Biomedical subjects

H Clarke Anderson

Publications and source records attributed to H Clarke Anderson.

11 recordsLinked to original sources

PubMed2006

Nature of phosphate substrate as a major determinant of mineral type formed in matrix vesicle-mediated in vitro mineralization: An FTIR imaging study.

Membrane-bound extracellular matrix vesicles play an important role in the de novo initiation and propagation of calcium-phosphate mineral formation in calcifying cartilage, bone, dentin, and in pathologic calcification. Characterization of the phase, composition, crystal size, and perfection provides valuable insight into the mechanism of the mineral deposition. In the present study, Fourier transform infrared imaging spectroscopy (FT-IRIS) was used to characterize the mineral phase generated during MV-mediated in vitro mineralization. FT-IRIS studies revealed that the mineral phase associated with MVs calcified in the presence of AMP and beta-GP was always found to be crystalline hydroxyapatite while with ATP only a small amount of immature mineral, most likely an amorphous or poorly crystalline hydroxyapatite, was observed. Low concentrations of pyrophosphate (PPi) (< or = 0.01 mM) showed apatitic mineral while high concentrations showed immature calcium pyrophosphate dihydrate (CPPD). The implications of these findings are that (a) hydrolysis of AMP or beta-GP, monophosphoester substrates of MV-5' AMPase (substrate: AMP) and TNAP (substrates: AMP, beta-GP), yields orthophosphate (Pi) which leads to the formation of mature crystalline, apatite mineral, while the hydrolysis of ATP, substrate for MV-TNAP or ATPase or NPP1, inhibits the formation of mature hydroxyapatite, and (b) pyrophosphate (PPi) has a bimodal effect on mineralization, i.e., at low PPi concentrations, alkaline phosphatase activity of matrix vesicles is able to hydrolyze PPi to orthophosphate and thus facilitates the formation of basic calcium phosphate mineral which subsequently transforms into apatitic mineral. We hypothesize that, at high PPi concentrations, PPi by itself or Pi released by partial PPi hydrolysis could act as inhibitors of alkaline phosphatase activity, thereby preventing complete hydrolysis of PPi to Pi, and thus resulting in the accumulation of calcium pyrophosphate dihydrate. Therefore, in order for physiological mineralization to proceed, a balance is required between levels of Pi and PPi.

Animals↗

PubMed2005

The role of matrix vesicles in growth plate development and biomineralization.

Skeletal cells control the initiation of mineralization in vivo and determine the selective distribution pattern of mineralization by releasing calcification-initiating, submicroscopic, extracellular matrix vesicles (MVs) at selected sites in the extracellular matrix. The overall objective of this review is to outline what is currently known about the mechanisms of MV biogenesis and mineral initiation, while emphasizing recent observations that enhance our understanding of these mechanisms. Data from studies on the general mechanism of biogenesis of outer membrane vesicles and the formation and function of non-skeletal matrix vesicles is presented to stimulate thought concerning the possible biological functions that these structures may share with MVs.

Alkaline Phosphatase↗

PubMed2005

Sustained osteomalacia of long bones despite major improvement in other hypophosphatasia-related mineral deficits in tissue nonspecific alkaline phosphatase/nucleotide pyrophosphatase phosphodiesterase 1 double-deficient mice.

We have shown previously that the hypomineralization defects of the calvarium and vertebrae of tissue nonspecific alkaline phosphatase (TNAP)-deficient (Akp2-/-) hypophosphatasia mice are rescued by simultaneous deletion of the Enpp1 gene, which encodes nucleotide pyrophosphatase phosphodiesterase 1 (NPP1). Conversely, the hyperossification in the vertebral apophyses typical of Enpp1-/- mice is corrected in [Akp2-/-; Enpp1-/-] double-knockout mice. Here we have examined the appendicular skeletons of Akp2-/-, Enpp1-/-, and [Akp2-/-; Enpp1-/-] mice to ascertain the degree of rescue afforded at these skeletal sites. Alizarin red and Alcian blue whole mount analysis of the skeletons from wild-type, Akp2-/-, and [Akp2-/-; Enpp1-/-] mice revealed that although calvarium and vertebrae of double-knockout mice were normalized with respect to mineral deposition, the femur and tibia were not. Using several different methodologies, we found reduced mineralization not only in Akp2-/- but also in Enpp1-/- and [Akp2-/-; Enpp1-/-] femurs and tibias. Analysis of calvarial- and bone marrow-derived osteoblasts for mineralized nodule formation in vitro showed increased mineral deposition by Enpp1-/- calvarial osteoblasts but decreased mineral deposition by Enpp1-/- long bone marrow-derived osteoblasts in comparison to wild-type cells. Thus, the osteomalacia of Akp2-/- mice and the hypomineralized phenotype of the long bones of Enpp1-/- mice are not rescued by simultaneous deletion of TNAP and NPP1 functions.

