**1. Introduction**

Heterotopic ossification (HO) is a disregulation of skeletal muscle homeostasis and regeneration, which results in mature bone formation in atypical locations. HO could develop in the skeletal muscles, and also in surrounding tissues such as fascia, tendons, skin, and subcutis [1]. HO can be acquired or have genetic origin. The most prevalent is acquired HO which can occur in response to a direct trauma, burn, or amputations. Similarly, iatrogenic trauma, caused by orthopedic surgery such as hip replacement, often triggers HO development [1,2]. Another acquired form of the disease is neurogenic HO (NHO) which is a frequent complication of central nervous system injury [3]. The knowledge about the molecular mechanisms that leads to HO formation and cell precursors engaged in this process is still limited. HO requires the presence of stem or progenitor cells which are able to follow the osteogenic program, although the identity of these cells remains unclear. Many different populations of progenitor cells could be possible precursors in the HO development. The animal studies suggest that progenitor cells can vary depending on the HO subtype. The studies using mouse HO models show that endothelial cells, mesenchymal cells, pericytes present in the skeletal muscles, tendons and connective tissue cells, or even

circulating stem/precursor cells could be a source of HO precursors [1,4,5]. It is also known, that trauma or micro-trauma, which leads to a local inflammatory response, delivers the signals to develop HO. Recent studies showed the role of immune response cells, especially monocytes/macrophages, at the early stages of trauma-induced HO development [6]. They confirm the importance of macrophages in the induction of neurogenic and genetic forms of HO [7]. Activated macrophages express osteoinductive signaling factors in the course of HO pathogenesis. Thus, the presence of the cells reflects increased secretion of HO promoting cytokines/chemokines such as interleukin 6 (IL6), IL10, transforming beta-1 growth factor (TGFβ1), and neurotrophin 3 (NT3). A significant dysregulation of macrophage immune checkpoints was proven in HO animal models [8–10]. Finally, both individual predisposition and risk factors also attribute to HO development [11].

Histologically HO formation is similar to the physiological bone fracture healing. During HO development initially soft tissue is infiltrated with the whole spectrum of inflammatory cells. Such infiltration is followed by enhanced fibroblast proliferation, neovascularization, differentiation of chondrocytes, and results in mature bone formation [12]. HO is formed mainly by endochondral ossification. However, an intramembranous mechanism can also be involved. Typically HO formation is characterized by a zonal bone development model called "eggshell calcification" [13]. HO consists mainly of mechanically weak woven bone with an irregular osteoblasts distribution, but mature lamellar bone with Haversian-like canals can be often found. The bone tissue gradually matures with the outer appearance of the cortical bone [14]. Primarily, HO occurs in soft tissues and has no connection with the skeletal bone, but when it grows in the volume, it can attach to the periosteum.

#### **2. Heterotopic Ossification as a Clinical Issue**

HO is a diverse pathologic process and its spectrum can range from mild, clinically irrelevant to severe cases. In most of the patients it is minor and symptomless. Unfortunately, in some patients, extensive HO located around joints can cause restrictions in the range of motion (ROM), resulting even in the total ankylosis of the joint. In this group of patients HO can be associated with a significant limitation of daily activities and disability [1].

#### *2.1. Traumatic HO*

HO lesions can occur in response to direct trauma, such as connective tissue injury, bone fractures, burns, amputations, and combat-related blast injuries [2]. Approximately 30% of all fractures and dislocations which were subjected to operative treatment can trigger HO formation. It was a recognized clinical problem, in the acetabular and proximal femur fractures and fractures or dislocations of the elbow [1,15]. HO is also a common complication of traumatic limb amputation, both in civilian (22.8%) and in a military setting (62.9%) [16]. After the isolated burn injury HO incidence is relatively low (1%–4%), but it can be underestimated due to the lack of routine x-ray screening of such patients [17,18]. The typical locations of burn-induced HO are the elbow (50.0%), glenohumeral joint (20.3%), and the hip joint (17.6%) [18].

Recognized risk factors for trauma-induced HO are young age, male sex, severe concomitant injuries, compound fractures, extensive surgical approaches, and postponed surgeries [19,20]. The presence of local wound infection is also a well-established factor associated with HO [21]. In the military setting, the high incidence of HO is associated with concomitant brain injury, multiple wounds, and the severity of the injury [2]. The extent of burns, local wound infection, and the duration of intensive therapy are the risk factors for the formation of burn-induced HO. The role of immobilization and iatrogenic paralysis is also under investigation [18,22].

As far as the symptoms are concerned it can be associated with reduced joint mobility, pain, and decreased limb function. In the upper extremity, HO can limit everyday activities such as eating, dressing, and personal grooming, while in the lower limb it can affect gait and cause limp and difficulties in sitting [15]. Patients with amputation associated HO may experience difficulties with prosthesis fitting. Other local complications can occur, such as ulcers, skin graft necrosis, and neurovascular

impairment [16]. The contractures and reduction of joint ROM in burn injuries are often caused by soft tissue scarring. However, HO should always be taken into consideration in differential diagnosis [18].

#### *2.2. Surgery-Induced HO*

HO is a well-described complication of orthopedic surgical procedures, typically joint replacements. The main indication for this kind of treatment is symptomatic, end-stage joint osteoarthritis. This type of HO usually involves the tissues where the surgical approach is performed, as unavoidable trauma is done to the muscle and fascia. It is most commonly described after a total hip replacement (THR) and cervical total disc arthroplasty (CTDA). It was also reported after the ankle, knee, and shoulder arthroplasty [23–25]. HO is radiologically present in every second patient after total hip replacement (40%–56%), and cervical total disc arthroplasty (44.6%–58.2%). However, high-grade HO occurs only in 2%–7% of total hip replacement and 11%–16% of cervical total disc arthroplasty patients [24–26].

The risk factors for the development of HO in patients undergoing total hip replacement are young age and male sex. The other predisposing conditions are bone and joint diseases such as ankylosing spondylitis, hypertrophic arthritis, and Paget's disease. The impact of the surgical approach, especially micro-invasive surgery (MIS) techniques, is being intensively discussed, but the results are still inconclusive [24,27,28]. Similarly, in patients after the cervical total disc arthroplasty, the male sex is an independent risk factor for HO development. Another important aspect is an artificial disc device type [29].

In the majority of patients suffering from surgery induced HO, small islands of the bone of no clinical significance are observed. However, extensive lesions can affect the biomechanical function of an endoprosthesis and block the movement in the affected joint. In total hip replacement patients, high-grade HO can significantly impact ROM, especially flexion, abduction, and external rotation, and affect the overall function of the hip [30]. In extreme cases, HO can require surgical intervention and excision (3.3%) [27]. In contrast to total hip replacement, in cervical total disc arthroplasty patients, severe HO does not affect patient-related pain, quality of life, or function [31].
