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Purpose: Autologous chondrocyte transplantation (ACT) is an established method in cartilage repair. Although long-term results show durable repair of isolated cartilage defects, some problems still remain. Since hypertrophy of the transplanted periosteum is a common problem, alternatives for periosteum are in demand. Periosteal grafts have been reported to stimulate neochondrogenesis via paracrine effects. The objective of this study was to evaluate the modulation of chondrocyte metabolism by periosteal grafts in vitro., Methods: Periosteal explants and articular chondrocytes obtained from slaughtered adult cattle were co-cultured in a newly established perfusion system. The experimental groups were: 1. monocultured chondrocytes; 2. chondrocytes cultured with synovial supernatants; 3. chondrocytes cultured with periosteal supernatants; 4. chondrocytes co-cultured with periosteal explants., Results: Chondrocyte proliferation, evaluated by measuring total DNA content, was prolongated by periosteal and synovial explants. Immunocytochemical staining of collagen type II was stronger in monoculture than in co-culture. Protein biosynthetic activity estimated by [³H]-proline incorporation, as well as extracellular matrix deposition for collagen type II, were reduced by periosteal and synovial explants. Additionally, co-culturing led to a decrease in aggrecan synthesis and release. The inhibiting effects were significantly stronger when cellular chondrocyte-periosteal cross-talk was made possible via paracrine effects., Conclusions: The results of our study suggest a catabolic effect of periosteal explants on isolated chondrocytes in vitro. Further investigations are necessary whether periosteum in ACT is dispensable.

Connexin 43 (Cx43) mediates osteocyte communication with other cells and with the extracellular milieu and regulates osteoblastic cell signaling and gene expression. We now report that mice lacking Cx43 in osteoblasts/osteocytes or only in osteocytes (Cx43(ΔOt) mice) exhibit increased osteocyte apoptosis, endocortical resorption, and periosteal bone formation, resulting in higher marrow cavity and total tissue areas measured at the femoral mid-diaphysis. Blockade of resorption reversed the increased marrow cavity but not total tissue area, demonstrating that endocortical resorption and periosteal apposition are independently regulated. Anatomical mapping of apoptotic osteocytes, osteocytic protein expression, and resorption and formation suggests that Cx43 controls osteoclast and osteoblast activity by regulating osteoprotegerin and sclerostin levels, respectively, in osteocytes located in specific areas of the cortex. Whereas empty lacunae and living osteocytes lacking osteoprotegerin were distributed throughout cortical bone in Cx43(ΔOt) mice, apoptotic osteocytes were preferentially located in areas containing osteoclasts, suggesting that osteoclast recruitment requires active signaling from dying osteocytes. Furthermore, Cx43 deletion in cultured osteocytic cells resulted in increased apoptosis and decreased osteoprotegerin expression. Thus, Cx43 is essential in a cell-autonomous fashion in vivo and in vitro for osteocyte survival and for controlling the expression of osteocytic genes that affect osteoclast and osteoblast function.

Various bone matrix proteins are produced during the process of osteogenesis. Many previous studies suggested that the topography of an implant surface might affect the expression of osteoblast-mediated cytokines. However, these earlier studies were performed using in vitro cell culture. This study investigated the influence of the surface topography of a titanium implant placed under the periosteum on the gene expression of bone morphogenic markers in rat. Six custom-made implants with a rough upper surface and 6 custom-made implants with a smooth machined upper surface were placed subcrestally with the upper surface facing up in the femurs of 6 adult male rats. Five rats were sacrificed 7 days after the implant placement, and the periosteum above the embedded implant was obtained and analyzed by quantitative real-time RT-PCR for the target genes: alkaline phosphatase (ALP), bone sialoprotein (BSP) and osteocalcin (OCN). The other rat was sacrificed at day 7, and both implants and the surrounding tissue were embedded in paraffin. For light microscopic observations, paraffin sections were stained with toluidine blue. Gene expression of ALP, BSP and OCN at the rough surface implant was significantly higher than that at the smooth machined surface implant. At day 7, both types of implant were covered with soft tissue, but a lower number of cells stained with toluidine blue was observed on the machined surface compared with on the rough surface. It is considered that rough surfaces may stimulate osteoblasts, and that ALP activity is increased indirectly. Furthermore, the two other markers were also increased by the rough surface in vivo, and different distributions of cellular and extracellular components on the upper surface of the implants were observed at day 7. These results suggest that a rough surface implant under the periosteum promotes higher gene expression of ALP, BSP and OCN in rat.

As the only known example of complete organ regeneration in mammals, deer antler in the growing or velvet phase is of major interest in developmental biology. This regeneration event initiates from self-renewing antler stem cells that exhibit pluripotency. At present, it remains unclear how the activation and quiescence of antler stem cells are regulated. Therefore, in the present study proteins that were differentially expressed between the antler stem cells and somatic cells (facial periosteum) were identified by a gel-based proteomic technique, and analysed using Ingenuity Pathway Analysis software. Several molecular pathways (PI3K/Akt, ERK/MAPK, p38 MAPK, etc.) were found to be activated during proliferation. Also expressed were the transcription factors POU5F1, SOX2, NANOG and MYC, which are key markers of embryonic stem cells. Expression of these proteins was confirmed in both cultured cells and fresh tissues by Western blot analysis. Therefore, the molecular pathways and transcription factors identified in the current study are common to embryonic and adult stem cells. However, expression of embryonic stem cell transcription factors would suggest that antler stem cells are, potentially, an intermediary stem cell type between embryonic and the more specialized tissue-specific stem cells like those residing in muscle, fat or from a hematopoietic origin. The retention of this embryonic, pluripotent lineage may be of fundamental importance for the subsequent regenerative capacity of antlers.

Objective: It is widely known that stress conditioning can protect microcirculation and induce the release of vasoactive factors for a period of several hours. Little, however, is known about the long-term effects of stress conditioning on microcirculation, especially on the microcirculation of the periosteum of the calvaria. For this reason, we used intravital fluorescence microscopy to investigate the effects of heat shock priming on the microcirculation of the periosteum over a period of several days., Methods: Fifty-two Lewis rats were randomized into eight groups. Six groups underwent heat shock priming of the periosteum of the calvaria at 42.5°C, two of them (n = 8) for 15 minutes, two (n = 8) for 25 minutes and two (n = 8) for 35 minutes. After 24 hours, a periosteal chamber was implanted into the heads of the animals of one of each of the two groups mentioned above. Microcirculation and inflammatory responses were studied repeatedly over a period of 14 days using intravital fluorescence microscopy. The expression of heat shock protein (HSP) 70 was examined by immunohistochemistry in three further groups 24 hours after a 15-minute (n = 5), a 25-minute (n = 5) or a 35-minute (n = 5) heat shock treatment. Two groups that did not undergo priming were used as controls. One control group (n = 8) was investigated by intravital microscopy and the other (n = 5) by immunohistochemistry., Results: During the entire observation period of 14 days, the periosteal chambers revealed physiological microcirculation of the periosteum of the calvaria without perfusion failures. A significant (p < 0.05) and continuous increase in functional capillary density was noted from day 5 to day 14 after 25-minute heat shock priming. Whereas a 15-minute exposure did not lead to an increase in functional capillary density, 35-minute priming caused a significant but reversible perfusion failure in capillaries. Non-perfused capillaries in the 35-minute treatment group were reperfused by day 10. Immunohistochemistry demonstrated an increase in cytoprotective HSP70 expression in the periosteum after a 15-minute and a 35-minute heat shock pretreatment when compared with the control group. The level of HSP70 expression that was measured in the periosteum after 25 minutes of treatment was significantly higher than the levels observed after 15 or 35 minutes of heat shock exposure., Conclusion: A few days after heat shock priming over an appropriate period of time, a continuous increase in functional capillary density is seen in the periosteum of the calvaria. This increase in perfusion appears to be the result of the induction of angiogenesis.

Background: Periosteal cells are important in embryogenesis, fracture healing, and cartilage repair and could provide cells for osteochondral tissue engineering., Questions/purpose: We determined whether a population of cells isolated from human periosteal tissue contains cells with a mesenchymal stem cell (MSC) phenotype and whether these cells can be expanded in culture and used to form tissue in vitro., Methods: We obtained periosteal tissue from six patients. Initial expression of cell surface markers was assessed using flow cytometry. Cells were cultured over 10 generations and changes in gene expression evaluated to assess phenotypic stability. Phenotype was confirmed using flow cytometry and colony-forming ability assays. Mineral formation was assessed by culturing Stro-1(-) and unsorted cells with osteogenic supplements. Three cell culture samples were used for a reverse transcription-polymerase chain reaction, four for flow cytometry, three for colony-forming assay, and three for mineralization., Results: Primary cultures, containing large numbers of hematopoietic cells were replaced initially by Stro-1 and ALP-expressing immature osteoblastic cell types and later by ALP-expressing cells, which lacked Stro-1 and which became the predominant cell population during subculture. Approximately 10% of the total cell population continued to express markers for Stro1(+)/ALP(-) cells throughout., Conclusions: These data suggest periosteum contains a large number of undifferentiated cells that can differentiate into neotissue and persist despite culture in noncell-specific media for over 10 passages., Clinical Relevance: Cultured periosteal cells may contribute to tissue formation and may be applicable for tissue engineering applications.

