Engineered osteoclasts as living treatment materials for heterotopic ossification therapy

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.

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BRD4 promotes heterotopic ossification through upregulation of LncRNA MANCR

Bone and Joint Research ◽

10.1302/2046-3758.1010.bjr-2020-0454.r1 ◽

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.

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Treatment of heterotopic ossification through remote ATP hydrolysis

Science Translational Medicine ◽

10.1126/scitranslmed.3008810 ◽

2014 ◽

Vol 6 (255) ◽

pp. 255ra132-255ra132 ◽

Cited By ~ 77

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.

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Biological and biomedical applications of collagenous biomaterials

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100161849 ◽

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.

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Ultra-strong bio-glue from genetically engineered polypeptides

Nature Communications ◽

10.1038/s41467-021-23117-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Chao Ma ◽

Jing Sun ◽

Bo Li ◽

Yang Feng ◽

Yao Sun ◽

...

Keyword(s):

Soft Tissues ◽

Covalent Bond ◽

Genetically Engineered ◽

Supramolecular Interactions ◽

Molar Ratio ◽

High Adhesion ◽

Strong Adhesion ◽

Order Of Magnitude

AbstractThe development of biomedical glues is an important, yet challenging task as seemingly mutually exclusive properties need to be combined in one material, i.e. strong adhesion and adaption to remodeling processes in healing tissue. Here, we report a biocompatible and biodegradable protein-based adhesive with high adhesion strengths. The maximum strength reaches 16.5 ± 2.2 MPa on hard substrates, which is comparable to that of commercial cyanoacrylate superglue and higher than other protein-based adhesives by at least one order of magnitude. Moreover, the strong adhesion on soft tissues qualifies the adhesive as biomedical glue outperforming some commercial products. Robust mechanical properties are realized without covalent bond formation during the adhesion process. A complex consisting of cationic supercharged polypeptides and anionic aromatic surfactants with lysine to surfactant molar ratio of 1:0.9 is driven by multiple supramolecular interactions enabling such strong adhesion. We demonstrate the glue’s robust performance in vitro and in vivo for cosmetic and hemostasis applications and accelerated wound healing by comparison to surgical wound closures.

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MyD88 Is Not Required for Muscle Injury-Induced Endochondral Heterotopic Ossification in a Mouse Model of Fibrodysplasia Ossificans Progressiva

Biomedicines ◽

10.3390/biomedicines9060630 ◽

2021 ◽

Vol 9 (6) ◽

pp. 630

Author(s):

Huili Lyu ◽

Cody M. Elkins ◽

Jessica L. Pierce ◽

C. Henrique Serezani ◽

Daniel S. Perrien

Keyword(s):

Heterotopic Ossification ◽

Muscle Injury ◽

Cell Types ◽

Fibrodysplasia Ossificans Progressiva ◽

Inflammatory Signaling ◽

Conditional Deletion ◽

Pathway Crosstalk ◽

Multiple Cell

Excess inflammation and canonical BMP receptor (BMPR) signaling are coinciding hallmarks of the early stages of injury-induced endochondral heterotopic ossification (EHO), especially in the rare genetic disease fibrodysplasia ossificans progressiva (FOP). Multiple inflammatory signaling pathways can synergistically enhance BMP-induced Smad1/5/8 activity in multiple cell types, suggesting the importance of pathway crosstalk in EHO and FOP. Toll-like receptors (TLRs) and IL-1 receptors mediate many of the earliest injury-induced inflammatory signals largely via MyD88-dependent pathways. Thus, the hypothesis that MyD88-dependent signaling is required for EHO was tested in vitro and in vivo using global or Pdgfrα-conditional deletion of MyD88 in FOP mice. As expected, IL-1β or LPS synergistically increased Activin A (ActA)-induced phosphorylation of Smad 1/5 in fibroadipoprogenitors (FAPs) expressing Alk2R206H. However, conditional deletion of MyD88 in Pdgfrα-positive cells of FOP mice did not significantly alter the amount of muscle injury-induced EHO. Even more surprisingly, injury-induced EHO was not significantly affected by global deletion of MyD88. These studies demonstrate that MyD88-dependent signaling is dispensable for injury-induced EHO in FOP mice.

