Matrix Vesicles in Osteomalacic Hypophosphatasia Bone Contain Apatite-Like Mineral Crystals

American Journal ofPathology, Vol. 151, No. 6, December 1997 Copyright X) American Societyfor Investigative Patbology

Short Communication Matrix Vesicles in Osteomalacic Hypophosphatasia Bone Contain Apatite-Like Mineral Crystals

H. Clarke Anderson,* Howard H. Hsu,* Hypophosphatasia is a heritable form of rickets and/or David C. Morris,* Kenton N. Fedde,t and osteomalacia due to defects within the gene that en- Michael P. Whytett codes the tissue-nonspecific (bone/liver/kidney) isoen- zyme of alkaline phosphatase (TNSALP).1 In bones, den- From the Department ofPathology and Laboratory Medicine,* tin and growth plate cartilage, alkaline phosphatase University ofKansas Medical Center, Kansas City, Kansas, and (ALP) has been known for many years to be associated the Division ofBone and Mineral Diseases,t Washington with calcification.2-5 Although the precise role of ALP in University School ofMedicine at the Barnes-Jewish Hospital, and promoting mineralization has long been debated, it is the Metabolic Research Unit,* Shriners Hospitalfor Children, St. widely agreed that the enzymatic action of ALP and of Louis, Missouri related phosphoesterases (eg, ATPase, inorganic pyro- phosphatase, etc) does function in calcification.6-9 This assertion concerning TNSALP is strongly supported by investigations of hypophosphatasia, in which diminished Hypophosphatasia, a heritable disease characterized TNSALP phosphohydrolase activity in bones and teeth is by deficient activity of the tissue nonspecific isoen- accompanied by a failure of hard tissue mineraliza- zyme of aLkaline phosphatase (TNSALP), results in tion.1011 rickets and osteomalacia. Although identification of Insight into the physiological role of ALP could come TNSALP gene defects in hypophosphatasia establishes from a better understanding of the mechanism by which a role of ALP in skeletal mineralization, the precise matrix vesicles initiate mineraliza- function remains unclear. The initial site of mineral- ALP-enriched (MVs) tion. MVs are submicroscopic, extracellular, membrane- ization (primary mineralization) normally occurs are at within the lumen of TNSALP-rich matrix vesicles invested bodies that selectively located initial sites in and dentin. We in- of calcification developing bone, growth plate carti- (MVs) of growth cartilage, bone, in vestigated whether defective calcification in hy- lage, and the dentin of teeth.612 They function calcifi- cation by generating the first crystals of hydroxyapatite is due to a paucity and/or a func- pophosphatasia (HAP) mineral within the protective confines of their in- tional failure of MVs to TNSALP secondary deficiency. vesting membranes.12 The phosphatases of MVs, includ- Nondecalcified autopsy bone and growth plate carti- and nucleoside pyro- lage from five patients with perinatal (lethal) hy- ing TNSALP, ATPase, triphosphate phosphohydrolase (NTPPase), are concentrated in pophosphatasia were studied by nondecalcified light MVs,13 and are attached to the MV membrane's outer and electron microscopy to assess MV numbers, size, surface.4,14 15 There is considerable evi- shape, and ultrastructure and whether hypophos- experimental dence indicating that these ectophosphatases can stim- phatasia MVs contain apatite-like mineral, as would ulate in vitro calcification by isolated mammalian the case these MVs retained their to con- be if ability MVs.2'7'8'9'16'17 The failure of skeletal mineralization ob- centrate calcium and phosphate internally despite a paucity of TNSALP in their investing membranes. We found that hypophosphatasia MVs are present in ap- Supported in part by grants 15958 and 15963 from the Shriners Hospitals proximately normal numbers and distribution and for Children, and by USPHS grant DE05262 from the National Institute of that they are capable of initiating internal mineraliza- Dental Research, National Institutes of Health. tion. There is retarded extravesicular crystal propaga- Accepted for publication September 11, 1997. tion. Thus, in hypophosphatasia the failure of bones D. C. Morris's current address: Glaxo Inc., Venture-V-104, 5 Moore to calcify appears to involve a block of the vectorial Drive, Research Triangle Park, NC 27709. spread of mineral from initial nuclei within MVs, Presented in part at the 18th Annual Meeting of the American Society outwards, into the matrix. We conclude that hy- for Bone and Mineral Research, Seattle, WA, Sept. 7-11, 1996. (J. Bone pophosphatasia MVs can concentrate calcium and Min. Res. 11:S254, 1996.) phosphate internally despite a deficiency of TNSALP Address reprint requests to Dr. H. Clarke Anderson, Department of activity. (AmJPathol 1997, 151:1555-1561) Pathology, University of Kansas Medical Center, Kansas City, KS 66160. 