Pharmacological activation of SIRT1 by metformin prevented trauma-induced heterotopic ossification through inhibiting macrophage mediated inflammation.

Trauma-induced heterotopic ossification (HO) is the aberrant extra-skeletal bone formation that severely incapacitates patient's daily life. Inflammation is the first stage of this progression, becoming an appealing target of early therapeutic intervention. Metformin, a widely used antidiabetic drug, also poses the therapeutic potential to modulate various inflammatory-related diseases. Therefore, this study aimed to investigate the preventive effect of metformin on trauma-induced HO progression, and unveil the underlying molecular mechanisms. A murine burn/tenotomy model was established to mimic trauma-induced HO in vivo. The anti-inflammation and anti-ossification effects of metformin were evaluated by histological staining and micro-CT. The inhibitory effects of metformin on macrophages activation in vitro were examined by ELISA and qRT-PCR. The underlying molecular mechanisms were further explored by immunofluorescence staining and western-blotting in vivo. Increased macrophages infiltration and inflammatory responses were found at early stage during HO progression. However, metformin dose-dependently attenuated the macrophage-mediated inflammatory responses both in vivo and vitro, which might account for the inhibitory effect of metformin on chondrogenesis and HO formation after trauma. Furthermore, elevated SIRT1 expression and decreased NF-κB p65 acetylation were found in the beneficial effects of metformin. Moreover, similar preventive effects were also found in SRT1720 HCI, a specific SIRT1 activator, while were remarkably reversed after the administration of EX527 (a specific SIRT1 inhibitor) with metformin. Taken together, our results provide a novel evidence that metformin can effectively attenuate trauma-induced HO by mitigating macrophage inflammatory responses through inhibiting NF-κB signaling via SIRT1-dependent mechanisms, which favors future therapeutic investigations for trauma-related disease.

Quercetin Attenuates Trauma-Induced Heterotopic Ossification by Tuning Immune Cell Infiltration and Related Inflammatory Insult.

Heterotopic ossification (HO) is one of the most intractable disorders following musculoskeletal injury and is characterized by the ectopic presence of bone tissue in the soft tissue leading to severe loss of function in the extremities. Recent studies have indicated that immune cell infiltration and inflammation are involved in aberrant bone formation. In this study, we found increased monocyte/macrophage and mast cell accumulation during early HO progression. Macrophage depletion by clodronate liposomes and mast cell stabilization by cromolyn sodium significantly impeded HO formation. Therefore, we proposed that the dietary phytochemical quercetin could also suppress immune cell recruitment and related inflammatory responses to prevent HO. As expected, quercetin inhibited the monocyte-to-macrophage transition, macrophage polarization, and mast cell activation in vitro in a dose-dependent manner. Using a murine burn/tenotomy model, we also demonstrated that quercetin attenuated inflammatory responses and HO in vivo. Furthermore, elevated SIRT1 and decreased acetylated NFκB p65 expression were responsible for the mechanism of quercetin, and the beneficial effects of quercetin were reversed by the SIRT1 antagonist EX527 and mimicked by the SIRT agonist SRT1720. The findings in this study suggest that targeting monocyte/macrophage and mast cell activities may represent an attractive approach for therapeutic intervention of HO and that quercetin may serve as a promising therapeutic candidate for the treatment of trauma-induced HO by modulating SIRT1/NFκB signaling.

Post-COVID-19 exacerbation of fibrodysplasia ossificans progressiva with multiple flare-ups and extensive heterotopic ossification in a 45-year-old female patient.

