Cell Biology, Cell Differentiation, and Stem Cells of the Skeletal System

Michael J. Zuscik, PhD

Lianping Xing, PhD

Brendan F. Boyce, MBChB

Francis Y. Lee, MD, PhD

Dr. Zuscik or an immediate family member serves as an unpaid consultant to Solarea BIO. Dr. Lee or an immediate family member has stock or stock options held in L&J BIO and has received research or institutional support from Musculoskeletal Transplant Foundation, National Institutes of Health (NIAMS & NICHD), and OREF. Neither of the following authors nor any immediate family member has received anything of value from or has stock or stock options held in a commercial company or institution related directly or indirectly to the subject of this chapter: Dr. Xing and Dr. Boyce.

INTRODUCTION

The skeleton and the bones that comprise it are formed during embryonic development through two distinct processes: endochondral and intramembranous ossification. These processes involve the commitment and differentiation of cells arising from both the mesenchymal and hematopoietic lineages, and a coordinated interaction of these cells, particularly chondrocytes, osteoblasts (OBs), osteocytes, and osteoclasts (OCs) (Figure 1), is required to regulate cartilage and bone formation and resorption (Figure 2). These coordinated interactions support development, growth, repair, and homeostasis of skeletal tissues. When they are disrupted, skeletal diseases manifest themselves.

Long bones of the appendicular skeleton are primarily formed by the process of endochondral ossification initiated in limb buds arising from the lateral plate mesoderm that comprises the trunk of an embryo. The buds are the epicenter of the formation of cartilage templates (cartilage anlagen), which provide a structural scaffold for the formation of each skeletal element leading up to fully formed and anatomically correct bones (Figure 3). Chondroblasts, the cells that produce the cartilaginous matrix of the anlagen, arise from mesenchymal stem cells (MSCs). In addition to chondro-lineage cells, MSCs are pluripotent cells possessing the ability to differentiate into numerous other musculoskeletal cell lineages including OBs, fibroblasts, adipocytes, striated muscle, and endothelial cells.¹ Chondroblasts in the central parts of the anlagen differentiate from early chondroprogenitors into hypertrophic chondrocytes, and the matrix around the hypertrophic chondrocytes calcifies to create the first mineralized tissues.¹ Some of these chondroprogenitors positioned near the proximal and distal ends of bones where joints will form differentiate into articular chondrocytes. Blood vessels grow into the calcified cartilage, followed by matrix degrading enzymes, including various metalloproteinases, which served to remove the mineralized cartilage at the midshaft of the developing bone. Parallel to this, the hypertrophic chondrocytes in the remodeling anlagen undergo apoptosis. As calcified cartilage is removed and chondrocytes die off, OBs that have mainly differentiated from MSCs in the limb bud and precursors circulating in the blood² replace it with new bone organized into rod- and plate-shaped structures called trabeculae. Some OBs residing on the new trabeculae or on the endosteal (ie, inside) surface of the shaft of the
long bone will encase themselves in unmineralized collagen-proteoglycan matrix known as osteoid that can bind calcium.³ The binding of calcium to osteoid results in hardening of the matrix, encasement of OBs, and their terminal differentiation into osteocytes. Osteocytes have numerous functions within and outside of bone, most important being their role as load sensors that translate mechanical stresses in bones into cellular signals that control bone remodeling/homeostasis to maximize bone strength in response to load-bearing requirements.⁴

FIGURE 1 Graphical images of the main skeletal cell types and where they reside. A femoral head is shown with the rough locations identified for the four key cells described in this chapter: chondrocytes, osteoblasts, osteocytes, and osteoclasts.

Growth of long bones perpendicular to their long axis (ie, radial growth) as well as the formation and expansion of flat bones such as the calvarial plates in the skull is supported by the process of intramembranous ossification. This process involves the direct conversion of mesenchymal tissue into bone and the differentiation of MSCs into OBs.⁵ Although this process does not involve a cartilage precursor, as in endochondral ossification, OBs participating in intramembranous bone formation can also encase themselves in mineralized osteoid and terminally differentiate into osteocytes.

