Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2013, Article ID 161976, 11 pages http://dx.doi.org/10.1155/2013/161976 Research Article Potential Osteoporosis Recovery by Deep Sea Water through Bone Regeneration in SAMP8 Mice Hen-Yu Liu,1,2 Ming-Che Liu,3,4 Ming-Fu Wang,5 Wei-Hong Chen,1,2 Ching-Yu Tsai,1,2 Kuan-Hsien Wu,1,2 Che-Tong Lin,6 Ying-Hua Shieh,7 Rong Zeng,8 and Win-Ping Deng1,2,9,10 1 Stem Cell Research Center, Taipei Medical University, Taipei, Taiwan 2 Graduate Institute of Biomedical Materials and Tissue Engineering, Taipei Medical University, Taipei, Taiwan 3 School of Dentistry, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan 4 Department of Urology, Taipei Medical University Hospital, Taipei, Taiwan 5 DepartmentofFoodandNutrition,ProvidenceUniversity,Taichung,Taiwan 6 Graduate Institute of Oral Rehabilitation Sciences, Taipei Medical University, Taipei, Taiwan 7 Department of Family Medicine, Taipei Medical University, Wan Fang Hospital, Taipei, Taiwan 8 Department of Orthopedic Surgery, The Affiliated Hospital, Guangdong Medical College, Zhanjiang 524001, China 9 Translational Research Laboratory, Cancer Center, Taipei Medical University, Taipei, Taiwan 10Cancer Center, Taipei Medical University Hospital, Taipei, Taiwan Correspondence should be addressed to Rong Zeng; [email protected] and Win-Ping Deng; [email protected] Received 3 January 2013; Revised 22 June 2013; Accepted 27 June 2013 Academic Editor: Wei-Chiang Lin Copyright © 2013 Hen-Yu Liu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The aim of this study is to examine the therapeutic potential of deep sea water (DSW) on osteoporosis. Previously, wehave established the ovariectomized senescence-accelerated mice (OVX-SAMP8) and demonstrated strong recovery of osteoporosis by stem cell and platelet-rich plasma (PRP). Deep sea water at hardness (HD) 1000 showed significant increase in proliferation of osteoblastic cell (MC3T3) by MTT assay. For in vivo animal study, bone mineral density (BMD) was strongly enhanced followed by the significantly increased trabecular numbers through micro-CT examination after a 4-month deep sea water treatment, and biochemistry analysis showed that serum alkaline phosphatase (ALP) activity was decreased. For stage-specific osteogenesis, bone marrow-derived stromal cells (BMSCs) were harvested and examined. Deep sea water-treated BMSCs showed stronger osteogenic differentiation such as BMP2, RUNX2, OPN, and OCN, and enhanced colony forming abilities, compared to the control group. Interestingly, most untreated OVX-SAMP8 mice died around 10 months; however, approximately 57% of DSW-treated groups lived up to 16.6 months, a life expectancy similar to the previously reported life expectancy for SAMR1 24 months. The results demonstrated the regenerative potentials of deep sea water on osteogenesis, showing that deep sea water could potentially be applied in osteoporosis therapy as a complementary and alternative medicine (CAM). 1. Introduction and osteoblasts also leads to increased bone resorption [6, 7]. In addition, senile osteoporosis with increased age and the Osteoporosis, a common disease caused by imbalanced bone reduction of growth hormone (GH) which stimulates renal- remodeling, is a global public health problem. Patients with synthesized 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) osteoporosis are often accompanied with pain, disability, and is also another cause of osteoporosis [8, 9]. Age-related bone decline in the quality of life, rise in mortality, and depletion of loss causes bone trabecular thinning as well as the loss of localhealthcarebudget[1–4]. The major type of osteoporosis cortical layer of bone tissue, and the cortex becomes porous occurred in postmenopausal women with an age range of which will increase the femoral neck fracture rate [10]. For 50–70 [5]. Bone density index rapidly decreased after the osteoporosis, supplemental dietary calcium and vitamin D menopause, due to sex hormone imbalance and the lack of may reduce the risk of fractures in postmenopausal women estrogen. The imbalance of homeostasis between osteoclasts [11]. Drugs, lifestyle changes, home safety, and hip protection 2 Evidence-Based Complementary and Alternative Medicine are viable approaches for the prevention of osteoporosis. 2.2. Cell Line and Cell Viability. Mouse preosteoblast cell line According to the World Health Organization Criteria (WHO) (MC3T3-E1, ATCC CRL-2593) was provided by Dr. Alexan- statistics, the penetration rate of osteoporosis in women over der T.H Wu (Taipei medical University, Taipei, Taiwan) and 4 the age of 65 is up to 35%; hence, the advanced treatment for wasculturedin96wellplates(1× 10 cells/well) in alpha min- osteoporosis has become emergent. imum essential medium (-MEM) supplemented with 10% Deep sea water (DSW) generally refers to sea water from a fetal bovine serum (FBS) or/and different concentration of depth of more than 200 meters (m). It could be characterized DSW (ranged from 500 to 2000 HD). MTT reagent was by its purity, abundant nutrients, and minerals. Currently, added into each well on day 3 of cell growth in culture, and DSW has been applied in the ground of food, agriculture, cos- cell viability was detected by Multiskan PC (Thermo Lab). 