**2. Immune cells and bone regeneration**

#### **2.1. Bone – a complex organ**

Bone is not simply a hard nonorganic material that functions as an anchor for muscles and tendons providing stability and form for our bodies and enabling movement through the interplay of our musculoskeletal system; it is also protecting vital organs, such as the brain, lungs, and heart, and it is a living organ regulating homeostasis. Additionally, it is an organ that is essential for our immune system, as these cells arise and/or mature from stem cells in the bone marrow, it is also an organ that interacts with our hormonal balance through a multitude of factors, including the hormone osteocalcin [35], and acts as a storage not only for calcium, phosphate, and magnesium but also for growth factors, as for example transforming growth factor-β (TGF-β).

**Figure 4. Bone is a complex organ**. A long bone can be divided into epi-, meta-, and diaphyseal regions. The epiphy‐ seal region contains the growth plate—the region of length growth of the bone. The epiphyseal zone is broad in young individuals and diminishes with age. Details are shown in a histological image where the transition from cartilage to trabecular bone is shown (A). Bone building cells are the osteoblasts. On the bone surface, they are arranged in pali‐ sade formation while synthesizing new bone matrix, the osteoid. They mature while they encase themselves in osteoid and finally mineralized bone matrix and become osteocytes (E). Osteoclasts on the other hand degrade bone; they are large multinucleated cells with a ruffled border directed at the bone surface (D). To emphasize the size difference, scale bars are enclosed in the image of the osteoclast and osteoblast. The bone marrow cavity is filled with bone marrow cells and a network of vessels (C). The vessel structure is explained more in detail with a cross section of long bone on the right-hand side. The cortical bone is covered by the endosteum on the inside and the periosteum on the outside. The periost is a rich source of cells, which are located in the stratum cambium (indicated in the histological out take), here visible by their dark nuclei. The stratum fibrosum covers the stratum cambium and is followed by a fascia and muscle closely adjacent to the bone (B). **Blood vessel structure in the bone marrow**: The bone is highly vascularized, next to a central vein and an artery system of sinusoids, arterials and transient zone vessels pervade the bone marrow cavity as indicated in the cross section of the bone on the right. **Osteon structure of lamellar bone**: The histological out take of the cross section shows the osteon structure of lamellar bone with its Haversian system. The bones are depicted as μCT 3D reconstruction images of mouse femura. Histological stainings are HE, hematoxylin eosin; MP, Movat pen‐ tachrome; and Ab, Alcian blue on paraffin- or plastic-embedded sections of long bone samples of mouse and sheep.

Bone healing is a complex process that involves a variety of different cells and signaling molecules, which originate not only from the bone, and here specifically from the periosteum, the cortical or cancellous bone, the endosteum and the bone marrow, but also from surround‐ ing muscle tissue (**Figure 4**). An important supplier for cells and signals is the vasculature and thus the blood as a carrier. Bone is a very well-vascularized organ. Osteons are tube-shaped structures within the bone with an open space for blood vessels, veins, and nerves in the center. Small capillaries are found in the bone marrow near the endosteum, which continue into arterioles and sinusoids (with fenestrated basal membranes) towards the center where a large artery and central sinusoid transverse longitudinally through the bone marrow space [36]. Through the vessel connectivity, any osseous injury is prone to be influenced by systemic effects and vice versa to influence the systemic homeostasis. For example, the callus formation of injured bone is heightened in patients with traumatic brain injury. In this case, systemic changes caused by the brain injury influence the bone healing, most likely due to a competition for nutrients between the two injury sites and an altered hormone homeostasis [37, 38]. Another systemic effect that is most likely communicated to the bone is a change in the inflammatory state of an injured person—a higher systemic inflammatory reactivity will disturb the bone healing process and prolong the healing time necessary to achieve bridging [39]. Upon fracture, the vascular system of the bone is disrupted at the injury site, and it is imperative that revascularization swiftly occurs in order for a successful healing process. Tissue formation relies on the supply through the vasculature with oxygen, nutrients, signaling molecules and cells [29, 31, 40–42]. Restoration of the vasculature also enables cell recruitment of circulating regenerative cells towards the fracture site [41–44].

