Recent advances in harnessing biological macromolecules for wound management: A review.

Wound dressings (WDs) are an essential component of wound management and serve as an artificial barrier to isolate the injured site from the external environment, thereby helping to prevent exogenous infections and supporting healing. However, maintaining a moist wound environment, providing protection from infection, good biocompatibility, and allowing for gas exchange, remain a challenge in device design. Functional wound dressings (FWDs) prepared from hybrid biological macromolecule-based materials can enhance efficacy of these systems for skin wound management. This review aims to provide an overview of the state-of-the-art FWDs within the field of wound management, with a specific focus on hybrid biomaterials, techniques, and applications developed over the past five years. In addition, we highlight the incorporation of biological macromolecules in WDs, the emergence of smart WDs, and discuss the existing challenges and future prospects for the development of advanced WDs.

Reduced Monocyte and Neutrophil Infiltration and Activation by P-Selectin/CD62P Inhibition Enhances Thrombus Resolution in Mice.

BACKGROUND: Venous thromboembolism is a major health problem. After thrombus formation, its resolution is essential to re-establish blood flow, which is crucially mediated by infiltrating neutrophils and monocytes in concert with activated platelets and endothelial cells. Thus, we aimed to modulate leukocyte function during thrombus resolution post-thrombus formation by blocking P-selectin/CD62P-mediated cell interactions. METHODS: Thrombosis was induced by inferior vena cava stenosis through ligation in mice. After 1 day, a P-selectin-blocking antibody or isotype control was administered and thrombus composition and resolution were analyzed. RESULTS: Localizing neutrophils and macrophages in thrombotic lesions of wild-type mice revealed that these cells enter the thrombus and vessel wall from the caudal end. Neutrophils were predominantly present 1 day and monocytes/macrophages 3 days after vessel ligation. Blocking P-selectin reduced circulating platelet-neutrophil and platelet-Ly6Chigh monocyte aggregates near the thrombus, and diminished neutrophils and Ly6Chigh macrophages in the cranial thrombus part compared with isotype-treated controls. Depletion of neutrophils 1 day after thrombus initiation did not phenocopy P-selectin inhibition but led to larger thrombi compared with untreated controls. In vitro, P-selectin enhanced human leukocyte function as P-selectin-coated beads increased reactive oxygen species production by neutrophils and tissue factor expression of classical monocytes. Accordingly, P-selectin inhibition reduced oxidative burst in the thrombus and tissue factor expression in the adjacent vessel wall. Moreover, blocking P-selectin reduced thrombus density determined by scanning electron microscopy and increased urokinase-type plasminogen activator levels in the thrombus, which accelerated caudal fibrin degradation from day 3 to day 14. This accelerated thrombus resolution as thrombus volume declined more rapidly after blocking P-selectin. CONCLUSIONS: Inhibition of P-selectin-dependent activation of monocytes and neutrophils accelerates venous thrombosis resolution due to reduced infiltration and activation of innate immune cells at the site of thrombus formation, which prevents early thrombus stabilization and facilitates fibrinolysis.

Comparison of Decellularized Human Dermal Scaffolds versus Bovine Collagen/Elastin Matrices for Engineering of Soft-Tissue Flaps.

