Injection of vascular endothelial growth factor into knee joints induces osteoarthritis in mice.

UNLABELLED: Osteoarthritis (OA) is a common joint disorder affecting circa 2% of the population. OBJECTIVES: It has been suggested that secretion of vascular endothelial growth factor (VEGF) could play a role in the chain of events leading to OA. METHODS: In the present study, healthy mice were injected intra-articularly with VEGF. RESULTS: Shortly after the administration of VEGF, synovial hyperplasia, increased calcification of the articular cartilage and bone sclerosis were observed. Consequently, cartilage degradation characteristic of OA was found. These changes were seen to a lesser degree in the opposite knees of VEGF-injected mice and did not occur in the control mice. CONCLUSIONS: The findings suggest an active role of VEGF in the pathogenesis of OA and render support to a possible role for subchondral bone sclerosis in the pathogenesis of cartilage degradation.

Differential effects of bone graft substitutes on regeneration of bone marrow.

BACKGROUND: This study used a rat tibial marrow ablation model to test the hypothesis that bone remodeling within the medullary canal varies with bone graft materials of different chemical compositions and structural properties, impacting marrow cavity restoration. Bone graft materials were selected based on their relative resorption or degradation in vivo and their osteogenic properties. METHODS: Following ablation of the right tibial marrow in male Sabra-strain rats, materials were implanted in the proximal marrow cavity: poly-D,L-lactide-co-glycolide 75 : 25 (PLGA); coralline-hydroxyapatite (HA), calcium-sulfate (CaSO4), collagen-HA-tricalcium phosphate granules, anorganic bovine bone mineral, demineralized bone matrix (DBM), 45S5 Bioglass (BG), PLGA with BG 50 : 50, PLGA : BG 80 : 20, and PLGA and PLGA:BG 50 : 50 plus bone marrow (BM). Control tibias were ablated but received no implants. At 2 (endosteal bone healing), 4 (marrow cavity remodeling) and 8 weeks (marrow restoration), six to eight animals per group were euthanized and tibias processed for histomorphometry of proximal and distal medullary canals. RESULTS: Control tibias showed primary bone in proximal and distal medullary canals at 2 weeks, with trabeculae surrounded by cellular marrow. At 4 and 8 weeks, control trabeculae were thinned and marrow had more fat cells. In the treated tibias, trabecular bone volume (TBV) varied with time and was material specific. Most implants supported comparable TBV at 2 weeks. Sites with CaSO4 or DBM exhibited decreased TBV with time whereas trabecular bone was retained in proximal tibias containing other materials, closely juxtaposed to the implants. TBV did not always correlate directly with implant volume, but changes in BM volume were correlated inversely with TBV. Addition of BM increased marrow restoration in sites containing PLGA; however, BM reduced restoration of marrow when added to PLGA : BG. Although the presence of implants in the proximal tibia resulted in retention of trabecular bone, there was a time-dependent reduction in TBV in distal canals; the rate and extent of the distal TBV reduction were implant dependent. CONCLUSIONS: Thus, although many materials can support bone formation in the marrow cavity, bone quality, quantity, and physical relationship to the implant, and its rate of resorption differ in a material-dependent manner, resulting in differences in the restoration of marrow. CLINICAL RELEVANCE: Bone graft materials should be selected not only for their ability to support new bone formation but also for their impact on the remodeling phase of bone healing.

Extracellular matrix vesicle distribution in primary mineralization two weeks after injury to rat tibial bone (ultrastructural tissue morphometry).

