Biophysical aspects of biomineralization

Abstract

During the process of endochondral bone formation, chondrocytes and osteoblasts mineralize their extracellular matrix (ECM) by promoting the synthesis of hydroxyapatite (HA) seed crystals in the sheltered interior of membrane-limited matrix vesicles (MVs). Several lipid and proteins present in the membrane of the MVs mediate the interactions of MVs with the ECM and regulate the initial mineral deposition and posterior propagation. Among the proteins of MV membranes, ion transporters control the availability of phosphate and calcium needed for initial HA deposition. Phosphatases (orphan phosphatase 1, ectonucleotide pyrophosphatase/phosphodiesterase 1 and tissue-nonspecific alkaline phosphatase) play a crucial role in controlling the inorganic pyrophosphate/inorganic phosphate ratio that allows MV-mediated initiation of mineralization. The lipidic microenvironment can help in the nucleation process of first crystals and also plays a crucial physiological role in the function of MV-associated enzymes and transporters (type III sodium-dependent phosphate transporters, annexins and Na⁺/K⁺ ATPase). The whole process is mediated and regulated by the action of several molecules and steps, which make the process complex and highly regulated. Liposomes and proteoliposomes, as models of biological membranes, facilitate the understanding of lipid-protein interactions with emphasis on the properties of physicochemical and biochemical processes. In this review, we discuss the use of proteoliposomes as multiple protein carrier systems intended to mimic the various functions of MVs during the initiation and propagation of mineral growth in the course of biomineralization. We focus on studies applying biophysical tools to characterize the biomimetic models in order to gain an understanding of the importance of lipid-protein and lipid-lipid interfaces throughout the process.

Keywords: Biomineralization; Hydroxyapatite; Lipid microenvironment; Liposome; Matrix vesicles; Proteoliposome.

Fig. 1

Proteoliposomes as matrix vesicles (MVs) biomimetic systems. The construction of MV models has the aim to provide an environment that would allow the initial nucleation of apatite inside of liposomes as well as to provide the surface nucleation that can also help in the propagation of apatite onto the extracellular matrix. The proteoliposomes internal nucleation can be initiated by orphan phosphatase 1 (PHOSPHO1) action producing inorganic phosphate (P_i) from the hydrolysis of phosphocoline (PC), which itself is derived from sphingomyelin (SM) by the action of sphingomyelin phosphodiesterase 3 (SMPD3) located in the inner surface of the MV membrane. In addition, protein transporters, such as annexin A5 (AnxA5) and phosphate transporter 1 (PiT-1), provide ions for nucleation inside the proteoliposomes. The events outside the vesicles are studied by the incorporation of the regulated proteins, tissue nonspecific alkaline phosphatase (TNAP) and ectonucleotide pyrophosphatase/phosphodiesterase (NPP1), which play a crucial role controlling appropriate inorganic pyrophosphate (PP_i)/P_i ratio. Furthermore, calcium channels accelerate the nucleational activity of calcium–phosphate–lipid complexes (CPLX) inside the MVs. AnxA5 also shows binding affinity to type II collagen and when present in the proteoliposome membrane can drive the vesicles’ attachment onto to collagenous extracellular matrix stimulating mineral propagation. Hydroxyapatite (HA)

Fig. 2

Use of atomic force microscopy to image and characterize the structure of liposomes and proteoliposomes harboring relevant proteins present in the membrane of MVs: a 9:1 DPPC:DPPS (molar ratio) proteoliposomes harboring TNAP (scale bar 250 nm), b DPPC proteoliposomes harboring Na⁺/K⁺ ATPase (scale bar 500 nm), c 9:1 DPPC:DPPS (molar ratio) proteoliposomes harboring AnxA5 (scale bar 250 nm).

Fig. 3

Different effects of osteogenic proteins on the morphology of GUV membrane composed of DOPC (2 mg/mL). a GUVs exhibiting spherical shape, b the AnxA5–membrane interaction with DOPC allowing changes between the internal and external media and subsequent loss of the optical contrast without membrane disruption, c the effect on GUV membrane morphology after TNAP interaction, showing excess area and filament formation. Magnification ×60, scale bars 20 μm