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RESEARCH PERSPECTIVE Current Insights on the Regenerative Potential of the Periosteum: Molecular, Cellular, and Endogenous Engineering Approaches Ce´line Colnot,1 Xinping Zhang,2 Melissa L. Knothe Tate3 1

INSERM U781, Universite´ Paris Descartes-Sorbonne Paris Cite´, Institut Imagine, Tour Lavoisier 2e`me e´tage, Hoˆpital Necker-Enfants Malades, 149 rue de Se`vres-75015 Paris, France, 2The Center for Musculoskeletal Research, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, New York 14642, 3Departments of Biomedical Engineering and Mechanical & Aerospace Engineering, Case Western Reserve University, 2071 Martin Luther King Drive, Cleveland, Ohio 44106-7207 Received 29 March 2012; accepted 5 June 2012 Published online 9 July 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.22181

ABSTRACT: 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. ß 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1869–1878, 2012 Keywords: periosteum; regenerative medicine; tissue engineering; bone biology; advanced materials

A workshop was held at the 2012 Orthopaedic Research Society Annual Meeting to present the science of the periosteum and its regenerative potential. While clinical reports of the periosteum’s remarkable regenerative capacity can be found in century old scientific literature, mechanistic investigations of its regenerative potential have been published mostly in the past decade. The periosteum comprises a complex, composite structure that provides a niche for pluripotent cells and a repository for molecular factors that modulate cell behavior. In addition, periosteum exhibits advanced, smart material properties that change depending on the mechanical, chemical, and biological state of the tissue. Hence, the aim of the workshop was to review molecular, cellular, and tissue scale approaches to elucidate the mechanisms underlying the periosteum’s regenerative potential and to discuss these approaches in light of translational therapies and engineering solutions inspired by the its remarkable regenerative capacity. An interactive panel discussion highlighted current hurdles to advancement, clinical translation of these insights, and current controversies in the field.

DEVELOPMENT OF THE PERIOSTEUM AND ITS ROLE DURING BONE REPAIR The periosteum is a thin tissue lining the outer surface of bone. From a structural perspective, the Correspondence to: Melissa L. Knothe Tate (T: 1-216-3685884; F: 1-216-3684969; E-mail: [email protected]) ß 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

periosteum is a bilayered membrane. The outer layer consists mostly of collagens, aligned with the longitudinal axis, and elastin,1–3 and is thought to serve a mostly structural (mechanical) role.4 The periosteum’s innermost layer (closest to the bone) consists mostly of progenitor cells that constantly build1 and repair bone.5–7 This tissue is highly vascularized, and its preservation is crucial for normal bone repair. Periosteum is rich in osteoblasts, which deposit new bone matrix in the outer cortex, and in osteoblast precursors. Although bone marrow-derived pluripotent cells have been mostly exploited in regenerative medicine to facilitate healing of orthopaedic injuries, the periosteum is now recognized as an attractive source of cells.8–10 Over the past decade, animal models have been developed to assess the mechanisms of skeletal stem/progenitor cell recruitment during bone repair and to test the therapeutic effects of skeletal stem/ progenitor cells. Several studies revealed that the endogenous regenerative potential of periosteum is high compared to bone marrow and other cell sources. Development During long bone development, all the populations of osteoblasts within periosteum and osteoblasts at the endosteal and trabecular bone surfaces within the marrow, are derived from the embryonic perichondrium.11 Each skeletal element is derived from a mesenchymal condensation that gives rise to a cartilage template surrounded by the perichondrium (Fig. 1). Ossification of these elements begins with vascular invasion of the JOURNAL OF ORTHOPAEDIC RESEARCH DECEMBER 2012

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perichondrium followed by the invasion of hypertrophic cartilage in the center of the cartilage template.13 The vascular invasion is followed by rapid removal of the calcified cartilage matrix and its replacement by bone and bone marrow. This process of endochondral ossification is highly regulated by several signaling pathways, including Hedgehog, bone morphogenetic protein (BMP), TGF-beta, PTH/PTHrP, FGF, Wnt, Notch, and VEGF, that act at the levels of chondrocytes, perichondrium, and blood vessels, allowing the synchronization of cell differentiation in these adjacent tissues.14–19 Many cell types participate in this process with some differentiating locally and others brought by blood vessels, but lineage analyses show that osteoblasts all come from the perichondrium.11 Osteoblasts precursors originating from the perichondrium migrate along with blood vessels to form the primary ossification center.20 Thus, during development, osteoblasts within periosteum and bone marrow are derived locally from the initial mesenchymal condensations, and more specifically the perichondrium, without systemic contribution from invading blood vessels.

