Effect of recombinant human bone morphogenetic protein-2 and Ling Zhi-8 on osteogenesis: a comparative study using a rabbit sinus model.

PURPOSE: This study evaluated the osteogenetic capability of Ling Zhi-8 (LZ-8; a protein purified from traditional Chinese medicine [lingzhi]) compared with recombinant human bone morphogenic protein-2 (rhBMP-2) in a standardized bony defect using a rabbit sinus model. MATERIALS AND METHODS: Twelve male New Zealand white rabbits (18 to 24 weeks old, 3.3 to 3.8 kg) were included in the study. Implants of normal saline 0.1 mg, rhBMP-2 0.1 mg, and LZ-8 0.1 mg were each mixed with a uniform biodegradable polyurethane-based material (Nasopore). The implants were inserted in a standardized bony defect of the nasal bone created by a 2.5-mm trephine bur. The rabbits were sacrificed at 1, 2, 4, and 8 weeks postoperatively. Volume computerized tomographic and histomorphometric examinations were used to evaluate the quantity and quality of regenerated bone. RESULTS: At postoperative week 4, radiography showed that the new bone volume was significantly larger in the rhBMP-2 group compared with the LZ-8 group (P = .041) and the control group (P = .015). Histomorphometrically, better wound healing of the rhBMP-2 group was found during the healing phase compared with the other 2 groups. CONCLUSION: The biomaterial implants using rhBMP-2 and LZ-8 had good biocompatibility and osteogenetic capabilities in the rabbit sinus model. Bone healing in rhBMP-2-treated defects was excellent and showed a significant difference compared with LZ-8. However, LZ-8-treated defects also exhibited bone regeneration, and this traditional Chinese medicine may possess osteogenic potential. Further investigations of the mechanism and application of this protein in osteogenesis are needed.

Bone regeneration by statin and low-intensity pulsed ultrasound (LIPUS) in rabbit nasal bone.

OBJECTIVES: To compare bone regeneration between local implantation of statin and low-intensity pulsed ultrasound (LIPUS), and the combination of statin with LIPUS in rabbit nasal bone using histological and immunohistochemical methods. STUDY DESIGN: Thirty-two adult male Japanese white rabbits (age: 12-16 weeks, weight: 2.5-3.0 kg) were used in this study. Two bone circular defects (5 mm in diameter) per rabbit were created in the nasal bone while preserving the nasal membrane. The two defects in each rabbit were filled with 2.5 mg/ml simvastatin in 0.2 ml water with an atelocollagen sponge (ACS) and ACS alone respectively. Sixteen rabbits (32 sides) received the LIPUS application; the remaining 16 rabbits (32 sides) did not. Therefore, the subjects composed of 4 groups, namely, (1) LIPUS + ACS + simvastatin (the LAS group), (2) LIPUS + ACS (the LA group), (3) ACS + simvastatin (the AS group) and (4) ACS alone (the A group). Four animals were killed in each period, at 1, 2, 4 and 8 weeks postoperative. The parts that had been operated on were removed and prepared for histological assessment. The expression of BMP-2 and the bone area ratio were evaluated using histological and immunohistochemical methods. RESULTS: Bone square in the LAS group was significantly larger than that in the AS group after 1 (P < 0.0001) and 2 week (P = 0.0113). The bone square in the LA group was significantly larger than that in the A group after 1 (P < 0.0001) and 2 weeks (P = 0.0090). However, there was no significant difference between the LAS and LA groups. In the number of cells that stained positive for BMP-2, the LAS group was significantly larger than that in the AS group after 1 (P < 0.0001) and 2 weeks (P = 0.0113). CONCLUSION: This study suggests that bone regeneration can be promoted by LIPUS alone and statin alone, respectively. However the combination use of LIPUS with statin does not differ from LIPUS alone or statin alone.

Ontogenetic changes of craniofacial complex in Turner syndrome patients treated with growth hormone.

