Multimodal analyses of immune cells during bone repair identify macrophages as a therapeutic target in musculoskeletal trauma.

Musculoskeletal traumatic injuries (MTI) involve soft tissue lesions adjacent to a bone fracture leading to fibrous nonunion. The impact of MTI on the inflammatory response to fracture and on the immunomodulation of skeletal stem/progenitor cells (SSPCs) remains unknown. Here, we used single cell transcriptomic analyses to describe the immune cell dynamics after bone fracture and identified distinct macrophage subsets with successive pro-inflammatory, pro-repair and anti-inflammatory profiles. Concurrently, SSPCs transition via a pro- and anti-inflammatory fibrogenic phase of differentiation prior to osteochondrogenic differentiation. In a preclinical MTI mouse model, the injury response of immune cells and SSPCs is disrupted leading to a prolonged pro-inflammatory phase and delayed resolution of inflammation. Macrophage depletion improves bone regeneration in MTI demonstrating macrophage involvement in fibrous nonunion. Finally, pharmacological inhibition of macrophages using the CSF1R inhibitor Pexidartinib ameliorates healing. These findings reveal the coordinated immune response of macrophages and skeletal stem/progenitor cells as driver of bone healing and as a primary target for the treatment of trauma-associated fibrosis. Summary: Hachemi et al. report the immune cell atlas of bone repair revealing macrophages as pro-fibrotic regulators and a therapeutic target for musculoskeletal regeneration. Genetic depletion or pharmacological inhibition of macrophages improves bone healing in musculoskeletal trauma.

Periosteal Skeletal Stem and Progenitor Cells in Bone Regeneration.

PURPOSE OF REVIEW: The periosteum, the outer layer of bone, is a major source of skeletal stem/progenitor cells (SSPCs) for bone repair. Here, we discuss recent findings on the characterization, role, and regulation of periosteal SSPCs (pSSPCs) during bone regeneration. RECENT FINDINGS: Several markers have been described for pSSPCs but lack tissue specificity. In vivo lineage tracing and transcriptomic analyses have improved our understanding of pSSPC functions during bone regeneration. Bone injury activates pSSPCs that migrate, proliferate, and have the unique potential to form both bone and cartilage. The injury response of pSSPCs is controlled by many signaling pathways including BMP, FGF, Notch, and Wnt, their metabolic state, and their interactions with the blood clot, nerve fibers, blood vessels, and macrophages in the fracture environment. Periosteal SSPCs are essential for bone regeneration. Despite recent advances, further studies are required to elucidate pSSPC heterogeneity and plasticity that make them a central component of the fracture healing process and a prime target for clinical applications.

Skeletal Stem/Progenitor Cells in Periosteum and Skeletal Muscle Share a Common Molecular Response to Bone Injury.

Bone regeneration involves skeletal stem/progenitor cells (SSPCs) recruited from bone marrow, periosteum, and adjacent skeletal muscle. To achieve bone reconstitution after injury, a coordinated cellular and molecular response is required from these cell populations. Here, we show that SSPCs from periosteum and skeletal muscle are enriched in osteochondral progenitors, and more efficiently contribute to endochondral ossification during fracture repair as compared to bone-marrow stromal cells. Single-cell RNA sequencing (RNAseq) analyses of periosteal cells reveal the cellular heterogeneity of periosteum at steady state and in response to bone fracture. Upon fracture, both periosteal and skeletal muscle SSPCs transition from a stem/progenitor to a fibrogenic state prior to chondrogenesis. This common activation pattern in periosteum and skeletal muscle SSPCs is mediated by bone morphogenetic protein (BMP) signaling. Functionally, Bmpr1a gene inactivation in platelet-derived growth factor receptor alpha (Pdgfra)-derived SSPCs impairs bone healing and decreases SSPC proliferation, migration, and osteochondral differentiation. These results uncover a coordinated molecular program driving SSPC activation in periosteum and skeletal muscle toward endochondral ossification during bone regeneration. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Mouse Periosteal Cell Culture, in vitro Differentiation, and in vivo Transplantationin Tibial Fractures.

