Overview of Orthobiologics for Articular Cartilage Repair



Overview of Orthobiologics for Articular Cartilage Repair


Erica G. Gacasan, MS

Robert L. Sah, MD, ScD


Dr. Sah or an immediate family member has stock or stock options held in GlaxoSmithKline, Johnson & Johnson, and Medtronic. Neither Erica Gacasan 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.



INTRODUCTION

The predilection of articular cartilage to be damaged, along with its biology and extracellular matrix (ECM) structure, renders it a target for orthobiologics. Throughout an adult’s life, articular cartilage normally functions as a key component of diarthrodial joints, withstanding years of repetitive loading and facilitating pain-free motion. However, with injury and aging, articular cartilage is often damaged; it then exhibits a limited intrinsic repair response and often deteriorates (Figure 1), making it an attractive candidate for orthobiologics.1 Orthobiologics address tissue repair, protection, healing, and regeneration based on individual or combination products comprising cells, biomaterials, and/or bioactive factors. Orthobiologics can be classified using one or more of the three classic components: cells, scaffolds, and signals. These components can be derived from naturally occurring substances, such as tissues or blood, or consist of synthesized or modified materials or cells, such as engineered proteins, gene therapies, and culture-expanded cells. Effective orthobiologic treatment strategies may induce repairs that range from the ideally regenerated normal adult articular cartilage to tissues that variably restore function.

An overview of orthobiologics for articular cartilage repair is provided. It begins with summaries of the hierarchal structure of articular cartilage, the biology of indwelling chondrocytes and their precursors, and its ECM composition, turnover, and function. It is important to review the three major components of cartilage orthobiologics: scaffolds, cells, and signals. In addition, within this context, established and emerging orthobiologic approaches to cartilage repair are described.


BRIEF BIOLOGY OF CARTILAGE

The biology of articular cartilage is fundamentally coupled through its ECM to its biomechanical and biotransport properties, which vary during growth, homeostasis, and disease. Articular cartilage is a component of joints that functions as an organ, with interactions and cross-talk between tissues, including articular cartilage, synovium, and underlying bone, mediated by synovial fluid (Figure 2). Chondrocytes, the indwelling cells of cartilage, elaborate and remodel the cartilage ECM, affecting their microenvironment. Conversely, chondrocytes are regulated by the milieu of insoluble (ECM), soluble (cytokines, growth factors, and extracellular vesicles), and mechanical factors (due to joint loading). Thus, chondrocytes interact reciprocally with their surrounding matrix and fluid to form and maintain the structure and function of cartilage tissue.

The articular surface is bathed in synovial fluid that affects cartilage biology and physiology. Synovial fluid is a dialysate of plasma that contains substances originating from the local joint tissues. Synovial fluid helps lubricate the articulating joint surfaces, due to its high concentration of hyaluronic acid and lubricin/proteoglycan 4 (PRG4) lubricant molecules. Synovial fluid also mediates regulation and degradation of articular cartilage through synovium-secreted cytokines and proteases. In general, synovial fluid acts as a transport medium to provide cartilage with basic nutrients, including carbohydrates such as glucose, lipids, proteins, vitamins, minerals, and water.2

Cartilage growth, remodeling, maturation, and morphogenesis typically occur concurrently during joint development. During postnatal growth, the overall size and volume of joints increase (Figure 3). In skeletally immature individuals, the covering of bone ends includes a layer of epiphyseal growth cartilage and articular cartilage, which are collectively known as the articular-epiphyseal cartilage complex.3,4 This complex grows before being either replaced by mineralized tissue via endochondral ossification or retained as articular cartilage. The overall growth of the joint involves expansion of the joint surface with concomitant thinning of the articular cartilage. The normal homeostatic balance between matrix synthesis and loss may become disrupted not only by direct damage or infiltrating enzymes but also by dysregulated chondrocytes, displaying an aberrant or development-like phenotype.5 Common regulatory pathways of matrix turnover in normal joint development and joint pathology can provide avenues for orthobiologic interventions.












