Up-regulation of stromal cell-derived factor-1alpha and its receptor CXCR4 expression accompanied with epithelial-mesenchymal transition in human oral squamous cell carcinoma.

Stromal cell-derived factor 1alpha (SDF-1alpha) and its receptor CXCR4 have been implicated in the tumorigenesis, proliferation, and lymph node metastasis of cancer. Here, we report that highly invasive squamous cell carcinoma (SCC) cells with a spindle cell morphology show a strong expression of both SDF-1alpha and CXCR4. CXCR4 expression and cell migratory activity were further up-regulated by treatment with SDF-1alpha or TGF-beta1 in these cells. When epithelial-mesenchymal transition (EMT) was induced by Snail over-expression in SCC cells with an epithelial phenotype, an increased expression of SDF-1alpha was observed. Furthermore, SDF-1alpha and TGF-beta1 up-regulated the expression of CXCR4 and cell migratory activity in these cells. These results indicate that SDF-1alpha and CXCR4 expressions are possible markers of highly-invasive SCC and regulated by EMT.

Effect of FGF-2 and melatonin on implant bone healing: a histomorphometric study.

Melatonin influences the release of growth hormone and cortisol in humans, and it was recently reported that it promoted bone formation. On the other hand, fibroblast growth factor-2 (FGF-2) was reported to facilitate the proliferation of osteoblasts. In the present study, we examined the effect of recombinant human FGF-2 and melatonin on the promotion of osteogenesis around titanium implants. Twenty-four 10-week-old female rats of the Wistar strain received titanium implants in both tibiae. In the experimental groups, 100 mg/kg body weight of melatonin was administered by intraperitoneal injection for 4 weeks after implantation and 10 microg of FGF-2 was locally injected around the implant sites 5 days after implantation. The control groups were administered saline only. In the control group, few newly formed bone could be seen around the implants. It was observed to be in direct contact with the implant surface, but otherwise unmineralized connective tissue was occasionally interposed. In the experimental group, newly formed bone was observed around the titanium implant. In addition, in contrast to the control group, abundant bone trabeculae were seen in the medullary canal region. Bone trabeculae were directly connected to existing cortical bone. These results strongly suggested that melatonin and FGF-2 have the potential to promote osseointegration.

Basic research on aw-AC/PLGA composite scaffolds for bone tissue engineering.

Recently, it has become important to develop effective material to be used as scaffolds for bone tissue engineering. Therefore, we fabricated new three-dimensional (3D) scaffolds consisting of biodegradable poly(D,L-lactide-co-glycolic acid)(PLGA)(75/25) with anti-washout type AC (aw-AC) particles. The aim of this study was to evaluate this new scaffold concerning its basic properties and biocompatibility. The obtained scaffolds were observed with scanning electron microscopy (SEM), and measured for porosity, shrinkage and biaxial compressive strengths. It was shown that PLGA with aw-AC composite scaffolds (aw-AC/PL) showed a greater strength and stability than PLGA scaffolds (PL). Also, the mass reduction of aw-AC/PL during incubation decreased compared to that of PL. The number of MC3T3-E1 cell in PL and aw-AC/PL was counted at 5 h, 1 week, and 2 weeks after cell seeding. As a result, aw-AC/PL exhibited a superior performance in terms of attachment and proliferation compared to PL. Histologically, aw-AC/PL showed an excellent response toward soft tissues. Therefore, it was shown that aw-AC/PL was more biocompatible than PL. In conclusion, it was strongly suggested that aw-AC/PL was more useful for cell transplantation than PL in bone tissue engineering.

The effects inhibiting the proliferation of cancer cells by far-infrared radiation (FIR) are controlled by the basal expression level of heat shock protein (HSP) 70A.

We developed a tissue culture incubator that can continuously irradiate cells with far-infrared radiation (FIR) of wavelengths between 4 and 20 microm with a peak of 7-12 microm, and found that FIR caused different inhibiting effects to five human cancer cell lines, namely A431 (vulva), HSC3 (tongue), Sa3 (gingiva), A549 (lung), and MCF7 (breast). Then, in order to make clear the control system for the effect of FIR, the gene expression concerned to the inhibition effect by FIR were analyzed. In consequence, basal expression level of HSP70A mRNA was higher in A431 and MCF7 cells than in the FIR-sensitive HSC3, Sa3, and A549 cells. Also, the over expression of HSP70 inhibited FIR-induced growth arrest in HSC3 cells, and an HSP70 siRNA inhibited the proliferation of A431 cells by irradiation with FIR. These results indicate that the effect of a body temperature range of FIR suppressing the proliferation of some cancer cells is controlled by the basal expression level of heat shock protein (HSP) 70A. This finding suggested that FIR should be very effective medical treatment for some cancer cells which have a low level of HSP70. Still more, if the level of HSP70 in any cancer of a patient was measured, the effect of medical treatment by FIR can be foreseen for the cancer.

