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Physiological mineralization is necessary for the formation of skeletal tissues and for their appropriate functions during adulthood. Pathological or ectopic mineralization of soft tissues, including articular cartilage and cardiovascular tissues, leads to morbidity and mortality. Recent findings suggest that the mechanisms and factors regulating physiological mineralization may be identical or similar to those regulating ectopic mineralization. Therefore, the purpose of this review is to describe the current knowledge of mechanisms and determinants that regulate physiological mineralization and how these determinants can be used to understand ectopic mineralization better.Recent findings have indicated that physiological and pathological mineralization are initiated by matrix vesicles, membrane-enclosed particles released from the plasma membrane of mineralization-competent cells. An understanding of how these vesicles initiate the physiological mineralization process may provide novel therapeutic strategies to prevent ectopic mineralization. In addition, other regulators (activators and inhibitors) of physiological mineralization have been identified and characterized, and evidence indicates that the same factors also contribute to the regulation of ectopic mineralization. Finally, programmed cell death (apoptosis) may be a contributor to physiological mineralization, and if occurring after tissue injury may induce ectopic mineralization and mineralization-related differentiation events in the injured area and surrounding areas.This review describes how the understanding of mechanisms and factors regulating physiological mineralization can be used to develop new therapeutic strategies to prevent pathological or ectopic mineralization events.

Purpose of review Our understanding of the causation of the chondrocalcinosis and other disorders characterized by ectopic mineralization is rapidly increasing, and genetic studies have contributed substantially to recent major advances in the field. This review will discuss what is known about the genetics of chondrocalcinosis and what we have learned from genetic studies to date. Recent findings Chondrocalcinosis is one of a family of conditions associated with ectopic mineralization. This family also includes disorders of mineralization of bone and spinal and other ligaments, and vascular calcification. There has been increasing evidence of the key role of transport and metabolism of inorganic pyrophosphate (PPi) in control of mineralization, and as the likely explanation for the association of a variety of genetic variants with chondrocalcinosis and ectopic mineralization elsewhere. This may be an overly simplistic view of this family of conditions, with recent evidence suggesting that, for example, ANKH variants may not all predispose to chondrocalcinosis by effects on PPi transport, but may also influence chondrocyte maturation. Summary Understanding the control of the process of mineralization and its tissue specificity are important steps in the search for rational therapies for these conditions.

Introduction: Pseudoxanthoma elasticum (PXE), a multisystem orphan disease, clinically affects the skin, the eyes and the cardiovascular system with considerable morbidity and mortality. The clinical manifestations reflect the underlying pathology consisting of ectopic mineralization of peripheral connective tissues. Areas covered: The diagnostic criteria of PXE include characteristic clinical findings, together with histopathology of accumulation of pleiomorphic elastic structures in the dermis with progressive mineralization, and the presence of mutations in the ABCC6 gene. PXE-like cutaneous changes can also be encountered in other ectopic mineralization disorders, including generalized arterial calcification of infancy (GACI) caused by mutations in the ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) gene. In some cases, overlapping clinical features of PXE/GACI, associated with mutations either in ABCC6 or ENPP1, have been noted. PXE demonstrates considerable inter- and intrafamilial hetero...

BACKGROUND: Pseudoxanthoma elasticum (PXE) is characterized by skin (papular lesions), ocular (subretinal neovascularisation) and cardiovascular manifestations (peripheral artery disease), due to mineralization and fragmentation of elastic fibres in the extracellular matrix (ECM). Caused by mutations in the ABCC6 gene, the mechanisms underlying this disease remain unknown. The knowledge on the molecular background of soft tissue mineralization largely comes from insights in vascular calcification, with involvement of the osteoinductive Transforming Growth Factor beta (TGFbeta) family (TGFbeta1-3 and Bone Morphogenetic Proteins [BMP]), together with ectonucleotides (ENPP1), Wnt signalling and a variety of local and systemic calcification inhibitors. In this study, we have investigated the relevance of the signalling pathways described in vascular soft tissue mineralization in the PXE knock-out mouse model and in PXE patients. METHODS: The role of the pro-osteogenic pathways BMP2-SMADs-RUNX2, TGFbeta-SMAD2/3 and Wnt-MSX2, apoptosis and ER stress was evaluated using immunohistochemistry, mRNA expression profiling and immune-co-staining in dermal tissues and fibroblast cultures of PXE patients and the eyes and whiskers of the PXE knock-out mouse. Apoptosis was further evaluated by TUNEL staining and siRNA mediated gene knockdown. ALPL activity in PXE fibroblasts was studied using ALPL stains. RESULTS: We demonstrate the upregulation of the BMP2-SMADs-RUNX2 and TGFbeta-2-SMAD2/3 pathway, co-localizing with the mineralization sites, and the involvement of MSX2-canonical Wnt signalling. Further, we show that apoptosis is also involved in PXE with activation of Caspases and BCL-2. In contrast to vascular calcification, neither the other BMPs and TGFbetas nor endoplasmic reticulum stress pathways seem to be perturbed in PXE. CONCLUSIONS: Our study shows that we cannot simply extrapolate knowledge on cell signalling in vascular soft tissue calcification to a multisystem ectopic mineralisation disease as PXE. Contrary, we demonstrate a specific set of perturbed signalling pathways in PXE patients and the knock-out mouse model. Based on our findings and previously reported data, we propose a preliminary cell model of ECM calcification in PXE