Figure 1. Development of the periosteum and its contribution to bone repair. Stages of long bone development including formation of the initial mesenchymal condensations, followed by the segregation of cartilage (pink) and perichondrium (blue), vascular invasion, and replacement of hypertrophic cartilage by bone and bone marrow. Osteoblasts within periosteum (blue), bone marrow, and endosteum (green) are derived from the embryonic perichondrium. In the adult, after bone injury, cells that form cartilage and bone in the fracture callus are recruited locally from periosteum, bone marrow, blood vessels (pericytes), and potentially other adjacent tissues such as muscle and fat. Cellular contribution from systemic sources is minimal (red dots). Figure modified from Ref. 12. [Color figure can be seen in the online version of this article, available at http://wileyonlinelibrary.com/journal/jor] JOURNAL OF ORTHOPAEDIC RESEARCH DECEMBER 2012

Repair In the adult, the stages of bone repair recapitulate the well-defined stages of bone formation during embryogenesis, except for the inflammatory response, which is critical for bone regeneration.21–26 During this initial phase of repair, skeletal stem cells are activated at the fracture site and differentiate into osteoblasts and chondrocytes that deposit the extracellular matrix necessary for bone bridging. Skeletal stem cells differentiate during the inflammatory phase of repair and are exposed to inflammatory cytokines, growth factors, and mechanical signals.27–35 How these cellular, molecular and mechanical signals influence the recruitment of skeletal stem cells remains largely unknown. Histologically, cells within the periosteum respond rapidly to these signals, as a periosteal reaction can be detected within 24–48 h post-injury.36,37 In the absence of stabilization, this periosteal reaction is particularly robust leading to the formation of a large callus and healing via endochondral ossification.36,37 When fractures are rigidly stabilized, cells in the periosteum are not stimulated as efficiently, and callus formation is minimal. In this mechanically stable environment, healing occurs essentially via intramembranous ossification.38 These differences in cellular response to mechanical signals reside mostly within the periosteum, as shown by the up-regulation of the BMP pathway within the periosteum during the first stage of healing in non-stabilized fractures.39,40 The periosteum contains skeletal progenitors that play an important role in bone repair; however, their identity and relative contribution to healing compared to other cell sources are not well defined. Many sources of skeletal progenitors have been proposed to participate in adult bone repair, including the local

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bone marrow, periosteum, soft tissues, and blood vessel walls, as well as cells brought to the injury site via blood vessels. Several approaches have been used to determine the potential recruitment of cells from distant sites during fracture repair, including parabiosis, bone marrow transplantation, and intravenous injection of cells in animal models.41–45 These approaches suggest that cells recruited systemically are minimal contributors to cartilage and bone, but give rise mostly to inflammatory cells and osteoclasts.46,47 The contribution of blood vessels themselves was addressed using genetic lineage tracing. Endothelial cells do not appear to transdifferentiate into skeletal progenitors involved in fracture healing as cartilage and bone within the callus are not derived from Tie2-expressing cells.48 Pericytes marked with smooth muscle actin, however, coincide with a population of endogenous mesenchymal progenitors and can largely contribute to fracture healing.49 The tissue origins of these mesenchymal progenitors are undetermined, yet they are likely derived from the local blood vessels around the fracture site. Other populations of mesenchymal stem cells expressing Mx1 exist within the bone marrow cavity and have osteogenic potential during the repair of calvarial defects.50 These types of studies provide new molecular tools to elucidate the mechanisms of skeletal stem cell recruitment during bone repair. Lineage analyses based on bone grafting have also revealed that periosteum largely contributes to cartilage and bone within the callus compared to bone marrow and endosteum.5–8,51,52 Cells within local marrow and endosteum form bone mostly within the marrow cavity, and do not migrate out of the marrow compartment to form callus.5 Thus, not only local tissues, but perhaps most importantly, the periosteum are key cellular contributors to bone repair (Fig. 1). The extent to which progenitors within marrow and periosteum exhibit distinct regenerative capacities has not been established. The tissue location may play an important role, as cells located in periosteum and marrow may not receive the same biological and mechanical signals upon injury. Understanding these differences may help define competent cell sources and ways to stimulate the regenerative capacities of mesenchymal stem cells from other tissues, such as muscle, fat, and umbilical cord. Research is underway to characterize the endogenous cell sources for bone repair, identify the molecular pathways controlling their recruitment, and apply this knowledge to bone tissue engineering approaches.10,53–58