Multi-institutional phase II trial of S-1 in patients with oral squamous cell carcinoma.

The aim of this study was to investigate the efficacy and safety of an oral fluoropyrimidine anticancer agent, S-1, in patients with oral squamous cell carcinoma. Patients with pathologically confirmed squamous cell carcinoma and at least one measurable lesion were enrolled. Oral administration of S-1 (40 mg/m2 twice daily) for 28 days was followed by a 14-day rest period. A total of 41 consecutive eligible patients were enrolled in the study between October 2002 and August 2004. The sites of the primary tumor were the gingiva (n=18), the tongue (n=12), the palate (n=5), the oral floor (n=4), the buccal mucosa (n=1), and the labial mucosa (n=1). A median of two cycles of treatment (range, 1-5) was administered. A complete response was achieved in nine patients and a partial response in eight patients, for an overall response rate of 41.5% (95% confidence interval, 26.4-56.5%). The 3-year survival rate was 76.4% (95% confidence interval, 62.8-90.0%). Although grade 3 anemia and anorexia occurred in two patients each (4.9%), and grade 3 neutropenia, thrombocytopenia, nausea, vomiting, stomatitis, and diarrhea in one patient each (2.4%), no grade 4 toxicities were observed. S-1 exhibits definite antitumor activity in patients with oral squamous cell carcinoma and is well tolerated.

Possible involvement of stem cell factor and endothelin-1 in the emergence of pigmented squamous cell carcinoma in oral mucosa.

We present here the clinical, morphological and immunohistochemical features of a pigmented squamous cell carcinoma (SCC) in the oral mucosa of the hard palate of a 76-year-old Japanese man. He underwent a partial resection of the maxilla subsequent to radiotherapy. The tumor was typical, moderately well-differentiated SCC but had many melanocytes (melanocytosis) within it. Immunohistochemical analysis for stem cell factor (SCF) and endothelin-1, both of which are known to stimulate proliferation and differentiation of melanocytes, revealed prominent expression of both factors in the neoplastic squamous cells of the pigmented SCC, while the non-pigmented oral SCC showed little sign of either factor. These findings strongly suggest that SCF and endothelin-1 secreted by neoplasmic squamous cells are involved in the emergence of a rare variant of oral SCC.

Histological evaluation of apatite cement containing atelocollagen.

Tissue response to apatite cement (AC) containing atelocollagen (AC (ate)) was evaluated using conventional AC (c-AC) as a control material. At one week, the only difference between AC (ate) and c-AC was found in the soft tissue response. With c-AC, a moderate inflammatory response was exhibited: small particles of c-AC were scattered in the cutaneous tissue and many foreign body giant cells were aggregated around the scattered c-AC, whereas AC (ate) showed only a slight inflammatory response with few foreign body giant cells. In terms of bone tissue response, difference between AC (ate) and c-AC was observed at four weeks. New bone formation was observed along the cement at the edge of the pre-existing cortical bone in both c-AC and AC (ate). However, in the case of AC (ate), more abundant and thicker new bone was formed along the cement in the bone marrow when compared with c-AC.

Maspin is involved in bone matrix maturation by enhancing the accumulation of latent TGF-beta.

