Periostin in Bone Regeneration
Bone regeneration is an efficient regenerative process depending on the recruitment and activation of skeletal stem cells that allow cartilage and bone formation leading to fracture consolidation. Periosteum, the tissue located at the outer surface of bone is now recognized as an essential player in the bone repair process and contains skeletal stem cells with high regenerative potential. The matrix composition of the periosteum defines its roles in bone growth, in cortical bone modeling and remodeling in response to mechanical strain, and in bone repair. Periostin is a key extracellular matrix component of the periosteum involved in periosteum functions. In this chapter, we summarize the current knowledge on the bone regeneration process, the role of the periosteum and skeletal stem cells, and Periostin functions in this context. The matricellular protein Periostin has several roles through all stages of bone repair: in the early days of repair during the initial activation of stem cells within periosteum, in the active phase of cartilage and bone deposition in the facture callus, and in the final phase of bone bridging and reconstitution of the stem cell pool within periosteum.
KeywordsPeriosteum Periostin Bone repair Stem cell
Following a fracture, bone regeneration is initiated. Bone tissue has a great capacity to regenerate, creating new bone which is indistinguishable from the uninjured bone, without leaving a scar. Although the steps of bone regeneration largely recapitulate those observed during bone development, bone regeneration is also regulated by other factors such as the inflammatory response and the mechanical environment that can influence the repair process [1, 2, 3]. The origins of skeletal stem cells and the factors regulating their functions in bone repair have been investigated using mouse models. While systemic recruitment of cells is minimal, bone marrow stromal cells/skeletal stem cells (BMSCs) have a local osteogenic potential and mostly have trophic and immunomodulatory roles within the bone marrow compartment. Periosteum is another local source of skeletal stem cells for bone repair [4, 5, 6].
The osteogenic potential of the periosteum has been highlighted since 1742 when Duhamel placed a periosteum on a silver ring around a bone and observed new bone formation coming from the periosteum . One century later, Dupuytren suggested that periosteum and bone marrow played a role in cartilage formation in the fracture callus . It is now well established that the periosteum plays many roles during bone development, growth, repair and aging, and its functions may be affected in some pathologies. The ECM composition of the periosteum is likely to influence its functions. The matricellular protein Periostin is expressed within periosteum and periodontal ligament and was first identified as Osteoblast-specific factor 2 (OSF-2) in a mouse osteoblastic calvarial cell line (MC3T3-E1) and in primary osteoblasts [9, 10, 11]. In bone, Periostin acts as a structural component of the matrix regulating collagen cross-linking and as a signaling molecule via interaction with integrin receptors . Functional analyses of periosteal cells and Periostin using Periostin KO mice have now revealed several roles of Periostin in bone regeneration.
6.2 Bone Regeneration after Fracture
Bone regeneration generally occurs through a combination of endochondral and intramembranous ossification. The regeneration process can be summarized into four critical stages: the inflammation phase (hematoma formation and skeletal stem cell recruitment), the soft callus phase (neo-angiogenesis and cartilage/bone formation), the hard callus phase (cartilage resorption and/or active bone deposition) and the remodeling phase [13, 14]. Following a fracture, cells coming from the local bone marrow and the vasculature form the hematoma and the inflammatory process is initiated . Pro-inflammatory mediators are secreted and initiate the repair cascade . Neutrophils are first mobilized at the fracture site and secrete interleukins and chemokines like IL-6 (interleukin-6) and CCL2 (chemokine ligand 2) [15, 17]. Interleukins-1β (IL-1β) , IL-6 [19, 20], IL-11 , IL-17 , IL-18 , IL-23  as well as Tumor Necrosis Factor-α (TNF-α)  promote angiogenesis and attract inflammatory cells like macrophages that remove necrotic tissues . The inflammatory phase of healing is generally completed after 7 days following a fracture in mice . In cases of bacterial infection, the inflammation phase persists and the healing process fails . The inflammatory cells, macrophages and platelets secrete growth factors like Platelet-Derived Growth Factor (PDGF), Vascular Endothelial Growth Factor (VEGF) and Bone Morphogenetic Proteins (BMPs) to initiate the recruitment of skeletal stem/progenitor cells at the fracture site that are activated around day 3 [13, 16, 25]. During the soft callus phase, skeletal stem cells differentiate into chondrocytes or directly into osteoblasts depending on the mechanical tensions. In the center of the callus, where the mechanical tensions are high, bone regeneration occurs via endochondral ossification while at the periphery, bone regeneration mainly occurs through intramembranous ossification. During endochondral ossification, skeletal stem cells first differentiate into chondrocytes, proliferate and secrete a cartilaginous matrix rich in Type II Collagen and Aggrecan. These cells are then replaced by osteoblasts that produce a bone matrix rich in Type I Collagen. During the hard callus phase, the cartilage matrix is mineralized. Chondrocytes undergo hypertrophy and secrete VEGF to allow vascular invasion of hypertrophic cartilage. Due to osteoclast activity, cartilage is resorbed and replaced by woven bone. While it was generally recognized that hypertrophic chondrocytes die by apoptosis allowing vascular invasion and endochondral ossification, recent studies have shown that some hypertrophic chondrocytes can transdifferentiate into osteoblasts as observed at the level of the growth plate [26, 27, 28, 29, 30]. The cascade of events during this transition between mineralized cartilage and new bone is strictly regulated and any disruption of this transition leads to delayed or impaired healing [31, 32]. During the remodeling phase of repair, primary bone in the callus is degraded by osteoclasts and replaced by lamellar bone to restore the anatomical structure of bone that supports mechanical loads . In mice, the replacement of cartilage by bone is complete by day 28 and the bone remodeling process carries on until after 2 months.