This study was designed to evaluate the effect of autologous bone marrow mesenchymal stem cells (MSCs) seeded into Gelfoam® on structural bone allograft healing. Thirty New Zealand white rabbits were divided into two groups. Segmental bone defect was created on diaphysis of the femur, and the defect was reconstructed with structural bone allograft. In experimental group, structural allograft was wrapped around by Gelfoam® containing autologous MSCs, whereas cells were not included in control group. At 4, 8, 12 weeks, the femur of rabbits underwent radiographic and histologic evaluation for bony union. Bone morphogenic protein-2 (BMP-2), BMP-4, BMP-7, vascular endothelial growth factor (VEGF), and receptor activator of nuclear factor-kappa B ligand (RANKL) were measured within the grafted periosteal tissue. Bony union was not achieved in both groups at 4 and 8 weeks. At 12 weeks, three out of five femurs in experimental group were united, but one out of five in control group was united. Mean Taira scores were significantly different between two groups. The expression of BMP-2 was significantly higher at 4, 8 weeks, the expressions of BMP-4 and BMP-7 were significantly higher at 8 and 12 weeks, and the expression of VEGF and RANKL were significantly higher at all time points in experimental group. Incorporation of the structural bone allograft could be enhanced if allograft is covered with Gelfoam® containing autologous MSCs. MSCs have influence on not only bone formation, but neo-angiogenesis, and bone resorption.

Objectives: The periosteum plays an important role in bone regeneration. However, the harvesting of autogenous periosteum is associated with disadvantages such as donor site morbidity and limited donor sources. This study uses an osteogenic predifferentiated cell sheet to fabricate a scaffold-free tissue-engineered periosteum (TEP)., Material and Methods: We generated an osteogenic predifferentiated cell sheet from rabbit bone marrow stromal cells (BMSCs) using a continuous culture system and harvested it using a scraping technique. Then, the in vitro characterization of the sheet was investigated using microscopy investigation, quantitative analysis of alkaline phosphatase (ALP) activity, and RT-PCR. Next, we demonstrated the in vivo osteogenic potential of the engineered sheet in ectopic sites together with a porous β-tricalcium phosphate ceramic. Finally, we evaluated its efficiency in treating delayed fracture healing after wrapping the cell sheet around the mandible in a rabbit model., Results: The engineered periosteum showed sporadic mineralized nodules, elevated ALP activity, and up-regulated gene expression of osteogenic markers. After implantation in the subcutaneous pockets of the donor rabbits, the in vivo bone-forming capability of the engineered periosteum was confirmed by histological examinations. Additionally, when wrapping the engineered periosteum around a mandibular fracture gap, we observed improved bone healing and reduced amounts of fibrous tissue at the fracture site., Conclusion: The osteogenic predifferentiated BMSC sheet can act as a scaffold-free TEP to facilitate bone regeneration. Hence, our study provides a promising strategy for enhancing bone regeneration in clinical settings., (© 2011 John Wiley & Sons A/S.)

Although androgen is considered an anabolic hormone, the consequences of androgen receptor (AR) overexpression in skeletally-targeted AR-transgenic lines highlight the detrimental effect of enhanced androgen sensitivity on cortical bone quality. A compartment-specific anabolic response is observed only in male and not in female AR3.6-transgenic (tg) mice, with increased periosteal bone formation and calvarial thickening. To identify anabolic signaling cascades that have the potential to increase bone formation, qPCR array analysis was employed to define expression differences between AR3.6-tg and wild-type (WT) periosteal tissue. Notably, categories that were significantly different between the two genotypes included axonal guidance, CNS development and negative regulation of Wnt signaling with a node centered on stem cell pathways. Further, fine mapping of AR3.6-tg calvaria revealed that anabolic thickening in vivo is not uniform across the calvaria, occurring only in frontal and in not parietal bones. Multipotent fraction 1 progenitor populations from both genotypes were cultured separately as frontal bone neural crest stem-like cells (fNCSC) and parietal bone mesenchymal stem-like cells (pMSC). Both osteoblastic and adipogenic differentiation in these progenitor populations was influenced by embryonic lineage and by genotype. Adipogenesis was enhanced in WT fNCSC compared to pMSC, but transgenic cultures showed strong suppression of lipid accumulation only in fNCSC cells. Osteoblastogenesis was significantly increased in transgenic fNCSC cultures compared to WT, with elevated alkaline phosphatase (ALP) activity and induction of mineralization and nodule formation assessed by alizarin red and von Kossa staining. Osteocalcin (OC) and ALP mRNA levels were also increased in fNCSC cultures from AR3.6-tg vs. WT, but in pMSC cultures ALP mRNA levels, mineralization and nodule formation were decreased in AR3.6-tg cells. Expression differences identified by array in long bone periosteal tissue from AR3.6-tg vs. WT were recapitulated in the fNCSC samples while pMSC profiles reflected cortical expression. These observations reveal the opposing effects of androgen signaling on lineage commitment and osteoblast differentiation that is enhanced in cells derived from a neural crest origin but inhibited in cells derived from a mesodermal origin, consistent with in vivo compartment-specific responses to androgen. Combined, these results highlight the complex action of androgen in the body that is dependent on the embryonic lineage and developmental origin of the cell. Further, these data these data suggest that the periosteum surrounding long bone is derived from neural crest., (Published by Elsevier Inc.)

Demineralized bone matrix (DBM) has been widely investigated as a biomaterial to promote new bone formation and is utilized clinically for bone repair and regeneration. We investigated gene expression patterns of osteogenic differentiation in human periosteal (HPO) cells cultured with demineralized bone matrix, using cDNA array technology. Osteogenic differentiation of HPO cells was determined using alkaline phosphatase assay. In order to examine differential gene expression during osteogenic differentiation, total RNA was isolated from HPO cells in the absence or presence of DBM on day seven and analyzed using osteogenesis cDNA gene array. The selected genes were verified using reverse transcriptase (RT)-PCR analysis. Human periosteal cells differentiated along an osteogenic lineage after treatment of DBM. The alkaline phosphatase activity assay showed that HPO cells differentiated into an osteogenic lineage. Gene expression of HPO cells treated with DBM for seven days was analyzed with cDNA array and RT-PCR analyses. Expression of biglycan, TGF-β1, and TGF-βR1 was upregulated, whereas collagen14A1 expression was downregulated, as confirmed by RT-PCR. Human periosteal cells expressed osteogenesis genes when treated with DBM. These findings provide new insight into the capability of demineralized bone matrix to modulate the osteogenic differentiation of human periosteal cells.

Antler development is triggered by interactions between antler stem cells resident in the antlerogenic periosteum (AP) and the niche cells in the upper portion of overlying skin mediated by diffusible molecules. These interactive cell populations are interposed by the lower portion of the skin and the subcutaneous loose connective tissue (SLCT). It is known that mechanical deletion of just the central AP (having an area equivalent to the size of a pedicle base) by cutting through the skin and SLCT effectively stimulates the marginal AP to initiate antler development. This study was designed to investigate whether the SLCT layer plays a role in antler development by acting as a physical barrier. The results showed that the marginal AP failed to give rise to an antler after the central AP was cryosurgically destroyed with the preservation of the collagen structure of the SLCT. Furthermore, antler development was significantly advanced when the collagen structures of the skin and SLCT layers were substantially attenuated by repeated sprays with liquid nitrogen while keeping the central AP intact. Therefore, we conclude that the interposing SLCT layer acts as a physical barrier between antler stem cells and the niche cell types, and that timing of antler development is primarily controlled by the permeability of the SLCT layer to the putative interactive diffusible molecules., (Copyright © 2011 Wiley-Liss, Inc., A Wiley Company.)

