Researchers in Oregon have engineered a material that replicates human bone tissue with an unprecedented level of precision, from its microscopic crystal structure to its biological activity.
They are using it to explore fundamental disease processes, such as the origin of metastatic tumors in bone, and as a treatment for large bone injuries.
“Essentially it is a miniaturized bone in a dish that we can produce in a matter of 72 hours or less,” says biomedical engineer Luiz Bertassoni, D.D.S., Ph.D., an assistant professor in the OHSU School of Dentistry and a member of CEDAR, the Cancer Early Detection Advanced Research Center in the OHSU Knight Cancer Institute.
Like real bone, the material has a 3-D mineral structure populated with bone cells, nerve cells and endothelial cells that self-organize into functioning blood vessels.
“What is remarkable is that researchers in our field have become used to cultivating cells within a protein mixture to approximate how cells live in the body.
But this is the first time anyone has been able to embed cells in minerals, which is what characterizes the bone tissue,” Bertassoni says.
And that’s what makes the new material promising as a model to study bone function, diseases, and bone regeneration.
“With this model system, you can start asking questions about how bone cells attract different types of cancers, how cancer cells move into bone, how bone takes part in the regulation of marrow function,” says Bertassoni.
“It can even be relevant to dissect the mechanisms that are leading to diseases such as leukemia.”
Bertassoni published the results in the journal Nature Communications with postdoctoral researcher Greeshma Nair, Ph.D., doctoral student Avathamsa Athirasala, M.Sc.Eng., and other co-authors.
“Being able to engineer truly bone-like tissues in the lab can also be transformative for regenerative medicine,” Bertassoni says, “since the current treatment for large bone fractures requires the removal of the patient’s own healthy bone so that it can be implanted at the site of injury.”
The recipe starts with the mixing of human stem cells into a solution loaded with collagen, a protein abundant in the matrix of bone tissue.
The collagen proteins link together, forming a gel embedded with the stem cells.
The researchers then flood the gel with a mixture of dissolved calcium and phosphate, the minerals of bone.
They add another key ingredient, the protein osteopontin derived from cow milk, to keep the minerals from forming crystals too quickly.
This additive, which clings to calcium and phosphate, also minimizes the minerals’ toxicity to cells.
The mixture diffuses through a network of channels about the width of a strand of DNA in the spongey collagen, and the dissolved minerals precipitate into orderly layers of crystals.
“We can reproduce the architecture of bone down to a nanometer scale,” says Bertassoni. “Our model goes through the same biophysical process of formation that bone does.”
In this calcified environment, stem cells develop into functioning bone cells, osteoblasts and osteocytes without the addition of any other molecules, as if they knew they were being embedded in an actual bone matrix.
Within days, the growing cells squeeze slender protrusions through spaces in their mineralized surroundings to connect and communicate with neighboring cells.
The bone-like engineered structure creates a microenvironment that is sufficient to cue stem cells that it’s time to mature into bone cells.
“It’s nature doing its job, and it’s beautiful,” Bertassoni says. “So, we went farther and decided to try it with the bone vasculature and innervation as well.”
Nerve cells added to the mixture formed interconnected networks that persisted after mineralization. Endothelial cells, likewise, formed networks of tubes that remained opened after mineralization.
To test the usefulness of the material as a disease model, the researchers implanted their engineered bone beneath the skin of laboratory mice.
After a few days, the lab-made blood vessels had connected with the vasculature in the mouse bodies.
When the researchers injected prostate cancer cells nearby, they found that tumor growth was three times higher in the mice given mineralized bone constructs than in those with the non-mineralized controls.
The team now is engineering versions with marrow cells growing within a surrounding of artificial bone for use as a model to study the initiation and development of blood cancers including the various forms of leukemia.
Also, they have tested the engineered bone-like material as a replacement for injured bone in animal models—with positive results that they expect to report in the near future.
In bone tissue engineering (BTE), 3-D printing is a reliable and customizable method used to repair bone defects by producing biomimetic tissue scaffolds.
In a recent study published online on Tissue Engineering Part A (Mary Ann Liebert, Inc), Qing Li and a team of scientists engineered a biomimetic substitute closest to the natural bone structure and composition for bone grafting.
For this work, they used two different types of hydroxyapatite (HA) materials: nanohydroxyapatite (nHA) and deproteinized bovine bone (DBB) dispersed into collagen (CoL) to prepare a bio-ink and engineer composites of nHA/CoL and DBB/CoL as 3-D printed scaffolds.