OBJECTIVE: The present study assessed changes of craniofacial complex in Turner syndrome (TS) patients treated with growth hormone (GH) during development. The objective was to examine the growth rate and pattern of craniofacial structures and to establish effects of GH on craniofacial development. MATERIALS AND METHODS: The study population consisted of 15 TS patients treated with GH aged 5-18.5 years (13.3 ± 4.4) and corresponding control group of 45 females aged 6.8-18.7 (11.4 ± 2.6). According to the stage of cervical vertebral maturation, subjects were categorized into pre-growth (5 TS and 15 controls) and growth (10 TS and 30 controls) subgroups. The cephalometric analysis comprised angular and linear variables, measured on lateral cephalometric radiographs. RESULTS: The mandibular corpus/anterior cranial base ratio increased significantly only in controls during development. In growth period, ramus/corpus ratio was significantly larger in TS group. SNA and SNB angles were significantly smaller in TS growth subgroup compared to corresponding controls. Among other variables, no statistically significant differences were revealed. CONCLUSIONS: In TS patients treated with GH, growth capacities of cranial base and maxilla are adequate which can be attributed to GH treatment. Shape of mandible is altered due to decreased growth of corpus and overdeveloped ramus. Both maxillary and mandibular retrognathism are becoming more expressed during development. CLINICAL RELEVANCE: Favorable influence of GH on craniofacial complex growth rate and altered growth pattern revealed in this study should be considered while planning both orthodontic treatment and retention.

Effects of induced precocious puberty on cranial growth in female Wistar rats.

This investigation examined the effects of pharmacologically induced precocious puberty on cranial growth in Wistar rats. Forty-eight female newborn Wistar rats were divided into two groups: a control group (C) and an experimental group (E), with four subgroups of six animals each. The time interval from birth until sacrifice differed between the subgroups, and was set at 30, 60, 90, and 120 days. An intramuscular single dose (300 µg) of steroid hormone danazol was administered on day 5 after birth, as a means of inducing precocious puberty. Alizarin (2 mg/100 g) was administered to three animals in each subgroup three days prior to sacrifice. Body mass and dates corresponding to the beginning of the oestrous cycle were recorded. Craniometric measurements were undertaken. Histological analysis using light and fluorescence microscopy was then carried out to qualitatively and quantitatively evaluate the spheno-occipital synchondrosis and to visualize bone deposition patterns. The results were analysed with a Student's t-test and analysis of variance. Precocious puberty was effectively induced and differences between groups denoted an earlier maturation in the experimental rats. In qualitative analysis, a significant increase of total synchondrosis width was noted only in group E60, in comparison with C60, and an increase in the E90 subgroup cortical bone width compared with the C90 subgroup. Histomorphometrically, a statistical difference between total width values of subgroups E60 (434.3 µm) and C60 (323.5 µm) was detected. However, body mass and macroscopic measurements did not show statistically significant differences. An appropriate model for studying bone growth associated with precocious puberty in Wistar female rats was not achieved using steroid hormone danazol, when evaluated at 30 day intervals.

Bone healing of critical-sized nasal defects in rabbits by statins in two different carriers.

OBJECTIVES: To evaluate bone healing following implantation of a statin with two different carriers in rabbit nasal bone using histological and immunohistochemical methods. MATERIALS AND METHODS: Twenty adult, male Japanese white rabbits (age: 12-16 weeks, weight: 2.5-3 kg) were used in this study. Five bone circular defects (5 mm in diameter) per rabbit were created in the nasal bone while preserving the nasal membrane. In the experimental groups, 2.5 mg/ml simvastatin dissolved in 0.2 ml water with hydrogel was implanted in one group, 2.5 mg/ml simvastatin dissolved in 0.2 ml water with an atelocollagen sponge (ACS) in the second group with, only the hydrogel in the third group and only an ACS in the fourth group. No material was implanted in the control group. Four animals were killed in each period, at 1, 2, 4, 8 and 12 weeks postoperatively. The parts that had been operated on were removed and prepared for histological assessment. The expression of bone morphogenetic proteins (BMP)-2 and the bone ration was evaluated using histological and immunohistochemical methods. RESULTS: No significant differences were observed between the simvastatin with hydrogel group and the simvastatin with ACS group at 1, 2, 4, 8 and 12 weeks postoperatively regarding expression of BMP-2, although the number of cells that stained positive for BMP-2 in both of the implanted groups increased significantly at 2 and 4 weeks postoperatively in comparison with the control group (P<0.0001). For new bone area ratio, there were no significant differences between the simvastatin with hydrogel groups and the simvastatin with ACS group after 2, 4, 8 and 12 weeks, although these groups showed higher value than control group (P<0.0001). CONCLUSION: This study suggests that both the simvastatin with hydrogel and simvastatin with ACS implants showed similar BMP-2 expression and new bone formation, and there were no significant differences between the two carriers.

