Development of limbs starts with embryonic-outhgrowths, first with front- and then with hind-limb buds. The regulation of limb-bud development, including cartilage differentiation, is highly complicated as it depends on activating or silencing many genes and other DNA fragments. A chemical methylation change of DNA, being involved in the regulation of DNA activity, is crucial for normal development. Because there are few studies on global DNA methylation dynamics during the limb-bud cartilage differentiation, researchers from the School of Medicine Zagreb and Hospital for Medical Rehabilitation of the Heart and Lung Diseases and Rheumatism – Thalassotherapia Opatija aimed to investigate this process during the early limb development of a mammal.
To avoid the research on living animals, researchers devised an original humane alternative. Tiny rat limb buds were isolated under the dissecting microscope and were grown in the natural three-dimensional system in the test tube (in vitro). Front- and hind limb-buds from gestation days (GD) 13 and 14 were cultivated in the chemically defined serum- and protein-free liquid medium, and in the undefined serum-supplemented medium. Cartilage differentiation progressed during 14 days equally well except in serum-free-cultivated GD13 hind-limb buds that lacked cartilage only on day three.
Presented in a nut-shell, researchers found that the global DNA methylation increase is associated with less developed limb-buds that strive towards cartilage differentiation. In contrast, they associated its decrease to originally more developed limb-buds. Results on the degree of global DNA methylation and cartilage-differentiation may be significant for novel strategies in regenerative medicine dealing with manufacturing cartilage grafts for bone or cartilage defects, as well as a better understanding of diseases such as osteoarthritis, cancer, or developmental anomalies.
Razvoj udova počinje s embrionalnim izraslinama, prvo s prednjim pupoljcima udova, a zatim sa stražnjim pupoljcima udova . Regulacija razvoja pupoljaka udova, uključujući diferencijaciju hrskavice, vrlo je komplicirana jer ovisi o aktiviranju ili utišavanju mnogih gena i drugih fragmenata DNK. Kemijska, metilacijska promjena DNK, uključena u regulaciju aktivnosti DNK, ključna je za normalan razvoj. Budući da postoji malo studija o dinamici globalne metilacije tijekom diferencijacije hrskavice pupoljaka ekstremiteta, istraživači Medicinskog fakulteta Zagreb i Bolnice za medicinsku rehabilitaciju bolesti srca, pluća i reumatizma – Thalassotherapia Opatija nastojali su istražiti ovaj proces tijekom ranog razvoja ekstremiteta sisavca.
Kako bi izbjegli istraživanje na živim životinjama, istraživači su osmislili originalnu humanu alternativu. U prirodnom trodimenzionalnom sustavu, sićušni pupoljci udova štakora izolirani pod mikroskopom za seciranje razvijali su se u epruveti (in vitro), na sučelju zrak-tekućina. Pupoljci prednjih i stražnjih udova iz 13. i 14. dana trudnoće (GD) uzgajani su u kemijski definiranom tekućem mediju bez seruma i proteina, kao i u nedefiniranom mediju obogaćenim serumom. Diferencijacija hrskavice je napredovala tijekom 14 dana jednako dobro svugdje osim u GD13 pupoljcima stražnjih udova uzgajanih u tekućem mediju bez seruma gdje samo trećeg dana nije bilo hrskavice.
Ukratko, istraživači su otkrili da je povećanje globalne metilacije DNA povezano s manje razvijenim pupoljcima udova koji teže diferencijaciji hrskavice. Nasuprot tome, povezali su njeno smanjenje s izvorno razvijenijim pupoljcima udova. Rezultati o stupnju globalne DNA metilacije i diferencijacije hrskavice mogu biti značajni za nove strategije u regenerativnoj medicini koje se bave proizvodnjom transplantata hrskavice za defekte kosti ili hrskavice, kao i za bolje razumijevanje bolesti kao što su osteoartritis, rak ili razvojne anomalije.
Reference: Mužić Radović V, Bunoza P, Marić T, Himelreich-Perić M, Bulić-Jakuš F, Takahashi M, Jurić-Lekić G, Sinčić N, Ježek D, Katušić-Bojanac A. Global DNA methylation and chondrogenesis of rat limb buds in a three-dimensional organ culture system. Bosn J of Basic Med Sci [Internet]. 2022Feb.21 [cited 2022Feb.27];. Available from: https://www.bjbms.org/ojs/index.php/bjbms/article/view/6584.
Editor: Merima Bukva, MPharm
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