NEW APPROACHES TOWARD UNDERSTANDING SKELETAL REPAIR AND REGENERATION Skeletal repair is a dynamic, well-orchestrated process that involves complex and spatiotemporally coordinated function of different cellular compartments and integrated molecular pathways. Understanding complex

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molecular and cellular interactions during healing represents a critical step toward developing effective treatment strategies for enhancing repair and reconstruction. Immediately following cortical bone injury, periosteum undergoes a series of changes to initiate endochondral and intramembraneous bone formation at the injury site. Both types of bone formation begin with intensive proliferation of periosteal progenitor cells. Cells near the cortical bone junction differentiate into chondroprogenitors, whereas cells at the periphery of the cortex furthest away from the junction adopt an osteogenic cell fate. The contribution of the periosteal progenitors to callus formation was examined using a segmental bone graft transplantation model in mice.6,58 By transplantation of a Rosa26A bone graft, the study demonstrated a predominant contribution of periosteal progenitors to both endochondral and intramembraneous bone formation at the initiation of repair. By tracking the LacZþve cell fate, the study further suggested a research paradigm in which activation, expansion, and differentiation of periosteal stem/progenitor cells act as an essential step to build a template for subsequent neovascularization, bone formation, and remodeling. Understanding this progenitor cell-driven initiation process not only provides mechanistic insight into endogenous regeneration capacity of periosteum, but could also offer information for optimizing tissue engineering constructs for fabrication of a periosteum substitute for repair and reconstruction. Bone Morphogenetic Proteins (BMPs) Using genetically modified mouse models, a number of molecular pathways have been discovered to play a critical role in the initiation of periosteum-mediated regeneration. Among them, BMP-2 appears to be at the apex of the signaling cascade that initiates the cellular proliferation and differentiation of periosteal progenitors during repair and regeneration. Genetic deletion of BMP-2 gene via Prx-1-Cre in limb mesenchyme condensation is dispensable for development of long bones. However, long bone lacking BMP-2 expression develops spontaneous fractures in adult animals. Most strikingly, deletion of BMP-2 completely abolishes fracture callus formation, suggesting a critical role of BMP-2 in the initiation of repair.59 In a similar study in which BMP-2 was knocked out at the initiation stage of healing in adult animals using a Tamoxifen inducible CreER mouse model,60,61 deletion of BMP-2 at the onset of healing completely abrogated both endochondral and intramembranous bone repair. Deletion of BMP-2 in periosteal progenitor cells not only blocked cellular differentiation, but also impaired proliferation and survival of the cells. Further tracking of the BMP-2 mutant cells in a chimeric periosteal callus showed that few mutant cells could differentiate into chondrocytes and osteoblasts, even when they were placed in a wild type host injury environment, indicating an essential role of endogenous BMP-2 JOURNAL OF ORTHOPAEDIC RESEARCH DECEMBER 2012

