The distribution of extracellular matrix vesicles in healing of rat tibial bone three days after intramedullary injury

The distribution of extracellular matrix vesicles on the third day of bone healing was studied by morphometric analysis of transmission electron micrographs. Detection and grouping of the vesicles was performed according to type, diameter, and distance from the calcified front. The different types were selected as follows: vesicles with electron-lucent contents ("empty"), vesicles with amorphous electron-opaque contents ("amorphic"), vesicles containing crystalline depositions ("crystal"), and vesicles containing crystalline structures with ruptured membranes ("rupture"). The majority of vesicles were between 0.07 micron and 0.12 micron in diameter and were located at less than 3 micron from the calcified front. The distribution of the "empty", "amorphic", "crystal", and "rupture" vesicles was 23.2%, 74%, 2.5%, and 0.3% respectively. Their sequence of arrangement according to diameter was as follows: "empty", "amorphic", "crystal", and "rupture", the empty vesicles constituting the smallest and the "rupture" the largest type. Distances from the calcified front were similar for the "empty", "amorphic", and "crystal" vesicles, while the "rupture" type was located nearest to the front. The present observations support the widely acknowledged hypothesis on the role of extracellular matrix vesicles in mineralization. It is thought that the secretion of "empty" vesicles from the cell is followed by intravascular accumulation of amorphous Ca and Pi to form a hydroxyapatite crystal that, in turn, ruptures the vesicle's membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in the distribution of extracellular matrix vesicles during healing of rat tibial bone (computerized morphometry and electron microscopy)

A study of the distribution of extracellular matrix vesicles during the first 3 weeks of healing of adult rat tibial bone was performed by transmission electron microscopy in combination with computerized morphometry. Bone injury comprised removal of the marrow followed by regeneration of the tissue via a phase of primary mineralization. A total number of 39,498 vesicles were traced on electron micrographs and sorted according to their diameters, distance from the calcified front and types. The different vesicular types were defined as follows: (a) vesicles with electron lucent contents, i.e. "empty", (b) vesicles with amorphous electron opaque contents, i.e. "amorphic", (c) vesicles containing crystalline depositions, i.e. "crystal", and (d) vesicles containing crystalline structures with ruptured membranes i.e. "rupture". The vesicles were studied on the days 3, 6, 14 and 21 after bone injury. Most of the vesicles were concentrated between diameters of 0.07 and 0.17 micron. Most of the vesicles were found in a distance less than 3 microns from the calcified front. The sequence of changes of distances from the calcified front and of the vesicular diameters were recorded as follows: "rupture", "crystal", "amorphic" and "empty", the "rupture" type being the closet to the front and of the largest diameter in each day. The results of the present study confirm the accepted hypothesis on calcification via extracellular matrix vesicles. It is thought that the cell secretes "empty" vesicles that accumulate Ca and Pi forming amorphous calcium phosphate that is then converted to hydroxyapatite. This is followed by rupture of the vesicular membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in extracellular matrix vesicles during healing of rat tibial bone: a morphometric and biochemical study

Primary mineral formation in woven bone has been associated with the production of extracellular matrix vesicles. Previous studies have demonstrated an increase in phospholipid: Ca:Pi complexes (CPLX) immediately prior to hydroxyapatite formation. Since matrix vesicles are enriched in phosphatidylserine and PS is the major phospholipid in CPLX, the present study examined whether the morphologic appearance of matrix vesicles and initial formation of crystals within them could be correlated to changes in their phospholipid composition and metabolism. Ablation of the tibial marrow in rats was used as the model since this procedure induces endosteal repair with primary mineralization. The morphologic appearance of the matrix vesicles was assessed by morphometric analysis at the electron microscopic level. Matrix vesicles were divided into 4 categories: empty, amorphic, crystal, and rupture. There was time dependent decrease in the number of empty and amorphic matrix vesicles which correlated with an increase in crystal and rupture type. Distance from the calcification front decreased as more rupture-type vesicles were noted. In a parallel set of experiments, matrix vesicle-enriched membranes (MVEM) were isolated from homogenates of endosteal tissue removed from the treated tibia as well as from the contralateral control. There was an increase at 6 days in MVEM alkaline phosphatase and phospholipase A2 specific activities in both limbs, the magnitude of response being significantly greater in the treated legs. The phospholipid composition of the MVEM changed with time. SPH was highest at day 3, PS was detectable only in day 6 and 14 samples, and PC exhibited a time dependent decrease.(ABSTRACT TRUNCATED AT 250 WORDS)     