Novel skeletogenic patterning roles for the olfactory pit.

The position of the olfactory placodes suggests that these epithelial thickenings might provide morphogenetic information to the adjacent facial mesenchyme. To test this, we performed in ovo manipulations of the nasal placode in the avian embryo. Extirpation of placodal epithelium or placement of barriers on the lateral side of the placode revealed that the main influence is on the lateral nasal, not the frontonasal, mesenchyme. These early effects were consistent with the subsequent deletion of lateral nasal skeletal derivatives. We then showed in rescue experiments that FGFs are required for nasal capsule morphogenesis. The instructive capacity of the nasal pit epithelium was tested in a series of grafts to the face and trunk. Here, we showed for the first time that nasal pits are capable of inducing bone, cartilage and ectopic PAX7 expression, but these effects were only observed in the facial grafts. Facial mesenchyme also supported the initial projection of the olfactory nerve and differentiation of the olfactory epithelium. Thus, the nasal placode has two roles: as a signaling center for the lateral nasal skeleton and as a source of olfactory neurons and sensory epithelium.

The inhibitory effects of macrolide antibiotics on bone remodeling in chronic rhinosinusitis.

OBJECTIVE: To investigate the effect of nasal mucosal inflammation on bone remodeling and the inhibitory effect of macrolide antibiotics on bone remodeling through the inhibition of receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). STUDY DESIGN AND SETTING: Human nasal fibroblasts were primary-cultured from nasal polyp. After interleukin (IL)-1beta stimulation of fibroblasts with or without macrolide pretreatment, real-time polymerase chain reaction for RANKL messenger RNA (mRNA) and enzyme-linked immunosorbent assay for M-CSF were performed at various intervals. Peripheral blood mononuclear cells (PBMCs) were cultured for 10 days with M-CSF only, M-CSF plus RANKL, or macrolide antibiotic plus M-CSF and RANKL. RESULTS: IL-1beta stimulation of nasal polyp fibroblasts induced expression of RANKL mRNA and secretion of M-CSF. Macrolide antibiotics reduced RANKL mRNA and M-CSF expression by nasal polyp fibroblasts in a dose-dependent manner, and inhibited osteoclastogenesis from PBMCs. CONCLUSION: Nasal fibroblasts stimulated with IL-1beta may take on the role of osteoblasts in osteoclastogenesis, which may be inhibited by macrolide antibiotics.

Effects of insulin-like growth factor-I on nasopremaxillary growth under different masticatory loadings in growing mice.

It is well accepted that reduced masticatory function induced by a diet with soft physical consistency causes alterations in the craniofacial morphology in growing animals. It is assumed that these alterations are associated with reduced proliferative activity of osteoblasts on the bone surface, indicating a significant role for mechanical stimuli mediated by various local growth factors including insulin-like growth factor-I (IGF-I). Here, the effects of IGF-I on the linear growth of nasal and premaxillary bones subjected to different masticatory loadings were examined. The length of the nasal bone and the width of the premaxilla were measured. These dimensions were significantly greater in mice fed a solid diet than in mice fed a granulated diet. In animals treated with IGF-I, the nasal bone length and premaxillary width increased significantly in a subgroup receiving a solid diet, but these changes were not found in a similar group fed a granulated diet. No statistically significant differences in these dimensions were found between solid-diet mice injected with saline and granulated-diet group injected with IGF-I. It is concluded that IGF-I induces nasal and premaxillary growth, and that its effect is enhanced or accelerated by increased mechanical masticatory loading.

Impact of nicotine on bone healing.