UNLABELLED: Maspin, a serine protease inhibitor, is expressed by formative osteoblasts. The repression of maspin expression in osteoblastic cells decreased the level of latent TGF-beta in the extracellular matrix, whereas the overexpression of maspin increased latent TGF-beta. These findings suggest that maspin plays an important role in bone matrix formation, particularly in the accumulation of latent TGF-beta. INTRODUCTION: Maspin is a serine protease inhibitor that exhibits tumor suppressive and anti-angiogenic activities. This study was performed to elucidate a possible role for maspin in bone formation. MATERIALS AND METHODS: We performed immunohistochemical analysis of the expression of maspin during endochondral ossification. We evaluated the expression of maspin mRNA and protein in ROS 17/2.8 cells and primary rat osteoblastic cells by RT-PCR, immunocytochemistry, and Western blot analysis. We also examined the accumulation of TGF-beta in the extracellular matrix of cultured ROS 17/2.8 cells after transfection with vectors expressing either maspin or maspin antisense. RESULTS: We observed expression of maspin by active osteoblasts in vivo. Rat osteoblastic cells also expressed maspin mRNA and protein in vitro. Moreover, the accumulation of latent TGF-beta in the extracellular matrix significantly decreased in cultures exposed to an anti-maspin antibody and when cells were transfected with a maspin antisense-expressing vector. In contrast, accumulation of latent TGF-beta in the extracellular matrix increased after transfection of cells with a vector expressing maspin. CONCLUSIONS: These findings suggest that maspin expressed in active osteoblasts plays an important physiological role during maturation of the bone matrix, and in particular, during the process of accumulation of latent TGF-beta in the extracellular matrix.

Melatonin at pharmacological doses enhances human osteoblastic differentiation in vitro and promotes mouse cortical bone formation in vivo.

Melatonin is known to regulate a variety of physiological processes including control of circadian rhythms, regulation of seasonal reproductive function, regulation of body temperature, and so forth. Accumulating evidence from in vitro and in vivo experiments using rodent and chicken has also suggested that melatonin may have an influence on skeletal growth and bone formation. However, little is known about the effects of melatonin on human osteoblasts, which thus remains to be elucidated. This study was performed to determine whether melatonin could affect the proliferation and differentiation of human osteoblasts in vitro and to demonstrate the possibility that melatonin could be applied as a pharmaceutical agent to shorten the treatment period of bone fracture, various osteotomies, and bone distraction. Reverse transcription-polymerase chain reaction and Western blot analysis showed that human osteoblasts expressed melatonin 1a receptor and that its expression levels decreased gradually with the age of the hosts. Melatonin stimulated the proliferation and alkaline phosphatase activity of human osteoblasts in a dose-dependent manner at the pharmacological concentrations. Melatonin also promotes gene expression of type I collagen, osteopontin, bone sialoprotein, and osteocalcin in a dose-dependent manner, and stimulated the mineralized matrix formation in vitro. Moreover, intraperitoneal administration of melatonin to mice increased the volume of newly formed cortical bone of femora. These results demonstrated that melatonin directly accelerated the differentiation of osteoblasts of human as well as rodent and chicken and also suggested that melatonin could be applied as a pharmaceutical agent to promote bone regeneration.

Involvement of Ets-1 transcription factor in inducing matrix metalloproteinase-2 expression by epithelial-mesenchymal transition in human squamous carcinoma cells.

Epithelial-mesenchymal transition (EMT) is a crucial event in cancer progression. We previously reported that EMT up-regulates matrix metalloproteinase-2 (MMP-2) expression in squamous cell carcinoma (SCC) cells. In this study, we showed that Tet Off-induced expression of Snail or SIP1, and treatment with TGF-beta1 induced EMT in terms of down-regulation of E-cadherin, and up-regulation of vimentin and MMP-2 expression with morphological changes. In SCC cells, SIP1 expression was induced by Snail and TGF-beta1, but Snail expression was not induced by SIP1 or TGF-beta1. However, expression of Snail but not SIP1 was strongly increased by TGF-beta1 in highly invasive SCC cells with mesenchymal phenotypes. Analysis of the MMP-2 promoter revealed that an Ets-1 binding site, located between position -1255 and -1248 relative to the transcriptional start site, was critical for the activation by Snail, SIP1 and TGF-beta1 in SCC cells. Induced expression of Snail and SIP1 resulted in the increased expression of Ets-1 and DNA-binding activities of nuclear proteins to the Ets-1-binding site and strong Ets-1 expression was detected in highly invasive SCC cells. Furthermore, overexpression of Ets-1 induced the promoter-activation and expression of MMP-2 without EMT. These results indicate that EMT induces Ets-1 expression, which activates the MMP-2 promoter, but Ets-1 by itself has no activity to induce EMT in SCC cells.

Proliferation and differentiation of cultured MC3T3-E1 osteoblasts on surface-layer modified hydroxyapatite ceramic with acid and heat treatments.

