Vpliv iz maščevja pridobljene stromalne vaskularne frakcije na obstojnost in kakovost presadka maščevja v predhodno obsevanem tkivu golih miši

Koren, Matic (author), Ihan Hren, Nataša (mentor)

Uvod: Prosti presadki maščevja za funkcionalno in estetsko rehabilitacijo bolnikov so lahko dostopni in imajo nizke stroške posega, težavo pa predstavlja nepredvidljivost njihove volumske obstojnosti po presaditvi na prejemno mesto, kar se v obsevanem tkivu še poslabša. Velike prostornine presadkov dodatno povečajo tveganje izgubo njihove prostornine. Stromalna vaskularna frakcija (SVF) je sestavljena iz številnih progenitornih celic in iz maščevja pridobjenih matičnih celic, dodane presadku maščevja lahko izboljšajo njihovo volumsko obstojnost. Namen in hipoteze: Namen študije je bil ovrednotiti vpliv stromalne vaskularne frakcije na obstojnost in kakovost velikega presadka maščevja v predhodno obsevanem tkivu, hipoteze so bile:. 1. Obstojnost presadka maščevja je v obsevanem tkivu statistično značilno slabša, kot v neobsevanem. 2. Obstojnost presadka maščevja se v neobsevanem in obsevanem tkivu statistično značilno izboljša ob dodatku iz maščevja pridobljene SVF. 3. Kakovost presadka maščevja je v obsevanem tkivu statistično značilno slabša, kot v neobsevanem. 4. Kakovost presadka maščevja se v neobsevanem in obsevanem tkivu statistično značilno izboljša ob dodatku iz maščevja pridobljene SVF. Metoda: V naši študiji smo uporabili živalski model, vključili smo 64 imunsko oslabljenih golih miši NU(NCr)-Foxn1nu. Polovico miši smo obsevali z rentgenskimi žarki v predelu hrbta. Maščevje smo med načrtovanim lipofilingom odvzeli človeškemu darovalcu in obdelali z ustreznimi postopki. Maščevje smo uporabili za lipofiling in za pripravo SVF. Celice v SVF smo označili s citosolnim barvilom CFSE (5-(in 6)-karboksifluorescein diacetatnim sukcinilmidilnim estrom. Miši smo razdelili v 6 skupin, 3 neobsevane brez (G2) in s SVF (G3) ter 3 obsevane brez (G5) in s SVF (G6). V neobsevani (G1) in obsevani (G4) skupini je bila po ena kontrolna skupina. V podkožje miši smo nad levo lopatico in križnico glede na dodeljenjo skupino vbrizgali presadek maščevja brez ali s SVF. Kontrolni skupini sta prejeli Ringerjevo raztopino. S tridimenzionalno kamero smo posneli izbokline zaradi lipofilinga na hrbtu miši pred vsaditvijo presadkov, nato čez 14 dni (T1), 3 mesece (T2) in 6 mesecev (T3) po vsaditvi. Pridobljene posnetke smo obdelali s programsko opremo (RapidForm 2016) in izračunali prostornine presadkov. Ob T3 smo živali žrtvovali in odvzeli presadke. Del smo jih shranili v 10% formalin za oceno histoloških lastnosti po točkovni metodi, opravili smo pregled presadkov pod stereomikroskopom za oceno deleža flourescence predhodno označenih celic. Pridobljene rezultate smo statistično obdelali s programom SPSS. Rezultati: Izgube prostornine presadkov so bile po 6 mesecih velike. V neobsevanih skupinah so izgube prostornine presadkov znašale od 80% do 85%, ob dodatku SVF pa od 50% do 75%. V obsevanih skupinah so izgube presadkov znašale 70%, ob dodatku SVF je bila izguba 50%. Ob T2 in T3 smo v obsevani in neobsevani skupini ugotavljali statistično značilno (p < 0.05) večjo prostornino presadkov v skupinah, ki so prejele SVF. Obsevanje se je izkazalo kot dejavnik, ki statistično značilno (p < 0.05) vpliva na obstojnost presadkov. Ob oceni histoloških značilnosti smo ugotovili, da dodatek SVF statistično značilno (p < 0.05) izboljša kakovost presadka maščevja v neobsevani skupini z razliko 1,5 točke. V obsevani skupini se kljub dodatku SVF kakovost statistično značilno (p < 0.05) ni izboljšala. Razlika točk je znašala 0,5. Ob primerjavi neobsevane in obsevane skupine smo ugotovili statistično značilno (p < 0.05) slabšo kakovost presadkov pri skupinah, ki so imele predhodno obsevano prejemno mesto. Delež vnetja je bil v skupini G3 statistično značilno (p < 0.05) manjši za 0,5 točke glede na G2. V skupini G6 je bil delež vnetja statistično značilno (p < 0.05) večji za 0,2 točke glede na G5. Pod stereomikroskopom smo ugotovili minimalni delež fluorescence predhodno obarvanih celic v neobsevani in obsevani skupini z dodatkom SVF. Zaključek: V naši raziskavi smo ugotovili, da dodatek SVF dolgoročno izboljša volumsko obstojnost velikih presadkov maščevja, bolj pomembno v obsevanem tkivu. Z ugotovitvami smo v celoti potrdili hipotezo 1 in 2. Hipotezi 3 in 4 smo delno potrdili.

