Nanoscale Scaffolds and Stem Cells Show Promise For Cartilage Repair

Tissue engineers have designed scaffolds for stem cells made from nanotubes that induce them to form cartilage. These nanofibers are tiny, artificial fiber scaffolds that are thousands of times smaller than a human hair. Cartilage formation has succeeded in laboratory and animal model systems.

Much more work is necessary before these scaffolds can be used inside a human body; the results of this present study hold promise for designing new techniques to help millions who endure joint pain.

Jennifer Elisseeff, Ph.D., Jules Stein Professor of Ophthalmology and director of the Translational Tissue Engineering Center at the Johns Hopkins University School of Medicine, said: “Joint pain affects the quality of life of millions of people. Rather than just patching the problem with short-term fixes, like surgical procedures such as microfracture, we’re building a temporary template that mimics the cartilage cell’s natural environment, and taking advantage of nature’s signals to biologically repair cartilage damage,”

Unfortunately, cartilage does not repair itself when damaged. For the last decade, Elisseeff’s research team has been trying to better understand the development and growth of cartilage cells (chondrocytes). Part of these studies has involved building scaffolds that mimics the environment inside the body that produces new cartilage tissue. The cartilage-making environment consists of a three-dimensional mix of protein fibers and gel. This matrix provides support to connective tissue throughout the body, and physical and biological cues for cells to grow and differentiate.

In the laboratory, the Elisseeff team created a nanofiber-based network that utilized a process called electrospinning. Electrospinning shoots a polymer stream onto a charged platform, to which a compound called chondroitin sulfate is added. Chondroitin sulfate is commonly found in many joint supplements. After characterizing the manufactured fibers, they made several different scaffolds from spun polymer or spun polymer plus chondroitin sulfate. Elisseeff and her colleagues then seeded the scaffolds with bone marrow stem cells from goats in order to test how well the scaffolds supported the growth of the stem cells.

The results showed that the scaffold-supported stem cells formed more voluminous, cartilage-like tissues that those grown without the manufactured scaffolds.

“The nanofibers provided a platform where a larger volume of tissue could be produced,” said Elisseeff, adding that 3-dimensional nanofiber scaffolds were more useful than the more common nanofiber sheets for studying cartilage defects in humans.

Next, Elisseeff and her group tested the ability of these nanotube scaffolds and the cartilage they produce in an animal model. They implanted the nanofiber scaffolds into damaged cartilage in the knees of rats. They compared the quality of the knee repair to damaged cartilage in knees that were not treated with any stem cells. The nanofiber scaffolds improved tissue development and repair. The nanofiber-implanted knees produced far more cartilage as measured by the production of collagen, which is a component of cartilage. The nanofiber scaffolds induced the production of larger quantities of a more durable type of collagen, which is typically absent in surgically repaired cartilage tissue. In rats, the limbs with damaged cartilage treated with nanofiber scaffolds generated a higher percentage of the more durable collagen (type 2) than those damaged areas that were left untreated.

“Whereas scaffolds are generally pretty good at regenerating cartilage protein components in cartilage repair, there is often a lot of scar tissue-related type 1 collagen produced, which isn’t as strong,” said Elisseeff. “We found that our system generated more type 2 collagen, which ensures that cartilage lasts longer. Creating a nanofiber network that enables us to more equally distribute cells and more closely mirror the actual cartilage extracellular environment are important advances in our work and in the field. These results are very promising.”

Platelet-Lysate Bioactive Scafold for Tissue Engineered Cartilage

Cartilage replacement at joints represents a tremendous challenge for regenerative medicine. While growing cartilage in culture is possible, scaling this technology up to generate enough high-quality articular cartilage (the kind of cartilage found at joints), is still a distinct challenge. To date, stem cell treatments can heal small breaches in cartilage, but reconstructing large lesions is still not possible. In general, cartilage at joints has very poor healing properties, and therefore, is a major challenge in orthopedics.

A major improvement in therapeutics is the use of a technique called “autologous chondrocyte implantation” or ACI. ACI involves the delivery of healthy cartilage-making cells (chondrocytes) from the patient’s own body after these cells have been grown and expanded in culture. In order to coax these cartilage-making cells to make cartilage, special scaffolds are used that provide a three-dimensional matrix upon which the chrondrocytes grow and form cartilage. These 3-D scaffolds are essential to keep the chondrocytes differentiated and making cartilage.

One of the most promising types of scaffolds for making cartilage are “bioactive 3D scaffolds.” These types of scaffolds can deliver growth factors and other molecules to the chrondrocytes and boost their growth and cartilage production.

In a recent publication, Andrei Moroz and colleagues in the Extracellular Matrix Laboratory at the Botucatu Institute of Biosciences, São Paulo State University, Brazil, have used mesenchymal stem cells (MSCs) from rabbit bone marrow and differentiated them into chondrocytes. This allowed them to use stem cells from bone marrow instead of harvesting cartilage from the joints, which can be very painful and deleterious to the joint. The main innovation in this paper was the use of a platelet-lysate-based 3D bioactive scaffold to support the chondrogenic differentiation and maintenance of MSCs.

