Objective To investigate the antioxidant and osteogenic induction capabilities of calcium phosphate nanoflowers (hereinafter referred to as nanoflowers) in vitro at different concentrations. Methods Nanoflowers were prepared using gelatin, tripolyphosphate, and calcium chloride. Their morphology, microstructure, elemental composition and distribution, diameter, and molecular constitution were characterized using scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, and energy-dispersive spectroscopy. Femurs and tibias were harvested from twelve 4-week-old Sprague Dawley rats, and bone marrow mesenchymal stem cells (BMSCs) were isolated and cultured using the whole bone marrow adherent method, followed by passaging. The third passage cells were identified as stem cells by flow cytometry and then co-cultured with nanoflowers at concentrations of 0, 0.4, 0.8, 1.2, 1.6, 2.0, 2.4, 2.8, 3.2, and 3.6 mg/mL. Cell counting kit 8 (CCK-8) assay was performed to screen for the optimal concentration that demonstrated the best cell viability, which was subsequently used as the experimental concentration for further studies. After co-culturing BMSCs with the screened concentration of nanoflowers, the biocompatibility of the nanoflowers was verified through live/dead cell staining, scratch assay, and cytoskeleton staining. The antioxidant capacity was assessed by using reactive oxygen species (ROS) fluorescence staining. The in vitro osteoinductive ability was evaluated via alkaline phosphatase (ALP) staining, alizarin red staining, and immunofluorescence staining of osteocalcin (OCN) and Runt-related transcription factor 2 (RUNX2). All the above indicators were compared with the control group of normally cultured BMSCs without the addition of nanoflowers. Results Scanning electron microscopy revealed that the prepared nanoflowers exhibited a flower-like structure; transmission electron microscopy scans discovered that the nanoflowers possessed a multi-layered structure, and high-magnification images displayed continuous atomic arrangements, with the nanoflower diameter measuring (2.00±0.25) μm; energy-dispersive spectroscopy indicated that the nanoflowers contained elements such as C, N, O, P, and Ca, which were uniformly distributed across the flower region; Fourier transform infrared spectroscopy analyzed the absorption peaks of each component, demonstrating the successful preparation of the nanoflowers. Through CCK-8 screening, the concentrations of 0.8, 1.2, and 1.6 mg/mL were selected for subsequent experiments. The live/dead cell staining showed that nanoflowers at different concentrations exhibited good cell compatibility, with the 1.2 mg/mL concentration being the best (P<0.05). The scratch assay results indicated that the cell migration ability in the 1.2 mg/mL group was superior to the other groups (P<0.05). The cytoskeleton staining revealed that the cell morphology was well-extended in all concentration groups, with no significant difference compared to the control group. The ROS fluorescence staining demonstrated that the ROS fluorescence in all concentration groups decreased compared to the control group after lipopolysaccharide induction (P<0.05), with the 1.2 mg/mL group showing the weakest fluorescence. The ALP staining showed blue-purple nodular deposits around the cells in all groups, with the 1.2 mg/mL group being significantly more prominent. The alizarin red staining displayed orange-red mineralized nodules around the cells in all groups, with the 1.2 mg/mL group having more and denser nodules. The immunofluorescence staining revealed that the expressions of RUNX2 and OCN proteins in all concentration groups increased compared to the control group, with the 1.2 mg/mL group showing the strongest protein expression (P<0.05). Conclusion The study successfully prepares nanoflowers, among which the 1.2 mg/mL nanoflowers exhibits excellent cell compatibility, antioxidant properties, and osteogenic induction capability, demonstrating their potential as an artificial bone substitute material.
ObjectiveTo review the application and research progress of in vivo bioreactor as vascularization strategies in bone tissue engineering. MethodsThe original articles about in vivo bioreactor that can enhance vascularization of tissue engineered bone were extensively reviewed and analyzed. ResultsThe in vivo bioreactor can be created by periosteum, muscle, muscularis membrane, and fascia flap as well as biomaterials. Using in vivo bioreactor can effectively promote the establishment of a microcirculation in the tissue engineered bones, especially for large bone defects. However, main correlative researches, currently, are focused on animal experiments, more clinical trials will be carried out in the future. ConclusionWith the rapid development of related technologies of bone tissue engineering, the use of in vivo bioreactor will to a large extent solve the bottleneck limitations and has the potential values for clinical application.
