Biofield Energy Enriched Vitamin D3 versus Vitamin D3 in Preventing Fractures and Bone Loss Using MG-63 Cells

Bone is a dynamic tissue, which continually adapting its structure during the development process. The aim of the study was to investigate the effect of Consciousness Energy Healing based vitamin D3 and DMEM medium on bone health parameters such as alkaline phosphatase enzyme (ALP) activity, collagen levels and bone mineralization using MG-63 cells. The test items (TI) i.e. vitamin D3 and DMEM medium were divided into two parts. The test samples received Consciousness Energy Healing Treatment by Lorraine Marie Hachfeld and samples were defined as the Biofield Energy Treated (BT) samples, while the other parts of each sample were denoted as the untreated test items (UT). Cell viability using MTT assay showed that cell viability was more than 79% with a safe and nontoxic profile on MG-63 cell line. The level of ALP was increased by 400%, 84%, and 179.4% at 0.1, 1, and 10μg/mL, respectively in the UT-DMEM+BT-TI, while 314.3% at 10μg/mL in the BT-DMEM+UT-TI group as compared with the untreated test item and DMEM group. BT-DMEM+BT-TI group showed an increased ALP level by 128% and 60.3% at 1 and 10μg/mL, respectively. Collagen was significantly increased by 180% and 128.1% at 0.1 and 1μg/mL, respectively in the UT-DMEM+BT-TI group, while 84.4% and 69.7% at 1 and 10μg/ mL, respectively in BT-DMEM+UT-TI group as compared with the untreated group. Furthermore, BT-DMEM+BT-TI group showed a significant increased collagen level by 123.4% and 184.8% at 1 and 10 μg/mL, respectively as compared with the untreated test item and DMEM group. The percent of bone mineralization was significantly increased by 385.1% at 0.1μg/mL in the UT-DMEM+BT-TI group, while 53.2%, 30.2%, and 163.9% at 0.1, 1, and 10μg/ mL, respectively in BT-DMEM+UT-TI group as compared with the untreated group. In addition, BT-DMEM+BT-TI group showed a significant increased bone mineralization by 283%, 65.1%, and 197.3% at 0.1, 1, and 10μg/mL, respectively as compared with the untreated group. The experimental data suggested that the Biofield Energy Treated vitamin D3 and DMEM would play an important role in the promotion and maintenance of strong and healthy bones, which improve quality of life. It regulates the osteoblast function, improves the bone mineralization, and calcium absorption in wide range of bone disorders along with wide range of adverse health conditions, comprising cancer and certain autoimmune diseases.


Biofield Energy Therapies, as Complementary and Alternative
Medicine (CAM) are the noninvasive therapies in which the renowned Biofield Energy practitioner explicitly interacts with the energy fields that surround the living systems, which would significantly, stimulates the healing capacity. Due to the significant outcomes, Biofield Energy Treatment or energy medicine emerged as one of the best alternative approach of treatment. CAM therapies are practiced worldwide, some are commonly known as Reiki, therapeutic touch, pranic healing, external qigong, Tai Chi, Johrei, Qi Gong, polarity therapy, yoga, deep breathing, chiropractic/ osteopathic manipulation, massage, meditation, mindfulness, Rolfing structural integration, homeopathy, movement therapy, progressive relaxation, hypnotherapy, acupressure, acupuncture, relaxation techniques, pilates, and some medicinal approaches to Ayurvedic medicine or traditional Chinese herbs [11]. Among CAM, The Trivedi Effect ® -Biofield Energy Healing Treatment has been reported to have significant scientific results and the outcomes have been published in peer-reviewed journals [12]. National Center for Complementary and Alternative Medicine (NCCAM), classified the Biofield therapies under the subcategory of Energy Therapies [13]. Consciousness Energy Healing Treatment has been reported with significant revolution in the material science [14][15][16], agricultural science [17,18], microbiology [19,20], bone health [21,22], biotechnology [23], increased bioavailability [24][25][26], enhanced skin health [27,28], used as a nutraceutical [29,30], cancer science [31,32], and human health and wellness.Thus, authors evaluated the in vitro effect of the Biofield Energy Treated vitamin D 3 as a test item for bone health using MG-63 cell line for major biomarkers.

Cell culture
Human bone osteosarcoma (MG-63) cell line was used in this experiment and was maintained with DMEM growth medium for routine culture, which was supplemented with 10% FBS. Growth conditions were maintained at 37°C, 5% CO 2 and 95% humidity and sub-cultured by trypsinisation followed by splitting of the cell suspension into fresh flasks and further supplementing with fresh cell growth medium. Before the start of the experiment, the growth medium of near-confluent cells was replaced with fresh phenolfree DMEM, supplemented with 10% charcoal-dextran stripped FBS (CD-FBS) and 1% penicillin-streptomycin [6].

