Previous studies have shown that human bone marrow–derived mesenchymal stromal cells have potential to accelerate and augment wound healing. However, in the clinic, it is difficult to properly culture and then use bone marrow stem cells. Human umbilical cord blood–derived mesenchymal stromal cells (hUCB-MSCs) recently have been commercialized for cartilage repair as a cell-based therapy product that uses allogeneic stem cells.
Presently, current cell therapy products for wound healing utilize fibroblasts. Is it possible that hUCB-MSCs are superior to fibroblasts for wound healing? Seung-Kyu Han and his colleagues from the Department of Plastic Surgery at the Korea University College of Medicine in Seoul, South Korea used a cell culture system to compare the ability of hUCB-MSCs and fibroblasts to heal wounds.
For their study, Han and others used diabetic mice and isolated fibroblasts from normal and diabetic mice. Then they tested the ability of these cells to heal skin wounds in the very mice from which they were isolated. A third group of diabetic mice with skin wounds were treated with hUCB-MSCs. A comparison of all three groups examined the cell proliferation, collagen synthesis and growth factor (basic fibroblast growth factor, vascular endothelial growth factor and transforming growth factor-β) production and compared them among the three groups.
The results showed that hUCB-MSCs produced significantly higher amounts of vascular endothelial growth factor and basic fibroblast growth factor in comparison to both fibroblast groups. Human UCB-MSCs were better than diabetic fibroblasts but healthy fibroblasts in collagen synthesis, and there were no significant differences in cell proliferation and transforming growth factor-β production. Human UCB-MSCs produced significantly higher amounts of VEGF and bFGF when compared with both fibroblasts.
These results suggest that Human UCB-MSCs might be a better source for diabetic wound healing than either allogeneic or autologous fibroblasts. Larger animal studies will be needed, but this particular study seems like a good start.
Within the knee-joint, on either side, is a cartilage shock absorber called the meniscus. Tears to this structure can cause pain and swelling in the knee and erosion of the meniscus can lead to bone-on-bone joints that abrade the bone and cause further inflammation and osteoarthritis. Because the meniscus is made of cartilage, and since cartilage can be grown in the laboratory, it should be possible, in theory, to make a new meniscus. Researchers at Columbia University Medical Center have succeeded in using 3-D printing to do that just.
The laboratory of Jeremy Mao used made personalized 3-D implants made from a scaffold infused with human growth factors. When implanted into the knee, these growth factors stimulate the body to regenerate the meniscus on its own. Mao and his coworkers successfully tested their treatment strategy in sheep. Their procedure could provide the first effective and long-lasting way to repair of damaged menisci, which occur in millions of Americans each year and can lead to debilitating arthritis. This work from the Mao lab was published in Science Translational Medicine.
“At present, there’s little that orthopedists can do to regenerate a torn knee meniscus,” said Mao, who is the Edwin S. Robinson Professor of Dentistry (in Orthopedic Surgery) at the Medical Center. “Some small tears can be sewn back in place, but larger tears have to be surgically removed. While removal helps reduce pain and swelling, it leaves the knee without the natural shock absorber between the femur and tibia, which greatly increases the risk of arthritis.”
Heavily damaged menisci can be replaced with a meniscal transplant that utilizes tissue from other parts of the body or from cadavers. Such transplants, however, have a low success rate and carries significant risks. Approximately one million meniscus surgeries are performed in the United States each year.
Mao and his colleagues began with MRI scans of the intact meniscus in the undamaged knee. Special computer software then converts these high-resolution scans in to a 3D image. Data from these images are then used to drive a 3D printer, which produces a scaffold in the exact shape of the meniscus, all the way down to a resolution of 10 microns, which is less than the width of a human hair. The scaffold takes about 30 minutes to print and is made from an organic polymer called polycaprolactone, which is the same biodegradable polymer used to make surgical sutures.
The printed scaffold is infused with two recombinant human proteins: connective growth factor (CTGF) and transforming growth factor β3 (TGFβ3). In earlier work, Mao’s team discovered that sequential delivery of these two proteins attracts resident stem cells from the body and induces them to form meniscal tissue.
In order for a meniscus to properly form, these growth factors must be released from specific areas of the scaffold and in a specific order. To accomplish this, the growth factors were encapsulated in two types of slow-dissolving polymeric microspheres. The first of these microspheres released CTGF, which stimulates the production of the outer meniscus. The second microspheres release TGFβ3, which induces the production of the inner meniscus. Finally, this protein-infused scaffold is inserted into the knee so that it can direct the generation of a new meniscus. When these printed, growth factor-infused scaffolds were implanted into the knees of sheep, the meniscus regenerated in approximately four to six weeks. The implanted, biodegradable scaffold eventually disintegrates.
