An innovative intramedullary bone graft harvesting concept as a fundamental component of scaffold-guided bone regeneration: A preclinical in vivo validation

Abstract

Background: The deployment of bone grafts (BGs) is critical to the success of scaffold-guided bone regeneration (SGBR) of large bone defects. It is thus critical to provide harvesting devices that maximize osteogenic capacity of the autograft while also minimizing graft damage during collection. As an alternative to the Reamer-Irrigator-Aspirator 2 (RIA 2) system - the gold standard for large-volume graft harvesting used in orthopaedic clinics today - a novel intramedullary BG harvesting concept has been preclinically introduced and referred to as the ARA (aspirator + reaming-aspiration) concept. The ARA concept uses aspiration of the intramedullary content, followed by medullary reaming-aspiration of the endosteal bone. This concept allows greater customization of BG harvesting conditions vis-à-vis the RIA 2 system. Following its successful in vitro validation, we hypothesized that an ARA concept-collected BG would have comparable in vivo osteogenic capacity compared to the RIA 2 system-collected BG.

Methods: We used 3D-printed, medical-grade polycaprolactone-hydroxyapatite (mPCL-HA, wt 96 %:4 %) scaffolds with a Voronoi design, loaded with or without different sheep-harvested BGs and tested them in an ectopic bone formation rat model for up to 8 weeks.

Results: Active bone regeneration was observed throughout the scaffold-BG constructs, particularly on the surface of the bone chips with endochondral bone formation, and highly vascularized tissue formed within the fully interconnected pore architecture. There were no differences between the BGs derived from the RIA 2 system and the ARA concept in new bone volume formation and in compression tests (Young's modulus, p = 0.74; yield strength, p = 0.50). These results highlight that the osteogenic capacities of the mPCL-HA Voronoi scaffold loaded with BGs from the ARA concept and the RIA 2 system are equivalent.

Conclusion: In conclusion, the ARA concept offers a promising alternative to the RIA 2 system for harvesting BGs to be clinically integrated into SGBR strategies.

The translational potential of this article: Our results show that biodegradable composite scaffolds loaded with BGs from the novel intramedullary harvesting concept and the RIA 2 system have equivalent osteogenic capacity. Thus, the innovative, highly intuitive intramedullary harvesting concept offers a promising alternative to the RIA 2 system for harvesting bone grafts, which are an important component for the routine translation of SGBR concepts into clinical practice.

Keywords: Bone graft; Bone regeneration; Intramedullary harvesting; Polycaprolactone; Scaffold; Voronoi.

Graphical abstract

Figure 1

Clinically relevant intramedullary harvesting methods for obtaining BGs from the sheep femur and illustration of the study design in terms of the experimental group composition. Please note the RA option was applied following the removal of bone marrow. The mPCL-HA Voronoi scaffolds alone as well as the mPCL-HA Voronoi scaffolds loaded with BGs obtained from the intramedullary canal of sheep femora were additionally loaded with 120 μL fibrin glue. ARA concept, aspirator + reaming-aspiration concept; RIA 2 system, Reamer-Irrigator-Aspirator 2 system; Sc, scaffold. Adapted from Ref. [21]. Partially created with BioRender.com.

Figure 2

Macroscopic assessments of in situ specimens and μCT quantification of total bone volume. During specimen retrieval, excellent tissue integration in all the experimental in vivo groups and particularly pronounced ingrowth of blood vessels from surrounding tissue (white triangles) was evidenced (A). In the groups loaded with different types of BG (ScRIA2, ScRA, and ScARA), representative images of reconstructed μCT data show homogenous distribution of the BG throughout the Voronoi scaffolds (B). Please note that the samples of the ScRIA2 and ScRA groups show similar total bone volume; in the ScARA group, about half of the total bone volume is observed compared to the ScRIA2 and ScRA groups (C). Scale bars: B, 1000 μm. Post hoc test Tukey method corrected p-values: * <0.05, ** <0.001.

Figure 3

Micro-computed tomography and histological and IHC analysis (inflammatory markers) of explanted mPCL-HA Voronoi scaffolds alone and loaded with different types of BG. Three-dimensional reconstruction of the μCT data of the sample groups after specimen collection (A1–A4). H&E overview (B1–B4) and IHC inflammatory markers (C1–E4). Dashed lines indicate original bone chips. CD68, Cluster of differentiation 68; H&E, hematoxylin and eosin; iNOS, inducible nitric oxide synthase; μCT, micro-computed tomography; MR, mannose receptor. Scale bars: B1–B4, 2000 μm; C1–E4, 50 μm.

Figure 4

Histological and IHC analysis of explanted specimens. H&E high magnification (A1–A4) and IHC bone formation, vascularization and osteoclastic markers (B1–F4). Dashed lines indicate original bone chips. COL I, collagen type I; COL II, collagen type II; H&E, hematoxylin and eosin; OC, osteocalcin; TRAP, tartrate-resistant acid phosphatase; vWF, von Willebrand factor. Scale bars: 50 μm.

Figure 5

Osteogenesis throughout the scaffold-alone and scaffold-BG constructs assessed with GT (A1–B4), CLSM (C1–C4), SHG (D1–D4) and SEM imaging (E1–E4). Please note the different findings of GT staining: green staining indicates remnants of bone chips, vascularization is indicated by yellow arrows (in B2–B4), red staining indicates osteoid deposits and light blue staining indicates woven bone formation (B1–B4). Dashed lines indicate original bone chips. GT, Goldner's trichrome; CLSM, confocal laser scanning microscopy; SHG, second-harmonic generation; SEM, scanning electron microscopy. Scale bars: A1–A2, 2000 μm; A3–A4, 1000 μm; B1–B4, 50 μm; C1–C4, 100 μm; D1, 200 μm; D2, 200 μm; D3, 250 μm; D4, 250 μm; E1–E4, 20 μm.

Figure 6

New bone formation and biomechanical analysis. New bone formation quantified from μCT imaging data (A). Exemplary stress–strain curves and sample photos following compression testing (B). Unconfined biomechanical compression testing under simulated physiological conditions showed no significant differences in the one-way ANOVA analysis between the experimental groups for the Young's modulus (C) and yield strength (D). CTRL, control scaffold (not implanted).