Introduction: Bone defects can be ascribed to trauma, tumors and genetic diseases, from which an increasing number of patients are suffering. Considerable efforts have been made to the development of artificially synthesized bone substitutes since the limitation of bone graft such as shortage of donors, donor site morbidity and immune rejection. However, many studies ignored the siginificance of vascularization for artificial tissues’ nutrient supply in their latter in vivo application, which could result in tissue necrosis[1]. Via piling of porous microspheres, we fabricated a novel multilevel-construction scaffold of which osteoblasts-loaded PLGA microspheres played a role as the base while hemangioblasts were incubated in the spaces among the spheres. On account of the porous microspheres as cell carriers, spaces of the scaffold were well allocated to allow osteoblasts and hemangioblasts to coexist for the final vascularized bone tissue construction.
Materials and Methods:: Biodegradable porous Poly(lactic-co-glycolic acid) (PLGA) microspheres were synthesized via solvent evaporation method[2], and ammonium bicarbonate was used as the pore-foaming agent. As the container, a PDMS device was prepared with a filter membrane on its bottom to ensure nutrient fluid exchange and at the same time block cells (Fig.1). Then osteoblasts were seeded into the porous microspheres before they were filled in the PDMS container. To be mentioned, as the cells grew and proliferated, the extracellular matrix were secreted and the microspheres were thus bound and combined into an integral whole. After incubating for a time, human umbilical vascular endothelial cells were added in the spaces between the microspheres, which were expected to form vascular tissues. Cell adhesion was observed under scanning electron microscope (SEM) while cell viability tests were performed using Live/Dead assay kit. To evaluate the osteogenesis and angiogenesis condition of the bionic tissue, methods such as alkaline phosphatase (ALP) activity assay, real-time PCR assay and immunofluorescence were carried out.
Figure 1. Schematic diagram of the tissue culture device.
Results and Discussion:: According to the scanning electron micrographs, cells showed the microspheres with similar parameters around 400μm and pore sizes about 60μm. Cell viability tests confirmed the material with low cytotoxicity and good biocompatibility. ALP activity assay, alizarin red staining, real-time PCR assay and immunofluorescence results showed smooth bone formation and high expression of growth factors relevent to osteogenesis and angiogenesis (eg. typeⅠcollagen, osteopontin, vascular endothelial growth factor, etc.), which meant the vascularized tissue-engineered bone was built successfully.
Conclusions: With well designed space allocation, different kinds of cells coexisted together harmously to form a integral whole. Our research suggests that porous PLGA microspheres could act as cell carriers for osteoblasts, and consequently pile up to construct vascularized bone tissue in the presence of hemangioblasts.
The National Basic Research Program of China (2012CB619100); The National Natural Science Foundation of China (51232002); The 111 project (B13039)
References:
[1] Santos M I, Reis R L. Vascularization in bone tissue engineering: physiology, current strategies, major hurdles and future challenges[J]. Macromolecular bioscience, 2010, 10(1): 12-27.
[2] Kim T K, Yoon J J, Lee D S, et al. Gas foamed open porous biodegradable polymeric microspheres[J]. Biomaterials, 2006, 27(2): 152-159.