scholarly journals Treatment of heterotopic ossification through remote ATP hydrolysis

2014 ◽  
Vol 6 (255) ◽  
pp. 255ra132-255ra132 ◽  
Author(s):  
Jonathan R. Peterson ◽  
Sara De La Rosa ◽  
Oluwatobi Eboda ◽  
Katherine E. Cilwa ◽  
Shailesh Agarwal ◽  
...  
Keyword(s):  
Heterotopic Ossification ◽  
Soft Tissues ◽  
Atp Hydrolysis ◽  
Burn Injury ◽  
In Vitro Assays ◽  
Osteogenic Potential ◽  
Heterotopic Bone ◽  
Heterotopic Bone Formation

Heterotopic ossification (HO) is the pathologic development of ectopic bone in soft tissues because of a local or systemic inflammatory insult, such as burn injury or trauma. In HO, mesenchymal stem cells (MSCs) are inappropriately activated to undergo osteogenic differentiation. Through the correlation of in vitro assays and in vivo studies (dorsal scald burn with Achilles tenotomy), we have shown that burn injury enhances the osteogenic potential of MSCs and causes ectopic endochondral heterotopic bone formation and functional contractures through bone morphogenetic protein–mediated canonical SMAD signaling. We further demonstrated a prevention strategy for HO through adenosine triphosphate (ATP) hydrolysis at the burn site using apyrase. Burn site apyrase treatment decreased ATP, increased adenosine 3′,5′-monophosphate, and decreased phosphorylation of SMAD1/5/8 in MSCs in vitro. This ATP hydrolysis also decreased HO formation and mitigated functional impairment in vivo. Similarly, selective inhibition of SMAD1/5/8 phosphorylation with LDN-193189 decreased HO formation and increased range of motion at the injury site in our burn model in vivo. Our results suggest that burn injury–exacerbated HO formation can be treated through therapeutics that target burn site ATP hydrolysis and modulation of SMAD1/5/8 phosphorylation.

scholarly journals Engineered osteoclasts as living treatment materials for heterotopic ossification therapy

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Wenjing Jin ◽  
Xianfeng Lin ◽  
Haihua Pan ◽  
Chenchen Zhao ◽  
Pengcheng Qiu ◽  
...  
Keyword(s):  
Heterotopic Ossification ◽  
Soft Tissues ◽  
Ectopic Calcification ◽  
Calcified Tissue ◽  
Restricted Activity ◽  
Calcium Minerals ◽  
Reliable Methods ◽  
Poor Adhesion

AbstractOsteoclasts (OCs), the only cells capable of remodeling bone, can demineralize calcium minerals biologically. Naive OCs have limitations for the removal of ectopic calcification, such as in heterotopic ossification (HO), due to their restricted activity, migration and poor adhesion to sites of ectopic calcification. HO is the formation of pathological mature bone within extraskeletal soft tissues, and there are currently no reliable methods for removing these unexpected calcified plaques. In the present study, we develop a chemical approach to modify OCs with tetracycline (TC) to produce engineered OCs (TC-OCs) with an enhanced capacity for targeting and adhering to ectopic calcified tissue due to a broad affinity for calcium minerals. Unlike naive OCs, TC-OCs are able to effectively remove HO both in vitro and in vivo. This achievement indicates that HO can be reversed using modified OCs and holds promise for engineering cells as “living treatment agents” for cell therapy.

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

2021 ◽  
Author(s):  
Lovorka Grgurevic ◽  
Rudjer Novak ◽  
Stela Hrkac ◽  
Grgur Salai ◽  
Simeon Grazio
Keyword(s):  
Heterotopic Ossification ◽  
Female Patient ◽  
Soft Tissues ◽  
Hereditary Disease ◽  
Fibrodysplasia Ossificans Progressiva ◽  
Heterotopic Bone ◽  
Heterotopic Bone Formation ◽  
Cytokine Analysis ◽  
Viral Illness ◽  
First Time

AbstractFibrodyplasia 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.

scholarly journals Melatonin Promotes Heterotopic Ossification Through Regulation of Endothelial-Mesenchymal Transition in Injured Achilles Tendons in Rats

2021 ◽  
Vol 9 ◽  
Author(s):  
Jie Zhang ◽  
Jiajun Tang ◽  
Jie Liu ◽  
Bo Yan ◽  
Bin Yan ◽  
...  
Keyword(s):  
Heterotopic Ossification ◽  
Healing Process ◽  
Marker Gene ◽  
Underlying Mechanism ◽  
Heterotopic Bone ◽  
Mesenchymal Transition ◽  
Gene And Protein Expression ◽  
Endothelial Mesenchymal Transition