Fibrodyplasia ossificans progressiva (FOP) is a rare hereditary disease, which has a variable course characterized by occasional flare-ups of heterotopic ossification (HO) in soft tissues that are followed by swelling, stiffness, pain and warmth. Here, we report for the first time a case of a 45-year-old female patient with known FOP recovering from COVID-19 with disease progression potentially linked with the viral illness. In December 2020 the patient contracted a mild form of COVID-19 infection without need for hospital admission. Since January 2021, the patient felt unwell, with occasional abdominal pain which progressively intensified. In March 2021 she presented with new onset of HO, complaining of pain, swelling and thickening sensation in the lower abdomen and left part of the neck. Computerized tomography (CT) and cytokine analysis were performed. CT scan revealed new heterotopic bone formation in multiple soft tissue areas of the neck indicating clear radiological progression. Radiotherapy, which has proven to be an efficient tool to control HO in this patient, was not able to halt HO formation after COVID-19 infection. Cytokine analysis of a plasma sample obtained during a flare-up after COVID-19 infection showed a significantly elevated pro-inflammatory cytokines compared to a flare-up panel prior to infection. Of the 23 analyzed levels of cytokines, a staggering number of 21 were above normal levels. This case is the first confirmation of uncontrolled post-COVID-19 effects in a FOP patient, which manifested with flare-ups followed by progressive HO, possibly caused by a thus far, never described form of post-COVID syndrome.

Inflammation in Fibrodysplasia Ossificans Progressiva and Other Forms of Heterotopic Ossification.

PURPOSE OF REVIEW: Heterotopic ossification (HO) is associated with inflammation. The goal of this review is to examine recent findings on the roles of inflammation and the immune system in HO. We examine how inflammation changes in fibrodysplasia ossificans progressiva, in traumatic HO, and in other clinical conditions of HO. We also discuss how inflammation may be a target for treating HO. RECENT FINDINGS: Both genetic and acquired forms of HO show similarities in their inflammatory cell types and signaling pathways. These include macrophages, mast cells, and adaptive immune cells, along with hypoxia signaling pathways, mesenchymal stem cell differentiation signaling pathways, vascular signaling pathways, and inflammatory cytokines. Because there are common inflammatory mediators across various types of HO, these mediators may serve as common targets for blocking HO. Future research may focus on identifying new inflammatory targets and testing combinatorial therapies based on these results.

Elevated plasma RANTES in fibrodysplasia ossificans progressiva - A novel therapeutic target?

Fibrodysplasia ossificans progressiva (FOP) is a rare hereditary disease caused by a mutation in the intracellular domain of the activin A receptor type I and is characterized by episodes (flare-ups) of progressive heterotopic endochondral ossification (HO) in the soft tissues. The mutation alone is not sufficient for the occurrence of HO since flare-ups are triggered by inflammation and activation of the innate immune system. A number of cellular and humoral mediators have been implicated in animal and in vitro models. Observations in humans support the inflammatory nature of the condition, but data on the involved mediators are variable. We hypothesize that for induction of flare-ups in patients with FOP increase in at least one of the pro-inflammatory cytokines is both essential and sufficient to trigger the entire process of the inflammatory cells influx resulting in the novel ectopic bone formation and we suggest that C-C motif ligand 5 (CCL5), a pro-inflammatory chemokine also known as Regulated on activation, normal T-cell expressed and secreted (RANTES), might be the key candidate. CCL5 is a chemoattractant for all cellular types implicated in HO and is produced by the cells of the tissue microenvironment at the sites of HO as well as by the pro-inflammatory cellular mediators. CCL5 induces ossification in cultured human pluripotent mesenchymal cells (hMSCs) and in the primary culture of monocytes from FOP patients (but not from their healthy relatives), stimulation with lipopolysaccharide induces CCL5 expression. Finally, in a pilot study we used a panel of 23 cytokines and chemokines to screen the plasma samples of three subjects: a female patient with FOP during a flare-up; a female patient with hyperostosis corticalis generalisata (van Buchem disease), another rare disease characterized by excessive bone formation at the sites where it regularly occurs that does not include inflammatory events; and a healthy woman without bone disorders. There appeared a rather clear-cut signal of a 2-fold higher level of CCL5 in the FOP patient vs. the healthy subject and the van Buchem patient. Evaluation of the hypothesis would require an international prospective study, with main motivation being the lack of a conclusive treatment as the major unmet need in FOP. A treatment targeting CCL5 receptor already exists and is used in HIV-infected patients.

