ROTATOR MANȘET TENDON HASARLARI İÇİN GDF-5 YÜKLÜ VE HİZALI NANOFİBERLERLE KATKILANDIRILMIŞ POLİ(BÜTİLEN ADİPAT-KO-TEREFTALAT) YAMALARIN GELİŞTİRİLMESİ: IN VİTRO ÇALIŞMALAR

Abstract

Injuries to the supraspinatus tendon of the rotator cuff are unable to heal spontaneously because of the limited intrinsic regenerative capacity of the affected tissue. Depending on the severity of the injury, physical therapy or surgical repair is recommended. However, current treatment approaches are unable to fully recapitulate the anisotropic architecture and biochemical composition of the native tendon. Tissue engineering has therefore emerged as a promising strategy for the repair and regeneration of tendon injuries. The aim of the present thesis was to develop growth/differentiation factor-5 (GDF-5)-incorporated bilayer anisotropic nanofibrous poly(butylene adipate-co-terephthalate) (PBAT) matrices for use as supraspinatus tendon patches and to evaluate their effects on the tenogenic differentiation of adipose-derived mesenchymal stem cells (ADMSCs) under dynamic culture conditions. In the first part of the thesis, a random nanofibrous matrix was initially fabricated by electrospinning a PBAT solution prepared using hexafluoroisopropanol (HFIP). Subsequently, aligned nanofibers were electrospun onto the random layer to produce bilayer PBAT matrices with different thicknesses. The collection times for both the random and aligned fiber layers were maintained equally at 4, 6, and 8 h, and the resulting matrices were designated as 4S, 6S, and 8S, respectively. Following physicochemical characterization, the mechanical properties of the matrices were evaluated before and after degradation. Based on cell viability, morphological, and gene expression analyses conducted under static culture conditions, the 4S group was identified as the most effective scaffold for promoting the tenogenic differentiation of ADMSCs, and all subsequent experiments were therefore performed using this group. In the second part of the thesis, the optimal dose of GDF-5 for inducing the tenogenic differentiation of ADMSCs was first determined. This dose was subsequently incorporated into the aligned fiber layer of the 4S scaffold during the electrospinning process to fabricate G-PBAT matrices. The G-PBAT matrices exhibited enhanced nanofiber alignment, reduced fiber diameter, and improved mechanical properties. The release profile of GDF-5 was evaluated by enzyme-linked immunosorbent assay (ELISA), demonstrating an initial burst release on the first day followed by a controlled and sustained release over a period of 20 days. Comprehensive evaluation of cell viability, tenocyte-like morphology, tendon-like mechanical properties, and tenogenic marker expression at both the gene and protein levels demonstrated that the G-PBAT matrix most effectively supported the tenogenic differentiation of ADMSCs. In the third part of the thesis, an experimental design based on response surface methodology was established by considering the 2–4% strain range accepted for the physiological stress–strain behavior of native tendon tissue, together with the loading frequencies and loading–rest durations reported in the literature for mechanically stimulated dynamic bioreactor studies. According to the determined parameters, preliminary experiments were conducted using a mechanically active dynamic bioreactor system capable of applying both uniaxial and biaxial tensile loading. Following mechanical stimulation, the morphological and mechanical properties of the samples were evaluated, and the elastic modulus values obtained from the mechanical analyses were defined as the response variable of the response surface model. The model analysis demonstrated that the embryonic-stage mechanical stimulation parameters were within the safe operating range, and these parameters were subsequently employed in the cell culture experiments. The embryonic-stage mechanical stimulation protocol was developed to mimic the biomechanical microenvironment present during embryonic tendon development, whereas the physical therapy-mimicking mechanical stimulation protocol was designed to model the mechanical loading applied during the non-surgical treatment of supraspinatus tendon injuries. Accordingly, the embryonic-stage mechanical stimulation protocol consisted of tensile loading at 1 Hz frequency and 3% strain for 4 h, followed by a 20 h resting period. In contrast, the physical therapy-mimicking mechanical stimulation protocol consisted of tensile loading at 0.1 Hz frequency and 4% strain, applied for 30 s per stimulation, nine times daily in three sessions with three repetitions per session. Cell culture studies performed using ADMSCs included cell viability, morphological, mechanical, gene expression, and protein expression analyses. The findings demonstrated that the synergistic effect of the controlled release of GDF-5 from the matrix and mechanical stimulation markedly enhanced the tenogenic differentiation and tendon maturation of ADMSCs. In the G-PBAT/P group, approximately 700-fold, 14-fold, and 300-fold increases were observed in the expression levels of tenomodulin (TNMD), collagen type III (COL3A1), and tenascin-C (TNC), respectively. Collectively, the findings obtained under dynamic culture conditions demonstrated that the combined application of G-PBAT matrices and physical therapy-mimicking mechanical stimulation effectively promoted the tenogenic differentiation and tendon maturation of ADMSCs. Within the scope of the present thesis, a bilayer anisotropic tendon patch was successfully developed to simultaneously mimic the structural organization, biochemical cues, and mechanical microenvironment of the posterior region of the supraspinatus tendon. Furthermore, the physical therapy-mimicking mechanical stimulation applied to the matrices was shown to establish a favorable microenvironment that supports functional tendon tissue formation. The findings of this study indicate that the developed tendon patch represents a promising tissue engineering strategy for supraspinatus tendon repair. Nevertheless, further in vivo studies are required to validate its clinical translation potential.