Characterization of the microstructural and biomechanical properties of the human fascia in type 2 diabetes mellitus

Ugwoke, Chiedozie Kenneth (avtor), Umek, Nejc (mentor)

Aims. This doctoral research investigated how type 2 diabetes mellitus (T2DM) remodels the extracellular matrix (ECM) of human deep fascia and whether such remodeling increases tissue rigidity. The specific objectives were: (1) to determine T2DM-related structural alterations across functionally diverse deep fasciae and to evaluate the feasibility of three-dimensional (3D) microscopy for depicting fascial microanatomy; (2) to characterize diabetes-associated biomolecular changes using infrared spectroscopy and targeted histochemistry; and (3) to quantify nanoscale mechanical properties with atomic force microscopy (AFM). Hypotheses. (I) T2DM is associated with increased remodeling of the fibrous and aqueous components of the deep fascia matrix. (II) Biochemical alterations in deep-fascia macromolecules in T2DM result in increased tissue rigidity. Methods. A multimodal, multiscale investigation was conducted on postmortem samples of the fascia lata (FL), thoracolumbar fascia (TLF), and plantar fascia (PF) from 18 male subjects with T2DM and 18 non-diabetic controls. Following histological staining for general morphology, feasibility of 3D microstructural visualisation was assessed using optical projection tomography, digital light sheet microscopy, and magnetic resonance microscopy. Label-free multiphoton microscopy, including second harmonic generation (SHG), two-photon excited fluorescence (TPEF), and fluorescence lifetime imaging microscopy (FLIM), was used to detect structural and molecular matrix remodeling. The biomolecular composition was profiled using attenuated total reflectance fourier transform infrared (ATR-FTIR) spectroscopy and chemometric analysis, supported with targeted histochemistry. Finally, the nanoscale tissue stiffness was measured using AFM on hydrated tissue sections. Results. General histology and 3D microscopy demonstrated a trilaminar organization in the deep fascia, with a thicker collagen-dense intermediate layer between less densely and homogenously organized superficial and deep layers, and confirmed significant region-specific architectural specializations. Multiphoton imaging showed preserved SHG forward-to-backward ratios across groups, indicating no gross alteration of fibrillar collagen architecture. In contrast, TPEF ratios were higher in T2DM for PF and FL, and FLIM revealed shorter mean fluorescence lifetime in PF, together consistent with a remodeled collagen microenvironment influenced by crosslinking, oxidative stress, and altered hydration. ATR-FTIR with chemometrics and targeted histochemistry resolved fascia-type dependent spectral components and demonstrated trends of disease-associated alteration in collagen-dominant and ground-substance signatures. AFM established regional variation of stiffness, and showed significant T2DM-associated increased Young’s modulus in PF and FL, independent of age and body mass index, with no detectable change in TLF. Conclusions. The integrated optical, spectroscopic, histochemical, and nanomechanical evidence substantiates Hypothesis I by demonstrating molecular-level collagen remodeling and decreased hyaluronan-rich ground-substance in T2DM. The data strongly support Hypothesis II by showing that these biochemical alterations are accompanied by increased nanoscale rigidity in PF and FL even if fibrillar order detected by SHG remains largely preserved. Collectively, the thesis establishes that deep-fascia remodeling in T2DM is anatomically specific and subtle, but detectable by label-free optical and spectral signatures, and mechanically consequential. The integrated, multiscale model provides quantitative benchmarks and translationally relevant biomarkers adaptable for risk stratification, and evaluation of mechanism-based interventions in diabetic fasciopathy.

• Jezik: Angleški jezik
• Ključne besede: Deep fascia, plantar fascia, fascia lata, thoracolumbar fascia, type 2 diabetes mellitus, extracellular matrix, multiphoton microscopy, fluorescence lifetime imaging, Fourier-transform infrared spectroscopy, atomic force microscopy, 3D microscopy.
• Vrsta gradiva: Doktorsko delo/naloga
• Organizacija: MF - Medicinska fakulteta
• Leto izida: 2026
• PID: 20.500.12556/RUL-178187
• Datum objave v RUL: 21.01.2026

Sekundarni jezik

• Jezik: Slovenski jezik
• Naslov: Opredelitev mikrostrukturnih in biomehanskih lastnosti človeške fascije pri sladkorni bolezni tipa 2

