Objective To investigate the effect of stretch on long non-coding RNA taurine upregulated gene 1 (TUG1)-mediated miR-545-3p/cannbinoida receptor 2 (CNR2) pathway regulating bone regeneration in the distraction area of rats during distraction osteogenesis. MethodsThirty-six 10-week-old male Sprague Dawley rats were randomly divided into 3 groups (n=12 in each group): group A (femoral fracture+injection of interfering RNA), group B (distraction osteogenesis+injection of interfering RNA), and group C (distraction osteogenesis+injection of TUG1). Groups A and B were injected with 60 μg of interfering RNA at the beginning of incubation period (immediate after operation), the beginning of distraction phase (7 days after operation), and the end of distraction phase (21 days after operation), and group C was injected with 60 μg of synthetic TUG1 in vivo interfering sequence at the same time. The general situation of rats in each group was observed during the experiment. The mineralization of fracture space or distraction area was observed by X-ray films at 21, 35, and 49 days after operation. At 49 days after operation, the samples of the distraction area were taken for HE staining to observe the mineralization, and real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expressions of osteoblast-related genes such as TUG1, miR-545-3p, CNR2, alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). Blood samples were collected from the abdominal aorta of the rats, and the expressions of ALP and C terminal telopeptide of type Ⅰ (CTX-Ⅰ) protein were detected by ELISA assay.Results The results of X-ray film and HE staining observations showed that osteogenesis in group C was superior to groups A and B at the same time point. The results of qRT-PCR showed that the relative mRNA expressions of TUG1, CNR2, ALP, OCN, and OPN in group C were significantly higher than those in group A and group B, and the relative mRNA expression of miR-545-3p in group C was significantly lower than that in group A and group B (P<0.05). The relative mRNA expressions of TUG1 and ALP in group B were significantly higher than those in group A, and the relative mRNA expression of miR-545-3p in group B was significantly lower than that in group A (P<0.05). There was no significant difference in the relative mRNA expressions of CNR2, OCN, and OPN between group A and group B (P>0.05). The results of ELISA showed that the expressions of ALP and CTX-Ⅰ protein were significantly higher in group C than in group A and group B, and in group B than in group A (P<0.05). ConclusionUnder the action of stretch, the expression of TUG1 in the femoral distraction area of rats increases, which promotes the expression of CNR2 by inhibiting the expression of miR-545-3P, which is helpful to the mineralization of the extension area and osteogenesis.
OBJECTIVE: To establish the animal models of mandibular distraction osteogenesis in rabbits and study its osteogenetic mechanism. METHODS: The right mandibles just anterior to the first molars of 12 rabbits were performed osteotomies, and the mandibles were positioned with distractors. The left mandibles were control group without operation. After 1 week, the distractors were stretched 0.9 mm every day for 10 days progressively. One day, 2, 4, 8 weeks after distraction, the mandibles were studied with gross measurement, X-ray, and histological examination. RESULTS: The right mandible were lengthened 8.3 mm on average without bone nonunion and deformity healing. It was observed that the gaps between the distracted bone edges were first occupied by fibrous tissue. Two weeks after distraction, it was found that the gaps were bridged by callus in X-ray, the new bone and the normal bone could not be differentiated clearly after 8 weeks. In histological sections, there were collagen bundles in early distraction, then those collagen bundles were calcificated and become trabeculaes. No Cartilage was found during distraction. CONCLUSION: It suggests that the rabbit mandible can be lengthened by distraction osteogenesis, and the new bone is formed by intramembranous ossification.
Objective To investigate the mode and influential factor of newbone formation following distraction osteogenesis in mandibular lengthening. Methods Corticotomy was performed on bilateral mandibles in twelve adult male goats. A custommade distractor was used to lengthen the mandible at a rate of 1mm/day for 10 days (total 10 mm elongation). Four goats were sampled respectivelyat 2, 4 and 8 weeks after completion of distraction. The lengthening mandibles were examined by roentgenography and histology. Results Newly formed callus was observed in the distraction gap after mandibular lengthening. The new bone exhibited intramembranous ossification generally, but cartilage islands could be found in the specimen that diastractor loosed. Conclusion The above findings indicate that the mode of new bone formation in mandibular lengthening following distraction osteogenesis appears to be intramembranous ossification and that endochondral ossification takes place in case distractor has loosened.
