Three-dimensional scaffold-free fusion culture: the way to enhance chondrogenesis of in vitro propagated human articular chondrocytes

Abstract

Cartilage regeneration based on isolated and culture-expanded chondrocytes has been studied in various in vitro models, but the quality varies with respect to the morphology and the physiology of the synthesized tissues. The aim of our study was to promote in vitro chondrogenesis of human articular chondrocytes using a novel three-dimensional (3-D) cultivation system in combination with the chondrogenic differentiation factors transforming growth factor beta 2 (TGF-β2) and L-ascorbic acid. Articular chondrocytes isolated from six elderly patients were expanded in monolayer culture. A single-cell suspension of the dedifferentiated chondrocytes was then added to agar-coated dishes without using any scaffold material, in the presence, or absence of TGF-β2 and/or L-ascorbic acid. Three-dimensional cartilage-like constructs, called single spheroids, and microtissues consisting of several spheroids fused together, named as fusions, were formed. Generated tissues were mainly characterized using histological and immunohistochemical techniques. The morphology of the in vitro tissues shared some similarities to native hyaline cartilage in regard to differentiated S100-positive chondrocytes within a cartilaginous matrix, with strong collagen type II expression and increased synthesis of proteoglycans. Finally, our innovative scaffold-free fusion culture technique supported enhanced chondrogenesis of human articular chondrocytes in vitro. These 3-D hyaline cartilage-like microtissues will be useful for in vitro studies of cartilage differentiation and regeneration, enabling optimization of functional tissue engineering and possibly contributing to the development of new approaches to treat traumatic cartilage defects or osteoarthritis.

Testing the potency of anti‐TNF‐α and anti‐IL‐1β drugs using spheroid cultures of human osteoarthritic chondrocytes and donor‐matched chondrogenically differentiated mesenchymal stem cells

“…The reason for selecting a scaffold‐free 3D culture over an alginate‐based system was based on superior chondrogenic re‐differentiation of primary chondrocytes achieved in hanging drops . In addition, tissue formation in hanging drops mimics the condensation process of MSCs, which is one of the earliest phases of the in vivo cartilage development …”

“…65 In addition, tissue formation in hanging drops mimics the condensation process of MSCs, which is one of the earliest phases of the in vivo cartilage development. 13 To avoid permanent de-differentiation of OACs which occurs at passage 5 (P5) in monolayer culture, 64 microsphreoids were prepared from passage 2 (P2) cells. The average diameter of microspheroids formed from chondrogenically differentiated MSCs (250 lm) was 28% smaller than that of microspheroids prepared from OACs (344 lm).…”

“…As 3D cell culture systems can mimic physiological tissue microenvironments, they could be used as predictive models for drug testing . For decades, scaffold‐free 3D tissues (spheroids), uniform in cell numbers and sizes, are being successfully generated by self‐assembly of cells seeded in hanging drops . Also, this cell culture procedure can be accomplished by using automated liquid handling systems, thereby enabling high‐throughput testing in preclinical drug discovery.…”

High seeding density of human chondrocytes in agarose produces tissue-engineered cartilage approaching native mechanical and biochemical properties

“…Further, successes in chondrocyte-based CTE studies in animal models will likely translate best for hACs over other human cell types. Relatively few studies have utilized hACs in CTE (Adkisson et al, 2010; Dehne et al, 2009; Dell’Accio et al, 2001; Fernandes et al, 2013; Kafienah et al, 2002; Lehmann et al, 2013; Saha et al, 2013; Sittinger et al, 1994; Wang et al, 2006; Wenger et al, 2006), due perhaps to logistical challenges of obtaining donor cells in sufficient numbers and encouraging them to produce sufficient matrix. Even fewer studies have characterized functional properties of human cartilage tissue constructs such as mechanical integrity (Murphy et al, 2015; O’Connell et al, 2015; Zhao et al, 2012).…”

Are surface antigens suited to verify the redifferentiation potential and culture purity of human chondrocytes in cell-based implants

“…In this study, redifferentiation was initiated using a bioresorbable polyglycolic acid-fibrin (PGA-fibrin) scaffold, which has been used before to verify articular chondrocyte redifferentiation obtained from living and deceased donors (Sittinger et al, 1996;Trimborn et al, 2012). It should be noted, however, that in contrast to growth factor-stimulated chondrogenesis using transforming growth factor beta (Lehmann et al, 2013) or bone morphogenetic proteins (Kaps et al, 2002), the increase in the expression of typical chondrogenic marker genes, e.g. collagen type II is less pronounced in 3D chondrocyte cultures without growth factor stimulation in vitro.…”