Induction of chondrogenic morphogenesis in tissue culture using different combinations of transforming growth factor-beta superfamily proteins in vitro

Xiong, Fei (2020): Induction of chondrogenic morphogenesis in tissue culture using different combinations of transforming growth factor-beta superfamily proteins in vitro. Dissertation, LMU München: Medizinische Fakultät

Abstract

BACKGROUND: The regeneration of cartilage has always been a challenge for tissue engineering. Constantly renewed insights into the role of transforming growth factor-beta (TGF-β) supergene family of proteins, which are vital in several fundamental biological processes in cartilage health and regeneration, has opened up new prospects for the treatment of cartilage-related diseases. In this study, the aim was to investigate what the effect of three different growth factors from the TGF-β supergene family specifically [bone morphogenetic protein 2 (BMP-2); TGF-β3; osteogenic protein 1 (OP-1)], alone but especially in varying combinations including application durations, would have on the induction of chondrogenesis in muscle tissue of rats. METHODS: Abdominal muscle tissue from rats was utilized. To monitor what the effect of morphogen presence would have on chondrogenesis, the “withdrawal study”, assessed two modes of stimulation. These were a continuous application of relevant morphogens and their combinations for the entire duration of the in vitro culture or a single application only for 48h. The detections were performed on day 7, 14 and 30 using immunohistochemistry (IHC), histological staining (alcian blue staining) and quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Aggrecan was treated as the target antigen in the IHC. The relative gene expression levels were analyzed to confirm the survival of the model and the chondrogenesis, including vascular endothelial growth factor A (VEGF-A), collagen type IV alpha 1 (Col4α1), sex-determining region Y (SRY)-box 9 (SOX9), aggrecan (ACAN), collagen type II alpha 1 (Col2α1), collagen type X alpha 1 (Col10α1), collagen type I alpha 1 (Col1α1) and alkaline phosphatase (ALP). RESULTS: The results of the qRT-PCR showed that the up-regulation in gene expression for the continuous experimental groups was more significant than that of the single 48h stimulation groups. The group with BMP-2 alone continuously presented the highest relative expression levels on day 7, in terms of the chondrogenic-related genes. Positive reactions were observed in the alcian blue staining and IHC with semi-quantitative histomorphometrical analysis showing a correlation to that of the gene expression patterns. CONCLUSIONS: Muscle tissue was proven to be a viable model in this chondrogenic induction study. The application of members of the TGF-β supergene family, alone or in combinations, induced chondrogenesis in this tissue model, with results suggesting that hyaline cartilage chondrogenesis was being developed based on the Col2α1 expression patterns. Although it was attempted to get a more economic-efficiency induction scheme using the withdraw-study in this project, it was shown that single stimulation of a growth factor was insufficient to evoke the relevant response, strongly suggesting that a continuous stimulation is necessary. However, the results in this regard have to be interpreted with care as it is clear that a single morphogen has a limited spatial and temporal effect where the presence of the appropriate corresponding complementary soluble signal(s) needs to be present at the correct time to ensure a proper and sustained biological reaction of specific pathways with time. This was exemplified by BMP-2 that on its own was able to initiate chondrogenesis, yet when added in combination with TGF-β3 and/or OP-1 was inhibited. However, while the BMP-2 initially stimulated chondrogenesis, it could not maintain the relevant reaction in the middle and late stages of chondrogenic induction, where TGF-β3 and OP-1 were necessary to maintain the cartilage tissue engineering reaction. Although limitations still exist, the experiments provide a crucial realization in the TGF-β supergene family tissue engineering prospect and deliver novel awareness and strategies in producing engineered hyaline cartilage for future clinical applications.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: BMP-2, TGF-β3, OP-1, chondrogenesis, morphogen combinations, muscle tissue
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 13. Februar 2020
• 1. Bericht­erstatter:in: Hausdorf, Jörg