Effects of functionally gradient calcium phosphate consisting of hydroxyapatite (HAP) and alpha-tricalcium phosphate (alpha-TCP) on proliferation and differentiation of osteoblasts were evaluated using MC3T3-E1 cells. There were no significant differences in the proliferation of MC3T3-E1 cells among HAP-alpha-TCP functionally gradient calcium phosphate, pure HAP, and cell culture plastic wells. mRNA expressions of type I collagen, alkaline phosphate, and osteocalcine were evaluated as indexes of initial; mid-stage, and late-stage osteoblastic differentiation. Basically, HAP-alpha-TCP functionally gradient calcium phosphate and pure HAP enhanced the expressions of the three markers when compared with that of cell culture plastic wells. For type I collagen and alkaline phosphate expressions, HAP-alpha-TCP functionally gradient calcium phosphate showed the same expression level as pure HAP. For osteocalcine expression, HAP-alpha-TCP functionally gradient calcium phosphate showed a higher level than pure HAP. We concluded, therefore, HAP-alpha-TCP functionally gradient calcium phosphate has good potential to be a bone filler material with high osteoconductivity.

Immortalization of human dental papilla, dental pulp, periodontal ligament cells and gingival fibroblasts by telomerase reverse transcriptase.

BACKGROUND: Human telomerase reverse transcriptase (hTERT) is catalytic subunit of human telomerase. METHODS: We studied the immortalization of a series of human dental and periodontal cells by ectopic expression of hTERT and co-expression of hTERT with human papilloma virus 16 (HPV16) or simian virus 40 (SV40). Differentiation abilities of the established cell lines were studied in terms of the mineralized matrix formation and gene expression. RESULTS: We established immortalized gingival fibroblasts by hTERT, dental papilla and periodontal ligament cells by hTERT and HPV16, and pulp cells by hTERT and SV40. The papilla and pulp cells showed mineralization and dentin sialophosphoprotein (DSPP) expression when cultured in the presence of beta-glycerophosphate. The immortalized periodontal ligament cells did not show mineralization or DSPP expression, although expressions of alkaline phosphatase, osteopontin and osteocalcin were detected. CONCLUSIONS: These cell lines will be useful tools for studying the repair and regeneration of dental and periodontal tissues and various diseases including odontogenic tumors.

The novel gene encoding a putative transmembrane protein is mutated in gnathodiaphyseal dysplasia (GDD).

Gnathodiaphyseal dysplasia (GDD) is a rare skeletal syndrome characterized by bone fragility, sclerosis of tubular bones, and cemento-osseous lesions of the jawbone. By linkage analysis of a large Japanese family with GDD, we previously mapped the GDD locus to chromosome 11p14.3-15.1. In the critical region determined by recombination mapping, we identified a novel gene (GDD1) that encodes a 913-amino-acid protein containing eight putative transmembrane-spanning domains. Two missense mutations (C356R and C356G) of GDD1 were identified in the two families with GDD (the original Japanese family and a new African American family), and both missense mutations occur at the cysteine residue at amino acid 356, which is evolutionarily conserved among human, mouse, zebrafish, fruit fly, and mosquito. Cellular localization to the endoplasmic reticulum suggests a role for GDD1 in the regulation of intracellular calcium homeostasis.

Effects of various sterilization methods on the setting and mechanical properties of apatite cement.

Sterilization capability is a necessary requirement for any material that is to be used in a medical application. Therefore, it is necessary for apatite cement (AC) to be sterilized. Because there is little information on the sterilization methods of AC, the aims of this investigation were to evaluate the effects of various sterilization methods, including steam, dry heat, ethylene oxide (EtO) gas, and gamma irradiation sterilizations, on the setting and mechanical properties of AC. In the case of steam sterilization, because AC powder aggregated before setting-time measurements, the setting time could not be measured. When the powder was sterilized by dry heat or EtO gas, the setting time was prolonged significantly and the wet diametral tensile strength (DTS) value decreased significantly. Therefore, sterilizations with steam, dry heat, or EtO gas were suggested to be inappropriate methods for AC. Accordingly, the following experiments focused on gamma sterilization. The setting time of AC was retarded with an increase in gamma irradiation dose. The wet DTS value decreased with the increase in gamma irradiation dose. There was no compositional change due to the gamma irradiation. The following tests were carried out in order to examine the effect of the gamma irradiation on the setting reaction of AC in detail. Tetracalcium phosphate [TTCP: Ca(4)(PO(4))(2)O] and dicalcium phosphate anhydrous (DCPA: CaHPO(4)) were separately irradiated, and the cements were produced with the use of irradiated powder and nonirradiated powder. Although the wet DTS value of AC produced from irradiated TTCP and nonirradiated DCPA decreased with increasing gamma irradiation dose, there was no significant difference. In contrast, the wet DTS value of AC produced from irradiated DCPA and nonirradiated TTCP significantly decreased with the increase in gamma irradiation dose. In conclusion, although the detailed mechanism of the delayed setting time and decreased DTS value was not clarified by the present study, it was found that gamma irradiation affected DCPA more than TTCP.

