Appearance Of Alkaline Phosphatase Activity Research Articles

Retracted: Mouse ES cells have a potential to differentiate into odontoblast‐like cells using hanging drop method

We examined whether mouse embryonic stem (ES) cells can differentiate into odontoblast-like cells without epithelial-mesenchymal interaction. Cells were cultured by the 'hanging drop' method using a collagen type-I scaffold (CS) combined with bone morphogenetic protein (BMP)-4 (CS/BMP-4). Expression of odontoblast-related mRNA and protein, and cell proliferation were performed by reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence staining and WST-1 assay, respectively. Cells potently expressed odontoblast-related cell marker mRNAs following induction of odontoblastic differentiation. Dentin sialophosphoprotein, a marker of mature odontoblasts, was strongly expressed in differentiated ES cells. The cells also acquired an odontoblast-like functional phenotype, as evidenced by the appearance of alkaline phosphatase activity and calcification. The cell-surface expression of α2, α6, αV and αVβ3 integrin proteins was rapidly upregulated in differentiated cells. Finally, anti-α2 integrin antibody suppressed the expression of odontoblastic markers in cells grown using this culture system, suggesting that α2 integrin expression in ES cells triggers their differentiation into odontoblast-like cells. Mouse ES cells cultured by the 'hanging drop' method are able to differentiate into cells with odontoblast-specific physiological functions and cell-surface integrin protein expression.

Multiwall carbon nanotube scaffolds for tissue engineering purposes

The use of scaffolds composed of a major fraction of multiwall carbon nanotubes (MWCNT, up to 89 wt.%) and a minor one of chitosan (CHI), and with a well-defined microchannel porous structure as biocompatible and biodegradable supports for culture growth is described. Cell adhesion, viability and proliferation onto the external surface of MWCNT/CHI scaffolds with C2C12 cell line (myoblastic mouse cell), which is a multipotent cell line able to differentiate towards different phenotypes under the action of some chemical or biological factors, has been evaluated in vitro and quantified by MTT assays. The evolution of the C2C12 cell line towards an osteoblastic lineage in presence of the recombinant human bone morphogenetic protein-2 (rhBMP-2) has also been studied both in vitro (e.g., following the appearance of alkaline phosphatase activity) and in vivo (e.g., by implantation of MWCNT/CHI scaffolds adsorbed with rhBMP-2 in muscle tissue and evaluation of the ectopic formation of bone tissue).

Hydroxyapatite accelerates differentiation and suppresses growth of MC3T3-E1 osteoblasts.

Hydroxyapatite describes both the natural mineral phase of bone as well as the widely used calcium-phosphate implant substitute. Given that hydroxyapatite is a major component of the in vivo surface with which osteoblasts interact, it is surprising that most studies examining the regulation of osteoblast growth and differentiation utilize plastic surfaces. Here we demonstrate that the phenotype of mouse MC3T3-E1 osteoblasts is significantly altered on hydroxyapatite compared with plastic surfaces. Specifically, alkaline phosphatase activity and messenger RNA levels, markers of early stages of osteoblast differentiation, are increased in osteoblasts cultured on hydroxyapatite. The precocious appearance of alkaline phosphatase activity on the hydroxyapatite surface suggests that osteoblast differentiation is activated earlier compared with plastic surfaces. Osteocalcin expression, a marker of late-stage differentiation, is also increased on hydroxyapatite and further demonstrates enhanced differentiation. Cell counts indicate that fewer osteoblasts are present on hydroxyapatite versus plastic surfaces 24 h after plating. Measurement of osteoblast attachment, apoptosis, and necrosis indicated no differences between surfaces. In contrast, the number of bromodeoxyuridine-incorporating cells was significantly decreased on hydroxyapatite compared with plastic surfaces. Taken together, our findings indicate that hydroxyapatite enhances osteoblast differentiation while also suppressing growth.

Segment-specific expression of alkaline phosphatase in the Tubifex embryo requires DNA replication and mRNA synthesis.

