Cyclooxygenase-2 activity is important in craniofacial fracture repair.

The aim of this study was to examine the effect of cyclooxygenase (COX)-2 on bone repair after craniofacial fracture in mice. A 4-mm fracture was created in the parietal bone of 8-week-old male COX-2 wild-type (COX-2(+/+)) and knockout (COX-2(-/-)) mice. Ribonucleic acid was extracted from the fractured bone and analysed. For morphological and histological analysis, the mice were killed 8 and 12 weeks after treatment, and sections were prepared. Three-dimensional computed tomography was performed, and the sections were stained with hematoxylin-eosin for histological examination. Expression of COX-2 messenger ribonucleic acid was induced in COX-2(+/+) mice, but not in COX-2(-/-) mice. Ossification at the fracture site was almost complete 12 weeks after fracture in COX-2(+/+) mice. In COX-2(-/-) mice, incomplete union had occurred at the fracture site. In both types of mice, the fracture site contained no cartilaginous tissue, and the callus formed from the periosteal side. These results suggest that COX-2 plays an important role in craniofacial fracture repair and that COX-2-selective non-steroidal anti-inflammatory drugs might interfere with fracture repair of the membranous viscerocranium in the clinical setting.

An immunolocalization study of tissue inhibitors of metalloproteinase-1 of bone graft healing on parietal bone.

This immunolocalization study was performed to investigate the temporal and spatial expression of tissue inhibitors of metalloproteinase (TIMP) 1 within endochondral and intramembranous bone grafts during the early stages of healing, in the hope of gaining a better understanding of the mechanisms of bone graft healing, which could influence the choice of bone graft used. Twenty-seven adult New Zealand White rabbits were used as the experimental model. Autogenous bone grafts taken from the cranial bone (intramembranous in origin) and the femur (endochondral in origin) were grafted into skull defects created on either side of the parietal suture. Rabbits were killed on days 1 to 9 postgrafting, and the bone graft alone was harvested for immunolocalization of TIMP-1. In endochondral bone grafts, TIMP-1 was expressed on days 1 to 3, followed by a period of absence until days 8 and 9. Intramembranous bone grafts did not express TIMP-1 until days 6 to 9. The timing and location of TIMP-1 expression coincided with osteogenesis, which indicates a role for TIMP-1 in preserving newly formed bone during the initial stages of graft healing. The differential temporal expression of TIMP-1 in endochondral and intramembranous bone grafts suggests that bone graft type plays an important role in influencing the healing process mediated by the host tissues. The earlier expression of TIMP-1 in endochondral bone grafts could be the reason for delayed vascularization of defects containing these grafts, whereas the delayed expression of TIMP-1 in intramembranous bone grafts could allow earlier vascularization of the intramembranous bone grafts.

Osteoblasts and osteocytes express MMP2 and -8 and TIMP1, -2, and -3 along with extracellular matrix molecules during appositional bone formation.

Our previous studies suggested that a part of bone extracellular matrix (ECM) molecules are degraded and remodeled during embryonic bone formation. In contrast, little is known about ECM remodeling in postnatal appositional bone formation. The present study was designed to investigate expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) during experimentally initiated appositional bone formation in rats. Expressions of ECM molecules, MMPs, and TIMPs were examined using in situ hybridization. Osteoblasts and osteocytes expressed MMP2 and -8, TIMP1, -2, and -3, as well as type I collagen, osteopontin, and osteocalcin in the course of the appositional bone formation, while they showed few transcripts of MMP13. The results indicated that while osteoblasts and osteocytes in the apposed bone produce ECM molecules, they degrade ECM molecules with MMPs and regulate the degradation by inhibiting the activity of MMPs using TIMPs. Osteoblasts and osteocytes may reorganize the ECM composition to mature the bone matrix in appositional bone formation.

Inhibition of prostaglandin synthesis leads to a change in adherence of mouse osteoclasts from bone to periosteum.

