Expansion of human mesenchymal stromal cells from fresh bone marrow in a 3D scaffold-based system under direct perfusion.

Mesenchymal stromal/stem cell (MSC) expansion in conventional monolayer culture on plastic dishes (2D) leads to progressive loss of functionality and thus challenges fundamental studies on the physiology of skeletal progenitors, as well as translational applications for cellular therapy and molecular medicine. Here we demonstrate that 2D MSC expansion can be entirely bypassed by culturing freshly isolated bone marrow nucleated cells within 3D porous scaffolds in a perfusion-based bioreactor system. The 3D-perfusion system generated a stromal tissue that could be enzymatically treated to yield CD45- MSC. As compared to 2D-expanded MSC (control), those derived from 3D-perfusion culture after the same time (3 weeks) or a similar extent of proliferation (7-8 doublings) better maintained their progenitor properties, as assessed by a 4.3-fold higher clonogenicity and the superior differentiation capacity towards all typical mesenchymal lineages. Transcriptomic analysis of MSC from 5 donors validated the robustness of the process and indicated a reduced inter-donor variability and a significant upregulation of multipotency-related gene clusters following 3D-perfusion--as compared to 2D-expansion. Interestingly, the differences in functionality and transcriptomics between MSC expanded in 2D or under 3D-perfusion were only partially captured by cytofluorimetric analysis using conventional surface markers. The described system offers a multidisciplinary approach to study how factors of a 3D engineered niche regulate MSC function and, by streamlining conventional labor-intensive processes, is prone to automation and scalability within closed bioreactor systems.

Spatial and temporal patterns of bone formation in ectopically pre-fabricated, autologous cell-based engineered bone flaps in rabbits.

Biological substitutes for autologous bone flaps could be generated by combining flap pre-fabrication and bone tissue engineering concepts. Here, we investigated the pattern of neotissue formation within large pre-fabricated engineered bone flaps in rabbits. Bone marrow stromal cells from 12 New Zealand White rabbits were expanded and uniformly seeded in porous hydroxyapatite scaffolds (tapered cylinders, 10-20 mm diameter, 30 mm height) using a perfusion bioreactor. Autologous cell-scaffold constructs were wrapped in a panniculus carnosus flap, covered by a semipermeable membrane and ectopically implanted. Histological analysis, substantiated by magnetic resonance imaging (MRI) and micro-computerized tomography scans, indicated three distinct zones: an outer one, including bone tissue; a middle zone, formed by fibrous connective tissue; and a central zone, essentially necrotic. The depths of connective tissue and of bone ingrowth were consistent at different construct diameters and significantly increased from respectively 3.1+/-0.7 mm and 1.0+/-0.4 mm at 8 weeks to 3.7+/-0.6 mm and 1.4+/-0.6 mm at 12 weeks. Bone formation was found at a maximum depth of 1.8 mm after 12 weeks. Our findings indicate the feasibility of ectopic pre-fabrication of large cell-based engineered bone flaps and prompt for the implementation of strategies to improve construct vascularization, in order to possibly accelerate bone formation towards the core of the grafts.

The osteogenicity of implanted engineered bone constructs is related to the density of clonogenic bone marrow stromal cells.

Reproducible osteogenicity is a key requirement for the clinical use of bone substitutes based on bone marrow stromal cells (BMSCs) and three-dimensional (3D) scaffolds. In this study we addressed whether a minimal cell density is required for ectopic osteogenicity of constructs generated using a recently developed perfusion system for seeding and culturing human BMSCs on 3D scaffolds. Cells from human bone marrow aspirates were directly seeded and expanded for 3 weeks within the pores of ceramic-based scaffolds, using a perfusion bioreactor. The resulting constructs were either implanted subcutaneously in nude mice, to determine their capacity to generate bone tissue, or digested to retrieve the expanded cells and assess their number, phenotype and clonogenic capacity. The final number of BMSCs in the constructs was correlated neither to the initial number of seeded cells, nor to the subsequent bone formation. Instead, the final number of clonogenic BMSCs in the constructs was positively correlated to the initial number of BMSCs seeded, and was significantly higher in osteogenic than in non-osteogenic constructs. These results indicate that clonogenic cells play a crucial role in determining the osteogenicity of engineered bone substitutes. Possible ways to quantify the density of clonogenic cells as a quality control parameter to predict potency of BMSC-based constructs are discussed.

Three-dimensional perfusion culture of human bone marrow cells and generation of osteoinductive grafts.

Three-dimensional (3D) culture systems are critical to investigate cell physiology and to engineer tissue grafts. In this study, we describe a simple yet innovative bioreactor-based approach to seed, expand, and differentiate bone marrow stromal cells (BMSCs) directly in a 3D environment, bypassing the conventional process of monolayer (two-dimensional [2D]) expansion. The system, based on the perfusion of bone marrow-nucleated cells through porous 3D scaffolds, supported the formation of stromal-like tissues, where BMSCs could be cocultured with hematopoietic progenitor cells in proportions dependent on the specific medium supplements. The resulting engineered constructs, when implanted ectopically in nude mice, generated bone tissue more reproducibly, uniformly, and extensively than scaffolds loaded with 2D-expanded BMSCs. The developed system may thus be used as a 3D in vitro model of bone marrow to study interactions between BMSCs and hematopoietic cells as well as to streamline manufacture of osteoinductive grafts in the context of regenerative medicine.