During embryogenesis of the oligochaete annelid Tubifex, segments VII and VIII specifically express mesodermal alkaline phosphatase (ALP) activity in the ventrolateral region. In this study, using specific inhibitors, we examined whether this segment-specific expression of ALP activity depends on DNA replication and RNA transcription. BrdU-incorporation experiments showed that presumptive ALP-expressing cells undergo the last round of DNA replication at 12-24 hr prior to emergence of ALP activity. When this DNA replication was inhibited by aphidicolin, ALP development was completely abrogated in the ventrolateral mesoderm. Similar inhibition of ALP development was also observed in alpha-amanitin-injected embryos. While injection of alpha-amanitin at 24 hr prior to the emergence of ALP activity exerted inhibitory effects on ALP development, injection at 14 hr was no longer effective. In contrast, ALP activity developed normally in cytochalasin-D-treated embryos in which cytokinesis was prevented from occurring for 36 hs prior to appearance of ALP activity. These results suggest that the segment-specific development of ALP activity in the Tubifex embryo depends on DNA replication and mRNA transcription, both of which occur long before the emergence of ALP activity.

Nuclear Plasticity and Timing Mechanisms of the Initiation of Alkaline Phosphatase Expression in Cytoplasm-Transferred Blastomeres of Ascidians

Egg cytoplasm containing endoderm determinants was transferred to presumptive-muscle or presumptive-epidermis blastomeres isolated from cleavage-stage embryos of the ascidian Halocynthia roretzi. We investigated three aspects of the expression of endoderm-specific alkaline phosphatase (ALP) activity. First, we examined whether ectopic ALP expression, an indication of ectopic endoderm formation, was promoted in cytoplasm-transferred blastomeres isolated at late-cleavage stage. The results showed that the cell fate was converted by the introduced cytoplasm, even in recipient blastomeres in which the cell fate was already restricted to muscle or epidermis, and in those where expression of the muscle- or epidermis-specific genes was already initiated. Next, we examined the formation of endoderm and other tissue in embryos by double staining for ALP and muscle- or epidermis-specific marker. Regions positive for ALP and positive for muscle or epidermis marker were mutually exclusive. These results suggested that muscle- or epidermis-specific genes that were already expressed in the recipient blastomeres were down-regulated in ectopically forming endoderm cells. This is evidence for nuclear plasticity during ascidian embryogenesis. In the last series of experiments, we investigated the timing of the appearance of ALP activity in cytoplasm-transferred embryos. In the partial embryos that were derived from various combination of recipient blastomeres and donor cytoplasm obtained from various staged eggs and embryos, the timing seemed to coincide with the time that starts when cell fusion for cytoplasmic transfer was done. Therefore, the clock that determines the timing of the initiation of ALP expression is likely to start at the moment of cell fusion. Several possible hypotheses for the timing mechanism are discussed.

Temporal and local appearance of alkaline phosphatase activity in early stages of guided bone regeneration. A descriptive histochemical study in humans.

Alkaline phosphatase (ALP) catalyzes the hydrolysis of phosphate esters and it seems to be a prerequisite for normal skeletal mineralization. Also, ALP is the most widely recognized marker of osteoblast phenotypes. By a tissue regenerative technique called Guided Bone Regeneration (GBR), it is possible nowadays to regenerate small bony defects. The aim of the present study was to investigate early events in bone healing and neogenesis by studying histochemically the temporal and local appearance of the marker Alkaline Phosphatase (ALP) in a GBR model system. Nine healthy volunteers (5 males, 4 females, mean age 31.7 years) participated in the experiment. After raising a mucoperiosteal flap from the mandibular second molar to the retromolar area in each volunteer, a hollow titanium test cylinder was placed into a congruent bony bed and the coronal end of the cylinder was closed with an ePTFE-membrane. Then the flap was adapted and sutured to obtain primary wound closure. After 2, 7 and 12 weeks, the regenerated tissue within the cylinders was harvested. Histologically, ALP activity was observed associated with the osteoid seams in the very basal part of the regenerate where new bone trabeculae were in the process of being formed. More coronally, large round cells seemed to secrete an ALP-positive substance since in the center of such cell clusters strong ALP activity located extracellularly was detected. In the present experiment, ALP seemed to have been an early sign of osteoblast secretion of a matrix which subsequently was determined to become osteoid. ALP activity was never seen isolated within connective tissue and away from bone. This is an indication that its source is linked to existing bone. The present study has documented for the first time the appearance of ALP activity in guided bone regenerations in humans. It has revealed that: 1) Osteogenesis in guided bone regeneration is preceded by localized, marked expression of ALP in an organized connective tissue environment. 2) Bone neogenesis is an early event in this experimental setup and may be detected already 2 weeks after wounding. 3) Expression of ALP and subsequent bone neogenesis is originating from and topographically linked to pre-existing bone structures.