1555 1556 Anderson et al AJP December 1997, Vol. 151, No. 6 served in hypophosphatasia could be due solely to a lack rum ALP activity and markedly elevated plasma pyridoxal- of intrinsic phosphatase activity within matrix vesicles. 5'-phosphate (PLP) levels were documented in cases 2 Additional pathogenetic problems could include 1) MV through 5. The normal pediatric range at the Metabolic agenesis leading to a failure of mineralization or 2) MV Research Unit (MRU), Shriners Hospital for Children, in St. defects in addition to low levels of TNSALP activity. Louis is 80 to 342 lU/L for serum ALP activity and 30 to 100 The detailed ultrastructure of MVs in hypophosphatasia nmol/L for plasma PLP concentration. bone has not been extensively studied. A report in 1985 describes the electron microscopic structure of hypophos- phatasia bone.e1 Here it was reported that MVs are present Case 1 in growth plate cartilage in approximately normal numbers. This 24-week-gestation fetus was delivered stillborn MVs of bone matrix are not illustrated in the report. The from a woman whose previous baby lived only 1 day, had authors state that "matrix vesicles of growth plate cartilage low serum ALP activity, clinical evidence of severe rickets and of metaphyseal bone do not appear to mineralize."18 and osteomalacia, and was diagnosed elsewhere as hav- Furthermore, in a 1996 report, a 21-week-gestation fetus ing hypophosphatasia. X-rays taken during this second with hypophosphatasia was examined by electron micros- pregnancy showed severe hypomineralization of all fetal copy and microprobe element analysis.19 These authors bones. Pathological examination of the tibia and femur reported that "there was almost no hydroxyapatite mineral- showed both bones to be essentially noncalcified. MVs ization in the matrix vesicles" of osteoid. were isolated from an unfixed, frozen sample of growth A definitive answer to the question of whether hypophos- plate by collagenase digestion and differential centrifu- phatasia MVs can initiate mineralization, despite their pre- gation as previously described.9 13 The specific activity sumed lack of ALP, can provide an important insight into the of ALP was measured in the isolated MVs from this pa- fundamental mechanism of skeletal calcification. The an- tient and from the growth plate of an age-matched still- swer to this question relates to whether ALP is essential for born fetus. The hypophosphatasia MVs were totally lack- initial MV calcification or whether the function of ALP can be ing in ALP activity (Table 1). assumed by other phosphatases present in the vesicle membrane, especially ATPase. Recent investigations have shown that ATP is more active as a substrate than ,B-glyc- Case 2 erophosphate in supporting in vitro MV calcification.1" Also, metabolic inhibitors of ALP activity only partially inhibit MV This boy was born 2 weeks after the expected date of calcification in vitro when ATP is supplied as substrate.17 confinement. The pregnancy had been uncomplicated, These findings suggest that 1) MV calcification is promoted but fetal distress was detected just before birth. Neonatal by several different phosphatases (one of which is TNSALP) weight was 3.2 kg. Apgar scores were 4 at 5 minutes and all working together, 2) that the specific ATPase of MVs may 5 at 10 minutes, and intubation and artificial ventilation be more important for initial calcification than TNSALP ac- were required. Physical examination showed deformities tivity, and 3) that, despite ALP deficiency, MVs remain ca- of his chest and the long bones of his extremities. At the pable of initiating calcification through the action of ATPase MRU, serum ALP was 5 IU/L and