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.
Bioactive glass (BG) has been widely used in the preparation of artificial bone scaffolds due to its excellent biological properties and non-cytotoxicity, which can promote bone and soft tissue regeneration. However, due to the brittleness, poor mechanical strength, easy agglomeration and uncontrollable structure of glass material, its application in various fields is limited. In this regard, most current researches mainly focus on mixing BG with organic or inorganic materials by freeze-drying method, sol-gel method, etc., to improve its mechanical properties and brittleness, so as to increase its clinical application and expand its application field. This review introduces the combination of BG with natural organic materials, metallic materials and non-metallic materials, and demonstrates the latest technology and future prospects of BG composite materials through the development of scaffolds, injectable fillers, membranes, hydrogels and coatings. The previous studies show that the addition of BG improves the mechanical properties, biological activity and regeneration potential of the composites, and broadens the application of BG in the field of bone tissue engineering. By reviewing the recent BG researches on bone regeneration, the research potential of new materials is demonstrated, in order to provide a reference for future related research.
Objective To investigate the feasibility of a new kind of porous β tricalcium phosphate (β-TCP) as a scaffold for the bone tissue engineering Methods The inverted phase contrast microscope was used to observe the growth of the marrow mesenchymal stem cells (MSCs) in the experimentalgroup and the control group at 10 days.In the experimental group, the MSCs were cultured with β-TCP(3 mm×3 mm×3 mm) in the 24-hole cultivation board, and in the control to control group, only MSCs were cultivated. The scanning electron microscope was used to observe growth of MSCs at 6 days. Cultivated with β-TCP at 3, 6, 9, 12 days, the MTT assay was used to judge the biocompatibility. The cytotoxicity was analyzed with the method that used the different density(100%， 50%， 10%， 1%，0%) leaching liquor gained from β-TCP to raise MSCs. MSCs were induced into the osteoblasts and were mixed with β-TCP, and the composite was used to repair a large radius bone defect in the rabbit. The specimens were made at 2,6,12 weeks. The histology imageology, and the radionuclide bone scan were used to analyze the bone formation. Results Some MSCs had a good adherence 4 hours after MSCs were inoculated and had a complete adherence at 12 hours. The cells were shaped like polyangle, spindle or converge monolayer after 8-10 days. The cells in the two groups had no difference. The cell adhesion was good, when observed by the inverted phase contrast microscope and the scanning electron microscope at 6 days. MTT showed that the absorbance (A)was not statistically different between the experimental group and the control group (P＞0.05); the different density leaching liquor had no cytotoxicity at the different time points. Histology, X-ray, and CT tomograph showed that itcould repair the large radius bone defect in the rabbit and its in vivo degradationrate was the same as the bone formation rate. Conclusion The new porous β-TCP has a unique three dimensional (3D) stereochemical structure and superordinary physicochemical property, and so it is a good scaffold for the bone tissue engineering.
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.
ObjectiveTo summarize the application status of hypoxia mimetic agents in bone tissue engineering.MethodsThe related literature about the hypoxia mimetic agents in bone tissue engineering was reviewed and analyzed. And the application status and progress of hypoxia mimetic agents in bone tissue engineering were retrospectively analyzed.ResultsHypoxia mimetic agents have the same effect as hypoxia in up-regulating the level of hypoxia inducible factor 1α (HIF-1α). The combination of hypoxia mimetic agents and scaffolds can up-regulate the level of HIF-1α in bone tissue engineering, thus promoting early vascularization and bone regeneration of the bone defect area, which provides a new idea for using bone tissue engineering to repair bone defect. At present, the commonly used hypoxia mimetic agents include iron chelating agents, oxoglutarate competitive analogues, proline hydroxylase inhibitors, etc.ConclusionHypoxia mimetic agents have a wide application prospect in bone tissue engineering, but they have been used in bone tissue engineering for a short time, more attention should be paid to their possible side effects. In the future research, the hypoxia mimetic agents should be developed in the direction of higher targeting specificity and safety, and the exact mechanism of hypoxia mimetic agents in promoting bone regeneration should be further explored.
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 develop a biodegradable implantable bone material with compatible mechanics with the bone tissue, providing a new biomaterial for clinical bone repair and regeneration. Methods Silk reinforced polycaprolactone composites (SPC) containing 20%, 40%, and 60% silk were prepared by layer-by-layer assembly and hot-pressing technology. Macroscopic morphology was observed and microstructure were observed by scanning electron microscopy, compressive mechanical properties were detected by compression test, surface wettability was detected by surface contact angle test, degradation of materials was observed after soaking in PBS for 180 days, and proliferation of MC3T3-E1 cells was detected by cell counting kit 8 assay. Six Sprague Dawley rats were subcutaneously implanted with polycaprolactone (PCL) and 20%-SPC, respectively. Masson staining was used to analyze the in vivo degradation behavior and vascularization effect within 180 days. Results The pore defects of the three SPC sections were relatively few. In the range of 20% to 60%, as the silk content increased and the PCL content decreased, the interlayer spacing of silk fabric decreased, and the fibers almost covered the entire cross-section. The compressive modulus and compressive strength of SPC showed an increasing trend, and the compressive modulus of 60%-SPC was slightly lower than that of 40%-SPC. There were significant differences in compressive modulus and compressive strength between the materials (P<0.05). In vitro simulated fluid degradation experiments showed that the mass loss of the three types of SPC after 180 days of degradation was within 5%, with the highest mass loss observed in 60%-SPC. The differences in mass loss between the materials were significant (P<0.05). As the silk content increased, the static water contact angle of each material gradually decreased, and all could promote the proliferation of MC3T3-E1 cells. The subcutaneous degradation experiment in rats showed that 20%-SPC began to degrade at 30 days after implantation, and material degradation and vascularization were significant at 180 days, which was in sharp contrast to PCL. Conclusion SPC has the mechanical and hydrophilic properties that are compatible with bone tissue. It maintains its mechanical strength for a long time in a simulated body fluid environment in vitro, and achieves dynamic synchronization of material degradation, tissue regeneration, and vascularization through the body’s immune regulation mechanism in vivo. It is expected to provide a new type of implant material for clinical bone repair.
