Joint damage, articular cartilage degeneration, and the research into methods of restoring the articulation surfaces are not new topics of interest. For centuries, clinicians and scientists have recognized the importance of cartilage damage, and learned about the normal form and function of articular cartilage, as well as the process of degradation and restoration. Unfortunately, only some of the real problems have been resolved.
Recently, tissue engineering and regenerative medicine have been introduced as a method of restoring tissue or organ function, and has already these concepts have been applied to cartilage repair. The possibility of modulating cells/tissues and applying this novel capability towards the restoration of articular cartilage function provides great promise and opportunity for progress in orthopaedics and traumatology, but to date, no concrete basis for full clinical implementation has been established.
This article investigates influences of cartilage tissue engineering and identifies areas in which outcomes can be improved by using a combination of basic and applied research. Identifying the effect and optimal characteristics of various basal culture media, serum supplementation, polypeptide growth factors, scaffolds, and autologous tissue-engineered cartilage implantation in animal models can be beneficial for both in vitro and clinical application. It is hoped that this information may allow researchers to improve the outcome of cartilage tissue engineering in patients.
Articular cartilage, the knee’s shock absorber, is an avascular, aneural, and alymphatic tissue that covers the end of the subchondral bones and is responsible for the distribution of mechanical loads over the articulating surface area, giving a near frictionless surface. Articular cartilage is made up of chondrocytes embedded in an extracellular matrix consisting of collagen, proteoglycan, noncollagenous proteins, and tissue fluid (Mankin 2000b). Chondrocytes represent only about 1% of the volume of the hyaline cartilage, but they are essential: Chondrocytes are responsible for replacing the degraded matrix molecules to maintain the biomechanical properties of the tissue. Although just a few millimetres in thickness, articular cartilage is nevertheless quite complex and consists of four zones, the superficial, transitional, deep, and calcified zones, each with its own distinct composition and structure.
Healthy articular cartilage has the ability to deform and enlarge its surface contact area to lessen the effects of the direct loads by decreasing applied stress (Buckwalter and Mankin 1997, Mankin 2000b). This unique biomechanical property is supported efficiently by the presence of aggrecan complexes that are entrapped within the complicated insoluble collagen network. Collagen is the main component that contributes to the shear strength of cartilage, as well as its tensile strength.
Although collagen types II, VI, IX, X, and XI can be found in articular cartilage, the major collagen component of normal cartilage is collagen type II. Collagen type II accounts for approximately 90-95% of the collagen found in the knee joint, and it has a high amount of bound carbohydrate groups to allow more interaction with water than some other types of collagen. The production of collagen type II by mesenchymal cells in culture is taken generally as evidence of the cartilage-specific phenotype. On the contrary, the production of collagen type I by monolayer cultured cells is an indication of the loss of specific cartilaginous phenotype in vitro, which normally refers to as dedifferentiation (Baek 2002).
Proteoglycans, which help distribute load at the bony ends of the joint, are the main source of resistance to solute transport and fluid flow in the cartilage matrix; they contribute significantly to cartilage mechanical stiffness. These proteoglycan strongly influence the mechanical function of cartilage and play a central role in the transduction of microphysical signals to chondrocytes during tissue compression (Quinn 2002). Proteoglycans are composed of approximately 95% polysaccharide and 5% protein. They are monomers or aggregates joined to hyaluronic acid filaments by means of specialized link proteins. The monomer consists of a central protein core that is bound covalently to multiple sulphated glycosaminoglycan (sGAG) chains and two types of oligosaccharides. The GAG chains are unbranched polysaccharides made from disaccharides of an amino sugar and another sugar. At least one component of the disaccharide has a negatively charged sulfate or carboxylate group. Therefore, the glycosaminoglycans tend to repel each other and other anions while attracting cations and facilitating interaction with water in the tissue.
Chondroitin sulfate is a glycosaminoglycan that is found naturally in the extracellular matrix of articular cartilage. It is composed of a long unbranched polysaccharide chain with a repeating disaccharide structure of N-acetylgalactosamino and glucuronic acid. Chondroitin sulfate proteoglycan, also known as aggrecan, is one of the large aggregates that is a major extracellular matrix component in cartilage. Hyaluronic acid, keratan sulfate, dermatan sulfate, and heparan sulfate are additional GAGs generally found in articular cartilage.
Because no blood vessels penetrate into the tissue, articular cartilage obtains its nutrients and oxygen from the synovial fluid. Cartilage exhibits a very limited capacity to regenerate and repair due to its avascular nature. Spontaneous repair is achieved only when the injured area penetrates the subchondral bone, result in bleeding and probably triggering a wound healing response from the bone surface. The ability of articular cartilage to repair is compromised by the lack of inflammatory wound healing response, as evidence by the inability of chondral or partial thickness injuries of the tissue to repair (Buckwalter and Mankin 1997, Mankin 2000a).