Background: Bisphosphonate associated osteonecrosis of the jaw (BRONJ) implies an impairment in oral hard- and soft tissue repair. An understanding of the signal transduction alterations involved can inform therapeutic strategies. Transforming growth factor β1 (TGFβ1) is a critical regulator of tissue repair; galectin-3 mediates tissue differentiation and specifically modulates periodontopathic bacterial infection. The aim of this study was to compare the expression of TGFβ1-related signaling molecules and Galectin-3 in BRONJ-affected and healthy mucosal tissues. To discriminate between BRONJ-specific impairments in TGFβ1 signaling and secondary inflammatory changes, the results were compared to the expression of TGFβ1 and Galectin-3 in mucosal tissues with osteoradionecrosis., Methods: Oral mucosal tissue samples with histologically-confirmed BRONJ (n = 20), osteoradionecrosis (n = 20), and no lesions (normal, n = 20) were processed for immunohistochemistry. Automated staining with an alkaline phosphatase-anti-alkaline phosphatase kit was used to detect TGFβ1, Smad-2/3, Smad-7, and Galectin-3. We semiquantitatively assessed the ratio of stained cells/total number of cells (labeling index, Bonferroni-adjustment)., Results: TGFβ1 and Smad-2/3 were significantly decreased (p < 0.032 and p(0.028, respectively) in the BRONJ samples and significantly increased (p < 0.04 and p <0.043, respectively) in the osteoradionecrosis samples compared to normal tissue. Smad-7 was significantly increased (p < 0.031) in the BRONJ group and significantly decreased (p < 0.026) in the osteoradionecrosis group. Galectin-3 staining was significantly (p < 0.025) increased in both the BRONJ and the osteoradionecrosis (p < 0.038) groups compared to the normal tissue group. However, Galectin-3 expression was significantly higher in the BRONJ samples than in the osteoradionecrosis samples (p < 0.044)., Conclusion: Our results showed that disrupted TGFβ1 signaling was associated with delayed periodontal repair in BRONJ samples. The findings also indicated that impairments in TGFβ1-signaling were different in BRONJ compared to osteoradionecrosis. BRONJ appeared to be associated with increased terminal osseous differentiation and decreased soft tissue proliferation. The increase in Galectin-3 reflected the increase in osseous differentiation of mucoperiosteal progenitors, and this might explain the inflammatory anergy observed in BRONJ-affected soft tissues. The results substantiated the clinical success of treating BRONJ with sequestrectomy, followed by strict mucosa closure. BRONJ can be further elucidated by investigating the specific intraoral osteoimmunologic status.

Cultured human periosteal sheets constitute a promising grafting material for periodontal tissue regenerative therapy. However, preparation of these sheets usually requires six weeks or longer, and this lengthy commitment and delay limits both clinical applicability and availability. The aim of this study is to develop an efficient, practical, cost-effective cryopreservation method for periosteal tissue segments (PTSs). Human PTSs were aseptically excised from alveolar bone and pre-cultured in Medium 199+10% fetal bovine serum (FBS) for the indicated number of days before they were slowly frozen down to -75°C in a commercial freezing vessel using medium containing 10% dimethyl sulfoxide (Me(2)SO) and various concentrations of FBS. After fast-thawing at 37°C, PTSs were again cultured, and their growth and responses to standard osteogenic induction were evaluated (vs. freshly excised PTSs). Proliferating cells were obtained at the highest levels from cryopreserved PTSs that were pre-cultured for 14 days before freezing. When a concentration of 50% or more FBS was included in the cryopreservation solution, cells migrated out more actively and grew faster. Importantly, osteoinduction up-regulated alkaline phosphatase (ALP) activity and osteoblastic marker mRNAs in cryopreserved PTS-derived sheets just as effectively as it did in native PTS-derived ones. These data suggest that pre-conditioned PTSs can be efficiently cryopreserved in a freezing solution containing high FBS by traditional manual cryopreservation methods without aid of a program freezer or more elaborate equipment., (Copyright © 2011 Elsevier Inc. All rights reserved.)

In an ovine femur model, proliferative woven bone fills critically sized defects enveloped by periosteum within 2 weeks of treatment with the one-stage bone-transport surgery. We hypothesize that mechanical loading modulates this process. Using high-definition optical strain measurements we determined prevailing periosteal strains for normal and surgically treated ovine femora subjected ex vivo to compressive loads simulating in vivo stance shifting (n = 3 per group, normal vs. treated). We determined spatial distribution of calcein green, a label for bone apposition in first the 2 weeks after surgery, in 15°, 30°, and 45° sectors of histological cross sections through the middle of the defect zone (n = 6 bones, three to four sections per bone). Finally, we correlated early bone formation to either the maximal periosteal strain or the net change in maximal periosteal strain. We found that treatment with the one-stage bone-transport surgery profoundly changes the mechanical environment of cells within the periosteum during stance shift loading. The pattern of early bone formation is repeatable within and between animals and relates significantly to the actual strain magnitude prevailing in the periosteum during stance shift loading. Interestingly, early bone apposition after the surgery correlates well to the maximal net change in strain (above circa 2000-3000 με, in tension or compression) rather than strain magnitude per se, providing further evidence that changes in cell shape may drive mechanoadaptation by progenitor cells. These important insights regarding mechanobiological factors that enhance rapid bone generation in critically sized defects can be translated to the tissue and organ scale, providing a basis for the development of best practices for clinical implementation and the definition of movement protocols to enhance the regenerative effect.

Bone modeling and remodeling has been the subject of extensive experimental studies. There have been several mathematical models proposed to explain the observed behavior, as well. A different approach is taken here in which the bone is treated from a macroscopic view point. In this investigation, a one-dimensional analytical model is used to shed light on the factors which play the greatest role in modeling or growth of cortical bone at the periosteal surface. It is presumed that bone growth is promoted when increased amounts of bone nutrients, such as nitric oxide synthase (NOS) or messenger molecules, such as prostaglandin E2 (PGE2), seep out to the periosteal surface of cortical bone and are absorbed by osteoblasts. The transport of the bone nutrients is assumed to be a strain controlled process. Equations for the flux of these nutrients are written for a one-dimensional model of a long bone. The obtained partial differential equation is linearized and solved analytically. Based upon the seepage of nutrients out of the bone, the effect of loading frequency, number of cycles and strain level is examined for several experiments that were found in the literature. It is seen that bone nutrient seepage is greatest on the tensile side of the bone; this location coincides with the greatest amount of bone modeling., (Copyright © 2010 Elsevier Inc. All rights reserved.)

We have shown that ischaemic preconditioning ameliorates both the local periosteal and the systemic leukocyte activation evoked by limb ischaemia-reperfusion. We hypothesized that the activation of chemosensitive afferent nerves by transient ischaemia contributes to the protective mechanisms of ischaemic preconditioning via a calcitonin gene-related peptide (CGRP)-dependent mechanism. In Sprague-Dawley rats, 60-min complete limb ischaemia was followed by 180 min of reperfusion. In further experiments, the CGRP analogue hCGRP (0.3 μg kg(-1)) or ischaemic preconditioning (2 × 10-min ischaemia/10-min reperfusion) was applied prior to the ischaemia-reperfusion insult. Ischaemic preconditioning was performed in three subgroups in which animals received the CGRP receptor antagonist CGRP(8-37) (30 μg kg(-1) h(-1)), the chemosensitive afferent nerve inactivator resiniferatoxin (3 × 15 μg kg(-1), sc), or vehicle. The effects of CGRP(8-37) and resiniferatoxin on ischaemia-reperfusion without ischaemic preconditioning were also evaluated. In the tibial periosteum of rats, intravital fluorescence microscopy and immunohistochemistry revealed significant attenuations of ischaemia-reperfusion-induced post-ischaemic leukocyte-endothelial interactions (rolling and adherence in the postcapillary venules) and tissue intracellular adhesion molecule expression following ischaemic preconditioning or hCGRP administration. Administration of CGRP(8-37) or pretreatment of animals with resiniferatoxin reversed the anti-inflammatory effects of limb ischaemic preconditioning, but failed to affect the microcirculatory consequences of ischaemia-reperfusion without ischaemic preconditioning. The results suggest that activation of the chemo- (capsaicin-) sensitive afferent nerves is involved in the mechanisms of microcirculatory anti-inflammatory protection provided by limb ischaemic preconditioning. Controlled activation of chemosensitive C-fibres or the CGRP receptors by the induction of ischaemic preconditioning or other means may furnish therapeutic benefit by ameliorating the periosteal microcirculatory consequences of tourniquet ischaemia.

