Bony Matrix Research Articles

Avian tenascin-W: Expression in smooth muscle and bone, and effects on calvarial cell spreading and adhesion in vitro

Tenascins are glycoproteins found primarily in the embryonic extracellular matrix. Here we have characterized the fourth and final member of the tenascin family in birds: tenascin-W. Avian tenascin-W has 3.5 epidermal growth factor-like repeats, 6 fibronectin type III domains, and a C-terminal fibrinogen-related domain. Immunohistochemistry reveals that avian tenascin-W is expressed transiently in developing smooth muscle, tendons, and ligaments, but the primary site of tenascin-W expression during development is in the extracellular matrix of bone and the cellular periosteum. In bony matrix, tenascin-W-coated fibrils partly overlap with fibrils that contain tenascin-C. The anti-tenascin-W also labels fibrils in cultures of osteogenic embryonic chicken calvarial cells. Primary calvarial cells cultured on purified tenascin-W become rounded, and fewer of these cells spread on fibronectin when tenascin-W is added to the medium when compared with calvarial cells cultured on fibronectin alone. Moreover, tenascin-W reduces the adhesion of calvarial cells to collagen type I in a shear force assay. We conclude that tenascin-W is likely to play a phylogenetically conserved role in developing bone and that it shares some of the basic anti-adhesive and matrix modulatory properties as tenascin-C.

Expression pattern of matrilins and other extracellular matrix proteins characterize distinct stages of cell differentiation during antler development

Deer antler regeneration is a uniquely intense and complex process, which involves chondrogenic and intramembranous ossification. Cell differentiation in the developing antler of red deer, Cervus elaphus, was characterized with extracellular matrix markers. Expression of the four matrilin genes was monitored by immunohistochemistry and in situ hybridization and compared to cartilage markers collagen II and cartilage link protein, the bone component collagen I, and the endothelial basement membrane constituent laminin. The mesenchyme layer at the very tip of the velvet antler was enriched in link protein, indicative of the role of hyaluronan in apical morphogenesis. Matrilin-2, formerly described as a component of hard and soft connective tissue matrices, was identified here also as a marker of cells with high differentiation potential: it is expressed predominantly by mesenchyme cells, prechondrocytes and preosteoblasts. In addition to matrilin-3, documented as a component of the bony extracellular matrix, expression of the other three matrilin genes was observed in osteoprogenitor cells and osteoblasts. A layer of presumed osteoprogenitor cells, which surrounded the perivascular channels, expressed all four matrilins and collagen I. As a consequence, all four matrilins, including matrilin-1, previously detected in the skeleton only in cartilage, were found associated to collagen I-rich structures in a thin layer bordering the columns of hypertrophic chondrocytes. Cells with similar morphology and expression pattern were identified in the periosteum. Altogether all cell types of the chondrogenic and osteogenic lineage that expressed the four matrilins were in a separate study [Faucheux, C., Nicholls, B.M., Allen, S., Danks, J.A, Horton, M.A., Price, J.S., 2004. Recapitulation of the parathyroid hormone-related peptide-Indian hedgehog pathway in the regenerating deer antler. Dev. Dyn. 231, 88-97] positive for parathyroid hormone-related peptide and its receptor.

Indian hedgehog synchronizes skeletal angiogenesis and perichondrial maturation with cartilage development

A null mutation in the morphogen Indian hedgehog (IHH) results in an embryonic lethal phenotype characterized by the conspicuous absence of bony tissue in the extremities. We show that this ossification defect is not attributable to a permanent arrest in cartilage differentiation, since Ihh(-/-) chondrocytes undergo hypertrophy and terminal differentiation, express angiogenic markers such as Vegf, and are invaded, albeit aberrantly, by blood vessels. Subsequent steps, including vessel expansion and persistence, are impaired, and the net result is degraded cartilage matrix that is devoid of blood vessels. The absence of blood vessels is not because the Ihh(-/-) skeleton is anti-angiogenic; in fact, in an ex vivo environment, both wild-type and Ihh mutant vessels invade the Ihh(-/-) cartilage, though only wild-type vessels expand to create the marrow cavity. In the ex vivo setting, Ihh(-/-) cells differentiate into osteoblasts and deposit a bony matrix, without benefit of exogenous hedgehog in the new environment. Even more surprising is our finding that the earliest IHH-dependent skeletal defect is obvious by the time the limb mesenchyme segregates into chondrogenic and perichondrogenic condensations. Although Ihh(-/-) cells organize into chondrogenic condensations similar in size and shape to wild-type condensations, perichondrial cells surrounding the mutant condensations are clearly faulty. They fail to aggregate, elongate and flatten into a definitive, endothelial cell-rich perichondrium like their wild-type counterparts. Normally, these cells surrounding the chondrogenic condensation are exposed to IHH, as evidenced by their expression of the hedgehog target genes, patched (Ptch) and Gli1. In the mutant environment, the milieu surrounding the cartilage -- comprising osteoblast precursors and endothelial cells -- as well as the cartilage itself, develop in the absence of this important morphogen. In conclusion, the skeletal phenotype of Ihh(-/-) embryos represents the sum of disturbances in three separate cell populations, the chondrocytes, the osteoblasts and the vasculature, each of which is a direct target of hedgehog signaling.