Mineralization of bone and tooth extracellular matrix (ECM) is a physiologic process, while soft tissue mineralization, also known as ectopic mineralization (calcification), is a pathologic condition. Vascular calcification is common in aging and also in a number of genetic and metabolic disorders. The calcific deposits in arteries complicate the prognosis and increase the morbidity in diseases such as atherosclerosis, diabetes and chronic kidney disease (CKD). To completely understand the pathophysiology of these lifethreatening diseases, it is critical to elucidate the molecular mechanisms underlying vascular calcification. Unveiling these mechanisms will eventually identify new therapeutic targets and also improve the management of the associated complications. In the current review, we discussed the common determinants of ECM mineralization, the mechanism of vascular calcification associated with several human diseases and outlined the most common therapeutic approaches to prevent its progression.

生後1年以上のニワトリの足の腱には, 石灰化物の沈着がみられる。さらに, 電子顕微鏡観察によれば, すでに生後100日以上の個体においてその徴候が認められる。本研究では, ニワトリ足腱の異所性石灰化部位の無機成分と有機マトリックス成分をプラズマ重合膜法によりオスミウムコートし, その走査電子顕微鏡観察を行った。その結果, コラーゲンマトリックスと石灰化物のそれぞれに, 新規の構造物を見いだした。第1に, 有機成分を溶解した後には長径が1.86±0.38μm (平均±標準偏差), 短径が1.05±0.16μm (平均±標準偏差) の楕円形の石灰化顆粒がみられ, それらはさらに融合して多数の細長いロッド状の構造を形成していた。さらに透過電顕観察では, 石灰化顆粒の表面には特徴的な67nmの周期的な縞模様が検出され, 本構造が生体内ではコラーゲン線維と接して存在していることが明らかとなった。一方, 脱灰処理後の腱のコラーゲン線維には, 2.8±0.38μmの周期的な波状構造が観察された。しかも, その一端のみが波状を呈する領域が観察され, その場合, 波状部分は必ず石灰化側に, 非波状部分は非石灰化側に位置していた。これらの結果は, 波状のコラーゲン線維構造が顆粒状の石灰化物生成を誘導している可能性を示唆している。

Biomineralization is a multifactorial and complex process, which results in the deposition of mineral crystals in the extracellular matrix of various tissues. Physiological mineralization is restricted to tissues, such as bones, teeth, and certain areas of cartilage. Pathological or ectopic mineralization can occur in many soft tissues, including articular cartilage, cardiovascular tissues, kidney, ligaments, and tendons, and can lead to serious problems. Therefore, the understanding of factors and mechanisms that regulate the mineralization process is essential for the development of novel therapeutic strategies to prevent or inhibit ectopic mineralization. This review will discuss some of the mechanisms and factors that regulate physiological mineralization and their potential roles in ectopic mineralization. Finally, potential therapeutic approaches for the treatment of ectopic mineralization are being discussed.

