Objective Tissue engineered bone (TEB) lacks of an effective and feasible method of storage and transportation. To evaluate the activity of osteogenesis and capabil ity of ectopic osteogenesis for TEB after freeze-dried treatment in vitro and in vivo and to explore a new method of preserving and transporting TEB. Methods Human bone marrow mesenchymal stem cells (hBMSCs) and decalcified bone matrix (DBM) were harvested from bone marrow and bone tissue of the healthy donators. TEB was fabricated with the 3rd passage hBMSCs and DBM, and they were frozen and dried at extremely low temperatures after 3, 5, 7, 9, 12, and 15 days of culture in vitro to obtain freeze-dried tissue engineered bone (FTEB). TEB and FTEB were observed by gross view and scanning electron microscope (SEM). Western blot was used to detect the changes of relative osteogenic cytokines, including bone morphogenetic protein 2 (BMP-2), transforming growth factor β1 (TGF-β1), and insul in-l ike growth factor 1 (IGF-1) between TEB and FTEB. The ectopic osteogenesis was evaluated by the methods of X-ray, CT score, and HE staining after TEB and FTEB were transplanted into hypodermatic space in athymic mouse. Results SEM showed that the cells had normal shape in TEB, and secretion of extracellular matrix increased with culture time; in FTEB, seeding cells were killed by the freeze-dried process, and considerable extracellular matrix were formed in the pore of DBM scaffold. The osteogenic cytokines (BMP-2, TGF-β1, and IGF-1) in TEB were not decreased after freeze-dried procedure, showing no significant difference between TEB and FTEB (P gt; 0.05) except TGF-β1 15 days after culture (P lt; 0.05). The ectopic osteogenesis was observed in TEB and FTEB groups 8 and 12 weeks after transplantation, there was no significant difference in the calcified level of grafts between TEB and FTEB groups by the analysis of X-ray and CT score. On the contrary, there was no ectopic osteogenesis in group DBM 12 weeks after operation. HE staining showed that DBM scaffold degraded and disappeared 12 weeks after operation. Conclusion The osteogenic activity of TEB and FTEB is similar, which provides a new strategy to preserve and transport TEB.
ObjectiveTo review the research progress of the co-culture system for constructing vascularized tissue engineered bone. MethodsThe recent literature concerning the co-culture system for constructing vascularized tissue engineered bone was reviewed, including the selection of osteogenic and endothelial lineages, the design and surface modification of scaffolds, the models and dimensions of the co-culture system, the mechanism, the culture conditions, and their application progress. ResultsThe construction of vascularized tissue engineered bone is the prerequisite for their survival and further clinical application in vivo. Mesenchymal stem cells (owning the excellent osteogenic potential) and endothelial progenitor cells (capable of directional differentiation into endothelial cell) are considered as attractive cell types for the co-culture system to construct vascularized tissue engineered bone. The culture conditions need to be further optimized. Furthermore, how to achieve the clinical goals of minimal invasion and autologous transplantation also need to be further studied. ConclusionThe strategy of the co-culture system for constructing vascularized tissue engineered bone would have a very broad prospects for clinical application in future.
