scholarly journals Direct Perfusion Improves Redifferentiation of Human Chondrocytes in Fibrin Hydrogel with the Deposition of Cartilage Pericellular Matrix

2021 ◽  
Vol 11 (19) ◽  
pp. 8923
Author(s):  
Alexandre Dufour ◽  
Frédéric Mallein-Gerin ◽  
Emeline Perrier-Groult
Keyword(s):  
Type I Collagen ◽  
Articular Chondrocytes ◽  
Type Ii Collagen ◽  
Type I ◽  
Pericellular Matrix ◽  
Fibrin Gels ◽  
Matrix Quality ◽  
The Matrix ◽  
Cartilage Injuries ◽  
Main Component

Articular cartilage has limited potential for self-repair, and cell-based strategies combining scaffolds and chondrocytes are currently used to treat cartilage injuries. However, achieving a satisfying level of cell redifferentiation following expansion remains challenging. Hydrogels and perfusion bioreactors are known to exert beneficial cues on chondrocytes; however, the effect of a combined approach on the quality of cartilage matrix deposited by cells is not fully understood. Here, we combined soluble factors (BMP-2, Insulin, and Triiodothyronine, that is, BIT), fibrin hydrogel, direct perfusion and human articular chondrocytes (HACs) to engineer large cartilage tissues. Following cell expansion, cells were embedded in fibrin gels and cultivated under either static or perfusion conditions. The nature of the matrix synthesized was assessed by Western blotting and immunohistochemistry. The stability of cartilage grafts and integration with native tissue were also investigated by subcutaneous implantation of human osteochondral cylinders in nude mice. Perfusion preconditioning improved matrix quality and spatial distribution. Specifically, perfusion preconditioning resulted in a matrix rich in type II collagen but not in type I collagen, indicating the reconstruction of hyaline cartilage. Remarkably, the production of type VI collagen, the main component of the pericellular matrix, was also increased, indicating that chondrocytes were connecting to the hyaline matrix they produced.

scholarly journals Mechanisms balancing skeletal matrix synthesis and degradation

2002 ◽  
Vol 364 (2) ◽  
pp. 329-341 ◽  
Author(s):  
Harry C. BLAIR ◽  
Mone ZAIDI ◽  
Paul H. SCHLESINGER
Keyword(s):  
Parathyroid Hormone ◽  
Type I Collagen ◽  
Bone Matrix ◽  
Type Ii Collagen ◽  
Type I ◽  
Parathyroid Hormone Related Protein ◽  
Bone Degradation ◽  
The Matrix ◽  
Acid Secreting ◽  
Matrix Removal

Bone is regulated by evolutionarily conserved signals that balance continuous differentiation of bone matrix-producing cells against apoptosis and matrix removal. This is continued from embryogenesis, where the skeleton differentiates as a solid mass and is shaped into separate bones by cell death and proteolysis. The two major tissues of the skeleton are avascular cartilage, with an extracellular matrix based on type II collagen and hydrophilic proteoglycans, and bone, a stronger and lighter material based on oriented type I collagen and hydroxyapatite. Both differentiate from the same mesenchymal stem cells. This differentiation is regulated by a family of related signals centred on bone morphogenic proteins. Fibroblast growth factors, Indian hedgehog and parathyroid hormone-related protein are important in determining the type of matrix and the relation of skeletal and non-skeletal structures. Removal of mineralized matrix involves apoptosis of matrix cells and differentiation of acid-secreting cells (osteoclasts) from macrophage precursors. Key regulators of matrix removal are signals in the tumour-necrosis-factor family. Osteoclasts dissolve bone by isolating a region of the matrix and secreting HCl and proteinases at that site. Successive cycles of removal and replacement allow growth, repair and remodelling. The signals for bone turnover are predominantly cell-membrane-associated, allowing very specific spatial regulation. In addition to its support function, bone is a reservoir of Ca2+, PO3-4 and OH−. Secondary modulation of mineral secretion and bone degradation are mediated by humoral signals, including parathyroid hormone and vitamin D, as well as the cytokines that also regulate the underlying cell differentiation.

