Osseointegration of zirconia and titanium implants in a rabbit tibiae model evaluated by microtomography, histomorphometry and fluorochrome labeling analyses.

OBJECTIVES: This study compares the osseointegration of machined-zirconia implants containing yttria (M-Y-TZP) with machined (M-Ti) and resorbable blast media (RBM-Ti) titanium implants. MATERIAL AND METHODS: M-Y-TZP, M-Ti and RBM-Ti implants were randomly placed in rabbit tibiae. Fluorochrome bone labels (tetracycline, alizarin and calcein) were administered at different time periods. After 8 weeks, osseointegration was evaluated in terms of bone-to-implant contact (BIC), new bone area (nBA), remaining cortical bone area (rBA) and temporal quantification of fluorochromes, using micro-CT and histomorphometric analyses. RESULTS: RBM-Ti implants showed higher resorption of the remaining cortical bone and bone formation (rBA = 36.9% and nBA = 38.8%) than M-Y-TZP implants (rBA = 48% and nBA = 26.5%). The BIC values showed no differences among the groups in the cortical region (mean = 52.2%) but in the medullary region, they were 0.45-fold higher in the RBM-Ti group (51.2%) than in the M-Y-TZP group (35.2%). In all groups, high incorporation of tetracycline was observed (2nd to 4th weeks), followed by alizarin (4th to 6th weeks) and calcein (6th to 8th weeks). In the cortical region, incorporation of tetracycline was similar between RBM-Ti (49.8%) and M-Y-TZP (35.9%) implants, but higher than M-Ti (28.2%) implants. Subsequently, alizarin and calcein were 1.1-fold higher in RBM-Ti implants than in the other implants. In the medullary region, no significant differences were observed for all fluorochromes. CONCLUSION: All implants favored bone formation and consequently promoted primary stability. Bone formation around the threads was faster in RBM-Ti and M-Y-TZP implants than in M-Ti implants, but limited bone remodeling with M-Y-TZP implants over time can have significant effects on secondary stability, suggesting caution for its use as an alternative substitute for titanium implants.

Tibial segmental bone defect treated with bone plate and cage filled with either xenogeneic composite or autologous cortical bone graft. An experimental study in sheep.

Tibia segmental defect healing in sheep were clinically, radiographically and histologically evaluated. Twelve young sheep aged four to five months were divided into two groups, G1 and G2. A 3.5 cm long segmental defect was created in the right tibial diaphysis with maintenance of the periosteum. The bone defects in both groups were stabilized with a bone plate combined with a titanium cage. In G1 the cage was filled with pieces of autologous cortical bone graft. In G2 it was filled with a composite biomaterial which consisted of inorganic bovine bone, demineralized bovine bone, a pool of bovine bone morphogenetic proteins bound to absorbable ultra-thin powdered hydroxyapatiteand bone-derived denaturized collagen. Except for one G1 animal, all of them showed normal limb function 60 days after surgery. Radiographic examination showed initial formation of periosteal callus in both groups at osteo-tomy sites, over the plate or cage 15 days postoperatively. At 60 and 90 days callus remodeling occurred. Histological and morphometric analysis at 90 days after surgery showed that the quantity of implanted materials in G1 and G2 were similar, and the quantity of new bone formation was less (p = 0.0048) and more immature in G1 than G2, occupying 51 +/- 3.46% and 62 +/- 6.26% of the cage space, respectively. These results suggest that the composite biomaterial tested was a good alternative to autologous cortical bone graft in this experimental ovine tibial defect. However, additional evaluation is warranted prior to its clinical usage.

Mistura de proteínas morfogenéticas ósseas, hidroxiapatita, osso inorgânico e colágeno envolta por membrana de pericárdio no preenchimento de defeito ósseo segmentar em coelhos / Mixture of bone morphogenetic protein, hydroxyapatite, inorganic bone and collagen interposed by pericardium barrier membrane in the filling of the segmental bone defect in rabbits