In a normal situation, articular cartilage can perform its required function for a lifetime, although some age-related, degenerative alterations occur. Unfortunately, cartilage defects are not a limited problem: Defects larger than 2 mm in diameter do not heal, and persistent defects frequently progress to joint degeneration or osteoarthritis.
Spontaneous repair of musculoskeletal tissue begins with an inflammatory response (Buckwalter and Mankin 1997, Mankin 2000a, Akeson 2001). Injured cells and platelets release mediators to promote vascular response to injury. Inflammatory cells help remove necrotic tissues and release mediators that stimulate migration and proliferation of chondrogenic mesenchymal cells. The occurrence of these events during inflammation is critical for initiation of effective tissue repair.
This response is not sufficiency present in the avascular articular cartilage surface; therefore, many superficial or partial cartilage injuries do not heal. In full-thickness defects, mesenchymal cells enter the injury site from the subchondral marrow. These cells provide repair, but do not consistently restore the injury with tissue that has the unique composition, structure, and material properties of normal articular cartilage.
The only cell type found in articular cartilage, the highly differentiated chondrocyte, has limited capacity for proliferation or migration because chondrocytes are encased within the tissue. In normal mature cartilage, chondrocytes synthesize sufficient matrix macromolecules to maintain the matrix, and they can increase their rate of matrix synthesis in response to injury or osteoarthritic changes (Hunziker 1999, Hunziker 2001). However, chondrocytes do not synthesize sufficient matrix to repair significant tissue defects, and the matrix macromolecules they synthesize change with increasing age.
Another factor that may limit the ability of mature cartilage to repair tissue defects is that the number of chondrocytes declines during aging, thus reducing the capacity of the tissue to repair itself. Natural healing often results in a repair that reconstitutes some of the form and function. This is not identical to full restoration of all tissue properties suggested in using the term "regeneration."
To date, a variety of treatment options exist to restore joint function. These include conservative measures such as functional adaptation, physiotherapy, and medication, and surgical intervention ranging from arthroscopic or minimally invasive procedures to whole tissue transplants, joint replacement and, more recently, tissue engineering. The selected method depends on the presenting complaint and findings at physical examination, diagnostic imaging, patient age, defect characteristics, and surgeon preferences.
Nutraceuticals such as chondroitin sulfate and glucosamine are available in the market for relief of osteoarthritis (McAlindon 2000a, McAlindon 2000b). However, the idea of dietary supplementation of the cartilage matrix proteins is not supported by information on long-term efficacy or level of pain relief (Johnson 2001a). Non-steroidal anti-inflammatory drugs (NSAIDs) are used to reduce pain and synovitis. Improvement of pain and function by NSAIDs was proven better than in placebo groups, but there was no lasting effect (Walker-Bone 2000). The adverse effects on gastrointestinal and renal function and blood coagulation are significant. Topical treatment with NSAIDs or capsaicin may be offered to patients with inadequate pain relief from systemic medications or those who cannot tolerate such therapy.
Intra-articular injections are used when signs of effusion or synovitis are present in osteoarthritic cases. Corticosteroids have been shown to facilitate significant pain relief and reduction of effusion for 4-6 weeks. The long-term effect is ambiguous due to confounding factors, such as surgical intervention or parallel treatments. Because of a negative effect on cartilage metabolism and acceleration of cartilage damage, it is suggested that intra-articular injections should not be the only therapeutic strategy.
Hyaluronic acid, a polysaccharide present in normal synovial fluid, provides lubrication and shock absorption in the knee joint. It plays an important role during embryonic joint formation in regulating cartilage cavitations. In osteoarthritis, there is a reduction in hyaluronic acid level. Randomized controlled trials of hyaluronic acid injections have shown pain relief superior to placebo and comparable to corticosteroids, although corticosteroids had longer-lasting effects. Approximately 20% of patients experience a flare up of complaints soon after hyaluronic acid injection (Henderson 1994). The exact placement and cost-effectiveness of this therapy within a treatment algorithm remains to be determined.
Future investigations should be carried out to test intra-articular substances to determine whether joint homeostasis may be normalized prior to treatment of a cartilage defect by tissue engineering.
Total knee replacement is one of the most successful surgical reconstructive procedures. A well-implanted, modern joint prosthesis replaces the degenerated osteoarthritic surface and will function properly for 15 to 20 years in a relatively sedentary older patient population. Unavoidably, at some point increasing symptoms will occur and signs of prosthetic loosening will be detected. With increased loading and more strenuous activities, the longevity of the joint replacement will decrease. Revision surgery can be a technical challenge, the outcome of which is considerably inferior to that of primary surgery. Therefore, it is generally accepted that total joint replacement is not a realistic treatment option for young patients.
