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Tumor-associated neutrophil (TAN) effects on glioblastoma (GBM) biology remain under-characterized. We show here that neutrophils with dendritic features-including morphological complexity, expression of antigen presentation genes, and the ability to process exogenous peptide and stimulate major histocompatibility complex (MHC)II-dependent T cell activation-accumulate intratumorally and suppress tumor growth in vivo. Trajectory analysis of patient TAN scRNA-seq identifies this "hybrid" dendritic-neutrophil phenotype as a polarization state that is distinct from canonical cytotoxic TANs, and which differentiates from local precursors. These hybrid-inducible immature neutrophils-which we identified in patient and murine glioblastomas-arise not from circulation, but from local skull marrow. Through labeled skull flap transplantation and targeted ablation, we characterize calvarial marrow as a contributor of antitumoral myeloid antigen-presenting cells (APCs), including TANs, which elicit T cell cytotoxicity and memory. As such, agents augmenting neutrophil egress from skull marrow-such as intracalvarial AMD3100, whose survival-prolonging effect in GBM we report-present therapeutic potential., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Tannerella forsythia is a major periodontal pathogen, and T. forsythia GroEL is a molecular chaperone homologous to human heat-shock protein 60. Interleukin-17 (IL-17) has been implicated in the pathogenesis of periodontitis and several systemic diseases. This study investigated the potential of T. forsythia GroEL to induce inflammatory bone resorption and examined the cooperative effect of IL-17 and T. forsythia GroEL on inflammatory responses. Human gingival fibroblasts (HGFs) and periodontal ligament (PDL) fibroblasts were stimulated with T. forsythia GroEL and/or IL-17. Gene expression of IL-6, IL-8, and cyclooxygenase-2 (COX-2) and concentrations of IL-6, IL-8, and prostaglandin E 2 (PGE 2 ) were measured by real-time reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assays, respectively. After stimulation of MG63 cells with T. forsythia GroEL and/or IL-17, gene expression of osteoprotegerin (OPG) was examined. After subcutaneous injection of T. forsythia GroEL and/or IL-17 above the calvaria of BALB/c mice, calvarial bone resorption was assessed by micro-computed tomography and histological examination. Tannerella forsythia GroEL induced IL-6 and IL-8 production in HGFs and PDL cells, and IL-17 further promoted IL-6 and IL-8 production. Both T. forsythia GroEL and IL-17 synergistically increased PGE 2 production and inhibited OPG gene expression. Calvarial bone resorption was induced by T. forsythia GroEL injection, and simultaneous injection of T. forsythia GroEL and IL-17 further increased bone resorption. These results suggest that T. forsythia GroEL is a novel virulence factor that can contribute to inflammatory bone resorption caused by T. forsythia and synergizes with IL-17 to exacerbate inflammation and bone resorption., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The immunomodulatory functions of monocytes are increasingly being recognized. Silicified collagen scaffolds (SCSs), produced by infiltrating collagen matrices with intrafibrillar amorphous silica, exhibit osteogenic and angiogenic potential and are promising candidates in tissue engineering. Here, we demonstrate that SCS promotes in situ bone regeneration and angiogenesis via monocyte immunomodulation. Increased numbers of TRAP-positive monocytes, nestin-positive bone marrow stromal cells (BMSCs) and CD31-positive and endomucin-positive new vessels can be identified from new bone formation regions in a murine calvarial defect model. In addition, sustained release of silicic acid by SCS stimulates differentiation of blood-derived monocytes into TRAP-positive cells, with increased expressions of SDF-1α, TGF-β1, VEGFa and PDGF-BB. These cytokines further promote homing of BMSCs and endothelial progenitor cells as well as neovascularization. Taken together, these novel findings indicate that SCSs possess the ability to enhance recruitment of progenitor cells and promote osteogenesis and angiogenesis by immunomodulation of monocytes., (Published by Elsevier Ltd.)

