Cartilage Regeneration Characteristics of Human and Goat Auricular Chondrocytes

Although cartilage regeneration technology has achieved clinical breakthroughs, whether auricular chondrocytes (AUCs) represent optimal seed cells to achieve stable cartilage regeneration is not clear. In this study, we systematically explore biological behaviors of human- and goat-derived AUCs during in vitro expansion as well as cartilage regeneration in vitro and in vivo. To eliminate material interference, a cell sheet model was used to evaluate the feasibility of dedifferentiated AUCs to re-differentiate and regenerate cartilage in vitro and in vivo. We found that the dedifferentiated AUCs could re-differentiate and regenerate cartilage sheets under the chondrogenic medium system, and the generated chondrocyte sheets gradually matured with increased in vitro culture time (2, 4, and 8 weeks). After the implantation of cartilage sheets with different in vitro culture times in nude mice, optimal neocartilage was formed in the group with 2 weeks in vitro cultivation. After in vivo implantation, ossification only occurred in the group with goat-regenerated cartilage sheet of 8 weeks in vitro cultivation. These results, which were confirmed in human and goat AUCs, suggest that AUCs are ideal seed cells for the clinical translation of cartilage regeneration under the appropriate culture system and culture condition.

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Fabrication of hyaline-like cartilage constructs using mesenchymal stem cell sheets

Scientific Reports ◽

10.1038/s41598-020-77842-0 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Hallie Thorp ◽

Kyungsook Kim ◽

Makoto Kondo ◽

David W. Grainger ◽

Teruo Okano

Keyword(s):

Chondrogenic Differentiation ◽

Cell Sheet ◽

Cartilage Regeneration ◽

Cell Sheets ◽

Chondrogenic Potential ◽

Cell Sheet Technology ◽

Cell Functionality ◽

Articular Cartilage Regeneration

AbstractCell and tissue engineering approaches for articular cartilage regeneration increasingly focus on mesenchymal stem cells (MSCs) as allogeneic cell sources, based on availability and innate chondrogenic potential. Many MSCs exhibit chondrogenic potential as three-dimensional (3D) cultures (i.e. pellets and seeded biomaterial scaffolds) in vitro; however, these constructs present engraftment, biocompatibility, and cell functionality limitations in vivo. Cell sheet technology maintains cell functionality as scaffold-free constructs while enabling direct cell transplantation from in vitro culture to targeted sites in vivo. The present study aims to develop transplantable hyaline-like cartilage constructs by stimulating MSC chondrogenic differentiation as cell sheets. To achieve this goal, 3D MSC sheets are prepared, exploiting spontaneous post-detachment cell sheet contraction, and chondrogenically induced. Results support 3D MSC sheets’ chondrogenic differentiation to hyaline cartilage in vitro via post-contraction cytoskeletal reorganization and structural transformations. These 3D cell sheets’ initial thickness and cellular densities may also modulate MSC-derived chondrocyte hypertrophy in vitro. Furthermore, chondrogenically differentiated cell sheets adhere directly to cartilage surfaces via retention of adhesion molecules while maintaining the cell sheets’ characteristics. Together, these data support the utility of cell sheet technology for fabricating scaffold-free, hyaline-like cartilage constructs from MSCs for future transplantable articular cartilage regeneration therapies.

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Hydrogels with tunable modulus regulate chondrocyte microaggregates growth for cartilage repair

Biomedical Materials ◽

10.1088/1748-605x/ac41fc ◽

2021 ◽

Author(s):

Jing Chen ◽

Peng An ◽

Hua Zhang ◽

Yansheng Zhang ◽

Hua Wei ◽

...

Keyword(s):

