Analysis of osteopontin in mouse growth plate cartilage by application of laser capture microdissection and RT-PCR.
*Reverse Transcriptase Polymerase Chain Reaction; Animals; Articular/chemistry/cytology; Cartilage; Chondrocytes/chemistry; Gene Expression; Growth Plate/*chemistry/cytology; Inbred C57BL; Laser Therapy; Messenger/metabolism; Mice; Microdissection/*methods; Newborn; Non-programmatic; Osteopontin; RNA; Sialoglycoproteins/*analysis/genetics; Tibia
Gene expression of osteopontin (OPN) has been investigated in mice by application of laser capture microdissection (LCM) and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. LCM permits individual cells to be isolated ("captured") from tissue sections for molecular analyses. In this study, chondrocytes were captured from growth plate zones in frozen sections of tibiae from 1-11-day-old postnatal mice. RNA was extracted from cells, DNAse-treated, and reverse-transcribed. cDNA was amplified by PCR and OPN mRNA was revealed on agarose gels. Whole cartilage and brain (a positive control) from the same animals also were examined. Reactions containing no RT were negative controls, and 18S rRNA standardized expressed message from captured cells. RT-PCR analysis of laser-captured whole cartilage showed a general qualitative loss of OPN mRNA as animal age increased. Youngest mice gave equivalent OPN expression over all laser-microdissected cartilage zones. For 7-11 day-old mice, OPN expression was qualitatively greatest in resting and lowest in hypertrophic regions of cartilage. Expression of OPN correlated with mineral in the tissue suggests that OPN functionally may inhibit normal vertebrate growth plate mineralization, and its loss with increasing tissue maturation appears permissive to mineral development.
Landis William J; Jacquet Robin; Hillyer Jennifer; Zhang Jean
Connective tissue research
2003
1905-06
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1080/03008200390152052" target="_blank" rel="noreferrer noopener">10.1080/03008200390152052</a>
Aspects of mineral structure in normally calcifying avian tendon.
Anatomic; Animals; Atomic Force/methods; Calcification; Collagen/chemistry/ultrastructure; Microscopy; Minerals/*chemistry; Models; Molecular; Non-programmatic; Physiologic; Tendons/*chemistry/*ultrastructure; Turkeys
Structural characteristics of normally calcifying leg tendons of the domestic turkey Meleagris gallopavo have been observed for the first time by tapping mode atomic force microscopy (TMAFM), and phase as well as corresponding topographic images were acquired to gain insight into the features of mineralizing collagen fibrils and fibers. Analysis of different regions of the tendon has yielded new information concerning the structural interrelationships in vivo between collagen fibrils and fibers and mineral crystals appearing in the form of plates and plate aggregates. TMAFM images show numerous mineralized collagen structures exhibiting characteristic periodicity (54-70 nm), organized with their respective long axes parallel to each other. In some instances, mineral plates (30-40 nm thick) are found interspersed between and in intimate contact with the mineralized collagen. The edges of such plates lie parallel to the neighboring collagen. Many of these plates appear to be aligned to form larger aggregates (475-600 nm long x 75-90 nm thick) that also retain collagen periodicity along their exposed edges. Intrinsic structural properties of the mineralizing avian tendon have not previously been described on the scale reported in this study. These data provide the first visual evidence supporting the concept that larger plates form from parallel association of smaller ones, and the data fill a gap in knowledge between macromolecular- and anatomic-scale studies of the mineralization of avian tendon and connective tissues in general. The observed organization of mineralized collagen, plates, and plate aggregates maintaining a consistently parallel nature demonstrates the means by which increasing structural complexity may be achieved in a calcified tissue over greater levels of hierarchical order.
Siperko L M; Landis W J
Journal of structural biology
2001
2001-09
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1006/jsbi.2001.4414" target="_blank" rel="noreferrer noopener">10.1006/jsbi.2001.4414</a>
Effect of high transcapillary pressures on capillary ultrastructure and permeability coefficients in dog lung.