The periosteum covering the outer surface of bone contains skeletal stem/progenitor cells that can efficiently form cartilage and bone during bone repair. Several methods have been described to isolate periosteal cells based on bone scraping and/or enzymatic digestion. Here, we describe an explant culture method to isolate periosteum-derived stem/progenitor cells for subsequent in vitro and in vivo analyses. Periosteal cells (PCs) isolated using this protocol express mesenchymal markers, can be expanded in vitro, and exhibit high regenerative potential after in vivo transplantation at a fracture site, suggesting that this protocol can be employed for PC production to use in new cell-based therapies.

Direct contribution of skeletal muscle mesenchymal progenitors to bone repair.

Bone regenerates by activation of tissue resident stem/progenitor cells, formation of a fibrous callus followed by deposition of cartilage and bone matrices. Here, we show that mesenchymal progenitors residing in skeletal muscle adjacent to bone mediate the initial fibrotic response to bone injury and also participate in cartilage and bone formation. Combined lineage and single-cell RNA sequencing analyses reveal that skeletal muscle mesenchymal progenitors adopt a fibrogenic fate before they engage in chondrogenesis after fracture. In polytrauma, where bone and skeletal muscle are injured, skeletal muscle mesenchymal progenitors exhibit altered fibrogenesis and chondrogenesis. This leads to impaired bone healing, which is due to accumulation of fibrotic tissue originating from skeletal muscle and can be corrected by the anti-fibrotic agent Imatinib. These results elucidate the central role of skeletal muscle in bone regeneration and provide evidence that skeletal muscle can be targeted to prevent persistent callus fibrosis and improve bone healing after musculoskeletal trauma.

Renal Capsule Transplantation to Assay Angiogenesis in Skeletal Development and Repair.

Renal capsule transplantation is a very helpful method to grow embryonic tissues or tumors in a vascular environment, allowing for long-term engraftment and biological analyses. This chapter describes the surgical procedure for the transplantation of embryonic skeletal elements in the renal capsule of adult mice and points out the manipulations that can be applied for assaying the role of angiogenesis during bone development and repair.

FGFR3 in Periosteal Cells Drives Cartilage-to-Bone Transformation in Bone Repair.

Most organs and tissues in the body, including bone, can repair after an injury due to the activation of endogenous adult stem/progenitor cells to replace the damaged tissue. Inherent dysfunctions of the endogenous stem/progenitor cells in skeletal repair disorders are still poorly understood. Here, we report that Fgfr3Y637C/+ over-activating mutation in Prx1-derived skeletal stem/progenitor cells leads to failure of fracture consolidation. We show that periosteal cells (PCs) carrying the Fgfr3Y637C/+ mutation can engage in osteogenic and chondrogenic lineages, but following transplantation do not undergo terminal chondrocyte hypertrophy and transformation into bone causing pseudarthrosis. Instead, Prx1Cre;Fgfr3Y637C/+ PCs give rise to fibrocartilage and fibrosis. Conversely, wild-type PCs transplanted at the fracture site of Prx1Cre;Fgfr3Y637C/+ mice allow hypertrophic cartilage transition to bone and permit fracture consolidation. The results thus highlight cartilage-to-bone transformation as a necessary step for bone repair and FGFR3 signaling within PCs as a key regulator of this transformation.

American Society for Bone and Mineral Research-Orthopaedic Research Society Joint Task Force Report on Cell-Based Therapies - Secondary Publication.

Cell-based therapies, defined here as the delivery of cells in vivo to treat disease, have recently gained increasing public attention as a potentially promising approach to restore structure and function to musculoskeletal tissues. Although cell-based therapy has the potential to improve the treatment of disorders of the musculoskeletal system, there is also the possibility of misuse and misrepresentation of the efficacy of such treatments. The medical literature contains anecdotal reports and research studies, along with web-based marketing and patient testimonials supporting cell-based therapy. Both the American Society for Bone and Mineral Research (ASBMR) and the Orthopaedic Research Society (ORS) are committed to ensuring that the potential of cell-based therapies is realized through rigorous, reproducible, and clinically meaningful scientific discovery. The two organizations convened a multidisciplinary and international Task Force composed of physicians, surgeons, and scientists who are recognized experts in the development and use of cell-based therapies. The Task Force was charged with defining the state-of-the art in cell-based therapies and identifying the gaps in knowledge and methodologies that should guide the research agenda. The efforts of this Task Force are designed to provide researchers and clinicians with a better understanding of the current state of the science and research needed to advance the study and use of cell-based therapies for skeletal tissues. The design and implementation of rigorous, thorough protocols will be critical to leveraging these innovative treatments and optimizing clinical and functional patient outcomes. In addition to providing specific recommendations and ethical considerations for preclinical and clinical investigations, this report concludes with an outline to address knowledge gaps in how to determine the cell autonomous and nonautonomous effects of a donor population used for bone regeneration. © 2020 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:485-502, 2020.