FIGURE 2 Illustrated model of osteoclast-Osteoblast (OC-OB) coupling during bone turnover. Active OCs excavate trenches in bony tissue, followed by OBs, which lay down new mineral at sites that have been resorbed. Key factors that drive myeloid precursor differentiation into OCs and osteoprogenitors into OBs are depicted.

A portion of new bone must be removed to accommodate the formation of bone marrow cavities during skeletal development and the removal of bone throughout life in a tight balance with OBs to support remodeling and homeostasis. The process of bone resorption in these contexts is supported by OCs, which resorb matrix on bone surfaces to create trenches that are subsequently filled with new bone produced by OBs (Figure 2). The OCs that perform this work arise from myeloid lineage hematopoietic cells that also give rise to macrophages.⁶ OCs are multinucleated cells formed by cytoplasmic fusion of these hematopoietic precursors in response to cues from various cytokines and growth factors.

With all of these in mind, this chapter will focus on describing the main cells that contribute to skeletal system development, growth, and maintenance: chondrocytes, OBs, osteocytes, and OCs (Figure 1). This is not to suggest that other cell types are not involved or critical to the skeletal system, but these groups of cells are the dominant players in bone and cartilage biology. The central factors and signaling pathways that regulate progenitor cell differentiation to these key lineages will also be presented as foundational information to establish the mechanisms underlying how these cells create and maintain the skeleton by carrying out the key functions that are specific to their differentiated phenotypes.

CHONDROCYTES

CHONDROCYTE DIFFERENTIATION

Chondrocytes undergo differentiation along two major distinct pathways. During endochondral ossification, cells undergo maturation, hypertrophy, and apoptosis, ultimately supporting matrix calcification. This pathway is used in the physes (ie, growth plates) of growing adolescent vertebrates and is recapitulated in bone repair following fracture. The second pathway of chondrocyte differentiation takes place in hyaline cartilage. In this context, cells are relatively quiescent, carrying out structural or load-bearing functions in the articular cartilages of joints. In either case, chondrogenesis is required to initiate differentiation of chondrocyte progenitors toward their fate in these related but functionally distinct tissues.

FIGURE 3 Illustrated summary of endochondral ossification. Each stage of endochondral ossification is depicted from mesenchymal stem cell (MSC) condensations in the limb bud through to the development of a mineralizing skeletal element with chondrocytes spanning the spectrum of differentiation from pre-hypertrophic to terminally differentiated cells. Colors denote cell differentiation stage. Blood supply is depicted in red.

DIFFERENTIATION OF CHONDROCYTES DURING ENDOCHONDRAL OSSIFICATION

In skeletal development, chondrogenesis begins with the aggregation and condensation of loose mesenchymal tissue to form anlagen comprised of cells that actively express various extracellular matrix and cell adhesion molecules, including aggrecan and the IIa splice variant of the type 2 collagen α1 chain.⁷ The appropriate temporal and spatial secretion of extracellular matrix and the associated steps of chondrocyte differentiation rely on the action of various morphogens/growth factors, their cognate receptors and the downstream signaling pathways, and transcription factors. The earliest events in the mesenchyme that participate in anlagen formation are initially driven by the morphogen Sonic Hedgehog (Shh) via its transmembrane receptors Patched 1 and 2 (Ptc1,2), leading to recruitment of another transmembrane protein Smoothened (Smo) and activation of Gli transcription factors.⁸ Activation of this signaling system leads to the induction of various targets including the master transcriptional regulator of chondrogenesis, Sox9,⁹ and NK3 homeobox 2 (Nkx3-2), another transcription factor necessary to maintain Sox9 expression in condensing cells.¹⁰

