Cartilage Repair and Regeneration

The Chondrocyte as an Anabolic/Catabolic Cell

Chondrocytes are highly specialized cells that differentiate from clusters of mesenchymal cells during skeletal embryogenesis. The chondrocyte synthesizes and secretes the components of the extracellular matrix, primarily proteoglycans and type II collagen. Most of the immature cartilage is temporary and is replaced by bone during epiphyseal development, whereas the regions nearest the synovial cavity remain as the permanent articular cartilage of the adult. During growth and development, immature cartilage undergoes cellular replication in both the superficial and deep zones. However, as skeletal maturity approaches, replication occurs only in the deep zone. Cell replication after skeletal maturity is rare. The cell content of articular cartilage is low, occupying no more than 10% of the tissue volume in humans. Cell density has been estimated as 10 ⁵ cells per cubic millimeter in newborns and 1/10 of that in adult cartilage. Values are higher in the superficial zone than in the deeper zone. Experimental animals have far greater cellularity. For example, adult rabbits have nearly 10-fold greater cell density than human cartilage and mice have 25-fold greater cell density. The general cellular morphology ranges from flattened and discoidal in the most superficial zones to ovoid in the deeper regions. The ovoid cells display enlarged Golgi bodies, a characteristic of cells actively secreting proteins, and cellular processes that extend into the adjacent pericellular matrix.

Chondrocytes are normally long-lived cells. They are not replaced by new cells as occurs in the turnover of other tissues. However, the capacity for cell division is manifest when the integrity of the matrix is compromised, as in osteoarthritis. Cartilage fibrillation is associated with necrosis in the superficial zone and with clusters of cells in deep zones. Metabolic studies show increased sulfate incorporation by the cells in the clusters surrounded by proteoglycan-poor matrix. If viewed as an attempt to repair, the cells in clusters are active in matrix synthesis but are not capable of matrix replacement at distances from the clusters. Thus, the overall content of proteoglycan is low in fibrillated cartilage.

Chondrocytes are embedded in an avascular matrix in which nutrients and waste products must diffuse. Oxygen tension is approximately 1/3 that measured between capillaries in soft tissues. On a per-cell basis, oxygen uptake is 1/50 that of kidney, but the rates of glycolysis between chondrocytes and kidney are comparable. Thus, chondrocytes engage in relatively anaerobic metabolism.

Chondrocytes are dynamic cells with anabolic and catabolic activity; they mediate both synthesis and degradation of the matrix. Proteoglycan metabolism has been studied extensively. As is typical for other cells, the protein components are synthesized in the cytoplasmic rough endoplasmic reticulum and sulfation of the polysaccharides occurs in the Golgi bodies. Ex vivo studies with radioactive tracer isotope of ³⁵ S-sulfate show incorporation into glycosaminoglycans by intermediate and deep cells and subsequent movement into the matrix. Type II collagen is synthesized and secreted as separate procollagen chains with extensions on its ends, converted to tropocollagen, and organized into fibrils with small amounts of type IX and type XI collagen. In contrast to the dense, thick, highly oriented collagen fibers of bone, cartilage fibrils are thin and cross-linked into an open meshwork. The fibrils contain variable amounts of noncollagenous macromolecules, notably decorin. The half-life of type II collagen is more than 200 years in humans. Thus, the major component of the native fibrils does not appear to be renewed or reparable in the normal setting. The collagen fibrillar network is under tension and serves to contain the glycosaminoglycans in a compressed state. It is difficult to imagine how that network could be turned over without compromising the mechanical integrity of the tissue or how denatured foci could be mended. Evidence shows damage to the fibrillar network in osteoarthritis. Osteoarthritis drastically affects the mechanical properties of cartilage. Excessive swelling of osteoarthritic samples in dilute salt solution is taken as evidence of the loss of resistance afforded by the fibrillar net to absorption of water by the polysaccharides. Although there appears to be little turnover of the fibrillar network, the entrapped proteoglycans undergo turnover that can be accelerated by local cytokines. Chondrocytes are responsible for maintaining the matrix environment in which they are encased and hence ensure the tissue's mechanical characterics. They are protected from osmotic and mechanical damage by the rigid pericellular matrix, called the chondron . Maintenance of the matrix involves degradation by proteinases and free radicals generated by the chondrocyte. Matrix metalloproteinases and aggrecanase catalyze the turnover of cartilage matrix in normal as well as in diseased cartilage. Because many of the matrix components in cartilage are specific to that tissue, there is great interest in developing and validating assays for their degradation products in plasma or synovial fluid as markers for turnover.

