Introduction: Osteoporosis and related bone fractures are a major global health issue. Several previous studies have reported that lower bone mineral density (BMD) or vertebral osteoporotic fractures are related to increased mortality [1-3]. Therefore, treatment of osteoporosis has been considered important in aged patients. Teriparatide (TPTD),which is one of the medicines used to treat osteoporosis, has shown significant effects on BMD by stimulating bone formation and reducing incidences of fragility fractures of the vertebral body[4-6]. However, treatment with TPTD cannotcompletely prevent osteoporotic fragility fractures in elderly osteoporotic patients.

Vitamin C (Ascorbic acid; Aa) deficiency may cause osteoporotic fragility fractures during TPTD treatment. Although Aa is essential for the differentiation of osteoblasts[7,8] it has been reported that the serum level of Aa decreases with age [9-12]. Thus, this age-related decrease in the serum level of Aa may have negative effects on TPTD treatment of osteoporosis. However, the effects of TPTD on BMD and bone strength underan Aa deficient condition are completely unknown.

In this study, to evaluate the short-term effects of TPTD on BMD and bone strength under an Aa-deficient condition, we used an animal model of Aa deficiency,osteogenic disorder Shionogi (ODS) rats. The exact purpose of the present study was to evaluate the effects of TPTD on the BMD of the femur and vertebrae,as well ason the bone strength at the mid-diaphysis and the distal metaphysis of the femurin theseAa-deficient ODS rats.

Materials and Methods

Animals

ODS rats (Clea Japan, Tokyo, Japan) are genetically deficient for the synthesis of Aa because theylack L-gulono-γ-lactone oxidase, and as a result develop body weight loss, scurvy, and osteoporosis [13,14]. When ODS rats are fed an Aa-free diet, their polypeptide hydroxyproline levels, which are known to be related to collagen synthesis[15] decrease below those in normal rats after one week and are about one-third of the normal level after two weeks[13,14]. When Aa is added to their drinking water, the scorbutic symptoms of these rats resolve in a few days. The rats were housed in a controlled environment at 22°C with a 12-hlight/dark cycle. They were pair-fed and allowed ad libitum access to water and standard ascorbic acid-free food (CE-2; Clea Japan) containing 1.14% calcium, 1.06% phosphorus, and 250 IU vitamin D3 per 100 g of food, as described previously[16,17]. Rats received 2 mg/mL Aa (Iwaki Pharma, Tokyo, Japan) in their drinking water for 16 weeks from birth. At 4 monthsof age, they received 0.5 mg/mL Aa in their drinking water[18] for 8 weeks to create an Aa-deficient condition.

Experimental design

After an 8-weekexperimental period of feeding with Aa-deficient (0.5 mg/mL) drinking water, the 6-month-old ODS rats were divided into the following 4 groups (n= 10 per group): (1) Aa-deficient group (Aa^- group), which was given 0.5 mg/mL Aa-deficient water and administered vehicle of TPTD;(2) Aa-supplemented group (Aa⁺ group), which was given 2 mg/mL Aa-supplemented water and administered vehicle of TPTD;(3) Aa-deficient and TPTD-administered group (Aa^- + T group), which was given Aa-deficient water and administered TPTD; and (4) Aa-supplemented and TPTD-administered group (Aa⁺ + T group), which was given Aa-supplemented water and administered TPTD. TPTD (Asahi Kasei Pharma Corporation, Shizuoka, Japan) was dissolved in saline containing 0.1% rat serum albumin and was administered subcutaneously once a week for 8 weeks (30 µg/kg bodyweight). After 8 weeks, these animals were euthanized under anesthesia with an intra-abdominal injection of ketamine (Sankyo, Tokyo, Japan) and xylazine (Zenoaq, Fukushima, Japan), and the bilateral femurs and lumbar vertebrae were harvested. All animal experimental protocols were approved by the Animal Committee of our institute, and all animal experiments conformed to the “Guidelines for Animal Experimentation” of our institute.

Preparation of bone

The right femur and the lumbar vertebrae were fixed in 10% neutral-buffered formalin until preparation for BMD measurement. The left femur was dissected of soft tissue, wrapped in gauze moistened with saline, and frozen to -80ºC until biomechanical testing.

Bone mineral density

BMD of the right total femurand of the lumbar vertebrae (L4-5) was measured using dual energy X-ray absorptiometry (QDR-4500, Hologic Inc.; Waltham, MA, USA). The BMD of the total femur was measured and the region of interest for BMD measurement at (L4-5) was determined manually.

