Top

Published in:

01-01-2022 | Fracture Healing | Original Article

A novel in vitro assay to study chondrocyte-to-osteoblast transdifferentiation

Authors: Miriam E. A. Tschaffon, Stefan O. Reber, Astrid Schoppa, Sayantan Nandi, Ion C. Cirstea, Attila Aszodi, Anita Ignatius, Melanie Haffner-Luntzer

Published in: Endocrine | Issue 1/2022

Abstract

Purpose

Endochondral ossification, which involves transdifferentiation of chondrocytes into osteoblasts, is an important process involved in the development and postnatal growth of most vertebrate bones as well as in bone fracture healing. To study the basic molecular mechanisms of this process, a robust and easy-to-use in vitro model is desirable. Therefore, we aimed to develop a standardized in vitro assay for the transdifferentiation of chondrogenic cells towards the osteogenic lineage.

Methods

Murine chondrogenic ATDC5 cells were differentiated into the chondrogenic lineage for seven days and subsequently differentiated towards the osteogenic direction. Gene expression analysis of pluripotency, as well as chondrogenic and osteogenic markers, cell–matrix staining, and immunofluorescent staining, were performed to assess the differentiation. In addition, the effects of Wnt3a and lipopolysaccharides (LPS) on the transdifferentiation were tested by their addition to the osteogenic differentiation medium.

Results

Following osteogenic differentiation, chondrogenically pe-differentiated cells displayed the expression of pluripotency and osteogenic marker genes as well as alkaline phosphatase activity and a mineralized matrix. Co-expression of Col2a1 and Col1a1 after one day of osteogenic differentiation indicated that osteogenic cells had differentiated from chondrogenic cells. Wnt3a increased and LPS decreased transdifferentiation towards the osteogenic lineage.

Conclusion

We successfully established a rapid, standardized in vitro assay for the transdifferentiation of chondrogenic cells into osteogenic cells, which is suitable for testing the effects of different compounds on this cellular process.

Available only for authorised users

E.J. Mackie, Y.A. Ahmed, L. Tatarczuch, K.S. Chen, M. Mirams, Endochondral ossification: how cartilage is converted into bone in the developing skeleton. Int J Biochem Cell Biol 40(1), 46–62 (2008). https://doi.org/10.1016/j.biocel.2007.06.009CrossRefPubMed

M. Pines, S. Hurwitz, The role of the growth plate in longitudinal bone growth. Poult Sci 70(8), 1806–1814 (1991). https://doi.org/10.3382/ps.0701806CrossRefPubMed

C. Phornphutkul, P.A. Gruppuso, Disorders of the growth plate. Curr Opin Endocrinol Diabetes Obes 16(6), 430–434 (2009). https://doi.org/10.1097/MED.0b013e328331dca2CrossRefPubMedPubMedCentral

L.J. Sandell, J.V. Sugai, S.B. Trippel, Expression of collagens I, II, X, and XI and aggrecan mRNAs by bovine growth plate chondrocytes in situ. J Orthop Res 12(1), 1–14 (1994). https://doi.org/10.1002/jor.1100120102CrossRefPubMed

V. Lefebvre, P. Smits, Transcriptional control of chondrocyte fate and differentiation. Birth Defects Res C Embryo Today 75(3), 200–212 (2005). https://doi.org/10.1002/bdrc.20048CrossRefPubMed

R.J. O’Keefe, L.S. Loveys, D.G. Hicks, P.R. Reynolds, I.D. Crabb, J.E. Puzas, R.N. Rosier, Differential regulation of type-II and type-X collagen synthesis by parathyroid hormone-related protein in chick growth-plate chondrocytes. J Orthop Res 15(2), 162–174 (1997). https://doi.org/10.1002/jor.1100150203CrossRefPubMed

T.A. Franz-Odendaal, B.K. Hall, P.E. Witten, Buried alive: how osteoblasts become osteocytes. Dev Dyn 235(1), 176–190 (2006). https://doi.org/10.1002/dvdy.20603CrossRef

E.E. Golub, Enzymes in mineralizing systems: state of the art. Connect Tissue Res 35(1-4), 183–188 (1996). https://doi.org/10.3109/03008209609029190CrossRefPubMed

T. Komori, Regulation of bone development and extracellular matrix protein genes by RUNX2. Cell Tissue Res 339(1), 189–195 (2010). https://doi.org/10.1007/s00441-009-0832-8CrossRefPubMed

10.