6.3 Role of Periosteum in Bone Repair
Due to its external localization on bone, the periosteum is extremely sensitive to mechanical stimuli. When mechanical tensions or increased loading are applied, new bone formation deriving from the periosteum is observed, as well as periosteal hypertrophy, DNA synthesis, and cell proliferation [39, 40, 41, 42]. During childhood and through adulthood, periosteal appositions allow bone to grow in width and cortical bone modeling/remodeling in concert with osteoclasts maintain bone structure in response to mechanical strain. In the context of fracture, it has been long known that removal/stripping of the periosteum impairs bone regeneration . In models of diaphyseal fracture in rats, disruption of the periosteum impairs early chondrogenesis [43, 44]. Transplantation of Rosa26-LacZ segmental grafts into critical bone defects or periosteum grafts into fractures showed that periosteum was a major contributor to cartilage and bone in the callus compared to bone marrow and endosteum [4, 45, 46, 47].
The periosteal response to the fracture can be observed histologically as early as 24 h following injury with a cellular proliferation leading to thickening of the periosteum near the fracture site. The size and the cellular content of the callus depend on the periosteal response to the mechanical environment. If the fracture is left unstabilized, the periosteal response to mechanical strain is very efficient, leading to bone regeneration via endochondral ossification. In the presence of mechanical stability or if the fracture is reduced, the periosteal response is less efficient leading to direct ossification [2, 32, 48]. These studies highlight the high regenerative potential of the periosteum. However, the absence of specific marker(s) to follow periosteum-derived cells (PCs) during bone regeneration makes it difficult to distinguish its endogenous potential compared to bone marrow. Therefore, many studies have aimed to isolate cells from the periosteum to study their in vitro and in vivo properties .
6.4 Periosteal Cells Express Periostin in Response to Bone Injury
Bone marrow stromal cells/skeletal stem cells (BMSCs) are the most described skeletal stem cells in the literature. In vitro, BMSCs can differentiate into osteoblasts, adipocytes and chondrocytes, and form colonies at low density. In vivo, the regenerative potential of BMSCs has been tested using cell transplantation in subcutaneous sites, where they form bone ossicles and support hematopoiesis . However, these ectopic sites do not recapitulate the environment of a fracture (i.e. inflammation and mechanical stimuli). Self-renewal was shown using clonal expansion in vitro followed by ectopic subcutaneous transplantation. Many studies report transplantation of BMSCs in bone defects or at injury sites, but few include lineage tracing to follow their fate. BMSCs rapidly disappear after transplantation and have poor osteogenic potential. BMSCs rather stimulate repair via their ability to secrete growth factors and inflammatory factors [50, 51]. Several lineage studies using Cre reporter mice have aimed to define markers for BMSCs and follow these cells during bone repair [52, 53, 54]. However, there is no clear evidence regarding the specificity of these markers that may also label cells in periosteum and other tissues. Indeed, periosteal cells (PCs) express markers described initially for BMSCs such as PDGFRα, Gremlin 1, Cxcl12 and Nestin. PCs and BMSCs exhibit similar differentiation characteristics in vitro, but PCs have increased clonogenicity and cell growth. After transplantation in fractures, PCs have a higher integration potential in cartilage and bone in the callus compared to BMSCs and persist within bone indicating the higher regenerative potential of PCs compared to BMSCs .
6.5 Periostin Is Required for Bone Repair
Matricellular proteins have diverse functions such as promoting cell adhesion that may induce cell migration, they interact with growth factors that facilitate cell-matrix interactions, and bridge inorganic matter and proteins of the ECM . Matricellular proteins are crucial regulators of cell phenotype, and consequently tissue function. While mice knockout models of structural ECM proteins generally induce severe phenotypes that can be lethal during development, gene deletion of matricellular proteins induces mild phenotypes that are exacerbated upon injury . Like other matricellular proteins, Postn gene deletion in mice does not lead to embryonic lethality . Postn KO mice show no apparent phenotype at birth, but exhibit growth retardation and weight loss within 1 month. The Postn KO bone phenotype is marked by the reduction of the size of the skull, ribs and cartilaginous growth plates and less trabecular bone in the limbs suggesting a role of Postn in bone development and homeostasis . The absence of Postn also leads to loss of alveolar bone in the tooth probably due to increased osteoclasts activity in the periodontium. Thus the phenotype of Postn KO mice might at least, in part, be due to feeding disabilities caused by lesions in the in periodontal ligament, and under soft diet, growth retardation was attenuated .