plasma PLP 29,529 or other non-ALP phosphatases. Our finding, described nmol/L. Serum calcium and phosphate levels were nor- below, of mineral in MVs in hypophosphatasia, supports the mal. Artificial ventilation was discontinued at 10 days of hypothesis that phosphatases other than ALP can initiate age. Autopsy showed severe generalized skeletal hy- MV calcification and that TNSALP plays an important role in pomineralization, limb deformities, and multiple fractures. the propagation of HAP crystals into the collagenous ex- travesicular matrix. Our objective was to examine hypomineralized matrix Case 3 zones in growth plate cartilage and in developing bone in This full-term, deformed newborn boy was not intu- severely affected hypophosphataia patients using non- bated. Radiographs at birth were consistent with severe decalcified tissue preparations and high-resolution trans- hypophosphatasia. At the MRU, serum ALP was 4 IU/L mission electron microscopy. By this approach, we as- and plasma PLP 6808 nmol/L. He died at 11 days of age. sessed whether MVs were present in approximately normal numbers and in a normal distribution and whether these vesicles were unmineralized, as suggested earli- er,18 19 or conversely, whether they showed ultrastruc- tural evidence of some initial mineral deposition either Table 1. Alkaline-Phosphatase-Specific Activity in Isolated Cells and Matrix Vesicles from Growth Plate of within or at their surfaces. Perinatal Hypophosphatasia (Case 1) versus from an Age-Matched Normal Infant ALP ALP Materials and Methods Sample normal hypophosphatasia Cases Cell fraction 9.8* <0.001 Ut Matrix vesicle fraction 55.7 <0.001U All five patients had clinical findings consistent with perina- *ALP units (AOD410/minutelmg) per mg of protein. tal (lethal) hypophosphatasia. Profoundly low levels of se- tLevel of detectability, 0.001 U. Matrix Vesicles of Hypophosphatasia Bone 1557 AJP December 1997, Vol. 151, No. 6

Case 4 a thick layer of nonmineralized osteoid. Thus, there was a marked mineral deficit in both cartilage and bone in all At birth, this boy was found to have poor formation of cases studied. the membranous bones of the skull (radiographs showed small plates of bone within the calvarium and other find- ings consistent with severe hypophosphatasia). Apgar Electron Microscopy of Growth Plate Cartilage scores were 9 at both 5 and 10 minutes. Birth weight was 2.9 kg. Hypercalcemia and hyperphosphatemia were MVs were identified in approximately normal numbers in also noted. Subsequently, he developed feeding difficul- all growth plates examined and were normally distributed ties and respiratory problems. Sequential radiographs within the matrix of longitudinal septa (Figure 2). The revealed multiple nondisplaced rib fractures. At the MRU, majority of growth plate MVs in hypophosphatasia were serum ALP was 5 IU/L and plasma PLP 5682 nmol/L. He devoid of mineral (Figure 2A). However, in all cases, died at 5.5 months of age. vesicles located in what would have been the normal calcifying zone of the growth plate contained needle-like crystals of apparent HAP (Figure 2B). These crystals Case 5 often were attached to the inner leaflet of the vesicle This girl was delivered by Caesarean section at 39 membrane. In all of the cases, the MV sap also contained weeks gestation and weighed 2.5 kg. Fetal ultrasound a diffuse, apparently noncrystalline, electron-dense ma- was abnormal during the third trimester of pregnancy. terial in addition to the needle-like deposits of apatite-like Apgar scores were 4 and 9 at 5 and 10 minutes, respec- crystals (Figure 2). tively. The skull was soft with enlarged fontanels. There were chest and limb deformities. Ventilatory support was necessary. Radiographs showed profound hypomineral- Electron Microscopy of Bone Matrix ization. At the MRU, serum ALP was <7 IU/L and plasma In the hypophosphatasia bone matrix, a calcification front PLP was 22,131 nmol/L. Urine phosphoethanolamine was (ie, a surface layer for the appositional growth of mineral) elevated. She expired at 2 weeks of age. was either not detected (cases 1 and 3) or was covered by thick layers of nonmineralized osteoid matrix (cases 2, Light and Electron Microscopy 4, and 5). In all cases, MVs were identified in