Biomechanical testing

Mechanical testing of the left femur was performed at room temperature using a materials testing machine (MZ500S; Maruto, Tokyo, Japan). For stabilization of the femur, the mid-diaphysis of the femur was placed on two supports of the test apparatus that were 20 mm apart. The load of a three-point bending test was applied in the anteroposterior direction midway between the two supports. Load-displacement curves were recorded at a crosshead speed of 5 mm/min (Figure 1). The breaking force (N), breaking energy (N.mm), maximum load (N), and stiffness (N/mm) were calculated using software for the measurement of cortical bone strength (CTR win. Ver. 1.05; System Supply, Nagano, Japan). After the three-point bending test, the distal part of the femur was evaluated using a ompression test. Load-displacement curves were recorded and the breaking force (N), breaking energy (N.mm), maximum load (N), and stiffness (N/mm) were calculated as a cancellous bone strength using the same software.

Statistical analyses

Results are expressed as means ± standard deviation. Inter-group differences were assessed by one-way analysis of variance (ANOVA). Following ANOVA, Scheffe’s multiple comparison method was used as a post-hoc test. Differences with a P value < 0.05 were considered statistically significant. All statistical analyses were performed using the Statistical Package for the Biosciences software (SPBS v9.54; Murata& Yano 2002)[19].

Results

The BMDs of the total femur and oflumbar vertebrae were significantly increased by treatment with Aa and/or TPTD (P value by ANOVA, P < 0.0001 and P < 0.002, respectively). The BMDs of both the femur and oflumbar vertebrae in the Aa⁺, Aa^- + T, and Aa⁺ + T groups were significantly higher than those of the Aa^- group (P = 0.001 ~ P = 0.01).TPTD treatment eitherwith or without Aa significantly increased (P = 0.004 ~ P < 0.0001) the BMDs of the total femur and of lumbar vertebrae compared with those of the Aa treatment (Aa⁺) group.Combined treatment with Aa and TPTD significantly increased (P = 0.01) the total femoral BMD compared with TPTD monotherapy (Aa^- + T group).On the other hand, there was no significant difference in the BMD of lumbar vertebrae between the Aa^- + T and the Aa⁺ + T group

In the compression test of the distal femoral metaphysis, breaking force (P = 0.003) and maximum load (P = 0.003) at the cancellous bone were significantly increased with Aa and/or TPTDtreatmentas assessed by ANOVA analysis. Thecombined treatment with TPTD and Aa significantly increased the breaking force and the maximum load compared with the Aa^- group (P = 0.01 and P = 0.007, respectively) and the Aa⁺ group (P = 0.016 and P = 0.036, respectively). Moreover, the combined treatment had an additive effecton breaking force compared tosingle administration of TPTD (Aa^- + T group) (P = 0.04). There was no significant difference in breaking energy or stiffnessbetween any ofthe groups.

Discussion

In the present study, we investigated the effects of Aa and TPTD on BMD and bone strength at cortical and cancellous lesionsof the femur in an Aa-deficient rat model, ODS rats. Under an Aa-deficient condition, Aa or TPTD treatment recovered BMD, but notbone strength. Combined treatment with TPTD and Aa for 8 weeks improved femoral and lumbar vertebralBMD and also bone strength at the mid-diaphysis and distal metaphysis of the femur.

It is well known that Aa is a necessary factor for the differentiation of osteoblasts, and contributes to bone formation and increases in bone volume[20]. Aa supplementation in ODS rats significantly increased the BMD of the femur and oflumbar vertebrae in the present study. Another animal study showed that Aa supplementation increased the BMD of ovariectomized rats [21]. In addition, several clinical studies have demonstrated that vitamin C intake is positively associated with bone mass [22-24]. Based on these results, Aa isconsidered animportant determinant of BMD.

TPTD significantly increased the BMD of the total femur and oflumbar vertebraeof ODS rats in the present study. This is the first study to report that TPTD recovers BMD under an Aa-deficient condition in an animal model. Compared to non-treated cells, Aa-treated osteoblasts display increased expression of transforming growth factor (TGF)-β, estrogen receptor (ER)-α, and osteopontin (OPN), which are regulators of bone formation [25]. Furthermore, Aa prevents theloss of osteoblast differentiation markers such as osteorix, osteocalcin, Runx2, and bone morphometric protein (BMP)-2 and stimulates bone formation [26]. On the other hand, TPTD has been considered to exert its anabolic effects on bone formation via downregulation of sclerostin expression in osteocytes and stimulation of the Wnt signaling pathway [27]. The mechanisms of the anabolic effects of Aa and TPTD on BMD might therefore be different, which would explain how TPTD could recover BMD under Aa-deficient conditions.