This work analyzes the data of contemporary scientific literature regarding the ectopic mineralization in the prostate gland, its pathogenetic features are considered.The scientific literature of recent decades gives grounds to assert that the processes of concrement formation in the prostate gland are influenced by many factors, pathological mineralization can be realized by different mechanisms. They include chronic inflammation, stagnation fractions in gland, reflux of urine from the urethra at intravesicle obstruction, malformation of prostate and seminal vesicles, specific inflammation, polymorphism of gene protein inhibitors of calcification. These mechanisms areinterconnected, each of them may participate in the overall development of concrement formation in the prostate.In recent years, due to improved instrumental diagnosis we observe a significant increase of the number of patients, who were found with pathogenic prostate gland bioliths, which requires more detailed and in-depth study of the mechanisms of mineral formation in the prostate., В настоящей работе на основании анализа данных современной научной литературы установлено, что процессы эктопического минералообразования в предстательной железе могут реализовываться посредством воздействия различных этиологических факторов. К ним относятся хроническое воспаление, застойные явления в железе, рефлюкс мочи из уретры при интравезикальной обструкции, мальформации предстательной железы и семенных пузырьков, специфическое воспаление, полиморфизм генов белков-ингибиторов кальцификации. Механизмы эктопической минерализации взаимосвязаны, каждый из них может принимать участие в общем развитии конкрементообразования в предстательной железе. В связи с улучшением инструментальной диагностики в последние годы произошло значительное увеличение количества пациентов, у которых были выявлены конкременты предстательной железы, что требует более детального и углубленного изучения причин и механизмов их развития, У роботі на основі аналізу даних сучасної наукової літератури встановлено, що процеси ектопічного мінералоутворення в передміхуровій залозі можуть реалізовуватися через вплив різних етіологічних факторів. До них належать хронічне запалення, застійні явища у залозі, рефлюкс сечі з сечівника при інтравезикулярній обструкції, мальформації простати та сім’яних міхурців, специфічне запалення, поліморфізм генів білків-інгібіторів кальцифікації. Механізми ектопічної мінералізації взаємопов'язані, кожний з них може брати участь у загальному розвитку конкрементоутворення в передміхуровій залозі. За останні роки, у зв’язку з покращенням інструментальної діагностики відбулося значне зростання числа хворих, у яких були виявлені патогенні біоліти передміхурової залози, що потребує більш детального і поглибленого вивчення механізмів мінералоутворення у передміхуровій залозі.

A patient is presented with severe cutis laxa of the abdomen. Molecular investigations, including sequencing of the fibulin-5 and elastin gene, failed to endorse the diagnosis of inherited cutis laxa. Ultrasonographical discovery of renal calcifications during his general work-up suggested a possible diagnosis of pseudo-xanthoma elasticum (PXE). A discrete yellowish reticular pattern in the anterior neck region was detected upon careful clinical examination. Clinically, the patient presented characteristics of both classic PXE (retinopathy, renal calcifications) and the PXE-like syndrome (cutis laxa beyond the flexural areas). Skin biopsy and ophthalmological examination confirmed the diagnosis of PXE. In addition, ultrastructural evaluation revealed calcium deposits in the periphery of elastic fibers, a typical observation in the PXE-like syndrome. Immunohistochemical experiments and ELISA tests for various inhibitors of calcification displayed results which were partly reminiscent of both PXE and the PXE-like syndrome. Molecular analysis revealed not only two ABCC6 mutations (related to PXE), but also a gain of function SNP in the GGCX gene, in which loss-of-function mutations cause the PXE-like syndrome. We conclude that the patients phenotype and--to a further extent--the PXE-like syndrome, are part of a spectrum of ectopic calcification disorders which are clinically and/or pathogenetically related to PXE. The molecular findings in this patient are however insufficient to explain the entire phenotype and suggest a role for multiple genetic factors in soft tissue mineralization.

Pseudoxanthoma elasticum (PXE) is a heritable multisystem disorder manifesting with ectopic calcification of peripheral connective tissues, caused by mutations in the ABCC6 gene. Alterations in vitamin K metabolism have been suggested to contribute to the pathomechanisms of the mineralization process. In this study we administered vitamin K or its glutathione conjugate (K3-GSH) into Abcc6 (-/-) mice which recapitulate features of PXE. Oral administration of vitamin K2 in dosages, which vastly exceed the amounts in control diet or the recommended amounts for humans, did not alter the ectopic mineralization in Abcc6 (-/-) mice. Similarly, intravenous administration of K3-GSH did not alter the degree of mineralization. Testing of vitamin K2, K3 and K3-GSH in an in vitro calcification system provided no evidence of mineralization inhibition. Collectively, our data suggest that vitamin K deficiency in the peripheral tissues is not a simple explanation for development of mineral deposits in PXE.