Severe Heterotopic Ossification in the Skeletal Muscle and Endothelial Cells Recruitment to Chondrogenesis Are Enhanced by Monocyte/Macrophage Depletion.

Altered macrophage infiltration upon tissue damage results in inadequate healing due to inappropriate remodeling and stem cell recruitment and differentiation. We investigated in vivo whether cells of endothelial origin phenotypically change upon heterotopic ossification induction and whether infiltration of innate immunity cells influences their commitment and alters the ectopic bone formation. Liposome-encapsulated clodronate was used to assess macrophage impact on endothelial cells in the skeletal muscle upon acute damage in the ECs specific lineage-tracing Cdh5CreERT2:R26REYFP/dtTomato transgenic mice. Macrophage depletion in the injured skeletal muscle partially shifts the fate of ECs toward endochondral differentiation. Upon ectopic stimulation of BMP signaling, monocyte depletion leads to an enhanced contribution of ECs chondrogenesis and to ectopic bone formation, with increased bone volume and density, that is reversed by ACVR1/SMAD pathway inhibitor dipyridamole. This suggests that macrophages contribute to preserve endothelial fate and to limit the bone lesion in a BMP/injury-induced mouse model of heterotopic ossification. Therefore, alterations of the macrophage-endothelial axis may represent a novel target for molecular intervention in heterotopic ossification.

NF-κB/MAPK activation underlies ACVR1-mediated inflammation in human heterotopic ossification.

BACKGROUND: Inflammation helps regulate normal growth and tissue repair. Although bone morphogenetic proteins (BMPs) and inflammation are known contributors to abnormal bone formation, how these pathways interact in ossification remains unclear. METHODS: We examined this potential link in patients with fibrodysplasia ossificans progressiva (FOP), a genetic condition of progressive heterotopic ossification caused by activating mutations in the Activin A type I receptor (ACVR1/ALK2). FOP patients show exquisite sensitivity to trauma, suggesting that BMP pathway activation may alter immune responses. We studied primary blood, monocyte, and macrophage samples from control and FOP subjects using multiplex cytokine, gene expression, and protein analyses; examined CD14+ primary monocyte and macrophage responses to TLR ligands; and assayed BMP, TGF-ß activated kinase 1 (TAK1), and NF-κB pathways. RESULTS: FOP subjects at baseline without clinically evident heterotopic ossification showed increased serum IL-3, IL-7, IL-8, and IL-10. CD14+ primary monocytes treated with the TLR4 activator LPS showed increased CCL5, CCR7, and CXCL10; abnormal cytokine/chemokine secretion; and prolonged activation of the NF-κB pathway. FOP macrophages derived from primary monocytes also showed abnormal cytokine/chemokine secretion, increased TGF-ß production, and p38MAPK activation. Surprisingly, SMAD phosphorylation was not significantly changed in the FOP monocytes/macrophages. CONCLUSIONS: Abnormal ACVR1 activity causes a proinflammatory state via increased NF-κB and p38MAPK activity. Similar changes may contribute to other types of heterotopic ossification, such as in scleroderma and dermatomyositis; after trauma; or with recombinant BMP-induced bone fusion. Our findings suggest that chronic antiinflammatory treatment may be useful for heterotopic ossification.

Immunological and morphological analysis of heterotopic ossification differs to healthy controls.