During the soft and hard callus phase of bone repair, Postn is also expressed in hypertrophic cartilage at the junction between cartilage and bone within the callus and in osteoblasts within new bone trabeculae . Postn inactivation may thus impact bone formation directly during bone repair as Postn has been shown to regulate osteoblast proliferation, adhesion and differentiation [11, 60]. Although we did not detect expression of Postn in TRAP-positive osteoclasts in the fracture callus (data not shown), Postn deposition in ECM may also indirectly influence osteoclast function . A conditional knockout approach may therefore be useful to determine the role of Postn in various cell populations during bone repair. At later stages of repair, fibrosis is observed in the callus of Postn KO mice (Fig. 6.3) . Postn has been shown to promote fibrosis in allergic and respiratory diseases [70, 71]. The cellular origin of this fibrosis and the specific role of Postn in fibrosis deposition and remodelling in the fracture callus remain to be established.
6.6 Periostin Regulates the Skeletal Stem Cell Niche Within Periosteum
In addition to regulating periosteal cell activation in the inflammatory phase of repair, and periosteal cell differentiation during the soft callus phase of repair, Postn also regulates the niche of periosteal cells during the remodelling phase of repair. This was first suggested by the expression of Postn in the inner layer of the newly formed periosteum . A niche for stem cells regulates their quiescence, self-renewal and differentiation potential in normal tissues and in response to injury. To demonstrate the presence of stem cells with self-renewing potential within periosteum, GFP labelled periosteal grafts were transplanted at the fracture site and graft contribution to repair was evaluated over 3 consecutive cycles of injuries. The results show that PCs within periosteum could be activated to participate in callus formation, reintegrate their niche in the newly formed periosteum at the end of the repair process and be reactivated after several injuries to contribute again to bone repair (Fig. 6.4, left panels) . To test whether the absence of Postn had an impact on the capacity of PCs to reintegrate their niche and reconstitute a pool of SSCs, GFP labelled periosteum grafts from Postn KO mice were transplanted in wild type hosts. Although the fracture callus was mostly wild type host-derived, bone regeneration was impaired around the Postn KO graft with no cartilage formation and local induction of fibrosis. Moreover, Postn KO PCs could not reintegrate their niche in the new periosteum at later days and did not contribute to cartilage and callus formation after a second injury (Fig. 6.4, right panels). PCs isolated from Postn KO mice show less clonogenicity as well as decreased osteogenic and adipogenic potential in vitro. Together these data indicate that Postn is required in the periosteal niche to reconstitute the pool of PCs at the end of the repair process. Interestingly, transplantation of Postn KO graft in Postn KO hosts caused an aggravation of the repair phenotype after a second injury, further supporting the role of Postn in re-establishing the stem cell pool in periosteum .
6.7 Clinical Applications Using Periosteal Cells and Periostin in Bone Repair
The increased regenerative capacities of PCs compared to BMSCs make the periosteum a good target to stimulate repair or an attractive source of cells for therapy. For clinical applications in orthopaedics, several concerns are raised regarding the use of SSCs such as their site and method of isolation, ex vivo expansion, mechanism of activation as well as their osteogenic potential and long term integration after transplantation. For cell-based approaches aiming to treat complex fractures or critical size defects, a two-step surgery to isolate PCs would be necessary. The resection methods as well as culture conditions could influence stem cell number and regenerative potential. Moreover, types of bones from which periosteum could be isolated and inter-individual differences (age, sex) could also impact PCs potential [72, 73]. Cell-based approaches using PCs need to be explored or these cells could be reactivated in their native environment (periosteal niche) using pharmacological drugs that would act on signalling pathways to activate these cells or to modulate the ECM niche.
The enhanced regenerative potential of PCs marked by an augmented molecular response to injury and Periostin up regulation is essential for adequate bone repair, PCs self-renewal and the reconstitution of the periosteal niche . Periostin could be used therapeutically to stimulate cell migration and contribution to repair in cell-based approaches. In a model of critical size defect in rabbit, Zhang and collaborators (2017) transplanted osteogenic-induced modified BMSCs and showed increased bone formation, bone mineral density and bone volume concomitant with increased Periostin/β-catenin protein expression. In vitro, Periostin increased ALP and alizarin red S production by BMSCs with increased levels of associated proteins (p-LRP-6, p-GSK3, β-catenin and Runx2) . This suggests that Periostin may be delivered to directly activate Wnt/β-catenin signaling to stimulate bone formation in the callus [75, 76]. In another study, recombinant Periostin stimulated human adipose tissue-derived stromal cells (hASC) proliferation and migration, and augmented hASC-mediated repair in a critical size calvarial defect model . The multiple roles of Periostin in regulating matrix composition and cellular functions during bone repair may be mediated by various isoforms, therefore the roles of these different isoforms need to be defined to target Periostin or select appropriate Periostin fragments for tissue engineering approaches in bone repair.
This work is supported by Osteosynthesis and Trauma Care Foundation, ANR-13-BSV1-001, ANR-18-CE14-0033 and NIH R01AR072707.
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