Potential therapeutic role of spermine via Rac1 in osteoporosis: Insights from zebrafish and mice.

Osteoporosis is a prevalent metabolic bone disease. While drug therapy is essential to prevent bone loss in osteoporotic patients, current treatments are limited by side effects and high costs, necessitating the development of more effective and safer targeted therapies. Utilizing a zebrafish ( Danio rerio) larval model of osteoporosis, we explored the influence of the metabolite spermine on bone homeostasis. Results showed that spermine exhibited dual activity in osteoporotic zebrafish larvae by increasing bone formation and decreasing bone resorption. Spermine not only demonstrated excellent biosafety but also mitigated prednisolone-induced embryonic neurotoxicity and cardiotoxicity. Notably, spermine showcased protective attributes in the nervous systems of both zebrafish embryos and larvae. At the molecular level, Rac1 was identified as playing a pivotal role in mediating the anti-osteoporotic effects of spermine, with P53 potentially acting downstream of Rac1. These findings were confirmed using mouse ( Mus musculus) models, in which spermine not only ameliorated osteoporosis but also promoted bone formation and mineralization under healthy conditions, suggesting strong potential as a bone-strengthening agent. This study underscores the beneficial role of spermine in osteoporotic bone homeostasis and skeletal system development, highlighting pivotal molecular mediators. Given their efficacy and safety, human endogenous metabolites like spermine are promising candidates for new anti-osteoporotic drug development and daily bone-fortifying agents.

Osteogenic Induction of Low-dose Ipriflavone on Bone Marrow Mesenchymal Stem Cells Extracted from Osteoporosis Rats.

OBJECTIVE: To investigate the effects of ipriflavone (IPF), a synthetic isoflavone plant oestrogen with a structure similar to that of oestradiol, on the osteogenic differentiation of bone mesenchymal stem cells (BMSCs). METHODS: BMSCs were derived from ovariectomised rats (rBMSCs-OVX) and then induced with or without IPF. Cell cytotoxicity, mineralisation in vitro and osteoblast-specific gene expression of BMSCs were studied. RESULTS: IPF at a concentration of 10-8, 10-7 and 10-6 mol/l exhibited no cytotoxic effect on the proliferation of BMSCs but increased alkaline phosphatase activity and osteoblast-specific gene expression. CONCLUSION: IPF enhances osteogenic differentiation of rBMSCs-OVX partly in vitro, thus its use offers a potential strategy for the treatment of osteoporosis.

The Crucial Role of Wntless in Osteogenesis and Odontogenesis.

Wnt signalling pathways have been the focus of intense research activity for decades due to their fundamental role in skeletal and dental development. Wntless, an exclusive chaperone protein for the exocytotis of Wnt ligands, was identified in 2006. In the last decade, the molecular biological studies of Wntless and its genetic studies in human and mice have highlighted the importance of this protein in mineralised tissues, including bone, cartilage and teeth. This article reviews recent developments and discrepancies in the role of Wntless in skeletal and dental development based on mutant phenotypes, as well as the underlying mechanism involved in its molecular and physiological regulation. We conclude that, though some controversial phenotypes exist due to different Cre line resources, Cre recombinase activity and detection time points, Wntless undeniably exerts a context- and stage-dependent regulatory function during the development and homeostasis of both skeletal and dental tissue.

[Advances in biomimetic modification of materials for oromaxillofacial bone regeneration and dental implant].

Oromaxillofacial hard tissue defects is still a difficult problem in clinical treatment. Regeneration of oromaxillofacial hard tissue based on tissue engineering technology has a good clinical application prospect. The functional modification of scaffolds is one of key factors that influence the outcome of tissue regeneration. The biomimetic design of biomaterials through simulating the natural structure and composition of oromaxillofacial hard tissue has gradually become a research hotspot due to its advantages of simplicity and efficiency. In this article, the biomimetic modification of biomaterials for oromaxillofacial hard tissue regeneration is reviewed, expecting to provide a new idea for the treatment of oromaxillofacial hard tissue defect.

