Osteopontin is associated with bioprosthetic heart valve calcification in humans

Calcification of non-osseous tissues such as heart valves or vessels is a major concern in clinical practice. The exact mechanism is still unknown. Numerous studies have shown that mineral deposits of crystalline hydroxyapatite within these tissues were associated with increased non-collagenous protein content. More recently osteopontin was found to be associated with calcification in living tissues such as vessels and native human aortic valves. The aim of this study was to determine whether or not non-collagenous proteins can also be found in non-living tissues such as glutaraldehyde-pretreatedporcine valves after implantation in humans. Thirty-eight glutaraldéhyde pretreated porcine bioprostheses were studied: 16 not implanted and 22 after 11 years of implantation in the aortic and mitral valve position in humans. In areas of calcification vizualized by Von Kossa staining and microradiography, immunostaining using polyclonal antibodies against calcium-bindingproteins showed osteopontin positive staining and no staining for osteocalcin, bone sialoprotein or osteonectin. In uncalcified areas and in non-implanted valves, staining for osteopontin or other calcium-binding proteins was negative. Western blot analysis of macroscopically calcified and uncalcified areas showed that several proteins were adsorbed in implanted valves and confirmed the presence of osteopontin in the calcified areas, while no immunolabelling was found in non-calcified areas, in uncalcified valves and in non-implanted valves. Thus the presence of osteopontin in the calcified areas of bioprosthetic heart valves implanted in human indicates that this protein is associated with bioprosthetic valvular calcification. Since these valves are made of non-living connective tissue, and no cell immunostained for osteopontin was found around the calcified area, this study suggests that a non-cellular mediated mechanism involving protein adsorption may play a role in bioprosthetic valvular calcification.     

An in vitro model quantifying the effect of calcification on the tissue–stent interaction in a stenosed aortic root

Transcatheter Aortic Valves rely on the tissue-stent interaction to ensure that the valve is secured within the aortic root. Aortic stenosis presents with heavily calcified leaflets and it has been proposed that this calcification also acts to secure the valve, but this has never been quantified. In this study, we developed an in vitro calcified aortic root model to quantify the role of calcification on the tissue-stent interaction. The in vitro model incorporated artificial calcifications affixed to the leaflets of porcine aortic heart valves. A self-expanding nitinol braided stent was deployed into non-calcified and artificially calcified porcine aortic roots and imaged by micro computed tomography. Mechanical tests were then conducted to dislodge the stent from the aortic root and it was found that, in the presence of calcification, there was a significant increase in pullout force (8.59 ± 3.68 N vs. 2.84 ± 1.55 N p = 0.045), stent eccentricity (0.05 ± 0.01 vs. 0.02 ± 0.01, p = 0.049), and coefficient of friction between the stent and aortic root (0.36 ± 0.12 vs. 0.09 ± 0.05, p = 0.018), when compared to non-calcified roots. This study quantifies for the first time the impact of calcification on the friction between the aortic tissue and transcatheter aortic valve stent, showing the role of calcification in anchoring the valve stent in the aortic root.     

Time course of osteopontin, osteocalcin, and osteonectin accumulation and calcification after acute vessel wall injury.

Although mineral deposits have long been described to be a prominent feature of atherosclerosis, the mechanisms of arterial calcification are not well understood. However, accumulation of the non-collagenous matrix bone-associated proteins, osteopontin, osteocalcin, and osteonectin, has been demonstrated in atheromatous plaques. The aim of this study was to evaluate the role of these proteins in arterial calcification and, more precisely, during the initiation of this process. A model of rapid aortic calcification was developed in rabbits by an oversized balloon angioplasty. Calcification was followed using von Kossa staining and osteopontin, osteocalcin, and osteonectin were identified using immunohistochemistry. The aortic injury was rapidly followed by calcified deposits that appeared in the media as soon as 2 days after injury and then accumulated in zipper-like structures. Osteonectin was not detected in calcified deposits at any time after injury. In contrast, osteopontin and osteocalcin were detected in 8- and 14-day calcified structures, respectively, but not in the very early 2-day mineral deposits. These results suggest that these matrix proteins, osteopontin, osteocalcin, and osteonectin, are not involved in the initiation step of the aortic calcification process and that the former two might play a role in the regulation of arterial calcification.     

Calcification of matrix vesicles in human aortic valve and aortic media.

