Fotios M Andreopoulos

Chronic wounds present a major burden to patients, health care professionals, and health care systems worldwide, yet treatment options remain limited and often ineffective. Although initially promising, growth factor-based therapies displayed limited and underwhelming effectiveness largely due to poor bioavailabilbioity and impaired receptor function within the chronic wound microenvironment. Here we demonstrate that chronic wounds exhibit elevated cholesterol synthesis, which disrupts growth factor signaling by sequestering receptors within lipid rafts. To address this, we developed a novel therapy combining growth factors with cyclodextrin in an ECM-mimetic scaffold, enabling localized cholesterol modulation and improved receptor accessibility. We demonstrate that this approach enhances growth factor bioavailability and functionality, creating a regenerative environment. In both human ex vivo and diabetic mouse wound models, this targeted co-delivery strategy significantly improved healing outcomes by stimulating angiogenesis and re-epithelialization, supporting a promising new direction for chronic wound therapy through localized metabolic modulation of the wound niche.

Current treatments for autoimmune diseases like Type 1 Diabetes (T1D) carry significant risks because they lack tissue specificity. A promising strategy is to achieve persistent presentation of relevant antigens (Ags) in non-inflamed sites by tolerogenic Ag-presenting cells (APCs) like fibroblastic reticular cells (FRCs). FRCs build lymph node (LN) reticula and act as immunomodulatory non-professional APCs. However, their therapeutic potential for Ag-specific immunomodulation for T1D remains unexplored. We engineered 3D FRC-based reticula using freeze-dried macroporous gelatin scaffolds with customizable pore diameters (small: <50 μm, medium: <200 μm, large: <300 μm) to evaluate FRC phenotype and FRC-T cell interactions, leveraging FRCs' ability to build dynamic LN reticula that expand and contract during inflammation. Our scaffolds promoted FRC viability, reticular formation, FRC phenotypic marker expression, and extracellular matrix secretion compared to 2D culture. GFP-Luciferase fusion (GLF)-expressing FRCs subcutaneously implanted in our scaffolds survived for at least 21 days regardless of pore size. Implantation in the vascularized fat pad led to graft rejection by day 14 in pre-diabetic NOD mice but not in immunodeficient NODscid. Our scaffolds outperformed clinically-used biologic gels, demonstrated the limitations of the NOD mouse model in longitudinal imaging of GLF
cell survival, and validated our scaffold-based FRC delivery approach for future therapeutic applications. Co-culture of T cells in scaffolds with FRCs presenting T1D Ags enabled Ag-specific T cell engagement with reduced cytotoxic and increased anergic and regulatory phenotypes. Our results validate the use of macroporous gelatin scaffolds to enhance FRC phenotypic markers, in vivo survival, and diabetogenic cell immunomodulation in vitro.

Orthopedic soft tissue injuries, such as those to the fibrocartilaginous meniscus in the knee, present a significant clinical challenge, impacting millions globally and often requiring surgical interventions that fail to fully restore mechanical function. Current bioengineered meniscal replacement options that incorporate synthetic and/or natural scaffolds have limitations in biomechanical performance and biological integration. This study introduces a novel scaffold fabrication approach, termed Hybrid Hydrogels Augmented via Additive Network Integration (HANI) with great potential for meniscal tissue engineering applications. HANI scaffolds combine cross-linked gelatin-based hydrogels with polycaprolactone (PCL) additive networks, created via Fused Deposition Modeling (FDM), to enhance mechanical strength and replicate the anisotropic properties of the meniscus. Custom Stereolithography (SLA)-printed molds ensure precise dimensional control and seamless incorporation of PCL networks within the hydrogel matrix. The mechanical evaluation of HANI scaffolds showed improvements in compressive stiffness, stress relaxation behavior, and load-bearing capacity, especially with circumferential and 3D PCL reinforcements, when compared to hydrogel scaffolds without additive networks. These findings highlight HANI’s potential as a cost-effective, scalable, and tunable scaffold fabrication approach for meniscal tissue engineering applications.