“This is a departure from classic tissue engineering, in which stems cells are harvested from the body, manipulated in the laboratory, and then returned to the patient—an approach that has met with limited success,” said Mao. “In contrast, we’re jumpstarting the process within the body, using factors that promote endogenous stem cells for tissue regeneration.”
“This research, although preliminary, demonstrates the potential for an innovative approach to meniscus regeneration,” said co-author Scott Rodeo, sports medicine orthopedic surgeon and researcher at Hospital for Special Surgery in New York City. “This would potentially be applicable to the many patients who undergo meniscus removal each year.”
Mao and others tested their procedure in 11 sheep. Even though they are four-legged creatures, sheep knees closely resemble that of humans, and therefore, as an excellent model system for orthopedic research. These animals were randomized to have part of their knee meniscus replaced with a protein-infused 3D scaffold (the treatment group) or a 3D scaffold that was not infused with growth factors (the nontreatment group). After three months, the treated animals all walked normally. A postmortem analysis of the treated animals demonstrated that the regenerated meniscus in the treatment group had structural and mechanical properties very similar to those of natural meniscus. Mao’s laboratory is now conducting studies to determine whether the regenerated tissue is long-lasting.
“We envision that personalized meniscus scaffolds, from initial MRI to 3D printing, could be completed within days,” said Mao. The personalized scaffolds will then be shipped to clinics and hospitals within a week. The researchers hope to begin clinical trials once funding is in place.
“These studies provide clinically valuable information on the use of meniscal regeneration in the knees of patients with torn or degenerate menisci,” said co-author Lisa Ann Fortier, professor of large animal surgery at Cornell University College of Veterinary Medicine in Ithaca, N.Y. “As a veterinary orthopedic surgeon-scientist on this multi-disciplinary team, I foresee the added bonus of having new techniques for treating veterinary patients with torn knee meniscus.”
A truly remarkable paper in the journal Advanced Healthcare Materials by Fergal J. O’Brien and his co-workers from the Tissue Engineering Research Group at the Royal College of Surgeons in Dublin, Ireland has examined a unique way to greatly speed up bone regeneration.
Mesenchymal stem cells from bone marrow (other locations as well) can differentiate into bone-making cells (osteoblasts) that will make architecturally normal bone under particular conditions. The use of mesenchymal stem cells and a variety of manufactured biomaterial matrices and administered growth factors enhance bone formation by mesenchymal stem cells (M. Noelle Knight and Kurt D. Hankenson, Adv Wound Care 2013; 2(6): 306–316; also see Marx RE, Harrell DB. Int J Oral Maxillofac Implants 2014 29(2)e201-9; and Kaigler D, et al., Cell Transplant 2013;22(5):767-77).
Protein growth factors tend to have rather short half-lives when applied to growth scaffolds. A better way to apply growth factors is to use the genes for these growth factors and apply them to “gene activated scaffolds.” Gene-activated scaffolds consist of biomaterial scaffolds modified to act as depots for gene delivery while simultaneously offering structural support and a matrix for new tissue deposition. A gene-activated scaffold can therefore induce the body’s own cells to steadily produce specific proteins providing a much more efficient alternative.
In this paper by O’Brien and his groups, the genes for two growth factors, VEGF and BMP2, were applied to a gene-activated scaffold that consisted of collagen-nanohydroxyapatite. VEGF drives the formation of new blood vessels, and this fresh vascularization, coupled with increase bone deposition, which is induced by BMP2, accelerated bone repair.
Mind you, the assays in the paper were conducted in cell culture systems. However, O’Brien and his colleagues implanted these gene-activated scaffolds with their mesenchymal stem cells into rats that had large gaps in their skulls. In this animal model system for bone repair, stem cell-mediated bone production, in addition to increased blood vessel formation accelerated bone repair in these animals. Tissue examinations of the newly-formed bone showed that bone made from gene-activated scaffolds with mesenchymal stem cells embedded in them made thicker, more vascularized bone than the other types of strategies.
This is not a clinical trial, but this preclinical trial shows that vascularization and bone repair by host cells is enhanced by the use of nanohydroxyapatite vectors to deliver a combination of genes, thus markedly enhancing bone healing.
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.
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.”
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
The regenerative medicine company CardioCell LLC has announced two new clinical trials in two different countries that utilize its allogeneic stem-cell therapy to treat subjects with acute myocardial infarction (AMI), which is a problem that faces more than 1.26 million Americans annually. The United States-based trial is a Phase IIa AMI clinical trial that is designed to evaluate the clinical safety and efficacy of the CardioCell Ischemia-Tolerant Mesenchymal Stem Cells or itMSCs. The second clinical trial in collaboration with the Ministry of Health in Kazakhstan is a Phase III AMI clinical trial on the intravenous administration of CardioCell’s itMSCs. This clinical trial is proceeding on the strength of the efficacy and safety of itMSCs showed in previous Phase II clinical trials.
