Preparation and evaluation of acellular sheep periostea for guided bone regeneration.

The objective of this study was to fabricate an acellular sheep periosteum and explore its potential application in guided bone regeneration. Sheep periosteum was collected and decellularized by a modified decellularization protocol. The effectiveness of cell removal was proved by hematoxylin and eosin and 4',6-diamidino-2-phenylindole staining, DNA quantitative test, and agarose gel electrophoresis. After decellularization, its microstructure was found to become more porous while the integrality of collagen fibers remained undamaged, and the contents of collagen and glycosaminoglycan were not decreased significantly. Biomechanical analysis showed that the elastic modulus was significantly declined, while the yield stress was not affected, probably due to the collagen integrality. In vitro study of CCK-8 assay demonstrated that the acellular periosteum not only had no toxic effect to the MC3T3-E1 cells, but benefited the cell proliferation to some degree. In vivo experiment of guided bone regeneration was performed using a rabbit cranial model. Micro-CT and histological results revealed that the acellular periosteum not only effectively prevented the ingrowth of fibrous connective tissues, but also potentially facilitated bone regeneration. In conclusion, acellular sheep periostea, with wider sources, less costs, and more convenient fabrication process, would have great potential in the employment for guided bone regeneration.

The effect of hydrochloric acid on microstructure of porcine (Sus scrofa domesticus) cortical bone tissue.

We evaluated the degradation of cortical bone tissue by hydrochloric acid (HCl) since intentional bone decalcification in a forensic context has not been studied on a histomorphological level. We used 70 pig metatarsal bones split into subsamples and immersed in one of three concentrations of acidic solutions (0.5M, 1M, 2M HCl) for two and four hours. We analyzed the cortical thicknesses on transversal cross-sections, thicknesses of the three histomorphologically distinct zones present in acid-immersed bones, and number and area of crystals present in one of the zones. Furthermore, we analyzed the ratio of calcium to phosphorus (Ca:P). We observed a division of the cortical bone cross section into three distinctive zones: demineralized matrix (DM) in the periosteal part of bone, middle contact zone (CZ), and mineralized matrix (MM) in the endosteal part of bone. With increasing acid concentration and time of immersion (from 0.5M HCl for 2h to 2M HCl for 4h), the thickness of DM increased by 67%, the thickness of CZ increased by 56%, and the thickness of MM decreased by 32%. The Ca:P ratio in the contact zone of acid-treated samples did not change significantly with changing acid concentration and time of immersion. The Ca:P ratio of the CZ decreased by 10% when compared to the Ca:P ratio of MM in acid-treated samples. Moreover, we observed crystals on the outer periosteal border of the DM zone, in the CZ, and in the MM Haversian/Volkmann's canals. The size and number of the crystals in the CZ of acid-treated bones increased with acid concentration and time of acid immersion. Moreover, we also observed significant differences in all analyzed properties between anatomical regions. Due to varying reactions to acid immersion among anatomical regions, bone micro-degradation should be observed separately for each region.

Anchoring structure of the calvarial periosteum revealed by focused ion beam/scanning electron microscope tomography.

An important consideration in regeneration therapy is the fact that the tissue surrounding an organ supports its function. Understanding the structure of the periosteum can contribute to more effective bone regeneration therapy. As a cellular source, the periosteum also assists bone growth and fracture healing; this further necessitates its direct contact with the bone. However, its anchoring strength appears to be inexplicably stronger than expected. In this study, we used focused ion beam/scanning electron microscope tomography to investigate ultrathin serial sections as well as the three dimensional ultrastructure of the periosteum to clarify the architecture of its anchoring strength, as such assessments are challenging using conventional methods. We discovered perforating fibres that arise from the bone surface at 30 degree angles. Additionally, the fibres across the osteoblast layer were frequently interconnected to form a net-like structure. Fibroblast processes were observed extending into the perforating fibres; their morphologies were distinct from those of typical fibroblasts. Thus, our study revealed novel ultrastructures of the periosteum that support anchorage and serve as a cellular source as well as a mechanical stress transmitter.

Bone regeneration performance of surface-treated porous titanium.

