{"id":20299,"date":"2018-06-25T11:50:47","date_gmt":"2018-06-25T11:50:47","guid":{"rendered":"http:\/\/biomedpharmajournal.org\/?p=20299"},"modified":"2020-04-23T06:50:32","modified_gmt":"2020-04-23T06:50:32","slug":"the-impact-of-bisphosphonates-on-the-osteoclast-cells-of-osteogenesis-imperfecta-patients","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol11no2\/the-impact-of-bisphosphonates-on-the-osteoclast-cells-of-osteogenesis-imperfecta-patients\/","title":{"rendered":"Impact of Bisphosphonates on the Osteoclast Cells of Osteogenesis Imperfecta Patients"},"content":{"rendered":"

Introduction<\/strong><\/p>\n

Osteogenesis imperfecta (OI) is a fairly rare disorder (one in 15-20000 births).1, 2\u00a0<\/sup>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<\/sup>\u00a0OI is characterized by fragility fractures, blue sclera, short stature, and hearing loss.4<\/sup>\u00a0Less commonly observed findings in OI are decreased pulmonary function, cardiac valvular regurgitation, chest wall deformities (pectus carinatum, barrel chest) and scoliosis.4<\/sup><\/p>\n

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<\/sup>\u00a0Among different types of OI, type II & III are relatively uncommon but are extremely aggressive and lethal.4<\/sup>\u00a0BPs are mainstay of therapy for OI. They are analogs of pyrophosphate, which inhibit the bone resorption by blocking the key enzyme farnesyl-pyrophosphate.6<\/sup>\u00a0In response to inhibition of prenylation of intracellular proteins by BPs, there is ultimately an increase in osteoclast apoptosis.7<\/sup><\/p>\n

BPs can also reduce number of osteoclasts inducing apoptosis in macrophages and osteoclast cells.8<\/sup>\u00a0In addition, BPs can directly inhibit the bone resorbing activity of osteoclasts.9<\/sup>\u00a0The mechanism by which BPs act directly on osteoclasts and osteoclast precursors has been reported to be partly due to inhibition of the mevalonate pathway.6<\/sup>\u00a0There is also a likely indirect effect via osteoblasts which have cellular link with osteoclast cells due to modulation of osteoblast secretion of soluble paracrine factors that influence osteoclast activity.10, 11<\/sup><\/p>\n

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.12-14<\/sup>\u00a0The 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<\/sup>\u00a0Zoledronic acid (ZOL) enhances osteoblastic activity and differentiation,16<\/sup>\u00a0which further increases the bone mass in OI patients.3<\/sup>\u00a0It 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<\/sup><\/p>\n

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.<\/p>\n

Material and Methods<\/strong><\/p>\n

Study Design<\/strong><\/p>\n

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 OI patients after obtaining an informed written consent from the parents of\u00a0 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).<\/p>\n

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 (><\/u> 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.<\/p>\n

Biochemical and Radiological Diagnosis<\/strong><\/p>\n

Total serum calcium, phosphate, alkaline phosphatase, were measured in the hospital laboratory by standard methods (Auto Analyzer Modular P 800; Roche Diagnostics). Plasma 25 hydroxyvitamin D, C terminal telopeptide (CTx) and procollagen type 1 amino terminal propeptide (P1NP) levels were also<\/span> 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%.<\/p>\n

Extractions and Immunostaining of Osteoclast Cells<\/strong><\/p>\n

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 FACS Diva 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 \u00a0\u00a0activity 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\u2019s demography, biochemistry, radiology & ZOL dose are summarized in Table 1.<\/p>\n

Statistical Analysis<\/strong><\/p>\n

Data are presented as the mean \u00b1 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.<\/p>\n

Result<\/strong><\/p>\n

Clinical findings of OI patients <\/strong><\/p>\n

The mean age of study participants (n=10) was 7 \u00b11.5 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\u00b10.24 mg\/dl, phosphate 4.72\u00b10.2 mg\/dl, alkaline phosphatase 295.3\u00b164.31 IU\/L, 25-hydroxyvitamin D 31.66\u00b14.32 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.<\/p>\n

