Autologous minced muscle grafts improve endogenous fracture healing and muscle strength after musculoskeletal trauma

Abstract

The deleterious impact of concomitant muscle injury on fracture healing and limb function is commonly considered part of the natural sequela of orthopedic trauma. Recent reports suggest that heightened inflammation in the surrounding traumatized musculature is a primary determinant of fracture healing. Relatedly, there are emerging potential therapeutic approaches for severe muscle trauma (e.g., volumetric muscle loss [VML] injury), such as autologous minced muscle grafts (1 mm³ pieces of muscle; GRAFT), that can partially prevent chronic functional deficits and appear to have an immunomodulatory effect within VML injured muscle. The primary goal of this study was to determine if repair of VML injury with GRAFT rescues impaired fracture healing and improves the strength of the traumatized muscle in a male Lewis rat model of tibia open fracture. The most salient findings of the study were: (1) tibialis anterior (TA) muscle repair with GRAFT improved endogenous healing of fractured tibia and improved the functional outcome of muscle regeneration; (2) GRAFT repair attenuated the monocyte/macrophage (CD45⁺CDllb⁺) and T lymphocyte (CD3⁺) response to VML injury; (3) TA muscle protein concentrations of MCP1, IL-10, and IGF-1 were augmented in a proregenerative manner by GRAFT repair; (4) VML injury concomitant with osteotomy induced a heightened systemic presence of alarmins (e.g., soluble RAGE) and leukocytes (e.g., monocytes), and depressed IGF-1 concentration, which GRAFT repair ameliorated. Collectively, these data indicate that repair of VML injury with a regenerative therapy can modulate the inflammatory and regenerative phenotype of the treated muscle and in association improve musculoskeletal healing.

Keywords: Inflammation; insulin‐like growth factor‐1; orthopedic trauma; regenerative medicine; skeletal muscle injury; volumetric muscle loss.

Figure 1

Autologous minced muscle grafts restores aggregation of mineralized content at the site of fracture following severe musculoskeletal trauma. (A) Digitalized fracture site renderings acquired by μ CT 28 days postinjury from tibia osteotomy (OST) and osteotomy + tibialis anterior (TA) muscle volumetric muscle loss (VML) injury with no repair (OST + VML) or minced graft repair of the VML injury (OST + VML + GRAFT) groups. (B) Quantified mineralized content from μ CT images at the fracture site. Values are mean ± SEM. Groups denoted with different letters are significantly different (P < 0.05), while those denoted with similar letters are statistically similar (P > 0.05).

Figure 2

Tibia mechanical properties are augmented by repair of volumetric muscle loss (VML) injury with autologous minced muscle grafts (GRAFT). Three point bending was performed on tibia isolated 28 days postinjury and analyzed for (A) maximal load and (B) stiffness. Values are mean ± SEM. Groups denoted with different letters are significantly different (P < 0.05), while those denoted with similar letters are statistically similar (P > 0.05).

Figure 3

Tibialis anterior muscle isometric torque is partially restored after minced muscle graft repair. In vivo isometric torque was assessed as a function of stimulation frequency in (A) contralateral and (B) injured limbs 28 days postinjury. Values are mean ± SEM. * All values from each group are < respective values from OST; ^† values are < OST + VML + GRAFT; P < 0.05

Figure 4

Autologous minced grafts promote muscle fiber regeneration. (A) Cross‐sections from the middle of the volumetric muscle loss (VML) defect region in tibialis anterior muscles harvested 28 days postinjury were stained with H&E and qualitatively analyzed. (B) Magnified regions specified by border outline (solid or dashed) from each respective muscle group. Scale bars = 100 μm.

Figure 5

Acute myeloid cellular response to tibialis anterior (TA) muscle volumetric muscle loss (VML) injury is damped by muscle graft repair. Flow cytometry was performed in cell isolates from the middle third of the TA muscle 3 and 14 days postinjury (time postinjury; TPI). (A) hemaptopoietic cells (CD45⁺), (B) Monocytes/Macrophages (CD45⁺ CD11b⁺), (C) M1 macrophages (CD45⁺ CD11b⁺ CD86⁺), and (D) M2 macrophages (CD45⁺ CD11b⁺ CD163⁺) were quantified per group. Values are mean ± SEM. Groups denoted with different letters are significantly different (P < 0.05), while those denoted with similar letters are statistically similar (P > 0.05).

Figure 6

Autologous muscle graft repair differentially modulates T lymphocyte infiltration at acute and intermediate times postinjury. Flow cytometry was performed in cell isolates from the middle third of the tibialis anterior (TA) muscle 3 and 14 days after injury (time postinjury; TPI). (A) lymphocytes (CD3⁺), (B) T helper (CD3⁺ CD4⁺), (C) T cytotoxic (CD3⁺ CD8⁺) lymphocytes were quantified per group. Values are mean ± SEM. Groups denoted with different letters are significantly different (P < 0.05), while those denoted with similar letters are statistically similar (P > 0.05).

Figure 7

Tibialis anterior (TA) muscle inflammatory and growth factor concentrations a modulated by volumetric muscle loss (VML) injury and muscle graft repair. (A) TNF‐α, (B) IL‐6, (C) MCP‐1, (D) IL‐10, and (E) IGF‐1 were measured within the middle third of the TA muscle 3 days postinjury. Values are mean ± SEM. Groups denoted with different letters are significantly different (P < 0.05), while those denoted with similar letters are statistically similar (P > 0.05).

Figure 8

Heightened alarmin protein and circulating immune cells induced by concomitant volumetric muscle loss (VML) injury are selectively attenuated by muscle graft repair. Blood was collected 3 days postinjury and analyzed for (A and B) serum alarmin proteins or (C and D) circulating immune cells. Values are mean ± SEM. Groups denoted with different letters are significantly different (P < 0.05), while those denoted with similar letters are statistically similar (P > 0.05).

Figure 9

Autologous minced grafts augment circulating IGF‐1 concentrations. IGF‐1 was measured in blood serum collected 3 days postinjury. Values are mean ± SEM. Groups denoted with different letters are significantly different (P < 0.05), while those denoted with similar letters are statistically similar (P > 0.05).