Objective To investigate the role and regulatory mechanism of ring finger protein 11 (RNF11) on Akt signaling pathway in the process of osteogenesis of bone marrow mesenchymal stem cells (BMSCs) to provide ideas for further clarifying its osteogenesis mechanism and its use in clinical treatment in the future. Methods BMSCs were isolated and cultured from fresh bone marrow of healthy donors and subcultured. The 4th generation cells were used in experiments after identification by flow cytometry, and osteogenic, chondrogenic, and adipogenic induction. BMSCs were cultured in osteogenic differentiation medium for 0-14 days. The degree of osteogenic differentiation was detected by Alizarin red staining and alkaline phosphatase (ALP) staining, and the protein expression of RNF11 was detected by Western blot. The 4th generation BMSCs were divided into blank control group (group A), empty lentivirus (Lv-NC) group (group B), and knockdown RNF11 (Lv-ShRNF11) group (group C). Osteogenesis was induced and cultured for 0-14 days. The expression of RNF11 protein was detected by Western blot, the degree of osteogenic differentiation was detected by Alizarin red staining and ALP staining, and the relative mRNA expressions of Runx2, osteocalcin (OCN), and osteopontin (OPN) were detected by real-time fluorescence quantitative PCR (qRT-PCR). The protein relative expressions of Akt, Smad1/5/8, and β-catenin signaling pathway were detected by Western blot, expressed as the ratio before and after phosphorylation. In order to study the effect mechanism of RNF11 on Akt signaling pathway, the 4th generation BMSCs were divided into Lv-NC transfection group (group A1), Lv-ShRNF11 transfection group (group B1), and Lv-ShRNF11 transfection supplemented with Akt signaling pathway activator SC79 group (group C1). The protein relative expressions of RNF11 and Akt signaling pathway were detected by Western blot, the related osteogenesis indexes were detected by Alizarin red staining, ALP staining, and qRT-PCR. ResultsThe flow cytometry, and osteogenic, chondrogenic, adipogenic induction culture identification showed that the isolated and cultured cells were BMSCs. The protein relative expression of RNF11 increased gradually with the extension of osteogenic differentiation time (P<0.05); after knockdown RNF11, Alizarin red and ALP stainings showed that the degree of osteogenic differentiation of BMSCs in group C were significantly lower than those in groups A and B, and qRT-PCR detection showed that the relative expression of Runx2, OCN, and OPN mRNA significantly decreased (P<0.05). The protein relative expressions of RNF11 and Akt signaling pathway significantly increased with the extensions of osteogenic differentiation time (P<0.05). After knockdown RNF11, the protein relative expression of Akt signaling pathway in group C was significantly lower than that in groups A and B (P<0.05), while Smad1/5/8 and β-catenin signaling pathway had no significant effect (P>0.05). Compared with group A1, the protein relative expression of RNF11 in groups B1 and C1 significantly decreased (P<0.05). Compared with groups A1 and C1, the protein relative expression of Akt signaling pathway in group B1 was significantly lower (P<0.05); Alizarin red and ALP stainings showed that the degree of osteogenic differentiation of BMSCs in group C1 were slightly lower than that of group A1 (P>0.05), but significantly higher than that of group B1 (P<0.05); qRT-PCR detection showed that the relative expressions of Runx2, OCN, and OPN mRNA in group C1 were slightly lower than those of group A1 (P>0.05), but were significantly higher than those of group B1 (P<0.05). ConclusionRNF11 promotes the differentiation of BMSCs into osteoblasts by positively regulating the activation level of Akt signaling pathway. RNF11 can be used as a potential target to improve the bone repair efficacy of BMSCs and treat bone metabolic diseases.