Table 1a: Clinical characteristics of OI patients with high dose (case 1-5) and low dose (case 6-10)<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
Characteristic<\/td>\nCase1<\/td>\nCase2<\/td>\nCase3 \u00a0\u00a0\u00a0 Case4 \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Case5<\/td>\nCase 6<\/td>\nCase7 Case 8 Case 9 Case 10<\/td>\n<\/tr>\n
Age (Years)<\/td>\n8<\/td>\n8<\/td>\n10 \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 7 \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 6<\/td>\n8<\/td>\n\u00a0\u00a0\u00a0 5 \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 9 \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 1 \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 9<\/td>\n<\/tr>\n
Gender<\/td>\nM<\/td>\nM<\/td>\nM \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 F \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 M<\/td>\nM<\/td>\nM \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 M \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 M \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 M<\/td>\n<\/tr>\n
Short stature<\/td>\nYes<\/td>\nYes<\/td>\nYes \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Yes \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Yes<\/td>\nYes<\/td>\nYes \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Yes \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Yes \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Yes<\/td>\n<\/tr>\n
Weight (kg)<\/td>\n9.3<\/td>\n10.2<\/td>\n\u00a09.4 \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a015.0\u00a0 \u00a0\u00a010.0<\/td>\n9.3<\/td>\n20\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 \u00a0\u00a012.6\u00a0 \u00a0\u00a0\u00a0\u00a015.0\u00a0 \u00a0\u00a0\u00a0\u00a012<\/td>\n<\/tr>\n
Blue Sclera<\/td>\n+<\/td>\n+<\/td>\n+<\/td>\n+<\/td>\n+<\/td>\n+<\/td>\n+<\/td>\n+<\/td>\n+<\/td>\n+<\/td>\n<\/tr>\n
Dentinogenesis imperfecta<\/td>\n+<\/td>\n+<\/td>\n+<\/td>\n+<\/td>\n+<\/td>\n+<\/td>\n+<\/td>\n+<\/td>\n–<\/td>\n+<\/td>\n<\/tr>\n
Number of fractures<\/td>\n\u00a06<\/td>\n7<\/td>\n5<\/td>\n\u00a09<\/td>\n\u00a05<\/td>\n\u00a07<\/td>\n6<\/td>\n5<\/td>\n\u00a02<\/td>\n\u00a05<\/td>\n<\/tr>\n
Scoliosis<\/td>\nYes<\/td>\nYes<\/td>\nYes<\/td>\nYes<\/td>\nNo<\/td>\nNo<\/td>\nYes<\/td>\nNo<\/td>\nNo<\/td>\nNo<\/td>\n<\/tr>\n
Leg deformities<\/td>\nYes<\/td>\nYes<\/td>\nYes<\/td>\nYes<\/td>\nYes<\/td>\nYes<\/td>\nYes<\/td>\nYes<\/td>\nYes<\/td>\nYes<\/td>\n<\/tr>\n
Total received dose<\/td>\n36<\/td>\n68<\/td>\n64<\/td>\n64<\/td>\n40<\/td>\n<30<\/td>\n<30<\/td>\n<30<\/td>\n<30<\/td>\n<30<\/td>\n<\/tr>\n
of ZOL (mg) \u00a0\u00a0Type of OI<\/td>\n\u00a0IV<\/td>\n\u00a0III<\/td>\n\u00a0IV<\/td>\n\u00a0IV<\/td>\n\u00a0VII<\/td>\n\u00a0\u00a0IV<\/td>\n\u00a0 IV<\/td>\n\u00a0IV<\/td>\n\u00a0I<\/td>\n\u00a0III<\/td>\n<\/tr>\n
\u00a0<\/span><\/p>\n

S.ca (8.7-10.2 mg\/dl)<\/td>\n

8.4<\/td>\n10<\/td>\n10.2<\/td>\n\u00a010.2<\/td>\n9.7<\/td>\n8.0<\/td>\n8.76<\/td>\n8.8<\/td>\n9.60<\/td>\n9.5<\/td>\n<\/tr>\n
S.Po4<\/sub> (2.7-4.5mg\/dl)<\/td>\n5.4<\/td>\n4.2<\/td>\n5<\/td>\n\u00a04.6<\/td>\n5.2<\/td>\n4.6<\/td>\n4.92<\/td>\n4.92<\/td>\n3.22<\/td>\n5.1<\/td>\n<\/tr>\n
ALP (40-129 IU\/L)<\/td>\n217<\/td>\n\u00a0216<\/td>\n135<\/td>\n\u00a0167<\/td>\n276<\/td>\n771.4<\/td>\n318.6<\/td>\n518.0<\/td>\n106<\/td>\n212<\/td>\n<\/tr>\n
25(OH)vit D<\/td>\n20.22<\/td>\n30.07<\/td>\n\u00a027.33<\/td>\n\u00a024.62<\/td>\n26.25<\/td>\n27.03<\/td>\n37.01<\/td>\n31.06<\/td>\n68.43<\/td>\n24.62<\/td>\n<\/tr>\n
(11.1-42.9 ng\/ml)<\/p>\n

