Optimization of Protocol for Isolation of Chondrocytes from Human Articular Cartilage

Cartilage ◽  
2019 ◽  
pp. 194760351987633 ◽  
Author(s):  
Suleiman Alhaji Muhammad ◽  
Norshariza Nordin ◽  
Paisal Hussin ◽  
Muhammad Zulfadli Mehat ◽  
Sheau Wei Tan ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Collagen Type ◽  
Maximum Yield ◽  
Cartilage Tissue Engineering ◽  
Cartilage Tissue ◽  
Population Doubling ◽  
Population Doubling Time ◽  
Human Articular Cartilage ◽  
Isolated Cells ◽  
Chondrocyte Phenotype

Objective Cartilage tissue engineering has evolved as one of the therapeutic strategies for cartilage defect, which relies on a large number of viable chondrocytes. Because of limited availability of cartilage and low chondrocytes yield from cartilage, the need for an improve isolation protocol for maximum yield of viable cells is a key to achieving successful clinical constructs. This study optimizes and compares different protocols for isolation of chondrocytes from cartilage. Design We employed enzymatic digestion of cartilage using collagenase II and trypsin. The chondrocytes yield, growth kinetics, aggrecan, and collagen type 2 (COL2) expression were evaluated. Collagen type 1 (COL1) mRNA expression was assessed to monitor the possibility of chondrocytes dedifferentiation. Results Chondrocyte yield per gram of cartilage was significantly higher ( P < 0.05) using collagenase II in Hank’s balanced salt solution (HBSS) compared with 0.25% trypsin. The number of chondrocyte yield per gram was higher in cartilage digested with collagenase in HBSS compared with Dulbecco’s modified Eagle medium/F12; however, the difference was not statistically significant. Chondrocytes seeded at lower densities had shorter population doubling time compared to those seeded at higher density. Protein and gene expression of chondrocyte phenotype indicates the expression of aggrecan and COL2. The expression of COL1 was significantly increased ( P < 0.05) in passage 3 compared with primary chondrocytes. The mRNA expression of chondrocyte phenotype was similar in primary and passaged one cells. Conclusions Collagenase in HBSS yield the highest number of viable chondrocytes and the isolated cells expressed chondrocyte phenotype. This protocol can be employed to generate large number of viable chondrocytes, particularly with limited cartilage biopsies.

scholarly journals Assessment of Native Human Articular Cartilage: A Biomechanical Protocol

Cartilage ◽  
2020 ◽  
pp. 194760352097324
Author(s):  
Wassif Kabir ◽  
Claudia Di Bella ◽  
Peter F.M. Choong ◽  
Cathal D. O’Connell
Keyword(s):  
Articular Cartilage ◽  
Poisson Ratio ◽  
Cartilage Tissue Engineering ◽  
Biomechanical Properties ◽  
Cartilage Tissue ◽  
Loading Frequency ◽  
Human Articular Cartilage ◽  
Compression Tests ◽  
Human Knee ◽  
Viscoelastic Parameters

Objectives Recapitulating the mechanical properties of articular cartilage (AC) is vital to facilitate the clinical translation of cartilage tissue engineering. Prior to evaluation of tissue-engineered constructs, it is fundamental to investigate the biomechanical properties of native AC under sudden, prolonged, and cyclic loads in a practical manner. However, previous studies have typically reported only the response of native AC to one or other of these loading regimes. We therefore developed a streamlined testing protocol to characterize the elastic and viscoelastic properties of human knee AC, generating values for several important parameters from the same sample. Design Human AC was harvested from macroscopically normal regions of distal femoral condyles of patients ( n = 3) undergoing total knee arthroplasty. Indentation and unconfined compression tests were conducted under physiological conditions (temperature 37 °C and pH 7.4) and testing parameters (strain rates and loading frequency) to assess elastic and viscoelastic parameters. Results The biomechanical properties obtained were as follows: Poisson ratio (0.4 ± 0.1), instantaneous modulus (52.14 ± 9.47 MPa) at a loading rate of 1 mm/s, Young’s modulus (1.03 ± 0.48 MPa), equilibrium modulus (7.48 ± 4.42 MPa), compressive modulus (10.60 ± 3.62 MPa), dynamic modulus (7.71 ± 4.62 MPa) at 1 Hz and loss factor (0.11 ± 0.02). Conclusions The measurements fell within the range of reported values for human knee AC biomechanics. To the authors’ knowledge this study is the first to report such a range of biomechanical properties for human distal femoral AC. This protocol may facilitate the assessment of tissue-engineered composites for their functionality and biomechanical similarity to native AC prior to clinical trials.

