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The presence of insufficient bone volume remains a major clinical problem for dental implant placement to restore the oral function. Gene-transduced stem cells provide a promising approach for inducing bone regeneration and enhancing osseointegration in dental implants with tissue engineering technology. Our previous studies have demonstrated that the hypoxia-inducible factor-1α (HIF-1α) promotes osteogenesis in rat bone mesenchymal stem cells (BMSCs). In this study, the function of HIF-1α was validated for the first time in a preclinical large animal canine model in term of its ability to promote new bone formation in defects around implants as well as the osseointegration between tissue-engineered bone and dental implants. A lentiviral vector was constructed with the constitutively active form of HIF-1α (cHIF). The ectopic bone formation was evaluated in nude mice. The therapeutic potential of HIF-1α-overexpressing canine BMSCs in bone repair was evaluated in mesi-implant defects of immediate post-extraction implants in the canine mandible. HIF-1α mediated canine BMSCs significantly promoted new bone formation both subcutaneously and in mesi-implant defects, including increased bone volume, bone mineral density, trabecular thickness, and trabecular bone volume fraction. Furthermore, osseointegration was significantly enhanced by HIF-1α-overexpressing canine BMSCs. This study provides an important experimental evidence in a preclinical large animal model concerning to the potential applications of HIF-1α in promoting new bone formation as well as the osseointegration of immediate implantation for oral function restoration.

The spectrum of F8 genetic variants identified in sporadic families was fairly diverse. The high prevalence of chimaeras in carriers suggests that more cautions should be taken in genetic counselling of sporadic haemophilia families.

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The present study investigated the detailed in vitro osteogenic differentiation process and in vivo bone regenerative property of human amniotic epithelial cells (hAECs). The in vitro osteogenic differentiation process of hAECs was evaluated by biochemical staining, real-time polymerase chain reaction, and immunofluorescence. Next, β-tricalcium phosphate (β-TCP) scaffolds alone or loaded with hAECs were implanted into the alveolar defects of rats. Micro-computed tomography evaluation and histologic studies were conducted. Our results validated the in vitro osteogenic capacity of hAECs by upregulation of Runx2, osterix, alkaline phosphatase, collagen I, and osteopontin, with positive biochemical staining for osteoblasts. An epithelial-mesenchymal transformation process might be involved in the osteogenic differentiation of hAECs by increased expression of transforming growth factor-β1. Our data also demonstrated that in vivo implantation of hAECs loaded on β-TCP scaffolds, not only improved bone regeneration by direct participation, but also reduced the early host immune response to the scaffolds. The presented data indicate that hAECs possess proper osteogenic differentiation potential and a modulatory influence on the early tissue remodeling process, making these cells a potential source of progenitor cells for clinical restoration of the alveolar defect.

Linkage analysis is a widely used strategy for genetic counselling in haemophilia A (HA) families. We attempted to develop more informative markers closely linked to factor VIII (FVIII) gene and establish a rapid multifluorescent polymerase chain reaction (PCR) method with these markers. Five extragenic (DXS15, DXS9901, G6PD, DXS1073 and DXS1108) and one intragenic (F8Civs13) markers were examined in 118 healthy individuals and 12 HA families which had been diagnosed before. Five extragenic markers were within an interval of about 1.5 Mb to FVIII gene and located on each side of the gene. The expected heterozygote rate (HR) of DXS15, DXS9901, G6PD, DXS1073, DXS1108 and F8Civs13 were 74.97%, 79.77%, 56.06%, 59.92%, 39.97% and 47.61%, while the observed HR were 88.24%, 82.35%, 21.57%, 62.75%, 35.29% and 52.94%. When six polymorphic markers were combined together, all the studied females were informative in at least one of these markers and 29.41% of them were detected informative in three markers with the highest frequency. The diagnostic rates of DXS15, DXS9901, G6PD, DXS1073, DXS1108 and F8Civs13 in 12 haemophilia families were 75.00%, 91.67%, 41.67%, 75.00%, 33.33% and 66.67% respectively. All the genetic diagnosis was consistent with the result we analysed before and no recombination was observed. Family 1 was given as an example in this study and was found to be informative in three polymorphic markers DXS15, DXS9901 and DXS1073. The patient's sister was detected the same allele as the proband, but her male fetus did not inherit the affected allele from her, which was consistent with the result of sequencing. It was demonstrated that the multifluorescent PCR method established in this study was convenient and efficient and can be applied to carrier detection and prenatal diagnosis in HA families.

. Female hemophilia A heterozygous for a de novo frameshift and a novel missense mutation of factor VIII. J Thromb Haemost 2006; 4: 1969-74. Summary. Background: Hemophilia A (HA) is an X-chromosome-linked recessive disorder. Aim: We report the case of a female HA patient with a moderate decrease of factor (F) VIII activity and antigen (FVIII:C 3.4%, FVIII:Ag 4.2%) and severe bleeding symptoms. Methods: The patient's father had mild FVIII deficiency (FVIII:C 6.9%, FVIII:Ag 7.4%), and her mother had normal FVIII activity. The von Willebrand disease antigen and von Willebrand factor ristocetin cofactor activity were normal in all family members. The genomic DNA was extracted from the peripheral blood lymphocytes of the patient and her family members. Long-distance polymerase chain reaction (PCR) was employed to screen for the intron 22 inversion of the FVIII coding gene (F8). The F8 coding sequence was amplified with PCR and sequenced with an automatic sequencer. Results: Two heterozygous mutations were identified in the patient: one a substitution of nucleotide 5981T by C that leads to a missense mutation Leu1975Pro, and the other an insertion of an ÔAÕ between nucleotides 3637 and 3638 (3637_3638insA) that shifts the reading frame and predicts a premature stop codon downward. The mutation Leu1975Pro was identified in the father's F8; however, 3637_3638insA was a de novo mutation that occurred in the patient's maternalderived F8. Real-time PCR was applied to analyze the level of ectopically F8 gene transcripts in the peripheral lymphocytes of family members. The ectopic transcripts of F8 of the patient were less abundant than the normal control (patient:normal control ratio 0.67), whereas her parents showed no significant difference from the normal control. Conclusion: The FVIII deficiency of the HA patient resulted from a de novo occurrence of a frameshift 3637_3638insA in her maternal-derived F8 and a novel missense mutation Leu1975Pro inherited from her father.

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