When surgeons need to replace bone in the head or face, they typically remove and then shape part of the patient’s fibula to fill in the space. But this can lead to leg trauma, and the relatively straight fibula doesn’t fit the curves of the face very well. Now, researchers at the Johns Hopkins University are using plastic 3-D printing materials instead, mixed with a dash of bone powder to stimulate and encourage new bone growth.
“Cells placed on plastic scaffolds need some instructional cues to become bone cells,” said Warren Grayson, PhD, associate professor of biomedical engineering at the university’s medical school and the study’s senior author. “The ideal scaffold is another piece of bone, but natural bones can’t usually be reshaped very precisely.”
The researchers have developed a composite material that combines the printability and strength of plastic with the biological information in natural bone. They began with polycaprolactone (PCL), which is biodegradable polyester that has been approved by the Food and Drug Administration for clinical use.
“PCL melts at 80° to 100°C (176° to 212°F), a lot lower than most plastics, so it’s a good one to mix with biological materials that can be damaged at higher temperatures,” said Ethan Nyberg, a graduate student on Grayson’s team.
While PCL is quite strong, it doesn’t support the formation of new bone well. The researchers then mixed it with increasing amounts of bone powder, made by pulverizing the porous bone inside cow knees after stripping it of cells.
“Bone powder contains structural proteins native to the body plus pro-bone growth factors that help immature stem cells mature into bone cells,” said Grayson. “It also adds roughness to the PCL, which helps the cells grip and reinforces the message of the growth factors.”
The team first tested the composite materials for printability. The blends with 5%, 30%, and 70% bone powder performed well. But the 85% bone powder had too little PCL “glue” to maintain clear lattice shapes, making it “like a chocolate chip cookie with too many chocolate chips,” said Nyberg. The 85% composite will be dropped from future experiments.
To see if these scaffolds encouraged bone formation, the researchers added human fat-derived stem cells taken during a liposuction procedure to scaffolds immersed in a nutritional broth lacking pro-bone ingredients.
After 3 weeks, cells grown on 70% bone powder scaffolds showed gene activity hundreds of times higher in 3 genes indicative of bone formation than cells grown on pure PCL scaffolds. Cells on scaffolds made with 30% bone powder showed large but less impressive increases in the same genes.
After the researchers added beta-glycerophosphate to the cells’ broth to enable their enzymes to deposit calcium, the cells on the 30% scaffolds produced about 30% more calcium per cell, while those on the 70% scaffolds produced more than twice as much calcium per cell, compared to those on pure PCL scaffolds.
The scaffolds were tested on mice with relatively large holes in their skulls made experimentally that were too large to heal without intervention. The mice with scaffold implants laden with stem cells had new bone growth over the hole during the 12 weeks of the experiment. Also, CT scans showed at least 50% more bone grew in scaffolds with the 30% or 70% powder, compared to those with pure PCL.
“In the broth experiments, the 70% scaffold encouraged bone formation much better than the 30% scaffold,” said Grayson, “but the 30% scaffold is stronger. Since there wasn’t a difference between the 2 scaffolds in healing the mouse skulls, we are investigating further to figure out which blend is best overall.”
In future studies, the researchers hope to test bone powder made from human bone since it is more widely used clinically. They also want to experiment with the designs of the scaffolds’ interior to make it less geometric and more natural. And, they plan to test additives that encourage new blood vessels to infiltrate the scaffolds, which will be necessary for thicker bone implants to survive.
The study, “Three-Dimensional Printing of Bone Extracellular Matrix for Craniofacial Regeneration,” was published by ACS Biomaterials Science & Engineering.
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