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In Vitro and In Vivo Effects of Tetrakisphosphonates on Bone Resorption, Tumor Osteolysis, Ectopic Calcification, and Macrophages

Journal of Pharmaceutical Sciences ◽

10.1021/js960429h ◽

1997 ◽

Vol 86 (3) ◽

pp. 283-289 ◽

Cited By ~ 9

Author(s):

Joel M. Van Gelder ◽

Eli Breuer ◽

Ada Schlossman ◽

Asher Ornoy ◽

Jukka Mönkkönen ◽

...

Keyword(s):

Bone Resorption ◽

Ectopic Calcification ◽

Tumor Osteolysis ◽

In Vivo Effects

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Cervical Spine Mechanism for Reproduction of the Biomechanical Behaviours of the Human Neck during Rotation-Traction Manipulation

Applied Bionics and Biomechanics ◽

10.1155/2017/5829048 ◽

2017 ◽

Vol 2017 ◽

pp. 1-14

Author(s):

Yuancan Huang ◽

Shuai Li ◽

Minshan Feng ◽

Liguo Zhu

Keyword(s):

Cervical Spine ◽

Soft Tissues ◽

Cervical Vertebrae ◽

High Rate ◽

Cervical Injury ◽

System A ◽

Mass Spring ◽

Electromagnetic Clutch

Rotation-traction (RT) manipulation is a commonly used physical therapy procedure in TCM (traditional Chinese medicine) for cervical spondylosis. This procedure temporarily separates the C3 and C4 cervical vertebrae from each other when a physician applies a jerky action while the neck is voluntarily turned by the patient to a specific position as instructed by the physician, where the cervical vertebrae are twisted and locked. However, a high rate of cervical injury occurs due to inexperienced physician interns who lack sufficient training. Therefore, we developed a cervical spine mechanism that imitates the dynamic behaviours of the human neck during RT manipulation. First, in vivo and in vitro experiments were performed to acquire the biomechanical feature curves of the human neck during RT manipulation. Second, a mass-spring-damper system with an electromagnetic clutch was designed to emulate the entire dynamic response of the human neck. In this system, a spring is designed as rectilinear and nonlinear to capture the viscoelasticity of soft tissues, and an electromagnetic clutch is used to simulate the sudden disengagement of the cervical vertebrae. Test results show that the mechanism can exhibit the desired behaviour when RT manipulation is applied in the same manner as on humans.

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PEG-modified gadolinium nanoparticles as contrast agents for in vivo micro-CT

Scientific Reports ◽

10.1038/s41598-021-95716-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Charmainne Cruje ◽

P. Joy Dunmore-Buyze ◽

Eric Grolman ◽

David W. Holdsworth ◽

Elizabeth R. Gillies ◽

...

Keyword(s):

Contrast Agent ◽

Contrast Agents ◽

Soft Tissues ◽

Three Dimensional ◽

Blood Pool ◽

Micro Ct ◽

Micro Computed Tomography ◽

Poly Ethylene

AbstractVascular research is largely performed in rodents with the goal of developing treatments for human disease. Micro-computed tomography (micro-CT) provides non-destructive three-dimensional imaging that can be used to study the vasculature of rodents. However, to distinguish vasculature from other soft tissues, long-circulating contrast agents are required. In this study, we demonstrated that poly(ethylene glycol) (PEG)-coated gadolinium nanoparticles can be used as a vascular contrast agent in micro-CT. The coated particles could be lyophilized and then redispersed in an aqueous solution to achieve 100 mg/mL of gadolinium. After an intravenous injection of the contrast agent into mice, micro-CT scans showed blood pool contrast enhancements of at least 200 HU for 30 min. Imaging and quantitative analysis of gadolinium in tissues showed the presence of contrast agent in clearance organs including the liver and spleen and very low amounts in other organs. In vitro cell culture experiments, subcutaneous injections, and analysis of mouse body weight suggested that the agents exhibited low toxicity. Histological analysis of tissues 5 days after injection of the contrast agent showed cytotoxicity in the spleen, but no abnormalities were observed in the liver, lungs, kidneys, and bladder.