Langerhans cell histiocytosis (LCH) is a proliferation of Langerhans cells intermixed with inflammatory cells, in particular eosinophils, that may manifest as a unisystem (unifocal or multifocal) or multisystem disease. We describe the clinical and histologic spectrum of LCH of the orbit and skull in our two cases. Both cases had unifocal erosive skull lesions with a history of trauma. Typical histologic features included numerous histiocytes with varying degrees of giant cell formation and scattered eosinophilic granulocytes. The presence of Langerhans cells was confirmed by CD1a and S100 immunohistochemistry. LCH has an excellent prognosis when treated with surgical resection, steroids and radiotherapy or chemotherapy. One of our patients is disease free at 7 year follow-up and one patient had regression of lesion on follow-up.

Wear particles induce periprosthetic inflammation and osteolysis through activation of nuclear factor kappa B (NF-κB), which up-regulates the downstream target gene expression for proinflammatory cytokines in macrophages. It was hypothesized that direct suppression of NF-κB activity in the early phases of this disorder could be a therapeutic strategy for preventing the inflammatory response to wear particles, potentially mitigating osteolysis. NF-κB activity can be suppressed via competitive binding with double stranded NF-κB decoy oligodeoxynucleotides (ODNs) that blocks this transcription factor from binding to the promoter regions of targeted genes. In this murine calvarial study, clinically relevant polyethylene particles (PEs) with/without ODN were subcutaneously injected over the calvarial bone. In the presence of PE particles, macrophages migrated to the inflammatory site and induced tumor necrosis factor alpha (TNF-α) and receptor activator of nuclear factor kappa B ligand (RANKL) expression, resulting in an increase in the number of osteoclasts. Local injections of ODN mitigated the expression of TNF-α, RANKL, and induced the expression of two anti-inflammatory, antiresorptive cytokines: interleukin-1 receptor antagonist and osteoprotegerin. Local intervention with NF-κB decoy ODN in early cases of particle-induced inflammation in which the prosthesis is still salvageable may potentially preserve periprosthetic bone stock., (© 2015 Wiley Periodicals, Inc.)

Recently, we have shown that anti-BMP2 monoclonal antibodies (mAbs) can trap endogenous osteogenic BMP ligands, which can in turn mediate osteodifferentiation of progenitor cells. The effectiveness of this strategy requires the availability of the anti-BMP-2 monoclonal antibodies antigen-binding sites for anti-BMP-2 monoclonal antibodies to bind to the scaffold through a domain that will leave its antigen-binding region exposed and available for binding to an osteogenic ligand. We examined whether antibodies bound to a scaffold by passive adsorption versus through Protein G as a linker will exhibit differences in mediating bone formation. In vitro anti-BMP-2 monoclonal antibodies was immobilized on absorbable collagen sponge (ACS) with Protein G as a linker to bind the antibody through its Fc region and implanted into rat calvarial defects. The biomechanical strength of bone regenerated by absorbable collagen sponge/Protein G/anti-BMP-2 monoclonal antibodies immune complex was compared to ACS/anti-BMP-2 monoclonal antibodies or ACS/Protein G/isotype mAb control group. Results demonstrated higher binding of anti-BMP-2 monoclonal antibodies/BMPs to C2C12 cells, when the mAb was initially attached to recombinant Protein G or Protein G-coupled microbeads. After eight weeks, micro-CT and histomorphometric analyses revealed increased bone formation within defects implanted with absorbable collagen sponge/Protein G/anti-BMP-2 monoclonal antibodies compared with defects implanted with absorbable collagen sponge/anti-BMP-2 monoclonal antibodies (p < 0.05). Confocal laser scanning microscopy (CLSM) confirmed increased BMP-2, -4, and -7 detection in sites implanted with absorbable collagen sponge/Protein G/anti-BMP-2 monoclonal antibodies in vivo. Biomechanical analysis revealed the regenerated bone in sites with Protein G/anti-BMP-2 monoclonal antibodies had higher mechanical strength in comparison to anti-BMP-2 monoclonal antibodies. The negative control group, Protein G/isotype mAb, did not promote bone regeneration and exhibited significantly lower mechanical properties (p < 0.05). Altogether, our results demonstrated that application of Protein G as a linker to adsorb anti-BMP-2 monoclonal antibodies onto the scaffold was accompanied by increased in vitro binding of the anti-BMP-2 mAb/BMP immune complex to BMP-receptor positive cell, as well as increased volume and strength of de novo bone formation in vivo., (© The Author(s) 2015.)