Objective To investigate the physicochemical properties, osteogenic properties, and osteogenic ability in rabbit model of femoral condylar defect of acellular dermal matrix (ADM)/dicalcium phosphate (DCP) composite scaffold. Methods ADM/DCP composite scaffolds were prepared by microfibril technique, and the acellular effect of ADM/DCP composite scaffolds was detected by DNA residue, fat content, and α-1, 3-galactosyle (α-Gal) epitopes; the microstructure of scaffolds was characterized by field emission scanning electron microscopy and mercury porosimetry; X-ray diffraction was used to analyze the change of crystal form of scaffold; the solubility of scaffolds was used to detect the pH value and calcium ion content of the solution; the mineralization experiment in vitro was used to observe the surface mineralization. Twelve healthy male New Zealand white rabbits were selected to prepare the femoral condylar defect models, and the left and right defects were implanted with ADM/DCP composite scaffold (experimental group) and skeletal gold® artificial bone repair material (control group), respectively. Gross observation was performed at 6 and 12 weeks after operation; Micro-CT was used to detect and quantitatively analyze the related indicators [bone volume (BV), bone volume/tissue volume (BV/TV), bone surface/bone volume (BS/BV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), bone mineral density (BMD)], and HE staining and Masson staining were performed to observe the repair of bone defects and the maturation of bone matrix. Results Gross observation showed that the ADM/DCP composite scaffold was a white spongy solid. Compared with ADM, ADM/DCP composite scaffolds showed a significant decrease in DNA residue, fat content, and α-Gal antigen content (P<0.05). Field emission scanning electron microscopy showed that the ADM/DCP composite scaffold had a porous structure, and DCP particles were attached to the porcine dermal fibers. The porosity of the ADM/DCP composite scaffold was 76.32%±1.63% measured by mercury porosimetry. X-ray diffraction analysis showed that the crystalline phase of DCP in the ADM/DCP composite scaffolds remained intact. Mineralization results in vitro showed that the hydroxyapatite layer of ADM/DCP composite scaffolds was basically mature. The repair experiment of rabbit femoral condyle defect showed that the incision healed completely after operation without callus or osteophyte. Micro-CT showed that bone healing was complete and a large amount of new bone tissue was generated in the defect site of the two groups, and there was no difference in density between the defect site and the surrounding bone tissue, and the osteogenic properties of the two groups were equivalent. There was no significant difference in BV, BV/TV, BS/BV, Tb.Th, Tb.N, and BMD between the two groups (P>0.05), except that the Tb.Sp in the experimental group was significantly higher than that in the control group (P<0.05). At 6 and 12 weeks after operation, HE staining and Masson staining showed that the new bone and autogenous bone fused well in both groups, and the bone tissue tended to be mature. Conclusion The ADM/DCP composite scaffold has good biocompatibility and osteogenic ability similar to the artificial bone material in repairing rabbit femoral condylar defects. It is a new scaffold material with potential in the field of bone repair.
Objective To investigate the effects of flow shear stress and mass transport on the construction of largescale tissue engineered bone using a perfusion bioreactor. Methods Bone marrow (20 mL) was harvested from the il iac crestof the healthy volunteer, and then hBMSCs were isolated, cultured and identified. The hBMSCs at passage 3 were seeded on the critical-size β-TCP scaffold and cultured in a perfusion bioreactor for 28 days. Different flow shear stress (1 ×, 2 × and 3 ×) and different mass transport (3, 6 and 9 mL/min) were exerted on the cells seeded on the scaffold by changing the viscosity of media or perfusion flow rate. The cell prol iferation and ALP activity of cells seeded on the scaffold were detected, and histology observation and morphology measurement of cell/scaffold complex were conducted. Results When the perfusion flow rabe was 3 mL/min, the cell viabil ity of 2 × group was higher than that of other groups (P lt; 0.05). When the flow shear stress was 3 ×, no significant differences were found among 3, 6 and 9 mL/min in cell viabil ity (P gt; 0.05). When the perfusion flow rate was 3 mL/min, the activity of ALP of 2 × and 3 × groups was higher than that of 1 × group (P lt; 0.05). When the flow shear stress was 3 ×, the activity of ALP of 6 mL/min group was the highest (P lt; 0.05). After 28 days of perfusion culture, the ECM of all the groups distributed throughout the scaffold, and the formation and mineral ization of ECM was improved with the increase of flow shear stress when the perfusion flow rate was 3 mL/min. However, the increase of perfusion flow rate decreased the mineral ization of ECM when the flow shear stress was 3 ×. Conclusion As two important fluid dynamics parameters affecting the construction of large-scale tissue engineered bone, the flow shear stress and the mass transport should be measured duringthe process of constructing large-scale tissue engineered bone so as to maximize their roles.
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.