Spontaneous redifferentiation of dedifferentiated human articular chondrocytes on hydrogel surfaces (original) (raw)
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Hydrogel optimization for cultured elastic chondrocytes seeded onto a polyglycolic acid scaffold
Journal of Biomedical Materials Research Part A, 2005
The purpose of this study was to compare the effect of different hydrogels on the production of tissue-engineered cartilage based on polyglycolic acid (PGA). Chondrocytes were isolated from adult sheep auricles. Alginate, Type I collagen, methylcellulose, and pluronic F127 hydrogels were evaluated, as were controls prepared without hydrogels. Proliferated chondrocytes were mixed with each hydrogel at 20 ϫ 10 6 cells/mL and seeded onto PGA (1 ϫ 1 ϫ 0.2 cm, n ϭ 60). The constructs were cultured with serum-free medium containing 5 ng/mL TGF- 2 and 5 ng/mL des(1-3)IGF-I in rotational bioreactors for up to 6 weeks. The cellular morphology, histology, and biochemistry were analyzed. Type I collagen, methylcellulose, and pluronic F127 displayed improved cartilage matrix deposition in terms of histology and biochemistry compared to alginate. It was not concluded that the combined seeding of chondrocytes and hydrogels on a PGA scaffold had significantly better effects than cell seeding without hydrogels. However, the histology and other useful findings in this ECM analyses suggested that Type I collagen and MC hydrogels were the best candidates for cartilage regeneration, because of their stimulation for chondrocyte proliferation in a three-dimensional culture as well as cartilage regeneration.
Cell Transplantation, 2001
Natural cartilage tissue has a limited self-regenerative capacity; thus, strategies to replenish the lost cartilage are desired in reconstructive and plastic surgery. Tissue-engineered cartilage using biodegradable polymeric scaffolds is one such approach gaining wide attention. We have earlier demonstrated the biocompatible nature and ability of chitosan-gelatin hydrogel to maintain differentiated populations of respiratory epithelial cells. The aim of the present study was to evaluate its suitability as a substratum for inducing chondrocyte growth and differentiation. Electron microscopic (SEM) analysis of freeze-dried hydrogels showed a highly porous morphology with interconnections as seen in cross section. Chondrocytes were observed to attach and exhibited a differentiated phenotype with proper cell-cell contact on three-dimensional freeze-dried hydrogels. When cultured on two-dimensional hydrogel films they showed higher growth rates (4-6%) compared with a polystyrene (TCPS) control until 6 days (p > 0.05), which slowed down after 10 days. Immunofluorescent microscopic studies revealed that chondrocytes on hydrogel films exhibited comparable expression of β1 integrin (CD29) to TCPS controls, indicating the ability of the hydrogel substrate to maintain normal expression of β1 integrin. RT-PCR analysis of chondrocytes grown on hydrogel films showed that chondrocytes express the mRNA for extracellular matrix proteins such as collagen type IIα1 (COL IIα1), COL III, COL IXα3. Expression of COL I was less prominent than COL II as indication of differentiation. Expression of COL X could not be detected, suggesting an absence of chondrocyte hypertrophy. Chondrocytes also showed weak mRNA expression of aggrecan, a cartilage-specific proteoglycan. All of these results point out the ability of the chitosan-gelatin hydrogel to induce the expression of mRNAs for cartilagespecific extracellular matrix proteins by nasal septal chondrocytes. This hydrogel needs to be further evaluated for its ability to support chondrocyte-specific marker expression to explore the possibility of forming a tissue resembling natural cartilage in vitro.
Methods in Molecular Biology, 2010
This chapter is intended to provide a summary of the current materials used in cell encapsulation technology as well as methods for evaluating the performance of cells encapsulated in a polymeric matrix. In particular, it describes the experimental procedure to prepare a hydrogel matrix based on natural polymers for encapsulating and culturing human articular chondrocytes with the interest in cartilage regeneration. Protocols to evaluate the viability, proliferation, differentiation, and matrix production of embedded cells are also described and include standard protocols such as the MTT and [3H] Thymidine assays, reverse transcription polymerase chain reaction (RT-PCR) technique, histology, and immunohistochemistry analysis. The assessment of cell distribution within the 3D hydrogel construct is also described using APoTome analysis.
