A Novel Cross-Linked Hyaluronic Acid Porous Scaffold for Cartilage Repair

Purpose An important feature of biomaterials used in cartilage regeneration is their influence on the establishment and stabilization of a chondrocytic phenotype of embedded cells. The purpose of this study was to examine the effects of a porous 3-dimensional scaffold made of cross-linked hyaluronic acid on the expression and synthesis performance of human articular chondrocytes. Materials and Methods Osteoarthritic chondrocytes from 5 patients with a mean age of 74 years were passaged twice and cultured within the cross-linked hyaluronic acid scaffolds for 2 weeks. Analyses were performed at 3 different time points. For estimation of cell content within the scaffold, DNA-content (CyQuant cell proliferation assay) was determined. The expression of chondrocyte-specific genes by embedded cells as well as the total amount of sulfated glycosaminoglycans produced during the culture period was analyzed in order to characterize the synthesis performance and differentiation status of the cells. Results Cells showed a homogenous distribution within the scaffold. DNA quantification revealed a reduction of the cell number. This might be attributed to loss of cells from the scaffold during media exchange connected with a stop in cell proliferation. Indeed, the expression of cartilage-specific genes and the production of sulfated glycosaminoglycans were increased and the differentiation index was clearly improved. Conclusions These results suggest that the attachment of osteoarthritic P2 chondrocytes to the investigated material enhanced the chondrogenic phenotype as well as promoted the retention.

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Human platelet-rich plasma improves the nesting and differentiation of human chondrocytes cultured in stabilized porous chitosan scaffolds

Journal of Tissue Engineering ◽

10.1177/2041731417697545 ◽

2017 ◽

Vol 8 ◽

pp. 204173141769754 ◽

Cited By ~ 6

Author(s):

Maria Sancho-Tello ◽

Sara Martorell ◽

Manuel Mata Roig ◽

Lara Milián ◽

MA Gámiz-González ◽

...

Keyword(s):

Type I Collagen ◽

Articular Chondrocytes ◽

Platelet Rich Plasma ◽

Cartilage Regeneration ◽

Cell Number ◽

Type I ◽

Type Ii ◽

Cell Counts ◽

Porous Chitosan ◽

Chitosan Scaffolds

The clinical management of large-size cartilage lesions is difficult due to the limited regenerative ability of the cartilage. Different biomaterials have been used to develop tissue engineering substitutes for cartilage repair, including chitosan alone or in combination with growth factors to improve its chondrogenic properties. The main objective of this investigation was to evaluate the benefits of combining activated platelet-rich plasma with a stabilized porous chitosan scaffold for cartilage regeneration. To achieve this purpose, stabilized porous chitosan scaffolds were prepared using freeze gelation and combined with activated platelet-rich plasma. Human primary articular chondrocytes were isolated and cultured in stabilized porous chitosan scaffolds with and without combination to activated platelet-rich plasma. Scanning electron microscopy was used for the morphological characterization of the resulting scaffolds. Cell counts were performed in hematoxylin and eosin–stained sections, and type I and II collagen expression was evaluated using immunohistochemistry. Significant increase in cell number in activated platelet-rich plasma/stabilized porous chitosan was found compared with stabilized porous chitosan scaffolds. Chondrocytes grown on stabilized porous chitosan expressed high levels of type I collagen but type II was not detectable, whereas cells grown on activated platelet rich plasma/stabilized porous chitosan scaffolds expressed high levels of type II collagen and type I was almost undetectable. In summary, activated platelet-rich plasma increases nesting and induces the differentiation of chondrocytes cultured on stabilized porous chitosan scaffolds.

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Chondrogenic differentiation induced by extracellular vesicles bound to a nanofibrous substrate

npj Regenerative Medicine ◽

10.1038/s41536-021-00190-8 ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Marta R. Casanova ◽

Hugo Osório ◽

Rui L. Reis ◽

Albino Martins ◽

Nuno M. Neves

Keyword(s):