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signaling in the initiation of periosteal callus formation. In addition to BMP-2, several key components of BMP family proteins and their corresponding receptors are found in the activated periosteum, including BMP-2, 3, 4, 5, 8, noggin, BMPRIA, BMPRII, and pSmad 1/5/8.40 The differential role of BMP isoforms in repair was examined using floxed mouse models that allowed conditional deletion of BMP isoforms via Prx-1Cre. BMP-4 and 6 were dispensable for repair whereas BMP-3 played a negative role for early periosteum development and potentially in postnatal repair and regeneration.62–64 Transgenic mice overexpressing BMP3 via the type I collagen promoter displayed spontaneous rib fractures as early as E17.0. The fractures were due to defects in differentiation of the periosteum and late hypertrophic chondrocytes resulting in thinner cortical bone with decreased mineralization. Hedgehog, Ihh Pathway Downstream of BMPs, the hedgehog pathway, in particular the Ihh pathway has long been suspected to play a role in periosteum-mediated endochondral bone repair.24,25,65–67 Both Shh and Ihh have been implicated in early embryonic development that suggests mesenchymal progenitor cell differentiation and selfrenewal.68 Ihh plays a key role in perichondrium development and collar bone formation. Embryonic deletion of Ihh disrupts collar bone formation and early osteoblast development.69–71 Ihh is abundantly expressed in prehypertrophic and hypertrophic chondrocytes in callus. Using in situ hybridization and Ptc-LacZ staining, a recent study from Zhang et al. further showed that Ihh was expressed in nascent cartilaginous tissues in periosteum callus adjacent to the bone surface at the initiation stage of healing. These hedgehog producing cells send out signals to the surrounding chondroprogenitors, osteoblast progenitors as well as cells associated with early invading vessels.72 To further determine the role of hedgehog pathway in postnatal periosteum-mediated repair and regeneration, Wang et al. isolated a unique population of mesenchymal progenitors from day 5 autograft periosteum. These isolated periosteal cells expressed mesenchymal progenitor cell markers: SSEA4, CD105, CD29, CD140b, and ScaI. They could further give rise to osteoblasts, chondrocytes and adipocyte in vitro. Compared with mesenchymal progenitors isolated from other tissues, such as adipose and bone marrow, these cells showed stronger responsiveness to both BMP-2 and hedgehog agonists. Overexpression of Shh N-terminal peptide, a hedgehog agonist, in these cells induced robust ectopic bone formation in nude mice. Further deletion of Smoothened1, a receptor that transduces all hedgehog signaling, using a Tamoxifen inducible CreER mouse model significantly reduced periosteal bone formation in the conditional knockout mice.72 These studies indicate an important role of hedgehog pathway in periosteum-mediated repair and regeneration. Further JOURNAL OF ORTHOPAEDIC RESEARCH DECEMBER 2012

studies are necessary to determine the potential use of hedgehog agonists in repair and in bone tissue engineering applications. Cyclooxygenase-2, COX-2 COX-2 is discovered as an inducible isoform of cyclooxygenase in the prostaglandin biosynthesis pathway. As an immediate early gene, COX-2 is induced by a variety of inflammatory cytokines and growth factors, including bone anabolic factors FGF, IGF, TGF-b, and BMP-2.73–76 COX-2 induction was localized in chondroprogenitors, proliferating chondrocytes, and osteoblasts, concomitant with the initiation of endochondral and intramembraneous bone formation in periosteum.51 Although deletion of COX-2 has no discernible effect on postnatal bone development, deletion of COX2 globally or specifically in mesenchyme or cartilage significantly impairs periosteal progenitor cell proliferation and delays subsequent endochondral and intramembranous repair.21,51 The critical role of COX-2 in repair was further illustrated in a study of fracture healing in aged mice. In comparison to young mice, aged mice elicited a mitigated induction of COX-2 during early endochondral bone formation. Treatment of aged mice with an agonist of prostaglandin receptor type 4 receptor (EP4) rescued the delayed endochondral bone formation,77 suggesting a beneficial effect of targeting EP receptor for improved healing. Wnt Signaling Pathway Wnt/b-catenin, a critical player in osteoblast differentiation and bone formation, has recently emerged as a potential therapeutic target for bone repair and fracture healing.78,79 The Wnt pathway plays a key role in bone and cartilage development. Activation of the Wnt pathway favors osteoblastic differentiation, but inhibits chondrogenesis.80,81 Multiple Wnt proteins and their modulators are expressed in periosteum.82 Although the detailed molecular actions of Wnt pathway on different phases of endochondral bone repair remain to be determined, genetic manipulation of Wnt signaling in mice show that inhibition of Wnt/ b-catenin could suppress early chondrogenesis but favor osteogenesis, leading to accelerated fracture repair.83,84 Consistently, delivery of a Wnt/b-catenin inhibitor DKK1 suppressed bone repair, whereas administration of a DKK1 neutralizing antibody improved repair and regeneration.82,85,86 Interestingly, several pathways known to stimulate fracture repair, including the BMP-2 and Hh pathways, enhance the Wnt/b-catenin pathway.87,88,72 In addition, intermittent PTH treatment strongly stimulated fracture healing in part by inducing canonical Wnt signaling.89 Prostaglandin E2, the major metabolite from COX-2 enzymatic activity, also activates canonical Wnt signaling via EP2 and EP4 receptors.90 How these pathways converge on Wnt/b-catenin to enhance repair has become the focus of intensive studies.