Cyclooxygenase-2 (COX-2) is thought to be an inducible enzyme, but increasing reports indicate that COX-2 is constitutively expressed in several organs. The status of COX-2 expression in bone and its physiological role remains undefined. Non-selective non-steroidal anti-inflammatory drugs (NSAIDs) and selective COX-2 inhibitors, which commonly suppress COX-2 activity, were reported to suppress osteoblast proliferation via Akt/FOXO3a/p27(Kip1) signaling, suggesting that COX-2 may be the key factor of the suppressive effects of NSAIDs on proliferation. Although Akt activation correlates with PTEN deficiency and cell viability, the role of COX-2 on PTEN/Akt regulation remains unclear. In this study, we hypothesized that COX-2 may be constitutively expressed in osteoblasts and regulate PTEN/Akt-related proliferation. We examined the localization and co-expression of COX-2 and p-Akt in normal mouse femurs and in cultured mouse (mOBs) and human osteoblasts (hOBs). Our results showed that osteoblasts adjacent to the trabeculae, periosteum and endosteum in mouse femurs constitutively expressed COX-2, while COX-2 co-expressed with p-Akt in osteoblasts sitting adjacent to trabeculae in vivo, and in mOBs and hOBs in vitro. We further used COX-2 siRNA to test the role of COX-2 in Akt signaling in hOBs; COX-2 silencing significantly inhibited PTEN phosphorylation, enhanced PTEN activity, and suppressed p-Akt level and proliferation. However, replenishment of the COX-2 enzymatic product, PGE2, failed to reverse COX-2-dependent Akt phosphorylation. Furthermore, transfection with recombinant human COX-2 (rhCOX-2) significantly reversed COX-2 siRNA-suppressed PTEN phosphorylation, but this effect was reduced when the enzymatic activity of rhCOX-2 was blocked. This finding indicated that the effect of COX-2 on PTEN/Akt signaling is not related to PGE2 but still dependent on COX-2 enzymatic activity. Conversely, COX-1 silencing did not affect PTEN/Akt signaling. Our findings provide new insight into bone physiology; namely, that COX-2 is constitutively expressed in osteoblasts in the dynamic bone growth area, which facilitates osteoblast proliferation via PTEN/Akt/p27(Kip1) signaling., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The advent of high-throughput measurements of gene expression and bioinformatics analysis methods offers new ways to study gene expression patterns. The primary goal of this study was to determine the time sequence for gene expression in a bone subjected to mechanical loading during key periods of the bone-formation process, including expression of matrix-related genes, the appearance of active osteoblasts, and bone desensitization. A standard model for bone loading was employed in which the right forelimb was loaded axially for 3 minutes per day, whereas the left forearm served as a nonloaded contralateral control. We evaluated loading-induced gene expression over a time course of 4 hours to 32 days after the first loading session. Six distinct time-dependent patterns of gene expression were identified over the time course and were categorized into three primary clusters: genes upregulated early in the time course, genes upregulated during matrix formation, and genes downregulated during matrix formation. Genes then were grouped based on function and/or signaling pathways. Many gene groups known to be important in loading-induced bone formation were identified within the clusters, including AP-1-related genes in the early-response cluster, matrix-related genes in the upregulated gene clusters, and Wnt/β-catenin signaling pathway inhibitors in the downregulated gene clusters. Several novel gene groups were identified as well, including chemokine-related genes, which were upregulated early but downregulated later in the time course; solute carrier genes, which were both upregulated and downregulated; and muscle-related genes, which were primarily downregulated., (© 2011 American Society for Bone and Mineral Research.)

Background: The adaptive nature of bone formation under mechanical loading is well known; however, the molecular and cellular mechanisms in vivo of mechanical loading in bone formation are not fully understood. To investigate both mechanisms at the early response against mechanotransduction in vivo, we employed a noninvasive 3-point bone bending method for mouse tibiae. It is important to investigate periosteal woven bone formation to elucidate the adaptive nature against mechanical stress. We hypothesize that cell morphological alteration at the early stage of mechanical loading is essential for bone formation in vivo., Principal Findings: We found the significant bone formation on the bone surface subjected to change of the stress toward compression by this method. The histological analysis revealed the proliferation of periosteal cells, and we successively observed the appearance of ALP-positive osteoblasts and increase of mature BMP-2, resulting in woven bone formation in the hypertrophic area. To investigate the mechanism underlying the response to mechanical loading at the molecular level, we established an in-situ immunofluorescence imaging method to visualize molecules in these periosteal cells, and with it examined their cytoskeletal actin and nuclei and the extracellular matrix proteins produced by them. The results demonstrated that the actin cytoskeleton of the periosteal cells was disorganized, and the shapes of their nuclei were drastically changed, under the mechanical loading. Moreover, the disorganized actin cytoskeleton was reorganized after release from the load. Further, inhibition of onset of the actin remodeling blocked the proliferation of the periosteal cells., Conclusions: These results suggest that the structural change in cell shape via disorganization and remodeling of the actin cytoskeleton played an important role in the mechanical loading-dependent proliferation of cells in the periosteum during bone formation.

Periosteal growth at human mandibular ramus is characterized by bone apposition at the posterior border and resorption at the anterior border. Molecular control of this regional variation is unclear. This study examined the expression of several molecules involved in bone apposition/resorption at these regions in vivo and in vitro. By using growing pigs as a model, the periosteal growth was assessed at the mandibular ramus by vital staining and histological observations. In parallel, periosteal tissues were harvested and pulverized for RNA and protein extraction. Periosteal cells were also isolated, expanded in osteogenic media, and subjected to a single dose of dynamic tensile strain (0, 5, or 10% magnitude at 0.5 Hz) to examine their responses to mechanical loading. Real-time RT-PCR and Western blot analyses were used to examine mRNA and protein expression from periosteal tissues and cultured cells. Histological observation confirmed an anterior-resorption/posterior-apposition pattern in the pig mandibular ramus. Both in vivo tissue and in vitro cells demonstrated greater mRNA expression of receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) ratio and bone morphogenetic protein 2 (BMP2) at the anterior region, while OPG expression at the anterior region was lower than the posterior region. In response to the application of a single dose of dynamic tensile strain, cultured periosteal cells appeared to change the expression profile of osteogenic markers but not that of RANKL/OPG and BMP2. These findings suggest that the unique regional variation of periosteal activity at the mandibular ramus is regulated by a differential expression of RANKL/OPG ratio (likely through differential induction of OPG) and BMP2., (2010 Wiley-Liss, Inc.)

While the essential role of periosteum in cortical bone repair and regeneration is well established, the molecular pathways that control the early osteogenic and chondrogenic differentiation of periosteal stem/progenitor cells during repair processes are unclear. Using a murine segmental bone graft transplantation model, we isolated a population of early periosteum-callus-derived mesenchymal stem cells (PCDSCs) from the healing autograft periosteum. These cells express typical mesenchymal stem cell markers and are capable of differentiating into osteoblasts, adipocytes, and chondrocytes. Characterization of these cells demonstrated that activation of the hedgehog (Hh) pathway effectively promoted osteogenic and chondrogenic differentiation of PCDSCs in vitro and induced bone formation in vivo. To determine the role of the Hh pathway in adult bone repair, we deleted Smoothened (Smo), the receptor that transduces all Hh signals at the onset of bone autograft repair via a tamoxifen-inducible RosaCreER mouse model. We found that deletion of Smo markedly reduced osteogenic differentiation of isolated PCDSCs and further resulted in a near 50% reduction in periosteal bone callus formation at the cortical bone junction as determined by MicroCT and histomorphometric analyses. These data strongly suggest that the Hh pathway plays an important role in adult bone repair via enhancing differentiation of periosteal progenitors and that activation of the Hh pathway at the onset of healing could be beneficial for repair and regeneration.

Periosteal tissue is a source of growth factors and of osteochondral progenitor cells which makes it suitable for implantation in chondral defects as known in autologous chondrocyte implantation. The aim of this study was to determine the interaction between periosteal tissue and articular chondrocytes with respect to catabolic effectors such as matrix metalloproteinases (MMPs) and IL-6. Human articular chondrocytes were cultured for up to 28 days as micromass pellets in coculture either with physical contact to periosteal explants or allowing paracrine interactions only. Expression, secretion, and activation of MMPs and IL-6 were analyzed in chondrocytes, periosteum, and culture supernatants. Both coculture conditions influence gene expression levels of MMPs and IL-6 in a time-, culture-, and tissue-dependent manner. Coculturing of periosteum with chondrocytes promotes gene expression and secretion of IL-6. In periosteum, physical contact inhibits MMP-2 and MMP-13 gene expression while paracrine coculture induces expression of IL-6, MMP-2, -7, and -13. Pro-MMP-2, -7, and -13 were detected in supernatants of all culture regimens whereas pro-MMP-9 was secreted from periosteum only. As a balanced amount of MMP activity is likely required to achieve sufficient integration of the regenerate tissue with the surrounding healthy cartilage, an exceeding expression of proteinases might result in degradation, hypertrophy or rejection of the graft., (© 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Use of recombinant insulin-like growth factor 1 (IGF-1) as a treatment for primary IGF-1 deficiency in children has become increasingly common. When untreated, primary IGF-1 deficiency may lead to a range of metabolic disorders, including lipid abnormalities, insulin resistance, and decreased bone density. To date, results of this therapy are considered encouraging; however, our understanding of the role played by IGF-1 during development remains limited. Studies on long-term treatment with recombinant IGF-1 in both children and animals are few. Here, we used two novel transgenic mouse strains to test the long-term effects of elevated circulating IGF-1 on body size and skeletal development. Overexpression of the rat igf1 transgene in livers of mice with otherwise normal IGF-1 expression (HIT mice) resulted in approximately threefold increases in serum IGF-1 levels throughout growth, as well as greater body mass and enhanced skeletal size, architecture, and mechanical properties. When the igf1 transgene was overexpressed in livers of igf1 null mice (KO-HIT), the comparably elevated serum IGF-1 failed to overcome growth and skeletal deficiencies during neonatal and early postnatal growth. However, between 4 and 16 weeks of age, increased serum IGF-1 fully compensated for the absence of locally produced IGF-1 because body weights and lengths of KO-HIT mice became comparable with controls. Furthermore, micro-computed tomography (microCT) analysis revealed that early deficits in skeletal structure of KO-HIT mice were restored to control levels by adulthood. Our data indicate that in the absence of tissue igf1 gene expression, maintaining long-term elevations in serum IGF-1 is sufficient to establish normal body size, body composition, and both skeletal architecture and mechanical function., ((c) 2010 American Society for Bone and Mineral Research.)

