrograph of developing 1st molar at 1st day ,showing positive brown intense reaction of COL-1 in dentin and

bone of CG (A) and less intense reaction in bone of RhG (B). (COL-1 stain, x 100)

A B

A B

Mansoura Journal of Dentistry 2014;1(3):142-145.

Ahmed R. Zaheret al.

Figure 4: Photomicrograph of developing 1st molar at 10th day ,showing positive brown intense reaction of COL-1 in dentin

and bone of both CG (A) and RhG (B). (COL-1 stain, x 100)

References

1.Tucker A, Sharpe P. The cutting-edge of mammalian development; how the embryo makes teeth. Nat Rev

Genet. 2002; 5:499508. 2.Kadler KE, Hill A, Canty-Laird EG. Collagen

fibrillogenesis: fibronectin, integrins, and minor collagens

as organizers and nucleators. Curr Opin Cell Biol. 2008;

20:495-501.

3.Habelitz S, Balooch M, Marshall SJ, Balooch G,

Marshall GW Jr. In situ atomic force microscopy of

partially demineralized human dentin collagen fibrils. J

Struct Biol. 2002; 138: 227-236.

4.Salsas-Escat R, Nerenberg PS, Stultz CM. Cleavage site

specificity and conformational selection in type I collagen degradation. Biochem. 2010; 49: 4147-4158.

5.Aletaha D, Neogi T, Silman AJ, Funovits J, Felson DT,

Bingham CO 3rd, et al. 2010 Rheumatoid arthritis

classification criteria: an American College of

Rheumatology/European League Against Rheumatism

collaborative initiative. Arthritis Rheum. 2010; 62: 2569-

2581.

6.Rantap-Dahlqvist S, de Jong BA, Berglin E, Hallmans

G, Wadell G, Stenlund H, et al. Antibodies against cyclic

citrullinated peptide and IgA rheumatoid factor predict the

development of rheumatoid arthritis. Arthritis Rheum. 2003; 48: 2741-2749.

7.Shahi MM, Rashidi MR, Mahboob S, Haidari F, Rashidi

B, Hanaee J. Protective effect of soy protein on collagen-

induced arthritis in rat. Rheumatol Int. 2012; 32: 2407-

2414.

8.Brucato A, Cimaz R, Caporali R, Ramoni V, Buyon J

Pregnancy outcomes in patients with autoimmune diseases

and anti-Ro/SSA antibodies. Clin Rev Allergy Immunol.

2011; 40: 27-41.

9.Bingham CO 3rd, Moni M. Periodontal disease and

rheumatoid arthritis: the evidence accumulates for

complex pathobiologic interactions. Curr Opin Rheumatol.

2013; 25: 345-353.

10.Boggess KA, Edelstein BL Oral health in women

during preconception and pregnancy: implications for birth

outcomes and infant oral health. Matern Child Health J.

2006; 10: S169-174.

11.Scott JR. Risks to the children born to mothers with autoimmune diseases. Lupus. 2002; 11: 655-660.

12.Ostensen M, Villiger PM The remission of rheumatoid

arthritis during pregnancy. Semin Immunopathol. 2007;

29: 185-191.

13.de Steenwinkel FD, Hokken-Koelega AC, de Ridder

MA, Hazes JM, Dolhain RJ. Rheumatoid arthritis during

pregnancy and postnatal catch-up growth in the offspring.

Arthr Rheumatol. 2014; 66: 1705-1711.

14.Fox E, Amaral D, Van de Water J. Maternal and fetal

antibrain antibodies in development and disease. Dev

Neurobiol. 2012; 72: 1327-1334. 15.Bauman MD, Iosif AM, Ashwood P, Braunschweig D,

Lee A, Schumann CM, et al. Maternal antibodies from

mothers of children with autism alter brain growth and

social behavior development in the rhesus monkey.

Translat psych. 2013; 3: e278.