The Ideal Recipe for Cartilage from Stem Cells


Researchers at Case Western Reserve and Harvard University will use a 5-year, $2-million NIH grant to build a microfactory that bangs out the optimal formula for joint cartilage. Such an end product could one day potentially benefit many of the tens of thousands of people in the United States who suffer from cartilage loss or damage.

Joint cartilage or articular cartilage caps the ends of long bones and bears the loads, absorbs shocks and, in combination with lubricating synovial fluid, helps knees, hips, shoulders, and other joints to smoothly bend, lift, and rotate. Unfortunately, this tissue has little capacity to regenerate, which means that there is a critical need for new therapeutic strategies.

Artificial substitutes cannot match real cartilage and attempts to engineer articular cartilage have been stymied by the complexities of directing stem cells to differentiate into chondrocytes and form the right kind of cartilage.

Stem cells are quite responsive to the environmental cues presented to them from their surroundings. What this research project hopes to determine are those specific cue that drive stem cells to differentiate into chondrocytes that make the right kind of cartilage with the right kind of microarchitecture that resembles natural, articular cartilage. To do this, they will engage in a systematic study of the effects of cellular micro-environmental factors that influence stem cell differentiation and cartilage formation.

Bone marrow- and fat-derived mesenchymal stem cells have been differentiated into cartilage-making chondrocytes in the laboratory. These two stem cell populations are distinct, however, and required different conditions in order to drive them to differentiate into chondrocytes. This research group, however, has designed new materials with unique physical properties, cell adhesive capabilities, and have the capacity to deliver bioactive molecules.

By controlling the presentation of these signals to cells, independently and in combination with mechanical cues, this group hopes to identify those most important cues for driving cells to differentiate into chondrocytes.

Ali Khademhosseini specializes in microfabrication and micro-and nano-scale technologies to control cell behavior. He and his team will develop a microscale high-throughput system at his laboratory that will accelerate the testing and analysis of materials engineered in another laboratory.

This research cooperative hopes to test and analyze more than 3,000 combinations of factors that may influence cell development, including differentiation, amounts of biochemicals, extracellular matrix properties, compressive stresses, and more. Khademhosseini and his colleagues hope to begin testing comditions identified from these studies in animal models by the of the grant term.

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.”

Bone Progenitor Cells Discovered – Might Help Children Who Need Corective Facial Surgery


In children, bone grow thicker and longer and get stronger and denser. When children reach adolescence, they know that the time has come to stop growing longer and stronger. However, even into adulthood, bones still retain the capacity to heal. Why the differences between adolescents and adults? This is a question that has long fed the imaginations of scientists.

Recently, a collaborative team of biomedical researchers from the University of Michigan, Kyoto University and Harvard University has made the answer to this question a little clearer.

Dr. Noriaki Ono, U-M assistant professor of dentistry, and his collaborators discovered that a certain subset of cartilage-making cells – cells known as chondrocytes – proliferate and differentiate into other bone cells that drive bone growth. These discoveries could lead to new treatments for children with facial deformities who normally have to wait until adulthood for corrective surgery. This study appeared in the journal Nature Cell Biology.

A long-held view is that bone-making chondrocytes die once children reach adolescence and their bones stop growing. However, in adults, bone still heal without the benefit of these bone-making chondrocytes. How does this occur? This question has generated a fair amount of disagreement between researchers.

Ono’s group discovered that some of these bone-making chondrocytes don’t die. Instead, they are transformed into other types of bone-growing and bone-healing cells.

“Up until now, the cells that drive this bone growth have not been understood very well. As an orthodontist myself, I have special interest in this aspect, especially for finding a cure for severe bone deformities of the face in children,” he said. “If we can find a way to make bones that continue to grow along with the child, maybe we would be able to put these pieces of growing bones back into children and make their faces look much better than they do.”

According to Ono, one of the challenges in bone and cartilage medicine is that resident stem cells haven’t really been identified. The only widely accepted idea is that certain stem cells like mesenchymal stem cells help bones heal and other help them grow, but the progenitor cells for these cell populations and what goes wrong with them in conditions such as osteoporosis remains mysterious.

Ono and his team used a technique called “fate mapping,” which labels cells genetically and them follows them throughout development. Ono and others came upon a specific precursor cell that gives rise to fetal chondrocytes, and all the other later bone-making cells and . By mesenchymal stromal cells later in life. Most exciting, Ono and his coworkers found a way to identify the cells responsible for growing bone. By identifying these cells, isolating them and even implanting them into the skull or long bones of a child with a bone deformity condition, the cells would make bone that would also grow with the child.