Validation of Digital Evaluation in Systematic Training on Tooth Preparation in Aesthetic Veneer Rehabilitation.

OBJECTIVE: To evaluate the outcome of systematic video training on tooth preparation in veneer restoration and the practicability of the application of the digital evaluation system of scan design and assessment software. METHODS: Ten residents were selected from a group enrolled on the first-year programme for the National Standard Training of Dentistry in the Department of Prosthodontics, College of Stomatology, Ninth People's Hospital Affliated with Shanghai Jiao Tong University, School of Medicine. First, each student prepared five teeth based on their knowledge and clinical experience, and then received systematic video training on veneer preparation. Before and after the training, the evaluation of the effects of training was conducted on the prepared teeth by measuring the continuity of the finishing line and tooth reduction amount automatically using the prepCheck 2.0 (Dentsply Sirona, Charlotte, NC, USA) CAD/CAM system. RESULTS: The results showed a significant difference in the quality of finishing line continuity pre- and post-training. Furthermore, the data for tooth reduction after training, which met standard values, improved remarkably, increasing from 32.40 ± 7.82% to 60.50 ± 5.48%. CONCLUSIONS: Video training could significantly enhance the quality of tooth preparation for veneers. Moreover, the digital evaluation system could serve as an ideal alternative for tooth preparation evaluation for preclinical students, offering new insights for clinical education.

Associations between the red blood cell distribution width and primary angle-closure glaucoma: a potential for disease prediction.

The red blood cell distribution width (RDW) is a simple and inexpensive laboratory parameter that can be linked to oxidative stress, inflammation and microvascular flow resistance. For this research, we performed a large-sample case-control study to describe the relationships between the RDW and primary angle-closure glaucoma (PACG). A total of 1191 PACG patients (422 males and 769 females), who were divided into mild, moderate and severe PACG groups, and 982 healthy controls (344 males and 638 females) were recruited between January 2008 and June 2018. Detailed eye and physical examinations were performed for each subject. Based on the laboratory results, the mean RDW was significantly higher (p < 0.001) in the PACG group (13.01 ± 0.82%) than in the control group (12.65 ± 0.53%). Moreover, the mean RDW level was lower (p < 0.05) in the mild PACG group than in the moderate and severe PACG groups. The Pearson correlation analyses showed significant positive correlations between the mean deviation and the RDW (r = 0.141, p < 0.001) and the intraocular pressure and the RDW (r = 0.085, p = 0.004). After adjusting for the confounding factors, the logistic regression analyses indicated that the odds ratio for the PACG group was 2.318 (p < 0.001, 95% confidence interval 1.997, 2.690) when compared to the control group. Additionally, an increased RDW was associated with the PACG severity, and this trend was also observed in the gender and age subgroups. In summary, the results of our study showed that an elevated RDW was associated with PACG and its severity. If future studies confirm this relationship, the use of an RDW assessment may help to predict the PACG severity in each patient in order to better customise effective prevention treatments.

Applications of Bioactive Ions in Bone Regeneration.

The repair of large bone defects remains a huge challenge for bone regenerative medicine. To meet this challenge, a number of bone substitutes have been developed over recent years to overcome the drawbacks of traditional autograft and allograft therapies. Thus, the improvement of the osteoinductive ability of these substitutes has become a major focus for research in the field of bone tissue engineering. It has been reported that some metallic ions play an important role in bone metabolism in the human body, and that bone repair could be enhanced by incorporating these ions into bone substitutes. Moreover, it is well documented that ions released from these substitutes such as magnesium, zinc, and strontium can increase the osteogenic and angiogenic properties of bone repair scaffolds. However, the mechanisms of action of these ions on cellular bioactivity are currently unclear. Therefore, in the present article, we highlight the recent use of bioactive ions in bone tissue engineering and discuss the effects of these ions on osteogenesis and neovascularisation.

