Muscle-Derived Stem Cells And Platelet-Rich Plasma Improve Cartilage Formation


Skeletal muscle contains a stem cell population called muscle derived stem cells or MDSCs that might have tremendous therapeutic potential. MDSCs have been isolated from skeletal muscle by means of their ability to adhere to culture flasks coated with collagen. Samples of muscle taken from a biopsy are mechanically mashed and then treated with enzymes the separate the cells. These cells are plated onto collagen-coated dishes and the cells either adhere quickly (fibroblasts and myoblasts), or slowly (MDSC-enriched fraction).

Skeletal muscle contains another cell population known as satellite cells. Satellite cells can divide and form muscle progenitor cells known as myoblasts that fuse to form myotubes. MDSCs, however, as distinct from satellite cells. They express different sets of genes: satellite cells typically express Pax7, whereas MDSCs are more heterogeneous but express Sca-1 consistently and often express CD34.

Studies in culture and in living animals have established that MDSCs can self-renew and differentiate into multiple lineages. They also have the potential to regenerate various adult tissues. See Usas A, et al Medicina (Kaunas) 2011;47:469–479; Cao B, et al Nat Cell Biol. 2003;5:640–646; Deasy BM, et al Blood Cells Mol Dis. 2001;27:924–933.

MDSCs also display a superior regenerative capacity relative to satellite cells following transplantation into mice with a form of rodent muscular dystrophy (mdx mice). MDSCs are at least partially invisible to the immune system. When transplanted into mdx mice and left for at least 3 months, no sign of immune rejection was detected.

The laboratory of Johnny Huard at the University of Pittsburgh has been genetically engineering MDSCs from mouse for use as cartilage making cells to treat rodents with osteoarthritis. In 2009, Huard’s group published an intriguing paper in the journal Arthritis and Rheumatism in which they genetically engineered MDSCs with two genes: Bone Morphogenetic Protein 4 (BMP-4) and Soluble Flt-1. If you are wondering what the heck these two genes encode, then you are not alone. BMP-4 is a secreted signaling protein that is very important for bone healing, but it also plays a central role in helping cartilage-making cells (chondrocytes) survive and divide. Flt-1 is one of the receptor proteins that binds the growth factor VEGF (vascular endothelial growth factor).  Normally, VEGF forms blood vessels and remodels existing blood vessels.  However, when it comes to cartilage, VEGF tends to cause cartilage to die back.  Therefore, Huard’s group used a soluble version of Flt-1, which scavenged the available VEGF in the environment and bound it up.

In their 2009 paper, Huard and others showed that BMP-4/soluble Flt-1-expressing MDSCs did a remarkable job of making new cartilage and repairing damage joint cartilage in rodents.  See Tomoyuki Matsumoto, et al ARTHRITIS & RHEUMATISM Vol. 60, No. 5, May 2009, pp 1390–1405.