Although heterotopic ossification (HO) has been reported to be a common complication of the posttraumatic healing process, the underlying mechanism remains unknown. Endothelial-mesenchymal transition (EndMT) is known to play a role in HO, and our recent study observed that neuroendocrine signals can promote HO by modulating EndMT. Melatonin, a neuroendocrine hormone secreted mainly by the pineal gland, has been documented to perform its function in the skeletal system. This study aimed at describing the expression of melatonin during the formation of HO in rat models of Achilles tendon injury and to further investigate its role in regulating EndMT in HO. Histological staining revealed the expression of melatonin throughout the formation of heterotopic bone in injured Achilles tendons, and the serum melatonin levels were increased after the initial injury. Double immunofluorescence showed that the MT2 melatonin receptor was notably expressed at the sites of injury. Micro-CT showed the enhancement of heterotopic bone volume and calcified areas in rats treated with melatonin. Additionally, our data showed that melatonin induced EndMT in primary rat aortic endothelial cells (RAOECs), which acquired traits including migratory function, invasive function and EndMT and MSC marker gene and protein expression. Furthermore, our data exhibited that melatonin promoted the osteogenic differentiation of RAOECs undergoing EndMT in vitro. Importantly, inhibition of the melatonin-MT2 pathway by using the MT2 selective inhibitor 4-P-PDOT inhibited melatonin-induced EndMT and osteogenesis both in vivo and in vitro. In conclusion, these findings demonstrated that melatonin promoted HO through the regulation of EndMT in injured Achilles tendons in rats, and these findings might provide additional directions for the management of HO.

A higher expression of P2X7 receptor at the site of heterotopic ossification promotes osteogenesis

2021 ◽  
Author(s):  
Shi Cheng ◽  
Pengbin Yin ◽  
Yi Li ◽  
Ming Chen ◽  
Duanyang Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Achilles Tendon ◽  
Heterotopic Ossification ◽  
P2x7 Receptor ◽  
Complex Disease ◽  
Burn Injury ◽  
Heterotopic Bone ◽  
Heterotopic Bone Formation ◽  
Inflammatory Condition ◽  
Ectopic Bone

Abstract Background Heterotopic ossification (HO) refers to a painful and complex disease. Adenosine triphosphate (ATP), as a key modulator of inflammation, is verified to promote the development of HO. However, the mechanism remains to be illustrated. The ionotropic P2X7 receptor (P2X7R) is an ATP-gated ion channel expressed in the majority of stem cells. Here, this paper hypothesizes that P2X7R may be activated by extracellular ATP and promote osteogenesis of stem cells under inflammatory condition, ending up in the formation of ectopic bone. Methods The tenotomy puncture and burn injury-induced HO model was constructed. The expression of P2X7R was found increasing at the site of injured Achilles tendon where HO occurs. Mesenchymal stem cells (MSCs) were cultivated under an inflammatory condition plus Bz-ATP treatment which mimicked a microenvironment of HO site. An induction in P2X7R expression was also observed along with an enhancement of osteogenesis. In addition, an inhibition of P2X7R expression by its specific antagonist successfully reversed the above process. Results P2X7R expression of the Achilles tendon and osteogenic capability of SCs is higher in HOG than in other two groups. Bz-ATP promoted osteogenesis under inflammation condition. BBG impeded the heterotopic bone formation in animal model. Conclusions P2X7R is a crucial mediator in ATP-signaling promotion of HO, blocking which may represent a potential therapeutic target for HO.

scholarly journals BRD4 promotes heterotopic ossification through upregulation of LncRNA MANCR

2021 ◽  
Vol 10 (10) ◽  
pp. 668-676
Author(s):  
Lei Liu ◽  
ZiHao Li ◽  
Siwen Chen ◽  
Haowen Cui ◽  
Xiang Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Formation ◽  
Achilles Tendon ◽  
Heterotopic Ossification ◽  
Soft Tissues ◽  
Quantitative Polymerase Chain Reaction ◽  
Gradient Centrifugation ◽  
Alizarin Red