Izvleček

Namen. Namena doktorske raziskave sta bila ugotoviti, kako sladkorna bolezen tipa 2 (T2DM) vpliva na zunajcelični matriks (ECM) človeške globoke fascije ter ali takšno preoblikovanje povzroči povečano togost tkiva. Specifični cilji so bili: (1) določiti spremembe v zgradbi fascije, povezane s T2DM, v funkcionalno različnih anatomskih regijah ter oceniti uporabnost tridimenzionalne mikroskopije za prikaz mikroanatomije fascije; (2) opredeliti biokemične spremembe, povezane s T2DM, z uporabo infrardeče spektroskopije in ciljno usmerjene histokemije; ter (3) kvantificirati nanomehanske lastnosti fascije z mikroskopijo na atomsko silo (AFM). Hipoteze. (H1) sladkorna bolezen tipa 2 povzroča povečano preoblikovanje vlaknaste in gelaste komponente zunajceličnega matriksa globoke fascije; (H2) biokemične spremembe v strukturnih makromolekulah fascije pri T2DM vodijo v povečano togost tkiva. Metode. Raziskavo smo izvedli na posmrtnih vzorcih fascije late (FL), torakolumbalne fascije (TLF) in plantarne fascije (PF), pridobljenih od 18 moških oseb s T2DM in 18 kontrol brez sladkorne bolezni. Po osnovni histološki analizi smo izvedli tridimenzionalno slikovno študijo z optično projekcijsko tomografijo, digitalno svetlobno listno mikroskopijo in magnetnoresonančno mikroskopijo. Spremembe v strukturi in kemiji matriksa smo spremljali z večfotonsko mikroskopijo brez barvil, ki je vključevala signal sekundarne harmonične generacije (SHG), dvofotonsko vzbujeno fluorescenco (TPEF) in mikroskopijo življenjskega časa fluorescence (FLIM). Biokemično sestavo smo analizirali z ATR-FTIR spektroskopijo in kemometrično dekompozicijo, podprto s ciljno histokemijo, nanomehanske lastnosti pa smo izmerili z AFM na hidriranih rezinah tkiva. Rezultati. Tridimenzionalno slikanje in histologija sta pokazala dosledno trilaminarno zgradbo fascije z debelo srednjo plastjo gostega kolagena in tanjšima površinsko ter globoko plastjo ohlapnejšega veziva. Večfotonska mikroskopija je pokazala ohranjeno fibrilarno urejenost kolagena, medtem ko so bile vrednosti TPEF višje in življenjski čas fluorescence krajši pri PF in FL v skupini s T2DM, kar kaže na spremenjeno kolagensko mikrookolje z večjim križnim povezovanjem, oksidativnim stresom in spremenjeno hidracijo. ATR-FTIR in histokemija sta potrdili zmanjšanje količine osnovne snovi, bogate z glikozaminoglikani, zlasti v PF in TLF. Mikroskopija na atomsko silo je pokazala izrazito povečano togost PF in FL pri T2DM, neodvisno od starosti in indeksa telesne mase, medtem ko sprememb v TLF nismo odkrili. Sklepi. Celoviti optični, spektroskopski, histokemični in mehanski podatki potrjujejo hipotezo H1, saj dokazujejo preoblikovanje kolagena in zmanjšanje količine osnovne snovi, bogate z glikozaminoglikani. Potrjujejo tudi hipotezo H2, saj dokazujejo povezavo med omenjenimi biokemičnimi spremembami in povečano lokalno togostjo fascije. Sladkorna bolezen tipa 2 tako povzroča anatomsko specifično in biokemično zaznavno preoblikovanje fascije, ki je mehansko pomembno in kvantitativno merljivo z neinvazivnimi optičnimi in mehanskimi pristopi. Raziskava vzpostavlja večmodalni in večnivojski model, uporaben za razvoj bioloških označevalcev tveganja, zgodnje odkrivanje in spremljanje mehanistično usmerjenih oblik zdravljenja diabetične fasciopatije.

• Ključne besede: Globoka fascija, plantarna fascija, fascija lata, torakolumbalna fascija, sladkorna bolezen tipa 2, zunajcelični matriks, večfotonska mikroskopija, mikroskopija življenjskega časa fluorescence, infrardeča spektroskopija, mikroskopija na atomsko silo.