American Society for Bone and Mineral Research-Orthopaedic Research Society Joint Task Force Report on Cell-Based Therapies.

Cell-based therapies, defined here as the delivery of cells in vivo to treat disease, have recently gained increasing public attention as a potentially promising approach to restore structure and function to musculoskeletal tissues. Although cell-based therapy has the potential to improve the treatment of disorders of the musculoskeletal system, there is also the possibility of misuse and misrepresentation of the efficacy of such treatments. The medical literature contains anecdotal reports and research studies, along with web-based marketing and patient testimonials supporting cell-based therapy. Both the American Society for Bone and Mineral Research (ASBMR) and the Orthopaedic Research Society (ORS) are committed to ensuring that the potential of cell-based therapies is realized through rigorous, reproducible, and clinically meaningful scientific discovery. The two organizations convened a multidisciplinary and international Task Force composed of physicians, surgeons, and scientists who are recognized experts in the development and use of cell-based therapies. The Task Force was charged with defining the state-of-the art in cell-based therapies and identifying the gaps in knowledge and methodologies that should guide the research agenda. The efforts of this Task Force are designed to provide researchers and clinicians with a better understanding of the current state of the science and research needed to advance the study and use of cell-based therapies for skeletal tissues. The design and implementation of rigorous, thorough protocols will be critical to leveraging these innovative treatments and optimizing clinical and functional patient outcomes. In addition to providing specific recommendations and ethical considerations for preclinical and clinical investigations, this report concludes with an outline to address knowledge gaps in how to determine the cell autonomous and nonautonomous effects of a donor population used for bone regeneration. © 2019 American Society for Bone and Mineral Research.

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.

Periosteum contains skeletal stem cells with high bone regenerative potential controlled by Periostin.

Bone regeneration relies on the activation of skeletal stem cells (SSCs) that still remain poorly characterized. Here, we show that periosteum contains SSCs with high bone regenerative potential compared to bone marrow stromal cells/skeletal stem cells (BMSCs) in mice. Although periosteal cells (PCs) and BMSCs are derived from a common embryonic mesenchymal lineage, postnatally PCs exhibit greater clonogenicity, growth and differentiation capacity than BMSCs. During bone repair, PCs can efficiently contribute to cartilage and bone, and integrate long-term after transplantation. Molecular profiling uncovers genes encoding Periostin and other extracellular matrix molecules associated with the enhanced response to injury of PCs. Periostin gene deletion impairs PC functions and fracture consolidation. Periostin-deficient periosteum cannot reconstitute a pool of PCs after injury demonstrating the presence of SSCs within periosteum and the requirement of Periostin in maintaining this pool. Overall our results highlight the importance of analyzing periosteum and PCs to understand bone phenotypes.

BMP signaling regulates satellite cell-dependent postnatal muscle growth.

Postnatal growth of skeletal muscle largely depends on the expansion and differentiation of resident stem cells, the so-called satellite cells. Here, we demonstrate that postnatal satellite cells express components of the bone morphogenetic protein (BMP) signaling machinery. Overexpression of noggin in postnatal mice (to antagonize BMP ligands), satellite cell-specific knockout of Alk3 (the gene encoding the BMP transmembrane receptor) or overexpression of inhibitory SMAD6 decreased satellite cell proliferation and accretion during myofiber growth, and ultimately retarded muscle growth. Moreover, reduced BMP signaling diminished the adult satellite cell pool. Abrogation of BMP signaling in satellite cell-derived primary myoblasts strongly diminished cell proliferation and upregulated the expression of cell cycle inhibitors p21 and p57 In conclusion, these results show that BMP signaling defines postnatal muscle development by regulating satellite cell-dependent myofiber growth and the generation of the adult muscle stem cell pool.

