In the January 5 post Important new mesenchymal stem cell therapies, I promised this post specifically devoted to research on use of stem cells for cartilage regeneration. It is a long and fairly thorough post with focus on regeneration of cartilage damage due to osteoarthritis, the toughest kind to deal with.
About cartilage and osteoarthritis
From Wikipedia, “Cartilage is a stiff yet flexible connective tissue found in many areas in the bodies of humans and other animals, including the joints between bones, the rib cage, the ear, the nose, the elbow, the knee, the ankle, the bronchial tubes and the intervertebral discs. It is not as hard and rigid as bone but is stiffer and less flexible than muscle. — Cartilage is composed of specialized cells called chondrocytes that produce a large amount of extracellular matrix composed of collagen fibers, abundant ground substance rich in proteoglycan, and elastin fibers. Cartilage is classified in three types, elastic cartilage, hyaline cartilage and fibrocartilage, which differ in the relative amounts of these three main components. — Unlike other connective tissues, cartilage does not contain blood vessels. The chondrocytes are supplied by diffusion, helped by the pumping action generated by compression of the articular cartilage or flexion of the elastic cartilage. Thus, compared to other connective tissues, cartilage grows and repairs more slowly.” Hyaline Cartilage lines the ends of bones in joints in the body where there is movement, such as in the elbow or knee. A synovial fluid bathes the joint continuously so as to provide a frictionless interface.
Osteoarthritis (OA) is classically thought to be a “wear and tear” disease where the cartilage gradually wears out, like brake shoes do in a car, leaving bones rubbing directly on bones. It can occur in the knees, hands, hips and the spinal areas of the lower back and neck. “In osteoarthritis of the spine, the spaces between the vertebrae narrow. Bone spurs often form. When bone surfaces rub together, the vertebral joints (facets) and areas around the cartilage become inflamed and painful. Gradually, your spine stiffens and loses flexibility. Once these changes appear on X-rays, osteoarthritis has already started(ref).” Osteoarthritis can create stiffness, be very painful, and be seriously debilitating.
The classical view is that osteoarthritis is an incurable disease of progressing age: “Osteoarthritis gradually worsens with time, and no cure exists. But osteoarthritis treatments can relieve pain and help you remain active. Taking steps to actively manage your osteoarthritis may help you gain control over your symptoms(ref).” According to this view, if the osteoarthritis situation gets bad enough your best recourse might be surgery such as total knee replacement.
The classical view of osteoarthritis is evolving in that it is increasingly being seen as a disease that can arise from multiple causes in younger as well as older people and in that there is an increasingly brighter prospect for cartilage regeneration, even of seriously damaged joints, without need for drastic surgery. Osteoarthritis can arise from mutated genes, traumatic injury or an operation as well as from wear and tear. It is not uncommon for people in their 40s and 50s to have a serious osteoarthritis problem. Osteoarthritis can frequently arise in young people and even in children, such as when associated with a mutation in the type II procollagen gene (COL2A1)(ref)(ref).
Osteoarthritis can create collateral damage besides loss of cartilage. In the case of the spine, for example, “Sometimes, the wear-and-tear of osteoarthritis puts pressure on the nerves leaving the spinal column. This can cause weakness and pain in the arms or legs. Osteoarthritis might also cause bone spurs to form in the spinal area. Osteoarthritis of the spine sometimes is called spinal spondylosis if the damage affects the facet joint and the disks in the spine(ref).”
Osteoarthritis involves a number of degenerative processes and any effective regenerative treatment must be able to deal with these. From the 2008 review article Technology Insight: Adult Mesenchymal Stem Cells for Osteoarthritis Therapy: “Much research into the pathophysiology of OA has focused on the loss of articular cartilage, caused by mechanical and oxidative stresses, aging or apoptotic chondrocytes. Articular chondrocytes within diseased cartilage synthesize and secrete proteolytic enzymes, such as matrix metalloproteinases and aggrecanases, which degrade the cartilaginous matrix. The proinflammatory cytokine interleukin 1 (IL-1) is the most powerful inducer of these enzymes and of other mediators of OA in articular chondrocytes. The induction of these factors leads to matrix depletion through a combination of accelerated breakdown and reduced synthesis. Other proinflammatory cytokines, such as tumor necrosis factor, are also involved in cartilage breakdown and, together with biomechanical factors implicated in OA etiopathophysiology, contribute to induction of the disease.”
