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Keywords:

  • lysyl hydroxylase;
  • procollagen-lysine;
  • 2-oxoglutarate 5-dioxygenase;
  • collagen fibrillogenesis;
  • mineralization;
  • MC3T3-E1

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Several MC3T3-E1 cell-derived clones expressing higher levels of LH2b were analyzed for their abilities to form collagen fibrils and mineralization. The clones all exhibited smaller collagen fibrils and defective matrix mineralization in vitro and in vivo, indicating a critical role of LH2b-catalyzed post-translational modifications of collagen in bone matrix formation and mineralization.

Introduction: We have recently shown that lysyl hydroxylase (LH) 2b, through its action on the telopeptidyl lysine residues of collagen, regulates collagen cross-linking pathway in the osteoblastic cell line, MC3T3-E1. To further elucidate the roles of LH2b in bone physiology, the effects of overexpression of LH2b on collagen fibrillogenesis and matrix mineralization were investigated.

Materials and Methods: Several MC3T3-E1-derived osteoblastic cell clones expressing higher levels of LH2b (S clones) and two controls (i.e., MC3T3-E1 cells and those transfected with an empty vector) were cultured. MALDI-TOF mass spectrometry was used to identify the LH2b. The collagen fibrillogenesis in the cultures was characterized by transmission electron microscopy, and the ability of these clones and cells to form mineralized matrix was analyzed by both in vitro and in vivo mineralization assays.

Results: The diameter of collagen fibrils in the S clone cultures was markedly smaller than that of the controls. The onset of matrix mineralization in the S clones was significantly delayed, and considerably fewer mineralized nodules were formed in their cultures in comparison with the controls. When transplanted into immunodeficient mice, the S clones failed to form mineralized matrices in vivo, whereas a bone-like mineralized matrix was well formed by the controls. The diameter of the collagen fibrils and the timing/extent of matrix mineralization in vitro were inversely correlated with the level of LH2b. In vitro cell differentiation was unaffected by the LH2b overexpression.

Conclusions: These results indicate a critical role of LH2b catalyzed post-translational modification of collagen (i.e., telopeptidyl lysine hydroxylation and subsequent cross-linking) in collagen matrix formation and mineralization in bone.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

COLLAGEN, A LARGE family of structurally related proteins, is the most abundant molecule in vertebrates. Type I collagen, a heterotrimeric molecule composed of two α1 chains and one α2 chain, is the major fibrillar component in most connective tissues including mineralized tissues. This molecule is composed of three domains: amino-terminal nontriple helical (N-telopeptide), triple helical, and carboxy-terminal nontriple helical (C-telopeptide) domains.(1) Collagen biosynthesis is characterized by a large number of post-translational modifications, many of which are unique to collagens.(2) One of the important post-translational modifications of type I collagen is hydroxylation of specific lysine (Lys) residues, because hydroxylysine (Hyl) serves as the only glycosylation site and it involves specific cross-linking pathways.(2) The Lys hydroxylation is catalyzed by lysyl hydroxylase (LH) in the presence of cofactors: Fe2+, 2-oxoglutarate, O2, and ascorbate.(2) The difference in the extent of Lys hydroxylation of collagen depends on genetic types, tissues, tissue's physiological conditions, and domains (helical versus nonhelical telopeptides) of a collagen molecule.(1)

Recently, three genes encoding for isoforms of LH (procollagen-lysine, 2-oxoglutarate, 5-dioxygenase; PLOD 1-3) have been cloned and characterized in mice,(3,4) humans,(5-8) and rats.(9) In addition, an alternative splice form of LH2, LH2b or LH2alt, with an additional 63-bp exon 13A, has been identified.(10) Our previous study showed that an increase in LH2 gene expression was associated with an increase in Lys hydroxylation of the nonhelical telopeptide domains of type I collagen molecule in human osteoblastic cells.(11) Furthermore, two recent reports indicated that LH2b (LH2alt) is the predominant form of LH2 expressed in bone and is a putative telopeptidyl LH.(12,13) Most recently, by overexpressing and suppressing the levels of LH2b in a MC3T3-E1 cell culture system, we have directly shown that the level of LH2b determines collagen cross-linking pathways likely through its action on the telopeptidyl Lys residues.(14)