Many factors cause craniofacial deformities. These types of deformities can place pressure on the brain, eyes, or other structures and prevent them from developing normally. For example, children with Goldenhar syndrome have underdeveloped facial tissues that can harm the developing jawbone. Another bone deformity called deformational plagiocephaly causes a child’s head to grow asymmetrically. Maybe the implantation of such cells can provide a way to restart the abnormally growing bones in these children.

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.

Stem Cell Treatments to Repair Cartilage Defects in the Knee


Erosions of the cartilage that covers the surfaces at the ends of our leg bones has motivated several laboratories to undertake clinical studies to test new techniques to heal lost cartilage, particularly at the knee. Many of these techniques have their share of drawbacks and advantages, but the number of clinical trials to deal with cartilage lesions of the knee are increasing. Unfortunately, more work remains to be done, but much more is known about several of these techniques than before. This article will summarize many of these techniques.

Microfracture is a procedure in which several small holes are drilled into the end of the bone to enhance the migration of mesenchymal stem cells from the bone marrow to the site of the cartilage defect. These MSCs then differentiate into chondrocytes and make cartilage that fills the lesion with new cartilage. Unfortunately, the cartilage made in these cases is fibrocartilage and not hyaline cartilage. Fibrocartilage lacks the biomechanical strength and durability of hyaline cartilage and it typically deteriorates 18-24 months after surgery. When used to treat large lesions, 20-50% of all cases develop intralesional osteophytes and the sclerotic bone increases the failure rate of autologous chrondrocyte implantation 3-7X. Thus microfractionation is only performed under very specific conditions and only in young patients, since this technique does not work in older patients.

Microfracture

Autologous Chondrocyte Implantation or ACI uses a full-thickness punch biopsy from a low-weight-bearing region of the joint taken during an arthroscopic surgery. This biopsy contains chondrocytes that are grown in cell culture to a population of about 12-48 million chondrocytes, which are troweled into the lesion during a second arthroscopic surgery. Clinical trials have established that ACI is safe and effective for large knee lesions. Peterson and others and Minas and others have established that even after 10 years, patients who have been treated with ACI show good relief of pain and increased knee function.

In the Peterson study, questionnaires were sent to 341 patients. 224 of 341 patients replied to the questionnaires, and of these respondents, 74% of the patients reported their status as better or the same as the previous years 10-20 years after the procedure (mean, 12.8 years).  92% were satisfied and would have ACI again.  Knee function and pain levels were significantly better after the procedure than before.  From this study, Peterson and others concluded that ACI is an effective and durable solution for the treatment of large full-thickness cartilage and osteochondral lesions of the knee-joint, and that the clinical and functional outcomes remain high even 10 to 20 years after the implantation.

Minas and others analyzed data from 210 patients treated with ACI who were followed for more than 10 years. ACI provided durable outcomes with a survivorship of 71% at 10 years and improved function in 75% of patients with symptomatic cartilage defects of the knee at a minimum of 10 years after surgery. A history of prior marrow stimulation as well as the treatment of very large defects was associated with an increased risk of failure.
In comparison studies by Bentley and others, ACI produced superior results to mosaicplasty (osteochondral transplantation or cylinders of bone drilled form low-weight-bearing parts of the knee that are implanted in a mosaic fashion into the knee).  In the Bentley study, 10 of 58 ACI patients had failed grafts after 10 years, but 23 of 42 mosaicplasty patients had failed cartilage repair.  According to studies by Based and others, and Saris and others, ACI is also superior to microfractionation in the repair of large cartilage lesions (>3 cubic cm), but seems to provide the same outcomes as microfracture for smaller lesions, according to Knudsen and others.  There are drawbacks to ACI.  The tissue flap used to seal the cartilage implant sometimes becomes pathologically enlarged.  Other materials have been used to seal the patch, such as hyaluronic acid, or collagen types I and III, but the use of these materials increases the expense of the procedure and the likelihood that the immune system will response to the covering.  Also, ACI outcomes vary to such an extent that the procedure is simply too unstandardized at the present time to be used consistently in the clinic.

Autologous Cartilage Implantation

In an attempt to standardize ACI, several orthopedic surgeons have tried to add a supportive scaffold of some sort to the chondrocytes harvested from the patient’s body.  Several studies in tissue culture have shown that chondrocytes not only divide better, but also keep their identities as chondrocytes better in a three-dimensional matrix (see Grigolo et al, Biomaterials (2002) 23: 1187-1195 and Caron et al, Osteoarthritis Cartilage (2012) 20; 1170-1178).  Therefore, ACI has given way to MACI or Matrix-Induced Autologous Chondrocyte Implantation, which seeds the chondrocytes on an absorbable porcine-derived mixed collagen (type I and III) prior to implantation.  The implant is then secured into the debrided cartilage lesion by means of a fibrin cover.