У роботі на основі аналізу даних сучасної наукової літератури встановлено, що процеси ектопічного мінералоутворення в передміхуровій залозі можуть реалізовуватися через вплив різних етіологічних факторів. До них належать хронічне запалення, застійні явища у залозі, рефлюкс сечі з сечівника при інтравезикулярній обструкції, мальформації простати та сім'яних міхурців, специфічне запалення, поліморфізм генів білків-інгібіторів кальцифікації. Механізми ектопічної мінералізації взаємопов'язані, кожний з них може брати участь у загальному розвитку конкрементоутворення в передміхуровій залозі. За останні роки, у зв'язку з покращенням інструментальної діагностики відбулося значне зростання числа хворих, у яких були виявлені патогенні біоліти передміхурової залози, що потребує більш детального і поглибленого вивчення механізмів мінералоутворення у передміхуровій залозі. При цитуванні документа, використовуйте посилання http://essuir.sumdu.edu.ua/handle/123456789/26170 В роботе на основе анализа данных современной научной литературы установлено, что процессы эктопического минералообразования в предстательной железе могут реализовываться по средствам влияния разних факторов. К ним принадлежат хроническое воспаление, застойные явления в железе, рефлюкс мочи из мочеточника при интравезикулярной обструкции, мальформации простаты и семенных пузырьков, специфическое воспаление, полиморфизм генов белков-ингибиторов кальцификации. Механизмы эктопической минерализации взаимосвязаны, каждый из них может участвовать в общем развитии конкрементоутворення в предстательной железе. За последние годы, в связи с улучшением инструментальной диагностики состоялся значительный рост числа больных, в которых были выявлены патогенные биолиты предстательной железы, которая нуждается в более детальном и углубленном изучении механизмов минералообразования в предстательной железе. При цитировании документа, используйте ссылку http://essuir.sumdu.edu.ua/handle/123456789/26170 This work analyzes the data of contemporary scientific literature regarding the ectopic mineralization in the prostate gland, its pathogenetic features are considered. The scientific literature of recent decades gives grounds to assert that the processes of concrement formation in the prostate gland are influenced by many factors, pathological mineralization can be realized by different mechanisms. They include chronic inflammation, stagnation fractions in gland, reflux of urine from the urethra at intravesicle obstruction, malformation of prostate and seminal vesicles, specific inflammation, polymorphism of gene protein inhibitors of calcification. These mechanisms are interconnected, each of them may participate in the overall development of concrement formation in the prostate. In recent years, due to improved instrumental diagnosis we observe a significant increase of the number of patients, who were found with pathogenic prostate gland bioliths, which requires more detailed and in-depth study of the mechanisms of mineral formation in the prostate. When you are citing the document, use the following link http://essuir.sumdu.edu.ua/handle/123456789/26170

Pseudoxanthoma elasticum (PXE) is a rare autosomal recessive disorder characterized by ectopic mineralization of connective tissue in the skin, eyes and vascular organs. Multiple small yellowish papules appear in the skin in the flexion areas of large joints, the neck and the periumbirical region. Calcified degenerated elastic fibers are observed upon pathological analysis. PXE is caused by mutations in the ABCC6 gene; however, it is not known how such mutations contribute to the symptoms of PXE [...]