J.J. Weng, Y. Su, Nuclear matrix-targeting of the osteogenic factor Runx2 is essential for its recognition and activation of the alkaline phosphatase gene. Biochim Biophys Acta 1830(3), 2839–2852 (2013). https://doi.org/10.1016/j.bbagen.2012.12.021CrossRefPubMed

11.

L. Claes, S. Recknagel, A. Ignatius, Fracture healing under healthy and inflammatory conditions. Nat Rev Rheumatol 8(3), 133–143 (2012). https://doi.org/10.1038/nrrheum.2012.1CrossRefPubMed

12.

L.C. Gerstenfeld, D.M. Cullinane, G.L. Barnes, D.T. Graves, T.A. Einhorn, Fracture healing as a post-natal developmental process: molecular, spatial, and temporal aspects of its regulation. J Cell Biochem 88(5), 873–884 (2003). https://doi.org/10.1002/jcb.10435CrossRefPubMed

13.

C.E. Farnum, N.J. Wilsman, Condensation of hypertrophic chondrocytes at the chondro-osseous junction of growth plate cartilage in Yucatan swine: relationship to long bone growth. Am J Anat 186(4), 346–358 (1989). https://doi.org/10.1002/aja.1001860404CrossRefPubMed

14.

G. Gibson, D.L. Lin, M. Roque, Apoptosis of terminally differentiated chondrocytes in culture. Exp Cell Res 233(2), 372–382 (1997). https://doi.org/10.1006/excr.1997.3576CrossRefPubMed

15.

V. Srinivas, J. Bohensky, I.M. Shapiro, Autophagy: a new phase in the maturation of growth plate chondrocytes is regulated by HIF, mTOR and AMP kinase. Cells Tissues Organs 189(1–4), 88–92 (2009). https://doi.org/10.1159/000151428CrossRefPubMed

16.

L. Wang, Q. Jie, L. Yang, Chondrocytes-osteoblast transition in endochondral ossification. Ann Joint 2(2) (2017).

17.

L. Yang, K.Y. Tsang, H.C. Tang, D. Chan, K.S. Cheah, Hypertrophic chondrocytes can become osteoblasts and osteocytes in endochondral bone formation. Proc Natl Acad Sci USA 111(33), 12097–12102 (2014). https://doi.org/10.1073/pnas.1302703111CrossRefPubMedPubMedCentral

18.

X. Zhou, K. von der Mark, S. Henry, W. Norton, H. Adams, B. de Crombrugghe, Chondrocytes transdifferentiate into osteoblasts in endochondral bone during development, postnatal growth and fracture healing in mice. PLoS Genet 10(12), e1004820 (2014). https://doi.org/10.1371/journal.pgen.1004820CrossRefPubMedPubMedCentral

19.

D.P. Hu, F. Ferro, F. Yang, A.J. Taylor, W. Chang, T. Miclau, R.S. Marcucio, C.S. Bahney, Cartilage to bone transformation during fracture healing is coordinated by the invading vasculature and induction of the core pluripotency genes. Development 144(2), 221–234 (2017). https://doi.org/10.1242/dev.130807CrossRefPubMedPubMedCentral

20.

R.Y. Kong, K.M. Kwan, E.T. Lau, J.T. Thomas, R.P. Boot-Handford, M.E. Grant, K.S. Cheah, Intron-exon structure, alternative use of promoter and expression of the mouse collagen X gene, Col10a-1. Eur J Biochem 213(1), 99–111 (1993). https://doi.org/10.1111/j.1432-1033.1993.tb17739.xCrossRefPubMed

21.

