Creating Cartilage

Cartilage is the shock absorber of the body. It allows two bones to move past each other without deleterious effects. Today, we walk on paved streets and carpeted buildings with stiff floors. Our cartilage takes a constant beating and as we age, it has a tougher and tougher time bouncing back.

As we age, the daily wear and tear eventually grinds this tissue down and movement can become painful and tedious. Osteoarthritis affects over 27 million Americans and it can cause pain, stiff joints, cracking sounds, inflammation and bone spurs.

Cartilage formation depends upon the activity of one cell type – the chondrocyte. With advancing age, chondrocytes divide less and less and eventually, they fall behind making new cartilage to repair the defects generated by everyday wear and tear. In the long-term, chondrocytes can respond to stress by simply dying-off.

Orthopedic specialists consider cartilage regeneration the holy grail of orthopedic medicine. Since people are living longer, orthopedists have taken to cleaning out damaged joints with arthroscopic surgery, braces to stabilize a wobbly gait, and artificial knees and hips to replace heavily damaged joints.

Now, stem cell technology has given the hope of actually re-making new cartilage in aging,arthritic patients. Bio-engineers are working hard to crack the nut of cartilage production. They have identified prominent proteins required to turn stem cells into chrondrocytes, and have also designed three-dimensional scaffolds upon which stem cells will grow and eventually make cartilage. Cartilage-forming cells seem to behave normally if they are constantly surrounded by molecules found in normal cartilage. Also the scaffolding derived from cartilage seems to provide many of the molecular prompts for cartilage behavior.

Much of this is still in the experimental phase, the “stem cell strategy” for re-synthesizing cartilage seems to be one of the best possibilities and clinical trials are in the works in Norway, Spain, Iran, Malaysia, and other places too.

Tissue engineering had its start in the 1970s, and then it comes to cartilage, it has certainly had its ups and downs. For example, the type of cartilage found at the ends of long bones is known as “hyaline cartilage” because of its slippery feel, glassy look, elastic properties and smooth texture. Hyaline cartilage is a terrific weight-bearing cap. Nevertheless attempts to make cartilage at joints has resulted in the production of “fibrocartilage.”

For example, surgeons have often treated arthritic joints by cleaning out bone spurs, scar tissue, and then drilling small holes into the ends of the bones. This causes stem cells to move into the joint and make new cartilage, but they make fibrocartilage instead of hyaline cartilage. Fibrocartilage is found at the place where our pelvic bones join, our intervertebral discs, and our jaw joint. It does not have the ability to resist impact forces the way hyaline cartilage does. Therefore, it erodes quickly at joints. John Sandy, a biochemist at Rush University Medical Center, Chicago put it this way, “Those stem cells that come out are confused. They’re not getting the right signals….So they hit the middle road.” In a study that examined microfracture surgery, two-thirds of athletes who had the procedure showed good results, but only half of those were able to play at their original level for several years.

Chondrocyte transplantation has also been attempted. A Cambridge, MA biotech company called Genzyme has an off-the-shelf product known as Carticel that takes thousands of live chondrocytes from healthy cartilage elsewhere in the body and cultures them to expand their numbers. The expanded chondrocytes are then injected into the affected site. This is known as autologous chondrocyte implantation and this procedure has outperformed microfracture surgery, at least in some studies. Unfortunately, some patients need follow-up surgery, and a nine-year study found that 50% of patients did not improve at all (see Christopher M. Revell, and Kyriacos A. Athanasiou, Success Rates and Immunologic Responses of Autogenic, Allogenic, and Xenogenic Treatments to Repair Articular Cartilage Defects. Tissue Eng Part B Rev. 2009 March; 15(1): 1–15). According to Wan-Ju Li, a tissue engineer at the University of Wisconsin, Madison, chondrocytes lose their ability to form cartilage if they are grown for too many generations in culture.

Can other cells be used to form cartilage? The answer is a clear “yes.” Stem cells from cartilage, tendons, and synovial membranes (the sac that surrounds the joint) can all form cartilage, as can stem cells from fat, and umbilical cord.

The next question is, “how do we coax these stem cells into making cartilage?” What works in culture dishes in the laboratory may not work inside a living joint, but certainly, getting it to work in the laboratory is the first place to start. Several compounds have been found that are definitely pro-cartilage molecules. These include a growth factor called TGF-beta, which jumps starts stems into the cartilage-forming program. However, as John Sandy explains, TGF-beta does not work alone because it will tend to drive cells to form fibrocartilage rather than hyaline cartilage.

The other growth factor needed to turn stem cells into hyaline cartilage-making chondrocytes is fibroblast growth factor-2 (FGF-2; see Andrew M. Handorf and Wan-Ju Li, Fibroblast Growth Factor-2 Primes Human Mesenchymal Stem Cells for Enhanced Chondrogenesis PLoS One 2011; 6(7): e22887). FGF-2 turns on a transcription factor in stem cells called Sox9 which switches on the production of type 2 collagen and aggrecan (two cartilage-specific proteins).

