Time Course of Immune Response and Immunomodulation During Normal and Delayed Healing of Musculoskeletal Wounds

Abstract

Single trauma injuries or isolated fractures are often manageable and generally heal without complications. In contrast, high-energy trauma results in multi/poly-trauma injury patterns presenting imbalanced pro- and anti- inflammatory responses often leading to immune dysfunction. These injuries often exhibit delayed healing, leading to fibrosis of injury sites and delayed healing of fractures depending on the intensity of the compounding traumas. Immune dysfunction is accompanied by a temporal shift in the innate and adaptive immune cells distribution, triggered by the overwhelming release of an arsenal of inflammatory mediators such as complements, cytokines and damage associated molecular patterns (DAMPs) from necrotic cells. Recent studies have implicated this dysregulated inflammation in the poor prognosis of polytraumatic injuries, however, interventions focusing on immunomodulating inflammatory cellular composition and activation, if administered incorrectly, can result in immune suppression and unintended outcomes. Immunomodulation therapy is promising but should be conducted with consideration for the spatial and temporal distribution of the immune cells during impaired healing. This review describes the current state of knowledge in the spatiotemporal distribution patterns of immune cells at various stages during musculoskeletal wound healing, with a focus on recent advances in the field of Osteoimmunology, a study of the interface between the immune and skeletal systems, in long bone fractures. The goals of this review are to (1) discuss wound and fracture healing processes of normal and delayed healing in skeletal muscles and long bones; (2) provide a balanced perspective on temporal distributions of immune cells and skeletal cells during healing; and (3) highlight recent therapeutic interventions used to improve fracture healing. This review is intended to promote an understanding of the importance of inflammation during normal and delayed wound and fracture healing. Knowledge gained will be instrumental in developing novel immunomodulatory approaches for impaired healing.

Keywords: delayed fracture healing; dysregulated inflammatory response; fracture healing; osteoimmunology; wound healing.

Figure 1

Classification of wounds/fracture and wound/fracture-healing outcomes.

Figure 2

Schematic illustration of the time course of immune cells and muscle cells during (A) normal and (B) delayed muscle healing/regeneration. The three phases of normal muscle healing are inflammatory phase: 0–7 days (yellow area), remodeling phase: 4–14 days (orange area), and regeneration and muscle growth phase: 14–28 days (blue area). Overlap of inflammatory and remodeling phases: 4–7 days (bright yellow area). Paired-box transcription factor 7 (Pax7) and myoblast determination protein (MyoD) are major players during muscle regeneration and are used as markers to indicate the activated (Pax7⁺MyoD⁺), differentiated (Pax7⁻MyoD⁺) and quiescent (Pax7⁺MyoD⁻) states of satellite cells. Delayed muscle regeneration is characterized by a prolonged inflammatory phase (yellow area) with continuous infiltration of macrophages (both M1 and M2), CD4⁺ helper T cells and CD8⁺ cytotoxic T cells; a short period of remodeling-like phase (orange area) and followed by fibrosis of the muscle wound area (green area). The time scale starts at the time of injury and extends through 46 days post-injury. M1 and M2 are the two different macrophage phenotypes pro- and anti- inflammatory, respectively; and 1 h denotes 1-h post-injury. This figure was created with BioRender.com.

Figure 3

Overview of the cellular and molecular events occurring during fracture healing. The interplay between stem cells, immune cells and skeletal cells (chondrocytes, osteoblasts, and osteoclasts) is required for efficient fracture healing. In long bone fractures repair occurs via two routes: primary healing mediated by intramembranous ossification and no callus/cartilage formation or via secondary healing mediated by endochondral ossification and with callus/cartilage formation. Typically, in primary healing the mesenchymal stem cells (MSCs) directly differentiate to osteoblasts, a process regulated by the transcription factor Runt-related transcription factor 2 (Runx2) and bone marrow resident macrophages/osteomacs. Whereas, in secondary healing MSCs differentiate into chondroblast and is regulated by the transcription factor SRY-related high mobility group-box gene 9 (Sox9) and M2 macrophages. Chondroblasts further differentiate to chondrocytes, which in turn differentiate to hypertrophic chondrocytes. Hypertrophic chondrocytes have a role in calcification of the cartilage matrix, angiogenesis, and vascular invasion. They differentiate into osteoblasts, via the induction of transcription factors like Runx2 and Sp7; TNFα; and other osteogenic mediators. Osteoblasts are bone forming cells and they mature into osteocytes which mineralize the bone matrix. During repair, Th17 cells and γδ T cells promote osteoblastogenesis via the secretion of cytokines like IL17F, and IL17A, TNFα and IL-6, respectively. Osteoblasts also regulate osteoclastogenesis via production of receptor activator of nuclear factor kappa-B (RANK) ligand/RANKL and osteoprotegerin (OPG). Osteoclasts are bone resorptive cells. They belong to hematopoietic stem cell (HSC) origin and are derived from macrophages and dendritic cells (DCs) in the presence of macrophage colony stimulating factor (M-CSF) and RANKL. CD4⁺ T helper (Th) cells also secrete RANKL and crosstalk with osteoclasts in order to regulate osteoclastic activity and vice versa. Functions of osteoclasts are highly regulated by the binding of their surface receptor RANK to either RANKL for activation or OPG for suppression. Regulatory T cells (Tregs) inhibit osteoblastogenesis and osteoclastogenesis, via secretion of IL4, IL10 and transforming growth factor β (TGFβ). Activated B cells activate osteoblastogenesis and osteoclastogenesis, while CD8⁺ T cells suppress osteoclastogenesis. The dotted arrows indicate indirect role; the solid arrows indicate direct role; and the double head arrow indicates cell to cell crosstalk. The red “T” lines indicate inhibition. This figure was created with BioRender.com.

Figure 4

Schematic illustration of the time course of inflammatory cells, immune cells and skeletal cells during (A) normal fracture healing and (B) delayed bone regeneration in conditions like (i) severe isolated fracture; (ii) polytrauma; and (iii) concomitant muscle loss. The three phases of normal fracture healing are Hematoma formation and inflammatory phase: 0–5 days (yellow area); Repair—Soft callus formation: 5–16 days (green area); and Hard callus formation: 6–21days (orange area); and Remodeling of hard callus to mature lamellar bone: 21–>63 days (blue area). Delayed bone healing is characterized by prolonged and dysregulated inflammatory phase which causes a significant delay to repair and remodeling of bone. The time scale starts at the time of injury and extends beyond 63 days post-injury. M1 and M2 are the two different macrophage phenotypes pro- and anti- inflammatory, respectively; 1 h denotes 1-h post-injury; and CD8⁺T_EMRA cells (CD8⁺ terminally differentiated effector memory T cells.