We describe a novel bioreactor system for tissue engineering of bone that enables cultivation of up to six tissue constructs simultaneously, with direct perfusion and imaging capability. The bioreactor was used to investigate the relative effects of initial seeding density and medium perfusion rate on the growth and osteogenic differentiation patterns of bone marrow-derived human mesenchymal stem cells (hMSCs) cultured on three-dimensional scaffolds. Fully decellularized bovine trabecular bone was used as a scaffold because it provided suitable ''biomimetic'' topography, biochemical composition, and mechanical properties for osteogenic differentiation of hMSCs. Trabecular bone plugs were completely denuded of cellular material using a serial treatment with hypotonic buffers and detergents, seeded with hMSCs, and cultured for 5 weeks. Increasing seeding density from 30Â10 6 cells/mL to 60Â10 6 cells/mL did not measurably influence the characteristics of tissue-engineered bone, in contrast to an increase in the perfusion rate from 100 lms À1 to 400 lms À1 , which radically improved final cell numbers, cell distributions throughout the constructs, and the amounts of bone proteins and minerals. Taken together, these findings suggest that the rate of medium perfusion during cultivation has a significant effect on the characteristics of engineered bone.
Objective The objective of the study was to investigate the combined effects of three sets of regulatory factors: cell pre-differentiation, soluble factors and medium perfusion on spatial control of human mesenchymal stem cell (hMSC) differentiation into cells forming the cartilaginous and bone regions in engineered osteochondral constructs. Design Bone-marrow derived hMSCs were expanded in their undifferentiated state (UD) or pre-differentiated (PD) in monolayer culture, seeded into biphasic constructs by interfacing agarose gels and bone scaffolds and cultured for 5 weeks either statically (S) or in a bioreactor (BR) with perfusion of medium through the bone region. Each culture system was operated with medium containing either chondrogenic supplements (C) or a cocktail (Ck) of chondrogenic and osteogenic supplements. Results The formation of engineered cartilage in the gel region was most enhanced by using undifferentiated cells and chondrogenic medium, whereas the cartilaginous properties were negatively affected by using pre-differentiated cells or the combination of perfusion and cocktail medium. The formation of engineered bone in the porous scaffold region was most enhanced by using pre-differentiated cells, perfusion and cocktail medium. Perfusion also enhanced the integration of bone and cartilage regions. Conclusions Pre-differentiation of hMSCs before seeding on scaffold was beneficial for bone but not for cartilage formation. The combination of medium perfusion and cocktail medium inhibited chondrogenesis of hMSCs. Perfusion improved the cell and matrix distribution in the bone region and augmented theintegration at the bone-cartilage interface. Osteochondral grafts can be engineered by differentially regulating the culture conditions in the two regions of the scaffold seeded with hMSCs (hydrogel for cartilage, perfused porous scaffold for bone).
Tissue engineering is expected to help us outlive the failure of our organs by enabling the creation of tissue substitutes capable of fully restoring the original tissue function. Degenerative joint disease, which affects one-fifth of the US population and is the country's leading cause of disability, drives current research of actively growing, functional tissue grafts for joint repair. Toward this goal, living cells are used in conjunction with bio-material scaffolds (serving as instructive templates for tissue development) and bioreactors (providing environmental control and molecular and physical regulatory signals). In this review, we discuss the requirements for engineering customized, anatomically-shaped, stratified grafts for joint repair and the challenges of designing these grafts to provide immediate functionality (load bearing, structural support) and long-term regeneration (maturation, integration, remodeling).
Direct-current electric fields mediate motility (galvanotaxis) of many cell types. In 3T3 fibroblasts, electric fields increased the proportion, speed and cathodal directionality of motile cells. Analogous to fibroblasts' spontaneous migration, we initially hypothesized that reorientation of microtubule components modulates galvanotaxis. However, cells with intact microtubules did not reorient them in the field and cells without microtubules still migrated, albeit slowly, thus disproving the hypothesis. We next proposed that, in monolayers wounded and placed in an electric field, reorientation of microtubule organizing centers and stable, detyrosinated microtubules towards the wound edge is necessary and/or sufficient for migration. This hypothesis was negated because field exposure mediated migration of unoriented, cathode-facing cells and curtailed migration of oriented, anode-facing cells. This led us to propose that ablating microtubule detyrosination would not affect galvanotaxis. Surprisingly, preventing microtubule detyrosination increased motility speed, suggesting that detyrosination inhibits galvanotaxis. Microtubules might enhance adhesion/de-adhesion remodeling during galvanotaxis; thus, electric fields might more effectively mediate motility of cells poorly or dynamically attached to substrata. Consistent with this hypothesis, incompletely spread cells migrated more rapidly than fully spread cells. Also, overexpression of PAK4, a Cdc42-activated kinase that decreases adhesion, enhanced galvanotaxis speed, whereas its lack decreased speed. Thus, electric fields mediate fibroblast migration via participation of microtubules and adhesive components, but their participation differs from that during spontaneous motility.
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