Three-Dimensional-Printed Bone Grafts for Simultaneous Bone and Cartilage Regeneration: A Promising Approach to Osteochondral Tissue Engineering

Abstract

Background/Objectives: A novel 3D-printed, bioresorbable bone graft, made of nanohydroxyapatite (nHAP) covered by poly(lactide-co-glycolide) (PLGA), showed strongly expressed osteoinductive properties in our previous investigations. The current study examines its application in the dual regeneration of bone and cartilage by combining with nHAP gel obtained by nHAP enrichment with hydroxyethyl cellulose, sodium hyaluronate, and chondroitin sulfate. Methods: In the in vitro part of the study, the mitochondrial activity and osteogenic and chondrogenic differentiation of stem cells derived from apical papilla (SCAPs) in the presence of nHAP gel were investigated. For the in vivo part of the study, three rabbits underwent segmental osteotomies of the lateral condyle of the femur, and defects were filled by 3D-printed grafts customized to the defect geometry. Results: In vitro study revealed that nHAP gel displayed significant biocompatibility, substantially increasing mitochondrial activity and facilitating the osteogenic and chondrogenic differentiation of SCAPs. For the in vivo part of the study, after a 12-week healing period, partial resorption of the graft was observed, and lamellar bone tissue with Haversian systems was detected. Histological and stereological evaluations of the implanted grafts indicated successful bone regeneration, marked by the infiltration of new bone and cartilaginous tissue into the graft. The existence of osteocytes and increased vascularization indicated active osteogenesis. The hyaline cartilage near the graft showed numerous new chondrocytes and a significant layer of newly formed cartilage. Conclusions: This study demonstrated that tailored 3D-printed bone grafts could efficiently promote the healing of substantial bone defects and the formation of new cartilage without requiring supplementary biological factors, offering a feasible alternative for clinical bone repair applications.

Keywords: 3D printing; animal model; bone reconstruction; cartilage regeneration; nanohydroxyapatite; segmental osteotomy; stem cells.

Figure A1

Schematic representation of the extruder design in a custom-made laboratory 3D printer: from technical reasons, the 3D printer was modified to include two extruders: 1—syringe extruder; 1.1—glass syringe; 1.2—external frame of the extruder 1; 1.3—electrical heater coil; 1.4—metal piston of the syringe; 1.5—syringe nozzle (0.8 mm in diameter); 1.6—gears of the piston drive; 1.7—piston drive motor with reduction; 1.8—HAP PLGA mixture; 2—PLGA extruder; 2.1—heater block of the PLGA extruder; 2.2—extruder cooler; 2.3—PLGA extruder motor drive with reduction; 2.4—PLGA filament 1.75 mm in diameter; 2.5—PLGA extruder nozzle 0.4 mm in diameter; 3—paste extruder attached to the head of the printer, with removed electrical heater coil, so the syringe with the metal piston can be visible. (Figure originates from the PhD thesis: Micic M. [2023]. Influence of the choice of protocols on the accuracy of three-dimensional medical models, surgical guides, and bone replacement. School of Medicine, University of Belgrade, National Repository of Dissertations in Serbia, https://nardus.mpn.gov.rs/bitstream/id/155980/Disertacija_14177.pdf. accessed on 1 April 2025).

Figure 1

Surgery planning procedure in the Autodesk 3D Max 2010 software. Numbers 1–3: The identification numbers of every experimental animal and 3D model of the lateral femur reconstruction graft with the fixation hole. 4: The 3D appearance of the rabbit’s femur. 5: The surgical guide for the resection of the lateral condyle (green) with the fixation holes for fixation screws (pink). 6: The planned resection plane (transparent red) of the lateral condyle.