Extracellular Vesicles ◽

Tissue Regeneration ◽

Collagen Type ◽

Articular Chondrocytes ◽

Cartilage Regeneration ◽

Type Ii ◽

Chondrogenic Potential ◽

Marrow Mesenchymal Stem Cells ◽

Chondrogenic Phenotype ◽

Medium Supplementation

AbstractExtracellular vesicles (EVs) are being increasingly studied owing to its regenerative potential, namely EVs derived from human bone marrow mesenchymal stem cells (hBM-MSCs). Those can be used for controlling inflammation, repairing injury, and enhancing tissue regeneration. Differently, the potential of EVs derived from human articular chondrocytes (hACs) to promote cartilage regeneration has not been thoroughly investigated. This work aims to develop an EVs immobilization system capable of selectively bind EVs present in conditioned medium obtained from cultures of hACs or hBM-MSC. For that, an anti-CD63 antibody was immobilized at the surface of an activated and functionalized electrospun nanofibrous mesh. The chondrogenic potential of bound EVs was further assessed by culturing hBM-MSCs during 28 days under basal conditions. EVs derived from hACs cultured under differentiation medium or from chondrogenically committed hBM-MSCs induced a chondrogenic phenotype characterized by marked induction of SOX9, COMP, Aggrecan and Collagen type II, and matrix glycosaminoglycans synthesis. Indeed, both EVs immobilization systems outperformed the currently used chondroinductive strategies. These data show that naturally secreted EVs can guide the chondrogenic commitment of hBM-MSCs in the absence of any other chemical or genetic chondrogenic inductors based in medium supplementation.

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Human Articular Chondrocytes Retain Their Phenotype in Sustained Hypoxia While Normoxia Promotes Their Immunomodulatory Potential

Cartilage ◽

10.1177/1947603518769714 ◽

2018 ◽

Vol 10 (4) ◽

pp. 467-479 ◽

Cited By ~ 11

Author(s):

Claire Mennan ◽

John Garcia ◽

Helen McCarthy ◽

Sharon Owen ◽

Jade Perry ◽

...

Keyword(s):

Articular Chondrocytes ◽

Quantitative Polymerase Chain Reaction ◽

Growth Factor Receptor ◽

Interferon Γ ◽

Human Articular Chondrocytes ◽

Pellet Culture ◽

3 Dimensional ◽

Hypoxic Conditions ◽

Chondrogenic Phenotype ◽

Immunomodulatory Potential

Objective To assess the phenotype of human articular chondrocytes cultured in normoxia (21% O2) or continuous hypoxia (2% O2). Design Chondrocytes were extracted from patients undergoing total knee replacement ( n = 5) and cultured in ~21% (normoxic chondrocytes, NC) and 2% (hypoxic chondrocytes, HC) oxygen in both monolayer and 3-dimensional (3D) pellet culture and compared with freshly isolated chondrocytes (FC). Cells were assessed by flow cytometry for markers indicative of mesenchymal stromal cells (MSCs), chondrogenic-potency and dedifferentiation. Chondrogenic potency and immunomodulatory gene expression was assessed in NC and HC by reverse transcription quantitative polymerase chain reaction. Immunohistochemistry was used to assess collagen II production following 3D pellet culture. Results NC were positive (>97%, n = 5) for MSC markers, CD73, CD90, and CD105, while HC demonstrated <90% positivity ( n = 4) and FC ( n = 5) less again (CD73 and CD90 <20%; CD105 <40%). The markers CD166 and CD151, indicative of chondrogenic de-differentiation, were significantly higher on NC compared with HC and lowest on FC. NC also produced the highest levels of CD106 and showed the greatest levels of IDO expression, following interferon-γ stimulation, indicating immunomodulatory potential. NC produced the highest levels of CD49c (>60%) compared with HC and FC in which production was <2%. Hypoxic conditions upregulated expression of SOX9, frizzled-related protein ( FRZB), fibroblast growth factor receptor 3 ( FGFR3), and collagen type II ( COL2A1) and downregulated activin receptor-like kinase 1 ( ALK1) in 3 out of 4 patients compared with normoxic conditions for monolayer cells. Conclusions Hypoxic conditions encourage retention of a chondrogenic phenotype with some immunomodulatory potential, whereas normoxia promotes dedifferentiation of chondrocytes toward an MSC phenotype with loss of chondrogenic potency but enhanced immunomodulatory capacity.

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In Vitro Evaluation of a Composite Gelatin–Hyaluronic Acid–Alginate Porous Scaffold with Different Pore Distributions for Cartilage Regeneration

Gels ◽

10.3390/gels7040165 ◽

2021 ◽

Vol 7 (4) ◽

pp. 165

Author(s):

Ssu-Meng Haung ◽

Yu-Ting Lin ◽

Shih-Ming Liu ◽

Jian-Chih Chen ◽

Wen-Cheng Chen

Keyword(s):