Alkaline Phosphatase↗

PubMed2004

A simple and non-radioactive technique to study the effect of monophosphoesters on matrix vesicle-mediated calcification.

A simple and non-radioactive technique based on O-cresolpthalein complexone assay was developed to study in vitro non-radioactive calcium ((40)Ca) deposition by isolated matrix vesicles. Using this technique, the effect of various phosphoester substrates including ATP, AMP and beta-GP on in vitro MV-calcification was studied. O-cresolpthalein complexone assay with non-radioactive calcium demonstrated that AMP or beta-GP were more effective in promoting calcium deposition by isolated MVs than ATP. The application of this non-radioactive technique, which is highly sensitive and simple, would offer a useful alternative approach to the routinely used radiometric biomineralization assay which employs radioactive (45)Ca.

Journal Article↗

PubMed2004

Differential expression of osteogenic factors associated with osteoinductivity of human osteosarcoma cell lines.

Differential expression of multiple osteogenic factors may be responsible for the different osteoinductivity of osteosarcoma cell lines. We compared in vivo osteoinductivity of human osteosarcoma cell lines (Saos-2 vs. U-2 OS) in nude mice, and their in vitro expression of various osteogenic factors of protein level by quantitative immunocytochemistry and mRNA level by RT-PCR and/or in situ hybridization. Saos-2 cells, but not U-2 OS, were osteoinductive in vivo. Significantly higher expression (independent t-test, all p < 0.005) of osteogenic factors were observed in Saos-2 cells compared with U-2 OS, which included bone morphogenetic proteins (particularly BMPs-2, 3, 4, and 7), transforming growth factor-beta (TGF-beta), BMP receptor (BMPR)-1A, receptor-regulated Smads (R-Smads), Smads 1, 2, and 5, and common-mediator Smad (Co-Smad), Smad 4. In contrast, U-2 OS cells expressed higher levels of inhibitory Smad 6 (I-Smad) protein than Saos-2 cells (p < 0.001). These results suggest that a combination of osteogenic factors (BMPs, TGF-beta, BMPRs, and R/Co-Smads) against I-Smad may play important roles in the Saos-2 cell osteoinductivity. This may have a clinical implication in selecting key osteogenic factors for combined therapy for bone defect diseases. The characterized cell lines can be used as positive and negative controls for the assessments of both in vitro and in vivo bone formation capabilities of designed tissues or biomaterials.

Animals↗

PubMed2004

Primary culture of rat growth plate chondrocytes: an in vitro model of growth plate histotype, matrix vesicle biogenesis and mineralization.

During endochondral ossification (EO), cartilage is replaced by bone. Chondrocytes of growth plate undergo proliferation, maturation, hypertrophy, matrix vesicle (MV) biogenesis and programmed cell death (PCD, apoptosis). The in vitro system presented here provides a potential experimental model for studying in vitro differentiation and MV biogenesis in chondrocyte cultures. Chondrocytes were obtained from collagenase-digested tibial and femoral growth plate cartilage of 7-week-old rachitic rats. The isolated chondrocytes were plated as monolayers at a density of 0.5 x 10(6) cells per 35-mm plate and grown for 17 days in BGJ(b) medium supplemented with 10% fetal bovine serum, 50 microg/ml ascorbic acid. Light microscopy revealed Sirius red-positive, apparent bone matrix in layers at the surfaces of cartilaginous nodules that developed in the cultures. The central matrix was largely alcian blue staining thus resembling cartilage matrix. Electron microscopy revealed superficial areas of bone like matrix with large banded collagen fibrils, consistent with type I collagen. Most of the central matrix was cartilaginous, with small fibrils, randomly arranged consistent with type II collagen. The presence of peripheral type I and central type II and type X collagen was confirmed by immunohistochemical staining. Immunohistochemistry with anti-Bone morphogenetic proteins 2, 4 and 6 showed that BMP expression is associated with maturing hypertrophic central chondrocytes, many of which were TUNEL positive and undergoing cell death with plasma membrane breaks, hydropic swelling and cell fragmentation. During early mineralization, small radial clusters of hydroxyapatite-like mineral were associated with matrix vesicles. Collagenase digestion-released MVs from the cultures showed a high specific activity for alkaline phosphatase and demonstrated a pattern of AMP-stimulated nonradioactive (40)Calcium deposition comparable to that observed with native MVs. These studies confirm that primary cultures of rat growth plate chondrocytes are a reasonable in vitro model of growth plate histotype, MV biogenesis and programmed cell death.