In the modern view, osteoarthritis is considered to result from an imbalance between dynamic anabolic and catabolic activities that are normally well balanced. The chondrocyte functions as the agent of these two processes. This is in contrast to bone tissue, for example, in which anabolic activities are ascribed to the osteoblast and catabolic or osteolytic activities to the osteoclast. Although normally interaction between the two cell types maintains skeletal mass constant within the remodeling process, imbalance occurs with aging, osteoporosis, and infection. The situation in cartilage is distinct. Evidence suggests that the earliest stages of osteoarthritis are balanced by an upregulation of the biosynthetic processes. Synovial fluid collects a byproduct of procollagen II processing, called chondrocalcin or C-propeptide , whose levels are elevated in traumatic and primary osteoarthritis.

Previously, cartilage was regarded to be immunologically privileged because of either the absence of transplantation antigens or the protective effect of the matrix. These states were invoked to explain the endurance of allografts in heterotopic sites. It is now appreciated that chondrocytes do display major transplantation antigens and that components of the matrix are weakly antigenic. In healthy intact cartilage, these determinants are probably shielded from antibodies because of steric hindrance caused by the proteoglycans in the matrix. Preservation of matrix integrity appears to be essential to prevent exposure of the cells and rejection of the tissue.

The heterogeneous group of inflammatory joint diseases involve underlying disturbances in immune regulation. In rheumatoid arthritis, the synovial lining is the initial target of inflammatory pathology. Proliferation of synovial lining cells and infiltration by lymphocytes and activated macrophages produce a tissue mass called the pannus . The pannus can invade and destroy the integrity of articular cartilage. Products of the pannus act as cytokine mediators of both chondrolysis and osteolysis. The major agents are interleukin-1 (IL-1) and tumor necrosis factor-α. These agents signal the cascade of release of IL-6, IL-8, IL-17, cyclooxygenase-2, and nitric oxide, all of which are actual or potential targets of pharmacologic management. In vitro models have been useful to describe the mechanisms by which these immunomodulatory cytokines change gene expression in chondrocytes, thus promoting chondrolysis.

Incidence of Cartilage Lesions

The true incidence of cartilage lesions and their natural history are unknown. It has been proposed that between 5% and 10% of acute knee hemarthroses after a work-related or sports injury is associated with an acute chondral injury. In a retrospective review of 31,516 knee arthroscopies, the prevalence of chondral lesions was 63%. However, isolated unipolar chondral defects in patients younger than 40 years were rare, occurring in only 5% of this patient population. Both clinical and experimental evidence showed that with time focal cartilage injuries will enlarge and progress to osteoarthritis.

Mechanical injury to articular cartilage during sporting injuries may occur with shearing forces secondary to disruption of the anterior cruciate ligament. Shearing osteochondral fractures occurring at the time of ligament disruption have been noted. Blunt injury to the joint surfaces may cause injury to and death of articular chondrocytes. If the articular chondrocyte cannot continue to synthesize and remodel its matrix macromolecules, the pericellular matrix will eventually degenerate. This may account for the high incidence of osteoarthritis encountered with anterior cruciate ligament injuries. Acutely the incidence of chondral injuries is approximately 2% but may approach 20% in the long run.