It has long been our hypothesis that the specific patterning of collagen cross-linking and molecular packing (i.e., an initial establishment of stoichiometric cross-linking and later the reduction of connectivity by dissociation of some bifunctional cross-links) is important to modulate the process of mineralization.(15-19) In this study, we investigated the effects of overhydroxylated telopeptidyl Lys and subsequent cross-linking induced by LH2b overexpression on collagen fibrillogenesis and subsequent mineralization.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The protocol of animal experiments was approved by Institutional Animal Care and Use Committee at the University of North Carolina at Chapel Hill.

Cell culture, transfection, and generation of S clones

The pcDNA3.1/V5-His/LH2b construct was generated, and MC3T3-E1-derived cell clones expressing higher levels of LH2b (S clones) were obtained as recently described.(14) MC3T3-E1 cells and those transfected with a pcDNA3.1/V5-His A vector (empty vector [EV]; Invitrogen, Carlsbad, CA, USA) alone were used as controls. The levels of LH2b synthesized were assessed by immunoprecipitation followed by Western blot analysis with antiV5 antibody (Invitrogen) against V5 epitope at C-terminal tag of LH2b fusion protein.(14) Three S clones that exhibited varied levels of LH2b overexpression (S0, S1, and S2; S0 exhibiting the lowest and S2 the highest level of LH2b overexpression among the three clones) were chosen for this study. The S1 and S2 clones were generated as described above and are the same clones recently reported.(14)

Collagen cross-link analysis

The collagen cross-link analysis for the cells and clones was performed in the same manner as reported recently.(1,14) Briefly, at 2 weeks of cell culture, cell layers/matrices were collected, washed with cold PBS and distilled water, lyophilized, and reduced with standardized Na3BH4. The reduced samples were hydrolyzed with 6N HCl in vacuo, after flushing with N2, at 105°C for 22 h, dried, dissolved in distilled water, and filtered. An aliquot of the hydrolysate was subjected to amino acid analysis to determine hydroxyproline (Hyp) content, and the hydrolysates with known amounts of Hyp were analyzed for cross-links on a cation-exchange column (AA-911; Transgenomic) linked to a fluorescence detector (FP1520; Jasco Spectroscopic) and a liquid scintillation analyzer (Packard Instrument). All cross-links and precursor aldehydes were analyzed as their reduced forms and were quantified as moles per mole of collagen.

Protein identification by mass spectrometry

To identify the V5-tagged LH2b protein, the S2 clone (the clone synthesizing the highest level of LH2b among the S clones tested) was plated onto 15-cm dishes containing α-MEM and 10% FBS (Sigma-Aldrich, St Louis, MO, USA). After confluence, the cell/matrix layer of each cell/clone was collected, lysed with a lysis buffer (150 mM NaCl, 20 mM Tris-HCl pH 7.5, 10 mM EDTA, 1% Triton X-100, 1% deoxycholate, 1 mM phenylmethylsulfonylfluoride, and 1.5% aprotinin), and precipitated with antiV5 antibody and rec-Protein A-Sepharose 4B conjugate beads (Zymed Laboratories, South San Francisco, CA, USA). These beads were washed with a lysis buffer, and the LH2b fusion protein was detached by heating the beads with the SDS-PAGE sample buffer. The samples were resolved in a 4-12% NuPAGE Bis-Tris gel (Invitrogen). After the electrophoresis was completed, the gel was fixed with 25% isopropanol/10% acetic acid and stained with 0.01% Coomassie Brilliant Blue (Sigma) in 10% acetic acid overnight. Gel bands were destained, carefully excised using a razor blade, and digested in a ProGest automated digester (Genomic Solutions, Ann Arbor, MI, USA). Peptides were extracted using a protocol developed by Wilm et al.(20) and modified as reported previously.(21) A 0.5-μl aliquot of the digest was spotted onto the MALDI target with the matrix, using a saturated solution of recrystallized α-cyano-4-hydroxycinnamic acid (HCCA; Sigma) in 10:10:80 ethanol: formic acid:water. Samples were analyzed on an ABI 4700 MALDI TOF/TOF mass spectrometer, internally calibrated with trypsin autoproteolysis peaks. The MS spectrum was searched against the NCBI database, using the on-line version of Protein Prospector (http://prospector.ucsf.edu/).