Primary mineralization on the 14th day of bone healing served as a model to study the distribution of extracellular matrix vesicles by means of transmission electron microscopy combined with computerized morphometry. Vesicles were traced on electron micrographs and classified according to diameter, distance from the calcified front, and type. The different types were determined as follows: electron-lucent vesicles ("empty"), vesicles with amorphous contents ("amorphic"), vesicles containing crystalline depositions ("crystal"), and vesicles with crystals and ruptured membranes ("rupture"). The majority of the vesicles measured between 0.02 and 0.07 micron and were located at a distance of less than 3 micron from the calcified front. They were distributed according to "empty", "amorphic", "crystal" and "rupture" type in concentrations of 10%, 31%, 51% and 8%, respectively. The diameters of the "rupture" vesicles were significantly larger than those of the "empty" and "amorphic" types. The sequence of their location, starting at the calcified front, ran as follows: "rupture", "crystal", "amorphic" and "empty", with the "rupture" type proximate to the front. According to the working hypothesis on calcification via extracellular matrix vesicles, it is thought that the "empty" vesicles are secreted by the cell and that subsequently amorphous Ca and Pi accumulate intravesicularly to form a hydroxyapatite crystal which, in turn, brings about rupture of the vesicle's membrane. The results of the present study support this theory and, additionally, show that the maturation process is accompanied by an increase of the vesicular diameter and by its approximation to the calcifying front.

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)

Uptake and biodistribution of technetium-99m-MD32P during rat tibial bone repair.

The present study was carried out in order to test the hypothesis that intravenously injected Tc-MDP separates into its technetium and methylene diphosphonate components in the bone, and that the technetium is preferentially taken-up by the newly-formed osteoid, while the methylene diphosphonate is taken up by the forming mineral. Uptake of Tc-MDP was studied in a rat model of primary bone formation following tibial bone marrow ablation. Each of five radiopharmaceuticals (99mTCO4, 99mTc-MDP, Tc-MD32P, 99mTc-MD32P or MD32P) was injected and their uptake was followed in the whole bone as well as in the organic and inorganic phases of the bone. Irrespective of the radionuclides injected, 99mTc was always taken-up preferentially by the organic phase, while the 32P was preferentially taken-up by the inorganic phase. When 99mTcO4 was injected, it was not taken up by the bone at all. These results indicate that the increased incorporation of 99mTc, when administered as 99mTc-MDP during bone healing, reflects an enhancement in the formation of the organic matrix and not of the calcification process. The study also suggests that the 99mTc-MDP dissociates into its technetium and methylene diphosphonate moieties, which are then adsorbed onto the organic and inorganic phases respectively.

Correlations between uptake of technetium, calcium, phosphate, and mineralization in rat tibial bone repair.

Technetium-99m-(99mTc) phosphates are extensively used for detection of bone formation and resorption. The present is a study of 99mTc incorporation during bone remodeling. Uptake of 99mTc-labeled phosphate was studied in an animal model of primary osteogenesis following tibial marrow injury and incorporation was correlated to that of calcium-47 (47Ca), phosphorus-32 (32P), and with matrix vesicle calcification. Isotope uptake on Day 6 in the whole bone was increased compared to controls. On this day, an increase in vesicular diameter and distance from the calcified front was previously observed. Technetium-99m-labeled phosphates were detected only in the organic phase. Phosphorus-32 and 47Ca were detected in both organic and inorganic phases. It is suggested that 99mTc serves as a specific marker to the anabolic phase of remodeling. Increased incorporation of 99mTc during bone healing indicates enhanced organic matrix formation and not calcification.

Inhibition of the inductive activity of demineralized bone matrix by different percutaneous implants.

Teflon tubes with either Gore-Tex (PTFE) or Dacron felt sleeves were implanted percutaneously in rats, in conjunction with demineralized bone matrix. This resulted in different inflammatory reactions, as well as in chondrogenesis and osteogenesis in the subcutaneous tissues. Although induction of osteogenesis by the demineralized bone matrix in the vicinity of the foreign material was inhibited, circumferential bone formation was highly reproducible. The prospect of utilizing demineralized bone matrix in order to enhance acceptance of percutaneous tubes is discussed.

Effect of glass-ceramic implants on primary calcification in rat tibial bone after injury.