Background: The aim of this study is to investigate the potential use of periosteum-derived cells (PCs) for tissue engineering in peri-implant defects., Methods: Bone marrow cells (BMCs) and PCs were harvested from seven adult beagle dogs, cultured in vitro, and phenotypically characterized with regard to their osteogenic properties. The animals were then subjected to teeth extraction, and 3 months later, two implant sites were drilled, bone dehiscences created, and dental implants placed. Dehiscences were randomly assigned to one of two groups: PCs (PCs + carrier) and BMCs (BMCs + carrier). After 3 months, the animals were sacrificed and the implants with adjacent hard tissues were processed for undecalcified sections. Bone-to-implant contact, bone fill within the limits of implant threads, and new bone area in a zone lateral to the implant were histometrically obtained., Results: In vitro, phenotypic characterization demonstrated that both cell populations presented osteogenic potential, as identified by the mineral nodule formation and the expression of bone markers. Histometrically, an intergroup analysis showed that both cell-treated defects had similar bone fill within the limits of implant threads and bone-to-implant contact (P >0.05), and although a trend toward higher new bone area values was found for the PC group, there was no significant difference between the experimental groups (P >0.05)., Conclusions: Periosteal and bone marrow cells presented a similar potential for bone reconstruction. As such, periosteum may be considered as an alternative source of osteogenic cells in implant dentistry.

We elucidated the localization of Thy-1-positive cells in the perichondrium of fetal rat limb bones to clarify the distribution of osteogenic cells in the process of endochondral ossification. We also examined the formation of calcified bone-like matrices by isolated perichondrial cells in vitro. At embryonic day (E) 15.5, when the cartilage primodia were formed, immunoreactivity for Thy-1 was detected in cells of the perichondrium adjacent to the zone of hypertrophic chondrocytes. At E17.5, when the bone collar formation and the vascular invasion were initiated, fibroblast-like cells at the sites of vascular invasion, as well as in the perichondrium, showed Thy-1 labeling. Double immunostaining for Thy-1 and osterix revealed that Thy-1 was not expressed in the osterix-positive osteoblasts. Electron microscopic analysis revealed that Thy-1-positive cells in the zone of hypertrophic chondrocytes came in contact with blood vessels. Perichondrial cells isolated from limb bones showed alkaline phosphatase activity and formed calcified bone-like matrices after 4 weeks in osteogenic medium. RT-PCR demonstrated that Thy-1 expression decreased as calcified nodules formed. Conversely, the expression of osteogenic marker genes Runx2, osterix, and osteocalcin increased. These results indicate that Thy-1 is a good marker for characterizing osteoprogenitor cells.

Objective: Periosteum is involved in bone growth and fracture healing and has been used as a cell source and tissue graft for tissue engineering and orthopedic reconstruction including joint resurfacing. Periosteum can be induced by transforming growth factor beta (TGF-beta) or insulin-like growth factor-I (IGF-I) alone or in combination to form cartilage. However, little is known about the interaction between IGF and TGF-beta signaling during periosteal chondrogenesis. The purpose of this study was to determine the effect of TGF-beta1 on IGF binding protein-4 (IGFBP-4) and the IGFBP-4 protease pregnancy-associated plasma protein-A (PAPP-A) expression in cultured periosteal explants., Design: Periosteal explants from rabbits were cultured with or without TGF-beta1. IGFBP-4 and PAPP-A mRNA levels were determined by real-time quantitative PCR. Conditioned medium was analyzed for IGFBP-4 and PAPP-A protein levels and IGFBP-4 protease activity., Results: TGF-beta1-treated explants contained lower IGFBP-4 mRNA levels throughout the culture period with a maximum reduction of 70% on day 5 of culture. Lower levels of IGFBP-4 protein were also detected in the conditioned medium from TGF-beta1-treated explants. PAPP-A mRNA levels were increased 1.6-fold, PAPP-A protein levels were increased threefold, and IGFBP-4 protease activity was increased 8.5-fold between 7 and 10days of culture (the onset of cartilage formation in this model) in conditioned medium from TGF-beta1-treated explants., Conclusions: This study demonstrates that TGF-beta1 modulates the expression of IGFBP-4 and PAPP-A in cultured periosteal explants., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

Bone repair is a complex phenomenon involving many cell types and signaling factors. Substantial evidence exists to suggest that stem cells originating from local osseous tissues, particularly the periosteum, can contribute to bone repair. However, there are situations where injury or post-surgical management can deplete the amount of, and/or access to these crucial progenitors. The fact that bone repair can still occur in these circumstances implicitly reflects the existence of compensatory secondary systems. One potential alternate source of osteoprogenitors is muscle, which is closely associated with bone and typically suffers trauma during an orthopedic insult. While muscle access is known to be beneficial to bone repair, this is conventionally credited to its high vascularity, and thus its contribution to the local blood supply. However, there is emerging evidence to suggest that progenitors from muscle may directly contribute to bone healing. Defining the role of muscle in bone formation and repair has significant clinical implications, particularly where promoting access to this tissue may enhance the repair outcome.

The periosteum and Sharpey's fibre extensions occupy the musculoskeletal interface and may be strategic in age-related deterioration. Because of its exceptionally powerful insertions the porcine mandible is an ideal model and its periosteal system was compared in 4 separate regions of adult young (1 year) and older (3 year) animals. These were examined by undecalcified histology, collagen immunohistochemistry and mineral histochemistry using polarization, epifluorescence and laser confocal microscopy; mineral ultrastructure was facilitated by chromium labelling with EDX microanalysis. Birefringent Sharpey's fibres were coarse (>8 microm) or fine and classified as horizontal (more common with age), oblique (most common in youth) or vertical (least common); in addition they were designated "superficial", "transcortical" and "intertrabecular" (the latter being deep, coarse and vertical). Their specific affinity for collagen type III FITC-labelled antibody demonstrated 3-dimensional arrays of bone-permeating fibres. With age at each region the cortical thickness rose (e.g. 4.9 mm to 9.3 mm), the periosteum thinned (e.g. 180-/+7 microm to 129-/+8 microm; p<0.001), and the periosteum: bone ratio diminished (e.g. 3.65-/+0.36 to 1.40-/+0.14; p<0.001) while Sharpey's fibres became fewer, fragmented, superficial and shortened (e.g. 226-/+27 microm to 55-/+6 microm; p<0.001). Accompanying was the sporadic encroachment of calcified particles, 1 microm diameter, in irregular periosteal aggregates or interlinked around Sharpey bundles (resembling calcifying turkey leg tendon). EDX microanalysis confirmed prominent chromium spectral peaks in the older periosteum only, coincident with chromium-labelled mineral "ghosts". It was concluded that the periosteum and Sharpey's fibres, deep-penetrating and complex in youth, partially hardens and regresses with age with implications for its functional properties.

Objective: To observe the effects of the three-period treatment theory of bone fracture in TCM (Traditional Chinese Medicine) on VEGF and VEGF mRNA expression in the issues of outer periosteum, endosteum and bone marrow of rabbits, and to explore the rationality of phasing method in TCM in treating fracture., Methods: 3 mm bone defection were made at lower one third part of both radius in 140 male healthy rabbits. The rabbits were randomly divided into four groups, including three-period treatment group (TTG), one-period treatment group(OTG), positive medicine treatment group(PTG) and model control group (MCG). The rabbits in TG were treated with three-period treatment, rabbits in OTG were treated with one-period treatment, rabbits in PTG were fed by Guzhe-Cuoshangsan (a Chinese patent medicine which was used to treat bone fracture), rabbits in model control group were given no prescription or drug but distilled water as same dose as that of other groups. At day 3, 6, 9, 14, 28, 42 and 56, five rabbits from every group were randomly selected and were killed by aeroembolism. The left radiuses were taken out as the research object. Immunohistochemistry stain and in situ hybridization stain were performed to examinate the VEGF and VEGF mRNA expression in the outer periosteum, endosteum and bone marrow., Results: The VEGF and VEGF mRNA expression of all TCM treatment groups were enhanced in the outer periosteum, endosteum and bone marrow at different time points in fracture healing. The VEGF and VEGF mRNA expression in the three tissues of TTG had the tendency of higher than that of the other groups at the most time points after operation., Conclusion: Treating fracture in stages has more predominant effect on the expression of VEGF and VEGF mRNA in the outer periosteum, endosteum and bone marrow than that of treating fracture with single prescription or drug.