Effect of seeding density on stability of the differentiated phenotype of pig articular chondrocytes in culture

1988 ◽  
Vol 89 (3) ◽  
pp. 373-378
Author(s):  
F.M. Watt
Keyword(s):  
Type I Collagen ◽  
Articular Chondrocytes ◽  
Type Ii Collagen ◽  
High Density ◽  
Seeding Density ◽  
Type I ◽  
Low Density ◽  
Keratan Sulphate ◽  
Chondrocyte Phenotype ◽  
Culture Cells

Articular chondrocytes are known to be phenotypically unstable in culture. One condition that has been reported to suppress dedifferentiation is cultivation at high density on tissue-culture plastic. The aim of the experiments described here was to study the effect of seeding density on chondrocyte proliferation and 35SO4 incorporation, and on the types of collagen and proteoglycan synthesized. I found that cells seeded at low or high density reached the same final density at confluence, and that 35SO4 incorporation, while initially higher (per cell) in high-density cultures, fell under both conditions, reaching the same low level after 3 weeks. The proportion of cells expressing keratan sulphate fell in low- but not high-density cultures and the decline was not prevented by inhibition of cell division. In all the cultures cells expressing keratan sulphate tended to have a rounded morphology. After 21 days in culture, chondrocytes grown at high density expressed predominantly large proteoglycans that aggregated with hyaluronic acid, whereas in low-density cultures a smaller, non-aggregating form was also present. By 21 days in culture cells at both high and low density were expressing type I collagen, although the high-density cells also had an extensive extracellular matrix of type II collagen. These observations support the conclusion that high seeding density stabilizes the chondrocyte phenotype to a greater extent than low seeding density. They also suggest that enhanced dedifferentiation at low density may be due to cell spreading, rather than to selective proliferation of a phenotypically unstable subpopulation of cells.

scholarly journals Marine Collagen Hydrolysates Promote Collagen Synthesis, Viability and Proliferation While Downregulating the Synthesis of Pro-Catabolic Markers in Human Articular Chondrocytes

2021 ◽  
Vol 22 (7) ◽  
pp. 3693
Author(s):  
Bastien Bourdon ◽  
Frédéric Cassé ◽  
Nicolas Gruchy ◽  
Pierre Cambier ◽  
Sylvain Leclercq ◽  
...  
Keyword(s):  
Dietary Supplements ◽  
Type I Collagen ◽  
Articular Chondrocytes ◽  
Type Ii Collagen ◽  
Preventive Therapy ◽  
Human Articular Chondrocytes ◽  
Cartilage Tissue ◽  
Type I ◽  
Repair Capacity ◽  
Collagen Hydrolysates

Cartilage is a non-innervated and non-vascularized tissue. It is composed of one main cell type, the chondrocyte, which governs homeostasis within the cartilage tissue, but has low metabolic activity. Articular cartilage undergoes substantial stresses that lead to chondral defects, and inevitably osteoarthritis (OA) due to the low intrinsic repair capacity of cartilage. OA remains an incurable degenerative disease. In this context, several dietary supplements have shown promising results, notably in the relief of OA symptoms. In this study, we investigated the effects of collagen hydrolysates derived from fish skin (Promerim®30 and Promerim®60) and fish cartilage (Promerim®40) on the phenotype and metabolism of human articular chondrocytes (HACs). First, we demonstrated the safety of Promerim® hydrolysates on HACs cultured in monolayers. Then we showed that, Promerim® hydrolysates can increase the HAC viability and proliferation, while decreasing HAC SA-β-galactosidase activity. To evaluate the effect of Promerim® on a more relevant model of culture, HAC were cultured as organoids in the presence of Promerim® hydrolysates with or without IL-1β to mimic an OA environment. In such conditions, Promerim® hydrolysates led to a decrease in the transcript levels of some proteases that play a major role in the development of OA, such as Htra1 and metalloproteinase-1. Promerim® hydrolysates downregulated HtrA1 protein expression. In contrast, the treatment of cartilage organoids with Promerim® hydrolysates increased the neosynthesis of type I collagen (Promerim®30, 40 and 60) and type II collagen isoforms (Promerim®30 and 40), the latter being the major characteristic component of the cartilage extracellular matrix. Altogether, our results demonstrate that the use of Promerim® hydrolysates hold promise as complementary dietary supplements in combination with the current classical treatments or as a preventive therapy to delay the occurrence of OA in humans.

scholarly journals The lncRNA, Nespas, Is Associated with Osteoarthritis Progression and Serves as a Potential New Prognostic Biomarker

Cartilage ◽  
2017 ◽  
Vol 10 (2) ◽  
pp. 148-156 ◽  
Author(s):  
Sujung Park ◽  
Myeungsoo Lee ◽  
Churl-Hong Chun ◽  
Eun-Jung Jin
Keyword(s):  
Noncoding Rna ◽  
Type I Collagen ◽  
Articular Chondrocytes ◽  
Type Ii Collagen ◽  
Human Chondrocytes ◽  
Type I ◽  
Caspase 1 ◽  
Osteoarthritis Progression ◽  
Polymerase Chain ◽  
Abnormal Lipid Metabolism

