Overcoming the detrimental impact of volumetric muscle loss on segmental fracture healing via the induced membrane technique

Abstract

Aims: Open fractures pose a substantial treatment challenge, with adjacent muscle loss being a major complication. The induced membrane (IM) technique has shown promise in treating complicated fractures. The aim of this study is to investigate the impact of adjacent muscle trauma on segmental fracture healing using recombinant human bone morphogenetic protein-2 (rhBMP-2) via the IM technique.

Methods: Skeletally mature male rats (n = 10 to 11 per group) underwent unilateral 3 mm segmental bone defects (SBD) of the tibial diaphysis or a composite tissue injury (CTI), which included a SBD along with volumetric muscle loss (VML). A polymethyl methacrylate (PMMA) spacer was formed within the SBD of each rat. After a four-week period, the PMMA spacer was removed, and the defect was treated with a rhBMP2-impregnated collagen sponge. Longitudinal micro-CT (µCT) imaging was conducted at baseline (Day 0) and at weeks 2, 4, 8, and 12 post-spacer removal to monitor fracture healing progress. At the 12-week postoperative mark, a comprehensive analysis was conducted, including endpoint µCT analysis, evaluation of neuromuscular function, tibia torsional testing, and histological examination.

Results: Longitudinal µCT scans revealed no differences in bone formation or bone mineral density (BMD) at any timepoint between the SBD and CTI groups. High-resolution µCT analysis at the endpoint also showed no variations in bone quality. Torsion testing confirmed that VML did not affect bone strength. Notably, CTI animals exhibited an irreversible reduction in muscle mass and neuromuscular function, which was not observed in the SBD group.

Conclusion: Introducing the additional challenge of VML alongside SBD did not hinder the effectiveness of the induced membrane technique in healing a critical-sized defect.

Fig. 1

Induced membrane (IM) technique for segmental bone defects (SBD) with and without volumetric muscle loss (VML). a) Schematic of the surgical steps involved in the IM technique for both SBD and SBD+VML. b) Timeline of the injury and surgical phases of the IM technique. SBDs are fixed with a polyether ether ketone (PEEK) plate and polymethyl methacrylate (PMMA) spacer immediately after injury. The spacer is removed four weeks later, revealing the induced membrane and bone void. rhBMP-2, recombinant human bone morphogenetic protein-2.

Fig. 2

Muscle function and mass compared after segmental bone defect (SBD) and composite tissue injuries (CTIs). a) Neuromuscular torque production, presented as normalized torque per body weight, in response to increasing stimulation frequency. b) Tibialis anterior (TA) muscle weights from SBD, CTI, and their respective contralateral limbs (cSBD, cCTI) at 12 weeks post-injury. c) Maximum torque normalized to TA muscle weight, measured from limbs harvested 12 weeks post-injury. Data were analyzed using analysis of variance followed by Holm-Šídák post hoc tests. Data are represented as means with error bars representing SDs.

Fig. 3

Longitudinal analysis of fracture healing in segmental bone defect (SBD) versus composite tissue injuries (CTI) using micro-CT (μCT). a) Representative μCT scans of SBD and CTI injuries at zero, two, four, eight, and 12 weeks post-injury. b) Changes in bone volume (BV) over the 12-week healing period for SBD and CTIs, following PMMA spacer removal. c) Mean bone mineral density (BMD) throughout the healing period.

Fig. 4

Ex vivo analysis of fracture healing in segmental bone defect (SBD) vs composite tissue injuries (CTIs) at 12 weeks using micro-CT (μCT). a) Representative μCT scans (X-ray and 3D reconstruction) of SBD and CTIs at 12 weeks post-injury. b) Changes in bone mineral density (BMD), bone volume (BV), bone surface (BS), and object number (Obj.N) for SBD and CTI injuries over the 12-week healing period. c) Analysis of trabecular bone structure parameters (trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular spacing (Tb.S), and degree of anisotropy) at the 12-week endpoint.

Fig. 5

Biomechanical testing of segmental bone defect (SBD) and composite tissue injury (CTI) tibial bones. a) Schematic of the torsion apparatus used to evaluate bone strength. b) Maximum torque at failure (lbs·in). c) Stiffness calculated as torque divided by angle at fracture (lbs·in/deg). Data are represented as mean (SD) of n = four to seven biological replicates.

Fig. 6

Histological evaluation of fracture healing in segmental bone defect (SBD) versus composite tissue injuries (CTIs). a) Haematoxylin and eosin (H&E) staining to assess cellular infiltration and tissue morphology. b) Safranin O/Fast Green staining to evaluate woven bone formation in the fracture callus. c) Mason’s Trichrome (MT) staining to assess mineralized tissue content and collagen fibre orientation.