Response of zonal chondrocytes to extracellular matrix-hydrogels
FEBS Letters, 2007
We investigated the biological response of chondrocytes isolated from different zones of articular cartilage and their cellular behaviors in poly (ethylene glycol)-based (PEG) hydrogels containing exogenous type I collagen, hyaluronic acid (HA), or chondroitin sulfate (CS). The cellular morphology was strongly dependent on the extracellular matrix component of hydrogels. Additionally, the exogenous extracellular microenvironment affected matrix production and cartilage specific gene expression of chondrocytes from different zones. CS-based hydrogels showed the strongest response in terms of gene expression and matrix accumulation for both superficial and deep zone chondrocytes, but HA and type I collagen-based hydrogels demonstrated zonal-dependent cellular responses.
Characterization of polydactyly chondrocytes and their use in cartilage engineering
Scientific Reports
treating cartilage injuries and degenerations represents an open surgical challenge. the recent advances in cell therapies have raised the need for a potent off-the-shelf cell source. Intra-articular injections of tGF-β transduced polydactyly chondrocytes have been proposed as a chronic osteoarthritis treatment but despite promising results, the use of gene therapy still raises safety concerns. In this study, we characterized infant, polydactyly chondrocytes during in vitro expansion and chondrogenic redifferentiation. Polydactyly chondrocytes have a steady proliferative rate and re-differentiate in 3D pellet culture after up to five passages. Additionally, we demonstrated that polydactyly chondrocytes produce cartilage-like matrix in a hyaluronan-based hydrogel, namely transglutaminase cross-linked hyaluronic acid (HA-tG). We utilized the versatility of tG cross-linking to augment the hydrogels with heparin moieties. The heparin chains allowed us to load the scaffolds with TGF-β1, which induced cartilage-like matrix deposition both in vitro and in vivo in a subcutaneous mouse model. this strategy introduces the possibility to use infant, polydactyly chondrocytes for the clinical treatment of joint diseases. Globally 1.2 million patients are affected by cartilage degeneration annually, a burden that will increase as population ages 1,2 and which accounts for one of the leading causes of disabilities worldwide 3. Additionally, it is estimated that up to 36% of athletes present focal cartilage defects 4 , while up to 69% of adults older than 50 years old show signs of cartilage anomalies in their knees 5. Articular cartilage has a very limited ability to regenerate due to its low cellularity and lack of vascularization. Consequently, cartilage injuries often lead to the development of post-traumatic osteoarthritis (OA) and frequently require surgical intervention 6. The limited capability of cartilage to heal has driven the development of cell-based and tissue engineering strategies 7 such as microfracture, autologous chondrocyte implantation (ACI) and matrix-assisted autologous chondrocyte implantation (MACI). ACI is so far the most effective, clinically approved technique to repair cartilage lesions 8. However, this technique has major limitations, which include fibrocartilage tissue formation 9,10 , lack of integration of the grafts, the requirement of multiple surgeries and high donor-to-donor variability 11. These latter drawbacks contribute to more than 20% of non-responders to ACI 12,13 and justify the need for a next-generation of chondrocyte implantation. The potential of infant and juvenile cartilage as non-immunogenic, off-the-shelf cell source with stable chondrogenic potential have been extensively investigated and exploited. Infant chondrocytes from deceased donors have been characterized and proposed as a cell source for scaffold-free articular cartilage repair 14,15 and disc regeneration techniques 16. Juvenile cells were shown not only to have an enhanced, inherent ability to synthesize cartilage matrix 14 , but also to exhibit immunosuppressive properties 17. Infant hip chondrocytes from donors with hip dysplasia and Perthes disease in polyglycolic acid (PGA)-fibrin scaffolds were shown to express higher levels of chondrogenic markers and lower levels of undesirable fibroblastic markers compared to adult cells 18. Clinically, the use of allogeneic, juvenile cartilage has been commercialized since 2007 as DeNovo ® NT Natural
Porous multi-layered composite hydrogel as cell substrate for in vitro culture of chondrocytes
International Journal of Polymeric Materials and Polymeric Biomaterials, 2020
A porous multi-layered composite hydrogel (MSC), made of poly(vinyl alcohol) (PVA) and hydroxyapatite (HA), suitable as substitute for damaged cartilage, has been modified by the production of pores and its cytocompatibility and ability to prevent chondrocyte dedifferentiation in in vitro cell culture systems have been evaluated. Pores resulted homogeneously distributed on all the hydrogel surface and bulk. The material was not able to compromise cell viability, proliferation and structure of both NIH3T3 mouse fibroblasts and human chondrocytes (HC), supported HC colonization and showed a good ability to stimulate the production of hyaline extracellular matrix (ECM).