The large surface area of highly porous titanium structures produced by additive manufacturing can be modified using biofunctionalizing surface treatments to improve the bone regeneration performance of these otherwise bioinert biomaterials. In this longitudinal study, we applied and compared three types of biofunctionalizing surface treatments, namely acid-alkali (AcAl), alkali-acid-heat treatment (AlAcH), and anodizing-heat treatment (AnH). The effects of treatments on apatite forming ability, cell attachment, cell proliferation, osteogenic gene expression, bone regeneration, biomechanical stability, and bone-biomaterial contact were evaluated using apatite forming ability test, cell culture assays, and animal experiments. It was found that AcAl and AnH work through completely different routes. While AcAl improved the apatite forming ability of as-manufactured (AsM) specimens, it did not have any positive effect on cell attachment, cell proliferation, and osteogenic gene expression. In contrast, AnH did not improve the apatite forming ability of AsM specimens but showed significantly better cell attachment, cell proliferation, and expression of osteogenic markers. The performance of AlAcH in terms of apatite forming ability and cell response was in between both extremes of AnH and AsM. AcAl resulted in significantly larger volumes of newly formed bone within the pores of the scaffold as compared to AnH. Interestingly, larger volumes of regenerated bone did not translate into improved biomechanical stability as AnH exhibited significantly better biomechanical stability as compared to AcAl suggesting that the beneficial effects of cell-nanotopography modulations somehow surpassed the benefits of improved apatite forming ability. In conclusion, the applied surface treatments have considerable effects on apatite forming ability, cell attachment, cell proliferation, and bone ingrowth of the studied biomaterials. The relationship between these properties and the bone-implant biomechanics is, however, not trivial.

PERIOSTIN: role in formation and maintenance of dental tissues.

The matricellular protein periostin is strongly expressed in collagen-rich connective tissues such as periodontal ligaments (PDLs), skeletal muscle, adipose tissue, tendons, skin, and bone. It is prominent in tumorigenesis, angiogenesis, and cardiac repair. It is localized in the periosteum and PDL, where it is seen in the cytoplasmic extensions of the PDL fibroblasts. It plays a key role in morphogenesis, postnatal development, and maintenance of the tooth, and related structures. It mediates and augments collagen fibrillogenesis, cell migration, adhesion, response to mechanical stress, and wound healing. It has been shown to be an integral regulator of periodontal disease pathogenesis and repair. This review focuses on the various functional aspects of periostin in dental connective tissue development and maintenance.

Biomechanical evaluation by AFM of cultured human cell-multilayered periosteal sheets.

We previously demonstrated that thicker periosteal sheets with enhanced cell layering maintain their component cells at relatively immature stages of differentiation but express a high in vivo osteogenic potential. As it has been recently proposed that stiff scaffolds provide a mechanical cue to various cell types that promotes differentiation, we postulated that the maintenance of immature cells in our periosteal sheets is due to the mechanical stiffness of the multilayered-cell architecture. To demonstrate the biomechanical characteristics of our periosteal sheets, we have determined their stiffnesses with atomic force microscopy (AFM) and evaluated the expression of extracellular matrix (ECM) components specifically by both immunocytochemistry and a complementary DNA microarray technology. Compared to osteoblastic Saos2 cells, the cytoskeletal fibers were developed more in the periosteal cells, but the periosteal cells in monolayer culture developed before either the cells in the peripheral or central regions of the periosteal sheets developed. However, the nanoindentation by AFM distinguished the central region from the peripheral region. The peak stiffness values of cells were ordered as follows: tissue culture polystyrene (1.66GPa)≫dispersed (9.99kPa)>central region (5.20kPa)>peripheral regions (3.67kPa). Similarly, the degree of development of α-smooth muscle actin (αSMA) filaments within cells was dispersed>central region>peripheral region. In conjunction with the abundantly deposited ECM in the periosteal sheets, these findings suggest that the order of cell stiffness may depend on the integration of the stiffness of individual ECM components and the extent of cytoskeletal fiber formation. Because recently published data have demonstrated that the optimal stiffness for osteogenic differentiation is 25-40kPa, it is plausible that the periosteal cells residing in the less-stiff multilayer regions could be maintained at relatively immature stages under the control of the stem-cell medium in vitro but start differentiating when exposed to the proper stiffness upon release from the culture conditions at the implantation site.