the inter- stices between large collagen fibrils (Figure 3A). In bone Nondecalcified growth plates with adjacent metaphyses matrix beneath osteoblasts, a few MVs were intact and and cortical bone were fixed in 2.5% glutaraldehyde contained needles of apatite-like mineral (Figure 3A), buffered with cacodylate, washed, dehydrated, and em- whereas the majority appeared to be in the early propa- bedded in epoxy resin by standard procedures.12 Light gation phase of mineralization6 in which MV membranes microscopy was performed on plastic-embedded, 1-,um- are penetrated by small radial clusters of apatite-like thick sections after staining with toluidine blue. In case 1, crystals that project into the intercollagenous spaces parallel samples of growth plate and long bone were also (Figure 3B). In the normal growth plate, mineral clusters embedded in paraffin, sectioned at 4 ,um thickness and grow by surface accumulation of apatite-like crystals until stained with hematoxylin and eosin (H&E). Decalcifica- they ultimately fuse to form confluent areas of mineral. tion was not necessary because of the hypomineralized However, the growth and ultimate fusion of these radial state of the bones and was not carried out in any of the clusters of mineral appeared to be retarded in hypophos- cases. Ultrathin sections were stained with uranyl acetate phatasia, thus allowing the persistence of large areas of and lead citrate12 and examined using a Phillips EM 300 unmineralized bone matrix. or a Zeiss electron microscope. Discussion Results Our studies confirm and clarify the association between Confirmation of the Presence of low ALP catalytic activity and the impairment of skeletal Hypomineralization in Growth Plates and Bone mineralization expressed in perinatal (lethal) hypophos- phatasia. Light microscopy showed that mineral was diminished or Hypophosphatasia is characterized biochemically by absent from the growth plate and markedly reduced in a deficiency of TNSALP activity.20.21 The TNSALP the perichondrial, metaphyseal, and cortical bone in all isozyme of ALP is encoded on chromosome 1 p 36.1- five cases (Figure 1). The tibial growth plate in cases 1 34.22 In hypophosphatasia, inorganic pyrophosphate and 4 was misshapen with irregular projections of uncal- (PPi) accumulates in the extracellular fluid (ECF) appar- cified epiphyseal cartilage extending downward into the ently because hydrolysis of PPi is impeded by a defi- metaphysis. The metaphyseal cortex was devoid of min- ciency of TNSALP activity. PPi, in supraphysiological eral (Figure 1B) in cases 2, 3, and 4. However, in these amounts, has been shown to retard HAP crystal growth in cases, the central bone matrix of medullary bone trabec- vitro23 and thus would be expected to inhibit the propa- ulae showed some calcification deep to the osteoid layer gation of bone mineral, once initial crystal nuclei have (Figure 1C). In all cases, bone surfaces were covered by formed. As discussed below, accumulation of PPi sur- 1558 Anderson et al AJP December 1997, Vol. 151, No. 6

Figure 1. Photomicrographs of nondecalcified growth plate cartilage and bone in hypophosphatasia. A: Growth plate (G P1) and trabecular spicules of subjacent metaphyseal bone. Mineral deposits, staining intensely black in this tetrachrome silver stain, are absent from the growth plate and greatly reduced in bone trabecula. The latter are covered by abnormally large amounts of pink-staining osteoid (Ost). Marrow cells, located between bone trabecula, stain faintly blue. Patient 4, costochondral junction; magnification, X580. B: Totally unmineralized cortical bone (C) with adjacent fibrous periosteum (P) from upper tibial metaphysis of patient 1. Nondecalcified section, stained with H&E; magnification, X 1450. C: Medullary bone spicules composed mostly of surface layers of osteoid (Ost) covering central deposits of mineralized lamellar bone (M). Nondecalcified section from rib of patient 4, toluidine blue stain; magnification, X980. Matrix Vesicles of Hypophosphatasia Bone 1559 AJP December 1997, Vol. 151, No. 6