A limited number of experimental animal studies and in vitro data confirm that nicotine impairs bone healing, diminishes osteoblast function, causes autogenous bone graft morbidity, and decreases graft biomechanical properties. Therefore, our long-term goal is to develop an effective therapy to reverse the adverse impact of nicotine from tobacco products. However, before accomplishing this goal, we had to develop an animal model. Our hypotheses were nicotine administration preceding and following autogenous bone grafting adversely affected autograft incorporation and depressed donor site healing in a characterized animal wound model. Hypothesis testing was accomplished in bilateral, 4-mm diameter parietal bone defects prepared in 60 Long-Evans rats (male, 35-day-old). A 4-mm diameter disk of donor bone was removed from the left parietal bone and placed in the contralateral defect. The donor site served as a spontaneously healing bone wound. The rats were partitioned equally among three doses of nicotine administered orally in the drinking water (12.5, 25, and 50 mg/L). For each dose, the duration and sequence of nicotine treatment followed four courses, including no nicotine and designated combinations of nicotine administration and abatement prior to and following osseous surgery. Experimental sites were recovered on 14 and 28 days postsurgery, responses quantitated, and data analyzed by analysis of variance and post hoc statistics (p < or = 0.05). We developed a convenient and effective osseous model, and the results validated our hypothesis that nicotine negatively impacts on bone healing.

Increased turnover of small proteoglycans synthesized by human osteoblasts during cultivation with ascorbate and beta-glycerophosphate.

The small proteoglycan decorin had been localized previously at the d-band in the gap zone of collagen fibrils in nonmineralizing tissues. In bone matrix this zone is proteoglycan free and is at least in some species the place where mineralization along collagen fibrils starts. To study the metabolism of the small proteoglycans decorin and biglycan under mineralizing conditions, osteoblasts from human nasal bone were cultured for several weeks in the presence or absence of beta-glycerophosphate and ascorbate. An immediate consequence of the treatment was a reduced expression of decorin, as judged by immune precipitation, whereas the biosynthesis of biglycan was not affected. Pulse-chase experiments were performed with osteoblasts embedded in floating type I collagen gels. In the presence of beta-glycerophosphate and ascorbate, a more rapid turnover of both proteoglycans was noted; the one of biglycan reached statistical significance. Indirect evidence for an enhanced rate of proteoglycan endocytosis was obtained. This effect was not seen in cultured skin fibroblasts. Thus, osteoblasts respond rapidly to mineralizing conditions with alterations of small proteoglycan biosynthesis and turnover.

Cleft beak induced by hydrocortisone in the chick is prevented by increased cell division after experimental reduction of amniotic fluid.

Hypoplasia of the medial nasal process has been reported in chick embryos on embryonic day (ED) 5, 24 h after their exposure to hydrocortisone (HC). As a result, the cleft beak occurs in 80-100% of specimens on ED 9. In order to analyze its influence on cell proliferation, HC was injected intra-amniotically into embryos on ED 4, and the mitotic index and number of BrdU-positive cells were evaluated 24 h later, both in the epithelium and mesenchyme of the medial nasal processes, on serial frontal histological sections. Two hours after BrdU administration, there were 50% of labeled mesenchymal cells in the embryos exposed to HC and only 23% in the control group. The mitotic index of mesenchymal cells was significantly lower in the HC group than in the controls. The epithelium showed no significant difference. HC seemed to prevent the mesenchymal cells from entering mitosis. The cleft beak in the embryos exposed to HC on ED 4 was totally eliminated by tearing open the amnion (amniotomia) and allowing fluid to leak out on ED 5. In some of specimens exposed to HC, the mitotic index was investigated at six time intervals from 15 to 120 min after amniotomia. A significant increase in the mitotic index was detected in the mesenchymal cells of the medial nasal processes during the first hour after amniotomia. Such a prompt increase of the mitotic activity may be hypothetically explained by release of the HC from its receptor binding as a consequence of outflow of the amniotic fluid together with the HC pool, and freeing of the mesenchymal cells, blocked in the G2 phase, to enter mitosis. As a result, the hypoplasia of the medial nasal process might be compensated and the development of the cleft beak prevented.

Binder's syndrome due to prenatal vitamin K deficiency: a theory of pathogenesis.

