Sfrp4 is required to maintain Ctsk-lineage periosteal stem cell niche function.

We have previously reported that the cortical bone thinning seen in mice lacking the Wnt signaling antagonist Sfrp4 is due in part to impaired periosteal apposition. The periosteum contains cells which function as a reservoir of stem cells and contribute to cortical bone expansion, homeostasis, and repair. However, the local or paracrine factors that govern stem cells within the periosteal niche remain elusive. Cathepsin K (Ctsk), together with additional stem cell surface markers, marks a subset of periosteal stem cells (PSCs) which possess self-renewal ability and inducible multipotency. Sfrp4 is expressed in periosteal Ctsk-lineage cells, and Sfrp4 global deletion decreases the pool of PSCs, impairs their clonal multipotency for differentiation into osteoblasts and chondrocytes and formation of bone organoids. Bulk RNA sequencing analysis of Ctsk-lineage PSCs demonstrated that Sfrp4 deletion down-regulates signaling pathways associated with skeletal development, positive regulation of bone mineralization, and wound healing. Supporting these findings, Sfrp4 deletion hampers the periosteal response to bone injury and impairs Ctsk-lineage periosteal cell recruitment. Ctsk-lineage PSCs express the PTH receptor and PTH treatment increases the % of PSCs, a response not seen in the absence of Sfrp4. Importantly, in the absence of Sfrp4, PTH-dependent increase in cortical thickness and periosteal bone formation is markedly impaired. Thus, this study provides insights into the regulation of a specific population of periosteal cells by a secreted local factor, and shows a central role for Sfrp4 in the regulation of Ctsk-lineage periosteal stem cell differentiation and function.

Refining the identity of mesenchymal cell types associated with murine periosteal and endosteal bone.

Single-cell RNA-seq has led to novel designations for mesenchymal cells associated with bone as well as multiple designations for what appear to be the same cell type. The main goals of this study were to increase the amount of single-cell RNA sequence data for osteoblasts and osteocytes, to compare cells from the periosteum to those inside bone, and to clarify the major categories of cell types associated with murine bone. We created an atlas of murine bone-associated cells by harmonizing published datasets with in-house data from cells targeted by Osx1-Cre and Dmp1-Cre driver strains. Cells from periosteal bone were analyzed separately from those isolated from the endosteum and trabecular bone. Over 100,000 mesenchymal cells were mapped to reveal 11 major clusters designated fibro-1, fibro-2, chondrocytes, articular chondrocytes, tenocytes, adipo-Cxcl12 abundant reticular (CAR), osteo-CAR, preosteoblasts, osteoblasts, osteocytes, and osteo-X, the latter defined in part by periostin expression. Osteo-X, osteo-CAR, and preosteoblasts were closely associated with osteoblasts at the trabecular bone surface. Wnt16 was expressed in multiple cell types from the periosteum but not in cells from endocortical or cancellous bone. Fibro-2 cells, which express markers of stem cells, localized to the periosteum but not trabecular bone in adult mice. Suppressing bone remodeling eliminated osteoblasts and altered gene expression in preosteoblasts but did not change the abundance or location of osteo-X or osteo-CAR cells. These results provide a framework for identifying bone cell types in murine single-cell RNA-seq datasets and suggest that osteoblast progenitors reside near the surface of remodeling bone.

Vitamin A enhanced periosteal osteoclastogenesis is associated with increased number of tissue-derived macrophages/osteoclast progenitors.