Several case studies have shown that MACI has substantial promise, but individual case studies are the weakest evidence available.  To prove its superiority over ACI or microfracture surgery, MACI must be compared in controlled studies.  In the few studies that have been conducted, the superiority of MACI remains unproven to date.  Patients who received MACI or ACI showed similar clinical outcomes in two studies (Bartlett and others, Journal of Bone and Joint Surgery (2005) 87: 640-645; and Zeifang et al, American Journal of Sports Medicine (2010) 38: 924-933), although those who received MACI showed a significantly lower tendency for the graft to enlarge.  MACI is clearly superior to microfracture surgery (Basad, et al., Knee Surgery, Sports Traumatology and Arthroscopy (2010) 18: 519-527), but longer-term studies are needed to establish the superiority of MACI over other treatment options.

A slight variation of the MACI theme is to embed the chondrocytes in a gel-like material called hyaluronic acid (HA).  HA-embedded chondrocytes have been shown to promote the formation of hyaline cartilage in patients (Maracci et al., Clinical Orthopedics and Related Research (2005) 435: 96-105).  Even though the outcomes are superior for patients treated with HA-MACI, the recovery period is longer (Kon E, et al., American Journal of Sports Science (2011) 39: 2549-2567).  MACI is available in Europe but not the US to date.  FDA approval is supposedly pending.  Long-term follow-up studies are required to establish the efficacy of this procedure.

Future prospects for treating knee cartilage lesions include culturing collagen-seeded chondrocytes for a longer period of time than the three days normally used for MACI.  During these longer culture periods, the seeded chondrocytes mature, and make their own scaffolds, which ensure higher-quality cartilage and better chondrocyte engraftment (see Khan IM and others, European Cell Materials (2008) 16: 26-39).  Alternatively, joint cartilage responds to stress by undergoing cell proliferating and increasing in density.  This response is due to the production of growth factors such as Transforming Growth Factor-β1 and -β3 (TGF-β1 and TGF-β3).  This motivated some enterprising tissue engineers to use recombinant forms of these growth factors to grow cartilage in bioreactors under high-stress conditions.  Such a strategy has given rise to NeoCart, a tissue-engineered product that has gone through Phase I and II trials and has been shown in two-year follow-up studies to be safe and more effective than microfracture surgery (Crawford DC and others, Journal of Bone and Joint Surgery, American Volume. 2012 Jun 6;94(11):979-89 and Crawford DC, and others, Am J Sports Med. 2009 Jul;37(7):1334-43).

Mesenchymal stem cells (MSCs) from bone marrow and other sites have also been used to successfully treat cartilage lesions.  These types of treatments are less expensive than ACI and MACI, and do not require two surgeries as do ACI and MACI.  The studies that have been published using a patient’s own MSCs have been largely positive, although some pain associated with the site of the bone marrow aspiration is a minor side effect (see Centeno and others, Pain Physician (2008) 11:343-353; Emadedin, et al., Arch Iran Med (2012) 15: 422-428; Wong RL, et al., Arthroscopy (2013) 29: 2020-2028).  Fat-based MSCs have been tested as potential cartilage-healers in elderly patients (Koh YG, et al., Knee Surgery, Sports Traumatology, and Arthroscopy (Dec 2013, published on-line ahead of print date).  While these initial results look promising,, fat-based, MSCs have only just begun to be tested for their ability to regenerate cartilage.  Fat-based MSCs show different properties than their bone-marrow counterparts, and it is by no means guaranteed that fat-based MSCs can regenerate cartilage as well as MSCs from bone marrow.

Fresh cartilage grafts from donors (aka – cartilage allografts) use transplanted cartilage that has been freshly collected from a donor.  Fresh cartilage allografts have had positive benefits for young, active patients and the grafts have lasted 1-25 years (Gross AE, et al., Clinical Orthopedics and Related Research (2008) 466: 1863-1870).  Particulate cartilage allografts takes minced cartilage and lightly digests it with enzymes to liberate some of the cartilage-synthesizing chondrocytes, and then pats this material into the cartilage lesion, where it is secured with a fibrin glue plug.  The cartilage provides an excellent matrix for the synthesis of new cartilage, and the chondrocytes make new cartilage while seeded onto this cartilage scaffold.  Clinical experience with this technique includes a two-year follow-up in which MRI evidence showed good filling of the lesions (Bonner KF, Daner W, and Yao JQ, Journal of Knee Surgery 2010 23: 109-114 and Farr J, et al., Journal of Knee Surgery 2012 25: 23-29).  A variation on this technique uses a harvested hyaline cartilage plug that is glued into an absorbable scaffold before transplantation into the cartilage lesion.  This procedure had the same safety profile as microfracture surgery, but resulted in better clinical outcomes, high quality cartilage, and fewer adverse side effects (Cole JB et al., American Journal of Sports Medicine 2011 39: 1170-1179).  A clinical trial that tested this procedure remains uncompleted after the company suspended the trial because of conflicts with the FDA (Clinical Trial NCT00881023).

