Orthopaedic Tissues
Sean V. Cahill, BA
Francis Y. Lee, MD, PhD
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 Mr. Cahill 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.
ABSTRACT
A sound understanding of musculoskeletal tissue biology and structure is fundamental to rapidly evolving orthopaedic practice. This chapter will begin by discussing the basic components of skeletal development and stem cell differentiation that underlie all orthopaedic tissues. We will summarize the pertinent elements of the cellular biology and structural organization of bone, joint, and connective tissue. Finally, we will discuss how physiologic and pathologic states such as advanced age, diabetes, and smoking affect the quality of these tissues.
Keywords: bone biology; cartilage; diabetes; mesenchymal stem cells; metastatic bone disease
Stem Cells, Differentiation, and Osteogenesis
Orthopaedic tissues are derived from pluripotent stem cells which become increasingly more specialized. Osteoblasts, chondrocytes, adipose cells, fibroblasts, and myocytes share the mesenchymal stem cell (MSC) as the common precursor, while osteoclasts are derived from the macrophage/monocyte lineage of hematopoietic stem cells.
Variable expression of transcription factors facilitates stem cell differentiation into terminal lineages to form orthopaedic tissues as cellular migration and ossification take place.1 Runx2 and osterix are essential for differentiation of the osteoblast lineage. Sox5, 6, and 9 are markers of chondrocyte development, with Sox9 having been identified as an essential regulator2 (Figure 1). The Wnt/β-catenin pathway is one of the most important signaling pathways for regulating bone formation, leading MSCs toward osteoblastic differentiation and suppressing adipose development.
During development, contact between mesenchymal cells and epithelial cells triggers preosteoblastic differentiation and intramembranous ossification, during which mesenchymal cells differentiate directly into periosteum and osteoblasts.1 During endochondral ossification (Figure 2), mesenchymal tissue develops into bone from a cartilage template.3 Chondrocytes proliferate and undergo hypertrophy and apoptosis, and the remaining matrix is mineralized and invaded by vasculature. Systemic factors, such as growth hormone and thyroid hormone, and local factors, such as Indian hedgehog and parathyroid hormone related peptide (PTHrP), promote and regulate these processes (Figure 2). Woven bone, secreted by osteoblasts, is eventually replaced by lamellar bone. After a rudimentary skeleton is formed, osteoblasts and chondrocytes undertake skeletal modeling to shape the skeleton and improve its strength and resilience.
Secondary ossification widens bones, with peripheral growth from the apophysis. In contrast to primary ossification, which begins in the embryonic stage and continues through adolescence, secondary ossification only begins during the postnatal period.
Although the classic, step-wise paradigm of mesenchymal differentiation marks the majority of skeletal formation from pluripotent stem cell to terminal tissue elements, recent research has identified that transdifferentiation, the process of one mature cell line becoming another, plays a role in the formation of bone.4 It has been shown that osteocytes possess the ability to undergo transdifferentiation into chondrocytes either directly or through a pluripotent, intermediate form. There has also been evidence of hematopoietic transdifferentiation, with osteoclasts formed from immune cells such as macrophages and B lymphocytes. A current area of musculoskeletal investigation, signaling pathway manipulation may prevent depletion of chondrocyte precursors in osteoarthritis and may serve as a potential therapy for fracture nonunions.
 Figure 1 Illustration demonstrating the major transcription factors and regulators of bone cell differentiation. Mesenchymal stem cells differentiate via a stepwise progression into chondrocytes, adipocytes, osteoblasts, osteocytes, tenocytes, and myocytes. Osteoclasts arise from the monocyte lineage of hematopoietic stem cells. A host of transcription factors, genes, and growth factors regulate differentiation. Activation via RANK-L and inhibition by osteoprotegerin (OPG), both expressed by preosteoblasts, are major regulators for osteoclastogenesis. RANK = Receptor activator of nuclear factor kappa-B, RANK-L = Receptor activator of nuclear factor kappa-B ligand. |
Bone, the Fundamental Orthopaedic Tissue
Cellular Biology
Bone is a rich, biologically active tissue. Osteoblasts, osteocytes, and osteoclasts maintain and renew the bony matrix and are involved in systemic processes such as mineral metabolism.
Mature osteoblasts contain abundant rough endoplasmic reticulum for collagen synthesis, as well as an extensive golgi apparatus. Osteoblasts synthesize bone through type I collagen secretion and production of osteoid (unmineralized matrix). Parathyroid hormone stimulation and Runx2 expression induce the expression of alkaline phosphatase, type 1 collagen, and bone sialoprotein II in the preosteoblast stage5 (Figure 1). Transcriptional activation of RUNX2 and osterix results in osteoblast differentiation, allowing for matrix mineralization and expression of other proteins such as osteocalcin to occur. Osteoblasts create a basic environment with alkaline phosphate that helps catalyze calcium-phosphate crystal deposition.
