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).     

rBMP represses Wnt signaling and influences skeletal progenitor cell fate specification during bone repair

Bone morphogenetic proteins (BMPs) participate in multiple stages of the fetal skeletogenic program from promoting cell condensation to regulating chondrogenesis and bone formation through endochondral ossification. Here, we show that these pleiotropic functions are recapitulated when recombinant BMPs are used to augment skeletal tissue repair. In addition to their well‐documented ability to stimulate chondrogenesis in a skeletal injury, we show that recombinant BMPs (rBMPs) simultaneously suppress the differentiation of skeletal progenitor cells in the endosteum and bone marrow cavity to an osteoblast lineage. Both the prochondrogenic and antiosteogenic effects are achieved because rBMP inhibits endogenous β‐catenin‐dependent Wnt signaling. In the injured periosteum, this repression of Wnt activity results in sox9 upregulation; consequently, cells in the injured periosteum adopt a chondrogenic fate. In the injured endosteum, rBMP also inhibits Wnt signaling, which results in the runx2 and collagen type I downregulation; consequently, cells in this region fail to differentiate into osteoblasts. In muscle surrounding the skeletal injury site, rBMP treatment induces Smad phosphorylation followed by exuberant cell proliferation, an increase in alkaline phosphatase activity, and chondrogenic differentiation. Thus different populations of adult skeletal progenitor cells interpret the same rBMP stimulus in unique ways, and these responses mirror the pleiotropic effects of BMPs during fetal skeletogenesis. These mechanistic insights may be particularly useful for optimizing the reparative potential of rBMPs while simultaneously minimizing their adverse outcomes. © 2010 American Society for Bone and Mineral Research     

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.     

Bone regeneration and limb lengthening

Regeneration of bone in the presence of stable fixation and the maintenance of the osteogenic tissue (marrow, endosteum, nutrient artery, and periosteum) required another factor to stimulation of bone regeneration: incremental distraction produces bone of both endosteal and periosteal origin. The soft tissues undergo to same growth phenomenon. The mechanism of ossification occurs without intermediate fibrocartilage.     

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. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1869–1878, 2012     

Perivascular Mesenchymal Progenitors for Bone Regeneration

Mesenchymal progenitor cells reside in all assayed vascularized tissues, and are broadly conceptualized to participate in homeostasis/renewal and repair. The application of mesenchymal progenitor cells has been studied for diverse orthopaedic conditions related to skeletal degeneration, regeneration, and tissue fabrication. One common niche for mesenchymal progenitors is the perivascular space, and in both mouse and human tissues, perivascular progenitor cells have been isolated and characterized. Of these “perivascular stem cells” or PSC, pericytes are the most commonly studied cells. Multiple studies have demonstrated the regenerative properties of PSC when applied to bone, including direct osteochondral differentiation, paracrine‐induced osteogenesis and vasculogenesis, and immunomodulatory functions. The confluence of these effects have resulted in efficacious bone regeneration across several preclinical models. Yet, key topics of research in perivascular progenitors highlight our lack of knowledge regarding these cell populations. These ongoing areas of study include cellular diversity within the perivascular niche, tissue‐specific properties of PSC, and factors that influence PSC‐mediated regenerative potential. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1221–1228, 2019.     

Healing of Membranous and Long Bone Defects

Different isolation models have been used for qualitative and quantitative studies on the individual roles of periosteum, cortical bone, endosteum and bone marrow in the repair of long bone defects. Corresponding skull defects were studied to evaluate also the dural osteogenetic capacity. A total of 53 operations were performed on the tibia and skull of 33 growing rabbits. The animals were killed 10–12 weeks after surgery. Ordinary rough histological methods were used for studying bone formation, and quantitative estimates were made using serial sections. The cortex, endosteum and bone marrow were demonstrated to play a minor part in the healing of tibial defects. Periosteum, on the other hand, had the most potent healing capacity. Calvarial periosteum, however, was found to be less bone producing and in that respect not to be superior to the dura. For a complete bony restoration combined periosteal and dural bone formation is necessary, also regarding the structural normalization.     

Early osteogenesis in compact bone isografts: A quantitative study of the contributions of the different graft cells

Summary Isografts of cortical bone were transplanted subcutaneously in the rat and the rate of osteogenesis 12 to 14 days later was assessed by measurement of⁸⁵Sr uptake and by histology. Some grafts were implanted complete whereas others had had one or more of their cellular components (viz. periosteum, endosteum, osteocytes, marrow) removed by mechanical or enzymatic pretreatment. From an analysis of the differences in osteogenesis between grafts devoid of different combinations of cellular components, the contribution of each component to osteogenesis was determined. The results indicate that the endosteal lining cells and marrow stroma together produce more than half of the new bone, the periosteal cells contribute about 30%, the osteocytes possibly make a small (10%) contribution, and the free, hemopoietic cells of the marrow make no significant contribution. Evidence about the relative contributions to osteogenesis of graft and host cells is reviewed and the possible osteogenetic role of bone marrow is discussed.     