Objective To evaluate the feasibility of poly-L-lactide(PLLA)/porcinederived xenogeneic bone(PDXB) composite as a scaffold for the bone tissue engineering. Methods The film and the scaffold of the PLLA-PDXB composite were respectively prepared by a solution casting method and a solution casting-particle leaching method. The composite film and scaffold were further treated by the surface alkaline hydrolysis. The surface morphology of the composite was observed by the scanning electron microscopy, and hydrophilicity degree of the composite was measured. The OCT-1 osteoblastlike cells were cultured and amplified in vitro as the seeding cells, which werethen implanted on the film and scaffold. The adherence rate, adherence shape,proliferating activity, and growing morphology of the OCT-1 osteoblastlikecells were observed on the film. Results The PDXB particle 50 μm in diameter on average had a similar phase structure to that of hydroxyapatite. But its Ca/P ratio was lower than that of hydroxyapatite. After the surface alkaline hydrolysis, the PDXB particle could be exposed on the surface of the PLLA-PDXB composite. The surface roughness and hydrophilicity of the PLLAPDXB composite were obviously enhanced. The cell adherence rate and the cell proliferation activity of the PLLAPDXB composite were higher than those of the pure PLLA material. The cells tended to grow on the exposed surface of the PDXB particles. The cells seeded on the composite scaffold could migrate to the inside of the composite scaffold and grew well. Conclusion The PLLA-PDXB composite has a good cell affinity, and this kind of composite can hopefullybecome a new scaffold material to be used in the bone tissue engineering.
ObjectiveTo study the effect of Schwann cells (SCs) promoting the function of nitric oxide (NO) secretion of bone marrow mesenchymal stem cells (BMSCs) derived endothelial cells so as to lay the experimental foundation for research of the effect of nerves on vessels during the process of tissue engineering bone formation. MethodsSCs were collected from 1-day-old Sprague Dawley (SD) rats,and identified through S100 immunohistochemistry (IHC).BMSCs were collected from 2-week-old SD rats and induced into endothelial cells (IECs),which were identified through von Willebrand factor (vWF) and CD31 immunofluorescence (IF).Transwell system was used for co-culture of SCs and IECs without contact as the experimental group,and simple culture of IECs served as the control group.The NO concentration in the medium was measured at 1,3,5,and 7 days after culture; the mRNA expressions of nitric oxide synthetase 2 (NOS2) and NOS3 were detected by real-time fluorescence quantitative PCR (RT-qPCR) at 1,3,7,and 10 days. ResultsSCs and IECs were identified through morphology and immunology indexes of S100 IHC,vWF and CD31 IF.Significant differences were found in the NO concentration among different time points in 2 groups (P<0.05); the NO concentration of the experimental group was significantly higher than that of the control group at the other time points (P<0.05) except at 3 days.NOS2 mRNA expression of the experimental group was significantly higher than that of the control group (P<0.05); difference was significant in the NOS2 mRNA expression among different time points in 2 groups (P<0.05).NOS3 mRNA expression of the experimental group was significantly higher than that of the control group at the other time points (P<0.05) except at 10 days.No significant difference was found in NOS3 mRNA expression among different time points in the experimental group (F=6.673,P=0.062),but it showed significant differences in the control group (F=36.581,P=0.000). ConclusionSCs can promote NO secretion of BMSCs derived endothelial cells,which is due to promoting the activity of NOS.
Objective To review the latest research progress of heme oxygenase 1 (HO-1), to thoroughly understand different functions of HO-1 and its influence on osteogenesis and angiogenesis of stem cells, and to analyze HO-1 application in bone tissue engineering. Methods Domestic and international literature on HO-1 in recent years was extensively reviewed and analyzed. Results The activity of HO-1 and its enzymatic products not only have the properties of anti-inflammatory, anti-apoptosis, and cytoprotection, but also can promote angiogenesis combined with other growth factors and protect the vessel which already exist. Moreover, HO-1 has an effect on the proliferation, paracrine signaling, osteoblastic differentiation, and anti-apoptosis of stem cells. Conclusion HO-1 can be used as a multi-function growth factor in bone tissue engineering, but more investigation should emphasis on synergistic effect of each function so as to improve bone repair.
