Researchers 3D print calcium phosphate graphene scaffolds for bone regeneration

A team of researchers from Carnegie Mellon University (CMU) and the University of Connecticut (UConn) has 3D printed novel calcium phosphate graphene (CaPG) scaffolds that could be used for bone regeneration applications in the future. 

The team sought to develop an alternative to traditional autogenous bone grafts that simply stabilize bone defects and injuries. The study saw the successful fabrication of a 3D bioprinted alternative that supports tissue regeneration at the defect site, and which possesses numerous desirable properties such as osteoinductivity, biological safety, a long shelf-life, and reasonable production costs. 

Biomimetic 3D printed CaPG matrix design. Image via Nature.
Biomimetic 3D printed CaPG matrix design. Image via Nature.

Challenges of 3D printing graphene

While graphene’s lightweight properties, electrical and thermal conductivity, and mechanical strength make it a desirable material for applications within biomedicine, energy generation, and microelectronics, much of graphene’s potential comes from deploying the material in its monolayer form. This therefore presents a significant challenge when trying to utilize the material for 3D printing.

Despite this, progress has been made to harness the material’s potential for additive manufacturing in recent years. For instance, Virginia Tech and Lawrence Livermore National Laboratory (LLNL) have developed a high-resolution 3D printing method that involves the dispersal of graphene within a gel to form a 3D printable resin, and the latter has also worked with the University of California Santa Cruz to produce graphene-based aerogel electrodes for energy storage devices. 

Elsewhere, graphene-oxide scaffolds that retain many of the monolayer material’s sought-after properties have been successfully 3D printed by Spain’s Institute of Ceramics and Glass and Aix-Marseille University, and University at Buffalo researchers have developed a 3D printed water-purifying aerogel that could be used within wastewater treatment plants. 

Most recently, researchers from the Harbin Institute of Technology 3D printed a graphene-oxide soft robot capable of moving backward and forward on its own when exposed to moisture.

Compatibility and osteogenic differentiation of hMSCs on 3DP-CaPG matrices. Image via Nature.
Compatibility and osteogenic differentiation of hMSCs on 3DP-CaPG matrices. Image via Nature.

3D printing CaPG scaffolds

For this study, the combined research team is exploring how graphene’s properties can be deployed within the medical sector for bone tissue regeneration applications. 

Existing biomaterial bone matrices aim to support tissue regeneration at the defect site or injury while degrading over time and being replaced with newly-grown bone. However, despite advances in this area there is still currently no material that contains all the desirable properties necessary to replace autogenous bone, the researchers said. 

Several materials have been explored in the past to fabricate a synthetic matrix that exhibits suitable osteoinductivity properties while also being biologically safe, having a long shelf-life, and being cost-effective to produce. Among these, materials containing graphene have received considerable attention for displaying excellent chemical, mechanical, and biological properties. 

Graphene-containing materials can promote the adhesion and growth of cells, with some evidence suggesting their osteogenic potential, and as such these materials are being increasingly applied as biomaterials for bone regenerative engineering applications. However, graphene oxide alone lacks the chemical cues necessary to initiate the regeneration of native bone at the site of injury. Additionally, the team observed that while graphene oxide flakes have excellent mechanical properties, bulk constructs of graphene oxide lack the water stability necessary to provide mechanical support for regenerating bone.

The CMU and UConn research group has previously carried out work in this area, having successfully created an intrinsically osteoinductive family of functional graphene-containing materials called phosphate graphenes that displayed potential for bone regeneration. 

For their latest study, they have successfully 3D printed a bone-mimicking CaPG material, through the addition of calcium, which they say is capable of facilitating the differentiation of stem cells into bone cells. 

Biocompatibility of the matrices in a mouse calvarial defect model. Image via Nature.
Biocompatibility of the matrices in a mouse calvarial defect model. Image via Nature.

A ‘paradigm’ in bone regeneration

The team used biofabrication firm Dimension Inx’s Direct Ink Writing (DIW) 3D printing method to print porous constructs of its CaPG material with a high weight fraction to enable a “remarkably high” graphenic content within the ink (around 90 percent). This enabled cellular access to the osteoconductive backbone and the controlled release of calcium and phosphate ions, meaning that the host’s response to the matrix was dominated by the functional graphenic content rather than the bioinert binder. 

The team sent its CaPG powder to Dimension Inx where large sheets of porous CaPG matrices were 3D printed. From the sheets, the team could readily cut out matrices with specific geometries for cellular and animal studies. The group noted the matrices could also be directly printed to match a specific bone defect. As such, the combination of 3D printing and the team’s CaPG material offers a customizable matric system for bone regenerative engineering.

The team studied the osteogenic potential of their 3D printed CaPG matrices both in vitro and in vivo. According to the study, the team’s 3D printed matrices demonstrated their bone formation capabilities in vivo with human mesenchymal stem cells (hMSCs) and in vivo in a bone defect model. 

For the in vivo test, CaPG was combined with bone marrow stromal cells (BMSCs) to instigate the formation of bone within the subcutaneous space of mice. The CaPG matrices were found to be capable of being resorption and biodegradation in vivo, which the team said is rare for a synthetic material and suggests promise as a resorbable osteoinductive matrix.

Additionally, the team did not observe any detrimental effects within vital organs during the study. Going forward, the researchers will conduct further developmental studies to improve the mechanical properties of their 3D printed CaPG matrices and confirm their long-term safety in vivo. 

For now, the group is confident that the 3D printed CaPG matrices have significant potential for future bone regeneration applications.

Further information on the study can be found in the paper titled: “Ultra-low binder content 3D printed calcium phosphate graphene scaffolds a resorbable, osteoinductive matrices that support bone formation in vivo,” published in the Nature journal. The study is co-authored by L. Daneshmandi, B. Holt, A. Arnold, C. Laurencin, and S. Sydlik.

Biodegradation and biodistribution of 3DP-CaPG matrices. Image via Nature.
Biodegradation and biodistribution of 3DP-CaPG matrices. Image via Nature.

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Featured image shows biomimetic 3D printed CaPG matrix design. Image via Nature.