Aims Acquired heterotopic ossification (HO) is a debilitating disease characterized by abnormal extraskeletal bone formation within soft-tissues after injury. The exact pathogenesis of HO remains unknown. It was reported that BRD4 may contribute to osteoblastic differentiation. The current study aims to determine the role of BRD4 in the pathogenesis of HO and whether it could be a potential target for HO therapy. Methods Achilles tendon puncture (ATP) mouse model was performed on ten-week-old male C57BL/6J mice. One week after ATP procedure, the mice were given different treatments (e.g. JQ1, shMancr). Achilles tendon samples were collected five weeks after treatment for RNA-seq and real-time quantitative polymerase chain reaction (RT-qPCR) analysis; the legs were removed for micro-CT imaging and subsequent histology. Human bone marrow mesenchymal stem cells (hBMSCs) were isolated and purified bone marrow collected during surgeries by using density gradient centrifugation. After a series of interventions such as knockdown or overexpressing BRD4, Alizarin red staining, RT-qPCR, and Western Blot (Runx2, alkaline phosphatase (ALP), Osx) were performed on hBMSCs. Results Overexpression of BRD4 enhanced while inhibition of Brd4 suppressed the osteogenic differentiation of hBMSCs in vitro. Overexpression of Brd4 increased the expression of mitotically associated long non-coding RNA (Mancr). Downregulation of Mancr suppressed the osteoinductive effect of BRD4. In vivo, inhibition of BRD4 by JQ1 significantly attenuated pathological bone formation in the ATP model (p = 0.001). Conclusion BRD4 was found to be upregulated in HO and Brd4-Mancr-Runx2 signalling was involved in the modulation of new bone formation in HO. Cite this article: Bone Joint Res 2021;10(10):668–676.

scholarly journals 3′-End Formation of Baculovirus Late RNAs

2000 ◽  
Vol 74 (19) ◽  
pp. 8930-8937 ◽  
Author(s):  
Jianping Jin ◽  
Linda A. Guarino
Keyword(s):  
Rna Polymerase ◽  
Atp Hydrolysis ◽  
Ternary Complexes ◽  
Polyadenylation Signal ◽  
In Vitro Assays ◽  
Rich Region ◽  
Viral Mrnas ◽  
Viral Rnas

ABSTRACT Baculovirus late RNAs are transcribed by a four-subunit RNA polymerase that is virus encoded. The late viral mRNAs are capped and polyadenylated, and we have previously shown that capping is mediated by the LEF-4 subunit of baculovirus RNA polymerase. Here we report studies undertaken to determine the mechanism of 3′-end formation. A globin cleavage/polyadenylation signal, which was previously shown to direct 3′-end formation of viral RNAs in vivo, was cloned into a baculovirus transcription template. In vitro assays with purified baculovirus RNA polymerase revealed that 3′ ends were formed not by a cleavage mechanism but rather by termination after transcription of a T-rich region of the globin sequence. Terminated RNAs were released from ternary complexes and were subsequently polyadenylated. Mutational analyses indicated that the T-rich sequence was essential for termination and polyadenylation, but the poly(A) signal and the GT-rich region of the globin polyadenylation/cleavage signal were not required. Termination was not dependent on ATP hydrolysis, indicating a slippage mechanism.

Biological and biomedical applications of collagenous biomaterials

1990 ◽  
Vol 48 (3) ◽  
pp. 856-857
Author(s):  
Yasushi P. Kato ◽  
Michael G. Dunn ◽  
Frederick H. Silver ◽  
Arthur J. Wasserman
Keyword(s):  
Biomedical Applications ◽  
Soft Tissues ◽  
Collagen Fibers ◽  
Bone Collagen ◽  
Cover Slip ◽  
Fibroblast Migration ◽  
Cellular Expression ◽  
Defect Repair

Collagenous biomaterials have been used for growing cells in vitro as well as for augmentation and replacement of hard and soft tissues. The substratum used for culturing cells is implicated in the modulation of phenotypic cellular expression, cellular orientation and adhesion. Collagen may have a strong influence on these cellular parameters when used as a substrate in vitro. Clinically, collagen has many applications to wound healing including, skin and bone substitution, tendon, ligament, and nerve replacement. In this report we demonstrate two uses of collagen. First as a fiber to support fibroblast growth in vitro, and second as a demineralized bone/collagen sponge for radial bone defect repair in vivo.For the in vitro study, collagen fibers were prepared as described previously. Primary rat tendon fibroblasts (1° RTF) were isolated and cultured for 5 days on 1 X 15 mm sterile cover slips. Six to seven collagen fibers, were glued parallel to each other onto a circular cover slip (D=18mm) and the 1 X 15mm cover slip populated with 1° RTF was placed at the center perpendicular to the collagen fibers. Fibroblast migration from the 1 x 15mm cover slip onto and along the collagen fibers was measured daily using a phase contrast microscope (Olympus CK-2) with a calibrated eyepiece. Migratory rates for fibroblasts were determined from 36 fibers over 4 days.