Aseptic loosening secondary to particle-associated periprosthetic osteolysis remains a major cause of failure of total joint replacements (TJR) in the mid- and long term. As sentinels of the innate immune system, macrophages are central to the recognition and initiation of the inflammatory cascade, which results in the activation of bone resorbing osteoclasts. Toll-like receptors (TLRs) are involved in the recognition of pathogen-associated molecular patterns and danger-associated molecular patterns. Experimentally, polymethylmethacrylate and polyethylene (PE) particles have been shown to activate macrophages via the TLR pathway. The specific TLRs involved in PE particle-induced osteolysis remain largely unknown. We hypothesized that TLR-2, -4, and -9 mediated responses play a critical role in the development of PE wear particle-induced osteolysis in the murine calvarium model. To test this hypothesis, we first demonstrated that PE particles caused observable osteolysis, visible by microCT and bone histomorphometry when the particles were applied to the calvarium of C57BL/6 mice. The number of TRAP positive osteoclasts was significantly greater in the PE-treated group when compared to the control group without particles. Finally, using immunohistochemistry, TLR-2 and TLR-4 were highly expressed in PE particle-induced osteolytic lesions, whereas TLR-9 was downregulated. TLR-2 and -4 may represent novel therapeutic targets for prevention of wear particle-induced osteolysis and accompanying TJR failure., (© 2013 Wiley Periodicals, Inc.)

Background and Objective: The interleukin (IL)-1 receptor antagonist (Ra) binds to IL-1 receptors and inhibits IL-1 activity. However, it is unclear whether the IL-1Ra plays a protective role in periodontal disease. The purpose of this study was to compare IL-1Ra knockout (KO) and wild-type (WT) mice in regard to proinflammatory cytokine production, osteoclast formation and bone resorption in response to periodontal bacterial lipopolysaccharide (LPS)., Material and Methods: Peritoneal macrophages (Mφs) were obtained from 13-wk-old IL-1Ra KO and WT mice. Peritoneal Mφs were cultured with or without 10 μg/mL of Aggregatibacter actinomycetemcomitans LPS for 24 h. The levels of IL-1alpha (IL-1α), IL-1beta (IL-1β), tumor necrosis factor-α (TNF-α) and IL-6 were measured in periotoneal Mφs supernatant fluid (PM-SF) using an ELISA. Bone marrow cells were obtained from the mice and stimulated with PM-SF for 9 d, then stained with TRAP. The frequency of TRAP-positive multinucleated giant cell formation was calculated based on a fusion index. PM-SF-stimulated calvarial bone resorption was analyzed using micro-computed tomography, and calvarial histological analysis was performed using hematoxylin and eosin and TRAP staining. The expression of cyclooxygenase-2 (Cox2), prostanoid receptor EP4 (Ep4) and Rank mRNAs in bone marrow cells were measured using real-time quantitative PCR, while prostaglandin E2 (PGE2 ) production was determined by ELISA., Results: The levels of IL-1α, IL-1β, TNF-α and IL-6 in IL-1Ra KO mice PM-SF stimulated with A. actinomycetemcomitans LPS were significantly increased by approximately 4- (p < 0.05), 5- (p < 0.05), 1.3- (p < 0.05) and 6- (p < 0.05) fold, respectively, compared with the levels in WT mice. Moreover, osteoclast formation, expression of Rank, Ep4 and Cox2 mRNAs and production of PGE2 were significantly increased by approximately 2- (p < 0.05), 1.6- (p < 0.05), 2.5- (p < 0.05), 1.6- (p < 0.05) and 1.9- (p < 0.05) fold, respectively, in IL-1Ra KO mice stimulated with A. actinomycetemcomitans LPS compared with WT mice., Conclusion: IL-1Ra regulates IL-1 activity and appears to reduce the levels of other inflammatory cytokines, including TNF-α and IL-6, while it also reduces expression of the EP4 receptor related to prostanoid sensitivity and osteoclast formation. These results suggest that IL-1Ra is an important molecule for inhibition of inflammatory periodontal bone resorption., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Activation of myeloid cells by orthopedic particulate debris is a key event in the pathogenesis of periprosthetic osteolysis and implant loosening after total joint replacement (TJR). Several lines of evidence implicate NACHT, LRR, and PYD domains-containing protein 3 (NALP3) inflammasome-mediated production of interleukin 1 beta (IL-1β) in the pathogenesis of clinical disorders ascribable to foreign particulate materials, including asbestos, silica, and urate crystals. Recent reports indicate that orthopedic polymer products and metallic particulates and ions may activate the same pathway. Here, we investigated the contribution of the NALP3 inflammasome to the pathogenesis of peri-implant osteolysis. Pharmaceutical and genetic perturbations of caspase-1 and inflammasome components were used to assess the role of the NALP3 inflammasome in IL-1β production and osteoclast formation by human monocytes and mouse macrophages in response to polymethylmethacrylate (PMMA) particle phagocytosis. The role of caspase-1 in a mouse calvarial model of particle-mediated osteolysis was assessed using µCT. Phagocytosis of PMMA particles induces caspase-1 dependent release of IL-1β from human monocytes and mouse macrophages. Importantly, using macrophages from mice deficient in components of the NALP3 inflammasome, we show PMMA-induced IL-1β production is strictly dependent on these components. Mice lacking caspase-1, the sole effector of the NALP3 inflammasome, show reduced orthopedic wear particle-induced calvarial osteolysis compared to wild-type controls. Absence of NALP3 inflammasome components fails to alter osteoclast formation in vitro. Our findings identify the NALP3 inflammasome as a critical mediator of orthopedic wear-induced osteolysis and as a viable therapeutic target for the treatment of periprosthetic osteolysis., (Copyright © 2012 Orthopaedic Research Society.)

