Traumatic bone injuries or deformities are among the most common conditions that require surgical intervention affecting patients in the United States today. Current treatment options suffer shortcomings, clearly demonstrating a clinical need for tissue replacement techniques. An ideal treatment consists of a scaffold that promotes regeneration, matches the chemical and mechanical properties of bone, and degrades with a timeline matching the healing process. However, this ideal scaffold has not been realized. Here, we present a degradable, graphene-based material that mimics the chemical and mechanical composition of bone, promotes differentiation of stem cells, and leads to the formation of new bone in an animal model.

Synthetic, resorbable scaffolds for bone regeneration have potential to transform the clinical standard of care. Here, we demonstrate that functional graphenic materials (FGMs) could serve as an osteoinductive scaffold: recruiting native cells to the site of injury and promoting differentiation into bone cells. By invoking a Lewis acid-catalyzed Arbuzov reaction, we are able to functionalize graphene oxide (GO) to produce phosphate graphenes (PGs) with unprecedented control of functional group density, mechanical properties, and counterion identity. In aqueous environments, PGs release inducerons, including Ca 2+ and PO 4 3− . Calcium phosphate graphene (CaPG) intrinsically induces osteogenesis in vitro and in the presence of bone marrow stromal cells (BMSCs), can induce ectopic bone formation in vivo. Additionally, an FGM can be made by noncovalently loading GO with the growth factor recombinant human bone morphogenetic protein 2 (rhBMP-2), producing a scaffold that induces ectopic bone formation with or without BMSCs. The FGMs reported here are intrinsically inductive scaffolds with significant potential to revolutionize the regeneration of bone.

Strong, biodegradable, and intrinsically osteoinductive implants have the capacity to revolutionize the treatment of severe bone injuries. Despite their potential, decades of research have failed to generate a synthetic material that incorporates all of the necessary characteristics of an ideal bone scaffold and cultivates an optimal environment for bone regeneration. Current treatments rely on surgical fixation of bone grafts or devices to impart structural stability at the site of injury. However, these therapies suffer limitations: suitable donor sites are at a premium for autografts, while allografts risk rejection. Metallic hardware produces good results but remains permanently (1, 2), which limits use in children and can damage surrounding tissue (3). Regenerative engineering strategies could ultimately produce superior degradable scaffolds that activate the body’s innate healing pathways and eliminate the need for grafts and nonresorbable prosthetics.

Bone spontaneously heals minor fractures; however, inherent healing mechanisms fail when tissue is severely damaged (4). To enhance the natural regenerative response, stem cell therapies hold tremendous potential (5), but the recruitment, retention, and differentiation of these cells at the site of injury remain a challenge (6, 7). To support cell-mediated regeneration, a resorbable synthetic scaffold that matches the chemical and mechanical properties of bone and retains and drives differentiation of stem cells could substantially improve the treatment of traumatic bone injuries. However, no synthetic scaffold material has been reported that provides mechanical stabilization, degradability, and cell instructive moieties.

To drive regenerative healing, a composite material that mimics the complexities of native tissue must be developed. One promising composite component is graphene oxide (GO), a nanostructured carbon material with mechanical strength (8), an aqueous degradation pathway (9), and tunable surface chemistry (10). Furthermore, cells readily adhere to and grow on graphenic materials (11), and GO offers a plethora of organic functionalities that can be exploited to derive functional graphenic materials (FGMs) (12). However, many reported functionalization strategies for creating FGMs lack the control and nuance required for biomimetic functionalization.

Herein, we report phosphate graphene (PG) as a biomimetic class of stem cell scaffolds for bone regeneration that show intrinsic osteoinductivity in vitro and in vivo. To produce this scaffold material, the Arbuzov reaction was borrowed from organic chemistry and applied to GO: by using a Lewis acid catalyst, reaction conditions allow for unprecedented control of PG composition, enabling the production of hydroxyapatite-like surfaces. Furthermore, this chemistry incorporates selectively labile bonds to enable the tunable, controlled release of “inducerons”: (13) ions that are known to direct osteogenic differentiation of stem cells without the use of growth factors. This material exhibits tunable mechanical properties and degradability and retains stem cells while instructing their osteogenic differentiation. Thus, PG is a promising scaffold material that could enable stem cell-driven bone regeneration.

The ideal scaffold for stem cell-driven bone regeneration encourages stem cell retention and differentiation, is mechanically sound and intrinsically ordered to support activity while the injury is healing, and degrades over a year to match the timeline of intrinsic bone healing. FGMs chemically derived from GO have an aqueous degradation pathway, intrinsic long-range order, and robust mechanical properties. Furthermore, recent research has shown GO to be compatible, support cell adhesion, and in some cases, promote osteogenic differentiation of stem cells. Here, we describe a method to create an osteomimetic PG scaffold. This synthetic approach gives us a graphenic scaffold with hydroxyapatite-like functionality at the interface and furthermore, is programmed to elute osteoinstructive inducerons, including Ca 2+ , Li + , Mg 2+ , and PO 4 3− ( 14 ). These simple signaling ions instruct stem cell differentiation; thus, this scaffold is designed to be intrinsically osteoinductive and degradable while offering mechanical stability derived from the strength of graphene.