scholarly journals A 3D Porous Gelatin-Alginate-Based-IPN Acts as an Efficient Promoter of Chondrogenesis from Human Adipose-Derived Stem Cells

2015 ◽  
Vol 2015 ◽  
pp. 1-17 ◽  
Author(s):  
Sorina Dinescu ◽  
Bianca Galateanu ◽  
Eugen Radu ◽  
Anca Hermenean ◽  
Adriana Lungu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Collagen Type ◽  
Cartilage Tissue Engineering ◽  
Superior Performance ◽  
Cartilage Tissue ◽  
Adipose Derived Stem Cells ◽  
Interpenetrating Network ◽  
Cell Scaffold ◽  
New Strategies

Cartilage has limited regeneration potential. Thus, there is an imperative need to develop new strategies for cartilage tissue engineering (CTE) amenable for clinical use. Recent CTE approaches rely on optimal cell-scaffold interactions, which require a great deal of optimization. In this study we attempt to build a novel gelatin- (G-) alginate- (A-) polyacrylamide (PAA) 3D interpenetrating network (IPN) with superior performance in promoting chondrogenesis from human adipose-derived stem cells (hADSCs). We show that our G-A-PAA scaffold is capable of supporting hADSCs proliferation and survival, with no apparent cytotoxic effect. Moreover, we find that after exposure to prochondrogenic conditions a key transcription factor known to induce chondrogenesis, namely, Sox9, is highly expressed in our hADSCs/G-A-PAA bioconstruct, along with cartilage specific markers such as collagen type II, CEP68, and COMP extracellular matrix (ECM) components. These data suggest that our G-A-PAA structural properties and formulation might enable hADSCs conversion towards functional chondrocytes. We conclude that our novel G-A-PAA biomatrix is a good candidate for prospectivein vivoCTE applications.

scholarly journals Characterization of Collagen Type I and II Blended Hydrogels for Articular Cartilage Tissue Engineering

2016 ◽  
Vol 17 (10) ◽  
pp. 3145-3152 ◽  
Author(s):  
Nelda Vázquez-Portalatı́n ◽  
Claire E. Kilmer ◽  
Alyssa Panitch ◽  
Julie C. Liu
Keyword(s):  
Tissue Engineering ◽  
Articular Cartilage ◽  
Collagen Type ◽  
Cartilage Tissue Engineering ◽  
Collagen Type I ◽  
Cartilage Tissue ◽  
Type I

scholarly journals Adipose-Derived Mesenchymal Stem Cell Chondrospheroids Cultured in Hypoxia and a 3D Porous Chitosan/Chitin Nanocrystal Scaffold as a Platform for Cartilage Tissue Engineering

2020 ◽  
Vol 21 (3) ◽  
pp. 1004 ◽  
Author(s):  
Veronica Zubillaga ◽  
Ana Alonso-Varona ◽  
Susana C. M. Fernandes ◽  
Asier M. Salaberria ◽  
Teodoro Palomares
Keyword(s):  
Tissue Engineering ◽  
Articular Cartilage ◽  
Collagen Type ◽  
Porous Scaffold ◽  
Cartilage Tissue Engineering ◽  
Cartilage Regeneration ◽  
Cartilage Tissue ◽  
Type I ◽  
Low Oxygen ◽  
Porous Chitosan

Articular cartilage degeneration is one of the most common causes of pain and disability in middle-aged and older people. Tissue engineering (TE) has shown great therapeutic promise for this condition. The design of cartilage regeneration constructs must take into account the specific characteristics of the cartilaginous matrix, as well as the avascular nature of cartilage and its cells’ peculiar arrangement in isogenic groups. Keeping these factors in mind, we have designed a 3D porous scaffold based on genipin-crosslinked chitosan/chitin nanocrystals for spheroid chondral differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs) induced in hypoxic conditions. First, we demonstrated that, under low oxygen conditions, the chondrospheroids obtained express cartilage-specific markers including collagen type II (COL2A1) and aggrecan, lacking expression of osteogenic differentiation marker collagen type I (COL1A2). These results were associated with an increased expression of hypoxia-inducible factor 1α, which positively directs COL2A1 and aggrecan expression. Finally, we determined the most suitable chondrogenic differentiation pattern when hASC spheroids were seeded in the 3D porous scaffold under hypoxia and obtained a chondral extracellular matrix with a high sulphated glycosaminoglycan content, which is characteristic of articular cartilage. These findings highlight the potential use of such templates in cartilage tissue engineering.

