Periosteal Stem Cells Research Articles

A hierarchical biomimetic periosteum combined immunomodulatory and osteogenic functions for bone regeneration

The periosteum plays an indispensable role in immunomodulation and osteogenesis during bone regeneration. The natural periosteum has a hierarchical structure, in which the outer fibrous layer is composed of fibrous tissue arranged in parallel, while disorganized nanofibers are found in the inner cambium layer. Herein, inspired by the nanostructure/function of the periosteum, we designed a collagen membrane aligned in an oriented manner (CMalig) to mimic the fibrous layer of the natural periosteum and thereby facilitate the M1-to-M2 phenotypic macrophage transition. The randomly oriented nanofiber membrane (CMrand) simulated the cambium layer and promoted the osteogenic differentiation of endogenous bone progenitor cells. CMalig induced the M2 polarization of RAW 264.7 cells in vitro, and numerous CD206+F4/80+ cells were observed at femur defect sites after CMalig treatment. CMrand promoted the osteogenic differentiation of periosteal stem cells and ectopic bone formation in a subcutaneous implantation model. These heterotopic ossifications were also verified in a rat Achilles tendon defect model. The biomimetic periosteum with a hierarchical structure significantly accelerated new bone formation in the rat femur defect model. These results suggest that the hierarchically structured biomimetic periosteum with immunomodulatory and osteogenic functions can be used as an alternative to periosteal autografts.

Wnt10b-overexpressing umbilical cord mesenchymal stem cells promote fracture healing via accelerated cartilage callus to bone remodeling

ABSTRACT The aim of this study was to investigate whether HUCMSCsWnt10b could promote long bone fracture healing. Commercially-available HUCMSCsEmp (human umbilical cord mesenchymal stem cells transfected with empty vector) in hydrogel, HUCMSCsWnt10b in hydrogel and HUCMSCsWnt10b with the Wnt signaling pathway inhibitor IWR-1 were transplanted into the fracture site in a rat model of femoral fracture. We found that transplantation of HUCMSCsWnt10b significantly accelerated bone healing in a rat model of femoral fracture. Meanwhile, three-point bending test proved that the mechanical properties of the bone at the fracture site in the HUCMSCWnt10b treatment group were significantly better than those of the other treatment groups. To understand the cellular mechanism, we explored the viability of periosteal stem cells (PSCs), as they contribute the greatest number of osteoblast lineage cells to the callus. In line with in vivo data, we found that conditioned medium from HUCMSCsWnt10b enhanced the migration and osteogenic differentiation of PSCs. Furthermore, conditioned medium from HUCMSCsWnt10b also induced endothelial cells to form capillary-like structures in a tube formation assay, which was blocked by SU5416, an angiogenesis inhibitor, suggesting that enhanced vessel formation and growth also contribute to accelerated hard callus formation. In summary, our study demonstrates that HUCMSCsWnt10b promote fracture healing via accelerated hard callus formation, possibly due to enhanced osteogenic differentiation of PSCs and vessel growth. Therefore, HUCMSCsWnt10b may be a promising treatment for long bone fractures.

Constructing the in vitro culture system of the sika deer (cervus nippon) antler periosteal cell to detect its function on antler regeneration.

Periosteum is essential for bone regeneration and damage repair in mammals. Most species of deer family (Cervidae) develop two kinds of special periosteum, antler periosteum and pedicle periosteum, both supporting the complete regeneration of antler. Antler is the bone organ with the fastest growth rate in mammals. Along with the fast growth of antler, its external tissues such as blood vessels, nerves and the covering skin also grow rapidly. Currently, it is still unclear whether antler periosteum contributes to the fast growth of antler and how. It is also unclear why the regenerative capacity of antler periosteum is weaker than that of pedicle periosteum. In this study, the in vitro culture system for antler periosteal cells (AnPC) was constructed for the first time using the mid-beam antler periostea during antler fast-growth period. According to our results, the cultured AnPC expressed classical MSC markers, consistent with the pedicle periosteal stem cells (PPSC). However, the fluorescence intensities of the MSC markers on AnPC were significantly weaker than those on PPSC. In addition, AnPC showed much lower proliferation rates than PPSC. The proliferation rates of the AnPC also gradually decreased after successive passages, while the proliferation rates of the pedicle periosteal stem cells remained unchanged. These findings may partially explain the weaker regenerative capacity of antler periosteum. Further comparative global gene analysis revealed clearly the different gene expressed patterns between AnPC and PPSC. AnPC may mainly function on promoting angiogenesis, nerve growth and intramembrane bone formation during antler regeneration, whereas PPSC may primarily be involved in androgen signaling receptor pathway and PI3K-Akt signaling pathway and function on maintaining stem cell renewal.