Lavage and debridement are performed to overcome mechanical impediment by removing loose or unstable cartilage and to limit the rate of degeneration (Fu 2005). Most literature suggests that this widely accepted operation is palliative, not curative, and symptomatic relief is achieved for a limited period (Minas and Nehrer 1997).
Abrasion arthroplasty (Johnson 2001b), drilling (Pridie 1959), and micro fracturing (Steadman 2001) are similar methods. After breaching the subchondral bone, the marrow cavity will open and provide a source of undifferentiated stem cells that have osteochondrogenic potential. Under the stimulus of movement and regulated load bearing, bone marrow cells in the clot on the surface of the exposed bone will form finite durable fibrocartilages. The techniques used vary considerably, which creates difficulty in comparing outcomes of such procedures. In animal experiments of subchondral perforation, the repaired tissue more closely resembled hyaline cartilage than that in large superficial defects. Most authors agree that these methods provide relief for 3-5 years. These arthroscopic techniques are currently accepted as initial treatment of choice for cartilage defects.
Osteotomy provides relief of symptoms and the possible prevention of further joint deterioration (Weisl 1980). This surgical intervention aims to decrease the load on the most severely damaged cartilage, to bring regions of the joint that remain intact into opposition with regions that lack articular cartilage, or to correct the misalignment or incongruency in the joint. A decrease in pain and functional restoration of the joint is seen in relation with local widening of the joint space and a decrease of stress on the damaged region. Negative predictive factors are older age, obesity, ligament instability, over- or under-correction, and severe degeneration. Even patients regarded as optimal candidates for osteotomy given these parameters show deterioration with time at long-term clinical follow up. Osteotomy is generally indicated in patients too young for arthroplasty, with angular deformities of more than 5° and limited walking distance with pain at rest.
Osteochondral techniques involve transplanting single or multiple autologous plugs from less loaded areas or using donor tissue (Bobic and Noble 2000). Autografts can only be harvested from a limited area to avoid morbidity at the donor side. Insufficient proof exists that this procedure does not induce arthritic deformation, as matrix integrity and joint homeostasis are disturbed. Allograft osteochondral transplants provide the advantage of matching size and geometry of the full defect. Clinical results in focal post-traumatic defects were described as good to excellent in 27 of 31 patients with a follow-up of 2 to 10 years (Meyers 1989). Allograft-related factors such as storage influences and disease transmission remain to be addressed.
Tissue Engineering and Regenerative Medicine
The National Institute of Health (NIH) defines tissue engineering and regenerative medicine as follows: “Tissue engineering and regenerative medicine is an emerging multidisciplinary field involving biology, medicine and engineering that is likely to revolutionize the ways to improve health and quality of life for millions of people worldwide by restoring, maintaining, or enhancing tissue and organ function.”
Tissue engineering research includes the following area:
- Cell biology, which includes enabling methodologies for cells growth and differentiation, and acquiring appropriate cells sources, such as autologous cells, allogeneic cells, stem cells, and genetically manipulated cells
- Biomaterials, which are needed to direct cell growth, differentiation, and organisation to form functional engineered tissue
- Biomolecules, which includes signalling molecules such as growth factors, differentiation factors, or angiogeneic factors that help enhance the formation of functional tissue
- Engineering design, which includes manipulation and selection of suitable microenvironment for cells growth using monolayer culture expansion, three-dimensional culture system, and bioreactors
- Biomechanical aspects of design, which includes characterisation of native tissue properties, identification of minimum mechanical requirement for engineered tissue, mechanical signals regulating engineered tissue, and efficacy and safety of the engineered tissue represent essential components
In addition, rapid progress in bioinformatics technology has enabled easy access to tissue engineering research and development. All information regarding gene and protein expression, sequencing, quantitative tissue analysis, digital tissue manufacturing, automated quality assurance systems, data mining tools, and clinical informatics interfaces has already been made available and reliable to help support tissue engineering growth.
Application in Knee Dysfunction
Patients seek medical care on a daily basis because of a recent trauma or joint dysfunction that can be related to articular cartilage damage. Many of these patients are young and active, and unfortunately handicapped to some extent because of their complaints. Cartilage defects larger than 2 mm in diameter do not heal, and the occurrence of cartilage defects this size is high (Mankin 2000a).
Cartilage damage most frequently occurs in the knee, due to, for instance, trauma, ligamentous instability, malalignment of the extremity, meniscectomy, or primary osteochondritis dissecans. The treatment of these cartilage defects forms a considerable challenge for orthopaedic surgeons. Initial treatment typically consists of symptomatic relief by rest, NSAIDs, and functional mobilization. In most cases, accompanying ligament or meniscal damage exists, and a solitary cartilage lesion is not diagnosed on primary evaluation. Further diagnostic measures, such as magnetic resonance imaging (MRI), may be indicated; arthroscopic intervention may even be necessary.