BACKGROUND: Formation of lamellar bone in non-osseus tissue is a pathological process called heterotopic ossification. It is the aim of this study to analyse the morphology and immunological status of patients with heterotopic ossification compared to individual healthy persons. METHODS: Human bone marrow and blood samples were obtained from 6 systemically healthy individuals and 4 patients during resection of heterotopic ossification from bone at hip arthroplasty. Bone was fragmented and treated with purified collagenase. Immunofluorescence surface staining was performed and analyzed with flow cytometry. Microcomputed tomography scanning was done performed at a resolution of 11 and 35 µm isometric voxel size respectively using a two different cone beam X-computer tomography systems and a microfocus X-ray tube. Subsequently the volume data was morphometrically analysed. RESULTS: The monocytes, stem cells, stroma cells and granulocytes progenitor cells were strongly reduced in the heterotopic ossification patient. Additionally a significant reduction of stromal stem cells cells and CD34 positive stem cells was observed. The frequency of NK-cells, B cells and T cells were not altered in the patients with heterotopic ossification compared to a healthy person. Micromorphometric parameters showed a lower content of mineralized bone tissue compared to normal bone. Mean trabecular thickness showed a high standard deviation, indicating a high variation in trabecular thickness, anisotropy and reducing bone strength. CONCLUSIONS: This work shows altered immunological distribution that is accompanied by a low decrease in bone volume fraction and tissue mineral density in the heterotopic ossification sample compared to normal bone. Compared to healthy subjects, this might reflect an immunological participation in the development of this entity.

Role of Stem Cells in Pathophysiology and Therapy of Spondyloarthropathies-New Therapeutic Possibilities?

Considerable progress has been made recently in understanding the complex pathogenesis and treatment of spondyloarthropathies (SpA). Currently, along with traditional disease modifying anti-rheumatic drugs (DMARDs), TNF-α, IL-12/23 and IL-17 are available for treatment of such diseases as ankylosing spondylitis (AS) and psoriatic arthritis (PsA). Although they adequately control inflammatory symptoms, they do not affect the abnormal bone formation processes associated with SpA. However, the traditional therapeutic approach does not cover the regenerative treatment of damaged tissues. In this regards, stem cells may offer a promising, safe and effective therapeutic option. The aim of this paper is to present the role of mesenchymal stromal cells (MSC) in pathogenesis of SpA and to highlight the opportunities for using stem cells in regenerative processes and in the treatment of inflammatory changes in articular structures.

Macrophage-derived oncostatin M contributes to human and mouse neurogenic heterotopic ossifications.

Neurogenic heterotopic ossification (NHO) is the formation of ectopic bone generally in muscles surrounding joints following spinal cord or brain injury. We investigated the mechanisms of NHO formation in 64 patients and a mouse model of spinal cord injury-induced NHO. We show that marrow from human NHOs contains hematopoietic stem cell (HSC) niches, in which mesenchymal stromal cells (MSCs) and endothelial cells provide an environment supporting HSC maintenance, proliferation, and differentiation. The transcriptomic signature of MSCs from NHOs shows a neuronal imprinting associated with a molecular network required for HSC support. We demonstrate that oncostatin M (OSM) produced by activated macrophages promotes osteoblastic differentiation and mineralization of human muscle-derived stromal cells surrounding NHOs. The key role of OSM was confirmed using an experimental model of NHO in mice defective for the OSM receptor (OSMR). Our results provide strong evidence that macrophages contribute to NHO formation through the osteogenic action of OSM on muscle cells within an inflammatory context and suggest that OSM/OSMR could be a suitable therapeutic target. Altogether, the evidence of HSCs in ectopic bones growing at the expense of soft tissue in spinal cord/brain-injured patients indicates that inflammation and muscle contribute to HSC regulation by the brain-bone-blood triad.

The traumatic bone: trauma-induced heterotopic ossification.