A deleterious effect of elevated levels of vitamin A on bone health has been reported in clinical studies. Mechanistic studies in rodents have shown that numbers of periosteal osteoclasts are increased, while endocortical osteoclasts are simultaneously decreased by vitamin A treatment. The present study investigated the in vitro and in vivo effect of all-trans retinoic acid (ATRA), the active metabolite of vitamin A, on periosteal osteoclast progenitors. Mouse calvarial bone cells were cultured in media containing ATRA, with or without the osteoclastogenic cytokine receptor activator of nuclear factor kappa B-ligand (RANKL), on plastic dishes or bone discs. Whereas ATRA did not stimulate osteoclast formation alone, the compound robustly potentiated the formation of RANKL-induced bone resorbing osteoclasts. This effect was due to stimulation by ATRA (half-maximal stimulation ∼3 nM) on the numbers of macrophages/osteoclast progenitors in the bone cell cultures, as assessed by mRNA and protein expression of several macrophage and osteoclast progenitor cell markers, such as macrophage colony-stimulating factor receptor, receptor activator of nuclear factor kappa B, F4/80, and CD11b, as well as by flow cytometry (FACS) analysis of CD11b+/F480+/Gr1- cells. The stimulation of macrophage numbers in the periosteal cell cultures was not mediated by increased macrophage colony-stimulating factor or interleukin-34. In contrast, ATRA did not enhance macrophages in bone marrow cell cultures. Importantly, ATRA treatment upregulated the mRNA expression of several macrophage-related genes in the periosteum of tibia in adult mice. These observations demonstrate a novel mechanism by which vitamin A enhances osteoclast formation specifically on periosteal surfaces.

Periosteum-derived podoplanin-expressing stromal cells regulate nascent vascularization during epiphyseal marrow development.

Bone marrow development and endochondral bone formation occur simultaneously. During endochondral ossification, periosteal vasculatures and stromal progenitors invade the primary avascular cartilaginous anlage, which induces primitive marrow development. We previously determined that bone marrow podoplanin (PDPN)-expressing stromal cells exist in the perivascular microenvironment and promote megakaryopoiesis and erythropoiesis. In this study, we aimed to examine the involvement of PDPN-expressing stromal cells in postnatal bone marrow generation. Using histological analysis, we observed that periosteum-derived PDPN-expressing stromal cells infiltrated the cartilaginous anlage of the postnatal epiphysis and populated on the primitive vasculature of secondary ossification center. Furthermore, immunophenotyping and cellular characteristic analyses indicated that the PDPN-expressing stromal cells constituted a subpopulation of the skeletal stem cell lineage. In vitro xenovascular model cocultured with human umbilical vein endothelial cells and PDPN-expressing skeletal stem cell progenies showed that PDPN-expressing stromal cells maintained vascular integrity via the release of angiogenic factors and vascular basement membrane-related extracellular matrices. We show that in this process, Notch signal activation committed the PDPN-expressing stromal cells into a dominant state with basement membrane-related extracellular matrices, especially type IV collagens. Our findings suggest that the PDPN-expressing stromal cells regulate the integrity of the primitive vasculatures in the epiphyseal nascent marrow. To the best of our knowledge, this is the first study to comprehensively examine how PDPN-expressing stromal cells contribute to marrow development and homeostasis.

BMP-9 Improves the Osteogenic Differentiation Ability over BMP-2 through p53 Signaling In Vitro in Human Periosteum-Derived Cells.

Bone morphogenetic proteins (BMPs) have tremendous therapeutic potential regarding the treatment of bone and musculoskeletal disorders due to their osteo-inductive ability. More than twenty BMPs have been identified in the human body with various functions, such as embryonic development, skeleton genesis, hematopoiesis, and neurogenesis. BMPs can induce the differentiation of MSCs into the osteoblast lineage and promote the proliferation of osteoblasts and chondrocytes. BMP signaling is also involved in tissue remodeling and regeneration processes to maintain homeostasis in adults. In particular, growth factors, such as BMP-2 and BMP-7, have already been approved and are being used as treatments, but it is unclear as to whether they are the most potent BMPs that induce bone formation. According to recent studies, BMP-9 is known to be the most potent inducer of the osteogenic differentiation of mesenchymal stem cells, both in vitro and in vivo. However, its exact role in the skeletal system is still unclear. In addition, research results suggest that the molecular mechanism of BMP-9-mediated bone formation is also different from the previously known BMP family, suggesting that research on signaling pathways related to BMP-9-mediated bone formation is actively being conducted. In this study, we performed a phosphorylation array to investigate the signaling mechanism of BMP-9 compared with BMP-2, another influential bone-forming growth factor, and we compared the downstream signaling system. We present a mechanism for the signal transduction of BMP-9, focusing on the previously known pathway and the p53 factor, which is relatively upregulated compared with BMP-2.

