Calcium-Phospholipid-Phosphate Complexes in Mineralizing Tissue

Abstract

Past work has shown that the acidic phospholipids are involved in tissue mineralization ( 1-3). Calcium-phospholipid-phosphate (Ca-PL-P04) complexes, containing the acidic phospholipids, phosphatidyl inositol (PI) and phosphatidyl serine (PS), were isolated from the long bones of developing animals (4). These complexes induced hydroxyapatite (HA) formation in metastable calcium phosphate solutions (5). The same study also showed that the uncomplexed acidic phospholipids when placed in metastable calcium phosphate solution, first formed a Ca-PL-P04 complex and then nucleated HA. The fact that more complexed lipids were found in the bones of developing as opposed to more mature animals further supports the thesis that such compounds are related to the onset of mineralization. The purpose of this study was to analyze further for complexed lipids in a number of hard and soft tissues in order to enlarge our view of the role of the Ca-PL-PO4 complexes in mineralization.Materials and methods. This investigaton was carried out on white New Zealand rabbits of ages 1 4 days (50-70 g), 4-6 weeks (0.9-1.4 kg), 12-16 weeks (1.5-1.8 kg), and older than 100 weeks (3.6-5.4 kg). The tissues studied were: cortex of long bones, epiphyseal regions of long bones (including articular cartilage, growth plate, and secondary ossification centers), skull bones (cranium and mandible), scapulae, marrow, adipose tissue, heart muscle, pooled leg muscles, ear cartilage, and incisors. All bones were scraped to remove the periosteum and marrow, ground in a liquid nitrogen-cooled colloid mill, washed in 0.05 M tris-hydroxy-methyl amino methane-HC1 (Tris) buffer at pH 7.4 to remove blood, and then were lyophilized. Noncalcified tissues were frozen and minced with dry ice and then lyophilized. The ash weight

In Vitro Modeling of Matrix Vesicle Nucleation

“…For example, if PS is added to preformed ACP, it markedly delayed its conversion to hydroxyapatite; however, if PS is present during the formation of ACP, its inhibitory effect is less pronounced and greatly alters the structure of the complex formed (35). Using a different approach, Boskey et al (18) induced formation of "Ca⅐acidic phospholipid⅐P i " complexes by incubating the lipids with a Ca 2ϩ -and P i -containing synthetic lymph for several hours, followed by washing with diethylether/ethanol to remove noncomplexed lipids and finally by washing with HCl, pH 5, to remove any noncomplexed mineral. Such complexes are generally stimulatory to mineral formation.…”

“…Early work had shown that acidic phospholipids, especially PS, are bound with Ca 2ϩ at sites of early mineralization (14,15). PS⅐Ca 2ϩ ⅐P i complexes (CPLXs), first described by Cotmore et al (16), have been found in calcifying tissues, such as tumors (17), bone (18), chondrocytes, and MVs (19). They have been shown to nucleate hydroxyapatite formation (20,21).…”

Analysis and Molecular Modeling of the Formation, Structure, and Activity of the Phosphatidylserine-Calcium-Phosphate Complex Associated with Biomineralization

“…PS-Ca 2ϩ -P i complexes (PS-CPLX) are present at the early stages of almost all calcifying tissues as follows: growth plate cartilage (14), tumors (15), bone (16), and especially MV (8). Synthetic PS-CPLX formed in the absence of Mg 2ϩ has been found to be a powerful nucleator that rapidly induces hydroxyapatite (HA) formation when incubated in SCL (3,4); however, when formed from Mg 2ϩ -and HCO 3 Ϫ -containing P i -rich buffers, PS-CPLXs are weak nucleators unless certain lipophilic proteins (17,18), such as the annexins (5,6), are also included. Annexin-A5, a major lipid-dependent Ca 2ϩ -binding protein of MV (19 -22), potently activates the nucleational activity of Mg 2ϩ -containing PS-CPLX (5).…”

“…2 is the presence of a nucleation core (1, 2) that contains lipid-calcium-phosphate complexes (CPLX) capable of inducing mineral formation when incubated in a synthetic cartilage lymph (SCL) (3)(4)(5)(6). A critical component of CPLX is the acidic phospholipid, phosphatidylserine (PS) (3,7,8), which is known to have a high affinity for Ca 2ϩ (9,10).…”

“…A critical component of CPLX is the acidic phospholipid, phosphatidylserine (PS) (3,7,8), which is known to have a high affinity for Ca 2ϩ (9,10). PS is largely confined to the inner leaflet of the MV membrane (11), and electron micrographs of calcifying MV show the electrondense calcium phosphate precipitate juxtaposed along the inner leaflet of the MV membrane (12).…”