The Impact of Age Upon Healing: Absolute Quantification of Osteogenic Genes in Calvarial Critical-Sized Defects.

BACKGROUND: The current study was performed to elucidate changes in growth factor expression over time in critical-sized calvarial defects in rats from infancy to skeletal maturity. MATERIALS AND METHODS: Critical-sized parietal defects of 5, 6, and 8 mm were created in postnatal day 6 (P6), postnatal day (P20), and postnatal day (P84) adult rats, respectively. Dura was harvested at 3, 7, or 14 days after surgery, and serial micro-computed tomography imaging was performed through 12 weeks postoperatively. Absolute quantitative polymerase chain reaction was performed for Bone Morphogenic Protein-2 (BMP-2), Fibroblast Growth Factor-2 (FGF-2), Insulin-like Growth Factor-1 (IGF-1), and Transforming Growth Factor-ß1 (TGF-ß). RESULTS: The P6 (6-d-old) rats showed the greatest difference in gene expression between the dura derived from the defect side and the dura derived from the control side, demonstrating significant differences in TGF-ß1, BMP-2, IGF-1, and FGF-2 at various time intervals. Absolute gene expression in the defect dura was highest in the P6 rats and declined with age. Significant differences were noted at limited time points in the P20 rats for TGF-ß1 and BMP-2 as well as in the P84 rats for TGF-ß1. TGF-ß1 was the only gene studied that showed significant differences at postoperative days 3, 7, and 14 in varying age groups. CONCLUSIONS: The P6 rats have a higher osteogenic potential accompanied by a more vigorous alteration in growth factor expression compared with the P20 or P84 rats. Decrease in BMP-2 and FGF-2 as well as relative increase in TGFß-1 messenger RNA were observed in healing defects. These data provide valuable insight into the mechanism of healing of critical-sized defects and may be of use to engineer factor-releasing implants to correct skull defects.

Calcification during bone healing in a standardised rat calvarial defect assessed by micro-CT and SEM-EDX.

OBJECTIVE: The study was designed to investigate the process of calcification during bone healing in a standardized rat calvarial bone defect model, measured by bone mineral density and the concentrations and distributions of calcium, phosphorus and carbon in the bone matrix. MATERIALS AND METHODS: A standard defect was made on the parietal bone of 12-week-old rats under anaesthesia. The rats were fixed in weeks 1, 2, 4 and 8,and the calvaria were resected and examined with microcomputed tomography, then frozen and sectioned for histology and analysed with energy-dispersive X-ray spectroscopy (EDX). Parietal bone of 12-week-old control rats was processed similarly. RESULTS: The mineral density of healing bone increased with time. The healing bone became thicker and denser with time in histology. The distributions of Ca and P expanded over the bone matrix, whereas that of C became localised and complemented that of C and P. The Ca/P concentration ratio increased, whereas the C/Ca and C/P ratios decreased in the healing bone matrix. CONCLUSION: Healing bone is immaturely calcified initially and proceeds calcification gradually, that is, as the bone volume increases, mineral increases in density and matures in quality, while organic components decrease.

Differences in gene expression between the otic capsule and other bones.

Our long term goal is to understand the molecular pathology of otosclerosis and to develop better forms of therapy. Toward this goal, the current study focused on characterizing the molecular factors responsible for the unique biological features of the otic capsule: its minimal rate of remodeling, and lack of healing capacity when fractured. We compared expression levels of 62 genes involved in bone metabolism between the adult murine otic capsule and the tibia and parietal bones; the latter exemplify bones formed by endochondral and intramembranous ossification, respectively. Gene expression levels were measured using real-time quantitative RT-PCR and analyzed using tools of bioinformatics. Expression patterns of key genes were verified with in situ hybridization. The molecular profile of the otic capsule was distinctly different from that of the tibia and parietal bone. Genes found to be most characteristic of the otic capsule were: osteoprotegerin (opg), bone morphogenetic protein receptor 1b (bmpr1b) and bone morphogenetic protein 3 (bmp3). Expression levels were high for opg and bmpr1b, and minimal for bmp3 within the otic capsule. We concluded that opg and bmpr1b likely play important roles in inhibition of remodeling within the otic capsule.

Two types of bone resorption lacunae in the mouse parietal bones as revealed by scanning electron microscopy and histochemistry.