is~ - ;R_R 's w fAU Figure 2. Electron micrographs of MVs in hypophosphatasia growth plate, hypertrophic zone. A: MVs in an early stage of calcification with electron-dense internal mineral content and a suggestion of early linear crystal formation within the two lower vesicles. Granules of condensed proteoglycan (PG) are seen attached to the outer surfaces of MVs. Case 1, tibia; magnification, X115,000. B: MVs in a hypophosphatasia growth plate, showing a more advanced state of calcification with dense, needle-like apatite deposits (arrows) located mostly within vesicles. Crystal rigidity is causing a flattening of the vesicle profiles. Narrow, cartilage-type collagen fibrils traverse the field in a random arrangement. Case 2; magnification, X115,000. rounding ALP-deficient MVs in hypophosphatasia may First, it was proposed by Robison in the 1920s that the help to explain the rickets and osteomalacia characteris- phosphoesterase activity of ALP may hydrolyze endoge- tic of this condition. nous organic phosphate esters, thus releasing or- A notable clinical feature of hypophosphatasia is its thophosphate (Pi) for incorporation into nascent calcium extremely variable severity, ranging from death in utero, phosphate mineral in the skeleton.5 This hypothesis per- associated with almost complete failure of skeletal min- sists despite some problems, including the fact that cells eralization, to only premature loss of teeth in adults, with- of a variety of tissues, including liver and kidney, also out bone symptoms.24 Six clinical phenotypes of hy- express abundant TNSALP activity but do not mineralize. pophosphatasia have been characterized based upon, in Despite this apparent paradox, it remains possible that part, the age of discovery of skeletal abnormalities.21'24 A mineralization is augmented in the specific setting of genetic explanation for the variable severity of hypophos- skeletal tissues by the unique presence of ALP carriers phatasia appears to originate from the variety of defects such as MVs, which are selectively located at the miner- that may occur in the TNSALP gene.25 To date, 13 mis- alization fronts of cartilage, bone, and dentin.6 sense mutations have been reported in severely affected Second, it may be that ALP hydrolyzes and thus elimi- patients.26 28 Homozygosity or compound heterozygos- nates certain organic phosphate ester inhibitors of mineral- ity of specific TNSALP gene defects can explain the ization.23 The most frequently cited example, PPi, is nor- profoundly low ALP activity that has been observed in mally present at micromolar levels in extracellular fluids and perinatal (lethal) cases.26-28 However, as yet, we do not has been shown to inhibit HAP crystal growth in vitro by know the precise molecular basis of the hypophosphata- coating preformed HAP nuclei and thus preventing epitaxial sia affecting any of the five cases reported here (Dr. HAP crystal growth.29 Thus, in hypophosphatasia, as the Paula Henthorn, personal communication). The associa- concentration of endogenous PPi reaches supraphysiologi- tion of genetic defects that diminish TNSALP activity with cal levels, excess PPi could impede the growth of nascent secondary failure of skeletal and tooth mineralization in HAP clusters. hypophosphatasia patients indicates that at least one Third, a recent suggestion relates to the fact that ALP biological function of ALP is to promote calcification of is normally anchored upon the outer surfaces of MV hard tissues. As reviewed below, three major hypotheses membranes by linkage to phosphatidylinositol15 and that have been advanced to explain how ALP can promote ALP binds to collagen types 11 and X.30 Accordingly, it mineralization .523,29 was suggested that ALP might serve as a bridge-like 1560 Anderson et al AJP December 1997, Vol. 151, No. 6

Figure 3. Electron micrographs of MVs in the poorly mineralized bone matrix of hypophosphatasia. A: MVs appear as rounded electron-dense profiles between broad, banded, type 1 collagen fibrils running diagonally across the field. The MV at lower right is in an early stage of calcification, having acquired electron-dense amorphous content plus a few profiles of apatite-like mineral. Its membrane is still faintly visible. Case 2, metaphysis; magnification, X220,000. B: MV of bone in detail showing an intact membrane and containing rigid, needle-like deposits of apatite-like mineral. Case 1, tibial metaphysis; magnification, X275,000.