骨内膜成骨的动物模型

目的 为了解骨内膜在骨折愈合不同阶段的功能变化及其调节机制。设计一个骨折愈合过程中骨内膜成骨的动物模型。方法 将３月龄雌性ＳＤ大鼠４０只随机分成４组,每组１０只,在左胫骨造成长５ｍｍ的环形骨膜缺损,用骨锉在骨膜缺损区造成长５ｍｍ的骨缺损,深达髓腔,约显露髓腔的１／６。结果 １４天时裸露的贿腔已开始出现骨性关闭的征象,２８天时髓腔完全由坚质骨关闭,而各时间点组的骨缺损处填充物均为颜痕样结缔组织。结论     

A Perspective: Engineering Periosteum for Structural Bone Graft Healing

Autograft is superior to both allograft and synthetic bone graft in repair of large structural bone defect largely due to the presence of multipotent mesenchymal stem cells in periosteum. Recent studies have provided further evidence that activation, expansion and differentiation of the donor periosteal progenitor cells are essential for the initiation of osteogenesis and angiogenesis of donor bone graft healing. The formation of donor cell-derived periosteal callus enables efficient host-dependent graft repair and remodeling at the later stage of healing. Removal of periosteum from bone autograft markedly impairs healing whereas engraftment of multipotent mesenchymal stem cells on bone allograft improves healing and graft incorporation. These studies provide rationale for fabrication of a biomimetic periosteum substitute that could fit bone of any size and shape for enhanced allograft healing and repair. The success of such an approach will depend on further understanding of the molecular signals that control inflammation, cellular recruitment as well as mesenchymal stem cell differentiation and expansion during the early phase of the repair process. It will also depend on multidisciplinary collaborations between biologists, material scientists and bioengineers to address issues of material selection and modification, biological and biomechanical parameters for functional evaluation of bone allograft healing.     

Bone Vasculature and Bone Marrow Vascular Niches in Health and Disease

Bone vasculature and bone marrow vascular niches supply oxygen, nutrients, and secrete angiocrine factors required for the survival, maintenance, and self-renewal of stem and progenitor cells. In the skeletal system, vasculature creates nurturing niches for bone and blood-forming stem cells. Blood vessels regulate hematopoiesis and drive bone formation during development, repair, and regeneration. Dysfunctional vascular niches induce skeletal aging, bone diseases, and hematological disorders. Recent cellular and molecular characterization of the bone marrow microenvironment has provided unprecedented insights into the complexity, heterogeneity, and functions of the bone vasculature and vascular niches. The bone vasculature is composed of distinct vessel subtypes that differentially regulate osteogenesis, hematopoiesis, and disease conditions in bones. Further, bone marrow vascular niches supporting stem cells are often complex microenvironments involving multiple different cell populations and vessel subtypes. This review provides an overview of the emerging vascular cell heterogeneity in bone and the new roles of the bone vasculature and associated vascular niches in health and disease. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).     

Bone repair induced by bone morphogenetic protein in ulnar defects in dogs

In dogs, resection of a length of the ulna equal to twice the diameter of the mid-shaft leaves a defect which consistently fails to unite. In response to an implant of 100 mg of bovine bone morphogenetic protein (BMP), the defect becomes filled by callus consisting of fibrocartilage, cartilage and woven bone within four weeks. The cartilage is resorbed and replaced by new bone in four to eight weeks. Woven bone is then resorbed, colonised by bone marrow cells and remodelled into lamellar bone. Union of the defect is produced by 12 weeks. Control defects filled with autogeneic cortical bone chips unite after the same period. In regeneration induced by bone morphogenetic protein (BMP) and in repair enhanced by bone graft, union depends upon the proliferation of cells within and around the bone ends. Our working hypothesis is that BMP induces the differentiation of perivascular connective tissue cells into chondroblasts and osteoprogenitor cells and thereby augments the process of bone regeneration from the cells already present in the endosteum and periosteum.     