Alkaline Phosphatase↗

PubMed2004

Localization of bone morphogenetic proteins (BMPs)-2, -4, and -6 within megakaryocytes and platelets.

In this study, we localized bone morphogenetic proteins (BMPs), proteins known to induce ectopic osteogenesis, within megakaryocytes and in lysates of human platelets. Immunohistochemistry localized BMP-2, -4, and -6 within marrow megakaryocytes of 5-week-old rat tibias. In situ hybridization was utilized to confirm the presence of BMP-2, BMP-4, and BMP-6 mRNA in 2- to 3-week-old mouse tibial marrow megakaryocytes. Finally, the presence of BMP-2, -4, and -6 was confirmed in human platelet lysates using Western blot technique. The expression and release of BMPs by megakaryocytes, within platelets and perhaps by secretion, may help to explain recent reports of excessive bone formation associated with increased numbers of marrow megakaryocytes in GATA-1- or NF-E2 gene-deficient mice. Also, excess local release of BMPs may provide an explanation for the bone overgrowth (osteosclerosis) seen in human patients suffering from a type of myelogenous leukemia characterized by increased numbers of marrow megakaryocytes.

Animals↗

PubMed2004

Impaired calcification around matrix vesicles of growth plate and bone in alkaline phosphatase-deficient mice.

The presence of skeletal hypomineralization was confirmed in mice lacking the gene for bone alkaline phosphatase, ie, the tissue-non-specific isozyme of alkaline phosphatase (TNAP). In this study, a detailed characterization of the ultrastructural localization, the relative amount and ultrastructural morphology of bone mineral was carried out in tibial growth plates and in subjacent metaphyseal bone of 10-day-old TNAP knockout mice. Alizarin red staining, microcomputerized tomography (micro CT), and FTIR imaging spectroscopy (FT-IRIS) confirmed a significant overall decrease of mineral density in the cartilage and bone matrix of TNAP-deficient mice. Transmission electron microscopy (TEM) showed diminished mineral in growth plate cartilage and in newly formed bone matrix. High resolution TEM indicated that mineral crystals were initiated, as is normal, within matrix vesicles (MVs) of the growth plate and bone of TNAP-deficient mice. However, mineral crystal proliferation and growth was inhibited in the matrix surrounding MVs, as is the case in the hereditary human disease hypophosphatasia. These data suggest that hypomineralization in TNAP-deficient mice results primarily from an inability of initial mineral crystals within MVs to self-nucleate and to proliferate beyond the protective confines of the MV membrane. This failure of the second stage of mineral formation may be caused by an excess of the mineral inhibitor pyrophosphate (PPi) in the extracellular fluid around MVs. In normal circumstances, PPi is hydrolyzed by the TNAP of MVs' outer membrane yielding monophosphate ions (Pi) for incorporation into bone mineral. Thus, with TNAP deficiency a buildup of mineral-inhibiting PPi would be expected at the perimeter of MVs.

Alkaline Phosphatase↗

PubMed2003

Matrix vesicles and calcification.

Matrix vesicles (MVs) are extracellular, 100 nM in diameter, membrane-invested particles selectively located at sites of initial calcification in cartilage, bone, and predentin. The first crystals of apatitic bone mineral are formed within MVs close to the inner surfaces of their investing membranes. Matrix vesicle biogenesis occurs by polarized budding and pinching-off of vesicles from specific regions of the outer plasma membranes of differentiating growth plate chondrocytes, osteoblasts, and odontoblasts. Polarized release of MVs into selected areas of developing matrix determines the nonrandom distribution of calcification. Initiation of the first mineral crystals, within MVs (phase 1), is augmented by the activity of MV phosphatases (eg, alkaline phosphatase, adenosine triphosphatase and pyrophosphatase) plus calcium-binding molecules (eg, annexin I and phosphatidyl serine), all of which are concentrated in or near the MV membrane. Phase 2 of biologic mineralization begins with crystal release through the MV membrane, exposing preformed hydroxyapatite crystals to the extracellular fluid. The extracellular fluid normally contains sufficient Ca2+ and PO4(3-) to support continuous crystal proliferation, with preformed crystals serving as nuclei (templates) for the formation of new crystals by a process of homologous nucleation. In diseases such as osteoarthritis, crystal deposition arthritis, and atherosclerosis, MVs initiate pathologic calcification, which, in turn, augments disease progression.

