RESUMO
Cell sheet technology has been widely used in bone tissue engineering and regenerative medicine. However, controlling the shape and volume of large pieces of engineered bone tissue remains impossible without additional suitable scaffolds. Three-dimensional (3D) printed titanium (Ti) alloy scaffolds are mostly used as implant materials for repairing bone defects, but the unsatisfactory bioactivities of traditional Ti-based scaffolds severely limit their clinical applications. Herein, we hypothesize that the combination of bone marrow mesenchymal stem cell (BMSC) sheet technology and 3D porous Ti-6Al-4V (PT) alloy scaffolds could be used to fabricate biomimetic engineered bone. First, various concentrations of BMSCs were directly cocultured with PT scaffolds to obtain complexes of osteoblastic cell sheets and scaffolds. Then, as an experimental control, an osteoblastic BMSC sheet was prepared by continuous culturing under osteogenic conditions for 2 weeks without passaging and used to wrap the scaffolds. The BMSC sheet was composed of several layers of extracellular matrix (ECM) and a mass of BMSCs. The BMSCs exhibited excellent adherent, proliferative and osteogenic potential when cocultured with PT scaffolds, which may be attributed to the ability of the 3D microstructure of scaffolds to facilitate the biological behaviors of cells, as confirmed by the in vitro results. Moreover, the presence of BMSCs and ECM increased the angiogenic potential of PT scaffolds by the secretion of VEGF. Micro-CT and histological analysis confirmed the in vivo formation of biomimetic engineered bone when the complex of cocultured BMSCs and PT scaffolds and the scaffolds wrapped by prepared BMSC sheets were implanted subcutaneously into nude mice. Therefore, the combination of BMSC sheet technology and 3D-printed PT scaffolds could be used to construct customized biomimetic engineered bone, offering a novel and promising strategy for the precise repair of bone defects.
RESUMO
Tumor resection and treatment of trauma-related regional large bone defects have major challenges in the field of orthopedics. Scaffolds that treat bone defects are the focus of bone tissue engineering. 3D printing porous titanium alloy scaffolds, prepared via electron beam melting technology, possess customized structure and strength. The addition of a growth factor coating to the scaffold introduces a specific form of biological activation. Vascular endothelial growth factor (VEGF) is key to angiogenesis and osteogenesis in vivo. We designed a porous titanium alloy scaffold/thermosensitive collagen hydrogel system, equipped with VEGF, to promote local osseointegration and angiogenesis. We also verified the VEGF release via thermosensitive collagen and proliferation and induction of the human umbilical vein endothelial cells (HUVECs) via the composite system in vitro. In vivo, using microscopic computed tomography (Micro-CT), histology, and immunohistochemistry analysis, we confirmed that the composite scaffold aids in angiogenesis-mediated bone regeneration, and promotes significantly more bone integration. We also discovered that the composite scaffold has excellent biocompatibility, provides bioactive VEGF for angiogenesis and osteointegration, and provides an important theoretical basis for the restoration of local blood supply and strengthening of bone integration.
RESUMO
Porous titanium alloy scaffold was widely used in treating bone defect caused by traumatic injury and osteomyelitis, which was incapable of self-healing. The implantation of scaffold produced stress shielding thereby forming osteolysis. The objective of this study was to analysis trabecular morphological features of osseointegrated bone. 14 New Zealand rabbits were divided into two groups, surgery group and healthy control group. 7 rabbits in surgery group were selected to perform 3D printed porous titanium alloy scaffold implantation surgery with preload at the defect of femoral condyle for osseointegration. The other 7 rabbits in control group were feed free. After 90 days healing, femoral condyles were extracted to perform micro-CT scanning with hydroxyapatite calibration phantom. Mean bone mineral density (BMD), bone volume fraction (BV/TV), BS/TV (bone surface area ratio), Tb.Th (thickness of trabeculae), Tb.N (number of trabeculae), Tb.Sp (trabecular separation) and DA (degree of anisotropy) were calculated from micro-CT images. The results revealed that osseointegration inside and at the surface of scaffolds worked well from grey values of micro-CT images. After 12 weeks healing, mean bone mineral densities (BMD) in surgery group and healthy control group were calculated as 800±20mg/cm3 and 980±90mg/cm3, respectively. This revealed that the strength of trabeculae in surgery group might lower than that in the healthy group. Trabecular morphological parameters test showed that trabecular morphological parameters at the surface of scaffolds in the surgery group deteriorated significantly. It was found from micro-CT images that ingrowth bone was filled with pores of scaffold. Overall, the effect of osseointegration was promoted through the change of mechanical micro-environment in the scaffold region. Overall, preload could improve osseointegration effect in the long-term after surgery. However, the trabecular morphology in the surgery group was deteriorated, which might bring secondary fracture risk again.
La malla de aleación de titanio poroso se usó ampliamente en el tratamiento de defectos óseos causados por lesiones traumáticas y osteomielitis. El implante de la malla generó una protección contra el estrés, formando así osteolisis. El objetivo de este estudio fue analizar las características morfológicas trabeculares del hueso osteointegrado. Se dividieron 14 conejos (Neozelandeses) en dos grupos, grupo cirugía y grupo control saludable. Se seleccionaron 7 conejos en el grupo de cirugía para realizar una implantación de mallas de aleación de titanio poroso, impresas en 3D con precarga en el defecto del cóndilo femoral para la osteointegración. Los 7 conejos restantes del grupo control se mantuvieron sin alimentación. Después de 90 días de curación, se extrajeron los cóndilos femorales para realizar una exploración por micro-CT con un espectro de calibración de hidroxiapatita. Se calcularon a partir de imágenes de micro-CTDensidad mineral ósea media (DMO), fracción de volumen óseo (BV / TV), BS / TV (relación de área de superficie ósea), Tb.Th (espesor de trabéculas), Tb.N (número de trabéculas), Tb.Sp (trabecular separación) y DA (grado de anisotropía). Los resultados revelaron que la osteointegración dentro y en la superficie de los andamios funcionó bien a partir de los valores grises de las imágenes de micro-CT. Después de 12 semanas de curación, las densidades medias de minerales óseos (DMO) en el grupo cirugía y en el grupo control sano se calcularon como 800 ± 20 mg/cm3 y 980 ± 90 mg/cm3, respectivamente. Esto reveló que la fuerza de las trabéculas en el grupo de cirugía podría ser menor que la del grupo sano. La prueba de parámetros morfológicos trabeculares mostró que en el grupo de cirugía, la superficie de las mallas, se deterioraron significativamente. Se descubrió a partir de imágenes de microCT que el hueso en crecimiento estaba lleno de poros de andamio. En general, el efecto de la osteointegración se promovió mediante el cambio del microambiente mecánico en la región de la malla. En general, la precarga podría mejorar el efecto de osteointegración a largo plazo después de la cirugía. Sin embargo, la morfología trabecular en el grupo de cirugía se deterioró, lo que podría traer un nuevo riesgo de fractura secundaria.