Most, if not all patients with a relevant cartilage defect are seen again in the outpatient clinic weeks to months after trauma and present with complaints of pain, persistent effusion, and intermittent locking of the joint. As mentioned, most cartilage defects do not heal, putting the patient at risk for osteoarthritis. No successful and lasting treatment method has been developed other than joint replacement surgery, leading to considerable patient morbidity. This has motivated an increasing interest in tissue engineering techniques for cartilage repair. Most tissue engineers aim to restore the articular cartilage surface either by repairing the defect so that functional demands can be met or by ideally achieving true regeneration of a hyaline articular cartilage, which will fulfill all biological and mechanical requirements for the remaining life span (Munirah 2007, Munirah 2008).
The goal of autologous cell or tissue transplantation is mainly to provide a durable regenerate tissue surface rather than short-term repair. For perichondrial and periosteal grafting, for example, the defect is cleaned and extended into the subchondral bone. Periosteum from the proximal tibia or perichondrium from a rib is placed at the bottom of the defect, with the cambium layer facing outward towards the joint. The mesenchymal stem cells trigger a chondrogenic process that restores both the cartilage surface and the subchondral bone.
To date, autologous chondrocyte implantation (ACI) is the most commercial strategy available, and it has been extensively investigated (Brittberg 1994, Brittberg 2000, Peterson 2000, Brittberg 2001, Peterson 2002, Peterson 2003). In this procedure, chondrocytes are harvested from a less weight-bearing area of the knee joint. Cells are culture-expanded for 3-4 weeks and then re-implanted under a periosteal flap sutured surrounding cartilage.
Both small and large animal models were used and results were similar: Good results were seen in 70-85% of subjects in short- to mid-term follow-up experiments evaluating the percentage of defect filling, quality of cartilage, and incorporation into surrounding defect. Outcomes decrease somewhat with longer follow-up (Peterson 2000, Peterson 2002, Peterson 2003).
Numerous human studies on cartilage defect repair using chondrocytes with or without matrices have been published, with varying results (Frankel 2005, Mardones 2005, Redman 2005, Munirah 2006a, Munirah 2006b, Ruszymah 2006, Jakobsen and Engebretsen 2007, Munirah 2007). A correlation has been seen between clinical results and the quality of the biopsy material. There are, however, several issues that limit the efficacy of this technique. For example, the cells may not survive and multiply in culture. The cartilage cells in culture typically undergo dedifferentiation to fibroblasts after several passages. The periosteal covering provides growth factors and contains cells that can contribute to the repair, but they also undergo calcification and hypertrophy (Peterson 2000).
Commercial interest is considerable. More than 16 biotechnology companies in Europe and in the United States are offering one or more options for cartilage repair, and patient registries maintained by industry document over 4,500 patients treated with ACI. First generation products comprise cultured cells to be implanted under a periosteal flap or collagen sheath.
The most established company for cartilage repair is the Genzyme Biosurgery, a division of Genzyme Corporation, USA. They have been known for years for the Carticel® (autologous cultured chondrocytes) for implantation, which was initially approved in the United States in 1997. Carticel is an autologous cellular product indicated for the repair of symptomatic cartilage defects of the femoral condyle (medial, lateral or trochlea), caused by acute or repetitive trauma in patients who have had an inadequate response to a prior arthroscopic or other surgical repair procedure, such s debridement, microfracture, drilling/abrasion arthroplasty, or osteochondral allograft/autograft.
With the emergence of tissue engineering research, second-generation products aim at implantation using a solid three-dimensional matrix, thereby helping to enable minimal invasive or even arthroscopic implantation. For example, the matrix-induced autologous chondrocyte implantation (MACI®; Verigen, Leverküsen, Germany) is a tissue engineering technique for the treatment of deep chondral lesions. Cultured chondrocytes are seeded on a collagen membrane that can be implanted into the defect using exclusively fibrin glue.
These features imply some surgical advantage with respect to the traditional ACI technique, such as the possibility of performing the procedure in articular sites, in which suturing the periosteal patch is impossible (Ronga 2004). However, only limited initial prospective scientific data are available. A realistic outcome of clinical cartilage repair can only be judged after 5-10 years or more, and this long-term requirement is in conflict with the short-term commercial interests of companies involved (Jakobsen and Engebretsen 2007). Obviously, such comparisons and subsequent analysis of cost effectiveness would be necessary to progress towards solid implementation of tissue engineering as a novel treatment strategy.
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