In the present study, combined treatment with Aa and TPTD showed an additive effect for increasingBMD at the cortical bone compared to the effect of TPTD single administration. Monotherapy of Aa or TPTD could not recover the cortical and cancellous bonestrength in Aa-deficient rats. However, combined therapy with Aa and TPTD improved the cortical and cancellous bone strength parameters of breaking force and maximum load. These results indicated that Aa deficiency may be one of the causes of incidental insufficiency fractures during treatment of aged osteoporotic patientswith TPTD.

Furthermore, the effect of combined TPTD and Aa supplementation on cancellous bone strength in the compression test at the distal femoral metaphysis was more obvious than that on cortical bone strength in the three-point bending test. Previous studiesin humans indicated that TPTD causes cortical porosity for up to Twety four months after its administration [28,29], and this change may have influenced the observed difference between cortical and cancellous bone. Saito et al. investigated the relationship between the parameter of bone strength and bone structural and material properties after TPTD administration. Their results suggested that the total amount of enzymatic cross-links of collagen, which hasan importantinfluence on bone quality, was a determinant that was significantly associated with increased maximum load. The effects of TPTD or Aa on bone quality under Aa-deficient conditions will be investigated in future studies.

Conclusion

Treatment with Aa or TPTD for Eight weeks increased the BMD of the total femur and oflumbar vertebrae in Six-month-old ODS rats. Combined treatment with Aa and TPTD increased the BMD of the femur and oflumbar vertebrae as well asbone strength at the mid diaphysis and distal metaphysis of the femur in ODS rats.

Acknowledgements

The authors would like to thank Ms. Matsuzawa for her support in performing the experiments.

Conflict of Interest

All authors have no conflict of interest to declare.

Table 1: Comparisons of the BMD of the rat groups using ANOVA with Scheffe’s multiple comparison method