[Effect of overexpression of transcription factor Runx2 and Osterix on osteogenic differentiation of endothelial cells].

PURPOSE: To explore the effect of overexpression of Runx2 and Osterix (OSX) genes on osteogenic differentiation of human umbilical vein endothelial cells (HUVECs). METHODS: Overexpressed Runx2 and OSX lentiviral vectors were transfected into HUVECs respectively. The osteogenic potential of transfected cells was identified by alkaline phosphatase (ALP) staining and ALP activity. Furthermore, real time-PCR, Western blot and immunofluorescence staining were performed to detect the expression of osteogenic genes and proteins in HUVECs. GraphPad Prism 6.01 software was used for statistical analysis. RESULTS: Overexpression of Runx2 gene was beneficial for osteogenic differentiation of HUVECs, while overexpression of osterix gene did not show osteogenic differential potential. Moreover, overexpression of Runx2 gene in HUVECs up-regulated the gene expression level of Runx2, OSX, ALP, bone sialoprotein (BSP), osteopontin (OPN), and osteocalcin (OCN), and up-regulated protein level of OPN and OCN. CONCLUTIONS: Overexpression of Runx2 could promote osteogenic differentiation of HUVECs.

Meta-analysis reveals gender difference in the association of liver cancer incidence and excess BMI.

Excess body weight has a positive association with risk of liver cancer, but the gender difference in the relationship between body mass index and liver cancer risk remains uncertainty. In this work, we performed meta-analysis for excess body weight and risk of liver cancer incidence to identify the gender difference. We searched the English-languages database and the Chinese literature databases to May 12, 2017. Overall, a total of 17 studies were included. Relative risks (RRs) with 95% confidence intervals was used to evaluate the strength of these associations. The RRs of liver cancer incidence for obese men and women were 2.04 (1.70-2.44) and 1.56 (1.37-1.78). The former one was significantly higher than the later one (P for interaction = 0.02). Notably, the RR of liver cancer incidence in non-Asian obese men was even higher than their counter part (2.31(1.85-2.91) vs. 1.56 (1.31-1.86), P for interaction = 0.01). Similar gender difference was observed in the dose-response curve. As example, at the point of BMI = 32 kg/m2, the RRs for men and women were 1.61 (1.45-1.79) and 1.41 (1.02-1.94) respectively. Findings from this meta-analysis indicate that obesity is associated with a higher risk of liver cancer incidence in men, especially in non-Asian men, which might partially contribute to the male dominance of liver cancer incidence.

Quantitative Proteomic Analysis of Rat Condylar Chondrocytes during Postnatal Development.

OBJECTIVE: To investigate differentially expressed proteins in rat mandibular condylar cartilage (MCC) chondrocytes caused by initial mastication for short postnatal periods. METHODS: Four groups of protein samples were extracted from primary cultured rat MCC chondrocytes, harvested from eigthy postnatal SD rats aged 1,7,14 and 28 days, with twenty in each group. Total proteins were labelled with isobaric tags for relative and absolute quantification (iTRAQ) reagents. Two-dimensional nano-high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption ionization-time-of-flight/ time-of-flight (MALDI-TOF/TOF) mass spectrometry analysis with iTRAQ technique were performed. All data were analysed by MASCOT software with the SWISSPROT protein database. Furthermore, bioinformatics and statistical analysis were performed to classify their cellular components, biological processes, molecular functions and metabolic pathway by the PANTHER database. RESULTS: In total, 137 differentially expressed proteins were identified during MCC growth and were assigned to one or more cellular components. According to the PANTHER analysis, a significant proportion of proteins are involved in the metabolic process, cellular process, biological regulation, developmental process and response to stimulus. The most extensive molecular function was 43% in catalytic activity. In addition, it was found that proteins in MCC chondrocytes change markedly on the growth stage of eruption of the teeth. CONCLUSION: This study provides an integrated perspective of molecular mechanisms regulating early normal postnatal growth and development of rat MCC at the protein level.