Aggregatibacter actinomycetemcomitans is a gram-negative facultative capnophile involved in pathogenesis of aggressive forms of periodontal disease. In the present study, we interrogated the ability of A. actinomycetemcomitans to stimulate innate immune signaling and cytokine production and established that A. actinomycetemcomitans causes bone loss in a novel rat calvarial model. In vitro studies indicated that A. actinomycetemcomitans stimulated considerable production of soluble cytokines, tumor necrosis factor-α, interleukin-6 and interleukin-10 in both primary bone marrow-derived macrophages and NR8383 macrophages. Immunoblot analysis indicated that A. actinomycetemcomitans exhibits sustained activation of all major mitogen-activated protein kinase (MAPK) pathways, as well as the negative regulator of MAPK signaling, MAPK phosphatase-1 (MKP-1), for at least 8 h. In a rat calvarial model of inflammatory bone loss, high and low doses of formalin-fixed A. actinomycetemcomitans were microinjected into the supraperiosteal calvarial space for 1-2 weeks. Histological staining and micro-computed tomography of rat calvariae revealed a significant increase of inflammatory and fibroblast infiltrate and increased bone resorption as measured by total lacunar pit formation. From these data, we provide new evidence that fixed whole cell A. actinomycetemcomitans stimulation elicits a pro-inflammatory host response through sustained MAPK signaling, leading to enhanced bone resorption within the rat calvarial bone., (© 2012 John Wiley & Sons A/S.)

Microvascular supply is of fundamental importance to the survival and integration of grafting. Since the autogenous bone is still the gold standard for osseous augmentation, the aim of this study was to analyze the initial osseous, angiogenic and inflammatory response and subsequent osseointegration after implantation of dentin and beta-tricalcium phosphate (ß-TCP) scaffolds into the calvaria chamber of balb/c mice comparing with bone. The vascularisation of perforated implants of dentin (n=8), ß-TCP (n=8) and isogenic calvarial bone (n=8) displaying pores similar in size and structure was analyzed in vivo using intravital fluorescence microscopy. In additional animals (n=24) the osseointegration of dentin, ß-TCP and bone implants was assessed by fluorochrome sequential labelling of growing bone for up to 12 weeks. Animals without implants served as controls. Intravital fluorescence microscopy revealed that implantation of bone substitutes caused an only mild inflammatory response. Comparable to isogenic bone both dentin and ß-TCP scaffolds were found nearly completely vascularized by day 22 and osseointegrated within 12 weeks. In conclusion, dentin and ß-TCP scaffolds are similar to isogenic bone in terms of inflammatory and neovascularization response, highlighting their potential utility in regeneration of bone defects., (Copyright © 2012. Published by Elsevier Inc.)