Calcification of human aortic valve and aortic media occurs regularly, increases with age, and is distinctively associated with a zone of lipid accumulation. Ultrastructurally, the accumulated lipids are seen as cellular degradation products derived from senescent and degenerate fibrocytes and smooth muscle cells. The products when deposited in the matrix are morphologically similar to the matrix vesicles described in other calcifying tissues, and serve as the initial site of calcification rather than collagen or elastic fibers. Scattered among the smaller and more typical matrix vesicles, there are seen frequently giant vesicle-like structures measuring several microns in diameter. Many of these large calcified bodies contain needle-shaped, radially arranged apatite crystal deposits. Some of the large calcifying bodies are bounded by folded structures suggesting a membrane component, at times obscured by a more dense floccular osmiophilic deposition. Alcianophilic apparent proteoglycan particles are also adherent to these large calcified bodies. The substance forming the large calcified bodies might be a complex of phospholipids derived from cell membrane and proteoglycan derived from ground substance, this combination possible serving as a nidus for calcification.     

Raloxifene attenuates Gas6 and apoptosis in experimental aortic valve disease in renal failure

Renal failure is associated with aortic valve calcification. Using our rat model of uremia-induced reversible aortic valve calcification, we assessed the role of apoptosis and survival pathways in that disease. We also explored the effects of raloxifene, an estrogen receptor modulator, on valvular calcification. Gene array analysis was performed in aortic valves obtained from three groups of rats (n = 7 rats/group): calcified valves obtained from rats fed with uremic diet, valves after calcification resolution following diet cessation, and control. In addition, four groups of rats (n = 10 rats/group) were used to evaluate the effect of raloxifene in aortic valve calcification: three groups as mentioned above and a fourth group fed with the uremic diet that also received daily raloxifene. Evaluation included imaging, histology, and antigen expression analysis. Gene array results showed that the majority of the altered expressed genes were in diet group valves. Most apoptosis-related genes were changed in a proapoptotic direction in calcified valves. Apoptosis and decreases in several survival pathways were confirmed in calcified valves. Resolution of aortic valve calcification was accompanied by decreased apoptosis and upregulation of survival pathways. Imaging and histology demonstrated that raloxifene significantly decreased aortic valve calcification. In conclusion, downregulation of several survival pathways and apoptosis are involved in the pathogenesis of aortic valve calcification. The beneficial effect of raloxifene in valve calcification is related to apoptosis modulation. This novel observation is important for developing remedies for aortic valve calcification in patients with renal failure.     

Differences in accumulation of elements in human cardiac valves

To elucidate changes of human cardiac valves with aging, the authors determined age-related changes of element contents in the four human cardiac valves by inductively coupled plasma-atomic emission spectrometry and attempted to examine the relationships in the element contents among the four cardiac valves. The subjects consisted of 10 men and 15 women, ranging in age from 65 to 102 yr. The accumulation of calcium and phosphorus was the highest in the aortic valve, and decreased in the order mitral, pulmonary, and tricuspid valves. The contents of calcium and phosphorus in the aortic valves corresponded to about 12 and 19 times the amounts of those in the tricuspid valves, in which the contents were very low. The contents of calcium and phosphorus in the aortic valves were about 2.5-fold the amounts of those in the mitral valves. An examination was attempted to determine whether or not there were relationships in element contents among the four cardiac valves. As for the aortic and mitral valves, there were no relationships in the contents of calcium and phosphorus between them, but there were relationships in the contents of sulfur and magnesium between them. Three out of 24 cases contained high contents of calcium and phosphorus in both the mitral and aortic valves, whereas 16 out of 24 cases contained high contents of calcium and phosphorus in the aortic valves alone, without the high contents in the mitral valves. Likewise, there were no relationships in the element contents, such as calcium, phosphorus, sulfur, and magnesium, between the mitral and pulmonary valves or between the mitral and tricuspid valves. It is suggested that the accumulation of calcium and phosphorus in the cardiac valve occurs independent of the other cardiac valves.     