When mouse parietal bones were incubated for 1 day in medium containing indomethacin (Ind), the number of tartrate-resistant acid phosphatase-positive osteoclasts (TRAP+OC) counted on the bone surface was drastically reduced. This reduction did not occur with calcitonin or if the endocranial membrane (periosteum) was removed prior to incubation with Ind. The aim of this work was to determine the mechanism involved. TRAP+OC were found to be increased on the endocranial membrane adjacent to the resorbing surface after Ind treatment, compared with cultures supplemented with parathyroid hormone (PTH) or prostaglandin E2 (PGE2). However, this increase accounted for only half of those lost from the bone surface. TRAP negative osteoclasts were also seen on the membrane and, to a lesser extent, on the bone. Increased TRAP specific activity could be extracted from the endocranial membranes of bones incubated with Ind compared with PGE2 controls. When bones that had been exposed to Ind were then cultured for 1 day in PGE2, an increase in TRAP+OC occurred. This increase was blocked by the removal of the endocranial membrane prior to incubation with PGE2. We conclude that when prostaglandin production ceases, TRAP+OC become less adherent to bone and more adherent to the endocranial membrane. Stimulators of bone resorption appear to reverse this process.

Interleukin-4 inhibits prostaglandin G/H synthase-2 and cytosolic phospholipase A2 induction in neonatal mouse parietal bone cultures.

We have shown previously that prostaglandin (PG) production in 7-day-old neonatal mouse calvarial cultures is regulated largely by changes in prostaglandin G/H synthase-2 (PGHS-2) expression and to a lesser extent by changes in arachidonic acid (AA) release. In this study, we examined the effects of interleukin-4 (IL-4), and its interactions with other cytokines and with parathyroid hormone (PTH), on mRNA levels of PGHS-2, PGHS-1, and cytosolic phospholipase A2 (cPLA2) and on medium protaglandin E2 (PGE2) levels in calvarial cultures. IL-1 and tumor necrosis factor-alpha (TNF-alpha), both at 1-100 ng/ml, and PTH at 0.1-10 nM increased PGHS-2 and cPLA2 mRNA and medium PGE2 levels dose-dependently after 4 h of treatment. IL-6 and IL-11 at 1-100 ng/ml did not affect mRNA or PGE2 levels. IL-4 at 1-100 ng/ml decreased PGHS-2 and cPLA2 mRNA and PGE2 levels in control as well as IL-1, TNF-alpha, and PTH-stimulated cultures. The inhibition of PGHS-2 and cPLA2 mRNA expression by IL-4 (10 ng/ml) was present at 1 h, reached a maximum at 4 h, and persisted for 24 h. The effects were maintained in the presence of cycloheximide. IL-4 also decreased PGHS-2 protein levels in control and IL-1-stimulated cultures. PGHS-1 mRNA levels were not stimulated by any of the factors studied nor inhibited by IL-4. IL-4 partially inhibited control and PTH-stimulated 45Ca release from prelabeled mouse calvariae at 4 days. However, neither the inhibition of resorption by IL-4 nor the stimulation by IL-1 and PTH were altered by indomethacin (1 microM). We conclude that (1) IL-1, TNF-alpha, and PTH, but not IL-6 nor IL-11, can increase the expression of PGHS-2, cPLA2, and PGE2 production in cultured mouse calvariae; (2) IL-4 inhibits PGE2 production in both control and stimulated calvarial cultures by inhibiting PGHS-2 and cPLA2; and (3) IL-4 has an inhibitory effect on bone resorption which is independent of PG production.

Effect of local injection of activin A on bone formation in newborn rats.

In order to examine the effect of activin A on the process of bone formation, activin A was injected onto the periosteum of parietal bone in newborn rats, and the effect was compared with that of transforming growth factor (TGF)-beta. The daily periosteal injection of activin A increased the thickness of both the periosteal and bone matrix layers in a dose- and time-dependent manner. A maximal effect was obtained with 5.0 micrograms/day activin A. The time course of the effect of activin A on the periosteal thickness was similar to that of TGF-beta 1. However, the effect of TGF-beta 1 was much more pronounced and was mainly on fibroblasts and inflammatory cells. The time course of the effect of activin A on the thickness of bone matrix layer was different from that of TGF-beta 1. The effect of TGF-beta 1 reached maximum (1.8-fold) within 3 days, whereas that of activin A did not develop until day 6 and reached maximum at the end of the 12-day injection period (1.4-fold). Histological examinations revealed that both TGF-beta 1 and activin A increased the number of alkaline phosphatase-positive osteoblastic cells. These results demonstrate that periosteal injection of activin A stimulates bone formation. In addition, although the possibility cannot be ruled out that the dramatic effect of TGF-beta 1 on the periosteal layer might have affected the delivery of TGF-beta 1 to the bone surface, these observations also suggest that the mode of action of activin A may be different from that of TGF-beta 1.(ABSTRACT TRUNCATED AT 250 WORDS)