Soft tissue calcification in the human body can be considered part of a process of continuous degeneration which we tend to designate as “aging”. Being an example of technological wit and superb bio-engineering second to none, even the decay of this corpus can hardly be considered a random or passive event. On the contrary, calcium precipitation is regulated quite tightly by an intruiging interplay between stimulatory proteins and inhibitory factors. Thus, it has been foreseen man not to be turned into a chalk pillar in his prime years, but rather to endure a much slower process of gradual mineralization. But when this brilliant regulatory opus starts failing, the reign of human pathology is entered, confronting the body with ectopic mineralization disorders. One of the archetypes of such disease is pseudoxanthoma elasticum or PXE, in which ectopic mineralization of elastic fibres causes skin, ocular and cardiovascular complications. Despite its identification more than two centuries ago, PXE has – as many genetic disorders – always been surrounded by a haze of mystery. It is the aim of this thesis to contribute to the clinical, molecular and histopathological characterization of this fascinating disease. Through careful characterization of the PXE patient cohort followed at the Ghent Center for Medical Genetics, we were able to emphasize important clinical features, such as stroke and peripheral artery disease, as well as identifying novel phenotypical features in patients and carriers, among which were abdominal calcifications and testicular microlithiasis. Also the question of a limited or subclinical phenotype in PXE carriers was addressed and we showed them to be more prone to cardiovascular disease, next to limited ophthalmological symptoms represented by comets and comet tails. In an exploratory pilot study among over 200 consecutive ischemic stroke patients, ABCC6 hotspot analysis yielded a significant increase in ABCC6 mutations compared to a healthy reference population. This signified another example of heterozygous carriers being prone to cardiovascular and/or cerebrovascular disease and introduced the ABCC6 gene in stroke research. In single and multi-center studies, this thesis contributed to the characterization and expansion of the ABCC6 mutation spectrum, as well as the exclusion of genotype-phenotype correlations. The applied molecular strategy for mutation analysis of the ABCC6 gene proved to be an efficient and cost-effective method, yielding the highest mutation detection rate so far. Also, the continuous discussion on the mode of inheritance and in particular the existence of an autosomal dominant form of PXE could be addressed constructively. Throughout the clinical follow-up of PXE patients, we applied novel fundus imaging techniques, such as autofluorescence and infrared imaging, with substantial improvement of the diagnostic capacities of limited or subtle lesions in fundo. Through collaborative efforts, the importance of electrophysiological abnormalities – subdivided in three retinopathy phenotypes – was brought to attention. Within the span of this PhD thesis, a novel phenotype was identified and characterized both clinically and molecularly. This novel autosomal recessive disorder was coined the PXE-like syndrome, because of its resemblance with classic PXE, and was proven to be caused by mutations in the GGCX gene. Encoding the gamma-carboxylase, an enzyme important in the vitamin K (VK)-cycle, this observation implicated VK and proteins depending on this vitamin – among which are several inhibitors of mineralization – in the pathogenesis of the PXE-like syndrome and hence PXE. Through various immunohistochemical and ELISA methods, VK-dependent inhibitors of calcification were shown to be inactive or defective in these syndromes, leading to ectopic mineralization in the PXE-like syndrome but also in PXE patients. These observations could be attributed to the GGCX mutations in the PXE-like syndrome. The observation of extremely low VK serum levels – an essential co-factor for protein carboxylation in the VK-cycle – in PXE patients explained why the VK-cycle is defective in PXE. The exact link with the impaired ABCC6 transporter remains unclear, although it is tempting to think of VK or one of its associated molecules as the substrate of ABCC6. Also, these findings hold out the prospect of VK suppletion as a treatment for PXE. As such, the findings summarized in this thesis have elaborated the clinical and molecular knowledge of PXE and related disorders, and have opened novel avenues for further fundamental and applied research in the field of ectopic mineralization. Above all, they have benefitted patients and their family though a more efficient molecular diagnosis, a more to-the- point follow-up and the prospect of a treatment for their burdensome disease.

ejd.2012.1718 Auteur(s) : Mikiko Michigami1, Hitoshi Kudo1, Akira Iwanaga2, Atsushi Utani2 utani@nagasaki-u.ac.jp 1 Department of Dermatology, Hyogo Prefectural Amagasaki Hospital, Hyogo, Japan 2 Department of Dermatology, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan Pseudoxanthoma elasticum (PXE) is an autosomal recessive disorder that causes ectopic mineralization of soft connective tissues and mainly affects the eyes, skin, and cardiovascular system [1]. The [...]

Bisphosphonates are non-biodegradable compounds characterized by a phosphorus-carbon-phosphorus bond. By substituting the hydrogens on the carbon atom, a variety of bisphosphonates can be synthesized, each with distinct physical-chemical, biologic, therapeutic, and toxicologic characteristics. Bisphosphonates have in general a strong affinity to calcium phosphates, both in vitro and in vivo. They inhibit bone resorption through a cellular mechanism that is not yet completely understood. When given in large amounts, some bisphosphonates can inhibit normal and ectopic mineralization through a physical-chemical mechanism. The main difference among the various compounds appears to be their potency in inhibiting bone resorption, which can vary from 1 to 10,000. The potential efficacy of bisphosphonates in osteoporosis has been investigated extensively in animals. In the growing rat, they induce an increase in intestinal absorption and body retention of calcium. Various types of experimental osteoporosis, such as induced by immobilization, ovariectomy, administration of corticosteroids, or low calcium diet, can be prevented. Bisphosphonates are rapidly cleared from plasma, with 20%-60% deposited in bone and the remainder excreted in the urine. The half-life in bone is, however, very long. The toxicity of bisphosphonates is low, probably because of their rapid plasma and soft tissue clearance. It varies greatly from compound to compound. Bisphosphonates are used successfully in diseases with increased bone turnover, such as Paget's disease of bone, tumoral bone disease, and recently, osteoporosis. Most results in osteoporosis have been obtained with etidronate and pamidronate. Both of these compounds, as well as other bisphosphonates, such as tiludronate, alendronate, and clodronate, inhibit bone loss and sometimes even increase bone mass.(ABSTRACT TRUNCATED AT 250 WORDS)

Pseudoxanthoma elasticum (PXE) is a genetic multisystem disorder characterized by ectopic mineralization of connective tissues with primary manifestations in the skin, retina and cardiovascular system, and a phenotypic spectrum highly variable.This article presents the case of a 46-year-old male patient with sporadic late-onset PXE, without severe systemic complications.