Heterotopic ossification (HO) is a common occurrence after multiple forms of extensive trauma. These include arthroplasties, traumatic brain and spinal cord injuries, extensive burns in the civilian setting, and combat-related extremity injuries in the battlefield. Irrespective of the form of trauma, heterotopic bone is typically endochondral in structure and is laid down via a cartilaginous matrix. Once formed, the heterotopic bone typically needs to be excised surgically, which may result in wound healing complications, in addition to a risk of recurrence. Refinements of existing diagnostic modalities, like micro- and nano-CT are being adapted toward early intervention. Trauma-induced HO is a consequence of aberrant wound healing, systemic and local immune system activation, infections, extensive vascularization, and innervation. This intricate molecular crosstalk culminates in activation of stem cells that initiate heterotopic endochondral ossification. Development of animal models recapitulating the unique traumatic injuries has greatly facilitated the mechanistic understanding of trauma-induced HO. These same models also serve as powerful tools to test the efficacy of small molecules which specifically target the molecular pathways underlying ectopic ossification. This review summarizes the recent advances in the molecular understanding, diagnostic and treatment modalities in the field of trauma-induced HO.

The involvement and possible mechanism of pro-inflammatory tumor necrosis factor alpha (TNF-α) in thoracic ossification of the ligamentum flavum.

Thoracic ossification of the ligamentum flavum (TOLF) is characterized by ectopic bone formation in the ligamentum flavum and is considered to be a leading cause of thoracic spinal canal stenosis and myelopathy. However, the underlying etiology is not well understood. An iTRAQ proteomics was used to reveal the involvement of inflammation factors in TOLF. TNF-α is a pro-inflammatory cytokine implicated in the pathogenesis of many human diseases. Protein profiling analysis showed that the protein level of TNF-α increased in the ossified ligamentum flavum of TOLF, which was confirmed by western blot. The effects of TNF-α on primary ligamentum flavum cells was examined. Cell proliferation assay demonstrated that primary cells from the ossified ligamentum flavum of TOLF grew faster than the control. Flow cytometry assay indicated that the proportions of cells in S phase of cell cycle of primary cells increased after TNF-α stimulation. To address the effect of TNF-α on gene expression, primary cells were derived from ligamentum flavum of TOLF patients. Culture cells were stimulated by TNF-α. RNA was isolated and analyzed by quantitative RT-PCR. G1/S-specific proteins cyclin D1 and c-Myc were upregulated after TNF-α stimulation. On the other hand, osteoblast differentiation related genes such as Bmp2 and Osterix (Osx) were upregulated in the presence of TNF-α. TNF-α activated Osx expression in a dose-dependent manner. Interestingly, a specific mitogen-activated protein kinase ERK inhibitor U0126, but not JNK kinase inhibitor SP600125, abrogated TNF-α activation of Osx expression. This suggests that TNF-α activates Osx expression through the mitogen-activated protein kinase ERK pathway. Taken together, we provide the evidence to support that TNF-α involves in TOLF probably through regulating cell proliferation via cyclin D1 and c-Myc, and promoting osteoblast differentiation via Osx.

The role of the adaptive immune system in burn-induced heterotopic ossification and mesenchymal cell osteogenic differentiation.

BACKGROUND: Heterotopic ossification (HO) is the pathologic process of extraskeletal bone formation. Although the exact etiology remains unknown, inflammation appears to catalyze disease progression. The goal of this study is to determine the impact of the adaptive immune system on HO. METHODS: HO was induced in 8-wk-old control C57BL/6 and immunocompromised Rag1tm1Mom (Rag1 KO) male mice deficient in B- and T-lymphocytes via combined Achilles tenotomy and burn injury. Microcomputed tomography quantified the extent of HO formation at the tenotomy site. Adipose-derived mesenchymal stem cells were harvested to evaluate osteogenic differentiation potential. RESULTS: Areas of developing HO demonstrated substantial enrichment of CD45 + leukocytes at 3 wk after injury. HO from Rag1 KO mice was substantially less mature with foci of cartilage and disorganized trabecular bone present 12 wk after injury. Rag1 KO mice formed 60% less bone compared to immunocompetent controls (4.67 ± 1.5 mm versus 7.76 ± 0.65 mm; P = 0.001). Tartrate-resistant acid phosphatase staining and immunofluorescent analysis of osteoprotegerin and nuclear factor kappa-light-chain-enhancer of activated B cells demonstrated no appreciable difference in osteoclast number or activation. Alizarin red staining in vitro demonstrated a significant decrease in osteogenic potential in immunocompromised mice compared to controls (29.1 ± 0.54 mm versus 12.1 ± 0.14 mm; P < 0.001). CONCLUSIONS: We demonstrate a prominent role for the adaptive immune system in the development of HO. In the absence of mature B- and T-lymphocytes, HO growth and development are attenuated. Furthermore, we demonstrate that mesenchymal populations from B- and T-cell deficient mice are inherently less osteogenic. This study identifies a potential therapeutic role for modulation of the adaptive immune system in the treatment of HO.