Results and Discussion

Processing into 3D Scaffolds with Osteomimetic Mechanical Properties. For applications as a load-bearing bone scaffold, the mechanical properties of the scaffold must approximate native bone for stabilization. Single-layer and bulk graphene-based composites have excellent mechanical properties (8, 24⇓–26); thus, the graphenic component of PG provides mechanical strength. To create a 3D scaffold, PG powders were processed into pellet constructs (SI Appendix, Fig. S1B) via hot pressing at moderate temperature and high pressure. Hot press processing did not disrupt the covalent phosphate functionalization of PG materials. Compared with unprocessed PG powders, PG pellets showed minimal changes in their TGA thermograms as well as their spectra from XPS, Raman spectroscopy, and FTIR spectroscopy (SI Appendix, Figs. S3–S9 and S11). TGA confirmed that the processing temperature did not exceed the onset temperature (T o ) of PG powders. XRD indicated a reduction in the intergallery spacing postprocessing that is likely a result of packing from high-pressure processing (SI Appendix, Fig. S10). Compressive universal testing and dynamic mechanical analysis were used confirm that the mechanical properties of PG scaffolds approximated those of bone. With the increased agility of the synthetic method, we were able to tune the chemical functionalization density to the properties of PG scaffolds to exhibit stiffness and strengths comparable with those of bone (27, 28): Young’s moduli (E) up to 1.8 GPa, compressive storage moduli (E′) up to 291 MPa, shear storage moduli (G′) up to 545 MPa, and ultimate compressive strengths (UCS) up to 300 MPa (Fig. 2 and SI Appendix, Fig. S12). PG pellets were also tough (U T ; up to 2,326 J⋅m−3⋅104). Furthermore, the compressive mechanical properties did not display a strain rate dependence (SI Appendix, Fig. S13), indicating that the scaffolds could withstand an array of loads associated with activities, such as walking and running, without compromising mechanical integrity. Critically, producing PG scaffolds with biomimetic mechanical properties will prevent stress shielding observed with metallic implants while providing support capable of withstanding large loads to protect the fragile healing interface. Fig. 2. Mechanical characterization of the PG scaffolds. (A) Compressive universal testing until failure at a 0.1-s−1 strain rate, (B) Young’s moduli (E), (C) UCS, and (D) toughness (U T ) of graphenic constructs. All data were calculated from the stress–strain curves; n = 3. Bars and the listed values are sample means, and error bars are sample SDs.

Aqueous Stability of PG Scaffolds. For application in vivo, the 3D PG scaffolds must maintain mechanical integrity in aqueous environments while bone is regenerated. In nonunion bone defects, it is expected that stabilization will be required over the course of a month. Thus, the compressive mechanical properties of pellets in aqueous conditions were evaluated over 28 d (SI Appendix, Fig. S14). PG scaffolds remained intact throughout the course of the experiment, except for LiPG, which lost mechanical integrity upon immersion in water. This is likely because the large intergallery spacing enabled water infiltration, accelerating disassembly of LiPG scaffolds. Hydrated PG scaffolds had E′ an order of magnitude lower than dry pellets; however, over 28 d in water, minimal changes were observed. In contrast, without the entanglement of the polyphosphate chains, GO pellets were unstable and rapidly dispersed in water (SI Appendix, Fig. S1C). FTIR, TGA, and XPS analyses of scaffolds after prolonged aqueous exposure revealed that the CaPG scaffold underwent significant chemical changes into a structure resembling reduced GO (SI Appendix, Figs. S3–S9). We consider this result to be especially promising, as it suggests that CaPG shares an aqueous degradation pathway with GO and that the large volume of literature studying the compatibility of GO is indicative of the compatibility of CaPG. Additional experiments are underway to confirm this hypothesis.

Elution of Inducerons. We hypothesized that, over the course of degradation, CaPG scaffolds were eluting osteoinductive Ca2+ and PO 4 3− ions during their aqueous degradation. We were able to quantitatively measure Ca2+ elution (29) from CaPG scaffolds in PBS, revealing a controlled delivery of up to 500 μM of Ca2+ per 1 mg of CaPG (SI Appendix, Figs. S15 and S16). The release profile of Ca2+ showed an initial burst release within the first day followed by a controlled release up to day 14. The final Ca2+ concentration was ∼10 mM, which is similar to the ideal concentration of ∼8 mM for osteogenic differentiation (30). After fitting the data to a mathematical model (31), we concluded that the elution of Ca2+ was diffusion controlled. Furthermore, PO 4 3− was also released from CaPG pellets with a burst release profile that reached equilibrium after the second day (SI Appendix, Figs. S15 and S16). CaPG scaffolds deliver up to 20 μM of PO 4 3− per 1 mg of CaPG. Since Ca2+ and PO 4 3− are known to induce osteogenic differentiation, this suggests that a degradable CaPG scaffold is a highly promising material for controlled release of inducerons to support osteogenic differentiation.