ObjectiveTo investigate the effects of silencing P75 neurotrophin receptor (P75NTR) and nerve growth factor (NGF) overexpression on the proliferative activity and ectopic osteogenesis ability of bone marrow mesenchymal stem cells (BMSCs) combined with demineralized bone matrix for heterotopic osteogenesis.MethodsBMSCs of Sprague Dawley (SD) rats were cultured and passaged by adherent isolation method. The third generation BMSCs were transfected with lentivirus mediated P75NTR gene silencing (group B), NGF overexpression gene (group C), P75NTR silencing and NGF overexpression double genes (group D), respectively, and untransfected cells as control (group A). After 7 days of transfection, the expression of fluorescent protein of the target gene was observed by fluorescence microscope; cell counting kit 8 method was used to detect the cells activity for 8 days after transfection; the expressions of P75NTR and NGF proteins in each group were detected by Western blot. The adhesion of BMSCs to demineralized bone matrix (DBM) was observed by inverted phase contrast microscope and scanning electron microscope after transfection of p75NTR silencing and NGF overexpression double genes. After transfection, BMSCs and DBM were co-cultured to prepare 4 groups of tissue engineered bone, which were respectively placed in the dorsal subcutaneous tissue of 8-week-old SD rats to construct subcutaneous ectopic osteogenesis model (n=6). HE staining was performed at 4 and 8 weeks after operation. ALP staining was used to observe the formation of calcium nodules at 8 weeks after operation. The expressions of Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and osteocalcin (OCN) were detected by real-time fluorescent quantitative PCR.ResultsAt 7 days after transfection, there was no fluorescence expression in group A, red fluorescence expression was seen in group B, green fluorescence expression in group C, and red-green compound fluorescence expression in group D. The fluorescence expression rate of target gene was about 70%. Western blot detection showed that the relative expression of P75NTR protein in groups A and C was significantly higher than that in groups B and D, and the relative expression of NGF protein in groups C and D was significantly higher than that in groups A and B (P<0.05). With the passage of time, the cell proliferation activity increased in all groups, especially in group D, which was significantly higher than that in group A at 3-8 days (P<0.05). The results of inverted phase contrast microscope and scanning electron microscope showed that BMSCs could adhere well to DBM. In the subcutaneous ectopic osteogenesis experiment, HE staining showed that at 4 and 8 weeks after operation, the more bone tissue was formed in group D than in the other 3 groups. ALP staining showed that group D had the highest ALP activity and better osteogenic expression. Compared with group A, the relative expressions of Runx2, ALP, and OCN mRNAs in group D were significantly higher than those in group A (P<0.05).ConclusionSilencing P75NTR and NGF overexpression double genes co-transfected BMSCs with DBM to construct tissue engineered bone has good ectopic osteogenic ability. By increasing NGF level and closing P75NTR apoptosis channel, it can not only improve cell activity, but also promote bone tissue regeneration.