The cells partaking in the bone healing process do not only originate from the bone itself, but they also migrate out of different cell sources, which contribute finally to the healing process. A rich cell source for cells contributing to bone healing after injury is the periosteum as well as the bone marrow from where cells are attracted to migrate towards the injury site [45–47]. The muscle surrounding the fractured bone is also a valuable source for growth factors and stem cells, promoting revascularization and thus the bone healing process [48].

On analyzing bone healing, it is important to keep in mind that there are several different compartments involved, including the bone itself, the medullary cavity, the surrounding muscle and connective tissue, the blood supply, the metabolism, and the immune system.

### **2.2. Fracture healing**

**2. Immune cells and bone regeneration**

Bone is not simply a hard nonorganic material that functions as an anchor for muscles and tendons providing stability and form for our bodies and enabling movement through the interplay of our musculoskeletal system; it is also protecting vital organs, such as the brain, lungs, and heart, and it is a living organ regulating homeostasis. Additionally, it is an organ that is essential for our immune system, as these cells arise and/or mature from stem cells in the bone marrow, it is also an organ that interacts with our hormonal balance through a multitude of factors, including the hormone osteocalcin [35], and acts as a storage not only for calcium, phosphate, and magnesium but also for growth factors, as for example transforming

**Figure 4. Bone is a complex organ**. A long bone can be divided into epi-, meta-, and diaphyseal regions. The epiphy‐ seal region contains the growth plate—the region of length growth of the bone. The epiphyseal zone is broad in young individuals and diminishes with age. Details are shown in a histological image where the transition from cartilage to trabecular bone is shown (A). Bone building cells are the osteoblasts. On the bone surface, they are arranged in pali‐ sade formation while synthesizing new bone matrix, the osteoid. They mature while they encase themselves in osteoid and finally mineralized bone matrix and become osteocytes (E). Osteoclasts on the other hand degrade bone; they are large multinucleated cells with a ruffled border directed at the bone surface (D). To emphasize the size difference, scale bars are enclosed in the image of the osteoclast and osteoblast. The bone marrow cavity is filled with bone marrow cells and a network of vessels (C). The vessel structure is explained more in detail with a cross section of long bone on the right-hand side. The cortical bone is covered by the endosteum on the inside and the periosteum on the outside. The periost is a rich source of cells, which are located in the stratum cambium (indicated in the histological out take), here visible by their dark nuclei. The stratum fibrosum covers the stratum cambium and is followed by a fascia and muscle closely adjacent to the bone (B). **Blood vessel structure in the bone marrow**: The bone is highly vascularized, next to a central vein and an artery system of sinusoids, arterials and transient zone vessels pervade the bone marrow cavity as indicated in the cross section of the bone on the right. **Osteon structure of lamellar bone**: The histological out take of the cross section shows the osteon structure of lamellar bone with its Haversian system. The bones are depicted as μCT 3D reconstruction images of mouse femura. Histological stainings are HE, hematoxylin eosin; MP, Movat pen‐ tachrome; and Ab, Alcian blue on paraffin- or plastic-embedded sections of long bone samples of mouse and sheep.

**2.1. Bone – a complex organ**

174 Advanced Techniques in Bone Regeneration

growth factor-β (TGF-β).

The fracture healing process itself is a strictly controlled complex process composed of consecutive and partly overlapping phases, which progress towards rebuilding bone integrity in form and function. Different cell types (immune cells, progenitor cells, and mesenchymal cells) [11] and their signaling molecules (cytokines, growth factors, and chemokines) [49] are partaking during a successful regenerative process.

Several growth factors involved in the healing cascade are currently under investigation to develop new therapeutic approaches to enhance bone healing: fibroblast growth factor [50], insulin-like growth factor [51], platelet-derived growth factor [52], transforming growth factorβ [53], vascular endothelial growth factor [50], and growth and differentiation factor 5 [54, 55]. However, the only growth factors so far clinically applied to further bone healing are bone morphogenetic protein 2 and 7 [56, 57].

