ObjectiveTo evaluate the biological effect on vascularization during bone repair of prevascularized porous β-tricalcium phosphate (β-TCP) tissue engineered bone (hereinafter referred to as prevascularized tissue engineered bone), which was established by co-culture of endothelial progenitor cells (EPCs) and bone marrow mesenchymal stem cells (BMSCs) based on tissue engineering technology. Methods EPCs and BMSCs were isolated from iliac bone marrow of New Zealand white rabbits by density gradient centrifugation and differential adhesion method. The cells were identified by immunophenotypic detection, BMSCs-induced differentiation, and EPCs phagocytosis. After identification, the third-generation cells were selected for subsequent experiments. First, in vitro tubule formation in EPCs/BMSCs direct contact co-culture (EPCs/BMSCs group) was detected by Matrigel tubule formation assay and single EPCs (EPCs group) as control. Then, the prevascularized tissue engineered bone were established by co-culture of EPCs/BMSCs in porous β-TCP scaffolds for 7 days (EPCs/BMSCs group), taking EPCs in porous β-TCP scaffolds as a control (EPCs group). Scanning electron microscopy and laser scanning confocal microscopy were used to observe the adhesion, proliferation, and tube formation of cells. Femoral condyle defect models of 12 New Zealand white rabbits were used for implantation of prevascularized tissue engineered bone as the experimental group (n=6) and porous β-TCP scaffold as the control group (n=6). The process of vascularization of β-TCP scaffolds were observed. The numbers, diameter, and area fraction of neovascularization were quantitatively evaluated by Microfill perfusion, Micro-CT scanning, and vascular imaging under fluorescence at 4 and 8 weeks. ResultsThe isolated cells were BMSCs and EPCs through identification. EPCs/BMSCs co-culture gradually formed tubular structure. The number of tubules and branches, and the total length of tubules formed in the EPCs/BMSCs group were significantly more than those in the EPCs group on Matrigel (P<0.05) after 6 hours. After implanting and culturing in porous β-TCP scaffold for 7 days, EPCs formed cell membrane structure and attached to the material in EPCs group, and the cells attached more tightly, cell layers were thicker, the number of cells and the formation of tubular structures were significantly more in the EPCs/BMSCs group than in the EPCs group. At 4 weeks after implantation, neovascularization was observed in both groups. At 8 weeks, remodeling of neovascularization occurred in both groups. The number, diameter, and area fraction of neovascularization in the experimental group were higher than those in the control group (P<0.05), except for area fraction at 4 weeks after implantation (P>0.05). ConclusionThe prevascularized tissue engineered bone based on direct contact co-culture of BMSCs and EPCs can significantly promote the early vascularization process during bone defects repair.
Objective To perfect the theory system of minimally invasive treatment for osteonecrosis of the femoral head (ONFH) with β tricalcium phosphate (β-TCP) bioceramic system and evaluate the effectiveness. Methods Eighteen New Zealand white rabbits aged 7-8 months were used to establish an animal model to verify the vascularization of porous β-TCP bioceramic rods. Micro-CT based three-dimensional reconstruction and fluorescence imaging were used to display the new blood vessels at 4, 8, and 12 weeks after operation. The inserting depth, number and diameter of vessels in the encapsulated area were analyzed. Nine pig femoral specimens were randomly divided into 3 groups (n=3): group A was normal femur; group B had cavity (core decompression channel+spherical bone defect in femoral head); in group C, mixed bioceramic granules were implanted to fill the defect in femoral head, and porous β-TCP bioceramic rod was implanted into decompression channel. The stiffness and yield load of specimens were analyzed by biomechanical test. A multicenter retrospective study was conducted to analyze 200 patients (232 hips) with femoral head necrosis treated with bioceramic system in 7 hospitals in China between January 2012 and July 2018. There were 145 males and 55 females, with an average age of 42 years (range, 17-76 years). According to the Association Research Circulation Osseous (ARCO) stage, 150 hips were in stage Ⅱ and 82 hips in stage Ⅲ. Postoperative imaging assessment was carried out regularly, and hip function was evaluated by Harris score. The effectiveness of ARCO stage Ⅱ and Ⅲ was also compared. Results Animal experiments showed that blood vessels could grow into the encapsulated area and penetrate it at 12 weeks. The inserting depth, number and diameter of blood vessels in the encapsulated area gradually increased, and there was significant difference between different time points (P<0.05). Biomechanical tests showed that the stiffness and yield load of specimens in groups B and C were significantly lower than those in group A, while the yield load in group B were significantly lower than that in group C (P<0.05). The stiffness in group C was restored to 41.52%±3.96% in group A, and the yield load was restored to 46.14%±7.85%. Clinical study showed that 200 patients were followed up 6-73 months, with an average of 22.7 months. At last follow-up, 12 patients (16 hips) underwent total hip arthroplasty, and the hip survival rate was 93.10%. According to the imaging evaluation, 184 hips (79.31%) were stable and 48 (20.69%) were worse. Harris score (79.3±17.3) was significantly higher than that before operation (57.3±12.0) (t=18.600, P=0.000). The excellent rate of hip function was 64.22% (149/232). The survival rate of hip joint, imaging score and Harris score of patients in ARCO stage Ⅱ were better than those in ARCO stage Ⅲ (P<0.05). Conclusion β-TCP bioceramic system can guide the abundant blood supply of greater trochanter and femoral neck to the femoral head to promote repair; it can partly restore the mechanical properties of the femoral head and neck in the early stage, providing a new minimally invasive hip-preserving method for patients with ONFH, especially for those in early stage.