ObjectiveTo investigate the heterotopic osteogenesis of tissue engineered bone using the co-culture system of vascular endothelial cells (VECs) and adipose-derived stem cells (ADSCs) as seed cells.MethodsThe partially deproteinized biological bone (PDPBB) was prepared by fibronectin combined with partially deproteinized bone (PDPB). The ADSCs of 18-week-old Sprague Dawley (SD) rats and VECs of cord blood of full-term pregnant SD rats were isolated and cultured. Three kinds of tissue engineered bone were constructed in vitro: PDPBB+VECs (group A), PDPBB+ADSCs (group B), PDPBB+co-cultured cells (VECs∶ADSCs was 1∶1, group C), and PDPBB was used as control group (group D). Scanning electron microscopy was performed at 10 days after cell transplantation to observe cell adhesion on scaffolds. Forty-eight 18-week-old SD rats were randomly divided into groups A, B, C, and D, with 12 rats in each group. Four kinds of scaffolds, A, B, C, and D, were implanted into the femoral muscle bags of rats in corresponding groups. The animals were killed at 2, 4, 8, and 12 weeks after operation for gross observation, HE staining and Masson staining histological observation, and the amount of bone collagen was measured quantitatively by Masson staining section.ResultsScanning electron microscopy showed that the pores were interconnected in PDPB materials, and a large number of lamellar protein crystals on the surface of PDPBB modified by fibronection were loosely attached to the surface of the scaffold. After 10 days of co-culture PDPBB and cells, a large number of cells attached to PDPBB and piled up with each other to form cell clusters in group C. Polygonal cells and spindle cells were mixed and distributed, and some cells grew along bone trabeculae to form cell layers. Gross observation showed that the granulation tissue began to grow into the material pore at 2 weeks after operation. In group C, a large number of white cartilage-like substances were gradually produced on the surface of the material after 4 weeks, and the surface of the material was uneven. At 12 weeks, the amount of blood vessels on the surface of group A increased, and the material showed consolidation; there was a little white cartilage-like material on the surface of group B, but the pore size of the material did not decrease significantly; in group D, the pore size of the material did not decrease significantly. Histological observation showed that there was no significant difference in the amount of bone collagen between groups at 2 weeks after operation (F=2.551, P=0.088); at 4, 8, and 12 weeks after operation, the amount of bone collagen in group C was significantly higher than that in other 3 groups, and that in group B was higher than that in group D (P<0.05); there was no significant difference between group A and groups B, D (P>0.05).ConclusionThe ability of heterotopic osteogenesis of tissue engineered bone constructed by co-culture VECs and ADSCs was the strongest.
Implant surface modified coating can improve its osteoinductivity, about which simple calcium phosphate coating has been extensively studied. But it has slow osteointegration speed and poor antibacterial property, while other metal ions added, such as nano zinc ion, can compensate for these deficiencies. This paper describes the incorporation form, the effect on physical and chemical properties of the material and the antibacterial property of nano zinc, and summarizes the material's biological property given by calcium ion, zinc ion and inorganic phosphate (Pi), mainly focusing on the influence of these three inorganic ions on osteoblast proliferation, differentiation, protein synthesis and matrix mineralization in order to present the positive function of zinc doped calcium phosphate in the field of bone formation.
The authors made a profound review on the development and the recent status of craniomaxillofacial surgery in China during past three decades. The emphases were placed on the following aspects: the modifications of the reconstructive procedure and minimal invasive mode, the researches on molecular genetic characteristics of the congenital craniofacial malformations, the clinical applications of three-dimensional digital computer-aided techniques (including three-dimensional printing and prefabricated template for precious osteotomies), the craniomaxillofacial defects reconstructing by using the distraction osteogenesis and osseous integrated titanium implant and prothesis, etc. Finally, the authors outlooked prospectively the future trends of the craniomaxillofacial surgery.
By using Urist s method four types of BMG from the long bones of the rabbit、 pig、sheep、 and human being were prepared. Each of them was implanted into the pectoralis and thigh muscles in 25 adult rats, respectiely. Two-eight weeks after implantation, the unoreaction and inductive osteogensis potential in the tissues were observed under mieroscope. The result showed that aBMG had inductive osteogenesis potential. However, rejection in varying digree existed around aBMG. It was important to further decrease the antingenicity digree exised around a BMG . and enhance its osteogennic potential before the possibility of its clinical application.
