The Impact of Bisphosphonates on the Osteoclast Cells of Osteogenesis Imperfecta Patients

Published by Oriental Scientific Publishing Company © 2018 This is an Open Access article licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-nc-sa/4.0/ ), which permits unrestricted Non Commercial use, distribution and reproduction in any medium, provided the original work is properly cited. The Impact of Bisphosphonates on the Osteoclast Cells of Osteogenesis Imperfecta Patients

Osteogenesis imperfecta is a fairly rare disorder (one in 15-20000 births) 1, 2 . The phenotypic spectrum ranges in severity from mild skeletal phenotypes with few to no fractures, to prenatal lethality. On molecular basis, OI is classified into 12 types 3 . OI is characterized by fragility fractures, blue sclera, short stature, and hearing loss 4 . Less commonly observed findings in OI are decreased pulmonary function, cardiac valvular regurgitation, chest wall deformities (pectus carinatum, barrel chest) and scoliosis 4 .
Although about 85-90% of cases are caused by structural or quantitative mutations in the collagen gene, recent studies show that this disorder is majorly due to defective collagen production 5 . Among different types of OI, types II & III are relatively uncommon but are extremely aggressive and lethal 4 . BPs are mainstay of therapy for OI. They are analogs of pyrophosphate, which inhibit the bone resorption by blocking the key enzyme farnesyl-pyrophosphate 6 . In response to inhibition of prenylation of intracellular proteins by BPs, there is ultimately an increase in osteoclast apoptosis 7 .
BPs can also reduce number of osteoclasts inducing apoptosis in macrophages and osteoclast cells 8 . In addition, BPs can directly inhibit the boneresorbing activity of osteoclasts 9 . The mechanism by which are BPs act directly on osteoclasts and osteoclast precursors has been reported to be partly due to inhibition of the mevalonate pathway 6 . There is also a likely indirect effect via osteoblasts which are bone forming cells and have cellular link with osteoclast cells due to modulation of osteoblast secretion of soluble paracrine factors that influence osteoclast activity 10,11 .
BPs, administered to children with OI, have been shown to increase bone volume by counteracting the high turnover cellular status of bone in classic OI [12][13][14] . The new bone still contains defective collagen. The hypothesis behind the treatment is that an increased bone mass (even of impaired quality) leads to moderate reduction in the fracture risk 15 . Highly potent BP-Zoledronic acid (ZOL) enhances osteoblastic activity and differentiation 16 , which further increases the bone mass in OI patients 3 . It is likely that BPs decrease the fracture rate but increase bone brittleness and also conceding the fact that BPs directly do not affect the defective collagen however they inhibit the bone resorption by osteoclasts 17,18 .
The Present study was planned to compare the frequency of caspase 3 expressing cells (suggestive of apoptosis) in precursor osteoclast cells of OI children who received ZOL (high dose vs. low dose) with age-matched controls.

study design
The Present study is a case series of 10 OI patients. The study was conducted from January 2014 to July 2014 at the Postgraduate Institute of Medical Education and Research, Chandigarh, India. Whole blood sample was taken from the OI patients after obtaining an informed written consent from the parents of the patients. The study was approved by institute ethics committee. Total 15 children participated in the study (n=10 OI patients, n=5 age matched, healthy controls).
Cumulative dose is defined as total dose of ZOL received by the patient. Out of the 10 OI children, 5 had received a low cumulative dose (<30mg) of ZOL while 5 had received a high cumulative dose (e"30 mg) over the last 3 years. The diagnosis of OI was suspected on basis of clinical features and radiological findings and was confirmed by mutational analysis.

Biochemical and radiological diagnosis
Total serum calcium, phosphate, alkaline phosphatase, were measured in the hospital laboratory by standard methods (Auto Analyzer Modular P 800; Roche Diagnostics). Plasma 25hydroxyvitamin D, C terminal telopeptide (CTx) and procollagen type 1 amino terminal propeptide (P1NP) levels were also measured by Elecsys and cobase immunoassay analyzers (ECLIA). The measuring range for CTx as per the package insert was 10-6000 pg/mL with an analytical sensitivity of 10 pg/mL and an intra assay CV of 17.9%. The measuring range for P1NP as per the package insert was 5-1200 ng/mL with an analytical sensitivity of 5 ng/ml and an intra assay CV of 4.1%.

extractions and immunostaining of osteoclast cells
Sample of whole blood was layered over Ficoll-Hypaque density gradient and direct immunostaining for Peripheral blood mononuclear cells (PBMC) was performed. Mononuclear cells were stained with monoclonal antihuman RANK-PE (9A725, Thermofisher, scientific) and caspase-3 NucView 488 antibodies. Cells were incubated for 30 minutes at room temperature, followed by washing using phosphate buffer saline (PBS) with 2% fetal bovine serum (FBS). The cells were finally acquired using flow cytometer (BD FACS CANTO-II, Becton Dickinson, CA, USA) and data were analyzed using FACSDiva software. Single color tubes were used for compensation. Cells were first gated on the basis of forward and side scatter (P1), followed by gating of RANK positive cells (P2). caspase-3 activity was assessed in these RANK positive precursor osteoclast cells. The frequency of caspase-3 expressing precursor osteoclasts and median fluorescence intensity (MFI) of caspase-3 were compared among subject groups. The OI patient's demography, biochemistry, radiology & ZOL dose are summarized in Table 1.

statistical analysis
Data are presented as the mean ± standard error of the mean (SEM). Data was checked for normality using KS test. Normally distributed data was compared using unpaired t test, while skewed data was compared using Mann Whitney test. All statistical analysis were performed using graph pad prism 5.0.

