OREF grant recipient explores articular cartilage 3D architecture in stem cell preparations as potential cartilage grafts
Current surgical interventions to repair articular cartilage often result in less than optimal outcomes because the new cartilage does not sufficiently replicate both the structural and functional properties of healthy tissue. The repairs generally fail to restore the gradient composition of the osteochondral tissue. That gradient runs from the smooth surface layer of cartilage, to the transitional layer of calcified cartilage, and into the subchondral bone.

To address this problem, Jeffrey T. Spang, MD, associate professor of orthopaedics at the University of North Carolina, Chapel Hill, is investigating a strategy to graft stem cells into damaged tissues. This grafting might guide cell differentiation, thereby promoting a more complete osteochondral tissue repair.

Dr. Spang explained, “Our treatment of articular cartilage injuries could be greatly improved. Currently, we don’t recreate the body’s natural function very well. Our scaffold and stem cell research is one step toward building tissue that does replicate that function.”

Dr. Spang’s research is supported by a 2015 Orthopaedic Research and Education Foundation/National Stem Cell Foundation (OREF/NSCF) Career Development Grant in Stem Cells and Regenerative Medicine.

How to train a stem cell
In preliminary research, Dr. Spang and his colleagues found that when stem cells were cultured in a cartilage-promoting medium, the introduction of extracellular calcium induced osteogenesis and inhibited chondrogenesis. The controlled levels of extracellular calcium essentially guided stem cell differentiation. The absence of calcium promoted cartilage growth.

In their investigations, the researchers chose adipose-derived stem cells because in a clinical application, the cells would be relatively easy to harvest from a patient’s own fat tissue. In the OREF/NSCF-funded study, the researchers conducted an in vitro experiment with both human and porcine adipose-derived stem cells. In the final year of the grant, the researchers will conduct an in vivo study in a pig model of articular cartilage damage.

One aim of the study is to use 3D-printing technology to create micro-porous 3D scaffolds. The scaffold acts as a framework to hold and organize stem cells. The scaffold is biodegradable and designed to be replaced by the extracellular matrix as tissue develops. The osteochondral scaffold’s zones of polycaprolactone with decellularized cartilage extracellular matrix and β-tricalcium phosphate seek to engineer cartilage and bone portions, respectively.

The novel strategy for recreating the gradient seen in osteochondral tissues is to create a scaffold with a calcium gradient built into its 3D-printed structure. The bottom layers are infused with calcium. As the scaffold degrades, the calcium is gradually released. A tidemark layer separates calcium-containing from non-calcium-containing scaffold material in the hopes of separating stem-cell differentiation into bone sections and cartilage sections.

To test this strategy, the scaffolds loaded with stem cells were placed in a growth medium and then assessed for the composition and organization of cells in the new tissue. “Our goal for the in vitro study is to demonstrate that the environment of each section of the scaffold helps cells differentiate into cartilage, calcified cartilage, or bone,” stated Dr. Spang.

Modeling the treatment
In the final year of the investigation, the researchers will test a model of articular cartilage damage in the knee with Sinclair miniature pigs because the joint size, cartilage thickness, and weight-bearing requirements of the porcine joint are similar to those of the human knee joint.

Two small surgically created defects in both knee joints of each of the seven pigs will serve as the injuries. Some defects will either be untreated or treated with an empty scaffold—the control groups. The test groups will be defects treated with an osteochondral plug, and a test group treated with the scaffold loaded with adipose-derived stem cells from the pigs.

“We’ll be looking at two things: how the scaffold performs and how the stem cells perform in that environment,” Dr. Spang explained. “With imaging and histological studies, we’ll look at the integration of the scaffolds into healthy tissue. And we’ll look at how the cells behaved. Are the cells in the cartilage portion making cartilage matrix? Are the cells in the bone end going toward bone? Is there mixing?”

He added, “The ability to create portions of bone and cartilage from the patient’s own stem cells, if we’re eventually proven successful, is going to add a valuable tool for correcting problems we don’t treat well right now.”

Preparing for the big leagues
This is not Dr. Spang’s first experience with grant funding. He also received a grant in 2007, during his fellowship in sports medicine at the University of Connecticut, Farmington. “That grant helped me understand what I could do as a clinician scientist and what I couldn’t do. I was able to start working with other people, collaborators who were more senior.”

He noted that this start helped him learn how to find common ground with collaborators—to find projects that excited all of them and projects that tapped into each of their areas of expertise.

Jay D. Lenn is a contributing writer for OREF. He can be reached at communications@oref.org.
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