Loss Modulus ◽

Femoral Condyle ◽

Cartilage Regeneration ◽

Cartilage Defects ◽

High Viability ◽

Chondrogenic Phenotype ◽

A Cell ◽

Weak Gel

Abstract Chondrocyte spheroids in 3D hydrogel are more beneficial to improve their survival and maintain chondrogenic phenotype comparing to dissociated chondrocytes. However, in-situ inducing cell into spheroids rather than encapsulating spheroids in a hydrogel remains a tremendous challenge because of the limitations of biochemical and viscoelastic controllability for hydrogel. Herein, a hydrogel consisting of photo-crosslinkable chitosan methacrylate (CHMA) and semi-interpenetrating polyvinyl alcohol (PVA) is developed as a cell-responsive matrix with controllable viscoelastic properties. The proposed CHMA-PVA precursor preferentially exhibits a weak gel-like state with a storage modulus of 16.9 Pa, loss modulus of 13.0 Pa and yielding stain of 1%, which could allow chondrocyte to vigorously move and assemble but hinder their precipitation before crosslinking. The chondrocytes could form microaggregates within 8 h in vitro and keep high viability. Moreover, subcutaneous implantation experiments demonstrate that the CHMA/PVA hydrogels are biocompatible and degrade within five weeks in vivo. The cell-free hydrogels are further placed in cylindrical cartilage defects in the rabbit femoral condyle and examined 8 weeks postoperatively. Gross, histological and immunohistochemical analyses reveal a significant acceleration for the cartilage regeneration. These findings suggest that this novel cell adhesion-responsive and histo-compatible hydrogel is promising for cartilage regeneration.

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Cell-Free Fat Extract Attenuate Osteoarthritis via Chondrocytes Regeneration and Macrophages Immunomodulation

10.21203/rs.3.rs-1242320/v1 ◽

2022 ◽

Author(s):

zhuoxuan jia ◽

Bijun Kang ◽

Yizuo Cai ◽

Chingyu Chen ◽

Zheyuan Yu ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Reactive Oxygen ◽

Cartilage Regeneration ◽

Cell Counting ◽

Low Grade ◽

Oxygen Species ◽

Qrt Pcr ◽

A Cell

Abstract Background: The prevalence of osteoarthritis (OA) is increasing, yet clinically effective and economical treatments are unavailable. We have previously proposed a cell-free fat extract (CEFFE) containing multiple cytokines, which possessed anti-apoptotic, anti-oxidative, and proliferation promotion functions, as a “cell-free” strategy. In this study, we aimed to evaluate the therapeutic effect of CEFFE in vivo and in vitro . Methods: In vivo study, sodium iodoacetate-induced OA rats were treated with CEFFE by intra-articular injections for 8 weeks. Behavioral experiments were performed every two weeks. Histological analyses, anti-type II collagen, and toluidine staining provided structural evaluation. Macrophage infiltration was assessed by anti-CD68 and anti-CD206 staining. In vitro study, the effect of CEFFE on macrophage polarization and secretory factors was evaluated by flow cytometry, immunofluorescence, and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The effect of CEFFE on cartilage regeneration was accessed by cell counting kit-8 assay and qRT-PCR. The generation of reactive oxygen species (ROS) and levels of ROS-related enzymes were investigated by qRT-PCR and western blotting. Results: In rat models with sodium iodoacetate (MIA)-induced OA, CEFFE increased claw retraction pressure while decreasing bipedal pressure in a dose-depend manner. Moreover, CEFFE promoted cartilage structure restoration and increased the proportion of CD206 + macrophages in the synovium. In vitro , CEFFE decreased the proportion of CD86 + cells and reduced the expression of pro-inflammatory factors in LPS + IFN-γ induced Raw 264.7. In addition, CEFFE decreased the expression of interleukin-6 and ADAMTs-5 and promoted the expression of SOX-9 in mouse primary chondrocytes. Besides, CEFFE reduced the intracellular levels of reactive oxygen species in both in vitro models through regulating ROS-related enzymes. Conclusions: CEFFE inhibits the progression of OA by promoting cartilage regeneration and limiting low-grade joint inflammation.

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Different pre-implantation phenotypes of bovine blastocysts produced in vitro

Reproduction ◽

10.1530/rep-18-0439 ◽

2019 ◽

pp. 163-178 ◽

Cited By ~ 2

Author(s):

Isabelle Hue ◽

Isabelle Dufort ◽

Anaïs Vitorino Carvalho ◽

Denis Laloe ◽

Nathalie Peynot ◽

...

Keyword(s):

