Bone Marrow Mesenchymal Stem Cells Spontaneously Make Cartilage After Blockage of VEGF Signaling


Bone marrow-derived mesenchymal stem cells (MSCs) can be induced to make cartilage by incubating the cells with particular growth factors.  Unfortunately, batches of MSCs show respectable variability from patient-to-patient.  Therefore the growth factor-dependent method suffers from poor efficacy, limited reproducibility from batch-to-batch, and the cell types that are induced are not always terribly stable.  Finding a better way to make cartilage would certainly be a welcome addition to regenerative treatments,

Cartilage that coats the ends of bones is known as articulate cartilage, and articular cartilage lacks blood vessels.  Therefore, is it possible that inhibiting blood vessel formation could conveniently push MSCs to differentiate into cartilage-making chondrocytes?

A new paper by Ivan Martin and Andrea Basil from the University Hospital Basel and their colleagues have used this very strategy to induce cartilage formation in MSCs from bone marrow.

Martin and others isolated MSCs from bone marrow aspirates from human donors.  These cultured human MSCs were then genetically engineered with modified viruses to express a receptor for soluble vascular endothelial growth factor (VEGF) that binds this growth factor, but fails to induce any intracellular signals.  Such a receptor that binds the growth factor but does not induce any biological effects is called a “decoy receptor,” and decoy receptors efficiently sequester or vacuum up all the endogenous VEGF.  VEGF is the major blood vessel-inducing growth factor and it is heavily expressed during development, by cancer cells, and during healing.

After expressing the decoy VEGF receptor in these human MSCs, these genetically engineered cells were grown on collagen sponges and then implanted in immunodeficient mice.  If the implanted MSCs were not genetically engineered to express decoy VEGF receptors, they induced for formation of vascularized fibrous tissue.  However, the implantation of genetically engineered MSCs that expressed the decoy VEGF receptor efficiently and reproducibly differentiated into chondrocytes and formed hyaline cartilage. This is significant because headline cartilage is the very type of cartilage found at articular surfaces where the ends of bones come together to form joints.

In vivo chondrogenesis. Histological staining with Safranin-O for glycosaminoglycans and immunohistochemistry for type II collagen of engineered tissue generated by naïve (control) or sFlk-1 MSCs after 4 (A) or 12 (B) weeks in vivo. Fluorescence staining with DAPI (in blue) and a specific anti-human nuclei antibody (in red) of constructs generated by control or sFlk-1 MSCs after 4 (A) or 12 (B) weeks in vivo. Scale bar = 100 µm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; MSC, bone marrow-derived mesenchymal stromal/stem cell.
In vivo chondrogenesis. Histological staining with Safranin-O for glycosaminoglycans and immunohistochemistry for type II collagen of engineered tissue generated by naïve (control) or sFlk-1 MSCs after 4 (A) or 12 (B) weeks in vivo. Fluorescence staining with DAPI (in blue) and a specific anti-human nuclei antibody (in red) of constructs generated by control or sFlk-1 MSCs after 4 (A) or 12 (B) weeks in vivo. Scale bar = 100 µm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; MSC, bone marrow-derived mesenchymal stromal/stem cell.

This articular cartilage was quite stable and showed no signs of undergoing the chondrocytes enlargement found in terminally differentiated cartilage that is ready to form bone.  This stability was maintained for up to 12 weeks.

In vivo cartilage stability. Immunohistochemistry for type X collagen, BSP, and MMP-13 on sections of hypertrophic cartilage generated in vitro by MSCs (as a positive control) and on sections of the cartilaginous constructs generated in vivo by sFlk1 MSCs 12 weeks after implantation. Scale bar = 50 µm. Abbreviations: BSP, bone sialoprotein; MMP-13, metalloproteinase-13; MSC, bone marrow-derived mesenchymal stromal/stem cell.
In vivo cartilage stability. Immunohistochemistry for type X collagen, BSP, and MMP-13 on sections of hypertrophic cartilage generated in vitro by MSCs (as a positive control) and on sections of the cartilaginous constructs generated in vivo by sFlk1 MSCs 12 weeks after implantation. Scale bar = 50 µm. Abbreviations: BSP, bone sialoprotein; MMP-13, metalloproteinase-13; MSC, bone marrow-derived mesenchymal stromal/stem cell.

Why did inhibition of VEGF signaling induce cartilage?  Inhibition of angiogenesis induced low oxygen tensions, which activated a growth factor called transforming growth factor-β.  Activation of the TGF-beta pathway robustly enhanced the formation of articular cartilage.

In vitro chondrogenesis at different oxygen tensions. Histological staining with Safranin-O and immunohistochemistry for type II collagen on constructs generated in vitro by naïve MSC cultured with (A) or without (B) TGFβ3 supplementation at 2% or 20% of oxygen tension. Scale bar = 50 µm. Expression levels of the mRNA for type II and X collagen, Gremlin-1, IHH TGFβ1 were quantified in pellets generated by naïve bone marrow-derived mesenchymal stromal/stem cells (C, D) cultured in the two different oxygen tensions. ∆Ct values were normalized to expression of the GAPDH housekeeping gene, and results are shown as mean ± SD (n = 6 samples/group from 3 independent experiments). ∗, p < .05, ∗∗∗, p < .001. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IHH, Indian hedgehog; TGFβ, transforming grown factor-β.
In vitro chondrogenesis at different oxygen tensions. Histological staining with Safranin-O and immunohistochemistry for type II collagen on constructs generated in vitro by naïve MSC cultured with (A) or without (B) TGFβ3 supplementation at 2% or 20% of oxygen tension. Scale bar = 50 µm. Expression levels of the mRNA for type II and X collagen, Gremlin-1, IHH TGFβ1 were quantified in pellets generated by naïve bone marrow-derived mesenchymal stromal/stem cells (C, D) cultured in the two different oxygen tensions. ∆Ct values were normalized to expression of the GAPDH housekeeping gene, and results are shown as mean ± SD (n = 6 samples/group from 3 independent experiments). ∗, p < .05, ∗∗∗, p < .001. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IHH, Indian hedgehog; TGFβ, transforming grown factor-β.