Trauma-induced heterotopic bone formation and the role of the immune system: A review.

Extremity trauma, spinal cord injuries, head injuries, and burn injuries place patients at high risk of pathologic extraskeletal bone formation. This heterotopic bone causes severe pain, deformities, and joint contractures. The immune system has been increasingly implicated in this debilitating condition. This review summarizes the various roles immune cells and inflammation play in the formation of ectopic bone and highlights potential areas of future investigation and treatment. Cell types in both the innate and adaptive immune system such as neutrophils, macrophages, mast cells, B cells, and T cells have all been implicated as having a role in ectopic bone formation through various mechanisms. Many of these cell types are promising areas of therapeutic investigation for potential treatment. The immune system has also been known to also influence osteoclastogenesis, which is heavily involved in ectopic bone formation. Chronic inflammation is also known to have an inhibitory role in the formation of ectopic bone, whereas acute inflammation is necessary for ectopic bone formation.

Granting immunity to FOP and catching heterotopic ossification in the Act.

The progressive transformation of one organ system into another is a fundamental signature of fibrodysplasia ossificans progressiva (FOP), the most catastrophic form of extraskeletal bone formation in humans. In all affected individuals, FOP is caused by heterozygous missense gain-of-function mutations in Activin receptor A type I (ACVR1), a bone morphogenetic protein (BMP) type I receptor. Loss of autoinhibition of the mutant receptor (mACVR1) results in dysregulated BMP pathway signaling, and is necessary for the myriad developmental features of FOP, but does not appear sufficient to induce the episodic flare-ups that lead to disabling post-natal heterotopic endochondral ossification (HEO) and that are a hallmark of the disease. Post-natal FOP flare-ups strongly implicate an underlying immunological trigger involving inflammation and the innate immune system. Recent studies implicate canonical and non-canonical TGFß/BMP family ligands in the amplification of mACVR1 signaling leading to the formation of FOP lesions and resultant HEO. BMP and Activin ligands that stimulate mACVR1 signaling also have critical regulatory functions in the immune system. Cross-talk between the morphogenetic and immunological pathways that regulate tissue maintenance and wound healing identifies potential robust therapeutic targets for FOP. Here we review current evidence for an immunological trigger for flare-ups and HEO in FOP, propose a working schema for the pathophysiology of observed phenomena, and highlight outstanding questions under investigation.

Herniated intervertebral disk induces hypertrophy and ossification of ligamentum flavum.