Repairing critical-sized rat calvarial defects with progenitor cell-seeded acellular periosteum: a novel biomimetic scaffold.

BACKGROUND: Many types of scaffolds have been used in bone tissue engineering, with none emerging as favorites. We propose the use of acellular periosteum as a biologic scaffold to allow for progenitor cell adherence, migration, and proliferation in vitro and to test the construct in vivo in a rat calvarial defect model. METHODS: Bovine periosteum was processed to remove all antigenic material (RTI Biologics), and its cambial layer was then seeded with adipose-derived stromal cells (ASCs) or periosteal-derived stromal cells (PSCs) and incubated for 14 days. Adherence required a fibronectin coat and was verified for both cell types via scanning electron microscopy and histology. Two 5-mm diameter calvarial defects were created in each of 19 rats. These were filled with xenograft bone chips and covered with acellular periosteum in combination with cells (ASC or PSC), growth factors (vascular endothelial growth factor, bone morphogenetic protein-2, or both), or alone (controls). Rats were killed 56 days postoperatively. Bone deposition was quantified by microcomputed tomography, and viability was determined histologically. Significance was determined through analysis of variance. RESULTS: Acellular allo-periosteum with a fibronectin coat permitted ASC and PSC adherence, migration, and proliferation in vitro. In the rat calvarial defects, the addition of stem cells (P < .001) and growth factors (P < .001) to the acellular periosteum increased de novo bone growth relative to controls. Although the stem cell source did not influence revitalization (P = .242), the combination of growth factors was more effective (P > .001) than either growth factor alone. The interaction indicated that the 2 cell types did not respond equally to growth factors (P = .039). CONCLUSION: Acellular allo-periosteum is a biomimetic scaffold that permits pleuripotent cell adherence, migration, and proliferation in vitro. The combination of acellular periosteum, xenograft bone, stem cells, and growth factors may prove a viable combination for cranial bone tissue engineering.

The effect of pore geometry on the in vitro biological behavior of human periosteum-derived cells seeded on selective laser-melted Ti6Al4V bone scaffolds.

The specific aim of this study was to gain insight into the influence of scaffold pore size, pore shape and permeability on the in vitro proliferation and differentiation of three-dimensional (3-D) human periosteum-derived cell (hPDC) cultures. Selective laser melting (SLM) was used to produce six distinct designed geometries of Ti6Al4V scaffolds in three different pore shapes (triangular, hexagonal and rectangular) and two different pore sizes (500 µm and 1000 µm). All scaffolds were characterized by means of two-dimensional optical microscopy, 3-D microfocus X-ray computed tomography (micro-CT) image analysis, mechanical compression testing and computational fluid dynamical analysis. The results showed that SLM was capable of producing Ti6Al4V scaffolds with a broad range of morphological and mechanical properties. The in vitro study showed that scaffolds with a lower permeability gave rise to a significantly higher number of cells attached to the scaffolds after seeding. Qualitative analysis by means of live/dead staining and scanning electron micrography showed a circular cell growth pattern which was independent of the pore size and shape. This resulted in pore occlusion which was found to be the highest on scaffolds with 500 µm hexagonal pores. Interestingly, pore size but not pore shape was found to significantly influence the growth of hPDC on the scaffolds, whereas the differentiation of hPDC was dependent on both pore shape and pore size. The results showed that, for SLM-produced Ti6Al4V scaffolds with specific morphological and mechanical properties, a functional graded scaffold will contribute to enhanced cell seeding and at the same time can maintain nutrient transport throughout the whole scaffold during in vitro culturing by avoiding pore occlusion.

Relating age and micro-architecture with apparent-level elastic constants: a micro-finite element study of female cortical bone from the anterior femoral midshaft.