Cartilage formation from MSCs was induced by blocking VEGF-mediated angiogenesis.  These results represent a remarkable advance in cartilage formation that can be used for regenerative treatments.  This cartilage formation was spontaneous and efficient and if it can be carried out with VEGF-inhibiting drugs rather than genetic engineering techniques, then we might have a transferable technique for making cartilage in the laboratory to treat osteoarthritis and other joint-based maladies.  Clinical trials will be required, but this is certainly an auspicious start.

Cartilage-Making Stem Cells from Joints


Chiharo Akazawa from the Tokyo Medical and Dental University and his colleagues have tested two types of mesenchymal stem cells from human patients for their ability to make bone, cartilage, or fat. Their tests illustrated what has been shown several time before; mesenchymal stem cells tend to differentiate into the tissues that most closely resemble their tissue of origin.

Akazawa and his colleagues previously discovered a way to effectively isolated mesenchymal stem cells from bone marrow, which is no small feat because mesenchymal stem cells (MSCs) are a minority of the cells in bone marrow (Mabuchi and others (2013), Stem Cell Reports 1: 152-165). In a recent paper in the journal PLoS ONE, Akazawa and others used this technique to isolate MSCs from bone marrow and from synovial membrane – the fluid-filled sac that encases joints. In large joints, this synovium is large and called a “bursa.”.

In culture, the bone marrow-derived MSCs from several different human donors showed a marked tendency to form bone, but they did not make good cartilage or fat. The synovial MSCs, on the other hand, did not do so well at making bone, but made very good fat and cartilage. These differentiation trends were observed in MSCs culture for several different human donors. All cells were collected during arthroscopic surgery.

Since the synovial membrane of patients suffering from osteoarthritis undergoes, increased cell division, it is possible that the number of stem cells also increases. Alternatively, using MSCs from healthy donors who do not have arthritis may be even more preferable. Nevertheless, MSCs from synovial membrane show excellent cartilage-making potential and they may be a suitable source of cell for cartilage regeneration.

Repairing Cartilage With Fat-Based Stem Cells May Be Feasible With New Technology


Head-to-head comparisons between bone marrow and fat stem cells have shown that bone marrow stem cells consistently outperform fat stem cells. As I have written in past posts, the present protocols for inducing cartilage from mesenchymal stem cells were developed using bone marrow stem cells. Therefore, the fact that bone marrow stem cells outperform fat stem cells with it comes to cartilage formation is no surprise.

In a study in New Zealand White rabbits, bone marrow stem cells outperformed fat stem cells when it came to the repair the cartilage defects in the knee joint. See Li Q, Tang J, Wang R, Bei C, Xin L, Zeng Y, Tang X. “Comparing the chondrogenic potential in vivo of autogeneic mesenchymal stem cells derived from different tissues.” Artif Cells Blood Substit Immobil Biotechnol. 2011 Feb;39(1):31-8. Here again, the system for chondrocyte differentiation system used was developed with, by, and for bone marrow mesenchymal stem cells. Thus the ability of these cells to outperform fat stem cells says nothing about the ability of fat-based mesenchymal stem cells to form cartilage in alternative culture systems.

Because fat-based stem cells are highly accessible and unlikely to be rejected by the immune system, there is a deep desire to efficiently convert fat-based stem cells into cartilage. Unfortunately, this task is not as straightforward as previously believed. As it turns out, fat-derived stem cells secrete molecules that actually inhibit cartilage formation. However, new research has found that if fat-based stem cells are pre-treated with antibodies that neutralize Vascular Endothelial Growth Factor (VEGF) and growing them in nutrients that are specifically designed to promote cartilage formation can counteract the effects of these molecules.

Chondrocytes make and maintain healthy cartilage. However, damage and diseases, such as osteoarthritis, can destroy cartilage and this can result in pain, compromising the patient’s mobility.

Professor Barbara Boyan, and her colleagues from the Georgia Institute of Technology showed that adipose (fat) stem cells (ASCs) secrete large amounts of factors. Some of these factors, especially the growth factor VEG, prevents cartilage regeneration and actually causes the death (apoptosis) of chondrocytes.

However, by treating ASCs with a media designed to encourage the differentiation of fat-based stem cells into cartilage cells reduced the amount of these secreted factors and prevented the growth of blood vessels. Also, the fat-based stem cells were treated with an antibody that neutralizes VEGF, and this pretreatment prevented chondrocyte death.

Professor Boyan said: “Non-treated ASCs actually impeded healing of hyaline cartilage defects, and although treating ASCs improved the situation they added no benefit compared to cartilage allowed to heal on its own. However we only looked at cartilage repair for a week after treatment. Other people have shown that two to six weeks is required before the positive effect of ASCs on cartilage regeneration is seen.”

Therefore, fat-based stem cells might be able to help repair damaged cartilage, and careful handling plus pre-treatment can help ensure a positive result.

See: “Adipose stem cells can secrete angiogenic factors that inhibit hyaline cartilage regeneration,” Christopher SD Lee et al.; Stem Cell Research & Therapy, 24 August 2012, 3:35, DOI:10.1186/scrt126

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.