Experimental design
The following groups were defined in the study, such as baseline control, vehicle control groups (0.05% DMSO with Biofield Energy Treated and untreated DMEM media), positive control group included rutin hydrate and the experimental test groups. The experimental test sample groups have combination of the Biofield Energy Treated and untreated vitamin D 3 /DMEM. Four major treatment groups on specified cells with Untreated-DMEM + Untreated-Test item (UT-TI), UT-DMEM + Biofield Energy Treated test item (BT-TI), BT-DMEM + UT-TI, and BT-DMEM + BT-TI.

Consciousness energy healing treatment strategies
The test sample and the DMEM were treated with the Biofield Energy (also known as The Trivedi Effect ® ) by a renowned Biofield Energy Healer, Lorraine Marie Hachfeld remotely for ~5 minutes. These test groups were divided as treated and untreated test samples. The renowned Biofield Energy Healer was remotely located in the Canada and on the other side the test samples were located in the Dabur Research Foundation, New Delhi, India, which were treated through the Healer's unique Energy Transmission process remotely. Lorraine Marie Hachfeld in this study never visited the laboratory in person, nor had any contact with the test item and medium. In addition, the experimental control group was treated with a sham healer, and sham healer did not have any knowledge about the Biofield Energy Treatment. The samples were stored under laboratory condition for experimental test setup.

Determination of non-cytotoxic concentration
MTT assay was used in order to test the cell viability in MG-63 cells for treated and untreated test samples. The details procedure Where, X=Absorbance of treated cells; R=Absorbance of untreated cells The percentage of cell viability corresponding to each treatment group was calculated by Equation (2): The concentration exhibiting ≥70% cell viability was defined as non-cytotoxic [33].

Assessment of alkaline phosphatase (ALP) activity
For the estimation of ALP activity of the Biofield Energy Treatment on the test items in MG-63 cells. The procedure of cell counting, plating, and treatment was followed as per Koster et al. [6,22]. The percent increase in ALP activity with respect to the untreated cells was calculated using Equation (3): Where, X=Absorbance of cells corresponding to positive control and test groups, R=Absorbance of cells corresponding to untreated cells

Assessment of collagen synthesis
For the estimation of collagen level in MG-63 cells, standard methods were used for the evaluation of the potential of Biofield Treated test items and the procedure in details was as per Koster et al. [6,22]with few modifications.The increase collagen level with respect to the untreated cells was calculated using Equation (4): Where, X=Collagen levels in cells corresponding to positive control and test groups, R=Collagen levels in cells corresponding to untreated cells

Assessment of bone mineralization by alizarin red s staining
For the evaluation of the percent alteration in bone mineralization after treatment of the Biofield Treated test items in MG-63 cells, and the details steps were followed according to Ansari et al. [29,30]. The percentage increase in bone mineralization compared to the untreated cells was calculated using Equation (5): Where, X=Absorbance in cells corresponding to positive control or test groups; R=Absorbance in cells corresponding to untreated group.

Statistical analysis
All the values were represented as percentage of respective parameters. For multiple group comparison, one-way analysis of variance (ANOVA) was used followed by post-hoc analysis by Dunnett's test.

Cell viability study using MTT
The results of the cell viability using MTT assay of the Biofield Energy Treated vitamin D 3 and DMEM in MG-63 cells are shown in Figure 1. All the results were compared with respect to rutin and untreated test samples at various concentrations for the estimation of percentage cell viability. The data showed that the test samples in combination found as nontoxic and safe (as evidence of cell viability approximately greater than 79%) across all the tested concentrations with range up to maximum of 100µg/mL. These safe concentrations are used for estimation of different bone health parameters in MG-63 cells.

Alkaline phosphatase (ALP) enzyme activity
The ALP activity results of the Biofield Energy Treated vitamin D 3 and DMEM on the level of ALP in MG-63 cells is shown in the the untreated test samples. The vehicle control group showed 17.4% increased level of ALP as compared with the untreated cells group. The positive control, rutin showed a significant increased value by 40.89%, 59.53%, and 60.38% at 0.001, 0.01, and 0.1µg/mL, respectively with respect to the untreated cells. The experimental test group's viz. untreated medium and Biofield Treated Test item (UT-DMEM+BT-TI) showed a significant increased level of ALP by 400%, 84%, and 179.4% at 0.1, 1, and 10µg/mL, respectively while Biofield Treated medium and untreated Test item (BT-DMEM+UT-TI) showed a significant increased ALP level by 314.3% at 10µg/ mL as compared with the untreated test item and DMEM group. However, the Biofield Energy Treated medium and Biofield Energy Treated Test item (BT-DMEM+BT-TI) showed a significant increased ALP level by 128% and 60.3% at 1 and 10µg/mL, respectively as compared with the untreated test item and DMEM group. ALP is one of the biomarker used to access the growth of bone and bone formation and resorption along with other biomarkers such as bone-specific alkaline phosphatase (BALP), osteocalcin (OC), procollagen type 1 N-terminal propeptide (P1NP) and procollagen type 1 C-terminal propeptide (P1CP). BALP, a bone-specific isoform of ALP present on the surface of osteoblasts defines the biosynthetic activity of the bone-forming cells. In addition, ALP level continuously decreased with age, that might results in diseases such as postmenopausal women, osteoporosis, bone cancers, Paget's disease of bone, healing fracture, bone growth, acromegaly, myelofibrosis, osteogenic sarcoma, or bone metastases, leukemia, and rarely myeloma. These bone health diseases can be overcome using some health supplements rich in calcium and vitamin D 3 [34][35][36]. Overall, the experimental data concluded that the Biofield Energy Healing Treatment in the test samples showed a significant improved level of the ALP used against various age related bone diseases. The experimental data well described that The Trivedi Effect ® -Energy of Consciousness Healing based vit D 3 and DMEM could be used to improve the ALP concentration in many bone disorders.