In Vivo Mineral Composition Evolution of a Synthetic CaCO3 Aragonite Used as Biomaterial for Osseous Substitution

In orthopedic surgery, grafts are used as a substitute for defective bones. The development of synthetic biomaterials has been greatly successful. In this work, the in vivo behavior of synthetic aragonite calcium carbonate as a biomaterial was studied. This material porous at 44% was synthesized in our research group by double decomposition in an aqueous environment of salts of calcium and carbonate. Animal experimentation has been achieved on ovine spongy bony site. Biopsies have been carried out according to periods of 1, 3, 6, and 12 months after implantation. Samples were the subject of biologic assessments and in vivo mineral composition evolution. The analysis of trace and major elements (Ca, Sr, and P) of the biomaterial implanted has been achieved by the proton induced x‐rays emission (PIXE) method. Cartographies of every element of the biomaterial, of the interface (biomaterial–bone), and finally, of the bony matrix were established. Results obtained highlight the centripetal migrations of phosphorus and the strontium, from the physiological environment toward the aragonite CaCO3 versus decreasing time of the calcium concentration in the biomaterial. The elements migration kinetics is inhomogeneous in the biomaterial–bone interface area. These results have been correlated with histological studies.

Mineralization kinetics of various implanted bioceramics

Proton induced X-ray emission was used for a multi-element analysis of three biomaterials implanted in ovine thighbone: one tri-tricalcic phosphate and two calcium carbonates with different percentages of porosity. The proton PIXE analysis method allowed the simultaneous quantification of several elements to get profiles of concentrations versus implantation delay. We focused on calcium and phosphorous which were major elements of the bony matrix and strontium, a trace element characteristic of the mineralization of the implants. Concentrations of calcium (constitutive of CaCO3 and TCP) and phosphorous (constitutive of TCP) were known and differed from the bony matrix concentrations. The analysis of each sample for various times after implantation (1, 3, 6 and 12 months) by PIXE permitted to observe the evolution of the concentration of Ca, P and Sr in the implant area and at the interface implant/bone and to compare the different biomaterials implanted.

Distinguishing the contributions of the perichondrium, cartilage, and vascular endothelium to skeletal development

During the initiation of endochondral ossification three events occur that are inextricably linked in time and space: chondrocytes undergo terminal differentiation and cell death, the skeletal vascular endothelium invades the hypertrophic cartilage matrix, and osteoblasts differentiate and begin to deposit a bony matrix. These developmental programs implicate three tissues, the cartilage, the perichondrium, and the vascular endothelium. Due to their intimate associations, the interactions among these three tissues are exceedingly difficult to distinguish and elucidate. We developed an ex vivo system to unlink the processes initiating endochondral ossification and establish more precisely the cellular and molecular contributions of the three tissues involved. In this ex vivo system, the renal capsule of adult mice was used as a host environment to grow skeletal elements. We first used a genetic strategy to follow the fate of cells derived from the perichondrium and from the vasculature. We found that the perichondrium, but not the host vasculature, is the source of both trabecular and cortical osteoblasts. Endothelial cells residing within the perichondrium are the first cells to participate in the invasion of the hypertrophic cartilage matrix, followed by endothelial cells derived from the host environment. We then combined these lineage analyses with a series of tissue manipulations to address how the absence of the perichondrium or the vascular endothelium affected skeletal development. We show that although the perichondrium influences the rate of chondrocytes maturation and hypertrophy, it is not essential for chondrocytes to undergo late hypertrophy. The perichondrium is crucial for the proper invasion of blood vessels into the hypertrophic cartilage and both the perichondrium and the vasculature are essential for endochondral ossification. Collectively, these studies clarify further the contributions of the cartilage, perichondrium, and vascular endothelium to long bone development.

