ObjectiveTo review the research progress of the role of seed cells and related cytokines in angiogenesis of the vascularized tissue engineered bone. MethodsThe latest literature of tissue engineered bone angiogenesis was reviewed, including the common source of seed cells, biological characteristics, transformation mechanism, related cytokines, and signaling pathways in re-vascularization. ResultsMicrosurgery technique, genetic technique, and co-culture system of vascularized tissue engineered bone have developed to a new level. Moreover, both the induction of introduced pluripotent stem cells and vascular endothelial growth factor-angiopoietins 1 transfected mesenchymal stem cells and endothelial progenitor cells have some advantages for bone regeneration and vascularization. However, all the techniques were not used in clinical practice. ConclusionUsing techniques of genetically modified seed cells, related cytokines, and scaffolds may have bright prospects for building vascularized tissue engineered bone.
Objective To study the osteogenic effects of a new type of peptides anchored aminated-poly-D, L-lactide acid (PA/PDLLA) scaffold in repairing femoral defect in rats. Methods The PDLLA scaffolds were treated by ammonia plasma and subsequent anchor of Gly-Arg-Gly-Asp-Ser (GRGDS) peptides via amide linkage formation. Thus PA/PDLLA scaffolds were prepared. The bone marrow was harvested from the femur and tibia of 4 4-week-old Sprague Dawley (SD) rats, and bone marrow mesenchymal stem cells (BMSCs) were isolated and cultured by whole bone marrow adherence method. BMSCs-scaffold composites were prepared by seeding osteogenic-induced BMSCs at passages 3-6 on the PA/PDLLA and PDLLA scaffolds. The right femoral defects of 8 mm in length were prepared in 45 adult male SD rats (weighing, 350-500 g) and the rats were divided into 3 groups (n=15) randomly. BMSCs-PA/PDLLA (PA/PDLLA group) or BMSCs-PDLLA (PDLLA group) composites were used to repair defects respectively, while defects were not treated as blank control (blank control group). General state of the rats after operation was observed. At 4, 8, and 12 weeks after operation, general, radiological, histological, micro-CT observations and real-time fluorescent quantitative PCR were performed. Results Two rats died after operation, which was added; the other rats survived to the end of the experiment. At each time point after operation, general and radiological observations showed more quick and obvious restoration in PA/PDLLA group than in PDLLA group; no bone repair was observed in blank control group. The X-ray scores were the highest in PA/PDLLA group, higher in PDLLA group, and the lowest in blank control group; showing significant difference in multiple comparison at the other time (P lt; 0.05) except between blank control group and PDLLA group at 4 weeks (P gt; 0.05). The X-ray scores showed an increasing trend in PDLLA group and PA/PDLLA group with time (P lt; 0.05). Histological and micro-CT observations showed the best osteogenesis in PA/PDLLA group, better in PDLLA group, and worst in blank control group. Comparison between groups had significant differences (P lt; 0.05) in bone mineral density, bone volume/total volume of range of interest, trabecular number, and structure model index. Significant differences (P lt; 0.05) were found in the expression levels of osteogenesis-related genes, such as osteocalcin, alkaline phosphatase, collagen type I, bone morphogenetic protein 2, and osteopontin when compared PA/PDLLA group with the other groups by real-time fluorescent quantitative PCR analysis. Conclusion The PA/PDLLA scaffolds can accelerate the repair of femoral defects in rats.
Objective To investigate the protocols of combined culture of human placenta-derived mesenchymal stem cells (HPMSCs) and human umbilical vein endothelial cells (HUVECs) from the same and different individuals on collagen material, to provide the. Methods Under voluntary contributions, HPMSCs were isolated and purified from human full-term placenta using collagenase IV digestion and lymphocyte separation medium, and confirmed by morphology methods and flow cytometry, and then passage 2 cells were cultured under condition of osteogenic induction. HUVECs were isolated from fresh human umbilical vein by collagenase I digestion and subcultured to purification, and cells were confirmed by immunocytochemical staining of von Willebrand factor (vWF). There were 2 groups for experiment. Passage 3 osteoblastic induced HPMSCs were co-cultured with HUVECs (1 ∶ 1) from different individuals in group A and with HUVECs from the same individual in group B on collagen hydrogel. Confocal laser scanning microscope was used to observe the cellular behavior of the cell-collagen composites at 1, 3, 5, and 7 days after culturing. Results Flow cytometry showed that HPMSCs were bly positive for CD90 and CD29, but negative for CD31, CD45, and CD34. After induction, alizarin red, alkaline phosphatase, and collagenase I staining were positive. HUVECs displayed cobble-stone morphology and stained positively for endothelial cell marker vWF. The immunofluorescent staining of CD31 showed that HUVECs in the cell-collagen composite of group B had richer layers, adhered and extended faster and better in three-dimension space than that of group A. At 7 days, the class-like microvessel lengths and the network point numbers were (6.68 ± 0.35) mm/mm2 and (17.10 ± 1.10)/mm2 in group A, and were (8.11 ± 0.62) mm/mm2 and (21.30 ± 1.41)/mm2 in group B, showing significant differences between the 2 groups (t=0.894, P=0.000; t=0.732, P=0.000). Conclusion Composite implant HPMSCs and HUVECs from the same individual on collagen hydrogel is better than HPMSCs and HUVECs from different individuals in integrity and continuity of the network and angiogenesis.
