Effect Of Combined Platelet Rich Plasma And Mesenchymal Stem Cell Derived Exosomes On Chondrocyte Gene Expression
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| Release | : 2017 |
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INTRODUCTION: Platelet-rich plasma (PRP) and mesenchymal stem cells (MSCs) serve as two of the most popular potential therapies to enhance cartilage regeneration. Platelets contain alpha granules that house a plethora of growth factors, cytokines, and other molecules that may potentiate tissue healing. Some studies posit that MSCs exert regenerative effects via paracrine signaling [1] which may be mediated by exosomes. Defined as extracellular vesicles released from the fusion of a cytosolic multivesicular body with the cell membrane, exosomes have been implicated in intracellular communication and molecular transport [2]. Early evidence suggests that MSC-derived exosomes possess chondroprotective and anti-inflammatory properties [3]. Similarly, PRP has been shown to increase chondrocyte proliferation and gene expression indicative of extracellular matrix (ECM) production [4]. However, no studies have examined the concomitant effects of PRP and MSC-derived exosomes on chondrocyte gene expression in both a neutral and inflammatory environment. It was hypothesized that chondrocytes cultured in a combination of PRP and exosomes would demonstrate enhanced gene expression suggestive of ECM synthesis and decreased inflammation than chondrocytes grown with PRP or exosomes in isolation.METHODS: Articular chondrocytes were harvested from articular surfaces of bovine ankle joints and cultured in DMEM-F12 medium with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic (AA) solution. MSCs were isolated from the bone marrow of Sprague-Dawley rats and cultured in low-glucose DMEM medium with 15% FBS and 1% AA. Both cell lines were incubated with 5% CO2 at 37u00b0C. PRP was obtained from one healthy donor using a Regen PRP kit. Following a previously published protocol [5], exosomes were obtained from MSCs as a 100K pellet. Chondrocytes were seeded at 50,000 cells/well into 24-well plates. They were partitioned into one control (media only) and five experimental conditions: exosomes alone (Exo), 1% unactivated PRP (1u), 5u, combined exosomes and 1% uPRP (Exo1), and Exo5. Exosomes were added to the appropriate treatment groups at a concentration of 10 u03bcg/ml. All conditions were further allocated into one of two environments: with or without IL-1u03b2 (10 ng/ml) to simulate an osteoarthritic or neutral environment respectively. Following 1 and 3 days, RNA was harvested, isolated, and reverse transcribed into cDNA. Quantitative RT-PCR was then performed (n = 3 per condition), using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the endogenous control, for the following target genes: aggrecan (ACAN), sex-determining region Y box 9 (SOX9), alpha-1 chain of type I collagen (Col1), alpha-1 chain of type II collagen (Col2), and matrix metalloproteinase 3 (MMP3), thioredoxin-interacting protein (TXNIP), and proliferating cell nuclear antigen (PCNA). The data were analyzed according to the 2-u0394u0394CT method. Because significant deviations from normality were observed, within-day comparisons across conditions and within-condition comparisons across days were analyzed with Kruskal-Wallis tests followed by Conover-Iman post hoc tests. Due to the large number of comparisons performed, the Benjamini-Hochberg procedure was employed to control the false discovery rate. Significance was determined if p
| Author | : Yaoliang Tang |
| Publisher | : Academic Press |
| Total Pages | : 287 |
| Release | : 2015-09-02 |
| Genre | : Science |
| ISBN | : 0128004975 |
Mesenchymal stem cell-derived exosomes are at the forefront of research in two of the most high profile and funded scientific areas – cardiovascular research and stem cells. Mesenchymal Stem Cell Derived Exosomes provides insight into the biofunction and molecular mechanisms, practical tools for research, and a look toward the clinical applications of this exciting phenomenon which is emerging as an effective diagnostic. Primarily focused on the cardiovascular applications where there have been the greatest advancements toward the clinic, this is the first compendium for clinical and biomedical researchers who are interested in integrating MSC-derived exosomes as a diagnostic and therapeutic tool. - Introduces the MSC-exosome mediated cell-cell communication - Covers the major functional benefits in current MSC-derived exosome studies - Discusses strategies for the use of MSC-derived exosomes in cardiovascular therapies