Introduction. In this article, we explored the hypothesis that the long noncoding RNA, Nespas, promotes osteoarthritis (OA) by supporting abnormal lipid metabolism in human chondrocytes. Materials and Methods. Human articular chondrocytes from osteoarthritis patients were used and expression level of Nespas were determined by real-time polymerase chain reaction. Introduction of Nespas and Nespas-associated genes/miRNAs were performed by using a lentiviral system. The effect of Nespas on mitochondrial function was determined by staining mitochondria and analyzing mitopotential and mitochondrial genes. Moreover, to identify the responsible molecules in Nespas-induced pathogenesis, profiling of peroxisomal genes and miRNAs were applied and interactome analysis was performed. Results. Highly elevated levels of Nespas and Acyl-CoA synthetase 6 (ACSL6) were observed in OA patients. Both Nespas overexpression and ACSL6 upregulation into human chondrocytes induced typical OA characteristics, such as downregulation of type II collagen; upregulation of type I collagen, metalloproteinase 13, and caspase-1 and -3; and dysfunction of mitochondria and peroxisome. Co-expression of Nespas and ACSL6 siRNA reduced caspase-1 and -3 levels. Moreover, Nespas overexpression significantly suppressed levels of miR-291a-3p, -196a-5p, -23a-3p, -24-3p, and let-7a-5p, and these miRs are known to potentially target ACSL6 according to ingenuity pathway analysis. We also confirmed that these miRs were significantly suppressed in human OA chondrocytes. Overexpression of miR-291a-3p, -196a-5p, -23a-3p, -24-3p, or let-7a-5p in the presence of Nespas suppressed levels of ACSL6, caspase-1 and -3. Discussion. Overall, we suggest that elevated Nespas level in OA are associated with OA pathogenesis by suppressing miRs targeting ACSL6 and subsequent ACSL6 upregulation.

scholarly journals Synthesis of cartilage matrix by mammalian chondrocytes in vitro. II. Maintenance of collagen and proteoglycan phenotype.

1982 ◽  
Vol 93 (3) ◽  
pp. 751-757 ◽  
Author(s):  
K E Kuettner ◽  
V A Memoli ◽  
B U Pauli ◽  
N C Wrobel ◽  
E J Thonar ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Articular Chondrocytes ◽  
Type Ii Collagen ◽  
Type I ◽  
Type Ii ◽  
Roller Bottle ◽  
The Media ◽  
Bovine Articular Chondrocytes ◽  
Associated Matrix

The in vitro phenotype of bovine articular chondrocytes is described. Chondrocytes plated at high density in roller-bottle and dish cultures were maintained in vitro. The major matrix macromolecules, collagen and proteoglycan, synthesized by these cells were characterized during the course of the culture period. The chondrocytes synthesized mainly Type II collagen, which was found predominantly in the cell-associated matrix. The media contained a mixture of Type II and Type III collagens. Type I collagen was detectable in neither the medium nor the cell-associated matrix. The proteoglycan monomers found in media and cell-associated matrix had the same hydrodynamic sizes as monomers synthesized by cartilage slices or those extracted from adult articular cartilage. The majority of proteoglycans synthesized by the cells were found in high molecular weight aggregates which were readily recovered from the media and were extractable from cell-associated matrix with low ionic strength buffers. The results demonstrate the long-term in vitro phenotypic stability of the bovine articular chondrocytes. The advantages of the in vitro system as a model for studying the effects of external agents, such as drugs and vitamins, are discussed.

scholarly journals Transcriptomics Study to Determine the Molecular Mechanism by which sIL-13Rα2-Fc Inhibits Caudal Intervertebral Disc Degeneration in Rats

2020 ◽  
Vol 2020 ◽  
pp. 1-13
Author(s):  
Xin Wang ◽  
Jianshi Tan ◽  
Junhao Sun ◽  
Pengzhong Fang ◽  
Jinlei Chen ◽  
...  
Keyword(s):  
Intervertebral Disc ◽  
Disc Degeneration ◽  
Intervertebral Disc Degeneration ◽  
Type I Collagen ◽  
Type Ii Collagen ◽  
Type I ◽  
Model Group ◽  
Expression Levels ◽  
Protein Levels ◽  
Collagen Protein

