Experimental Study Of Generation Of Tissue Engineered Bone Flap With A Vascular Pedicle And Its Repair Of Bone Defects In Beagle Dogs

Objective:The technique of bone tissue engineering has been reported to generate bone tissue and repair the medium and small size bone defect in small mammals. As the reconstruction of large bone defects or the poor recipient bed vascularity in large mammals, engineered bone formation are often incomplete due to insufficient blood supply in the initial phase after implantation. Insufficient vascularization can lead to nutrient deficiencies and/or hypoxia deeper in tissue-engineered constructs. Moreover, nutrient and oxygen gradients will be present in the outer regions of the constructs, which could result in improper cell integration or cell death and thus decreased tissue formation. To establish an effective blood supply for promoting tissue engineering bone formation and shortening the healing time of bone is crucial in the laboratorial study and clinical applications of bone tissue engineering.For this purpose, we carried out the experimental study of generation of tissue engineered bone flap with a vascular pedicle and its repair of bone defects in beagle dogs according to clinical operation of fibular transplantation with a vascular pedicle. Besides, the influences of BMSCs, vascularization and microenvironment on tissue engineered bone formation were discussed after a long term observation using Computed Tomography (CT) and histology analysis.Methods:1. In vitro preparation of cell-CHA constructsBone marrow of beagle dogs were harvested from iliac aspirations and Bone Mesenchymal Stem Cells (BMSCs) were isolated by a Ficoll-Paque Plus solution and then expanded in an osteogenic culture medium for 2 weeks. The osteogenic characteristics of induced BMSCs were evaluated by Alkaline phosphatase, Alizarin red staining and immunocytochemistry staining of osteopontin (OPN), osteocalcin (OCN). BMSCs were seeded on the coral hydroxyapatite (CHA) scaffold with a prefabricated inclined groove and the cell-CHA constructs were 3D cultured for 1 week before implantation. Scanning electron microscope and confocal laser electronic microscope were used to detect the growth of BMSCs.2. Generation of vascularized engineered bone flap and its repair of bone defectThe cell-CHA constructs were implanted into spatium intramuscular of the hind limb of the beagle dogs with arteria saphena and saphenous vein embedded in the prefabricated groove. Vascularized engineering bone flaps were analyzed by real time contrast-enhanced B model ultrasonic inspection, angiography and computed tomography (CT), vascular corrosion cast and histology assays at 4 weeks implantation. Then the critical-size fibular bone defects were repaired by the engineered bone constructs. The engineered constructs were divided into 4 groups in both ectopic and orthotopic environments:Vascularized Scaffold Cells group (VSC, n=5), Scaffold Cells group (SC, n=5), Vascularized Scaffold group (VS, n=5), Scaffold group (S, n=5). Engineered bone formation and CHA scaffold degradation were evaluated by CT and histology assays at 1,3,6, and 9 months after implantation.3. Study of the influences of BMSCs, prevascularization and microenvironment on tissue engineered bone formation with coral hydroxyapatite (CHA) scaffold.Because there is a linear relationship between CT values (Hounsfield unit, HU) and bone density, the engineered bone formation was calculated by the relative CT value: The mean CT value of VSC groups minus that of VS groups, the mean CT value of SC groups minus that of S groups. Staged engineered bone formation was calculated by the subtraction of the relative CT value at different time points. Scaffold degradation was evaluated by the mean CT value of VS and S groups, and staged scaffold degradation also calculated by the subtraction of the mean CT value at different time points as well. To analyze the influences of BMSCs, prevascularization and microenvironment on tissue engineered bone formation and CHA scaffold degradation, different CT values from different groups were compared using Independent-Samples T Test by SPSS Statistics 17.0 software and mean differences were considered to be statistically significant at p< 0.05.Results:1. Osteogenic differentiation of BMSCs and cell-CHA constructs culturingThe osteogenic markers (ALP, OCN and OPN) were highly expressed in the second passage of BMSCs cultured in osteogenic medium about 14 days, in which the deposition of calcium was also detected. After seeding on the CHA, SEM and CLSM images showed the cells attached and proliferated well with abundant production of extracellular matrix.2. Vascularization of cell-CHA constructs at 4 weeks post-implantationAn organized vascular network and blood signal were observed in the construct region of VSC group after 4 weeks implantation. Abundant capillaries were seen in different sites of the construct.3. Engineered bone formation in ectopic environmentHistology of VSC group showed that there were small amounts of bone-like tissue after 3 months implantation in the spatium intramuscular of hind leg of beagle dogs. Engineered bone tissue gradually increased