Biomaterials-aided mandibular reconstruction using in vivo bioreactors.

Large mandibular defects are clinically challenging to reconstruct due to the complex anatomy of the jaw and the limited availability of appropriate tissue for repair. We envision leveraging current advances in fabrication and biomaterials to create implantable devices that generate bone within the patients themselves suitable for their own specific anatomical pathology. The in vivo bioreactor strategy facilitates the generation of large autologous vascularized bony tissue of customized geometry without the addition of exogenous growth factors or cells. To translate this technology, we investigated its success in reconstructing a mandibular defect of physiologically relevant size in sheep. We fabricated and implanted 3D-printed in vivo bioreactors against rib periosteum and utilized biomaterial-based space maintenance to preserve the native anatomical mandibular structure in the defect site before reconstruction. Nine weeks after bioreactor implantation, the ovine mandibles were repaired with the autologous bony tissue generated from the in vivo bioreactors. We evaluated tissues generated in bioreactors by radiographic, histological, mechanical, and biomolecular assays and repaired mandibles by radiographic and histological assays. Biomaterial-aided mandibular reconstruction was successful in a large superior marginal defect in five of six (83%) sheep. Given that these studies utilized clinically available biomaterials, such as bone cement and ceramic particles, this strategy is designed for rapid human translation to improve outcomes in patients with large mandibular defects.

Hyperstimulation of CaSR in human MSCs by biomimetic apatite inhibits endochondral ossification via temporal down-regulation of PTH1R.

In adult bone injuries, periosteum-derived mesenchymal stem/stromal cells (MSCs) form bone via endochondral ossification (EO), whereas those from bone marrow (BM)/endosteum form bone primarily through intramembranous ossification (IMO). We hypothesized that this phenomenon is influenced by the proximity of MSCs residing in the BM to the trabecular bone microenvironment. Herein, we investigated the impact of the bone mineral phase on human BM-derived MSCs' choice of ossification pathway, using a biomimetic bone-like hydroxyapatite (BBHAp) interface. BBHAp induced hyperstimulation of extracellular calcium-sensing receptor (CaSR) and temporal down-regulation of parathyroid hormone 1 receptor (PTH1R), leading to inhibition of chondrogenic differentiation of MSCs even in the presence of chondroinductive factors, such as transforming growth factor-ß1 (TGF-ß1). Interestingly rescuing PTH1R expression using human PTH fragment (1-34) partially restored chondrogenesis in the BBHAp environment. In vivo studies in an ectopic site revealed that the BBHAp interface inhibits EO and strictly promotes IMO. Furthermore, CaSR knockdown (CaSR KD) disrupted the bone-forming potential of MSCs irrespective of the absence or presence of the BBHAp interface. Our findings confirm the expression of CaSR in human BM-derived MSCs and unravel a prominent role for the interplay between CaSR and PTH1R in regulating MSC fate and the choice of pathway for bone formation.

Primary cilia are necessary for Prx1-expressing cells to contribute to postnatal skeletogenesis.

Although Prx1 (also known as PRRX1)-expressing cells and their primary cilia are critical for embryonic development, they have yet to be studied in the context of postnatal skeletogenesis owing to the lethality of mouse models. A tamoxifen-inducible Prx1 model has been developed, and we determined that expression directed by this promoter is highly restricted to the cambium layers in the periosteum and perichondrium after birth. To determine the postnatal role of these cambium layer osteochondroprogenitors (CLOPs) and their primary cilia, we developed models to track the fate of CLOPs (Prx1CreER-GFP;Rosa26tdTomato) and selectively disrupt their cilia (Prx1CreER-GFP;Ift88fl/fl). Our tracking studies revealed that CLOPs populate cortical and trabecular bone, the growth plate and secondary ossification centers during the normal program of postnatal skeletogenesis. Furthermore, animals lacking CLOP cilia exhibit stunted limb growth due to disruptions in endochondral and intramembranous ossification. Histological examination indicates that growth is stunted due to limited differentiation, proliferation and/or abnormal hypertrophic differentiation in the growth plate. Collectively, our results suggest that CLOPs are programmed to rapidly populate distant tissues and produce bone via a primary cilium-mediated mechanism in the postnatal skeleton.