Extracellular matrix (ECM) underlies a complicated multicellular architecture that is subjected to significant forces from mechanical environment. Although various components of the ECM have been enumerated, mechanisms that evolve the sophisticated ECM architecture remain to be addressed. Here we show that periostin, a matricellular protein, promotes incorporation of tenascin-C into the ECM and organizes a meshwork architecture of the ECM. We found that both periostin null mice and tenascin-C null mice exhibited a similar phenotype, confined tibial periostitis, which possibly corresponds to medial tibial stress syndrome in human sports injuries. Periostin possessed adjacent domains that bind to tenascin-C and the other ECM protein: fibronectin and type I collagen, respectively. These adjacent domains functioned as a bridge between tenascin-C and the ECM, which increased deposition of tenascin-C on the ECM. The deposition of hexabrachions of tenascin-C may stabilize bifurcations of the ECM fibrils, which is integrated into the extracellular meshwork architecture. This study suggests a role for periostin in adaptation of the ECM architecture in the mechanical environment.

Human jaw periosteum-derived cells (JPCs) represent an alternative cell source to bone marrow-derived mesenchymal stem cells for tissue engineering applications in the oral and maxillofacial surgery. In this study we investigated how far the presence or expression of human mesenchymal stem cell antigen-1/tissue non-specific alkaline phosphatase (MSCA-1/TNAP) and LNGFR (CD271) can be utilized to select and enrich the osteogenic progenitor cell fraction from the entire JPC population. Depending on their mineralization capacity, we classified the human isolated JPCs into mineralizing (mJPCs) and non-mineralizing JPCs (nmJPCs). Flow cytometric analyses revealed that undifferentiated mJPCs expressed MSCA-1/TNAP at significant higher levels than nmJPCs at day 5 and 10 of osteogenesis. Western blot analyses showed increased MSCA-1/TNAP expression levels in mJPCs during osteogenesis, whereas in nmJPCs MSCA-1/TNAP expression remained undetectable. Using the MSCA-1 and LNGFR specific antibodies, we separated the positive and negative fractions from the entire mJPC population. In order to analyse the mineralization capacity of the MSCA-1(+) and LNGFR(+) cell subsets, we quantified the calcium deposition in both subpopulations in comparison to the respective negative subpopulations. The MSCA-1(+)/TNAP(+) cell fraction showed a significant higher osteogenic capacity compared to the MSCA-1-/TNAP- cell fraction whereas the LNGFR(+/-) cell fractions did not differ in their osteogenic potential. Our findings suggest that MSCA-1 may represent a promising osteogenic marker for mJPC., (Copyright © 2010 S. Karger AG, Basel.)

In female mice, estrogen receptor-alpha (ERalpha) mediates the anabolic response of bone to mechanical loading. Whether ERalpha plays a similar role in the male skeleton and to what extent androgens and androgen receptor (AR) affect this response in males remain unaddressed. Therefore, we studied the adaptive response of in vivo ulna loading in AR-ERalpha knockout (KO) mice and corresponding male and female single KO and wild-type (WT) littermates using dynamic histomorphometry and immunohistochemistry. Additionally, cultured bone cells from WT and AR KO mice were subjected to mechanical loading by pulsating fluid flow in the presence or absence of testosterone. In contrast with female mice, ERalpha inactivation in male mice had no effect on the response to loading. Interestingly, loading induced significantly more periosteal bone formation in AR KO (+320%) and AR-ERalpha KO mice (+256%) compared with male WT mice (+114%) and had a stronger inhibitory effect on SOST/sclerostin expression in AR KO versus WT mice. In accordance, the fluid flow-induced nitric oxide production was higher in the absence of testosterone in bone cells from WT but not AR KO mice. In conclusion, AR but not ERalpha activation limits the osteogenic response to loading in male mice possibly via an effect on WNT signaling., (2010 American Society for Bone and Mineral Research)

Periostin (gene Postn) is a secreted extracellular matrix protein involved in cell recruitment and adhesion and plays an important role in odontogenesis. In bone, periostin is preferentially expressed in the periosteum, but its functional significance remains unclear. We investigated Postn(-/-) mice and their wild type littermates to elucidate the role of periostin in the skeletal response to moderate physical activity and direct axial compression of the tibia. Furthermore, we administered a sclerostin-blocking antibody to these mice in order to demonstrate the influence of sustained Sost expression in their altered bone phenotypes. Cancellous and cortical bone microarchitecture as well as bending strength were altered in Postn(-/-) compared with Postn(+/+) mice. Exercise and axial compression both significantly increased bone mineral density and trabecular and cortical microarchitecture as well as biomechanical properties of the long bones in Postn(+/+) mice by increasing the bone formation activity, particularly at the periosteum. These changes correlated with an increase of periostin expression and a consecutive decrease of Sost in the stimulated bones. In contrast, mechanical stimuli had no effect on the skeletal properties of Postn(-/-) mice, where base-line expression of Sost levels were higher than Postn(+/+) and remained unchanged following axial compression. In turn, the concomitant injection of sclerostin-blocking antibody rescued the bone biomechanical response in Postn(-/-) mice. Taken together, these results indicate that the matricellular periostin protein is required for Sost inhibition and thereby plays an important role in the determination of bone mass and microstructural in response to loading.

Although skeletal pain can have a marked impact on a patient's functional status and quality of life, relatively little is known about the specific populations of peripheral nerve fibers that drive non-malignant bone pain. In the present report, neonatal male Sprague-Dawley rats were treated with capsaicin or vehicle and femoral fracture was produced when the animals were young adults (15-16 weeks old). Capsaicin treatment, but not vehicle, resulted in a significant (>70%) depletion in the density of calcitonin-gene related peptide positive (CGRP(+)) sensory nerve fibers, but not 200 kDa neurofilament H positive (NF200(+)) sensory nerve fibers in the periosteum. The periosteum is a thin, cellular and fibrous tissue that tightly adheres to the outer surface of all but the articulated surface of bone and appears to play a pivotal role in driving fracture pain. In animals treated with capsaicin, but not vehicle, there was a 50% reduction in the severity, but no change in the time course, of fracture-induced skeletal pain-related behaviors as measured by spontaneous flinching, guarding and weight bearing. These results suggest that both capsaicin-sensitive (primarily CGRP(+) C-fibers) and capsaicin-insensitive (primarily NF200(+) A-delta fibers) sensory nerve fibers participate in driving skeletal fracture pain. Skeletal pain can be a significant impediment to functional recovery following trauma-induced fracture, osteoporosis-induced fracture and orthopedic surgery procedures such as knee and hip replacement. Understanding the specific populations of sensory nerve fibers that need to be targeted to inhibit the generation and maintenance of skeletal pain may allow the development of more specific mechanism-based therapies that can effectively attenuate acute and chronic skeletal pain.

Background: Estrogen deficiency contributes to postmenopausal osteoporosis. Periosteum might be a potential target of estrogen, but the underlying mechanism at gene level is far from being elucidated. The objective of this study was to investigate the correlation between estrogen and fatty acid synthase (FAS) expression in periosteum., Methods: Human periosteum cells were cultured in vitro. Expressed genes in the substrated cDNA library were verified using semi-quantitative PCR and real-time PCR. The expression of FAS in periosteum of ovarectomized (OVX) SD rats was investigated., Results: FAS gene was most significantly expressed in the subtracted cDNA library of periosteal cells screened by semi-quantitative PCR. Low FAS expression was verified by real-time PCR in the estrogen exposed human periosteum rather than in the control. The estradiol levels were (20.81 +/- 12.62) pg/ml, (19.64 +/- 4.35) pg/ml and (13.47 +/- 1.84) pg/ml in the sham group, the control, and the OVX group, respectively. The estradiol levels in the OVX group was significantly lower (P = 0.0386). The FAS gene expression in periosteum in the OVX group, sham group, and control group was 3.09 +/- 1.97, 1.33 +/- 0.47 and 1.51 +/- 1.32, respectively. The gene expression in the OVX group was significantly higher (P = 0.0372)., Conclusion: Estrogen modulates FAS gene expression in in vitro human perisoteum as well as in in vivo rat periosteum.

Background: Although hyperphosphatemia is one of the few established risk factors for cardiovascular mortality in patients on dialysis, the relationship between peritoneal dialysis (PD) prescription and dialytic phosphate removal is largely unexplored., Methods and Patients: We analyzed 24-hour clearances (n = 60) together with peritoneal equilibration tests (PETs) (n = 52) performed in children and adolescents (n = 35) on automated PD., Results: Dialytic phosphate clearance was more closely correlated with 2-hour and 4-hour dialysate-to-plasma ratio (D/P) of phosphate in the PETs (r = 0.44 and r = 0.52, both p < 0.0001) than with 2-hour and 4-hour D/P creatinine (r = 0.26 and r = 0.27, both p < 0.05). Dialytic 24-hour phosphate clearance was independently predicted by total fluid turnover (partial R(2) = 0.48, p < 0.001), the number of cycles (r = 0.52, p < 0.001), 2-hour D/P phosphate (partial R(2) = 0.07, p = 0.001), dwell time (partial R(2) = 0.05, p = 0.01), and achieved ultrafiltration (partial R(2) = 0.05, p = 0.005). 4-hour D/P phosphate and 24-hour phosphate clearance were significantly lower in hyperphosphatemic children (3.38 +/- 1.17 vs 4.56 +/- 1.99 L/1.73 m(2)/day, p < 0.05), whereas creatinine equilibration and clearance rates were not distinctive., Conclusion: Dialytic phosphate removal is an important modifiable determinant of phosphate control in automated PD. It strongly depends on total dialysate turnover and the prescribed number of cycles and is more adequately predicted by phosphate than by creatinine equilibration characteristics. Due to the deleterious effects of hyperphosphatemia, dialytic phosphate removal should be monitored routinely.