AMIC or Autologous Matrix-Induced Chondrogenesis is a cell-free treatment option in which the cartilage lesion is cleaned and filled subjected to microfracture, after which the lesion is filled with a mixed collagen matrix that is glued or stitched to the cartilage lesion.  The MSCs released by the microfracture procedure now move into a scaffold-laden cartilage lesion that induces the formation of hyaline cartilage.  This technique appears to aid the filling of full-thickness cartilage defects, and follow-up examinations have revealed that after 5 years, patients showed substantial improvements in knee function, pain relief and MRI analyses of knee cartilage showed high-quality cartilage in repaired lesion (Kusano T, et al., Knee Surgery, Sports Traumatology, and Arthroscopy 2012 20: 2109-2115; Gille J, et al., Archives of Orthopedic Trauma Surgery 2013 133: 87-93; Gille J, et al., Knee Surgery, Sports Traumatology, and Arthroscopy 2010 18: 1456-1464).

These are just a few of the new treatments of cartilage lesions of the knee and other joints.  As you can see, all of this will lead to greater repair of knee lesions and it is all being done without embryonic stem cells or destroying embryos.

ALK2 Manipulation Increases Bone Formation in Fat-Based Stem Cells


Mesenchymal stem cells possess a cell-surface protein called ALK2. ALK2 acts as a receptor for bone-inducing growth factors. ALK2, for example, is expressed in cartilage and if mesenchymal stem cells express a constantly-active form of ALK2, known as caALK2, these cells are driven to become cartilage-making cells (known as chondrocytes).

Can this receptor be used to drive bone formation? It turns out that manipulating ALK2 can drive fat-based stem cells (ASCs) to become bone making cells that ultimately improve bone tissue engineering. Researchers from the laboratory of Benjamin Levi at Massachusetts General Hospital, Boston, Massachusetts have fiddled with ALK2 in mesenchymal stem cells to for formation of bone from ASCs, and to enhance bone regeneration in a living animal.

To do this, Levi’s team genetically manipulated mice so that they expressed a form of ALK2 that was constantly turned on known as caALK2. The fat-based MSCs were then isolated and analyzed for their ability to make bone in culture. caALK ASCs were much more responsive to bone-inducing growth factors. These cells also expressed a whole host of bone-specific genes (e.g., Alp, Runx2, Ocn, Ops) after seven days. Since the caALK2 MSCs did so well in culture, they were then tested in mice with skull defects. Bone formation was significantly higher in mice treated with caALK2-expressing ASCs than those treated with normal ASCs.

Thus, Levi’s laboratory has shown that by treating mice with fat-based stem cells that express a constitutively active ALK2 receptor showed significantly increased bone formation. This increased bone formation can also be harnessed to improve skull healing in mice with bone defects.

Mesenchymal Stem Cells Repair Cartilage Defects in Cynomolgus Monkeys


Repairing cartilage defects in the knee represents one of the primary goals of orthopedic regenerative medicine. Cartilage that covers the joints, otherwise known as articular cartilage, has a limited capacity for repair, which leads to further degeneration of the cartilage when it is damaged if it remains untreated. A number of surgical options for treating cartilage defects include microfracture, osteochondral grafting, and cell-based techniques such as autologous chondrocyte implantation (ACI). Each of these procedures have been used in clinical settings. Unfortunately cartilage injuries treated with microfracturing deteriorate with time, since the cartilage made by microfracturing has a high proportion of softer. less durable fibrocartilage.  Also osteochondral grafting suffers from a lack of lateral integration between host and donor cartilage.

Alternatively, tissue engineering has shown some promise when it comes to the healing of cartilage defects.  Mesenchymal stem cells (MSCs) are multipotent progenitor cells that have the ability to differentiate into several different cell lineages including cartilage-making chondrocytes.  MSCs have theoretical advantages over implanted chondrocytes when it come to healing potential.  MSCs have the ability to proliferate without losing their ability to differentiate into mature chondrocytes and produce collagen II and aggrecan. In the short-term, bone marrow-derived MSCs combined with scaffolds have been successful in cartilage repair using animal models such as rabbits (Dashtdar H, et al., J Orthop Res 2011; 29: 1336-42) sheep (Zscharnack M, et al., Am J Sports Med 2010; 38: 185769) and horses (Wilke MM, et al.,l J Orthop Res 2007; 25: 9132).  