The relationship between extracellular matrix vesicles and the calcifying front on the 21st day after injury to rat tibial bone

The relationship between extracellular matrix vesicles and the calcifying fronts was examined by studying vesicular diameters and types. Transmission electron microscopy combined with computerized morphometry three weeks after injury to the tibial bone in rats was used. The different vesicle types were defined as: (1) vesicles with electron lucent contents referred to as empty; (2) vesicles with amorphous electron opaque contents, called amorphic; (3) vesicles containing crystalline depositions, called crystalline; and (4) vesicles containing crystalline structures with ruptured membranes, referred to as ruptured. The diameters of most vesicles ranged between 0.07 and 0.17 micron. More than 95% of the vesicles were located less than 2 micron from the calcified front. The vesicles were distributed among the categories as follows: empty, 9.6%; amorphic, 19.3%; crystal, 39.2%; and ruptured, 31.9%, respectively. The diameters of the crystalline and ruptured vesicles were significantly larger than those of the empty and amorphic types. The ruptured type had the largest diameters. The sequence of distances from the calcified front was recorded as follows: ruptured, crystalline, amorphic, and empty, with the ruptured and crystalline types being the closest to the front. This study supports the accepted theory on matrix vesicle mineralization. The cell is responsible for secretion of empty vesicles that accumulate amorphous Ca and Pi to form a hydroxyapatite crystal. This is followed by rupture of the vesicular membrane. The propagation of the process is accompanied by an increase in the vesicular diameter and its approximation to the calcifying front.     

Primary mineralization and extracellular matrix vesicles in rat bone after administration of glass-ceramic implants

Summary Glass-ceramic implants were administered into cavities prepared in rat femoral shaft. Electron microscope examination revealed formation of collagenous rich matrix in the implant-bone interface. Features typical to primary mineralization as well as bone and implant resorption were present in the interface. Primary mineralization was characterized by the occurrence of active forming cells, extracellular matrix vesicles and calcifying calcospheritic structures. Intensive primary mineralization in association with the implants indicates that glass-ceramic may be stimulative to ossification, allowing favourable tissue-implant relationship.

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Zusammenfassung Glaskeramikimplantate wurden in passende Bohrlöcher des Rattenfemurschafts eingesetzt. Die elektronenmikroskopische Untersuchung ergab die Bildung einer kollagenreichen Matrix an der Grenze zwischen Implantat und Knochen. Typische Erscheinungen der primären Mineralisation und der Resorption von Implantat und Knochen wurden an der Implantat-Knochengrenze festgestellt. Die primäre Mineralisation war gekennzeichnet durch aktive knochenbildende Zellen, extrazelluläre Matrixvesikel und kalzifizierende globuläre Strukturen. Die intensive primare Mineralisation in der Umgebung der Implantate spricht dafür, daß die Glaskeramik die Verknöcherung fördern und eine günstige Beziehung zwischen Gewebe und Implantat zustande kommen kann.

