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      Bioreactor for mobilization of mesenchymal stem/stromal cells into scaffolds under mechanical stimulation: Preliminary results

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          Abstract

          Introduction

          Articular cartilage (AC) is a viscoelastic tissue with a limited regenerative capability because of the lack of vasculature. Mechanical stimulation contributes to the homeostasis of functional AC since it promotes the delivery of nutrients, cytokines and growth factors between the distant chondrocytes. We hypothesized that biomechanical stimulation might enhance mobilization of endogenous mesenchymal stem/stromal cells (MSCs) from neighboring niches as the bone marrow.

          Aim

          This study aimed to introduce a bioreactor for inducing mobilization of MSCs from one compartment to another above by mechanical stimulation in vitro.

          Methods

          A novel mechanical system for evaluating mobilization of cells in a 3D context in vitro is presented. The system consists of a compression bioreactor able to induce loading on hydrogel-based scaffolds, custom-made software for settings management and data recording, and image based biological evaluation. Intermittent load was applied under a periodic regime with frequency of 0.3 Hz and unload phases of 10 seconds each 180 cycles over 24 hours. The mechanical stimulation acted on an alginate scaffold and a cell reservoir containing MSCs below it. The dynamic compression exerted amplitude of 200 μm as 10% strain regarding the original height of the scaffold.

          Results

          The bioreactor was able to stimulate the scaffolds and the cells for 24.4 (±1.7) hours, exerting compression with vertical displacements of 185.8 (±17.8) μm and a force-amplitude of 1.87 (±1.37; min 0.31, max 4.42) N. Our results suggest that continuous mechanical stimulation hampered the viability of the cells located at the cell reservoir when comparing to intermittent mechanical stimulation (34.4 ± 2.0% vs. 66.8 ± 5.9%, respectively).

          Functionalizing alginate scaffolds with laminin-521 (LN521) seemed to enhance the mobilization of cells from 48 (±21) to 194 (±39) cells/mm 3 after applying intermittent mechanical loading.

          Conclusion

          The bioreactor presented here was able to provide mechanical stimulation that seemed to induce the mobilization of MSCs into LN521-alginate scaffolds under an intermittent loading regime.

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          Most cited references43

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          Biomaterials for articular cartilage tissue engineering: Learning from biology.

          Articular cartilage is commonly described as a tissue that is made of up to 80% water, is devoid of blood vessels, nerves, and lymphatics, and is populated by only one cell type, the chondrocyte. At first glance, an easy tissue for clinicians to repair and for scientists to reproduce in a laboratory. Yet, chondral and osteochondral defects currently remain an open challenge in orthopedics and tissue engineering of the musculoskeletal system, without considering osteoarthritis. Why do we fail in repairing and regenerating articular cartilage? Behind its simple and homogenous appearance, articular cartilage hides a heterogeneous composition, a high level of organisation and specific biomechanical properties that, taken together, make articular cartilage a unique material that we are not yet able to repair or reproduce with high fidelity. This review highlights the available therapies for cartilage repair and retraces the research on different biomaterials developed for tissue engineering strategies. Their potential to recreate the structure, including composition and organisation, as well as the function of articular cartilage, intended as cell microenvironment and mechanically competent replacement, is described. A perspective of the limitations of the current research is given in the light of the emerging technologies supporting tissue engineering of articular cartilage.
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            Role of Chondrocytes in Cartilage Formation, Progression of Osteoarthritis and Cartilage Regeneration

            Articular cartilage (AC) covers the diarthrodial joints and is responsible for the mechanical distribution of loads across the joints. The majority of its structure and function is controlled by chondrocytes that regulate Extracellular Matrix (ECM) turnover and maintain tissue homeostasis. Imbalance in their function leads to degenerative diseases like Osteoarthritis (OA). OA is characterized by cartilage degradation, osteophyte formation and stiffening of joints. Cartilage degeneration is a consequence of chondrocyte hypertrophy along with the expression of proteolytic enzymes. Matrix Metalloproteinases (MMPs) and A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS) are an example of these enzymes that degrade the ECM. Signaling cascades involved in limb patterning and cartilage repair play a role in OA progression. However, the regulation of these remains to be elucidated. Further the role of stem cells and mature chondrocytes in OA progression is unclear. The progress in cell based therapies that utilize Mesenchymal Stem Cell (MSC) infusion for cartilage repair may lead to new therapeutics in the long term. However, many questions are unanswered such as the efficacy of MSCs usage in therapy. This review focuses on the role of chondrocytes in cartilage formation and the progression of OA. Moreover, it summarizes possible alternative therapeutic approaches using MSC infusion for cartilage restoration.
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              Clonal culturing of human embryonic stem cells on laminin-521/E-cadherin matrix in defined and xeno-free environment.