Gene Expression ◽

Survival Rate ◽

Recovery Rate ◽

Culture System ◽

Primitive Streak ◽

Culture Time ◽

Defined Media ◽

Microarray Analyses

Embryo transfer in cattle is performed with blastocysts produced in vivo or in vitro using defined media. However, outdated systems such as those that use serum and co-culture remain of interest for research purposes. Here, we investigated the effect of additional culture time on in vitro-produced embryos. Specifically, we compared embryos that formed a blastocoel at different times after fertilisation to those that stayed in culture for up to two additional days with respect to their development in vivo after temporary transfer to oestrus-synchronised recipients. A pre-transfer set (D6, D6+1, D6+2, D7, D7+1, D8) was examined using microarray analyses and correlated with a post-transfer set that included two different days of transfer (D6-T6, D6+2-T8, D7+1-T8, D8-T8). All surviving conceptuses reached primitive-streak stages and filamentous sizes similarly to in vivo (D18) or in vitro controls (D7/T7). The recovery rate differed between D6 and D8 embryos that were immediately transferred (58 vs 25%). With an intermediate survival rate (33%), the D6 embryos with two additional days in culture produced nine times more IFN-tau (IFNT) at D18 than the D6 embryos that were immediately transferred. At the end of culture, D6 and D6+2 embryos displayed the highest number of gene expression differences. Despite a mortality of 40–60%, no signature was detectable in any of the transferred groups that would account for the embryos’ fates. Initially reputed to be beneficial in producing more blastocysts, our culture system of B2 medium plus serum and co-culture generated blastocysts that were distinct from those developed in vivo (D7).

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Development of early larval stages of Taenia ovis in vitro using a cell monolayer

Parasitology ◽

10.1017/s0031182000055013 ◽

1980 ◽

Vol 81 (1) ◽

pp. 35-40 ◽

Cited By ~ 8

Author(s):

S. B. Lawrence ◽

D. D. Heath ◽

S. N. Parmeter ◽

P. J. Osborn

Keyword(s):

Culture System ◽

Cell Monolayer ◽

Developmental Phase ◽

Larval Stages ◽

A Cell ◽

Taenia Ovis ◽

Developmental Phases

SUMMARYAn in vitro culture system incorporating a cell monolayer was used to induce most activated oncospheres of Taenia ovis to enter post-oncospheral development. Seven distinct developmental phases were recognized during the 23 days required for development to reach a stage similar to that achieved after 14 days in vivo. Secretions or tegumental substance were released during each developmental phase.

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Fundamental Technologies and Recent Advances of Cell-Sheet-Based Tissue Engineering

International Journal of Molecular Sciences ◽

10.3390/ijms22010425 ◽

2021 ◽

Vol 22 (1) ◽

pp. 425

Author(s):

Chikahiro Imashiro ◽

Tatsuya Shimizu

Keyword(s):

Tissue Engineering ◽

Ex Vivo ◽

Cell Sheet ◽

Tissue Cell ◽

Culture Methods ◽

Recent Advances ◽

A Cell ◽

Cell Sheet Technology

Tissue engineering has attracted significant attention since the 1980s, and the applications of tissue engineering have been expanding. To produce a cell-dense tissue, cell sheet technology has been studied as a promising strategy. Fundamental techniques involving tissue engineering are mainly introduced in this review. First, the technologies to fabricate a cell sheet were reviewed. Although temperature-responsive polymer-based technique was a trigger to establish and spread cell sheet technology, other methodologies for cell sheet fabrication have also been reported. Second, the methods to improve the function of the cell sheet were investigated. Adding electrical and mechanical stimulation on muscle-type cells, building 3D structures, and co-culturing with other cell species can be possible strategies for imitating the physiological situation under in vitro conditions, resulting in improved functions. Finally, culture methods to promote vasculogenesis in the layered cell sheets were introduced with in vivo, ex vivo, and in vitro bioreactors. We believe the present review that shows and compares the fundamental technologies and recent advances for cell-sheet-based tissue engineering should promote further development of tissue engineering. The development of cell sheet technology should promote many bioengineering applications.

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Ultrastructure of melanocyte-keratinocyte interactions and pigment transfer in vitro

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100084120 ◽

1978 ◽

Vol 36 (2) ◽

pp. 650-651

Author(s):

Raul I. Garcia ◽

Evelyn A. Flynn ◽

George Szabo

Keyword(s):

Direct Injection ◽

Skin Pigmentation ◽

Freeze Fracture ◽

Pigment Granule ◽

Time Lapse ◽

Ultrastructural Level ◽

Lanthanum Tracer ◽

A Cell

Skin pigmentation in mammals involves the interaction of epidermal melanocytes and keratinocytes in the structural and functional unit known as the Epidermal Melanin Unit. Melanocytes(M) synthesize melanin within specialized membrane-bound organelles, the melanosome or pigment granule. These are subsequently transferred by way of M dendrites to keratinocytes(K) by a mechanism still to be clearly defined. Three different, though not necessarily mutually exclusive, mechanisms of melanosome transfer have been proposed: cytophagocytosis by K of M dendrite tips containing melanosomes, direct injection of melanosomes into the K cytoplasm through a cell-to-cell pore or communicating channel formed by localized fusion of M and K cell membranes, release of melanosomes into the extracellular space(ECS) by exocytosis followed by K uptake using conventional phagocytosis. Variability in methods of transfer has been noted both in vivo and in vitro and there is evidence in support of each transfer mechanism. We Have previously studied M-K interactions in vitro using time-lapse cinemicrography and in vivo at the ultrastructural level using lanthanum tracer and freeze-fracture.