First-generation cartilage regeneration using implanted chondrocytes
The objective of cartilage regeneration in the case of a joint where the cartilage is seriously compromised by OA is to induce new cartilage to grow in the joint, modeling and organizing itself correctly in the process so as to restore the original functionality of the joint. The area of therapy is sometimes called cartilage tissue engineering. Hyaline cartilage is formed by chondrocytes, specialized cells that reside in and “produce and maintain the cartilaginous matrix, which consists mainly of collagen and proteoglycans.” So, first-generation attempts at joint cartilage regeneration have been focused on introduction of new chondrocytes into osteoarthritis-compromised joints. The method has become known as ACT (or ACI), standing for Autologous Chondrocyte Transplantation (or implantation) and its use goes back to 1987. “In ACT, a cartilage biopsy is taken from the patient and articular chondrocytes are isolated. The cells are then expanded after several passages in vitro and used to fill the cartilage defect. Since its introduction, ACT has become a widely applied surgical method with good to excellent clinical outcomes. More recently, classical ACT has been combined with tissue engineering and implantable scaffolds for improved results. However, there are still major problems associated with the ACT technique which relate mainly to chondrocyte de-differentiation during the expansion phase in monolayer culture and the poor integration of the implants into the surrounding cartilage tissue(ref).”
Results of ACT, when it could be applied, were often not bad. According to a 2000 publication reported on a number of ACT papers, regarding one study “The Swedish clinical experience with ACT now extends to more than 800 patients, including 200 patients with a minimum follow-up of 2 years. — Treatment of chondral and osteochondral lesions with ACT appears to produce new tissue similar in histologic and mechanical characteristics to hyaline cartilage, resulting in good clinical outcomes in more than 75% of patients. Results are best in lesions of the distal femur, including multiple defects. Patellar lesions require strict attention to alignment, and trochlear results are size-dependent.” Regarding another paper “The multicenter results presented in this paper appear to parallel the Swedish experience and demonstrate a durable repair out to 36 months. ACT appears to be an efficacious and safe treatment for full-thickness chondral lesions of the femoral condyles and trochlea.” However regarding a third paper “Osteochondral grafts improve symptoms, but may increase risk of osteoarthritis.”
“Currently, more than 12,000 ACIs are documented. Different studies showed a permanence of clinical results that were gained in a period of about 10 years [14–16]. Despite good clinical results, some disadvantages hamper the prevalence of ACI: (a) the nonuniform spatial distribution of chondrocytes and the lack of initial mechanical stability, (b) the suture of the periosteal flap into the surrounding healthy cartilage and the necessity of a perifocal solid cartilage shoulder that limits ACI to the treatment of small defects and excludes the treatment of OA diseased cartilage, and (c) the arthrotomic surgery(ref).”
The last decade showed the emergence of Arthroscopic treatment regimens for osteoarthritis of the knee, with some degree of success. “Arthroscopic treatment included joint insufflation, lysis of adhesions, anterior interval release, contouring of cartilage defects to a stable rim, shaping of meniscus tears to a stable rim, synovectomy, removal of loose bodies, and removal of osteophytes that affected terminal extension. — CONCLUSIONS: This arthroscopic treatment regimen can improve function and activity levels in patients with moderate to severe osteoarthritis. Of 69 patients, 60 (87%) patients had a satisfactory result. However, in this group of 60, 11 patients needed a second procedure, resulting in a 71% satisfactory result after 1 surgery.” There was also some degree of success reported in implanting scaffolds in osteoarthritis-damaged knees and using a modified form of ACT to regenerate knee cartilage(). “Tissue regeneration was found even when implants were placed in joints that had already progressed to osteoarthrosis. Cartilage injuries can be effectively repaired using tissue engineering, and osteoarthritis does not inhibit the regeneration process.”
However, ACT has several limitations and often can’t be used when severe cartilage loss is due to degenerative arthritis or osteoarthritis. An excellent summary of the limits of ACT can be found in the write-up of a clinical trial of a second-generation stem cell therapy. “this treatment requires the extraction of chondrocytes directly from the patient and thus causes trauma in healthy articular cartilage. Also, this type of treatment cannot be applied to large lesions, nor is the efficacy satisfactory in patients over the age of 40 whose cellular activation levels are low. Thus, autologous chondrocyte transplant is rather limited in the number of cells harvested and their activation level and is therefore restricted in terms of treatment site, severity of the condition, and the size of lesion. The current technology allows the application of treatments in local cartilage defects but not in degenerative arthritis or rheumatoid arthritis. The technology needs to be taken up to another level in order to benefit such prevalent arthritic disorders. Treatments using stem cells do not cause damage to healthy articular cartilage as they don’t require the harvesting of healthy cartilage tissues from the patients. Moreover, the number of successfully cultured cells is larger due to the excellent proliferation capability of stem cells and thus, mass supply is possible(ref).”