BACKGROUND: Free flap-based soft-tissue reconstruction comes at the price of donor-site morbidity. The arteriovenous loop (AVL) technique can overcome this issue by allowing for the de novo generation of axially vascularized soft-tissue flaps from vein grafts embedded into different matrices. Application of the AVL technique has been limited by insufficient long-term volume retention and poor tissue stability. The authors investigated the suitability of a novel human dermal scaffold to improve volume retention and tissue stability. METHODS: AVLs were created in 28 immunocompetent rats and embedded in either decellularized human dermal scaffolds (experimental group, n = 14) (Epiflex) or bovine collagen/elastin matrices (control group, n = 14) (MatriDerm) in subcutaneous polytetrafluoroethylene chambers. The weight and volume of engineered tissues, the extent of angiogenesis, and the proportion of proliferating cells were compared between groups on postoperative days (PODs) 21 and 28 by means of immunohistochemistry and micro-computed tomography. RESULTS: On POD 28, both groups displayed homogeneous microvascular networks on histopathology and micro-computed tomography. Mean microvessel counts and surface areas and the percentage of proliferating cells did not differ between the groups. However, the experimental human scaffold group displayed significantly smaller volume loss and significantly less tissue degradation compared with bovine matrix controls (volume retention, 102% ± 5% versus 27% ± 7% on POD 21, and 79% ± 12% versus 12% ± 7% on POD 28, respectively; P < 0.0001). CONCLUSION: Compared with bovine matrices, decellularized human scaffolds allow for superior volume retention and tissue stability of de novo engineered soft-tissue AVL flaps in rats. CLINICAL RELEVANCE STATEMENT: AVLs allow for the de novo generation of vascularized soft-tissue flaps. However, insufficient long-term volume retention is still an issue. The authors' study shows that decellularized human matrices guarantee superior volume stability of de novo grown soft-tissue flaps in rats.

Medical 3D printing with polyjet technology: effect of material type and printing orientation on printability, surface structure and cytotoxicity.

Due to its high printing resolution and ability to print multiple materials simultaneously, inkjet technology has found wide application in medicine. However, the biological safety of 3D-printed objects is not always guaranteed due to residues of uncured resins or support materials and must therefore be verified. The aim of this study was to evaluate the quality of standard assessment methods for determining the quality and properties of polyjet-printed scaffolds in terms of their dimensional accuracy, surface topography, and cytotoxic potential.Standardized 3D-printed samples were produced in two printing orientations (horizontal or vertical). Printing accuracy and surface roughness was assessed by size measurements, VR-5200 3D optical profilometer dimensional analysis, and scanning electron microscopy. Cytotoxicity tests were performed with a representative cell line (L929) in a comparative laboratory study. Individual experiments were performed with primary cells from clinically relevant tissues and with a Toxdent cytotoxicity assay.Dimensional measurements of printed discs indicated high print accuracy and reproducibility. Print accuracy was highest when specimens were printed in horizontal direction. In all cytotoxicity tests, the estimated mean cell viability was well above 70% (p < 0.0001) regardless of material and printing direction, confirming the low cytotoxicity of the final 3D-printed objects.

Biodegradable, Self-Reinforcing Vascular Grafts for In Situ Tissue Engineering Approaches.

Clinically available small-diameter synthetic vascular grafts (SDVGs) have unsatisfactory patency rates due to impaired graft healing. Therefore, autologous implants are still the gold standard for small vessel replacement. Bioresorbable SDVGs may be an alternative, but many polymers have inadequate biomechanical properties that lead to graft failure. To overcome these limitations, a new biodegradable SDVG is developed to ensure safe use until adequate new tissue is formed. SDVGs are electrospun using a polymer blend composed of thermoplastic polyurethane (TPU) and a new self-reinforcing TP(U-urea) (TPUU). Biocompatibility is tested in vitro by cell seeding and hemocompatibility tests. In vivo performance is evaluated in rats over a period for up to six months. Autologous rat aortic implants serve as a control group. Scanning electron microscopy, micro-computed tomography (µCT), histology, and gene expression analyses are applied. TPU/TPUU grafts show significant improvement of biomechanical properties after water incubation and exhibit excellent cyto- and hemocompatibility. All grafts remain patent, and biomechanical properties are sufficient despite wall thinning. No inflammation, aneurysms, intimal hyperplasia, or thrombus formation are observed. Evaluation of graft healing shows similar gene expression profiles of TPU/TPUU and autologous conduits. These new biodegradable, self-reinforcing SDVGs may be promising candidates for clinical use in the future.

Silk Fibroin/Wool Keratin Composite Scaffold with Hierarchical Fibrous and Porous Structure.