Expansion of CD14(+)CD16(+) monocytes is related to acute leukemia.

OBJECTIVE: Aim to investigate the proportion of CD14(+)CD16(+) monocytes and understand the pathogenesis of this monocyte subset in acute leukemia. METHODS: Flow cytometry was utilized to study the phenotype expression of CD14(+)CD16(+) monocytes and CD3(+) T lymphocytes in peripheral blood derived from patients with acute leukemia. All the data were analyzed by SPSS 13.0 software. RESULTS: The proportion of CD14(+)CD16(+) monocytes including both intermediate and non-classical monocytes, increased significantly in patients with acute leukemia and changed negatively or positively according to the disease process. Meanwhile, the proportion of CD14(+)CD16(+) monocytes was inversely correlated with absolute number of CD4(+) T lymphocytes, ratio of CD4(+)/CD8(+) T cells, and positively correlated with the proportion of neutrophil granulocytes. CONCLUSIONS: The proportion of CD14(+)CD16(+) monocytes (especially the intermediate subpopulation) is related to the progression of acute leukemia, and the expansion of this monocyte subset could indicate the severity of the disease.

[Repair of calvarial defect using a tissue-engineered bone with simvastatin-loaded ß-tricalcium phosphate scaffold and adipose derived stem cells in rabbits].

PURPOSE: The osteogenic-angiogenic differentiation effects of simvastatin (Sim) were explored on adipose tissue-derived stem cells (ASCs). A tissue-engineered bone with simvastatin loaded ß-tricalcium phosphate (ß-TCP) scaffold and ASCs was constructed to repair the calvarial defect in rabbits. METHODS: ASCs were obtained from the groin of rabbits. After 14 days of osteogenic inducing culture, sufficient cells were expanded for the following experiments. Cell counting was conducted to ASCs in osteogenic inducing medium containing 0, 0.01, 0.1 and 1 µmol/L simvastatin. Concentrations of 0.05 and 0.1 µmol/L simvastatin were administrated to ASCs for real-time PCR of angiogenesis-osteogenesis related genes like RUNX2, OPN, OCN, and VEGF on day 1, 7. ALP staining was performed on day 7, Alizarin red staining for calcium deposits was carried out on day 14. Bilateral critical-sized defects were created on 12 New Zealand rabbits. Four groups of tissue-engineered bone were randomly allocated to them. Group A: ß-tricalcium phosphate (ß-TCP) (n=6); group B: ß-TCP/Cell (n=6); group C: ß-TCP/Sim (n=6); group D: ß-TCP/Cell/Sim (n=6). Specimens were decalcified and stained by HE 8 weeks after operation. The data was statistically analyzed using SPSS 17.0 software package. RESULTS: The use of simvastatin with the concentration of 0.05 µmol/L enhanced the expression of angiogenic-osteogenic related genes like RUNX2, OPN, OCN, and VEGF. ALP activity and von Kossa were significantly stronger in osteogenic inducing medium containing 0.05 µmol/L simvastatin. The new bone formation area of ß-TCP/Cell/Sim group at 8-week after implantation was significantly larger than the other groups. CONCLUSIONS: 0.05 µmol/L simvastatin enhances the angiogenic-osteogenic differentiation of ASCs. Simvastatin loaded ß-TCP scaffold and ASCs successfully repair the calvarial defect in rabbits. These results indicate a promising future in application of simvastatin for bone regeneration.

Osteogenic potential of bone marrow stromal cells derived from streptozotocin-induced diabetic rats.