Utilizing a suitable combination of lactide and glycolide in a copolymer would optimize the degradation rate of a scaffold upon implantation in situ. Moreover, 3D printing technology enables customizing the shape of the scaffold to biometric data from CT and MRI scans. A previous in vitro study has shown that novel 3D-printed poly-lactic-co-glycolic acid (PLGA) scaffolds had good biocompatibility and mechanical properties comparable with human cancellous bone, while they could support proliferation and osteogenic differentiation of osteoblasts. Based on the previous study, this study evaluated PLGA scaffolds for bone regeneration within a rabbit model. The scaffolds were implanted at two sites on the same animal, within the periosteum and within bi-cortical bone defects on the iliac crest. Subsequently, the efficacy of bone regeneration within the implanted scaffolds was evaluated at 4, 12 and 24 weeks post-surgery through histological analysis. In both the intra-periosteum and iliac bone defect models, the implanted scaffolds facilitated new bone tissue formation and maturation over the time course of 24 weeks, even though there was initially observed to be little tissue ingrowth within the scaffolds at 4 weeks post-surgery. Hence, the 3D-printed porous PLGA scaffolds investigated in this study displayed good biocompatibility and are osteoconductive in both the intra-periosteum and iliac bone defect models.

Background: In some pediatric ankle and wrist injuries, the clinical picture is suggestive of a fracture despite negative standard radiographs., Objective: The goal of this prospective study was to determine the effectiveness of high-resolution ultrasound (US) in differentiating radiographically occult fractures from sprains., Methods: During a period of 3 years children aged 2 to 16 years, who sustained an acute ankle and wrist injury suggestive of being a fracture on clinical examination, but with negative radiograph, were referred for high-resolution US. Follow-up radiographs were obtained at 2 to 3 weeks., Results: Fifty-eight children (41 children with ankle injury and 17 children with wrist injury) were examined. In 41 patients, US did not reveal fractures, and in 15, small fractures were detected. All patients with negative US studies had negative follow-up x-rays. In 13 patients with positive US, the follow-up radiographs demonstrated a periosteal reaction. In 2 children (1 in each group) in whom a fracture line identified by US was in the depth of the metaphyseal bone, the follow-up radiogram demonstrated an area of increased bone density. In 2 children, 1 in each group, US diagnosed fractures that were not confirmed by follow-up radiograms., Conclusion: Ultrasound is effective in the detecting radiographically silent fractures of the pediatric ankle and wrist. Ultrasound may be used as an adjunct to radiography in clinically suspicious but radiographically negative ankle and wrist injuries., Level of Evidence: 1.

Periostin-like-factor (PLF), an isoform related to Periostin, is expressed in bone, heart, and vascular smooth muscle cells. PLF was detected by immunostaining in mesenchymal cells in the periosteum and in osteoblasts lining trabecular bone, suggesting that PLF has a role in osteogenesis. PLF has a signal peptide and is also secreted from osteoblasts in vitro. To study the function of PLF in osteogenesis, we assessed the effect of PLF on osteoblast proliferation and differentiation in vitro and bone formation in vivo. First, to examine whether PLF regulates osteoblast proliferation in vitro, the CyQUANT cell proliferation assay was performed. PLF over-expression by adenovirus resulted in a significantly higher rate of cell proliferation compared to controls. This finding suggests that PLF promotes osteoblast proliferation in vitro. Second, to test whether PLF mediates osteoblast differentiation in vitro, differentiation markers of osteoblasts, were assessed, including alkaline phosphatase staining and activity, von Kossa staining and calcium deposition. Over-expression of PLF resulted in higher expression and activity of alkaline phosphatase and higher amounts of mineralization and calcium deposition compared to controls. These data suggest that PLF promotes osteoblast differentiation in vitro. Third, to investigate the role of PLF in bone formation in vivo, PLF adenovirus was injected into 6-week-old rat femur bone marrow. Over-expression of PLF resulted in increased bone formation within the marrow cavity. Lastly, in a model of fracture healing, PLF expression is robustly upregulated in callus osteoblasts at post-fracture days 7 and 14. Taken together, these findings suggests that PLF induces bone formation in vivo. We conclude that PLF stimulates bone formation in vivo possibly by promoting osteoblast proliferation and differentiation.

Work-related musculoskeletal disorders (WMSDs), also known as overuse injuries, account for a substantial proportion of work injuries and workers' compensation claims in the United States. However, the pathophysiological mechanisms underlying WMSDs are not well understood, especially the early events in their development. In this study we used an animal model of upper extremity WMSD, in which rats perform a voluntary repetitive reaching and pulling task for a food reward. This innovative model provides us an opportunity to investigate the role of molecules which may be used either as markers of early diagnosis of these disorders, and/or could be targeted for therapeutic purposes in the future. Periostin-like-factor (PLF), and Periostin were examined in this study. Both belong to a family of vitamin K-dependent gamma carboxylated proteins characterized by the presence of conserved Fasciclin domains and not detected in adult tissues except under conditions of chronic overload, injury, stress or pathology. The spatial and temporal pattern of PLF and Periostin localization was examined by immunohistochemistry and western blot analysis in the radius and ulna of animals performing a high repetition, high force task for up to 12 weeks and in controls. We found that PLF was present primarily in the cellular periosteum, articular cartilage, osteoblasts, osteocytes and osteoclasts at weeks 3 and 6 in all distal bone sites examined. This increase coincided with a transient increase in serum osteocalcin in week 6, indicative of adaptive bone formation at this time point. PLF immunoexpression decreased in the distal periosteum and metaphysis by week 12, coincided temporally with an increase in serum Trap5b, thinning of the growth plate and reduced cortical thickness. In contrast to PLF, once Periostin was induced by task performance, it continued to be present at a uniformly high level between 3 and 12 weeks in the trabeculae, fibrous and cellular periosteum, osteoblasts and osteocytes. In general, the data suggest that PLF is located in tissues during the early adaptive stage of remodeling but not during the pathological phase and therefore might be a marker of early adaptive remodeling.

Our recent clinical studies have demonstrated that autologous implantation of human cultured periosteal (hCP) sheets in combination with porous hydroxylapatite (HAp) particles at the site of periodontal bone defects strikingly facilitates tissue regeneration. To better understand how the hCP sheet functions at the implantation site, we have now examined its biochemical and morphological characteristics in vitro and its ectopic osteoinductivity in nude mice. Cultured human periosteal tissue segments produced periosteal cells that migrated out from the central region within 4-8 days and grew more rapidly with longer culture times. Alkaline phosphatase activity increased in parallel with actual osteoblastic induction. Cytokine array assays demonstrated that osteoblastic induction downregulated IL-6 and thrombopoietin, but upregulated IL-8, IL-13, IGF-I and IGFBP-2 in hCP sheets. When differentiated hCP sheets were implanted alone, areas of osteoid and mineralized tissue were formed within 2 weeks, but non-induced, immature hCP sheets did not produce much mineralization. These findings suggest that mature hCP sheets potentially function not only as seeds of ectopic bone formation without the need of synthetic tissue scaffolds, but also as living drug-delivery systems, to influence cells near implantation sites by producing several important cytokines. These two major characteristics indicate that a mature hCP sheet is a promising osteoinductive biomaterial, even without conventional scaffolds for periodontal regenerative therapy.

An anabolic response driven by osteoblasts is critical for the process of bone healing. Current evidence suggests that these osteoblasts may arise from multiple tissue types and cell lineages. Stem cells present in the bone marrow, periosteum, local soft tissues, vasculature, and/or circulation have been shown to have osteogenic potential. Transplanted cells from these sources have also been shown to incorporate into induced ectopic bone or repaired bone. While these experiments demonstrate the latent capacity of different lineages to assume an osteoblastic phenotype under pro-osteogenic conditions, the actual contribution of the different lineages to various repair situations in vivo remains unclear. This review explores the data arising from different bone formation and repair models. We propose a model suggesting that cells arising from the local tissues, particularly muscle cells, may play an important role in fracture repair under situations where the periosteal and/or bone marrow progenitor populations are depleted.

