(H-305) Patient-Specific Cryogel with Mineral-Inducing Additives as a Craniofacial Bone Graft Alternative

Introduction:: Bone grafting is a critical procedure employed to address various congenital, traumatic, and pathological craniofacial defects. However, current limitations of traditional bone grafting techniques, such as donor site morbidity, inadequate tissue integration, and limited graft availability, necessitate the exploration of innovative alternatives to meet the growing clinical demand. Cryogel scaffolds are formed through a process of freezing and thawing a cross-linked polymer solution, leading to a highly porous and sponge-like structure that facilitates cellular infiltration and angiogenesis (Figure 1A). Moreover, the integration of natural materials, such as chitosan and gelatin, enhances the scaffold's biocompatibility, biodegradability, and non-toxicity. Recent work by our group has demonstrated the feasibility of forming cryogels within 3D printed cylindrical geometries without disrupting physical properties; however, these scaffolds can also be customized to replicate bone defect sites using patient-specific molds. The current conventional method for mandible reconstruction involves a bone graft accompanied by a surrounding titanium mesh, with an optional bone morphogenetic proteins (BMP) slurry. Thus, this study aims to develop a scaffold that emulates this standard practice using bioactive materials (Figure 1B). We hypothesize that patient-specific cryogels with mineral-inducing additives within 3D printed meshes will improve osteogenic potential, stimulate bone regeneration, and enhance graft integration within the host tissue.


Materials and Methods:: 3D printed lattices were generated using MSLattice and edited in Blender. The lattices were printed on a SLA printer using plastic resin. These printed lattices were infiltrated with a pre-crosslinked chitosan-gelatin cryogel solution and frozen at -20°C for 18 hours. Plain cryogel samples without the 3D printed lattice served as controls. Following formation, the combined 3D printed lattice with cryogel scaffolds were lyophilized for 12 hours and evaluated for pore morphology using scanning electron microscopy (SEM) and ImageJ. Swell testing was performed to assess the scaffolds' expansion and liquid retention capabilities. Mechanical properties were identified by testing the scaffolds until failure on an Instron. Significance was tested using ANOVA with Tukey post hoc (P < 0.05). To further demonstrate patient-specific feasibility, patient CT scans (N=3) with mandibular defects (e.g., Figure 1C) were imported into Blender. Virtual correction of bone structure was used to create a proof-of-concept mold for anatomically relevant scaffold formation (Figure 1D). The 3D lattice/cryogel combinations were formed within the molds and assessed as previously described.

Cryogels are currently being fabricated to contain three different additives (BMP-2, hydroxyapatite, and CaSiO₃), which have been shown to increase mineralization in vitro. These cryogel solutions will be formed in plastic meshes (Figure 1B) to emulate the mesh that is currently employed in corrective mandibular surgeries; pore morphology and swell kinetics will be assessed as described above. MG-63 (osteoblast-like) cells will be seeded on each cryogel additive group to compare infiltration and proliferation, as well as mineralization potential of each additive.

Results, Conclusions, and Discussions:: SEM analysis of the cryogel combined with the plastic lattice possessed pore size similar to the control cryogel with no significant changes (P < 0.05). Although the mean pore sizes were slightly below the optimal range for bone regeneration (100-200 μm), they were comparable to the control cryogel, which supports cell infiltration. All cryogels exhibited maximum swelling capacity after four minutes, but the presence of the 3D printed lattice reduced the swelling capacity compared to the control cryogel. This suggests that the 3D printed lattice may hinder nutrient absorption and reduce the number of infiltrating cells in the overall scaffold in vivo. Additionally, the mechanical testing indicated that the incorporation of a 3D printed lattice significantly increased the compressive modulus of the combined scaffold compared to plain cryogel. Results were similar for cylindrical scaffolds and patient specific scaffolds. Ongoing work is focused on mineralization potential of the mineral-inducing additives as well as cell infiltration and proliferation.

Developing an alternative to bone grafts for complex critical-size defects is desirable. Cryogels serve as an ideal scaffold for this purpose due to their macroporous structure and biocompatible properties. The incorporation of the 3D printed lattice added several benefits over the standard control cryogel, such as proof of concept for patient-specific geometry, significant increases in mechanical strength, and similar porosity to plain cryogels. However, these combined scaffolds did show decreased elasticity and swelling capacity. By adjusting the 3D printing parameters, it is possible to apply these combined scaffolds to different bone areas (e.g., cortical vs. trabecular) or types of loading (e.g., transverse vs. longitudinal). Ongoing work involves creating the combined cryogel and 3D printed scaffold inside the patient-specific mold and testing the incorporation of various mineral-inducing additives (BMP-2, hydroxyapatite, and CaSiO₃), along with cellular infiltration/proliferation. The resulting construct will provide an optimized method for improving osteogenic potential and bone regeneration in complex craniofacial defects.

Acknowledgements (Optional): : Thank you to Izzy LaBombard and Kelli Kehoe for assistance in operating the 3D printer, Dr. Van Citters’ Lab for access to the Instron compression testing machine, and Mason Jacques for assistance with using Blender.

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