A. Ruscitto, M.M. Morel, C.J. Shawber, G. Reeve, M.K. Lecholop, D. Bonthius, H. Yao, M.C. Embree, Evidence of vasculature and chondrocyte to osteoblast transdifferentiation in craniofacial synovial joints: Implications for osteoarthritis diagnosis and therapy. FASEB J 34(3), 4445–4461 (2020). https://doi.org/10.1096/fj.201902287RCrossRefPubMed

22.

S.A. Wong, K.O. Rivera, T. Miclau 3rd, E. Alsberg, R.S. Marcucio, C.S. Bahney, Microenvironmental regulation of chondrocyte plasticity in endochondral repair—a new frontier for developmental engineering. Front Bioeng Biotechnol 6, 58 (2018). https://doi.org/10.3389/fbioe.2018.00058CrossRefPubMedPubMedCentral

23.

A. Yamaguchi, K. Sakamoto, T. Minamizato, K. Katsube, S. Nakanishi, Regulation of osteoblast differentiation mediated by BMP, Notch, and CCN3/NOV. Jpn Dent Sci Rev 44, 48–56 (2008). https://doi.org/10.1016/j.jdsr.2007.11.003CrossRef

24.

Z Liu, H Chen, J Zhou. In: S Choi (Ed.) Apoptosis regulator BAX. Encyclopedia of Signaling Molecules. Springer New York: New York, NY, 2016; 1–6.

25.

X. Qin, Q. Jiang, K. Nagano, T. Moriishi, T. Miyazaki, H. Komori, K. Ito, K.V. Mark, C. Sakane, H. Kaneko, T. Komori, Runx2 is essential for the transdifferentiation of chondrocytes into osteoblasts. PLoS Genet 16(11), e1009169 (2020). https://doi.org/10.1371/journal.pgen.1009169CrossRefPubMedPubMedCentral

26.

A. Houben, D. Kostanova-Poliakova, M. Weissenbock, J. Graf, S. Teufel, K. von der Mark, C. Hartmann, Beta-catenin activity in late hypertrophic chondrocytes locally orchestrates osteoblastogenesis and osteoclastogenesis. Development 143(20), 3826–3838 (2016). https://doi.org/10.1242/dev.137489CrossRefPubMedPubMedCentral

27.

S.A. Wong, D. Hu, T. Shao, E. Niemi, E. Barruet, B. M. Morales, O. Boozarpour, T. Miclau, E. C. Hsiao, M. Nakamura, C. S. Bahney, R. S. Marcucio, β-catenin signaling regulates cell fate decisions at the transition zone of the chondro-osseous junction during fracture healing. Preprint at bioRxiv https://doi.org/10.1101/2020.03.11.986141 (2020).

28.

L.I. Wolff, C. Hartmann, A second career for chondrocytes-transformation into osteoblasts. Curr Osteoporos Rep 17(3), 129–137 (2019). https://doi.org/10.1007/s11914-019-00511-3CrossRefPubMed

29.

R.L. Huang, Y. Yuan, G.M. Zou, G. Liu, J. Tu, Q. Li, LPS-stimulated inflammatory environment inhibits BMP-2-induced osteoblastic differentiation through crosstalk between TLR4/MyD88/NF-kappaB and BMP/Smad signaling. Stem Cells Dev 23(3), 277–289 (2014). https://doi.org/10.1089/scd.2013.0345CrossRefPubMed

30.

O. Reikeras, H. Shegarfi, J.E. Wang, S.E. Utvag, Lipopolysaccharide impairs fracture healing: an experimental study in rats. Acta Orthop 76(6), 749–753 (2005). https://doi.org/10.1080/17453670510045327CrossRefPubMed

31.

F. Descalzi Cancedda, C. Gentili, P. Manduca, R. Cancedda, Hypertrophic chondrocytes undergo further differentiation in culture. J Cell Biol 117(2), 427–435 (1992). https://doi.org/10.1083/jcb.117.2.427CrossRefPubMed

32.