The bone healing process can be roughly divided according to the healing steps into an inflammatory phase, a soft callus phase, and a hard callus phase (**Figure 5**). Upon closer observation, however, it becomes apparent that the healing process is more complicated than that. A more in-depth sequence of the healing cascade would be hematoma phase, proinflam‐ matory phase, hypoxic phase, anti-inflammatory phase, revascularization phase, organized connective tissue phase, cartilage phase, hypertrophic cartilage phase, revascularization phase, cartilage mineralization phase, woven bone formation phase and remodeling phase [58].

**Figure 5.** Fracture healing cascade: On closer examination, the inflammatory phase can be divided into at least six con‐ secutive and partly overlapping phases showing the transition from the hematoma (red blood cells with some lympho‐ cytes with dark stained nuclei) towards fibrocytes in the organized connective tissue (hematoxylin–eosin staining, different magnifications and an immunohistological staining for alpha smooth muscle for the revascularization phase). Soft callus phase can be divided into three phases (Movat pentachrome staining and Safranin van Kossa staining for the revascularization). The hard callus phase is divided into cartilage mineralization, woven bone formation and re‐ modeling (Movat pentachrome staining).

Due to the complexity of the bone healing cascade with the multitude of different cell types involved and the plethora of tightly interacting and simultaneously highly controlled signaling molecules aiming to rebuild an organ consisting of periosteum, cortical bone, endosteum, and bone marrow in a way that optimally withstands the ruling mechanical strains, the process of bone regeneration is so far not understood. Therefore, research is compelled to use heuristic approaches to gain a more in-depth understanding and in conclusion develop new treatment approaches for patients in need.

#### **2.3. Osteoimmunology**

For a long time, bone homeostasis was explained with the balanced interaction of bone-forming osteoblasts and bone resorbing osteoclasts (**Figure 4**), however, this simple concept has changed. The interconnectivity of the skeletal system and the immune system has come into the focus of current research, consecutively leading to the founding of the new research field of "osteoimmunology." This new research field aims to elucidate the complex interactions between these two systems in health and disease and already more and more knowledge has accumulated [59–63], enabling us to consider new treatment possibilities for regeneration in general and also specifically for bone [64]. The opportunity to control the inflammatory cascade to stimulate successful bone healing has now been confirmed [32–34, 65].

Both cell systems, the skeletal system and the immune system, originate in the bone marrow. They share progenitor cells (e.g. osteoclasts/macrophages) and signaling pathways, and due to their colocalization, which often cross react with each other. This is apparent for example when considering the RANK/RANKL/OPG system, the system controlling osteoclast differ‐ entiation/activity and thus bone resorption. Activated T cells and osteoblasts are able to express the membrane-bound and the soluble form of RANKL (receptor activator of nuclear factor kappa-B ligand) promoting osteoclastogenesis. B cells and osteoblasts produce and secrete OPG (osteoprotegerin), a decoy receptor blocking the RANK-RANKL ligation, thus inhibiting osteoclastogenesis [59, 62, 66]. This example illustrates that immune cells are involved in bone homeostatic processes directing either bone resorption or bone apposition.

Due to the interdependency of the two systems, any considered treatment option of immune modulation must take into account that by affecting the immune system the skeletal systems could also be targeted unintentionally.

#### **2.4. The initial inflammatory phase**

55]. However, the only growth factors so far clinically applied to further bone healing are bone

The bone healing process can be roughly divided according to the healing steps into an inflammatory phase, a soft callus phase, and a hard callus phase (**Figure 5**). Upon closer observation, however, it becomes apparent that the healing process is more complicated than that. A more in-depth sequence of the healing cascade would be hematoma phase, proinflam‐ matory phase, hypoxic phase, anti-inflammatory phase, revascularization phase, organized connective tissue phase, cartilage phase, hypertrophic cartilage phase, revascularization phase, cartilage mineralization phase, woven bone formation phase and remodeling phase