SURGICAL AND ENGINEERING APPROACHES TO ELUCIDATE AND UNLEASH THE REGENERATIVE POWER OF THE PERIOSTEUM The periosteum is a composite tissue1,2 that provides a niche for pluripotent osteochondroprogenitor cells and exhibits a remarkable capacity to generate bone de novo within critical sized defects. Surgeons have harnessed this regenerative capacity for more than a century.91–93 Recent studies have focused on the mechanisms underlying periosteum’s remarkable regenerative capacity, and in particular the role of mechanical and mechanically modulated signals in this process. Periosteum’s Regenerative Capacity A recently described case study94 demonstrating the capacity of the periosteum in situ to regenerate a several inch segment of resected fibula provided the

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inspiration for a one stage bone transport procedure,95 which has provided a new platform to elucidate the role of specific biological and mechanical fractures in critical-sized long bone defect repair in large mammals.96,97 In this procedure (Fig. 2A), a solid, reamed intramedullary nail fills the medullary cavity, stabilizing the femur. Proximal to the defect, the periosteum is carefully lifted and peeled back, maintaining the blood supply but disrupting the Sharpey’s fibers that anchor the periosteum to the underlying bone. Osteotomy then produces a periosteum denuded bone segment, which is transported and docked distally to fill the original defect. The periosteum is then sutured closed in situ around the newly created defect zone and to the denuded bone segment, forming a sleeve around the haematoma or autologous graft filled defect.95 When treated this way, de novo bone

Figure 2. One stage bone transport model to harness the regenerative capacity of the periosteum provides an in vivo platform to elucidate the role of specific biological and mechanical factors in critical-sized long bone defect healing. (A) Surgical procedure, where (A1) the periosteum proximal to the defect is peeled back, and an osteotomy is performed, after which the denuded bone segment is transported distally and docked (A2), filling the original defect zone and creating a new, more proximal defect. The periosteum is then sutured in place in situ, forming a sleeve around the new, haematoma-filled defect. The entire construct is stabilized by an interlocked intramedullary nail. (B) Addition of factors to the periosteal sleeve model, including morcelized autologous bone graft from the iliac crest and bone chips adhering in situ to the inside of the periosteal sleeve, results in better bone infilling and defect healing than the baseline control. Groups with adherent bone chips but without morcelized graft showed the best healing at 16 weeks. (C) Addition of factors to the periosteum substitute model show an improvement in healing with increasing numbers of factors added, including periosteum-derived cells seeded on collagen membranes and the inclusion of periosteal strips. (D) The periosteum substitute implant exhibits a modular design for inclusion of desired periosteal factors and/or pharmaceuticals. Figure modified from Refs. 96,102. [Color figure can be seen in the online version of this article, available at http://wileyonlinelibrary.com/journal/jor] JOURNAL OF ORTHOPAEDIC RESEARCH DECEMBER 2012