• Language: Slovenian
• Keywords: maščevje, stromalna vaskularna frakcija, obsevanje
• Work type: Doctoral dissertation
• Organization: MF - Faculty of Medicine
• Year: 2024
• PID: 20.500.12556/RUL-156131
• Publication date in RUL: 10.05.2024

Secondary language

• Language: English
• Title: Adipose tissue derived Stromal Vascular Fraction Influence on Survival and Quality of Fat Transplant in Nude Mice Irradiated Tissue

Abstract

Background: Free fat grafts are suitable for functional and aesthetic rehabilitation because they are readily available and procedure costs are low. The main downfall is unpredictable volume loss after transplantation. When the recipient bed is irradiated before transplantation, the final result is even more unpredictable. Large-volume fat grafts a have higher volume loss. The stromal vascular fraction (SVF) is a mixture of different progenitor cells and adipose-derived stem cells (ASCs). Adding SVF to fat grafts could improve their volume stability in the long term. Objectives and hypothesis: In our study, we wanted to evaluate the influence of SVF on quantity and quality of large-volume fat grafts in irradiated tissue. At the beginninig of our research we had 4 hypotheses: 1. Fat graft volume loss is worse in irradiated tissue compared to a healthy recipient bed. 2. SVF added to free fat graft improves volume stability in irradiated and healthy tissue. 3. The quality of fat graft in irradiated tissue is worse compared to healthy tissue. 4. The quality of free fat graft in irradiated and healthy tissue could be improved by adding SVF. Methods and materials: This study was conducted in a nude mouse model with fatty tissue transplanted from a human donor. The sample comprised 64 immunodeficient and hairless nude NU(NCr)-Foxn1nu mice. Half of the mice had irradiated skin on their backs. The fatty tissue for grafts was collected from human donor. Lipoaspirate was processed with a standard procedure. One part of fatty tissue was used for SVF production, the other part as a fat graft. Cells in the SVF were dyed with CFSE (5-(in 6)-carboxyfluorescein diacetate succinilmidyl ester. Mice were divided into 6 groups. Three groups were irradiated. They were divided into a control group (G4), a group without SVF (G5) and with SVF (G6). The same division was done in the non-irradiated group; control group (G1) and the group without SVF (G2) and with SVF (G3). The free fat grafts were injected into the subcutaneous tissue above the left scapula and sacrum. The bulges on the mice's backs were scanned with a three-dimendionsional (3D) camera. Scans of the grafted area were done 14 days (T1), 3 months (T2) and 6 months (T3) after application. Volumes were calculated with appropriate software (RapidForm 2006). After 6 months, the animals were sacrificed and fat grafts were collected for histological evaluation. After harvesting fat grafts with added SVF, their fluorescence was evaluated with a stereomicroscope. Statistical analysis was done with SPSS software. Results: The fat graft volume loss after 6 months was high. In non-irradiated groups, volume loss was from 80 % to 85 %. Grafts with SVF had volume loss from 50 % to 75 %. In irradiated groups, volume loss was up to 70 % of the initial volume. When we added SVF to fat grafts, volume loss was 50 % of the initial volume. At T2 and T3 we found a statistically significant (p < 0.05) higher volume retention in groups with SVF. Irradiation had a statistically significant (p < 0.05) negative influence on long-term fat graft volume retention. The SVF had a statistically significant (p < 0.05) positive influence on the quality of fat grafts in groups without previous irradiation. The difference between the groups was 1.5 points. In irradiated groups, SVF did not statistically significantly (p < 0.05) improve the quality of fat grafts. The difference between groups, was 0.5 points. Comparing non-irradiated and irradiated groups, we found a statistically significantly (p < 0.05) better result in non-irradiated tissue. The inflammation rate was statistically significantly (p < 0.05) lower in the non-irradiated group with SVF compared to the group with fat graft only. The difference was 0.5 points. In irradiated groups, inflammation appears at a higher rate in groups with added SVF. The difference was 0.2 points. Fluorescence of dyed SVF appeared at very low rates. Conclusion: In our study, we confirmed the positive effect of SVF on volume stability of large-volume fat grafts. The effect is especially significant in irradiated tissue. We entirely confirmed hypotheses 1 and 2. Hypotheses 3 in 4 were partially confirmed.

• Keywords: adipose tissue, stromal vascular fraction, irradiation