Research on Runx2 gene induced differentiation of human amniotic mesenchymal stem cells into ligament fibroblasts in vitro and promotion of tendon-bone healing in rabbits / 中国修复重建外科杂志

OBJECTIVE@#To investigate whether the Runx2 gene can induce the differentiation of human amniotic mesenchymal stem cells (hAMSCs) to ligament fibroblasts in vitro and promote the tendon-bone healing in rabbits.@*METHODS@#hAMSCs were isolated from the placentas voluntarily donated from healthy parturients and passaged, and then identified by flow cytometric identification. Adenoviral vectors carrying Runx2 gene (Ad-Runx2) and empty vector adenovirus (Ad-NC) were constructed and viral titer assay; then, the 3rd generation hAMSCs were transfected with Ad-Runx2 (Ad-Runx2 group) or Ad-NC (Ad-NC group). The real-time fluorescence quantitative PCR and Western blot were used to detect Runx2 gene and protein expression to verify the effectiveness of Ad-Runx2 transfection of hAMSCs; and at 3 and 7 days after transfection, real-time fluorescence quantitative PCR was further used to detect the expressions of ligament fibroblast-related genes [vascular endothelial growth factor (VEGF), collagen type Ⅰ, Fibronectin, and Tenascin-C]. The hAMSCs were used as a blank control group. The hAMSCs, hAMSCs transfected with Ad-NC, and hAMSCs were mixed with Matrigel according to the ratio of 1 : 1 and 1 : 2 to construct the cell-scaffold compound. Cell proliferation was detected by cell counting kit 8 (CCK-8) assay, and the corresponding cell-scaffold compound with better proliferation were taken for subsequent animal experiments. Twelve New Zealand white rabbits were randomly divided into 4 groups of sham operation group (Sham group), anterior cruciate ligament reconstruction group (ACLR group), anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-NC-scaffold compound group (Ad-NC group), and anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-Runx2-scaffold compound group (Ad-Runx2 group), with 3 rabbits in each group. After preparing the ACL reconstruction model, the Ad-NC group and the Ad-Runx2 group injected the optimal hAMSCs-Matrigel compunds into the bone channel correspondingly. The samples were taken for gross, histological (HE staining and sirius red staining), and immunofluorescence staining observation at 1 month after operation to evaluate the inflammatory cell infiltration as well as collagen and Tenascin-C content in the ligament tissues.@*RESULTS@#Flow cytometric identification of the isolated cells conformed to the phenotypic characteristics of MSCs. The Runx2 gene was successfully transfected into hAMSCs. Compared with the Ad-NC group, the relative expressions of VEGF and collagen type Ⅰ genes in the Ad-Runx2 group significantly increased at 3 and 7 days after transfection ( P<0.05), Fibronectin significantly increased at 3 days ( P<0.05), and Tenascin-C significantly increased at 3 days and decreased at 7 days ( P<0.05). CCK-8 detection showed that there was no significant difference ( P>0.05) in the cell proliferation between groups and between different time points after mixed culture of two ratios. So the cell-scaffold compound constructed in the ratio of 1∶1 was selected for subsequent experiments. Animal experiments showed that at 1 month after operation, the continuity of the grafted tendon was complete in all groups; HE staining showed that the tissue repair in the Ad-Runx2 group was better and there were fewer inflammatory cells when compared with the ACLR group and the Ad-NC group; sirius red staining and immunofluorescence staining showed that the Ad-Runx2 group had more collagen typeⅠ and Ⅲ fibers, tending to form a normal ACL structure. However, the fluorescence intensity of Tenascin-C protein was weakening when compared to the ACLR and Ad-NC groups.@*CONCLUSION@#Runx2 gene transfection of hAMSCs induces directed differentiation to ligament fibroblasts and promotes tendon-bone healing in reconstructed anterior cruciate ligament in rabbits.

Prenatal diagnosis for two families affected with cleidocranial dysplasia due to novel RUNX2 variants / 中华医学遗传学杂志

Objective@#To analyze variants of RUNX2 gene in two pedigrees affected with cleidocranial dysplasia and provide prenatal diagnosis for them.@*Methods@#For the two probands, the coding sequences of the RUNX2 gene were analyzed with PCR and bidirectional Sanger sequencing. To verify the results, peripheral blood samples were collected from their parents and 100 healthy controls. For family 1, umbilical cord blood was also collected for prenatal genetic diagnosis.@*Results@#In family 1, the proband and the fetus both carried a heterozygous c. 578G>C (p.Arg193Pro) mutation. For family 2, the proband was found to carry a heterozygous c. 909C>A (p.Tyr303X) mutation. The same mutations were not found among their parents and 100 healthy controls. Neither mutation was reported previously.@*Conclusion@#Variants of the RUNX2 gene probably underlie the cleidocranial dysplasia in both pedigrees. The results enabled prenatal diagnosis for the affected family.

