Calvaria Bone Research Articles

Local Tolerance and Biodegradability of a Novel Artificial Dura Mater Graft Following Implantation Onto a Dural Defect in Rabbits.

Dura mater defects are a common problem following neurosurgery. Dural grafts are used to repair these defects; among them are biodegradable polymeric synthetic grafts. ArtiFascia is a novel synthetic and fibrous Dural graft, composed of poly(l-lactic-co-caprolactone acid) (PLCL) and poly(d-lactic-co-caprolactone acid). In this study, the biodegradability and local tolerance of ArtiFascia was evaluated in rabbits and compared with a bovine collagen matrix as a reference control. ArtiFascia implantation resulted in the formation of neo-dura at the site of implantation and recovery of the dural damage and the calvaria bone above. The implanted graft was completely absorbed after 12 months and the remaining macrophages were morphologically consistent with the anti-inflammatory M2-like phenotype, which contributes to tissue healing and are not pro-inflammatory. The site of the drilled skull bone had a continuous smooth surface, without exuberant tissue or inflammation and a newly formed trabecular bone formation indicated the healing process of the bone. These results support the local tolerability and biodegradability of ArtiFascia when used as a dural graft in rabbits. This study suggests that PLCL-based grafts including ArtiFascia are safe and effective to repair Rabbit Dura.

Long-term evaluation of alendronate treatment on the healing of calvaria bone defects in rats. Biochemical, histological and immunohistochemical analyses

ObjectiveThe aim of this study was to investigate the effects of the long-term alendronate administration on bone healing in defects created in rat calvarias. Materials and methodsFemale Wistar rats were randomly distributed into 2 groups: Control (CTL): animals received saline solution once a week; and Alendronate (ALD): rats underwent alendronate treatment (1 mg/kg/weekly). After 120 days from the commencement of treatment, a critical size defect was created in all animals, and 10 animals from each group were sacrificed at 5, 10, 15, 20, 25, 30, 45 and 60-days after the defect creation. On the day of sacrifice, urine and blood samples were collected for determination of the serum levels of bone resorption and formation markers by enzyme linked immunosorbent assay, and the urinary concentration of deoxypyridinoline. Bone mineral density (BMD) in the femurs, descriptive histology, tartrate-resistant acid-phosphatase staining and immunohistochemical analyzes were assessed in the calvaria. ResultsAlendronate group showed increased BMD compared to the test group. The concentration of C-terminal telopeptide of type I collagen and deoxypyridinoline decreased significantly, and the concentration of aminoterminal propeptide of procollagen type 1 and osteocalcin were significant lower in the alendronate group. Immunohistochemical analysis showed significant downregulation in the inducible nitric oxide synthase, runt-related transcription factor-2, cathepsin-K and receptor activator of nuclear factor kappa-B ligand expression in the alendronate group. Vascular endothelial growth factor and osteopontin were upregulated in the later periods of alendronate group. ConclusionsOur results suggest that long-term treatment with alendronate did not compromise the repair processing of critical size defects in rat.

Marine collagen scaffolds and photobiomodulation on bone healing process in a model of calvaria defects.

Collagen from marine esponges has been used as a promising material for tissue engineering proposals. Similarly, photobiomodulation (PBM) is able of modulating inflammatory processes after an injury, accelerating soft and hard tissue healing and stimulating neoangiogenesis. However, the effects of the associated treatments on bone tissue healing have not been studied yet. In this context, the present study aimed to evaluate the biological temporal modifications (using two experimental periods) of marine sponge collagen or sponging (SPG) based scaffold and PBM on newly formed bone using a calvaria bone defect model. Wistar rats were distributed into two groups: SPG or SPG/PBM and euthanized into two different experimental periods (15 and 45 days post-surgery). A cranial critical bone defect was used to evaluate the effects of the treatments. Histology, histomorfometry and immunohistological analysis were performed. Histological findings demonstrated that SPG/PBM-treated animals, 45 days post-surgery, demonstrated a higher amount of connective and newly formed bone tissue at the region of the defect compared to CG. Notwithstanding, no difference among groups were observed in the histomorphometry. Interestingly, for both anti-transforming growth factor-beta (TGF-β) and anti-vascular endothelial growth factor (VEGF) immunostaining, higher values for SPG/PBM, at 45 days post-surgery could be observed. It can be concluded that the associated treatment can be considered as a promising therapeutical intervention.