STUDY DESIGN: In vitro experiment using degenerated human ligamentum flavum (LF) and herniated intervertebral disk (IVD). OBJECTIVES: To investigate the role and effect of degenerated and herniated IVDs on LF hypertrophy and ossification. SUMMARY OF BACKGROUND DATA: Spinal stenosis is caused, in part, by hypertrophy and ossification of the LF, which are induced by aging and degenerative process. It is well known that degenerated IVDs spontaneously produce inflammatory cytokines. Therefore, we hypothesized that degenerated IVD may affect adjacent LF through secreted inflammatory cytokines. METHODS: LF and herniated lumbar IVD tissues were obtained during surgical spinal procedures. LF fibroblasts were isolated by enzymatic digestion of LF tissue. LF cell cultures were treated with disk supernatant from herniated IVDs. Secreted cytokines from IVD tissue culture were detected by enzyme-linked immunosorbent assay. After analysis of cytotoxicity, DNA synthesis was measured. Reverse transcription-polymerase chain reaction for mRNA expressions of types I, II, III, V, and XI collagen and osteocalcin, and histochemical stains were performed. RESULTS: Supernatant from tissue culture of herniated IVD showed increased production of interleukin-1α, interleukin-6, tumor necrosis factor-α, prostaglandin E2, and nitric oxide compared with disk tissue culture from traumatic condition. There was no cytotoxicity in LF cells treated with disk supernatant from herniated IVDs. There was significant increase in DNA synthesis, upregulation in mRNA expression of types III, XI collagen and osteocalcin, whereas variable expression pattern of type I and V, and strong positive stains for Von Kossa and alkaline phosphatase in LF cultures with disk supernatant. CONCLUSIONS: Degenerated and herniated IVDs provide an important pathomechanism in hypertrophy and ossification of the LF through inflammatory cytokines.

Exaggerated inflammatory environment decreases BMP-2/ACS-induced ectopic bone mass in a rat model: implications for clinical use of BMP-2.

OBJECTIVE: Numerous recent reports have observed a low osteoinductive efficacy property of bone morphogenetic protein-2 (BMP-2) and disappointing long-term outcomes in clinical cases. An alternative hypothesis, that these observations are caused by an exaggerated inflammatory environment, needs experimental evidence. METHOD: Thirty-seven Sprague Dawley (SD) rats were administrated with Lipopolysaccharide (LPS) injections and BMP-2/absorbable collagen sponge (ACS) implantation to respectively mimic pre-operative and post-operative inflammatory responses. Blood samples and BMP-2/ACS implants were analyzed by enzyme-linked immunosorbent assay (ELISA), real-time polymerase chain reaction (PCR), micro-computed tomography (µCT) and histological examination. RESULTS: LPS injections and BMP-2/ACS implantation provoked a significant elevation of inflammatory cytokines in serum and an obvious infiltration of inflammatory cells around BMP-2/ACS implants. The bone volume, mineral content and mineral density of the BMP-2/ACS implants from LPS-injected rats were significantly decreased, indicating that attenuated BMP-2-induced bone mass might be associated with down-regulated bone formation activity and up-regulated bone resorption activity. Furthermore, histological examination of the rhBMP-2/ACS implants showed a decreased expression of osteocalcin (OCN) and an increased number of osteoclasts in LPS-injected rats at 8 weeks; the expression level of bone turnover markers in serum and BMP-2/ACS implants revealed inhibited osteoblastogenesis activity and activated osteoclastogenesis activity in LPS-injected rats. Among the top three elevated pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) showed a suppressive effect on BMP-2-induced osteoblastic differentiation in vitro. CONCLUSION: These data indicate that an exaggerated inflammatory environment may decrease BMP-2/ACS-induced bone mass in vivo by suppressing BMP-2-induced osteoblastic differentiation and by increasing the number or activity of osteoclasts. The negative role of exaggerated inflammation deserves consideration for future clinical use of BMP-2 in inducing bone regeneration.

Inflammatory cytokines induce fibrosis and ossification of human ligamentum flavum cells.