CardioCell’s itMSCs are exclusively licensed from CardioCell’s parent company Stemedica Cell Technologies Inc. Normally, when mesenchymal stem cells from fat, bone marrow, or some other tissue source are grown in the laboratory, the cells are provided with normal concentrations of oxygen. However, CardioCell itMSCs are grown under low oxygen or hypoxic conditions. Such growth conditions more closely mimic the environment in which these stem cells normally live in the body. By growing these MSCs under these low-oxygen conditions, the cells become tolerant to low-oxygen conditions (ischemia-tolerant), and if transplanted into other low-oxygen environments, they will flourish rather than die.
Another advantage of itMSCs for regenerative treatments over other types of MSCs is that itMSCs secrete higher levels of growth factors that induce the formation of new blood vessels and promote tissue healing. These clinical trials have been designed to help determine if CardioCell’s itMSC-based therapies stimulate a regenerative response in acute heart attack patients.
“CardioCell’s new Phase IIa AMI study is built on the excellent safety data reported in previous Phase I clinical trials using our unique, hypoxically grown stem cells,” says Dr. Sergey Sikora, Ph.D., CardioCell’s president and CEO. “We are also pleased to report that the Ministry of Health in Kazakhstan is proceeding with a Phase III CardioCell-therapy study following its Phase II study that was highly promising in terms of efficacy and safety. Our studies target AMI patients who have depressed left ventricular ejection fraction (LVEF), which makes them prone to developing extensive scarring and therefore to the development of chronic heart failure. CardioCell hopes our itMSC therapies will inhibit the development of extensive scarring and, thus, the occurrence of chronic heart failure in these patients.”
The United States-based Phase IIa clinical trial will take place at Emory University, Sanford Health and Mercy Gilbert Medical Center. The CardioCell Phase IIa AMI trial is a double-blinded, multicenter, randomized study designed to assess the safety, tolerability and preliminary clinical efficacy of a single, intravenous dose of allogeneic mesenchymal bone-marrow cells infused into subjects with ST segment-elevation myocardial infarction (STEMI).
“While stem-cell therapy for cardiovascular disease is nothing new, CardioCell is bringing to the field a new, unique type of stem-cell technology that has the possibility of being more effective than other AMI treatments,” says MedStar Heart Institute’s Director of Translational and Vascular Biology Research and CardioCell’s Scientific Advisory Board Chair Dr. Stephen Epstein. “Evidence exists demonstrating that MSCs grown under hypoxic conditions express higher levels of molecules associated with angiogenesis and healing processes. There is also evidence indicating they migrate with greater avidity to various cytokines and growth factors and, most importantly, home more robustly to ischemic tissue. Studies like those underway using CardioCell’s technology are designed to determine if we can evoke a more potent healing response that will reduce the extent of myocardial cell death occurring during AMI and thereby decrease the amount of scar tissue resulting from the infarct. A therapy that could achieve this would have a major beneficial impact in reducing the occurrence of chronic heart failure.”
Kazakhstan’s National Scientific Medical Center is conducting a Phase III AMI clinical trial using CardioCell’s itMSCs, which are sponsored by local licensee Altaco. This clinical trial is entitled, “Intravenous Administration of itMSCs for AMI Patients,” and is proceeding based on a completed Phase II efficacy and safety study. However, the results of this previous Phase II study are preliminary because the sample group was so small. Despite these limitations, the findings demonstrated statistically significant elevation (more than 12 percent over the control group) in the ejection fraction of the left ventricle of the heart in patients who had received itMSCs. Also, a significant reduction in inflammation was also observed, as ascertained by lower CRP (C-reactive protein) levels in the blood of treated patients in comparison to control groups. Thus, Dr. Daniyar Jumaniyazov, M.D., Ph.D., principal investigator in Kazakhstan clinical trials states: “In our clinical Phase II trial for patients with AMI, treatment using itMSCs improved global and local myocardial function and normalized systolic and diastolic left ventricular filling, as compared to the control group. We are encouraged by these results and look forward to confirming them in a Phase III study.”