Coating biopolymer nanofibers with carbon nanotubes accelerates tissue healing and bone regeneration through orchestrated cell- and tissue-regulatory responses

Tailoring the surface of biomaterial scaffolds has been a key strategy to modulate the cellular interactions that are helpful for tissue healing process. In particular, nanotopological surfaces have been demonstrated to regulate diverse behaviors of stem cells, such as initial adhesion, spreading and lineage specification. Here, we tailor the surface of biopolymer nanofibers with carbon nanotubes (CNTs) to create a unique bi-modal nanoscale topography (500nm nanofiber with 25nm nanotubes) and report the performance in modulating diverse in vivo responses including inflammation, angiogenesis, and bone regeneration. When administered to a rat subcutaneous site, the CNT-coated nanofiber exhibited significantly reduced inflammatory signs (down-regulated pro-inflammatory cytokines and macrophages gathering). Moreover, the CNT-coated nanofibers showed substantially promoted angiogenic responses, with enhanced neoblood vessel formation and angiogenic marker expression. Such stimulated tissue healing events by the CNT interfacing were evidenced in a calvarium bone defect model. The in vivo bone regeneration of the CNT- coated nanofibers was significantly accelerated, with higher bone mineral density and up-regulated osteogenic signs (OPN, OCN, BMP2) of in vivo bone forming cells. The in vitro studies using MSCs could demonstrate accelerated adhesion and osteogenic differentiation and mineralization, supporting the osteo-promoting mechanism behind the in vivo bone forming event. These findings highlight that the CNTs interfacing of biopolymer nanofibers is highly effective in reducing inflammation, promoting angiogenesis, and driving adhesion and osteogenesis of MSCs, which eventually orchestrate to accelerate tissue healing and bone regeneration process. STATEMENT OF SIGNIFICANCE: Here we demonstrate that the interfacing of biopolymer nanofibers with carbon nanotubes (CNTs) could modulate multiple interactions of cells and tissues that are ultimately helpful for the tissue healing and bone regeneration process. The CNT-coated scaffolds significantly reduced the pro-inflammatory signals while stimulating the angiogenic marker expressions. Furthermore, the CNT-coated scaffolds increased the bone matrix production of bone forming cells in vivo as well as accelerated the adhesion and osteogenic differentiation of MSCs in vitro. These collective findings highlight that the CNTs coated on the biopolymer nanofibers allow the creation of a promising platform for nanoscale engineering of biomaterial surface that can favor tissue healing and bone regeneration process, through a series of orchestrated events in anti-inflammation, pro-angiogenesis, and stem cell stimulation.

Histomorphometric Comparison of 2 types of FDBA and DFDBA bone powders manufactured by Faravardehaye Peyvandi Iran Company in treatment of defects in rabbit calvaria bone

Histomorphometric Comparison of 2 types of FDBA and DFDBA bone powders manufactured by Faravardehaye Peyvandi Iran Company in treatment of defects in rabbit calvaria bone

Regulation of bone metabolism by megakaryocytes in a paracrine manner

Megakaryocytes (MKs) play key roles in regulating bone metabolism. To test the roles of MK-secreted factors, we investigated whether MK and promegakaryocyte (pro-MK) conditioned media (CM) may affect bone formation and resorption. K562 cell lines were differentiated into mature MKs. Mouse bone marrow macrophages were differentiated into mature osteoclasts, and MC3T3-E1 cells were used for osteoblastic experiments. Bone formation was determined by a calvaria bone formation assay in vivo. Micro-CT analyses were performed in the femurs of ovariectomized female C57B/L6 and Balb/c nude mice after intravenous injections of MK or pro-MK CM. MK CM significantly reduced in vitro bone resorption, largely due to suppressed osteoclastic resorption activity. Compared with pro-MK CM, MK CM suppressed osteoblastic differentiation, but stimulated its proliferation, resulting in stimulation of calvaria bone formation. In ovariectomized mice, treatment with MK CM for 4 weeks significantly increased trabecular bone mass parameters, such as bone volume fraction and trabecular thickness, in nude mice, but not in C57B/L6 mice. In conclusion, MKs may secrete anti-resorptive and anabolic factors that affect bone tissue, providing a novel insight linking MKs and bone cells in a paracrine manner. New therapeutic agents against metabolic bone diseases may be developed from MK-secreted factors.