Avaliou-se o uso de biomaterial de origem bovina na regeneração de defeitos ósseos segmentares empregando-se 12 coelhos, fêmeas, da raça Norfolk, com idade de seis meses e pesos entre 3 e 4,5kg. Realizou-se falha segmentar bilateral de um centímetro de comprimento na diáfise do rádio, com inclusão do periósteo. No membro direito, o defeito foi delimitado por membrana de pericárdio liofilizada, contendo em seu interior mistura de proteínas morfogenéticas ósseas adsorvidas a hidroxiapatita, colágeno liofilizado e osso inorgânico. No membro esquerdo, o defeito não recebeu tratamento. Radiografias foram obtidas ao término do procedimento cirúrgico e aos sete, 30, 60, 90, 120 e 150 dias de pós-operatório. Após eutanásia de seis coelhos aos 60 dias e seis aos 150 dias de pós-cirúrgico, os resultados radiográficos e histológicos mostraram que a regeneração óssea foi inibida nos defeitos segmentares tratados com o biomaterial.

Biomaterials of bovine origin in regenerating segmental bone defects were evaluated. Twelve six-month old Norfolk rabbits, weighting 3 to 4.5kg were used. A 1cm long segmental defect was created in the radial diaphysis, including the periosteum, of both forelimbs. In the right forelimb, the defect was filled using a mixture of bone morphogenic proteins adsorbed to hydroxyapatite, agglutinant of lyophilized collagen in granules and anorganic cortical bone in granules delimited by a pericardial membrane. In the left forelimb, the defect did not receive treatment and served as a control. Radiographies were taken immediately after surgery and at seven, 30, 60, 90, 120 and 150 days post-operatively. Six rabbits were euthanized at 60 days and the other six at 150 days post-surgery for histological evaluation. Radiographic and histological results revealed that bone regeneration was inhibited in the segmental defects receiving biomaterials.

Bone morphogenetic proteins: from structure to clinical use

Bone morphogenetic proteins (BMPs) are multi-functional growth factors belonging to the transforming growth factor ß superfamily. Family members are expressed during limb development, endochondral ossification, early fracture, and cartilage repair. The activity of BMPs was first identified in the 1960s but the proteins responsible for bone induction were unknown until the purification and cloning of human BMPs in the 1980s. To date, about 15 BMP family members have been identified and characterized. The signal triggered by BMPs is transduced through serine/threonine kinase receptors, type I and II subtypes. Three type I receptors have been shown to bind BMP ligands, namely: type IA and IB BMP receptors and type IA activin receptors. BMPs seem to be involved in the regulation of cell proliferation, survival, differentiation and apoptosis, but their hallmark is their ability to induce bone, cartilage, ligament, and tendon formation at both heterotopic and orthotopic sites. This suggests that, in the future, they may play a major role in the treatment of bone diseases. Several animal studies have illustrated the potential of BMPs to enhance spinal fusion, repair critical-size defects, accelerate union, and heal articular cartilage lesions. Difficulties in producing and purifying BMPs from bone tissue have prompted the attempts made by several laboratories, including ours, to express these proteins in the recombinant form in heterologous systems. This review focuses on BMP structure, molecular mechanisms of action and significance and potential applications in medical, dental and veterinary practice for the treatment of cartilage and bone-related diseases.

Bone morphogenetic proteins: from structure to clinical use.

Bone morphogenetic proteins (BMPs) are multi-functional growth factors belonging to the transforming growth factor ss superfamily. Family members are expressed during limb development, endochondral ossification, early fracture, and cartilage repair. The activity of BMPs was first identified in the 1960s but the proteins responsible for bone induction were unknown until the purification and cloning of human BMPs in the 1980s. To date, about 15 BMP family members have been identified and characterized. The signal triggered by BMPs is transduced through serine/threonine kinase receptors, type I and II subtypes. Three type I receptors have been shown to bind BMP ligands, namely: type IA and IB BMP receptors and type IA activin receptors. BMPs seem to be involved in the regulation of cell proliferation, survival, differentiation and apoptosis, but their hallmark is their ability to induce bone, cartilage, ligament, and tendon formation at both heterotopic and orthotopic sites. This suggests that, in the future, they may play a major role in the treatment of bone diseases. Several animal studies have illustrated the potential of BMPs to enhance spinal fusion, repair critical-size defects, accelerate union, and heal articular cartilage lesions. Difficulties in producing and purifying BMPs from bone tissue have prompted the attempts made by several laboratories, including ours, to express these proteins in the recombinant form in heterologous systems. This review focuses on BMP structure, molecular mechanisms of action and significance and potential applications in medical, dental and veterinary practice for the treatment of cartilage and bone-related diseases.