**Figure 5.** Fracture healing cascade: On closer examination, the inflammatory phase can be divided into at least six con‐ secutive and partly overlapping phases showing the transition from the hematoma (red blood cells with some lympho‐ cytes with dark stained nuclei) towards fibrocytes in the organized connective tissue (hematoxylin–eosin staining, different magnifications and an immunohistological staining for alpha smooth muscle for the revascularization phase). Soft callus phase can be divided into three phases (Movat pentachrome staining and Safranin van Kossa staining for the revascularization). The hard callus phase is divided into cartilage mineralization, woven bone formation and re‐

Due to the complexity of the bone healing cascade with the multitude of different cell types involved and the plethora of tightly interacting and simultaneously highly controlled signaling molecules aiming to rebuild an organ consisting of periosteum, cortical bone, endosteum, and bone marrow in a way that optimally withstands the ruling mechanical strains, the process of bone regeneration is so far not understood. Therefore, research is compelled to use heuristic approaches to gain a more in-depth understanding and in conclusion develop new treatment

For a long time, bone homeostasis was explained with the balanced interaction of bone-forming osteoblasts and bone resorbing osteoclasts (**Figure 4**), however, this simple concept has changed. The interconnectivity of the skeletal system and the immune system has come into the focus of current research, consecutively leading to the founding of the new research field of "osteoimmunology." This new research field aims to elucidate the complex interactions

morphogenetic protein 2 and 7 [56, 57].

176 Advanced Techniques in Bone Regeneration

modeling (Movat pentachrome staining).

approaches for patients in need.

**2.3. Osteoimmunology**

[58].

Vessels are disrupted and bleeding occurs upon injury and the fracturing of bone. The infiltrating blood coagulates and forms the initial hematoma in the fracture gap. The formation of a fracture hematoma in the early healing phase is an indispensable step for successful healing because it develops an angiogenic and osteogenic potential [29, 67]. The removal of the early fracture hematoma can delay bone healing as it has been demonstrated in animal studies, where the transplantation of a fracture hematoma can lead to ectopic bone formation [68, 69], demonstrating its osteogenic potential. The coagulation process and a simultaneous proin‐ flammatory reaction are phylogenetically connected [70]. During evolution, the closure of a breached outer shell and the defense against possible pathogenic intruders were performed by one cell, the amebocytes, capable of clotting and a defensive immune response. This connection has survived evolutionary diversification of the clotting system and the immune system—both reactions still occur simultaneously upon bleeding. The amebocytes can still be found today in living fossils, such as the horse shoe crab [70]. Their immune response is so potent that it is used to monitor endotoxin levels within solutions by pharmaceutical compa‐ nies. The limulus amebocyte lysate (LAL) test is capable of detecting contaminations as low as one part per trillion [71]. In evolutionary younger organisms, this highly effective immune cell is being replaced by a whole array of immune cells, which can be divided into an innate immunity and an adapted immunity, the latter is only found in vertebrates (**Figure 6**). Each of these is composed of various different cells: macrophages, neutrophils/granulocytes, mast cells, natural killer cells, dendritic cells and the complement system belong to the innate immune system, whereas T and B cells and the humoral immunity belong to the adaptive immune system. The cells of the adaptive immune system provide their host with a long lasting and protective immunity by maturing from naïve T and B cells to effector cells, when they come in contact with their cognate antigen, and in some cases to memory cells, which allow a rapid immune response upon recurrent infection with an antigen previously encountered by the host. It has to be pointed out that the immune system is not only a barrier for extracellular microbes but also a regulatory system for body homeostasis. The immune system senses alteration in the environment, for instance damaged or aged cells [72, 73], expressing Toll-like receptors and other pattern-recognition receptors (PRRs).

**Figure 6.** Diversity of cells of the immune system. Cells from the bone marrow give rise to the immune cells of the innate and adaptive immune system and also to the osteoblasts and osteoclasts of the skeletal system.