MSCs from adult rabbit bone marrow were isolated, characterized, and grown in 60 microliters of platelet lysate from rabbit blood. Platelets are very small cells from circulating blood that assist in the formation of clots that staunch bleeding after a blood vessel in damaged. Platelets are easy to isolate from circulating blood and the rabbit platelet-lysate clot scaffold was maintained is a standard tissue culture medium (Dulbecco’s Modified Eagle Medium Nutrient Mixture F-12) that was supplemented with other molecules known to induce cartilage formation in MSCs. After three weeks in culture, the MSCs were examined in detail. Not only were they nice and round, but they were filled with cartilage-specific molecules, and clumped together like chondrocytes.

According to this research group, they are on to something with this platelet-lysate bioactive scaffold. It provided a suitable system for culturing MSCs and allowed them to make lots of cartilage. The scaffold also was easy to make, and maintained the MSCs in a cartilage-making state without causing cell death or stressing the cells. Therefore, it might provide an alternative to autologous chondrocyte implantation. The next steps in this research will be to use this engineered cartilage to repair damaged joints to see if the cartilage made by cells embedded in platelet-lysate 3D bioactive scaffolds can act as functional cartilage.

For the article see Andrei Moroz, et al., Platelet lysate 3D scaffold supports mesenchymal stem cell chondrogenesis: An improved approach in cartilage tissue engineering.  Platelets. 2012.

Induced Pluripotent Stem Cells Repair Spinal Cord Injury in Mice

Induced pluripotent stem cells (iPSCs), are made from adult cells by means of genetic engineering and share many, though not all, of the characteristics of embryonic stem cells. The regenerative capacity of iPSCs is truly remarkable, but there are definitely safety concerns with them. The procedure that makes iPSCs from adult cells drives them to divide quickly and often. These cell divisions produce high rates of genetic mutations, some of which are of little consequence, and others that are. Also prolonged culture of iPSCs can select for cells that acquire cancer-causing mutations. Laboratory tests have established that iPSCs have a capability to cause tumors in laboratory animals that at least equals that of embryonic stem cells.

Nevertheless, some labs have designed protocols to screen iPSC lines for tumor-causing or non-tumor-causing lines. Also, iPSCs have been successfully used in therapeutic experiments in laboratory animals without generating tumors. Therefore, iPSCs might be closer to therapeutic use than we think.

With this comes a fascinating publication from the Laboratory of Molecular Neuroscience in the Graduate School of Biological Sciences at the Nara Institute of Science and Technology in Ikoma, Japan; specifically from the laboratory of Kinichi Nakashima. In this experiment, workers in Nakashima’s laboratory used iPSCs that were made from mouse adult cells to make neural stem cells (NSCs).

NSCs are found in the central nervous system and they replace cells in the central nervous system or augment the central nervous system in response to learning and memory or things like that. NSCs are not a monolithic cell population, since some NSCs have the ability to make specific populations of neurons (the cells responsible for neural impulses), while others form glial cells (the cells that support and maintain the neurons).

Nakashima’s laboratory has designed a highly efficient protocol for converting iPSCs into NSCs. They predicted that these NSCs would represent a much less mixed population. Nakashima surmised that such NSCs would almost certainly do a better job of repairing a spinal cord injury. Therefore, led by Yusuke Fujimoto, his colleagues produced several iPSC lines and converted them into NSCs. They called these cell lines “neuroepithelial-like stem cells from human iPS cells” or hiPS-lt-NES cells.

Characterization of these cells in culture showed that they were a homogeneous population that differentiated into many different types of spinal-specific neurons and glial cells. Next, as predicted by Nakashima, Fujimoto and his colleagues transplanted these hiPS-lt-NES cells into the spinal cords of mice that had suffered spinal cord injuries.

The results were remarkable. The transplanted hiPS-lt-NES cells differentiated into neural cells in the spinal cord and promoted functional recovery of hind limb motor function. This is a remarkable finding, but perhaps the transplanted cells only secreted growth factors that helped heal the spine and played no real role in regenerating the spinal cord. Nakashima was not satisfied with this result.

To determine if the transplanted cells were actually regenerating the spinal cord, Fujimoto and the rest of his laboratory workers used two different tracers and also killed off the transplanted cells. The nerve cell tracers showed that the transplanted cells and nerve cells that were already in the spinal cord formed the new neural networks and connections to restore normal hind limb function. Neurons native to the spinal cord and the newly introduced neurons hat were formed from transplanted hiPS-lt-NES cells reconstructed the corticospinal tract by forming proper connections with other neurons and integrating neuronal circuits. Then, when they deliberately killed off the transplanted cells, no neural regeneration occurred. Thus the transplanted hiPS-lt-NES cells not only contributed to the regeneration of the spinal cord and its neural circuits, but they initiated and drove the process.

These fascinating findings suggest a new way to treat spinal cord injury and it does not require the killing of embryos.