Modulation of chondrogenic differentiation of human mesenchymal stem cells in jellyfish collagen scaffolds by cell density and culture medium

Abstract

Studies on tissue-engineering approaches for the regeneration of traumatized cartilage focus increasingly on multipotent human mesenchymal stem cells (hMSCs) as an alternative to autologous chondrocytes. The present study applied porous scaffolds made of collagen from the jellyfish Rhopilema esculentum for the in vitro chondrogenic differentiation of hMSCs. Culture conditions in those scaffolds differ from conditions in high-density pellet cultures, making a re-examination of these data necessary. We systematically investigated the influence of seeding density, basic culture media [Dulbecco's modified Eagle's medium (DMEM), α-minimum essential medium (α-MEM)] with varying glucose content and supplementation with fetal calf serum (FCS) or bovine serum albumin (BSA) on the chondrogenic differentiation of hMSCs. Gene expression analyses of selected markers for chondrogenic differentiation and hypertrophic development were conducted. Furthermore, the production of cartilage extracellular matrix (ECM) was analysed by quantification of sulphated glycosaminoglycan and collagen type II contents. The strongest upregulation of chondrogenic markers, along with the highest ECM deposition was observed in scaffolds seeded with 2.4 × 10 cells/cm after cultivation in high-glucose DMEM and 0.125% BSA. Lower seeding densities compared to high-density pellet cultures were sufficient to induce in vitro chondrogenic differentiation of hMSCs in collagen scaffolds, which reduces the amount of cells required for the seeding of scaffolds and thus the monolayer expansion period. Furthermore, examination of the impact of FCS and α-MEM on chondrogenic MSC differentiation is an important prerequisite for the development of an osteochondral medium for simultaneous osteogenic and chondrogenic differentiation in biphasic scaffolds for osteochondral tissue regeneration. Copyright © 2015 John Wiley & Sons, Ltd.

Biphasic Scaffolds from Marine Collagens for Regeneration of Osteochondral Defects

“…To develop an osteochondral medium supporting both osteogenic and chondrogenic differentiation of MSC (additionally to the stimuli, which are exerted by the scaffold matrix) it is not sufficient just to combine the single media 1:1. In a previous study we have analyzed the impact of FCS on the chondrogenic differentiation of hMSC in monophasic scaffolds of jellyfish collagen [ 25 ] and demonstrated, that even small amounts of FCS (2%) in the culture medium significantly decrease the mRNA expression of chondrogenic markers. In addition, reduction of glucose content caused a decreased mRNA expression of chondrogenic markers as well as a decreased extracellular matrix production of the chondrogenically differentiated cells.…”

“…The supernatants were used for collagen II ELISA, while the pellet was further processed for sGAG determination. Collagen II ELISA was performed as already published [ 25 ]. Briefly, each 50 µL of the supernatants was added to wells which were precoated with primary antibody (Mouse Anti-Chick Collagen II Capture Antibody (clone 35; Chondrex, Redmont, WA, USA) diluted 1:500 in PBS).…”

“…sGAG content from the pellets was quantified as previously described [ 25 ]. Briefly, 1 mL of papain digestion solution (containing 125 µg/mL papain, 5 mM EDTA (both from Sigma), 100 mM Na 2 HPO 4 , and 5 mM cystein (both from Carl Roth, Karlsruhe, Germany) in deionized water) were added to each pellet.…”

Reconstruction of Anterior Tracheal Defects Using a Bioengineered Graft in a Porcine Model

“…Decellularized bovine dermal matrices (4 mm thick, Surgimend, Integra Life Sciences, Billerica, MA) were seeded with hMSCs (sixth passage at 1.0 Â 10 6 cells/cm 2 density by manual pipetting evenly distributing cells on the surface of ECM. The growth media were supplied with a formulated MSC Chondrogenic Differentiation Medium (PromoCell) according to manufacturer's protocol [22].…”

Combined Jellyfish Collagen Type II, Human Stem Cells and Tgf-β3 as a Therapeutic Implant for Cartilage Repair

“…Indeed, collagen is one of the materials most used in the field of cartilage tissue engineering, due to its biodegradability, biocompatibility, low immunogenicity and cell-adhesive properties. Nevertheless, collagen type II is the main represented collagen of the hyaline cartilage tissue, and recently, an unexpected source of non-mammalian collagen emerged (jellyfish marine collagen type II) and leaves open the possibility to overcome the limitations attributed to mammalian collagen use, such as risks of infections (bovine spongiform encephalopathy), initiation of arthritis by inflammation, or rejections due to religious reasons [7][8][9]. This new source of marine collagen type II has shown to possess a superior impact on chondrogenesis compared to collagen I scaffold [8].…”