Osteogenesis inducers promote distinct biological responses in aortic and mitral valve interstitial cells

Both aortic and mitral valves calcify in pathological conditions; however, the prevalence of aortic valve calcification is high whereas mitral valve leaflet calcification is somewhat rare. Patterns of valvular calcification may differ due to valvular architecture, but little is known to that effect. In this study, we investigated the intrinsic osteogenic differentiation potential of aortic versus mitral valve interstitial cells provided minimal differentiation conditions. For the assessment of calcification at the cellular level, we used classic inducers of osteogenesis in stem cells: β-glycerophosphate (β-Gly), dexamethasone (Dex), and ascorbate (Asc). In addition to proteomic analyses, osteogenic markers and calcium precipitates were evaluated across treatments of aortic and mitral valve cells. The combination of β-Gly, Asc, and Dex induced aortic valve interstitial cells to synthesize extracellular matrix, overexpress osteoblastic markers, and deposit calcium. However, no strong evidence showed the calcification of mitral valve interstitial cells. Mitral cells mainly responded to Asc and Dex by cell activation. These findings provide a deeper understanding of the physiological properties of aortic and mitral valves and tendencies for calcific changes within each valve type, contributing to the development of future therapeutics for heart valve diseases.     

Age-related changes of element contents in human mitral and tricuspid valves

To examine age-related changes of human cardiac valves, mitral and tricuspid valves were analyzed by inductively coupled plasma-atomic emission spectrometry. The subjects for mitral valves consisted of 12 men and 8 women, ranging in age from 52 to 96 yr. The subjects for tricuspid valves consisted of 11 men and 6 women, ranging in age from 52 to 93 yr. Furthermore, 16 of the samples of the cardiac valves were derived from the same subjects. The contents of calcium, phosphorus, and magnesium in the mitral valves increased progressively with advancing age and reached maximum in the 80s in regard to calcium and phosphorus and maximum in the 90s in regard to magnesium. The maximum average amounts corresponded to about three times the average contents in the 60s. In contrast, the content of sulfur in the mitral valves remained constant between the 50s and 90s. Regarding tricuspid valve, the contents of calcium, phosphorus, and magnesium scarcely increased with advancing age, except for one subject who died of chronic renal failure. Histological observations of the mitral valves revealed that deposits of calcium were present in both the elastic fibers and its degenerative tissues of the mitral valve. The present study indicates that the accumulation of calcium, phosphorus, and magnesium occurs progressively in the mitral valve with aging, but does not occur in the tricuspid valve.     

Thermal behavior of bone and synthetic hydroxyapatites submitted to magnesium interaction in aqueous medium

The thermal behavior of the products obtained from magnesium interaction with powdered femoral bone and carbonate containing synthetic hydroxyapatite under conditions of pH fluctuation in aqueous medium has been investigated. The products, heat treated at different temperatures from 100 to 1300 degrees C, have been characterized by infrared spectroscopy and X-ray diffraction technique. The results show that the interaction with magnesium ion destabilizes the apatitic structure and favours its thermal conversion into beta-tricalcium phosphate (beta-TCP). The replacement of magnesium with calcium in the beta-TCP crystal lattice hinders its subsequent thermal conversion into the alpha form. The influence of magnesium on the thermal stability is much more evident for carbonate-containing synthetic hydroxyapatite than for bone apatite.     

Ultrasonic Decalcification of Calcified Cardiac Valves and Annuli

Heavily calcified annuli increase the incidence of complications after prosthetic valve replacement—heart block, separation of the aorta or the atrium from the ventricle, late aneurysm formation, paravalvular leak, and haemolysis. An ultrasonic calculus-disintegrator has been developed to remove calcific deposits. The instrument is portable, robust, easily sterilized, inexpensive, and provides nebulized water at the ultrasonic tip which keeps the tissues cool, helps to break up the calculus by cavitation, and washes the calcific debris into the sucker. Preliminary trial on excised calcific valves showed the ultrasound instrument to be capable of removing most forms of calcification. In clinical prosthetic replacement of valves it enabled good clearance of the annulus to be performed in six out of seven cases, in one of which earlier operation had been unsuccessful because of calcification. Two elderly patients with pure calcific aortic stenosis were successfully treated by debridement of the aortic valve with ultrasound.     