Post-natal maturation of acinar cells of the guinea pig pancreas: an ultrastructural morphometric study.

The morphological maturation of the acinar cells of the guinea pig pancreas during post-natal development was characterized morphometrically by determining the intracytoplasmic accumulation of rough endoplasmic reticulum (RER) and zymogen granules. The following results were obtained for the period analysed, i.e., from 2 to 70 days of post-natal life: (a) the acinar cell volume increased by 210% (P < 0.01); (b) the mostly cisternal RER occupied more than 30% of the cytoplasm at any age studied and their total volume and surface in the cell were increased by 300 and 534% (P < 0.01), respectively; (c) maturation in the morphological pattern of the RER was observed; (d) the mean number of zymogen granules per cell increased from 261 at 2 days to 422 at 70 days (P < 0.01), while their mean diameter increased from 0.52 to 0.94 micron (P < 0.01) during the same period; (e) these increases in granule number and size were responsible for a 500% (P < 0.01) increase in total volume from 2 to 70 days and for a 304% increase (P < 0.01) in total surface from 2 to 35 days; (f) the RER and the zymogen granules together occupied 44, 54, 55 and 57% of the cytoplasm at 2, 14, 35 and 70 days of age, respectively. We conclude that although the pancreatic acinar cells of the guinea pig are morphologically well differentiated at 2 days of age, with the cytoplasm already showing a large amount of RER and zymogen granules, they are still immature. Morphological maturation of the acinar cell occurs during the first months of post-natal life and is characterized by a substantial gain in cell volume and intracytoplasmic accumulation of RER and zymogen granules, which significantly increase of both their absolute volume and total surface, with a higher growth rate being observed during the period from 2 to 14 days of post-natal life.

Allometric analysis of the postnatal development of the Syrian golden hamster pancreas.

The objective of the present study was to analyse allometrically the growth of the Syrian golden hamster pancreas during days 2 to 70 of postnatal development. Body and pancreatic mass were determined, followed by stereological determination of the absolute volume of each morphological compartment of the pancreas. The marked pancreatic growth, by 4360%, was due to an increase in the absolute volume of all morphological compartments, mainly the acini which showed an increase of 10 431%. Bivariate allometric analysis of pancreatic mass and morphological compartmental volume in relation to body mass gain showed: (1) a biphasic pattern for pancreatic mass, acinar volume, excretory duct volume and stromal volume, with the first phase being observed from 2 to 21 days of age and the second from 21 to 70 days of age, with allometry coefficients of 1.537-0.513, 1.770-0.543, 1.651-0.506 and 0.967-0.258, respectively, and (2) a monophasic pattern from 2 to 70 days for intercalated duct volume and islet volume, with allometry coefficients of 0.913 and 1.727, respectively. These results show that during the growth of the pancreas in relation to that of the body some structures - acini and excretory ducts - follow the growth pattern of the organ, while others - intercalated ducts and islets - show a different pattern. This may be related to the genetic growth characteristics of each compartment itself or to some relationship between compartments during some stage of the ontogenetic development of this organ.

Hyperplasia of myoepithelial cells expressing calponin during atrophy of the rat parotid gland induced by duct ligation.

The number and location of myoepithelial cells in the rat parotid gland submitted to surgical ligation of its main excretory duct were studied through immunohistochemical labelling for calponin. These cells were labelled by the streptavidin-biotin method using anti-calponin primary antibody, and their number was determined during each step of glandular atrophy, i.e., at time zero (control) and 1, 7, 15, 21, 30 and 60 days after ligation. Morphological analysis showed a gradual decrease and fibrosis of the glandular lobules accompanied by disappearance of the acini and the occurrence of duct-like structures. The expression of calponin was observed in all specimens analysed, being restricted to myoepithelial cells. Labelling revealed the distribution of myoepithelial cells around the acini and intercalated ducts in the control group and around duct-like structures later during the course of atrophy. Quantitative analysis demonstrated significant increase in the number of myoepithelial cells up to day 7 post-ligation, followed by gradual increases which, however, were not statistically significant. These results suggest that myoepithelial cells proliferate intensely up to day 7 post-ligation, an event that coincides with a higher rate of disappearance of acinar cells. After this period, the elevatednumber of cells observed at the end of the previous period is maintained.

