Vitamin K2 Modulates Vitamin D‐Induced Mechanical Properties of Human 3D Bone Spheroids In Vitro

Abstract

Rotational culture promotes primary human osteoblasts (hOBs) to form three‐dimensional (3D) multicellular spheroids with bone tissue‐like structure without any scaffolding material. Cell‐based bone models enable us to investigate the effect of different agents on the mechanical strength of bone. Given that low dietary intake of both vitamin D and K is negatively associated with fracture risk, we aimed to assess the effect of these vitamins in this system. Osteospheres of hOBs were generated with menaquinone‐4 (MK‐4; 10μM) and 25‐hydroxyvitamin D3 [25(OH)D3; 0.01μM], alone and in combination, or without vitamins. The mechanical properties were tested by nanoindentation using a flat‐punch compression method, and the mineralized extracellular bone matrix was characterized by microscopy. The in vitro response of hOBs to MK‐4 and 25(OH)D3 was further evaluated in two‐dimensional (2D) cultures and in the 3D bone constructs applying gene expression analysis and multiplex immunoassays. Mechanical testing revealed that 25(OH)D3 induced a stiffer and MK‐4 a softer or more flexible osteosphere compared with control. Combined vitamin conditions induced the same flexibility as MK‐4 alone. Enhanced levels of periostin (p < 0.001) and altered distribution of collagen type I (COL‐1) were found in osteospheres supplemented with MK‐4. In contrast, 25(OH)D3 reduced COL‐1, both at the mRNA and protein levels, increased alkaline phosphatase, and stimulated mineral deposition in the osteospheres. With the two vitamins in combination, enhanced gene expression of periostin and COL‐1 was seen, as well as extended osteoid formation into the central region and increased mineral deposition all over the area. Moreover, we observed enhanced levels of osteocalcin in 2D and osteopontin in 3D cultures exposed to 25(OH)D3 alone and combined with MK‐4. In conclusion, the two vitamins seem to affect bone mechanical properties differently: vitamin D enhancing stiffness and K2 conveying flexibility to bone. These effects may translate to increased fracture resistance in vivo. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Three-Dimensional Co-Culture System of Human Osteoblasts and Osteoclast Precursors from Osteoporotic Patients as an Innovative Model to Study the Role of Nutrients: Focus on Vitamin K2

“…Although it the anabolic effect of vitamin K2 on an osteoblast cell line cultured in a 2-D system has been well established [ 41 , 42 , 43 , 44 ], recently, Schröder and colleagues [ 45 ] investigated the effect of vitamin K2 using human 3-D bone spheroids (osteospheres), which, unlike our approach, were derived from healthy primary hOBs. The hypothesis that the responsiveness of the hOBs/hOCs 3D co-culture system we developed can predict the patient’s responsiveness to a specific treatment is an important challenge that requires further investigation.…”

The Role of Vitamin K in Humans: Implication in Aging and Age-Associated Diseases

“…In clinical studies, combined administration of VK and vitD, plus calcium, improved BMD, bone quality and decrease fracture risk, demonstrating a positive synergistic effect on bone health [57,71]. In a group of 181 healthy postmenopausal women, between 50 and 60 years, after 3 years of daily treatment with VK1, in addition to vitD, calcium, magnesium, and zinc, the bone loss at the site of the femoral neck was significantly reduced compared to the placebo group [72].…”

Vitamin D Enhanced the Osteogenic Differentiation of Cell Spheroids Composed of Bone Marrow Stem Cells

“…The cells were grown in an incubator at 37 osteoblasts through vitamin D receptors and membrane-binding protein [9]. Low dietary intake of vitamin D is reported to be negatively associated with fracture risk [7].…”

“…Addition of both vitamin D and osteogenic factors resulted in an osteoblast phenotype which expresses alkaline phosphatase activity, secretes osteocalcin, and deposits calcium [6]. Mechanical testing showed that vitamin D induced a stiffer osteosphere compared with control [7]. Vitamin D enhanced cell responses of osteoblasts on the titanium surfaces [8].…”