Periosteal stem cell microenvironment compositional and elasticity changes in the vicinity of bone fracture

Bone regeneration after fracture is a complex biological process involving an initial acute inflammatory response, followed by the recruitment of various types of skeletal stem cells (SSCs) that proliferate and differentiate into chondrogenic and osteogenic cells. SSCs have been discovered in the bone marrow, the growth plate, and the periosteum. Functionally distinct and highly regenerative, periosteal skeletal stem cells (pSSCs, also known as PSCs) have been recently identified, but the extracellular matrix (ECM) mechanobiology that direct PSC activation in fracture is poorly understood.

Mechanosensitive Piezo1 is crucial for periosteal stem cell-mediated fracture healing.

The biomechanical environment plays a dominant role in fracture healing, and Piezo1 is regarded as a major mechanosensor in bone homeostasis. However, the role of Piezo1 in fracture healing is not yet well characterized. In this study, we first delineated that Piezo1 is highly expressed in periosteal stem cells (PSCs) and their derived osteoblastic lineage cells and chondrocytes. Furthermore, downregulation of Piezo1 in callus leads to impaired fracture healing, while activation by its specific agonist promotes fracture healing through stimulation of PSC-modulated chondrogenesis and osteogenesis, along with accelerated cartilage-to-bone transformation. Interestingly, vascular endothelial growth factor A is upregulated after Yoda1 treatment of PSCs, indicating an indirect role of Piezo1 in angiogenesis. Mechanistically, activation of Piezo1 promotes expression of Yes-associated protein (YAP) and its nuclear localization in PSCs, which in turn increases the expression and nuclear localization of β-catenin. In detail, YAP directly interacts with β-catenin in the nucleus and forms a transcriptional YAP/β-catenin complex, which upregulates osteogenic, chondrogenic and angiogenic factors. Lastly, Yoda1 treatment significantly improves fracture healing in a delayed union mouse model generated by tail suspension. These findings indicate that Piezo1 is a potential therapeutic target for fracture delayed union or nonunion.

What's New in Musculoskeletal Basic Science.

What's New in Musculoskeletal Basic Science.

Connective Tissue Growth Factor From Periosteal Tartrate Acid Phosphatase-Positive Monocytes Direct Skeletal Stem Cell Renewal and Fate During Bone Healing.

The periosteum is critical for bone healing. Studies have shown that the periosteum contains periosteal stem cells (PSCs) with multidirectional differentiation potential and self-renewal ability. PSCs are activated in early fracture healing and are committed to the chondrocyte lineage, which is the basis of callus formation. However, the mechanism by which PSCs are activated and committed to chondrocytes in bone regeneration remains unclear. Here, we show that tartrate acid phosphatase (TRAP)-positive monocytes secrete CTGF to activate PSCs during bone regeneration. The loss function of TRAP-positive monocytes identifies their specific role during bone healing. Then, the secreted CTGF promotes endochondral ossification and activates PSCs in mouse bone fracture models. The secreted CTGF enhances PSC renewal by upregulating the expression of multiple pluripotent genes. CTGF upregulates c-Jun expression through αVβ5 integrin. Then, c-Jun transcription activates the transcription of the pluripotent genes Sox2, Oct4, and Nanog. Simultaneously, CTGF also activates the transcription and phosphorylation of Smad3 through αVβ5 integrin, which is the central gene in chondrogenesis. Our study indicates that TRAP-positive monocyte-derived CTGF promotes bone healing by activating PSCs and directing lineage commitment and that targeting PSCs may be an effective strategy for preventing bone non-union.

Stem cell therapies in tendon-bone healing.

Tendon-bone insertion injuries such as rotator cuff and anterior cruciate ligament injuries are currently highly common and severe. The key method of treating this kind of injury is the reconstruction operation. The success of this reconstructive process depends on the ability of the graft to incorporate into the bone. Recently, there has been substantial discussion about how to enhance the integration of tendon and bone through biological methods. Stem cells like bone marrow mesenchymal stem cells (MSCs), tendon stem/progenitor cells, synovium-derived MSCs, adipose-derived stem cells, or periosteum-derived periosteal stem cells can self-regenerate and potentially differentiate into different cell types, which have been widely used in tissue repair and regeneration. Thus, we concentrate in this review on the current circumstances of tendon-bone healing using stem cell therapy.

