Stem Cell-Based Cartilage Regeneration Could Decrease Knee and Hip Replacements


Work by Chul-Won Ha, director of the Stem Cell and Regenerative Medicine Institute at Samsung Medical Center and his colleagues illustrates the how stem cell treatments might help regrow cartilage in patients with osteoarthritis or have suffered from severe hip or knee injuries.

A 2011 report from the American Academy of Orthopedic Surgeons showed that approximately one million patients in the US alone (645,000 hips and 300,000 knees) have had joint replacements in the U.S. alone. Most joint replacements occur with few complications, artificial joints can only last for a certain period of time and some will even eventually require replacement. Also these procedures require extensive rehabilitation and are, in general, quite painful. A goal for regenerative medicine is the regenerate the cartilage that was worn away to prevent bones from eroding each other and obviate the need for artificial joint replacement procedures.

Extensive research from the past two decades from a whole host of laboratories in the United States, Europe, and Japan have shown that mesenchymal stem cells (MSCs) have the ability to make cartilage, and might even have the capability to regenerate cartilage in the joint of a living organism. MSCs have the added benefit of suppressing inflammation, which is a major contributor to the pathology of osteoporosis. Additionally, MSCs are also relatively easy to isolate from tissues and store.

“Over the past several years, we have been investigating the regeneration potential of human umbilical cord blood- derived MSCs in a hyaluronic acid (HA) hydrogel composite. This has shown remarkable results for cartilage regeneration in rat and rabbit models. In this latest study we wanted to evaluate how this same cell/HA mixture would perform in larger animals,” said Ha.

Ha collaborated with researchers from Ajou University, which is also in Seoul, and Jeju University in Jeju, Korea. Ha and his team used pigs as their model system, which is a better system than rodents for such research.

The stem cells for this project were isolated from human umbilical cord blood that was obtained from a cord blood bank. They isolated MSCs from the umbilical cord blood and grew them in culture to establish three different human Umbilical Cord Blood MSC lines. Then they pelleted the cells and mixed them with the HA solution and applied them to the damaged knee joints of pigs.

“After 12 weeks, there was no evidence of abnormal findings suggesting rejection or infection in any of the six treated pigs. The surface of the defect site in the transplanted knees was relatively smooth and had similar coloration and microscopic findings as the surrounding normal cartilage, compared to the knees of a control group of animals that received no cells. The borderline of the defect was less distinct, too,” said the study’s lead investigator, Yong-Beom Park, who is a colleague of Ha’s at the SungKyunKwan University’s Stem Cell and Regenerative Medicine Institute.

“This led us to conclude that the transplantation of hUCB-MSCs and 4 percent HA hydrogel shows superior cartilage regeneration, regardless of the species. These consistent results in animals may be a stepping stone to a human clinical trial in the future,” Dr. Ha noted.

“These cells are easy to obtain, can be stored in advance and the number of potential donors is high,” said Anthony Atala, M.D., Editor of STEM CELLS Translational Medicine and Director of the Wake Forest Institute for Regenerative Medicine. “The positive results in multiple species, including the first study of this treatment in large animals, are certainly promising for the many patients requiring treatments for worn and damaged cartilage.”

Non-Randomized Stem Cell Study for Knee Osteoarthritis Yields Positive Results


A peer-reviewed study that was neither placebo-controlled nor randomized, but did examine 840 patients, has shown that the use of a patient’s own bone marrow stem cells are both safe and effective.

Christopher Centeno and his colleagues, who pioneered the Regenexx protocol, use live-imaging to guide the application of stem cells to the site in need of healing. Centeno and others have established several clinics around the United States that utilize the Regenexx system, and the data published in this paper came from these clinics, in addition to Chris Centeno’s own clinic in the Denver, Colorado area.

In this study, patients self-rated their lower extremity functional using a lower extremity functional scale (LEFS), and their knee pain by using a numerical pain scale (NPS). Patients had bone marrow extracted through a bone marrow aspiration. These bone marrow cells were isolated and concentrated, and then prepared for reinvention. In addition, platelet rich plasma (PRP) and platelet lysate (PL) were prepared from the patient’s own blood and these, with the bone marrow cells, were injected into the knee under guided imaging. The frequency and types of adverse events (AE) were also recorded by the physicians.

Some of these patients had fat overlaid on their knee lesions in addition to their bone marrow cells. Of the 840 procedures that were performed, 616 had treatment without additional fat, and 224 had treatment with the fat graft. This was to determine if the use of fat, with its resident stem cell population, augmented healing of the arthritic knee.

