Reconstruction of large craniofacial bone defects resulting from cerebral trauma remains a significant challenge. Current reconstructive surgeries using autologous or allogeneic grafts face limitations such as high costs, limited bone sources, and potential donor site complications. Readily available substitutes like titanium mesh and polyether ether ketone suffer from drawbacks including poor stretchability, weak osteointegration, tissue friction, and high Young's modulus, which can restrict intracranial tissue and prevent immediate implantation post-craniectomy. Furthermore, the use of additional devices in traditional cranioplasty, such as skull locks or bone nails, increases surgical complexity and cost. Therefore, a functional implant that can be immediately fixed at the defect site after craniectomy, adapting to the intracranial microenvironment and recruiting endogenous stem cells (ESCs) for cranial tissue regeneration, is highly desirable. Craniofacial bones primarily form through intramembranous ossification, where mesenchymal cells differentiate directly into osteoblasts, eventually developing into a hybrid organic/inorganic cancellous bone structure. This structure and composition may provide crucial environmental signals for stem cell migration, infiltration, proliferation, and differentiation. Cranial bone formation is closely linked to capillary development, creating highly vascularized tissue to supply oxygen and nutrients for skeletal development, integration, and homeostasis. The interplay between osteogenesis and angiogenesis is crucial for cranial bone formation. However, current strategies often fail to produce highly vascularized new bone due to structural instability, poor adhesion to surrounding tissues in wet environments, and limited functional cell settlement. Hydrogel-based adhesives have been explored for tissue sealing and implant coating to improve adhesion and cell retention. However, limitations remain in adhesion in wet environments and inefficient cell recruitment. Strategies to enhance angiogenesis include scaffold optimization, angiogenic growth factor delivery, and the use of potent cell sources like stem cells or vascular cells. Yet, ex vivo stem cell expansion and growth factor delivery are hindered by limited stem cell availability, high costs, clinical translation challenges, and regulatory hurdles. The ideal approach involves instantly fixable scaffolds mimicking the osteogenic niche, interacting with host tissue to rapidly achieve homeostasis by recruiting ESCs and promoting angiogenesis and osteogenesis.

The literature extensively documents the challenges associated with skull defect repair. Current methods, including autologous and allogeneic bone grafting, face limitations in terms of availability, cost, and potential complications at the donor site. Synthetic materials such as titanium mesh and polyetheretherketone (PEEK) offer alternatives but often lack sufficient biocompatibility, osteointegration, and flexibility to conform to the complex cranial anatomy. These limitations highlight the need for biocompatible, readily implantable, and self-adaptive scaffolds that can effectively promote bone regeneration. Existing studies have explored the use of hydrogels, growth factors, and stem cells to enhance bone regeneration, but these approaches often face challenges in terms of clinical translation and cost-effectiveness. The authors' work builds upon these efforts by focusing on a novel approach that combines the advantages of a biomimetic scaffold with inherent self-adhesive properties and the body's natural regenerative capacity.

The researchers designed a hybrid cross-linked scaffold (HCLS) mimicking the natural bony extracellular matrix. The scaffold is composed of dopamine-modified hyaluronic acid (HAD), type I collagen (Col I), and microhydroxyapatite (µHAp). Dopamine modification of hyaluronic acid serves as a “bridge” to chelate Ca2+ from µHAp and bind Col I through Michael addition, creating strong chemical bonds between the organic and inorganic phases. This approach aims to improve mechanical strength, stability, and adhesion. The study involved a comprehensive characterization of the HCLS using various techniques such as scanning electron microscopy (SEM), X-ray diffraction (XRD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), X-ray photoelectron spectroscopy (XPS), Fourier-transform infrared spectroscopy (FTIR), and dynamic mechanical analysis (DMA). These analyses assessed the scaffold's microstructure, chemical composition, thermal stability, mechanical properties, and calcium chelation capacity. In vitro studies evaluated the HCLS's biocompatibility and its effects on bone marrow stromal cells (BMSCs) and macrophages. BMSCs were cultured on the HCLS to assess proliferation, osteogenic differentiation, and the expression of osteogenesis-related genes (Runx2, osteocalcin, osteopontin). Macrophages were co-cultured with the HCLS to analyze their polarization (M1 vs. M2 phenotypes) and cytokine production. In vivo studies employed rabbit (9 mm defect) and beagle dog (15 mm defect) cranial defect models. The HCLS was implanted into the defects, and the bone regeneration process was evaluated at different time points (4 and 12 weeks) using micro-computed tomography (micro-CT), histological analysis (H&E, Masson's trichrome), immunofluorescence staining (CD31, BMP-2, Runx2, osteocalcin, VEGF), and gene expression analysis (qPCR). A subcutaneous implantation model in nude mice was also used to assess ectopic bone formation. Finally, transcriptomic analysis was performed to identify differentially expressed genes and signaling pathways involved in osteogenesis, angiogenesis, and stem cell recruitment in response to the HCLS.