The development of cell-based biomaterial alternatives holds significant promise in tissue engineering applications, but it requires accurate mechanical assessment. Herein, we present the development of a novel 3D-printed confined compression apparatus, fabricated using clear resin, designed to cater to the unique demands of biomaterial developers. Our objective was to enhance the precision of force measurements and improve sample visibility during compression testing. We compared the performance of our innovative 3D-printed confined compression setup to a conventional setup by performing stress relaxation testing on hydrogels with variable degrees of crosslinking. We assessed equilibrium force, aggregate modulus, and peak force. This study demonstrates that our revised setup can capture a larger range of force values while simultaneously improving accuracy. We were able to detect significant differences in force and aggregate modulus measurements of hydrogels with variable degrees of crosslinking using our revised setup, whereas these were indistinguishable with the convectional apparatus. Further, by incorporating a clear resin in the fabrication of the compression chamber, we improved sample visibility, thus enabling real-time monitoring and informed assessment of biomaterial behavior under compressive testing.

Background: Arteriovenous fistula (AVF) postoperative stenosis is a persistent healthcare problem for hemodialysis patients. We have previously demonstrated that fibrotic remodeling contributes to AVF non-maturation and lysyl oxidase (LOX) is upregulated in failed AVFs compared to matured. Herein, we developed a nanofiber scaffold for the periadventitial delivery of beta-aminopropionitrile (BAPN) to determine whether unidirectional periadventitial LOX inhibition is a suitable strategy to promote adaptive AVF remodeling in a rat model of AVF remodeling. Methods: Bilayer poly (lactic acid) ([PLA)-]- poly (lactic-co-glycolic acid) ([PLGA)] scaffolds were fabricated with using a two-step electrospinning process to confer directionality. BAPN-loaded and vehicle control scaffolds were wrapped around the venous limb of a rat femoral-epigastric AVF during surgery. AVF patency and lumen diameter were followed monitored using Doppler ultrasound surveillance and flow was measured before euthanasia. AVFs were harvested after 21 days for histomorphometry and immunohistochemistry. AVF compliance was measured using pressure myography. RNA from AVF veins was sequenced to analyze changes in gene expression due to LOX inhibition. Results: Bilayer periadventitial nanofiber scaffolds extended BAPN release compared to the monolayer design (p < 0.005) and only released BAPN in one direction. Periadventitial LOX inhibition led to significant increases in AVF dilation and flow after 21 days. Histologically, BAPN trended toward increased lumen and significantly reduced fibrosis compared to control scaffolds (p < 0.01). Periadventitial BAPN reduced downregulated markers associated with myofibroblast differentiation including SMA, FSP-1, LOX, and TGF-beta while increasing the contractile marker MYH11. RNA sequencing revealed differential expression of matrisome genes. Conclusion: Periadventitial BAPN treatment reduces fibrosis and promotes AVF compliance. Interestingly, the inhibition of LOX leads to increased accumulation of contractile VSMC while reducing myofibroblast-like cells. Periadventitial LOX inhibition alters the matrisome to improve AVF vascular remodeling.

Periadventitial biomaterials have been employed for nearly three decades to promote adaptive venous remodeling following hemodialysis vascular access creation in preclinical models and clinical trials. These systems are predicated on the combination of scaffolds, hydrogels, and/or particles with therapeutics (small molecules, proteins, genes, and cells) to prevent venous stenosis and subsequent maturation failure. Periadventitial biomaterial therapies have evolved from simple drug delivery vehicles for traditional drugs to more thoughtful designs tailored to the pathophysiology of access failure. The emergence of tissue engineering strategies and gene therapies are another exciting new direction. Despite favorable results in experimental and preclinical studies, no periadventitial therapy has been clinically approved to improve vascular access outcomes. After conducting an exhaustive review of the literature, we identify the seminal studies and clinical trials that utilize periadventitial biomaterials and discuss the key features of each biomaterial format and their respective shortcomings as they pertain to access maturation. This review provides a foundation from which clinicians, surgeons, biologists, and engineers can refer to and will hopefully inspire thoughtful, translatable treatments to finally address access failure.