Background: More and more studies have shown Angelica sinensis' (AS) therapeutic action on chronic inflammatory diseases in recent years. We investigated effects of aqueous extract of AS on inflammatory cytokines release and wear debris particles-induced osteolysis., Materials and Methods: Ultra high molecular weight polyethylene (UHMWPE) particles were used to induce inflammation in RAW264.7 cell and C57BL/J6 mice. AS extract was obtained through a series of purification steps, and divided into high dose group and low dose group during the research of cell culture, tissue culture, and animal treatment. After 72 h culture with optimal particles, supernatants were collected for cytokine analysis. Calvaria were harvested from the mice model after 10 d treatment with the AS extract. Six calvaria of each group were cultured into medium for 72 h for analyzing cytokine generated in vivo. Histologic analyses and micro-computed tomography (micro-CT) scan were used to determine osteoclastogenesis and inflammatory bone resorption., Results: Concentration of tumor necrosis-alpha (TNF-α) and interleukin-1beta (IL-1β) was significantly attenuated by AS extract both in vitro and in vivo. The osteolysis area and the osteoclast numbers were decreased from 0.406 ± 0.0799 to 0.117 ± 0.0103 mm(2), and from 22.7 ± 5.0 to 11.3 ± 1.8, respectively (P < 0.01). Compared with the control group, the protection effects of AS extract was further confirmed with data of the more accurate 3-dimension micro-CT reconstruction., Conclusions: This study suggests a potential resolution of inhibiting wear debris particles-induced inflammatory bone resorption, as well as a possible way of inhibiting aseptic loosening after joint replacement surgery., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The bone and immune systems are closely interconnected. The immediate inflammatory response after fracture is known to trigger a healing cascade which plays an important role in bone repair. Toll-like receptor 4 (TLR4) is a member of a highly conserved receptor family and is a critical activator of the innate immune response after tissue injury. TLR4 signaling has been shown to regulate the systemic inflammatory response induced by exposed bone components during long-bone fracture. Here we tested the hypothesis that TLR4 activation affects the healing of calvarial defects. A 1.8 mm diameter calvarial defect was created in wild-type (WT) and TLR4 knockout (TLR4(-/-)) mice. Bone healing was tested using radiographic, histologic and gene expression analyses. Radiographic and histomorphometric analyses revealed that calvarial healing was accelerated in TLR4(-/-) mice. More bone was observed in TLR4(-/-) mice compared to WT mice at postoperative days 7 and 14, although comparable healing was achieved in both groups by day 21. Bone remodeling was detected in both groups on postoperative day 28. In TLR4(-/-) mice compared to WT mice, gene expression analysis revealed that higher expression levels of IL-1β, IL-6, TNF-α,TGF-β1, TGF-β3, PDGF and RANKL and lower expression level of RANK were detected at earlier time points (≤ postoperative 4 days); while higher expression levels of IL-1β and lower expression levels of VEGF, RANK, RANKL and OPG were detected at late time points (> postoperative 4 days). This study provides evidence of accelerated bone healing in TLR4(-/-) mice with earlier and higher expression of inflammatory cytokines and with increased osteoclastic activity. Further work is required to determine if this is due to inflammation driven by TLR4 activation.

Osteoclasts are resident cells of the bone that are primarily involved in the physiological and pathological remodeling of this tissue. Mature osteoclasts are multinucleated giant cells that are generated from the fusion of circulating precursors originating from the monocyte/macrophage lineage. During inflammatory bone conditions in vivo, de novo osteoclastogenesis is observed but it is currently unknown whether, besides increased osteoclast differentiation from undifferentiated precursors, other cell types can generate a multinucleated giant cell phenotype with bone resorbing activity. In this study, an animal model of calvaria-induced aseptic osteolysis was used to analyze possible bone resorption capabilities of dendritic cells (DCs). We determined by FACS analysis and confocal microscopy that injected GFP-labeled immature DCs were readily recruited to the site of osteolysis. Upon recruitment, the cathepsin K-positive DCs were observed in bone-resorbing pits. Additionally, chromosomal painting identified nuclei from female DCs, previously injected into a male recipient, among the nuclei of giant cells at sites of osteolysis. Finally, osteolysis was also observed upon recruitment of CD11c-GFP conventional DCs in Csf1r(-/-) mice, which exhibit a severe depletion of resident osteoclasts and tissue macrophages. Altogether, our analysis indicates that DCs may have an important role in bone resorption associated with various inflammatory diseases.

