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.