Markers for Identification of Postnatal Skeletal Stem Cells In Vivo.

The adult skeleton contains stem cells involved in growth, homeostasis, and healing. Mesenchymal or skeletal stem cells are proposed to provide precursors to osteoblasts, chondrocytes, marrow adipocytes, and stromal cells. We review the evidence for existence and functionality of different skeletal stem cell pools, and the tools available for identifying or targeting these populations in mouse and human tissues. Lineage tracing and single cell-based techniques in mouse models indicate that multiple pools of stem cells exist in postnatal bone. These include growth plate stem cells, stem and progenitor cells in the diaphysis, reticular cells that only form bone in response to injury, and injury-responsive periosteal stem cells. New staining protocols have also been described for prospective isolation of human skeletal stem cells. Several populations of postnatal skeletal stem and progenitor cells have been identified in mice, and we have an increasing array of tools to target these cells. Most Cre models lack a high degree of specificity to define single populations. Human studies are less advanced and require further efforts to refine methods for identifying stem and progenitor cells in adult bone.

Ablation of Ephrin B2 in Col2 Expressing Cells Delays Fracture Repair.

Ephrin B2 is critical for endochondral bone development. In this study, we investigated its role in fracture repair by deleting ephrin B2 in type II collagen (Col.2) expressing cells. We used a nonstable tibia fracture model to evaluate fracture repair at 3 sites: intramembranous bone formation, endochondral bone formation, and intramedullary bone formation. We observed that during fracture repair, deletion of ephrin B2 impaired periosteal stem cell activation, inhibited their proliferation, decreased their survival, and blocked their differentiation into osteoblasts and chondrocytes. In addition, deletion of ephrin B2 decreased vascular endothelial growth factor production as well as vascular invasion into the fracture site. These changes led to reduced cartilage to bone conversion in the callus with decreased new bone formation, resulting in impaired fracture repair. Our data indicate that ephrin B2 in Col2-expressing cells is a critical regulator of fracture repair, pointing to a new and potentially targetable mechanism to enhance fracture repair.

Periosteum progenitors could stimulate bone regeneration in aged murine bone defect model.

Periosteal stem cells are critical for bone regeneration, while the numbers will decrease with age. This study focused on whether Prx1+ cell, a kind of periosteal stem cell, could stimulate bone regeneration in aged mice. Four weeks and 12 months old Prx1CreER‐GFP; Rosa26tdTomato mice were used to reveal the degree of Prx1+ cells participating in the femoral fracture healing procedure. One week, 8 weeks, 12 and 24 months old Prx1CreER‐GFP mice were used to analyse the real‐time distribution of Prx1+ cells. Twelve months old C57BL/6 male mice (n = 96) were used to create the bone defect model and, respectively, received hydrogel, hydrogel with Prx1− mesenchymal stem cells and hydrogel with Prx1+ cells. H&E staining, Synchrotron radiation‐microcomputed tomography and mechanical test were used to analyse the healing results. The results showed that tdTomato+ cells were involved in bone regeneration, especially in young mice. At the same time, GFP+ cells decreased significantly with age. The Prx1+ cells group could significantly improve bone regeneration in the murine bone defect model via directly differentiating into osteoblasts and had better osteogenic differentiation ability than Prx1− mesenchymal stem cells. Our finding revealed that the quantity of Prx1+ cells might account for decreased bone regeneration ability in aged mice, and transplantation of Prx1+ cells could improve bone regeneration at the bone defect site.

Allogenic Bone Graft Enriched by Periosteal Stem Cell and Growth Factors for Osteogenesis in Critical Size Bone Defect in Rabbit Model: Histopathological and Radiological Evaluation.

Background & Objective:This study aimed to investigate the effect of decellularized allogeneic bone graft enriched by periosteal stem cells (PSCs) and growth factors on the bone repair process in a rabbit model, which could be used in many orthopedic procedures.Methods:In this experimental study, a critical size defect (CSD) (10 mm) was created in the radial diaphysis of 40 rabbits. In group A, the defect was left intact with no medical intervention. In group B, the defect was filled by a decellularized bone graft. In group C, the defect was implanted by a decellularized bone graft enriched with platelet growth factors. In group D, the defect was treated by a decellularized bone graft seeded by periosteal mesenchymal stem cells (MSCs). Also, in group E, the defect was filled by a decellularized bone graft enriched with platelet growth factors and periosteal MSCs. Radiological evaluation was done on the first day and then in the second, fourth, and eighth weeks after the operation. The specimens were harvested on the 28th and 56th postoperative days and evaluated for histopathological criteria.Results:The radiologic and microscopic analysis of the healing process in bone defects of the treated groups (C, D, and E) revealed more advanced repair criteria than those of groups A and B significantly (P<0.05).Conclusion:Based on this study, it appears that implantation of concentrated PSCs in combination with growth factors and allogeneic cortical bone graft is an effective therapy for the repair of large bone defects.

