Cranial bone defects from trauma, congenital conditions, or surgery are challenging to treat due to the skull's limited regeneration. Traditional methods like autografts and allografts have drawbacks, including donor site issues and poor integration. 3D-printed scaffolds provide a patient-specific alternative, improving bone regeneration and integration. This review evaluates advancements in 3D-printed scaffolds for cranial bone regeneration, focusing on fabrication techniques, material innovations, and structural optimization while assessing their preclinical and clinical potential. A systematic literature search (2014-2024) was conducted using PubMed and other databases. Studies addressing scaffold properties such as porosity, pore interconnectivity, and mechanical stability were included, while non-cranial scaffold studies were excluded. Advances in 3D printing have enabled patient-specific scaffolds with optimized architecture to enhance bone regeneration, mechanical support, and nutrient transport. Bioceramics, polymers, and composites mimic native bone properties, while bioactive coatings further improve osteogenesis. However, limited clinical translation and insufficient customization remain challenges. Further preclinical and clinical trials are crucial to overcoming barriers in mechanical optimization and patient-specific scaffold fabrication, bridging the gap between research and clinical applications.

A polysaccharide gel containing covalently bound amikacin, a broad-spectrum antibiotic, was produced by using epichlorohydrin-activated hydroxyethyl starch (HES). The structure of the polymers was analyzed by 13C and 1H nuclear magnetic resonance (13C NMR and 1H NMR) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The sites of covalent attachment of amikacin to the epoxypropyl substituent and the HES backbone were determined. The antibacterial activity of the polymer was evaluated in vitro using the agar well diffusion method with the Staphylococcus aureus P209 strain. It was demonstrated that the polymer retained activity in the presence of bacterial amylase, which is released upon bacterial attack. The gel was applied for coating pores and surfaces of a biocomposite material based on a xenogenic bovine bone matrix. In vivo experiments showed the effectiveness of utilizing amikacin-containing biocomposite bone-substitute materials in the treatment of experimental osteomyelitis in rats using objective histological control and X-ray tomography.

Reconstruction of critical-size bone defects (CSDs) in the craniomaxillofacial (CMF) region remains challenging. Scaffold-based bone-engineered constructs have been proposed as an alternative to the classical treatments made with autografts and allografts. Scaffolds, a key component of engineered constructs, have been traditionally viewed as biologically passive temporary replacements of deficient bone lacking intrinsic cues to promote osteogenesis. Nowadays, scaffolds are functionalized, giving rise to bioactive scaffolds promoting bone regeneration more effectively than conventional counterparts. This review focuses on the three approaches most used to bioactivate scaffolds: (1) conferring microarchitectural designs or surface nanotopography; (2) loading bioactive molecules; and (3) seeding stem cells on scaffolds, providing relevant examples of in vivo (preclinical and clinical) studies where these methods are employed to enhance CSDs healing in the CMF region. From these, adding bioactive molecules (specifically bone morphogenetic proteins or BMPs) to scaffolds has been the most explored to bioactivate scaffolds. Nevertheless, the downsides of grafting BMP-loaded scaffolds in patients have limited its successful translation into clinics. Despite these drawbacks, scaffolds containing safer, cheaper, and more effective bioactive molecules, combined with stem cells and topographical cues, remain a promising alternative for clinical use to treat CSDs in the CMF complex replacing autografts and allografts.

Human adult dental pulp stem cells (hDPSC) and stem cells from human exfoliated deciduous teeth (SHED) hold promise in bone regeneration for their easy accessibility, high proliferation rate, self-renewal and osteogenic differentiation capacity. Various organic and inorganic scaffold materials were pre-seeded with human dental pulp stem cells in animals, with promising outcomes in new bone formation. Nevertheless, the clinical trial for bone regeneration using dental pulp stem cells is still in its infancy. Thus, the aim of this systematic review and meta-analysis is to synthesise the evidence of the efficacy of human dental pulp stem cells and the scaffold combination for bone regeneration in animal bone defect models.