During fracture healing, both the cells of the innate and the adaptive immunity are involved, and immune cells play essential roles during all the fracture healing phases [74–77]. The initial inflammatory reaction ensuing upon hematoma formation initiates the healing cascade and thus can significantly affect the healing outcome [33, 34]. This initial inflammatory reaction is characteristic for bone, tightly controlled and different from other tissue healing with scar formation [32]. In fracture repair, the anti-inflammatory signaling is up-regulated between 24 and 36 hours after injury to terminate the proinflammatory reaction needed to attract necessary cells to the injury side [32, 33]. In parallel, the angiogenic signaling is up-regulated to initiate the essential revascularization process. The timely down-regulation of the initial proinflam‐ matory reaction has been shown to be important as a prolonged proinflammatory reaction delays the bone healing process [29, 33].

come in contact with their cognate antigen, and in some cases to memory cells, which allow a rapid immune response upon recurrent infection with an antigen previously encountered by the host. It has to be pointed out that the immune system is not only a barrier for extracellular microbes but also a regulatory system for body homeostasis. The immune system senses alteration in the environment, for instance damaged or aged cells [72, 73], expressing Toll-like

**Figure 6.** Diversity of cells of the immune system. Cells from the bone marrow give rise to the immune cells of the

During fracture healing, both the cells of the innate and the adaptive immunity are involved, and immune cells play essential roles during all the fracture healing phases [74–77]. The initial inflammatory reaction ensuing upon hematoma formation initiates the healing cascade and thus can significantly affect the healing outcome [33, 34]. This initial inflammatory reaction is characteristic for bone, tightly controlled and different from other tissue healing with scar formation [32]. In fracture repair, the anti-inflammatory signaling is up-regulated between 24

innate and adaptive immune system and also to the osteoblasts and osteoclasts of the skeletal system.

receptors and other pattern-recognition receptors (PRRs).

178 Advanced Techniques in Bone Regeneration

The complexity of the initial immune reaction becomes even more apparent when considering cytokines expressed by immune cells during the different stages of the bone healing cascade. Tumor necrosis factor-α (TNF-α) has been reported to peak 24 hours after injury and return to baseline levels afterwards. During the remodeling phase, TNF-α shows a second expression peak during normal bone healing [64]. It is suggested that the first wave is due to activated tissue-resident cells, like macrophages, triggered through PRRs, and the second wave directly and indirectly by activated T cells. Looking closer into the role of this factor during bone healing is has been shown that too little, but also too much TNF-α leads to a delay in bone healing [78– 80]. This demonstrates that the cytokine pattern has to be tightly controlled during the regenerative healing cascade to lead to a satisfactory healing outcome. Interleukin (IL)-17 is another cytokine that has been acknowledged to influence bone formation. On one hand, this cytokine has been reported to enable osteoblast formation [81], thus supporting bone forma‐ tion; on the other hand, in the context of osteoporosis treatment, evidence occurred that IL-17 furthered osteoclastogenesis [82], thus supporting bone degradation. Contradictory reports can also be found for IL-6, which enhances fracture healing [83, 84] but reduces the mechanical strength of noninjured bone [85]. The microenvironment seems to be highly important for determination of the effect the cytokines have on the bone healing process, a fact that indicates the difficulties in using inflammatory cytokines to improve bone healing. The balanced immune response is highly important for a successful bone regenerative cascade [32, 33, 67].

Upon injury and disruption of the blood vessels, the nutrient and oxygen supply as well as the transport of metabolic waste is interrupted. The early tissue in the fracture gap consisting of the hematoma becomes hypoxic because oxygen is no longer provided by the vasculature. Therefore, cells trapped in the hematoma have to switch towards an anaerobic energy supply. The use of the remaining glucose in glycolysis to produce adenosine triphosphate (ATP), the energy molecule of the cellular metabolism, without the consecutive citrate cycle, results in lactate, an acid that consecutively lowers the pH value during the initial healing phase. Simultaneously, the sodium and potassium concentrations rise. These conditions present a milieu that is difficult for some cells, such as progenitor cells [86]. However, innate immune cells are well equipped to deal with these conditions and thus can be seen as the first responders to an injury. They express a range of cytokines that attract scavenger cells to clear the detritus that ensued upon tissue disruption and also direct the cells needed for the regenerative process towards the injury side. They readily switch from an aerobic energy supply towards an anaerobic and are often activated upon injury. Not only macrophages but also some T cell subsets are the most important actors during this first response [87, 88]. Hypoxia is a strong inducer of hypoxia inducible factor 1α (HIF1α), a transcription factor that is important for revascularization, cell migration, energy metabolism and growth factor expression, and therefore involved in the regenerative bone healing cascade [89]. HIF1α is expressed by most innate and adaptive immune cells, including macrophages and lymphocytes; they stabilize HIF1α and are being influenced by HIF1α in their immune cell function [90].