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This study was supported in part by a grant from The Government of Israel, The Ministry of Health by grant MT 0250 Ministerium für Forschung und Technologie, Bonn, West Germany, and by E. Leitz Wetzlar GmbH, Wetzlar, West Germany     

Serum bone markers after intramedullary fixed tibial fractures

Serum levels of bone markers were measured prospectively for 1 year in 30 adult patients with an intramedullary fixed tibial fracture. In a double blinded design, half of the patients received low intensity ultrasound. All fractures healed, although in seven of 30 the healing was delayed more than 6 months. There was no significant difference in radiologic healing time between the ultrasound group (median, 113 days) and the placebo group (median, 112 days). The marker for bone resorption, crosslinked telopeptide, peaked at 1 to 4 weeks, whereas markers for bone formation peaked at 10 to 16 weeks for bone specific alkaline phosphatase and osteocalcin. Crosslinked telopeptide was lower at 1 week in patients treated with ultrasound than in those receiving placebo. Patients with delayed healing did not differ in crosslinked telopeptide compared with patients with normal healing. There were no differences in bone formation markers between patients who received ultrasound or placebo. Patients with delayed healing had lower levels of bone specific alkaline phosphatase between 4 and 7 weeks than did patients with normal healing, although no such differences were seen for osteocalcin. The results indicate that low intensity ultrasound might slow bone resorption, although there is no visible effect on bone formation. Patients with delayed healing had adequate bone resorption but slower early bone formation than did patients with normal healing.     

Severe heterotopic bone formation in the knee after tibial intramedullary nailing.

The case of a 48-year-old man who developed severe heterotopic bone within and under his patella tendon after closed intramedullary (IM) nailing of his tibia is reported. The patient had a concomitant closed head injury at the time of his tibia fracture. The tibial nailing was done through a patella tendon splitting approach. The patella tendon splitting approach may predispose patients to clinically significant heterotopic bone. Approaches medial or lateral to the tendon are recommended, particularly in patients at risk for heterotopic bone formation.     

Dynamic changes in bone cells and extracellular matrix vesicles during healing of alveolar bones in rats : An ultrastructural and biochemical study

The changes In ultrastructure, quantities and enzymatic activity of cells and extracellular matrix vesicles were studied during alveolar bone healing after tooth extraction in rats. Biochemical studies revealed a marked decrease In protein content of the isolated fractions of vesicles immediately after the surgical procedure. Transmission electron microscopy confirmed that an extensive tissue disruption occurred at that stage. These changes were accompanied by decreased activities of cellular and vesicular alkaline phosphatase and of different ATPases. The recovery of enzymatic activity on the 9th day was expressed in cells and vesicles. The different stages of healing were characteristic of primary calcification comprising active osteoblasts, matrix vesicles and calcifying nodules. It is suggested that the amounts and enzymatic activities of extracellular matrix vesicles may serve as a marker system to evaluate the different stages of primary mineralization.     

Ultrastructural and biochemical characterization of extracellular matrix vesicles in healing alveolar bone sockets: Preliminary indications for the presence of contractile proteins

Extracellular matrix vesicles were identified and isolated from healing bone sockets. The diameters of these trilaminar membrane-invested organelles was 0.05 to OAS μm. Ca²⁻, Mg²⁺-, and K⁺ (EDTA)-mediated ATPases; alkaline and acid phosphatase; and pyrophosphatase activities of isolated matrix vesicles were compared with the membrane and cellular fractions. The vesicle fraction showed high alkaline phosphatase activity, which correlated with Mg²⁺-, and Ca²⁺-mediated ATPases. Pyrophosphatase activity was highest in the vesicle fraction as well. The presence of K⁺ (EDTA)-mediated ATPase in all the fractions indicated the occurrence of myosin. These findings were supported by electrophoretic patterns which revealed the presence of another contractile protein-actin.     

