Generation of osteoinductive grafts by three-dimensional perfusion culture of human bone marrow cells into porous ceramic scaffolds

摘要

The main aims of this thesis were (i) to identify and develop a system that could beudreproducibly used to streamline manufacture of osteoinductive grafts based on human bone marrowudstromal cells (BMSC) in the context of regenerative medicine, (ii) to characterize the developedudsystem in order to identify key elements responsible for its reproducible and efficient performance,udand (iii) to extend its use to a sheep cell source, thus opening the way to test the osteoinductivity ofudorthotopic implants in a large animal model.udBone Marrow Stromal Cells (BMSC), which are typically defined by their capacity to adhereudon plastic [1] and form a fibroblastic colony (CFU-f) [2], represent a very low fraction (approximatelyud0.01%) among the nucleated cells of the bone marrow. Therefore, to obtain a sufficient number ofudcells for bone tissue engineering applications, BMSC are typically first selected and expanded inudmonolayer (2D) prior to loading into 3D scaffolds. However, 2D-expansion causes BMSC toudprogressively lose their early progenitor properties and differentiation potential [3-5], and to decreaseudtheir capability to form colonies and to induce bone tissue formation upon ectopic implantation [3],udplacing several potential limits on their clinical utility. To bypass the process of 2D-expansion and itsudassociated limitations, we used an innovative bioreactor-based approach to seed, expand, anduddifferentiate BMSC directly in a 3D ceramic scaffold [6]. Nucleated cells, freshly isolated from a boneudmarrow aspirate, were introduced into the bioreactor system and perfused through the pores of 3Dudceramics for five days, then further cultured under perfusion for an additional two weeks. Using theuddeveloped procedure, BMSC could be seeded and extensively expanded within the 3D environment ofudthe ceramic pores. Interestingly, we found that the 3D-generated constructs contained bothudhemopoietic cells and BMSC, whose relative fractions could be modulated by appropriate mediaudsupplements, and that a consistent fraction of expanded BMSC was clonogenic. In contrast, followingudthe typical 2D-expansion, cells of the hemopoietic lineage could not be maintained, and, consistentlyudwith previous studies, only a minor fraction of expanded BMSC was still clonogenic. When constructsudwere ectopically implanted in nude mice, those engineered in the bioreactor reproducibly generatedudbone tissue that was uniformly distributed throughout the scaffold volume and filled up to 60% of theudceramic pores. In marked contrast, when similar numbers of 2D-expanded BMSC were loaded intoudceramic scaffolds and implanted, bone was infrequently generated, and even in the mostududosteoinductive constructs, it was localized to peripheral regions, filling only 10% of the ceramic poreudvolume [6].udConsidering the need of reproducibility or at least of predictability in the osteoinductive abilityudof the constructs for their standardized clinical use, in order to validate the possibility of extending theuduse of the developed bioreactor-based approach for generating osteoinductive grafts of clinicallyudrelevant size, we then investigated whether a minimum cell density was required for the reproducibleudbone tissue formation. Based on the established association between the higher clonogenicity ofudBMSC expanded in the 3D-system and the more reproducible and extensive osteoinductivity of theudresulting constructs, as compared to those based on 2D-expanded BMSC, we demonstrated thatudpresence or absence of bone in the constructs following ectopic implantation is related not to the totaludnumber of implanted BMSC, but to the number of CFU-f present in the construct at the time ofudimplantation. In particular, we identified an apparent threshold in the amount of CFU-f discriminatingudbetween osteoinductive and not osteoinductive constructs.udThe developed bioreactor-based approach has been validated in a heterotopic model. Beforeudenvisioning a clinical trial in human, a study in a large animal model is needed to validate the safetyudand the surgical feasibility of the overall procedure. Thus, in the perspective of testing our noveludapproach for repairing experimental bone defects in a sheep model, it was first necessary to validateudour system using ovine BMSC. We demonstrated that osteoinductive constructs can be generated byudperfusing 3D ceramic scaffolds with the nucleated cell fraction of ovine bone marrow aspirates [7].udOngoing studies in the context of an EU-funded Project are aimed at testing the capability of theudgenerated constructs to repair large bone defects in sheep (i.e. defects around titanium implantsudinserted into trabecular bone of the proximal humerus, and postero-lateral spinal fusion in lumbarregion).