Induced Periosteum-Mimicking Membrane with Cell Barrier and Multipotential Stromal Cell (MSC) Homing Functionalities.

The current management of critical size bone defects (CSBDs) remains challenging and requires multiple surgeries. To reduce the number of surgeries, wrapping a biodegradable fibrous membrane around the defect to contain the graft and carry biological stimulants for repair is highly desirable. Poly(ε-caprolactone) (PCL) can be utilised to realise nonwoven fibrous barrier-like structures through free surface electrospinning (FSE). Human periosteum and induced membrane (IM) samples informed the development of an FSE membrane to support platelet lysate (PL) absorption, multipotential stromal cells (MSC) growth, and the prevention of cell migration. Although thinner than IM, periosteum presented a more mature vascular system with a significantly larger blood vessel diameter. The electrospun membrane (PCL3%-E) exhibited randomly configured nanoscale fibres that were successfully customised to introduce pores of increased diameter, without compromising tensile properties. Additional to the PL absorption and release capabilities needed for MSC attraction and growth, PCL3%-E also provided a favourable surface for the proliferation and alignment of periosteum- and bone marrow derived-MSCs, whilst possessing a barrier function to cell migration. These results demonstrate the development of a promising biodegradable barrier membrane enabling PL release and MSC colonisation, two key functionalities needed for the in situ formation of a transitional periosteum-like structure, enabling movement towards single-surgery CSBD reconstruction.

Rat sinus mucosa- and periosteum-derived exosomes accelerate osteogenesis.

Guided bone regeneration (GBR) is commonly used for alveolar bone augmentation. The paracrine mechanism in the field of bone tissue engineering has been emphasized in recent years and exosomes are considered to have the potential of promoting osteogenesis. We aimed to study the influence of sinus mucosa and periosteum on bone regeneration through paracrine stimulation, especially via exosomes, and compare the differences between them. Here, we report that conditioned medium (CM) from sinus mucosa-derived cells (SMCs) and periosteum-derived cells (PCs) and the isolated exosomes enhanced the proliferation, migration and osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BM-MSCs) in vitro. A rat model of femoral bone defects was used to demonstrate that the exosomes derived from SMCs (SMC-Exos) and PCs (PC-Exos) can accelerate bone formation in vivo. Furthermore, we present a preliminary discussion of the possible functional components involved in the effects of SMC-Exos and PC-Exos on bone regeneration. In conclusion, these results demonstrated that the sinus mucosa and periosteum can accelerate osteogenesis through paracrine effects and the exosomes play important roles in this process.

Donor-matched comparison of chondrogenic progenitors resident in human infrapatellar fat pad, synovium, and periosteum - implications for cartilage repair.

Purpose: There is a clinical need to better characterize tissue sources being used for stem cell therapies. This study focuses on comparison of cells and connective tissue progenitors (CTPs) derived from native human infrapatellar fatpad (IPFP), synovium (SYN), and periosteum (PERI). Materials and Methods: IPFP, SYN, PERI were harvested from twenty-eight patients undergoing arthroplasty. CTPs were quantitatively characterized using automated colony-forming-unit assay to compare total nucleated cell concentration-[Cell], cells/mg; prevalence-(PCTP), CTPs/million nucleated cells; CTP concentration-[CTP], CTPs/mg; proliferation and differentiation potential; and correlate outcomes with patient's age and gender. Results: [Cell] did not differ between IPFP, SYN, and PERI. PCTP was influenced by age and gender: patients >60 years, IPFP and SYN had higher PCTP than PERI (p < 0.001) and females had higher PCTP in IPFP (p < 0.001) and SYN (p = 0.001) than PERI. [CTP] was influenced by age: patients <50 years, SYN (p = 0.0165) and PERI (p < 0.001) had higher [CTP] than IPFP; patients between 60 and 69 years, SYN (p < 0.001) had higher [CTP] than PERI; patients >70 years, IPFP (p = 0.006) had higher [CTP] than PERI. In patients >60 years, proliferation potential of CTPs differed significantly (SYN>IPFP>PERI); however, differentiation potentials were comparable between all three tissue sources. Conclusion: SYN and IPFP may serve as a preferred tissue source for patients >60 years, and PERI along with SYN and IPFP may serve as a preferred tissue source for patients <60 years for cartilage repair. However, the heterogeneity among the CTPs in any given tissue source suggests performance-based selection might be useful to optimize cell-sourcing strategies to improve efficacy of cellular therapies for cartilage repair.

