The trans-well coculture of human synovial mesenchymal stem cells with chondrocytes leads to self-organization, chondrogenic differentiation, and secretion of TGFβ.
BACKGROUND
Synovial mesenchymal stem cells (SMSC) possess a high chondrogenic differentiation potential, which possibly supports natural and surgically induced healing of cartilage lesions. We hypothesized enhanced chondrogenesis of SMSC caused by the vicinity of chondrocytes (CHDR).


METHODS
Human SMSC and CHDR interactions were investigated in an in-vitro trans-well monolayer coculture over a time period of up to 21 days. Protein expression was analyzed using histology, immunostaining, or enzyme-linked immunosorbent assay. Additionally, mRNA expression was assessed by quantitative PCR.


RESULTS
After 7 days, phase-contrast microscopy revealed cell aggregation of SMSC in coculture with CHDR. Afterwards, cells formed spheres and lost adherence. However, this phenomenon was not observed when culturing SMSC alone. Fluorescence labeling showed concurrent collagen type II expression. Addition of transforming growth factor beta (TGFβ) to the cocultures induced SMSC aggregation in less time and with higher intensity. Additionally, alcian blue staining demonstrated enhanced glycosaminoglycan expression around SMSC aggregates after 1 and 2 weeks. Although TGFβ mRNA was expressed in all SMSC, the protein was measured with constantly increasing levels over 21 days only in supernatants of the cocultures. Considering the enhanced mRNA levels following supplementation with TGFβ, a positive feedback mechanism can be supposed. In line with the development of a chondrogenic phenotype, aggrecan mRNA expression increased after 7 and 14 days in the cocultures with and without TGFβ. Coculture conditions also amplified collagen type II mRNA expression after 2 weeks without and already after 1 week with TGFβ. There was no difference in collagen type I and type X expression between SMSC alone and the coculture with CHDR. Expression of both collagens increased following addition of TGFβ. mRNA data correlated with the intensity of immunofluorescence staining.


CONCLUSIONS
Paracrine effects of CHDR induce a chondrogenic phenotype in SMSC possibly mimicking joint homeostasis. Coculture approaches may lead to a better understanding of cellular interactions with potential implications for cartilage repair procedures.
Department of Orthopedics and Trauma Surgery, Albert-Ludwigs University Medical Center Freiburg, Freiburg, Germany.
Eva Johanna
EJ
Kubosch
Department of Orthopedics and Trauma Surgery, Albert-Ludwigs University Medical Center Freiburg, Freiburg, Germany.
Emanuel
E
Heidt
Department of Orthopedics and Trauma Surgery, Albert-Ludwigs University Medical Center Freiburg, Freiburg, Germany.
Anke
A
Bernstein
Department of Orthopedics and Trauma Surgery, Albert-Ludwigs University Medical Center Freiburg, Freiburg, Germany.
Katharina
K
Böttiger
Department of Orthopaedics and Traumatology, Odense University Hospital, Sdr. Boulevard 29, 5000, Odense C, Denmark. hagen.schmal@freenet.de.|Department of Clinical Research, University of Southern Denmark, Odense, Denmark. hagen.schmal@freenet.de.
Hagen
H
Schmal
D000328:Adult; D000368:Aged; D053674:Aggrecans / Q000235:genetics / Q000378:metabolism; D019645:Arthroplasty, Replacement, Knee; D002449:Cell Aggregation; D002454:Cell Differentiation; D019902:Chondrocytes / Q000378:metabolism / Q000473:pathology*; D018920:Coculture Techniques; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D024043:Collagen Type II / Q000235:genetics / Q000378:metabolism; D015194:Diffusion Chambers, Culture; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D017746:Joint Capsule / Q000378:metabolism / Q000473:pathology / Q000601:surgery; D007719:Knee Joint / Q000378:metabolism / Q000473:pathology / Q000601:surgery; D008297:Male; D059630:Mesenchymal Stem Cells / Q000378:metabolism / Q000473:pathology*; D008875:Middle Aged; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D016212:Transforming Growth Factor beta / Q000235:genetics* / Q000378:metabolism
C508319:ACAN protein, human; D053674:Aggrecans; D024042:Collagen Type I; D024043:Collagen Type II; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta
Chondrocytes; Chondrogenesis; Coculture; Differentiation; Synovial mesenchymal stem cell; Synovium