ObjectiveTo investigate the mechanism of early vascularization of the tissue engineered bone in the treatment of rabbit radial bone defect by local injection of angiopoietin 2 (Ang-2).MethodsForty-eight New Zealand white rabbits were established unilateral 1.5 cm long radius defect models. After implantation of hydroxyapatite/collagen scaffolds in bone defects, the rabbits were randomly divided into 2 groups: control group (group A) and Ang-2 group (group B) were daily injected with 1 mL normal saline and 1 mL saline-soluble 400 ng/mL Ang-2 at the bone defect within 2 weeks after operation, respectively. Western blot was used to detect the expressions of autophagy related protein [microtubule associated protein 1 light chain 3 (LC3), Beclin-1], angiogenesis related protein [vascular endothelial growth factor (VEGF)], and autophagy degradable substrate protein (SQSTMl/p62) in callus. X-ray films examination and Lane-Sandhu X-ray scoring were performed to evaluate the bone defect repair at 4, 8, and 12 weeks after operation. The rabbits were sacrificed at 12 weeks after operation for gross observation, and the angiogenesis of bone defect area was observed by HE staining.ResultsWestern blot assay showed that the relative expressions of LC3-Ⅱ/LC3-Ⅰ, Beclin-1, and VEGF in group B were significantly higher than those in group A, and the relative expression of SQSTMl/p62 was significantly lower than that in group A (P<0.05). Radiographic and gross observation of specimens showed that only a few callus were formed in group A, the bone defect was not repaired; more callus were formed and complete repair of bone defect was observed in group B. The Lane-Sandhu scores in group B were significantly higher than those in group A at 4, 8, and 12 weeks after operation (P<0.05). HE staining showed that the Harvard tubes in group B were well arranged and the number of new vessels was significantly higher than that in group A (t=–11.879, P=0.000).ConclusionLocal injection of appropriate concentration of Ang-2 may promote early vascularization and bone defect repair of tissue engineered bone in rabbits by enhancing autophagy.
Objective To investigate the effect of icarin/attapulgite/collagen type Ⅰ/polycaprolactone (ICA/ATP/Col Ⅰ/PCL) composite scaffold in repair of rabbit tibia defect. Methods The ICA/20%ATP/Col Ⅰ/PCL (scaffold 1), ICA/30%ATP/Col Ⅰ/PCL (scaffold 2), 20%ATP/Col Ⅰ/PCL (scaffold 3), and 30%ATP/Col Ⅰ/PCL (scaffold 4) composite scaffolds were constructed by solution casting-particle filtration method. The structure characteristics of the scaffold 2 before and after cross-linking were observed by scanning electron microscopy, and the surface contact angles of the scaffold 2 and the scaffold 4 were used to evaluate the water absorption performance of the material. The in vitro degradation test was used to evaluate the sustained-release effect of the scaffold 2. Thirty male Japanese white rabbits, weighing (2.0±0.1) kg, were randomly divided into groups A, B, C, D, and E, 6 in each group. After making a 1 cm- diameter bilateral tibial defects model, group A was the defect control group without any material implanted. Groups B, C, D, and E were implanted with scaffolds 3, 4, 1, and 2 at the defect sites, respectively. At 4, 8, and 12 weeks after operation, the repairing effects of 4 scaffolds were observed by gross observation, histological observation of HE and Masson staining, and immunohistochemical staining of osteogenic specific transcription factor (runt-related transcription factor 2, RUNX2), osteogenic related transcription factor [Osterix (OSX), Col Ⅰ, osteopontin (OPN)]. Results Scanning electron microscopy observation showed that the scaffolds were all porous. The structure of the material was loose before and after cross-linking. The surface contact angle showed that the scaffold was hydrophobic, and the scaffold 2 was more hydrophobic than scaffold 4. The sustained-release effect in vitro showed that the drug could be released in a micro and long-term manner. In the animal implantation experiment, the gross observation showed that the defects were significantly smaller in groups D and E than in groups A, B, and C at 4 and 12 weeks after operation. HE and Masson staining showed that the defect of group A was full of connective tissue at 4 weeks after operation, a large number of fibers were seen in groups B and C, and the new bone formation was observed in groups D and E. The increase of new bone was observed in each group at 8 weeks after operation. The defect of group A was still dominated by connective tissue at 12 weeks after operation, and a small amount of new bone tissue was observed in groups B and C, and a large number of new bone tissue was observed in groups D and E, especially in group E, and most of the materials degraded. Immunohistochemical staining showed that the expressions of RUNX2 and OSX in the new tissues of groups D and E were significantly higher than those of the other groups at 4 weeks after operation. The expression of RUNX2 decreased at 8 and 12 weeks after operation. After 8 weeks and 12 weeks, the expressions of Col Ⅰand OPN increased than in 4 weeks. And the expressions of Col Ⅰ and OPN in the new tissues of groups D and E were significantly more than those of the other groups. Conclusion ICA/ATP/Col I/PCL composite scaffolds have good porosity and biocompatibility, can promote bone formation, and have good bone regeneration and repair effect.
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.
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.