Acute Megakaryoblastic Leukemia With Diffuse Periosteal Reaction of Bilateral Lower Extremities.

We report the case of a 3-year-old girl diagnosed with acute megakaryoblastic leukemia, who presented after >1 year of bilateral leg pain. At times the pain was severe enough to prevent ambulation, prompting visits to her primary care provider. However, it was not until acute respiratory failure occurred with subsequent hospitalization in the pediatric intensive care unit that severe anemia and thrombocytopenia were discovered and the diagnosis of acute myeloid leukemia was made. Bilateral lower extremity swelling was noted on admission and radiographs showed diffusely abnormal appearance of the long bones of her lower extremities with periosteal reaction and echogenic debris in the subperiosteal space, thought to represent leukemic cells. This case highlights the importance of recognizing atypical signs and symptoms of myelodysplastic syndrome progressing to acute leukemia in the context of abnormal bone pain and radiographic changes.

Platelet-Rich Fibrin Extract: A Promising Fetal Bovine Serum Alternative in Explant Cultures of Human Periosteal Sheets for Regenerative Therapy.

In 2004, we developed autologous periosteal sheets for the treatment of periodontal bone defects. This regenerative therapy has successfully regenerated periodontal bone and augmented alveolar ridge for implant placement. However, the necessity for 6-week culture is a limitation. Here, we examined the applicability of a human platelet-rich fibrin extract (PRFext) as an alternative to fetal bovine serum (FBS) for the explant culture of periosteal sheets in a novel culture medium (MSC-PCM) originally developed for maintaining mesenchymal stem cells. Small periosteum tissue segments were expanded in MSC-PCM + 2% PRFext for 4 weeks, and the resulting periosteal sheets were compared with those prepared by the conventional method using Medium199 + 10% FBS for their growth rate, cell multilayer formation, alkaline phosphatase (ALP) activity, and surface antigen expression (CD73, CD90, and CD105). Periosteal sheets grew faster in the novel culture medium than in the conventional medium. However, assessment of cell shape and ALP activity revealed that the periosteal cells growing in the novel medium were relatively immature. These findings suggest that the novel culture medium featuring PRFext offers advantages by shortening the culture period and excluding possible risks associated with xeno-factors without negatively altering the activity of periosteal sheets.

Quality Analysis of Minerals Formed by Jaw Periosteal Cells under Different Culture Conditions.

Previously, we detected a higher degree of mineralization in fetal calf serum (FCS) compared to serum-free cultured jaw periosteum derived osteoprogenitor cells (JPCs). By Raman spectroscopy, we detected an earlier formation of mineralized extracellular matrix (ECM) of higher quality under serum-free media conditions. However, mineralization potential remained too low. In the present study, we aimed to investigate the biochemical composition and subsequent biomechanical properties of the JPC-formed ECM and minerals under human platelet lysate (hPL) and FCS supplementation. JPCs were isolated (n = 4 donors) and expanded under FCS conditions and used in passage five for osteogenic induction under both, FCS and hPL media supplementation. Raman spectroscopy and Alizarin Red/von Kossa staining were employed for biochemical composition analyses and for visualization and quantification of mineralization. Osteocalcin gene expression was analyzed by quantitative PCR. Biomechanical properties were assessed by using atomic force microscopy (AFM). Raman spectroscopic measurements showed significantly higher (p < 0.001) phosphate to protein ratios and in the tendency, lower carbonate to phosphate ratios in osteogenically induced JPCs under hPL in comparison to FCS culturing. Furthermore, higher crystal sizes were detected under hPL culturing of the cells. With respect to the ECM, significantly higher ratios of the precursor protein proline to hydroxyproline were detected in hPL-cultured JPC monolayers (p < 0.001). Additionally, significantly higher levels (p < 0.001) of collagen cross-linking were calculated, indicating a higher degree of collagen maturation in hPL-cultured JPCs. By atomic force microscopy, a significant increase in ECM stiffness (p < 0.001) of FCS cultured JPC monolayers was observed. The reverse effect was measured for the JPC formed precipitates/minerals. Under hPL supplementation, JPCs formed minerals of significantly higher stiffness (p < 0.001) when compared to the FCS setting. This study demonstrates that hPL culturing of JPCs leads to the formation of an anorganic material of superior quality in terms of biochemical composition and mechanical properties.