Osteocytes, which comprise over 90% of all bone cells in adults, are differentiated from osteoblasts.6 Our understanding of osteocytes has dramatically increased over the past decade, especially our understanding of the mechanisms of response to mechanical loading and their role in bone metabolism. The process of the osteoblast becoming embedded in bone lacunae induces changes in genetic expression to induce osteocyte differentiation, including participation in mineralization regulation and development of dendritic processes.6 The expression of membrane type 1 matrix metalloproteinase is necessary for canaliculi formation, through which
osteocytes form an extensive intercellular network of dendritic processes that directly communicate via gap junctions.7 The lacunar-canalicular system contains circulating fluid that provides osteocytes with oxygen and nutrients and allows osteocytes to sense acute deformation of the bone matrix, inducing release of anabolic factors to increase bone mass in response to strain.
osteocytes form an extensive intercellular network of dendritic processes that directly communicate via gap junctions.7 The lacunar-canalicular system contains circulating fluid that provides osteocytes with oxygen and nutrients and allows osteocytes to sense acute deformation of the bone matrix, inducing release of anabolic factors to increase bone mass in response to strain.
 Figure 2 Illustration demonstrating the anatomy and ossification of long bone. The major regions of long bone include the epiphysis (nearest to the joint), physis, metaphysis, and diaphysis. Blood supply via nutrient arteries is derived from the periosteum. Endochondral ossification occurs at the physis, during which chondrocytes undergo proliferation, hypertrophy, and apoptosis. The matrix left behind is mineralized and invaded by blood vessels. Growth factors, including growth hormone, thyroid hormone, and fibroblast growth factor 3 (FGF3), promote osteogenesis. Indian hedgehog and PTHrP create a feedback loop to modulate and regulate chondrocyte proliferation and hypertrophy. |
Osteocytes are the master regulators of bone metabolism. In addition to sensing fluid microcurrents and responding to bone matrix strain, osteocytes produce sclerostin and Dkk1, which are potent Wnt inhibitors and therefore key negative regulators of osteoblastogenesis.6 Sclerostin and Dkk1 monoclonal antibodies are under investigation as a potential means of increasing bone mass and improving fracture healing, especially in osteoporosis and diabetes.8
Osteoclasts, derived from hematopoietic stem cells, appear as large, multinucleated cells housed in Howship lacunae, microscopic grooves on the bone surface. The ruffled border seals the bone surface, creating a closed microenvironment in which degradation products such as acid (produced by carbonic anhydrase and H+-ATPase) and cathepsin K degrade the bone matrix.9
Osteoclast development and differentiation is tightly linked to the osteoblastic lineage, and dysregulation leads to bone pathology (Figure 1). Commitment to the osteoclastic lineage from hematopoietic precursors is induced by macrophage colony-stimulating factor (M-CSF), c-fos transcription factor, and RANK expression.10 RANK-ligand (RANK-L), expressed by osteoblasts, is required for preosteoclast differentiation into osteoclasts. Osteoprotegerin (OPG), produced by cells of the osteoblast lineage as well as some hematopoietic cells, serves as a decoy receptor for RANK-L and competes with osteoclast RANK receptor; increased OPG decreases osteoclast differentiation and activation. Systemic factors help to regulate RANK-L and OPG production, including tumor necrosis factor-α (TNF-α), a catabolic factor, and parathyroid hormone (PTH) and estrogen, anabolic factors.