Estudo histológico dos enxertos ósseos homólogos humanos

Devido ao crescente uso dos enxertos homólogos humanos (aloenxertos) nas cirurgias ortopédicas reconstrutivas, há a necessidade do completo conhecimento de suas características biomecânicas e histológicas. Este estudo compara, quanto às características histológicas, os enxertos de cadáver colhidos de côndilo femoral a fresco, aos criopreservados a menos 80° C por trinta dias. São comparados vinte espécimes em cada grupo, quanto aos seguintes parâmetros histológicos: viabilidade celular, presença de vascularização, necrose, manutenção da matriz óssea, processo inflamatório, remodelação óssea e fibrose. Após análise estatística utilizando o método de Fisher (p<0,05), o estudo conclui que apenas a viabilidade celular apresenta mudança significativa após a criopreservação. Os enxertos ósseos não necessitam de células viáveis para sua utilização. Logo, o processo de criopreservação é um método útil para o armazenamento dos aloenxertos em bancos de tecidos não inviabilizando seu emprego futuro nas cirurgias ortopédicas.

Histology of tooth attachment tissues in the Late Cretaceous mosasaurid Platecarpus

ABSTRACT We present new data on the tooth attachment histology of the Late Cretaceous marine lizard Platecarpus (Mosasauridae). Examination of thin sections of a right dentary reveals the presence of a woven-fiber bone matrix that forms the margins and floor of the tooth alveolus; this bony matrix is traditionally identified as bone of attachment. We identify it as alveolar bone based on its histologic and topologic similarities to archosaurian and mammalian alveolar bone. We also identify a cribiform plate, a structure usually associated with the periodontal ligament. Parallel fibers present in multiple, non-resorbed generations of alveolar bone are tentatively identified as remnants of mineralized portions of collagen fiber bundles, or Sharpey's fibers. Along the sides of the dentine root we identify a thin layer of acellular cementum. The acellular cementum is surrounded by an enormous mass of cellular cementum tissue that fills the alveolus. This cementum mass is composed of two histologically distinct forms: (1) a loosely organized cellular cementum ground matrix; (2) a laminar form surrounding the vascularization (cementeons) that we term osteocementum. Mosasaurs possess the attachment tissues that are used to diagnose thecodont ankylosis. Mosasaur thecodonty is derived within the Mosasauroidea (aigialosaurs + mosasaurs).

Processing the Complete Canine Tooth

This paper describes a unique, reproducible method for the processing, sectioning, and staining of sections of the canine tooth intact with the bony matrix and gingiva. A 10% trifluoroacetic acid solution was employed to de-enamelize the tooth; de-enamelization was assessed using X-rays. This method supports sectioning through the complete tooth, a process which frequently requires re-infiltration steps, and generates nearly 2,000 individual sections. An overoxidized hematoxylin was employed which yielded staining that distinguished between the incremental lines in the dentin portion of the tooth. Masson's Trichrome stain demonstrated excellent preservation of structures, including dentin, cementum, dental pulp, periodontal membrane. vessels, nerves, and alveolar bone. (The J Hisrorechnol 25:137, 2002)Submitted May 7, 2002; Accepted with revisions August 8, 2002

Processing the Complete Canine Tooth

This paper describes a unique, reproducible method for the processing, sectioning, and staining of sections of the canine tooth intact with the bony matrix and gingiva. A 10% trifluoroacetic acid solution was employed to de-enamelize the tooth; de-enamelization was assessed using X-rays. This method supports sectioning through the complete tooth, a process which frequently requires re-infiltration steps, and generates nearly 2,000 individual sections. An overoxidized hematoxylin was employed which yielded staining that distinguished between the incremental lines in the dentin portion of the tooth. Masson's Trichrome stain demonstrated excellent preservation of structures, including dentin, cementum, dental pulp, periodontal membrane. vessels, nerves, and alveolar bone. (The J Hisrorechnol 25:137, 2002)Submitted May 7, 2002; Accepted with revisions August 8, 2002

Osteocalcin and osteonectin immunoreactivity in extraskeletal osteosarcoma: A study of 28 cases