| Author | : Gunjan Gupta |
| Publisher | : |
| Total Pages | : 137 |
| Release | : 2009 |
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Mesenchymal stem cells (MSCs) have been considered as a promising cell source for cartilage tissue regeneration owing to their intrinsic musculoskeletal differentiation potential. A successful MSC-derived hyaline cartilage tissue should be structurally and functionally similar to native cartilage. During embryonic development morphogens have been shown to play a critical role in cartilage tissue formation. In this study we thus investigated the effect of chondrocyte-MSC interactions in the form of soluble morphogens on chondrogenic differentiation of MSCs using a co-culture system, wherein the hMSCs-laden hydrogels and chondrocytes were not in physical contact but can still exchange cell secreted factors. Healthy chondrocytes and osteoarthritic (OA) chondrocytes were able to induce chondrogenesis of hMSCs as analyzed by biochemical assays, gene analysis, and histology. We further explored the role of PTHrP and TGF-[beta]1 on chondrogenesis of hMSCs and found that in their combined presence, hMSCs had an upregulated expression of cartilage specific markers such as collagen type II and aggrecan and enhanced extracellular matrix production. Even in the absence of TGF-[beta]1 co-culture could induce chondrogenesis. A detailed analysis indicated that TGF-[beta]1 promotes proliferation and chondrogenic differentiation of MSCs, while PTHrP regulates hypertrophy. Mass spectrometry analysis underlined the crucial role of cross-talk on cell secreted morphogens. Extracellular histones inducing growth hormone production formed a major part of the morphogens unique to a co-culture system which is known to promote chondrogenesis. Our experimental observations along with numerical analysis indicated the crucial role of diffusion-mediated morphogenic concentration on chondrogenesis of MSCs.
| Author | : Alexander J. Neumann |
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| Total Pages | : |
| Release | : 2013 |
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Adult articular cartilage has a limited repair capacity. This leads to an increasing demand for optimised repair techniques. Furthermore, current procedures to regenerate articular cartilage fail to achieve sufficient results. Previous work within our group suggested that combination of functional tissue engineering and gene transfer represents a promising alternative approach. In this thesis, different viral gene transfer methods were investigated and optimised. A clinically relevant three dimensional transduction model was developed. These results were directly implemented in further work aiming to investigate the combined effect of multiaxial mechanical stimulation and adenoviral-mediated over-expression of bone morphogenetic protein 2 on human chondroprogenitor cell chondrogenesis and progression towards hypertrophy. Two cell sources were investigated, namely human mesenchymal stem cells and human articular cartilage progenitor cells. The combined approached enhanced human mesenchymal stem cell chondrogenesis. Yet, it was not possible to completely prevent progression towards hypertrophy. For human articular cartilage progenitor cells, over-expression of bone morphogenetic protein 2 did enhance their chondrogenic differentiation potential. However, mechanical stimulation alone, in the absence of exogenous growth factors, led to stable chondrogenic induction without signs of hypertrophic differentiation. This suggests these cells should be further investigated. Additionally, the potential of Dorsomorphin, as possible agent to block hypertrophic differentiation by inhibition of bone morphogenetic protein signalling, was investigated in a fibrin polyurethane composite system, using human mesenchymal stem cells. As opposed to the pellet culture model, application of Dorsomorphin led to a cytotoxic effect which decreased the general differentiation potential. Finally, the chondrogenic potential of the two cell types was directly compared, using the pellet culture model. Under serum-free conditions, human articular cartilage progenitor cells were not able to undergo chondrogenesis. The reasons for this remain to be elucidated. The combined results of the thesis can help to develop a novel one-step procedure to treat articular cartilage defects.