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Biogenesis, Biologic Function and Clinical Potential of Exosomes in Different Diseases

Applied Sciences ◽

10.3390/app10134428 ◽

2020 ◽

Vol 10 (13) ◽

pp. 4428 ◽

Cited By ~ 1

Author(s):

Amany Magdy Beshbishy ◽

Saad Alghamdi ◽

ThankGod E. Onyiche ◽

Muhammad Zahoor ◽

Nallely Rivero-Perez ◽

...

Keyword(s):

Soft Tissues ◽

Protein Quality ◽

Therapeutic Effects ◽

Exosome Biogenesis ◽

Wide Range ◽

Potential Therapeutic Application ◽

Intercellular Movement ◽

Clinical Potential

Exosomes are extracellular vesicles (EVs) belonging to the nanovesicles family that function as signaling molecules between cells. After their first description in the late 1960s, interest in their potential as a research target has steadily increased. They are small secreted organelles with a single membrane that are well enriched in lipids, proteins, nucleic acids, and glycoconjugates. Exosomes take part in a larger communication network in which cells communicate between one another by DNA shuttling, proteins, RNA, and membrane-bound factors. The machinery of protein quality control occurs through the process termed “exosome biogenesis”. Furthermore, the pathway involved in intercellular movement of vesicles is vital in various aspects of human health and diseases. Due to their inherent properties, exosomes are currently being developed as potential therapeutic agents in a wide range of diseases including infectious and non-infectious diseases. Exosomes and other EVs sourced from Mesenchymal stem cells (MSCs) have been shown in different studies to possess therapeutic effects in diverse disease models either in vivo or in vitro. Some mechanisms and/or pathways that MSC-derived exosomes use to illustrate their therapeutic effect against some diseases have also been summarized. This review aims to highlight the recent findings and potential therapeutic application of exosomes in different diseases such as autoimmune, cardiovascular, obesity, neural, soft tissues, bone, and cartilage.

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Induced Osteogenesis in Plants Decellularized Scaffolds

Scientific Reports ◽

10.1038/s41598-019-56651-0 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 5

Author(s):

Jennifer Lee ◽

Hyerin Jung ◽

Narae Park ◽

Sung-Hwan Park ◽

Ji Hyeon Ju

Keyword(s):

Three Dimensional ◽

3D Culture ◽

Specific Staining ◽

3D Culture System ◽

Calcified Tissue ◽

Decellularized Scaffolds ◽

Cellular Markers ◽

Rat Calvarial Defect

AbstractA three-dimensional (3D) culture system that closely replicates the in vivo microenvironment of calcifying osteoid is essential for in vitro cultivation of bone-like material. In this regard, the 3D cellulose constructs of plants may well serve as scaffolds to promote growth and differentiation of osteoblasts in culture. Our aim in this study was to generate bone-like tissue by seeding pluripotent stem cells (hiPSCs), stimulated to differentiate as osteoblasts in culture, onto the decellularised scaffolds of various plants. We then assessed expression levels of pertinent cellular markers and degrees of calcium-specific staining to gauge technical success. Apple scaffolding bearing regular pores of 300 μm seemed to provide the best construct. The bone-like tissue thus generated was implantable in a rat calvarial defect model where if helped form calcified tissue. Depending on the regularity and sizing of scaffold pores, this approach readily facilitates production of mineralized bone.

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