Objective: To investigate the molecular mechanisms underlying particle-induced osteolysis, we focused on osteopontin (OPN), a cytokine and cell-attachment protein that is associated with macrophage chemoattractant and osteoclast activation., Methods: We compared OPN protein levels in human periprosthetic osteolysis tissues with those in osteoarthritis (OA) synovial tissues. To investigate the functions of OPN during particle-induced osteolysis in vivo, titanium particles were implanted onto the calvaria of OPN-deficient mice and their wild-type (WT) littermates. Mice were killed on day 10 and evaluated immunohistologically. The effects of OPN deficiency on the secretion of inflammatory cytokines were examined using cultured bone marrow-derived macrophages (BMMs). BMMs from OPN-deficient and WT mice were cultured with titanium particles for 12 hours, and the concentrations of inflammatory cytokines in the conditioned media were measured by enzyme-linked immunosorbent assay., Results: Expression of OPN protein was enhanced in human periprosthetic osteolysis tissues as compared with OA synovial tissues. In the particle-induced model of osteolysis of the calvaria, bone resorption was significantly suppressed by OPN deficiency via inhibition of osteoclastogenesis, whereas an inflammatory reaction was observed regardless of the genotype. Results of immunostaining indicated that OPN protein was highly expressed in the membrane and bone surface at the area of bone resorption in WT mice. When BMMs were exposed to titanium particles, the concentration of proinflammatory cytokines, such as tumor necrosis factor alpha, interleukin-1alpha (IL-1alpha), IL-1beta, and IL-6, as well as chemotactic factors, such as monocyte chemoattractant protein 1 and macrophage inflammatory protein 1alpha, in the conditioned medium were significantly reduced by OPN deficiency. Whereas phagocytic activity of BMMs was not attenuated by OPN deficiency, phagocytosis-mediated NF-kappaB activation was impaired in OPN-deficient BMMs. These data indicated that OPN was implicated in the development of particle-induced osteolysis via the orchestration of pro-/antiinflammatory cytokines secreted from macrophages., Conclusion: OPN plays critical roles in wear debris-induced osteolysis, suggesting that OPN is a candidate therapeutic target for periprosthetic osteolysis.

Baculovirus efficiently transduces human mesenchymal stem cells (hMSCs) and transplantation of hMSCs transduced with a bone morphogenetic protein 2-expressing baculovirus (Bac-CB) into nude mice results in ectopic bone formation. To attest the clinical potential of baculovirus in bone regeneration, hereby we explored whether the hMSCs genetically modified by Bac-CB were tolerant in immunocompetent rats and further healed the critical-sized calvarial bone defect. The histological and computed tomographic studies demonstrated that Bac-CB-engineered hMSCs promoted the cell differentiation and new bone formation in the immunocompetent rats. Immunohistochemical staining revealed that the transplanted human cells remained detectable at 1 and 4 weeks posttransplantation, attesting the immunoprevileged properties of hMSCs. In the recipients, the donor cells aggregated and appeared osteoblast like at later stages, which paralleled the infiltration of macrophages, CD3(+), and CD8(+) T cells into the graft. Administration of immunosuppressive drugs prolonged the cell survival and improved the bone regeneration, yet it failed to entirely abolish the immune response and complete the bone healing. Our data altogether implicate the potential of Bac-CB for hMSCs engineering and calvarial bone repair, but the use of hMSCs cannot overcome the immunological barrier.