A and B, Macroscopic (A) and histologic (B) evaluation of representative joints from rats injected with muscle-derived stem cells (MDSCs) transduced with soluble Flt-1 (sFlt-1) and bone morphogenetic protein 4 (BMP-4 [B4]) (sFlt-1/BMP-4–MDSC), MDSCs transduced with vascular endothelial growth factor (VEGF) and BMP-4 (VEGF/BMP-4–MDSC), MDSCs transduced with BMP-4 alone (BMP-4–MDSC), nontransduced MDSCs (MDSC), or phosphate buffered saline (PBS) alone, 4 and 12 weeks after transplantation. Four weeks after transplantation, the sFlt-1/BMP-4–MDSC and BMP-4–MDSC groups macroscopically and histologically showed smooth joint surface with well-repaired articular cartilage and Safranin O–positive hyaline-like cartilage (red staining in B). However, the other groups showed marked arthritic progression, synovial hypertrophy, and osteophyte formation (arrows). Twelve weeks after transplantation, although the sFlt-1/BMP-4–MDSC group still showed well-repaired articular cartilage, the other groups exhibited more severe arthritis compared with 4 weeks. (Original magnification  100.) C, Semiquantitative histologic scores for all groups, 4 and 12 weeks following transplantation. The sFlt-1/BMP-4–MDSC group had the lowest (best) scores of all groups. Bars show the mean and SEM.   P   0.05 versus all other groups;   P   0.05 versus the VEGF/BMP-4–MDSC, MDSC, and PBS groups.
A and B, Macroscopic (A) and histologic (B) evaluation of representative joints from rats injected with muscle-derived stem cells (MDSCs) transduced with soluble Flt-1 (sFlt-1) and bone morphogenetic protein 4 (BMP-4 [B4]) (sFlt-1/BMP-4–MDSC), MDSCs transduced with vascular endothelial growth factor (VEGF) and BMP-4 (VEGF/BMP-4–MDSC), MDSCs transduced with BMP-4 alone (BMP-4–MDSC), nontransduced MDSCs (MDSC), or phosphate buffered saline (PBS) alone, 4 and 12 weeks after transplantation. Four weeks after transplantation, the sFlt-1/BMP-4–MDSC and BMP-4–MDSC groups macroscopically and histologically showed smooth joint surface with well-repaired articular cartilage and Safranin O–positive hyaline-like cartilage (red staining in B). However, the other groups showed marked arthritic progression, synovial hypertrophy, and osteophyte formation (arrows). Twelve weeks after transplantation, although the sFlt-1/BMP-4–MDSC group still showed well-repaired articular cartilage, the other groups exhibited more severe arthritis compared with 4 weeks. (Original magnification  100.) C, Semiquantitative histologic scores for all groups, 4 and 12 weeks following transplantation. The sFlt-1/BMP-4–MDSC group had the lowest (best) scores of all groups. Bars show the mean and SEM.   =P<0.05 versus all other groups;  =P<0.05 versus the VEGF/BMP-4–MDSC, MDSC, and PBS groups.
In another paper that came out in January of this year, Huard has used platelet-rich plasma with his engineered MDSCs to determine with platelet-rich plasma (PRP) can increase the cartilage-making activity of engineered MDSCs.

Since PRP has been reported to promote the synthesis of collagen and cell proliferation, and increase cartilage repair, it is possible that, when paired with the right stem cells, PRP can enhance cartilage repair.  To test this suspicion, MDSCs expressing BMP-4 and sFlt1 were mixed with PRP and injected into the knees of rats whose immune system did not work properly that had osteoarthritis.  Osteoarthritis can be chemically induced in rats rather easily, and the rats were treated with MDSCs expressing BMP-4 and sFlt1 or MDSCs expressing BMP-4 and sFlt1 plus PRP.  Tissue assessments of the arthritic joints were performed 4 and 12 weeks after cell transplantation.  Other tests conducted in culture determined the cell proliferation, adhesion, migration and cartilage-making capacities of cells in culture.

The results showed that addition of PRP to MDSCs expressing BMP-4 and sFlt1 significantly improved joint cartilage repair at week 4 compared to MDSCs expressing BMP-4 and sFlt1 alone.  The joints showed higher numbers of cells producing type II collagen and lower levels of chondrocyte cell death were observed by MDSCs expressing BMP-4 and sFlt1 and mixed with PRP.  In culture, the addition of PRP promoted proliferation, adhesion and migration of the MDSCs.  When pellets of cells were induced to make cartilage in culture, PRP tended to increase the number of type II collagen producing cells.

From this, Huard and his colleagues concluded that PRP can promote the cartilage-repairing capacities of MDSCs that express BMP-4 and sFlt1.  This enhancement involves the promotion of collagen synthesis, the suppression of chondrocyte cell death, and by enhancing the integration of the transplanted cells in the repair process.

See Mifune Y, et al Osteoarthritis Cartilage. 2013 Jan;21(1):175-85. doi: 10.1016/j.joca.2012.09.018.