Compatibility and Osteoconductivity in Vitro. We prepared and characterized aqueous dispersions of PG materials and demonstrated that they are cytocompatible and did not alter subcellular compartments using NIH-3T3 fibroblasts, RAW 264.7 macrophages, and human mesenchymal stem cells (hMSCs) (SI Appendix, Figs. S17–S20) (32). Cells adhere to and grow on PG substrates (SI Appendix, Fig. S19) and pellets (Fig. 3), demonstrating osteoconductivity. Fig. 3. hMSCs adhere to and grow on 3D scaffolds of PG materials. (A) Whole-pellet images (top view) and (B) higher-magnification images of hMSCs cultured on PG pellets for 7 d and then labeled for nuclei (blue), F-actin (green), and mitochondria (red).

Osteoinduction in Vitro. The efficacy of a bone regeneration scaffold can be enhanced if the material promotes osteogenic differentiation of hMSCs. Excitingly, CaPG encourages expression of an osteoblastic phenotype in hMSCs in vitro (Fig. 4). Alkaline phosphatase (ALP) is highly expressed in osteoblasts (33) and is a quantitative stain correlating with the level of osteoblastic expression. hMSCs exposed to CaPG and cultured in growth media (designed to maintain multipotency with no added growth factors) exhibited a 240% increase in ALP expression (Fig. 4 A and D). Compared with hMSCs cultured in osteogenic media (commercially available media designed to maximize osteogenic differentiation using growth factors), cells exposed to CaPG had similar expression levels. A similar result was obtained when assaying for Alizarin Red S (ARS), which labels calcium deposits that are indicative of mineralization from cells displaying an osteogenic phenotype (34): hMSCs exposed to CaPG had a 170% increase in the intensity of the ARS labeling (Fig. 4 B and E). Other PGs and GO showed intermediate increases in these levels; however, the ability of CaPG to intrinsically induce osteogenesis to the same extent as growth factors is particularly remarkable. Fig. 4. CaPG induces osteogenic differentiation of hMSCs. (A) ALP expression of hMSCs exposed to PG materials for 10 d for growth media and 21 d for osteogenic media. ANOVA F values: 13.48 for growth media and 12.83 for osteogenic media. (B) ARS of hMSCs exposed to PG materials for 28 d quantified from ARS absorbance. ANOVA F value: 3.66 for growth media and 8.44 for osteogenic media. *n < 3 due to off-center localization of the majority of hMSCs (SI Appendix, SI Text). (C) Gene expression after 14 d of exposure to PG materials quantified from qRT-PCR using the 2–ΔΔCt method compared with two reference genes. Note that the no treatment (NT) growth media sample is the calibrator. ANOVA F value: 1.88 for BMP-2, 3.30 for COL1A1, and 1.37 for RUNX-2. (D–F) Representative whole-well and higher-magnification images of (D) ALP expression (red); (E) ARS expression (red); and (F) nuclei (blue), F-actin (green), and phase contrast (gray). Bars are sample means, and error bars are sample SDs except for C, for which they are propagated SEs; n = 3 for all except for ALP growth media, for which n = 4. Lines between bars indicate a two-tailed P value < 0.05 from Sidak post hoc test. qRT-PCR was used to quantify the expression of important osteogenic genes of hMSCs exposed to PG materials: collagen type I alpha 1 (COL1A1), bone morphogenetic protein 2 (BMP-2), and runt-related transcription factor 2 (RUNX-2). Small nuclear ribonucleoprotein D3 (SNRPD3) and proteasome subunit beta 2 (PSMB2) were used as reference genes due to their constant levels of expression (35). Cellular exposure to either PG materials or osteogenic media for 14 d resulted in relatively small changes in the expression of the genes examined compared with the no treatment growth media control (Fig. 4C). At this time point, hMSCs were expressing ALP, a phenotypic marker of osteoblasts, indicating that hMSCs were already committed to the osteoblastic lineage. Since it is during the early stages of osteogenic differentiation that the potent growth factor BMP-2 serves to activate RUNX-2, which is considered the principle osteogenic master switch (36⇓–38), it is likely that peak expression of these osteogenic genes has already passed. Type I collagen is a target of RUNX-2 (36⇓–38), and optical imaging confirmed that, after 14 d, cells were confluent and already generated a substantial amount of extracellular matrix. Overall, in vitro, CaPG was best able to induce differentiation of hMSCs into an osteoblastic phenotype. Even in growth media designed to preserve multipotency, CaPG resulted in significant osteogenic differentiation of hMSCs that was similar to that observed for hMSCs cultured in osteogenic media with growth factors tailored to induce osteogenesis. This superior differentiation likely results from controlled release of Ca2+ and PO 4 3− inducerons, structural mimicry of natural bony apatite, and mechanical stiffness. Thus, CaPG shows promise as a cell-instructive, intrinsically osteoinductive material.