Using histomorphometry and transmission electron microscopy the distribution of extracellular matrix vesicles around bone-bonding and non-bonding glass-ceramic implants in tibial bone healing was studied. Grouping of vesicles was performed according to type, diameter and distance to the calcified front. Most vesicles were 0.06-0.22 microns in diameter in both implants tested. The mean vesicular distance of matrix vesicles to the calcified front was statistically significantly lower in non-bonding material compared with KG Cera. The sequence of vesicle arrangement according to diameter was: 'empty', 'amorphous', 'crystal', 'rupture'. Empty vesicles were the smallest and rupture vesicles the largest. Distances of vesicles to the calcified front showed the same sequence of arrangement, while the rupture type was located nearest to the front. The findings support the widely acknowledged hypothesis on the role of matrix vesicles in mineralization. It was shown that mineralizing tissue around bone-bonding glass-ceramics contains more matrix vesicles, which are distributed further from the front with a lower degree of calcification. The parameters studied could serve as standard criteria for the evaluation of implant materials.

The early host and material response of bone-bonding and non-bonding glass-ceramic implants as revealed by scanning electron microscopy and histochemistry.

The interface of bone-bonding and non-bonding glass-ceramics in the femur of of rats with the concomitant material and host response has been investigated by scanning electron microscopy (SEM) and histochemistry after transverse fractures in the interface level. During wound healing around these implants, four overlapping phases could be distinguished: (1) blood clot formation, (2) formation of organization tissue, (3) formation of primary bone and calcification, and (4) remodelling which lasts from months to years. This sequence of healing was disturbed around metal ions containing non-bonding implants, as shown by a longer settlement of acid phosphatase positive macrophages and a disturbed calcification at the surface of the material. Only bone-bonding implants developed considerable changes in surface morphology due to leaching and corrosion phenomena. A preferential leaching of the glass moiety, starting at the phase transition between glass and ceramic, contributes to the production of surface elevations which provide adhesion points for fibres and fibrils. Subsequent mineralization of inserting fibres contributes to a tensile strength at the interface. These findings are essential for further understanding of bone-bonding mechanisms and for further development of surface-reactive materials.

Effect of titanium implants on primary mineralization following 6 and 14 days of rat tibial healing.

The effect of pure commercial titanium implants on the process of primary mineralization was studied. This was examined by insertion of titanium implants into rat tibial bone after ablation. The effects of the titanium were studied through the behaviour of extracellular matrix vesicles (MV). Methods of morphometric analysis at the TEM level were applied. The insertion of titanium implants was followed by an increase in the number of MV as well as vesicular diameter and by a decrease in vesicular distance from the calcified front when compared to normal healing. These results suggest that the process of MV maturation around titanium implants was delayed when compared to normal primary bone formation during bone healing. The delay in mineralization was compensated by an increase in vesicle production, resulting in an enhancement of primary mineralization by the titanium.

Uptake and biodistribution of 99mtechnetium methylene-[32P] diphosphonate during endosteal healing around titanium, stainless steel and hydroxyapatite implants in rat tibial bone.