Homogenized elastic properties are often assumed for macro-finite element (FE) models used in orthopaedic biomechanics. The accuracy of material property assignments may have a strong effect on the ability of these models to make accurate predictions. For cortical bone, most macro-scale FE models assume isotropic elastic material behaviour and do not include variation of material properties due to bone micro-architecture. The first aim of the present study was to evaluate the variation of apparent-level (homogenized) orthotropic elastic constants of cortical bone with age and indices of bone micro-architecture. Considerable age-dependent differences in porosity were noted across the cortical thickness in previous research. The second aim of the study was to quantify the resulting differences in elastic constants between the periosteum and endosteum. Specimens were taken from the anterior femoral midshaft of 27 female donors (age 53.4 +/- 23.6 years) and micro-FE (gFE) analysis was used to derive orthotropic elastic constants. The variation of orthotropic elastic constants (Young's moduli, shear moduli, and Poisson's ratios) with various cortical bone micro-architectural indices was investigated. The ratio of canal volume to tissue volume, Ca.V/TV, analogous to porosity, was found to be the strongest predictor (r2(ave) = 0.958) of the elastic constants. Age was less predictive (r2(ave) = 0.385) than Ca.V/TV. Elastic anisotropy increased with increasing Ca.V/TV, leading to lower elastic moduli in the transverse, typically less frequently loaded, directions. Increased Ca.V/TV led to a more substantial reduction in elastic constants at the endosteal aspect than at the periosteal aspect. The results are expected to be most applicable in similar midshaft locations of long bones; specific analysis of other sites would be necessary to evaluate elastic properties elsewhere. It was concluded that Ca.V/TV was the most predictive of cortical bone elastic constants and that considerable periosteal-endosteal variations in these constants can develop with bone loss.

Separation of newly formed bone from older compact bone reveals clear compositional differences in bone matrix.

In long bone diaphyses, woven bone forms first and then transitions into a more mineralized compact bone tissue. Prior evidence suggests that the non-collagenous protein composition of woven bone may be distinct from that of more mature bone tissue, particularly with respect to a diverse group of phosphorylated, extracellular matrix proteins. To critically test this hypothesis, we developed an in situ approach to isolate newly formed bone from more mature bone within the same long bone, and combine this anatomical approach with Western blotting to make relative comparisons of 7 phosphorylated matrix proteins important for bone physiology and biomineralization. Interestingly, 75 kDa bone sialoprotein (BSP), 63 kDa osteopontin, and the 75 kDa form of bone acidic glycoprotein-75 (BAG-75) were enriched in primary bone as opposed to more mature cortical bone, while osteonectin, fetuin A, matrix extracellular phosphoglycoprotein (MEPE) and dentin matrix protein-1 (DMP-1) appeared to be equally distributed between these two bone tissue compartments. Analyses also revealed the presence of larger sized forms of osteopontin (and to a lesser degree BSP) mostly in newly formed bone, while larger forms of BAG-75 were mostly detected in more mature cortical bone. Smaller sized forms of DMP-1 and BAG-75 were detected in both newly formed and more mature bone tissue extracts, and they are likely the result of proteolytic processing in vivo. Intact DMP-1 (97 kDa) was only detected in unmineralized matrix extracts. These findings indicate that newly formed bone exhibits a non-collagenous matrix protein composition distinct from that of more mature compact bone even within the same long bone, and suggest that the temporal fate of individual non-collagenous proteins is variable in growing bone.

Morphoscopic analysis of experimentally produced bony wounds from low-velocity ballistic impact.

Understanding how bone behaves when subjected to ballistic impact is of critical importance for forensic questions, such as the reconstruction of shooting events. Yet the literature addressing microscopic anatomical features of gunshot wounds to different types of bone is sparse. Moreover, a biomechanical framework for describing how the complex architecture of bone affects its failure during such impact is lacking. The aim of this study was to examine the morphological features associated with experimental gunshot wounds in slaughtered pig ribs. We shot the 4th rib of 12 adult pigs with .22 mm subsonic bullets at close range (5 cm) and examined resultant wounds under the light microscope, scanning electron microscope SEM and micro tomograph µCT. In all cases there was a narrow shot channel followed by spall region, with evidence of plastic deformation with burnishing of the surface bone in the former, and brittle fracture around and through individual Haversian systems in the latter. In all but one case, the entrance wounds were characterized by superficially fractured cortical bone in the form of a well-defined collar, while the exit wounds showed delamination of the periosteum. Inorganic residue was evident in all cases, with electron energy dispersive spectroscopy EDS confirming the presence of carbon, phosphate, lead and calcium. This material appeared to be especially concentrated within the fractured bony collar at the entrance. We conclude that gunshot wounds in flat bones may be morphologically divided into a thin burnished zone at the entry site, and a fracture zone at the exit.