conduit across which initial mineral, which forms within clusters. Thus, the defect in skeletal mineralization in MVs, may proliferate and spread to involve adjacent col- hypophosphatasia appears to occur after crystal initia- lagen fibrils. This hypothesis does not require ALP cata- tion, during propagation of HAP from MVs into the sur- lytic activity to explain mineral propagation into the ma- rounding collagenous matrix. Our studies confirm the trix. In hypophosphatasia where the TNSALP molecules observation of Ornoy et al18 by showing the presence of may be present at MV surfaces but rendered inactive by approximately normal numbers of MVs in hypophos- mutations inhibiting catalytic activity, the mineral-bridg- phatasia in a normal distribution. However, in contrast to ing activity of ALP could remain intact. However, it re- the conclusions of Ornoy et al18 and Terada et al,19 we mains a possibility that TNSALP gene mutations could found that hypophosphatasia MVs are capable of initiat- also alter the dimeric or tetrameric structure of TNSALP at ing internal mineralization, at least in the near-term or MV surfaces and thereby impede mineralization by phys- neonatal patients we studied. The presence of non-ALP ical disruption of TNSALP bridging. phosphohydrolases within hypophosphatasia MVs, eg, Clearly, a combination of more than one of the above ATPase, PPiase, 5'-AMPase, etc, may account for this pathogenic mechanisms may lead to the defective min- ability of hypophosphatasia MVs to initiate mineralization eralization characteristic of hypophosphatasia. Not only despite a deficiency of ALP catalytic activity. may defective ALP catalytic activity fail to hydrolyze PPi, thus providing insufficient Pi to support initial mineral growth, but also the resulting buildup of unhydrolyzed PPi Acknowledgments in the extracellular fluid could inhibit the outward prolifer- ation of HAP from initial mineralization sites. Plasma pyridoxal 5'-phosphate (PLP) was measured Regarding the role of MVs in the pathogenesis of hy- courtesy of Dr. Stephen P. Coburn, at the Ft. Wayne pomineralization in hypophosphatasia, our observations Developmental Center, Ft. Wayne, IN. Mr. Paul Moylan suggest that, in this inborn error of metabolism, MVs was responsible for tissue preparation for transmission retain the capability to generate nascent HAP mineral electron microscopy. Matrix Vesicles of Hypophosphatasia Bone 1561 AJP December 1997, Vol. 151, No. 6

References 17. Hsu HHT, Anderson HC: A role for ATPase in the mechanisms of ATP-dependent Ca and phosphate deposition by isolated rachitic 1. Weiss MJ, Henthorn PS, Lafferty MK, Slaughter C, Raducha M, Harris matrix vesicles. Int J Biochem Cell Biol 1995, 27:1349-1356 H: Isolation and characterization of a cDNA encoding a human liver/ 18. Ornoy A, Adomian GE, Rimoin DL: Histologic and ultrastructural bone/kidney-type alkaline phosphatase. Proc Natl Acad Sci USA studies on the mineralization process in hypophosphatasia. Am J 1986, 83:7182-7186 Med Genet 1985, 22:743-758 2. Anderson HC, Sajdera SW: Calcification of rachitic cartilage to study 19. Terada S, Suzuki N, Ueno H, Uchid K, Kohama T: A congenital lethal matrix vesicle function. Fed Proc 1976, 35:148-153 form of hypophosphatasia: histologic and ultrastructural study. Acta 3. Gutman AB, Yu TF: A concept of the role of enzymes in endochondral Obstet Gynecol Scand 1996, 75:502-505 calcification. Metabolic Interrelations, vol 2. New York, Josiah Macy Jr 20. Caswell AM, Whyte MP, Russell RGG: Hypophosphatasia and the Foundation 1950, pp 167-190 extracellular metabolism of inorganic pyrophosphate: clinical and 4. Matsuzawa T, Anderson HC: Phosphatases of epiphyseal cartilage laboratory aspects. Crit Rev Clin Lab Sci 1991, 28:175-232 studied with electron microscopic