A 3D-Printed β-TCP–Cryogel Composite Scaffold to Enhance Bone–Vascular Crosstalk in Mandibular Defects

Introduction: : Mandibular bone defects present significant clinical challenges due to their complex geometry, need for mechanical stability, and critical requirement for robust vascularized regeneration. Large mandibular defects often exceed the body’s natural healing capacity, requiring grafts that not only support bone regrowth but also rapidly reestablish a functional blood supply. Vascularization is essential not only for delivering oxygen, nutrients, and signaling molecules, but also for recruiting bone-forming cells to the defect site. Without sufficient vascular integration, implanted scaffolds risk necrosis and incomplete regeneration. To address these limitations, we have developed 3D-printed β-tricalcium phosphate (β-TCP) lattices with tunable architecture and mechanical properties suited for craniofacial applications. These lattices were infiltrated with chitosan-gelatin cryogels that mimic the bone extracellular matrix, supporting cell growth and mineralization. Osteogenic–angiogenic crosstalk was evaluated through in vitro co-cultures of human mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs) on three scaffold designs: cryogel only, β-TCP lattice only, and a cryogel-infused β-TCP lattice composite. Cellular viability and signaling were assessed using fluorescent imaging, gene/protein expression, and functional (e.g., metabolic) assays. For in vivo translation, ongoing work is focused on comparing bone regeneration and neovascularization across the scaffold types via micro-computed tomography (micro-CT), histology, and immunohistochemistry. We hypothesize that cryogel-infused β-TCP lattice composites will enhance vascularized bone regeneration by integrating the mechanical strength of ceramic lattices with the bioactivity of cryogels.

Materials and

Methods: : Composite Scaffold

Design:
Building on our previous work, a gyroid lattice was fabricated via digital light processing (DLP) 3D-printing using a commercial base resin (Tethon) mixed with 45% w/w β-TCP powder and sodium dodecyl sulfate. Composite scaffolds were created by infiltrating the lattices with a 4% chitosan–gelatin cryogel, as previously published. Following fabrication, scaffolds were evaluated for compressive strength, swelling ratio, and pore size (N=3/group). Additionally, composite scaffolds were customized to fit patient-specific CT scans, with each composite tailored for a unique mandibular defect (N=3/lattice).

In Vitro Co-Culture Studies:
Scaffolds were seeded with either HUVECs, MSCs, or a HUVEC/MSC co-culture and cultured on one of three scaffold types (cryogel, β-TCP lattice, or cryogel–β-TCP composite) for 3, 7, or 14 days. Mono-cultures were seeded with 50,000 cells each, while co-cultures were seeded with a total of 50,000 cells at a 1:4 MSC:HUVEC ratio. Outcome analyses included Live/Dead imaging, metabolic activity assays, immunofluorescence staining, alkaline phosphatase activity, Alizarin Red staining, qPCR, ELISA, and Western blotting.

Animal Study:
Ongoing in vivo studies involve implanting cryogel–β-TCP composites into Sprague-Dawley rat mandibular defects (5×5×3 mm; N=6/group/timepoint). Experimental groups include empty defect, cryogel only, β-TCP only, and cryogel–β-TCP composite scaffolds. Animals will be euthanized at 4 and 8 weeks for analysis by micro-CT for bone volume and mineral density, histological assessment (H&E, Trichrome) to evaluate bone formation, and immunohistochemical staining (Runx2, CD31, OCN) for vascular and osteogenic factors.

Results, Conclusions, and Discussions:: Scaffold

Design:
Gyroid lattices were successfully fabricated into β-TCP scaffolds via DLP printing. Further, cryogel infiltrated β-TCP lattice composite scaffolds (Fig. 1A) had an average compressive modulus of 0.18 MPa, a relative swelling ratio of 500%, and an average pore diameter of 111 µm. The swelling ratio is significantly lower than that of cryogels alone, while the pore diameter shows no statistically significant difference. Through patient-specific CT fabrication, scaffolds were shown to maintain structural integrity and anatomical fit as evaluated by a maxillofacial surgeon, supporting translational feasibility (Fig. 2).

In Vitro Co-Culture Studies:
Immunofluorescence staining showed that the composite scaffolds supported high cell viability and function, indicated by abundant cells, well-spread actin filaments, and continuous CD31 expression outlining cell junctions (Fig. 1B). MSC–HUVEC co-cultures on composite scaffolds exhibited enhanced osteogenic and angiogenic marker expression with modified alkaline phosphatase activity and calcium deposition, as well as Runx2, VEGF-A, and OCN expression. Ongoing work is evaluating ELISA and Western blot data to verify an increased secretion of VEGF and BMP-2, as well as higher expression of osteogenic proteins, indicating osteo-angiogenic crosstalk promoted by the composite scaffold environment.

Animal Study:
Building on unpublished proof-of-concept studies demonstrating the regenerative potential of cryogel scaffolds (only) in a rat calvarial defect model (Fig. 3), ongoing work has transitioned to a mandibular defect model to incorporate the composite scaffold in a complex geometry. This current work is focused on assessing in vivo bone regeneration through micro-CT, as well as histological and immunohistochemical analyses to evaluate new bone formation and vascularization. Based on in vitro results and early in vivo data, the composite scaffolds are anticipated to promote greater regeneration than either material when used individually.


Conclusions:
This work demonstrates that infiltrating 3D-printed β-TCP lattices with cryogel results in mechanically stable, biologically favorable scaffolds capable of promoting vascularized bone regeneration. In vitro studies confirmed enhanced MSC–HUVEC co-culture viability, signaling, and differentiation on composite scaffolds. Ongoing animal studies aim to validate these findings in a complex, clinically relevant mandibular defect model. Overall, these results support cryogel–β-TCP composites as a promising platform for clinical mandibular bone repair.

Acknowledgements and/or References (Optional):: Acknowledgements:
Thank you to Dartmouth Biomedical Engineering Center for access to their Instron compression testing machine.
Thank you to Dr. Linqing Li's group for collaboration on this project.


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