Severe muscle trauma triggers heightened and prolonged local musculoskeletal inflammation and impairs adjacent tibia fracture healing

Abstract

Objectives: Complicated fracture healing is often associated with the severity of surrounding muscle tissue trauma. Since inflammation is a primary determinant of musculoskeletal health and regeneration, it is plausible that delayed healing and non-unions are partly caused by compounding local inflammation in response to concomitant muscle trauma.

Methods and results: To investigate this possibility, a Lewis rat open fracture model [tibia osteotomy with adjacent tibialis anterior (TA) muscle volumetric muscle loss (VML) injury] was interrogated. We observed that VML injury impaired tibia healing, as indicated by diminished mechanical strength and decreased mineralized bone within the fracture callus, as well as continued presence of cartilage instead of woven bone 28 days post-injury. The VML injured muscle presented innate and adaptive immune responses that were atypical of canonical muscle injury healing. Additionally, the VML injury resulted in a perturbation of the inflammatory phase of fracture healing, as indicated by elevations of CD3(+) lymphocytes and CD68+ macrophages in the fracture callus at 3 and 14d post-injury, respectively.

Conclusions: These data indicate that heightened and sustained innate and adaptive immune responses to traumatized muscle are associated with impaired fracture healing and may be targeted for the prevention of delayed and non-union following musculoskeletal trauma.

Figure 1

Traumatized muscle impairs bone healing after open fracture. Three-point bending mechanical testing and µCT imaging of tibiae were performed at 28 days post-injury. A) Contralateral tibia maximum load and stiffness were similar among groups and no significant difference was observed between contralateral and affected tibia of the SHAM group. Significant differences were detected among affected limb groups. † affected OST vs. contralateral OST & affected SHAM; * affected OST+VML vs. contralateral OST+VML & affected SHAM and OST groups (p-values <0.05). B) Representative µCT images of OST and OST+VML tibiae are presented from which callus volume was calculated; * p<0.05. Values are means ± SEM; n=4 – 6 per group. C) Histology of tibia of affected limb. Top, middle, and bottom rows: H&E, Masson’s Trichrome, and Safrinin O. Scale bars = 200 µm.

Figure 2

VML injury leads to increased cellular infiltration to TA muscle. The TA muscle was isolated at 3, 14, and 28 days post-trauma from SHAM, OST, and OST+VML groups. Tissue sections were stained with H&E. Representative images of TA cross-sections and magnified regions (inset black boxes) displaying overall muscle morphology and cellular recruitment were collected. Black arrows denote degenerating muscle fibers. n=4 per group. Black scale bars=1.0 mm and white scale bars=100 µm.

Figure 3

Increased expression of genes involved in innate and adaptive immune responses following complex musculoskeletal injury. Clustergram analysis displaying gene expression values in TA muscle tissue from naïve, OST, and OST+VML groups 3 d post-trauma as determined by PCR array (PARN-052Z, SABiosciences). n=3 per group. Magnitude of gene expression is depicted by color. Green represents reduced expression, black represents no change, and red represents increased expression according to the average value of the gene in all samples.

Figure 4

Heightened and prolonged recruitment of innate immune cells displaying mixed M/M2 phenotype within VML injured muscle. The TA muscle was isolated from OST, OST+VML, and specified control groups at indicated times post-injury (TPI) for A) immunofluorescence, B-E) flow cytometric, and F) transcriptional analyses. A) For immunofluorescence analysis tissue sections were stained for laminin, CD68, and nuclei (DAPI); n=4 per group. Scale bars=50 μm. B) For flow cytometric analysis, the middle third of the TA muscle that contains the defect and neighboring area was collected, digested, and cellular content isolated. Muscle-derived cells were labeled with antibodies to identify myeloid cells and analyzed by flow cytometry. To identify pro- and anti-inflammatory myeloid cells, populations were initially gated (red polygon) to exclude cell debris and doublets. Selected single cell populations were gated based on CD45 expression, followed by CD11b to identify myeloid cells. Pro- and anti-inflammatory myeloid cells were subsequently gated (black bar) based on their expression of CD86 or CD163, respectively. Gates were established by fluorescence-minus-one plus isotype controls. Relative cell numbers of C) CD45+CD11b+ myeloid cells, D) CD45+CD11b+CD86+ pro-inflammatory myeloid cells, and E) CD45+CD11b+CD163+ antiinflammatory myeloid cells were determined. Dashed-lines represent average cell numbers detected within contralateral control tissue. Data are normalized per gram of tissue and presented as mean ± SEM. n=4 per group. *P<0.05 versus OST group. (F) Gene expression of M1 (CCR7 and iNOS) and (F) M2-like (ARG1, CD163, and MRC1) macrophage markers were determined in TA muscle was isolated from OST and OST+VML groups at 3 and 14 days post-trauma by qPCR. Tissue isolated from contralateral TA muscle was used to normalize data. Data are presented as mean ± SEM. *P<0.05 versus OST group.

Figure 5

VML injury triggers increased recruitment of T helper and cytotoxic lymphocytes to muscle defect region. The TA muscle was isolated from OST, OST+VML, and specified control groups at indicated times points for flow cytometric analysis. A) To identify T lymphocytes (red polygon), samples were gated to exclude debris and doublets (as previously described), followed by gating for CD3 expression. CD3⁺ cells were gated based on CD4 and CD8α expression to identify T helper and cytotoxic lymphocytes, respectively. Cells were analyzed by flow cytometry for B) CD3⁺CD4⁺ and C) CD3⁺CD8⁺ expression. Dashed-lines represent average cell numbers detected within contralateral control tissue. Data are normalized per gram of tissue and presented as mean ± SEM. n=4 per group. *P<0.05 versus OST group.

Figure 6

VML injury directly influences immune responses in the adjacent fracture callus. Affected tibiae from OST and OST+VML groups were harvested 3 and 14d post-injury for immunohistological analysis of CD68⁺ macrophage and CD3⁺ T-lymphocyte infiltration in the fracture callus. A & C) Representative images from the callus region for each group are presented. Scale bars=100 µm. (B & D) The number of CD68⁺ and CD3⁺ cells present in the callus was quantified from immunohistological sections. *p<0.05 vs. OST & OST+VML within the respective time point. Values are means ± SEM; n=3 per group.