Cell population growth in the rat parotid gland during postnatal development.

The growth kinetics of different cell populations in the rat parotid was studied. The evolution of the frequency and absolute number of each cell type was determined morphometrically by a particle-counting method and the evolution of the [(3)H]thymidine labeling indices of the same cell types was determined by autoradiography. The data obtained for the evolution of cell number in each gland compartment, i.e. acini, intercalated ducts, striated ducts and stroma, were adjusted by exponential equations, permitting estimation of the effective cell accumulation rate in the compartment for each population, i.e. the mean population duplication time (T(D)). In addition, the cell production rate in each gland compartment was determined using the mean labeling index for the period studied and a mathematical estimation of the mean cell generation time (T(G)), assuming an exponential growth pattern for the acinar, intercalated duct and striated duct populations during the period from 5 to 20 days of postnatal development. Analysis of the relation between effective cell accumulation (T(D)) and presumed cell production (labeling index and T(G)) for each intralobular parenchymal compartment of the rat parotid during this period suggests that the proliferative activity of the acinar cell population was sufficient to guarantee marked growth of its compartment and provided cells that presumably dedifferentiated into intercalated duct cells, whereas cells produced in the intercalated duct compartment migrated to, and differentiated into, cells of the striated duct compartment.

Morphometric dimensions of the mouse parotid glands of both sexes.

The goal of this research was to evaluate the morphometric dimensions of the different structures of male and female albino mouse parotid glands. The following morphometric dimensions were evaluated for the acini, intercalated ducts, striated ducts, excretory ducts and stroma: volume density, total compartmental volume, surface density, total external surface, surface-to-volume ratio, cell volume and absolute number of cells. Analysis of the results showed that the parotid gland mass was 43.7% greater (P < 0.01) in the male mice than in the females. This difference was due to the fact that the compartmental volumes of the acini, intercalated ducts and striated ducts were markedly higher in the male mice, 57.6% (P < 0.01), 253.1% (P < 0.01) and 91.1% (P < 0.05), respectively. The higher volume of the acinar morphological compartment was due to the total number of cells and average cell volume being higher in the male mice, 24.8% P < 0.01) and 47.7% (P < 0.01), respectively. Based on the results obtained, it was concluded that there are morphological differences between male and female parotid glands. These differences are detectable through morphometry, mainly in the morphological acinar and intercalated ducts compartments, which are more developed in male mice.

Rabbit bone marrow response to bovine osteoinductive proteins and anorganic bovine bone.

The effects caused by the implantation of bioabsorbable hydroxyapatite (HA) bound to a pool of bone morphogenetic proteins (BMPs) and other bone noncollagenous hydrophobic proteins mixed with anorganic bovine bone inside rabbit bone marrow were assessed. Within the interior of hollow cylindric titanium prototypes, the following biomaterials were inserted: (1) test group: HA containing a pool of BMPs and noncollagenous hydrophobic proteins mixed with anorganic bovine bone; (2) control group: HA without any protein mixed with anorganic bovine bone; and (3) negative control group: blood clot. The cylinders were placed surgically into the medial portion of the tibiae of 7 rabbits in a manner that allowed the biomaterials to contact just the bone marrow. Morphometric analysis showed that: (1) the biomaterials containing the protein mixture resulted in significantly less new bone than the biomaterials without such a mixture; (2) the group without the protein pool formed larger amounts of bone within the cylinder when compared to the negative control (blood clot only); and (3) the biomaterials containing the protein pool did not show any difference in relation to the negative control. It was concluded that a pool of BMPs and other bone noncollagenous hydrophobic proteins had an inhibitory effect on osteogenesis, and that the biomaterials without a protein pool formed a favorable substrate to bone formation.

Morphometric dimensions of Syrian golden hamster pancreas of both sexes.