Objective To investigate the effect of simvastatin on inducing endothel ial progenitor cells (EPCs) homing and promoting bone defect repair, and to explore the mechanism of local implanting simvastatin in promoting bone formation. Methods Simvastatin (50 mg) compounded with polylactic acid (PLA, 200 mg) or only PLA (200 mg) was dissolved in acetone (1 mL) to prepare implanted materials (Simvastatin-PLA material, PLA material). EPCs were harvested from bone marrow of 2 male rabbits and cultured with M199; after identified by immunohistochemistry, the cell suspension of EPCs at the 3rd generation (2 × 106 cells/mL) was prepared and transplanted into 12 female rabbits through auricular veins(2 mL). After 3 days, the models of cranial defect with 15 cm diameter were made in the 12 female rabbits. And the defects were repaired with Simvastatin-PLA materials (experimental group, n=6) and PLA materials (control group, n=6), respectively. The bone repair was observed after 8 weeks of operation by gross appearance, X-ray film, and histology; gelatin-ink perfusion and HE staining were used to show the new vessels formation in the defect. Fluorescence in situ hybridization (FISH) was performed to show the EPCs homing at the defect site. Results All experimental animals of 2 groups survived to the end of the experiment. After 8 weeks in experimental group, new bone formation was observed in the bone defect by gross and histology, and an irregular, hyperdense shadow by X-ray film; no similar changes were observed in control group. FISH showed that the male EPC containing Y chromosome was found in the wall of new vessels in the defect of experimental group, while no male EPC containing Y chromosome was found in control group. The percentage of new bone formation in defect area was 91.63% ± 4.07% in experimental group and 59.45% ± 5.43% in control group, showing significant difference (P lt; 0.05). Conclusion Simvastatin can promote bone defect repair, and its mechanism is probably associated with inducing EPCs homing and enhancing vasculogenesis.
ObjectiveTo objectively evaluate the effectiveness of the ventricular fold pull-down combined with strip myofascial flap to repair laryngeal defect after early glottic carcinoma operation with glottic morphological parameters and voice parameters. MethodsBetween January 2008 and December 2012, 47 patients with early glottic carcinoma and anterior commissure involvement underwent partial laryngectomy. All patients were male, aged from 60 to 75 years (mean, 68.5 years). The disease duration was 4-11 months (mean, 7.2 months). According to American Joint Committee on Cancer (AJCC) TNM criteria, 28 cases were classified as T1aN0M0, 14 cases as T1bN0M0, and 5 cases as T2N0M0. Laryngeal defect after resection of tumor was repaired by ventricular fold pull-down combined with strip myofascial flap. At 1 day before operation and at 1 year after operation, multilayer spiral CT was used to scan larynx, to measure and compare the anteroposterior diameter of vocal area, the distance between both sides of the vocal process, and the thickness of soft tissue of vocal area, and the effect of combined soft tissue flap was objectively assessed in laryngeal morphology reconstruction. The actual voice parameters[including F0, Jitter, Shimmer, normalized noise energy (NNE), and maximum phonatory time (MPT)] were tested and compared, and the effect of the combined soft tissue flap on postoperative laryngeal pronunciation was evaluated. ResultsPostoperative pathological examination revealed well-differentiated squamous cell carcinoma in 38 cases, and moderately-differentiated squamous cell carcinoma in 9 cases; no tumor was found in the resection margin. Healing of neck incision was obtained in all patients at 7-9 days after operation. Forty-four cases were decannulated at 9-11 days after operation and the remaining 3 cases were decannulated at 3 weeks after operation. Oral feeding usually started in all cases at 3-4 days after operation. All patients were followed up 1 year. At 1 year after operation, the anteroposterior diameter of vocal area was significantly reduced when compared with preoperative one (t=15.161, P=0.000); the distance between both sides of the vocal process and the thickness of soft tissue of vocal area had no significant changes (P > 0.05). Compared with preoperative ones, there were significant differences in Shimmer, NNE, and MPT (P < 0.05), but no significant difference was found in F0 and Jitter (P > 0.05) at 1 year after operation. ConclusionVentricular fold pull-down combined with strip myofascial flap can repair laryngeal defect effectively after partial laryngectomy and maintain the effective airway after operation. It not only has no effect on postoperative laryngeal morphology, but also can be used as new laryngeal voice vibration body.