The swift up-regulation of a proinflammatory reaction upon injury activates immune cells, which are capable to withstand the unfavorable environment and initiate the healing cascade through a very specific and highly controlled release of cytokines. Hypoxia is an important trigger for the transcription factor HIF1α that in turn initiates gene expression to instigate revascularization. For this process to succeed, effective anti-inflammatory signaling has to begin to terminate the initial proinflammatory reaction. During this initial phase, the track for a successful healing is thus determined, and it becomes apparent that a skewed first reaction leads to a delayed healing by consecutively retarding the following healing steps.

#### **2.5. Challenging immune constraints**

The interdependency of the immune and skeletal system indicates that there is a change in the interaction as the immune system changes with the advancement of age. Due to the memory function of the adaptive immunity in vertebrates, the naïve T and B cell population diminishes upon aging, whereas the compartment of memory T and B cells grows. More and more lymphocytes encounter their antigens and the library of known pathogens enlarges. Recent studies could show that CD8 positive terminally differentiated memory and effector cells (CD8+ TEMRA cells) have a negative impact on bone healing and osteogenic differentiation of stem cells [91, 92]. Elderly people with a longer exposure time to antigens thus are prone to experience delayed healing.

Mice, a common laboratory animal to investigate bone healing, are mostly kept under sterile conditions. If these animals are housed under less sterile conditions, their immune cell composition changes so that after 4 weeks of semi-sterile housing the percentage of memory and effector (CD8+) T cells was markedly enhanced. If bone healing is compared between sterile raised mice and those exposed mice, our group could show that the regenerative capacity was reduced [91, 93]. This is an important aspect that should be kept in mind during future research questions, which are analyzed in mice.

Nonsteroidal anti-inflammatory drugs (NSAIDs) offer pain relief and are commonly used also on fracture patients. As the name already indicates, these selective cyclooxygenase-2 (COX-2) inhibitors have anti-inflammatory functions. After reviewing the importance of the initial inflammatory reaction, the question arises whether this pain medication could delay fracture healing or not. Indeed there are numerous reports that state that NSAIDs delay healing [94– 98]. The effect, however, depends on the dose and time frame of application and seems to be more pronounced in older nonselective anti–COX-2 agents [99]. Clinically, NSAIDs are a valuable alternative to opioids (painkillers directly addressing the nervous system) and still remain in use also in fracture patients for short-term pain relief.

Several diseases have also been reported to delay bone healing through a changed immune response. Diabetic-related delay of fracture healing has been linked to higher TNF-α levels [100]. A weakened immune response in diabetic patients results in a dampened chemotactic function and defective macrophage activity—two factors that are needed in a successful bone healing cascade [101]. A systemic disease with a high impact on the immune system is human immunodeficiency virus (HIV), and these patients have a bone phenotype with a high prevalence of osteoporosis and fragility fractures [102]. The impact on fracture healing, however, is unclear and difficult to determine due to the highly active antiretroviral therapy that these patients receive [102, 103]. Transplant patients receiving severe immune suppressive medication also show a higher risk for fractures and delayed healing outcomes. In contrast to these examples – where the immune system is weakened – conditions where a patient has a heightened immune answer or is already in a chronic proinflammatory systemic state, such as rheumatoid and arthritis patients, the prolonged proinflammatory reaction can result in delays in fracture healing [104–106].

Currently, the patient's immune status is not being evaluated when a fracture treatment is considered. However, this could help in the future to stratify patients who would benefit from an immune modulatory intervention to prevent a delay in fracture healing. This would especially be true in elderly patients because being bed-ridden for longer periods of time enhances frailty considerably.