Mesenchymal Progenitors and the Osteoblast Lineage in Bone Marrow Hematopoietic Niches

The bone marrow cavity is essential for the proper development of the hematopoietic system. In the last few decades, it has become clear that mesenchymal stem/progenitor cells as well as cells of the osteoblast lineage, besides maintaining bone homeostasis, are also fundamental regulators of bone marrow hematopoiesis. Several studies have demonstrated the direct involvement of mesenchymal and osteoblast lineage cells in the maintenance and regulation of supportive microenvironments necessary for quiescence, self-renewal and differentiation of hematopoietic stem cells. In addition, specific niches have also been identified within the bone marrow for maturing hematopoietic cells. Here we will review recent findings that have highlighted the roles of mesenchymal progenitors and cells of the osteoblast lineage in regulating distinct stages of hematopoiesis.     

Role of altered signal transduction in heterotopic ossification and fibrodysplasia ossificans progressiva

Heterotopic ossification is a pathologic condition in which bone tissue is formed outside of the skeleton, within soft tissues of the body. The extraskeletal bone that forms in these disorders is normal; the cellular mechanisms that direct cell fate decisions are dysregulated. Patients with fibrodysplasia ossificans progressiva (FOP), a rare human genetic disorder of extensive and progressive heterotopic ossification, have malformations of normal skeletal elements, identifying the causative gene mutation and its relevant signaling pathways as key regulators of skeletal development and of cell fate decisions by adult stem cells. The discovery that mildly activating mutations in ACVR1/ALK2, a bone morphogenetic protein (BMP) type I receptor, is the cause of FOP has provided opportunities to identify previously unknown functions for this receptor and for BMP signaling and to develop new diagnostic and therapeutic strategies for FOP and other more common forms of heterotopic ossification, as well as tissue engineering applications.     

Cellular aspects of bone regeneration: role of bone marrow periostium

Bone regeneration is only possible if stem cells give rise to progenitors of osteoblasts, chondroblasts or chondroidocytes. Stem cells and osteogenic progenitors were evidenced in bone marrow while only progenitors can be found in periosteum. Bone marrow stem cells did show an amazing plasticity and some cells of the bone surrounding tissues such as perivascular cells, adipocytes, muscle cells or even circulating cells are able to transdifferentiate in osteoblasts when submitted to an osteogenic environment. We have shown that the destruction of both bone marrow and periost impairs the bone healing. It indicates that the periost and bone marrow destruction removes the predetermined osteogenic cells and the informative factors able to induce the transdifferenciation of the cells contained in the peri-osseous tissues.     

Bone lengthening in rabbits by callus distraction. The role of periosteum and endosteum

The histology and mechanics of leg lengthening by callus distraction were studied in 27 growing rabbits. Tibial diaphyses were subjected to subperiosteal osteotomy, held in a neutral position for 10 days and then slowly distracted at 0.25 mm/12 hours, using a dynamic external fixator. Radiographs showed that the gap became filled with callus having three distinct zones. Elongation appeared to occur in a central radiolucent zone; this was bounded by two sclerotic zones. Histologically, the radiolucent zone consisted of longitudinally arranged cartilage and fibrous tissue while the sclerotic zones were formed by fine cancellous bone. New bone occasionally contained islands of cartilage, suggesting it had been formed by endochondral ossification. After completion of distraction, the two sclerotic zones fused, shrank and were eventually absorbed, leaving tubular bone with a new cortex. When the periosteum had been removed at the operation, callus formation was markedly disturbed and there was failure of bone lengthening. Scraping of endosteum, in contrast, did not have a pronounced effect. These results suggest that the preservation of periosteum is essential if bone lengthening by callus distraction is to succeed, and that preservation of the periosteum is more important than careful corticotomy.     

Multipotent progenitors resident in the skeletal muscle interstitium exhibit robust BMP-dependent osteogenic activity and mediate heterotopic ossification

Heterotopic ossification is a debilitating condition that can result from traumatic injury, surgery, or genetic disease. We investigated the cellular origins of heterotopic skeletogenesis in the mouse using lineage tracing and bioassays of heterotopic ossification based on intramuscular transplantation. We identified, characterized, and purified a tissue-resident stem/progenitor cell population that exhibits robust osteogenic potential and represents a major cell-of-origin for heterotopic ossification. These progenitors reside in the interstitium of skeletal muscle and other tissues, and are distinct from the endothelium, which does not exhibit osteogenic activity in response to bone morphogenetic protein 2 (BMP2) stimulation. Intramuscular transplantation, together with clonal analysis in culture, revealed that these progenitors are multipotent, exhibiting the capacity for both BMP-dependent skeletogenic differentiation and spontaneous adipogenic differentiation. Identifying the cells-of-origin responsible for heterotopic ossification provides a potential therapeutic target to treat, mitigate, or prevent this disabling condition.     