The HCLS demonstrated superior mechanical properties compared to control scaffolds, exhibiting high tensile strength and flexible deformation, even in the presence of blood. The scaffold exhibited strong adhesion to various substrates, including bone and tissue, enabling immediate fixation without additional devices. In vitro, the HCLS effectively promoted BMSC proliferation and osteogenic differentiation, with significantly increased expression of osteogenesis-related genes. The HCLS also guided macrophage polarization towards the M2 phenotype, associated with anti-inflammatory responses and enhanced angiogenesis. In vivo studies using rabbit and beagle dog cranial defect models showed significant bone regeneration. After 12 weeks, the HCLS achieved 97% bone cover in rabbits (9 mm defect) and 72% in beagle dogs (15 mm defect). Micro-CT analysis confirmed significant increases in bone volume, bone mineral density, and trabecular number in the HCLS group compared to controls. Histological analyses revealed well-integrated new bone tissue with abundant blood vessels, indicating successful osteogenesis and angiogenesis. The in vivo results confirmed the in vitro findings of M2 macrophage polarization, and the expression levels of BMP-2 and VEGF were significantly increased. Transcriptomic analysis further elucidated the mechanism underlying the HCLS's efficacy. The results revealed upregulation of genes and pathways related to M2 macrophage activation, ESC recruitment (CXCL12, LAMA4, LAMA2, and SRGN), angiogenesis (PGF, IGF1, PDGFRA, and ADAMTS3), and osteogenesis (PI3K-Akt signaling pathway). The results indicated that the HCLS effectively creates a biomimetic microenvironment that promotes cell adhesion, proliferation, differentiation, and angiogenesis, leading to rapid and substantial bone regeneration.

The findings demonstrate a novel, cell-free scaffold capable of facilitating rapid and extensive cranial bone regeneration. The HCLS's unique design, combining the adhesive properties of dopamine, the structural support of collagen and hydroxyapatite, and the biocompatibility of hyaluronic acid, creates a highly biomimetic environment that effectively recruits and guides endogenous stem cells, macrophages, and blood vessels. This approach addresses the limitations of current methods by eliminating the need for exogenous growth factors or cells, simplifying the surgical procedure, and reducing costs. The results highlight the potential of this technology to provide a safe and effective treatment for large cranial bone defects, improving patient outcomes and reducing the burden on healthcare systems. The significant bone regeneration observed in both rabbit and beagle dog models, albeit with better results in rabbits, suggests the clinical potential of the scaffold. Future optimization might focus on tailoring the mechanical properties to match the specific demands of different anatomical locations and patient populations.

This study presents a promising cell-free, instantly fixable, and self-adaptive scaffold for cranial bone regeneration. The HCLS effectively promotes bone formation through its unique biomimetic design and ability to orchestrate endogenous stem cell recruitment, macrophage polarization, and angiogenesis. The results from rabbit and beagle dog models demonstrate the scaffold’s potential for clinical translation, offering a simplified, cost-effective alternative for treating large cranial defects. Future work could focus on optimizing scaffold design for larger defects and different species, and exploring potential applications beyond cranial reconstruction.

While the HCLS shows great promise, some limitations exist. The study primarily focuses on preclinical models (rabbits and beagle dogs), and further research is needed to translate these findings to human clinical trials. The slightly lower efficacy observed in the beagle dog model compared to the rabbit model suggests that optimizing scaffold design to accommodate species-specific skull mechanics is important. Long-term studies are necessary to assess the durability and longevity of the bone regeneration achieved using the HCLS.