The present study describes a silk microfiber reinforced meniscus scaffold (SMRMS) with hierarchical fibrous and porous structure made from silk fibroin (SF) and wool keratin (WK) using electrospinning and freeze-drying technology. This study focuses on the morphology, secondary structure, mechanical properties, and water absorption properties of the scaffold. The cytotoxicity and biocompatibility of SMRMS are assessed in vivo and in vitro. The scaffold shows hierarchical fibrous and porous structure, hierarchical pore size distribution (ranges from 50 to 650 µm), robust mechanical properties (compression strength can reach at 2.8 MPa), and stable biodegradability. A positive growth condition revealed by in vitro cytotoxicity testing indicates that the scaffold is not hazardous to cells. In vivo assessments of biocompatibility reveal that only a mild inflammatory reaction is present in implanted rat tissue. Meniscal scaffold made of SF/WK composite shows a potential application prospect in the meniscal repair engineering field with its development.

Woven Vascular Stent-Grafts with Surface Modification of Silk Fibroin-Based Paclitaxel/Metformin Microspheres.

In-stent restenosis caused by tumor ingrowth increases the risk of secondary surgery for patients with abdominal aortic aneurysms (AAA) because conventional vascular stent grafts suffer from mechanical fatigue, thrombosis, and endothelial hyperplasia. For that, we report a woven vascular stent-graft with robust mechanical properties, biocompatibility, and drug delivery functions to inhibit thrombosis and the growth of AAA. Paclitaxel (PTX)/metformin (MET)-loaded silk fibroin (SF) microspheres were self-assembly synthesized by emulsification-precipitation technology and layer-by-layer coated on the surface of a woven stent via electrostatic bonding. The woven vascular stent-graft before and after coating drug-loaded membranes were characterized and analyzed systematically. The results show that small-sized drug-loaded microspheres increased the specific surface area and promoted the dissolution/release of drugs. The stent-grafts with drug-loaded membranes exhibited a slow drug-release profile more for than 70 h and low water permeability at 158.33 ± 17.56 mL/cm2·min. The combination of PTX and MET inhibited the growth of human umbilical vein endothelial cells. Therefore, it was possible to generate dual-drug-loaded woven vascular stent-grafts to achieve the more effective treatment of AAA.

Chorion-derived extracellular matrix hydrogel and fibronectin surface coatings show similar beneficial effects on endothelialization of expanded polytetrafluorethylene vascular grafts.

The endothelium plays an important regulatory role for cardiovascular homeostasis. Rapid endothelialization of small diameter vascular grafts (SDVGs) is crucial to ensure long-term patency. Here, we assessed a human placental chorionic extracellular matrix hydrogel (hpcECM-gel) as coating material and compared it to human fibronectin in-vitro. hpcECM-gels were produced from placental chorion by decellularization and enzymatic digestion. Human umbilical vein endothelial cells (HUVECs) were seeded to non-, fibronectin- or hpcECM-gel-coated expanded polytetrafluorethylene (ePTFE) SDVGs. Coating efficiency as well as endothelial cell proliferation, migration and adhesion studies on grafts were performed. hpcECM-gel depicted high collagen and glycosaminoglycan content and neglectable DNA amounts. Laminin and fibronectin were both retained in the hpcECM-gel after the decellularization process. HUVEC as well as endothelial progenitor cell attachment were both significantly enhanced on hpcECM-gel coated grafts. HUVECs seeded to hpcECM-gel depicted significantly higher platelet endothelial cell adhesion molecule-1 (PECAM-1) expression in the perinuclear region. Cell retention to flow was enhanced on fibronectin and hpcECM-gel coated grafts. Since hpcECM-gel induced a significantly higher endothelial cell adhesion to ePTFE than fibronectin, it represents a possible alternative for SDVG modification to improve endothelialization.

Graphene Oxide Coating Improves the Mechanical and Biological Properties of Decellularized Umbilical Cord Arteries.