Background. Intervertebral disc degeneration is related to tissue fibrosis. ADAMTS can degrade the important components of the ECM during the process of intervertebral disc degeneration, ultimately resulting in the loss of intervertebral disc function. sIL-13Rα2-Fc can inhibit fibrosis and slow down the degeneration process, but the mechanism involved remains unclear. Objective. To determine the mechanism by which sIL-13Rα2-Fc inhibits ECM degradation and reduces intervertebral disc tissue fibrosis using a transcriptomics analysis. Methods. A rat model of caudal intervertebral disc degeneration was established, and Sirius red staining was used to observe the pathological changes in the caudal intervertebral disc. Transcriptome sequencing was employed to assess the gene expression profiles of the intervertebral disc tissues in the model group and the sIL-13Rα2-Fc-treated group. Differentially expressed genes were identified and analyzed using GO annotation and KEGG pathway analyses. Real-time fluorescence quantitative PCR was used to verify the expression levels of candidate genes. The levels of GAG and HA were quantitatively assessed by ELISA, and the levels of collagen I and collagen II were analyzed by western blotting. Results. Sirius red staining showed that in the model group, the annulus fibrosus was disordered, the number of breaks increased, and the type I collagen protein levels increased, whereas in the sIL-13Rα2-Fc group, the annulus fibrosus was ordered, the number of breaks decreased, and the type II collagen protein levels increased. In comparison with the model group, we identified 58 differentially expressed genes in the sIL-13Rα2-Fc group, and these were involved in 35 signaling pathways. Compared with those in the model group, the mRNA expression levels of Rnux1, Sod2, and Tnfaip6 in the IL-13Rα2-Fc group were upregulated, and the mRNA expression levels of Aldh3a1, Galnt3, Fgf1, Celsr1, and Adamts8 were downregulated; these results were verified by real-time fluorescence quantitative PCR. TIMP-1 (an ADAMTS inhibitor) and TIMP-1 combined with the sIL-13Rα2-Fc intervention increased the levels of GAG and HA, inhibited the expression of type I collagen, and promoted the expression of type II collagen. Conclusion. Adamts8 may participate in the degradation of ECM components such as GAG and HA and lead to an imbalance in the ECM of the intervertebral disc, resulting in intervertebral disc degeneration. sIL-13Rα2-Fc promoted anabolism of the ECM and increased the levels of ECM components by inhibiting the expression of Adamts8, thus maintaining the dynamic equilibrium of the ECM and ultimately delaying intervertebral disc degeneration.

Type VI collagen expression is upregulated in the early events of chondrocyte differentiation

Development ◽  
1993 ◽  
Vol 117 (1) ◽  
pp. 245-251
Author(s):  
R. Quarto ◽  
B. Dozin ◽  
P. Bonaldo ◽  
R. Cancedda ◽  
A. Colombatti
Keyword(s):  
Suspension Culture ◽  
Type I Collagen ◽  
Type Ii Collagen ◽  
Type I ◽  
Type Ii ◽  
Type Vi Collagen ◽  
Full Spectrum ◽  
Collagen Expression ◽  
Hypertrophic Chondrocyte

Dedifferentiated chondrocytes cultured adherent to the substratum proliferate and synthesize large amounts of type I collagen but when transferred to suspension culture they decrease proliferation, resume the chondrogenic phenotype and the synthesis of type II collagen, and continue their maturation to hypertrophic chondrocyte (Castagnola et al., 1986, J. Cell Biol. 102, 2310–2317). In this report, we describe the developmentally regulated expression of type VI collagen in vitro in differentiating avian chondrocytes. Type VI collagen mRNA is barely detectable in dedifferentiated chondrocytes as long as the attachment to the substratum is maintained, but increases very rapidly upon passage of the cells into suspension culture reaching a peak after 48 hours and declining after 5–6 days of suspension culture. The first evidence of a rise in the mRNA steady-state levels is obtained already at 6 hours for the alpha 3(VI) chain. Immunoprecipitation of metabolically labeled cells with type VI collagen antibodies reveals that the early mRNA rise is paralleled by an increased secretion of type VI collagen in cell media. Induction of type VI collagen is not the consequence of trypsin treatment of dedifferentiated cells since exposure to the actin-disrupting drug cytochalasin or detachment of the cells by mechanical procedures has similar effects. In 13-day-old chicken embryo tibiae, where the full spectrum of the chondrogenic differentiation process is represented, expression of type VI collagen is restricted to the articular cartilage where chondrocytes developmental stage is comparable to stage I (high levels of type II collagen expression).(ABSTRACT TRUNCATED AT 250 WORDS)

Cytoskeleton and thyroglobulin expression change during transformation of thyroid epithelium to mesenchyme-like cells