Objective To observe the histological structure and cytocompatibility of novel acellular bone matrix (ACBM) and to investigate the feasibility as a scaffold for bone tissue engineering. Methods Cancellous bone columns were harvested from the density region of 18-24 months old male canine femoral head, then were dealt with high-pressure water washing, degreasing, and decellularization with Trixon X-100 and sodium deoxycholate to prepare the ACBM scaffold. The scaffolds were observed by scanning electron microscope (SEM); HE staining, Hoechst 33258 staining, and sirius red staining were used for histological analysis. Bone marrow mesenchymal stem cells (BMSCs) from canine were isolated and cultured with density gradient centrifugation; the 3rd passage BMSCs were seeded onto the scaffold. MTT test was done to assess the cytotoxicity of the scaffolds. The proliferation and differentiation of the cells on the scaffold were observed by inverted microscope, SEM, and live/dead cell staining method. Results HE staining and Hoechst 33258 staining showed that there was no cell fragments in the scaffolds; sirius red staining showed that the ACBM scaffold was stained crimson or red and yellow alternating. SEM observation revealed a three dimensional interconnected porous structure, which was the microstructure of normal cancellous bone. Cytotoxicity testing with MTT revealed no significant difference in absorbance (A) values between different extracts (25%, 50%, and 100%) and H-DMEM culture media (P gt; 0.05), indicating no cytotoxic effect of the scaffold on BMSCs. Inverted microscope, SEM, and histological analysis showed that three dimensional interconnected porous structure of the scaffold supported the proliferation and attachment of BMSCs, which secreted abundant extracellular matrices. Live/dead cell staining results of cell-scaffold composites revealed that the cells displaying green fluorescence were observed. Conclusion Novel ACBM scaffold can be used as an alternative cell-carrier for bone tissue engineering because of thoroughly decellularization, good mircostructure, non-toxicity, and good cytocompatibility.
Objective To explore the osteogenesis and angiogenesis effect of bone marrow mesenchymal stem cells (BMSCs) derived osteoblasts and endothelial cells compound with chitosan/hydroxyapatite (CS/HA) scaffold in repairing radialdefect in rats. Methods The BMSCs were isolated from Sprague Dawley rats and the 3rd generation of BMSCs were induced into osteoblasts and endothelial cells. The endothelial cells, osteoblasts, and mixed osteoblasts and endothelial cells (1 ∶ 1) were compound with CS/HA scaffold in groups A, B, and C respectively to prepare the cell-scaffold composites. The cell proliferation was detected by MTT. The rat radial segmental defect model was made and the 3 cell-scaffolds were implanted, respectively. At 4, 8, and 12 weeks after transplantation, the graft was harvested to perform HE staining and CD34 immunohistochemistry staining. The mRNA expressions of osteopontin (OPN) and osteoprotegerin (OPG) were detected by RT-PCR. Results Alkal ine phosphatase staining of osteoblasts showed that there were blue grains in cytoplasm at 7 days after osteogenic induction and the nuclei were stained red. CD34 immunocytochemical staining of the endothelial cells showed that there were brown grains in the cytoplasm at 14 days after angiogenesis induction. MTT test showed that the proliferation level of the cells in 3 groups increased with the time. HE staining showed that no obvious osteoid formation, denser microvessel, and more fibrous tissue were seen at 12 weeks in group A; homogeneous osteoid which distributed with cord or island, and many osteoblast-l ike cells were seen in groups B and C. The microvessel density was significantly higher in groups A and C than group B at 3 time points (P lt; 0.05), and in group A than in group C at 12 weeks (P lt; 0.05). The OPN and OPG mRNA expressions of group A were significantly lower than those of groups B and C at 3 time points (P lt; 0.05). In groups B and C, the OPN mRNA expressions reached peak t8 and 12 weeks, respectively, and OPG mRNA expressions reached peak at 4 weeks. Conclusion BMSCs derived steoblasts and endothelial cells (1 ∶ 1) compound with CS/HA porous scaffold can promote bone formation and vascularization in bone defect and accelerate the healing of bone defect.
