Bone Healing Reimagined: Electroactive Nanocomposite Hydrogels with Nanohydroxyapatite Decorated Carbon Nanotubes

Introduction:

Bone tissue engineering (BTE) is advancing biomaterials to address graft limitations. Injectable hydrogels provide a biocompatible alternative, delivering bioactive cues and cells to bone defect sites. By incorporating nanomaterials, nanocomposite hydrogels further enhance mechanical strength, bioactivity, and therapeutic potential for minimally invasive bone regeneration (1, 2). In this study, we developed a methacrylated gellan gum hydrogel functionalized with nano-hydroxyapatite-coated carbon nanotubes (GGMA-nHApCNT) that closely mimics the bone extracellular matrix, replicating the natural self-assembly of hydroxyapatite on bone collagen fibrils, to be applied as an injectable system for bone regeneration.

Methods:

GGMA was synthesized via glycidyl methacrylate reaction with low-acyl gellan gum and confirmed by NMR (3). CNTs were functionalized through Ca²⁺-mediated nHAp nucleation followed by PO₄³⁻ addition (4), with characterization via Confocal Raman spectroscopy, XRD, and XPS. Formulations of 1% w/v GGMA, 1% w/v GGMA - 0.5% w/v CNT, and 1% w/v GGMA - 0.5% w/v nHApCNT were prepared and assessed for its injectability. Hydrogels were formed via cationic gelation and analyzed for rheology, structural integrity, bioactivity, antioxidant and antibacterial properties, angiogenic potential, electrical conductivity, and hemocompatibility. Human adipose-derived stem cells were encapsulated and cultured for 21 days under basal and osteogenic conditions, and assessed for cell viability, proliferation, metabolic activity, ALP activity, and gene expression quantification.

Results:

NMR and spectroscopic analyses confirmed successful methacrylation of gellan gum and nHAp formation on CNTs. While CNTs alone reduced hydrogel stability, nHAp functionalization restored integrity to levels comparable to pristine GGMA hydrogels. Micro-CT analysis showed that both CNT and nHApCNT reduced pore wall thickness, but only CNT increased pore size and porosity. Bioactivity assays demonstrated robust mineralization, with GGMA-nHApCNT hydrogels exhibiting significantly enhanced calcium deposition over 21 days. Both GGMA-nHApCNT and GGMA-CNT hydrogels displayed antibacterial activity and retained antioxidant properties, though a slight decrease was observed in GGMA-nHApCNT. Conductivity measurements confirmed that all hydrogels exhibited some conductivity, with GGMA-nHApCNT showing the highest. Injectability assessments indicated that CNTs increased the injection force from 6.1 N up to 6.6 N, while nHApCNT reduced it to 5.6 N, suggesting improved injectability and reduced shear-induced cell death. CAM assays revealed that GGMA-nHApCNT hydrogels promoted greater blood vessel infiltration. Cell studies showed high viability, enhanced spreading, and osteogenic marker expression, despite reduced metabolic activity over 21 days. Hemocompatibility tests confirmed negligible hemolysis in GGMA and GGMA-nHApCNT, in contrast to GGMA-CNT.

Discussion:

Our results demonstrate that integrating in situ nucleated nano-hydroxyapatite (nHAp) on carbon nanotubes (CNTs) within a methacrylated gellan gum (GGMA) hydrogel creates a biomimetic scaffold with multiple advantages for bone regeneration. nHAp functionalization mitigates the mechanical instability and enlarged pore sizes caused by CNTs alone, promoting a balanced porous architecture that enhances biomineralization, as shown by increased calcium deposition. Injectability assessments revealed that while CNTs increased injection force from 6.1N to 6.6N, nHApCNT reduced it to 5.6N, improving injectability and reducing shear-induced cell death. Conductivity measurements confirmed all hydrogels exhibited some conductivity, with GGMA-nHApCNT demonstrating the highest, suggesting potential benefits for bone tissue engineering. Additionally, GGMA-nHApCNT hydrogels maintain antibacterial and antioxidant properties while promoting high cell viability, enhanced cell spreading, and osteogenic marker upregulation. They also exhibit improved angiogenic responses, as evidenced by increased vascular infiltration in CAM assays.

Conclusions:

The novel GGMA-nHApCNT nanocomposite hydrogels exhibit enhanced mechanical stability, robust biomineralization, and superior osteoinductive performance. Additionally, the improved injectability of GGMA-nHApCNT, with low injection force, suggests better delivery efficiency and lower shear-induced cell damage, further supporting its suitability for minimally invasive applications. Moreover, the enhanced conductivity of GGMA-nHApCNT hydrogels highlights their potential for applications where electrical conductivity may influence cellular responses, particularly in bone tissue engineering. This multifunctional hydrogel system presents a promising platform for bone regeneration, overcoming current limitations in scaffold-based therapies and providing valuable insights for the design of advanced biomaterials in regenerative medicine.