Osseous Healing in Surgically Prepared Bone Defects Using Different Grafting Materials: An Experimental Study in Pigs.

Regeneration of large jaw bone defects still remains a clinical challenge. To avoid incomplete bone repair, bone grafts have been advocated to support the healing process. This study comparatively evaluated new bone formation among a synthetic graft substitute, a human bone derivative, and a bovine xenograft. Materials were placed in 3 out of the 4 bone cavities, while 1 deficit was left empty, serving as a control, in mono-cortical defects, surgically prepared in the porcine calvaria bone. Animals were randomized in 2 groups and euthanized at 8 and 12 weeks. Harvested tissue specimens were qualitatively evaluated by histology. New bone formation was quantitatively measured by histomorphometry. Maximum new bone formation was noticed in defects grafted with beta-tricalcium phosphate b-TCP compared to the other bone substitutes, at 8 and 12 weeks post-surgery. Bovine and human allograft induced less new bone formation compared to empty bone cavity. Histologic analysis revealed that b-TCP was absorbed and substituted significantly, while bovine and human allograft was maintained almost intact in close proximity with new bone. Based on our findings, higher new bone formation was detected in defects filled with b-TCP when compared to bovine and human graft substitutes.

EPO could be regulated by HIF-1 and promote osteogenesis and accelerate bone repair

Bone defects caused by many factors prompt further study of pathological process and restoration methods. This study was aimed to clarify the effect of erythropoietin on the repair of bone defect. We added the designated concentration of rhEPO to endothelial progenitor cells and marrow stromal cells, then detected its osteogenic and angiogenesis effects. The results showed that rhEPO promoted the proliferation of EPC and ST2 by promoting the mitosis without affecting cell apoptosis. The protein and mRNA levels of angiogenesis and osteogenic related factors exhibited higher expressions. Additionally, rhEPO encapsulated in PLGA scaffolds accelerated the new bone formation in rat calvaria bone defect model. Since the centre of bone defect was hypoxia environment, we cultured EPC and ST2 under hypoxia. SiRNA and an inhibitor of HIF-1 were used to interfere HIF-1, then the following changes of VEGF and EPO were detected. The results showed that all the factors were upregulated under the hypoxia environment. The expression of VEGF at protein and mRNA level decreased as HIF-1 was inhibited or interfered from 6 h, while the mRNA expression of EPO from 6 h and changed significantly at protein level from 12 h. Therefore, EPO is a promising factor for further studies.

Influence of Green Tea Extract with Different Concentrations of Epigallocatechin Gallate on Calvaria Bone Repair of Ovariectomized Rats

Influence of Green Tea Extract with Different Concentrations of Epigallocatechin Gallate on Calvaria Bone Repair of Ovariectomized Rats

Exosomes from conditioned media of bone marrow-derived mesenchymal stem cells promote bone regeneration by enhancing angiogenesis.

Growth factors in serum-free conditioned media from human bone marrow-derived mesenchymal stem cells (MSC-CM) are known to be effective in bone regeneration. However, the secretomes in MSC-CM that act as active ingredients for bone regeneration, as well as their mechanisms, remains unclear. Exosomes, components of MSC-CM, provide the recipient cells with genetic information and enhance the recipient cellular paracrine stimulation, which contributes to tissue regeneration. We hypothesized that MSC-CM-derived exosomes (MSC-Exo) promoted bone regeneration, and that angiogenesis was a key step. Here, we prepared an MSC-Exo group, MSC-CM group, and Exo-antiVEGF group (MSC-Exo with angiogenesis inhibitor), and examined the osteogenic and angiogenic potential in MSCs. Furthermore, we used a rat model of calvaria bone defect and implanted each sample to evaluate bone formation weekly, until week 4 after treatment. Results showed that MSC-Exo enhanced cellular migration and osteogenic and angiogenic gene expression in MSCs compared to that in other groups. In vivo, early bone formation by MSC-Exo was also confirmed. Two weeks after implantation, the newly formed bone area was 31.5 ± 6.5% in the MSC-Exo group while those in the control and Exo-antiVEGF groups were 15.4 ± 4.4% and 8.7 ± 1.1%, respectively. Four weeks after implantation, differences in the area between the MSC-Exo group and the Exo-antiVEGF or control groups were further broadened. Histologically, notable accumulation of osteoblast-like cells and vascular endothelial cells was observed in the MSC-Exo group; however, fewer cells were found in the Exo-antiVEGF and control groups.In conclusion, MSC-Exo promoted bone regeneration during early stages, as well as enhanced angiogenesis. Considering the tissue regeneration with transplanted cells and their secretomes, this study suggests that exosomes might play an important role, especially in angiogenesis.