#### **2.6. Specific immune cell subsets that have been identified as important players in the bone regenerative process**

In fracture healing, immune cells from the innate immune system and from the adaptive immune system are involved with specific and essential roles. Main cell types of the adaptive immunity are B and T cells with highly specific antigen receptors. Another important aspect of the adaptive immune system is its memory that enables its fast reaction towards recurring pathogen invasion. Adaptive immune cells can be activated not only through their antigen receptors, but also probably more important for the bone healing process through signals released by the innate immune system. From the innate immune system, especially macro‐ phages have been in the current focus of osteoimmunology.

#### *2.6.1. Macrophages*

innate and adaptive immune cells, including macrophages and lymphocytes; they stabilize

The swift up-regulation of a proinflammatory reaction upon injury activates immune cells, which are capable to withstand the unfavorable environment and initiate the healing cascade through a very specific and highly controlled release of cytokines. Hypoxia is an important trigger for the transcription factor HIF1α that in turn initiates gene expression to instigate revascularization. For this process to succeed, effective anti-inflammatory signaling has to begin to terminate the initial proinflammatory reaction. During this initial phase, the track for a successful healing is thus determined, and it becomes apparent that a skewed first reaction

The interdependency of the immune and skeletal system indicates that there is a change in the interaction as the immune system changes with the advancement of age. Due to the memory function of the adaptive immunity in vertebrates, the naïve T and B cell population diminishes upon aging, whereas the compartment of memory T and B cells grows. More and more lymphocytes encounter their antigens and the library of known pathogens enlarges. Recent studies could show that CD8 positive terminally differentiated memory and effector cells (CD8+ TEMRA cells) have a negative impact on bone healing and osteogenic differentiation of stem cells [91, 92]. Elderly people with a longer exposure time to antigens thus are prone to

Mice, a common laboratory animal to investigate bone healing, are mostly kept under sterile conditions. If these animals are housed under less sterile conditions, their immune cell composition changes so that after 4 weeks of semi-sterile housing the percentage of memory and effector (CD8+) T cells was markedly enhanced. If bone healing is compared between sterile raised mice and those exposed mice, our group could show that the regenerative capacity was reduced [91, 93]. This is an important aspect that should be kept in mind during

Nonsteroidal anti-inflammatory drugs (NSAIDs) offer pain relief and are commonly used also on fracture patients. As the name already indicates, these selective cyclooxygenase-2 (COX-2) inhibitors have anti-inflammatory functions. After reviewing the importance of the initial inflammatory reaction, the question arises whether this pain medication could delay fracture healing or not. Indeed there are numerous reports that state that NSAIDs delay healing [94– 98]. The effect, however, depends on the dose and time frame of application and seems to be more pronounced in older nonselective anti–COX-2 agents [99]. Clinically, NSAIDs are a valuable alternative to opioids (painkillers directly addressing the nervous system) and still

Several diseases have also been reported to delay bone healing through a changed immune response. Diabetic-related delay of fracture healing has been linked to higher TNF-α levels [100]. A weakened immune response in diabetic patients results in a dampened chemotactic function and defective macrophage activity—two factors that are needed in a successful bone

HIF1α and are being influenced by HIF1α in their immune cell function [90].

leads to a delayed healing by consecutively retarding the following healing steps.

**2.5. Challenging immune constraints**

180 Advanced Techniques in Bone Regeneration

experience delayed healing.

future research questions, which are analyzed in mice.

remain in use also in fracture patients for short-term pain relief.

