American Journal of Clinical and Experimental Medicine 2018; 6(1): 1-9 http://www.sciencepublishinggroup.com/j/ajcem doi: 10.11648/j.ajcem.20180601.11 ISSN: 2330-8125 (Print); ISSN: 2330-8133 (Online) Impact of Biofield Energy Healing Treated Vitamin D on 3 Human Osteoblast Cell Line (MG-63) for Bone Health William Dean Plikerd1, Mahendra Kumar Trivedi1, Alice Branton1, Dahryn Trivedi1, Gopal Nayak1, Mayank Gangwar2, Snehasis Jana2, * 1Trivedi Global, Inc., Henderson, USA 2Trivedi Science Research Laboratory Pvt. Ltd., Bhopal, India Email address: *Corresponding author To cite this article: William Dean Plikerd, Mahendra Kumar Trivedi, Alice Branton, Dahryn Trivedi, Gopal Nayak, Mayank Gangwar, Snehasis Jana. Impact of Biofield Energy Healing Treated Vitamin D on Human Osteoblast Cell Line (MG-63) for Bone Health. American Journal of Clinical and 3 Experimental Medicine. Vol. 6, No. 1, 2018, pp. 1-9. doi: 10.11648/j.ajcem.20180601.11 Received: November 28, 2017; Accepted: December 8, 2017; Published: January 10, 2018 Abstract: Bone disorders are largely associated with increased morbidity, mortality, and substantial economic and health costs. Vitamin D play an important role for calcium absorption and bone mineralization, which can improve the patients' quality of life. The current study aimed to evaluate the potential of The Trivedi Effect®- Biofield Energy Healing on vitamin D3 and DMEM as test item (TI) on bone cell differentiation using human osteoblast cell line (MG-63, Osteosarcoma). Bone health biomarkers such as alkaline phosphatase enzyme (ALP) activity, collagen levels and bone mineralization were evaluated. The test items were treated with The Trivedi Effect® by William Dean Plikerd and divided as Biofield Energy Treated (BT) and untreated (UT) test items. Cell viability using MTT data showed that the test items were found to be safe. ALP level was significantly increased by 346.4% (at 50 µg/mL), 375.3% (at 100 µg/mL), and 343.2% (at 100 µg/mL) in the UT-DMEM+BT-TI, BT-DMEM+UT-TI, and BT-DMEM+BT-TI groups, respectively as compared to the untreated group. Collagen content was significantly increased by 336.2% and 237.2% in the UT-DMEM+BT-TI at 10 and 50 µg/mL, respectively while 399.3% (10 µg/mL) and 200% (0.1 µg/mL) increased collagen in the BT-DMEM+UT-TI and BT- DMEM+BT-TI groups, respectively. Moreover, the percent of bone mineralization was significantly increased in UT- DMEM+BT-TI, BT-DMEM+UT-TI, and BT-DMEM+BT-TI groups by 416.9% (10 µg/mL), 460.7% (0.1 µg/mL), and 441.7% (10 µg/mL) respectively as compared with the untreated test item and DMEM group. Thus, Biofield Energy Treated vitamin D3 and DMEM would play an important role to control the osteoblast function, improves bone mineralization, and calcium absorption in many bone disorders. Moreover, the bone health parameters such as collagen, calcium and ALP were significantly improved and can be used as supplement to improve bone health. Based on the outstanding results, it is assumed that the Biofield Energy Treated vitamin D3 could be a powerful alternative dietary sources and supplements to fight against various bone related diseases including low bone density and osteoporosis, osteogenesis imperfecta, Paget’s disease of bone, rickets, osteomalacia, bone and/or joint pain, increased frequency of fractures, deformed bones, osteoma, chondrodystrophia fetalis, hormonal imbalance, stress, aging, bone loss and fractures. Keywords: The Trivedi Effect®, Osteosarcoma Cells (MG-63), Alizarin Red S Staining, ALP, Collagen, Bone Mineralization Moreover, it has significant anti-inflammatory, anti-arthritic, 1. Introduction anti-osteoporosis, anti-stress, anti-aging, anti-apoptotic, wound healing, anti-cancer, anti-psychotic, and anti-fibrotic Vitamin D has multiple effects which regulate the roles. Vitamin D receptors (VDRs) are widely present in functions in different organs such as brain, lungs, liver, most of the body organs like brain, heart, lungs, kidney, liver, kidneys, heart, immune, skeletal, and reproductive systems. 