This study was registered in PROSPERO (CRD2021274976), and PRISMA guideline was followed to include the relevant full-text papers using exclusion and inclusion criteria. Data were extracted for the systematic review. Quality assessment and the risk of bias were also carried out using the CAMARADES tool. Quantitative bone regeneration data of the experimental (scaffold + hDPSC/SHED) and the control (scaffold-only) groups were also extracted for meta-analysis.

Forty-nine papers were included for systematic review and only 27 of them were qualified for meta-analysis. 90% of the included papers were assessed as medium to low risk. In the meta-analysis, qualified studies were grouped by the unit of bone regeneration measurement. Overall, bone regeneration was significantly higher (p < 0.0001) in experimental group (scaffold + hDPSC/SHED) compared to the control group (scaffold-only) (SMD: 1.863, 95% CI 1.121-2.605). However, the effect is almost entirely driven by the % new bone formation group (SMD: 3.929, 95% CI 2.612-5.246) while % BV/TV (SMD: 2.693, 95% CI - 0.001-5.388) shows a marginal effect. Dogs and hydroxyapatite-containing scaffolds have the highest capacity in % new bone formation in response to human DPSC/SHED. The funnel plot exhibits no apparent asymmetry representing a lack of remarkable publication bias. Sensitivity analysis also indicated that the results generated in this meta-analysis are robust and reliable.

This is the first synthesised evidence showing that human DPSCs/SHED and scaffold combination enhanced bone regeneration highly significantly compared to the cell-free scaffold irrespective of scaffold type and animal species used. So, dental pulp stem cells could be a promising tool for treating various bone diseases, and more clinical trials need to be conducted to evaluate the effectiveness of dental pulp stem cell-based therapies.

Repair of critical-size bone defects remains a considerable challenge in the clinic. The most critical cause for incomplete healing is that osteoprogenitors cannot migrate to the central portion of the defects. Herein, stem cells from exfoliated deciduous teeth (SHED) with the properties of easy attainability and low immunogenicity were loaded into gelatin/bioactive glass (GEL/BGM) scaffolds to construct GEL/BGM + SHED engineering scaffolds. An in vitro study showed that BGM could augment the osteogenic differentiation of SHED by activating the AMPK signaling cascade, as confirmed by the elevated expression of osteogenic-related genes, and enhanced ALP activity and mineralization formation in SHED. After implantation in the critical bone defect model, GEL/BGM + SHED scaffolds exhibited low immunogenicity and significantly enhanced new bone formation in the center of the defect. These results indicated that GEL/BGM + SHED scaffolds present a new promising strategy for critical-size bone healing.

Chronic periodontitis can lead to alveolar bone resorption and eventually tooth loss. Stem cells from exfoliated deciduous teeth (SHED) are appropriate bone regeneration seed cells. To track the survival, migration, and differentiation of the transplanted SHED, we used super paramagnetic iron oxide particles (SPIO) Molday ION Rhodamine-B (MIRB) to label and monitor the transplanted cells while repairing periodontal bone defects.

We determined an appropriate dose of MIRB for labeling SHED by examining the growth and osteogenic differentiation of labeled SHED. Finally, SHED was labeled with 25 μg Fe/ml MIRB before being transplanted into rats. Magnetic resonance imaging was used to track SHED survival and migration in vivo due to a low-intensity signal artifact caused by MIRB. HE and immunohistochemical analyses revealed that both MIRB-labeled and unlabeled SHED could promote periodontal bone regeneration. The colocalization of hNUC and MIRB demonstrated that SHED transplanted into rats could survive in vivo. Furthermore, some MIRB-positive cells expressed the osteoblast and osteocyte markers OCN and DMP1, respectively. Enzyme-linked immunosorbent assay revealed that SHED could secrete protein factors, such as IGF-1, OCN, ALP, IL-4, VEGF, and bFGF, which promote bone regeneration. Immunofluorescence staining revealed that the transplanted SHED was surrounded by a large number of host-derived Runx2- and Col II-positive cells that played important roles in the bone healing process.

SHED could promote periodontal bone regeneration in rats, and the survival of SHED could be tracked in vivo by labeling them with MIRB. SHED are likely to promote bone healing through both direct differentiation and paracrine mechanisms.