Perfusion electrodeposition of calcium phosphate on additive manufactured titanium scaffolds for bone engineering.

A perfusion electrodeposition (P-ELD) system was reported to functionalize additive manufactured Ti6Al4V scaffolds with a calcium phosphate (CaP) coating in a controlled and reproducible manner. The effects and interactions of four main process parameters - current density (I), deposition time (t), flow rate (f) and process temperature (T) - on the properties of the CaP coating were investigated. The results showed a direct relation between the parameters and the deposited CaP mass, with a significant effect for t (P=0.001) and t-f interaction (P=0.019). Computational fluid dynamic analysis showed a relatively low electrolyte velocity within the struts and a high velocity in the open areas within the P-ELD chamber, which were not influenced by a change in f. This is beneficial for promoting a controlled CaP deposition and hydrogen gas removal. Optimization studies showed that a minimum t of 6 h was needed to obtain complete coating of the scaffold regardless of I, and the thickness was increased by increasing I and t. Energy-dispersive X-ray and X-ray diffraction analysis confirmed the deposition of highly crystalline synthetic carbonated hydroxyapatite under all conditions (Ca/P ratio=1.41). High cell viability and cell-material interactions were demonstrated by in vitro culture of human periosteum derived cells on coated scaffolds. This study showed that P-ELD provides a technological tool to functionalize complex scaffold structures with a biocompatible CaP layer that has controlled and reproducible physicochemical properties suitable for bone engineering.

Evaluation of immunocompatibility of tissue-engineered periosteum.

Tissue-engineered periosteum (TEP) and 'intramembranous ossification' may be an alternative approach to bone tissue engineering. In the previous study we attained successful bone defect reparation with homemade TEP in an allogenic rabbit model. But its allogenic immunocompatibility remained unknown. In this study TEP was constructed by seeding osteogenically induced mesenchymal stem cells of rabbit onto porcine small intestinal submucosa (SIS). A mixed lymphocyte reaction (MLR) was applied to evaluate the in vitro immunogenicity. The ratio of CD4(+)/CD8(+) T-lymphocytes was tested kinetically to evaluate the systematic reaction of the TEP allograft, and a histological examination was performed to investigate local inflammation and ectopic osteogenesis. MLR indicated that TEP had a higher in vitro immunostimulation than SIS (p < 0.05). The ratios of CD4(+)/CD8(+) lymphocytes increased in both TEP and SIS implanted groups in 2 weeks, followed by a decrease to a normal level from 2 to 4 weeks. Histological examination revealed modest lymphocyte infiltration for no more than 2 weeks. Moreover, subcutaneous ectopic ossification was observed in TEP allograft animals (8/12). Our findings imply that TEP has a certain immune reaction for the allograft, but it is not severe enough to impact osteogenesis in the allogenic rabbit model.

Importancia relativa de las modificaciones del contenido mineral óseo y del área en la determinación del descenso de la densidad mineral ósea del cuello femoral en la posmenopausia / Decrease in femoral neck bone mineral density after menopause: relative importance of changes in bone mineral content and area