C. Hegert, J. Kramer, G. Hargus, J. Muller, K. Guan, A.M. Wobus, P.K. Muller, J. Rohwedel, Differentiation plasticity of chondrocytes derived from mouse embryonic stem cells. J Cell Sci 115(Pt 23), 4617–4628 (2002). https://doi.org/10.1242/jcs.00171CrossRefPubMed

33.

K. Ishizeki, M. Takigawa, T. Nawa, F. Suzuki, Mouse Meckel’s cartilage chondrocytes evoke bone-like matrix and further transform into osteocyte-like cells in culture. Anat Rec 245(1), 25–35 (1996). 10.1002/(SICI)1097-0185(199605)245:1<25:AID-AR5>3.0.CO;2-ECrossRef

34.

A.J. Kahn, D.J. Simmons, Chondrocyte-to-osteocyte transformation in grafts of perichondrium-free epiphyseal cartilage. Clin Orthop Relat Res 129, 299–304 (1977). https://doi.org/10.1097/00003086-197711000-00042CrossRef

35.

L. Song, R.S. Tuan, Transdifferentiation potential of human mesenchymal stem cells derived from bone marrow. FASEB J 18(9), 980–982 (2004). https://doi.org/10.1096/fj.03-1100fjeCrossRefPubMed

36.

C.S. Bahney, D.P. Hu, A.J. Taylor, F. Ferro, H.M. Britz, B. Hallgrimsson, B. Johnstone, T. Miclau, R.S. Marcucio, Stem cell-derived endochondral cartilage stimulates bone healing by tissue transformation. J Bone Miner Res 29(5), 1269–1282 (2014). https://doi.org/10.1002/jbmr.2148CrossRefPubMed

Title: A novel in vitro assay to study chondrocyte-to-osteoblast transdifferentiation
Authors: Miriam E. A. Tschaffon
Stefan O. Reber
Astrid Schoppa
Sayantan Nandi
Ion C. Cirstea
Attila Aszodi
Anita Ignatius
Melanie Haffner-Luntzer
Publication date: 01-01-2022
Publisher: Springer US
Keyword: Fracture Healing
Published in: Endocrine / Issue 1/2022
Print ISSN: 1355-008X
Electronic ISSN: 1559-0100
DOI: https://doi.org/10.1007/s12020-021-02853-4

ACC 2024 Congress Coverage

Heart Failure Video

At a glance: The ONWARDS insulin icodec trials

Springer Medicine

A novel in vitro assay to study chondrocyte-to-osteoblast transdifferentiation

Abstract

Purpose

Methods

Results

Conclusion

ACC 2024 Congress Coverage

Year in Review: Heart failure and cardiomyopathies

Semaglutide benefits people with type 2 diabetes and obesity-related heart failure

Incentivised to exercise

New intervention for STEMI-related cardiogenic shock

» View more highlights on the ACC 2024 congress hub page

At a glance: The ONWARDS insulin icodec trials

Springer Medicine

Abstract

Purpose

Methods

Results

Conclusion

Please log in to get access to this content

Other articles of this Issue 1/2022

Analysis of serum microRNA in exosomal vehicles of papillary thyroid cancer

Surgical outcome of transsphenoidal surgery in Cushing’s disease: a case series of 1106 patients from a single center over 30 years

NCOA3 is a critical oncogene in thyroid cancer via the modulation of major signaling pathways

A contemporary clinical approach to genetic testing for heritable hyperparathyroidism syndromes

Is the ghost of brown tumor back again? Features of hypercalcaemic primary hyperparathyroidism we must not forget

Chronic angiotensin receptor activation promotes hepatic triacylglycerol accumulation during an acute glucose challenge in obese-insulin-resistant OLETF rats

ACC 2024 Congress Coverage

Year in Review: Heart failure and cardiomyopathies

Semaglutide benefits people with type 2 diabetes and obesity-related heart failure

Incentivised to exercise

New intervention for STEMI-related cardiogenic shock

» View more highlights on the ACC 2024 congress hub page