2 William Dean Plikerd et al.: Impact of Biofield Energy Healing Treated Vitamin D on Human Osteoblast Cell Line 3 (MG-63) for Bone Health pancreas, large and small intestines, muscles, reproductive, bone mineralization and hence is an important marker for nervous system, etc. [1]. VDRs influence cell-to-cell osteogenic activity [8]. Similarly, active osteoblasts communication, normal cell growth, cell differentiation, cell synthesize and extrude collagen, which plays an important cycling and proliferation, hormonal balance, role in the formation of bone extracellular matrix by neurotransmission, skin health, immune and cardiovascular providing strength and flexibility. Collagen fibrils formed an functions. Bone-related health issues become a major arrays of an organic matrix known as Osteoid [9]. Likewise, problem among the population from village to the cities. calcium phosphate is deposited in the Osteoid and gets Vitamin D plays a vital role in preserving a healthy mineralized (combination of calcium phosphate and mineralized skeleton of most of the vertebrates including hydroxyapatite) and provides rigidity to the bone [10]. Thus, humans. Cod liver oil, irradiation of other foods including these parameters are very essential in order to study the bone plants, sunlight, etc. are found to be effective against bone health in cell lines. Authors evaluated the in vitro effect of related disorders, which lead to discovering the active the Biofield Energy Treated vitamin D as a test item, a 3 principle- vitamin D [1]. The role of vitamin D has been well Complementary and Alternative Medicine (CAM) on bone defined not only for improving the bone mineralization but health using MG-63 cell line for major biomarkers. also with increased bone resorption, aging, inflammation and Within the burgeoning ground of CAM therapies, Biofield overall quality of life. Vitamin D is synthesized in the skin Energy Treatment or energy medicine, is emerging with 3 by sunlight and once formed it sequentially metabolized in significant benefits in various scientific fields. The effects of the liver and kidney to 1,25-dihydroxyvitamin D (calcitriol, the CAM therapies have great potential, which include the vitamin D hormone) [2]. Calcitriol play an important role external qigong, Johrei, Reiki, therapeutic touch, polarity in maintaining the normal level of calcium and phosphorus, therapy, pranic healing, deep breathing, yoga, Tai Chi, Qi promotes bone mineralization, induce or repress the genes Gong, chiropractic/osteopathic manipulation, meditation, responsible for conserving the mineral homeostasis and massage, special diets, homeopathy, progressive relaxation, skeletal integrity, and inhibit hypertension, kidney damage, guided imagery, acupressure, acupuncture, relaxation cardiovascular and immune disorders (such as Lupus, techniques, hypnotherapy, healing touch, movement therapy, Addison Disease, Graves’ Disease, Hashimoto Thyroiditis, pilates, Rolfing structural integration, mindfulness, Multiple Sclerosis, Myasthenia Gravis, Anemia, Sjogren Ayurvedic medicine, traditional Chinese herbs and medicines Syndrome, Systemic Lupus Erythematosus, Diabetes, in biological systems both in vitro and in vivo [11]. Biofield Alopecia Areata, Fibromyalgia, Vitiligo, Psoriasis, Energy Healing Treatment (The Trivedi Effect®) contain a Scleroderma, Chronic Fatigue Syndrome and Vasculitis), and putative bioenergy, which is channeled by a renowned the secondary hyperparathyroidism [3]. Vitamin D practitioners from a distance. Biofield Energy Healing as a insufficiency and deficiency is the major health problem, CAM showed a significant results in biological studies [12]. which causes metabolic bone disease in the young and However, the National Center for Complementary and elderly populations [4]. Fortified foods have a variable Alternative Medicine (NCCAM), well-defined Biofield amount of vitamin D and most of the foods do not contain therapies in the subcategory of Energy Therapies [13]. The vitamin D, which can be fulfilled using some supplements. In Trivedi Effect®- Consciousness Energy Healing Treatment order to avoid the bone related disorders such as has been reported with significant revolution in the osteomalacia, exacerbate osteoporosis, hyperparathyroidism, physicochemical properties of metals, chemicals, ceramics immune disorders, etc. calcium 1000-1500 mg/day along and polymers [14-17], improved agricultural crop yield, with vitamin D supplement around 400 IU/day is very productivity, and quality [18-20], transformed antimicrobial important for maintaining the good bone health [5]. characteristics