Chondrogenesis is a process that is important for the creation of chondrocytes both during embryogenesis as well as in adult life (e.g., during skeletal tissue repair). The process begins with the aggregation and condensation of loose mesenchyme. Factors such as the bone morphogenetic proteins (BMPs) are known to play critical roles in the compaction of mesenchymal cells and the shaping of the condensations (1
). During this early step in chondrogenesis, the condensing mesenchyme expresses various ECM and cell adhesion molecules, including the IIa splice form of type II collagen [Col2a1(IIa)] (2
), N-cadherin (Ncad) (4
), N-cam (Ncam1) (5
), and tenascin C (Tnc) (6
), while also broadly expressing an important transcription factor, SRY-box 9 (Sox9) (Figure A, i, and Figure B). The Sox family of transcription factors has various roles during chondrogenesis and chondrocyte differentiation, although Sox9 is the primary determinant during the early stages of chondrogenesis (7
). As the mesenchyme differentiates into chondrocytes, the cells begin to produce an ECM rich in the IIb splice form of type II collagen [Col2a1(IIb)] and aggrecan (Agc). During development, following early chondrocyte differentiation, the cells rapidly proliferate, enlarging the cartilage templates that preform individual skeletal elements (Figure A, i and ii, and Figure C). Cells near the center of each growing element eventually withdraw from the cell cycle, initiating the process of hypertrophic differentiation (Figure A, iii). During the process of hypertrophic differentiation (i.e., maturation), chondrocytes enlarge, terminally differentiate, mineralize, and ultimately undergo apoptosis. As the chondrocytes die and degrade, their residual cartilage matrix serves as a scaffold for further mineral deposition and bone formation and turnover by invading osteoblasts and osteoclasts. In parallel, the degraded cartilage becomes vascularized by surrounding blood vessels to establish the bone marrow cavity (Figure A, iv).
Cellular events and molecular markers of chondrogenesis, chondrocyte differentiation, and AC development and maintenance.
The cartilage GPs located at each end of a developing long bone are generated through a continual process of chondrocyte proliferation, differentiation, and removal. During this process, distinct zones of cells are both morphologically and molecularly identifiable. In the embryo, cells near the distal ends of cartilage elements, periarticular chondrocytes, appear round in shape and express early chondrocyte lineage markers such as Sox9, Col2a1(IIb), Agc1,
and low levels of FGF receptor 3 (Fgfr3
), as well as specific downstream targets of the indian hedgehog (Ihh) signaling pathway (10
) (Figure A, ii, and Figure C). As these cells proliferate and undergo the early steps of maturation, they flatten and form columns parallel to the axis of longitudinal growth. The flat columnar chondrocytes, known to be the most proliferative cells in the cartilage element, express low levels of Runx2
and Osterix (Osx
) and high levels of Fgfr3, Nkx3.2
, and Ptc1
. Eventually, these columnar cells begin the process of hypertrophy and withdraw from the cell cycle. The prehypertrophic chondrocytes enlarge slightly and initiate expression of Ihh,
parathyroid hormone–related protein receptor (PTHrP-R
), as well as increase expression of alkaline phosphatase (AP
), and the important regulatory transcription factors Runx2
, which aid in chondrocyte differentiation as well as being required for mineralization of the cartilage (14
). As hypertrophy proceeds, the cells continue to enlarge, generate a mineralized matrix, and further enhance their expression of type X collagen (Col10a1
, and several growth factors that coordinate chondrocyte proliferation and differentiation. These factors are critical for signaling to the surrounding perichondrial cells to induce these cells to differentiate into osteoblast lineage cells that further mineralize the matrix after endochondral ossification is complete. Both hypertrophic chondrocytes and the more terminally differentiated hypertrophic chondrocytes located in the center of the cartilage produce high levels of Vegfa
, which is thought to aid in vascularization of the dying cartilage (17
). Only the most terminally differentiated hypertrophic chondrocytes express the matrix degrading enzyme Mmp13
). MMP13 is an enzyme that controls degradation of the cartilage matrix, a process that precedes mineralization by osteoblasts, that is required for creation of the bone marrow space, and that supports vascular invasion, which provides the cells that will populate the bone marrow (Figure A, iii and iv, and Figure C). As a point of reference, Figure C depicts a summary of the genetic program in chondrocytes undergoing hypertrophic differentiation.
During early postnatal development, epiphyseal chondrocytes (immature chondrocytes located in the center of the epiphyses of long bones) undergo maturation similar to the chondrocyte differentiation process described above that occurs during embryonic skeletogenesis. These cells differentiate, hypertrophy, undergo apoptosis, and are replaced by invading vasculature and osteoblasts, creating the secondary center of ossification (labeled 2° in Figure ). The secondary center of ossification separates the only two areas of remaining cartilage within individual long bones of the adult skeleton: the AC and the mature GP cartilage (Figure A, v). When chondrogenesis and chondrocyte maturation occur in the adult, such as during fracture repair, no secondary centers of ossification are formed, nor is a novel limb generated, but the processes progress essentially the same way and hypertrophic chondrocytes are ultimately required for analogous purposes — the initiation of mineralization and the induction of vascular invasion.
In the mouse, the development of AC begins during embryogenesis at sites of synovial joint formation. These joints develop through processes including patterning of the joint site, interzone formation, cavitation, and morphogenesis (reviewed in refs. 19
). It has been demonstrated that articular chondrocytes are formed from interzone cells during development. Following embryonic joint formation and postnatal growth, the adult skeleton maintains the cellularity and phenotype of AC via mechanisms largely unknown, whereas GP cartilage completely erodes following adolescent growth in humans. Adult AC is maintained as four distinct cellular zones: the superficial zone (SZ), the intermediate zone (IZ), the radial zone (RZ), and the zone of calcified cartilage (Figure B). The SZ consists of 1–2 layers of flattened chondrocytes expressing proteoglycan 4 (Prg4
) (also known as SZ protein and lubricin), Sox9, Col2a1(IIb)
, Agc1, Tnc
, and low levels of cartilage intermediate layer protein (Cilp
). Chondrocytes of the IZ are round in appearance and express many of the same molecules as the SZ chondrocytes, although they do not express Prg4
and express higher levels of Cilp
. Below the IZ reside the RZ chondrocytes and the zone of calcified cartilage. The RZ chondrocytes express markers of chondrocyte differentiation and hypertrophy such as Col10a1
. Each of the AC regions is normally maintained throughout adulthood unless stress related injury, inflammation, or a genetic defect leads to the loss of either the signals required to maintain these cells or the signals required to inhibit excessive differentiation of these cells. Disruption or impairment of the signals that inhibit excessive differentiation is believed to provide the basis for diseases such as OA.