Loss of Gi G-Protein-Coupled Receptor Signaling in Osteoblasts Accelerates Bone Fracture Healing.

G-protein-coupled receptors (GPCRs) are key regulators of skeletal homeostasis and are likely important in fracture healing. Because GPCRs can activate multiple signaling pathways simultaneously, we used targeted disruption of G(i) -GPCR or activation of G(s) -GPCR pathways to test how each pathway functions in the skeleton. We previously demonstrated that blockade of G(i) signaling by pertussis toxin (PTX) transgene expression in maturing osteoblastic cells enhanced cortical and trabecular bone formation and prevented age-related bone loss in female mice. In addition, activation of G(s) signaling by expressing the G(s) -coupled engineered receptor Rs1 in maturing osteoblastic cells induced massive trabecular bone formation but cortical bone loss. Here, we test our hypothesis that the G(i) and G(s) pathways also have distinct functions in fracture repair. We applied closed, nonstabilized tibial fractures to mice in which endogenous G(i) signaling was inhibited by PTX, or to mice with activated G(s) signaling mediated by Rs1. Blockade of endogenous G(i) resulted in a smaller callus but increased bone formation in both young and old mice. PTX treatment decreased expression of Dkk1 and increased Lef1 mRNAs during fracture healing, suggesting a role for endogenous G(i) signaling in maintaining Dkk1 expression and suppressing Wnt signaling. In contrast, adult mice with activated Gs signaling showed a slight increase in the initial callus size with increased callus bone formation. These results show that G(i) blockade and G(s) activation of the same osteoblastic lineage cell can induce different biological responses during fracture healing. Our findings also show that manipulating the GPCR/cAMP signaling pathway by selective timing of G(s) and G(i) -GPCR activation may be important for optimizing fracture repair.

Role of muscle stem cells during skeletal regeneration.

Although the importance of muscle in skeletal regeneration is well recognized clinically, the mechanisms by which muscle supports bone repair have remained elusive. Muscle flaps are often used to cover the damaged bone after traumatic injury yet their contribution to bone healing is not known. Here, we show that direct bone-muscle interactions are required for periosteum activation and callus formation, and that muscle grafts provide a source of stem cells for skeletal regeneration. We investigated the role of satellite cells, the muscle stem cells. Satellite cells loss in Pax7(-/-) mice and satellite cell ablation in Pax7(Cre) (ERT) (2/) (+) ;DTA(f/f) mice impaired bone regeneration. Although satellite cells did not contribute as a large source of cells endogenously, they exhibited a potential to contribute to bone repair after transplantation. The fracture healing phenotype in Pax7(Cre) (ERT) (2/) (+) ;DTA(f/f) mice was associated with decreased bone morphogenetic proteins (BMPs), insulin-like growth factor 1, and fibroblast growth factor 2 expression that are normally upregulated in response to fracture in satellite cells. Exogenous rhBMP2 improved bone healing in Pax7(Cre) (ERT) (2/) (+) ;DTA(f/f) mice further supporting the role of satellite cells as a source of growth factors. These results provide the first functional evidence for a direct contribution of muscle to bone regeneration with important clinical implications as it may impact the use of muscle flaps, muscle stem cells, and growth factors in orthopedic applications.

Cellular and molecular bases of skeletal regeneration: what can we learn from genetic mouse models?