When the LEFS scores before and after the Regenexx procedure were compared, an increase of 7.9 and 9.8 in the two groups (out of 80) was observed. The mean NPS score decreased from 4 to 2.6 and from 4.3 to 3 in the two groups. AE rates were 6% and 8.9% in the two groups. An examination of these data showed that pre- and posttreatment improvements were statistically significant. However, the differences between the fat- and fat+ groups were statistically insignificant.

The patients in this study suffered from osteoarthritis. Consequently, they were experiencing significant knee pain and many were candidates for a knee replacement. Many of these patients were able to avoid knee replacement by undergoing the Regenexx procedure.

The study concluded that there was no advantage of adding fat to the joint over the bone marrow cells. Safety in both groups (with and without fat) was excellent compared to knee replacement.

This study used data from patients who were part of the Regenexx registry. Therefore, this study was not a randomized, controlled study, like the kind that are used to test drugs. Randomized controlled trials are being conducted by Centeno and his colleagues at the various Regenexx centers. A knee osteoarthritis study is being studied in Chicago, another study regarding shoulder rotator cuff tears, and a third study examining ACL tears are in progress.

Cartilage Repair Using Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Embedded in Hyaluronic Acid Hydrogel in a Minipig Model


Cartilage shows lousy regenerative capabilities. Fortunately, it is possible to regenerate cartilage with human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) that have been embedded in a hyaluronic acid (HA) hydrogel composite. In fact, such a combination has shown remarkable results in rat and rabbit models.

In this present study, published in Stem Cells Translational Medicine, Yong-Geun Park and his colleagues from SungKyunKwan University School of Medicine, in Seoul, South Korea sought to confirm the efficacy of this protocol in a in a pig model using three different hUCB-MSC cell lines.

Park and his coworkers generated full-thickness cartilage injuries in the trochlear groove of each knee in 6 minipigs. Three weeks later, an even larger cartilage defect, 5 mm wide by 10 mm deep, was created, followed by an 8-mm-wide and 5-mm-deep boring. In short, the knee cartilages of these minipigs were very messed up.

Trochlear-groove

To these knee cartilages, a mixture (1.5 ml) of hUCB-MSCs (0.5 × 107 cells per milliliter) and 4% HA hydrogel composite were troweled into was then cartilage defects of the right knee. The left knee served as an untreated control. Each cell line was used in two minipigs.

Macroscopic findings of the osteochondral defects of the porcine knees. At 12 weeks postoperatively, the defects of both knees had produced regenerated tissues that were pearly white and firm. These new tissues, which resembled articular cartilage, appeared adherent to the adjacent cartilage and had restored the contour of the femoral condyles (smooth articular surfacewithout depression). The regenerated tissue of the control knee (left knee) looked fibrillated. Grossly, no differencewas seen in the quality of the repaired tissue in the transplanted knee (right knee) among the three groups with different cell lines. (A): Group A. (B): Group B. (C): Group C. Abbreviations: HA, hyaluronic acid; hUCB-MSCs, human umbilical cord blood-derived mesenchymal stem cells.
Macroscopic findings of the osteochondral defects of the porcine knees. At 12 weeks postoperatively, the defects of both
knees had produced regenerated tissues that were pearly white and firm. These new tissues, which resembled articular cartilage, appeared adherent to the adjacent cartilage and had restored the contour of the femoral condyles (smooth articular surface without depression). The regenerated tissue of the control knee (left knee) looked fibrillated. Grossly, no difference was seen in the quality of the repaired tissue in the transplanted knee (right knee) among the three groups with different cell lines. (A): Group A. (B): Group B. (C): Group C. Abbreviations: HA, hyaluronic acid; hUCB-MSCs, human umbilical cord blood-derived mesenchymal stem cells.

12 weeks after surgery, the pigs were sacrificed, and the degree of subsequent cartilage regeneration was evaluated by gross and more detailed microscopic analysis of the knee tissue. The transplanted knee showed superior and more complete joint-specific (hyaline) cartilage regeneration compared with the control knee. The microscopic characteristics of the knee cartilage showed that those animals that received the hUCB-MSCs had greater rates of cell proliferation and cells that differentiated into cartilage-making cells.