Objective To explore the application effect of 3D printed heart models in the training of young cardiac surgeons, and evaluate their application value in surgical simulation and skill improvement. MethodsEight young cardiac surgeons were selected form West China Hospital as the trainees. Before training, the Hands-On Surgical Training-Congenital Heart Surgery (HOST-CHS) operation scores of the 8 cardiac surgeons were obtained after operating on 2 pig heart models of ventricular septal defect (VSD). Subsequently, simulation training was conducted on a 3D printed peri-membrane VSD heart model for 6 weeks, once a week. After the training, all trainees completed 2 pig heart VSD repair surgeries. The improvement of doctors’ skills was evaluated through survey questionnaires, HOST-CHS scores, and operation time after training. ResultsBefore the training, the average HOST-CHS score of the 8 trainees was 52.2±6.3 points, and the average time for VSD repair was 54.7±7.1 min. During the 6-week simulation training using 3D printed models, the total score of HOST-CHS for the 8 trainees gradually increased (P<0.001), and the time required to complete VSD repair was shortened (P<0.001). The trainees had the most significant improvement in scores of surgical cognition and protective awareness. The survey results showed that trainees were generally very satisfied with the effectiveness of 3D model simulation training. Conclusion The 3D printed VSD model demonstrates significant application advantages in the training of young cardiac surgeons. By providing highly realistic anatomical structures, 3D models can effectively enhance surgeons’ surgical skills. It is suggested to further promote the application of 3D printing technology in medical education, providing strong support for cultivating high-quality cardiac surgeons.
Icariin（ICA） is one of the main active ingredients in the Berberidaceae family Epimedium. It makes a variety of biological activities, such as promoting bone formation, antibacterial and anti-inflammatory, and regulating immunity. Periodontitis is a chronic inflammatory disease that is present in the soft and hard tissues of the periodontium. The ultimate goals of its treatment are the reconstruction of periodontal tissues and bone defect repairing. At present, conventional treatment of periodontitis fails to achieve the ideal periodontal tissue regeneration. In recent years, the rapid development of tissue engineering technology has brought new ideas for the treatment of periodontal disease and bone defect repairing. Because of its anti-inflammatory and osteogenic effects, ICA has great potential for the treatments of periodontitis and bone defect repairing. This paper summarizes the effect and the molecular mechanism of ICA in the treatment of periodontitis and bone defect repairing, and discusses its application prospect as a drug for periodontal adjuvant therapy. This paper aims to provide a theoretical basis for the research and application of ICA in periodontitis treatment and bone defect repairing.
Objective To explore the reliability and effectiveness of prediction of the pedicle length of the proximally-based anterolateral thigh (pALT) flap which was used to repair the defects following the resection of various malignant tumors using computed tomographic angiography (CTA). Methods The clinical data of 12 patients who met the selection criteria by using pALT flap to repair wounds left after malignant tumor resection between June 2015 and December 2020 were retrospectively analyzed. There were 5 males and 7 females; the age ranged from 16 to 80 years, with an average age of 54.4 years. After tumor resection, the soft tissue defect ranged from 15 cm×5 cm to 30 cm×12 cm; defect sites included 4 cases of lower abdomen, 3 cases of groin, 2 cases of thigh, and 3 cases of buttocks. Preoperative CTA was used to obtain the location information of the descending branch of the lateral femoral circumflex artery and its perforators by maximum density projection, and the length of the pedicle of pALT flap was estimated. Fasciocutaneous flap (5 cases) or myocutaneous flap (7 cases) were cut during operation to repair the defect, and the size of flap ranged from 20 cm×7 cm to 30 cm×12 cm. The donor site of thigh was directly sutured (11 cases) or repaired with skin graft (1 case). Bland-Altman analysis was used to detect the consistency between the pALT flap vascular pedicle length estimated by CTA and the pALT flap vascular pedicle length actually obtained during operation. ResultsOne case had distal blood supply disturbance of the flap and was repaired with skin graft after debridement; the remaining 11 flaps survived. All donor and recipient incisions healed by first intention. All 12 cases were followed up 1-12 months, with an average of 4.3 months. One patient died of pelvic tumor recurrence at 6 months after operation, and no tumor recurrence was found in the other patients. Preoperative CTA estimated that the length of pALT flap vascular pedicle was 9.3-24.7 cm, with an average of 14.7 cm; the actual length of pALT flap vascular pedicle was 9.5-25.0 cm, with an average of 14.8 cm. Bland-Altman analysis showed that there was no significant difference between the pALT flap vascular pedicle length estimated by CTA before operation and the pALT flap vascular pedicle length actually obtained during operation, and the average difference was 0.1 (95% consistency limit: –0.89, 0.74), indicating that they had good consistency. ConclusionCTA can be accurately used to localize the perforator and predict the possible pedicle length of the pALT flap. When performing a pALT flap surgery, preoperative CTA is helpful for surgeons to make a preliminary assessment of the difficult of the operation. The time for exploration of perforators and dissection of the vascular pedicle, and complications can be reduced, and the safety of the operation can be improved.