Effects of neutral sodium hydrogen phosphate on the setting property and hemostatic ability of hydroxyapatite putty as a local hemostatic agent for bone.

Hydroxyapatite (HAP) putty has been reported to have good hemostatic ability and excellent biocompatibility. Although the setting reaction of HAP putty and resulting transformation to HAP is the reason for this excellent biocompatibility, it also limits the handling period. In this study, the relationship between the setting reaction of HAP putty and its hemostatic ability was investigated, where neutral sodium hydrogen phosphate concentration and post-preparation time were used as indexes. A higher concentration of neutral sodium hydrogen phosphate was found to result in decreased hemostatic ability. The hemostatic ability of HAP putty decreased with time after its preparation; thus it was important to use HAP putty within 10 min after its preparation. The adhesive strength of HAP putty to bone increased with time. Therefore reliable hemostasis can be expected with HAP putty. Although the limited handling time is a drawback, HAP putty is thought to have a good potential as a hemostatic agent-it is highly reliable, and has a high hemostatic ability with excellent biocompatibility.

Effects of apatite cements on proliferation and differentiation of human osteoblasts in vitro.

Although apatite cement (AC) and sintered hydroxyapatite (s-HAP) are known to show good osteoconductivity, it is not clear whether or not the degree of their osteoconductivity is the same. In addition, it has not been clarified whether or not it is dependent on the type of AC; conventional AC (c-AC), fast-setting AC (fs-AC) or anti-washout AC (aw-AC). The aim of this study was to investigate the effects of ACs on cultured human osteoblasts, as they may provide a useful index of osteoconductivity. Human osteoblasts were cultured on the surfaces of ACs and s-HAP, and were evaluated with respect to cell attachment, proliferation, and differentiation. We found that ACs and s-HAP showed similar cell attachment. No significant difference between ACs and s-HAP was found with respect to the proliferation of osteoblasts. In contrast, we found that the differentiation of osteoblasts was enhanced on the surface of ACs compared with that of s-HAP. However, there was no difference among the types of AC. We therefore concluded that AC may show better osteoconductivity than s-HAP, and that osteoconductivity of AC may be similar, regardless of the type of AC.

Down-regulation of Wnt-4 and up-regulation of Wnt-5a expression by epithelial-mesenchymal transition in human squamous carcinoma cells.

Gene expression of Wnt-1, 2, 3, 4, 5a, 6 and 7a was analyzed by RT-PCR in eleven squamous cell carcinoma (SCC) cell lines and compared with that in two normal oral keratinocyte strains. There appeared to be an inverse relationship between Wnt-4 and Wnt-5a expressions, i.e., Wnt-4 was not expressed in HOC719-NE, HOC313 or TSU cells, while Wnt-5a was strongly expressed only in these cells. These cell lines showed decreased expression of E-cadherin and elevated expression of vimentin accompanied with strong expressions of Snail and deltaEF1, which have been reported to be transrepressors of E-cadherin and to trigger epithelial-mesenchymal transition (EMT), suggesting associations of Wnt-4 with epithelial phenotype and Wnt-5a with mesenchymal phenotype of SCC cells. To study whether the expressions of these Wnt genes are regulated by EMT, we transfected a Snail-expression vector into A431 and OM-1 cells, which express Wnt-4 but not Wnt-5a. The stably Snail-overexpressing clones showed spindle morphology, increased expression of vimentin and decreased expression of E-cadherin accompanied with augmented expression of deltaEF1. In these clones, down-regulation of Wnt-4 and up-regulation of Wnt-5a were clearly observed. These results indicated that Wnt-4 and Wnt-5a are oppositely affected by EMT, and down-regulation of Wnt-4 and up-regulation of Wnt-5a are possible markers of the malignant phenotype of human SCC.

Basic fibroblast growth factor and epidermal growth factor reverse impaired ulcer healing of the rabbit oral mucosa.