La densidad mineral ósea (DMO, g/cm²) del cuello femoral (CF) está determinada por el contenido mineral óseo (CMO, g) y el área (A, cm²) de la región escaneada. En el presente trabajo nos propusimos estudiar el comportamiento relativo del CMO y del A en la determinación del descenso de la DMO en las etapas tempranas y mas tardías de la menopausia. Se evaluó la DMO del CF de 191 mujeres peri y posmenopáusicas. Las mismas fueron divididas en grupo 1: ≤ 60 años de edad y grupo 2: > 60 años. Se analizó el efecto de la edad sobre la DMO, el CMO y el A en toda la población y en ambos grupos mediante análisis de regresión univariado y se compararon las diferencias de las medias de las tres variables de ambos grupos. Resultados: DMO 0,897±0,12 vs. 0,80±0,11 y CMO 4,2±0,7 vs. 3,87±0,53 fueron significativamente mayores (p<0,01) en el grupo 1 que en el grupo 2 respectivamente, mientras que el A fue menor en el grupo 1 que en el grupo 2: 4,69±0,3 vs. 4,81±0,3 (p<0.01). La DMO, el CMO y el A correlacionaron con la edad en toda la población: r = -0,49; -0,34 y 0,26 respectivamente (todas p < 0,01). La DMO se correlacionó significativamente con la edad, r = -0,38 y -0,31 en los grupos 1 y 2 respectivamente, mientras que el CMO solamente lo hizo en el grupo 1, r = -0,27 y A solamente en el grupo 2, r = 0,3. CMO y A no correlacionaron con la edad en grupo 2 y grupo 1 respectivamente. Conclusiones: la pérdida de masa ósea (disminución del CMO) y la expansión perióstica (aumento del A) son respectivamente los principales determinantes de la caída de la DMO del CF en las etapas temprana y tardía de la menopausia.

Effects of local and whole body irradiation on the appearance of osteoblasts during wound healing in tooth extraction sockets in rats.

Irradiation before tooth extraction delays wound healing in the alveolar socket. This study examined the influences of local and whole body irradiation before tooth extraction on appearance of osteoblasts in the alveolar bone of rat maxillary first molars because bone formation is observed at the initial phase of wound healing. Several osteoblasts were generated 3 days after tooth extraction, and the number of cells increased day by day. Morphological studies showed there were little differences between local irradiation and non-irradiated controls. In contrast, the extraction wound in the whole body irradiation group showed delayed healing, and there was poor granulation tissue and very few osteoblasts at the bottom of the socket. An ultrastructural study showed that the osteoblasts in the extraction socket of whole body irradiation rats were smaller, and had poorly developed organelles. Injection of bone marrow cells to whole body-irradiated animals immediately after tooth extraction partially restored the number of osteoblasts. New periosteal bone formations outside of sockets showed little delay in the whole body irradiation group. These findings suggest that bone formation in the wound healing of extraction socket requires bone marrow cells from hematopoietic organs such as the bone marrow as well as local sources around the alveolar socket, during the initial phase of wound healing.

Ultrastructural study of tissues surrounding replanted teeth and dental implants.

OBJECTIVES: The aim of this study was to describe the ultrastructure of the dentogingival border at replanted teeth and implants. MATERIAL AND METHODS: Wistar rats (8 weeks old) were divided into groups for replantation and implantation experiments. In the former, the upper right first molars were extracted and then immediately replanted. In the latter, pure titanium implants were used. All tissues were fixed, demineralized and embedded in epoxy resin for ultrastructural observations. RESULTS: One week after replantation, the junctional epithelium was lost, and the oral sulcular epithelium covered the enamel surface. The amount of the epithelium increased in 2 weeks, and resembled the junctional epithelium, and the internal basal lamina and hemidesmosomes were formed in 4 weeks. One week after implantation, peri-implant epithelium was formed, and in 2 and 4 weeks, this epithelium with aggregated connective tissue cells were observed. In 8 weeks, the peri-implant epithelium receded, and aligned special cells with surrounding elongated fibroblasts and bundles of collagen fibers appeared to seal the implant interface. CONCLUSION: In replantation of the tooth, the internal basal lamina remained at the surface of the enamel of the replanted tooth, which is likely to be related to regeneration of the junctional epithelium and the attachment apparatus at the epithelium-tooth interface. Following implantation, a layer of cells with characteristics of connective tissue cells, but no junctional epithelium and attachment apparatus, was formed to seal the site of the implant.

Periosteal cell pellet culture system: a new technique for bone engineering.