The flat bones of the skull (calvaria) develop by balanced cell proliferation and differentiation in the calvarial sutures and the bone tips. As the brain grows and the calvaria expand, cells within the sutures must remain undifferentiated to maintain suture patency, but osteoprogenitors also need to be recruited into the osteogenic fronts. The exact identity of calvarial osteoprogenitors is currently not known. We used immunomagnetic cell sorting to isolate Sca-1+ and Sca-1(-) cells from fetal mouse calvaria and determined their differentiation potential in in vitro differentiation asssays and in vivo subcutaneous transplantations. Cells within the Sca-1+ cell fraction have a higher adipogenic potential, whereas cells within the Sca-1(-) cell fraction have a higher osteogenic and chondrogenic potential. The Sca-1(-) fraction retains its chondrogenic potential after in vitro expansion but not its osteogenic potential. The Sca-1+ fraction does not retain its adipogenic potential after in vitro expansion. Subcutaneous transplantation resulted in islands of bone and cartilage in implants that had been seeded with Sca-1(-) cells. In conclusion, immunomagnetic cell sorting with Sca-1 antibodies can be used to separate a Sca-1+ cell fraction with adipogenic potential from a Sca-1(-) cell fraction with osteogenic and chondrogenic potential. Isolation of pure populations of calvarial adipoprogenitors, osteoprogenitors, and chondroprogenitors will be beneficial for cellular studies of calvarial development, adipogenesis, osteogenesis, and chondrogenesis. Calvaria-derived osteogenic cell populations may be useful in craniofacial tissue regeneration and repair.

Background: The purpose was to analyse the clinical and radiological findings, and management approaches used in 30 consecutive cases of traumatic epidural haematoma of nonarterial origin treated at one centre., Method: Medical records for 30 patients surgically treated for epidural haematoma of nonarterial origin between 1997 and 2003 were reviewed. Epidural haematoma of nonarterial origin was diagnosed based on computed tomography (CT) and the bleeding source was confirmed intra-operatively. Admission status, outcome, fracture location, haematoma location/size/volume, and additional intracranial pathology were among the data noted. Two groups were formed for analysis: venous sinus bleeding (group 1) and other venous sources (group 2)., Findings: The 30 cases accounted for 25% of the total number of traumatic epidural haematomas (n = 120) treated during the same period. The epidural haematomas of nonarterial origin locations were transverse sigmoid sinus (n = 11; 36.7%), superior sagittal sinus (n = 6; 20%), venous lakes (n = 5; 16.6%), diploë (n = 5; 0.16%), arachnoid granulations (n = 2; 6.7%), petrosal sinus (n = 1; 3.3%). There were 12 postoperative complications in 9 patients: recurrence (n = 4; 13.3% of the 30 total), pneumonia (n = 4; 13.3%), meningitis (n = 2; 6.7%), hydrocephalus (n = 1; 3.3%) and subdural effusion (n = 1; 3.3%). All recurrence cases were re-explored. Six (20%) patients died. Glasgow Outcome Scale (GOS) scores (mean follow-up 13.3 +/- 7.8 months) revealed 22 (73.3%) patients with favourable results (GOS 4-5) and 8 (26.7%) had poor results (GOS 1-3)., Conclusions: Cases of epidural haematoma of nonarterial origin differ from the more common arterial-origin epidural haematomas with respect to lesion location, surgical planning, postoperative complications, and outcome. Epidural haematoma of nonarterial origin should be suspected if preoperative CT shows a haematoma overlying a dural venous sinus or in the posterior fossa and convexity. The sinus-origin group had a high frequency of fractures which crossed the sinuses, and this might be diagnostically and surgically useful in such cases.

Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) greatly induces osteoclast formation and stimulates bone resorption of mouse calvaria in culture. We examined the effects of the two cytokines on the collagenolysis and bone resorption by induction of matrix metalloproteinases (MMPs). The cells were analyzed using zymographic analysis. It was shown that the mouse calvarial osteoblasts constitutively synthesize progelatinase-A (MMP-2). Interleukin-1beta markedly enhanced the messenger RNAs (mRNAs) expression of MMP-2 (gelatinase A), but slightly MMP-9 (gelatinase B), which associated with increases in bone matrix degradation. Both pro- and active-forms of MMP-2 were detected in the conditioned medium collected from calvarial cultures, and IL-1beta markedly stimulated both pro- and active-forms of the MMP-2. The expression of MMP-2 mRNAs could be detected, and they were markedly enhanced by IL-1beta on days 1 and 2. These results demonstrate that the potency of induction of MMP-2 by IL-1beta and TNF-alpha is closely linked to the respective bone-resorbing activity, suggesting that MMP-2-dependent degradation of bone matrix plays a key role in bone resorption induced by these cytokines. On the other hand, when the mouse osteoblasts were stimulated with parathyroid hormone, 1,25(OH)2D3, mononuclear cell conditioned medium (MCM) and IL-1 as bone resorption agents, collagenolysis was increased by producing the active gelatinase. Interleukin-1 in stimulating bone resorption was examined using fetal mouse long bone organ culture. Interleukin-1 stimulated bone resorption and produced marked resorption when present simultaneously. Furthermore, treatment of indomethacin and dexamethasone clearly abolished the responses of IL-1alpha and IL-1beta.