The effects of myokines on osteoclasts and osteoblasts

Recently, muscle has received much attention as an endocrine organ regulating other biological targets, including the pancreas, liver, and adipose tissue. Although there is a possibility that muscle-secreting factors biochemically affect bone metabolism in a paracrine manner, the net effects of myokines on the biology of osteoclasts and osteoblasts, particularly on bone mass in vivo, have not yet been thoroughly investigated. Therefore, we performed in vitro as well as animal experiments using conditioned media (CM) collected from C2C12 myoblast and myotube cultures to better understand the interactions between muscle and bone. Compared with non-CM (i.e., control) and myoblast CM, myotube CM markedly inhibited in vitro bone resorption through the suppression of osteoclast differentiation and resorptive activity of individual osteoclasts. Consistently, the expressions of osteoclast differentiation markers, such as tartrate-resistant acid phosphatase (Trap) and calcitonin receptor (Ctr), decreased with myotube CM. Myotube CM significantly stimulated preosteoblast viability and migration and reduced apoptosis, thereby resulting in an increase in calvaria bone formation. Importantly, systemic treatment with myotube CM for 4 weeks increased bone per tissue volume by 30.7% and 19.6% compared with control and myoblast CM, respectively. These results support the hypothesis that muscle plays beneficial roles in bone health via secretion of anabolic factors, in addition to mechanical stimuli, and importantly indicate that muscle-derived factors can be potential therapeutic targets against metabolic bone diseases.

In situ prevascularization designed by laser-assisted bioprinting: effect on bone regeneration

Vascularization plays a crucial role in bone formation and regeneration process. Development of a functional vasculature to improve survival and integration of tissue-engineered bone substitutes remains a major challenge. Biofabrication technologies, such as bioprinting, have been introduced as promising alternatives to overcome issues related to lack of prevascularization and poor organization of vascular networks within the bone substitutes. In this context, this study aimed at organizing endothelial cells in situ, in a mouse calvaria bone defect, to generate a prevascularization with a defined architecture, and promote in vivo bone regeneration. Laser-assisted bioprinting (LAB) was used to pattern Red Fluorescent Protein-labeled endothelial cells into a mouse calvaria bone defect of critical size, filled with collagen containing mesenchymal stem cells and vascular endothelial growth factor. LAB technology allowed safe and controlled in vivo printing of different cell patterns. In situ printing of endothelial cells gave rise to organized microvascular networks into bone defects. At two months, vascularization rate (vr) and bone regeneration rate (br) showed statistically significant differences between the ‘random seeding’ condition and both ‘disc’ pattern (vr = +203.6%; br = +294.1%) and ‘crossed circle’ pattern (vr = +355%; br = +602.1%). These results indicate that in vivo LAB is a valuable tool to introduce in situ prevascularization with a defined configuration and promote bone regeneration.

Implant survival rate in calvarial bone grafts: A retrospective clinical study with 10 year follow-up.

In this study, we present medium- and long-term data on implant survival in a cohort of patients with severe maxillary atrophy rehabilitated using reconstructive implant site development with calvarial bone grafts. We obtained clinical records from patients treated with implant rehabilitation supported by calvaria bone grafts in the Oral Surgery Department of IRCSS San Raffaele (Milan, Italy). Implant and prosthetic survival and success rates were retrospectively evaluated. Graft survival and postoperative complications were also assessed. A total of 207 implants placed in 32 patients were evaluated for a mean period of 10.0 years from loading. After 10 years, the cumulative survival rate was 97.10%, the implant success rate was 92.75%, and the prosthetic complication rate was 9.76%. A graft survival percentage of 96.88% was observed, and postoperative complications occurred in 28.13% of cases. The 10-year survival rate and prosthetic complications for patients treated with implant rehabilitation supported by calvarial bone grafts are excellent, as implant loss was relatively rare, although limited subjects were available for the 10-year follow-up.