Introduction: Arteriovenous fistula (AVF) nonmaturation is associated with postoperative fibrosis and an increase in lysyl oxidase (LOX) expression. Objective: The purpose of this study was to determine whether unidirectional periadventitial delivery of β-aminoproprionitrile (BAPN) using bilayer poly lactic-co-glycolic acid nanofiber scaffolds is a feasible approach to reduce AVF fibrosis and encourage adaptive vascular remodeling. Methods: AVFs were created in thirty Sprague Dawley rats (200-350 g) of both sexes by an end-to-side anastomosis of the epigastric vein to the femoral artery. BAPN-loaded (b-BAPN) (n=10) or vehicle (b-VEH) (n=10) scaffolds (5 mm x 5 mm) were wrapped around the juxta-anastomotic zone of the epigastric vein immediately after anastomosis. AVFs without treatment were used as an additional control (n=10). Changes in lumen diameter were followed-up weekly using ultrasound. Flow was calculated using the pulse-wave velocity and lumen diameter. AVFs were collected at 21 days for histomorphometric analysis and to assess fibrosis. Results: b-BAPN significantly increased dilation of AVFs at day 14 (185.9%) compared to b-VEH (135.5%; p=0.0006) and control AVFs (113.6%; p<0.0001). The treatment (214.2%) had a significant difference compared to b-VEH (135.5%; p<0.0001) and AVF controls (122%; p<0.0001) at day 21 as well. In addition, blood flow was significantly enhanced in the treatment group (38.13 mL/min) versus b-VEH (9.436 mL/min; p=0.0002) and AVF controls (16.11 mL/min; p<0.0001). Histomorphometric analysis confirmed the ultrasound findings. Treatment with b-BAPN significantly increased lumen area compared to control AVFs (354,127 μm 2 vs. 95,580 μm 2 ; p=0.0048). While there was no difference in intimal growth (p=0.5460), b-BAPN significantly reduced luminal occlusion with respect to control AVFs (0.0611 vs. 0.2067; p=0.0002). Finally, b-BAPN (49.48%) significantly reduced the fibrosis percent area compared to b-VEH (72.14%; p=0.0016) and control AVFs (74.5%; p=0.0007). Conclusion: Periadventitial LOX inhibition prevents AVF postoperative fibrosis and promotes adaptive vascular remodeling.

AbstractAlthough impaired keratinocyte migration is a recognized hallmark of chronic wounds, the molecular mechanisms underpinning impaired cell movement are poorly understood. Here, we demonstrate that both diabetic foot ulcers (DFUs) and venous leg ulcers (VLUs) exhibit global deregulation of cytoskeletal organization in genomic comparison to normal skin and acute wounds. Interestingly, we found that DFUs and VLUs exhibited downregulation of ArhGAP35, which serves both as an inactivator of RhoA and as a glucocorticoid repressor. Since chronic wounds exhibit elevated levels of cortisol and caveolin-1 (Cav1), we posited that observed elevation of Cav1 expression may contribute to impaired actin-cytoskeletal signaling, manifesting in aberrant keratinocyte migration. We showed that Cav1 indeed antagonizes ArhGAP35, resulting in increased activation of RhoA and diminished activation of Cdc42, which can be rescued by Cav1 disruption. Furthermore, we demonstrate that both inducible keratinocyte specific Cav1 knockout mice, and MβCD treated diabetic mice, exhibit accelerated wound closure. Taken together, our findings provide a previously unreported mechanism by which Cav1-mediated cytoskeletal organization prevents wound closure in patients with chronic wounds.

The meniscus is crucial in maintaining the knee function and protecting the joint from secondary pathologies, including osteoarthritis. Although most of the mechanical properties of human menisci have been characterized, to our knowledge, its dynamic shear properties have never been reported. Moreover, little is known about meniscal shear properties in relation to tissue structure and composition. This is crucial to understand mechanisms of meniscal injury, as well as, in regenerative medicine, for the design and development of tissue engineered scaffolds mimicking the native tissue. Hence, the objective of this study was to characterize the dynamic and equilibrium shear properties of human meniscus in relation to its anisotropy and composition. Specimens were prepared from the axial and the circumferential anatomical planes of medial and lateral menisci. Frequency sweeps and stress relaxation tests yielded storage (G′) and loss moduli (G″), and equilibrium shear modulus (G). Correlations of moduli with water, glycosaminoglycans (GAGs), and collagen content were investigated. The meniscus exhibited viscoelastic behavior. Dynamic shear properties were related to tissue composition: negative correlations were found between G′, G″ and G, and meniscal water content; positive correlations were found for G′ and G″ with GAG and collagen (only in circumferential samples). Circumferential samples, with collagen fibers orthogonal to the shear plane, exhibited superior dynamic mechanical properties, with G′ ~70 kPa and G″ ~10 kPa, compared to those of the axial plane ~15 kPa and ~1 kPa, respectively. Fiber orientation did not affect the values of G, which ranged from ~50 to ~100 kPa.