Stirred flow bioreactor modulates chondrocyte growth and extracellular matrix biosynthesis in chitosan scaffolds

Abstract

The aim of this study is to show the favorable effect of simple dynamic culture conditions on chondrogenesis of previously expanded human chondrocytes seeded in a macroporous scaffold with week cell-pore walls adhesion. We obtained enhanced chondrogenesis by the combination of chitosan porous supports with a double micro- and macro-pore structure and cell culture in a stirring bioreactor. Cell-scaffold constructs were cultured under static or mechanically stimulated conditions using an intermittent stirred flow bioreactor during 28 days. In static culture, the chondrocytes were homogeneously distributed throughout the scaffold pores; cells adhered to the scaffold pore walls, showed extended morphology and were able to proliferate. Immunofluorescense and biochemical assays showed abundant type I collagen deposition at day 28. However, the behavior of chondrocytes submitted to mechanical stimuli in the bioreactor was completely different. Mechanical loading influenced cell morphology and extracellular matrix composition. Under dynamic conditions, chondrocytes kept their characteristic phenotype and tended to form cell aggregates surrounded by a layer of the main components of the hyaline cartilage extracellular matrix, type II collagen, and aggrecan. An enhanced aggrecan and collagen type II production was observed in engineered cartilage constructs cultured under stirred flow compared with those cultured under static conditions.

3D Dynamic Culture of Rabbit Articular Chondrocytes Encapsulated in Alginate Gel Beads Using Spinner Flasks for Cartilage Tissue Regeneration

“…On the other hand, mechanical stimulation is beneficial in the chondrogenesis [ 27 ]. Dynamic culture in spinner flasks has been suggested to be able to apply beneficial mechanical stimulation on chondrocytes [ 21 , 22 ]. Therefore, in the present study, whether combining 3D and dynamic culture would be beneficial for maintaining the chondrocyte phenotype during in vitro culture was proposed.…”

“…On the other hand, it was reported that in response to the hydrostatic pressure, chondrocytes encapsulated in alginate gel beads secreted more glycosaminoglycans (GAG), a characteristic cartilaginous ECM component [ 20 ]. Additionally, fluid shear force in a spinner flask was implicated in stimulating ECM production by chondrocytes seeded in chitosan scaffolds [ 21 ]. Recently, Lee et al found that culture of cellular aggregates of dedifferentiated chondrocytes in a spinner flask was able to induce the redifferentiation [ 22 ].…”

Scaffolds for 3D Cell Culture and Cellular Agriculture Applications Derived From Non-animal Sources

“…On the other hand, the emergence of recombinant collagen extracted from plants has important advantages as a scaffold in its own right or if used to functionalize the surfaces of the materials above. Furthermore, these materials have been modified, either as microspheres or bulk, to possess the porosity necessary for diffusion of nutrients through dynamic or static bioreactors (Oh et al, 2009;Wu et al, 2011;García Cruz et al, 2012;Varley et al, 2017;Huang et al, 2018;Specht et al, 2018). In order to scale an animal-free product with similarities to that of native animal tissue, the need for fetal bovine serum, costeffective engineering processes, antibiotic dependence, scaffold development, and cell line(immortalized vs. primary and cell co-culture) needs to be addressed (Specht et al, 2018;Stephens et al, 2018;Lynch and Pierrehumbert, 2019).…”

Human platelet-rich plasma improves the nesting and differentiation of human chondrocytes cultured in stabilized porous chitosan scaffolds

“…5 All specimens were coated with a conductive layer of sputtered gold. An accelerating voltage of 20 kV was used to obtain micrographs.…”

“… 3 Different strategies can improve the chondrogenic ability of chitosan, such as its conjugation to transforming growth factor-beta (TGF-β; alone or encapsulated in microparticles), association with hydrogels including type II collagen or platelet-rich plasma (PRP), or the use of specific bioreactors. 4 – 6 …”