Cartilage homeostasis is sensitive to mechanical stimuli, depending on magnitude, duration, and nature, in part through receptors that are responsive to mechanical stimulation. In vivo, articular cartilage is subjected to time-averaged static and time-varying dynamic cyclic compression, articulation-inducing shear stress, and resultant hydrostatic pressure, and compressive, shear, and tensile strain.6 Mechanosensitive receptors can transmit mechanical information from the ECM to the chondrocyte and may activate or repress a variety of mechanotransduction pathways that alter the balance between catabolic and anabolic activity.7 Catabolic activation may occur through both reduced loading and overloading as well as chronic pathologic joint loading. Net cartilage degradation and thinning are induced by high levels of peak stress, high strain rates, and long-term
pathologic mechanical loading (eg, joint malalignment) as well as prolonged immobilization.6












Hierarchy of Cartilage Structure

The primary load-bearing structure of synovial joints is the osteochondral unit, consisting of articular cartilage, calcified cartilage, and underlying subchondral bone. The calcified cartilage is a thin tissue layer between the articular cartilage and subchondral bone and is bounded histologically by the tidemark, between the uncalcified hyaline cartilage and calcified cartilage, and the cement line, between the calcified cartilage and subchondral bone. The calcified cartilage provides load transfer, structural integration, and a barrier to solute transport between the uncalcified cartilage and the subchondral bone.8

The cartilage matrix has zonal, regional, and site variation that facilitates tissue function under complex loading and changes with growth (Figure 4). The main
ECM components of articular cartilage include large aggregates of the proteoglycan aggrecan and fibrils of predominantly type II collagen, which contribute to the cartilage’s unique biomechanical properties. Aggrecan associates with hyaluronan and link protein to form large multimolecular aggregates (˜109 Da) that are effectively retained within the cartilage. Because of the large number of polyanionic glycosaminoglycan chains on aggrecan, and the restraining and space-filling nature of the dense collagen network, the aggregates provide a high negative fixed charge density and osmotic swelling pressure within the tissue, with a high water content and compressive resistance.9,10 In contrast to its dense matrix and compared with other tissues, normal adult articular cartilage has a relatively low density of cells and is also avascular, alymphatic, and aneural.

Articular cartilage exhibits depth-wise variation, with (1) superficial, (2) middle, and (3) deep zones, as well as (4) calcified cartilage. Within the superficial zone, chondrocytes are flattened and aligned parallel to the surface, collagen fibrils run parallel to the articular surface, and aggrecan content is relatively low. With depth, cell density decreases, chondrocytes exhibit a larger and more rounded hypertrophic appearance, collagen orientation is increasingly perpendicular to the articular surface, and the matrix becomes progressively aggrecan rich.

The cartilage matrix surrounding chondrocytes also exhibits distinctive arrangements at increasing distances from the cell surface. The pericellular matrix lies immediately around the cell and is the zone where molecules that interact with cell surface receptors are located. Slightly further from the cell is the territorial matrix, and even further is the interterritorial matrix. The types of collagen and the collagen-binding proteins that form the matrices are different in each zone.11 Furthermore, the compositional, structural, and functional properties of articular cartilage vary across joint surfaces, consistent with modulation by the extent and pattern of joint loading.12


Chondrocyte Biology and Lineage

Articular chondrocyte biology varies with depth from the articular surface, in health and disease. For example, superficial zone chondrocytes are a major source of the synovial fluid lubricant, PRG4. Furthermore, gene expression profiles may help to elucidate stages of disease by reflecting the evolving expression of anabolic and catabolic genes during different phases of maturation and disease.13 In normal adult articular cartilage, the greatest differences in gene expression occurs between the superficial and deep zones, which reflects the differing functional roles of the superficial zone, in maintaining the low-friction articulating surface, and the deep zone, where an enhanced biosynthetic capacity is necessary to form and maintain the dense cartilaginous matrix.14,15