Figure 2

Three-dimensional modeling of the desired porosity: (A)—Forming the planar maze structure in X- and Y-axis directions in software and stacking it in Z direction (green and blue maze layers); (B)—Extruding maze structure in Z-axis direction to form a 3D structure and stacking the formed structures on each other in Z-axis direction using Autodesk 3D Max (green and blue maze layers); (C)—After removing the outer walls of the stacked maze structure in Autodesk 3D Max, the 3D model of the bone construct structure with 50% porosity and trabecular thickness of 225 μm is given in transparent form.

Figure 3

Virtual planning of the surgery procedure: (A)—surgical guide (SG) (green) placement on the rabbit femur (white), cutting plane (transparent red) predicted by the position of the SG, fixation screws (blue) for the SG; (B)—SG (green) fixed on the femur (white), ensuring the precise cut, lateral condyle of the femur is removed; (C)—SG (green) still in place of the planned defect site, 3D graft (pale pink) is placed and fixed with the fixation screw (blue).

Figure 4

Mitochondrial activity of grade concentrations of nHAP gel and control, after 24 h, ns—not significant.

Figure 5

Chondrogenic and osteogenic differentiation of SCAPs after 7 days of culturing in induction mediums, enriched with 0.125% of nHAP gel, and control. Magnification 40×; * p < 0.05.

Figure 6

Longitudinal section of lateral femur condyle with implanted 3D graft, H&E, magnification 20×; locations of graft (red arrows), chondrocytes (black arrows), and fibroblasts (yellow arrows).

Figure 7

Cartilaginous tissue in longitudinal sections of the lateral femur condyles: (a) control and (b) with 3D graft, H&E, magnification 50×; chondrocytes (red arrows) and lacunae with fragments of implanted material (black arrows).

Figure 8

Cartilaginous tissue in the longitudinal section of the lateral femur condyle: (a) control and (b) with 3D graft, stained with immunohistochemical technique Picrosirius red, magnification 50×; chondrocytes (red arrows), fibroblasts (yellow arrows), and fragments of graft (black arrows).

Figure 9

(a) Longitudinal section of the articular femur surface with 3D graft: newly formed irregular cartilage (blue arrow) and regular cartilage in contact with bone tissue (white arrow), immunohistochemical Goldner stain, magnification 10×; (b) longitudinal section of the articular femur surface with 3D graft: regular cartilage structure (black arrow) and newly formed irregular cartilage structure (yellow arrows), Toluidine blue stain, magnification 10×.

Figure 10

Longitudinal section of a femur with 3D graft, stained with immunohistochemical technique Picrosirius red, magnification 20×: osteocytes (white arrows), fibroblasts (black arrows), and location of implanted material (red arrows).

Figure 11

Cross-section of femur with implanted 3D graft, H&E, magnification 100×: osteocytes (white arrows), fibroblasts with cytoplasmic extensions (red arrow), magnified junction of femur bone tissue and 3D graft (black arrow and black square), and location of implanted graft (yellow arrows).

Figure 12

Cross-section of lateral femur condyle with implanted graft, stained with immunohistochemical technique Picrosirius red, magnification 100×: osteocytes (yellow arrows) and location of implanted material (black arrows).

Figure 13

Cross-section of lateral femur condyle in control (a) and with implanted graft (b), H&E, magnification 200×: osteocytes (white arrows) and blood vessels (yellow arrows).

Figure 14

Immunoreactivity to osteocalcin. Representative micrographs of femur tissue stained with an immunohistochemical technique for the visualization of osteocalcin: longitudinal section of femur without an implant (a) and femur with an implanted graft (b). White arrows indicate the sites of immunoreaction of bone tissue to osteocalcin, magnification 100×.

Figure 15

Collagen deposition in bone tissue. Representative micrographs of bone tissue stained with histochemical technique Picrosirius red: control bone (a) and bone with implanted graft (b), 100× magnification.

Figure 16

Relative gene expression (RGE) levels of chondrogenic (ITM2A, FOXC1, and SOX9) and osteogenic (BMP4, RUNX2, and ALP) markers in tissues with implanted material compared to control; ns—not significant, * p < 0.05, and ** p < 0.01.