There is evidence that vitamin K-deficiency during human pregnancy can be caused by the therapeutic use of warfarin or phenytoin. The pregnancy histories of three cases of Binder's syndrome are reported. One was associated with warfarin exposure, one with phenytoin exposure and one with alcohol abuse. It is proposed that Binder's syndrome can be caused by prenatal exposure to agents that cause vitamin K-deficiency. Sprague-Dawley rats were treated from postnatal day 1 to 12 weeks with daily doses of warfarin (100 mg/kg) and concurrent vitamin K1 (10 mg/kg). This regimen creates a net extra-hepatic vitamin K-deficiency. The treated rats developed with a distinct facial appearance characterized by a markedly reduced snout. Histological examination showed that the normally non-calcified septal cartilage was extensively calcified. It is proposed that normal growth of the septal cartilage is necessary for the development of the profile of the nose and midface and that normal growth will only take place while the septal cartilage is uncalcified.

Differential and stage dependent effects of retinoic acid on chondrogenesis and synthesis of extracellular matrix macromolecules in chick craniofacial mesenchyme in vitro.

Retinoic acid (RA) is well known to be a potent teratogen and induces a variety of facial defects in vivo, but at concentration levels lower than those that cause facial defects, RA seems to play an important role in normal facial development. In a previous study, we demonstrated the ability of RA to stimulate chondrogenesis in vitro in HH stage 23/24 chick mandibular (MND) but not frontonasal (FNP) mesenchyme cultured in a serum-free medium. The present study furthers these results by examining the effects of RA on chondrogenesis of chick facial mesenchyme at earlier embryonic stages and the effects on cell proliferation and synthesis of specific extracellular matrix macromolecules at stage 23/24. MND and FNP cells were cultured as micromasses for 4 days in defined media. As described previously, chondrogenesis in stage 23/24 MND cells was significantly enhanced by concentrations of RA of 0.1-1 ng/ml; however, at all earlier stages examined (18 to 22) RA at these concentrations had no significant effect. Higher concentrations of the retinoid inhibited chondrogenesis in MND cultures from all stages tested. Cells of the FNP from all stages displayed no significant change in chondrogenesis below 1 ng/ml RA and a dose dependent inhibition at higher concentrations. Thus RA's promotional effects in the face are not only tissue specific (MND), but also stage-dependent (HH 23/24). The specific effects of RA on matrix production and cell proliferation of stage 23/24 MND and FNP cells was examined by analysis of 35S sulfate, 3H thymidine and 3H proline incorporation. Analysis of 35S sulfate incorporation into sulfated proteoglycans confirmed that concentrations of RA of 0.1-1 ng/ml stimulated cartilage matrix production in MND but not FNP cultures. Above this level of RA, 35S sulfate incorporation was reduced in both. Likewise, 3H proline incorporation into collagenous protein, and to a lesser extent non-collagenous proteins, was stimulated by low levels of RA in MND, but not FNP cultures. Higher concentrations of the retinoid in either MND or FNP cultures did not lower collagen production, undoubtedly due to stimulation of non-chondrogenic cells within the population. This indicates that levels of RA as high as 100 ng/ml cause phenotypic change rather than cell death. This last point is corroborated by the analysis of 3H thymidine uptake in the cultures which was only transiently modified in most. The data indicate that cell proliferation occurred even in the presence of high RA levels.

[Destruction of the nasal skeleton by cocaine sniffing]. / Destructie van het neusskelet door cocaïne snuiven.

An ill-understood, severely ulcerating inflammation of the nose in a 27-year-old female patient turned out to be due to cocaine abuse. The course of the disease process in the hospital, the differential diagnosis and the therapy of this cocaine-induced osteocartilaginous necrosis are described.

Bilateral nasal bone osteophytosis associated with short-term oral isotretinoin therapy for cystic acne vulgaris.

Bilateral 2.5 and 3.0 mm nasal bone osteophytes developed five weeks following the initiation of oral isotretinoin therapy (50 mg daily) for severe cystic acne vulgaris in a healthy 30-year-old white woman who had undergone uneventful rhinoplasty 12 years earlier. Histologically mature bone fragments were removed at surgery. Vitamin A and its analogs have been reported to cause hyperostosis of the vertebrae and long bones, but no known reports link them to nasal bone changes. Clinically significant nasal bone osteophytosis may be another adverse reaction to oral isotretinoin therapy.