Tribological loading of cartilage and chondrogenic cells.

Novel cartilage regeneration therapies often look promising in-vitro but fail when implanted in vivo. One of the possible reasons for this discrepancy is the simplified, static in-vitro chondrogenesis models typically used. Complex mechanical stimulation plays a key role in physiological cartilage and chondrogenic cell metabolism, including the development of cartilage structure, yet it is routinely lacking during in-vitro studies. Multiaxial load bioreactors are becoming more widespread and offer advantages over more simple loading devices. Within this article, we highlight some of the important findings from in-vitro assays and key aspects relating to tribological loading of cartilage and chondrogenic cells.

A multi-well bioreactor for cartilage tissue engineering experiments.

Cartilage tissue engineering necessitates the right mechanical cues to regenerate impaired tissue. For this reason, bioreactors can be employed to induce joint-relevant mechanical loading, such as compression and shear. However, current articulating joint bioreactor designs are lacking in terms of sample size and usability. In this paper, we describe a new, simple-to-build and operate, multi-well kinematic load bioreactor and investigate its effect on the chondrogenic differentiation of human bone marrow-derived stem cells (MSCs). We seeded MSCs into a fibrin-polyurethane scaffold and subsequently exposed the samples to a combination of compression and shear for 25 days. The mechanical loading activates transforming growth factor beta 1, upregulates chondrogenic genes, and increases sulfated glycosaminoglycan retention within the scaffolds. Such a higher-throughput bioreactor could be operated in most cell culture laboratories, dramatically accelerating and improving the testing of cells, new biomaterials, and tissue-engineered constructs.

Isolation and In Vitro Chondrogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stromal Cells.

Bone marrow-derived mesenchymal stromal cells (BM-MSC) are widely studied in the field of cartilage regeneration due to their capacity to differentiate into chondrocytes under specific in vitro culture conditions. This chapter describes the isolation of MSC from bone marrow aspirate, their expansion in monolayer, and the chondrogenic differentiation in pellet culture.

The Dimethylmethylene Blue Assay (DMMB) for the Quantification of Sulfated Glycosaminoglycans.

The 1,9-dimethylmethylene blue (DMMB) assay enables the detection of sulfated glycosaminoglycans (sGAGs). This assay can be used to quickly quantify the sGAG content in a large number of samples using spectrophotometry. While this widespread assay appears straightforward, there are certain pitfalls that need to be considered.

Co-culture of Human Articular Chondrocytes Seeded in Polyurethane Scaffolds and Human Mesenchymal Stromal Cells Encapsulated in Alginate Beads.

Co-culturing is an essential method for unravelling the importance of cross talk and cellular interaction. This chapter describes the preparation of an indirect co-culture technique based on encapsulation of chondrocytes and mesenchymal stromal cells in polyurethane scaffolds and alginate beads, respectively. This way, both cell populations can communicate through paracrine effects in the absence of cell-cell contact. Due to the mechanical properties of polyurethane, this model can be employed in mechanobiology studies. The resulting engineered cultures can provide a more realistic environment, recreating the complex joints' microenvironment and physiology.

Optimization of loading protocols for tissue engineering experiments.

Tissue engineering (TE) combines cells and biomaterials to treat orthopedic pathologies. Maturation of de novo tissue is highly dependent on local mechanical environments. Mechanical stimulation influences stem cell differentiation, however, the role of various mechanical loads remains unclear. While bioreactors simplify the complexity of the human body, the potential combination of mechanical loads that can be applied make it difficult to assess how different factors interact. Human bone marrow-derived mesenchymal stromal cells were seeded within a fibrin-polyurethane scaffold and exposed to joint-mimicking motion. We applied a full factorial design of experiment to investigate the effect that the interaction between different mechanical loading parameters has on biological markers. Additionally, we employed planned contrasts to analyze differences between loading protocols and a linear mixed model with donor as random effect. Our approach enables screening of multiple mechanical loading combinations and identification of significant interactions that could not have been studied using classical mechanobiology studies. This is useful to screen the effect of various loading protocols and could also be used for TE experiments with small sample sizes and further combinatorial medication studies.

Mesenchymal Stromal Cell Differentiation for Generating Cartilage and Bone-Like Tissues In Vitro.

In the field of tissue engineering, progress has been made towards the development of new treatments for cartilage and bone defects. However, in vitro culture conditions for human bone marrow mesenchymal stromal cells (hBMSCs) have not yet been fully defined. To improve our understanding of cartilage and bone in vitro differentiation, we investigated the effect of culture conditions on hBMSC differentiation. We hypothesized that the use of two different culture media including specific growth factors, TGFß1 or BMP2, as well as low (2% O2) or high (20% O2) oxygen tension, would improve the chondrogenic and osteogenic potential, respectively. Chondrogenic and osteogenic differentiation of hBMSCs isolated from multiple donors and expanded under the same conditions were directly compared. Chondrogenic groups showed a notable upregulation of chondrogenic markers compared with osteogenic groups. Greater sGAG production and deposition, and collagen type II and I accumulation occurred for chondrogenic groups. Chondrogenesis at 2% O2 significantly reduced ALP gene expression and reduced type I collagen deposition, producing a more stable and less hypertrophic chondrogenic phenotype. An O2 tension of 2% did not inhibit osteogenic differentiation at the protein level but reduced ALP and OC gene expression. An upregulation of ALP and OC occurred during osteogenesis in BMP2 containing media under 20% O2; BMP2 free osteogenic media downregulated ALP and also led to higher sGAG release. A higher mineralization was observed in the presence of BMP2 during osteogenesis. This study demonstrates how the modulation of O2 tension, combined with tissue-specific growth factors and media composition can be tailored in vitro to promote chondral or endochondral differentiation while using the same donor cell population.