Ultrastructural study of the long-term development of two experimental cutaneous calcinoses (topical calciphylaxis and topical calcergy) in the rat

Summary Skin calcification induced by topical calciphylaxis was provoked by a subcutaneous injection of iron chloride in rats previously sensitized by dihydrotachysterol. A cutaneous topical calcergy was induced by an injection of potassium permanganate. An electron-microscopical study of the long-term evolution of both these models of calcification was made. After the initial stages, mineralization of the connective tissue continued by a secondary nucleation process without matrix vesicles. The mineral composed of needle-like structures, apatite in nature, was mainly deposited between and around collagen fibrils, and showed various arrangements in calcified plaques. Intrafibrillar calcification was rarely observed and appeared only in the later stages. The extension of calcified deposits then stopped. Finally, there was a fragmentation of the mineralized area which was progressively surrounded by uncalcified collagen fibrils. A demineralization process, caused by cells such as macrophages and multinucleated giant cells, rather than a resorption of the calcified deposits, was noted. It is important to emphasize that, in both models of ectopic calcification, an evolution toward ectopic ossification was never observed, which is perhaps due to the absence of extensive resorption mechanisms.     

Accumulation of elements in the arteries and cardiac valves of Thai with aging

To elucidate whether the extent of element accumulation in the arteries and cardiac valves with aging was different between different races, the authors investigated the accumulation of elements in the arteries and cardiac valves of the Thai with aging and the relationships among elements in the cardiac valves. After ordinary dissection at Chiang Mai University was finished, 16 arteries and 4 cardiac valves were resected and element contents were determined by inductively coupled plasma-atomic emission spectrometry. In the 16 arteries, the average content of calcium was the highest in the site of the abdominal aorta ramifying into the common iliac arteries, and it decreased in the order internal iliac, coronary, abdominal aorta, common iliac, external iliac, superior mesenteric, inferior mesenteric, thoracic aorta, brachial, radial, common carotid, subclavian, ulnar, axillary, renal, and internal thoracic arteries. The average contents of phosphorus and magnesium in respective arteries were parallel with the average contents of calcium, except for the coronary artery. In comparison with the arteries of the Japanese, the trend of calcium accumulation in the arteries of the Thai was almost similar to that in the arteries of the Japanese, except for the coronary artery and thoracic aorta. The calcium accumulation in the coronary artery was much higher in the Thai than in the Japanese, whereas that in the thoracic aorta was lower in the Thai than in the Japanese. Regarding elements in the cardiac valves, the calcium content increased remarkably in the seventies in the aortic valve and in the nineties in the pulmonary valve, but it hardly increased in both the mitral and tricuspid valves with aging. The average content of calcium was the highest in the aortic valve, and it decreased in the order pulmonary, tricuspid, and mitral valves. Regarding the relationship among elements in the aortic valves, it was found that there were extremely significant direct correlations among the contents of calcium, phosphorus, and magnesium, whereas there were significant direct correlations between zinc and either calcium or phosphorus contents. Although significant correlations were found between sulfur and the other element contents in the aortic valves of the Japanese, no significant correlations were found between them in the aortic valves of the Thai. In the mitral valves, extremely or very significant direct correlations were found among the contents of calcium, phosphorus, magnesium, and sulfur, with some exceptions that there were no significant correlations between phosphorus and either magnesium or sulfur contents. In addition, no significant correlation was found in the calcium content between the aortic valve and coronary artery in the same individuals.     

Techniques of aortic valve repair

Similar to mitral repair, newer methods of aortic valve reconstruction are achieving excellent outcomes with an 85% to 90% freedom from valve-related complications at 10 years. The goal of this review is to illustrate these newer and more stable techniques of aortic valve repair. Most patients with aortic insufficiency from either trileaflet or bicuspid aortic valves are candidates for repair, in addition to selected patients with mixed aortic stenosis/insufficiency and aortic root aneurysms. Initially, aggressive commissural annuloplasty is performed to reduce measured valve diameter to 19 to 21 mm. Leaflet prolapse is corrected with plication stitches placed in the free edge of each leaflet adjacent to the Nodulus Arantius. In this regard, the leaflet free edge functions as the chorda tendinea of the aortic valve, and shortening with plication stitches raises the leaflet to a proper "effective height." Leaflet defects are augmented with gluteraldehyde-fixed autologous pericardium, and mild-to-moderate strategically placed spicules of calcium are removed with the cavitron ultrasonic surgical aspirator. Using these methods, most insufficient aortic valves, and many with mixed lesions, can be satisfactorily repaired. Six cases are illustrated in this review, spanning the spectrum of pathologies from annular dilatation without leaflet defects, to standard congenital bicuspid valve with prolapse, to trileaflet prolapse, to unusual bicuspid pathology with calcification, to a moderately calcified trileaflet valve with mixed lesions, and to aortic root aneurysms with severe aortic insufficiency. All valves were repaired using the techniques described above with trivial residual leak and minimal gradients. All repairs have been followed with yearly echocardiography, and valve reconstruction with these methods is now quite stable with excellent late outcomes. Most insufficient aortic valves now can undergo stable repair with minimal late valve-related complications. Greater application of aortic valve repair seems indicated.     

