Effect of reduced oxygen tension and long-term mechanical stimulation on chondrocyte-polymer constructs

Abstract

We have investigated the influence of long-term confined dynamic compression and surface motion under low oxygen tension on tissue-engineered cell-scaffold constructs. Porous polyurethane scaffolds (8 mm x 4 mm) were seeded with bovine articular chondrocytes and cultured under normoxic (21% O(2)) or hypoxic (5% O(2)) conditions for up to 4 weeks. By means of our joint-simulating bioreactor, cyclic axial compression (10-20%; 0.5 Hz) was applied for 1 h daily with a ceramic ball, which simultaneously oscillated over the construct surface (+/-25 degrees; 0.5 Hz). Culture under reduced oxygen tension resulted in an increase in mRNA levels of type II collagen and aggrecan, whereas the expression of type I collagen was down-regulated at early time points. A higher glycosaminoglycan content was found in hypoxic than in normoxic constructs. Immunohistochemical analysis showed more intense type II and weaker type I collagen staining in hypoxic than in normoxic cultures. Type II collagen gene expression was slightly elevated after short-term loading, whereas aggrecan mRNA levels were not influenced by the applied mechanical stimuli. Of importance, the combination of loading and low oxygen tension resulted in a further down-regulation of collagen type I mRNA expression, contributing to the stabilization of the chondrocytic phenotype. Histological results confirmed the beneficial effect of mechanical loading on chondrocyte matrix synthesis. Thus, mechanical stimulation combined with low oxygen tension is an effective tool for modulating the chondrocytic phenotype and should be considered when chondrocytes or mesenchymal stem cells are cultured and differentiated with the aim of generating cartilage-like tissue in vitro.

Physical Stimulation of Chondrogenic Cells In Vitro: A Review

“…In terms of stabilization of the chondrogenic phenotype, an additive effect of hypoxia, combined with compression and shear, has been observed [96]. Different approaches have been taken to engineer tissues that mimic the organization of natural articular cartilage [49].…”

Three-dimensional cultures of osteogenic and chondrogenic cells: A tissue engineering approach to mimic bone and cartilage in vitro

“…The great advantage of the polyurethane scaffolds for cartilage tissue engineering is their mechanical properties that allow the application of mechanical stimulation. The importance of mechanical stimulation in developing cartilage models has been confirmed by applying a dynamic compression to the scaffolds; this treatment led to an increase of proteoglycan and Type II collagen expression, thus suggesting its active role in maintaining the chondrocytic phenotype (Wernike et al, 2008). A further increase of these chondrogenic markers was observed coupling a dynamic compression with a low oxygen tension in the 3D cultures of osteogenic and chondrogenic cells medium (Wernike et al, 2008).…”

“…The importance of mechanical stimulation in developing cartilage models has been confirmed by applying a dynamic compression to the scaffolds; this treatment led to an increase of proteoglycan and Type II collagen expression, thus suggesting its active role in maintaining the chondrocytic phenotype (Wernike et al, 2008). A further increase of these chondrogenic markers was observed coupling a dynamic compression with a low oxygen tension in the 3D cultures of osteogenic and chondrogenic cells medium (Wernike et al, 2008). Thus, these results further confirmed the importance of the combination between mechanical stimulation and low oxygen tension as a tool for modulating chondrocyte phenotype in order to obtain a cartilage-like tissue.…”

Tissue Engineering von Knorpelersatzgewebe – Mechanische Stimulation in der In-vitro-Kultivierung von humanen Chondrozyten

“…Vor allem auf dedifferenzierte Chondrozyten hat ein niedriger, den physiologischen Gelenkverhältnissen entsprechender Sauerstoff-Gehalt differenzierende Wirkung im Sinne einer gesteigerten Typ-II-Kollagen-Synthese und Biosyntheseaktivität (Domm et al, 2002). Auch eine geringere Typ-I-Kollagen-Synthese konnte für die Kultivierung unter Hypoxie-und Stimulationsbedingungen nachgewiesen werden (Wernike et al, 2008). Die Kultivierung der Chondrozyten in der vorliegenden Studie fand unter Luftverhältnissen mit entsprechend 21 Vol-% Sauerstoff statt.…”