BACKGROUND: The therapies for refractory ulcers on the oral mucosa are symptomatic and very unsatisfactory. We hypothesized that application of growth factors might be able to achieve successful remission of the lesion. We evaluated the effects of systemic administration and topical application of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on impaired wound healing of ulcers in the rabbit gingiva. METHODS: Almost uniform round ulcers could be created on the gingiva of the rabbits by chemical injury with acetic acid. When the submandibular glands were removed or i.v. injection of cisplatin (CDDP) and peplomycin sulfate was performed before ulcer formation, healing of the ulcers took longer than in untreated rabbits. To ascertain whether or not human EGF and bFGF affect rabbit cells, we first examined the effects of EGF and bFGF on the proliferation of the cells derived from rabbit gingiva. We then applied EGF or bFGF in these impaired healing models. RESULTS: EGF and bFGF promoted proliferation of the fibroblasts, and EGF also promoted proliferation of the keratinocytes isolated from gingival tissue of rabbits in vitro. Systemic injections of EGF and bFGF in rabbits, which had their submandibular glands removed, and topical application of bFGF accelerated healing of ulcers created in rabbits injected with CDDP and peplomycin sulfate. The ability of bFGF to promote the healing of ulcers was much greater than that of EGF. CONCLUSION: Basic FGF may be effective for refractory oral mucosal lesions.

Increased invasion and matrix metalloproteinase-2 expression by Snail-induced mesenchymal transition in squamous cell carcinomas.

Loss of E-cadherin expression is a major characteristic of highly invasive and metastatic cancers. Epithelial-mesenchymal transition (EMT) has been advocated to be a causative mechanism for the suppression of E-cadherin and tumor progression. Snail is a zinc finger transcription factor that triggers the EMT and is one of the recently identified E-cadherin repressors. The reverse correlation of Snail and E-cadherin expressions has been reported in many types of human cancers including squamous cell carcinoma (SCC). In this study, we showed that three E-cadherin negative SCC cell lines had a fibroblastic morphology, strong expressions of vimentin, a mesenchymal marker gene, and Snail. Compared to other E-cadherin positive SCC cells, these cells showed higher invasive ability and expression of MMP-2, a matrix degrading enzyme which has been demonstrated to be highly expressed in invasive cancer cells. Over-expression of Snail in A431 cells resulted in the loss of E-cadherin expression, the change of their morphology to fibroblastic, and the up-regulation of vimentin gene expression, indicating that an EMT was induced by Snail. Furthermore, these cells became more invasive and showed higher levels of MMP-2 activity and its gene expression. Luciferase analysis demonstrated that the MMP-2 promoter activity was induced by Snail transfection and the promoter region from -262 to -411 relative to the transcriptional start site was necessary for this induction. These results indicate that Snail is a new inducer of MMP-2 expression and suggest that the EMT contributes to the increased invasion not only through the inhibition of cell-cell adhesion but also the up-regulation of MMP-2 expression in SCC cells.

Autosomal dominant gnathodiaphyseal dysplasia maps to chromosome 11p14.3-15.1.

Gnathodiaphyseal dysplasia (GDD) is a syndrome characterized by bone fragility, sclerosis of tubular bones, and cemento-osseous lesions of jawbones. Although some cases of this syndrome exist in families with autosomal dominant inheritance, the underlying gene has never been identified. We analyzed a large four-generation family with GDD by linkage analysis using genomic DNA from nine affected and six nonaffected family members. A genome-wide search using a set of highly polymorphic microsatellite markers showed evidence for linkage to chromosome 11p14.3-15.1. Two-point linkage analysis of microsatellite markers spanning this locus resulted in a maximum logarithm of odds (LOD) score of 2.70 with a recombination fraction (theta) of 0 at D11S1755, D11S1759, and D11S915, and a maximum LOD score of 3.01 at D11S4114 was obtained in multipoint linkage analysis. Haplotype analysis detected no recombination between GDD and six closely linked markers (D11S928, D11S1755, D11S4114, D11S1759, D11S915, and D11S929) and established the candidate interval of 8.7 cM on chromosome 11p for GDD. Although GDD has been considered to be a variation of osteogenesis imperfecta (MIM 166260), our results indicate that this syndrome is a new and distinct disease entity from other systemic bone diseases. Furthermore, these genetic markers are useful for presymptomatic diagnosis of GDD in some families and for identification of the GDD gene.