Type 1 diabetes mellitus (T1DM) is associated with a series of bone complications, which are still a great challenge in the clinic. Bone marrow stromal cells (BMSCs) are crucial to bone remodeling and are attractive candidates for tissue engineering. Hence, we aimed to investigate whether impaired functions of BMSCs play a role in the pathogenesis of bone complications associated with T1DM. BMSCs were isolated from normal and streptozotocin-induced diabetic rats, and their proliferation and osteogenic differentiation ability were analyzed. Diabetic BMSCs demonstrated reduced proliferation ability, osteoblast gene expression, alkaline phosphatase activity and mineralization. Nude mice transplanted with diabetic BMSCs in a calcium phosphate cement scaffold exhibited reduced new bone formation, as detected by hematoxylin and eosin staining and immunohistochemistry. These changes may be partially related to impaired insulin and insulin-like growth factor 1 (IGF-1) signaling. Weak gene expression of insulin receptor (IR), IGF-1, insulin-like growth factor 1 receptor (IGF-1R), and insulin receptor substrate-1 (IRS-1) was observed in the diabetic BMSCs compared with normal BMSCs, together with decreased protein level of IGF-1, IGF-1R, IRS-1 and phosphorylated extracellular signal-regulated kinase. Therefore, impaired proliferation and osteogenic potential of BMSCs may be responsible for bone complications related to T1DM, mediated partially by impaired insulin and IGF-1 signaling. These findings may provide a new target with which to devise strategies for therapy.

[Cloning and expression of human interleukin-32 and studies on its bioactivity].

AIM: To clone human interleukin-32(hIL-32) gene and express it in E.coli efficiently. METHODS: The gene of hIL-32 was amplified by RT-PCR from human peripheral blood mononuclear cells (PBMC) which stimulated with Con A for 60 h. The PCR product was inserted into the pMD18-T vector. The hIL-32 cDNA confirmed by sequencing was inserted into expression vector pET-30a(+) and expressed in E.coli BL21(DE3) strain. The hIL-32 protein expression was induced by IPTG and assayed by SDS-PAGE and coomassie blue stain. The recombination protein was identified by Western blot and its biological activity was analyzed. RESULTS: DNA sequencing confirmed that the cloned cDNA was identical to the published sequence of hIL-32 that the nucleotide sequence of this gene was 567 bp. The recombinant plasmid pET30a-hIL32 was transformed into E.coli BL21(DE3) strain for expression. An expected 28 kDa protein of hIL-32 was found mainly in the induced host strains by SDS-PAGE and coomassie blue stain. The 28 kDa protein was recognized by anti-IL-32 antibody in western blot. The purified recombination protein could induce the producing of IL-6 in the human PBMC. CONCLUSION: We have successfully cloned the gene and expressed the protein of hIL-32 and the expressed protein has specific bioactivity.

[Effect of HPV 16 E5 on E6 and E7 gene induced human immortalized oral epithelial cell].

PURPOSE: To investigate the influence of HPV16 E5 on E6 and E7 gene in human oral immortalized epithelial cells (HIOEC). METHODS: The pLEGFP-E5 was transferred into HIOEC cell; Deletion mutation expression vectors were constructed and transferred into HIOEC; RT-PCR was used to detect the gene expression in HIOEC; The expression of E6 and E7 mRNA was detected by real time-PCR;Cell proliferation of HIOEC was evaluated by MTT. All statistical analysis was performed by using SPSS 13.0 software package. RESULTS: The deletion mutation expression vector of E5 was successfully constructed and applied for further experiments; E5 and the deletion mutation genes were transferred into HIOEC successfully; HPV16 E5 promoted cell proliferation of HIOEC and made the increased expression raised of E6 and E7 genes; However, the deletion mutation had no significant effect on HIOEC. CONCLUSIONS: HPV16 E5 is transferred into HIOEC successfully. E5 may promote cell proliferation and upregulate the mRNA expression of E6 and E7 genes.

[Construction and detection of gene expression vector of the Von Hippel-Lindau in bone marrow stromal cells].