The morphometric dimensions of the various structures of the pancreas of adult Syrian golden hamsters, of both sexes, were evaluated using stereological methods. The average body mass of the animals used was 133.8 +/- 2.45 g and 140.6 +/- 7.98 g for the males and females, respectively, and the pancreatic mass, 389.9 +/- 14.88 and 409.7 +/- 21.42 mg, respectively. The analysis of variance of the obtained data showed that: a) the acini, intercalated ducts and stroma did not present statistically significant differences in any of the dimensions evaluated, with the exception of the nucleus volume of the acinar cells which was 8.5% larger in the female (P < 0.05); b) the excretory ducts exhibited surface density, total external surface, surface-to-volume ratio, and absolute cell number, 18%, 33%, 14%, and 44%, respectively, larger in the females (P < 0.05); and c) the pancreatic islets of the females exhibited volume density, total volume and absolute cell number, 20%, 27% and 27%, respectively, larger than those of the males (P < 0.05).

Congenital fistulae of the lower lip in Van der Woude syndrome: a histomorphological study.

OBJECTIVE: To evaluate a significant number of cases of fistulae of the lower lip with the light microscope in order to investigate the histological variation and to attempt to define the histopathological pattern of the lesion. METHODS: Congenital fistulae of the lower lips of 17 patients with Van der Woude syndrome were analyzed by light microscopy. RESULTS AND CONCLUSION: The walls of most of the fistulae consisted of stratified nonkeratinized squamous epithelium and a lamina propria of dense connective tissue with areas of lymphohistiocytic inflammatory infiltrates. Bundles of striated muscle fibers, blood vessels, nerves, adipose tissue, and mixed acinar glands were observed. These glands surrounded the entire wall of the lesion, and their excretory ducts opened into the lumen of the fistula, explaining the clinical observation of elimination of mucous secretion through the opening of the fistula. Two microform cases of fistula were analyzed that presented only a depression in the epithelium at the site corresponding to the opening of the fistula.

Morphometric and allometric study of the mouse exocrine pancreas growth during the postnatal life.

Postnatal mouse pancreas growth was submitted to allometric analysis by the Wald nonparametric method, modified by Bartlett. The body and gland mass were obtained and the total cell number and the absolute compartmental volumes of acini, ducts and stroma were evaluated by morphometric methods. The allometric coefficients were calculated for the growths between the following parameters: a) pancreatic mass and body mass; b) acinar compartmental volume and body mass; c) ductal compartmental volume and body mass; d) stroma volume and body mass; e) total cell number and body mass; and f) acinar volume and stroma volume. The results of these analysis showed that the pancreatic mass, total cell number and stroma volume exhibited statistically significant allometric growths with a monophasic pattern and allometric coefficients of 1.56, 1.27 and 1.29, respectively, for the periods of 2 to 70, 2 to 28 and 2 to 70 days of age; while the growth of compartmental acinar volume in relation to body mass and compartmental stroma volume was biphasic. In the first case, the 1st phase occurred between 2 to 14 days (K = 1.09) and the 2nd phase between 14 to 70 days (K = 1.44) and in the second case, the 1st and 2nd phases occurred, respectively, between 2 to 28 days (K = 1.31) and 28 to 70 days (K = 0.79) of age. The growth of ductal volume in relation to body mass was also biphasic with a 1st phase between 2 to 14 days (K = 0.88) and a 2nd phase between 14 to 70 days (K = 1.07). These results permitted us to conclude that the growth of the mouse pancreas is allometrically associated with the growth of body mass.

Postnatal development of the rat sublingual glands. A morphometric and radioautographic study.

The postnatal development of rat sublingual glands was analyzed by morphometric and radioautographic studies. The absolute number of each cell type was evaluated by the Aherne II morphometric method for cell counting and labeling indices of these cell types were determined in radioautographs from animals injected with 3H-thymidine. The quantitative cell population kinetic studies were accompanied by morphologic analysis of the modifications in each gland structure. The data concerning evolution of number of each cell type were submitted to analysis by least squares fit-exponential curve. The exponential equations duplication times for the acinar, serous demilune, intercalated duct, striated duct and stroma cells from 2 to 30 days of age were 7.5, 9.0, 10.8 and 9.5 days, respectively. On the other hand, the mean labeling indices for the same cell types during the same period were 9.5%, 5.8%, 7.2%, 3.3% and 4.3%, respectively. Thus, the intercalated duct cells exhibited the second highest labeling index and the slowest growth rate, while the striated duct cells showed the lowest labeling index and the third highest duplication time. The fact that the striated duct cell labeling index does not explain the relatively short duplication time of these cells, suggests that cells from other neighboring morphologic compartments, probably from intercalated duct, migrate and differentiate into striated ducts cells.

