Phosphatidylserine controls calcium phosphate nucleation and growth on lipid monolayers: A physicochemical understanding of matrix vesicle-driven biomineralization

Abstract

Bone biomineralization is an exquisite process by which a hierarchically organized mineral matrix is formed. Growing evidence has uncovered the involvement of one class of extracellular vesicles, named matrix vesicles (MVs), in the formation and delivery of the first mineral nuclei to direct collagen mineralization. MVs are released by mineralization-competent cells equipped with a specific biochemical machinery to initiate mineral formation. However, little is known about the mechanisms by which MVs can trigger this process. Here, we present a combination of in situ investigations and ex vivo analysis of MVs extracted from growing-femurs of chicken embryos to investigate the role played by phosphatidylserine (PS) in the formation of mineral nuclei. By using self-assembled Langmuir monolayers, we reconstructed the nucleation core - a PS-enriched motif thought to trigger mineral formation in the lumen of MVs. In situ infrared spectroscopy of Langmuir monolayers and ex situ analysis by transmission electron microscopy evidenced that mineralization was achieved in supersaturated solutions only when PS was present. PS nucleated amorphous calcium phosphate that converted into biomimetic apatite. By using monolayers containing lipids extracted from native MVs, mineral formation was also evidenced in a manner that resembles the artificial PS-enriched monolayers. PS-enrichment in lipid monolayers creates nanodomains for local increase of supersaturation, leading to the nucleation of ACP at the interface through a multistep process. We posited that PS-mediated nucleation could be a predominant mechanism to produce the very first mineral nuclei during MV-driven bone/cartilage biomineralization.

Keywords: Biomineralization; Calcium phosphate; Langmuir monolayers; Matrix vesicles; Phosphatidylserine.

Figure 1.

Characterization of MVs extracted from chicken embryo femurs: (a) AFM images by height distribution and (b) 3D projection of the isolated MVs on mica. (c) ALP specific activity for cell lysate and MVs fractions (μmol pNP⁻/min/mg protein). Values are presented as mean ± standard error and statistical significance was assessed with t-tests (*p < 0.05). (d) TEM images of the MVs after 24 h of mineralization in SCL (2 mM CaCl₂ and 3.42 mM NaH₂PO₄) at 37°C. (e) Enlarged TEM image of a pair of mineralized-vesicles and their respective amorphous-like SAED and (f) the respective EDS spectrum indicating the presence of Ca and P. The presence of Na and Cl is also observed due to the large amount of NaCl in the SCL and Cu is from de TEM grid.

Figure 2.

π -A isotherms of DPPC (black line), DPPC:DPPS (8:2, molar ratio) (red line) and DPPS (blue line) monolayers in subphase containing m-SCL solution, at 25°C. Inset depicts the C_s⁻¹ vs π curves for the monolayers. Right panel: chemical structures of DPPC and DPPS lipids.

Figure. 3.

DPPS monolayers control calcium phosphate nucleation at the air-liquid interface. (a) PM-IRRAS spectra in the 1150–950 cm⁻¹ range for the DPPS monolayer in subphase composed of the m-SCL mineralizing buffer (pH 7.4), at 25°C and π = 30 mN/m. Spectra were obtained at different time intervals for a period of 240 min. Blue box in the spectra highlighted the region between 1045–1010 cm⁻¹ assigned to the absorption bands of evolving inorganic phosphate group. (b) FTIR spectrum in the region of ν₃ PO₄^3- absorption obtained for the material collected from the monolayer of DPPS after 240 min of mineralization. (c) TEM images and SAED (inset) of DPPS monolayers transferred to Cu-grids after 240 min. (d) Potentiometric measurement of [Ca²⁺]_free in the subphase below the DPPS monolayer. (c) Ex-situ analysis of surface ζ-potential of DPPS monolayers in absence of Ca²⁺ ions and after different mineralization time points (blue dots).

Figure 4.

Morphology of DPPS-enriched monolayers after mineralization. TEM images and their respective SAED electron diffraction patterns for the monolayers of DPPC:DPPS (8:2) molar ratio, transferred after 240 min of mineralization at 25°C. For the mixed DPPC:DPPS monolayer, the presence of nanometric complexes (~ 5 nm) indicated by the green arrow aggregates into larger structures (purple arrow). It is observed that these initially amorphous complexes crystallize after 12 h (red arrow in the SAED pattern). The formation of micrometric aggregates and a complete rupture of the transferred monolayer is observed after 24 h.

Figure 5.

Mineralization mediated by Langmuir monolayers composed of lipids extracted from MVs. (a) PM-IRRAS spectra in the region between 1150–950 cm⁻¹ for the monolayer formed by the lipid extract of MVs, in subphase composed of the m-SCL mineralizing buffer (pH 7.4), at 25°C and π = 30 mN/m. Spectra were obtained at different time intervals for a period of 240 min. Blue boxes in the spectra highlight the region between 1045–1010 cm⁻¹ assigned to the absorption bands of evolving inorganic phosphate group. (b) TEM images for the monolayer formed by the lipid extract of MVs transferred after 4 hours of mineralization at 25°C.

Figure 6.

Role of MVs in the nucleation of calcium phosphates and in the mineralization of collagen fibrils. Mineral-competent cells release MVs harboring an enzymatic machinery capable of controlling the phosphate/pyrophosphate ratio required to trigger mineralization. These vesicles are rich on phosphatidylserine (PS) in their lumen, creating a highly negatively charged interface for nucleation and stabilization of calcium phosphate complexes. These complexes evolve to the formation of ACP, the very first mineral phase in the MVs. Vesicles filled with ACP could directly infiltrate within the collagen fibrils scaffold and then transform to platelet-like crystals. This direct amorphous-apatite transformation has been proposed in vitro using confined polymer domains (Lotsari et al., 2018) and similar structures (i.e. mineralized globules) has been observed in the collagen scaffold of zebra fish fin tails (Mahamid et al., 2010) and avian leg tendon (Zou et al., 2020). Then, MVs could breakdown and release their components to the mineralizing front, either by mechanical stress or actions of phospholipases (Wu et al., 2002). Phospholipases are highly active enzymes in the growth plate (Mebarek et al., 2013). This process could release phospholipid-mineral complexes (associated or not with proteins) to direct the infiltration of biomineral precursor phase in the gap region of collagen through a mechanism similar to the proposed for non-collagenous proteins (Nudelman et al., 2010). Alternatively, these phospholipid-mineral complexes could interact with non-collagenous proteins and then be directed to the collagen matrix for mineralization.