Morfometrijska analiza zglobne hrskavice u Bmp3-/- miševa koristeći mikro CT

master's thesis

• Title (english): Morphometric analysis of articular cartilage in Bmp3-/- using micro CT
• Author: Matej Nekić
• Mentor: Donatella Verbanac (mentor)
• Committee member: Donatella Verbanac (predsjednik povjerenstva)
• Committee member: Roberta Petlevski (član povjerenstva)
• Committee member: Igor Erjavec (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; Zagreb
• Defense date and country: 2023-07-20, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Medical Biochemistry

Abstract

BMP3 je najviše zastupljeni koštani morfogenetski protein u demineraliziranoj kosti i djeluje kao negativni regulator gustoće kosti. U ovome radu istražen je utjecaj BMP3 na zglobnu hrskavicu, koristeći morfometrijsku analizu mikro CT uređajem, čija nedestruktivna i neinvazivna priroda omogućuje analizu većeg broja uzoraka u izrazito kraćem vremenu naspram dugotrajnih i destruktivnih histoloških metoda. Nedostatak mikro CT metode u vizualizaciji mekih tkiva riješen je uspostavom protokola za kontrastiranje hrskavice fosfotungstičnom kiselinom (PTA). U istraživanju su korišteni WT i Bmp3-/- miševi oba spola čiji je genotip određen umnažanjem DNA izolirane iz uzoraka vršaka repova lančanom reakcijom polimeraze (PCR) s korištenim početnicama za Bmp3 gen i reporterski gen LacZ te popratnu agaroznu gel elektroforezu. 36 uzoraka bedrenih kostiju miševa sakupljeno je i kontrastirano te snimljeno ex vivo koristeći Skyscan 1076 (Bruker, Belgija) mikro CT uređaj, nakon čega je napravljena rekonstrukcija i morfometrijska analiza zglobne hrskavice distalne bedrene kosti na medijalnim i lateralnim kondilima, kojom su kvantificirane vrijednosti hrskavične površine (CS), hrskavičnog volumena (CV) te njihovog omjera (CS/CV) za oba kondila. Potvrdna analiza napravljena je histokemijskim bojanjem po protokolu H&E i toluidinom. Morfometrijskom analizom i statističkom obradom podataka utvrđeno je da WT miševi imaju veći volumen zglobne hrskavice (CV) od Bmp3-/- miševa na oba analizirana kondila, dok analiza površine hrskavice (CS) nije pokazala statistički značajnu razliku između Bmp3-/- i WT miševa na medijalnom kondilu, kao ni na lateralnom kondilu. Omjer CS/CV nije pokazao statistički značajnu razliku između Bmp3-/- i WT na analiziranim medijalnim i lateralnim kondilima, gdje normalizacija hrskavičnog volumena s hrskavičnom površinom nije rezultirala značajnim razlikama između genotipova miševa. Na histološkim presjecima jasno je vidljivo da je zglobna hrskavica WT miševa deblja s većom staničnosti od Bmp3-/- miševa. Rezultati istraživanja su pokazali da, iako nedostatak BMP3 ima pozitivan učinak na kost, BMP3 je potreban za normalan razvoj i homeostazu hrskavice.

Abstract (english)

BMP3 is the most abundant bone morphogenetic protein in demineralized bone and acts as a negative regulator of bone density. In this thesis, the influence of BMP3 on articular cartilage was investigated, with morphometric analysis using a micro CT device, whose non-destructive and non-invasive nature enables the analysis of a large number of samples in a significantly shorter time span compared to long-term and destructive histological methods. A disadvantage of the micro CT method in the visualization of soft tissues was solved by establishing a protocol for contrasting cartilage with phosphotungstic acid (PTA). In this experiment, WT and Bmp3-/- mice of both sexes were used, whose genotype was determined by amplification of DNA isolated from tail tip samples using polymerase chain reaction (PCR) with primers for the Bmp3 gene and the reporter gene LacZ and concomitant agarose gel electrophoresis. 36 samples of mouse femurs were collected and contrasted and recorded ex vivo using a Skyscan 1076 (Bruker, Belgium) micro CT device, after which reconstruction and morphometric analysis of articular cartilage of the distal femur on the medial and lateral condyles was carried out. The values of cartilage surface (CS), cartilage volume (CV) and their ratio (CS/CV) for both condyles were quantified. Confirmatory analysis was carried out by histochemical staining using H&E protocol and toluidine. Morphometric and statistical analysis revealed that WT mice have a higher articular cartilage volume (CV) than Bmp3-/- mice on both analyzed condyles, while cartilage surface (CS) analysis did not show a statistically significant difference between Bmp3-/- and WT mice on the medial condyle nor the lateral condyle. The CS/CV ratio did not show a statistically significant difference between Bmp3-/- and WT on the analyzed medial and lateral condyles, where normalization of cartilage volume with cartilage surface did not result in significant differences between the two analyzed murine genotypes. On histological sections, it is clearly visible that the articular cartilage of WT mice is thicker and with higher cellularity than the articular cartilage of Bmp3-/- mice. The obtained experimental results showed that, although BMP3 deficiency has a positive effect on bone, BMP3 is critical and required for normal cartilage development and homeostasis.

• Keywords: koštani morfogenetski protein 3; BMP3; mikro CT; hrskavica; morfometrijska analiza
• Keywords (english): bone morphogenetic protein 3; MP3; micro CT; cartilage; morphometric analysis
• Language: croatian
• URN:NBN: urn:nbn:hr:163:827341
• Study programme: Title: Medical biochemistry - integrated undergraduate and graduate - university study; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: sveučilišni magistar medicinske biokemije i laboratorijske medicine (sveučilišni magistar medicinske biokemije i laboratorijske medicine)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2024-03-21 13:28:57