1. Ensrud KE, Thompson DE, Cauley JA, Nevitt MC, Kado DM, et al. (2000) Prevalent vertebral deformities predict mortality and hospitalization in older women with low bone mass. Fracture Intervention Trial Research Group. J Am GeriatrSoc 48: 241-249.
2. Nguyen ND, Center JR, Eisman JA, Nguyen TV(2007) Bone loss, weight loss, and weight fluctuation predict mortality risk in elderly men and women. J Bone Miner Res 22: 1147-1154.
3. Suzuki T, Yoshida H (2010) Low bone mineral density at femoral neck is a predictor of increased mortality in elderly Japanese women. Osteoporosis Int21: 71-79.
4. Neer RM, Arnaud CD, Zanchetta JR, Prince R, Gaich GAet al. (2001) Effect of parathyroid hormone (1-34) on fractures and bone mineral density in postmenopausal women with osteoporosis. N Engl J Med344: 1434-1441.
5. Miyauchi A, Matsumoto T, Sugimoto T, Tsujimoto M, Warner MR (2010) Effects of teriparatide on bone mineral density and bone turnover markers in Japanese subjects with osteoporosis at high risk of fracture in a 24-month clinical study:  12-month, randomized, placebo-controlled, double-blind and 12-month open-label phases. Bone47: 493-502.
6. Nakamura T, Sugimoto T, Nakano T, Kishimoto H, Ito Met al.(2012) Randomized Teriparatide [human parathyroid hormone (PTH) 1-34] Once-Weekly Efficacy Research (TOWER) trial for examining the reduction in new vertebral fractures in subjects with primary osteoporosis and high fracture risk. J ClinEndocrinolMetab97: 3097-3106.
7. Sudo H, Kodama HA, Amagai Y, Yamamoto S, Kasai S (1983) In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria. J Cell Biol 96: 191-198.
8. Sugimoto T, Nakada M, Fukase M, Imai Y, Kinoshita Y et al. (1986) Effects of ascorbic acid on alkaline phosphatase activity and hormone responsiveness in the osteoblastic osteosarcoma cell line UMR-106. Calcif Tissue Int 39: 171-174.
9. Murata A, Kang KJ, Miyata S, Fujii J, Tamai H et al. (1995) Impaired vitamin C status of hospitalized elderly patients and its improvement by daily multivitamin supplementation. Vitamin69: 85-92.
10. Richardson T, Ball L, Rosenfeld T (2002) Will an orange a day keep the doctor away? Postgrad Med J78: 292-294.
11. Oeffinger KC (1993) Scurvy:  more than historical relevance. Am Fam Physician48: 609-613.
12. Falch J, Mowé M, Bøhmer T (1998) Low levels of serum ascorbic acid in elderly patients with hip fracture. Scand J Clin Lab Invest58: 225-228.
13. Horio F, Ozaki K, Yoshida A, Makino S, Hayashi Y (1985) Requirement for ascorbic acid in a rat mutant unable to synthesize ascorbic acid.JNutr115: 1630-1640.
14. Mori S, Murai T, Takeuchi Y, Toyama M, Makino S et al. (1990) Dose response of N-butyl-N-(4-hydroxybutyl)nitrosamine on urinary bladder carcinogenesis in mutant ODS rats lacking L-ascorbic acid synthesizing ability. Cancer Lett49: 139-145.
15. Krane SM, Munoz AJ, Harris ED Jr (1970) Urinary polypeptides related to collagen synthesis. J Clin Invest49: 716-729.
16. Suzuki K, Miyakoshi N, Kasukawa Y, Sato K, Itoi E (2003) Effect of combined treatment of insulin and human parathyroid hormone (1-34) on cancellous bone mass and structure in streptozotocin-induced diabetic rats. Bone 33: 108-114.
17. Tamura Y, Miyakoshi N, Itoi E, Abe T, Kudo T et al. (2001) Long-term effects of withdrawal of bisphosphonate incadronate disodium (YM175) on bone mineral density, mass, structure, and turnover in the lumber vertebrae of ovariectomized rats. J Bone Miner Res16: 541-549.
18. Alcantara-Martos T, Delgado-Martinez AD, Vega MV, Carrascal MT, Munuera-Martinez L (2007) Effect of vitamin C on fracture healing in elderly Osteogenic Disorder Shionogi rats. J Bone Joint Surg Br89: 402-407.
19. Murata K, Yano E (2002) Medical statistics for evidence-based medicine with SPBS user’s guide. Nankodo, Tokyo.
20. Morton DJ, Barrett-Connor EL, Schneider DL (2001) Vitamin C supplement use and bone mineral density in postmenopausal women. J Bone Miner Res16:  135-140.
21. Arsian A, Orkun S, Aydin G, Keles I, Tosun A et al. (2011) Effects of ovariectomy and ascorbic acid supplement on oxidative stress parameters and bone mineral density in rats. Libyan J Med 6.
22. Kim DE, Cho SH, Park HM, Chang YK(2016) Relationship between bone mineral density and dietary intake of β-carotene, vitamin C, zinc and vegetables in postmenopausal Korean women:  a cross-sectional study. J Int Med Res Sep 23.
23. Sugiura M, Nakamura M, Ogawa K, Ikoma Y, Yano M(2016) High Vitamin C Intake with High Serum β-Cryptoxanthin Associated with Lower Risk for Osteoporosis in Post-Menopausal Japanese Female Subjects: Mikkabi Cohort Study. J Nutr Sci Vitaminol62: 185-191.
24. KimYA, Kim KM, Lim S, Choi SH, Moon JH et al. (2015) Favorable effect of dietary vitamin C on bone mineral density in postmenopausal women (KNHANES IV, 2009):  discrepancies regarding skeletal sites, age, and vitamin D status. OsteoporosInt26: 2329-2337.
25. Park JK, Lee EM, Kim AY, Lee EJ, Min CW et al. (2012) Vitamin C deficiency accelerates bone loss inducing an increase in PPAR-γ expression in SMP30 knockout mice. Int J ExpPathol93: 332-340.
26. Zhu LL, Cao J, Sun M, Yuen T, Zhou R et al. (2012) Vitamin C prevents hypogonadal bone loss. PloS One7: e47058.
27. Silva BC, Bilezilian JP (2015) Parathyroid hormone:  anabolic and catabolic actions on the skeleton. CurrOpinPharmacol22: 41-50.
28. Jiang Y, Zhao JJ, Mitlak BH, Wang O, Genant HK et al. (2003) Recombinant human parathyroid hormone (1-34) [teriparatide] improves both cortical and cancellous bone structure. J Bone Miner Res18: 1932-1941.
29. Hodsman AB, Kisiel M, Adachi JD, Fraher LJ, Watson PH (2000) Histomorphometric evidence for increased bone turnover without change in cortical thickness or porosity after 2 years of cyclical hPTH(1-34) therapy in women with severe osteoporosis. Bone27: 311-318.