at genetic level [21-23], biotechnology [24- Various in vitro studies have readily demonstrated the role 26], skin health [27, 28], nutraceuticals [29, 30], cancer of bone health using cell lines and its resorbing effects using research [31, 32], and human health and wellness. three important key biomarkers, such as alkaline phosphatase Based on the significant outcomes of Biofield Energy (ALP), collagen and calcium. MG-63 cell line derived from Treatment and vital role of vitamin D on bone health, authors 3 juxtacortical osteosarcoma, which represents an immature sought to evaluate the impact of the Biofield Energy Treatment osteoblast phenotype and undergoes temporal development in (The Trivedi Effect®) on vitamin D as test sample for bone 3 long term culture. The response of MG-63 cells to 1,25- health activity with respect to the assessment of different bone dihydroxyvitamin D (1,25 (OH) 2D ) administration has health parameters like ALP, collagen content, and bone 3 3 been studied to be similar to normal human osteoblast cells mineralization using standard in vitro assays in MG-63 cells. [6]. Hence, MG-63 cell line is widely used for studying the potential of any test compounds to improve the bone health 2. Material and Methods [7]. The formation of new bone involves a complex series of events including the proliferation and differentiation of 2.1. Chemicals and Reagents osteoblasts, and eventually the formation of a mineralized Rutin hydrate was purchased from TCI, Japan, while extracellular matrix. ALP is a phenotypic marker for the vitamin D (denoted as test item) and L-ascorbic acid were early differentiation and maturation of osteoblasts. ALP 3 obtained from Sigma-Aldrich, USA. Fetal bovine serum increases the local concentration of inorganic phosphate for American Journal of Clinical and Experimental Medicine 2018; 6(1): 1-9 3 (FBS) and Dulbecco's Modified Eagle's Medium (DMEM) untreated samples were kept in similar sealed conditions for were purchased from Life Technology, USA. Antibiotics experimental study. solution (penicillin-streptomycin) was procured from 2.5. Determination of Non-Cytotoxic Concentration HiMedia, India, while 3-(4, 5-diamethyl-2-thiazolyl)-2, 5- diphenyl-2H-tetrazolium) (MTT), Direct Red 80, and The cell viability was performed by MTT assay in human ethylene diamine tetra acetic acid (EDTA) were purchased bone osteosarcoma cell line (MG-63). The cells were counted from Sigma, USA. All the other chemicals used in this and plated in 96 well plates at the density corresponding to 5 experiment were analytical grade procured from India. X 103 to 10 X 103 cells/well/180 µL of cell growth medium. The above cells were incubated overnight under growth 2.2. Cell Culture conditions and allowed the cell recovery and exponential Human bone osteosarcoma cell line -MG-63 was used as growth, which were subjected to serum stripping or test system in the present study. The MG-63 cell line was starvation. The cells were treated with the test item, DMEM, maintained in DMEM growth medium for routine culture and positive control. The untreated cells were served as supplemented with 10% FBS. Growth conditions were baseline control. The cells in the above plate (s) were maintained as 37°C, 5% CO and 95% humidity and incubated for a time point ranging from 24 to 72 hours in 2 subcultured by trypsinisation followed by splitting the cell CO incubator at 37°C, 5% CO , and 95% humidity. 2 2 suspension into fresh flasks and supplementing with fresh Following incubation, the plates were taken out and 20 µL of cell growth medium. Three days before the start of the 5 mg/mL of MTT solution were added to all the wells experiment (i.e., day -3), the growth medium of near- followed by additional incubation for 3 hours at 37°C. The confluent cells was replaced with fresh phenol-free DMEM, supernatant was aspirated and 150 µL of DMSO was added supplemented with 10% charcoal dextran stripped FBS (CD- to each well to dissolve formazan crystals. The absorbance of FBS) and 1% penicillin-streptomycin [33]. each well was read at 540 nm using Synergy HT micro plate reader, BioTek, USA [34]. The percentage cytotoxicity at 2.3. Experimental Design each tested concentrations of the test substance were calculated using the following equation (1): The experimental groups consisted of