Measurement of collagen fibril diameter

MC3T3-E1 cells, S (S0-S2) and EV clones were cultured in 3.5-cm culture dishes, containing α-MEM, 10% FBS, 50 μg/ml ascorbic acid, and 2 mM β-glycerophosphate, for 2 weeks. This is when the mineralized matrix formation begins in the cultures of MC3T3-E1 cells and EV clone. The cell layers/matrices were washed with PBS, fixed with 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4, for 1 h, and cut into small pieces (∼2 × 2 mm2). The samples were stained with 1% osmium tetraoxide in 0.1 M cacodylate buffer for 1 h at room temperature. After rinsing with distilled water, the samples were dehydrated, embedded with resin, sectioned (70 nm thickness), and stained with uranyl acetate and lead citrate. The sections were observed under transmission electron microscope (Tecnai 12; Philips), and the images were digitally acquired at a magnification of 11,000 using montage 3 × 3 (Gatan's Digital Micrograph software; Gatan, Warrendale, PA, USA). For each sample, the diameter of randomly selected 500 collagen fibrils was measured using Scion Image software (Scion, Frederick, MD, USA).

In vitro mineralization assay and mRNA expression of osteoblastic markers

MC3T3-E1 cells, S clones, and the EV clone were cultured in 3.5-cm culture dishes in α-MEM/10% FBS containing 50 μg/ml ascorbic acid and 2 mM β-glycerophosphate for 1, 2, 3, and 4 weeks. At the end of each week, cell layers/matrices were washed with PBS twice, fixed with 100% methanol, and stained with 1% Alizarin red S (Sigma). To assess the potential effects of the LH2b overexpression on osteoblastic cell differentiation, after 2 weeks of culture, total RNA was collected from each cell type, and RT-PCR with specific primers for the following osteoblastic markers was performed: type I collagen α1 chain (COLIA1), osteocalcin (OCN), bone sialoprotein (BSP), core binding factor α subunit 1 (CBFA1), and osterix (OSX). GAPDH was used as an internal control. The sequences of the primers are shown in Table 1. All reactions were performed using the HotStarTaq DNA polymerase (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol. The PCR products were analyzed by 3% agarose gel electrophoresis.

Table Table 1.. Sequences of the Primers Used to Detect Osteoblastic Markers
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In vivo mineralization assay