Once signals downstream of Shh initiate Sox9 upregulation in early development, its tight regulation by a series of other signaling pathways sets the pace for appropriate progression of chondrogenesis. Deceleration of chondroprogenitor entry into the chondrocyte lineage is in part facilitated by the Notch receptor family which comprises four different single-pass transmembrane receptors consisting of extracellular, transmembrane, and intracellular domains that determine cell fate. Notch receptor activation by its agonist ligands Delta-like 1, 3 and 4 (Dll1, 3 and 4) and Jagged 1 and 2 (Jag1 and 2) inhibits Sox9 via the Notch-activated transcription factor, RBP-Jκ, and the downstream activation of various other transcriptional regulators including HES1 and HES5. Chondrogenesis ensues only following reduced signaling on the Notch pathway (ie, reduced transcriptional repression of Sox9 by RBP-Jκ).¹¹ Once this Notch repression is overcome, several key pathways strongly drive Sox9 upregulation, expansion of chondrogenesis, and entry into the chondrocyte hypertrophic program. These include fibroblast growth factors (FGFs),¹² hypoxic conditions, and the concomitant upregulation of hypoxia-inducible factor 1α (HIF-1α),¹³ and transforming growth factor-β (TGFβ).¹⁴ Bone morphogenic proteins (BMPs), members of the TGFβ superfamily, also possess potent chondrogenic activity. Consistent with this, blockade of BMP signaling by overexpressing the soluble decoy receptor Noggin, which competes away BMPs from their cognate receptors, inhibits cartilage formation.¹⁵ Overall, these signaling pathways work in concert with each other to modulate MSC/progenitor cell commitment to the chondrocyte lineage. These events are critical to the expansion and conversion of anlagen into mineralized skeletal elements (Figure 3).

DIFFERENTIATION OF CHONDROCYTES IN HYALINE CARTILAGE

Articular cartilage development begins during embryogenesis at sites of synovial joint formation through a sequential series of steps that include patterning of the joint site, interzone formation, cavitation, and morphogenesis.¹⁶ Articular chondrocytes are formed from interzone cells and, unlike chondrocytes participating in endochondral ossification in the epiphyseal plate which are removed completely following adolescent growth, these chondrocytes persist. The tissue left behind
postnatally and in adulthood is articular cartilage, which is maintained as four distinct cellular zones from the surface to the underlying bone: superficial, intermediate, radial, and calcified cartilage. The superficial zone consists of one to two cell layers of flattened chondrocytes expressing Proteoglycan 4 (Prg4) (also known as superficial zone protein or lubricin), Sox9, type 2 collagen Col2a1(IIb), aggrecan (Acan), tenascin C (Tnc) and low levels of cartilage intermediate layer protein (Cilp).¹⁶ Chondrocytes of the intermediate zone are round and express many of the same molecules as the superficial zone except for Prg4, although they have higher levels of Cilp. Radial and calcified cartilage zone chondrocytes express markers of chondrocyte differentiation and hypertrophy, including, notably, type 10 collagen (Col10a1).¹⁶ Each zone is maintained throughout adulthood unless stress-inducing injury, inflammation, or genetic defects lead to loss of the signals required to maintain the appropriate matrix homeostatic activity of the chondrocytes and prevent their inappropriate terminal hypertrophy. Central among these cytokine signals that suppress inappropriate hypertrophic differentiation is the TGF-β/Smad signaling pathway.¹⁷ When this pathway and others that decelerate or prevent chondrocyte hypertrophy are disrupted or impaired, chondrocytes become hypertrophic, increasing their expression of matrix degrading enzymes such as matrix metalloproteinase (MMP)-9 and MMP-13 and members of the A Disintegrin And Metalloprotease with Thrombospondin Motifs (ADAMTS) family.¹⁸ Coupled with enzyme-driven progressive loss of biomechanically appropriate articular cartilage matrix is the terminal maturation of chondrocytes, which make Col10a1 and ultimately apoptose. These matrix and cellular changes lead to degenerative diseases such as osteoarthritis.¹⁸