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completely bridges the defect after 16 weeks, even in the absence of the medullary cavity, which is filled by the nail. Interestingly, filling the periosteumenveloped defect with graft retards the time for infilling by periosteum (Fig. 2B).93,95 Slowing of the healing response may be attributed to, on the one hand, an increased resistance to cellular egression and mass transport from the periosteum to the defect. On the other hand, the bone graft filling the defect must be resorbed prior to vasculogenesis and new bone apposition, requiring additional healing time.93 Clinical reports and recent experiments indicate that mechanical loading enhances the regenerative capacity of the periosteum. In studies using the previously described experimental platform, prevailing mechanical loads and proximity to the periosteum were hypothesized to modulate early bone generation in the defect zone and late measures of healing and remodeling of autograft in the denuded bone transport segment.93,95,96 Quantifying the area (a measure of bone quantity) and concentration (a measure of bone quality) of calcein green fluorochrome, which was administered in the first 2 weeks after surgery, allowed for correlation of new bone formation to both loading history and proximity to the periosteum. The laser confocal microscope was used as a spectroscope to measure the intensity of the fluorochrome signal as a function of distance from the periosteum; the intensity of fluorescence in a single focal plane indicates the concentration or amount of mineral chelation by the fluorochrome.93 Further, the major and minor centroidal axes of the long bone cross-section indicate axes about which the bone is most and least resistant to bending loads, respectively. Using these measures to compare groups treated with and without packed morcelized bone graft, the amount of early bone formation was significantly higher along the bone axis most resistant to bending loads (major centroidal axis), but the quality of early bone formed (density as measured by intensity or concentration of mineralized tissue) was higher along the bones axis least resistant to bending loads (minor centroidal axis). Finally, the spatial distribution of new bone formed in the first weeks after surgery correlated significantly with the distance from the periosteum and prevailing mechanical loads.93 Interestingly, although the periosteum itself regenerates in the denuded bone segment after the one stage bone transport procedure, the thickness of the regenerated periosteum did not correlate to prevailing mechanical loads.98 Based on the aforementioned studies, factors inherent to the periosteum, including the ingression of pluripotent cells from the periosteum, drive the process. Although correlation does not equal causation, ongoing studies aim to elucidate mechanistic relationships between mechanical loading, transport of cells and molecular factors, and de novo generation of tissue in critical sized bone defects. JOURNAL OF ORTHOPAEDIC RESEARCH DECEMBER 2012

The regenerative capacity of the periosteum exhibits great clinical promise for treatment of non-unions and tissue defects occurring due to tumor resection, infection, trauma, and congenital defects. The approach exemplified by the one stage bone transport procedure has been implemented successfully in limited clinical cases where other treatment modalities were not feasible.100 Engineering of substitute periosteum is a promising area of research that benefits from both top down perspectives and bottom up approaches to recreate the multiscale structure–function relationships embodied by the smart material, using nature’s engineering paradigms (Fig. 2C and D).101–105 Mechanical and Permeability Properties of Periosteum An understanding of periosteum’s mechanical properties and the local mechanical environment of its progenitor cells is important to understand and harness periosteum’s mechanobiology and regenerative capacity. In a series of recent studies, Knothe Tate’s lab showed that periosteum is hypoosmolaric (swells in phosphate buffered saline), pre-stressed, and anisotropic; periosteum shrinks twice as much in the axial than in the circumferential direction when released from the underlying bone, which is indicative of pre-stress in the tissue.103,104 Furthermore, the elastic modulus is ten times greater in the axial direction and exhibits strain stiffening at loading rates corresponding to trauma. These anisotropic properties are expected to profoundly influence bone mechanobiology, during development, growth, and healing, in both health and disease.103 The multiscale permeability properties of periosteum are intriguing. Periosteum tissue exhibits barrier properties, such as swelling under isotonic conditions and physiological pH.103,105 Furthermore, periosteum permeability is modulated by stress and is directionally dependent as well as site specific. For example, the permeability of the ovine femur is significantly more permeable in the bone ! muscle direction when pre-stress is maintained throughout testing. When periosteum pre-stress is not maintained,105 it is more permeable in the muscle ! bone direction. At a cellmolecular scale, recent Western blot experiments show that periosteum derived cells express proteins for zona occludens 1 (ZO-1, a tight junction protein) and N-cadherin (an adherens junction protein), both of which are necessary for the formation of tight junctions, which confer barrier properties to tissues.106 Prevailing stress also appears to modulate periosteum behavior. High resolution optical strain mapping of the local mechanical milieu of cells within the periosteum shows that bone generation in defects correlates to regions of native, intact periosteum experiencing the greatest net change in strain.99 Top Down and Bottom Up Approaches to Engineering Periosteum The spatial arrangement of cells and temporal presentation of other periosteal (biochemical) factors is critical to