              Lack of robust methods for establishment and expansion of pluripotent human embryonic stem (hES) cells still hampers development of cell therapy. Laminins (LN) are a family of highly cell-type specific basement membrane proteins important for cell adhesion, differentiation, migration and phenotype stability. Here we produce and isolate a human recombinant LN-521 isoform and develop a cell culture matrix containing LN-521 and E-cadherin, which both localize to stem cell niches in vivo. This matrix allows clonal derivation, clonal survival and long-term self-renewal of hES cells under completely chemically defined and xeno-free conditions without ROCK inhibitors. Neither LN-521 nor E-cadherin alone enable clonal survival of hES cells. The LN-521/E-cadherin matrix allows hES cell line derivation from blastocyst inner cell mass and single blastomere cells without a need to destroy the embryo. This method can facilitate the generation of hES cell lines for development of different cell types for regenerative medicine purposes.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: SoftwareRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: MethodologyRole: SupervisionRole: Writing – review & editing
                Role: Data curationRole: MethodologyRole: SoftwareRole: Writing – review & editing
                Role: MethodologyRole: SoftwareRole: Writing – review & editing
                Role: SoftwareRole: Writing – review & editing
                Role: SoftwareRole: Writing – review & editing
                Role: Funding acquisitionRole: InvestigationRole: Writing – review & editing
                Role: ConceptualizationRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: InvestigationRole: SupervisionRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: InvestigationRole: Project administrationRole: SupervisionRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                10 January 2020
                2020
                : 15
                : 1
                : e0227553
                Affiliations
                [1 ] Department for Experimental Orthopaedics and Trauma Surgery, Orthopaedics and Trauma Surgery Centre (OUZ), Medical Faculty Mannheim of the University of Heidelberg, Mannheim, Baden Württemberg, Germany
                [2 ] Medical Research Centre (ZMF), Medical Faculty Mannheim of the University of Heidelberg, Mannheim, Baden Württemberg, Germany
                [3 ] Institute of General Mechanics, RWTH Aachen University, Aachen, Nordrhein-Westfalen, Germany
                [4 ] Institute of Transfusion Medicine and Immunology, FlowCore Mannheim, German Red Cross Blood Service of Baden Württemberg-Hessen, Medical Faculty Mannheim of the University of Heidelberg, Mannheim, Baden Württemberg, Germany
                Università degli Studi della Campania, ITALY
                Author notes

                Competing Interests: An application for a Model Utility Protection for the bioreactor (Nr. 2019 103 387.8) has been done on June 17th, 2019. This work has been partially presented in a poster for the German Congress of Orthopaedics (DKOU 2019) in October 2019. The authors have transparently informed to PLOS ONE editorial. In our opinion, these submissions do not alter our adherence to PLOS ONE policies on sharing data and materials.

                Author information
                http://orcid.org/0000-0001-8112-4301
                Article
                PONE-D-19-08540
                10.1371/journal.pone.0227553
                6953860
                31923210
                3e955979-1a54-4aaa-b89a-552e20c5fc56
                © 2020 Gamez et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 25 March 2019
                : 20 December 2019
                Page count
                Figures: 6, Tables: 0, Pages: 17
                Funding
                Funded by: Ministerium für Wissenschaft, Forschung und Kunst Baden-Württemberg (DE)
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001659, Deutsche Forschungsgemeinschaft;
                Award ID: INST 35/1314-1 FUGG
                Funded by: funder-id http://dx.doi.org/10.13039/501100001659, Deutsche Forschungsgemeinschaft;
                This work has been funded by the Ministry of Science, Research and the Arts of the State of Baden-Württemberg (MWK-BW) Germany, and the Medicine Faculty Mannheim of the University of Heidelberg, the German Research Foundation (DFG) within the funding program Open Access Publishing by the MWK-BW and by Ruprecht-Karls-Universität Heidelberg for the publication financial support. The authors gratefully acknowledge the data storage service SDS@hd supported by the Ministry of Science, Research and the MWK-BW and the DFG through grant INST 35/1314-1 FUGG. CG is funded through the Cooperative Research-Training Group: Tissue Analytics for Stem Cell Based Diagnostics and Therapy by the MWK-BW, Germany. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Engineering and Technology
                Mechanical Engineering
                Pistons
                Biology and Life Sciences
                Anatomy
                Biological Tissue
                Connective Tissue
                Cartilage
                Medicine and Health Sciences
                Anatomy
                Biological Tissue
                Connective Tissue
                Cartilage
                Physical Sciences
                Physics
                Classical Mechanics
                Compression
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Stem Cells
                Mesenchymal Stem Cells
                Biology and Life Sciences
                Anatomy
                Biological Tissue
                Connective Tissue
                Cartilage
                Chondrocytes
                Medicine and Health Sciences
                Anatomy
                Biological Tissue
                Connective Tissue
                Cartilage
                Chondrocytes
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Connective Tissue Cells
                Chondrocytes
                Biology and Life Sciences
                Anatomy
                Biological Tissue
                Connective Tissue
                Connective Tissue Cells
                Chondrocytes
                Medicine and Health Sciences
                Anatomy
                Biological Tissue
                Connective Tissue
                Connective Tissue Cells
                Chondrocytes
                Research and Analysis Methods
                Bioassays and Physiological Analysis
                Cell Analysis
                Cell Viability Testing
                Biology and Life Sciences
                Anatomy
                Musculoskeletal System
                Skeletal Joints
                Medicine and Health Sciences
                Anatomy
                Musculoskeletal System
                Skeletal Joints
                Biology and Life Sciences
                Physiology
                Physiological Processes
                Tissue Repair
                Medicine and Health Sciences
                Physiology
                Physiological Processes
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