CTX\u00a0 (299\u00b1 137 pg\/ml)<\/td>\n

0.236<\/td>\n0.316<\/td>\n0.199<\/td>\n0.258<\/td>\n0.314<\/td>\n0.937<\/td>\n0.379<\/td>\n1.04<\/td>\n0.276<\/td>\n0.486<\/td>\n<\/tr>\n
P1NP (15.13- 58.5 ng\/ml) 129.8 \u00a0\u00a0\u00a0 143.5 \u00a0\u00a0\u00a0 59.37 \u00a0\u00a0\u00a0 116.2 \u00a0\u00a0\u00a0\u00a0 132 \u00a0\u00a0\u00a0\u00a0\u00a0 460.0 \u00a0\u00a0\u00a0 114.4 \u00a0\u00a0\u00a0\u00a0 439 \u00a0\u00a0\u00a0\u00a0\u00a0 219.2 \u00a0\u00a0\u00a0\u00a0 128<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Radiological Findings of the OI Patients<\/strong><\/p>\n

Radiographs of the patients on high dose of ZOL showed the presence of \u201czebra stripe sign\u201d: 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.<\/p>\n\n\n\n
\"Figure<\/td>\nFigure 1:<\/strong> Radiograph of lower limb in 8 year old boy showed the presence of generalized osteosclerosis ,Zebra strip sign and dense metaphyses.<\/strong><\/p>\n

 <\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Induction of Apoptosis in Osteoclasts<\/strong><\/p>\n

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\u00a0 low dose group and high dose group of OI patients.<\/p>\n\n\n\n
\u00a0\"Figure<\/td>\nFigure 2a: Percentage of Caspase-3 % of\u00a0 osteoclast cells in each group, presented as dots.<\/strong><\/p>\n

 <\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

The results are expressed as the mean \u00b1standard error of the mean (n=5 for each group.*P<0.016\u00a0 (control vs high dose), P<0.55 (control vs low dose ) and **P<0.005 (Low\u00a0 dose vs high dose)<\/p>\n\n\n\n
\u00a0\"Figure<\/td>\nFigure 2b: MFI (median) caspase-3 of\u00a0\u00a0 osteoclast cells in each group, presented as dots.<\/strong><\/p>\n

 <\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

The results are expressed as the mean \u00b1standard error of the mean (n=5 for each group.*P<0.008 (control vs high dose), P<0.99 (control vs low dose ) and **P<0.005 (Low\u00a0 dose vs high dose)<\/p>\n\n\n\n
\u00a0\"Figure<\/td>\nFigure 2c: Representative flow cytometric plots showing the gating strategy for the analysis of RANK+Caspase+ cells.<\/strong><\/p>\n

 <\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Firstly cells were gated on the basis of FSC and SSC followed by gating of RANK+ cells (P2) which is mainly expressed by osteoclasts cells. Further these osteoclasts cells were analyzed for Caspases activity (P3)<\/p>\n

Discussion<\/strong><\/p>\n

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.\u00a0 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<\/sup><\/p>\n

Recent studies have shown that malocclusion depends upon the severity of the OI type.20<\/sup>\u00a0In 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 months21<\/sup> 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<\/sup>\u00a0The 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<\/sup>\u00a0In present study we have demonstrated that precursor osteoclast apoptosis was increased as shown by high caspases3 positive precursor osteoclast cells and high percentage of apopto precursor osteoclasts in OI patients on ZOL therapy as compared to the control group. In a study by Hughes et al<\/em>, three BPs (risedronate, pamidronate, and clodronate) were shown to cause a 4- to 24-fold increase in the proportion of apoptotic osteoclasts in vitro.24<\/sup>\u00a0Of the three compounds, risedronate, the most potent inhibitor of bone resorption in vivo, was the strongest inducer of osteoclast apoptosis in vitro.24<\/sup>\u00a0In another study by Rogers et al<\/em>, also 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<\/sup>\u00a0Since 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.<\/p>\n

This kind of \u201cacquired osteopetrosis\u201d has been previously reported by Whyte et al<\/em> in a child of OI treated with pamidronate.26<\/sup>\u00a0In our study participants the dose of ZOL was more than five times the dose usually prescribed in pediatric population. Though the metaphyseal sclerotic \u201cbanding\u201d 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.<\/p>\n

