International Journal of Medical Laboratory

Background and Aims: The current study was conducted to compare the expression levels of collagen type Π and X during chondrogenesis of human adipose-derived mesenchymal stem cells (hADMSCs) pellet and micromass cultures. 
Materials and Methods: Extracted hADMSCs were cultured until three passages and then transferred to pellet and micromass cultures in the experimental groups of day 7 and day14. For pellet and micromass cultures, aliquots of 5×10⁵ cells/ml were centrifuged and respectively cultured in the conical tubes and droplets (12.5 µl) of the 24-well plates containing chondrogenic medium. Realtime-polymerase chain reaction technique was performed for gene expression levels.
Results: Increased expression of collagen type Π was shown in micromass day14 compared to micromass day 7, pellet day 14 (p<0.01) and pellet day 7 (p<0.001). Also, an increased expression of collagen type Π was seen in micromass day 7 and pellet day 14 compared to pellet day 7 (p< 0.05). Expression of collagen type X increased in pellet day 14 compared to micromass on days 7 and 14 (p<0.001, p<0.01) and pellet day14 compared to pellet day7 (p< 0.05). An increased expression of collagen type X was shown in pellet day 7 compared to micromass on days 7 and 14 (p<0.05).
Conclusions: According to the results, higher expression of collagen type Π and lower expression of collagen type X in micromass cultures that are prepared by cell suspension play a better role during cellular condensation that leads to the formation of large nodules exhibiting cartilage-like morphology, suggests a higher efficiency for micromass cultures.

Background and Aims: It has been proven that human mesenchymal stem cells (MSCs) conditioned medium (hMSCs-CM) can influence human embryonic stem cells (hESCs) chondrogenic differentiation. In this study, we hypothesized that conditioned medium (CM) from hESCs-derived MSCs in a sequential 3D-2D culture system could facilitate the induction of chondrogenesis in hESCs.
Materials and Methods: CM was collected from Yazd2 (hESCs; 46, XY) derived MSCs confluent cultures and stored at -20 °C. Yazd4 hESC line (46, XX) was induced for differentiation using EB formation as 3D culture into SD (spontaneously differentiation) and CM groups (differentiation using conditioned medium) for four days. Cell culture continued in a 2D (monolayer) culture system for both groups till day 14. Ultimately, chondrogenic differentiation was assessed by Alcian blue and masson′s trichrome staining at 4 and 14 days of differentiation, and quantitative real-time polymerase chain reaction (PCR) for NANOG, MEOX1, SOX5, SOX6, SOX9, ACAN, COL2A1 and RUNX2 genes for SD and CM groups on days 0 and 4.
Results:  The gene expression profile for chondrogenic genes in the CM group was significantly more than the SD group (p< 0.05). Furthermore, chemical staining assessment illustrated a significant GAG and collagen II difference between the CM and SD groups at days 4 and 14 (p< 0.05).
Conclusions: Our findings would pave the way for creating an in vitro human chondrogenesis model for further studies in the developmental biology of articular cartilage tissue, which lend itself to cell-based therapy to cure joint diseases such as osteoarthritis.

Introduction: Due to the lack of repair of cartilage lesions, tissue engineering tries to produce cartilage using cells, scaffolds, and growth factors. Pomegranate seed extract (PSE) and unsaponifiable soybean/ avocado (ASU) are plant compounds that effectively maintain the extracellular cartilage matrix. In this study, we tried to investigate the effects of PSE on the process of chondrogenesis and compared it with the effects of ASU in this process.
Materials and Methods: Human adipose-derived stem cells (hADSCs) were transferred to fibrin scaffolds in the third passage in three groups (control, PSE, and ASU) for chondrogenic differentiation. After 14 days, Western blotting evaluated the samples for cell survival by (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) (MTT) assay production of collagen type II and collagen type X.
Results: Evaluation of MTT results in different groups showed that the survival rate in the two groups of PSE and ASU was significantly lower than the control group (p ≤ 0.05). Quantitative western blotting showed that the production of collagen type II protein in both groups significantly increased compared to the control group (p ≤ 0.05). Quantitative analysis of collagen type X protein production showed that the production of this protein in the PSE group was significantly reduced compared to the ASU group.
Conclusion: PSE and ASU are two important factors in inducing chondrogenesis in hADSCs in fibrin scaffold. ASU's impact on chondrogenesis of hADSCs in fibrin scaffold is greater than that of PSE.