Extraskeletal osteosarcoma (EOSA), a rare malignant soft tissue tumor, is by definition unattached to the skeleton and composed of malignant cells of osteoblastic phenotype which produce osseous matrix (ie, neoplastic bone). Because of its location, it can mimic other soft tissue tumors, and its matrix can be mistaken for hyalinized collagen. Antiosteocalcin (OC) and antiosteonectin (ON), antibodies against two abundant human bone proteins, are explored in the diagnosis of EOSA. Twenty-eight cases coded as EOSA (n = 24) or probable EOSA (n = 4) were identified from the Soft Tissue Registry of the Armed Forces Institute of Pathology (Washington DC). All cases had paraffin blocks available for immunohistochemistry. OC and ON (Biodesign International, Kennebunk, ME, clones OC1 and OST1) immunostaining for tumor cells and matrix was graded on a four-tiered grading system: 1 = focal (<50%) weak staining; 2 = focal strong staining; 3 = diffuse (≥50%) weak staining; and 4 = diffuse strong staining. Patient ages ranged from 9 to 80 years, with a mean age of 57 years. There were 9 female patients and 19 male patients. The tumor sizes ranged from 1.5 to 15 centimeters, with a mean size of 5.8 centimeters. Locations included the lower extremity (n = 14), trunk (n = 9), upper extremity (n = 4), and head and neck (n = 1). Subtypes included 12 osteoblastic, 4 fibroblastic, 2 chondroblastic, 2 well differentiated, 1 telangiectatic, 1 small cell, and 6 giant cell rich EOSAs; the latter resembled giant cell rich malignant fibrous histiocytomas with neoplastic bone formation. All tumors had both neoplastic cells and bony tumor matrix to evaluate. OC was 82% sensitive for EOSA neoplastic cells (1 to 4+), with immunostaining of neoplastic cells away from bone in 91% of cases, and 75% for bony tumor matrix (2 to 4+). ON was 93% sensitive for EOSA neoplastic cells (2 to 4+), yet only 39% for bony tumor matrix (1 to 4+). In 100% giant cell rich EOSA, neoplastic cells were positive for OC and ON (2 to 4+). OC showed 100% specificity for osteoblasts as it was nonreactive in all nonbone cells. ON was not specific for osteoblasts but consistently immunostained other cell types in our EOSA tumors: fibroblasts (100%), pericytes (96%), endothelial cells (92%), chondrocytes ( 5 5 ), basal layer of skin epithelium ( 1 4 ); nerves ( 2 2 ), and osteoblastic giant cells (64%). ON also stained several other cell types in normal and neoplastic tissues in our battery of preliminary stainings; OC was negative in all nonosteoblastic tissues and tumors. Both OC and ON were specific for osteoid matrix as they were nonreactive in both collagen and cartilage matrix. OC is a sensitive and specific marker for bone cells and would be helpful in identifying EOSA, even in the absence of neoplastic bone on small biopsies. ON and OC (more sensitive) will both distinguish malignant bone from collagen and cartilage matrix, essential to the diagnosis of EOSA.

Coronal suture response to distraction osteogenesis in rabbits with delayed-onset craniosynostosis.

Recent studies have identified a subpopulation of persons with craniosynostosis who exhibit progressive or delayed-onset synostosis and mild cranial vault deformities. These persons may be good candidates for nonextirpation distraction osteogenesis. The present studies were designed to determine force-displacement parameters and assess the effects of distraction osteogenesis on coronal suture growth and morphologic characteristics in a rabbit model with congenital, delayed-onset craniosynostosis. Data were collected from a total of 178 rabbits: 71 normal controls; 16 normal controls with distraction; 72 with delayed-onset coronal suture synostosis; and 19 with delayed-onset coronal suture synostosis and distraction. At 10 days of age, all rabbits had amalgam markers placed on both sides of the coronal suture. In the force-displacement study, force-displacement distractors were placed across the coronal suture and distracted acutely for 1.0 mm at 42 days of age. Force-displacement curves for the coronal suture were best described by a third-order polynomial regression equation for both normal and synostosed groups. Significant differences (P < 0.05) were found in the mean force necessary to distract a normal suture 1 mm in distance (13.72 kg) compared with a suture with delayed-onset synostosis (48.39 kg). A significant (P < 0.05) relationship was also found between the extent of synostosis and the distractive force in rabbits with delayed-onset synostosis. In the distraction study, internal distractors were fixed across the coronal suture at 25 days of age and percutaneously and intermittently activated at an average of 0.11 mm/day for 42 days (4.54 mm total). Serial radiographs were taken at 10, 25, 42, and 84 days of age. Results revealed that rabbits with delayed-onset synostosis and distraction had significantly (P < 0.01) more coronal suture growth rates compared with rabbits with delayed-onset synostosis and no distraction. Coronal sutures were harvested at 84 days of age for qualitative histologic examination. Normal, distracted coronal sutures showed widened sutural ligaments and thin, active osteogenic fronts. In contrast, distracted coronal sutures from rabbits with delayed-onset synostosis showed narrowed sutural ligaments, thickened and blunt osteogenic fronts, and increased collagen and bony matrix deposition compared with controls. Results suggest that distraction osteogenesis without corticotomy may be a treatment alternative in persons with progressive, delayed-onset synostosis. However, these preliminary data also suggest that distractive forces may accelerate or stimulate osteogenesis differentially in persons with craniosynostosis, possibly through an underlying genetic disorder of bone and cytokine regulation. These differential osteogenic responses to distraction, if validated clinically, will need to be taken into account when planning distraction rate and rhythm protocols for patients with craniosynostosis.

