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.
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 Med2010; 38: 1857–69) and horses (Wilke MM, et al.,l J Orthop Res2007; 25: 913–2).
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.
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.
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.
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.
Making cartilage from fat-based stem cells would be so much more attractive if we didn’t have to use exogenous sources of growth factors. Nevertheless, fat-based stem cells remain quite attractive as a source of cartilage since these cells can be grown in culture to large numbers and can also be readily differentiated into chondrocytes if they are stimulated with the growth factor transforming growth factor-β1 (TGF-β1). Using exogenous TGF-β1, however, has side undesirable effects. Is there another way?
Maybe. A new study by Loran Solorio and Eben Alsberg at Case Western Reserve University has used a culture medium containing TGF-β1-loaded microspheres to make cartilage from fat-based stem cells in culture. This technique can make cartilage without any exogenous growth factors, since all growth factors required for cartilage production are found within the culture system.
In this study, Solorio and Alsberg used exogenous TGF-β1 to induce cartilage formation in fat-based stem cells that were grown in sheets. These sheets of cells made cartilage after 3 weeks. Once it was clear that their experimental system worked well, they used TGF-β1-loaded gelatin microspheres to deliver the growth factor. By tweaking the quantity of microspheres and the concentration of TGF-β1 required for this to work, Solorio and Alsberg showed that the use of TGF-β1-loaded microspheres could induce cartilage formation as well as exogenous TGF-β1. Staining for cartilage-specific molecules and detailed microscopic observation of the cartilage showed that it was indeed, good, solid cartilage.
This publication is the first demonstration of the self-assembly of fat-derived stem cells into high-density cell sheets capable of forming cartilage in the presence of TGF-β1-releasing microspheres. The incorporation of these microspheres might bypass the need for extended culture of the stem cells, potentially allowing stem cells sheets to be implanted more rapidly into defects to regenerate cartilage in a living organism.
According to a new study published in the January issue of the Journal of Bone and Joint Surgery(JBJS), a single stem cell injection after meniscus knee surgery can provide pain relief and aid in meniscus regrowth.
In the US alone, over one million knee arthroscopy procedures are performed each year. These surgeries are usually prescribed to treat tears to the wedge-shaped piece of cartilage on either side of the knee called the “meniscus.” The meniscus acts as an important shock absorber between the thighbone (femur) and the shinbone (tibia) at the knee-joint.
This novel study, “Adult Human Mesenchymal Stem Cells (MSC) Delivered via Intra-Articular Injection to the Knee, Following Partial Medial Meniscectomy,” examined 55 patients who had undergone a surgical removal or all or part of a torn meniscus (known as a partial medial meniscectomy). Each patient was randomly assigned to one of three treatment groups: Groups A, B and C. The 18 patients in group A received a “low-dose” injection of 50 million stem cells within seven to 10 days after their meniscus surgery. Another 18 patients in group B received a higher dose of 150 million stem cells seven to ten days after their knee surgery. The controls group consisted of 19 patients who received injections of sodium hyaluronate only (no stem cells). All patients were evaluated to determine the safety of the procedure, the degree of meniscus regeneration (i.e. with MRI and X-ray images), the overall condition of the knee-joint, and the clinical outcomes through two years. Most of the patients enrolled in this study had some arthritis, but patients with severe (level three or four) arthritis, were excluded from the study.
Most of the patients who had received stem cell treatments reported a significant reduction in pain. 24 percent of the patients in one MSC group and 6 percent of the other showed at least a 15 percent increase in meniscal volume at one year. Unfortunately, there was no additional increase in meniscal volume at year two.
“The results demonstrated that high doses of mesenchymal stem cells can be safely delivered in a concentrated manner to a knee-joint without abnormal tissue formation,” said lead study author C. Thomas Vangsness, Jr., MD. “No one has ever done that before.” In addition, “the patients with arthritis got strong improvement in pain” and some experienced meniscal regrowth.
The key findings of this study are that there no abnormal (ectopic) tissue formation or “clinically important” safety issues identified. Also, 24 percent of the patients in the low-dose injection group (A) and six percent of the high-dose injection group (B) at one year showed “significantly increased meniscal volume,” as determined by an MRI, and this increase did not continue into the second year, but remained stable (should future studies try a second injection of MSCs?). Third, none of the patients in the control group (non-MSC group) showed significant meniscus regrowth. Finally, patients with osteoarthritis experienced a reduction in pain in the stem cell treatment groups, but there was no reduction in pain in the control (non-MSC group).
“The results of this study suggest that mesenchymal stem cells have the potential to improve the overall condition of the knee joint,” said Dr. Vangsness. “I am very excited and encouraged” by the results. With the success of a single injection, “it begs the question: What if we give a series of injections?”
The biotechnology company InGeneron will test its patented Transpose RT system in a clinical study that examined the ability of regenerative cells from a patient’s own fat to enhance cartilage healing after knee surgery.
Qualified patients are being recruited through the Fondren Orthopedic Group in Houston. According to the American Orthopedic Society for Sports Medicine, over 4 million knee arthroscopies are performed worldwide each year. Damaged knee cartilage is very difficult to treat and can lead to chronic pain and long-term disability.
Robert Burke, who is serving as the principal investigator of this clinical study, is an orthopedic surgeon with the Fondren Orthopedic Group in Houston. Burke thinks that stem cells taken from a patient’s own fat may enhance cartilage healing. He studied adding patient-derived regenerative cells to the knee during arthroscopic surgery for particular patients, and compared them to patients who had arthroscopic surgery without added fat-derived stem cells.
Arthroscopic surgery is a common procedure is commonly used to treat damaged cartilage, and the patients who had received arthroscopic surgery were randomly chose to either receive fat-derived stem cells or not receive them. Burke, will then monitor these patients for the next 12 months after surgery to determine if the added cells improved cartilage healing.
According to Burke, “Articular cartilage, the smooth surface covering the joints at the ends of bones, has no good way of healing on its own. The body doesn’t create enough new cartilage of the same type to repair the damage.” Better treatments would use various techniques to help the body make new cartilage.
“Stem cells and other regenerative cells that we can obtain fat have the potential to do that,” said Burke. Such regenerative cells can divide and mature to form several types of cells and tissues. and are found in multiple places in the body. Fat that lies just below the skin is one of the easiest places to obtain stem cells.
The InGeneron Transpose RT System uses a small amount of fat, which is removed and processed to separate out the regenerative cells. The separated adipose tissue-derived mesenchymal stem cells are then immediately placed into the area of damaged cartilage during knee surgery. Once in the knee, these cells may divide to make new cartilage cells.
This kind of biological activity has been seen in laboratory studies and veterinary medicine. However, Burke’s study will be one of the first to test the technology in humans for treating cartilage damage. Like other types of stem cell-based therapies, the treatment is not currently licensed for human use in the United States but it is registered in Europe, Mexico, and other countries. Following the Texas Medical Board’s rules about the use of stem cells for treatment, this study is under the supervision of the research review board at Texas Orthopedic Hospital, where all of the patients will undergo surgeries.
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).
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.”
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.
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.
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.