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.<\/p>\n

BPs are known to increase bone mineral density in OI patients,27<\/sup>\u00a0however high dose can cause osteopetrosis.26<\/sup>\u00a0We have studied caspase3 activity in precursor osteoclasts. The study can be reproduced in a large number of patients, with analysis of entire apoptosis pathway.<\/p>\n

Conclusion<\/strong><\/p>\n

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\u00a0of 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 focused clinical trials so that its adverse effects on bones of OI patients can be prevented.<\/p>\n

Acknowledgement<\/strong><\/p>\n

The study was supported by the Indian Council of Medical Research Senior Research Fellow award to Ms. V. Dhiman (ICMR SRF Fund # 45\/10\/2014-HUM-BMS). Mahinder Wadhwa and Ravi Garg for helping in interpretation of flow cytometer result.<\/p>\n

Reference<\/strong><\/p>\n

    \n
  1. Stoll C, Dott B, Roth M.P, Alembik Y. Birth prevalence rates of skeletal dysplasias. Clinical genetics.<\/em> 1989;35(2):88-92.
    \n    CrossRef<\/a><\/li>\n
  2. Forlino A, Marini J.C. Osteogenesis imperfecta. Lancet<\/em>. 2016;387(10028):1657-71.
    \n    CrossRef<\/a><\/li>\n
  3. Marom R, Lee Y.C, Grafe I, Lee B. Pharmacological and biological therapeutic strategies for osteogenesis imperfecta. American <\/em>journal of medical genetics Part C, Seminars in medical genetics. 2016;172(4):367-83.
    \n    CrossRef<\/a><\/li>\n
  4. Forlino A, Cabral W.A, Barnes A.M, Marini J.C. New perspectives on osteogenesis imperfecta. Nature reviews Endocrinology.\u00a0<\/em>2011;7(9):540-57.
    \n    CrossRef<\/a><\/li>\n
  5. Marini J.C, Blissett A.R. New genes in bone development: what’s new in osteogenesis imperfecta. The Journal of clinical endocrinology and metabolism.\u00a0<\/em>2013;98(8):3095-103.
    \n    CrossRef<\/a><\/li>\n
  6. Huang X, Huang S, Guo F, Xu F, Cheng P, Ye Y, et al. Dose-dependent inhibitory effects of zoledronic acid on osteoblast viability and function in vitro. Molecular medicine reports<\/em>. 2016;13(1):613-22.
    \n    CrossRef<\/a><\/li>\n
  7. Boyce B.F. Advances in the regulation of osteoclasts and osteoclast functions. Journal of dental research.\u00a0<\/em>2013;92(10):860-7.
    \n    CrossRef<\/a><\/span><\/li>\n
  8. Arnett T.R, Orriss I.R. Metabolic properties of the osteoclast. Bone.\u00a0<\/em>2017.<\/li>\n
  9. Murakami H, Takahashi N, Tanaka S, Nakamura I, Udagawa N, Nakajo S, et al. Tiludronate inhibits protein tyrosine phosphatase activity in osteoclasts. Bone<\/em>. 1997;20(5):399-404.
    \n    CossRef<\/a><\/li>\n
  10. Martin T.J, Udagawa N. Hormonal regulation of osteoclast function. Trends in endocrinology and metabolism: TEM<\/em>. 1998;9(1):6-12.
    \n    CrossRef<\/a><\/li>\n
  11. Martin T.J, Ng K.W. Mechanisms by which cells of the osteoblast lineage control osteoclast formation and activity. Journal of cellular biochemistry.\u00a0<\/em>1994;56(3):357-66.
    \n    CrossRef<\/a><\/li>\n
  12. Pizones J, Plotkin H, Parra-Garcia J.I, Alvarez P, Gutierrez P, Bueno A, et al. Bone healing in children with osteogenesis imperfecta treated with bisphosphonates. Journal of pediatric orthopedics. <\/em>2005;25(3):332-5.
    \n    CrossRef<\/a><\/li>\n
  13. Antoniazzi F, Zamboni G, Lauriola S, Donadi L, Adami S, Tato L. Early bisphosphonate treatment in infants with severe osteogenesis imperfecta. The <\/em>Journal of pediatrics.\u00a0<\/em>2006;149(2):174-9.
    \n    CrossRef<\/a><\/li>\n