Platelet-Rich Plasma Enhances the Clinical Outcomes of Microfracture Surgery in Older Patients


Osteoarthritis occurs when the cartilage that covers the opposing bones at a joint erodes away and the bare opposing bones smash into each other causing the bone to crack, fragment and chip. The result is extensive inflammation of the joint and further destruction of the bone, which prompts a knee replacement.

Because knee replacement surgeries are so painful and because they only last about two decades at the most, replacing the lost cartilage is a better option. One surgical treatment for osteoarthritis is microfracture surgery. Microfracture surgery involves the drilling of small holes in the tips of the bones of the joint to serve as conduits for stem cells in the bone to come to the surface and make cartilage.

Unfortunately, there are some problems with microfacture surgery, the most prominent of which is that it works better in younger patients than in older patients. Patients older than 40 years old show a precipitous drop in success after microfracture surgery. Thus, finding some way to increase the activity of cartilage production by endogenous stem cells would be a welcome finding for orthopedic surgeons.

Platelet-rich plasma (PRP) has been used to augment the cartilage-making activities of mesenchymal stem cells from bone marrow. Therefore, some surgeons from South Korea decided to try adding PRP to the knees of patients who had just had microfracture surgery. They examined 49 patients with early arthritis. All of these patients were subjected to arthroscopic microfracture surgery for a cartilage lesion that was less than four cubic centimeters in size. These patients were all between the ages of forty to fifty years old, which means that they were outside the age range for successful microfracture surgery.

These 49 patients were randomly divided into two groups. The first group was a control group of 25 patients that only had arthroscopic microfracture surgery. The second group consisted of 24 patients and they had arthroscopic microfracture surgery and injections of PRP into the knee. 10 patients from each group had follow-up arthroscopies four to six months after the procedure to determine the extent of cartilage restoration. Further evaluations were also done 2 years after the procedure.

The results? There were significant improvements in clinical results between preoperative evaluation and postoperative at 2 years post surgery in both groups (p = 0.017). However in the group that received PRP injections plus microfracture surgery the results were significantly better than those of the control group. These patients had better range of motion and less pain (p = 0.012). In the 2nd look arthroscopies, the cartilage of the patients that received PRP and microfracture surgery was harder and showed increased elasticity than the cartilage of patients that received only microfracture surgery.

The conclusion of these authors: “The PRP injection with arthroscopic microfracture would be improved the results in early osteoarthritic knee with cartilage lesion in 40-50 years old, and the indication of this technique could be extended to 50 years.” (Lee GW et al., “Is platelet-rich plasma able to enhance the results of arthroscopic microfracture in early osteoarthritis and cartilage lesion over 40 years of age? European Journal of Orthopedic Surgery. 2012 Jul 5., epub ahead of publication)  If PRP could improve the outcomes of microfracture surgery, then maybe such a technique could extend the groups of patients who are successfully served by this procedure.

While this is an exciting result, we must temper our excitement with the realization that this is a small study and MRIs were not used to measure cartilage thickness. Therefore, while this study is useful and frankly, ingenious, it has its limitations.

Glucosamine, Chondroitin and Delaying Osteoarthritis


I have a confession to make. I have been taking 1200 mgs of glucosamine sulfate for the past 5-6 years for my knee cartilage. I do not presently have osteoarthritis, but I am trying to stave it off by taking this supplement.

Does this supplement work? That’s hard to say for certain because the studies disagree. There are theoretical reasons to suspect that glucosamine would help with cartilage deposition. Cartilage is very rich in a group of sticky, sugary compounds called “glycosaminoglycans,” which have the unfortunate acronym of GAGs. GAGs consist of repeating two-sugar motifs, and the building block for the vast majority of these two-sugar motifs is glucosamine. Therefore, glucosamine is a main building block of a prominent component of cartilage.