To confirm the results of in vitro mineralization assay, the capability of cells to form mineralized matrices in vivo was further evaluated by the method reported previously.(22,23) Briefly, MC3T3-E1 cells, EV, and S clones were cultured as described above (without ascorbic acid and β-glycerophosphate) until near confluence. The cells were released by trypsin, centrifuged, and resuspended in 1 ml of α-MEM/10% FBS. For a single transplant, 2 × 106 cells were mixed with 40 mg hydroxyapatite/tricalcium phosphate (HA/TCP) ceramic powder (Zimmer, Warsaw, IN, USA). Cells and the ceramic carrier were incubated at 37°C for 90 minutes with slow rotation (25 rpm) and centrifuged briefly. The pelleted HA/TCP powder with adherent cells was consecutively mixed with 15 μl each of mouse fibrinogen (3.3 mg/ml solution in PBS) and mouse thrombin (25 U/ml in 2% CaCl2, both from Sigma) to stabilize the transplant. The resulting fibrin clot with ceramic powder and attached cells were transplanted subcutaneously into 8- to 12-week-old female beige mice (NIH-bg-nu-xidBR; Harlan Sprague-Dawley, Indianapolis, IN, USA). Mice were anesthetized by intraperitoneal injection with a combination of ketamine (Fort Dodge Animal Health, Fort Dodge, IA, USA) at 140 mg/kg body weight and xylazine (The Butler Company, Columbus, OH, USA) at 7 mg/kg body weight. Single 1-cm-long skin incisions were made on the dorsal surface of each mouse, and four subcutaneous pockets per mouse were created by blunt dissection. A single transplant was placed in each pocket, and incisions were closed with surgical staples. The transplants were harvested at 9 and 11 weeks after transplantation, cut in half, fixed in 10% buffered formalin (Sigma), demineralized in 10% formic acid, and processed for routine histological examination (H&E staining). The stained sections were evaluated and photographed under a light microscope (Olympus America, Melville, NY, USA).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Generation of the S clones

The immunoprecipitation/Western blot analysis of three S clones (S0, S1, S2) that exhibited varied levels of LH2b overexpression and the controls are shown in Fig. 1. The S2 clone exhibited the highest levels of LH2b, and the S0 exhibited the lowest level of LH2b, whereas there were no positive bands of exogenous LH2b in the controls.

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Figure FIG. 1. Detection of V5-tagged LH2b protein by immunoprecipitation-Western blot analysis using antiV5 antibody. The immunoreactive bands at ∼97 kDa (V5-tagged LH2b) are seen only in the S clones (S0-2).20

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Collagen cross-link pattern

The characteristics of collagen cross-link pattern of MC3T3-E1 cells and EV, S1, and S2 clones that have been reported(14) were verified. The cross-link analysis of S0 clone exhibited the pattern typical of those seen in other S clones (i.e., a higher ratio of dihydroxylysinonorleucine [DHLNL] to hydroxylysinonorleucine [HLNL] and an increased pyridinoline in comparison with MC3T3-E1 cells/EV clone [5.58 versus 3.72/3.08 and 0.04 mol/mol of collagen versus 0.02/0.02, respectively]).(14) The levels of DHLNL to HLNL ratio in the S clones corresponded to those of LH2b overexpression (S0 < S1 < S2).(14)

Identification of LH2b by mass spectrometry

Peptide mass fingerprinting data obtained from an in-gel digestion of V5-tagged LH2b identified five peptides that matched mouse LH2b. The peptide sequences and the mass error (in ppm) are shown in Table 2.

Table Table 2.. Mass Spectrometric Confirmation of V5-Tagged LH2b
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Ultrastructural analysis of collagen fibrils

Typical cross-sectional views of collagen fibrils obtained from cultures of S clones (S0, S1, S2) and the controls (MC3T3-E1 cells and EV clone) are shown in Fig. 2. The MC3T3-E1 cells had a mean diameter of 57.85 ± 5.63 nm, with a range of 41-70 nm, which is similar to that of the EV clone (mean, 52.72 ± 4.76 nm; range, 41-64 nm). In all S clones, the mean fibril diameters and their ranges were significantly smaller than those of controls. Among the S clones, S2 showed the smallest fibril diameter (mean, 26.83 ± 3.36 nm; range, 19-36 nm) followed by S1 clones (mean, 32.59 ± 6.36 nm; range, 19-53 nm) and S0 (mean, 36.92 ± 7.00 nm; range, 23-54 nm). Therefore, the fibril diameter and distribution range were inversely correlated with the levels of LH2b overexpression of S clones.