Assessment of collagen synthesis
The collagen level among Biofield Energy Treated vit D 3 and DMEM was estimated at various safe concentrations and the data suggested significant increased collagen level with. The results are presented as % values with respect to the untreated cells in Figure 3. The rutin hydrate showed a significant increased value of collagen by 40.55%, 45.70%, and 58.59% at 0.01, 0.1, and 1µg/ mL, respectively. Besides, the experimental test groups such as UT-DMEM+BT-TI showed a significant increased collagen level by 180%, 128.1%, and 38.6% at 0.1, 1, and 10µg/mL, respectively while BT-DMEM+UT-TI group showed a significant increased collagen level by 84.4% and 69.7% at 1 and 10µg/mL, respectively as compared with the untreated test item and DMEM group. However, BT-DMEM+BT-TI group showed a significant increased collagen level by 44.4%, 123.4%, and 184.8% at 0.1, 1, and 10µg/ mL, respectively as compared with the untreated test item and DMEM group. The data suggested that the overall the collagen level was increased after Biofield Energy Treatment as compared with the untreated test samples. Collagen play important role in bones and joints, it is extremely important fibrous protein present in the connective tissue. Further, it was reported that collagen synthesis decreases with age and leads to high chance of bone, joints, muscle injuries. Collagen type I is the most abundant protein, which form the strength in bone health [37]. Thus, some reduced collagen synthesis results in serious bone diseases such as the type of bone loss experienced in osteoporosis, which can be overcome using various supplementations [38]. The experimental data suggest that Biofield Energy Treated vit D 3 would be the best form of supplement in order to retain the bone health irrespective to age. The data showed a significant improved level of collagen compared with the untreated group. Biofield Energy Treated vit D 3 (The Trivedi Effect ® ) demonstrated a significant improved level of collagen for bone health, which can be used to decrease the aging process and bone inflammation.
Vitamin D and calcium has been well-recognized with its significant role in bone health by normal bone formation and normal mineralization. Poor bone mineralization results in various bone disorders such as osteoporosis or other bone diseases. Bone mineralization and remodeling would renew the skeleton along with sequential involvement of the bone resorption and formation at the same spatial location. In addition, bone remodeling affects bone material assets such as microdamage, mineralization, and collagen cross-linking. Both these processes affect the bone mineral density (BMD), quality of bone, and structural abnormalities [39,40]. The present study was conducted to check the alteration in percentage of bone mineralization in Biofield Energy Treated test samples with respect to the untreated test samples. Biofield Energy Treated vit D 3 and DMEM groups showed a significant improved bone mineralization on MG-63 cell line. The results are presented in term of percentage change of bone mineralization among different experimental groups in Figure 4. The positive control, rutin group showed a significant increased value of bone mineralization by 47.98%, 59.73%, and 139.02% at 5, 10, and 25µg/mL, respectively. The experimental data among test group's viz. UT-DMEM+BT-TI showed a significant increased bone mineralization by 385.1%, 28.7%, and 19.7% at 0.1, 1, and 10µg/mL, respectively while BT-DMEM+UT-TI group showed a significantly increased bone mineralization by 53.2%, 30.2%, and 163.9% at 0.1, 1, and 10 µg/ mL, respectively as compared with the untreated test item and DMEM group. However, BT-DMEM+BT-TI group showed a significant increased bone mineralization by 283%, 65.1%, and 197.3% at 0.1, 1, and 10µg/mL, respectively as compared with the untreated test item and DMEM group. The experimental test groups showed that Biofield Energy Healing Treatment significantly improved the rate of bone mineralization compared with the untreated groups.

Conclusion
Biofield Energy Treated vitamin D 3 and DMEM was studied for bone health parameters. MTT study for cell viability data showed that a significant improved cell viability with more than 79% among all the tested groups. The level of ALP was increased by 400%, 84%, and 179.4% at 0.1, 1, and 10µg/mL, respectively in the UT-DMEM+BT-TI, while 314.3% at 10µg/mL in the BT-DMEM+BT-TI group as compared with the untreated test item and DMEM group. BT-DMEM+BT-TI group showed an increased ALP level by 128% and 60.3% at 1 and 10µg/mL, respectively. The