What about chrondroitin? Chondroitin is a GAG that usually comes attached to a protein. This complex of GAG + backbone protein is called a “proteoglycan.” The chondroitin you get in the store is a repeating polymer of a two-sugar motif, and this complex molecule is either degraded in your digestive system by bacteria, or by our own gastrointestinal tract.  The degradation and absorption of chondroitin probably varies considerably from person to person.  If chondroitin is absorbed then the building blocks of chondroitin can potentially help build cartilage, since chondroitin-containing proteoglycans are important structural components of cartilage.  There is also the possibility that chondroitin precursors prevent the breakdown of cartilage.

Chondroitin_sulfate-over

In 2006, a good-sized study called the GAIT study was published in the New England Journal of Medicine (Clegg, D.O. et al. (2006). Glucosamine, chondroitin sulfate, and the two in combination for painful knee osteoarthritis. New Eng. J. Med. 354(8):795-808). In this study, 1583 patients with symptomatic knee osteoarthritis were randomly assigned to different treatment subgroups. These groups were:

a) chondroitin sulphate alone (400 mg 3x a day)
b) glucosamine hydrochloride alone (500 mg 3x a day)
c) combined glucosamine hydrochloride/chondroitin sulphate (same doses but combined)
d) celecoxib (Celebrex®) (200 mg per day)
e) placebo (inactive dummy tablet)

Daily dosages for glucosamine and chondroitin were 1500 mgs and 1200 mgs, respectively. The efficacious dosage for these supplements have yet to be determined. Therefore, these dosages are a best guess. Celecoxib was included as a positive control for the GAIT study, since celecoxib is FDA approved for the management of osteoarthritis pain. Therefore, investigators therefore expected participants in this group to experience some pain relief, which would serve to validate the results of the GAIT study.

The GAIT study found that when patients were divided into two groups based on pain levels, 1,229 had mild pain and 354 had moderate to severe pain. With regard to the effectiveness of these supplements, neither glucosamine nor chondroitin sulphate either on their own or in combination were effective in reducing pain. However, when only those patients with moderate to severe pain was analyzed the combination of glucosamine and chondroitin sulphate was effective for pain relief. Unfortunately, no cartilage thickness studies were performed to determine if the supplements augment cartilage thickness. The GAIT study was publicly funded, and therefore, accusations of conflict of interest could not be used to discredit this study.

in 2005, results from the GUIDE study were presented at the 2005 Annual Meeting of the American College of Rheumatology. This study was funded by glucosamine manufacturers and examined of pain and mobility in 318 osteoarthritis sufferers between the ages of 45 and 75 at 13 European hospitals. Participants in this study were divided into three groups:

a) glucosamine sulphate in soluble powder form 1500mg daily
b) acetaminophen (e.g. Tylenol® and paracetamol) 3000mg daily
c) placebo

In addition, subjects in all three groups were allowed to take ibuprofen as needed as a ‘rescue’ for pain relief.

The GUIDE study found that glucosamine sulphate and acetaminophen were more effective in reducing pain than placebo. Patients who took glucosamine sulphate experienced greater pain relief than patients on acetaminophen.

The GUIDE and GAIT studies were positive for glucosamine and chondroitin, but there are negative studies too. In October 2004, Jolanda Cibere and others published a study in the journal Arthritis Care and Research in which they gave glucosamine or a placebo to arthritis suffers and then discontinued them. 42% of the patients receiving the placebo experienced a disease flare-up and 45% of the glucosamine-receiving patients experienced a flare-up. Also, the time to disease flare was not significantly different in the glucosamine compared with placebo group. Thus Cibere and others concluded that “this study provides no evidence of symptomatic benefit from continued use of glucosamine sulfate.”

The bottom line on all this is the glucosamine and chondroitin perform inconsistently in controlled studies. When poor-quality studies are excluded, glucosamine seems to delay arthritis. The highly respected Cochrane Library published a summary of human clinical trials with glucosamine and when the poor-quality trials were excluded, Towheed and his colleagues concluded that glucosamine provided relief of the symptoms of arthritis and also, based on X-rays, helped delay the onset of osteoarthritis.

However, the European Food Safety Authority reviewed over 60 articles on glucosamine and came to a completely different conclusion. In 2012, the EFSA concluded that “The Panel concludes that a cause and effect relationship has not been established between the consumption of glucosamine and maintenance of normal joint cartilage in individuals without osteoarthritis.”