A comparative study on the occurrence and activity of extracellular matrix vesicles in young and adult rat maxillary bone

Summary The occurrence of matrix vesicles in the maxillary bone of normal young and adult rats was demonstrated by both ultrastructural and enzymatic studies. Transmission electron microscopy revealed that the vesicles are invested in trilaminar membranes. Occasionally, crystals were found both within the vesicles and in the surrounding matrix. Separated fractions of vesicles, membranes, and cells were studied biochemically. The amounts of vesicular protein and enzymatic activity per gram bone in the adult rats were significantly lower than in the young. This coincides with the higher number of vesicles observed in the TEM specimens of young rats when compared to that in the old ones. The specific activities of all enzymes examined in the three bone fractions obtained from both young and adult rats were similar. This indicates that no significant difference exists in the enzymatic characteristics of matrix vesicles from the maxillary bone of young and adult rats.     

A comparative study on the occurrence and activity of extracellular matrix vesicles in young and adult rat maxillary bone

The occurrence of matrix vesicles in the maxillary bone of normal young and adult rats was demonstrated by both ultrastructural and enzymatic studies. Transmission electron microscopy revealed that the vesicles are invested in trilaminar membranes. Occasionally, crystals were found both within the vesicles and in the surrounding matrix. Separated fractions of vesicles, membranes, and cells were studied biochemically. The amounts of vesicular protein and enzymatic activity per gram bone in the adult rats were significantly lower than in the young. This coincides with the higher number of vesicles observed in the TEM specimens of young rats when compared to that in the old ones. The specific activities of all enzymes examined in the three bone fractions obtained from both young and adult rats were similar. This indicates that no significant difference exists in the enzymatic characteristics of matrix vesicles from the maxillary bone of young and adult rats.     

Occurrence of actin-like protein in extracellular matrix vesicles

Summary Preliminary indications of the occurrence of actin and myosin in crude matrix vesicle preparations have been reported previously. In the present study extracellular matrix vesicles from rat alveolar bone were isolated. They were further purified by a sucrose density gradient. SDS-polyacrylamide gel electrophoresis of the purified vesicles revealed the presence of a polypeptide with a molecular weight of 43 K daltons and with electrophoretic mobility identical to that of blood platelet actin. The limited proteolysis of both 43 K dalton vesicular polypeptide and actin byStaphylococcus aureus-V8-protease revealed three fragments with identical electrophoretic mobility. In addition, the vesicular preparations inhibited the activity of DNase I, a property typical of actin monomers. Filamentous material extracted from matrix vesicles showed ultrastructural features of F-actin. Reaction of this material with heavy meromyosin resulted in arrowhead formation, which is characteristic of acto-heavy meromyosin. The occurrence of actin in extracellular matrix vesicles may account for their budding from the osteoblastic plasma membrane, their possible motility in the matrix, and maintenance of the spherical shape.     

Interobserver and intraobserver variation in the assessment of the healing of tibial fractures after intramedullary fixation

The reliability of the radiological assessment of the healing of tibial fractures remains undetermined. We examined the inter- and intraobserver agreement of the healing of such fractures among four orthopaedic trauma surgeons who, on two separate occasions eight weeks apart, independently assessed the radiographs of 30 patients with fractures of the tibial shaft which had been treated by intramedullary fixation. The radiographs were selected from a database to represent fractures at various stages of healing. For each radiograph, the surgeon scored the degree of union, quantified the number of cortices bridged by callus or with a visible fracture line, described the extent and quality of the callus, and provided an overall rating of healing. The interobserver chance-corrected agreement using a quadratically weighted kappa (kappa) statistic in which values of 0.61 to 0.80 represented substantial agreement were as follows: radiological union scale (kappa= 0.60); number of cortices bridged by callus (kappa = 0.75); number of cortices with a visible fracture line (kappa= 0.70); the extent of the callus (kappa = 0.57); and general impression of fracture healing (kappa = 0.67). The intraobserver agreement of the overall impression of healing (kappa = 0.89) and the number of cortices bridged by callus (kappa = 0.82) or with a visible fracture line (kappa = 0.83) was almost perfect. There are no validated scales which allow surgeons to grade fracture healing radiologically. Among those examined, the number of cortices bridged by bone appears to be a reliable, and easily measured radiological variable to assess the healing of fractures after intramedullary fixation.     