The role of transforming growth factor-beta, insulin-like growth factor I, and basic fibroblast growth factor in distraction osteogenesis of the mandible.

Distraction osteogenesis is a viable method for regenerating large amounts of bone. In contrast to fracture healing, the mode of bone formation in distraction osteogenesis is primarily intramembranous ossification. The basic biology of the process is still not well understood. The growth factor cascade is likely to play an important role in distraction. This study examines the growth factor cascade in a lengthened ovine mandible model. Twenty-four animals were divided into four groups with varying rates of distraction (1, 2, 3, and 4 mm/day). A unilateral distractor at the angle of the mandible was used. The mandibles were lengthened to 24 mm and fixed for a period of 5 weeks, after which the animals were killed. The sections were probed for transforming growth factor-beta, basic fibroblast growth factor, and insulin-like growth factor I. The growth factors studied were present in all four groups. Transforming growth factor-beta, basic fibroblast growth factor, and insulin-like growth factor I were present in both the bony matrix of the sections and the cytoplasm of the cells, osteoblasts, and a small number of mesenchymal cells. The sections obtained from groups distracted at faster rates showed stronger presence of the growth factors examined by more intense staining. In fracture healing, the localization of transforming growth factor-beta in stage I of healing corresponded with the precise region of intramembranous ossification in stage II. Diffuse presence of transforming growth factor-beta throughout the lengthened region corresponded with the process of intramembranous ossification observed in distraction. In fracture healing, insulin-like growth factor I and basic fibroblast growth factor have been shown to promote proliferation and differentiation of osteoblasts from precursor cells. The intense presence of insulin-like growth factor I and basic fibroblast growth factor in the distracted region may account for osteoblast proliferation and formation from precursor mesenchymal cells. Mechanical strain has been shown to increase the expression of transforming growth factor-beta and insulin-like growth factor I. Distraction may serve as a source of mechanical strain, which may explain, in part, the expression of these growth factors, particularly in the faster groups.

Osteosarcoma of bone

The definition of osteosarcoma requires that a malignant tumor of bone produce osteoid or bony matrix. With this as a basic definition, osteosarcoma of bone can be divided into several clinicopathologic entities based on clinical, roentgenographic, and pathologic features. The tumors can be broadly divided into those arising within the bone and those arising on the surface of bone. Most intraosseous osteosarcomas are high-grade malignant tumors that occur in children and adolescents. A small number occur in older patients, and they may be related to a preexisting condition, such as Paget's disease or radiation. The site of the lesion has prognostic importance. Osteosarcoma of the jaws is associated with an especially good prognosis, whereas the same kind of tumor involving the skull has a very poor prognosis. Most osteosarcomas of the surface of bone are well or moderately differentiated and are associated with an excellent prognosis.

