Cell-Free Bioactive Scaffold Supports Cartilage Regeneration in Large Animal Joints

Northwestern University scientists have developed a cell-free bioactive material comprising a complex network of molecular components that work together as a scaffold to mimic cartilage’s natural environment in the body. In a newly reported study, the team used the material to successfully support high-quality cartilage regeneration in the knee joints of sheep. After applying the bioactive hybrid scaffold material to the animals’ damaged knee joint cartilage, the researchers observed evidence of enhanced repair within six months, including the growth of new cartilage containing the natural biopolymers (collagen II and proteoglycans), which enable pain-free mechanical resilience in joints.

The team suggests that with more work the new material could potentially in future be used to prevent full knee replacement surgeries, treat degenerative diseases like osteoarthritis, and repair sports-related injuries such as anterior cruciate ligament (ACL) tears.

“Cartilage is a critical component in our joints,” said Northwestern’s Samuel I. Stupp, PhD, who led the study. “When cartilage becomes damaged or breaks down over time it can have a great impact on people’s overall health and mobility. The problem is that, in adult humans, cartilage does not have an inherent ability to heal. Our new therapy can induce repair in a tissue that does not naturally regenerate. We think our treatment could help address a serious, unmet clinical need.”

A pioneer of regenerative nanomedicine, Stupp is a Board of Trustees Professor of Materials Science and Engineering, Chemistry, Medicine and Biomedical Engineering at Northwestern, where he is the founding director of the Simpson Querrey Institute for BioNanotechnology and its affiliated center, the Center for Regenerative Nanomedicine. Stupp has appointments in the McCormick School of Engineering, Weinberg College of Arts and Sciences, and Feinberg School of Medicine. Jacob Lewis, PhD, a former student in Stupp’s laboratory, is the paper’s first author.

Stupp, together with Lewis, and colleagues reported in PNAS on their study in the large animal model. Their paper is titled, “A bioactive supramolecular and covalent polymer scaffold for cartilage repair in a sheep model.” In their report the scientists concluded, “These results demonstrate the potential of the hybrid biomimetic scaffold as a niche to favor cartilage repair in mechanically active joints using a clinically relevant large-animal model.”

Developing scaffolds that support growth of articular cartilage is an important therapeutic goal since this tissue lacks intrinsic ability to regenerate in adult humans, the authors explained. “Regeneration of hyaline cartilage in human-sized joints remains a clinical challenge, and it is a critical unmet need that would contribute to longer healthspans.” However, they pointed out, experimental scaffolds often fail in large-animal preclinical models possibly due to the high mechanical forces encountered. “Injectable scaffolds for cartilage repair that integrate both bioactivity and sufficiently robust physical properties to withstand joint stresses offer a promising strategy.”

The newly reported study follows recently published work from the Stupp laboratory in which the team used “dancing molecules” to activate human cartilage cells to boost the production of proteins that build the tissue matrix. Instead of using dancing molecules, the new study evaluated a hybrid biomaterial also developed in Stupp’s lab. The new biomaterial comprises two components, one a bioactive peptide—a bioactive peptide amphiphile (PA) supramolecular polymer—that binds to transforming growth factor beta-1 (TGFβ-1), which is an essential protein for cartilage growth and maintenance. The second component is a modified hyaluronic acid (HA)—a natural polysaccharide present in cartilage and the lubricating synovial fluid in joints.

“Many people are familiar with hyaluronic acid because it’s a popular ingredient in skincare products,” Stupp said. “It’s also naturally found in many tissues throughout the human body, including the joints and brain. We chose it because it resembles the natural polymers found in cartilage.”

Stupp’s team integrated the bioactive peptide and chemically modified hyaluronic acid particles to drive the self-organization of nanoscale fibers into bundles that mimic the natural architecture of cartilage. “We report here on a hybrid biomaterial that combines a bioactive peptide amphiphile supramolecular polymer that specifically binds the chondrogenic cytokine transforming growth factor β-1 (TGFβ-1) and crosslinked hyaluronic acid microgels that drive formation of filament bundles, a hierarchical motif common in natural musculoskeletal tissues,” they stated.

The goal was to create an attractive scaffold for the body’s own cells to regenerate cartilage tissue. Using bioactive signals in the nanoscale fibers, the material encourages cartilage repair by the cells, which populate the scaffold. “Importantly, the material we report is fully injectable and shapable, unlike other high-toughness materials for cartilage repair reported previously,” the investigators added.

To evaluate the material’s effectiveness in promoting cartilage growth the researchers tested it in sheep with cartilage defects in the stifle joint, a complex joint in the hind limbs that is similar to the human knee. This work was carried out in the laboratory of Mark Markel, PhD, in the School of Veterinary Medicine at the University of Wisconsin–Madison.

According to Stupp, testing in a sheep model was vital. Much like humans, sheep cartilage is stubborn and incredibly difficult to regenerate. Sheep stifles and human knees also have similarities in weight bearing, size, and mechanical loads. “A study on a sheep model is more predictive of how the treatment will work in humans,” Stupp said. “In other smaller animals, cartilage regeneration occurs much more readily.”

For their study, the researchers injected the thick, paste-like material into cartilage defects, where it transformed into a rubbery matrix. Not only did new cartilage grow to fill the defect as the scaffold degraded, but in comparison with controls, the repaired tissue was consistently of higher quality. “These injectable scaffolds showed improved mechanical resilience and formed bundled supramolecular filaments, a structural motif observed in native cartilage,” the team continued. “We found that the hybrid scaffold placed in osteochondral defects in sheep—an animal model whose joints experience similar loading to humans—induced repair of hyaline cartilage in mechanically loaded joint surfaces.”

In the future, Stupp imagines the new material could be applied to joints during open-joint or arthroscopic surgeries. The current standard of care is microfracture surgery, during which surgeons create tiny fractures in the underlying bone to induce new cartilage growth.

“The main issue with the microfracture approach is that it often results in the formation of fibrocartilage—the same cartilage in our ears—as opposed to hyaline cartilage, which is the one we need to have functional joints,” Stupp said. “By regenerating hyaline cartilage, our approach should be more resistant to wear and tear, fixing the problem of poor mobility and joint pain for the long term while also avoiding the need for joint reconstruction with large pieces of hardware.”

The team suggests that their system could also be used to deliver additional growth factors as well as TGFβ-1. “This could be achieved by including additional binding peptides or by including agonist peptides that mimic other growth factors in order to further enhance chondrogenic bioactivity,” they wrote. “Given that the PA/HA scaffold investigated here is injectable and cell-free, but still capable of enhancing cartilage repair under high-mechanical loads, we envision using supramolecular and covalent polymer hybrids to arthroscopically deliver bioactive PA-based systems for regeneration in mechanically challenging joints.”