In a recent study, Kazumasa Ogasawara and Yoshitaka Matsusue and their colleagues from Shiga University of Medical Science in Shiga, Japan, tested the ability of expanded bone marrow-derived MSCs that had been placed in a collagen scaffold to improve healing of cartilage defects in cynomolgus macaques (type of monkey).  Before this study, there were no previous studies using MSCs from primates for cartilage repair.  The monkey MSCs were shown to properly differentiate into fat, bone, or cartilage in culture, and then were transplanted into the injured cartilage in the cynomolgus macaque.  The efficacy of these cells were ascertained at 6, 12, and 24 weeks after transplantation.

In culture, the cynomolgus MSCs were able to differentiate into fat, bone, and cartilage.

Characteristics of bone marrow-derived MSCs. Panel (a) demonstrates the colony-forming properties of MSCs isolated from bone marrow of cynomolgus macaques using the present protocol (arrows). Bar: 1 cm. Panel (b) shows the adipogenetic properties of MSC-derived cells from staining of lipid droplets with oil red O (arrowheads). Bar: 20 μm. Panel (c) confirms the osteoblastic properties of MSC-derived cells with alkaline phosphatase staining (arrowheads). Bar: 30 μm. Panel (d) confirms the chondrogenetic properties from immunostaining of type-II collagen. Type-II collagen-positive matrix is stained red. Bar: 0.5 mm. Read More: http://informahealthcare.com/doi/full/10.3109/17453674.2014.958807.
Characteristics of bone marrow-derived MSCs. Panel (a) demonstrates the colony-forming properties of MSCs isolated from bone marrow of cynomolgus macaques using the present protocol (arrows). Bar: 1 cm. Panel (b) shows the adipogenetic properties of MSC-derived cells from staining of lipid droplets with oil red O (arrowheads). Bar: 20 μm. Panel (c) confirms the osteoblastic properties of MSC-derived cells with alkaline phosphatase staining (arrowheads). Bar: 30 μm. Panel (d) confirms the chondrogenetic properties from immunostaining of type-II collagen. Type-II collagen-positive matrix is stained red. Bar: 0.5 mm.
Read More: http://informahealthcare.com/doi/full/10.3109/17453674.2014.958807.

Upon transplantation into cartilage defects in the knee cartilage of cynomolgus monkeys, MSCs were compared with collagen gel devoid of MSCs.  The knees that received the transplantations did not show any signs of irritation, bone spurs or infection.  All of the animals had so-called “full-thickness cartilage defects,” and those in the non-treated group showed cartilage defects that did not change all that much.  The cartilage defects of the gel group had sharp edges at 6 weeks that were thinly covered with reparative tissue by 12 weeks, and at 24 weeks, the defect was covered with thick tissue, but the central region of the defects often remained uncovered, with a hollow-like deformity.  In the cartilage defects of those animals treated with MSCs plus the collagen gel, the sharp edges of the defects were visible at 6 weeks after the operation, but at 12 weeks, the defects were evenly covered with yellowish reparative tissue.  At 24 weeks, the defects were covered with watery hyaline cartilage-like tissue that was very similar to the neighboring naïve cartilage.

Macroscopic observations of the repaired defects in the 3 groups at 6 weeks (a, d, g), 12 weeks (b, e, h), and 24 weeks (c, f, i) after implantation. Scale bar: 5 mm. Arrow in (d): the sharp edge of the defect is visible at 6 weeks in the gel group. Arrow in (f): a hollow-like deformity remains in the central region of the defect, despite thick coverage by the reparative tissue. Arrow in (g): the sharp edge of the defect is also visible in the MSC group at 6 weeks. Read More: http://informahealthcare.com/doi/full/10.3109/17453674.2014.958807.
Macroscopic observations of the repaired defects in the 3 groups at 6 weeks (a, d, g), 12 weeks (b, e, h), and 24 weeks (c, f, i) after implantation. Scale bar: 5 mm. Arrow in (d): the sharp edge of the defect is visible at 6 weeks in the gel group. Arrow in (f): a hollow-like deformity remains in the central region of the defect, despite thick coverage by the reparative tissue. Arrow in (g): the sharp edge of the defect is also visible in the MSC group at 6 weeks.
Read More: http://informahealthcare.com/doi/full/10.3109/17453674.2014.958807.

When evaluated at the tissue level, Ogasawara and Matsusue and others used a stain called toluidine blue to visualize the amount of cartilage made by each treatment.  As you can see in the picture below, the non-treated group didn’t do so well.  In the full-thickness defect the region below the cartilage was filled with amorphous stuff 6 weeks after the procedure, and at 12 weeks, amorphous stuff faintly stained with toluidine blue, which reflects the conversion of the amorphous stuff into bone.  At 24 weeks, bone tissue reappeared below the cartilage zone, even though the bone did not look all that normal (no trabecular structure but woven bone-like structure).