Osteal macrophages promote in vivo intramembranous bone healing in a mouse tibial injury model

Bone‐lining tissues contain a population of resident macrophages termed osteomacs that interact with osteoblasts in vivo and control mineralization in vitro. The role of osteomacs in bone repair was investigated using a mouse tibial bone injury model that heals primarily through intramembranous ossification and progresses through all major phases of stabilized fracture repair. Immunohistochemical studies revealed that at least two macrophage populations, F4/80⁺Mac‐2^?/lowTRACP^? osteomacs and F4/80⁺Mac‐2^hiTRACP^? inflammatory macrophages, were present within the bone injury site and persisted throughout the healing time course. In vivo depletion of osteomacs/macrophages (either using the Mafia transgenic mouse model or clodronate liposome delivery) or osteoclasts (recombinant osteoprotegerin treatment) established that osteomacs were required for deposition of collagen type 1⁺ (CT1⁺) matrix and bone mineralization in the tibial injury model, as assessed by quantitative immunohistology and micro–computed tomography. Conversely, administration of the macrophage growth factor colony‐stimulating factor 1 (CSF‐1) increased the number of osteomacs/macrophages at the injury site significantly with a concurrent increase in new CT1⁺ matrix deposition and enhanced mineralization. This study establishes osteomacs as participants in intramembranous bone healing and as targets for primary anabolic bone therapies. © 2011 American Society for Bone and Mineral Research.     

Molecular changes in extracellular matrix turnover after renal ischaemia-reperfusion injury

BACKGROUND: Renal ischaemia-reperfusion (IR) injury is an inevitable consequence of transplantation and contributes to later graft fibrosis. This study aimed to elucidate the possible mechanisms by studying the expression of genes associated with extracellular matrix (ECM) turnover. METHODS: Male Wistar rats underwent laparotomy, clamping of the right renal pedicle for 45 min, and left nephrectomy. Control animals underwent left nephrectomy only, or had no operation. Animals were killed at 8, 16 and 24 weeks and messenger RNA was extracted from renal tissue. Genes of interest were amplified and then quantified in an enzyme-linked immunosorbent assay system with levels expressed as a ratio to a known housekeeping gene (GAPDH). RESULTS: Experimental animals developed progressive proteinuria from 16 weeks onwards. At 8 weeks after IR injury, gene levels of matrix metalloproteinase (MMP) 2, an ECM-degrading enzyme, were significantly increased. Levels then fell progressively. This was associated with increasing expression of tissue inhibitor of metalloproteinases (TIMP) 1, an inhibitor of MMP-2, and of transforming growth factor (TGF) beta, a profibrotic cytokine, by 24 weeks following injury. CONCLUSION: These results suggest that, after an initial phase of increased ECM turnover following IR injury, the balance turns towards one of reduced degradation. This is likely to be an important mechanism in the subsequent development of fibrosis.     

Loss of bone strength after intramedullary nailing: Torsion tests of tibial osteotomies in rabbits

Rigid intramedullary nailing was used in 75 rabbits to stabilize a transverse osteotomy of the midshaft of the tibia. In 36 additional rabbits intramedullary nailing was performed without osteotomy. No additional external immobilization was used postoperatively. After removal of the nail the mechanical strength of the tibiofibular bones was tested torsiometrically in 30 osteotomized and 18 non-osteotomized animals from 3 to 24 weeks after the operation. At 3 weeks the torsional load fractured all osteotomized bones through the osteotomy line. At later stages a spiral fracture occurred either crossing or close to the osteotomy area, usually distal to the tibiofibular junction. The increase in mechanical strength of the osteotomized bones reached a maximum at 6 weeks and then decreased. The strength of the non-osteotomized nailed bones also decreased slightly. The results suggest that rigid intramedullary nailing, although providing good conditions for early consolidation of experimental osteotomy, leads secondarily to deterioration of the mechanical properties of tubular bone.