The aesthetic reconstruction of functional alveolar bone is challenging after bone loss due to trauma, inflammation and surgery.
Materials scientists can combine tissue engineering and biomaterials to promote alveolar bone regeneration; an increasingly popular theme in regenerative medicine.
The past decade has seen significant advances in 3-D printing technology for customized bone defect repair with successful clinical translation using metal laser printers.
The merits of 3-D printing largely include low complication risk, short operational time and good molding during surgery.
The biodegradable properties of materials can guide bone regeneration in situ
Among the available techniques, low temperature 3-D bioprinting (LT-3DP) is optimal for alveolar bone reconstruction, as it can generate a specific 3-D blueprint of the patient’s bone defects for reconstruction.
The LT-3DP system can engineer multiple polymer-mineral composites with enhanced material properties.
The resulting 3-D porous scaffolds can mimic bone architecture for conductive cell-matrix interactions and allow efficient blood vessel growth for advanced BTE.
The polymer material nHA selected in the study is a good candidate to substitute for natural bone, due to its high osteoconductive activity.
The natural DBB is an alternative xenogenous bone material, morphologically and structurally similar to human cancellous bone.
In the present study, Li et al. successfully replicated a previously established protocol for DBB preparation.
Type I collagen (CoL-1) is the most abundant structural protein in the human body and was therefore best suited to generate composite polymers in the proposed experimental setup for constructs with enhanced biomechanical properties.
Li et al. prepared and categorized the bio-inks to three groups as CoL, nHA/CoL and DBB/CoL in the study. To engineer the composite scaffolds, the scientists used the 3-D bioplotter (EnvisionTEC, Germany).
After printing the 3-D scaffold, they constructed a grid-like microarchitecture with pore widths of 600 µm. The internal honeycomb microstructure represented typical characteristics of cancellous bone.
The materials scientists first tested the physical and chemical properties of the two material composite scaffolds (nHA/CoL and DBB/CoL), followed by their biocompatibility and osteogenic impact on bone marrow stem cell (human BMSC) differentiation during cell-material interactions.
The scientists used standard methods of materials characterization such as X-ray photoelectron spectroscopy(XPS), X-ray powder diffraction (XRD) and Fourier Transform Infrared spectroscopy (FTIR) to understand the consistency of the two composites, as well as their diversity relative to chemical bonds and crystal phases.
They used scanning electron microscopy (SEM) to obtain imaging results, which showed different surface morphologies of the HA crystals and scaffolds as factors affecting the internal porous structure of the product.
Specifically, the Young’s modulus of the nHA/CoL group (7.9 ± 0.3 MPa) was higher than both CoL group (3.5 ± 0.4 MPa) and the DBB/CoL group (4.5 ± 0.7 MPa), indicating higher stiffness of the nHA/CoL composite scaffolds.
For biofunctionalization studies, Li et al. showed that the two composite scaffolds equally supported cell proliferation via immunofluorescence staining.
For this, they used fluorescently tagged antibodies to stain and microscopically identify hBMSCs growth on 3-D printed surface structures.
During cell culture, the scientists used an osteogenic induction medium (OM) and proliferation medium (PM).
The alkaline phosphatase (ALP) staining method used to determine stem cell differentiation, indicated the expression of ALP in the OM group, but not in the PM group.
The scientists conducted real-time polymerase chain reaction (RT-PCR) after extracting total RNA from the stem cells (hBMSCs). The results showed the expression levels of the osteogenesis-related genes of interest examined in the study.
In comparison with the PM groups, the genes related to early and late osteogenic differentiation; Runt-related transcription factor RUNX2, SRY related HMG box gene 9 SOX9, osteocalcin OCN and CoL1A1 in the OM group significantly increased after 7 days. In this way, the researchers demonstrated osteogenesis and increased effects of extracellular matrix formation for hBMSCs cultured on the bioplotted 3-D scaffolds to confirm surface biocompatibility.
Li et al. showed that the physicochemical and biological properties of the 3-D bioprinted scaffolds containing nHA/CoL or DBB/CoL were well suited as bone substitute materials (BSM) in bone tissue engineering (BTE). The ability to easily print 3-D customizable scaffolds can have potential for translational research from the bench to preclinical studies and to the clinic in the future.
More information:Nature Communications (2019). DOI: 10.1038/s41467-019-11455-8
Journal information: Nature Communications
Provided by Oregon Health & Science University
