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.

Epigenetic Alterations Are Linked to Cell Fate Decisions in Mesenchymal Stem Cells

Stem cell scientists at the University of California, Los Angeles (UCLA) have discovered that the activity of particular enzymes that modify the structure of chromatin is linked to the differentiation of mesenchymal stem cells into particular cell types.

A study in the laboratory from the laboratory of Cun-Yu Wang, who is a professor at the UCLA School of Dentistry, determined that even though the DNA sequences of the genomes of stem cells are not altered, the structure of their DNA is. Furthermore, these alterations to the DNA structure are inherited by the progeny of those cells. Changes in DNA that affect the appearance or behavior of cells that are not changes in the sequence of DNA are known as epigenetic changes and these epigenetic changes can greatly affect the differentiation of stem cells.

Wang’s research team discovered that two enzymes, KDM4B and KDM6B, promote the differentiation of mesenchymal stem cells (MSCs) into bone cells. KDM4B and KDM6B are “histone demethylases,” which is a fancy way of saying that they remove specific chemical groups known as “methyl groups” (—CH3) from histone proteins. Histone proteins help package DNA into a compact structure known chromatin. Chemical modification of these histones influences the nature of that chromatin. The addition of acetyl groups (-CH2-COO-) tends to make the chromatin rather loose and easily accessed by gene expression machinery. However, the addition of methyl groups tends to tighten the chromatin up and shut down gene expression. Demethylation or removing methyl groups from histones tends to take tight chromatin and loosen it up so that new gene expression is possible.

The conversion of MSCs into bone cells is stimulated by a group of growth factors called “Bone Morphogen Proteins” (BMPs). To this end, Wang and his colleagues used BMP-4 & -7 to induce the bone fate in MSCs. They discovered that BMP-4 & -7 induces expression of histone demethylases KDM4B and KDM6B. Then they determined the targets of these enzymes within MSCs. They found that KDM4B and KDM6B remove methyl groups from Histone H3 (specifically, H3K27me3 and H3K9me3). These epigenetic alterations mark the cells for a bone-making fate.

To more fully nail down the function of these histone demethylases, Wang and his crew depleted KDM4B or KDM6B in human MSCs. They found that reduction of these enzymes greatly reduced bone-cell differentiation and increased fat cell differentiation. KDM6B increases the expression of HOX genes expression by removing H3K27me3, which promotes bone differentiation. KDM4B jacked-up the expression of a gene called DLX by removing H3K9me3. DLX suppresses fat cell differentiation.

Wang and his colleagues found that in mice, the presence of H3K27me3 and H3K9me3 in MSCs are very highly correlated with an increase in osteoporosis or the aging of bone marrow in mice. Importantly, Wang’s colleagues showed that concentrations of H3K27me3- and H3K9me3-positive MSCs in mouse bone marrow were significantly elevated in female mice that had had their ovaries removed. In these mice, bone deposition was reduced and fat-making was highly active. The fact that BMP-4/7 signaling culminates in the removal of these methyl groups is an indication that KDM4B and KDM6B may represent novel therapeutic targets for metabolic bone disease.

Wang cautioned: “Through our recent discoveries on the lineage decisions of human bone marrow stem cells, we may be more effective in utilizing these stem cells for regenerative medicine for bone diseases such as osteoporosis, as well as for bone reconstruction. However, while we know certain genes must be turned on in order for the cells to become bone-forming cells, as opposed to fat cells, we have only a few clues as to how these genes are switched on.”

However, with respect to his work with older mice, Dr. Wang was more sanguine: “Interestingly, in our aged mice, as well as osteoporotic mice, we observed a higher amount of silencing histone methyl groups which were normally removed by the enzymes KDM4B and KDM6B in young and healthier mice. And since these enzymes can be easily modified chemically, they may become potential therapeutic targets in tissue regeneration and treatment for osteoporosis.”

Dr. No-Hee Park, the dean of the UCLA School of Dentistry said this about Wang’s findings: “The discovery that Dr. Wang and his team have made has considerable implications for craniofacial bone regeneration and treatment for osteoporosis. As a large portion of our population reaches an age where osteoporosis and gum disease could be major health problems, advancements in aging-treatment are very valuable.”

Mesenchymal Stem Cell Treatment Prevent Post-Fracture Arthritis in Mice

Researchers at Duke University Medical Center have found a promising stem cell therapy that might prevent osteoarthritis after joint injury.

Joint injuries tend to raise the odds of contracting a type of arthritis known as post-traumatic arthritis (PTA). There are no available therapies that delay the onset or progression of arthritis after joint-related injuries.

However, a research team at the Duke University Health System has discovered a promising therapeutic approach for PTA that utilizes a particular type of stem cell known as a mesenchymal stem cell (MSC). MSCs have the ability to quell PTA, since these particular stem cells have the ability to suppress inflammation. Additionally, MSCs have the ability to readily differentiate into cartilage-making, bone-making, fat-making, and smooth muscle-making cells, which makes them prim candidates to regenerate damaged joints, since these stem cells have beneficial properties in other regions of the body.

This therapeutic approach was investigated in mice that had suffered the type of bone fractures (intraarticular fractures) that would usually lead to PTA. Injections of 10,000 MSCs into the joints of mice that had suffered intraarticular fractures decreased inflammation and greatly increase bone deposition during healing. Furthermore, the mice that had MSC injections into the joints did not develop PTA while those that received injections of saline into their joints largely did develop PTA. This study could potentially help produce a therapy that would be used after joint injury and before the patient suffers the initial signs of significant osteoarthritis.