Although bone repairs through a very efficient regenerative process in 90% of the patients, many factors can cause delayed or impaired healing. To date, there are no reliable biological parameters to predict or diagnose bone repair defects. Orthopedic surgeons mostly base their diagnoses on radiographic analyses. With the recent progress in our understanding of the bone repair process, new methods may be envisioned. Animal models have allowed us to define the key steps of bone regeneration and the biological and mechanical factors that may influence bone healing in positive or negative ways. Most importantly, small animal models such as mice have provided powerful tools to apprehend the genetic bases of normal and impaired bone healing. The current review presents a state of the art of the genetically modified mouse models that have advanced our understanding of the cellular and molecular components of bone regeneration and repair. The review illustrates the use of these models to define the role of inflammation, skeletal cell lineages, signaling pathways, the extracellular matrix, osteoclasts and angiogenesis. These genetic mouse models promise to change the field of orthopedic surgery to help establish genetic predispositions for delayed repair, develop models of non-union that mimic the human conditions and elaborate new therapeutic approaches to enhance bone regeneration.

Renal capsule transplantations to assay skeletal angiogenesis.

Renal capsule transplantation is a very helpful method to grow embryonic tissues or tumors in a vascular environment, allowing long-term engraftment and biological analyses. This chapter describes the surgical procedure for the transplantation of embryonic skeletal elements in the renal capsule of adult mice and points out the manipulations that can be applied for assaying the role of angiogenesis during bone development.

Delayed bone regeneration is linked to chronic inflammation in murine muscular dystrophy.

Duchenne muscular dystrophy (DMD) patients exhibit skeletal muscle weakness with continuous cycles of muscle fiber degeneration/regeneration, chronic inflammation, low bone mineral density, and increased risks of fracture. Fragility fractures and associated complications are considered as a consequence of the osteoporotic condition in these patients. Here, we aimed to establish the relationship between muscular dystrophy and fracture healing by assessing bone regeneration in mdx mice, a model of DMD with absence of osteoporosis. Our results illustrate that muscle defects in mdx mice impact the process of bone regeneration at various levels. In mdx fracture calluses, both cartilage and bone deposition were delayed followed by a delay in cartilage and bone remodeling. Vascularization of mdx fracture calluses was also decreased during the early stages of repair. Dystrophic muscles are known to contain elevated numbers of macrophages contributing to muscle degeneration. Accordingly, we observed increased macrophage recruitment in the mdx fracture calluses and abnormal macrophage accumulation throughout the process of bone regeneration. These changes in the inflammatory environment subsequently had an impact on the recruitment of osteoclasts and the remodeling phase of repair. Further damage to the mdx muscles, using a novel model of muscle trauma, amplified both the chronic inflammatory response and the delay in bone regeneration. In addition, PLX3397 treatment of mdx mice, a cFMS (colony stimulating factor receptor 1) inhibitor in monocytes, partially rescued the bone repair defect through increasing cartilage deposition and decreasing the number of macrophages. In conclusion, chronic inflammation in mdx mice contributes to the fracture healing delay and is associated with a decrease in angiogenesis and a transient delay in osteoclast recruitment. By revealing the role of dystrophic muscle in regulating the inflammatory response during bone repair, our results emphasize the implication of muscle in the normal bone repair process and may lead to improved treatment of fragility fractures in DMD patients.

MMP9 regulates the cellular response to inflammation after skeletal injury.

Like other tissue injuries, bone fracture triggers an inflammatory response, which plays an important role in skeletal repair. Inflammation is believed to have both positive and negative effects on bone repair, but the underlying cellular mechanisms are not well understood. To assess the role of inflammation on skeletal cell differentiation, we used mouse models of fracture repair that stimulate either intramembranous or endochondral ossification. In the first model, fractures are rigidly stabilized leading to direct bone formation, while in the second model, fracture instability causes cartilage and bone formation. We compared the inflammatory response in these two mechanical environments and found changes in the expression patterns of inflammatory genes and in the recruitment of inflammatory cells and osteoclasts. These results suggested that the inflammatory response could influence skeletal cell differentiation after fracture. We then exploited matrix metalloproteinase 9 (MMP9) that is expressed in inflammatory cells and osteoclasts, and which we previously showed is a potential regulator of cell fate decisions during fracture repair. Mmp9(-/-) mice heal stabilized fractures via endochondral ossification, while wild type mice heal via intramembranous ossification. In parallel, we observed increases in macrophages and T cells in the callus of Mmp9(-/-) compared to wild type mice. To assess the link between the profile of inflammatory cells and skeletal cell fate functionally, we transplanted Mmp9(-/-) mice with wild type bone marrow, to reconstitute a wild type hematopoietic lineage in interaction with the Mmp9(-/-) stroma and periosteum. Following transplantation, Mmp9(-/-) mice healed stabilized fractures via intramembranous ossification and exhibited a normal profile of inflammatory cells. Moreover, Mmp9(-/-) periosteal grafts healed via intramembranous ossification in wild type hosts, but healed via endochondral ossification in Mmp9(-/-) hosts. We observed that macrophages accumulated at the periosteal surface in Mmp9(-/-) mice, suggesting that cell differentiation in the periosteum is influenced by factors such as BMP2 that are produced locally by inflammatory cells. Taken together, these results show that MMP9 mediates indirect effects on skeletal cell differentiation by regulating the inflammatory response and the distribution of inflammatory cells, leading to the local regulation of periosteal cell differentiation.