ObjectiveTo explore the effectiveness and method of Ilizarov technology for the treatment of infected forearm nonunion. MethodsBetween January 2004 and March 2014, 19 patients with infected forearm nonunion were treated, including 12 males and 7 females with a mean age of 37.4 years (range, 18-62 years). The injury causes included traffic accident in 11 patients, falling from height in 4 patients, and machine twist injury in 4 patients. The patients had received surgical treatment for 1-5 times (mean, 2.7 times). Bone defects located at the radius in 10 cases, at the ulna in 7 cases, and at the radius and ulna in 2 cases. The mean time of chronic infection was 8.3 months (range, 4-16 months). The mean length of the bone defects after debridement was 3.54 cm (range, 2.2-7.5 cm). Under the guidance of C-arm fluoroscope, the Orthofix unilateral external fixator was used to fix. Distraction was performed at 7-10 days after operation, and X-ray film was taken regularly to detect the osteogenesis. ResultsThe mean external fixation time was 6.5 months (range, 3-12 months), and the mean external fixation index was 1.72 months/cm (range, 1.14-2.15 months/cm). All patients were followed up for 35.4 months on average (range, 24-55 months). The bone union time was 3-11 months (mean, 6 months); and no recurrence of infection was observed. At last follow-up, the mean wrist range of motion (ROM) were 52.78° (range, 42-55°) in flexion and 46.53° (range, 40-60°) in extension; the mean elbow ROM were 139.23° (range, 130-150°) in flexion and 3.57° (range, 0-20°) in extension; and the mean forearm ROM were 76.68° (range, 68-90°) in pronation and 81.75° (range, 72-90°) in supination. ConclusionIlizarov technology for infected forearm nonunion can acquire satisfactory clinical results. Radical debridement is the key to control bone infection.
Objective To investigate the effectiveness of tunnel osteogenesis technique combined with locking plate in the treatment of aseptic non-hypertrophic nonunion of femoral shaft. MethodsThe clinical data of 23 cases of aseptic non-hypertrophic nonunion of femoral shaft treated with tunnel osteogenesis technique combined with locking plate between January 2017 and December 2020 were retrospectively analysed. There were 17 males and 6 females with an average age of 41.4 years (range, 22-72 years). There were 22 cases of closed fracture and 1 case of open fracture. The types of internal fixation at admission included intramedullary nail in 14 cases and steel plate in 9 cases. The number of nonunion operations received in the past was 0 to 1; the duration of nonunion was 6-60 months, with an average of 20.1 months. Among them, there were 17 cases of aseptic atrophic nonunion of the femoral shaft and 6 cases of dystrophic nonunion. Twenty-two cases were fixed with 90° double plates and 1 case with lateral single plate. The operation time, theoretical blood loss, hospitalization stay, nonunion healing, and postoperative complications were recorded. Harris hip function score, Lysholm knee function score, lower extremity function scale (LEFS), and short-form 36 health survey scale (SF-36) were used at last follow-up to evaluate hip and knee functions. Visual analogue scale (VAS) score was used to evaluate the relief of pain at 1 day after operation and at last follow-up. ResultsThe average operation time was 190.4 minutes, the average theoretical blood loss was 1 458.4 mL, and the average hospitalization stay was 8.2 days. All the 23 patients were followed up 9-26 months, with an average of 18.2 months. The healing time of nonunion in 22 patients was 3-12 months, with an average of 5.6 months. There were 8 cases of limb pain, 8 cases of claudication, 6 cases of limitation of knee joint movement, and 2 cases of limitation of hip joint movement. At last follow-up, the imaging of 1 patient showed that the nonunion did not heal, accompanied by pain of the affected limb, lameness, and limitation of knee joint movement. At 1 day after operation, the VAS score of 23 patients was 6.5±1.8, the pain degree was good in 7 cases, moderate in 12 cases, and poor in 4 cases; at last follow-up, the VAS score was 0.9±1.3, the pain degree was excellent in 21 cases and good in 2 cases, which were significantly better than those at 1 day after operation (t=12.234, P<0.001; Z=–5.802, P<0.001). At last follow-up, the average Harris hip function score of 22 patients with nonunion healing was 94.8, and the good rate was 100%; Lysholm knee function score averaged 94.0, and the excellent and good rate was 90.9%; LEFS score averaged 74.6; SF-36 physical functioning score averaged 85.0 and the mental health score averaged 83.6. ConclusionTunnel osteogenesis technique combined with locking plate in the treatment of aseptic non-hypertrophic nonunion of femoral shaft has a high healing rate and fewer complications, which can effectively relieve pain and improve lower limb function and quality of patients’ life.