Strategies to Modulate the Redifferentiation of Chondrocytes

Because of the low self-healing capacity of articular cartilage, cartilage injuries and degenerations triggered by various diseases are almost irreversible. Previous studies have suggested that human chondrocytes cultured in vitro tend to dedifferentiate during the cell-amplification phase and lose the physiological properties and functions of the cartilage itself, which is currently a critical limitation in the cultivation of cartilage for tissue engineering. Recently, numerous studies have focused on the modulation of chondrocyte redifferentiation. Researchers discovered the effect of various conditions (extracellular environment, cell sources, growth factors and redifferentiation inducers, and gene silencing and overexpression) on the redifferentiation of chondrocytes during the in vitro expansion of cells, and obtained cartilage tissue cultured in vitro that exhibited physiological characteristics and functions that were similar to those of human cartilage tissue. Encouragingly, several studies reported positive results regarding the modulation of the redifferentiation of chondrocytes in specific conditions. Here, the various factors and conditions that modulate the redifferentiation of chondrocytes, as well as their limitations and potential applications and challenges are reviewed. We expect to inspire research in the field of cartilage repair toward the future treatment of arthropathy.

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Calcium/Cobalt Alginate Beads as Functional Scaffolds for Cartilage Tissue Engineering

Stem Cells International ◽

10.1155/2016/2030478 ◽

2016 ◽

Vol 2016 ◽

pp. 1-12 ◽

Cited By ~ 22

Author(s):

Stefano Focaroli ◽

Gabriella Teti ◽

Viviana Salvatore ◽

Isabella Orienti ◽

Mirella Falconi

Keyword(s):

Tissue Engineering ◽

Bone Morphogenetic Proteins ◽

Transforming Growth Factor ◽

Cartilage Tissue Engineering ◽

Biomechanical Properties ◽

Alginate Beads ◽

Cartilage Tissue ◽

Self Healing ◽

Tissue Replacement

Articular cartilage is a highly organized tissue with complex biomechanical properties. However, injuries to the cartilage usually lead to numerous health concerns and often culminate in disabling symptoms, due to the poor intrinsic capacity of this tissue for self-healing. Although various approaches are proposed for the regeneration of cartilage, its repair still represents an enormous challenge for orthopedic surgeons. The field of tissue engineering currently offers some of the most promising strategies for cartilage restoration, in which assorted biomaterials and cell-based therapies are combined to develop new therapeutic regimens for tissue replacement. The current study describes thein vitrobehavior of human adipose-derived mesenchymal stem cells (hADSCs) encapsulated within calcium/cobalt (Ca/Co) alginate beads. These novel chondrogenesis-promoting scaffolds take advantage of the synergy between the alginate matrix and Co+2ions, without employing costly growth factors (e.g., transforming growth factor betas (TGF-βs) or bone morphogenetic proteins (BMPs)) to direct hADSC differentiation into cartilage-producing chondrocytes.

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An efficient polymer cocktail-based transportation method for cartilage tissue, yielding chondrocytes with enhanced hyaline cartilage expression during in vitro culturing

10.1101/2021.07.14.452414 ◽

2021 ◽

Author(s):

Shojiro Katoh ◽

Hiroshi Yoshioka ◽

Shoji Suzuki ◽

Hiroyuki Nakajima ◽

Masaru Iwasaki ◽

...

Keyword(s):