In summary, without a basic knowledge of embryology and development of human hair many abnormalities that occur in early postnatal life cannot be classified. Classification of abnormalities is necessary if specific genetic or biochemical causes are to be looked for and prognosis and genetic counseling accurately assessed.

A case of renal osteodystrophy with gross rim calcification of a parathyroid gland demonstrated radiographically in vivo is presented. The etiology of such ectopic mineralization is unknown, although subcapsular hemorrhage into the parathyroid glands may have contributed.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T18:52:46Z No. of bitstreams: 1 S0100-879X2006000500006.pdf: 799666 bytes, checksum: e8b560b21187e64c3fa3f68c4e9efcff (MD5) Made available in DSpace on 2013-08-22T18:52:46Z (GMT). No. of bitstreams: 1 S0100-879X2006000500006.pdf: 799666 bytes, checksum: e8b560b21187e64c3fa3f68c4e9efcff (MD5) Previous issue date: 2006-05-01 Made available in DSpace on 2013-09-30T18:00:37Z (GMT). No. of bitstreams: 2 S0100-879X2006000500006.pdf: 799666 bytes, checksum: e8b560b21187e64c3fa3f68c4e9efcff (MD5) S0100-879X2006000500006.pdf.txt: 28452 bytes, checksum: 6daf63a33ab513f8a6628495b20f6f68 (MD5) Previous issue date: 2006-05-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:17:48Z No. of bitstreams: 2 S0100-879X2006000500006.pdf: 799666 bytes, checksum: e8b560b21187e64c3fa3f68c4e9efcff (MD5) S0100-879X2006000500006.pdf.txt: 28452 bytes, checksum: 6daf63a33ab513f8a6628495b20f6f68 (MD5) Made available in DSpace on 2014-05-20T13:17:48Z (GMT). No. of bitstreams: 2 S0100-879X2006000500006.pdf: 799666 bytes, checksum: e8b560b21187e64c3fa3f68c4e9efcff (MD5) S0100-879X2006000500006.pdf.txt: 28452 bytes, checksum: 6daf63a33ab513f8a6628495b20f6f68 (MD5) Previous issue date: 2006-05-01 Endochondral calcification involves the participation of matrix vesicles (MVs), but it remains unclear whether calcification ectopically induced by implants of demineralized bone matrix also proceeds via MVs. Ectopic bone formation was induced by implanting rat demineralized diaphyseal bone matrix into the dorsal subcutaneous tissue of Wistar rats and was examined histologically and biochemically. Budding of MVs from chondrocytes was observed to serve as nucleation sites for mineralization during induced ectopic osteogenesis, presenting a diameter with Gaussian distribution with a median of 306 ± 103 nm. While the role of tissue-nonspecific alkaline phosphatase (TNAP) during mineralization involves hydrolysis of inorganic pyrophosphate (PPi), it is unclear how the microenvironment of MV may affect the ability of TNAP to hydrolyze the variety of substrates present at sites of mineralization. We show that the implants contain high levels of TNAP capable of hydrolyzing p-nitrophenylphosphate (pNPP), ATP and PPi. The catalytic properties of glycosyl phosphatidylinositol-anchored, polidocanol-solubilized and phosphatidylinositol-specific phospholipase C-released TNAP were compared using pNPP, ATP and PPi as substrates. While the enzymatic efficiency (k cat/Km) remained comparable between polidocanol-solubilized and membrane-bound TNAP for all three substrates, the k cat/Km for the phosphatidylinositol-specific phospholipase C-solubilized enzyme increased approximately 108-, 56-, and 556-fold for pNPP, ATP and PPi, respectively, compared to the membrane-bound enzyme. Our data are consistent with the involvement of MVs during ectopic calcification and also suggest that the location of TNAP on the membrane of MVs may play a role in determining substrate selectivity in this micro-compartment. Universidade de São Paulo Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto Departamento de Química The Burnham Institute Universidade Estadual Paulista Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal Departamento de Tecnologia Universidade Estadual Paulista Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal Departamento de Tecnologia