cells in baseline control, vehicle control groups (0.05% DMSO with Biofield % Cytotoxicity = (1-X/R)*100 (1) Energy Treated and untreated DMEM), positive control group (rutin hydrate) and experimental test groups. The Where, X = Absorbance of treated cells; R = Absorbance experimental groups included the combination of the Biofield of untreated cells Energy Treated and untreated vitamin D /DMEM. It The percentage cell viability corresponding to each 3 consisted of four major treatment groups on specified cells treatment was obtained using the following equation (2): with Untreated-DMEM + Untreated-Test item (UT-TI), UT- % Cell Viability = 100 -% Cytotoxicity (2) DMEM + Biofield Energy Treated test item (BT-TI), BT- DMEM + UT-TI, and BT-DMEM + BT-TI. The concentrations exhibiting ≥70% Cell viability was considered as non-cytotoxic. 2.4. Consciousness Energy Healing Treatment Strategies 2.6. Assessment of Alkaline Phosphatase (ALP) Activity The test item and DMEM were divided into two parts. One part each of the test item and DMEM was treated with the The cells were counted using an hemocytometer and plated Biofield Energy by a renowned Biofield Energy Healer (also in a 24-well plate at the density corresponding 1 x 104 known as The Trivedi Effect®) and coded as the Biofield cells/well in phenol free DMEM supplemented with 10% Energy Treated item, while the second part did not receive CD-FBS. Following respective treatments, the cells in the any sort of treatment and was defined as the untreated above plate were incubated for 48 hours in CO incubator at 2 samples. This Biofield Energy Healing Treatment was 37°C, 5% CO , and 95% humidity. After 48 hours of 2 provided by William Dean Plikerd remotely for ~5 minutes. incubation, the plate was taken out and processed for the Biofield Energy Healer was remotely located in the USA, measurement of ALP enzyme activity. The cells were washed while the test samples were located in the research laboratory with 1X PBS and lysed by freeze thaw method i.e., of Dabur Research Foundation, New Delhi, India. This incubation at -80°C for 20 minutes followed by incubation at Biofield Energy Treatment was administered for 5 minutes 37°C for 10 minutes. To the lysed cells, 50 µL of substrate through the Healer’s unique Energy Transmission process solution i.e., 5 mM of p-nitrophenyl phosphate (pNPP) in 1M remotely to the test samples under laboratory conditions. The diethanolamine and 0.24 mM magnesium chloride (MgCl ) 2 Biofield Energy Healer, in this study never visited the solution (pH 10.4) was added to all the wells followed by laboratory in person, nor had any contact with the test item incubation for 1 hour at 37°C. The absorbance of the above and medium. Further, the control group was treated with a solution was read at 405 nm using Synergy HT micro plate sham healer for comparative purposes. The sham healer did reader (Biotek, USA). The absorbance values obtained were not have any knowledge about the Biofield Energy normalized with substrate blank (pNPP solution alone) Treatment. After that, the Biofield Energy Treated and absorbance values [33]. The percentage increase in ALP 4 William Dean Plikerd et al.: Impact of Biofield Energy Healing Treated Vitamin D on Human Osteoblast Cell Line 3 (MG-63) for Bone Health enzyme activity with respect to the untreated cells (baseline 2.8. Assessment of Bone Mineralization by Alizarin Red S group) was calculated using equation (3): Staining % Increase = [(X-R)/R)]*100 (3) The MG-63 cells were counted using an hemocytometer and plated in 24-well plate at the density corresponding to 10 Where, X = Absorbance of cells corresponding to positive x 103 cells/well in phenol free DMEM supplemented with control and test groups 10% CD-FBS. Following respective treatments, the cells in R = Absorbance of cells corresponding to baseline the above plate were incubated for 48 hours in CO incubator 2 group (untreated cells) at 37°C, 5% CO , and 95% humidity to allow cell recovery 2 and exponential growth. Following overnight incubation, the 2.7. Assessment of Collagen Synthesis above cells will be subjected to serum stripping for 24 hours. The MG-63 cells were counted using an hemocytometer and The cells will be then be treated with non-cytotoxic plated in 24-well plate at the density corresponding to 10 x 103 concentrations