A silanized hydroxypropyl methylcellulose hydrogel for the three-dimensional culture of chondrocytes
Biomaterials, 2005
Articular cartilage has limited intrinsic repair capacity. In order to promote cartilage repair, the amplification and transfer of autologous chondrocytes using three-dimensional scaffolds have been proposed. We have developed an injectable and self-setting hydrogel consisting of hydroxypropyl methylcellulose grafted with silanol groups (Si-HPMC). The aim of the present work is to assess both the in vitro cytocompatibility of this hydrogel and its ability to maintain a chondrocyte-specific phenotype. Primary chondrocytes isolated from rabbit articular cartilage (RAC) and two human chondrocytic cell lines (SW1353 and C28/I2) were cultured into the hydrogel. Methyl tetrazolium salt (MTS) assay and cell counting indicated that Si-HPMC hydrogel did not affect respectively chondrocyte viability and proliferation. Fluorescent microscopic observations of RAC and C28/I2 chondrocytes double-labeled with cell tracker green and ethidium homodimer-1 revealed that chondrocytes proliferated within Si-HPMC. Phenotypic analysis (RT-PCR and Alcian blue staining) indicates that chondrocytes, when three-dimensionnally cultured within Si-HPMC, expressed transcripts encoding type II collagen and aggrecan and produced sulfated glycosaminoglycans. These results show that Si-HPMC allows the growth of differentiated chondrocytes. Si-HPMC therefore appears as a potential scaffold for threedimensional amplification and transfer of chondrocytes in cartilage tissue engineering. r
Acta Biomaterialia, 2013
New systematic approaches are necessary to determine and optimize the chemical and mechanical scaffold properties for hyaline cartilage generation using the limited cell numbers obtained from primary human sources. Peptide functionalized hydrogels possessing continuous variations in physico-chemical properties are an efficient three-dimensional platform for studying several properties simultaneously. Herein, we describe a polyethylene glycol dimethacrylate (PEGDM) hydrogel system possessing a gradient of arginine-glycine-aspartic acid peptide (RGD) concentrations from 0 mM to 10 mM. The system is used to correlate primary human osteoarthritic chondrocyte proliferation, phenotype maintenance and extracellular matrix (ECM) production to the gradient hydrogel properties. Cell number and chondrogenic phenotype (CD14:CD90 ratios) were found to decline in regions with higher RGD concentrations, while regions with lower RGD concentrations maintained cell number and phenotype. Over three weeks of culture, hydrogel regions containing lower RGD concentrations experience an increase in ECM content compared to regions with higher RGD concentrations. Variations in actin amounts and vinculin organization were observed within the RGD concentration gradients that contribute to the differences in chondrogenic phenotype maintenance and ECM expression.
Matrix Biology, 2010
This study aimed to elucidate the role of charge in mediating chondrocyte response to loading by employing synthetic 3D hydrogels. Specifically, neutral poly(ethylene glycol) (PEG) hydrogels were employed where negatively charged chondroitin sulfate (ChS), one of the main extracellular matrix components of cartilage, was systematically incorporated into the PEG network at 0%, 20% or 40% to control the fixed charge density. PEG hydrogels were employed as a control environment for extracellular events which occur as a result of loading, but which are not associated with a charged matrix (e.g., cell deformation and fluid flow). Freshly isolated bovine articular chondrocytes were embedded in the hydrogels and subject to dynamic mechanical stimulation (0.3 Hz, 15% amplitude strains, 6 hours) and assayed for nitric oxide production, cell proliferation, proteoglycan synthesis, and collagen deposition. In the absence of loading, incorporation of charge inhibited cell proliferation by ~75%, proteoglycan synthesis by ~22-50% depending on ChS content, but had no affect on collagen deposition. Dynamic loading had no effect on cellular responses in PEG hydrogels. However, dynamically loading 20% ChS gels inhibited nitrite production by 50%, cell proliferation by 40%, but stimulated proteoglycan and collagen deposition by 162% and 565%, respectively. Dynamic loading of 40% ChS hydrogels stimulated nitrite production by 62% and proteoglycan synthesis by 123%, but inhibited cell proliferation by 54% and collagen deposition by 52%. Upon removing the load and culturing under free swelling conditions for 36 hrs, the enhanced matrix synthesis observed in the 20% ChS gels was not maintained suggesting that loading is necessary to stimulate matrix production. In conclusion, extracellular events associated with a charged matrix has a dramatic affect on how chondrocytes respond to mechanical stimulation within these artificial 3D matrices suggesting that streaming potentials and/or dynamic changes in osmolarity may be important regulators of chondrocytes while cell deformation and fluid flow appear to have less of an effect.