*Blood-Air Barrier; *Pulmonary Circulation; Animals; Blood Pressure; Capillaries/cytology/*physiology/*ultrastructure; Dogs; Electron; Endothelium; Female; Lung/blood supply/*ultrastructure; Male; Microscopy; Non-programmatic; Osmotic Pressure; Respiratory Mucosa/ultrastructure; Vascular/ultrastructure
To determine the correlation between ultrastructural and physiological changes in blood-gas barrier function in lungs transiently exposed to very high vascular pressures, we increased capillary transmural pressure (Ptm) of 6 canine isolated perfused left lower lung lobe preparations (high-pressure group) to 80.3 Torr for 3.8 min and then determined the capillary filtration (K(fc)) and osmotic reflection (sigma(d)) coefficients at a Ptm of 19.1 Torr in the ventilated lung lobes. This was followed by perfusion fixation of the lobes at a Ptm of 20.5 Torr for ultrastructural analysis. These data were compared with those obtained in six lobes in which Ptm was not transiently elevated before K(fc), sigma(d), and ultrastructural evaluation. K(fc) was higher [0.249 +/- 0.042 (SE) vs. 0.054 +/- 0.009 g. min(-1). Torr(-1). 100 g(-1); P \textless 0.01] and sigma(d) was lower (0.52 +/- 0.07 vs. 0.85 +/- 0.08; P \textless 0.01) in the high-pressure group. In contrast, although endothelial and epithelial breaks were occasionally observed in some experiments, their incidence was not increased in the high-pressure group. These data suggest that the increased transvascular water and protein flux occurred through pathways of a size not resolvable by electron microscopy after vascular perfusion-fixation at a Ptm of 20.5 Torr.
Maron M B; Fu Z; Mathieu-Costello O; West J B
Journal of applied physiology (Bethesda, Md. : 1985)
2001
2001-02
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1152/jappl.2001.90.2.638" target="_blank" rel="noreferrer noopener">10.1152/jappl.2001.90.2.638</a>
Experimental use of fibrin glue to induce site-directed osteogenesis from cultured periosteal cells.
*Fibrin Tissue Adhesive; *Osteogenesis; Animals; Bone and Bones/chemistry/cytology/diagnostic imaging; Cattle; Cells; Cultured; Experimental; Implants; Injections; Mice; Non-programmatic; Nude; Osteopontin; Periosteum/*cytology; Radiography; Sialoglycoproteins/analysis
The purpose of this study was to determine whether a combination of fibrin glue and cultured periosteal cells will result in new bone formation at heterotopic sites in nude mice. Growing cells and developing matrices surrounding periosteal explants from the diaphyses of radii of newborn calves were minced and mixed with fibrin glue in a syringe. The cell/matrix-fibrin glue admixture was then injected into the subcutaneous space on the dorsum of athymic nude mice. After 12 weeks of implantation, gross morphology and histologic investigations showed newly formed bone structures in all cell/matrix-fibrin glue admixtures, but none in fibrin glue injected alone and used as control samples. Osteopontin, a protein important in bone development, was identified by a Western blot assay of the cell/matrix-fibrin glue composite. This study supports the feasibility of initiating site-directed formation of bone structures at heterotopic tissue sites by means of injection of cultured periosteal cells and matrix in a fibrin glue carrier.
Isogai N; Landis W J; Mori R; Gotoh Y; Gerstenfeld L C; Upton J; Vacanti J P
Plastic and reconstructive surgery
2000
2000-03
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1097/00006534-200003000-00019" target="_blank" rel="noreferrer noopener">10.1097/00006534-200003000-00019</a>
The structure and function of normally mineralizing avian tendons.
Animals; Biomechanical Phenomena; Birds/*anatomy & histology/*physiology; Minerals/*metabolism; Non-programmatic; Tendons/anatomy & histology/*cytology/*physiology
The leg tendons of certain avian species normally calcify. The gastrocnemius, or Achilles, tendon of the domestic turkey, Meleagris gallopavo, is one such example. Its structure and biomechanical properties have been studied to model the adaptive nature of this tendon to external forces, including the means by which mineral deposition occurs and the functional role mineralization may play in this tissue. Structurally, the distal rounded, thick gastrocnemius bifurcates into two smaller proximal segments that mineralize with time. Mineral deposition occurs at or near the bifurcation, proceeding in a distal-to-proximal direction along the segments toward caudal and medial muscle insertions of the bird hip. Mineral formation appears mediated first by extracellular matrix vesicles and later by type I collagen fibrils. Biomechanical analyses indicate lower tensile strength and moduli for the thick distal gastrocnemius compared to narrow, fan-shaped proximal segments. Tendon mineralization here appears to be strain-induced, the muscle forces causing matrix deformation leading conceptually to calcium binding through the exposure of charged groups on collagen, release of sequestered calcium by proteoglycans, and increased diffusion. Functionally, the mineralized tendons limit further tendon deformation, reduce tendon strain at a given stress, and provide greater load-bearing capacity to the tissue. They also serve as important and efficient elastic energy storage reservoirs, increasing the amount of stored elastic energy by preventing flexible type I collagen regions from stretching and preserving muscle energy during locomotion of the animals.
Landis William J; Silver Frederick H
Comparative biochemistry and physiology. Part A, Molecular & integrative physiology
2002
2002-12
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1016/s1095-6433(02)00248-9" target="_blank" rel="noreferrer noopener">10.1016/s1095-6433(02)00248-9</a>