Objective To compare the effect between vascularization osteogenesis and membrane guided osteogenesis in the bone repair by the tissue engineered bone with pedicled fascial flap packing autologous red bone marrow (ARBM), so as to provide a reference for the bone defect repair in cl inic. Methods The tissue engineered bone was constructed with ARBM and the osteoinductive absorbing recombinant human materials with recombinant human bone morphogenetic protein 2. Sixty New Zealand rabbits (aged 4-5 months, weighing 2.0-2.5 kg) were randomly divided into group A (n=16), group B (n=22), and group C (n=22). The complete periosteum defect model of 1.5 cm in length was prepared in right ulnar bone, then the tissue engineered bone was implanted in the bone defect area in group A, the tissue engineered bonewith free fascial flap in group B, and the tissue engineered bone with pedicled fascial flap in group C. At 4, 8, 12, and 16 weeks, the tissue of bone defect area was harvested from 4 rabbits of each group for the general, histological, and immunohistochemical staining observations; at 8, 12, and 16 weeks, 2 rabbits of groups B and C, respectively were selected to perform ink perfusion experiment by axillary artery. Results The general observation showed that the periosteum-l ike tissues formed in the fascial flap of groups B and C, chondroid tissues formed in group B, new bone formed in group C, and the fibrous and connective tissues in group A at 4 and 8 weeks; a few porosis was seen in group A, more new bone in group B, and bone stump formation in group C at 12 and 16 weeks. Histological observation showed that there were few new blood vessels and new bone trabeculae in groups A and B, while there were large amounts of new blood vessels and mature bone trabeculae in group C at 4 and 8 weeks. There were a few new blood vessels and new bone trabeculae in group A; more blood vessels, significantly increased mature trabeculae, and the medullary cavity formation in group B; and gradually decreased blood vessels, the mature bone structure formation, and the re-opened medullary cavity in group C at 12 and 16 weeks. The immunohistochemical staining observation showed that the levels of CD105, CD34, and factor VIII were higher in group C than in groups A and B at different time points.The bone morphometry analysis showed that the trabecular volume increased gradually with time in 3 groups after operation; the trabecular volume in group C was significantly more than those in groups A and B at different time points (P lt; 0.05); and there was significant difference between groups A and B (P lt; 0.05) except the volume at 4 weeks (P gt; 0.05). The vascular image analysis showed that the vascular regenerative area ratio in group C was significantly higher than those in groups A and B at different time points (P lt; 0.05). The ink perfusion experiment showed that the osteogenic zone had sparse ink area with no obvious change in group B, while the osteogenic zone had more intensive ink area and reached the peak at 8 weeks, then decreased in group C. Conclusion The tissue engineered bone with pedicled fascial flap packing ARBM has the vascularization osteogenesis effect at early stage, but the effect disappears at late stage gradually when the membrane guided osteogenesis is main.