In the gel group, cartilage-like tissue is seen at 6 weeks, and at 12 weeks, the faintly stained layer covered the cartilage defect. At 24 weeks, the defect was covered with the cartilage-like stuff, even though the central region had only a little cartilage, as ascertained by toluidine blue staining.  The bone underneath the cartilage looked crummy and there was excessive growth of cartilage into the region underneath the cartilage layer.

In the MSC group, the bone underneath the cartilage healed normally, and at 12 weeks, the boundary between the articular cartilage and the bone layer beneath it had reappeared.  At 24 weeks, the thickness of the toluidine blue-stained cartilage layer was comparable to that of the neighboring naïve cartilage.

Even though the gel group showed most cartilage-rich tissue covering the defect, this was due to the formation of excessive cartilage extruding through the abnormal lower bone layer.  Despite the lower amount of new cartilage produced, the MSC group showed better-quality cartilage with a regular surface, seamless integration with neighboring naïve cartilage, and reconstruction of the bone underneath the cartilage layer.

Histological findings after toluidine blue staining in the 3 groups at 6 weeks (a, d, g), 12 weeks (b, e, h), and 24 weeks (c, f, i) after implantation. Scale bar: 2 mm. Dotted line in (a): amorphous reparative tissue filling the subchondral region. Arrowheads in (b): faint toluidine blue staining that reflects involvement of endochondral ossification. Arrowhead in (c): toluidine blue-negative reparative tissue covering the defect. Dotted line in (c): reconstructed subchondral bone consisting of woven bone-like structure. Arrowhead in (d): toluidine blue-positive cartilaginous tissue. Arrowhead in (e): thin faintly toluidine blue-positive layer covering the defect. Arrowhead in (f): the unstained central region of the cartilaginous layer covering the defect. Arrow in (f): excessive cartilage extruding through the deficient tidemark. Dotted line in (g): woven bone-like subchondral bone already re-appearing at 6 weeks. Arrowhead in (h): reconstructed tidemark distinctly discriminating the articular cartilage from the subchondral bone.
Histological findings after toluidine blue staining in the 3 groups at 6 weeks (a, d, g), 12 weeks (b, e, h), and 24 weeks (c, f, i) after implantation. Scale bar: 2 mm. Dotted line in (a): amorphous reparative tissue filling the subchondral region. Arrowheads in (b): faint toluidine blue staining that reflects involvement of endochondral ossification. Arrowhead in (c): toluidine blue-negative reparative tissue covering the defect. Dotted line in (c): reconstructed subchondral bone consisting of woven bone-like structure. Arrowhead in (d): toluidine blue-positive cartilaginous tissue. Arrowhead in (e): thin faintly toluidine blue-positive layer covering the defect. Arrowhead in (f): the unstained central region of the cartilaginous layer covering the defect. Arrow in (f): excessive cartilage extruding through the deficient tidemark. Dotted line in (g): woven bone-like subchondral bone already re-appearing at 6 weeks. Arrowhead in (h): reconstructed tidemark distinctly discriminating the articular cartilage from the subchondral bone.

This protocol has been nicely optimized by Ogasawara and Matsusue and their research team.  From these data, they conclude:  “Application in larger defects is certainly in line with future clinical use. If MSCs—under optimized conditions—turn out to be superior to chondrocyte implantation in experimental cartilage repair, the procedure should be introduced to clinical practice after well-controlled randomized clinical trials.”  Hopefully, clinical trials will commence before long.  This procedure uses a patient’s own MSCs, and if such a procedure could reduce or delay the number of knee replacements, then it would surely be a godsend to clinicians and patients alike.

Human Infra-patellar Fat Pad-Derived Stromal Cells Show Great Cartilage-Making Potential, Which is Enhanced By Connective Tissue Components


With age and overuse, our knees wear out and we sometimes need an artificial one. The cartilage shock absorber at the ends of our bones simply does not regenerate very well, and this results in large problems when we get older.

Is there an effective way to regenerate cartilage? Stem cells do have the ability to make cartilage, but finding the right stem cell and delivering enough of them to make a difference remains a challenge.

To that end, Tang-Yuan Chu and his colleagues from Tzu Chi University and the Buddhist Tzu Chi General Hospital in Hualien, Taiwan have discovered that stem cells from the fat pad that surrounds the knee appear to be one of the best sources of cartilage-making cells for the knee.

The infra-patellar fat pad or IFP contains a stem cell population called infra-patellar fat pad-derived stromal cells or IFPSCs. These IFPSCs were isolated by Chu and his colleagues from patients who were undergoing arthroscopic surgery. When Chu and others grew these cells in culture, the IFPSCs grew robustly for two weeks. The culture protocol was a standard one and no special requirements were required. In fact, after two weeks, the IFPSCs grew to more than 10 million cells on the third passage.

