**5.** *In vivo* **GO effects during bone regeneration therapies**

Regeneration of large bone defects requires development of bioactive scaffolds with distinct properties of promoting stem cells osteogenic differentiation and inducing the *in vivo* new bone formation. There are just few studies with graphene-based composite materials, which demonstrated potential to stimulate osteogenesis *in vivo* (**Table 1**).



**Table 1.** Platforms to study *in vivo* bone regeneration therapies using graphene-based biomaterials.

Up to date, there is a small number of *in vivo* studies investigating the ability of graphenebased nanomaterial platforms to induce and support production of functional *de novo* bone tissue when practical approaches in bone regenerative medicine require it. Although the implications and benefits for patients experiencing bone defects are of great importance, research toward validation of novel bioimplantable materials designed for bone repair advances in small steps due to safety and ethical requirements. Graphene and its derivatives hold great promise for the synthesis of efficient osteoinductive materials and in-depth research looking at the interplay between graphene effects and molecular pathways active in bone formation will contribute to bringing graphene from bioengineering labs to clinical practice.

### **Acknowledgements**

**Material** *In vivo* **model Post-**

160 Advanced Techniques in Bone Regeneration

Rabbit calvarial defects

Mouse calvarial defects

Rabbit calvarial defects

2, 4, and 8 weeks

Nanocomposites of reduced graphene oxide (rGO) and hydroxyapatite (HAp) (rGO/HAp

GO-coated titanium

Graphene-oxidemodified β-tricalcium phosphate (β-TCP-GRA) bioceramics

implants

NCs)

**implant analysis** **Biological effects References**

[56]

[57]

[50]

and tumor necrosis factor-α (TNF-α) showed no specific inflammatory responses in the HAp grafts and

**•** relative micro-CT values for new bone formation were 11.68 ± 8.99, 609.30 ± 308.58 and 1157.83 Å ± 224.52 in the

**•** new bone density (%)in the control, HAp grafts, and rGO/HAp grafts were 17.66 (±8.81), 26.80 (±8.32), and

**•** conclusion: graphene-based composite materials have

alkaline phosphatase (ALP) activity in bone-forming

**•** substance P (SP), which is known to recruit mesenchymal stem cells (MSCs), was co-delivered using Ti or GO-coated Ti to further promote bone formation **•** GO-coated Ti induced dual delivery of BMP-2 and SP and increased new bone formation on Ti implanted in the mouse calvaria compared with other groups

**•** micro-CT analysis showed significantly increased new bone formation in the β-TCP-GRA group compared with

**•** the volume of the newly formed bone (BV/TV ratio) of the β-TCP-GRA group (26.12 ± 4.44% and 44.83 ± 10.82%) was significantly higher compared with control (16.64 ± 4.57% and 30.41 ± 4.10%) at weeks 4 and 8 post-implant;

**•** trabecular number (Tb.N) in the β-TCP-GRA group (0.39 ± 0.065 and 0.63 ± 0.102) was significantly higher compared with control (0.25 ± 0.049 and 0.41 ± 0.05) at

**•** histomorphometrically analysis of the mineralization area expressed as percentage resulting from the fluorochrome labeling with tetracycline (TE) at 2 weeks,

**•** at 4 weeks, and calcein (CA) at 6 weeks after the implantation surgery, showed increased % of

4 weeks **•** relative mRNA expression levels of interleukin 6 (IL-6)

control, Hap grafts, and rGO/HAp grafts

52.85 (±12.04), respectively

the β-TCP group;

weeks 4 and 8 post-implant;

alizarin red (AL)

potentials to stimulate osteogenesis

cells in vitro compared with bare Ti

8 weeks **•** BMP-2 delivery using GO-coated Ti found out a higher

rGO/HAp grafts

The authors acknowledge the sources of funding that supported their own studies in the field of graphene-based composites for bone tissue engineering: a grant of the Romanian National Authority for Scientific Research, Executive Agency for Higher Education, Research, Devel‐ opment and Innovation, Project Number PN-II-PCCA-140/2012. Additionally, the studies were financed by the Institute for Research of the University of Bucharest (ICUB), through "Scholarships for Excellence in Research for young researchers, 2015 competition" project.