ObjectiveTo investigate the effects of micro-fracture and insul in-l ike growth factor 1 (IGF-1) in treatment of articular cartilage defect in rabbits. MethodsTwenty-four New Zealand white rabbits (aged, 4-6 months; weighing, 2.5-3.5 kg) were randomly divided into 4 groups (n=6):micro-fractures and recombinant human IGF-1 (rhIGF-1) treatment group (group A), micro-fracture control group (group B), rhIGF-1 treatment control group (group C), and blank control group (group D). Full thickness articular cartilage defects of 8 mm×6 mm in size were created in the bilateral femoral condyles of all rabbits. The micro-fracture surgery was performed in groups A and B. The 0.1 mL rhIGF-1 (0.01 μg/μL) was injected into the knee cavity in groups A and C at 3 times a week for 4 weeks after operation, while 0.1 mL sal ine was injected in groups B and D at the same time points. At 4, 12, and 24 weeks, the gross, histological, and immunohistochemical observations were performed, and histological score also was processed according to Wakitani's score criteria. The collagen contents in the repair tissues and normal patellofemoral cartilage were detected by the improved hydroxyproline (HPR) method at 24 weeks. Electron microscope was used to observe repair tissues of groups A and B at 24 weeks. Results All animals were survival at the end of experiment. At 24 weeks after operation, defect was repaired with time, and the repair tissue was similar to normal cartilage in group A; the repair tissue was even without boundary with normal cartilage in group B; and the repair tissue was uneven with clear boundary with normal cartilage in groups C and D. Histological staining showed that the repair tissues had no difference with normal cartilage in group A; many oval chondrocytes-l ike cells and l ight-colored matrix were seen in the repair tissues of group B; only a few small spindle-shaped fibroblasts were seen in groups C and D. Moreover, histological scores of group A were significantly better than those of groups B, C, and D (P<0.05) at 4, 12, and 24 weeks. Electron microscope observation showed that a large number of lacuna were seen on the surface of repair tissue in group A, and chondrocytes contained glycogen granules were located in lacunae, and were surrounded with the collagen fibers, which was better than that in group B. Collagen content of the repair tissue in group A was significantly higher than that in groups B, C, and D (P<0.05), but it was significantly lower than that of normal cartilage (P<0.05). Conclusion Combination of micro-fracture and rhIGF-1 for the treatment of full thickness articular cartilage defects could promote the repair of defects by hyaline cartilage.
ObjectiveTo review the methods of improving the mechanical properties of hydrogels and the research progress in bone tissue engineering. MethodsThe recent domestic and foreign literature on hydrogels in bone tissue engineering was reviewed, and the methods of improving the mechanical properties of hydrogels and the effect of bone repair in vivo and in vitro were summarized. ResultsHydrogels are widely used in bone tissue engineering, but their mechanical properties are poor. Improving the mechanical properties of hydrogels can enhance bone repair. The methods of improving the mechanical properties of hydrogels include the construction of dual network structures, inorganic nanoparticle composites, introduction of conductive materials, and fiber network reinforcement. These methods can improve the mechanical properties of hydrogels to various degrees while also demonstrating a significant bone repair impact. ConclusionThe mechanical properties of hydrogels can be effectively improved by modifying the system, components, and fiber structure, and bone repair can be effectively promoted.
ObjectiveTo investigate the feasibility of tissue engineered periosteum (TEP) constructed by porcine small intestinal submucosa (SIS) and bone marrow mesenchymal stem cells (BMSCs) of rabbit to repair the large irregular bone defects in allogenic rabbits. MethodsThe BMSCs were cultivated from the bone marrow of New Zealand white rabbits (aged, 2 weeks-1 month). SIS was fabricated by porcine proximal jejunum. The TEP constructed by SIS scaffold and BMSCs was prepared in vitro. Eighteen 6-month-old New Zealand white rabbits whose scapula was incompletely resected to establish one side large irregular bone defects (3 cm×3 cm) model. The bone defects were repaired with TEP (experimental group,n=9) and SIS (control group,n=9), respectively. At 8 weeks after operation, the rabbits were sacrificed, and the implants were harvested. The general condition of the rabbits was observed; X-ray radiography and score according to Lane-Sandhu criteria, and histological examination (HE staining and Masson staining) were performed. ResultsAfter operation, all animals had normal behavior and diet; the incision healed normally. The X-ray results showed new bone formation with normal bone density in the defect area of experimental group; but no bone formation was observed in control group. The X-ray score was 6.67±0.32 in experimental group and was 0.32±0.04 in control group, showing significant difference (t=19.871,P=0.001). The general observation of the specimens showed bone healing at both ends of the defect, and the defect was filled by new bone in experimental group; no new bone formed in the control group. The histological staining showed new bone tissue where there were a lot of new vessels and medullary cavity, and no macrophages or lymphocytes infiltration was observed in the defect area of experimental group; only some connective tissue was found in the control group. ConclusionTEP constructed by porcine SIS and BMSCs of rabbit can form new bone in allogenic rabbit and has the feasibility to repair the large irregular bone defects.