Farshid Guilak, Ph.D., director of orthopedic research at Duke and senior author of the study, summarized the results of this study: “The stem cells were able to prevent post-traumatic arthritis.” The study was published on August 10 in the journal Cell Transplantation.

Another track investigated by Guilak’s lab was so-called “superhealer” strains of laboratory mice that tend to heal from bone fractures much faster than other strains. Guilak and his colleagues examined if MSCs from the MRL/MpJ (MRL) “superhealer” mouse strain would increase bone healing when compared to MSCs from C57BL/6 (B6) mice. This was to determine if the exceptional regenerative abilities of MRL mice was due to their MSCs. Unfortunately, Guilak and colleagues discovered that B6 MSCs did the job just as well, and, in fact, a little better than MSCs from MRL mice. Thus, even though they thought that superhealer mice bred for their super-healing properties would probably fare better than typical mice, in turned out that they were wrong.

“We decided to investigate two therapies for the study, said lead author Brian Diekman, Ph.D., who works as a postdoctoral research fellow in the Guilak lab. “We thought that stem cells from so-called superhealer mice would be superior at providing protection, and instead, we found that they were no better than stem cells from typical mice. We thought that maybe it would take stem cells from superhealers to gain an effect as strong as preventing arthritis after a fracture, but we were surprised – and excited – to learn that regular stem cells work just as well.”

Interestingly, certain people appear to fall into the superhealer category. They bounce back quickly and heal well naturally after a fracture, while other people eventually form cases of arthritis at the fractured joint, said Guilak, who is a professor of orthopedic surgery and biomedical engineering at Duke University.

“The ability of the superhealer mice to have superior healing after a fracture may go beyond the properties of their stem cells and be some beneficial factor, like a growth factor, that we don’t know about yet,” Guilak said.

Diekman said the team looked at markers of inflammation and saw that the stem cells affected the inflammatory environment of the joint after fracture.

“The stem cells changed the levels of certain immune factors, called cytokines, and altered the bone healing response,” said Diekman, who is also with the Duke Department of Biomedical Engineering.

According to Guilak, very few studies have purified stem cells to the degree they were purified for this study. The MSCs used in this study were from bone marrow, and they are not directly involved in the production of blood cells, even though they do play an important support role for blood cell-making stem cells in bone marrow (see C. Shi. Immunology 2012 136(2): 133-8 & also see here).

Diekman said that one of the challenges in the field is devising a precise protocol for identifying, isolating and purifying MSCs from bone marrow, since these stem cells tend to be rather rare in bone marrow.
“We found that by placing the stem cells into low-oxygen conditions, they would grow more rapidly in culture so that we could deliver enough of them to make a difference therapeutically,” Diekman said.

Stem Cell Helps for Patients with Head and Mouth Injuries

Craniofacial tissue regeneration, particularly bone regeneration has advanced remarkably in the past decade. In fact, facial bone re-growth with stem cells has proven less invasive and more effective than traditional bone regeneration treatments.

A partnership between researchers at the University of Michigan School of Dentistry, the Michigan Center for Oral Health Research and the Ann Arbor-based Aastrom Biosciences Inc. generated a clinical trial that involved 24 patients whose injuries required jawbone reconstruction.

In this clinical trial, patients received either experimental tissue repair cells or traditional guided bone regeneration therapy. The experimental cells in this trial are under development by Aastrom Biosciences and are called “ixmyelocel-T.” Ixmyelocel-T is a patient-specific stem cell from a patient’s bone marrow. It is a mixture of several different types of bone marrow-based stem cells.

Principle investigator and assistant professor at the U of M School of Dentistry Darnell Kaigler explained his rationale for his clinical trial: In patients with jawbone deficiencies who also have missing teeth, it is very difficult to replace the missing teeth so that they look and function naturally. This technology and approach could potentially by used to restore areas of bone loss so that missing teeth can be replaced with dental implants.”

This treatment is best suited for individuals with large defects. Such defects, that result from trauma, disease, or birth defects are very complex, since they involve several different tissue types (bone, gum, and skin). This makes them very challenging to treat. Since ixmyelocel-T is made from the patient’s own stem cells, it generates something completely living and compatible with the patient’s immune system rather than something man-made.

To date, the results from this trial have been very promising. Six-twelve weeks after treatment, patients in this clinical trial receive dental implants, and those who were treated with tissue repair cells had greater bone density and faster bone repair than those who received the traditional guide bone regeneration therapy. Additionally, the group who had received the tissue replacement therapy required fewer secondary bone grafting upon receiving their implants.

The cells for this therapy were extracted from bone marrow aspirations from the hip. Aastrom uses a proprietary system to process and grow the bone marrow stem cells, but once they were ready, they were placed into various areas of the mouth and jaw.

The next step in this research is to use more clinical trials with larger number of patients. Unfortunately, these stem cell treatments are probably 5-10 years away from FDA approval and regular use.

William Giannobile, who is the director of the Michigan Center for Oral Health Research and also the chair of the U-of-M Department of Periodontics and Oral Medicine, is one of the co-principal investigators on this project.