Bone Marrow Necrosis

Bone Marrow Necrosis

In VitroDifferentiation Potential of the Periosteal Cells from a Membrane Bone, the Quadratojugal of the Embryonic Chick

The quadratojugal (QJ) is a neural crest-derived membrane bone in the maxillary region of the avian head.In vivoits periosteum undergoes both osteogenesis to form membrane bone and chondrogenesis to form secondary cartilage. This bipotential property, which also exists in some other membrane bones, is poorly understood. The present study used cell culture to investigate the differentiation potential of QJ periosteal cells. Three cell populations were enzymatically released from QJ periostea and plated at different densities. Cell density greatly affected phenotypic expression and differentiation pathways. We found two culture conditions that favored osteogenesis and chondrogenesis, respectively. In micromass culture, the periosteal cells produced a layer of osteogenic cells that expressed alkaline phosphatase (APase) and secreted bony extracellular matrix (ECM). In contrast, low-density monolayer culture elicited chondrogenesis. Cells with pericellular refractile ECM and round shape appeared at 7 to 8 days and formed colonies later. The chondrogenic phenotype of these cells was confirmed by immunolocalization of type II collagen and Alcian blue staining of ECM. This result demonstrated that a fully expressed chondrogenic phenotype can be achieved from membrane bone periosteal cells in primary monolayer culture. Chondrogenesis requires a cell density lower than confluence and cannot be initiated in confluent cultures. Among the three cell populations, those cells from the outer layer have the highest growth rate and require the lowest initial plating density (below 5 × 103cells/ml) to achieve chondrogenesis. Cells from the inner layer have the slowest growth rate and chondrify at the highest initial density (below 5 × 104cells/ml). Chondrocytes from all populations express distinct phenotypic markers—APase and type I collagen—from initial chondrogenesis, but are not hypertrophic morphologically. Furthermore, the fact that chondrocytes arise within the same colony as APase-positive polygonal cells suggests that chondrocytes may differentiate from precursors related to the osteogenic cell lineage. This cell culture approach mimics secondary cartilage and membrane bone formationin vivo.

Endocrine Aspects of Growth Deficiency in OI

Growth deficiency is the most common secondary feature of osteogenesis imperfecta. It is unrelated to fracture history and appears to be due to the growth failure of the defective bony matrix. There are characteristic growth curves for different types of OI. We have been investigating the endocrine features of this disorder, in which the skeletal target tissue synthesizes defective matrix. We review the results of our evaluation of the growth hormone axis in 28 children with short stature and OI and of our pilot study to stimulate OI bone to increased growth rates. Our current focus is on the effect of growth hormone treatment on linear growth, bony mineral and bony matrix in OI.

Effects of the decrease of magnesium (MG) concentration in the dialysed fluid on the erythrocyte MG, PLASMATIC PARATHOHORMONE (PTH) and bony matrix

Effects of the decrease of magnesium (MG) concentration in the dialysed fluid on the erythrocyte MG, PLASMATIC PARATHOHORMONE (PTH) and bony matrix

Fetal Fracture Healing in a Lamb Model

A large animal model to assess fetal fracture repair and the ability to close excisional bony defects is presented. Incisional and excisional ulnar fractures were made in 14 midgestation fetal lambs, harvested at serial time points, and subjected to high-resolution low-kilovolt magnification radiographs, magnetic resonance imaging scans, and histologic analysis. Fetal fracture healing was characterized by early closure of excisional defects and rapid fracture healing with minimal or no soft-tissue inflammation or callus formation. Magnetic resonance imaging scans of the fractures revealed a characteristic pattern compatible with the histologic findings, namely, minimal inflammation in soft tissue adjacent to the fracture site. Histologic and magnification radiographic findings indicated that complete bony repair occurred within 21 days in incisional defects and within 40 days in excisional defects. In both cases, healed fetal bone resembled normal bone matrix. Excisional defects, including periosteum, of greater than three times the width of the bony cortex closed rapidly with virtually normal-appearing bony matrix and with minimal or no callus formation.

Study on the microstructures of skull fracture

The authors observed the edges of skull fractures under the scanning electron microscope. Many microfractures can be found along the main fracture lines. The width of the microfractures varies from 5 to 100 μm. They may be located either in the external compact bone or between the outer compact bone and diplöe, or in the diplöe. Those within the diplöe form transverse, longitudinal or spiral fractures of bony trabeculae. Collagenous fibre bunches in the bony matrix may be divided or broken and the different layers of collagenous fibre may be separated. The blood vessels inside the skull may be crosscut or longitudinally torn by the fracture lines. Sometimes the torn blood vessels may be drawn out of the Haversian canals. In antemortem fractures, the fibrin networks and red blood cells can be easily found in the depths of those cracks. The possible mechanism of formation of the microfractures and the possibility of differentiation between ante- and post-mortem fractures are discussed.