ObjectiveTo investigate the formation of nanostructure on cuttlefish bone transformed hydroxyapatite (CB-HA) porous ceramics and the effects of different nanostructures on the osteoblasts adhesion, proliferation, and alkaline phosphatase (ALP) expression.MethodsThe cuttlefish bone was shaped as plate with diameter of 10 mm and thickness of 2 mm, filled with water, and divided into 4 groups. The CB-HA in groups 1-4 were mixed with different phosphorous solutions and then placed in an oven at 120℃ for 24 hours. In addition, the samples in group 4 were further sintered at 1 200℃ for 3 hours to remove nanostructure as controls. The chemical composition of CB-HA were analyzed by X-ray diffraction spectroscopy, Fourier transform infrared spectrum, and inductively coupled plasma (ICP). The physical structure was analyzed using scanning electron microscopy, specific surface tester, and porosity tester. The MC3T3-E1 cells of 4th generation were co-cultured with 4 groups of CB-HA. After 1 day, the morphology of the cells was observed under scanning electron microscopy. After 1, 3, and 7 days, the cell proliferation was analyzed by MTT assay. After 7 and 14 days, the ALP expression was measured by pNPP method.ResultsX-ray diffraction spectrum showed that the four nanostructures of CB-HA were made of hydroxyapatite. The infrared absorption spectrum showed that the infrared absorption peak of CB-HA was consistent with hydroxyapatite. ICP showed that the ratio of calcium to phosphorus of all CB-HA was 1.68-1.76, which was consistent with hydroxyapatite. Scanning electron microscopy observation showed that the nanostructure on the surface of CB-HA in groups 1-3 were large, medium, and small cluster-like structures, respectively, and CB-HA in group 4 had no obvious nanostructure. There were significant differences in the specific surface areas between groups (P<0.05). There was no significant difference in the porosity between groups (P>0.05). Compared with group 4, groups 1-3 have more pores with pore size less than 50 nm. After co-cultured with osteoblasts, scanning electron microscopy observation and MTT assay showed that the cells in groups 2 and 3 adhered and proliferated better and had more ALP expression than that in groups 1 and 4 (P<0.05).ConclusionThe size of cluster-like nanostructure on the surface of CB-HA can be controlled by adjusting the concentration of ammonium ions in the phosphorous solution, and the introduction of small-sized cluster-like nanostructure on the surface of CB-HA can significantly improve the cell adhesion, proliferation, and ALP expression of the material which might be resulted from the enlarged surface area.
Objective To investigate the ectopic bone formation of the chitosan/phosphonic chitosan sponge combined with human umbil ical cord mesenchymal stem cells (hUCMSCs) in vitro. Methods Phosphorous groups were introduced in chitosan molecules to prepare the phosphonic chitosan; 2% chitosan and phosphonic chitosan solutions were mixed at a volume ratio of 1 ∶ 1 and freeze-dried to build the complex sponge, and then was put in the simulated body fluid for biomimetic mineral ization in situ. The hUCMSCs were isolated by enzyme digestion method from human umbil ical cord and were cultured. The chitosan/phosphonic chitosan sponge was cultured with hUCMSCs at passage 3, and the cell-scaffoldcomposite was cultured in osteogenic medium. The growth and adhesion of the cells on the scaffolds were observed by l ight microscope and scanning electron microscope (SEM) at 1 and 2 weeks after culturing, respectively. The cell prol iferation was detected by MTT assay at 1, 2, 3, 4, 5, and 6 days, respectively. Bilateral back muscles defects were created on 40 New Zealand rabbits (3-4 months old, weighing 2.1-3.2 kg, male or female), which were divided into groups A, B, and C. In group A, cellscaffold composites were implanted into 40 right defects; in group B, the complex sponge was implanted into 20 left defects; and in group C, none was implanted into other 20 left defects. The gross and histological observations were made at 4 weeks postoperatively. Results The analysis results of phosphonic chitosan showed that the phosphorylation occurred mainly in the hydroxyl, and the proton type and chemical shifts intensity were conform to its chemical structure. The SEM results showed that the pores of the chitosan/phosphonic chitosan sponge were homogeneous, and the wall of the pore was thinner; the coating of calcium and phosphorus could be observed on the surface of the pore wall after mineral ized with crystal particles; the cells grew well on the surface of the chitosan/phosphonic chitosan sponge. The MTT assay showed that the chitosan/phosphonic chitosan sponge could not inhibit the prol iferation of hUCMSCs. The gross observation showed that the size and shape of the cell-scaffold composite remained intact and texture was toughened in group A, the size of the complex sponge gradually reducedin group B, and the muscle defects wound healed with a l ittle scar tissue in group C. The histological observation showed that part of the scaffold was absorbed and new blood vessels and new bone trabeculae formed in group A, the circular cavity and residual chitosan scaffolds were observed in group B, and the wound almost healed with a small amount of lymphocytes in group C. Conclusion The chitosan/phosphonic chitosan sponge has good biocompatibil ity, the tissue engineered bone by combining the hUCMSCs with chitosan/phosphonic chitosan sponge has the potential of the ectopic bone formation in rabbit.