scholarly journals In vitro study of cartilage tissue engineering using human adipose-derived stem cells induced by platelet-rich plasma and cultured on silk fibroin scaffold

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Imam Rosadi ◽  
Karina Karina ◽  
Iis Rosliana ◽  
Siti Sobariah ◽  
Irsyah Afini ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Mrna Expression ◽  
Silk Fibroin ◽  
Platelet Rich Plasma ◽  
Cartilage Tissue Engineering ◽  
Cartilage Tissue ◽  
Silk Fibroin Scaffold

Abstract Background Cartilage tissue engineering is a promising technique for repairing cartilage defect. Due to the limitation of cell number and proliferation, mesenchymal stem cells (MSCs) have been developed as a substitute to chondrocytes as a cartilage cell-source. This study aimed to develop cartilage tissue from human adipose-derived stem cells (ADSCs) cultured on a Bombyx mori silk fibroin scaffold and supplemented with 10% platelet-rich plasma (PRP). Methods Human ADSCs and PRP were characterized. A silk fibroin scaffold with 500 μm pore size was fabricated through salt leaching. ADSCs were then cultured on the scaffold (ADSC-SS) and supplemented with 10% PRP for 21 days to examine cell proliferation, chondrogenesis, osteogenesis, and surface marker expression. The messenger ribonucleic acid (mRNA) expression of type 2 collagen, aggrecan, and type 1 collagen was analysed. The presence of type 2 collagen confirming chondrogenesis was validated using immunocytochemistry. The negative and positive controls were ADSC-SS supplemented with 10% foetal bovine serum (FBS) and ADSC-SS supplemented with commercial chondrogenesis medium, respectively. Results Cells isolated from adipose tissue were characterized as ADSCs. Proliferation of the ADSC-SS PRP was significantly increased (p < 0.05) compared to that of controls. Chondrogenesis was observed in ADSC-SS PRP and was confirmed through the increase in glycosaminoglycans (GAG) and transforming growth factor-β1 (TGF-β1) secretion, the absence of mineral deposition, and increased surface marker proteins on chondrogenic progenitors. The mRNA expression of type 2 collagen in ADSC-SS PRP was significantly increased (p < 0.05) compared to that in the negative control on days 7 and 21; however, aggrecan was significantly increased on day 14 compared to the controls. ADSC-SS PRP showed stable mRNA expression of type 1 collagen up to 14 days and it was significantly decreased on day 21. Confocal analysis showed the presence of type 2 collagen in the ADSC-SS PRP and positive control groups, with high distribution outside the cells forming the extracellular matrix (ECM) on day 21. Conclusion Our study showed that ADSC-SS with supplemented 10% PRP medium can effectively support chondrogenesis of ADSCs in vitro and promising for further development as an alternative for cartilage tissue engineering in vivo.

scholarly journals Metabolomic Profiling and Mechanotransduction of Single Chondrocytes Encapsulated in Alginate Microgels

2020 ◽  
Author(s):  
Jacob P. Fredrikson ◽  
Priyanka Brahmachary ◽  
Ebru Erdoğan ◽  
Zach Archambault ◽  
Ronald K. June ◽  
...  
Keyword(s):  
Single Cell ◽  
Dynamic Compression ◽  
Cartilage Tissue Engineering ◽  
Cell Encapsulation ◽  
Cartilage Tissue ◽  
Human Articular Cartilage ◽  
Mechanical Loads ◽  
Single Cell Encapsulation

AbstractHuman articular cartilage is comprised of two main components, the extracellular matrix (ECM) and the pericellular matrix (PCM). The PCM helps to protect chondrocytes in the cartilage from mechanical loads, but in patients with osteoarthritis, the PCM is weakened resulting in increased chondrocyte stress. As chondrocytes are responsible for cartilage synthesis and maintenance, it is important to understand how mechanical loads affect cellular responses of chondrocytes. Many studies have examined the chondrocyte response to in vitro mechanical loading by embedding in stiff agarose. However, these experiments are mostly performed in the absence of PCM which may obscure important responses to mechanotransduction. Here, we demonstrate that drop-based microfluidics allows culture of single chondrocytes in alginate microgels for cell-directed PCM synthesis that closely mimics the in vivo microenvironment. Chondrocytes form PCM over 10 days in these single cell microenvironments. Single cell microgels and monolayer controls were encapsulated in high stiffness agarose to mimic the cartilage PCM. After physiological dynamic compression in a custom-built bioreactor, microgels exhibited distinct metabolomic profiles from both uncompressed and monolayer controls. These results demonstrate the potential of single cell encapsulation in alginate microgels to advance cartilage tissue engineering and basic chondrocyte mechanobiology.