Particle Size ◽

Hyaluronic Acid ◽

Pore Size ◽

Porous Scaffold ◽

Musculoskeletal Diseases ◽

Cartilage Regeneration ◽

Hydrogel Scaffold ◽

Growth Environment ◽

Hydrogel Scaffolds ◽

Proliferation And Differentiation

Although considerable achievements have been made in the field of regenerative medicine, since self-repair is not an advanced ability of articular cartilage, the regeneration of osteochondral defects is still a challenging problem in musculoskeletal diseases. Cartilage regeneration aims to design a scaffold with appropriate pore structure and biological and mechanical properties for the growth of chondrocytes. In this study, porous scaffolds made of gelatin, hyaluronic acid, alginate, and sucrose in different proportions of 2 g (SL2) and 4 g (SL4) were used as porogens in a leaching process. Sucrose with particle size ranges of 88–177 μm (Hμ) and 44–74 μm (SHμ) was added to the colloid, and the individually cross-linked hydrogel scaffolds with controllable pore size for chondrocyte culture were named Hμ-SL2, Hμ-SL4, SHμ-SL2 and SHμ-SL4. The perforation, porosity, mechanical strength, biocompatibility, and proliferation characteristics of the hydrogel scaffold and its influence on chondrocyte differentiation are discussed. Results show that the addition of porogen increases the porosity of the hydrogel scaffold. Conversely, when porogens with the same particle size are added, the pore size decreases as the amount of porogen increases. The perforation effect of the hydrogel scaffolds formed by the porogen is better at 88–177 μm compared with that at 44–74 μm. Cytotoxicity analysis showed that all the prepared hydrogel scaffolds were non-cytotoxic, indicating that no cross-linking agent residues that could cause cytotoxicity were found. In the proliferation and differentiation of the chondrocytes, the SHμ-SL4 hydrogel scaffold with the highest porosity and strength did not achieve the best performance. However, due to the compromise between perforation pores, pore sizes, and strength, as well as considering cell proliferation and differentiation, Hμ–SL4 scaffold provided a more suitable environment for the chondrocytes than other groups; therefore, it can provide the best chondrocyte growth environment for this study. The development of hydrogels with customized pore properties for defective cartilage is expected to meet the requirements of the ultimate clinical application.

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Müllerian Inhibiting Substance Is Required for Germ Cell Proliferation during Early Gonadal Differentiation in Medaka (Oryzias latipes)

Endocrinology ◽

10.1210/en.2007-1535 ◽

2007 ◽

Vol 149 (4) ◽

pp. 1813-1819 ◽

Cited By ~ 47

Author(s):

Eri Shiraishi ◽

Norifumi Yoshinaga ◽

Takeshi Miura ◽

Hayato Yokoi ◽

Yuko Wakamatsu ◽

...

Keyword(s):

Cell Proliferation ◽

Germ Cell ◽

Germ Cells ◽

Sex Differentiation ◽

Somatic Cells ◽

Oryzias Latipes ◽

Cell Number ◽

Gonadal Differentiation ◽

Loss Of Function ◽

Germ Cell Proliferation

Müllerian inhibiting substance (MIS) is a glycoprotein belonging to the TGF-β superfamily. In mammals, MIS is responsible for the regression of Müllerian ducts in the male fetus. However, the role of MIS in gonadal sex differentiation of teleost fish, which have no Müllerian ducts, has yet to be clarified. In the present study, we examined the expression pattern of mis and mis type 2 receptor (misr2) mRNAs and the function of MIS signaling in early gonadal differentiation in medaka (teleost, Oryzias latipes). In situ hybridization showed that both mis and misr2 mRNAs were expressed in the somatic cells surrounding the germ cells of both sexes during early sex differentiation. Loss-of-function of either MIS or MIS type II receptor (MISRII) in medaka resulted in suppression of germ cell proliferation during sex differentiation. These results were supported by cell proliferation assay using 5-bromo-2′-deoxyuridine labeling analysis. Treatment of tissue fragments containing germ cells with recombinant eel MIS significantly induced germ cell proliferation in both sexes compared with the untreated control. On the other hand, culture of tissue fragments from the MIS- or MISRII-defective embryos inhibited proliferation of germ cells in both sexes. Moreover, treatment with recombinant eel MIS in the MIS-defective embryos dose-dependently increased germ cell number in both sexes, whereas in the MISRII-defective embryos, it did not permit proliferation of germ cells. These results suggest that in medaka, MIS indirectly stimulates germ cell proliferation through MISRII, expressed in the somatic cells immediately after they reach the gonadal primordium.