Periosteum: The forgotten source of osteoprogenitors

Bone is one of the few tissues capable of authentic regenerative repair. However, despite advances in surgical technique, orthopaedic hardware and our understanding of fracture biology, inadequate bone repair remains a major concern in both veterinary and human medicine. Cell-based technologies provide opportunities to utilize the osteogenic capacities of Mesenchymal Stem Cells (MSC) to augment bone repair. Much of the research on MSC biology has focused on cells derived from the bone marrow/endosteal compartment; however, osteoprogenitor cells (OPC) also reside in the periosteum. Periosteum develops as a fibro-cellular envelope surrounding developing skeletal elements. The inner, or cambium layer of periosteum, includes committed OPCs directly adjacent the bone surface, and a distinct sub-population of progenitors within the periosteal mid-substance that retain both chondrogenic and osteogenic capacities. During skeletogenesis, periosteal OPCs are responsible for appositional intramembranous bone formation that increases the radial diameter of long bones. Of critical importance, periosteal stem cells are the predominant cell population responsible for generating the cartilaginous or ‘soft’ callus that provides intermediate stabilization and a scaffold for subsequent callus ossification by endochondral ossification; the primary mechanism of bone repair. In recent experiments using isolates from ‘donor-matched’ periosteum and bone marrow, we have found that the basal osteogenic capacity of equine OPCs is considerably less than that of bone marrow-derived MSCs. Periosteal OPCs require exogenous Bone Morphogenetic Protein (BMP) for robust osteogenesis, a finding consistent with the clinical responses of bone to recombinant BMP protein. Perhaps more surprising, the osteogenic capacity of adult (2-10 years of age) OPCs is comparable to those of young foals’, although the cell yield is considerably greater from foal specimens. In light of the vital importance of callus formation for successful fracture healing of most, further research on the biology and clinical manipulation of periosteal OPCs is highly warranted.

Identification of Functionally Distinct Mx1+αSMA+ Periosteal Skeletal Stem Cells.

The periosteum is critical for bone maintenance and healing. However, the invivo identity and specific regulatory mechanisms of adult periosteum-resident skeletal stem cells are unknown. Here, we report animal models that selectively and durably label postnatal Mx1+αSMA+ periosteal stem cells (P-SSCs) and establish that P-SSCs are a long-term repopulating, functionally distinct SSC subset responsible forlifelong generation of periosteal osteoblasts. P-SSCs rapidly migrate toward an injury site, supply osteoblasts and chondrocytes, and recover new periosteum. Notably, P-SSCs specifically express CCL5 receptors, CCR3 and CCR5. Real-time intravital imaging revealed that the treatment with CCL5 induces P-SSC migration invivo and bone healing, while CCL5/CCR5 deletion, CCR5 inhibition, or local P-SSC ablation reduces osteoblast number and delays bone healing. Human periosteal cells express CCR5 and undergo CCL5-mediated migration. Thus, the adult periosteum maintains genetically distinct SSC subsets with a CCL5-dependent migratory mechanism required for bone maintenance and injury repair.

Response of human periosteal cells to degradation products of zinc and its alloy.

Zinc (Zn) and its alloys are proposed as promising resorbable materials for osteosynthesis implants. Detailed studies should be undertaken to clarify their properties in terms of degradability, biocompatibility and osteoinductivity. Degradation products of Zn alloys might affect directly adjacent cellular and tissue responses. Periosteal stem cells are responsible for participating in intramembranous ossification during fracture healing. The present study aims at examining possible effects emanating from Zn or Zn-4Ag (wt%) alloy degradation products on cell viability and osteogenic differentiation of a human immortalized cranial periosteal cell line (TAg cells). Therefore, a modified extraction method was used to investigate the degradation behavior of Zn and Zn-4Ag alloys under cell culture conditions. Compared with pure Zn, Zn-4Ag alloy showed almost fourfold higher degradation rates under cell culture conditions, while the associated degradation products had no adverse effects on cell viability. Osteogenic induction of TAg cells revealed that high concentration extracts significantly reduced calcium deposition of TAg cells, while low concentration extracts enhanced calcium deposition, indicating a dose-dependent effect of Zn ions. Our results give evidence that the observed cytotoxicity effects were determined by the released degradation products of Zn and Zn-4Ag alloys, rather than by degradation rates calculated by weight loss. Extracellular Zn ion concentration was found to modulate osteogenic differentiation of TAg cells. These findings provide significant implications and guidance for the development of Zn-based alloys with an optimized degradation behavior for Zn-based osteosynthesis implants.