Microscopic findings of the regenerating osteochondral defects on porcine articular cartilage (safranin O and fast green staining). At 12 weeks postoperatively, the surface of the repairing tissue in the control knee (left knee) was poorly stained for glycosaminoglycan. In the transplanted knee (right knee), both the regenerated tissue and the adjacent cartilage to which it had become adherent exhibited the normal orthochromatic staining properties with safranin O. (A): Group A. (B): Group B. (C): Group C. Scale bars = 2 mm. Abbreviations: HA, hyaluronic acid; hUCB-MSCs, human umbilical cord blood-derived mesenchymal stem cells.
Microscopic findings of the regenerating osteochondral defects on porcine articular cartilage (safranin O and fast green staining). At 12 weeks postoperatively, the surface of the repairing tissue in the control knee (left knee) was poorly stained for glycosaminoglycan. In the transplanted knee (right knee), both the regenerated tissue and the adjacent cartilage to which it had become adherent exhibited the normal orthochromatic staining properties with safranin O. (A): Group A. (B): Group B. (C): Group C. Scale bars = 2 mm. Abbreviations: HA, hyaluronic acid; hUCB-MSCs, human umbilical cord blood-derived mesenchymal stem cells.

These data show consistent cartilage regeneration using composites of hUCB-MSCs and HA hydrogel in a large animal model. These experiments could be a stepping stone to a human clinical trial in the future that treats osteoarthritis of the knees with hUCB-MSCs embedded in HA hydrogel.

High-Quality Cartilage Production from Pluripotent Stem Cells


High-quality cartilage has been produced from pluripotent stem cells by workers in the laboratory of Sue Kimber and her team in the Faculty of Life Sciences at The University of Manchester. Such success might be used in the future to treat the painful joint condition osteoarthritis.

Kimber and her colleagues used strict laboratory conditions to grow and transform embryonic stem cells into cartilage cells known as chondrocytes.

Professor Kimber said: “This work represents an important step forward in treating cartilage damage by using embryonic stem cells to form new tissue, although it’s still in its early experimental stages.” Kimber’s research was published in Stem Cells Translational Medicine.

During the study, the team analyzed the ability of embryonic stems cells to become cartilage precursor cells. Kimber and her colleagues then implanted these pre-chrondrocytes into cartilage defects in the knee joints of rats. After four weeks, the damaged cartilage was partially repaired and following 12 weeks a smooth surface, which looked very similar to normal cartilage, was observed. More detailed studied of this newly regenerated cartilage demonstrated that cartilage cells from embryonic stem cells were still present and active within the tissue.

Developing and testing this protocol in rats is the first step in generating the information required to run such a study in people with arthritis. Before such a clinical trial can be run, more data will need to be collected in order to check that this protocol is effective and that there are no toxic side-effects.

However, Kimber and her coworkers say that this study is very promising as not only did this protocol generate new, healthy-looking cartilage but also importantly there were no signs of any side-effects such as growing abnormal or disorganized, joint tissue or tumors. Further work will build on this finding and demonstrate that this could be a safe and effective treatment for people with joint damage.

Chondrocytes created from adult stem cells are being used on an experimental basis, but, to date, they cannot be produced in large amounts, and the procedure is expensive.

With their huge capacity to proliferate, pluripotent stem cells such as embryonic stem cells and induced pluripotent stem cells can be manipulated to form almost any type of mature cell. Such cells offer the possibility of high-volume production of cartilage cells, and their use would also be cheaper and applicable to a greater number of arthritis patients, the researchers claim.

“We’ve shown that the protocol we’ve developed has strong potential for developing large numbers of chondrogenic cells appropriate for clinical use,” added Prof Kimber. “These results thus mark an important step forward in supporting further development toward clinical translation.”

Osteoarthritis affects more than eight million people in the UK alone, and is a major cause of disability. It and occurs when cartilage at the ends of bones wears away causing joint pain and stiffness.

Director of research at Arthritis Research UK Dr Stephen Simpson added: “Current treatments of osteoarthritis are restricted to relieving painful symptoms, with no effective therapies to delay or reverse cartilage degeneration. Joint replacements are successful in older patients but not young people, or athletes who’ve suffered a sports injury.

“Embryonic stem cells offer an alternative source of cartilage cells to adult stem cells, and we’re excited about the immense potential of Professor Kimber’s work and the impact it could have for people with osteoarthritis.”

Human articular cartilage defects can be treated with nasal septum cells


A report from collaborating research teams from the University and the University Hospital of Basel specifies that cells isolated from the nasal septum cartilage can adapt to the environment the knee and repair articular cartilage defects. The ability of nasal cartilage cells to self-renew and adapt to the joint environment is associated with the expression of genes know as HOX genes. This research was published in the journal Science Translational Medicine in combination with reports of the first patients treated with their own nasal cartilage.

Lesions in articular or joint-specific cartilage is a degenerative that tends to occur in older people or younger athletes who engage in impact-heavy sports. Sometimes people who have experienced accidents can also suffer from cartilage lesions. Cartilage lesions present several challenges for orthopedic surgeons to repair. These surgeries are often complicated, and the recovery times are also long. However, Prof. Ivan Martin, professor of tissue engineering, and Prof. Marcel Jakob, Head of Traumatology, from the Department of Biomedicine at the University and the University Hospital of Basel have presented a new treatment option for cartilage lesions that includes the use of nasal cartilage cells to replace cartilage cells in joints.