To treat bone loss that is induced by disease or wounds, bone grafts are commonly used. In dentistry, guided tissue regeneration is effective in the treatment of periodontal diseases. However, bone resorption after implantation is a major problem with the bone graft and guided tissue regeneration technique. This study examines a cell pellet culture system without exogenous scaffolds for bone regeneration. First, we examined the effect of ascorbic acid on cells. Transmission electron microscopic observation revealed that cells formed a three-dimensional structure of multiple cell layers after 5 weeks of culturing in medium containing 50 microg/ ml ascorbic acid with the medium changed every 7 days. A single cell pellet was produced by centrifuging cells that were gathered from 10 tissue culture dishes. Van Gieson staining and collagen type I immunostaining showed that the pellet contained collagen fibers and cells that adhered to the collagen fibers. Several of these cell pellets were implanted subcutaneously on the backs of nude mice for 6 weeks. Histology and immunohistochemistry results indicated new bone formation, vascular invasion, and insular areas of calcification. Bone tissue was surrounded by osteoblasts. The appearance of new bone formation is similar to that seen in intramembranous ossification. The present pellet system is reliable and might solve problems of bone resorption after implantation.

A light and electron microscopic study of the limb long bones perichondral ossification in the quail embryo (Coturnix coturnix japonica).

The perichondral ossification of the limb long bones in the quail embryo is investigated, in this study, by means of light and electron microscopy. Longitudinal sections of the humerus, radius, ulna, femur, tibia and fibula stained with haematoxylin-eosin were examined by the light microscope. Ultrathin cross sections were selected for the electron microscope as well. Light microscopic analysis showed that the ossification began at the same time in the long bones of the wing and leg. At the embryonic day 6, all the cartilaginous rudiments consisted of three zones. The central zone composed of hypertrophic chondrocytes, a second zone on either side of the central zone, which consisted of flattened cells and a third zone, which represented the epiphyseal region. A thin sheath of osteoid and a bi-layered perichondrium-periosteum surrounded the central zone of the cartilaginous rudiments of the long bones. The perichondrium consisted of a layer of osteoblasts, in contact with the cartilage, and a layer of fibroblasts. At the embryonic day 7, the thickness of the calcified osteoid ring increased and a vasculature appeared between the layer of osteoblasts and the layer of fibroblasts. At the embryonic day 8, a second sheath of periosteal bone began to be formed. Concurrently, vascular and perivascular elements began to invade the cartilage. The ossification spread towards the distal ends of both the diaphysis. At the electron microscopic level, the osteoblasts of the perichondium showed cytoplasmatic characteristics of cells involved in protein synthesis. The perichondral ossification is the first hallmark of the osteogenesis in the long bones. The observations reported above, are in accordance with previous studies in the chick embryo.

Vascular changes in the periosteum of congenital pseudarthrosis of the tibia.

The etiology and the pathogenesis of congenital pseudarthrosis of the tibia (CPT) are still unknown. The affected tibia exhibits insufficient mechanical strength and osteogenetic capability. CPT is frequently associated with neurofibromatosis type 1 (NF1; von Recklinghausen's disease); however, both diseases have not yet been linked pathogenetically. This study presents the pathomorphologic findings of CPT under special consideration of NF1. Therefore, samples from patients operated on for CPT (n = 4) with (n = 3) and without (n = 1) neurofibromatosis were investigated by light microscopy, immunohistochemistry, and electron microscopy. The most striking finding in all patients was thickened periosteum with accumulation of nerval cells surrounding small arteries, causing subtotal or complete obliteration. In conclusion, impaired vascularization can result in decreased osteogenic capabilities. The similarity of ultrastructural findings in the abnormal periosteum and in skin neurofibromas of neurofibromatosis patients may indicate a pathogenetic association of both diseases.

Immunolocalization of vascular endothelial growth factor during heterotopic bone formation induced from grafted periosteum.

Vessel invasion is an important step in cartilage replacement that leads to bone formation, and vascular endothelial growth factor (VEGF) has been implicated as a key player in this process. Although grafted periosteum undergoes endochondral ossification, little is known about the role of VEGF in this process. In the current study the authors investigated by immunohistochemical, histochemical, and ultrastructural techniques the localization of VEGF during bone formation in periosteal grafts. At day 14 after grafting the tibias of Japanese white rabbits, periosteal cells in the grafted tissue had differentiated into chondrocytes to form cartilage. Some chondrocytes were immunopositive for VEGF expression, and subsequent vessel invasion occurred predominantly in these VEGF-positive areas. At day 45, the cartilage invaded by blood vessels had been replaced by newly formed bone. These findings suggest that VEGF is associated with the process of blood vessel invasion into cartilage before bone replacement in endochondral ossification from grafted periosteum.