Discovery of a periosteal stem cell mediating intramembranous bone formation

Bone consists of separate inner endosteal and outer periosteal compartments, each with distinct contributions to bone physiology and each maintaining separate pools of cells owing to physical separation by the bone cortex. The skeletal stem cell that gives rise to endosteal osteoblasts has been extensively studied; however, the identity of periosteal stem cells remains unclear1–5. Here we identify a periosteal stem cell (PSC) that is present in the long bones and calvarium of mice, displays clonal multipotency and self-renewal, and sits at the apex of a differentiation hierarchy. Single-cell and bulk transcriptional profiling show that PSCs display transcriptional signatures that are distinct from those of other skeletal stem cells and mature mesenchymal cells. Whereas other skeletal stem cells form bone via an initial cartilage template using the endochondral pathway4, PSCs form bone via a direct intramembranous route, providing a cellular basis for the divergence between intramembranous versus endochondral developmental pathways. However, there is plasticity in this division, as PSCs acquire endochondral bone formation capacity in response to injury. Genetic blockade of the ability of PSCs to give rise to bone-forming osteoblasts results in selective impairments in cortical bone architecture and defects in fracture healing. A cell analogous to mouse PSCs is present in the human periosteum, raising the possibility that PSCs are attractive targets for drug and cellular therapy for skeletal disorders. The identification of PSCs provides evidence that bone contains multiple pools of stem cells, each with distinct physiologic functions. A periosteal stem cell specialized in intramembranous bone formation has been identified and was found to be essential for normal bone development and fracture healing.

Discovery of a periosteal stem cell mediating intramembranous bone formation.

Bone is comprised of separate inner endosteal and outer periosteal compartments, each with distinct contributions to bone physiology and each maintaining separate pools of cells due to physical separation by the bone cortex. While the skeletal stem cell giving rise to endosteal osteoblasts has been extensively studied, the identification of a periosteal stem cell has been elusive1–5. Here, we identify a periosteal stem cell (PSC) present in the long bones and calvarium of mice that displays clonal multipotency, self-renewal and sits at the apex of a differentiation hierarchy. Single cell and bulk transcriptional profiling show that PSCs display distinct transcriptional signatures in comparison with both other skeletal stem cells and mature mesenchymal cells. While other skeletal stem cells form bone via an initial cartilage template using the endochondral pathway4, PSCs form bone via a direct intramembranous route, providing a cellular basis for the divergence between intramembranous versus endochondral developmental pathways. However there is plasticity in this division, as PSCs acquire endochondral bone formation capacity in response to injury. Genetic blockade of the ability of PSCs to give rise to bone-forming osteoblasts results in selective impairments in cortical bone architecture and defects in fracture healing. A cell analogous to PSCs is present in the human periosteum, raising the possibility that PSCs are attractive targets for drug and cellular therapy for skeletal disorders. Moreover, the identification of PSCs provides evidence that bone contains multiple pools of stem cells, each with distinct physiologic functions.

Aging periosteal progenitor cells have reduced regenerative responsiveness to bone injury and to the anabolic actions of PTH 1-34 treatment