of the test samples and positive control. After cells/well in phenol free DMEM supplemented with 10% CD- 3-7 days of incubation with the test samples and positive FBS. Following respective treatments, the cells in the above control, the plates were taken out cell layers and processed plate were incubated for 48 hours in CO incubator at 37°C, further for staining with Alizarin Red S dye. The cells were 2 5% CO , and 95% humidity. After 48 hours of incubation, the fixed in 70% ethanol for 1 hour, after which Alizarin Red 2 plate was taken out and the amount of collagen accumulated in solution (40 µm; pH 4.2) was added to the samples for 20 MG-63 cells corresponding to each treatment was measured by minutes with shaking. The cells were washed with distilled Direct Sirius red dye binding assay. In brief, the cell layers water to remove unbound dye. For quantitative analysis by were washed with PBS and fixed in Bouin’s solution (5% absorbance evaluation, nodules were solubilized with 10% acetic acid, 9% formaldehyde and 0.9% picric acid) for 1 hours cetylpyridinium chloride for 15 minutes with shaking. at room temperature (RT). After 1 hour of incubation, the Absorbance was measured at 562 nm using Biotek Synergy above wells were washed with milliQ water and air dried. The HT micro plate reader [33]. The percentage increase in bone cells were then stained with Sirius red dye solution for 1 hour mineralization with respect to the untreated cells (baseline at RT followed by washing in 0.01 N HCl to remove unbound group) was calculated using the following equation (5): dye. The collagen dye complex obtained in the above step was % Increase = [(X-R)/R]*100 (5) dissolved in 0.1 N NaOH and absorbance was read at 540 nm using Biotek Synergy HT micro plate reader. The level of Where, X = Absorbance in cells corresponding to positive collagen was extrapolated using standard curve obtained from control or test groups; R = Absorbance in cells corresponding purified Calf Collagen Bornstein and Traub Type I (Sigma to baseline (untreated) group. Type III) [33]. The percentage increase in collagen level with respect to the untreated cells (baseline group) was calculated 2.9. Statistical Analysis using equation (4): All the values were represented as percentage of respective % Increase = [(X-R)/R]*100 (4) parameters. For multiple group comparison, one-way analysis of variance (ANOVA) was used followed by post- Where, X = Collagen levels in cells corresponding to hoc analysis by Dunnett’s test. Statistically significant values positive control and test groups were set at the level of p≤0.05. R = Collagen levels in cells corresponding to baseline group (untreated cells) 3. Results and Discussion 3.1. MTT Assay-Non-Cytotoxic Effect of the Test Item Figure 1. Effect of the test item on MG-63 cell lines for cell viability after 72 hours using the MTT assays. VC: Vehicle control (DMSO-0.05%), UT: Untreated; BT: Biofield Treated; TI: Test Item. American Journal of Clinical and Experimental Medicine 2018; 6(1): 1-9 5 The cell viability of the test samples were studied in MG- cell line (Figure 2). The positive control, rutin showed a 63 cells. The obtained results were compared with respect to significant increased value by 38.78%, 43.61%, and 80.92% rutin at defined concentrations for the estimation of at 0.01, 0.1, and 1 µg/mL, respectively with respect to the percentage cell viability. The cell viability results are untreated cells. The experimental test group’s viz. untreated graphically presented in Figure 1. The results of percentage medium and Biofield Treated Test item (UT-DMEM+BT- cell viability in all the tested cell lines showed the cell TI) showed a significant increase in ALP level by 38.8%, viability range of 70% to 114% in different test item groups 346.4%, and 44.7% at 10, 50, and 100 µg/mL, respectively with DMEM, while for rutin group showed more than 85% while Biofield Treated medium and untreated Test item cell viability (Figure 1). These data suggests that the test item (BT-DMEM+UT-TI) showed a significant increased ALP along with DMEM groups were found safe at all the tested level by 50.8% and 375.3% at 50 and 100 µg/mL, concentrations range up to maximum of 100 µg/mL against respectively as compared with the untreated test item and the tested MG-63 cells. DMEM group. However, the