The natural transitional growth of immature epiphyseal cartilage to adult articular cartilage provides a paradigm for regenerative strategies of orthobiologics. Mature articular chondrocytes arise from multiple pools of progenitor cells from the superficial zone and perichondral tissues. Synovial joint formation involves descendants of mesenchymal stem cells (MSCs) from the interzone area of embryonic limbs, which give rise to joint tissues. Articular cartilage maturation is driven mainly by increase in cell volume, cellular rearrangement, and matrix deposition, with limited cellular proliferation in the neonatal stages16,17 (Figure 5). In mice, superficial zone PRG4+ cells expressing stem cell markers facilitate appositional and interstitial growth of articular cartilage and entirely reconstitute adult cartilage during growth and maturation.18 PRG4+ cells found in the synovial lining proliferate in response to injury and may contribute to healing.17 A population of cells positive for progenitor markers in the perichondral groove of Ranvier have also been identified and contribute appositionally to the articular cartilage from the joint periphery.19 In mature tissue, MSCs from bone marrow and adipose tissue are capable of differentiating into chondrocytes and may contribute to cartilage repair. Thus, lineage-tracing studies have substantial implications for how orthobiologics may target endogenous and exogenous cell populations to repair or regenerate cartilage.


ECM Composition and Homeostasis

The ECM of the cartilage is maintained at levels that depend on synthesis and accumulation of structural molecules, as balanced by their degradation and loss. The composition and functional properties of cartilage change during maturation and with pathology and reflects several biologic and physical processes.

Collagen biosynthesis and assembly follows the normal pathway for a secreted protein. Many different types of collagen molecules are expressed in articular cartilage, but the backbone polymeric template during development is a copolymer of collagens II, IX, and XI. After skeletal growth has ceased, the rate of type II collagen synthesis by articular chondrocytes drops dramatically, with an estimated turnover time of 400 years for human femoral head cartilage.20 The classic concept of collagen fibril degradation is through an initial cleavage of the collagen molecules (type I, II, or III) by collagenase into three-fourths-length and one-fourth-length fragments.21

Secreted aggrecan replaces those that are damaged by mechanical loading, removed by aggrecanases and/or proteases, or transported through the cartilage tissue via its migration down its concentration gradient, eventually exiting through the cartilage surface.22,23,24 Other proteoglycans, including syndecans, glypican, small leucine-rich proteoglycans, decorin, biglycan, and perlecan, among others, are expressed during chondrogenesis and aid in assembly and maintenance of the cartilage ECM.25







The ECM contains a variety of matrix-degrading enzymes that are present in precursor and active forms. The activation of stress-induced and inflammation-induced signaling and transcriptional and posttranscriptional events results in the release of the chondrocytes from growth arrest, imbalanced homeostasis, and chondrocyte activation with aberrant expression of inflammation-related genes.7 The ECM acts as a depot, storing growth factors and other signaling molecules. Changes in physiologic conditions can trigger protease activity that enables the release of such depots and activation of catabolism and inflammation, which can be modulated by targeted delivery of drugs and other signaling factors.26


Tissue Composition and Pathophysiology

The therapeutic effect of orthobiologic interventions depends on the residence time of the intervention within the joint space, if implemented by intra-articular injection, as well as penetration and residence time within the cartilage tissue itself. Soluble therapeutics (eg, growth factors and cytokines) are subject to convective and diffusive effects within the joint tissues.

Retention of solutes within the cartilage is affected by loading, ECM content, and integrity, as well as electrostatic interactions between the tissue and solute. Transport occurs relatively rapidly in areas of low matrix density, such as near the articular surface, but is also affected by
mechanical loading and residence time within the synovial fluid and cartilage.27

Synovial fluid is an ultrafiltrate of plasma with additional molecules secreted by local cell populations. The composition of synovial fluid is normally in dynamic equilibrium and reflects a balance between cellular secretion and loss due to transport through the synovial capillaries, synovial interstitium, and the lymphatic drainage system.28,29 The residence time of molecules within the synovial fluid depends on molecular mass and shape as well as joint state, including inflammation, vascular permeability, and synovial thickening.29,30 The half-life of drugs in the synovial fluid after injection is as short as ˜1 hour (eg, acetaminophen).31 However, residence time can be higher with increased molecular size, increased viscous and aggregating properties, and enhanced interaction with joint tissues, including the synovium and cartilage.32