de novo bone tissue generation.23–27 Two recent bottom up approaches to engineering substitute periosteum comprise replicating the periosteum and its anisotropic mechanical and transport properties as well as biochemical synthetic and cell biological approaches to engineer architectures emulating periosteum’s cellular and molecular organization.102,106,107 In the first, an implant cum delivery device was created and tested successfully in an ovine critical sized defect model, enabling controlled and directional delivery of periosteal factors to the defect zone.102 Furthermore, implementation of directional delivery implants designed as periosteum substitutes show that periosteum-derived cells and other biologic factors intrinsic to periosteum play key roles for infilling of critical sized defects. In the second approach, solid supported lipid bilayers were engineered to provide a novel substrate for the culture of periosteum derived cells and as a novel platform for bottom-up engineering and synthesis of periosteum substitutes.106 In summary, a number of mechanobiological factors facilitate de novo tissue building by the periosteum as well as surgical and engineering approaches to unleash the power of the periosteum for trauma and reconstruction surgery (Fig. 2). While top down approaches to understanding the endogenous capacity of the periosteum to heal defects in situ and in vivo are important to understand nature’s own healing and developmental paradigms, bottom up approaches to engineer substitute periosteum, or smart materials mimicking the properties of periosteum may open up novel treatment modalities to address the currently intractable clinical problem of creating bone and/or other tissues where there is none.

CONCLUSION Bone repair is a dynamic process beginning with the recruitment of skeletal stem/progenitor cells during the inflammatory phase of repair, followed by cell differentiation, extracellular matrix deposition, and remodeling. In humans, bone repair occurs spontaneously providing that the fractures are properly reduced. However, under certain conditions, due to extreme trauma, infection, or the health status of the patient, healing may be impaired leading to delayedunion or non-union. Orthopaedic interventions to treat these skeletal repair defects aim to mechanically support repair or better stimulate the endogenous healing response and therefore mainly rely on the intrinsic regenerative capacities of bone. When these regenerative capacities are compromised, more efficient therapeutic approaches are needed. Although BMPs and other molecules can augment bone repair therapeutically, demand is growing for cell-based therapies and novel, advanced materials-based implants to deliver cells and healing factors. The need for additional sources of cells is particularly evident in case of severe trauma, cancer treatment, and reconstructive surgery. Skeletal developmental and degenerative diseases, such as

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osteogenesis imperfecta and osteoporosis, could also benefit from cell-based therapy. Many efforts are now focused on the design of new scaffolds and materials to create a biocompatible environment and means to modulate spatiotemporal delivery of skeletal stem/ progenitor cells. To date, most of these engineering approaches take advantage of bone marrow-derived cells that are easily accessible and have been extensively described in the literature. This review has highlighted the potential of the periosteum and its resident cells as a new source of inspiration and raw material for novel regenerative medicine and tissue engineering approaches.

ACKNOWLEDGMENTS The ORS Workshop, ‘‘Current Insights on the Regenerative Potential of the Periosteum: Molecular, Cellular, and Endogenous Engineering Approaches’’ was organized by Dr. Melissa Knothe Tate and sponsored by the ORS Women’s Leadership Forum. CC’s work is supported by Institut National de la Sante´ et de la Recherche Me´dicale ATIP-AVENIR program, Sanofi, FP7 Marie Curie, Osteosynthesis, and Trauma Care Foundation and NIH-NIAMS R01 AR053645. XZ’s study is supported by grants from the Musculoskeletal Transplant Foundation (XPZ), NYSTEM N08G-495 (XPZ) and N09G346 (XPZ), and the National Institutes of Health (R21 DE021513 to XPZ, RC1AR058435 to XPZ, AR051469 to XPZ, and AR048681 to RJO and XPZ). MKT’s research studies are supported through grants from the National Institutes of Health, National Science Foundation, AO Foundation, Alexander von Humboldt Foundation, the Coulter Case Translational Research Partnership, and the Christopher Columbus Foundation—U.S. Chamber of Commerce.

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