Characterization of Porcine Aortic Valvular Interstitial Cell ‘Calcified’ Nodules

Valve interstitial cells populate aortic valve cusps and have been implicated in aortic valve calcification. Here we investigate a common in vitro model for aortic valve calcification by characterizing nodule formation in porcine aortic valve interstitial cells (PAVICs) cultured in osteogenic (OST) medium supplemented with transforming growth factor beta 1 (TGF-β1). Using a combination of materials science and biological techniques, we investigate the relevance of PAVICs nodules in modeling the mineralised material produced in calcified aortic valve disease. PAVICs were grown in OST medium supplemented with TGF-β1 (OST+TGF-β1) or basal (CTL) medium for up to 21 days. Murine calvarial osteoblasts (MOBs) were grown in OST medium for 28 days as a known mineralizing model for comparison. PAVICs grown in OST+TGF-β1 produced nodular structures staining positive for calcium content; however, micro-Raman spectroscopy allowed live, noninvasive imaging that showed an absence of mineralized material, which was readily identified in nodules formed by MOBs and has been identified in human valves. Gene expression analysis, immunostaining, and transmission electron microscopy imaging revealed that PAVICs grown in OST+TGF-β1 medium produced abundant extracellular matrix via the upregulation of the gene for Type I Collagen. PAVICs, nevertheless, did not appear to further transdifferentiate to osteoblasts. Our results demonstrate that ‘calcified’ nodules formed from PAVICs grown in OST+TGF-β1 medium do not mineralize after 21 days in culture, but rather they express a myofibroblast-like phenotype and produce a collagen-rich extracellular matrix. This study clarifies further the role of PAVICs as a model of calcification of the human aortic valve.     

Carotenoids Co-Localize with Hydroxyapatite,Cholesterol, and Other Lipids in Calcified Stenotic Aortic Valves. Ex Vivo Raman Maps Compared to Histological Patterns

Unlike its application for atherosclerotic plaque analysis, Raman microspectroscopy was sporadically used to check the sole nature of bioapatite deposits in stenotic aortic valves, neglecting the involvement of accumulated lipids/lipoproteins in the calcific process. Here, Raman microspectroscopy was employed for examination of stenotic aortic valve leaflets to add information on nature and distribution of accumulated lipids and their correlation with mineralization in the light of its potential precocious diagnostic use. Cryosections from surgically explanted stenotic aortic valves (n=4) were studied matching Raman maps against specific histological patterns. Raman maps revealed the presence of phospholipids/triglycerides and cholesterol, which showed spatial overlapping with one another and Raman-identified hydroxyapatite. Moreover, the Raman patterns correlated with those displayed by both von-Kossa-calcium- and Nile-blue-stained serial cryosections. Raman analysis also provided the first identification of carotenoids, which co-localized with the identified lipid moieties. Additional fit concerned the distribution of collagen and elastin. The good correlation of Raman maps with high-affinity staining patterns proved that Raman microspectroscopy is a reliable tool in evaluating calcification degree, alteration/displacement of extracellular matrix components, and accumulation rate of different lipid forms in calcified heart valves. In addition, the novel identification of carotenoids supports the concept that valve stenosis is an atherosclerosis-like valve lesion, consistently with their previous Raman microspectroscopical identification inside atherosclerotic plaques.Key words: Valve calcification, stenosis, carotenoids, lipids, Raman microspectroscopy     

Calcification of Spirostomum ambiguum

SYNOPSIS. A method is described for cultivation of large numbers of Spirostomum ambiguum , calcified intracellularly with hydroxyapatite (bone salt deposits). The structure and activity of living animals at various stages of calcification is described and also illustrated with unstained and silver impregnated ciliates. A suitable modification of von Kossa's method for localization of insoluble phosphate deposits is given.     