cytochemical methods. J Histo- 21. Whyte MP: Hypophosphatasia and the role of alkaline phosphatase in chem Cytochem 1971, 19:801-808 skeletal mineralization. Endocr Rev 1994, 15:439-461 5. Robison R, Rosenheim AH: Calcification of hypertrophic cartilage in 22. Swallow DM, Porvey S, Parkar M: Mapping for the gene coding for the vitro. Biochem J 1934, 28:684-698 human liver/bone/kidney isozyme of alkaline phosphatase to chromo- 6. Anderson HC, Morris DC: Mineralization. Handbook Exp Pharmacol some 1. Ann Hum Genet 1986, 50:229-235 1993, 107:267-298 23. Fleisch H, Bisaz S: Mechanism of calcification: inhibitory role of 7. Bellows CG, Rubin JE, Heersch JNM: Initiation and progression of pyrophosphate. Nature 1962, 195:911 mineralization of bone nodules formed in vitro: the role of alkaline 24. Whyte MP, Walkenhorst DA, Fedde KN, Henthorn PS, Hill CS: phosphatase and organic phosphate. Bone Miner 1991, 14:27-40 Hypophosphatasia: levels of bone alkaline phosphatase immunore- 8. Fallon MD, Whyte MP, Teitelbaum SL: Stereospecific inhibition of activity in serum reflect disease severity. J Clin Endocrinol Metab alkaline phosphatase by L-tetramisole prevents in vitro cartilage cal- 1996, 81:2142-2148 cification. Lab Invest 1980, 43:489-494 25. Fedde KN, Henthorn PS, Whyte MP: Aberrant properties of alkaline 9. Hsu HHT, Anderson HC: A simple and defined method to study phosphatase in patient fibroblasts correlate with clinical expressivity calcification by isolated matrix vesicles: effect of ATP and vesicle in severe forms of hypophosphatasia. J Clin Endocrinol Metab 1996, phosphatase. Biochem Biophys Acta 1977, 500:162-172 81:2587-2594 10. McCance RA, Fairweather DV, Barrett AM, Morrison AB: Genetic, 26. Henthorn PS, Raducha M, Fedde KN, Lafferty MA, Whyte MP: Differ- clinical, biochemical and pathological features of hypophosphatasia. ent missense mutations at the tissue-nonspecific alkaline phos- o J Med 1956, 25:523-538 phatase gene locus in autosomal recessively inherited forms of mild 11. Whyte MP, Magill HL, Fallon MD, Herrod HG: Infantile and severe hypophosphatasia. Proc Natl Acad Sci USA 1992, 89: hypophosphatasia: normalization of circulating bone alkaline phos- 9924-9928 phatase activity followed by skeletal remineralization. J Pediatr 1986, 27. Ozona K, Yamagata M, Michigami T, Nakaiima S, Sakai N, Satomu- 108:82-88 rak, Yasui N, Okada S, Nakayama M: Identification of novel missense 12. Anderson HC: Vesicles associated with calcification in the matrix of mutations (Phe 310 Leu and Gly 439 Ang) in a neonatal case of epiphyseal cartilage. J Cell Biol 1969, 41:59-72 hypophosphatasia. J Clin Endocrinol Metab 1996, 81:4458-4461 13. Ali SY, Sajdera SW, Anderson HC: Isolation and characterization of 28. Weiss MJ, Cole DEC, Ray K, Whyte MP, Lafferty MS, Mulivor RA, calcifying matrix vesicles from epiphyseal cartilage. Proc Natl Acad Harris H: A missense mutation in the human liver/bone/kidney alkaline Sci USA 1970, 67:1513-1520 phosphatase gene causing a form of lethal hypophosphatasia. Proc 14. Akisaka T, Gay CV: Ultrastructural localization of calcium-activated Natl Acad Sci USA 1988, 85:7666-7669 adenosine triphosphatase (Ca+-ATPase) in growth plate cartilage. 29. Termine JD, Conn KM: Inhibition of apatite formation by phosphory- J Histochem Cytochem 1985, 33:925-932 lated metabolites and macromolecules. Calcif Tissue Res 1976, 22: 15. Low JG: Biochemistry of the glycosyl-phosphatidylinositol membrane 149-157 protein anchors. Biochem J 1987, 224:1-13 30. Wu LNY, Genge BR, Lloyd GC, Wuthier RE: Collagen binding proteins 16. Ali SY, Evans L: The uptake of 45Ca calcium ions by matrix vesicles in collagenase-released matrix vesicles from cartilage. J Biol Chem isolated from calcifying cartilage. Biochem J 1973, 134:647-650 1991, 266:1195-1203