In 2009, in the Journal, Arthroscopy, Vangsness, Spiker, and Erickson came to a somewhat blasé conclusion, “glucosamine sulfate, glucosamine hydrochloride, and chondroitin sulfate have individually shown inconsistent efficacy in decreasing OA pain and improving joint function.”

The long and the short of it is that these supplements might work. Furthermore, my best guess at this point is that they probably work better for some people than for others. So should you take glucosamine or even chondroitin? All our information at this point says that it is safe to do so. No serious or even moderate side effects have been observed by taking these supplements. Secondly, they might work for some people. How do know if you are one of them? By taking the supplement.

I realize that this post is probably very unsatisfying to many of you, but some are very enthusiastic about glucosamine and chondroitin, and I think that this enthusiasm needs to be tempered by a hard dose of reality.  There is much we simply do not know at this time about the efficacy of these supplements, and more work needs to be done before we can say anything definitive about them.   A recent study shows that large doses of chondroitin (1200 mgs) are effective at reducing symptoms in patients with osteoarthritis of the knee, but given the vagaries of chondroitin absorption (see above), it is unlikely that we can make any hard and fast conclusions about it.

One more note about these supplements.  Several studies have shown that the quality of over-the-counter glucosamine vary considerably.  Be careful what you buy and from whom you buy your supplements.  Consumer Reports has shown that some supplements are even spiked with prescription drugs!  So caveat emptor and do not believe the marketer’s own statements about their supplements.

A Co-culture System Makes Better Cartilage for Tissue Replacement


At joints, the bones are covered with cartilage to act as a shock absorber. Articular cartilage, or cartilage at joints, is usually characterized by very low friction, high wear resistance, but very abilities to regenerate. Articular cartilage is responsible for much of the compressive resistance and load bearing qualities of joints, and without it, even activities as simple and walking is too painful. Osteoarthritis is a condition that results from cartilage failure, and limits the range of joint motion, increases the bone damage and also causes a respectable amount of pain. When the cartilage of the articular surface erodes, the bone is exposed and grinding of the bone creates bone spurs, extensive inflammation and pain.

Treating osteoarthritis requires that one make new cartilage that has similar properties as articular cartilage. Unfortunately, mesenchymal stem cells that are differentiated into cartilage making cells (chondrocytes) and implanted into the knee tend to make fibrocartilage, which is different than the hyaline cartilage that composes articular cartilage. Fibrocartilage does not possess the high-wear resistance characteristics of hyaline cartilage and it tends to erode rather rapidly after formation. Therefore, directing mesenchymal stem cells (MSCs) to form proper cartilage is a genuine challenge.

A paper that appear in Stem Cell Translational Medicine from Gilda A. Barabino, who is a faculty member at the Wallace H. Coulter Department of Biomedical Engineering at Georgia Institute of Technology and Emory University, examines a technique to coax MSCs to make articular cartilage.

As Barbino points out, traditional protocols that direct MSCs to differentiate into chondrocytes uses culture systems of MSCs that have been treated with various growth factors, such as transforming growth factor-β. Unfortunately, these culture systems tend to fall short in meeting the needs of clinical applications, largely because they yield terminally differentiated cells that enlarge and then form bone.

In this study Barbino and her co-workers co-cultured bone marrow-derived MSCs with juvenile articular chondrocytes. The rationale is that the MSCs would receive just the right growth factors in just the right concentrations and at the right time to drive MSC cartilage formation. Physical contact between cells can also do a better job of driving them to differentiate into various cells types rather than simply treating them with growth factors.

Barbino and others discovered that an initial chondrocyte/MSC ratio of 63:1 worked the best and the MSCs form chondrocytes that had the right cells shape, behavior, and characteristics of articular chondrocytes.