4 metic, and medical field due to its high contents of unique For the cell counting assay, MC3T3 (1 × 10 ) were seeded minerals including sodium (Na), magnesium (Mg), calcium in 6 cm dish containing DSW-derived medium (hardness of ∘ (Ca), potassium (K), zinc (Zn), and vanadium (V) [12]. 1000 mg/L)and incubated at 37 Cin5%CO2 atmosphere. DSW has been reported to stimulate both osteoblastogenesis After 72 hrs, MC3T3 were collected, and the cell numbers and osteoclastogenesis in bone turnover [13]. NaCl from were counted by trypsin and ethylenediaminetetraacetic acid DSW also improves the biochemical properties of bone. treatment. DSW combined with soluble silicon as natural material could promote cell proliferation of osteoblast and enhance the osteogenesis-related gene expression in animal studies [13]. 2.3. Experimental Animals. All the animal experiment pro- The DSW utilized in this study is drawn from the Pacific tocol was approved by the Institutional Animal Care and Ocean at a depth of 662 m and a distance of 5 kilometers Use Committee of Taipei medical University. SAMP8 mice (km) off the coast of Hualien County, Taiwan [14, 15], which were ovariectomized (OVX) at 4 months of age to induce contains abundant amounts of trace elements, including high osteoporosis and then used in this experiments. Mice were concentrations of four essential minerals: Mg (96200 mg/L), grouped into the following (six mice per group): control K (10800 mg/L), Na (9010 mg/L), and Ca (39 mg/L). group (CTRL, receiving PBS) and DSW group (receiving Our previous study has demonstrated that transplanta- DSW). Both mice were suggested for daily water uptake tion of platelet-rich plasma- (PRP-) treated NIH3T3-G cells which is based on a 22 g mouse consuming 5.2 mL water per into OVX-SAMP8 mice significantly reversed osteoporosis. day. We also showed that PRP could not only increase bone regenerationbutalsoreducebonemarrowadiposityinthe 2.4.SerumALP,Mg,andCaAnalyses. All mice were sacri- osteoporotic mice [16–18]. In this study, we investigated the ficed and the extracted blood specimens were obtained at possibility of the treatment of osteoporosis by DSW. For 4 months. The plasma concentrations for ALP, Mg, and Ca osteoporotic model, SAMP8 mice received bilateral salpingo- were then determined by a photometric method according oophorectomy at 4 months old and subsequently fed with to the manufacturer’s instructions (Fuji Dri-Chem Clinical DSW for 15 days. The effects of DSW on bone regeneration Chemistry Analyzer FDC 3500). were then analyzed by bone mineral density, micro-CT, bone structure with HE stain, and the activities of isolated bone marrow stromal cells. Here, we demonstrated that 2.5. Bone Mineral Density and Micro-CT Analysis. Dual- DSW not only induced bone regeneration but also strongly energy X-ray absorptiometry (DEXA) analysis was used for recovered bone loss in OVX-SAMP8 mice. In addition, we the measurement of bone mass in the spine, left/right knee, have observed that DSW would be effective in prevention of and left/right femurs. BMD (XR-36; Norland Corp.; host osteoporosis and might be a complementary and alternative software revision 2.5.3, scanner software revision 2.0.0) was medicine (CAM). performed 4 months after DSW treatment. All mice were sac- rificed and their femurs and tibia bones were collected at 4 months for detecting the trabecular bone and bone volume by 2. Materials and Methods micro-CT (Skyscan-1076, Skyscan, Belgium). For trabecular bone analysis and 3D imaging, construction was operated ∘ 2.1. Characterization of Deep Sea Water (DSW). DSW was at 50 KV, 200 uA, 0.4 of rotation step, 0.5 mm AI filter, and obtained from the Pacific Ocean at a depth of 662 m 9 m/pixel of scan resolution. Each group contains 6 animals. [14, 15]. The obtained DSW was subjected to filtration to remove microorganism and virus and then concentrated. 2.6. Histological Analysis. For histological determination, The elements contained in concentrated DSW were mea- bone sample was fixed in 10% formalin and decalcified in 14% sured by inductively coupled plasma mass spectroscopy. EDTA for 3 days. Bone sections (10 m) were subjected to As shown in Supplemental Table 1, the concentrated DSW paraffin-embeddingandthenstainedwithhematoxylinand contained high amounts of several essential minerals such eosin (H&E) staining to detect the trabecular bone in the as magnesium (Mg), potassium (K), sodium (Na), and cal- bone tissue. cium (Ca) (see Supplementary Material availbale online at http://dx.doi.org/10.1155/2013/161976). The final hardness of concentrated DSW was determined as 400,000 mg/L by using 2.7.