Bone Matrix↗

PubMed2002

Tissue-nonspecific alkaline phosphatase and plasma cell membrane glycoprotein-1 are central antagonistic regulators of bone mineralization.

Osteoblasts mineralize bone matrix by promoting hydroxyapatite crystal formation and growth in the interior of membrane-limited matrix vesicles (MVs) and by propagating the crystals onto the collagenous extracellular matrix. Two osteoblast proteins, tissue-nonspecific alkaline phosphatase (TNAP) and plasma cell membrane glycoprotein-1 (PC-1) are involved in this process. Mutations in the TNAP gene result in the inborn error of metabolism known as hypophosphatasia, characterized by poorly mineralized bones, spontaneous fractures, and elevated extracellular concentrations of inorganic pyrophosphate (PP(i)). PP(i) suppresses the formation and growth of hydroxyapatite crystals. PP(i) is produced by the nucleoside triphosphate pyrophosphohydrolase activity of a family of isozymes, with PC-1 being the only member present in MVs. Mice with spontaneous mutations in the PC-1 gene have hypermineralization abnormalities that include osteoarthritis and ossification of the posterior longitudinal ligament of the spine. Here, we show the respective correction of bone mineralization abnormalities in knockout mice null for both the TNAP (Akp2) and PC-1 (Enpp1) genes. Each allele of Akp2 and Enpp1 has a measurable influence on mineralization status in vivo. Ex vivo experiments using cultured double-knockout osteoblasts and their MVs demonstrate normalization of PP(i) content and mineral deposition. Our data provide evidence that TNAP and PC-1 are key regulators of the extracellular PP(i) concentrations required for controlled bone mineralization. Our results suggest that inhibiting PC-1 function may be a viable therapeutic strategy for hypophosphatasia. Conversely, interfering with TNAP activity may correct pathological hyperossification because of PP(i) insufficiency.

Alkaline Phosphatase↗

PubMed2002

Selective synthesis of bone morphogenetic proteins-1, -3, -4 and bone sialoprotein may be important for osteoinduction by Saos-2 cells.

An ability to induce new bone formation at a required site would represent a considerable advance in bone repair and tissue engineering. It has been shown that the healing of critical-size bone defects in rats can be augmented by extracts of Saos-2 cells. These human osteosarcoma cells uniquely contain a bone-inducing activity, whereas other human osteosarcoma cells, e.g., U-2 OS cells, cannot replicate the osteoinductive capacity. To understand the necessary components of the Saos-2 bone-inducing activity, this study compared osteoinductive Saos-2 cells with non-osteoinductive U-2 OS cells with respect to the synthesis of bone morphogenetic proteins (BMPs)-1, -2, -3, -4, -5, -6, and -7 and the non-collagenous matrix proteins bone sialoprotein (BSP), osteonectin (ON), osteopontin (OPN), and osteocalcin (OC). The main differences were abundant synthesis of BMP-1/tolloid, BMP-3, -4, and BSP by Saos-2 cells, but absence or reduced synthesis in U-2 OS cells. BMP-2 and -7 were present in low amounts in both cell types, while BMP-5 and -6 were more abundant in U-2 OS cells, suggesting that these BMPs were of lesser importance for the osteoinductivity of Saos-2 cells. However, a relatively high expression of BMP-3 and -4, together with BMP-1/tolloid, may be important for the osteoinductive capacity of Saos-2 cells. The inability of U2-OS cells to induce bone, despite expressing most of the BMPs, may be due to an insufficiency of tolloid, BMP-3 or -4, BSP, and/or other unknown factors. A better understanding of the necessary components of the Saos-2 cell bone-inducing agent may, in future, lead to clinically useful Saos-2 cell products for bone repair and tissue engineering.

Animals↗