after 6 months implantation and well-developed Haversian system were seen after 9 months implantation with little residual scaffold remaining. In SC group, only the osteoid were seen after 3 months implantation and there were no significant bone formation until 6 months implantation, engineered bone tissue increased after 9 months implantation with much residual scaffold remaining. In both VS and S groups, the majority of CHA scaffold pores filled with fibrous connective tissue and a large number of inflammatory cells, and CHA scaffolds gradually degraded without any bone formation.4. Engineered bone formation in orthotopic environmentHistology and CT imaging confirmed that the critical-size fibular defects were repaired by engineered bone in VSC and SC groups after 9 months implantation, but became nonunion in VS and S groups. Engineered bone formation in VSC group showed maturer than that in SC groups.5. CT evaluation of influences of BMSCs on engineered bone formationIn both ectopic and orthotopic environments, the mean CT value showed significantly different in VSC and SC groups comparing with VS and S groups respectively, which indicated that engineered bone formation in BMSCs groups (VSC and SC groups) gradually increased and there only CHA scaffolds gradually degradation were seen in VS and S groups after implantation.6. CT evaluation of influences of vascularization on engineered bone formationIn both ectopic and orthotopic environments, the relative CT value in VSC group showed significantly higher than that in SC group at different time points, which indicated that engineered bone formation were enhanced in vascularized group. Morever, staged relative CT value in the early 3 months was significantly different between VSC and SC groups.7. CT evaluation of influences of vascularization on CHA scaffold degradationIn both ectopic and orthotopic environments, the mean CT value in VS group showed significantly lower than that in S group at every time points (except for 1 month), which indicated that CHA scaffold degradation were faster in vascularized group. Morever, staged mean CT value in the early 3 months was significantly different between VS and S groups.8. CT evaluation of influences of microenvironment on engineered bone formationIn SC groups, the relative CT value in bone defect microenvironment showed significantly higher than that in spatium intramuscular microenvironment at every time points, which indicated that engineered bone formation were enhanced in orthotopic environment. Morever, staged relative CT values in the early and the third 3 months were significantly different between ectopic and orthotopic environments.9. CT evaluation of influences of microenvironment on CHA scaffold degradationIn S groups, the mean CT value in bone defect microenvironment showed significantly lower than that in spatium intramuscular microenvironment at every time points, which indicated that CHA scaffold degradation were faster in orthotopic environment. Morever, the three staged mean CT values were significantly different between ectopic and orthotopic environments.Conclusions:We successfully generate tissue engineered bone flap with a vascular pedicle, in which an organized vascular network were observed after 4 weeks implantation. Small amounts of bone-like tissue formed after 3 months implantation and mature bone formation with well-developed Haversian system were seen after 9 months implantation. Ectopic engineered bone formation in the group with a vascular pedicle was more rapid than that in the group without vascular pedicle, which has not been reported.BMSCs-CHA constructs with a vascular pedicle after 4 weeks implantation were transplanted to repair the critical-size fibular bone defects, in which strategy more significant engineered bone formation were detected in a long term observation. The approach of prevascularization of BMSCs-CHA construct provide sufficient blood supply in the initial phase after implantation, which can be used to repair local bone defects directly and also be applied in a distant transplantation by microsurgical techniques. Morever, vascularized engineered bone grafts transplantation is a better treatment strategy for the poor recipient bed vascularity or the reconstruction of large bony defects than nonvascularized grafts.In this study, ectopic and orthotopic engineered bone construction in beagle dogs established a large animal model for tissue engineered bone study. CT evaluation was used to investigate influences of BMSCs, vascularization and microenvironment on tissue engineered bone formation with CHA scaffold. BMSCs are proved to play an essential role in tissue engineered bone formation and repair of critical-size bone defect. Both vascularization and orthotopic environment enhance engineered bone formation and scaffold degradation during the first 3 months implantation. The results imply that once engineered bone form within the first 3 months construction, the engineering process could continue smoothly whatever strategy applied to construct engineered bone. Future research should focus on exploring more efficient methods to promote engineered bone formation within the first 3 months.