Lipopolysaccharide (LPS) was recognized by CD14, which may be an important mediator in the deleterious effects of LPS on the periodontal destruction. To investigate the roles of CD14 molecules on LPS-induced soft tissue inflammation and bone destruction, the tissues of CD14-deficient mice were examined histopathologically following a local injection of either Salmonella minnesota or Porphyromonas gingivalis LPS. In the first group, 12 mice received a local injection of 500 microg of purified P. gingivalis LPS and six mice were injected with saline to the calvaria as controls. In the second group 13 mice were injected subcutaneously on the laterally abdominal skin with 50 microg of S. minnesota LPS and three mice were injected with PBS. Mice were sacrificed at day 5. After histological preparation, the tissue sections of calvaria and soft tissue specimen were stained with tartrate-resistant acid phosphatase (TRAP) marker for osteoclast and macrophage. The soft tissue sections were also stained with hematoxylin & eosin (H&E). Resorption surface and osteoclast index were measured to quantify bone resorption. Necrotic area and inflammatory cell numbers were estimated to assess the situation of local inflammation. Our results indicated that LPS-induced bone resorption is inhibited in CD14-deficient mice. An increase in the number of total inflammatory cells was noticed in both CD14-deficient mice and wild-type mice; however, the cell numbers were less in CD14-deficient mice than those in wild-type mice (two- to three-fold decrease). Therefore, we conclude that the LPS-stimulated bone resorption is mainly via CD14 receptor but the LPS-induced soft tissue inflammation appears to be partially dependent on the receptor.

Objective: To evaluate the potential of viral interleukin-10 (vIL-10) gene therapy as an approach to prevent wear debris-induced inflammation, osteoclastogenesis, and bone resorption as it relates to periprosthetic osteolysis in patients with total joint replacements., Methods: Replication-defective adenovirus vectors expressing vIL-10 (AdvIL-10) or LacZ (AdLacZ) target genes were used to transduce fibroblast-like synoviocytes (FLS) in vitro, and the effects of these cells on wear debris-induced proinflammatory cytokine production and receptor activator of nuclear factor kappaB ligand + macrophage colony-stimulating factor splenocyte osteoclastogenesis were assessed by enzyme-linked immunosorbent assay and tartrate-resistant acid phosphatase assay. The effects of AdvIL-10 administration on wear debris-induced osteolysis in vivo were analyzed using the mouse calvaria model, in which AdLacZ was used as the control., Results: In the presence of AdLacZ-infected FLS, titanium particle-stimulated macrophages exhibited a marked increase in secretion of tumor necrosis factor alpha (TNFalpha) (6.5-fold), IL-6 (13-fold), and IL-1 (5-fold). Coculture with AdvIL-10-transduced FLS suppressed cytokine secretion to basal levels, while addition of an anti-IL-10 neutralizing antibody completely blocked this effect. The vIL-10-transduced FLS also inhibited osteoclastogenesis 10-fold in an anti-IL-10-sensitive manner. In vivo, titanium implantation resulted in a 2-fold increase in osteoclasts (P < 0.05) and in a 2-fold increase in sagittal suture area (P < 0.05). This increase over control levels was completely blocked in mice receiving intraperitoneal injections of AdvIL-10, all of whom had measurable serum vIL-10 levels for the duration of the experiment. Immunohistochemistry demonstrated reduced cyclooxygenase 2 and TNFalpha expression in AdvIL-10-infected animals., Conclusion: This study demonstrates that gene delivery of vIL-10 inhibits 3 processes critically involved in periprosthetic osteolysis: 1) wear debris-induced proinflammatory cytokine production, 2) osteoclastogenesis, and 3) osteolysis.