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STUDIES IN THE BIOLOGY OF METALS

Journal of Experimental Medicine ◽

10.1084/jem.48.5.659 ◽

1928 ◽

Vol 48 (5) ◽

pp. 659-665 ◽

Cited By ~ 15

Author(s):

Frederick S. Hammett ◽

Vilma L. Wallace

Keyword(s):

Cell Proliferation ◽

Chick Embryo ◽

Body Length ◽

Rapid Growth ◽

Cell Number ◽

Point Of View ◽

Lead Ion ◽

Head Region ◽

Differential Development

Our study of the effect of the lead ion on the development of the chick embryo has brought out the following facts: 1. Gross growth is retarded. 2. Somite growth is retarded to a degree greater than that exhibited by body length and width. 3. The head and optic anlagen are regions of particular sensitivity. Their differential development is markedly inhibited. From the purely biological point of view these results are in line with the findings of Child (10) and his school as to the sensitivity of the head end of rapidly growing organisms to harmful influences, and with those of Stockard (11) as to the peculiar sensitivity of the optic anlagen. It is almost too well known to need repetition that the head region and the somites of embryos are specific areas of intense growth by increase in cell number. Therefore, turning from the general to the particular, the differential retardation of these regions which is caused by lead, is evidence justifying the conclusion that it is areas of rapid growth by cell proliferation which are selectively inhibited by this metallic ion.

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Digital Light Processing Bioprinted Human Chondrocyte-Laden Poly (γ-Glutamic Acid)/Hyaluronic Acid Bio-Ink towards Cartilage Tissue Engineering

Biomedicines ◽

10.3390/biomedicines9070714 ◽

2021 ◽

Vol 9 (7) ◽

pp. 714

Author(s):

Alvin Kai-Xing Lee ◽

Yen-Hong Lin ◽

Chun-Hao Tsai ◽

Wan-Ting Chang ◽

Tsung-Li Lin ◽

...

Keyword(s):

United States ◽

Tissue Engineering ◽

Hyaluronic Acid ◽

Glutamic Acid ◽

Cellular Proliferation ◽

Cartilage Tissue Engineering ◽

Cartilage Regeneration ◽

The United States ◽

Cartilage Injury ◽

Cartilage Tissue

Cartilage injury is the main cause of disability in the United States, and it has been projected that cartilage injury caused by osteoarthritis will affect 30% of the entire United States population by the year 2030. In this study, we modified hyaluronic acid (HA) with γ-poly(glutamic) acid (γ-PGA), both of which are common biomaterials used in cartilage engineering, in an attempt to evaluate them for their potential in promoting cartilage regeneration. As seen from the results, γ-PGA-GMA and HA, with glycidyl methacrylate (GMA) as the photo-crosslinker, could be successfully fabricated while retaining the structural characteristics of γ-PGA and HA. In addition, the storage moduli and loss moduli of the hydrogels were consistent throughout the curing durations. However, it was noted that the modification enhanced the mechanical properties, the swelling equilibrium rate, and cellular proliferation, and significantly improved secretion of cartilage regeneration-related proteins such as glycosaminoglycan (GAG) and type II collagen (Col II). The cartilage tissue proof with Alcian blue further demonstrated that the modification of γ-PGA with HA exhibited suitability for cartilage tissue regeneration and displayed potential for future cartilage tissue engineering applications. This study built on the previous works involving HA and further showed that there are unlimited ways to modify various biomaterials in order to further bring cartilage tissue engineering to the next level.

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Cell proliferation and migration on collagen substrata in vitro

Journal of Cell Science ◽

10.1242/jcs.41.1.159 ◽

1980 ◽

Vol 41 (1) ◽

pp. 159-175

Author(s):

S.L. Schor

Keyword(s):

Cell Proliferation ◽

Cell Migration ◽

Collagen Gel ◽

Collagen Fibres ◽

3 Dimensional ◽

Cell Proliferation And Migration ◽

Native Collagen ◽

And Migration ◽

Collagen Gel Matrix ◽

Proliferation And Migration

Quantitative data are presented regarding cell proliferation and migration on (a) collagen films (b) the surface of 3-dimensional gels of native collagen fibres and (c) within the 3-dimensional collagen gel matrix, as part of a study of the effects of the extracellular matrix on cell behaviour. The nature of the collagen environment was found to influence the proliferation of certain cell types, but not of others. For example, HeLa cells proliferate at approximately the same rate and reach the same saturation cell densities on all of the collagen substrata, while human skin fibroblasts grow more slowly within the 3-dimensional collagen gel matrix compared with cells either on the gel surface or on collagen films. The 3-dimensional gels of native collagen fibres may also be used to study cell migration on the gel surface, as well as cell migration (or ‘infiltration’) from the gel surface into the 3-dimensional collagen matrix. Two methods have been used to obtain quantitative information concerning cell infiltration into the collagen gel, one involving the selective removal of cells from the gel surface, while the other relies on direct microscopic examination. Of the cells examined to date, epithelial cells (both normal and tumour) do not show infiltrative behaviour, while both normal and virally transformed fibroblasts, as well as tumour cells of non-epithelial origin (e.g. melanoma), do infiltrate into the collagen gel matrix, at rates which vary considerably according to cell type.

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