  14. Biggin A, Munns C.F. Long-Term Bisphosphonate Therapy in Osteogenesis Imperfecta. Current osteoporosis reports.\u00a0<\/em>2017;15(5): 412-8.
    \n    CrossRef<\/a><\/li>\n
  15. Dwan K, Phillipi C.A, Steiner R.D, Basel D. Bisphosphonate therapy for osteogenesis imperfecta. The Cochrane database of systematic reviews.CD005088. 2016;10.
    \n    CrossRef<\/a><\/li>\n
  16. Viereck V, Emons G, Lauck V, Frosch KH, Blaschke S, Grundker C., et al. Bisphosphonates pamidronate and zoledronic acid stimulate osteoprotegerin production by primary human osteoblasts. Biochemical and biophysical research communications.<\/em> 2002;291(3):680-6.
    \n    CrossRef<\/a><\/li>\n
  17. Misof B.M, Roschger P, Baldini T, Raggio C.L, Zraick V, Root L., et al. Differential effects of alendronate treatment on bone from growing osteogenesis imperfecta and wild-type mouse. Bone<\/em>. 2005;36(1):150-8.
    \n    CrossRef<\/a><\/li>\n
  18. Van Beek E.R, Lowik C.W, Papapoulos S.E. Bisphosphonates suppress bone resorption by a direct effect on early osteoclast precursors without affecting the osteoclastogenic capacity of osteogenic cells: the role of protein geranylgeranylation in the action of nitrogen-containing bisphosphonates on osteoclast precursors. Bone.\u00a0<\/em>2002;30(1):64-70.
    \n    CrossRef<\/a><\/li>\n
  19. Bhadada S.K, Santosh R, Bhansali A, Upreti V, Dutta P. Osteogenesis imperfecta. The Jianournal of the Association of Physics of India.\u00a0<\/em>2009;57:33-6.<\/li>\n
  20. Jabbour Z, Al-Khateeb A, Eimar H, Retrouvey J.M, Rizkallah J, Glorieux F.H., et al. Genotype and malocclusion in patients with osteogenesis imperfecta. Orthodontics & craniofacial research.\u00a0<\/em>2018.<\/li>\n
  21. Sato A, Ouellet J, Muneta T, Glorieux F,H, Rauch F. Scoliosis in osteogenesis imperfecta caused by COL1A1\/COL1A2 mutations – genotype-phenotype correlations and effect of bisphosphonate treatment. Bone.\u00a0<\/em>2016;86:53-7.
    \n    CrossRef<\/a><\/li>\n
  22. Rauch F, Travers R, Plotkin H, Glorieux F.H. The effects of intravenous pamidronate on the bone tissue of children and adolescents with osteogenesis imperfecta. The Journal of clinical investigation.\u00a0<\/em>2002;110(9):1293-9.
    \n    CrossRef<\/a><\/li>\n
  23. Adams A.L, Adams J.L, Raebel M.A, Tang B.T, Kuntz J.L, Vijayadeva V., et al. Bisphosphonate Drug Holiday and Fracture Risk: A Population-Based Cohort Study. Journal of bone and mineral research<\/em>: the official journal of the American Society for Bone and Mineral Research. 2018.<\/li>\n
  24. Hughes D.E, Wright K.R, Uy H.L, Sasaki A, Yoneda T, Roodman G.D, et al. Bisphosphonates promote apoptosis in murine osteoclasts in vitro and in vivo. h\u00a0Journal of bone and mineral researc:.<\/em>\u00a0the official journal of the American Society for Bone and Mineral Research. 1995;10(10):1478-87.<\/li>\n
  25. Rogers M.J, Crockett J.C, Coxon .FP, Monkkonen J. Biochemical and molecular mechanisms of action of bisphosphonates<\/em>. Bone<\/em>. 2011;49(1):34-41.
    \n    CrossRef<\/a><\/li>\n
  26. Whyte M.P, Wenkert D, Clements K.L, McAlister W.H, Mumm S. Bisphosphonate-induced osteopetrosis. The New England. journal of medicine.\u00a0<\/em>2003;349(5):457-63.
    \n    CrossRef<\/a><\/li>\n
  27. Glorieux F.H, Bishop N.J, Plotkin H, Chabot G, Lanoue G, Travers R. Cyclic administration of pamidronate in children with severe osteogenesis imperfecta. The New England journal of medicine.\u00a0<\/em>1998;339(14):947-52.
    \n    CrossRef<\/a><\/li>\n<\/ol>\n","protected":false},"excerpt":{"rendered":"

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