When grown in cell culture, cartilage cells extracted from the nasal septum (also known as nasal chondrocytes) have a remarkable ability to generate new cartilage tissue after their growth in culture. In an ongoing clinical study, the Basal research group have taken small biopsies (6 millimeters in diameter) from the nasal septa of seven of 25 patients below the age of 55 years. After isolating the cartilage cells from these cartilage samples, they cultured these cells and expanded them and applied them to a three-dimensional scaffold in order to engineer a cartilage graft with a specific size (30 x 40 millimeters).

Martin and his colleagues used these very cartilage grafts to treat the cartilage lesions in human patients. After removing the damaged cartilage tissue from the knee of several patients, their knees were treated with the engineered, tailored tissue from their noses.

Two previous experiments demonstrated the potential efficacy of this procedure. First, a previous clinical study conducted in cooperation with plastic surgeons and the Basel group used the same method to successfully reconstruct nasal wings affected by tumors.

Secondly, a preclinical study with goats whose knees were implanted with nasal cartilage cells showed that these cells were not only compatible with the knee-joint, but also successfully reconstituted the joint cartilage. Lead author of this study, Karoliina Pelttari, and her colleagues were quite surprised that the implanted nasal cartilage cells, which originate from a completely different set of embryonic cell types than the knee-joint were compatible. Nasal septum cells develop from neuroectodermal cells, which also form the nervous system and their self-renewal capacity is attributed to their lack of expression of some homeobox (HOX) genes. However, these same HOX genes are expressed in articular cartilage cells that are formed by mesodermal cells in the embryo.

“The findings from the basic research and the preclinical studies on the properties of nasal cartilage cells and the resulting engineered transplants have opened up the possibility to investigate an innovative clinical treatment of cartilage damage,” says Prof. Ivan Martin about the results. Several studies have confirmed that human nasal cells maintain their capacity to grow and form new cartilage despite the age of the patient. This means that older people could also benefit from this new method, as could patients with large articular cartilage defects.

The primary target of the ongoing clinical study at the University Hospital of Basel is to confirm the safety, efficacy and feasibility of nasal cartilage grafts transplanted into joints, the clinical effectiveness of this procedure, from the data presently in hand, is highly promising.

Mesenchymal Stem Cells from Fat Relieve Arthritis Pain for Up to Two Years


Regeneus is an Australian regenerative that has developed an experimental treatment for arthritis called HiQCells.  HiQCell is a stem cell treatment made from the patient’s own adipose (fat) tissue, and is subsequently injected into an affected joint or tendon. Regeneus has tested their HiQCell treatment in an independent clinical study that examined the efficacy of injections of HiQCells into the joints of patients with osteoarthritis of the knee.  The study examined 40 patients with knee osteoarthritis.  Half of these patients received the placebo and half HiQCell in a double-blinded study.  When asked about their pain levels six months after the procedure, patients in the placebo and HiQCell group ~45% of patients reported less pain and by 12 months after treatment 55% of patients in both groups reported less pain.  Thus both treatments relieved pain to a similar degree.  However, when the progression of the disease was examined, a very different result was observed.  As osteoarthritis progresses, some of the breakdown products of joint cartilage appear in urine and blood.  By collecting urine and blood samples from osteoarthritis patients, the progression of the disease can be readily tracked.  Blood and urine testing showed significantly less cartilage breakdown in the HiQCell group and significantly more breakdown in the patients in the placebo group who had advanced cartilage damage. Thus, even though the patients who received placebo had about the same level of pain reduction over the six-month period, it seems that their cartilage breakdown progressed at a faster rate.

Now a follow-up examination of these and other subjects who participated in this initial clinical study has revealed something surprising.  According to Regeneus, as of July 21, 2014, from a collection of 386 patients: 1) Pain has continued to decrease two years post-treatment; 2) One year after treatment, 63 of 86 patients reported more than a 30% reduction in pain;
3) Two years after treatment, 14 of 17 patients reported more than a 30 % reduction in pain and 14 patients experienced an average pain reduction of 84% at two years post-treatment; 4) Patients also reported significant improvements from pre-treatment in knee-function, sleep quality and reduced pain medications.  Finally, it is clear from these results that HiQCell is a safe therapy and well tolerated by patients, since the frequency and severity of adverse effects of patients who received HiQCell treatments were no different from those received the placebo.