In a study performed by Repo and Finlay, blunt force to articular chondrocytes in excess of 25 MPa reproducibly resulted in death of articular chondrocytes. Hence there appears to be a threshold to which articular chondrocytes can withstand blunt trauma. This may be an important factor in understanding articular cartilage degeneration after injury and may be an important technical factor during new repair techniques, such as osteochondral graft transfers. Large impaction forces needed to introduce osteochondral grafts to recipient sites may result in injury and cell death to the cartilage cap of the osteochondral grafts, leading to failed long-term results.

Magnetic resonance imaging scans demonstrated bone bruises after blunt injuries sustained during work-related and sporting activities. Arthroscopic biopsy studies of cartilage overlying bone bruises demonstrated superficial chondrocyte death and matrix dehydration. Cartilage cell death is proposed to arise directly from the blunt trauma exceeding this threshold.

The natural history of osteoarthritis itself is unknown. A Swedish longitudinal study notes radiographic progression of osteoarthritis in the knee occurs over a 20-year time course when greater than 50% joint space narrowing is present at initial evaluation (Ahlbäck stages 2–4). However, only 60% of patients with Ahlbäck stage 0 (peripheral osteophytes and a normal joint space) or Ahlbäck stage 1 (<50% joint space narrowing) at initial presentation will progress radiographically. Not all radiographic osteoarthritis will progress.

In the United States, more than 450,000 total knee replacements are performed annually. The disability and economic hardship encountered by osteoarthritis are substantial. This is especially true if a cartilage injury occurs at a young age when socioeconomic productivity and recreational activities are especially affected. The problem arises from the unique structure, function, and repair mechanisms of articular cartilage.

Cartilage Injuries and Repair

Articular cartilage is devoid of a nerve supply. Cartilage covers and protects the richly innervated subchondral bone plate from stimulation. Once articular cartilage is damaged, pain can result from contact of the subchondral bone plate. If a healing response does not develop, load will be borne by the shoulders of the chondral defects in addition to the exposed subchondral bone. This situation will result in overload and breakdown of the shoulders of the defect, with progressive enlargement of the defect. The opposing articulation would be exposed to a bare bone surface with resultant erosive degradation of its cartilage surface. The resultant bone-on-bone articulation is by definition osteoarthritis. Symptoms from direct stimulation of the subchondral bone plate or from indirect stimulation to the bone via an attached cartilage flap may occur. Breakdown products from the cartilage along with liberated enzymes may cause effusions in the joint, capsular distention, or synovitis as other mechanisms of pain. As the subchondral bone plate hardens, secondary vascular venous congestion in the medullary cavity results and may cause deep aching pain.

Cartilage repair would be beneficial in the short term to alleviate symptoms and in the long term to prevent progressive breakdown of the articulations of the joint and the development of osteoarthritis. Thus, the goal of cartilage repair is to produce a tissue that will fill the defect, integrate with the adjacent articular cartilage and subchondral bone plate, have the same viscoelastic mechanical properties, and maintain its matrix over time without breakdown. That is, the goal is to restore the osteochondral functional unit with a repair tissue that approaches regeneration.

Clinical and experimental evidence shows that damage involving the articular cartilage surface and confined to the cartilage undergoes little restoration. Cartilage has little intrinsic ability to heal. Chondrocytes in mature articular cartilage rarely divide and their density declines with age. In contrast, lesions that extend to the subchondral marrow may heal clinically. Therefore, a cell source for cartilage regeneration or repair must arise from the underlying subchondral bone marrow, the adjacent synovial tissue, or an exogenous source.

Absence of blood supply and endogenous source of new cells contribute to cartilage’s incapacity for repair. The typical wound healing response of hemorrhage, fibrin clot formation, and mobilization of cells and growth factors is absent. The only spontaneous repair reaction may occur at the edge of superficial articular cartilage lesions. Articular cartilage is isolated from the subchondral bone marrow cells by the dense subchondral bone and cartilage matrix.

Cartilage repair is dependent on the mobilization of cells derived from the subchondral bone marrow, which include multipotential cells, osteoblasts, chondroblasts, fibroblasts, and hematoprogenitor cells. Therefore, the repair tissue that results may be variable based on the predominant cell line that proliferates and its modulation by local growth factors, cytokines, and the local mechanical environment.

