Time-dependent processes in stem cell-based tissue engineering of articular cartilage

Abstract

Articular cartilage (AC), situated in diarthrodial joints at the end of the long bones, is composed of a single cell type (chondrocytes) embedded in dense extracellular matrix comprised of collagens and proteoglycans. AC is avascular and alymphatic and is not innervated. At first glance, such a seemingly simple tissue appears to be an easy target for the rapidly developing field of tissue engineering. However, cartilage engineering has proven to be very challenging. We focus on time-dependent processes associated with the development of native cartilage starting from stem cells, and the modalities for utilizing these processes for tissue engineering of articular cartilage.

Figure 1. Sequence of events during native chondrogenesis

The different stages are represented schematically, showing the temporal patterns of growth, differentiation and transcription factors as well as changes in cell morphology. The extracellular matrix (ECM) proteins that distinguish the different stages are indicated below by the gradients of expression. Darker zones in the gradients correlate to higher levels of expression.

Figure 2. Articular cartilage stratification zones correlate to the cell differentiation stages

Chondrocytes in the most superficial or tangential zone (Zone I) are small, immature and flattened, and collagen type II fibrils are arranged tangentially to the surface. Transitional zone (Zone II) is composed of proliferating spherical chondrocytes, less strongly bound in the ECM with collagen fibers oriented slightly perpendicularly to the surface. In the thickest, deep radial zone (Zone III), chondrocytes are the largest, with hypertrophic phenotype and usually arranged in lacunae larger than in the previous zone. Both chondrocytes and collagen fibers are oriented in vertical columns perpendicular to the subchondral plate. Below are the tidemark, a basophilic line which straddles the boundary between calcified and uncalcified cartilage; calcified cartilage where the chondrocytes undergo apoptosis and the subchondral bone.

Figure 3. Stages in chondrogenic differentiation of embryonic and iPS cells

Pluripotent cell types (ESC and iPS) have to differentiate into multipotent MSCs in order to form precartilage condensation required for efficient further differentiation into chondrocytes. Chondrocytes, as fully differentiated cells, have lower differentiation potential compared to fibroblasts which can still be induced to differentiation as well as to conversion to iPS cells.

Figure 4. Proposed steps in the biomimetic protocol for cartilage tissue engineering

Step 1: Directed differentiation of hESCs or iPS into MSCs-like cells (if the starting cell type is already MSC-like then this step is to be omitted). Step 2: Molecular stimulation of proliferation where MSC-like cells should be induced to undergo aggregation and subsequent condensation and determination. Starting with Step 2, the cells should be in 3D environment i.e in hydrogels or scaffolds to achieve the most similar native-like physical environment. Step 3: The cells are presumably at the level of immature chondrocytes, and molecular induction of full chondrogenesis should be implemented. Step 4: Mechanical stimulation. Step 5: Hydrogel degradation (see details in text). Step 6: Long-term mechanical conditioning.