To understand the bone resorption process on the basis of the morphology of bone resorption lacunae, the inner surface of parietal bones in juvenile mice was exposed with a treatment of ultrasonic waves or NaOCl treatment and examined by scanning electron microscopy (SEM). The bone resorption lacunae were divided into two types (I and II) according to differences in morphological features of their walls; the wall of type I lacunae was covered with loose collagen fibrils, while that of type II lacunae was smooth with almost no fibrillar structures. Collagen fibrils in type I lacunae treated with ultrasonic waves differed in appearance from those treated with NaOCl; the collagen fibrils were thin and displayed a smooth surface in type I lacunae treated with ultrasonic waves, while they were thick and showed a rough surface in those treated with NaOCl-probably because superficial uncalcified collagen fibrils were digested with the chemical. The results indicated that type I lacunae occupied 77% of all of the bone resorption lacunae treated with ultrasonic waves, but 51% of those treated with NaOCl. This finding led to the idea that type I lacunae can be subdivided into two: lacunae (Ia), covered with partially calcified fibrils as well as superficial uncalcified fibrils; and lacunae (Ib), covered only with uncalcified fibrils. The presence of uncalcified fibrils in the bone resorption lacunae was further confirmed by backscattered electron (BSE) imaging of SEM. Histochemistry for acid phosphatase or immuno-histochemistry for cathepsin B or carbonic anhydrase in combination with SEM revealed that type I lacunae were located under osteoclasts but type II lacunae were not. These findings indicate that type I lacunae are in the process of bone resorption by osteoclasts, while type II lacunae are in the final stage of bone resorption and free from osteoclasts. Bone resorption may thus proceed in the order of Ia, Ib, and II.

Transcriptional regulation of collagenase-3 by interleukin-1 alpha in osteoblasts.

Interleukin-1 (IL-1)alpha is an autocrine/paracrine agent of the skeletal tissue and it regulates bone remodeling. Collagenase-3 or matrix metalloproteinase (MMP)-13 is expressed in osteoblasts and its expression is modulated by several cytokines including IL-1alpha. Because the molecular mechanism of increased synthesis of collagenase-3 in bone cells by IL-1alpha is not known, we investigated if collagenase-3 expression by IL-1alpha in osteoblasts is mediated by transcriptional or post-transcriptional mechanisms. Exposure of rat osteoblastic cultures (Ob cells) to IL-1alpha at concentrations higher than 0.5 nM increased the synthesis of collagenase-3 mRNA up to eightfold and the secretion of immunoreactive protein up to 21-fold. The effects of IL-1alpha on collagenase-3 were time- and dose-dependent. Although prostaglandins stimulate collagenase-3 expression, stimulation of collagenase-3 in Ob cells by IL-1alpha was not mediated through increased biosynthesis of prostaglandins. The half-life of collagenase-3 mRNA from control and IL-1alpha-treated Ob cells was similar suggesting that the stabilization of collagenase-3 mRNA did not contribute to the increase in collagenase-3. However, IL-1alpha stimulated the rate of transcription of the collagenase-3 gene by twofold to fourfold indicating regulation of collagenase-3 expression in Ob cells at the transcriptional level. Stimulation of collagenase-3 by IL-1alpha in osteoblasts may in part mediate the effects of IL-1alpha in bone metabolism.

Material properties of the inner and outer cortical tables of the human parietal bone.

Even though the cranial vault functions as protection for the brain and as a support structure for facial and masticatory functions, little is known about its mechanical properties or their variations. The cranial vault bone is interesting because of its maintenance in spite of low functional strains, and because calvarial bone cells are often used in cell culture studies. We measured thickness, density, and ash weight, and ultrasonically determined elastic properties throughout the cortices of 10 human parietal bones. The results are unique for studies of the cranial vault because: 1) measurements focused specifically on the cortical components, 2) the orientations of the axes of maximum stiffness were determined before measurement of elastic properties, and 3) two related measurements (bone density and percent ash weight) were compared. Results showed that the periosteal cortical plate (outer table) and the endosteal cortical plate (inner table) had significant differences in material properties. The outer table was on average thicker, denser, and stiffer than the inner table, which had a higher ash weight percentage. Within each table there were significant differences in thicknesses, ash weight percentages, and E(2)/E(3) anisotropies among sites. Few sites on either table had significant orientations of the axes of maximum stiffness. Despite this apparent randomness in orientation, almost all sites exhibited anisotropies equivalent to other parts of the skeleton.