Autogenous graft: the use of calvarium bone graft as an option of a late orbital defect reconstruction

Background: The use of Autogenous Calvarium Graft (ACG) is indicated for both primary and secondary congenital deformities of the craniofacial area and for the acute treatment of facial trauma. One of the adhibition of ACG is in the correction of orbital fractures, which represent 20 to 40% of the maxillofacial injuries, having a satisfactory result in the reduction of posttraumatic enophthalmos.

The Obliteration of Noncritical Size Bone Defects With Bone Dust or Bone Replacement Material (Bioactive Glass S53P4).

Bone dust (BD) harvested during operation may be suitable as an autologous obliteration material for noncritical size defects. Bioactive glass (BA) can be an alternative. To treat noncritical size defects, BD and BA are commonly used for obliteration techniques. However, the optimal harvesting method and parameters for BD have not been examined. In this study, we analyzed the osseoregenerative potential of both materials. Thirteen female merino sheep (7-yr old) underwent surgery on the frontal calvaria. Three defects were inserted. The first defect was considered a reference and remained unfilled, the second defect was filled with BD from the calvaria bone, and the third defect was filled with BA S53P4. The animals were sacrificed after 3 weeks. To evaluate bone regeneration, we used digital volume tomography, bone density measurement, fluorochrome sequence labeling, and histological analysis. All analyses showed quantitative and qualitative bone regeneration 3 weeks after operation. The control blank defect showed significantly less new bone growth than the BD-filled defect. Moreover, bone regeneration occurred from the surrounding bone and showed only a defect bridge in the BD-filled defect. The BA completely filled the defect and had the highest density although the same amount of new mineralized bone generated as in the reference. BD and BA seemed to be suitable bone replacement materials for obliteration techniques because they completely filled the defects. Thus, BD harvested under standardized conditions provided a higher level of osteoreparation potential for the generation of woven bone and establishment of defect bridges.

Histomorphometric Comparison of 2 types of FDBA and DFDBA bone powders manufactured by Hamanand Saz Baft Kish Company in treatment of defects in rabbit calvaria bone

Histomorphometric Comparison of 2 types of FDBA and DFDBA bone powders manufactured by Hamanand Saz Baft Kish Company in treatment of defects in rabbit calvaria bone

Mouse Wnt1-CRE-RosaTomato Dental Pulp Stem Cells Directly Contribute to the Calvarial Bone Regeneration Process.

Stem cells endowed with skeletogenic potentials seeded in specific scaffolds are considered attractive tissue engineering strategies for treating large bone defects. In the context of craniofacial bone, mesenchymal stromal/stem cells derived from the dental pulp (DPSCs) have demonstrated significant osteogenic properties. Their neural crest embryonic origin further makes them a potential accessible therapeutic tool to repair craniofacial bone. The stem cells' direct involvement in the repair process versus a paracrine effect is however still discussed. To clarify this question, we have followed the fate of fluorescent murine DPSCs derived from PN3 Wnt1-CRE- RosaTomato mouse molar (T-mDPSCs) during the repair process of calvaria bone defects. Two symmetrical critical defects created on each parietal region were filled with (a) dense collagen scaffolds seeded with T-mDPSCs, (b) noncellularized scaffolds, or (c) no scaffold. Mice were imaged over a 3-month period by microcomputed tomography to evaluate the extent of repair and by biphotonic microscopy to track T-mDPSCs. Histological and immunocytochemical analyses were performed in parallel to characterize the nature of the repaired tissue. We show that T-mDPSCs are present up to 3 months postimplantation in the healing defect and that they rapidly differentiate in chondrocyte-like cells expressing all the expected characteristic markers. T-mDPSCs further maturate into hypertrophic chondrocytes and likely signal to host progenitors that form new bone tissue. This demonstrates that implanted T-mDPSCs are able to survive in the defect microenvironment and to participate directly in repair via an endochondral bone ossification-like process. Stem Cells 2019;37:701-711.

Low-level laser therapy enhances the number of osteocytes in calvaria bone defects of ovariectomized rats.