Turnover of synovial fluid constituents is driven, in part, by size-dependent clearance through the lymphatic and capillary systems. Clearance of particles from the joint may occur through the lymphatics for larger species, and through venous capillaries for smaller species.33 In the rabbit model, after anterior cruciate ligament transection, the molecular weight distribution of injected hyaluronan within the knee shifted toward a predominance of low-molecular-weight species (<1,000 kDa) and a lower hyaluronan residence time.34 Synovial permeability also depends on disease state. Increased synovial inflammation in patients with various forms of arthritis is associated with increases in vascular and lymphatic permeability, synovial hyperplasia, and cellular infiltration.29,34,35 Thus, strategies to increase retention time of therapeutic solutes within the context of an inflammatory joint environment may be important for effective orthobiologic interventions.

Within the cartilage, fluid flows produced by mechanical loading induce convection of solutes.27 Furthermore, as the cartilage degrades, the collagen network is disrupted, proteoglycans are lost from the tissue, and tissue permeability increases. Increased permeability of diseased cartilage enables enhanced transport of therapeutic agents within the cartilage, and allows for transport of large molecules, but can reduce retention times within the tissue.

The dense polyanionic ECM of articular cartilage not only provides the tissue with its essential load-bearing properties but also governs the regulation of chondrocytes by soluble factors. Because of the net negative fixed charge density within the cartilage, solute partitioning within the tissue is hindered, or enhanced, by the molecular size and charge of the solute.36 According to effects of steric exclusion and Donnan equilibrium, the distribution as well as the diffusion coefficients of a particular solute decreases with increasing molecular weight and size and is sensitive to variations in fixed charge density.37 These physicochemical principles affect the access of solutes to chondrocytes and may be the basis of biologic therapies. For instance, cationic carrier molecules may enhance the transport of biologically active molecules into the cartilage.32 However, cationic molecules have the potential to neutralize tissue fixed charge, which could counteract the natural load-bearing mechanism of cartilage and affect chondrocyte behavior.38


OVERVIEW OF THERAPEUTIC OPTIONS: CELLS, SCAFFOLDS, AND SIGNALS

Therapeutic approaches and developments target particular forms and stages of articular cartilage damage. Focal chondral and osteochondral defects are injuries or areas of degeneration that are limited to a defined area. Focal defects may occur because of acute traumatic injury or may be idiopathic. Defects can present as (1) partial-thickness chondral defects where damage is restricted to the chondral layer, (2) full-thickness chondral defects extending to the calcified cartilage or subchondral bone, and (3) osteochondral defects that affect both the cartilage and underlying bone (Figure 6). Defects may present as softened or partially eroded tissue and are classified according to size and geometry.39 Conversely, osteoarthritis is characterized by widespread joint degeneration due to progressive loss of articular cartilage and pathophysiologic bone and cartilage remodeling including fibrocartilage formation, sclerosis of subchondral bone, and osteophyte formation.40,41 Repair of cartilage and osteochondral injuries involve not only replacement or regeneration of the bulk cartilage tissue but also integration with appropriate interfaces for the various types of defect repair.

Cartilage repair strategies can be classified according to the type of therapeutic modality, either mechanical or biologic, as well as the method in which the therapy is delivered (Table 1). Mechanically directed treatment strategies aim to correct abnormal joint loading and joint malalignment or provide tissues or tissue substitutes that promptly restore mechanical (ie, load bearing) function. Conversely, regenerative therapies enable a biologic response that aims to regenerate the tissue or establish a reparative microenvironment that restores normal structure and function, and which has a longer time horizon to achieve functional repair.

Therapeutic interventions, and their evaluation, can be delivered, or assessed, at the tissue, organ, and whole-body levels (Figure 7). Although most orthobiologic interventions are delivered directly to the joint by means of intra-articular injection or surgical procedures, soluble signals may be administered orally or intravenously. In addition, therapeutic efficacy may be assessed from a holistic perspective (ie, surveys and questionnaires regarding patient-reported measures of pain and function),42 as well as locally at multiple scales by clinical
imaging of the joint and biopsy of the joint tissues.43 Biochemical analysis of fluids, including urine, blood, and synovial fluid, may also be used to identify by-products of joint degeneration and assess the metabolic state of the body or joint.