When the ability of IFPSCs to form cartilage-making cells (chondrocytes) were compared with mesenchymal stem cells from bone marrow, fat and umbilical cord connective tissue (Wharton’s jelly), the IFPSCs showed a clear superiority to these other cells types, and differentiated into chondrocytes quite effectively.

Next, Chu and his crew cultured the IFPSCs on a material called hyaluronic acid (HA). HA is a common component of the synovial fluid that helps lubricate our larger joints and in connective tissue, and basement membranes upon which epithelial cells sit.

Hyaluronic Acid

When grown on 25% HA, the IFPSCs were better at making bone or fat than IFPSCs grown on no HA. Furthermore, when grown on 25% HA, IFPSCs showed a four-fold increase in their ability to form chondrocytes. The HA also did not affect the ability of the cells to divide.

In conclusions, these IFPSCs seem to possess a strong potential to differentiate into chondrocytes and regenerate cartilage. Also, this ability is augmented in a growth environment of 25% HA. Certainly some preclinical trials with laboratory animal are due. Wouldn’t you say?

Source: Dah-Ching Ding; Kun-Chi Wu; Hsiang-Lan Chou; Wei-Ting Hung; Hwan-Wun Liu; Tang-Yuan Chu. Human infra-patellar fat pad-derived stromal cells have more potent differentiation capacity than other mesenchymal cells and can be enhanced by hyaluronan.  Cell Transplantation, http://dx.doi.org/10.3727/096368914X681937.

Orthopedic Regeneration With a Combination of Stem Cells, Gene Therapy, and Tissue Engineering


A Duke University research team has combined synthetic scaffolding materials with gene delivery techniques to generate replacement cartilage precisely where it’s needed in the body.

The ingenious strategy utilized by this research project circumvents the need for large quantities of growth factors, which are expensive and difficult to apply after implantation. The Duke team led by Farshid Guilak, director of orthopedic research at Duke University Medical Center, used gene therapy to make stem cells that synthesize their own growth factors.

In brief, Guilak and his collaborators used genetically engineered viruses to transfer genes to stem cells embedded in a synthetic matrix. Upon infection, the stem cells grew and differentiated as needed, but the scaffolding provided the necessary structural cues for the stem cells to move to the proper configuration and form cartilage with the proper shape and biomechanical properties.

Guilak has devoted several years to developing biodegradable synthetic scaffolds that mimic the mechanical properties of cartilage. After testing many different scaffolds, he settled on a 3D woven poly(ε-caprolactone) scaffold, which is completely biodegradable and provides an excellent structural matrix for the synthesis of cartilage.  However, an additional challenge for engineering good cartilage is to coax stem cells embed themselves in the scaffold while differentiating into cartilage-making cells, known as chondrocytes, after the scaffold has been implanted into a living organism.

One widely used strategy is to treat the stem cells with growth factors to induce chrondrocyte formation and cartilage production. Such cartilage can be implanted after it has been grown in the laboratory. However, this approach has some inherent limitations.

Guilak explained that “a major limitation in engineering tissue replacements has been the difficulty in delivering growth factors to the stem cells once they are implanted in the body.” Guilak continued: “There’s a limited amount of growth factor that you can put into the scaffolding, and once it’s released, it’s all gone. We need a method for long-term delivery of growth factors, and that’s where the gene therapy comes in.”

To tackle this perennial problem, Guilak tapped a talented colleague of his, Charles Gersbach, an assistant professor of biomedical engineering, who happens to also be a gene therapy expert.

Gersbach looked at the tissue engineering problem in an entirely new way and suggested that if the mountain will not come to Mohammed (that is to say if the growth factors cannot be given to stem cells after implantation), then Mohammed should grow his own mountain (the stem cells should be genetically engineered to make their own growth factors). Unfortunately, the conventional gene therapy methods are too complex to be commercially feasible. Typically, stem cells are collected, infected with genetically modified viruses that introduces new genes into them, grown to large numbers, and applied to synthetic cartilage scaffolds and implanted into the patient. Sounds like a headache? That’s because it is.

Fortunately, Gersbach had a slick gene therapy trick up his lab coat sleeve: “There are a few challenges with that process, one of them being that there are way too many extra steps,” said Gersbach. “So we turned to a technique I had previously developed that affixes the viruses that deliver the new genes onto a material’s surface.”

A microscopic view using electron microscopy of human stem cells and viral gene carriers adhering to the fibers of a polymer scaffold.  Photo source:  http://www.pratt.duke.edu/news/gene-therapy-might-grow-replacement-tissue-inside-body.
A microscopic view using electron microscopy of human stem cells and viral gene carriers adhering to the fibers of a polymer scaffold. Photo source: http://www.pratt.duke.edu/news/gene-therapy-might-grow-replacement-tissue-inside-body.