[Effects of the localized thermal enhancement on new bone formation following mechanical expansion of the rat sagittal suture].

The purpose of this study was to investigate the effects of localized thermal enhancement on new bone formation in rats. Sixty four male Wistar rats of 52-day old were used as the experimental animal. The sagittal suture of the rat was expanded with the force of 75 g by means of an expansion appliance. After the force application for 2 days, the expansion appliance was fixed with a composite resin. The infrared ray was applied to the expanded sagittal suture for 20 minutes a day and the temperature, just above the sagittal suture, was measured with a biothermometer. The local heat was given for 5-days. The experimental animals were divided into 4 groups based on the increase of temperature: Control, 1 degree C, 2.5 degrees C and 4 degrees C groups. The sagittal suture of each rat was examined by both light and fluorescent microscopies. And histochemistry for acid phosphatase and tartrate-resistant acid phosphatase (TRAP) were performed. The results obtained were as follows: 1) In control and 1 degree C groups, little reaction of acid phosphatase and TRAP were observed in the sutural tissue except for the marrow and the neighboring area of the capillary vessels. 2) In 2.5 degrees C and 4 degrees C groups, an increased absorptive area of the bone and an active reaction of acid phosphatase were found in the sutural area. 3) In only 1 degree C group, an increased new bone formation was evident in the sutural area. Results obtained suggested that the degree of new bone formation depended on the temperature of tissues concerned.

Regeneration of cranial suture and bone plate lesions in rabbits. Implications for positioning of osteotomies.

The positioning of osteotomies in intramembranous cranial bone was studied by exploring the pattern of bone regeneration in growth areas (the sutural region) as compared to that of the bone plate proper. Trephine defects in the left coronal suture area and the right parietal bone were produced in fifty-nine young rabbits. A pilot study to refine operative and analytical methods comprised 22 animals. The experiments were terminated at one, three, and six weeks after surgery. The bone regenerative response was assessed by x-ray planimetry, plain microscopy, enzyme histochemistry, and fluorescent labelling. Only minor divergences in healing capacity between the two defects were found. No adverse effects on the growth process were indicated. As to clinical management, the findings suggest that osteotomies designed to traverse sutural areas will, under normal circumstances, regenerate in a similar manner and rate to adjoining bone plates.

Morphological relationships between osteoclasts and bone resorption surfaces on mouse parietal bones.

Parietal bones from mice 1-20 weeks of age were histochemically stained for detection of acid-phosphatase activity and then observed by the light microscope to evaluate the distribution and shape of osteoclasts on the inner surface of their bones. After microscopic examination, the same bones were macerated by NaOCl to both remove organic materials and expose the mineralized surface. The inner surface was then examined by scanning electron microscopy and the observations were compared with the light micrographs of the areas where osteoclasts were located. The bone resorption areas were identified as well-demarcated rough areas, and corresponded to the areas where osteoclasts were distributed. In young mice, osteoclasts observed in the bone resorption areas, which were composed of accumulations of irregular concavities, were mainly polygonal or round in shape. In adult mice, elongated osteoclasts with longer or shorter cytoplasmic processes were predominant; the bone concavities were also elongated and gathered in a flame-like pattern. The findings suggest that osteoclasts change shape according to their resorptive activities and that the activities differ between growing bones and those where growth has ceased, probably in relation to the modeling and remodeling of the bone.

The morphologic and biochemical effects of tensile force application to the interparietal suture of the Sprague-Dawley rat.