Clinical findings of oi patients
The mean age of study participants (n=10) was 7 ±1.5 years. All (n=10) of them had short stature, blue sclera and 9 had dentinogenesis imperfecta. 6 of them had OI type IV, 2 had type III and one each had types I and II. Biochemical parameters namely, serum calcium were 9.3±0.24 mg/dl, phosphate 4.72±0.2 mg/dl, alkaline phosphatase 295.3±64.31 IU/L, 25-hydroxyvitamin D 31.66±4.32 ng/ml were in the normal range. In comparison to healthy controls, bone turnover markers were significantly suppressed in high and low dose ZOL treated group as given in Table 1.

radiological findings of the oi patients
Radiographs of the patients on high dose of ZOL showed the presence of "zebra stripe sign": indicative of cyclical ZOL therapy & also presence of dense metaphyses; suggestive of acquired osteosclerosis (Figure 1). There were various deformities in the bone as well as thickened cortices.

induction of apoptosis in osteoclasts
We observed that the OI subjects who received high dose of ZOL had significantly increased frequency of caspase-3 expressing precursor osteoclast cells (median; 99.73%) as compared to those who received low dose (median; 53.92%) (p <0.0159), as well as control subjects (median; 35.96%) (p=0.005) (Figure 2a). As expected, the expression of caspase-3 in precursor osteoclasts was also higher in these subjects as compared to the low dose group (p <0.0079) and controls (p <0.005) value control vs high ( Figure  2b). Thus, we observed significant difference in caspase-3 expression between the low dose group and high dose group of OI patients.

disCussion
We have reported that high dose of ZOL acid caused suppression of bone turnover markers and osteosclerosis of growing skeleton. We also demonstrated radiological changes, namely, presence of dense metaphyses in the patients on  high dose which occurred after therapy .We also demonstrated significantly increased expression of caspase-3 activity in osteoclasts isolated from PMBCs of ZOL (>30 mg) treated OI children compared to healthy controls. Among the 10 OI patients, 6 had OI type IV, 2 had type III and one each had types I and II. Out of these, types I and IV are relatively mild, with the patient remaining ambulatory, while types II and III have a more severe phenotype. All patients had fragility fractures, and blue sclera and 9 had dentinogenesis imperfecta. None of the patients had hearing loss. In a study from North India, of 20 OI patients, dentinogenesis imperfecta and blue sclera were seen in 50% of the patients. Similar to our study, all patients had fractures while none of them had hearing loss 19 .
Recent studies have shown that malocclusion depends upon the severity of the OI type 20 . In our study, we found that all patients had dentinogenesis imperfecta. The recommended dose of ZOL for children is 0.05 mg/kg body weight to be repeated every six months 21 but as we observed that doses as high as 16 mg per year had been received by our patients. The fact that bone turnover was slowed down was evident by the fact that the bone turnover markers (CTX, P1NP) were suppressed in the OI patients on prolonged ZOL therapy compared to the controls. Similar suppression of bone turnover in OI patients on pamidronate therapy was also shown in a previous study 22 . The fact that BPs accumulate in the bone and residual levels are measurable even after years of therapy further raises concern for their safety, particularly in growing skeleton. Hence, drug holidays have been advocated for patients receiving BPs therapy for osteoporosis 23 .
In present study we have demonstrated that precursor osteoclast apoptosis was increased as shown by high capases3 positive precursor osteoclast cells and high percentage of apoptotic precursor osteoclasts in OI patients on ZOL therapy as compared to the control group. In a study by Hughes et al, three BPs (risedronate, pamidronate, and clodronate) were shown to cause a 4-to 24-fold increase in the proportion of apoptotic osteoclasts in vitro. Of the three compounds, risedronate, the most potent inhibitor of bone resorption in vivo, was the strongest inducer of osteoclast apoptosis in vitro 24 . In another study by Rogers et al, it was demonstrated that BPs induce osteoclast apoptosis, in part by inhibiting the activity of enzymes in the mevalonate pathway and promoting caspase cleavage of mammalian sterile 20-like (Mst) kinase 1 25 . Since in normal bone remodeling, bone resorption and formation are coupled to each other, this osteoclast apoptosis is expected to ultimately decrease bone formation as well.
This kind of "acquired osteopetrosis" has been previously reported by Whyte et al in a child of OI treated with pamidronate 26 . In our study participants the dose of ZOL was more than five times the dose usually prescribed in pediatric population. Though the metaphyseal sclerotic "banding" seen on radiology is an expected finding in patients of OI on cyclic BPs therapy, but it should resolve after completion of therapy. However, it persisted in our patients even after cessation of therapy.
Limitations of study include small sample size and lack of base line data of bone markers for comparison. Nonetheless our study gives a new direction to work upon and warrants detailed studies for looking at mechanistic details.
BPs are known to increase bone mineral density in OI patients 27 , however high dose can cause osteopetrosis 26 . We have studied caspace 3 activity in precursor osteoclasts. The study can be reproduced in a large number of patients, with analysis of entire apoptosis pathway.

ConClusion
Overzealous use of ZOL in OI children suppresses bone turnover markers (CTx,P1NP) and also causes osteosclerosis. High dose ZOL inhibits osteoclastic activity (increased expression of caspase 3 activity) which in turn predisposes to atypical fractures and delayed fracture healing. This is an exploratory study that highlights that the dosing of BPs needs to be standardised with the help of focussed clinical trials so that its adverse effects on bones of OI patients can be prevented.