Elevated levels of transforming growth factors beta 2 and beta 3 in lambdoid sutures from children with persistent plagiocephaly.

OBJECTIVE: To analyze the pertinent history and physical findings specific to the subset of patients with a progressive posterior skull deformity, requiring surgery to correct their deformity. PATIENTS: Since the Academy of Pediatrics issued its recommendation on supine positioning of infants to prevent sudden infant death syndrome (SIDS) in 1992, 73 children have presented to the University of Virginia Craniofacial Anomalies Clinic with posterior-skull deformities. The majority were successfully managed with conservative therapy, but in six patients, the deformity was severe and persistent, requiring surgical correction. All six children were older (7.5-12 mo), presenting with more severe morphologic appearances and a higher incidence of associated neurodevelopmental delay. Three had family backgrounds of isolated craniosynostosis. METHODS: Characteristics of these patients were examined to determine why they may have differed from those that responded to conservative management. Immunohistochemical staining of their lambdoid sutures was performed. RESULTS: Significantly increased staining for TGF-beta 2 and TGF-beta 3, potent stimulators of bone cell growth and differentiation, was seen in all 'affected' sutures from the flattened side of the skull, compared to unaffected sutures from the protruding side of the skull-a pattern similar to that seen during normal bony obliteration of calvarial sutures. CONCLUSION: The majority of patients with posterior plagiocephaly associated with positioning responded to conservative management, while a small subset of patients with persistent posterior skull deformation required surgical intervention. A genetic basis for the latter patients' persistent plagiocephaly, rather than positioning, cannot be ruled out. Genetics, prolonged external pressure against the sutures, or a combination of these factors may lead to permanently raised levels of growth factors in 'affected' sutures.

Subperiosteal implantation of octacalcium phosphate (OCP) stimulates both chondrogenesis and osteogenesis in the tibia, but only osteogenesis in the parietal bone of a rat.

BACKGROUND: It is not known whether long bones and calvaria have distinct biological characteristics. Octacalcium phosphate (OCP), which is a precursor phase of the hydroxyapatite, has been reported to stimulate bone formation if implanted in the subperiosteal region of mouse calvaria. The present study was designed to investigate how the long bone and the calvarium respond to OCP implantation and to compare their biological characteristics. METHODS: The synthetic OCP was implanted into the subperiosteal region of rat tibiae and parietal bones being mixed with bovine type I collagen treated by pepsin (Atelocollagen). The biological response was examined histologically and immunohistochemically for collagen matrix phenotypes of types I and II to identify bone and cartilage formation. RESULTS: Both chondrogenesis and osteogenesis were initiated in the tibia 1 week after implantation of OCP and most of the cartilage was replaced by bone at week 2. However, the parietal bone did not show osteogenesis responding to OCP implantation until week 3, and no cartilage formation was associated with the osteogenesis. CONCLUSIONS: The present study demonstrated the distinct characteristics of biological response to OCP implantation between the long bone and the calvarium in terms of whether or not cartilage formation is involved in the stimulated osteogenesis by OCP, and in terms of timing of the stimulated chondrogenesis and/or osteogenesis, i.e., the parietal bone takes more time to respond to OCP implantation than the tibia.

Distribution profiles of fluoride in three different kinds of rat bones.

The study was performed to reveal the detailed distribution profiles of fluoride in three different kinds of rat bone--humerus, vertebral arch, and parietal bone--and to compare this with the histological appearance of each bone type. Two groups of Wistar rats were provided water ad libitum containing 0 and 100 ppm fluoride, respectively, for 24 weeks. The fluoride distribution profiles across the bone of the three different bones from the outer to the inner surface were determined by means of an abrasive micro-sampling technique. In control animals, both humerus and parietal bones showed higher concentrations at the periosteal and endosteal surfaces, while the vertebral arch showed additional high levels in the middle (containing trabecular bone) of the tissue. In exposed animals, fluoride levels increased greatly in all three bone types. The vertebral and parietal fluoride distribution profiles were relatively unchanged, although humerus fluoride increased from periosteum to endosteum. The differences in fluoride distribution profiles were apparently related to the histological appearances of these bones. The surface area of bone available and the extent of vascularity appear to affect fluoride uptake.

Transforming growth factor beta 1-induced cellular heterogeneity in the periosteum of rat parietal bones.