Biofield Energy Treated medium and Biofield Energy Treated Test item (BT- 3.2. Assessment of Test Items Effects on Alkaline DMEM+BT-TI) showed a significant increased ALP level Phosphatase (ALP) Enzyme Activity by 343.2% at 100 µg/mL as compared with the untreated test item and DMEM group. Overall, all the experimental The effect of the Biofield Energy Treated test item and test groups showed a significant improved level of ALP at DMEM on the ALP level showed a significantly increased the tested concentrations. at various experimental test item concentrations on MG-63 Figure 2. Effect of the test items on MG-63 cell line for the level of Alkaline Phosphatase (ALP) enzyme activity. VC: Vehicle control (DMSO-0.05%), UT: Untreated; BT: Biofield Treated; TI: Test Item. Bone ALP enzyme is very important for bone the collagen level at various experimental tested mineralization and calcification. It is defined as the phenotypic concentrations. The results in term of% increase in collagen clinical marker detection of osteoblasts differentiation and synthesis are presented in Figure 3. The positive control, maturation. ALP increases the local concentration of inorganic rutin showed a significant increased value of collagen by phosphate for bone mineralization and osteogenic activity. 46.6%, 30.3%, and 39.7% at 5, 10, and 25 µg/mL, ALP is reported to improve the local concentration of respectively. The experimental test group’s viz. UT- inorganic phosphate, a mineralization promoter along with DMEM+BT-TI showed a significant increased collagen level inhibition of extracellular pyrophosphate concentrations, an by 336.2%, 237.2%, and 36.9% at 10, 50, and 100 µg/mL, inhibitor of mineral formation. The Trivedi Effect®-Energy of respectively while BT-DMEM+UT-TI group showed a Consciousness Healing based vit D and DMEM reported to significant increased collagen level by 399.3%, 28.1%, and 3 improve the ALP concentration as compared with the 48.2% at 10, 50, and 100 µg/mL, respectively as compared untreated groups. Thus, it might improve the ALP expression, with the untreated test item and DMEM group. However, which is a good predictor of neotissue mineralization and BT-DMEM+BT-TI group showed a significant increased could provide beneficial therapeutic prospects for the treatment collagen level by 200%, 110.2%, 71.3%, and 97.2% at 0.1, of various bone diseases [35]. 10, 50, and 100 µg/mL, respectively as compared with the untreated test item and DMEM group. Overall, all the 3.3. Effect of Test Items on Collagen Synthesis experimental Biofield Energy Treated test item and DMEM groups showed a significant improved level of collagen at all The effect of the Biofield Energy Treated vit D3 and the tested concentrations compared with the untreated group. DMEM on the collagen level showed a significant increase in 6 William Dean Plikerd et al.: Impact of Biofield Energy Healing Treated Vitamin D on Human Osteoblast Cell Line 3 (MG-63) for Bone Health Figure 3. Effect of the test item on MG-63 cell lines for collagen level. VC: Vehicle control (DMSO-0.05%), UT: Untreated; BT: Biofield Treated; TI: Test Item. Collagen is the main component in bone for bone tissue results of bone mineralization among different groups has regeneration, enhanced bone apatite formation and is been presented in Figure 4. The positive control, rutin considered as the most abundant protein [36]. Collagen showed a significant increased value of bone mineralization defines the bone strength, which not only depends on the by 48%, 59.7%, and 139.0% at 5, 10, and 25 µg/mL, quantity of bone tissue but also on its quality, which is respectively. The experimental test group’s viz. UT- categorized by the geometry and the shape of bones, collagen DMEM+BT-TI showed a significant increased bone content, minerals, microarchitecture of the trabecular bones, mineralization by 416.9% at 10 µg/mL, while BT- and its turnover [37]. Thus it can be concluded that Biofield DMEM+UT-TI group showed a significant increased bone Energy (The Trivedi Effect®) Treated vit D and DMEM mineralization by 460.7%, 49.4%, 23.1%, and 20.6% at 0.1, 3 would be an important source to improve the level of 10, 50, and 100 µg/mL, respectively as compared with the collagen and its mineralization process against different untreated test item and DMEM