PURPOSE: To construct siRNA-VHL expression vector and detect the effect of VHL gene interference on BMSCs. METHODS: According to the dog's VHL gene sequences, four pairs of siRNA oligo were designed and synthesized. Using vector cloning kit reorganization, four pairs of double-stranded siRNA were inserted into the expression vector (pcDNA™ 6.2-GW/EmGFPmiR) and 4 siRNA expression plasmids (SR144-1,SR144-2,SR144-3,SR144-4) were constructed. With the vector universal primers, colony PCR was screened. The positive clones were sequenced to verify whether the sequence of insert fragments in recombinant clones was consistent with oligo sequences designed or not. Interference vector transiently transfected the BMSCs. qPCR and Western blot were used to detect the gene silencing effect. In order to improve transfection efficiency of siRNA-VHL as well as the effect of the VHL gene silencing, pLenti-mi-VHL was constructed. RESULTS: Through sequencing the plasmids cloned, the fragment sequences inserted in recombinant clones were consistent with the designed oligo sequences. After 24 h and 48 h transfection of BMSCs cells by plasmids, SR144-4 showed the best effect of interference by qPCR and Western blot. Through comparing the sequencing results, the inserted fragment sequences were completely correct and the pLenti-mi-VHL was successfully constructed. CONCLUSION: The siRNA-VHL expression vector for BMSCs is successfully constructed and applicable for further experiments. Supported by Research Fund of Science and Technology Commission of Shanghai Municipality (Grant No.9411954800) and Foundation for Open Project from Shanghai Key Laboratory of Stomatology (Grant No.S30206).

[The experimental study on porous calcium phosphate cement with bone marrow stromal cells for bone tissue engineering].

OBJECTIVE: To observe the biocompatibility of new biomaterials porous calcium phosphate (CPC) and ectopic bone formation of CPC with bone marrow stromal cells (BMSCs). METHODS: The BMSCs were cultured from Beagle dog and combined with the porous CPC with the best concentration after transfect green fluorescent protein (GFP). The adhesion and growth of BMSCs on CPC were observed under inversion, fluorescence and scanning electron microscopy. The ectopic bone formation were observed at the 8th week after CPC and BMSCs were implanted subcutaneously into nude mice. RESULTS: When BMSCs with CPC were cultured at the 1st day, cells were climbing out from CPC with normal morphology. At the 7th day cells can be seen protruding pseudopods, secretion of matrix. Bone formation could be seen histomorphologically at the 8th week. CONCLUSION: Porous CPC has good biocompatibility and is an ideal scaffold material for bone tissue engineering.

Evidence for calcifying nanoparticles in gingival crevicular fluid and dental calculus in periodontitis.

BACKGROUND: Calcifying nanoparticles (CNPs), also known as nanobacteria, can produce carbonate apatite on their cell walls and initiate pathologic calcification. The objective of this study was to determine whether CNPs are present in the gingival crevicular fluid (GCF) from subjects with periodontal disease and whether they can induce the pathologic calcification of primary cultured human gingival epithelial cells. METHODS: GCF and dental calculus samples were collected from 10 subjects with gingivitis and 10 subjects with chronic periodontitis. CNPs in GCF and calculus filtrates were detected with nanocapture enzyme-linked immunosorbent assay kits. The CNPs in cultures of dental calculus filtrates were also identified using immunofluorescence staining, transmission electron microscopy (TEM), and chemical analysis. Pathologic changes in the CNP-treated gingival epithelial cells were observed with TEM, alizarin red staining, and disk-scanning confocal microscopy. RESULTS: CNPs were found in GCF samples from two subjects with chronic periodontitis. Based on chemical analysis, the surface-associated material from CNPs isolated and cultured from calculus has a composition similar to dental calculus. The pathologic calcification of CNP-treated gingival epithelial cells was also observed. CONCLUSIONS: Self-replicating calcifying nanoparticles can be cultured and identified from dental calculus. This raises the issue of whether CNPs contribute to the pathogenesis of periodontitis.

Sonic hedgehog alleviates the inhibitory effects of high glucose on the osteoblastic differentiation of bone marrow stromal cells.