Cultured Cells ◽

Hyaline Cartilage ◽

Three Dimensional ◽

Cartilage Tissue ◽

Human Cartilage ◽

Tissue Samples ◽

Chondrocyte Implantation ◽

Initial Processing ◽

Regenerative Therapies

Chondrocytes are used in cell-based therapies such as autologous chondrocyte implantation (ACI) and matrix-associated cartilage implantation (MACI). To transport the cartilage tissue to the laboratory for in vitro culturing, phosphate-buffered saline (PBS), Euro-Collins solution (ECS) and Dulbecco Modified Eagle Medium (DMEM) are commonly employed at 4-8 deg C. In this study, eight samples of human cartilage biopsy tissues from elderly patients with severe osteoarthritis undergoing arthroscopy, which would otherwise have been discarded, were used. The cartilage tissue samples were compared to assess the cell yield between two transportation groups: i) a thermo-reversible gelation polymer (TGP) based method without cool preservation (~25 deg C) and ii) ECS transport at 4 deg C. These samples were subjected to in vitro culture in a two-dimensional (2D) monolayer for two weeks and subsequently in a three-dimensional (3D) TGP scaffold for six weeks. The cell count obtained from the tissues transported in TGP was higher (0.2 million cells) than those transported in ECS (0.08 million cells) both after initial processing and after in vitro culturing for 2 weeks in 2D (18 million cells compared with 10 million cells). In addition, mRNA quantification demonstrated significantly higher expression of Col2a1 and SOX-9 in 3D-TGP cultured cells and lower expression of COL1a1 in RT-PCR, characteristic of the hyaline cartilage phenotype, than in 2D culture. This study confirms that the TGP cocktail is suitable for both the transport of human cartilage tissue and for in vitro culturing to yield better-quality cells for use in regenerative therapies.

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Senolytic Peptide FOXO4-DRI Selectively Removes Senescent Cells From in vitro Expanded Human Chondrocytes

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.677576 ◽

2021 ◽

Vol 9 ◽

Author(s):

Yuzhao Huang ◽

Yuchen He ◽

Meagan J. Makarcyzk ◽

Hang Lin

Keyword(s):

Untreated Control ◽

Cell Number ◽

Cartilage Tissue ◽

Population Doubling ◽

Control Group ◽

Chondrocyte Implantation ◽

Cartilage Injuries ◽

Pre Treatment ◽

Articular Cartilage Injuries

Autologous chondrocyte implantation (ACI) is a procedure used to treat articular cartilage injuries and prevent the onset of post-traumatic osteoarthritis. In vitro expansion of chondrocytes, a necessary step in ACI, results in the generation of senescent cells that adversely affect the quality and quantity of newly formed cartilage. Recently, a senolytic peptide, fork head box O transcription factor 4-D-Retro-Inverso (FOXO4-DRI), was reported to selectively kill the senescent fibroblasts. In this study, we hypothesized that FOXO4-DRI treatment could remove the senescent cells in the expanded chondrocytes, thus enhancing their potential in generating high-quality cartilage. To simulate the in vitro expansion for ACI, chondrocytes isolated from healthy donors were expanded to population doubling level (PDL) 9, representing chondrocytes ready for implantation. Cells at PDL3 were also used to serve as the minimally expanded control. Results showed that the treatment of FOXO4-DRI removed more than half of the cells in PDL9 but did not significantly affect the cell number of PDL3 chondrocytes. Compared to the untreated control, the senescence level in FOXO4-DRI treated PDL9 chondrocytes was significantly reduced. Based on the result from standard pellet culture, FOXO4-DRI pre-treatment did not enhance the chondrogenic potential of PDL9 chondrocytes. However, the cartilage tissue generated from FOXO4-DRI pretreated PDL9 cells displayed lower expression of senescence-relevant secretory factors than that from the untreated control group. Taken together, FOXO4-DRI is able to remove the senescent cells in PDL9 chondrocytes, but its utility in promoting cartilage formation from the in vitro expanded chondrocytes needs further investigation.

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Exposure of human cartilage tissue to low concentrations of blood for a short period of time leads to prolonged cartilage damage: An in vitro study

Arthritis & Rheumatism ◽

10.1002/art.22304 ◽

2006 ◽

Vol 56 (1) ◽

pp. 199-207 ◽

Cited By ~ 69

Author(s):

Nathalie W. D. Jansen ◽

Goris Roosendaal ◽

Johannes W. J. Bijlsma ◽

Jeroen DeGroot ◽

Floris P. J. G. Lafeber

Keyword(s):

Cartilage Damage ◽

In Vitro Study ◽

Vitro Study ◽

Cartilage Tissue ◽

Human Cartilage ◽

Low Concentrations ◽

Short Period

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Dual Delivery of TGF-β3 and Ghrelin in Microsphere/Hydrogel Systems for Cartilage Regeneration