Clinical Repair for Full-Thickness Cartilage Defects

The spectrum of repair tissue is clinically variable, depending on the clinical technique used as well as intrinsic and local factors. Repair tissue may be fibrous tissue, transitional tissue, fibrocartilage, hyaline cartilage, articular cartilage, bone, or a mixture of these tissues. Fibrous tissue consists of fibrocytes and a type I collagen fibrous matrix. Transitional tissue consists of ovoid cells that may produce proteoglycans as well as a fibrous matrix. The matrix may stain positively with safranin O for proteoglycan production. Fibrocartilage consists of round chondrocyte-appearing cells with a type I collagen fibrous matrix. Hyaline cartilage consists of chondrocytes in a matrix of type II collagen and proteoglycans, with a hyaline, ground-glass appearance by light microscopy. The cellular and matrix organization may be different than normal articular cartilage. Articular cartilage resembles normal articular cartilage. Articular cartilage is essentially a regenerating tissue with articular chondrocytes, arranged in the usual palisading columns found in normal articular cartilage, with markers of normal articular cartilage matrix including type II collagen, proteoglycans, etc. A mixture of all these components may be present at a single repair site. The predominant repair tissue type will determine the long-term outcome of the patient. If the majority of the repair tissue is hyaline or articular cartilage, the viscoelastic properties found with a type II collagen framework and proteoglycans will give a durable repair and usually a superior clinical result. Fibrocartilage and fibrous repairs consist of type I collagen, which is usually not as strong as type II collagen and often contains short-chain proteoglycans. Fibrocartilage and fibrous repairs do not maintain a high negative charge density, are soft, and break down ( Fig. 2.1 ).

Factors that may influence the quality of repair tissue noted clinically include acuteness of injury, age, size of defect, ligament stability, axial alignment, and presence or absence of the meniscus. In a study conducted by Nehrer and associates ( Table 2.1 ), failed repair tissues were analyzed after three techniques of marrow stimulation: drilling, abrasion, and microfracture. The repair tissues retrieved were composed predominantly of fibrous and fibrocartilaginous tissues. The tissues were soft and degenerating. They had poor mechanical viscoelastic properties even though they filled the defects. The repair tissues clinically failed by 2.5 years after treatment, with an average defect size greater than 3 cm ² . Cartilage defects that were treated by perichondrial grafting had an excellent clinical result early postoperatively. By 4 to 5 years postoperatively, however, the repair tissue had undergone enchondral ossification. The perichondrial chondrocytes had features of hypertrophic chondrocytes, notably type X collagen, a precursor to mineralization ( Fig. 2.2 ). Although those grafts had a high percentage of hyaline cartilage, they also contained bone. The autologous chondrocyte implantation grafts that failed did so early (<6 months after implantation) as a result of trauma as the grafts were growing. At this early stage of repair, a high percentage of the repair tissue was fibrous or transitional in nature. Mature autologous chondrocyte implant grafts (>2 years after implantation) may have excellent clinical outcomes, with hyaline cartilage repair having firm viscoelastic properties ( Fig. 2.3 ).

Intrinsic repair in the acute situation is possible by marrow stimulation repair if chondral injury involves the underlying subchondral bone. However, appropriate rehabilitation after injury is critical ( Table 2.2 ). The factors previously described delineating the possibility of a successful repair are important. Rehabilitation after such an injury is also important. However, there may be a critical size of subchondral bone involvement that will result in cystic degeneration rather than repair. This occurs specifically in osteochondritis dissecans or when lesions are deep. A study with experimental osteochondral defects greater than 8 mm in diameter and deep in adult goats demonstrated cystic enlargement of the defects rather than repair. Therefore, repair of osteochondral defects is more complex. A staged reconstruction using autogenous tissues is required. Other options include allogeneic osteochondral reconstruction or autologous-derived tissue engineering solutions ( Fig. 2.4 ).