Mutations Preventing Regulated Exon Skipping in MET Cause Osteofibrous Dysplasia.

The periosteum contributes to bone repair and maintenance of cortical bone mass. In contrast to the understanding of bone development within the epiphyseal growth plate, factors that regulate periosteal osteogenesis have not been studied as intensively. Osteofibrous dysplasia (OFD) is a congenital disorder of osteogenesis and is typically sporadic and characterized by radiolucent lesions affecting the cortical bone immediately under the periosteum of the tibia and fibula. We identified germline mutations in MET, encoding a receptor tyrosine kinase, that segregate with an autosomal-dominant form of OFD in three families and a mutation in a fourth affected subject from a simplex family and with bilateral disease. Mutations identified in all families with dominant inheritance and in the one simplex subject with bilateral disease abolished the splice inclusion of exon 14 in MET transcripts, which resulted in a MET receptor (MET(Δ14)) lacking a cytoplasmic juxtamembrane domain. Splice exclusion of this domain occurs during normal embryonic development, and forced induction of this exon-exclusion event retarded osteoblastic differentiation in vitro and inhibited bone-matrix mineralization. In an additional subject with unilateral OFD, we identified a somatic MET mutation, also affecting exon 14, that substituted a tyrosine residue critical for MET receptor turnover and, as in the case of the MET(Δ14) mutations, had a stabilizing effect on the mature protein. Taken together, these data show that aberrant MET regulation via the juxtamembrane domain subverts core MET receptor functions that regulate osteogenesis within cortical diaphyseal bone.

Identification of a Recognizable Progressive Skeletal Dysplasia Caused by RSPRY1 Mutations.

Skeletal dysplasias are highly variable Mendelian phenotypes. Molecular diagnosis of skeletal dysplasias is complicated by their extreme clinical and genetic heterogeneity. We describe a clinically recognizable autosomal-recessive disorder in four affected siblings from a consanguineous Saudi family, comprising progressive spondyloepimetaphyseal dysplasia, short stature, facial dysmorphism, short fourth metatarsals, and intellectual disability. Combined autozygome/exome analysis identified a homozygous frameshift mutation in RSPRY1 with resulting nonsense-mediated decay. Using a gene-centric "matchmaking" system, we were able to identify a Peruvian simplex case subject whose phenotype is strikingly similar to the original Saudi family and whose exome sequencing had revealed a likely pathogenic homozygous missense variant in the same gene. RSPRY1 encodes a hypothetical RING and SPRY domain-containing protein of unknown physiological function. However, we detect strong RSPRY1 protein localization in murine embryonic osteoblasts and periosteal cells during primary endochondral ossification, consistent with a role in bone development. This study highlights the role of gene-centric matchmaking tools to establish causal links to genes, especially for rare or previously undescribed clinical entities.

Blastema formation and periosteal ossification in the regenerating adult mouse digit.

While mammals cannot regenerate amputated limbs, mice and humans have regenerative ability restricted to amputations transecting the digit tip, including the terminal phalanx (P3). In mice, the regeneration process is epimorphic and mediated by the formation of a blastema comprised of undifferentiated proliferating cells that differentiate to regenerate the amputated structures. Blastema formation distinguishes the regenerative response from a scar-forming healing response. The mouse digit tip serves as a preclinical model to investigate mammalian blastema formation and endogenous regenerative capabilities. We report that P3 blastema formation initiates prior to epidermal closure and concurrent with the bone histolytic response. In this early healing response, proliferation and cells entering the early stages of osteogenesis are localized to the periosteal and endosteal bone compartments. After the completion of stump bone histolysis, epidermal closure is completed and cells associated with the periosteal and endosteal compartments blend to form the blastema proper. Osteogenesis associated with the periosteum occurs as a polarized progressive wave of new bone formation that extends from the amputated stump and restores skeletal length. Bone patterning is restored along the proximal-distal and medial digit axes, but is imperfect in the dorsal-ventral axis with the regeneration of excessive new bone that accounts for the enhanced regenerated bone volume noted in previous studies. Periosteum depletion studies show that this compartment is required for the regeneration of new bone distal to the original amputation plane. These studies provide evidence that blastema formation initiates early in the healing response and that the periosteum is an essential tissue for successful epimorphic regeneration in mammals.