Second -generation cartilage regeneration using mesenchymal stem cells
Problems and limitations of ACT have led to interest in a better alternative as outlined in the March 2009 publication Mesenchymal stem cells in connective tissue engineering and regenerative medicine: applications in cartilage repair and osteoarthritis therapy. Animal experiments have demonstrated that under appropriate signaling conditions, mesenchymal stem cells (MSCs) differentiate into chondrocytes and can produce hyaline cartilage replacing that lost in injured sites. I have discussed a number of attractive properties of mesenchymal stem cells in the post Important new mesenchymal stem cell therapies. These properties are highly relevant to cartilage regeneration, including freedom from an immune or inflammatory response, donor independence, easy duplication in-vitro, homing capability and, particularly, that MSCs seem to be the body’s own natural means for cartilage renewal(ref).
I mentioned the 2007 publication Chondrogenic potential of human adult mesenchymal stem cells is independent of age or osteoarthritis etiology, indicating that neither the ability to collect sufficient numbers of MSCs nor the capability of those MSCs to differentiate into chondrocytes is affected by age of the person contributing the MSCs or whether or not they have osteoarthritis. “No correlation of age or OA etiology with the number of mononuclear cells in BM, MSC yield, or cell size was found. Proliferative capacity and cellular spectrum of the harvested cells were independent of age and cause of OA.” So, a patient’s own MSCs can be used for cartilage regeneration, even if the patient has osteoarthritis which destroyed the original cartilage. Despite this publication’s conclusions, some researchers are still concerned as to whether MSCs from an old sick person are as good as ones from a young healthy person. I personally wonder whether the telomere lengths of MSCs from old people are equal to those from younger people, and whether the epigenetic markers of MSCs from sick people allow those cells to be good candidates for tissue regeneration.
The 2009 publication Tissue engineering in the rheumatic diseases is an excellent treatise covering both the first and second generation tissue engineering approaches for osteoarthritis cartilage damage and I suggest it for anyone wishing to go into further depth than possible in this post. “Inflammatory conditions in the joint hamper the application of tissue engineering during chronic joint diseases. Here, most likely, cartilage formation is impaired and engineered neocartilage will be degraded. Based on the observations that mesenchymal stem cells (a) develop into joint tissues and (b) in vitro and in vivo show immunosuppressive and anti-inflammatory qualities indicating a transplant-protecting activity, these cells are prominent candidates for future tissue engineering approaches for the treatment of rheumatic diseases.” The following selective quotes are from the same paper.
“Diseases like rheumatoid arthritis (RA) or degenerative arthritis (osteoarthritis, OA) are accompanied by a progressive reduction of extracellular matrices (ECMs) in joint cartilage and bone and, eventually, loss of joint function and excessive morbidity. Current pharmacological treatment of RA focuses on alleviating symptoms and/or modifying the disease process. Despite recent success in controlling pain and inflammation, marginal cartilage regeneration has been observed.” The last comment is important: there is a natural process of cartilage regeneration, though usually inadequate. The passage goes on “Obviously, suppression of inflammation is not sufficient to restore joint structure and function. Probably, cartilage repair may be achieved only by triggering local cartilage tissue responses leading to recovery of chondrocyte remodeling. An imbalance in joint cartilage, subchondral bone, and synovial membrane remodeling is one important characteristic of OA.”
“Besides clinically applied tissue-specific chondrocytes, undifferentiated mesenchymal stem cells (MSCs) are of special interest as cell candidates. In particular, bone marrow MSCs are comprehensively characterized and represent promising candidates . They are easy to isolate and expand, they differentiate into various tissues like cartilage  and bone , and therefore they are able to regenerate osteochondral defects. Additionally, as they target diseased organs and secrete many bioactive factors, such as immunosuppressives for T cells facilitating their allogeneic use, they serve as vehicles capable of presenting proteins with therapeutic effects.“
“In this regard, secreted bioactive factors provide a regenerative environment, referred to as trophic activity, stimulating, for instance, mitosis and differentiation of tissue-intrinsic repair or stem cells (reviewed in ). Because of their anti-inflammatory and immunosuppressive properties, MSCs have been used as agents in autoimmune diseases (ADs) and have been applied in arthritis animal models (reviewed in ).”
The 2008 review article Technology Insight: Adult Mesenchymal Stem Cells for Osteoarthritis Therapy also provides a good summary of the reasons for basing therapies for large areas damaged by osteoarthritis on use of MSCs. “Unlike chondrocytes, the use of MSCs is not hindered by the limited availability of healthy articular cartilage or an intrinsic tendency of the cells to lose their phenotype during expansion. The use of MSCs also obviates the need for a cartilage biopsy and, thereby, avoids morbidity caused by damage to the donor-site articular surface.”
Mesenchymal stem cell delivery modes for tissue regeneration
A number of approaches have been suggested for obtaining and delivering MSCs to a site requiring regeneration(ref).
