%0 Generic
%A Hammersen, Tim Dieter
%C Heidelberg
%D 2023
%F heidok:33902
%R 10.11588/heidok.00033902
%T Physicochemical stimuli to enhance the quality of human engineered cartilage: the role of osmolarity and calcium
%U https://archiv.ub.uni-heidelberg.de/volltextserver/33902/
%X Due to the low regenerative capacity of articular cartilage, regenerative approaches are needed to treat cartilage defects and to restore the function of the tissue in the joint. However, a general drawback of current cartilage replacement tissues is an insufficient deposition of its main molecular components, type II collagen and proteoglycan. As a result, the tissue cannot withstand the demanding mechanical conditions in the joint. Recent studies of our group achieved an acute stimulation of cartilage matrix synthesis by a defined mechanical loading protocol which depended on the tissue’s glycosaminoglycan (GAG)-content and its associated fixed charge density (FCD). However, to what extend mechano-induced physicochemical sub-stimuli contribute to cartilage matrix production remains unclear. Identifying the decisive sub-parameter that contributes to load-induced stimulation of cartilage matrix synthesis would provide an easily applicable stimulus to optimize the quality of cartilage replacement tissues. Due to the essential role of osmotic pressure for cartilage function, hyperosmotic challenge appears as an important sub-parameter of the loading-response. However, the contribution of acute hyperosmotic pressure to cartilage homeostasis is unclear and models that take a cartilage typical FCD into consideration are required. Interestingly, long-term maturation of animal chondrocytes under hyperosmotic conditions enhanced the matrix content of engineered cartilage but this was so far never investigated for human 3D-cultured chondrocytes. Thus, the aim of this this study was to elucidate whether acute hyperosmotic stimulation, as a sub-parameter of mechanical compression, regulates cartilage matrix synthesis in a human engineered cartilage model at low and high FCD. In parallel, it was investigated whether long-term hyperosmotic stimulation can enhance the matrix synthesis and deposition of cartilage replacement tissue. To achieve these aims, human engineered cartilage was pre-matured for 3 or 35 days to develop a cartilage-like matrix of low or high FCD. Acute hyperosmotic stimulation on day 3 and on day 35 for 3 to 24 hours indicated that the known mechano-response markers ERK1/2, p38, NFAT5, FOS and FOSB are also immediate osmo-response markers, irrespective of the FCD content of the tissue. Opposite to previous results from mechanical loading studies, a downregulation of pro-chondrogenic SOX9 protein and BMP pathway activity indicated an anti-chondrogenic effect of short-term hyperosmotic stimulation on chondrocytes. However, this did not lead to changes in cartilage matrix synthesis at low and at high FCD. Thus, although acute hyperosmotic stimulation and mechanical compression partly triggered similar response pathways, short-term hyperosmotic pressure was no major player to influence the regulation of cartilage matrix synthesis.  In the context of long-term hyperosmotic stimulation, previous studies suggested a role of the extracellular calcium microenvironment for cartilage matrix synthesis and deposition. Since articular chondrocytes (AC) and mesenchymal stromal cells (MSC) are often used cell types for the design of cartilage replacement tissues, the response of both cell types to long-term hyperosmotic stimulation was investigated using extracellular calcium. Interestingly, the here obtained data revealed that long-term hyperosmotic calcium stimulation for 35 days compromised cartilage matrix formation in AC-based cartilage replacement tissue but promoted the cartilage matrix formation in neocartilage generated from MSC. Investigation of pro- and anti-chondrogenic signaling pathways after long-term calcium stimulation indicated a specific induction of catabolic S100A4 and PTHLH expression in AC. Stimulating AC with recombinant human PTHrP1-34 peptide partly reproduced the calcium-mediated reduction of cartilage matrix deposition, suggesting a role of PTHrP for impaired cartilage matrix formation. Importantly, the inverse regulation of GAG synthesis in AC and MSC-derived chondrocytes was calcium-specific and not caused by general hyperosmotic effects. Long-term extracellular calcium stimulation, therefore, provides a novel means to enhance the cartilage matrix content of MSC-based engineered cartilage whereas such conditions should be avoided during AC neocartilage formation.  Overall, this study provides important information on the role of physicochemical stimuli for cartilage matrix formation in human engineered cartilage. It was demonstrated that acute hyperosmotic pressure was no effective stimulus to influence cartilage matrix synthesis, and further studies are now needed to determine the contribution other load-induced sub-parameter for GAG synthesis. Furthermore, this study indicated that long-term high extracellular calcium treatment provides a novel means to enhance the quality of MSC-based cartilage replacement tissue by stimulating GAG synthesis and GAG deposition. For the application in osteochondral tissue engineering approaches, this implies that MSC should be the first choice for cartilage matrix deposition in the vicinity of resorbable calcified bone replacement materials. However, studies are now needed to confirm the here observed effects of soluble extracellular calcium using resorbable bone replacement material as a potential calcium source.