Viable Osteogenic Cells Are Obligatory for Tissue-Engineered Ectopic Bone Formation in Goats

Abstract

In this study we investigated the bone-forming capacity of tissue-engineered (TE) constructs implanted ectopically in goats. As cell survival is questionable in large animal models, we investigated the significance of vitality, and thus whether living cells instead of only the potentially osteoinductive extracellular matrix are required to achieve bone formation. Vital TE constructs of porous hydroxyapatite (HA) covered with differentiated bone marrow stromal cells (BMSCs) within an extracellular matrix (ECM) were compared with identical constructs that were devitalized before implantation. The devitalized implants did contain the potentially osteoinductive ECM. Furthermore, we evaluated HA impregnated with fresh bone marrow and HA only. Two different types of HA granules with a volume of approximately 40 microm were investigated: HA70/800, a microporous HA with 70% interconnected macroporosity and an average pore size of 800 microm, and HA60/400, a smooth HA with 60% interconnected macropores and an average size of 400 microm. Two granules of each type were combined and then treated as a single unit for cell seeding, implantation, and histology. The tissue-engineered samples were obtained by seeding culture-expanded goat BMSCs on the HA and subsequently culturing these constructs for 6 days to allow cell differentiation and ECM formation. To devitalize, TE constructs were frozen in liquid nitrogen according to a validated protocol. Fresh bone marrow impregnation was performed perioperatively (4 mL per implant unit). All study groups were implanted in bilateral paraspinal muscles. Fluorochromes were administered at three time points to monitor bone mineralization. After 12 weeks the units were explanted and analyzed by histology of nondecalcified sections. Bone formation was present in all vital tissue-engineered implants. None of the other groups showed any bone formation. Histomorphometry indicated that microporous HA70/800 yielded more bone than did HA60/400. Within the newly formed bone, the fluorescent labels showed that mineralization had occurred before 5 weeks of implantation and was directed from the HA surface toward the center of the pores. In conclusion, tissue-engineered bone formation in goats can be achieved only with viable constructs of an appropriate scaffold and sufficient BMSCs.

Bone Marrow-Derived Cell Concentrates Have Limited Effects on Osteochondral Reconstructions in the Mini Pig

“…42 It could be demonstrated that allogenic green fluorescence-labeled BMCC survive for approximately 11 weeks in mice, but the amount of new bone formation synthesized by these cells was very limited. 36,37 Transplanted cells usually face a hostile immune system, they lack grown physiological connections with the host tissues, and the supply with nutrition and other factors is problematic. Moreover, the transplanted cells compete directly with mumerous host cell types that are recruited from the organism for the healing process.…”

Relevance of Osteoinductive Biomaterials in Critical‐Sized Orthotopic Defect

“…29,30 Twelve weeks after the implantations, each animal was sacrificed by an overdose of pentobarbital (Euthesaat, Organon, Oss, The Netherlands) and potassium chloride.…”

In vivo bioluminescence imaging study to monitor ectopic bone formation by luciferase gene marked mesenchymal stem cells

“…To regenerate bone tissue in vivo, viable MSCs are required, but whether they directly differentiate or retain proliferative capacity is unclear. 17 We applied BLI to an ectopic implantation model to investigate the fate of MSCs in vivo. BLI has proven to be a powerful technology for noninvasive imaging in animal models of oncological diseases and in bone tissue engineering studies 18 and is often used to measure cell proliferation on the basis of luciferase gene expression.…”