CHONDROCYTE FUNCTION

Chondrocytes can be grown and studied in culture, with numerous 2-D and 3-D culturing conditions that support the chondro-differentiated phenotype. Isolation of chondrocytes can be accomplished by the digestion of cartilage with collagenase or combinations of enzymes including collagenase, hyaluronidase, and trypsin. Chondrocytes in monolayer culture tend to dedifferentiate into type 1 collagen-expressing fibroblast-like cells and lose their ability to express Col2a1 and other matrix components such as proteoglycans.¹⁸ When cultured in a suspension culture or in a three-dimensional gel made of collagen, agar, or alginate, the cells will maintain their chondrocytic phenotype,¹⁹ emphasizing the importance of cell-matrix interactions in controlling gene expression, chondrocyte differentiation, and maintaining the chondrocyte phenotype.

The predominant matrix protein in cartilage is type II collagen, which is composed of a single chain forming a triple helix.²⁰ Like type I collagen, it is secreted as triple helical proprotein, which is cleaved extracellularly by proteinases.²¹ The other major organic component of the matrix is proteoglycan, which includes several proteins containing covalently bound glycosaminoglycan side chains.²² The major proteoglycan is aggrecan, which consists of a protein core and chondroitin sulfate and keratan sulfate side chains. The proteoglycans confer many of the unique mechanical properties of cartilage, including its ability to absorb repetitive compressive mechanical loads without damage. Aggrecan molecules form aggregates with hyaluronic acid and a link glycoprotein. In addition, cartilage contains small proteoglycans, such as decorin and biglycan. In addition to type II collagen and aggrecan, there are a series of minor collagens that also contribute to the chondrocytic phenotype. These include type VI, IX, X, and XI collagens.²⁰ Type VI collagen is a pericellular matrix protein, whereas type IX is a collagen molecule with a proteoglycan moiety. Type IX collagen molecules coat the outer surface of type II collagen fibrils and interact with the matrix proteoglycan via proteoglycan moieties. This is thought to serve as an interconnection between the collagen and proteoglycan matrix. Type XI collagen is localized within the type II fibrils and may regulate the diameters of fibrils.

Several chondrocyte phenotypic markers are specific to the differentiation pathway of the chondrocyte. For instance, type X collagen is only expressed by hypertrophic chondrocytes and is a specific marker for this phenotype.¹⁸^,²² In addition, these cells express high levels of alkaline phosphatase, in contrast to the minimal expression observed in chondrocytes that are not committed to maturation. Chondrocytes committed to the endochondral calcification pathway also express and respond to several growth factors, including several BMPs²¹ and the cell signaling protein Indian hedgehog (IHH),²³ which promote chondrocyte maturation, and parathyroid hormone-related protein (PTHrp)²⁴ which inhibits it via inhibition of RUNX2 by cyclic AMP response element binding protein (CREBP). Differential expression of these genes in articular and epiphyseal plate chondrocytes demonstrates the critical role of regulatory gene products that may operate as determinants of chondrocytic phenotypes. Figure 4 summarizes the key signaling, matrix and phenotypic genes expressed, and chondrocyte functional activities at each stage of chondrocyte differentiation, and Table 1 compiles information about the key pathways that impact chondrocyte differentiation and function.

OSTEOBLASTS

OB DIFFERENTIATION

OB formation from MSCs, which occurs in endochondral ossification and intramembranous bone formation, is a two-step process involving (1) lineage commitment of MSCs to osteogenic precursors followed by (2) differentiation of pre-OBs and their maturation into OBs. Several critical signaling pathways and transcription factors are involved. Key regulators include the BMPs and their signaling pathways, the
transcription factor RUNX2, the Wingless (Wnt)-β-catenin signaling pathway, and the Notch pathway. A broad literature documents the central roles of each of these factors and signaling cascades; these concepts are summarized below and in Table 1, and their net effects in OB differentiation are summarized in Figure 5.

FIGURE 4 Pictorial representation of Chondrocyte differentiation. The key stages of chondrocyte differentiation from mesenchymal stem cells (MSCs) into terminally hypertrophic chondrocytes are depicted. Listed for each differentiation stage are the central factors, phenotypic genes, and important cellular activities.

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