The HiQCell Joint Registry established by Regeneus is the first of its kind in that the patients who participate in this study are subjected to long-term follow-up and undergo stem cell therapy using the patient’s own fat-derived stem cells. The HiQCell study has been approved by a human research ethics committee.  These 386 patients included in the Joint Registry will continue to be followed for up to 5 years with analysis updated regularly.

Professor Graham Vesey, CEO of Regeneus, comments: “The registry data is demonstrating that HiQCell has a therapeutic benefit for longer than 2 years. We are now also beginning to see very encouraging data from patients that have had cells frozen for future injections. This combination of the long-term effect from HiQCell and the successful storage of cells for repeat injections in the future, means that HiQCell can be used to treat joint pain for many years. This is particularly important for patients that are too young for joint replacement or are simply looking to delay joint replacement.”

Improving Cartilage Production By Stem Cells


To repair cartilage, surgeons typically take a piece of cartilage from another part of the injured joint and patch the damaged area, this procedure depends on damaging otherwise healthy cartilage. Also, such autotransplantation procedures are little protection against age-dependent cartilage degeneration.

There must be a better way. Bioengineers want to discover more innovative ways to grow cartilage from patient’s own stem cells. A new study from the University of Pennsylvania might make such a wish come true.

This research, comes from the laboratories of Associate professors Jason Burdick and Robert Mauck.

“The broad picture is trying to develop new therapies to replace cartilage tissue, starting with focal defects – things like sports injuries – and then hopefully moving toward surface replacement for cartilage degradation that comes with aging. Here, we’re trying to figure the right environment for adult stem cells to produce the best cartilage,” said Burdick.

Why use stem cells to make cartilage? Mauck explained, “As we age, the health and vitality of cartilage cells declines so the efficacy of any repair with adult chondrocytes is actually quite low. Stem cells, which retain this vital capacity, are therefore ideal.”

Burdick and his colleagues have long studied mesenchymal stem cells (MSCs), a type of adult stem cell found in bone marrow and many other tissues as well that can differentiate into bone, cartilage and fat. Burdick’s laboratory has been investigating the microenvironmental signals that direct MSCs to differentiate into chondrocytes (cartilage-making cells).

chondrocytes
chondrocytes

A recent paper from Burdick’s group investigated the right conditions for inducing fat cell or bone cell differentiation of MSCs while encapsulated in hydrogels, which are polymer networks that simulate some of the environmental conditions as which stem cells naturally grow (see Guvendiren M, Burdick JA. Curr Opin Biotechnol. 2013 Mar 29. pii: S0958-1669(13)00066-9. doi: 10.1016/j.copbio.2013.03.009). The first step in growing new cartilage is initiating cartilage production or chondrogenesis. To do this, you must convince the MSCs to differentiate into chondrocytes, the cells that make cartilage. Chondrocytes secrete the spongy matrix of collagen and acidic sugars that cushion joints. One challenge in promoting MSC differentiation into chondrocytes is that chondrocyte density in adult tissue is rather low. However, cartilage production requires that the chondrocytes be in rather close proximity.

Burdick explained: “In typical hydrogels used in cartilage tissue engineering, we’re spacing cells apart so they’re losing that initial signal and interaction. That’s when we started thinking about cadherins, which are molecules that these cells used to interact with each other, particularly at the point they first become chondrocytes.”

Desmosomes can be visualized as rivets through the plasma membrane of adjacent cells. Intermediate filaments composed of keratin or desmin are attached to membrane-associated attachment proteins that form a dense plaque on the cytoplasmic face of the membrane. Cadherin molecules form the actual anchor by attaching to the cytoplasmic plaque, extending through the membrane and binding strongly to cadherins coming through the membrane of the adjacent cell.
Desmosomes can be visualized as rivets through the plasma membrane of adjacent cells. Intermediate filaments composed of keratin or desmin are attached to membrane-associated attachment proteins that form a dense plaque on the cytoplasmic face of the membrane. Cadherin molecules form the actual anchor by attaching to the cytoplasmic plaque, extending through the membrane and binding strongly to cadherins coming through the membrane of the adjacent cell.

In order to simulate this microenvironment, Burdick and his collaborators and colleagues used a peptide sequence that mimics these cadherin interactions and bound them to the hydrogels that were then used to encapsulate the MSCs.

According to Mauck, “While the direct link between cadherins and chondrogenesis is not completely understood, what’s known is that if you enhance these interactions early during tissue formation, you can make more cartilage, and, if you block them, you get very poor cartilage formation. What this gel does is trick the cells into think it’s got friends nearby.”

See L Bian, et al., PNAS 2013; DOI:10.1073/pnas.1214100110.

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.