Molecules ◽

10.3390/molecules26195732 ◽

2021 ◽

Vol 26 (19) ◽

pp. 5732

Author(s):

Jianjing Lin ◽

Li Wang ◽

Jianhao Lin ◽

Qiang Liu

Keyword(s):

Growth Factors ◽

Chondrogenic Differentiation ◽

Transforming Growth Factor ◽

Double Emulsion ◽

Cartilage Regeneration ◽

Cartilage Tissue ◽

Self Healing ◽

Tissue Formation ◽

Extraction Technology

Articular cartilage (AC) damage is quite common, but due to AC’s poor self-healing ability, the damage can easily develop into osteoarthritis (OA). To solve this problem, we developed a microsphere/hydrogel system that provides two growth factors that promote cartilage repair: transforming growth factor-β3 (TGF-β3) to enhance cartilage tissue formation and ghrelin synergy TGF-β to significantly enhance the chondrogenic differentiation. The hydrogel and microspheres were characterized in vitro, and the biocompatibility of the system was verified. Double emulsion solvent extraction technology (w/o/w) is used to encapsulate TGF-β3 and ghrelin into microspheres, and these microspheres are encapsulated in a hydrogel to continuously release TGF-β3 and ghrelin. According to the chondrogenic differentiation ability of mesenchymal stem cells (MSCs) in vitro, the concentrations of the two growth factors were optimized to promote cartilage regeneration.

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Silencing Smad7 Potentiates BMP2-induced Chondrogenic Differentiation and Inhibits Endochondral Ossification in Human Synovial Derived Mesenchymal Stem Cells

10.21203/rs.3.rs-96609/v1 ◽

2020 ◽

Author(s):

pengcheng xiao ◽

Zhenglin Zhu ◽

Chengcheng Du ◽

Yongsheng Zeng ◽

junyi Liao ◽

...

Keyword(s):

Tissue Engineering ◽

Stem Cells ◽

Chondrogenic Differentiation ◽

Endochondral Ossification ◽

Cartilage Tissue Engineering ◽

Cartilage Tissue ◽

Blue Staining ◽

Alcian Blue Staining ◽

Cartilage Injuries

Abstract Background: Cartilage injuries pose formidable challenges for effective clinical management. Autologous stem cell-based therapies and transgene-enhanced cartilage tissue engineering may open new avenues for the treatment of cartilage injuries. Bone morphogenetic protein 2 (BMP2) is a promising chondrogenic growth factors for transgene-enhanced cartilage tissue engineering. However the BMP2 is failed to maintain a stable chondrogenic phenotype as it also induces robust endochondral ossification. Recently, human synovial derived mesenchymal stem cells (hSMSCs) arouse interested through the poor differentiation potential into osteogenic lineage. Smad7, a protein to antagonizes TGF-β/BMP signaling pathway has been discovered significant in the endochondral ossification. In the present study ,we further explore the effect of downregulate Smad7 in BMP2-induced chondrogenic differentiation of hSMSCs. Methods: hSMSCs were isolated from synovium of human knee joint through adhesion growth. In vitro and in vivo chondrogenic differentiation models of hSMSCs were constructed . Transgenes of BMP2, silencing Smad7 and Smad7 were expressed by adenoviral vectors. The osteogenic differentiation was detected by alkaline phosphatase staining, alizarin red staining. The chondrogenic differentiation was detected by alcian blue staining. Gene expression was determined by reverse transcription and quantitative real-time PCR (RT-qPCR), Immunofluorescence and immunohistochemistry. The subcutaneous stem cell implantation model was established and evaluated by micro-CT , h＆e staining, alcian blue staining and immunohistochemistry assay.Results: Compared to other MSCs, hSMSCs performed less of capability to osteogenic differentiation. But the occurrence of endochondral ossification is still inevasible during BMP2 induced cartilage formation. We found that silencing Smad7 enhanced the BMP2-induced chondrogenic differentiation of hSMSCs in vitro. Also, it leading to much less of hypertrophic differentiation. The subcutaneous stem cells implantation assays demonstrated silencing Smad7 potentiates BMP2-induced cartilage formation and inhibits endochondral ossification. Conclusion: This study strongly suggests that application of hSMSCs , cell scaffolds and silencing Smad7 can potentiate BMP2-induced chondrogenic differentiation and inhibit endochondral ossification. Thus, inhibit the expression of Smad7 in BMP2-induced hSMSCs differentiation may be a new strategy for cartilage tissue engineering.