Upregulation of inflammatory gene transcripts in periosteum of chronic migraineurs: Implications for extracranial origin of headache.

OBJECTIVE: Chronic migraine (CM) is often associated with chronic tenderness of pericranial muscles. A distinct increase in muscle tenderness prior to onset of occipital headache that eventually progresses into a full-blown migraine attack is common. This experience raises the possibility that some CM attacks originate outside the cranium. The objective of this study was to determine whether there are extracranial pathophysiologies in these headaches. METHODS: We biopsied and measured the expression of gene transcripts (mRNA) encoding proteins that play roles in immune and inflammatory responses in affected (ie, where the head hurts) calvarial periosteum of (1) patients whose CMs are associated with muscle tenderness and (2) patients with no history of headache. RESULTS: Expression of proinflammatory genes (eg, CCL8, TLR2) in the calvarial periosteum significantly increased in CM patients attesting to muscle tenderness, whereas expression of genes that suppress inflammation and immune cell differentiation (eg, IL10RA, CSF1R) decreased. INTERPRETATION: Because the upregulated genes were linked to activation of white blood cells, production of cytokines, and inhibition of NF-κB, and the downregulated genes were linked to prevention of macrophage activation and cell lysis, we suggest that the molecular environment surrounding periosteal pain fibers is inflamed and in turn activates trigeminovascular nociceptors that reach the affected periosteum through suture branches of intracranial meningeal nociceptors and/or somatic branches of the occipital nerve. This study provides the first set of evidence for localized extracranial pathophysiology in CM. Ann Neurol 2016;79:1000-1013.

The periosteal microcirculation in health and disease: An update on clinical significance.

Apart from its nutritive functions, the periosteum critically affects bone regeneration via its stem/osteoprogenitor cell content. Normal healing after bone fractures, trauma-orthopedic interventions and invasive dental procedures is critically linked to the reestablishment of the periosteal microcirculation, but the reconstruction, replacement or repair of lost tissues may also be performed with autologous periosteum. Besides the initiation of cell differentiation during bone repair and remodeling processes, the periosteum together with the endosteum plays significant roles in the pathogenesis of both hormone-related and trauma-induced osteoporotic alterations in the bone metabolism. Nevertheless, the axial bones, and in particular the jawbones, and the appendicular bones display differences not only in their blood supply and fracture healing characteristics, but also in respect of the development of osteoporosis and their reactions to treatment modalities (i.e. bisphosphonates). These reactions may also be linked to the differences in periosteal microcirculatory reactions. The present overview summarizes the relevant data of microcirculatory studies focusing on the periosteal reactions in different anatomical locations together with the optimal background methodologies, study models and the most significant observations.

Investigation of osteogenic activity of primary rabbit periosteal cells stimulated by multi-axial tensile strain.

Periosteum-derived cells was indicated to respond to mechanical force and have stem cell potential capable of differentiating into multiple tissue. Investigation of osteogenic activity under mechanical stimulation is important to understand the therapeutic conditions of fracture healing. In this work, a cell culture platform was developed for respectively providing isotropic and anisotropic axial strain. Primary rabbit periosteal cells were isolated and cultured in the chamber. Multi-axial tensile strain was received and osteogenic activity was investigated by mRNA expressions of CBFA1 and OPN. The highest mRNA expression was found in moderate strain (5-8%) under anisotropic axial strain. These results provided important foundation for further in vivo studies and development of tailor-made stretching rehabilitation equipment.