Osteogenic response to hydroxyapatite-fibrin implants in maxillofacial bone defects.

Bone formation in hydroxyapatite-fibrin implants has been reported several times. However, available studies refer to experimental animals, or are limited to short periods after implantation. We report the results of histological, histochemical and ultrastructural studies carried out 2.5-8 yr after implantation of non-resorbable, porous hydroxyapatite (HA) and fibrin glue in human maxillofacial bones. Prominent ossification was found in all cases, with the presence of normally structured spongy bone. HA granules were embedded in the calcified bone matrix. They had not elicited inflammatory reactions and did not induce bone resorption. Ossification was preceded by the appearance of alkaline phosphatase activity on fibroblast-like cells, and by the formation of dense collagenous layers, similar to osteoid borders, on the surface of HA granules. The early phases of the calcification process occurred in these borders, with the appearance of calcification nodules adjacent to alkaline phosphatase-positive osteoblast-like cells. A remodeling process similar to that occurring in normal bones was found in the newly formed bone. These results justify the conclusion that HA-fibrin implants lead to the formation of long-lasting bone that does not differ from that of the normal maxillofacial skeleton. Mixing the HA granules with fibrin has the advantage of creating an easily mouldable material which can be adapted to any skeletal surface and stays in place after surgery.

Antigenicity of pro-osteocalcin in hard tissue: The authenticity to visualize osteocalcin-producing cells

A polyclonal antibody against rat osteocalcin propeptide (anti-pro-OC) has been prepared and used in immunohistochemical studies to demonstrate cells expressing osteocalcin (OC). With the anti-pro-OC antibody, predominant immunoreaction was observed in cuboidal osteoblasts along the bone formation surface, and in odontoblasts and cementoblasts of the tooth. The appearance of pro-OC antigenicity was well consistent with the expression of OC mRNA in the cells as demonstrated by in situ hybridization. Matrices around these cells showed no apparent pro-OC antigenicity, which contrasted remarkably with the immunoreaction with an antibody against the N-terminal sequence of mature rat OC (anti-OC-N). Although the anti-OC-N antibody positively reacted with OC-producing cells, the intensity of the immunoreaction was much weaker than that obtained with the anti-pro-OC antibody. Furthermore, we examined the appearance of pro-OC antigenicity in maxillary alveolar bone during experimental tooth movement in rats. When the maxillary first molar was moved to the mesial direction with a closed coil spring, the bone remodeling phase in the distal alveolar bone surface changed from dominantly resorptive to formative. After the application of orthodontic force, pro-OC-positive osteoblasts appeared on the distal alveolar bone surface (tension side), according to the appearance of alkaline phosphatase activity on the bone surface. Thus, the use of the anti-pro-OC antibody is a practical means to demonstrate cells actively expressing OC and could be an alternative to in situ hybridization.

Infantile hypophosphatasia: Normalization of circulating bone alkaline phosphatase activity followed by skeletal remineralization: Evidence for an intact structural gene for tissue nonspecific alkaline phosphatase

After a 3-month course of weekly intravenous infusions of pooled normal plasma in an attempt at enzyme replacement therapy, we observed gradual and prolonged normalization of circulating alkaline phosphatase (AP) activity in a boy with infantile hypophosphatasia. During this 4-month period, when hypophosphatasemia had been corrected, electrophoretic and heat denaturation studies suggested that the AP in serum was skeletal in origin. Serial radiographic and histologic studies of bone demonstrated skeletal remineralization and the appearance of AP activity in osteoblasts and chondrocytes after the infusions. Considerable clinical improvement coincided with the skeletal remineralization. Our observations indicate that in one patient with infantile hypophosphatasia the structural gene for the tissue-nonspecific (bone/liver/kidney) AP isoenzyme was intact and could be expressed with marked physiologic effect. Infantile hypophosphatasia may result from absence or inactivation of a circulating factor(s) that regulates the expression of the gene for tissue nonspecific AP.