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Deep Sequencing MicroRNAs from Extracellular Membrane Vesicles Revealed the Association of the Vesicle Cargo with Cellular Origin

International Journal of Molecular Sciences ◽

10.3390/ijms21031141 ◽

2020 ◽

Vol 21 (3) ◽

pp. 1141

Author(s):

Uyen Thi Trang Than ◽

Dominic Guanzon ◽

James A Broadbent ◽

Tony J Parker ◽

David I Leavesley

Keyword(s):

Target Genes ◽

Membrane Vesicles ◽

Cellular Origin ◽

Fibroblast Migration ◽

Potential Applications ◽

Keratinocyte Cell ◽

Cell Sources ◽

Cell Type Specific ◽

Extracellular Environment ◽

Generation Sequencing

Extracellular membrane vesicles (EVs) have emerged as potential candidates for diagnostics and therapeutics. We have previously reported that keratinocytes release three types of EVs into the extracellular environment. Importantly, those EVs contain a large number of microRNAs (miRNAs) as cargo. In this study, we examined the expression level of keratinocyte-derived EV miRNAs, their target genes and potential functions. Next generation sequencing results showed that over one hundred miRNAs in each EV subtype exhibited greater than 100 reads per million (RPM), indicating a relatively high abundance. Analysis of the miRNAs with the highest abundance revealed associations with different keratinocyte cell sources. For instance, hsa-miR-205 was associated with the HaCaT cells whereas hsa-miR-21, hsa-miR-203, hsa-miR-22 and hsa-miR-143 were associated with human primary dermal keratinocytes (PKCs). Additionally, functional annotation analysis of genes regulated by those miRNAs, especially with regard to biological processes, also revealed cell-type-specific associations with either HaCaTs or PKCs. Indeed, EV functional effects were related to their parental cellular origin; specifically, PKC-derived EVs influenced fibroblast migration whereas HaCaT-derived EVs did not. In addition, the data in this current study indicates that keratinocyte-derived EVs and/or their cargoes have potential applications for wound healing.

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Pathway Analysis Hints Towards Beneficial Effects of Long-Term Vibration on Human Chondrocytes

Cellular Physiology and Biochemistry ◽

10.1159/000491006 ◽

2018 ◽

Vol 47 (4) ◽

pp. 1729-1741 ◽

Cited By ~ 3

Author(s):

Ronald Lützenberg ◽

Kendrick Solano ◽

Christoph Buken ◽

Jayashree Sahana ◽

Stefan Riwaldt ◽

...

Keyword(s):

Gene Expression ◽

Microarray Analysis ◽

Human Chondrocytes ◽

Cartilage Tissue ◽

Tunel Staining ◽

Human Cartilage ◽

Rhodamine Phalloidin ◽

The Impact

Background/Aims: Spaceflight negatively influences the function of cartilage tissue in vivo. In vitro human chondrocytes exhibit an altered gene expression of inflammation markers after a two-hour exposure to vibration. Little is known about the impact of long-term vibration on chondrocytes. Methods: Human cartilage cells were exposed for up to 24 h (VIB) on a specialised vibration platform (Vibraplex) simulating the vibration profile which occurs during parabolic flights and compared to static control conditions (CON). Afterwards, they were investigated by phase-contrast microscopy, rhodamine phalloidin staining, microarray analysis, qPCR and western blot analysis. Results: Morphological investigations revealed no changes between CON and VIB chondrocytes. F-Actin staining showed no alterations of the cytoskeleton in VIB compared with CON cells. DAPI and TUNEL staining did not identify apoptotic cells. ICAM-1 was elevated and vimentin, beta-tubulin and osteopontin proteins were significantly reduced in VIB compared to CON cells. qPCR of cytoskeletal genes, ITGB1, SOX3, SOX5, SOX9 did not reveal differential regulations. Microarray analysis detected 13 differentially expressed genes, mostly indicating unspecific stimulations. Pathway analyses demonstrated interactions of PSMD4 and CNOT7 with ICAM. Conclusions: Long-term vibration did not damage human chondrocytes in vitro. The reduction of osteopontin protein and the down-regulation of PSMD4 and TBX15 gene expression suggest that in vitro long-term vibration might even positively influence cultured chondrocytes.