group. However, BT- orthopedic diseases. DMEM+BT-TI group showed a significant increased bone mineralization by 441.7%, 17.9%, and 16.7% at 10, 50, and 3.4. Effect of Test Items on Bone Mineralization 100 µg/mL, respectively as compared with the untreated test item and DMEM group. Overall, all the experimental The Biofield Energy Treated test item and DMEM were Biofield Energy Treated test item and DMEM groups showed studied on bone mineralization and data showed a significant a significant improved level of bone mineralization at the increase in the bone mineralization process at various tested concentrations. experimental tested concentrations on MG-63 cell line. The Figure 4. Effect of the test item on MG-63 cell line for bone mineralization. VC: Vehicle control (DMSO-0.05%), UT: Untreated; BT: Biofield Treated; TI: Test Item. Vitamin D and other minerals has been reported to have be treated by Biofield Energy Treated Vitamin D [38]. 3 significant control on osteoblast function along with the bone Various bone disorders have reported that calcium extracellular matrix mineralization. However, it is the major supplementation restores the bone mineralization [39, 40]. regulator of calcium homeostasis, while vitamin D receptors Calcium phosphate is deposited in the ostoid and gets play a major role in bone mineralization. Thus, it might be mineralized (combination of calcium phosphate and expecting that calcium and vitamin D deficiency diseases can hydroxyapatite) and provides rigidity to the bone. American Journal of Clinical and Experimental Medicine 2018; 6(1): 1-9 7 4. Conclusions Ulcerative Colitis, Alzheimer’s Disease, Atherosclerosis, Dermatitis, Diverticulitis, Dermatomyositis, Graves’ Disease, The experimental results showed a significant effect of Hashimoto Thyroiditis, Multiple Sclerosis, Myasthenia Biofield Energy Healing Treatment on vitamin D3 and Gravis, Pernicious Anemia, Aplastic Anemia, Sjogren DMEM for bone health in MG-63 cell line. Cell viability test Syndrome, Systemic Lupus Erythematosus, Diabetes, using MTT assay showed a significant improved the cell Hepatitis, Irritable Bowel Syndrome, Parkinson’s Disease, viability with more than 80% cell viability among different stress etc. with a safe therapeutic index to improve overall test groups, while Biofield Energy Treated test vitamin D3 health and quality of life. and DMEM also improved the cell viability as compared with the untreated group. Cell viability test using MTT assay Abbreviations showed that the test items were safe and nontoxic in all the tested concentrations. After Biofield Energy Healing MG-63: Human Bone Osteosarcoma Cells, ALP: Alkaline Treatment, the concentration of ALP was significantly phosphatase, CAM: Complementary and alternative increased by 343.2% and 375.3% in the BT-DMEM+BT-TI medicine, NHIS: National Health Interview Survey, NCCIH: and BT-DMEM+UT-TI groups, respectively at 100 µg/mL, National Center of Complementary and Integrative Health, while 346.4% increased ALP was reported in the UT- DMEM: Dulbecco's modified eagle's medium, FBS: Fetal DMEM+BT-TI group at 50 µg/mL as compared to the bovine serum, UT: Untreated, BT: Biofield Energy Treated, untreated group. In addition, the level of collagen was TI: Test Item; FBS: Fetal bovine serum; EDTA: Ethylene significantly increased by 336.2% and 237.2% in the UT- diamine tetra acetic acid. DMEM+BT-TI group at 10 and 50 µg/mL, respectively, while 399.3% (10 µg/mL) and 200% (0.1 µg/mL) increased Acknowledgements collagen in the BT-DMEM+UT-TI and BT-DMEM+BT-TI groups, respectively as compared to the untreated group. Authors are grateful to Dabur Research Foundation, Trivedi Similarly, the percent of bone mineralization was Global, Inc., Trivedi Science, Trivedi Testimonials, and Trivedi significantly increased by 416.9% at 10 µg/mL in the UT- Master Wellness for their support throughout the work. DMEM+BT-TI group, while BT-DMEM+UT-TI group showed increased bone mineralization by 49.4%, 23.1%, and 20.6% at 10, 50, and 100 µg/mL, respectively as compared References with the untreated group. In addition, bone mineralization in MG-63 cells was increased by 441.7% in the BT- [1] Holick MF (1996) Vitamin D and bone health. J Nutr 126: DMEM+BT-TI group at 10 µg/mL, 460.7% at 0.1 µg/mL in 1159S-64S. 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