Early evaluation of intraosseous implant success and failure is critical, but, until now, there have been no reliable systems of measurement. The present study assessed whether the use of 99mtechnetium methylene-[32P]diphosphonate (99mTcMD32P), a marker for both bone formation and mineralization, can indicate if an implant is bone-bonding or non-bonding. Moreover, this study examined how bone-bonding (titanium and hydroxyapatite) and non-bonding (stainless steel) implants affected the normal healing of bone after marrow ablation, as measured by uptake of 99mTc and 32P. Titanium, hydroxyapatite and stainless steel implants were placed in the right tibiae of Sabra strain rats following ablation of the marrow, and 99mTcMD32P was injected 18 h before harvest. AT 3, 6, 14, 21 and 42 d (and in some experiments, on days 28 and 35) post-injury, the treated and contralateral tibiae were removed and cleaned of soft tissue. The uptake of 99mTc and 32P was measured in the whole bone, as well as in its organic and inorganic phases. Effects of the implants were assessed by comparing the treated to the untreated tibia in each rat. The distribution of 99mTc and 32P varied with each implant. After the insertion of titanium, increased 99mTc uptake was seen in whole bone and in the inorganic and organic phases at days 6-14. 32P uptake in whole bone and in the inorganic phase increased only at day 6, and 32P uptake was decreased in the organic phase at that time. In tibiae implanted with hydroxyapatite, 99mTc and 32P uptake was seen in the whole bone at days 6 and 14. While 99mTc uptake was increased in both the organic and inorganic phases, 32P uptake into the organic phase was decreased at both day 6 and day 14. In tibiae implanted with stainless steel, effects were observed only on day 6. The increased 99mTc uptake in whole bone reflected increases in both the organic and mineral phases. Increased 32P uptake was observed in whole bone as well, due to an increase in the 32P uptake in the mineral phase only; incorporation of 32P in the organic phase was comparable to that found in the contralateral limb. The results of this study indicate that implants alter bone healing, as indicated by the uptake of 99mTc and 32P in the different bone compartments. Moreover, decreased 32P uptake by the organic phase in the presence of bone-bonding implants suggests that cleavage of 99mTcMD32P into its technetium and methylene diphosphonate moieties was inhibited, perhaps as a function of the onset of calcification in the newly synthesized osteoid. The effect of the implants on bone healing was observed on days 6-14, when active bone formation and mineralization were occurring, supporting the hypothesis that these materials events associated with initial calcification. Uptake of 99mTc varies as a function of time, and uptake of 32P varies with time and distribution in the mineral or organic phase of bone, suggesting that these parameters may be useful as indicators of bone-bonding.

Dentinoenamel junction area of a resorbing permanent incisor studied by means of scanning electron microscopy.

The surfaces of enamel and dentin that had become separated from each other presumably in the region of the dentinoenamel junction were examined. The enamel surface at the junction was scalloped; the scallops were made of enamel rods. The dentinal surface showed a mirror image picture, tubular in nature. Both surfaces were granulated, which may indicate that apatite crystals from the two tissues intermingle there.

Chronic tympanomastoiditis with formation of calculi.

Numerous calculi of varying sizes and shapes were discovered in the mastoid, antrum, and attic during mastoid surgery for persistent chronic suppurative inflammation of the middle ear cleft with retroauricular and external meatal fistulas in an 8-year-old boy. Light microscopic and scanning electron microscopic examinations and x-ray microanalysis revealed the calculi to consist of masses of crystals containing calcium-oxalate and calcium- and magnesium-phosphate. No signs of tuberculosis were found. Since the patient had multiple dysplasias, including ipsilateral macrotia, in addition to chronic otitis media and mastoiditis, it is suggested that the formation of the calculi may have been due to congenital predisposition.

The use of monoclonal anti-CEA antibody immunohistochemistry in detecting the origin of oral cavity metastasis.

A 75-year-old woman presented with a painless swelling in the right mandibular retromolar area and numbness of the left lower lip. Radiographic examination of the mandible demonstrated an osteolytic lesion of the ascending ramus. Biopsy revealed adenocarcinoma of obscure origin. Staining of the specimen with a monoclonal antibody specific to colon carcinoma revealed its origin. On subsequent examinations, a primary tumor in the rectosigmoid region with extensive lung, liver and skeletal metastases were diagnosed. This unusual case of colonorectal carcinoma, presenting as a metastatic lesion of the mandible, was readily diagnosed by a novel immunohistochemical technique that utilizes highly specific monoclonal antibodies.

Woven bone formation around implants and the effect of bacterial infection.

Several implant materials used in dental and orthopedic surgery were placed in rat tibial bones to study their effects on mineralization. The implants consisted of bone bonding and non-bonding materials. Changes in mineralization were defined by morphometric analysis of matrix vesicle distribution at the implant interface and in normal bone healing following marrow injury. Bone-bonding materials induced an increase in matrix vesicle activity. This finding was supported by study of the biochemical changes in the same model that manifested high correlations to the morphometrical observations with regard to enhancement or delay of primary mineralization. In addition, the study of healing using nuclear methods indicated that implants alter bone healing as shown by the different uptakes of 99mTc and 32P in the different bone compartments. Decreased 32P uptake by the organic phase in the presence of bone-bonding implants suggested that cleavage of 99mTc-MD32P into its technetium and methylene diphosphonate moieties was inhibited by administration of implants. Further studies on the effect of bacterial infection on the peri-implant tissues revealed a decrease in woven bone formation due to infection.