Other booster compounds that increase the cartilage-making profiles of stem cells include a synthetic molecule called kartogenin (Kristen Johnson, et al., A Stem Cell–Based Approach to Cartilage Repair. Science 11 May 2012: Vol. 336 no. 6082 pp. 717-721). Kartogenin inhibits a stem cell protein called filamin A and this unleashes all kinds of cartilage-specific processes in the stem cells. Kartogenin has taken the cartilage camp by storm, and Joan Marini of the National Institutes of Health in Bethesda, MD and Antonella Forlino of the University of Pavia in Italy wrote in the June 28 edition of the New England Journal of Medicine: “Stimulating the differentiation of one’s own stem cells by means of an easily deliverable chemical compound would be more advantageous than using conventional drilling and microfracture techniques.”

Another pro-chondrocyte protein is vimentin, which helps cells assume a round shape. According to Ricky Tuan, tissue engineer at the University of Pittsburg, vimentin pushes bone marrow stem cells into nice round cells that look like chondrocytes. Even more vimentin pushes the cells to make type 2 collagen (cartilage-specific; see Bobick B. E., Tuan R. S., Chen F. H. (2010) The intermediate filament vimentin regulates chondrogenesis of adult human bone marrow-derived multipotent progenitor cells. J. Cell. Biochem 109, 265–276).

Thus with the right blend of compounds, good hyaline cartilage can be made, but according to Ming Pei, an orthopedic surgeon and cell biologist at West Virginia University in Morgantown, making proper hyaline cartilage probably comes down to using the right stem cell. Pei thinks that stem cells from the synovial membrane have an advantage over other stem cells when it comes to cartilage making because of a substance they make.

Pei’s research team mixed the matrix made by synovial stem cells with FGF-2 in a low-oxygen environment. When they added other synovial stem cells those cells ramped up their cartilage making capabilities (Pei M, He F, Kish VL. Expansion on extracellular matrix deposited by human bone marrow stromal cells facilitates stem cell proliferation and tissue-specific lineage potential. Tissue Eng Part A. 2011 Dec;17(23-24):3067-76). These conditions seem to provide a safe place or stem cells to become chondrocytes and make cartilage. Such safe places for stem cells are called “stem cell niches.” Pei’s niche seems to be optimized for stem cells to make cartilage.

In order to get the chondrocytes to make cartilage that sticks together, they need to be in a niche that closely enough resembles their native niche. To mimic this niche, Li and Tuan started to build synthetic scaffolds that they could seed with stem cells. Such matrices definitely improved cartilage production by stem cells. According to Pei, “It’s easy to fabricate and there’s no batch-to-batch difference.”

Li notes that the polymer is designed to degrade after six-twelve months in the body and they have all the strength and mechanical properties to keep the stem cells together until they make a matrix of their own. In 2009, Tuan and Li tested their scaffold by seeding it with human stem cells so that it would create a patch that was inserted into pigs that had suffered cartilage damage. They used two types of cells to seed the matrices – stem cells and mature chondrocytes. After implanting the matrices, those that had been seeded with stem cells make hyaline cartilage, but those seeded with mature chondrocytes made fibrocartilage, after six months. Li reported, “It was glassy cartilage with good mechanical properties.”

As an alternative scaffold, scientists are also using scaffolds made from cartilage procured from cadavers. According to Pei, natural cartilage scaffolds have advantages that synthetic scaffolds do not have, such as chondrocyte-inducing molecules embedded in them.

Other laboratories are using matrices made from fibrin (the stuff blood clots are made from) and seeding with platelets, which are rich in TGF-beta. Doctors at Cairo University in Egypt seeded this scaffold with stem cells that were then implanted into the knees of five patients, all of whom reported improvements after one year.

Regardless of the exact scaffold that is used, Li is buoyantly confident that a stem cell-based strategy will result in making cartilage in the joints of aging patients.

If a treatment is found for osteoarthritis, the next question becomes, “when should the treatment be offered?” David Felson, a rheumatologist at Boston University School of Medicine. notes that knee injuries increase the likelihood a person will suffer from osteoarthritis sixfold. Felson’s research seems to indicate that such injuries “probably account for a great majority of osteoarthritis.”

Early detection is probably not practical, since most people ignore their injuries. Some patients can have bones rubbing together long before they start to experience the pain of osteoarthritis.

However, perhaps chemical markers can help detect the early signs of joint trouble. Carla Scanzello, who works at Rush University Medical Center as a rheumatologist reported that inflammatory molecules that gradually destroy cartilage leave chemical tells that can be detected and might provide a way to detect the early signs of joint damage before symptoms appear (See Carla R. Scanzello, et al., Synovial inflammation in patients undergoing arthroscopic meniscectomy: molecular characterization and relationship with symptoms. Arthritis Rheum. 2011 February; 63(2): 391–400).

Stem cell treatments might also reduce the number of patients who need artificial joint replacements. An artificial hip or knee can last 10-15 years. IF you are older, that is usually not a problem, if you are younger, that becomes a problem. According to Li, there are technical problems to be worked out, but the largest hurdles have been largely conquered, what remains is largely engineering questions. His goal is to eventually make joint replacement a thing of the past and turn orthopedic surgeons into stem cell scientists.