Next, Barbino and her team grew the MSCs in a three-dimensional agarose system. Three-dimensional systems are generally thought to more realistically recapitulate the cartilage-making system present at joints. In this 3-D culture system, when co-cultured with juvenile articular chondrocytes, bone marrow MSCs develop into robust neocartilage that was structurally and mechanically stronger than the same cultures that only contained chondrocytes.

There was another advantage to this culture system; cultured MSCs that are induced to form cartilage tend to cease all expression of a surface protein called CD44, which is an important regulator in cartilage biology. However, when cultured in the 3-D culture, the MSCs retained the expression of CD44, which suggests that these co-cultured MSCs, which cultured in a 3-D culture system form chondrocytes that make superior articular cartilage, but retain CD44, which allows cartilage maintenance.

This shows that making articular cartilage from MSCs is probably possible and only requires the right culture system. Also, co-culturing MSCs with articular chondrocytes in a 3-D culture system might be one of the better culture systems for developing clinically relevant cartilage for tissue replacements.

Induced Pluripotent Stem Cells Make Cartilage


Induced pluripotent stem cells (iPSCs) are made from adult cells through genetic engineering techniques that drive terminally-differentiated adult cells to revert into embryonic-like cells. iPSCs have the capacity to form any cell type in the adult body, and they may represent the future of regenerative medicine when it comes to treatment of some diseases.

On the 30th of October, 2012, scientists from Durham NC reported that they were able to make cartilage from iPSCs. The cartilage made by iPSCs was not simply the fibrous cartilage found in the ribs and between the connection at the pelvis, but the whitish, hyaline cartilage found at weight-bearing joints. Hyaline cartilage acts as a shock absorber at the hip and knee joints and has proven difficult to make in culture.

According the Farshid Guilak, professor of orthopedics surgery at Duke University Medical Center and senior author of this study: “This technique of creating pluripotent stem cells is a way to take adult cells and convert them so that they have the properties of embryonic stem cells.”

Dr. Guilkak continued, “Adult stem cells are limited in what they can do and embryonic stem cells have ethical issues.” What this research shows in a mouse model is that ability to create an unlimited supply of stem cells that can turn into any type of tissue – in this case cartilage, which has no ability to regenerate itself.

Hyaline cartilage, which is found at articular surfaces (the surfaces between joints, allows us to walk and climb stairs. However, the everyday wear-and-tear or an injury can degrade the cartilage, leaving bones to grind against bones. the result is bone fragmentation, extensive inflammation and pain (osteoarthritis), and the replacement of that joint with an artificial joint. Articular cartilage has a very limited ability to repair itself and damage and osteoarthritis are the leading causes of impairment in older people.

Guilak’s research group, led by postdoctoral research fellow, Brian Diekman, is an alternative to other procedures presently in use, which include the application of stem cells from bone marrow or fat to the damaged cartilage.

The main challenge in using iPSCs was differentiate the cells so that they provided a relatively pure population of cartilage-making cells (chondrocytes). To hone their protocol for making and selecting chondrocytes from iPSCs, Diekman devised a technique that caused only those iPSCs that had differentiated into mature chondrocytes to glow a fluorescent green color. This provided a tag that Diekman and his colleagues used to sort the mature chondrocytes from the other cells.

The isolated chondrocytes made beautiful cartilage that had all the strength and resilience of nature cartilage. As noted by Diekman, “This was a multi-step approach, with the initial differentiation, the sorting, and then proceeding to make the tissue (cartilage in this case). What this shows is that iPSCs can be used to make high quality cartilage, either for replacement tissue or as a way to study disease and potential treatments.”

According to Diekman and Guilak, the next step in this research is to use human iPSCS to test and ultimately refine their cartilage-growing protocol. Guilak summarized his work with these words: “The advantage of this technique is that we can grow a continuous supply of cartilage in a dish. In addition to cell-based therapies, iPSC technology can also provide patient-specific cell and tissue models that could be used to screen for drugs to treat osteoarthritis.”

This work was published in the Proceedings of the National Academy of Sciences USA, 2012, DOI: 10.1073/pnas.1210422109.