Evidence of nanobacterial-like structures in calcified human arteries and cardiac valves

Mechanisms mediating vascular calcification remain incompletely understood. Nanometer scale objects hypothesized to be a type of bacteria (nanobacteria) are associated with calcified geological specimens, human kidney stones, and psammona bodies in ovarian cancer. Experiments were designed to evaluate human vascular tissue for the presence of similar nanometer-scale objects. Calcified human aneurysms (n = 8), carotid plaques (n = 2), femoral arterial plaques (n = 2), and cardiac valves (n = 2) and noncalcified aneurysms from patients with bicuspid aortic valve disease (n = 2) were collected as surgical waste from the Heart Hospital of Austin, Austin, Texas, and Mayo Clinic, Rochester, Minnesota. Whole mounts or adjacent sections from each specimen were examined by electron microscopy, stained for calcium phosphate, or stained with a commercially available antibody (8D10). Filtered (0.2 microm) homogenates of aneurysms were cultured and costained with 8D10 antibody followed by PicoGreen to detect DNA or incubated with [3H]uridine. Staining for calcium phosphate was heterogeneously distributed within all calcified tissues. Immunological staining with 8D10 was also heterogeneously distributed in areas with and without calcium phosphate. Analysis of areas with positive immunostaining identified spheres ranging in size from 30 to 100 nm with a spectral pattern of calcium and phosphorus (high-energy dispersive spectroscopy). Nanosized particles cultured from calcified but not from noncalcified aneurysms were recognized by a DNA-specific dye and incorporated radiolabeled uridine, and, after decalcification, they appeared via electron microscopy to contain cell walls. Therefore, nanometer-scale particles similar to those described as nanobacteria isolated from geological specimens and human kidney stones can be visualized in and cultured from calcified human cardiovascular tissue.     

Ionescu-Shiley pericardial xenograft valve: Hemodynamic evaluation and early clinical follow-up of 326 patients

Dissatisfaction with the hemodynamic characteristics of available porcine valves prompted a clinical trial of the Ionescu-Shiley percardial xenograft (ISPX) valve. Three hundred fifty-six ISPX valves were implanted consecutively in 326 patients. Operative mortality was 2.6% (2/75) for aortic valve replacement alone and 7.7% (12/155) for aortic valve replacements that included reoperations and combined procedures such as mitral commissurotomy, annuloplasty, and coronary artery bypass. Operative mortality for all patients who underwent mitral valve replacement was 9.5% (14/147). The mean peak systolic gradient pressure in the aortic position was 5.4 mm Hg overall and 4.27 mm Hg with the size 19 mm valve. There were no embolic episodes in patients who received the ISPX valve in the aortic position. The available data indicate that the rate of peripheral embolism with the ISPX valve compares favorably with that of porcine valves. Considering its hemodynamic advantage, if the longterm durability of the full-orifice Ionescu-Shiley pericardial xenograft valve continues to be confirmed by follow-up studies, it is our opinion that it is the biologic valve of choice.     

Evidence for a serum factor that initiates the re-calcification of demineralized bone

The present studies show for the first time that demineralized bone re-calcifies rapidly when incubated at 37 degrees C in rat serum: re-calcification can be demonstrated by Alizarin Red and von Kossa stains, by depletion of serum calcium, and by uptake of calcium and phosphate by bone matrix. Re-calcification is specific for the type I collagen matrix structures that were calcified in the original bone, with no evidence for calcification in periosteum or cartilage. Re-calcification ceases when the amount of calcium and phosphate introduced into the matrix is comparable to that present in the original bone prior to demineralization, and the re-calcified bone is palpably hard. Re-calcified bone mineral is comparable to the original bone mineral in calcium to phosphate ratio and in Fourier transform infrared and x-ray diffraction spectra. The serum activity responsible for re-calcification is sufficiently potent that the addition of only 1.5% serum to Dulbecco's modified Eagle's medium causes bone re-calcification. This putative serum calcification factor has an apparent molecular mass of 55-150 kDa and is inactivated by trypsin or chymotrypsin. The serum calcification factor must act on bone for 12 h before re-calcification can be detected by Alizarin Red or von Kossa staining and before the subsequent growth of calcification will occur in the absence of serum. The speed, matrix-type specificity, and extent of the serum-induced re-calcification of demineralized bone suggest that the serum calcification factor identified in these studies may participate in the normal calcification of bone.