The purpose of this study was to correlate the histologic and biochemical responses of the interparietal suture to a range of tensile forces. Stainless steel spring implants, calibrated to generate expansive forces from 50 to 250 g, were placed across the interparietal suture in 85 female Sprague-Dawley rats. After experimental periods from 2 hours to 14 days, the interparietal sutures were evaluated by radiography, histology, and biochemistry. An in vivo/in vitro system was used for the biochemical analysis; total protein, proline incorporated, percent collagen, and alkaline phosphatase activity were measured. The radiographs and histology showed that in vivo suture expansion was achievable with 50 to 70 g of force, but the heavier forces showed greater sutural opening, more cellular proliferation, and more bone formation. This increased biologic response by the heavier forces was substantiated by an increase in sutural protein and alkaline phosphatase activity but not in percent collagen. It was concluded that changes in the total protein content of the suture were not primarily caused by proliferation of osteogenic cells and fibroblasts but due to an influx of transudate. In contrast, the increase in incorporation of 3H-proline and alkaline phosphatase activity correlated with the observance of bone formation. This study indicated a positive correlation between the magnitude of tensile forces and osteogenic response.

The preosteoclast and its cytodifferentiation into the osteoclast: ultrastructural and histochemical studies of rat fetal parietal bone.

In order to elucidate the cytological features of preosteoclasts and the process of their differentiation into osteoclasts, fetal rat parietal bone was examined using light microscopy, organ culture, electron microscopy and histochemical methods. Parietal bones of rat fetuses from 15 to 21 days of gestational age were examined light microscopically. A solid bone plate was found in 19 day old fetuses, but not multinucleated giant cells were observed in either the ecto- or endocranial periosteal surfaces. They were first observed at the endocranial periosteal surface in 20 day old fetuses, and increased in number in 21 day old fetuses. Parietal bones of fetuses from 15 to 19 days of age were cultured and the possible occurrence of preosteoclasts prior to the appearance of osteoclasts was examined. During organ culture, eosinophilic multinucleated cells appeared in the parietal bones from 17, 18 and 19 day old fetuses, and increased in those from 19 day old fetuses. Electron microscope observation of the parietal bones in 19 day old fetuses revealed moderate numbers of mononuclear cells identified as preosteoclasts (Scott, 1967) principally among the osteoblasts and preosteoblasts at the endocranial periosteal surface. Preosteoclasts with ill-developed cell organelles tended to be located between blood vessels and active osteoblasts, and sometimes located close to the bone surface with only the thin cytoplasmic processes of adjacent osteoblasts intervening. On the other hand, well-developed preosteoclasts tended to be located close to flattened osteoblasts and came into direct contact with the exposed mineralized bone between them. Preosteoclasts were not clustered together but were usually found in contact with adjacent osteoblasts and/or preosteoblasts. Membrane fusion between a preosteoclast and a flattened osteoblastic cell was observed. Multinucleated cells were principally preosteoclastic in appearance but some were both osteoclastic and osteoblastic. The multinucleated cells with ruffled borders identified as active osteoclasts increased in number over a particular time span. The cytochemical localizations of ALPase, ACPase and peroxidase activities in the preosteoclasts resembled those in the osteoclasts but differed from the osteoblasts and preosteoblasts with respect to the ALPase activity. An intense peroxidase activity was detected only in monocytes and neither in preosteoclasts nor in osteoclasts. These results suggest that the cytodifferentiation of preosteoclasts into osteoclasts may be induced by their direct contact to the mineralized bone surface exposed by detachment of osteoblasts, and that the detached osteoblasts may also serve as either an inducer or a constituent of the forming osteoclasts.

Characteristics of growing bone surfaces.

Both bone-forming and bone-destructing cells may appear in different forms. This conclusion has been drawn as a result of compared observations from histomorphologic, histochemical and vital staining studies. The bone-forming cells may be cuboidal and situated in one or more rows along the bone surfaces, or may be flat. The type of cell reflects the rate of bone formation. The bone-resorbing cells may be big multinucleated cells or small mononucleated cells. The latter cell type is well-known from the literature although not referred to in textbooks of histology. Flat bone-forming cells and small mononucleated bone-resorbing cells resemble each other in the light microscope. Studies of bone growth with the aid of histomorphologic descriptions of cell types have to take this fact into account. Otherwise, erroneous conclusions may easily be drawn.