This new study combines Gersbach’s gene therapy technique—dubbed biomaterial-mediated gene delivery—to induce those human mesenchymal stem cells embedded in Guilak’s synthetic cartilage scaffolding to produce growth factor proteins (in particular a molecule called transforming growth factor β3  or TGF-β3). Based on the results of their experiments, the technique works and that the resulting synthetic, composite cartilage-like material is at least as good biochemically and biomechanically as if the growth factors were introduced in the laboratory.

“We want the new cartilage to form in and around the synthetic scaffold at a rate that can match or exceed the scaffold’s degradation,” said Jonathan Brunger, a graduate student who has spent time in both Guilak’s and Gersbach’s laboratories developing and testing the new technique. “So while the stem cells are making new tissue (in the body), the scaffold can withstand the load of the joint. In the ideal case, one would eventually end up with a viable cartilage tissue substitute replacing the synthetic material.”

This particular study examines cartilage regeneration, but Guilak and Gersbach hope that their technique could be applied to the regeneration of many different kinds of tissues, especially orthopaedic tissues such as tendons, ligaments and bones. Also, because the platform comes ready to use with any stem cell, it presents an important step toward commercialization.

“One of the advantages of our method is getting rid of the growth factor delivery, which is expensive and unstable, and replacing it with scaffolding functionalized with the viral gene carrier,” said Gersbach. “The virus-laden scaffolding could be mass-produced and just sitting in a clinic ready to go. We hope this gets us one step closer to a translatable product.”

Citation: “Scaffold-mediated lentiviral transduction for functional tissue engineering of cartilage.” Brunger, J.M., Huynh, N.P.T., Guenther, C.M., Perez-Pinera, P., Moutos, F.T., Sanchez-Adams, J., Gersbach C.A., and Guilak F. PNAS Plus, 2014. DOI: 10.1073/pnas.1321744111/-/DCSupplemental

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.

Silk and Cellulose as Scaffolds for Stem Cell-Mediated Cartilage Repair


When two bones come together, they grind each other into oblivion. This results in inflammation, joint swelling and pain, and scar tissue accumulation, which eventually results in the immobilization of the joint. To prevent this, bone are capped at their ends with a layer of hyaline cartilage that acts as a shock absorber. However, cartilage regenerates poorly and the wear and tear on cartilage, particularly at the knee, causes it to degenerate. The loss of the cartilage cap at the end of long bones causes osteoarthritis . The only way to mitigate the damage of osteoarthritis is to replace the knee with a prosthetic knee-joint.

Stem cells can make a significant contribution to the regeneration of lost cartilage. The Centeno/Schultz group near Denver, Colorado has been using bone marrow-derived mesenchymal stem cells to treat patients for over a decade with positive results. However, finding a way to grow large amounts of cartilage in culture that is the right shape for transplantation has proven difficult.

One way to mitigate this issue is the use of scaffolds for the cartilage-making cells that pushes them into a three-dimensional arrangement that forces them to make cartilage that mimics the cartilage found in a living organism. However a problem with scaffolds is finding the right material for the scaffold.

A recent publication has formed scaffolds from naturally occurring fibers such as cellulose and silk. By blending silk and cellulose fibers together, researchers at the University of Bristol have made a very inexpensive and easily manufactured scaffold for cartilage production.

Silk scaffold
Silk scaffold

When mixed with stem cells, cartilage and silk coax connective tissue-derived stem cells to differentiate into chondrocytes or cartilage-making cells. In the silk/cellulose scaffold, the chondrocytes secrete the extracellular matrix molecules characteristic of joint-specific cartilage.

Wael Kafienah, lead author of this work from the University of Bristol’s School of Cellular and Molecular Medicine, said, “The blend seems to provide complex chemical and mechanical cues that induce stem cell differentiation into preliminary form of chondrocytes without need for biochemical induction using expensive soluble differentiation factors. Kafienah continued: “This new blend can cut the cost for health providers and makes progress towards effective cell-based therapy for cartilage repair a step closer.”

To make the blended silk/cellulose scaffolds, Kafienah and his colleagues used ionic fluids, which effectively dissolve polymers like cellulose and silk, but are also much more environmentally benign in comparison to the organic solvents normally used to process silk and cellulose.

Presently, the U of Bristol team to trying to fabricate three-dimensional scaffolds that can be safely and easily implanted into patients for future clinical studies. Before human clinical studies are commenced, however, they must first be extensively tested in animals and also, the nature of the interactions between the scaffold and the stem cells that drive the cells to form cartilage must be better understood.

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.