Objective To investigate the application potential of alginate-strontium (Sr) hydrogel as an injectable scaffold material in bone tissue engineering. Methods The alginate-Sr/-calcium (Ca) hydrogel beads were fabricated by adding 2.0wt% alginate sodium to 0.2 mol/L SrCl2/CaCl2 solution dropwise. Microstructure, modulus of compression, swelling rate, and degradability of alginate-Sr/-Ca hydrogels were tested. Bone marrow mesenchymal stem cells (BMSCs) were isolated from femoral bones of rabbits by flushing of marrow cavity. BMSCs at passage 5 were seeded onto the alginate-Sr hydrogel (experimental group) and alginate-Ca hydrogel (control group), and the viability and proliferation of BMSCs in 2 alginate hydrogels were assessed. The osteogenic differentiation of cells embeded in 2 alginate hydrogels was evaluated by alkaline phosphate (ALP) activity, osteoblast specific gene [Osterix (OSX), collagen type I, and Runx2] expression level and calcium deposition by fluorescent quantitative RT-PCR and alizarin red staining, Von Kossa staining. The BMSCs which were embeded in alginate-Ca hydrogel and cultured with common growth medium were harvested as blank control group. Results The micromorphology of alginate-Sr hydrogel was similar to that of the alginate-Ca hydrogel, with homogeneous pore structure; the modulus of compression of alginate-Sr hydrogel and alginate-Ca hydrogel was (186.53 ± 8.37) and (152.14 ± 7.45) kPa respectively, showing significant difference (t=6.853, P=0.002); there was no significant difference (t=0.737, P=0.502) in swelling rate between alginate-Sr hydrogel (14.32% ± 1.53%) and alginate-Ca hydrogel (15.25% ± 1.64%). The degradabilities of 2 alginate hydrogels were good; the degradation rate of alginate-Sr hydrogel was significantly lower than that of alginate-Ca hydrogel on the 20th, 25th, and 30th days (P lt; 0.05). At 1-4 days, the morphology of cells on 2 alginate hydrogels was spherical and then the shape was spindle or stellate. When three-dimensional cultured for 21 days, the DNA content of BMSCs in experimental group [(4.38 ± 0.24) g] was significantly higher than that in control group [(3.25 ± 0.21) g ] (t=8.108, P=0.001). On the 12th day after osteogenic differentiation, the ALP activity in experimental group was (15.28 ± 1.26) U/L, which was significantly higher than that in control group [(12.07 ± 1.12) U/L] (P lt; 0.05). Likewise, the mRNA expressions of OSX, collagen type I, and Runx2 in experimental group were significantly higher than those in control group (P lt; 0.05). On the 21th day after osteogenic differentiation, alizarin red staining and Von Kossa staining showed calcium deposition in 2 groups; the calcium nodules and phosphate deposition in experimental group were significantly higher than those in control group (P lt; 0.05). Conclusion Alginate-Sr hydrogel has good physicochemical properties and can promote the proliferation and osteogenic differentiation of BMSCs, so it is an excellent injectable scaffold material for bone tissue engineering.
ObjectiveTo investigate the feasibil ity of the domestic porous tantalum as scaffold material of bone tissue engineering by observing the expressions of osteogenesis related factors of MG63 cells co-cultured with domestic porous tantalum. MethodsMG63 cells were cultured with porous tantalum scaffolds (group A), with porous tantalum leaching solution (group B), and with MEM as control group (group C). The cell adhesion of group A was observed on the scaffolds at 3, 5, and 7 days after culture by scanning electron microscopy (SEM); immunohistochemistry and Western blot methods were used to detect the expressions of Runt-related transcri ption factor 2 (Runx-2), osteocalcin (OC), and fibronectin (FN). ResultsAt 3 days after culture, the cells of group A adhered the surface and pore of the porous tantalum scaffolds, with sparse cell arrangement and less protuberances; at 5 days after culture, adjacent cells connected to be a flat each other, which covered the surface and pore of the scaffold; at 7 days after culture, cells secreted plenty of extracellular matrix, covering most of the material surface. The expressions of Runx-2, OC, and FN were positive in 3 groups; darker staining of the cytoplasm was observed in group A, the expressions were significantly higher in group A than in other 2 groups. The results of immunohistochemistry and Western blot showed that the expressions of Runx-2 and OC were significantly increased in group A when compared with those in groups B and C (P < 0.05), but no significant difference was found between groups B and C (P > 0.05). The expression of FN had no significant difference among 3 groups (P > 0.05). ConclusionDomestic porous tantalum could promote MG63 cells adhesion and growth, and may promote the expressions of Runx-2 and OC, so it can be used as a scaffold material of bone tissue engineering.
Objective To review the research progress of the osteogenic effect of strontium (Sr) and its application in the orthopaedics. Methods The recent literature concerning the osteogenic effect of Sr and its application in orthopaedics at home and abroad was extensively reviewed, and the research and development were summarized. Results Both in vivo and in vitro studies showed that Sr could enhance bone formation and inhibit bone resorption. Clinically, Sr was applied for treatment of osteoporosis, composite biomaterials in tissue engineering, and treatment of bone tumors and bone metastases. Conclusion Sr is one important combined element of alternative materials in bone tissue engineering, and can strengthen the mechanical and biological properties of the bone replacement material, so it has some development potential in bone tissue engineering.