Development ◽  
1988 ◽  
Vol 102 (3) ◽  
pp. 605-622 ◽  
Author(s):  
G. Greenburg ◽  
E.D. Hay
Keyword(s):  
Type I Collagen ◽  
Collagen Gel ◽  
Type I ◽  
Basal Cells ◽  
Collagen Gels ◽  
Expression Change ◽  
Cell Functions ◽  
The Matrix ◽  
3D Matrix ◽  
Mesenchymal Transformation

In considering the mechanism of transformation of epithelium to mesenchyme in the embryo, it is generally assumed that the ability to give rise to fibroblast-like cells is lost as epithelia mature. We reported previously that a definitive embryonic epithelium, that of the anterior lens, gives rise to freely migrating mesenchyme-like cells when suspended in type I collagen matrices. Here, we show that a highly differentiated epithelium that expresses cytokeratin changes to a vimentin cytoskeleton and loses thyroglobulin during epithelial-mesenchymal transformation induced by suspension in collagen gel. Using dispase and collagenase, we isolated adult thyroid follicles devoid of basal lamina and mesenchyme, and we suspended the follicles in 3D collagen gels. Cells bordering the follicle lumen retain epithelial polarity and thyroid phenotype, but basal cell surface organization is soon modified as a result of tissue multilayering and elongation of basal cells into the collagenous matrix. Cytodifferentiation, determined by thyroglobulin immunoreactivity, is lost as the basal epithelial cells move into the matrix after 3–4 days in collagen. By TEM, it can be seen that the elongating cells acquire pseudopodia, filopodia and mesenchyme-like nuclei and RER. Immunofluorescence examination of intermediate filaments showed that freshly isolated follicles and follicles cultured on planar substrata react only with anticytokeratin. However, all of the mesenchyme-like cells express vimentin and they gradually lose cytokeratin. These results suggest that vimentin may be necessary for cell functions associated with migration within a 3D matrix. The mesenchymal cells do not revert to epithelium when grown on planar substrata and the transformation of epithelium to mesenchyme-like cells does not occur within basement membrane gels. The results are relevant to our understanding of the initiation of epithelial-mesenchymal transformation in the embryo and the genetic mechanisms controlling cell shape, polarity and cytoskeletal phenotype.

Stress-Dependent Effects of Platelet-Derived Growth Factor-BB on Fibroblast Migration and Traction Differ in Collagen and Fibrin

2000 ◽  
Author(s):  
David I. Shreiber ◽  
Paul A. J. Enever ◽  
Robert T. Tranquillo
Keyword(s):  
Type I Collagen ◽  
Cell Behavior ◽  
Environmental Cues ◽  
Type I ◽  
Collagen Gels ◽  
Fibrin Gels ◽  
Fibroblast Migration ◽  
Tissue Equivalents ◽  
Stress Dependent ◽  
Statistical Conclusion

Abstract We used our novel assays of cell behavior in tissue equivalents to study the dose-response effects of PDGF-BB on RDF migration and traction in mechanically stressed and stress-free type I collagen and fibrin gels. PDGF-BB increased fibroblast migration significantly in all assays, but the effects on traction depended on the presence of stress and the nature of the ECM. PDGF-BB decreased fibroblast traction in stressed collagen gels, but increased traction in stress-free gels. No statistical conclusion could be inferred for stressed fibrin gels, and increasing PDGF-BB decreased traction in stress-free fibrin gels. These results demonstrate the complex response of fibroblasts to environmental cues, and point to opportunities to orchestrate cell behavior to affect the outcome of wound healing.

In situ hybridization reveals differential spatial distribution of mRNAs for type I and type II collagen in the chick limb bud

Development ◽  
1988 ◽  
Vol 103 (1) ◽  
pp. 111-118 ◽  
Author(s):  
C.J. Devlin ◽  
P.M. Brickell ◽  
E.R. Taylor ◽  
A. Hornbruch ◽  
R.K. Craig ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Limb Development ◽  
Type I Collagen ◽  
Type Ii Collagen ◽  
Limb Bud ◽  
Type I ◽  
Type Ii ◽  
Mrna Accumulation ◽  
Rna Probes

During limb development, type I collagen disappears from the region where cartilage develops and synthesis of type II collagen, which is characteristic of cartilage, begins. In situ hybridization using antisense RNA probes was used to investigate the spatial localization of type I and type II collagen mRNAs. The distribution of the mRNA for type II collagen corresponded well with the pattern of type II collagen synthesis, suggesting control at the level of transcription and mRNA accumulation. In contrast, the pattern of mRNA for type I collagen remained more or less uniform and did not correspond with the synthesis of the protein, suggesting control primarily at the level of translation or of RNA processing.

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