STUDY DESIGN: In vitro experiment using degenerated human ligamentum flavum (LF) and various inflammatory cytokines. OBJECTIVES: To examine the effect of inflammatory cytokines on LF cells and to identify their roles in the pathogenesis of LF hypertrophy and ossification. SUMMARY OF BACKGROUND DATA: Spinal stenosis is caused, in part, by hypertrophy and ossification of the LF, which are induced by the degenerative processes (ie, increased collagen synthesis and chondroid metaplasia) of ligament fibroblasts. Degenerated intervertebral disk spontaneously produces inflammatory cytokines, which might affect the adjacent LF through local milieu of the spinal canal. METHODS: The interlaminar portion of the LF was collected during surgical spinal procedures in 15 patients (age range, 49-78 y) with lumbar spinal stenosis. LF fibroblasts were isolated by enzymatic digestion of LF tissue. LF cell cultures were treated with various inflammatory cytokines: interleukin (IL)-1α, IL-6, tumor necrosis factor-α (TNF-α), prostaglandin E2 (PGE2), and nitric oxide (NO). Cytotoxicity was analyzed by MTT assays. DNA synthesis was measured with H-thymidine incorporation, and mRNA expression of types I, III, V, and XI collagen and osteocalcin were performed by reverse transcription-polymerase chain reaction. Histochemical stains such as Von Kossa were also performed to detect bone nodule formation. RESULTS: There was no cytotoxicity in the LF cells treated with each cytokine. There were significant increases in DNA synthesis and upregulated mRNA expression of types I, V, XI collagen and osteocalcin in LF cultures treated with various cytokines. LF cultures treated with IL-6, TNF-α, PGE2, and NO showed positive Von Kossa staining, indicating bone nodule formation from LF cells. CONCLUSIONS: Inflammatory cytokines (IL-6, TNF-α, PGE2, and NO) seem to play a crucial role in hypertrophy and ossification of LF. Degenerated, herniated intervertebral disks, and facet arthrosis may influence LF through inflammatory cytokines and cause hypertrophy and ossification of LF.

Inflammatory cytokine and chemokine expression is associated with heterotopic ossification in high-energy penetrating war injuries.

OBJECTIVE: Heterotopic ossification (HO) develops frequently after modern high-energy penetrating war injuries. The purpose of this prospective study was to identify and characterize the unique cytokine and chemokine profile associated with the development of HO as it pertained to the systemic inflammatory response after penetrating combat-related trauma. METHODS: Patients with high-energy penetrating extremity wounds were prospectively enrolled. Surgical debridement along with the use of a pulse lavage and vacuum-assisted-closure device was performed every 48-72 hours until definitive wound closure. Wound bed tissue biopsy, wound effluent, and serum were collected before each debridement. Effluent and serum were analyzed for 22 relevant cytokines and chemokines. Tissue was analyzed quantitatively for bacterial colonization. Correlations between specific wound and patient characteristics were also analyzed. The primary clinical outcome measure was the formation of HO as confirmed by radiographs at a minimum of 2 months of follow-up. RESULTS: Thirty-six penetrating extremity war wounds in 24 patients were investigated. The observed rate of HO in the study population was 38%. Of the 36 wounds, 13 (36%) demonstrated HO at a minimum follow-up of 2 months. An elevated injury severity score was associated with the development of HO (P = 0.006). Wound characteristics that correlated with the development of HO included impaired healing (P = 0.005) and bacterial colonization (P < 0.001). Both serum (interleukin-6, interleukin-10, and MCP-1) and wound effluent (IP-10 and MIP-1α) cytokine and chemokine bioprofiles were individually associated and suggestive of the development of HO (P < 0.05). CONCLUSIONS: A severe systemic and wound-specific inflammatory state as evident by elevated levels of inflammatory cytokines, elevated injury severity score, and bacterial wound colonization is associated with the development of HO. These findings suggest that the development of HO in traumatic combat-related wounds is associated with a hyper-inflammatory systemic response to injury. LEVEL OF EVIDENCE: Prognostic Level II. See Instructions for Authors for a complete description of levels of evidence.

[Calcification and ossification of lungs].

The different types of lung calcification and ossification including idiopathic and developing after various diseases are in the article. We have described rare lung pathology - pulmonary alveolar microlithiasis. We have drawn attention to the possible mechanisms of lung calcification and ossification development.