| Author | : Fengjuan Lu |
| Publisher | : Open Dissertation Press |
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| Release | : 2017-01-26 |
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| ISBN | : 9781361276341 |
This dissertation, "Potential of Bone Marrow and Umbilical Cord Derived Mesenchymal Stem Cells in Intervertebral Disc Repair" by Fengjuan, Lu, 吕凤娟, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Introduction: Intervertebral disc (IVD) degeneration is suggested to begin from the nucleus pulposus (NP). Evidence from various studies highlights mesenchymal stem cells (MSC), in most cases using bone marrow derived MSC, as a potential stem cell source for NP regeneration. However MSC can be isolated from many sources with various characteristics. There are indications that fetal or close to fetal tissue sources contain MSC with relatively undifferentiated phenotype with respect to MSC from adult sources. Moreover, umbilical cord (C)-MSC may have better chondrogenic differentiation potential than bone marrow (B)-MSC. We hypothesize CMSC are different from BMSC, and more efficient than BMSC in stimulating NP regeneration. Methods: MSC were isolated from human bone marrow and umbilical cord with corresponding ethical approval. BMSC and CMSC were characterized for cell surface marker expression profile and differentiation potential.. RT-PCR of interest genes in NP cells isolated from scoliosis and degenerate discs was performed to search for NP degeneration indicators. Conditioned media (CM) was collected from confluent MSC monolayer, and used for stimulation of four batches of degenerated NP cells isolated from human degenerative intervertebral discs. Cell proliferation and cytotoxicity were assessed by MTT assay. Proteoglycan content were measured by DMMB assay. Gene expression of a series of degeneration related molecules including ACAN, SOX9, CDH2, CD55, KRT19, KRT18, FBLN1 and MGP, and fibrosis related molecules, including MMP12, HSP47, COL1A1, COL3A1 and FN1, of NP cells in MSC-CM were determined by real- time RT-PCR. All results were normalized to the control cells in basal medium. The expression of discogenic, chondrogenic and osteogenic markers on BMSC and CMSC were compared by RT-PCR. Results and Conclusion: CMSC were similar to BMSC and fulfilled the minimum criteria of MSC, however the expression of CD146, CD106 and Stro-1 was different, and BMSC had a spontaneous osteogenesis tendency while CMSC expressed chondrogenic marker even without TGF-beta stimulation. BMSC demonstrated a paracrine effect on modulating human degenerated NP cells towards a non-degenerative phenotype in stimulating cell proliferation, slightly enhancing proteoglycan production, upregulating KRT19 while downregulating MMP12. Compared with BMSC, a higher paracrine effect of CMSC was disclosed in modulating the phenotype of NP cells in all aspects tested, and an intrinsic higher expression on CMSC of 'potential NP markers', including KRT19, KRT18 and CD55, but lower expression of osteogenic markers, including RUNX2 and ALPL, was revealed, which indicate a higher potential of CMSC for future clinical application to treat IVD degeneration diseases. KRT19 and MMP12 were also confirmed to be the highest differentially expressed candidate genes between cultured scoliosis and degenerated human NP cells, indicating a high indicator potential of NP degeneration. Furthermore, a subpopulation was detected in the degenerated NP cells that possessed macrophage-like phenotype and activities, which may play a role in the pathogenesis of
| Author | : Lucas G. Chase |
| Publisher | : Springer Science & Business Media |
| Total Pages | : 458 |
| Release | : 2012-12-12 |
| Genre | : Science |
| ISBN | : 1627032002 |
Over the past decade, significant efforts have been made to develop stem cell-based therapies for difficult to treat diseases. Multipotent mesenchymal stromal cells, also referred to as mesenchymal stem cells (MSCs), appear to hold great promise in regards to a regenerative cell-based therapy for the treatment of these diseases. Currently, more than 200 clinical trials are underway worldwide exploring the use of MSCs for the treatment of a wide range of disorders including bone, cartilage and tendon damage, myocardial infarction, graft-versus-host disease, Crohn’s disease, diabetes, multiple sclerosis, critical limb ischemia and many others. MSCs were first identified by Friendenstein and colleagues as an adherent stromal cell population within the bone marrow with the ability to form clonogenic colonies in vitro. In regards to the basic biology associated with MSCs, there has been tremendous progress towards understanding this cell population’s phenotype and function from a range of tissue sources. Despite enormous progress and an overall increased understanding of MSCs at the molecular and cellular level, several critical questions remain to be answered in regards to the use of these cells in therapeutic applications. Clinically, both autologous and allogenic approaches for the transplantation of MSCs are being explored. Several of the processing steps needed for the clinical application of MSCs, including isolation from various tissues, scalable in vitro expansion, cell banking, dose preparation, quality control parameters, delivery methods and numerous others are being extensively studied. Despite a significant number of ongoing clinical trials, none of the current therapeutic approaches have, at this point, become a standard of care treatment. Although exceptionally promising, the clinical translation of MSC-based therapies is still a work in progress. The extensive number of ongoing clinical trials is expected to provide a clearer path forward for the realization and implementation of MSCs in regenerative medicine. Towards this end, reviews of current clinical trial results and discussions of relevant topics association with the clinical application of MSCs are compiled in this book from some of the leading researchers in this exciting and rapidly advancing field. Although not absolutely all-inclusive, we hope the chapters within this book can promote and enable a better understanding of the translation of MSCs from bench-to-bedside and inspire researchers to further explore this promising and quickly evolving field.
