Enhancement of calcification by osteoblasts cultured on hydroxyapatite surfaces with adsorbed inorganic polyphosphate

Abstract

Inorganic polyphosphate has been expected to accelerate bone regeneration. However, there are limited evidences to prove that polyphosphate adsorbed on the surface of a hydroxyapatite plate enhances calcification of cultured osteoblasts. In this study, we examined the effect of polyphosphate adsorbed onto the surface of a hydroxyapatite plate on the attachment, proliferation, differentiation, and calcification of osteoblasts. After hydroxyapatite plates were soaked in solutions of polyphosphate, the plate surfaces were analyzed by scanning electron microscopy and toluidine blue staining to confirm adsorption of polyphosphate. The hydroxyapatite plates were further subjected to the measurements of surface roughness, water contact angle, and the binding capacity of calcium ions. Cell culture experiments were carried out using MC3T3-E1 pre-osteoblastic cells. It was found that soaking a hydroxyapatite plate in a polyphosphate solution gave rise to an increase in surface roughness and reduction in water contact angle in a concentration-dependent manner, suggesting the adsorption of polyphosphate onto the surface of a hydroxyapatite plate. It was further observed that surface-adsorbed polyphosphate exhibited an inhibitory effect on cell adhesion and proliferation. In contrast, cell differentiation was promoted on hydroxyapatite plates with adsorbed polyphosphate, when assessed from expression of differentiation marker genes including alkaline phosphatase, osteopontin, and osteocalcin. In addition, calcification of the culture was enhanced on hydroxyapatite plates with relatively low density of adsorbed polyphosphate. Our results as a whole provided an evidence to show that there is a narrow window with regard to the surface density of adsorbed polyphosphate for the enhancement of osteoblast calcification.

Cellular calcification induced by inorganic polyphosphate involves ATP depletion and opening of the mitochondrial permeability transition pore (mPTP)

“…The PolyP promotes osteocalcification and bone mineralization in both mouse and human osteoblastic cells [7][8][9][10]. Regarding the effect of polyP on cell calcification, Kawazoe et al [9] reported that it promotes cell calcification in murine osteoblastic MC3T3-E1 cells, while Morita et al [11] reported that inorganic polyphosphate-adsorbed hydroxyapatite enhances bone regeneration, and cellular calcification of MC3T3-E1 cells is enhanced on the surface of polyP-adsorbed hydroxyapatite [12]. Kawazoe also reported that in the calcification of MC3T3-E1 cells, polyP is utilized as a phosphate source and that this is accompanied by an increase in polyphosphatase activity [9].…”

Critical and diverse roles of phosphates in human bone formation

“…133 In addition to its role as a substrate, polyP has also been shown to have a direct influence on biomineralisation pathways by inducing the expression of bone formation markers OPN, OC and ALP, leading to the mineralisation of pre-osteoblast MC3T3 cells. 105,134 Increased ALP expression has also been shown in SaOS-2 cells, and an increase in intracellular calcium was seen when calcium-polyP was used as a substrate, but not with Ca, Pi, or polyP alone. 135 As well as a role in bone formation, polyP has been shown to increase cartilage production, but for a short time before it is degraded by the polyphosphatases released by chondrocytes.…”

Progress and Applications of Polyphosphate in Bone and Cartilage Regeneration

“…Compared with HA, the polyP/HA hybrid particles, which had bioactivity similar to that of Ca-polyP nanoparticles, could enhance the metabolic activity of SaOS-2 cells and human MSCs and upregulate the gene expression of COL-1 and ALP, providing a better design and improved selection for bone tissue engineering materials. PolyP adsorbed on the surface of an HA plate can also enhance the calcification of the cultured osteoblasts [90]. Wang et al [91] confirmed that the mixing of amorphous calcium carbonate particles with amorphous Ca-polyP particles can enhance the proliferation rate of human MSCs in in vitro studies.…”