Transient gamma-secretase inhibition accelerates and enhances fracture repair likely via Notch signaling modulation

Approximately 10% of skeletal fractures result in healing complications and non-union, while most fractures repair with appropriate stabilization and without pharmacologic intervention. It is the latter injuries that cannot be underestimated as the expenses associated with their treatment and subsequent lost productivity are predicted to increase to over $74 billion by 2015. During fracture repair, local mesenchymal stem/progenitor cells (MSCs) differentiate to form new cartilage and bone, reminiscent of events during skeletal development. We previously demonstrated that permanent loss of gamma-secretase activity and Notch signaling accelerates bone and cartilage formation from MSC progenitors during skeletal development, leading to pathologic acquisition of bone and depletion of bone marrow derived MSCs. Here, we investigated whether transient and systemic gamma-secretase and Notch inhibition is capable of accelerating and enhancing fracture repair by promoting controlled MSC differentiation near the fracture site. Our radiographic, microCT, histological, cell and molecular analyses reveal that single and intermittent gamma-secretase inhibitor (GSI) treatments significantly enhance cartilage and bone callus formation via the promotion of MSC differentiation, resulting in only a moderate reduction of local MSCs. Biomechanical testing further demonstrates that GSI treated fractures exhibit superior strength earlier in the healing process, with single dose GSI treated fractures exhibiting bone strength approaching that of un-fractured tibiae. These data further establish that transient inhibition of gamma-secretase activity and Notch signaling temporarily increases osteoclastogenesis and accelerates bone remodeling, which coupled with the effects on MSCs likely explains the accelerated and enhanced fracture repair. Therefore, we propose that the Notch pathway serves as an important therapeutic target during skeletal fracture repair.     

Subchondral Bone Reaction Associated with Chondral Defect and Attempted Cartilage Repair in Goats

Repair of cartilage damage with autologous chondrocyte transplantation (ACT) has become popular in clinical use during the past few years. Although clinical results have mostly been successful, several unanswered questions remain regarding the biological mechanism of the repair process. The aim of this study was to develop a goat model for ACT. The repair was not successful due to the graft delamination, but we characterize the subchondral changes seen after the procedure. A chondral lesion was created in 14 goat knees, operated on 1 month later with ACT, and covered with periosteum or a bioabsorbable poly-L/D-lactide scaffold. After 3 months, only two of the five lesions repaired with ACT showed partly hyaline-like repair tissue, and all lesions (n = 4) with the scaffold failed. Even though the lesions did not extend through the calcified cartilage, the bone volume and collagen organization of bone structure were decreased when assessed by quantitative polarized light microscopy. There was a significant loss of bone matrix and distortion of the trabecular structure of subchondral bone, which extended several millimeters into the bone. The subchondral bone demonstrated strong hyaluronan staining in the bone marrow and cartilaginous areas with signs of endochondral ossification, suggesting structural remodeling of the bone. The goat model used here proved not to be an optimal model for ACT. The changes in subchondral bone may alter the biomechanical properties of the subchondral plate and thus the long-term survival of the repair tissue after ACT.     

MT1‐MMP and Type II Collagen Specify Skeletal Stem Cells and Their Bone and Cartilage Progeny

Skeletal formation is dependent on timely recruitment of skeletal stem cells and their ensuing synthesis and remodeling of the major fibrillar collagens, type I collagen and type II collagen, in bone and cartilage tissues during development and postnatal growth. Loss of the major collagenolytic activity associated with the membrane‐type 1 matrix metalloproteinase (MT1‐MMP) results in disrupted skeletal development and growth in both cartilage and bone, where MT1‐MMP is required for pericellular collagen dissolution. We show here that reconstitution of MT1‐MMP activity in the type II collagen‐expressing cells of the skeleton rescues not only diminished chondrocyte proliferation, but surprisingly, also results in amelioration of the severe skeletal dysplasia associated with MT1‐MMP deficiency through enhanced bone formation. Consistent with this increased bone formation, type II collagen was identified in bone cells and skeletal stem/progenitor cells of wildtype mice. Moreover, bone marrow stromal cells isolated from mice expressing MT1‐MMP under the control of the type II collagen promoter in an MT1‐MMP‐deficient background showed enhanced bone formation in vitro and in vivo compared with cells derived from nontransgenic MT1‐MMP‐deficient littermates. These observations show that type II collagen is not stringently confined to the chondrocyte but is expressed in skeletal stem/progenitor cells (able to regenerate bone, cartilage, myelosupportive stroma, marrow adipocytes) and in the chondrogenic and osteogenic lineage progeny where collagenolytic activity is a requisite for proper cell and tissue function.