【Abstract】 Objective To construct tissue engineered skeletal muscle in vivo using glial cell derived neurotrophic factor (GDNF) genetically modified myoblast (Mb) on acellular collagen sponge with hypoglossal nerve implantation, and to observe whether structural or functional connection could be established between engineered tissue and motor nerve or not. Methods Mbs were isolated from 7 male Lewis rats at age of 2 days, cultured and genetically modified by recombinant adenovirus carrying GDNF cDNA (MbGDNF). Calf skin-derived acellular collagen sponge was used as scaffold; cell adhesion was detected by scanning electron microscope after 24 hours. Hypoglossal nerve was implanted into Mb-scaffold complex (Mb group, n=27) or MbGDNF-scaffold complex (MbGDNF group, n=27) in 54 female Lewis rats at age of 8 weeks. HE staining was performed at 1, 6, and 12 weeks postoperatively, and immunohistochemistry staining and fluorescence in situ hybridization were used. Results MbGDNF could highly expressed GDNF gene. Mb and MbGDNF could adhere to the scaffold and grew well. HE staining showed tight junctions between implant and peripheral tissue with new muscle fiber and no distinguished line at 12 weeks in 2 groups. Immunohistochemistry staining showed that positive cells of myogenin and slow skeletal myosin were detected, as well as positive cells of actylcholine receptor α1 at 1, 6, and 12 weeks. The positive cells of Y chromosome decreased with time. At 1, 6, and 12 weeks, the positive neurons were 261.0 ± 6.6, 227.3 ± 8.5, and 173.3 ± 9.1, respectively in MbGDNF group, and were 234.7 ± 5.5, 196.0 ± 13.5, and 166.7 ± 11.7, respectively in Mb group; significant differences were found between 2 groups at 1 and 6 weeks (P lt; 0.05), no significant difference at 12 weeks (P gt; 0.05). Conclusion Connection can be established between engineered tissue and implanted hypoglossal nerve. Recombinant GDNF produced by MbGDNF might play a critical role in protecting central motor neurons from apoptosis by means of retrograde transportation.
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.
Objective To investigate the osteogenesis effects of angiopoietin 1 (Ang-1) gene transfected bone marrow mesenchymal stem cells (BMSCs) seeded on β tricalcium phosphate (β-TCP) scaffolds (tissue engineered bone) with platelet-rich plasma (PRP). Methods BMSCs were isolated from bone marrow tissue of rabbits. The Ang-1 gene was transfected into the BMSCs at passage 2 by lentivector, which were seeded on β-TCP scaffolds with PRP (0.5 mL) after 48 hours of transfection. Bilateral radial segmental bone defects (15 mm in length) were created in 20 3-month-old New Zealand rabbits. Then the tissue engineered bone with the Ang-1 gene transfected BMSCs (experimental group) and untransfected BMSCs (control group) were implanted into the defects in the right and left radius, respectively. X-ray, histology, immunohistochemistry, and biomechanics observations were done at 2, 4, 8, and 12 weeks after operation. Results In vitro, the transfected rate was over 90% and RT-PCR showed that the Ang-1 expression were significantly increased after transfection. The X-ray films showed that some callus formed at 4 weeks, partial bony union was observed at 8 weeks, and complete union at 12 weeks in experimental group; and bone union was not observed at 12 weeks in control group. HE staining showed that capillary appeared at 8 weeks and more capillaries were observed in new bone at 12 weeks in experimental group; only a few capillaries were observed at 12 weeks in control group. At 8 and 12 weeks, the microvascular density were (50.1 ± 7.8) /mm2 and (66.1 ± 3.5) /mm2 in experimental group and were 0 and (30.3 ± 7.2)/mm2 in control group, showing significant differences between 2 groups at 12 weeks (Z= —2.107, P=0.031). Immunohistochemistry examination showed that the positive cells can be found at 8 weeks in experimental group. And the biomechanical analysis showed that maximum loads of experimental group were significantly higher than those of control group in three-point bending test and compression test at 12 weeks (P lt; 0.05). Conclusion The tissue engineered bone with PRP and Ang-1 can increase the osteogenic properties by enhancing capillary regeneration, thus it can be used to repair radial segmental bone defects of rabbit.
Objective To review the recent progress of the researches in construction of tissue engineered osteochondral composites, and to discuss the challenges in construction of tissue engineered osteochondral composites. Methods The recent literature on the construction of tissue engineered osteochondral composites was extensively reviewed and analyzed. Results The studies on the construction of tissue engineered osteochondral composites are relatively more in vivo, the current focus is that different tissues derived mesenchymal stem cells are widely used to be seed cells; single-phase scaffold has been limited, studies on biphase scaffold and triphase scaffold are new trends; the design and performance of bioreactor need to be further optimized in the future. Conclusion The construction of tissue engineered osteochondral composites will be a promising method for the treatment of cartilage defects.
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.