| Author | : Barbara Zavan |
| Publisher | : Humana Press |
| Total Pages | : 288 |
| Release | : 2016-07-25 |
| Genre | : Science |
| ISBN | : 3319332996 |
This book focuses on the basic aspects of dental stem cells (DSCs) as well as their clinical applications in tissue engineering and regenerative medicine. It opens with a discussion of classification, protocols, and properties of DSCs and proceeds to explore DSCs within the contexts of cryopreservation; epigenetics; pulp, periodontal, tooth, bone, and corneal stroma regeneration; neuronal properties, mesenchymal stem cells and biomaterials; and as sources of hepatocytes for liver disease treatment. The fifteen expertly authored chapters comprehensively examine possible applications of DSCs and provide invaluable insights into mechanisms of growth and differentiation. Dental Stem Cells: Regenerative Potential draws from a wealth of international perspectives and is an essential addition to the developing literature on dental stem cells. This installment of Springer’s Stem Cell Biology and Regenerative Medicine series is indispensable for biomedical researchers interested in bioengineering, dentistry, tissue engineering, regenerative medicine, cell biology and oncology.
| Author | : Manuela E. Gomes |
| Publisher | : Academic Press |
| Total Pages | : 471 |
| Release | : 2015-08-08 |
| Genre | : Science |
| ISBN | : 0128016000 |
Tendon Regeneration: Understanding Tissue Physiology and Development to Engineer Functional Substitutes is the first book to highlight the multi-disciplinary nature of this specialized field and the importance of collaboration between medical and engineering laboratories in the development of tissue-oriented products for tissue engineering and regenerative medicine (TERM) strategies. Beginning with a foundation in developmental biology, the book explores physiology, pathology, and surgical reconstruction, providing guidance on biological approaches that enhances tendon regeneration practices. Contributions from scientists, clinicians, and engineers who are the leading figures in their respective fields present recent findings in tendon stem cells, cell therapies, and scaffold treatments, as well as examples of pre-clinical models for translational therapies and a view of the future of the field. - Provides an overview of tendon biology, disease, and tissue engineering approaches - Presents modern, alternative approaches to developing functional tissue solutions discussed - Includes valuable information for those interested in tissue engineering, tissue regeneration, tissue physiology, and regenerative medicine - Explores physiology, pathology, and surgical reconstruction, building a natural progression that enhances tendon regeneration practices - Covers recent findings in tendon stem cells, cell therapies, and scaffold treatments, as well as examples of pre-clinical models for translational therapies and a view of the future of the field
| Author | : Mudasir Bashir Gugjoo |
| Publisher | : Springer Nature |
| Total Pages | : 347 |
| Release | : 2020-11-09 |
| Genre | : Science |
| ISBN | : 9811560374 |
This book focuses on mesenchymal stem cells (MSCs) of animal origin, including their isolation, characterization, and clinical applications. After briefly discussing the historical development of the field of stem cell research, it describes the basic properties and nature of stem cells particularly in relation to MSCs. In turn, it reviews materials and methods used to isolate MSCs from various sources, culture expansion, characterization and long-term storage. It also explores the therapeutic efficacy, immunomodulation and anti-inflammatory, and differentiation properties of MSCs. Importantly, the book discusses the applications of genetic engineering to enhance the efficacy and potential of MSCs in regenerative medicine. The book largely addresses the potential applications of mesenchymal stem cells in therapies for important species of domesticated animals including sheep, goats, cattle, buffalo, cats, dogs and horses. Finally, the book presents an abridgement of challenges and future prospects of stem cell research and application in medicine, in general and veterinary sciences, in particular.
| Author | : Isabel Andia |
| Publisher | : MDPI |
| Total Pages | : 178 |
| Release | : 2019-12-18 |
| Genre | : Medical |
| ISBN | : 3039218603 |
This Special Issue on “Blood-Derived Products for Tissue Repair and Regeneration” reveals the evolution and diversity of platelet rich plasma (PRP) technologies, which includes experimental research on novel formulations, the creation of combination therapies, and the exploration of potential modifiers of PRPs, as well as efficacy of PRP therapies in clinical veterinary and human applications. Scientist and clinicians are now starting to develop different treatments based on their reinterpretation of the traditional roles of platelets and plasma, and the current Issue has provided a forum for sharing research and ways of understanding the associated medicinal benefits from different points of view. The research interest in this area has covered different medical disciplines, such as ophthalmology, dentistry, orthopedics, and sports medicine.
