UNIVERSITY PARK, Pa. — An interdisciplinary team of engineers and chemists at Penn State has laid the groundwork to 3D print spheroids — tiny clusters of living cells — capable of regenerating bone tissue in response to severe trauma or infections.
By introducing different strands of genetic information into undifferentiated, commercially sourced stem cells, the team has demonstrated that bioprinting, which layers the fundamental building blocks of an organ tissue, can create cell clusters optimized to support bone tissue regeneration. The bioprinted spheroids are not just better at helping bone tissue heal, the researchers reported, but they also facilitate the successful formation of new blood vessels within generated tissue. The team verified their findings, available online now in Chemical Engineering Journal , through experiments in the lab and in mouse models.
Bioprinting spheroids can be used for a host of applications, including creating accurate biological models to test the impacts of experimental drugs. However, co-corresponding author on the paper Daniel Hayes , head of the Department of Biomedical Engineering at Penn State, explained that using these cells for regenerative medicine is not a straightforward process. Researchers are tasked with creating networks of cells that can have vastly different functions from a single fundamental baseline.
“Everything in your body is made up of a combination of different cell types, meaning we have to find a way of differentiating cells from one another as they mature when forming these tissues,” said Hayes, who also holds the Dorothy Foehr Huck and J. Lloyd Huck Chair in Nanotherapeutics and Regenerative Medicine. “This project is an attempt to build spheroids that could be used to help reconstruct these complex cellular structures”
Bioprinting usually involves encapsulating living cells in a scaffold, which is traditionally a jelly-like material known as hydrogel. The scaffold essentially acts like a 3D matrix with the living cells maturing and growing into complex tissues inside. However, facilitating vascularization — the process of forming new blood vessels in a tissue — has proven difficult when using spheroids to generate bone tissue.
“Without vascularization, conventional tissue generations techniques cannot adequately regenerate bone,” explained co-corresponding author Ibrahim Ozbolat , professor of engineering science and mechanics, of biomedical engineering, of neurosurgery and Huck Institutes of the Life Sciences Chair in 3D Bioprinting and Regenerative Medicine. “We need vascularization to support the thick bonds found in bone tissue.”
Ozbolat said that building tissues requires extremely precise, coordinated cellular networks, meaning it can be hard to organize spheroids in the patterns needed to spur successful tissue generation. Aspiration-assisted bioprinting is an advanced approach pioneered by Ozbolat and his research team that picks up individual spheroids and places them at specific locations within a scaffold, according to Ozbolat.
“We want the spheroids to be the same distance apart so that we have uniform regeneration,” Ozbolat explained. “Aspiration-assisted bioprinting allows us to precisely position spheroids in a scaffold, meaning we can control the exact location of these spheroids to print a host of different complex tissue types, including lung or pancreas cells.”
Using this technique, the team can create scaffolds that not only support natural healing but also help artificially generated tissue more effectively integrate into the body.
“There are two pieces to it: We're printing progenitor cells that will eventually help form vascularized bone, and we hope these same cells will also help drive that natural regenerative process,” Hayes said. “You wouldn't pursue this treatment for a normal bone break. This is for someone who's had substantial trauma or loss of bone due to cancer or an infection.”
Beginning with blank, undifferentiated stem cells, the team transfected, or introduced, foreign genetic information derived from two specific strands of microRNA — tiny molecules of genetic information that can serve as a switch to enable or disable a particular characteristic of a cell. The two strands used, miR-148b and miR-210, can help spur bone growth and vascularization, respectively. After giving the cells a few days to culture, the researchers assembled the cell clusters into spheroids. Using these spheroids, they produced several varieties of scaffolds — some containing only spheroids transfected with one microRNA strand, and others containing a combination of spheroids transfected with both strands and arranged in an alternating pattern.
“We take these undifferentiated stem cells, and use these microRNA molecules to push them down a differentiation pathway optimized for either tissue growth or, in this case, vascularization before they have a chance to fully mature,” Hayes explained. “We introduce them into the spheroid, and then they get printed into the gel so that as they mature, they differentiate and start to form a regular, sustainable tissue.”
After printing the spheroids into the scaffold, the team allowed the cell clusters to culture and differentiate for 28 days. They then analyzed the genetic markers exhibited by each cluster to see how each microRNA strand impacted the spheroid’s characteristics. Alongside this, the team tested their spheroids over six weeks in a sample of immunodeficient mice with bone tissue damage.
In mice that received no treatment, bone tissue regenerated to cover about 35% of the affected area after six weeks, while mice treated with a control spheroid containing just scaffold exhibited tissue regeneration covering 93% of the affected area, indicating that the team’s bioprinting procedure can play a substantial role in spurring tissue regeneration, Ozbolat said.
Upon adding spheroids, the team observed that using a combination of microRNA strands in the scaffold led to more effective bone tissue development and vascularization, with more overall bone coverage reported at the end of the six weeks. Interestingly, the combinational group showed higher expressions of CD31 — a protein marking the inner lining of blood vessels — compared to the non-transfected and specialized groups. Although the researchers noted that the relationship is still not entirely understood, this increased coverage indicates that spheroids with differing cellular characteristics may cooperate better with one another to spur bone, vascular and overall tissue growth.
The team plans to continue their work with a particular focus on investigating this co-development relationship in larger models, as well as investigating how vascularization may adversely affect bone growth in printed tissue.
“As these techniques move closer towards clinical application, clinicians will need a great deal of fundamental understanding provided by researchers to best apply these new technologies,” Ozbolat said. “There’s a huge opportunity now to establish that knowledge, though, as these materials are commercially available and scalable.”
Other co-authors on the work include Miji Yeo, a former postdoctoral fellow at Ozbolat’s lab who is now an assistant professor at CHA University; Myoung Hwan Kim, a postdoctoral fellow at Johns Hopkins University who received her doctorate in biomedical engineering at Penn State; Vaibhav Pal, a chemistry doctoral candidate; Nazmiye Celik, a postdoctoral fellow at Johns Hopkins University who received her doctorate in engineering science and mechanics at Penn State; Amar Yeware, a postdoctoral scholar at Penn State during the time of research who is now a cell and gene therapy scientist at the University of Wisconsin School of Medicine; and Logan Haugh, a chemical and biomolecular engineering doctoral candidate at the University of Pennsylvania who received their bachelors in biomedical engineering from Penn State.
This work was supported by the U.S. National Science Foundation (NSF) under award numbers 2033673 and 1914885 and the National Institutes of Health’s National Institute of Dental and Craniofacial Research under award numbers RDE024790A , R01DE028614 and R01EB034566 . The content is solely the responsibility of the authors and does not necessarily represent the official views of the NSF or the National Institutes of Health.
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Chemical Engineering Journal
Experimental study
Animals
Bioprinting of miRNA-induced spheroids for vascularized, heterocellular bone regeneration
19-Jun-2026