CardioCell’s treatment is the first to apply itMSC therapies for cardiovascular indications like AMI, chronic heart failure and peripheral artery disease. Manufactured by CardioCell’s parent company Stemedica and approved for use in clinical trials, itMSCs are manufactured under Stemedica’s patented, continuous-low-oxygen conditions and proprietary media, which provide itMSCs’ unique benefits: increased potency, safety and scalability. itMSCs differ from competing MSCs in two key areas. itMSCs demonstrate increased migratory ability towards the place of injury, and they show increased secretion of growth and transcription factors (e.g., VEGF, FGF and HIF-1), as demonstrated in a peer-reviewed publication (Vertelov et al., 2013). This can potentially lead to improved regenerative abilities of itMSCs. In addition, itMSCs have significantly fewer HLA-DR receptors on the cell surface than normal MSCs, which might reduce the propensity to cause immune responses. As another benefit, itMSCs are highly scalable. A single donor specimen can currently yield about 1 million patient treatments, and this number is expected to grow to 10 million once full robotization of Stemedica’s facility is complete.
Using stem cells to grow three-dimensional structures, such as organs or damaged body parts, requires that scientists have the ability to control the growth and behavior of those cells. Also, adapting such a technology to an off-the-shelf kind of process so that it does not cost an arm and a leg is also important.
A research project by scientists from Atlanta, Georgia has used gelatin-based microparticles to deliver growth factors to specific areas of aggregates of stem cells that are differentiating. This localized delivery of growth factors provides spatial control of cell differentiation, which enables the creation of complex, three-dimensional tissues. The local delivery of growth factors also decreases the amount of growth factor used and, consequently, the cost of the procedure.
This particular microparticle technique was used on mouse embryonic stem cells and it proved to provide better control over the kinetics of cell differentiation since it delivered that promote cell differentiation or inhibit it.
Todd McDevitt, associate professor in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University, said, “By trapping these growth factors within microparticle materials first, we are concentrating the signal they provide to the stem cells. We can then put the microparticle materials physically inside the multicellular aggregate system that we use for differentiation for the stem cells. We have good evidence that this technique can work, and that we can use it to provide advantages in several areas.”
The differentiation of stem cells is largely controlled by external cues, including protein growth factors that direct cell proliferation, and differentiation that are available in the three-dimensional environment in which the cells live. In most experiments, stem cells are grown in liquid culture and growth factor is equally accessible to the growth factors. This makes the cultures quite homogeneous. But delivering the growth factors via microparticles gives better control of the spatial and temporal presentation of these growth factors to the stem cells. This gives scientists the means to make heterogeneous structures from stem cell cultures.
When embryonic stem cells grow in culture, they tend to clump together. When the growth medium is withdrawn or if growth factors that induce differentiation are added, the cells form an “embryoid body” that is stuff with cells differentiating into all kinds of cell types. When McDevitt and his co-workers added microparticles with the growth factors BMP4 (bone morphogen protein 4) or Noggin (which inhibits BMP4 signaling), they centrifuged the cells and found that the microparticles found their way into the interior of the embryoid bodies.
When they examined the embryoid bodies, with confocal microscopy they found that BMP4 directed the cells to make mesodermal and endodermal derived cell types. However, because the microparticles were in direct contact with the cells, they needed 12 times less growth factor than was required by solution-based techniques.
“One of the major , in a practical sense, is that we are using much less growth factor,” said McDevitt. “From a bioprocessing standpoint, a lot of the cost involved in making stem cell products is related to the cost of the molecules that must be added to make the stem cells differentiate.”
Beyond more focuses signaling, the microparticles also provided localized control that was not available through other techniques. It allowed researchers to create spatial differences in the aggregates and this is an important possible first step toward forming more complex structures with different tissue types such as vascularization and stromal cells.
“To build tissues, we need to be able to take stem cells and use them to make many cell types which are grouped together in particular spatial patterns,” explained Andres M. Bratt-Leal, the paper’s first author and a former graduate student in McDevitt’s lab. “This spatial patterning is what gives the ability to perform higher order functions.”
Once the stem cell aggregates were made and treated with growth factor-endowed microparticles, McDevitt and his colleagues saw spheres of cells with differentiating cells.
“We can see the microparticles had effects on one population that were different from the population that didn’t have the particles,” said McDevitt. “This may allow us to emulate aspects of how development occurs. We can ask questions about how tissues are naturally patterned. With this material incorporation we have the ability to better control the environment in which these cells develop.”
The microparticles could provide better control over the kinetics of cell differentiation; slowing it down with molecules that antagonize differentiation or speed up with other molecules that promote stem cell differentiation.
Despite the fact that McDevitt and his colleagues used mouse embryonic stem cells in this paper, he and his co-workers are already testing this technology on human embryonic stem cells, and the results have been comparable.
“Our findings will provide a significant new tool for tissue engineering, bioprocessing of stem cells and for better studying early development processes such as axis formation in embryos,” said Bratt-Leal. “During development, particular tissues are formed by gradients of signaling molecules. We can now better mimic these signal gradients using our system.”