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Analysis of the Dynamics of the Electric Capacitance of a Cell Monolayer at the Late Stages of Caco-2 cell Differentiation

Biotekhnologiya ◽

10.21519/0234-2758-2019-35-6-87-90 ◽

2019 ◽

Vol 35 (6) ◽

pp. 87-90

Author(s):

S.V. Nikulin ◽

V.A. Petrov ◽

D.A. Sakharov

Keyword(s):

Impedance Spectroscopy ◽

Cell Monolayer ◽

Microfluidic Chips ◽

Russian Science ◽

Electric Capacitance ◽

A Cell ◽

Static Conditions ◽

Late Stages

The real-time monitoring of electric capacitance (impedance spectroscopy) allowed obtaining evidence that structures which look like intestinal villi can be formed during the cultivation under static conditions as well as during the cultivation in microfluidic chips. It was shown in this work via transcriptome analysis that the Hh signaling pathway is involved in the formation of villus-like structures in vitro, which was previously shown for their formation in vivo. impedance spectroscopy, intestine, villi, electric capacitance, Hh The study was funded by the Russian Science Foundation (Project 16-19-10597).

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Drosophila Hormone Receptor 38 Functions in Metamorphosis: A Role in Adult Cuticle Formation

Genetics ◽

10.1093/genetics/149.3.1465 ◽

1998 ◽

Vol 149 (3) ◽

pp. 1465-1475 ◽

Cited By ~ 3

Author(s):

T Kozlova ◽

G V Pokholkova ◽

G Tzertzinis ◽

J D Sutherland ◽

I F Zhimulev ◽

...

Keyword(s):

Pupal Stage ◽

Transcription Unit ◽

P Element ◽

Specific Response ◽

Orphan Receptors ◽

Mrna Isoforms ◽

A Cell ◽

In Vivo Analysis

Abstract DHR38 is a member of the steroid receptor superfamily in Drosophila homologous to the vertebrate NGFI-B-type orphan receptors. In addition to binding to specific response elements as a monomer, DHR38 interacts with the USP component of the ecdysone receptor complex in vitro, in yeast and in a cell line, suggesting that DHR38 might modulate ecdysone-triggered signals in the fly. We characterized the molecular structure and expression of the Dhr38 gene and initiated an in vivo analysis of its function(s) in development. The Dhr38 transcription unit spans more than 40 kb in length, includes four introns, and produces at least four mRNA isoforms differentially expressed in development; two of these are greatly enriched in the pupal stage and encode nested polypeptides. We characterized four alleles of Dhr38: a P-element enchancer trap line, l(2)02306, which shows exclusively epidermal staining in the late larval, pre-pupal and pupal stages, and three EMS-induced alleles. Dhr38 alleles cause localized fragility and rupturing of the adult cuticle, demonstrating that Dhr38 plays an important role in late stages of epidermal metamorphosis.

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Adipose and Muscle Cell Co-Culture System: A Novel In Vitro Tool to Mimic the In Vivo Cellular Environment

Biology ◽

10.3390/biology10010006 ◽

2020 ◽

Vol 10 (1) ◽

pp. 6

Author(s):

Palaniselvam Kuppusamy ◽

Dahye Kim ◽

Ilavenil Soundharrajan ◽

Inho Hwang ◽

Ki Choon Choi

Keyword(s):

Drug Development ◽

Culture System ◽

Cell Types ◽

Culture Cell ◽

In Vitro Techniques ◽

Culture Systems ◽

Complex Interactions ◽

Cellular Environment

A co-culture system allows researchers to investigate the complex interactions between two cell types under various environments, such as those that promote differentiation and growth as well as those that mimic healthy and diseased states, in vitro. In this paper, we review the most common co-culture systems for myocytes and adipocytes. The in vitro techniques mimic the in vivo environment and are used to investigate the causal relationships between different cell lines. Here, we briefly discuss mono-culture and co-culture cell systems and their applicability to the study of communication between two or more cell types, including adipocytes and myocytes. Also, we provide details about the different types of co-culture systems and their applicability to the study of metabolic disease, drug development, and the role of secretory factors in cell signaling cascades. Therefore, this review provides details about the co-culture systems used to study the complex interactions between adipose and muscle cells in various environments, such as those that promote cell differentiation and growth and those used for drug development.

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