Although osteoclast differentiation is tightly regulated by the osteoblastic lineage, osteoclast activity is also influenced by hormones (Figures 1 and 5). Resorptive
activity is increased by vitamin D, PTH, PTHrP, and prolactin and decreased by estrogen, calcitonin, and transforming growth factor beta (TGF-β). Cytokines also regulate osteoclast activity. Interleukin-17 (IL-17) has been shown to decrease bone resorption; IL-6 and TNF-α increase resorption.10
activity is increased by vitamin D, PTH, PTHrP, and prolactin and decreased by estrogen, calcitonin, and transforming growth factor beta (TGF-β). Cytokines also regulate osteoclast activity. Interleukin-17 (IL-17) has been shown to decrease bone resorption; IL-6 and TNF-α increase resorption.10
Marrow adipocytes have long considered inert place holders in the marrow space of long bone, ribs, sternum, and vertebrae. However, marrow fat has been increasingly recognized as an important, active element of the bone cell milieu and is considered a distinct type of adipocyte (as opposed to white, brown, or beige fat found elsewhere in the body). As discussed in a 2017 study, it has been shown that marrow adipocytes increase with age and are tightly correlated with reduced bone mass.11 These regulatory effects on bone mass are thought to be induced through modulation of PPARγ and RUNx2 proteins.12 Thus, marrow adipocytes and associated signaling pathways have become sought-after targets for bone disease therapies. In addition to bone metabolism, it is thought that marrow adipocytes influence other cell populations within the marrow and negatively affect hematopoiesis.11
Matrix Composition
The cellular components produce and maintain a mineralized bone matrix, which serves as the functional component of bone. The extracellular matrix is heterogeneous and structured. Mineral and organic components together impart strength and rigidity (Tables 1 and 2). The composition of bone varies with age, gender, and ethnicity. Health status can also affect matrix composition, and alterations of the matrix underlie diseases such as osteogenesis imperfecta and osteoporosis13 (Table 3).
The mineral calcium hydroxyapatite, Ca10(PO4)6(OH)2, is the most abundant substance in bone, 60% to 70% of its mineral composition. Other abundant minerals include sodium, magnesium, and bicarbonate.13 The organic component of bone, stabilizes the extracellular matrix, facilitates calcification and mineralization, and provides tensile strength. Type I collagen is the dominant organic substance of bone, comprising 90% of total protein and the second most abundant substance following hydroxyapatite. The collagen triple helix, characterized by glycine-X-Y repeating sequence, is highly cross-linked, providing elasticity. Fibronectin, another important structural protein of the bony matrix, helps develop and maintain the structure of the collagen network. Noncollagenous proteins, such as proteoglycans and osteocalcin, also play various roles14 (Table 1).
Bone Metabolism
As forces from daily activity impart microscopic damage, osteoclasts and osteoblasts work in concert to remove old bone and replace it with new bone to maintain strength and integrity. This process of remodeling is ongoing, working to replace approximately 10% of the skeleton every year and replacing the entire bone mass every 10 years.15
Remodeling is a tightly regulated process, as osteogenesis is intimately coupled to osteoclastogenesis. Remodeling imbalances can result in systemic disease such as osteoporosis as well as local bone destruction as in cancer metastasis. Bone loading affects the rate of bone remodeling as stated by Wolff law that mechanical stress results in greater bone density and strength. Systemic and hormonal factors, such as parathyroid hormone and 1,25-dihydroxy vitamin D, modulate remodeling by inhibiting bone resorption and promoting differentiation of osteoblasts and osteoclasts.16 Direct communication between osteoblasts and osteoclasts is also achieved through release of local factors from the bone matrix itself. As osteoblasts degrade the bony matrix, proteins including TGF-β, platelet-derived growth factor, and fibroblast growth factor are released to stimulate osteoblasts and thus enhance bone formation. Micro-RNA modulation has also been identified as a significant regulator of bone remodeling.17
Anatomy and Structure
Long bones allow for mechanical motion of the extremities and are formed by endochondral ossification (Figure 2). The epiphysis, covered by articular cartilage, forms joint surfaces. The physis, is the location of endochondral ossification, and is located between the epiphysis and the metaphysis. The apophysis, a feature of both long and flat bone, is an area of secondary ossification.
Periosteum is a thin, membranous tissue that surrounds both long and flat bones, and endosteum is the corresponding inner surface. The periosteum contains a rich vasculature that provides primary blood supply to bone, making it essential for fracture healing. It is also the site of muscle and tendon attachment.
Cortical bone is dense, compact tissue which functions to provide mechanical rigidity. Haversian canals run parallel to the diaphysis along the mechanical axis of bone. These spaces house nerves and microvasculature that supply the bone tissue. Laminae are discreet, concentric sheets of bone that surround the Haversian canal. Volkmann canals allow for communication between periosteal vessels and Haversian system.
Osteocytes are housed in lacunae and their dendritic processes communicate via small canaliculi. Cutting cones comprise the remodeling unit of cortical bone, with osteoclasts forming a canal along the longitudinal axis of bone and osteoblasts following to close the gaps.18
Osteocytes are housed in lacunae and their dendritic processes communicate via small canaliculi. Cutting cones comprise the remodeling unit of cortical bone, with osteoclasts forming a canal along the longitudinal axis of bone and osteoblasts following to close the gaps.18
Table 1 Bone and Cartilage Components and Associations With Orthopaedic Conditions | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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