Macrophages are an important part of the innate immune system; they are among the first responders in case of an injury. Not only do they prevent pathogen invasion, but they also help in clearing ensuing cell debris [107]. However, their role in bone healing is even more complex and even today we have not yet unraveled their participation completely. Tissueresident macrophages have been determined as key players in the orchestration of the recovery process towards a re-establishment of tissue integrity [108]. It was only in 1992 that it was recovered that macrophages are capable of a phenotype change from a proinflammatory type towards a prohealing phenotype [109]. The proinflammatory phenotype is named M1 or classically activated macrophage, and the second phenotype is termed M2 or alternatively activated macrophage. Since then, these "M2" macrophages have been associated with the resolution of wound healing *in vivo* in chronic leg ulcers [110], atherosclerotic lesions [111], traumatic spinal cord injury [112] and inflammatory renal disease [113]. It turned out that the M2 population is more divers and therefore subclassifications have been introduced: M2a (anti-inflammatory), M2b (immune-regulatory) and M2c (remodeling) [114]. In bone healing, the prominent macrophage phenotype during the initial phase is M1. Upon attenuating of the proinflammatory phase, the macrophage phenotype changes towards the M2 phenotype [77]. In a proof of concept study in mice, we were able to show that an induction of the M2 phenotype early in the fracture healing cascade can enhance bone healing [77].

#### *2.6.2. Regulatory T cells*

The T cell population is highly divers and probably pleiotropic as well as interchangeable. Among the T cells, there seem to be subpopulations supporting the fracture healing process and also other subpopulations, which have negative effects on the healing process. CD4+ and CD8+ T cell subsets have been addressed in this context. CD4+ T cells have been shown to increase osteogenic differentiation in human mesenchymal stem cell cultures in *in vitro* assays using their conditioned medium, whereas this effect was missing when observing CD8+ T cells [115]. The osteogenic effect of CD4+ T cells was further supported through their positive effects during wound healing [116], however without a more specific determination of the responsible CD4+ T cell subset. In later studies, regulatory T cells came more and more into the focus as a CD4+ T cell subset with positive effects on bone healing. Mice with an increased percentage of regulatory T cells showed higher bone mass and decreased bone resorption when compared to wild type mice [117, 118]. Regulatory T cells support osteoblast differentiation and have a negative impact on osteoclast differentiation and function [119]. In a skull defect model in mice, it was possible to enhance bone healing through the addition of regulatory T cells in combi‐ nation with applied autologous bone graft [120]. Currently under investigation is the possi‐ bility of a direct interaction of regulatory T cells and bone-forming cells or their progenitor cells, the mesenchymal stromal/stem cells. This interaction is supported by the fact that mesenchymal stromal/stem cells, as osteoblast precursors, and regulatory T cells use similar suppression mechanisms for an immune response [121]. The direct interaction between regulatory T cells and bone-forming cells as well as mesenchymal stromal/stem cells could proceed through coordination of the CD39-CD73-(adenosine)-ADOR pathway. This puriner‐ gic signaling would potentiate the differentiation of mesenchymal stromal/stem cells and thus facilitate bone regeneration [122]. Another direct interaction between osteoblasts and regula‐ tory T cells could be the induction of IDO (indoleamine 2,3-dioxygenase) and HO-1 (heme oxygenase-1) by regulatory T cells [123] or the fact that regulatory T cells can inhibit CD40L and thus regulating the RANKL-OPG balance in favor of osteoblast differentiation [124].

#### *2.6.3. T helper 17 cells*

The lead cytokine expressed by Th17 (T helper 17) is IL-17. The dual effect of IL-17 on osteoclasts and osteoblasts has been mentioned before. However, these cells are of interest as novel therapeutics targeting IL-12, IL-23, IL-17, and IL-17 receptor and which are now used to successfully treat psoriasis by either repressing Th17 differentiation (IL-12/IL-23) or by directly targeting IL-17. Psoriasis has two manifestations, one in skin (psoriasis vulgaris) and one in bone (psoriasis arthritis), and the immune modulatory treatment shows positive results in both [125]. Th-17 cell differentiation is induced by IL-1β, IL-6 and TGF-β [126, 127], with TGF-β being responsible for an increase in responsiveness of Th17 cells to IL-23. IL-23 is necessary for stabilization, survival and proliferation of Th17 cells [128]. This IL-23/Th17 axis is the target of the immune modulatory therapies currently introduced. For example, a cytokine neutralizing antibody against the p40 subunit of IL-23 inhibiting Th17 differentiation and survival, which in consequence lowers IL-17 concentrations, underwent clinical trials [129, 130].