Alkaline phosphatase and gamma-glutamyl transpeptidase as polarization markers during the organization of LLC-PK1 cells into an epithelial membrane.

Confluent monolayers of LLC-PK1 cells, an epithelial cell derived from a normal pig kidney, contain high levels of alkaline phosphatase and gamma-glutamyl transpeptidase activities. Inhibition studies show a close similarity between alkaline phosphatase and the so-called liver-bone-kidney isoenzyme. Nearly complete recovery of both activities in the microsomal fraction demonstrates the membrane-bound characteristics of these enzymes. Histochemical localization of the enzymes activities on the apical membrane of LLC-PK1 cells confirms this observation. The activity of both enzymes decreases to very low levels when the cells are in exponential growth. Confluent monolayers of LLC-PK1 cells plated at saturation density with cells that were in active growth show a progressive increase in the activity of alkaline phosphatase and gamma-glutamyl transpeptidase. This increase is dependent on the synthesis de novo of RNA and protein and supports the conclusion that the activity of both enzymes is regulated at the transcriptional level. The development of these enzymes is delayed with respect to the development of the occluding junctions. This delay and the direct appearance of alkaline phosphatase activity in the apical membrane indicate that the different components of this membrane are inserted after the limits of the membrane have been established by the synthesis and assembly of the occluding junctions. Alkaline phosphatase activity of confluent monolayers can be further induced by reducing the concentration of phosphate in the medium. When the junctions are dissociated by incubating the monolayers in Ca2+-free medium, the alkaline phosphatase activity migrates freely beyond the limits of the apical membrane until it covers the entire cell surface. Both enzymes are synthesized even in the absence of contact between the cells, suggesting that the occluding junctions in LLC-PK1 monolayers are involved in the polarized distribution, but not in the modulation, of the synthesis of these enzymes. In addition, the progressive decrease in enzyme synthesis that is obtained by reducing the cell-substratum adhesion supports the idea that it is the cell-to-substrate and not the cell-to-cell interaction which is involved in the modulation of these enzymatic markers of the apical membrane.

The effect of oestrogen on the nasal respiratory mucosa. An experimental histopathological and histochemical study.

An experimental histopathological and histochemical work carried out in thirty guinea-pigs of an average weight of 475 gm. receiving oestrogen in the form of ethynyl oestradiol in a dose of 10 microgram/animal/day and aiming at a study of the effects of oestrogen on the respiratory nasal mucosa. The histopathalogical lesions of the respiratory nasal mucosa were in the form of squamous metaplasia and spongiosis of the lining epithelium, with oedema of the underlying corium, glandular hyperplasia submucosal cellular infiltration, increased vascularity and some vascular changes in the form of endothelial proliferation with intimal thickening. Histochemical enzymatic alterations were in the form of increased succinic dehydrogenase activity in the epithelium as well as in the hyperplastic submucous glands, intensified reaction of the acid phosphatase in the cells of the corium, and the appearance of alkaline phosphatase activity in the apical parts of the cells lining the glands, indicating increased secretory activity. All the changes obtained in the histopathological and histochemical studies can be attributed to hormonal stimulation of the nasal mucosa.

Early morphogenetic events in normal and mutant skin development in the chick embryo and their relationship to alkaline phosphatase activity

The relationship between the appearance of alkaline phosphatase activity and the presence of dermal condensations in the dorsal skin of the chick embryo was investigated. A histological analysis of scaleless dorsal skin has revealed that this tissue does not develop any dermal condensations. Alkaline phosphatase activity appears and is evenly distributed in these skins. This enzymatic activity is restricted to the dermal condensations in genetically normal skin. β-Aminoproprionitrile and penicillamine were found to inhibit the formation of dermal condensation in genetically normal skins. Alkaline phosphatase appeared in these experimentally altered skins. The activity was uniformly distributed throughout the tissue. It is concluded that the appearance of alkaline phosphatase activity is not dependent on the formation of discrete dermal condensations.