To assess the influence of high extracellular glucose levels on the osteogenic differentiation of bone marrow stromal cells (BMSCs) and to determine if Sonic hedgehog (Shh) protein can alleviate those effects. BMSCs were incubated with NG (normal glucose), NG+Shh (200 ng/ml Shh in normal glucose), NG+Shh+Gan (200 ng/ml Shh and 5 micromol/L GANT61 in normal glucose), HG (high glucose), HG+Shh (200 ng/ml Shh in high glucose), and HG+Shh+Gan (200 ng/ml Shh and 5 micromol/L GANT61 in high glucose). The expression levels of Shh signaling pathway genes Patched 1 (PTCH1) and osteogenesis-related genes were tested, which included bone morphogenetic protein 4 (BMP4), runt-related transcription factor 2 (Runx2), and osteopontin (OPN). Alkaline phosphatase (ALPase) activity and mineralized matrix formation were also investigated. Immunofluorescent staining of Gli1 was tested for Shh signaling activation. We found that recombinant Shh in normal-glucose medium could promote osteogenic differentiation of BMSCs, while inhibiting Shh signaling by GANT61 could antagonize this differentiation. Besides that high glucose impaired the Shh signaling as well as osteogenic differentiation of BMSCs, reactivation of Shh signal pathway by addition of Shh protein could mitigate the inhibition while further deactivation by Shh inhibitor GANT61 could retain their osteogenic inhibitions. The above data suggest that Shh pathway activity is involved in the HG condition mediated osteoblastic differentiation deficiency for BMSCs and that recombinant Shh could alleviate this inhibitory effect.

An ectopic study of tissue-engineered bone with Nell-1 gene modified rat bone marrow stromal cells in nude mice.

BACKGROUND: Tissue engineering techniques combined with gene therapy have been recently used to improve osteogenesis. NEL-like molecule-1 (Nell-1), a novel growth factor, has been reported to have specificity for osteochondral lineage. The study assessed the osteogenic differentiation of rat bone marrow stromal cells (bMSCs) after Nell-1 gene modification and examined its ectopic bone formation ability in a nude mice model with tissue engineering technique. METHODS: bMSCs obtained from Fischer 344 rats were transduced with either AdNell-1 (Nell-1 group) or Ad-beta-galactosidase (AdLacZ, LacZ group) or left untransduced (untransduced group). The expression of Nell-1 protein was determined by Western blotting and transfer efficiency was assessed. mRNA expressions of osteopontin (OP), bone sialoprotein (BSP) and osteocalcin (OC) were assessed by real-time PCR 0, 3, 7, 14, and 21 days after gene transfer. Alkaline phosphatase (ALP) activity was measured and von Kossa test was also conducted. Finally, with a tissue engineering technique, gene transduced bMSCs, combining with beta-tricalcium phosphate (beta-TCP) at a concentration of 2 x 10(7) cells/ml, were implanted at subcutaneous sites on the back of nude mice. Four weeks after surgery, the implants were evaluated with histological staining and computerized analysis of new bone formation. RESULTS: Under current transduction conditions, gene transfer efficiency reached (57.9 +/- 6.8)%. Nell-1 protein was detected in Nell-1 group but not in untransduced group and LacZ group. Induced by Nell-1, BSP and OP expression were increased at intermediate stage and OC expression was increased at later stage. ALP activity and the number of calcium nodules were highest in Nell-1 group. Four weeks after implanted into nude mice subcutaneously, the percentage of new bone area in Nell-1 group was (18.1 +/- 5.0)%, significantly higher than those of untransduced group (11.3 +/- 3.2)% and LacZ group (12.3 +/- 3.1)% (P < 0.05). CONCLUSIONS: This study has demonstrated the ability of Nell-1 to induce osteogenic differentiation of rat bMSCs in vitro and to enhance bone formation with a tissue engineering technique. The results suggest that Nell-1 may be a potential osteogenic gene to be used in bone tissue engineering.