Isolated jaw periosteum-derived cells (JPCs) comprise a morphologically heterogeneous population. There are no known specific surface markers that are able to distinguish between progenitors and cells of other tissue types. The aim of our study was to identify differentiation markers as predictors of JPC mineralization capacity. JPCs underwent osteogenic differentiation after cultivation in osteogenic medium containing known activators. By FACS analysis, we found the low affinity nerve growth factor receptor (LNGFR-CD271) to be induced during the first five days of osteogenesis and that it was expressed at higher levels in mineralizing JPCs (mJPCs) in comparison to non-mineralizing JPCs (nmJPCs). Similar results were obtained by semi-quantitative immunohistochemical stainings and western blot analyses. Quantitative real-time PCR results showed significantly higher LNGFR and alkaline phosphatase transcript levels in mJPCs compared to nmJPCs. LNGFR is a differentiation marker that distinguishes between mineralizing JPCs and non-mineralizing JPCs during the first phase of osteogenesis and can therefore be considered an early surface marker of osteogenic capacity in vitro., (Copyright (c) 2009 S. Karger AG, Basel.)

Our previous studies have shown that on human periosteal cells, low-intensity pulsed ultrasound (LIPUS) has an immediate stimulatory effect whereas extracorporeal shockwaves (ESW) have an delayed stimulatory effect. Therefore, we hypothesized that a combined ESW and LIPUS treatment might provide additive or synergistic effects on periosteal cells, by using ESW to trigger a biological activity while using LIPUS to maintain the stimulated activity. Human periosteal cells were subjected to a single session of ESW treatment on day 0 and/or daily LIPUS treatments or no treatment (control). The cell viability, proliferation, and alkaline phosphatase activity on day 6 and day 18 as well as matrix mineralization on day 35 were measured. Results revealed that LIPUS alone had early positive effects on the activities on day 6 only. In contrast, ESW alone had an early destructive effect but exerted delayed stimulatory effects on the cellular activities on day 18. The combined treatment of ESW plus LIPUS produced effects that were comparable to the ESW treatment alone. Although these findings suggest that ESW and LIPUS stimulate the periosteal cells in two different ways and at different times, their additive or synergistic effects could not be proven.

Background and Objective: The types of collagens available today as biomaterials are purified from animal tissues. A major growing concern, however, is their safety, since there are risks of viral and prion contamination and of unknown and potentially zoonotic infectious diseases. The present study aimed to assess, using immunohistochemistry, the effects of recombinant human growth/differentiation factor-5 (rhGDF-5) combined with recombinant human collagen I (rhCI) on bone formation in murine calvariae., Material and Methods: Composite rhGDF-5-rhCI or rhCI alone was injected subcutaneously into murine calvariae. After 3, 7 or 14 days, tissues were examined radiologically, histologically and immunohistochemically. The production of vascular endothelial growth factor (VEGF) by primary osteoblasts, periosteal cells and connective tissue fibroblasts isolated enzymatically from neonatal murine calvariae was also assessed., Results: A protrusion was observed on the calvariae at the site injected with rhGDF-5/rhCI composite. Its mineral density was shown to be different from that of the existing bone by two-dimensional microcomputed tomography. Type II collagen-positive staining was restricted to newly formed tissues. Thus, the newly formed tissues seemed to be bone- and cartilage-like tissues. A number of vessels with positively stained cells for Von Willebrand factor were detected in the newly formed tissues. The rhGDF-5 enhanced VEGF production in cultured connective tissue fibroblasts. Sry-related HMG box 9 (Sox9)-positive cells were detected in the hypertrophic periosteum, and penetrated into the newly formed tissues., Conclusions: These results suggest that rhCI seems to allow the release of rhGDF-5 and that rhGDF-5-rhCI composite induces endochondral ossification via Sox9 expression and angiogenesis in murine calvariae.

The aim of this study was to establish the potential of human periosteum-derived cells from elderly patients as a cell source for cartilage tissue engineering by optimizing culture conditions for both proliferation and differentiation. Periosteum was obtained from the tibiae of nine patients. Biopsies were prepared for routine histological examination. Periosteum-derived cells were allowed to grow out from the remaining tissue, and were expanded in minimum essential medium containing D-valine (MEM-DV). Fetal bovine serum (FBS) or substitutes, fibroblast growth factor-2 (FGF-2), insulin-like growth factor-1 (IGF-1) and non-essential amino acids were added to study proliferation. For differentiation of cells, serum-free medium was used supplemented with one or more isoforms of transforming growth factor-beta (TGFbeta) and/or IGF-1. Samples were analysed for expression of collagens type I, II and X by competitive RT-PCR, immunohistochemically, and histologically using Alcian blue staining. In all samples the cambium layer could hardly be detected. Periosteum-derived cells proliferated in serum-containing MEM-DV. Optimal proliferation was found when this medium was supplemented with 100 ng/ml FGF-2 and non-essential amino acids. Chondrogenesis was detected in 59% of micromasses that were cultured with TGFbeta isomers, and in 83% of the samples cultured in media to which two TGFbeta isoforms were added. Periosteum from elderly humans (mean age 66, range 41-76 years) has chondrogenic potential and remains an attractive cell source for cartilage tissue engineering. By expanding cells in MEM-DV, the selection of progenitor cells might be favoured, which would result in a higher cartilage yield for tissue engineering applications., ((c) 2008 John Wiley & Sons, Ltd.)

Biomaterial, an essential component of tissue engineering, serves as a scaffold for cell attachment, proliferation, and differentiation; provides the three dimensional (3D) structure and, in some applications, the mechanical strength required for the engineered tissue. Both synthetic and naturally occurring calcium phosphate based biomaterial have been used as bone fillers or bone extenders in orthopedic and reconstructive surgeries. This study aims to evaluate two popular calcium phosphate based biomaterial i.e., hydroxyapatite (HA) and tricalcium phosphate/hydroxyapatite (TCP/HA) granules as scaffold materials in bone tissue engineering. In our strategy for constructing tissue engineered bone, human osteoprogenitor cells derived from periosteum were incorporated with human plasma-derived fibrin and seeded onto HA or TCP/HA forming 3D tissue constructs and further maintained in osteogenic medium for 4 weeks to induce osteogenic differentiation. Constructs were subsequently implanted intramuscularly in nude mice for 8 weeks after which mice were euthanized and constructs harvested for evaluation. The differential cell response to the biomaterial (HA or TCP/HA) adopted as scaffold was illustrated by the histology of undecalcified constructs and evaluation using SEM and TEM. Both HA and TCP/HA constructs showed evidence of cell proliferation, calcium deposition, and collagen bundle formation albeit lesser in the former. Our findings demonstrated that TCP/HA is superior between the two in early bone formation and hence is the scaffold material of choice in bone tissue engineering., (Copyright 2007 Wiley Periodicals, Inc.)

The purpose of this study was to examine the expression of vascular endothelial growth factor (VEGF) during osteoblastic differentiation of cultured human periosteal-derived cells. Periosteal tissues were obtained from mandible during surgical extraction of lower impacted third molars. Periosteal-derived cells were introduced into cell culture. After passage 3, the periosteal-derived cells were further cultured for 42 days in an osteogenic-inductive culture medium containing dexamethasone, ascorbic acid, and beta-glycerophosphate. The alkaline phosphatase activity in the cultured periosteal-derived cells increased rapidly up to day 14, followed by decrease in activity. The Runx2 protein was expressed at day 7 and day 14, and its expression was not observed thereafter. Both VEGF(165) and VEGF(121) were expressed strongly at days 35 and 42 of culture, particularly during the later stages of differentiation. Alizarin red S-positive nodules were first observed on day 14 and then increased in number during the entire culture period. Osteocalcin and VEGF were first detected in the culture medium on day 14, and their levels increased thereafter in a time-dependent manner. These results suggest that VEGF secretion from cultured human periosteal-derived cells increases along with the mineralization process of the extracellular matrix.

Glucocorticoid (GC) excess causes a great increase in fracture risk, but the effects of GC excess on cortical bone geometry are unknown. The present study was performed to examine the effects of GC excess on cortical bone geometry in both premenopausal and postmenopausal women. Ninety-six women receiving oral GC treatments and 10 women with Cushing syndrome (CS) were each compared to age-matched control subjects using peripheral quantitative computed tomography. Total area, periosteal circumference, and polar strength strain index (SSIp) were significantly lower in GC-treated patients compared with control subjects in premenopausal women but not in postmenopausal women. Moreover, cortical area and thickness as well as periosteal circumference and SSIp were significantly lower in patients with CS compared to controls in premenopausal women but not in postmenopausal women. Total area, cortical area, cortical thickness, periosteal circumference, as well as SSIp were significantly lower in GC-treated patients with vertebral fractures compared to those without vertebral fractures in premenopausal women but not in postmenopausal women. In conclusion, endogenous or exogenous GC excess affects bone geometry of forearms of premenopausal, but not postmenopausal, women. These effects of GC excess on bone geometry may provide a strength loss mechanism beneath increased vertebral fracture risk.