Stereological study of acinar growth in the rat parotid gland induced by isoproterenol.

The growth of the rat parotid gland induced by daily treatment with isoproterenol (IPR) for 2 weeks was investigated by stereological methods applied to light microscopy. After 7 days of treatment, the glandular mass presented a 286% growth, with the first 3 days being the period of greatest growth. Total acinar volume exhibited a 363% increase during the period from 0 to 7 days, while acinar-cell volume presented a 468% growth from 0 to 5 days of treatment. On the other hand, total acinar-cell number did not increase during the study period. Thus, under the conditions used, IPR-stimulated gland growth was essentially hypertrophic. However, a significant increase in the number of bipolar and multipolar mitoses was also observed, especially on the third and fifth days of treatment. As no increase in acinar-cell number occurred during growth, the presence of these mitoses suggests that cell death occurred during gland growth. On this basis, bipolar mitoses may occur to replace cells that probably degenerated during treatment, whereas multipolar mitoses may lead to the occurrence of polyploidy.

Relationship between lower-lip fistulae and cleft lip and/or palate in Von der Woude syndrome.

OBJECTIVE: This study was conducted to evaluate the relationship between fistulae of the lower lip and cleft lip and/or palate in patients with Van der Woude syndrome. METHODS: The medical records of 11,000 patients with cleft lip and/or palate registered at the Cleft Lip-Palate Research and Rehabilitation Hospital, University of São Paulo, Bauru were reviewed. Of these patients, 133 (1.2%) presented with Van der Woude syndrome. RESULTS: Of the 133 patients, 88 (66.2%) exhibited full clefts, 22 (16.5%) only cleft lip, and 23 (17.3%) only cleft palate. The lower-lip fistulae observed in these 133 patients were bilateral symmetric in 66 (49.7%), bilateral asymmetric in 42 (31.6%), microform in 19 (14.3%), median in 5 (3.8%), and unilateral in 1 (0.7%). CONCLUSION: This population sample appears to exhibit the previously published tendency for bilateral, unilateral, or mixed-type congenital fistulae to be associated with cleft lip with or without cleft palate, while so-called microforms or conic elevations are almost exclusively associated with cleft palate.

Allometric study of the postnatal development of the rat sublingual glands.

The authors studied the rat sublingual glands growth in the period of 2 to 40 days of the postnatal life. The allometric coefficients for the gland mass growth and morphometrically evaluated volume of different gland components in relation to body mass growth, and for the parenchymal volume growth in relation to stroma volume growth, were calculated by the Wald non parametric method, modified by Bartlett. The allometric analysis showed that the gland mass, the mucous cells volume, the serous cells volume, the duct volume and the stroma volume exhibited statistically significant allometric growths with monophasic pattern and allometric coefficient of 0.93, 1.11, 0.76, 0.86 and 1.00, respectively. The analysis of the confidence intervals for these various k values, permitted to conclude that the differential growth of the gland mass is isometric, of the mucous cells volume is positive, of the serous cells and duct are negative and of the stroma volume is isometric.

Stereological study of the sexual dimorphism in mouse submandibular glands.

The sexual dimorphism of the mouse submandibular glands was studied by stereological methods. Albino mice of both sexes aged 140 days were used. Their average weight was 34.7 g por males and 26.0 g for females. The following morphometric dimensions were evaluated for the acini, intercalated ducts, convoluted granular tubules, striated ducts, excretory ducts and stroma: volume density, total volume, surface density, total external surface, surface-to-volume ratio, nuclear and cellular volume, and absolute number of cells. We also determined the mean diameters and estimated the total length of striated ducts and granular tubules in the gland. Analysis of the results showed that sexual dimorphism is present in all the morphological compartments of the mouse submandibular glands.

Autoradiographic evaluation of the cell cycle parameters of the various cells categories of the parotid, submandibular and sublingual glands of the suckling rat.

The evolution of the percentage of radioactive mitosis after a single thymidine-H3 injection, was determined for the various cell categories of the parotid, submandibular and sublingual glands of rats at 5th and 15th day of postnatal age. Estimates of the lengths of the S and the G2 + M/2 phases of the cell cycle were thus obtained, and averaged 9.8 and 2.7 hours, respectively, with extreme values of 9.3-11.2 and 1.6-3.2 hours.