scholarly journals A Synthetic, Closed-Looped Gene Circuit for the Autonomous Regulation of RUNX2 Activity during Chondrogenesis

2021 ◽  
Author(s):  
Biming Wu ◽  
Gurcharan Kaur ◽  
Thomas Lanigan ◽  
Rhima M Coleman
Keyword(s):  
Cartilage Tissue Engineering ◽  
Cartilage Tissue ◽  
Matrix Synthesis ◽  
Gene Circuit ◽  
Chondrocyte Phenotype ◽  
Chondrocyte Maturation ◽  
Autonomous Regulation ◽  
Chondroprogenitor Cells ◽  
Matrix Accumulation

The transcription factor RUNX2 is a key regulator of chondrocyte phenotype during development, making it an ideal target for prevention of undesirable chondrocyte maturation in cartilage tissue engineering strategies. Here, we engineered an autoregulatory gene circuit (cisCXp-shRunx2) that negatively controls RUNX2 activity in chondrogenic cells via RNA interference initiated by a tunable synthetic Col10a1-like promoter (cisCXp). The cisCXp-shRunx2 gene circuit is designed based on the observation that induced RUNX2 silencing after early chondrogenesis enhances the accumulation of cartilaginous matrix in 2D ATDC5 model. We show that the cisCXp-shRunx2 initiates RNAi of RUNX2 in maturing chondrocytes in response to the increasing intracellular RUNX2 activity without interfering with early chondrogenesis in ATDC5 cells. The induced loss of RUNX2 activity in turn negatively regulates the gene circuit itself. Furthermore, the efficacy of RUNX2 suppression from cisCXp-shRunx2 can be controlled by modifying the sensitivity of cisCXp promoter. Long-term 3D cultures of reprogrammed ATDC5 cells had increased matrix accumulation compared to naive cells. Overall, our results demonstrate that the negative modulation of Runx2 activity with our autoregulatory gene circuit can reduce the effects of RUNX2 activity and enhance matrix synthesis in chondroprogenitor cells.

scholarly journals Efficacy of a Thermally Triggered Injectable Hydrogel CS/Col /PLA/GP and BMSCs for Cartilage Tissue Engineering

2020 ◽  
Author(s):  
Mingjing Li ◽  
Fan Li
Keyword(s):  
Tissue Engineering ◽  
Mrna Expression ◽  
Cartilage Tissue Engineering ◽  
Knee Joints ◽  
Cartilage Tissue ◽  
Cartilage Defects ◽  
Normal Cartilage ◽  
Rabbit Knee ◽  
Safranin O ◽  
Thermally Triggered