A in Vivo Assay for Standardizing Heparin Preparations

1979 ◽  
Vol 41 (03) ◽  
pp. 576-582
Author(s):  
A R Pomeroy
Keyword(s):  
In Vitro Assays ◽  
British Pharmacopoeia ◽  
In Vitro Method ◽  
Standard Preparation ◽  
Reliable Estimation ◽  
Assay Procedure ◽  
Accuracy And Precision ◽  
Heparin Preparation

SummaryThe limitations of currently used in vitro assays of heparin have demonstrated the need for an in vivo method suitable for routine use.The in vivo method which is described in this paper uses, for each heparin preparation, four groups of five mice which are injected intravenously with heparin according to a “2 and 2 dose assay” procedure. The method is relatively rapid, requiring 3 to 4 hours to test five heparin preparations against a standard preparation of heparin. Levels of accuracy and precision acceptable for the requirements of the British Pharmacopoeia are obtained by combining the results of 3 to 4 assays of a heparin preparation.The similarity of results obtained the in vivo method and the in vitro method of the British Pharmacopoeia for heparin preparations of lung and mucosal origin validates this in vivo method and, conversely, demonstrates that the in vitro method of the British Pharmacopoeia gives a reliable estimation of the in vivo activity of heparin.

Potentially Thrombogenic Materials in Factor IX Concentrates

1975 ◽  
Vol 33 (03) ◽  
pp. 617-631 ◽  
Author(s):  
H. S Kingdon ◽  
R. L Lundblad ◽  
J. J Veltkamp ◽  
D. L Aronson
Keyword(s):  
Human Plasma ◽  
Antithrombin Iii ◽  
Factor Ix ◽  
In Vitro Assays ◽  
Substantial Agreement ◽  
Coefficient Of Correlation

SummaryFactor IX concentrates manufactured from human plasma and intended for therapeutic infusion in man have been suspected for some time of being potentially thrombogenic. In the current studies, assays were carried out in vitro and in vivo for potentially thrombogenic materials. It was possible to rank the various materials tested according to the amount of thrombogenic material detected. For concentrates not containing heparin, there was substantial agreement between the in vivo and in vitro assays, with a coefficient of correlation of 0.77. There was no correlation between the assays for thrombogenicity and the antithrombin III content. We conclude that many presently available concentrates of Factor IX contain substantial amounts of potentially thrombogenic enzymes, and that this fact must be considered in arriving at the decision whether or not to use them therapeutically.

In-silico Studies of Isolated Phytoalkaloid Against Lipoxygenase: Study Based on Possible Correlation

2018 ◽  
Vol 21 (3) ◽  
pp. 215-221
Author(s):  
Haroon Khan ◽  
Muhammad Zafar ◽  
Helena Den-Haan ◽  
Horacio Perez-Sanchez ◽  
Mohammad Amjad Kamal
Keyword(s):  
In Silico ◽  
Docking Studies ◽  
In Vivo Studies ◽  
In Vitro Assays ◽  
Chronic Obstructive ◽  
Lipoxygenase Inhibitors ◽  
Lipoxygenase Inhibition ◽  
In Silico Studies

Aim and Objective: Lipoxygenase (LOX) enzymes play an important role in the pathophysiology of several inflammatory and allergic diseases including bronchial asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, rheumatoid arthritis and chronic obstructive pulmonary disease. Inhibitors of the LOX are believed to be an ideal approach in the treatment of diseases caused by its over-expression. In this regard, several synthetic and natural agents are under investigation worldwide. Alkaloids are the most thoroughly investigated class of natural compounds with outstanding past in clinically useful drugs. In this article, we have discussed various alkaloids of plant origin that have already shown lipoxygenase inhibition in-vitro with possible correlation in in silico studies. Materials and Methods: Molecular docking studies were performed using MOE (Molecular Operating Environment) software. Among the ten reported LOX alkaloids inhibitors, derived from plant, compounds 4, 2, 3 and 1 showed excellent docking scores and receptor sensitivity. Result and Conclusion: These compounds already exhibited in vitro lipoxygenase inhibition and the MOE results strongly correlated with the experimental results. On the basis of these in vitro assays and computer aided results, we suggest that these compounds need further detail in vivo studies and clinical trial for the discovery of new more effective and safe lipoxygenase inhibitors. In conclusion, these results might be useful in the design of new and potential lipoxygenase (LOX) inhibitors.

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