1. Get the MSCs from the patient or some other source (another person or umbilical cord blood), multiply them and inject them directly into the site requiring regeneration, e.g. intra-articular injection, perhaps with hyaluronic acid,
2. Same but first implanting a biodegradable synthetic or natural scaffold, perhaps employing a collagen hydrogel,
3. Attraction of autologous MSCs within the body using microfractures, an established technique, and
4. Attraction of autologous MSCs within the body using scaffolds and stem-cell attracting and differentiating factors.
Microfractures and mobilizing the body’s MSCs
Microfracturing was the first MSC cartilage regeneration technique and has been used clinically for years with some success. “Finally, it should be mentioned that ACI treatment is still controversial. In a prospective randomized controlled trial (level of evidence: therapeutic level I), no significant advantage for the complex ACI compared with standard self-repair-stimulating microfracture could be measured after 2 and 5 years .” Standard self-repair-stimulating microfracture is a technique in which microfractures are deliberately introduced in compromised cartilage to stimulate the body’s own cartilage regeneration capabilities. As I understand the procedure, a surgeon drills a number of small holes (2mm in diameter) in the cartilage down the point where there is bleeding from the bone marrow. The theory is that the holes provide access to the cartilage of MSCs from the bone marrow. See Chondral Resurfacing of Articular Cartilage Defects in the Knee with the Microfracture Technique. The results of applying this technique are not too shabby. “At the time of the latest follow-up, knee function was rated good to excellent for thirty-two patients (67%), fair for twelve patients (25%), and poor for four (8%). — Microfracture repair of articular cartilage lesions in the knee results in significant functional improvement at a minimum follow-up of two years.”
Apparently, the microfractures mobilize the body’s own mesenchymal stem cells to regenerate cartilage. It appears that this process can be enhanced by adding a bit of hyaluronic acid. “Following microfracture in rabbit knee cartilage defects, application of hyaluronic acid gel resulted in regeneration of a thicker, more hyaline-like cartilage(ref).” The technique is quite different than removing stem cells from the bone marrow of a patient, growing them in culture and then re-introducing them into the body where the repair is needed. It depends instead on a) stimulating a part of the body needing cartilage regeneration to send out signals that naturally mobilizes a patient’s own MSCs to multiply, swarm to the cartilage site and do the regeneration job that is needed, and b) providing easy physical access for the stem cells to reach the cartilage area involved.
The future direction
A distinct possibility is that similar results can be achieved in the future without a need for microfractures, using chemotactic agents, substances that can get MSCs moving to the right place and differentiating into cartilage tissue. “So, the next generation of tissue engineering focuses on in situ approaches . Here, for joint repair, scaffolds combined with chemotactic molecules and joint tissue formation-stimulating factors are transplanted, resulting in the in situ recruitment of bone marrow MSCs to the defect sites of degenerated cartilage and bone and their subsequent use for factor-guided joint repair. — Although MSC migration factors and their mechanisms are not known yet, molecules such as chemokines , bone morphogenetic proteins and platelet-derived growth factor , and hyaluronan  have been shown to have a dose-dependent chemotactic effect(ref).“ Recent research shows that transforming growth factor TGF-Î²3 has a capacity to mobilize and initiate the differentiation of the body’s MSCs(ref)(ref).
As to clinical investigations of using MSCs for OA cartilage damage, there is the trial Autologous Transplantation of Mesenchymal Stem Cells (MSCs) and Scaffold in Full-Thickness Articular Cartilage. It is a small study started in August 2998 and originally scheduled for completion in May 2010. Another relevant clinical trial just posted and now enlisting participants is Study to Compare the Efficacy and Safety of Cartistem® and Microfracture in Patients With Knee Articular Cartilage Injury or Defect. “The purpose of the study is to assess and compare the safety and efficacy of the allogeneic-unrelated umbilical cord blood-derived mesenchymal stem cell product (Cartistem®) to that of a microfracture treatment in patients with articular cartilage defect or injury. — This clinical trial for the stem cell therapies is essential because treatment of cartilage defects with umbilical cord blood-derived mesenchymal stem cells, known to have the highest level of activity among all adult stem cells, opens the possibility of articular cartilage regeneration even for aged patients and patients with large lesions unable to benefit from existing treatments.”
While MSCs offer great promise for tissue regeneration based on both theoretical understandings and experiments with animal models, many questions regarding human use remain incompletely resolved, e.g. use of allogeneic vs. autologous cells, mode of implantation and use of scaffolds, use of chemotactic and messenger molecules, and even nagging issues of whether age and arthritic status of donors are really irrelevant. In any event, now in 2010 the myth that nothing basic can be done about OA cartilage deterioration has lost credibility. Clinical trials of MSCs are getting underway and even more can be expected to be launched soon. My own guess is that in 5-10 years tissue-engineering regeneration of osteoarthritic and other forms of cartilage damage will be in widespread clinical use. It is the dawn of the age or tissue engineering and regenerative medicine.