Association between intermittent administration of parathyroid hormone 1-34 and ectopic calcification in rats

The present study was conducted to determine the association between parathyroid hormone 1-34 administration and ectopic calcification in rats with glucocorticoid induced osteoporosis. A total of 18 rats were used in the current study. Osteoporosis was induced in all rats via dexamethasone administration, then rats were randomly distributed into Control and Forteo groups and were sacrificed 4 weeks after initiation of drug administration. Hemi-mandibles were decalcified followed by routine histological analysis. Among the Forteo group rats, three rats displayed the presence of ectopic calcification. True pulp stone, intra-pulpal calcified structure with entrapped cells and intra periodontal bone-like calcified structure with entrapped cells were observed while no ectopic calcification was noticed in the control group.

Ectopic calcification is pathologic deposition of minerals within soft tissues as dental pulp or periodontal ligaments (PDL) 1,2 .Pulpal ectopic calcification may manifest as generalized, linear calcification, or as circumscribed calcification (also known as pulp stones or denticles).Pulp stones can be seen free within the pulp tissues, partially associated with dentin wall or completely embedded in dentin.They may manifest as false concentric calcification or true pulp stones 3 .The etiology of pulp calcification may be idiopathic 4 , although it may also be associated with pulp injury or degeneration 5 , orthodontic or physical forces [6][7][8] or chemical stimuli 9,10 .Its incidence tends to increase with age 3,11 .
Parathyroid hormone (PTH) is a naturally occurring hormone, important for calcium homeostasis 12,13 .Its level in the blood dictates its effect on the skeletal system, with bone catabolic effect upon chronic increase in PTH level associated with hyperparathyroidism 13,14 and bone anabolic effect upon administration of small intermittent dosage 15,16 .PTH secreted by the parathyroid gland (native PTH) is a polypeptide chain composed of 84 amino acids (PTH 1-84).While PTH 1-34, is a fragment of PTH molecule "synthetised through recombinant DNA technology using a strain of Escherichia coli bacteria" 17,18 .

Methods
This research was conducted as a part of a study examining the effect of PTH 1-34 on microarchitecture of alveolar process of osteoporotic rats.

Animals
In the current research, 18 male Wistar rats of the species Rattus norvegicus weighing 175-200 gm, aged between 3 to 4 months were used.The animals were acquired from and maintained in the Animal House, Faculty of Medicine, Cairo University under the care of a specialized veterinarian.Each animal was kept in a separate cage.They were maintained under controlled temperature at 25±2°C with 12 h light/dark cycle and had ad libitum access to standard rats' chow and water.This study was approved by the Research Ethics committee faculty of Dentistry, Cairo University (approval number 151028).

Osteoporosis induction and treatment
Osteoporosis was induced in all experimental animals (n=18) by five weekly doses, of 7 mg/kg body weight dexamethasone sodium phosphate (Decadron ® 4 mg/ml, Eipico Egypt), administered intramuscularly 30,31 .The animals were then randomly distributed by random sequence generator program (randomizer.org)into two groups each including 9 animals, matching of the animals with the numbers was done blindly through the primary investigator.Animals received either a daily subcutaneous injection of 60 μg/kg body weight PTH 1-34 (Forteo ® ; Eli lilly Pharmaceuticals) (n=9) 32 or an equal volume of saline (control group) (n=9).Drugs were administered in the early morning hours (8-9 am).The body weight of the animals was measured weekly, and drug dosages were adjusted accordingly.Animals were euthanized with an intra-cardiac overdose of sodium thiopental (80 mg/kg) 4 weeks after initiation of Forteo administration.Mandibles were dissected and separated into two halves, only one hemi-mandible from each rat was utilized for histological examination.The experimental unit was the hemimandible of rats.The primary investigator was blinded.
Hemi-mandibles (n=18) were fixed in 10% calcium formol solution for 48 hours.The specimens were then washed and soaked in 10% EDTA for 4-5 weeks for decalcification.After decalcification was completed, the specimens were dehydrated in ascending grades of alcohol, cleaned in xylol, and then embedded in paraffin blocks.Next, 6-μm-thick paraffin sections were cut and mounted on a clean glass slide, then stained with haematoxylin and eosin stain 31 .The specimens were examined using Leica DM300 light microscopic (Leica Microsystems, Inc., Switzerland).Histological examination was done through blinded primary investigator.Dental pulp and surrounding periodontal ligaments of all teeth within hemimandible of both experimental groups (n=18) were examined for the presence of ectopic calcification.