A stabilized tibia fracture model was used in young (8-week old) and aged (1-year old) mice to define the relative bone regenerative potential and the relative responsiveness of the periosteal progenitor population with aging and PTH 1-34 (PTH) systemic therapy. Bone regeneration was assessed through gene expressions, radiographic imaging, histology/histomorphometry, and biomechanical testing. Radiographs and microCT showed increased calcified callus tissue and enhanced bone healing in young compared to aged mice. A key mechanism involved reduced proliferation, expansion, and differentiation of periosteal progenitor cell populations in aged mice. The experiments showed that PTH increased calcified callus tissue and torsional strength with a greater response in young mice. Histology and quantitative histomorphometry confirmed that PTH increased callus tissue area due primarily to an increase in bone formation, since minimal changes in cartilage and mesenchyme tissue area occurred. Periosteum examined at 3, 5, and 7days showed that PTH increased cyclin D1 expression, the total number of cells in the periosteum, and width of the periosteal regenerative tissue. Gene expression showed that aging delayed differentiation of both bone and cartilage tissues during fracture healing. PTH resulted in sustained Col10a1 expression consistent with delayed chondrocyte maturation, but otherwise minimally altered cartilage gene expression. In contrast, PTH 1-34 stimulated expression of Runx2 and Osterix, but resulted in reduced Osteocalcin. β-Catenin staining was present in mesenchymal chondroprogenitors and chondrocytes in early fracture healing, but was most intense in osteoblastic cells at later times. PTH increased active β-catenin staining in the osteoblast populations of both young and aged mice, but had a lesser effect in cartilage. Altogether the findings show that reduced fracture healing in aging involves decreased proliferation and differentiation of stem cells lining the bone surface. While PTH 1-34 enhances the proliferation and expansion of the periosteal stem cell population and accelerates bone formation and fracture healing, the effects are proportionately reduced in aged mice compared to young mice. β-Catenin is induced by PTH in early and late fracture healing and is a potential target of PTH 1-34 effects.

A Comparative Morphometric Analysis of Biodegradable Scaffolds as Carriers for Dental Pulp and Periosteal Stem Cells in a Model of Bone Regeneration

Bone regeneration and bone fixation strategies in dentistry utilize scaffolds containing regenerating-competent cells as a replacement of the missing bone portions and gradually replaced by autologous tissues. Mesenchymal stem cells represent an ideal cell population for scaffold-based tissue engineering. Among them, dental pulp stem cells (DPSCs) and periosteal stem cells (PeSCs) have the potential to differentiate into a variety of cell types including osteocytes, suggesting that they can be used with this purpose. However, data on bone regeneration properties of these types of cells in scaffold-based tissue engineering are yet insufficient.In this study, we evaluated temporal dynamic bone regeneration (measured as a percentage of bone volume on the total area of the defect) induced by DPSCs or PeSCs when seeded with different scaffolds to fill critical calvarial defects in SCID Beige nude mice. Two commercially available scaffolds (granular deproteinized bovine bone with 10% porcine collagen and granular β;-tricalcium phosphate) and one not yet introduced on the market (a sponge of agarose and nanohydroxyapatite) were used. The results showed that tissue-engineered constructs did not significantly improve bone-induced regeneration process when compared with the effect of scaffolds alone. In addition, the data also showed that the regeneration induced by β;-tricalcium phosphate alone was higher after 8 weeks than that of scaffold seeded with the 2 stem cell lines. Altogether these findings suggest that further studies are needed to evaluate the potential of DPSCs and PeSCs in tissue construct and identify the appropriate conditions to generate bone tissue in critical-size defects.

Semipermanent volumization by an absorbable filler: onlay injection technique to the bone.

Background:Hyaluronic acid (HA) fillers have become the most popular tool for wrinkle treatment and volumization, although HA is generally absorbed within 6–12 months and requires repeated treatments to maintain the effects.Methods:HA was injected onto the bone for volumization with a small 30-gauge needle to examine the long-lasting effects. Of the 63 Japanese patients with 97 treated sites followed up more than 12 months, 51 had HA injections for cosmetic purposes and 12 were treated for reconstructive volumization of facial deformity such as localized scleroderma and postsurgical bony deformity. Treated sites included the forehead, temple, nasal root, mentum, tear trough, and infraorbital sulcus.Results:After long-term follow-up (12–93 months, mean = 21.6), persistent volumizing effects were observed in most patients. In fact, 86.6% of the treated sites showed >50% volume retention and 49.5% showed >75% retention. Magnetic resonance imaging analyses revealed that the injected space was well maintained, capsulated, and filled with heterogeneous content. Magnetic resonance imaging quantitative T2 maps indicated that much of the injected HA was replaced with other materials. Together with clinical inspection, these findings suggest that onlay injection of HA on the bone induced formation of capsule, fibrosis, and/or calcification/ossification, which contributed to persistent volumization.Conclusions:Semipermanent volumizing effects can be achieved by HA injection if the target area has an underlying bony floor. Periosteal stem cells may be activated by HA injection and may contribute to persistent volumizing effects. This treatment may be a much less invasive alternative to fat or bone grafting.