The outcome of teeth with periapical periodontitis treated with nonsurgical endodontic treatment: a computerized morphometric study.

OBJECTIVES: This study investigated the prognosis for successful endodontic treatment and the correlation between the size of the periapical lesion, the quality of the root canal treatment, and the type of coronal restoration. METHOD AND MATERIALS: Periapical radiographs of 319 teeth with periapical periodontitis were studied. The area of each lesion was measured before treatment and 1 to 12 years after completion of the endodontic treatment. The measurements were performed using computerized morphometry. RESULTS: In 65.2% of the teeth, the size of the lesion decreased, while in 34.8% of teeth, there was an increase. Lesions larger than 10 mm2 had a greater tendency for healing. CONCLUSION: No significant correlation was found between the quality of root canal treatment, the type of the coronal restoration, and the success rate of the endodontic treatment.

Adaptation of Class II Vitremer restorations with and without primer: a morphometric study.

PURPOSE: The aim of this study was to assess the adaptation of Vitremer with and without primer and compare to that of Z100 with Scotchbond Multi-Purpose. METHODS: Fifty-seven Class II cavities were prepared in 32 extracted or exfoliated primary molars. The cavities were randomly assigned to one of three groups and restored as follows: group A, Vitremer with primer (20 preps); group B, Vitremer without primer (19 preps); and group C, Z100 with Scotchbond Multi-Purpose (18 preps). The restored teeth were thermocycled, embedded in acrylic resin, and sectioned. At least three 1-mm thick sections were obtained from each restoration. Adaptation of the materials was assessed by computerized quantitative morphometry using an image analysis system. In addition to the margin, the entire contact length between the tooth and the restorative material was measured. Voids were recorded separately for the base and cavity margins, and the percentage of defected length was calculated. At least three sections of each restoration were assessed. The section with the worst results was selected as the representative of the restoration. RESULTS: Margin defects were present in 14% of all the restorations, equally distributed between the three groups (A, 10%; B, 16%; C, 17%). A significant difference was found between groups B and C when the percentage of defects in the base was assessed. CONCLUSIONS: Vitremer without primer presented considerably fewer voids when compared with Z100/Scotchbond Multi-Purpose. Although no difference in margin defects could be observed between the three groups a better adaptation to the cavity base was seen in the Vitremer restorations without primer. This finding might be of clinical importance and should be tested in other in-vitro and in-vivo studies.

[The styloid process elongation syndrome (Eagle syndrome): a case report].

Eagle syndrome is an aggregate of symptoms that includes recurrent throat pain, foreign body sensation, dysphagia, and/or facial pain as a direct result of an elongated styloid process or calcified stylohyoid ligament. The etiology is poorly understood and several theories have been proposed. The pathophysiological mechanism of symptoms is debated as well. Diagnosis is made both radiographically and by physical examination. Treatment of Eagle syndrome is either surgical or non surgical. A case report of temporomandibular joint pain that has been finally diagnosed as Eagle syndrome is presented and discussed.

[Salivary tests as a diagnostic tool in oral disease management].

Saliva plays an important role in maintaining the oral and the dental health, by lubricating the mucosae and protecting the teeth from caries attack. Furthermore, saliva participates in the taste sensation and recognition processes and is a central component in the first stage of the food digestion. Saliva collection, either in the pure or in the whole form, is a relatively easy procedure. Since the collection is non invasive, it is not harmful to the patient and has no complications. The paper discusses the use of saliva in the diagnosis of oral and systemic diseases and in monitoring the levels of molecules like hormones and medicines. The experience accumulated in our department in that field is also presented.