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Kartogenin Enhances Chondrogenic Differentiation of MSCs in 3D Tri-Copolymer Scaffolds and the Self-Designed Bioreactor System

Biomolecules ◽

10.3390/biom11010115 ◽

2021 ◽

Vol 11 (1) ◽

pp. 115

Author(s):

Ching-Yun Chen ◽

Chunching Li ◽

Cherng-Jyh Ke ◽

Jui-Sheng Sun ◽

Feng-Huei Lin

Keyword(s):

Tissue Engineering ◽

Chondrogenic Differentiation ◽

Cartilage Damage ◽

Cartilage Tissue Engineering ◽

3D Culture ◽

Cartilage Tissue ◽

Great Promise ◽

Clinical Consequence ◽

Human Cartilage

Human cartilage has relatively slow metabolism compared to other normal tissues. Cartilage damage is of great clinical consequence since cartilage has limited intrinsic healing potential. Cartilage tissue engineering is a rapidly emerging field that holds great promise for tissue function repair and artificial/engineered tissue substitutes. However, current clinical therapies for cartilage repair are less than satisfactory and rarely recover full function or return the diseased tissue to its native healthy state. Kartogenin (KGN), a small molecule, can promote chondrocyte differentiation both in vitro and in vivo. The purpose of this research is to optimize the chondrogenic process in mesenchymal stem cell (MSC)-based chondrogenic constructs with KGN for potential use in cartilage tissue engineering. In this study, we demonstrate that KGN treatment can promote MSC condensation and cell cluster formation within a tri-copolymer scaffold. Expression of Acan, Sox9, and Col2a1 was significantly up-regulated in three-dimensional (3D) culture conditions. The lacuna-like structure showed active deposition of type II collagen and aggrecan deposition. We expect these results will open new avenues for the use of small molecules in chondrogenic differentiation protocols in combination with scaffolds, which may yield better strategies for cartilage tissue engineering.

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Chitosan Woven Meshes: Influence of Threads Configuration on Mechanical, Morphological, and Physiological Properties

Polymers ◽

10.3390/polym13010047 ◽

2020 ◽

Vol 13 (1) ◽

pp. 47

Author(s):

Henrique Nunes da Silva ◽

Milena Costa da Silva ◽

Flavia Suzany Ferreira dos Santos ◽

José Alberto Campos da Silva Júnior ◽

Rossemberg Cardoso Barbosa ◽

...

Keyword(s):

Sodium Hydroxide Solution ◽

Tensile Tests ◽

Coagulation Bath ◽

Wet Spinning ◽

Physiological Properties ◽

Potential Applications ◽

Spinning Process ◽

The Individual ◽

Typical Skin

This study aimed to develop meshes from the weaving of mono- and multifilament wet-spun chitosan (CS), for possible biomedical applications. In the wet-spinning process, CS solution (4% w/v) was extruded in a coagulation bath containing 70% sodium hydroxide solution (0.5 M), and 30% methanol was used. The multifilament thread was prepared by twisted of two and three monofilaments. CS threads obtained were characterized by tensile tests and scanning electron microscopy (SEM). Moreover, it was verified from the morphological tests that threads preserve the characteristics of the individual filaments and present typical “skin-core” microstructure obtained by wet spinning. CS woven meshes obtained were evaluated by optical microscopy (OM), tensile test, swelling degree, and in vitro enzymatic biodegradation. Mechanical properties, biodegradation rate, and amount of fluid absorbed of CS woven meshes were influenced by thread configuration. Hydrated CS meshes showed a larger elastic zone than the dry state. Therefore, CS woven meshes were obtained with modular properties from thread configuration used in weaving, suggesting potential applications in the biomedical field, like dressings, controlled drug delivery systems, or mechanical support.

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