We examined the osteogenesis process in transforming growth factor beta 1 (TGF-beta 1)-treated neonatal and adult rats, aiming to investigate the age difference in the effect of TGF-beta 1 on mesenchymal cell differentiation. Recombinant human (rh) TGF-beta 1 (20 and 200 ng) was injected onto the outer periostea of the right side of the parietal bone of each rat once a day for 1-12 days starting at the age of either 1 day or 12 weeks. On the day after the final injection, the calvaria was excised and evaluated histologically. In the neonates, the 12-day treatment with rhTGF-beta 1 increased the number of osteoprogenitor cells, resulting in intramembranous ossification. In the adult rats, rhTGF-beta 1 induced differentiation of chondrocytes. Cartilage masses were surrounded by mesenchymal cells, which would differentiate into chondrocytes. The cartilage matrix was partially calcified, with chondrocytes buried therein. In the calcified matrix, marrow cavities containing some multinuclear osteoclasts were formed. These findings indicate that rhTGF-beta 1 stimulated the differentiation of mesenchymal cells into chondrocytes and produced the cartilaginous matrix. rhTGF-beta 1 induced intramembranous ossification of the parietal bone in neonatal rats, and it induced enchondral ossification in adults. This result suggests that the different responses of mesenchymal cells in the periosteum to rhTGF-beta 1 may depend on the age of the animals used: namely, they may reflect the respective osteogenic stages of modeling and remodeling.

Solitary infantile myofibromatosis of bone. An immunohistochemical and ultrastructural study.

A rare case of solitary infantile myofibromatosis of bone in an 11-month-old boy is reported. Radiographically the lesion of parietal bone was round, well-circumscribed, and osteolytic with a sclerotic rim. Histologically the tumor was made up of nodules that were hyalinized or cellular and containing plump, spindle-shaped cells that were intermediate in appearance between fibroblasts and smooth-muscle cells, arranged in short bundles or whorls. Another typical feature was the presence of distended, cleft-shaped vascular spaces around the nodules. The microscopic features of this tumor were consistent with those of infantile myofibromatosis of other sites, such as the skin and deep soft tissue. The tumor cells showed immunoreactivities for vimentin and alpha-smooth muscle actin. Microfilaments with dense bodies were observed in the fibroblast-like tumor cells. In addition, many tumor cells stained for collagen type IV and were covered by incomplete external laminae, indicating infantile myofibromatosis has more advanced smooth-muscle differentiation than conventional fibromatosis.

Intracellular localization and translocation of 1 alpha, 25-dihydroxyvitamin D3 receptor in osteoblasts.

The intracellular localization and translocation of the 1 alpha,25-dihydroxyvitamin D3 receptor (1 alpha,25(OH)2D3 receptor) in osteoblasts of mouse parietal bone and MC3T3-E1 cells were examined immunocytochemically using a rat monoclonal antibody to 1 alpha,25(OH)2D3 receptor. In osteoblasts of parietal bones in vivo, immunoreactivity for 1 alpha,25(OH)2D3 receptor was detected not only in the nuclei but also in lysosomal structures, and also sparsely in the cytoplasmic matrix. The transport of 1 alpha,25(OH)2D3 receptor was investigated immunocytochemically after incubation with 1 alpha,25(OH)2D3. In osteoblasts of parietal bones, after 1 min incubation with 10(-8) M 1 alpha,25(OH)2D3, the perinuclear cytoplasm showed intense immunoreactivity for 1 alpha,25(OH)2D3 receptor. After 10 min incubation, immunoreactivity was intense in the nuclei, especially in the heterochromatin. In MC3T3-E1 cells, after 1 min incubation with 1 alpha,25(OH)2D3, immunoreactivity for 1 alpha,25(OH)2D3 receptor was found in the form of a fibrillar structure extending radially to the periphery of the cells. The immunostaining pattern of anti-1 alpha,25(OH)2D3 receptor was similar to the distribution of microtubules stained with anti-beta-tubulin antibody. After 10 min incubation, the nuclei showed intense immunoreactivity for 1 alpha,25(OH)2D3 receptor. Incubation with colchicine dissociated the fibrillar structures and inhibited the intranuclear localization of the 1 alpha,25(OH)2D3 receptor. These findings suggest that the 1 alpha,25(OH)2D3 receptor is located in the nuclei, in lysosomal structures and also sparsely in the cytoplasmic matrix of osteoblasts in vivo, and that the receptor is transported to the perinuclear cytoplasm via microtubules to be then translocated into the nucleus, especially into the heterochromatin.