The influence of a pulsed magnetic field on the expression of runt-related transcription factor 2 in rats after ovary resection / 中华物理医学与康复杂志

Objective To observe the influence of a pulsating electromagnetic field (PEMF) on the expression of runt-related transcription factor 2 (Runx2) in ovariectomized rats so as to explore the possibility of using PEMFs to treat osteoporosis.Methods Sixty female Sprague-Dawley rats were randomly divided into four groups of 15-a control group (Sham group),an ovary resection group (OVX group),a resection group treated with alendronate sodium (OVX+ALN group) and a resection group exposed to a PEMF (PEMFs group).Both ovaries were resected to induce osteoporosis,except in the sham group where the adipose tissues around the ovaries were cut bilaterally without resection.After the operation,the ALN group were given 2 mg/kg of alendronate by gavage daily for 30 days,while the PEMFs group was exposed to a 3.8 mT electromagnetic field pulsing at 8 Hz for 40 min twice daily for 30 days.There was no intervention in the other two groups.The animals were sacrificed using intra-peritoneal injection of sodium pentobarbital and the density of their femurs was measured.The expression of Runx2 protein and Runx2 mRNA were detected using western blotting and quantitative PCR.Results The average bone density,Runx2 protein level and Runx2 mRNA level of the PEMFs group were all significantly higher than those of the OVX group,but there was no significant difference between the PEMFs group and ALN group averages.Conclusion PEMF exposure can upregulate the expression of Runx2 protein and Runx2 mRNA in female rats after ovary resection.This may be one of the mechanisms by which PEMFs treat osteoporosis.

The influence of a pulsed magnetic field on the expression of runt-related transcription factor 2 in rats after ovary resection / 中华物理医学与康复杂志

Objective To observe the influence of a pulsating electromagnetic field (PEMF) on the expression of runt-related transcription factor 2 (Runx2) in ovariectomized rats so as to explore the possibility of using PEMFs to treat osteoporosis.Methods Sixty female Sprague-Dawley rats were randomly divided into four groups of 15-a control group (Sham group),an ovary resection group (OVX group),a resection group treated with alendronate sodium (OVX+ALN group) and a resection group exposed to a PEMF (PEMFs group).Both ovaries were resected to induce osteoporosis,except in the sham group where the adipose tissues around the ovaries were cut bilaterally without resection.After the operation,the ALN group were given 2 mg/kg of alendronate by gavage daily for 30 days,while the PEMFs group was exposed to a 3.8 mT electromagnetic field pulsing at 8 Hz for 40 min twice daily for 30 days.There was no intervention in the other two groups.The animals were sacrificed using intra-peritoneal injection of sodium pentobarbital and the density of their femurs was measured.The expression of Runx2 protein and Runx2 mRNA were detected using western blotting and quantitative PCR.Results The average bone density,Runx2 protein level and Runx2 mRNA level of the PEMFs group were all significantly higher than those of the OVX group,but there was no significant difference between the PEMFs group and ALN group averages.Conclusion PEMF exposure can upregulate the expression of Runx2 protein and Runx2 mRNA in female rats after ovary resection.This may be one of the mechanisms by which PEMFs treat osteoporosis.

Manifestation and treatment in a cleidocranial dysplasia patient with a RUNX2 (T420I) mutation / 대한악안면성형재건외과학회지

Cleidocranial dysplasia is an autosomal dominant heritable skeletal disorder. The characteristic features of cleidocranial dysplasia (CCD) may include hypoplasia of the clavicle, delayed closure of frontanelles, late tooth eruption, and other skeletal disorders. This case report describes clinical and radiographic manifestations at the age of 11 and 29 of a CCD patient, investigates the mutation of core-binding factor A1 (CBFA1) based on gene analysis, and illustrates successful oral reconstruction with fixed prosthesis and dental implant after the extraction of multiple teeth.