Solid-supported lipid bilayers to drive stem cell fate and tissue architecture using periosteum derived progenitor cells.

A challenge to mimicking nature's "bottom up" approach to generate tissue is the coordination of cellular self-assembly and emergent phenotype. Here we create a biosynthetic platform to mimic native cell-cell interactions and to drive emergent tissue behavior by human multipotent cells from the periosteal niche, i.e. PDCs, whose regenerative capacity is equal or greater to those from the bone marrow niche. Western blots showed that human PDCs express proteins for both N-cadherin, a hallmark of mesenchymal condensation, as well as for ZO-1, a tight junction membrane protein conferring epithelial barrier membrane properties. Hence, we functionalized a solid supported lipid bilayer (SLB) membrane with recombinant N-cadherin and investigated the short term phenotype of PDCs seeded on unfunctionalized and N-cadherin functionalized SLBs compared to that of PDCs seeded on glass coverslips. After 24 h, SLB functionalization promoted aggregation of PDCs seeded at high density (35,000 cells/cm(2)), with no significant concomitant changes in transcription of N-cadherin (CDH2) as measured by rtPCR. In contrast, cells seeded on unfunctionalized SLBs remained non-adherent but showed a significant upregulation in transcription of N-cadherin. Furthermore, culture of PDCs at high density on N-cadherin functionalized SLBs was negatively correlated with expression of ZO-1, while culture on unfunctionalized SLBs was positively correlated with the expression of the tight junction membrane protein. High density seeding on N-cadherin functionalized and unfunctionalized SLBs places PDCs in distinct cellular contexts and relates to emergent behavior typical for mesenchymal condensation. These studies demonstrate a biosynthetic in vitro cell culture platform to elucidate and guide emergent tissue architectures by PDCs.

Current insights on the regenerative potential of the periosteum: molecular, cellular, and endogenous engineering approaches.

While century old clinical reports document the periosteum's remarkable regenerative capacity, only in the past decade have scientists undertaken mechanistic investigations of its regenerative potential. At a Workshop at the 2012 Annual Meeting of Orthopaedic Research Society, we reviewed the molecular, cellular, and tissue scale approaches to elucidate the mechanisms underlying the periosteum's regenerative potential as well as translational therapies engineering solutions inspired by its remarkable regenerative capacity. The entire population of osteoblasts within periosteum, and at endosteal and trabecular bone surfaces within the bone marrow, derives from the embryonic perichondrium. Periosteal cells contribute more to cartilage and bone formation within the callus during fracture healing than do cells of the bone marrow or endosteum, which do not migrate out of the marrow compartment. Furthermore, a current healing paradigm regards the activation, expansion, and differentiation of periosteal stem/progenitor cells as an essential step in building a template for subsequent neovascularization, bone formation, and remodeling. The periosteum comprises a complex, composite structure, providing a niche for pluripotent cells and a repository for molecular factors that modulate cell behavior. The periosteum's advanced, "smart" material properties change depending on the mechanical, chemical, and biological state of the tissue. Understanding periosteum development, progenitor cell-driven initiation of periosteum's endogenous tissue building capacity, and the complex structure-function relationships of periosteum as an advanced material are important for harnessing and engineering ersatz materials to mimic the periosteum's remarkable regenerative capacity.