Abstract BackgroundArticular cartilage has limited self-repair ability. Tissue engineering is considered to be one of the most promising therapeutic approaches. Chitosan (CS) based hydrogels are the most widely used scaffolds which still need improvement. The purpose of this study was to investigate the efficacy of a thermally triggered injectable chitosan / type II collagen / polylactic acid / sodium β-glycerophosphate (CS/Col/PLA/GP) hydrogel and bone marrow mesenchymal stem cells (BMSCs) for the treatment of cartilage defects in rabbit knee joints. Material/MethodsThe CS-based hydrogels consisting of CS, Col II, PLA and GP were fabricated by chemical cross-linking method. The gel forming time and elastic modulus of these hydrogels were measured. We tested the viability, proliferation and differentiation of rabbit BMSCs cultured in the hydrogels by fluorescence staining, CCK-8 and PCR method. The hydrogels combined with or without BMSCs were injected into cartilage defects in rabbit knee joints and the materials were collected at 8 weeks after surgery. The repair effect of cartilage defects was evaluated based on gross observation, HE, safranin O and immunohistochemical staining. ResultsThe CS/Col/PLA/GP hydrogel was liquid at room temperature and gelled after 7.5±0.41min at 37°C. CS/Col /PLA/GP hydrogel had a modulus of 8.90 ± 0.12 kPa while CS/GP and CS/Col/GP hydrogels had the modulus of 4.07 ± 0.24 kPa and 4.93 ± 0.09 kPa. The results of Live/Dead cell viability assay reveal that most of BMSCs remained alive in the hydrogels. CCK-8 assay shows that the number of cells in CS/Col /PLA/GP hydrogel was significantly higher in comparison to the other groups on days 2 and 3 of cell culture (p<0.05). Aggrecan mRNA expression in the CS/Col /PLA/GP gel was the highest (p<0.05). Sox9 mRNA expression in the CS/Col /GP group was the highest, in which CS/Col /PLA/GP hydrogel was higher than the CS/GP hydrogel(p<0.05). Furthermore, CS/Col/PLA/GP and CS/Col /GP hydrogels showed higher COL2A1 mRNA expression in comparison to CS/GP constructs (p<0.05). In vivo studies showed that approximately 90% of the cartilage defects of rabbits treated by the hydrogel and BMSCs were repaired with hyaline-like tissue without obvious inflammation response. HE, safranin O, and immunohistochemical staining showed that the hyaline like cartilage was formed in cartilage defects, and the collagen content in the new generated cartilage was similar to the normal cartilage. The neocartilage was thinner than the surrounding normal cartilage, but it exhibited integration with adjacent healthy tissue. The abundant well-defined chondrocytes were aligned in several apparent chondrocyte clusters in the new generated cartilage.ConclusionsThe thermo-sensitive injectable CS/Col/PLA/GP composite hydrogel has better ability to promote survive, proliferation and chondrogenic differentiation of seeded BMSCs as compared against CS/Col/GP and CS/GP hydrogels. Combined with BMSCs to repair cartilage defects of rabbit knee joints, they can effectively reduce the cartilage defect area, and the new generated cartilage is comparable to normal cartilage structure. In addition, abundant availability and simple fabrication process also make CS/Col/PLA/GP composite hydrogel a suitable candidate scaffold in cartilage tissue engineering.

Suppression of discoidin domain receptor 1 expression enhances the chondrogenesis of adipose-derived stem cells

2015 ◽  
Vol 308 (9) ◽  
pp. C685-C696 ◽  
Author(s):  
Shun-Cheng Wu ◽  
Hsu-Feng Hsiao ◽  
Mei-Ling Ho ◽  
Yung-Li Hung ◽  
Je-Ken Chang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Articular Cartilage ◽  
Collagen Type ◽  
Cartilage Tissue Engineering ◽  
Cartilage Tissue ◽  
Adipose Derived Stem Cells ◽  
Type Ii ◽  
Collagen Type Ii ◽  
Matrix Deposition ◽  
Discoidin Domain Receptor 1

Effectively directing the chondrogenesis of adipose-derived stem cells (ADSCs) to engineer articular cartilage represents an important challenge in ADSC-based articular cartilage tissue engineering. The discoidin domain receptor 1 (DDR1) has been shown to affect cartilage homeostasis; however, little is known about the roles of DDR1 in ADSC chondrogenesis. In this study, we used the three-dimensional culture pellet culture model system with chondrogenic induction to investigate the roles of DDR1 in the chondrogenic differentiation of human ADSCs (hADSCs). Real-time polymerase chain reaction and Western blot were used to detect the expression of DDRs and chondrogenic genes. Sulfated glycosaminoglycan (sGAG) was detected by Alcian blue and dimethylmethylene blue (DMMB) assays. Terminal deoxy-nucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was used to assess cell death. During the chondrogenesis of hADSCs, the expression of DDR1 but not DDR2 was significantly elevated. The depletion of DDR1 expression in hADSCs using short hairpin RNA increased the expression of chondrogenic genes (SOX-9, collagen type II, and aggrecan) and cartilaginous matrix deposition (collagen type II and sGAG) and only slightly increased cell death (2–8%). DDR1 overexpression in hADSCs decreased the expression of chondrogenic genes (SOX-9, collagen type II, and aggrecan) and sGAG and enhanced hADSC survival. Moreover, DDR1-depleted hADSCs showed decreased expression of the terminal differentiation genes runt-related transcription factor 2 (Runx2) and matrix metalloproteinase 13 (MMP-13). These results suggest that DDR1 suppression may enhance ADSC chondrogenesis by enhancing the expression of chondrogenic genes and cartilaginous matrix deposition. We proposed that the suppression of DDR1 in ADSCs may be a candidate strategy of genetic modification to optimize ADSC-based articular cartilage tissue engineering.

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