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Figure FIG. 2. Cross-section of the collagen fibrils observed under TEM and their diameter distribution based on total number of 500 fibrils. All of the S clones produced smaller collagen fibril diameters than those of the controls. The S2 clone, highest LH2b level, produced the smallest collagen fibrils. MC, MC3T3-E1 cells; S0, S1, S2, MC3T3-E1-derived cell clones expressing higher levels of LH2b; EV, MC3T3-E1 cells transfected with an empty pcDNA3.1/V5-His A vector.20

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In vitro mineralization assay and mRNA expression of osteoblastic markers

The results of in vitro mineralization assay are shown in Fig. 3. The mineralization pattern of two controls, MC3T3-E1 cells and EV clone, was essentially identical to each other. In both cases, the formation of mineralized nodules was already evident at 2 weeks of culture, and the number and size of mineralized nodules increased thereafter. In contrast to these controls, the matrix mineralization was markedly impaired in the S clones. None of them formed mineralized nodules at 2 weeks of culture. At 4 weeks, only a few and small mineralized nodules were observed in the cultures of S0 and S1. In the case of S2 clone, the clone synthesizing the highest level of LH2b, no mineralized nodules were formed even at this stage (Fig. 3).

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Figure FIG. 3. Alizarin red S staining of mineralized nodules at 1-4 weeks of cell culture from MC3T3-E1 cell, S clones, and EV clone. S0 and S1 clones formed fewer mineralized nodules than the controls and the S2 clone did not form any mineralized nodule when cultured for up to 4 weeks.20

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The mRNA expression of osteoblastic markers, including COLIA1, OCN, BSP, CBFA1, and OSX were similar among S clones and comparable with those of controls (Fig. 4), indicating that cell differentiation was unaffected in S clones.

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Figure FIG. 4. The mRNA expression of osteoblastic markers at 2 weeks of culture. There are no significant differences in the COLIA1, OCN, BSP, CBFA1, and OSX expression among the S clones.20

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In vivo mineralization assay

Typical H&E-stained histological sections of the transplants loaded with the control cells and clones obtained at 9 (Fig. 5A) and 11 (Fig. 5B) weeks of transplantation are shown. At 9 weeks of transplantation, there were some bone-like matrices including lacunae housing osteocyte-like cells in the transplants of both MC3T3-E1 cells and the EV clone. At 11 weeks, some mineralized matrices in both groups became more continuous and thicker with significant numbers of osteocyte-like cells. In contrast to the transplants of human bone marrow stromal cells,(22) no bone marrow-like tissues were observed in the transplants. In contrast to the control groups, the transplants of all three S clones showed no mineralized matrix even at 11 weeks of transplantation. In these transplants, only highly cellular fibrous tissues were observed between ceramic carriers (Fig. 5).

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Figure FIG. 5. H&E staining of mineralized matrix from in vivo transplantation. After (A) 9 and (B) 11 weeks of transplantation, MC3T3-E1 cells and the EV clone formed a comparable bone-like mineralized matrix with osteocyte-like cells embedded in it (arrows). S clones failed to form any bone-like matrix and only formed a fibrous tissue (arrowheads). *Carrier.20

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

In bone, a specific spatial relationship between the two predominant components (i.e., collagen fibrils and mineral) is critical for the tissue's mechanical functions,(24) and the collagen fibrils apparently regulate the manner of mineral deposition and growth.(25,26) A number of indirect evidence indicate that the covalent intermolecular cross-linking that is formed at the edge of the hole zones of the fibril, the putative nucleation sites, plays a crucial role in collagen mineralization.(15, 19, 27, 28) However, direct evidence is still lacking.

A critical factor to determine the collagen cross-linking pattern is the extent of Lys hydroxylation in the telopeptides of the molecule. Depending on the type of aldehyde (i.e., Lysald or Hylald) formed in the telopeptides and the amino acids that are then paired, several distinct cross-linking pathways are developed, which are known to be tissue-specific.(29) Several recent studies support the notion that LH2b may function as a telopeptidyl LH, thus important for determining cross-linking pattern.(11-13) To directly prove the role of LH2b in cross-linking, very recently we generated, without affecting cell differentiation, several MC3T3-E1 cell-derived clones that stably express higher or lower levels of LH2b and showed that the LH2b modulates cross-linking pathways through its action on the telopeptidyl Lys residues.(14) Thus, we now have the tool to directly investigate the effects of overhydroxylation of telopeptidyl Lys and subsequent cross-linking on collagen fibrillogenesis and matrix mineralization.