Results
Upon histological examination of the Forteo group specimens, six rats showed normal pulp and periodontal ligaments with no ectopic calcifications, while ectopic calcifications were detected in three specimens (Dataset 1) 33 .Where true pulp stone with pre-dentin and dentin surrounding a central cavity lined by cells was detected in one specimen (Figure 1a).Another specimen showed the presence of intra-pulpal calcified structure with entrapped cells (Figure 1b).Meanwhile, one specimen displayed the presence of intra-periodontal bone-like calcified structure with entrapped cells (Figure 1c).On the other hand, no ectopic calcification was perceived in the control group specimens (Dataset 1) 33 , which showed normal pulp and periodontal ligaments (n=9) (Figure 1d).increases in odontoblast-like cells proliferation upon short PTH exposure and increases in their apoptosis upon longer exposure 36 .
Wang et al. demonstrated the ability of PTH to induce human PDL stem cells to differentiate into osteoblasts, which was associated with increased alkaline phosphatase activity and increased mineralization capacity 37 .Moreover, Li et al. described the ability of PTH 1-34 to induce the formation of calcified nodule in cementoblast cell line, which was attributed to the ability of the drug to increase cementoblast activity, alkaline phosphatase level and subsequently calcification 38 .
The stimulatory effect of PTH 1-34 on odontoblast, cementoblast and osteoblasts function can help explain the findings of the current research.

Recommendations
Further research studying the effect of different dosage schemes of PTH 1-34, administered for different time periods, on dental pulp and odontoblast cells is recommended.

Open Peer Review
Current Referee Status: The current study performed by the authors is an interesting one which aims to determine the association between parathyroid hormone 1-34 administration and ectopic calcification formation in pulp and periodontal ligaments of osteoporotic rats.
Results revealed that rats receiving PTH 1-34 showed ectopic calcifications in their pulp and periodontal ligaments.
The study is well organized and the authors well documented their work in particular the images captured from rats of each group.However I have some minor remarks as follows: Aim: I suggest that the aim should include that the investigations were carried on dental structures (Pulp and periodontal ligaments)

Results:
I suggest that the histology of the calcified areas to be thoroughly examined to determine if its histology resembles dentin, bone or cementum in each area.

Discussion:
More information is needed to determine the mechanism by which the PTH 1-34 is involved in stimulation of odontoblast, cementoblast and osteoblasts functions.

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate?If applicable, is the statistical analysis and its interpretation appropriate?Not applicable Are all the source data underlying the results available to ensure full reproducibility?Yes Are the conclusions drawn adequately supported by the results?Yes No competing interests were disclosed.

Competing Interests:
I have read this submission.I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias You can publish traditional articles, null/negative results, case reports, data notes and more The peer review process is transparent and collaborative Your article is indexed in PubMed after passing peer review Dedicated customer support at every stage For pre-submission enquiries, contact research@f1000.com Radwan IA, Sedky Korany N and Adel Ezzat B. How to cite this article: Association between intermittent administration of parathyroid 2018, :1553 (doi: hormone 1-34 and ectopic calcification in rats [version 1; referees: 1 approved]

Figure 1 .
Figure 1.Light microscope images of the Forteo-treated group.(a) A true pulp stone with dentin, pre dentin and central cavity lined by cells (original magnification, x100 (left) and x400 (right)).(b) Intrapulpal calcification with entrapped cells (original magnification, x100 (left) and x400 (right)).(c) Intra-periodontal ectopic calcification with entrapped cells surrounded with disorganized periodontal ligaments (original magnification, x400).(d) Light microscope image of the control group showing normal pulp and periodontal ligaments with no ectopic calcification (original magnification x 100).
Biology Department, Faculty of Dentistry, Modern Sciences and Arts University, Cairo, Egypt

Dataset 1. Images captured from each mouse in each group not shown in Figure 1 https://dx.doi.org/10.5256/f1000research.16298.d218523 Discussion
35imaraes et al. observed increased dentin deposition rate and elevated level of serum alkaline phosphatase in PTH 1-34 treated rats35.In a subsequent research, Guimaraes et al. elucidated that PTH 1-34 can regulate odontoblast like cells via protein kinase A-and protein kinase C-dependent pathways, with