BackgroundOsteoporosis can make bone repair difficult. Low‐level laser therapy (LLLT) has been shown to be a promising tool for bone neoformation. This study aimed to analyze the effect of LLLT on calvaria bone defects of ovariectomized rats using stereology.MethodsFifty‐four Wistar rats were subjected to bilateral ovariectomy, and bone defects were created in calvaria after 150 days. The animals were divided into nine groups (n = 6 per group), and 24 hours after the bone defects were created they received 3, 6 or 12 sessions of LLLT at 0, 20 or 30 J/cm2, using a 780‐nm low‐intensity GaAlAs laser. One‐way ANOVA followed by Tukey's post hoc test was used for data processing. A difference of P < 0.05 was considered statistically significant. The parameters evaluated were osteocyte density (Nv ost), total osteocyte number (Nto ost), trabecular surface density (Sv t), and trabecular surface area (Sa t).ResultsData obtained showed that Nto ost, Sv t, and Sa t in group G2 rats were significantly different from G1 (0 J/cm2) (P < 0.05). Compared to group G4, G5 presented higher values for the parameters Sv t and Sa t, and G6 presented significantly higher values for almost all the analyzed parameters (Nv ost, Nto ost, Sv t, and Sa t) (P < 0.05). Compared to group G7, G8 showed a higher value only for the parameter Sa t, and G9 showed significantly higher values for parameters Nv ost, Nto ost, Sv t, and Sa t.ConclusionWe conclude that LLLT stimulated bone neoformation and contributed to an increase in the total number of osteocytes, especially with a laser energy density of 30 J/cm2 given for 6 and 12 sessions.

Aligned electrospun cellulose scaffolds coated with rhBMP-2 for both in vitro and in vivo bone tissue engineering

Physical properties of scaffolds such as nanofibers and aligned structures have been reported to exert profound effects on the growth and differentiation of stem cells due to their homing-effect features and contact guidance. However, the biological function of aligned nanofibers utilized as bone-scaffold has not been rigorously characterized. In the present study, aligned electrospun cellulose/CNCs nanocomposite nanofibers (ECCNNs) loaded with bone morphogenic protein-2 (BMP-2) were used for the first time to investigate (1) in vitro osteogenic differentiation of human mesenchymal stem cells (BMSCs) and (2) in vivo collagen assembly direction and cortical bone regeneration. Aligned ECCNNs scaffolds loaded with BMP-2 possess good biological compatibility. The growth orientation of BMSCs followed the underlying aligned nanofiber morphology, accompanied with increased alizarin red stain, alkaline phosphatase (ALP) activity and calcium content in vitro while, a rabbit calvaria bone defect model was used in an in vivo study with micro CT and histology analyses.

DPP-4 inhibitor impedes lipopolysaccharide-induced osteoclast formation and bone resorption in vivo

ObjectivesDipeptidyl peptidase 4 (DPP-4) inhibition is a new therapeutic strategy for type 2 diabetic patients. DPP-4 has been reported to enhance inflammation. However, the effect of DPP-4 inhibition on inflammation remains unknown. Lipopolysaccharide (LPS) is a strong inducer of inflammation and osteoclast formation. In this study, we investigated in vivo effects of DPP-4 inhibition on LPS-induced osteoclast formation and bone resorption, as well as in vitro effects of DPP-4 inhibition on RANKL-induced osteoclastogenesis and TNF-α-induced osteoclastogenesis. MethodsLPS with or without a DPP-4 inhibitor was subcutaneously injected into mouse calvaria for 5 days. Histological sections of calvaria were stained for tartrate-resistant acid phosphatase, and osteoclast numbers were determined. The ratio of calvaria bone resorption was evaluated via microfocal computed tomography reconstruction images. ResultsOsteoclast number and bone resorption were significantly lower in mice that underwent LPS and DPP-4 inhibitor co-administration than in those that underwent LPS administration alone. Moreover, RANKL, TNF-α, and M-CSF expression was reduced in the LPS and DPP-4 inhibitor co-administration group. In vitro, there were no direct effects of DPP-4 inhibitor or DPP-4 on RANKL- and TNF-α-induced osteoclastogenesis, or on LPS-induced RANKL expression in stromal cells. Nevertheless, macrophages from LPS and DPP-4 inhibitor co-administered mice exhibited lower TNF-α expression than macrophages from LPS-only mice. Notably, TNF-α expression was not reduced in LPS and DPP-4 inhibitor co-treated macrophages in vitro, compared with macrophages treated with LPS alone.