The collagen fibril diameters of the S clones were significantly smaller than those of the controls, and the diameter was inversely correlated to the levels of LH2b. Many factors have been proposed to control the collagen fibril diameter, including the presence of minor fibrillar collagens such as types V(30,31) and III collagen,(32) fibril-associated collagens with interrupted triple helices (FACITs),(33) small leucine-rich proteoglycans (SLRPs),(34-36) and post-translational modifications of type I collagen itself.(37,38) At this point, it can not be concluded whether the formation of markedly smaller fibrils seen in the S clones is caused by the direct effects of overhydroxylated telopeptidyl Lys/cross-linking of collagen on its fibrillogenesis or to more indirect effects (i.e., altered interaction of those overmodified collagen with other modulatory molecules). However, considering the facts that the major catalysis sites of LH2b are in the nonhelical telopeptides and that the interaction sites of type I collagen with other types of collagen/matrix molecules reside mainly in the helical domain, the former could be the case. It has been reported that the nonhelical telopeptides of collagen molecule, although they represent only a few percent of the whole molecule, regulate the self-assembly process of fibrillogenesis.(39-41) The N-telopeptide seems to be required for initial nucleation or priming for fibril growth, whereas the C-telopeptide is required for lateral growth of the fibril. At this point, it is not clear if LH2b catalyzes Lys hydroxylation in both N- and C-telopeptides or if it has preferred sites. In either case, the overhydroxylated Lys residues in the telopeptides may change the microenvironment of these important domains by adding hydrophilicity, resulting in small fibrils. A potential effect of overhydroxylated cross-linking on limiting lateral molecular packing/growth is not known. More detailed investigations such as the quantitative analysis of cross-links and their precursors at specific molecular loci are needed to obtain insights into the molecular packing structure of the collagen fibrils formed by the S clones.

The results of both in vitro and in vivo mineralization assays clearly show that matrix mineralization is severely impaired in S clones. Because cell differentiation evaluated by the mRNA expression of osteoblastic markers is not affected in vitro (Fig. 4), the mineralization defects seen in the S clones are likely caused by the changes in extracellular matrix. Likely, the defective fibrils (Fig. 2) caused by overhydroxylation of the telopeptidyl Lys and subsequent cross-linking of collagen are detrimental to collagen matrix mineralization. Those fibrils may not serve as a functional 3D template to accommodate and support minerals within the fibrils. These results of mineralization assay seem to be somewhat inconsistent with our previous(11) and other studies(13,42) indicating that the LH2b expression is associated with or necessary for proper mineralization. A possible explanation is that the tight regulation of LH2b expression in terms of timing and expression level during osteoblast differentiation and matrix mineralization is important to form such functional collagen fibrils for mineralization. Another possibility, although less likely, is that the interaction of collagen overmodified by LH2b with putative initiators (e.g., BSP, biglycan)(43,44) or inhibitors (e.g., decorin)(36,45) of mineralization may be altered, resulting in marked impairment of mineralization. A recent study indicating that decorin may bind to collagen at the sites close to where cross-linking occurs(46) may support this possibility.

In conclusion, this study shows that excessive post-translational modifications of collagen induced by LH2b overexpression have profound effects on collagen fibrillogenesis, leading to impaired matrix mineralization in vitro and in vivo. This underscores the importance of such post-translational modifications of collagen in matrix mineralization.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The authors thank Halrold E Mekeel for assistance in transmission electron microscopic study and Dr Carol Parker for valuable analysis in mass spectrometry. We also thank Drs Arabella Leet and Pamela Gehron Robey for providing HA/TCP for cell transplantation. This study was supported by NASA Grant NAG2-1596 and NIH Grant DE10489.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
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