The lack of small-diameter vascular grafts (inner diameter <5 mm) to substitute autologous grafts in arterial bypass surgeries has a massive impact on the prognosis and progression of cardiovascular diseases, the leading cause of death globally. Decellularized arteries from different sources have been proposed as an alternative, but their poor mechanical performance and high collagen exposure, which promotes platelet and bacteria adhesion, limit their successful application. In this study, these limitations were surpassed for decellularized umbilical cord arteries through the coating of their lumen with graphene oxide (GO). Placental and umbilical cord arteries were decellularized and perfused with a suspension of GO (C/O ratio 2:1) with ∼1.5 µm lateral size. A homogeneous GO coating that completely covered the collagen fibers was obtained for both arteries, with improvement of mechanical properties being achieved for umbilical cord decellularized arteries. GO coating increased the maximum force in 27%, the burst pressure in 29%, the strain in 25%, and the compliance in 10%, compared to umbilical cord decellularized arteries. The achieved theoretical burst pressure (1960 mmHg) and compliance (13.9%/100 mmHg) are similar to the human saphenous vein and mammary artery, respectively, which are used nowadays as the gold standard in coronary and peripheral artery bypass surgeries. Furthermore, and very importantly, coatings with GO did not compromise the endothelial cell adhesion but decreased platelet and bacteria adhesion to decellularized arteries, which will impact on the prevention of thrombosis and infection, until full re-endothetialization is achieved. Overall, our results reveal that GO coating has an effective role in the improvement of decellularized umbilical cord artery performance, which is a huge step toward their application as a small-diameter vascular graft.

S-nitroso human serum albumin as a nitric oxide donor in drug-eluting vascular grafts: Biofunctionality and preclinical evaluation.

Currently available synthetic small diameter vascular grafts reveal low patency rates due to thrombosis and intimal hyperplasia. Biofunctionalized grafts releasing nitric oxide (NO) in situ may overcome these limitations. In this study, a drug-eluting vascular graft was designed by blending polycaprolactone (PCL) with S-nitroso-human-serum-albumin (S-NO-HSA), a nitric oxide donor with prolonged half-life. PCL-S-NO-HSA grafts and patches were fabricated via electrospinning. The fabrication process was optimized. Patches were characterized in vitro for their morphology, drug release, biomechanics, inflammatory effects, cell proliferation, and expression of adhesion molecules. The selected optimized formulation (8%PCL-S-NO-HSA) had superior mechanical/morphological properties with high protein content revealing extended NO release (for 28 days). 8%PCL-S-NO-HSA patches significantly promoted endothelial cell proliferation while limiting smooth muscle cell proliferation. Expression of adhesion molecules (ICAM-1, VCAM-1) and pro-inflammatory macrophage/cytokine markers (CD80, IL-1α, TNF-α) was significantly reduced. 8%PCL-S-NO-HSA patches had superior immunomodulatory properties by up-regulating anti-inflammatory cytokines (IL-10) and M2 macrophage marker (CD163) at final time points. Grafts were further evaluated in a small rodent model as aortic implants up to 12 weeks. Grafts were assessed by magnetic resonance imaging angiography (MRI) in vivo and after retrieval by histology. All grafts remained 100 % patent with no signs of thrombosis or calcification. 8%PCL-S-NO-HSA vascular grafts supported rapid endothelialization, whereas smooth muscle cell proliferation was hampered in earlier phases. This study indicates that 8%PCL-S-NO-HSA grafts effectively support long-term in situ release of bioactive NO. The beneficial effects observed can be promising features for long-term success of small diameter vascular grafts. STATEMENT OF SIGNIFICANCE: Despite extensive research in the field of small diameter vascular graft replacement, there is still no appropriate substitute to autografts yet. Various limitations are associated with currently available synthetic vascular grafts such as thrombogenicity and intimal hyperplasia. Therefore, developing new generations of such conduits has become a major focus of research. One of the most significant signaling molecules that are involved in homeostasis of the vascular system is nitric oxide. The new designed nitric-oxide eluting vascular grafts described in this study induce rapid surface endothelialization and late migration of SMCs into the graft wall. These beneficial effects have potential to improve current limitations of small diameter vascular grafts.

Graphene-based materials: the key for the successful application of pHEMA as a blood-contacting device.

Thrombosis and infection are the leading causes of blood-contacting device (BCD) failure, mainly due to the poor performance of existing biomaterials. Poly(2-hydroxyethyl methacrylate) (pHEMA) has excellent hemocompatibility but the weak mechanical properties impair its use as a bulk material for BCD. As such, pHEMA has been explored as a coating, despite the instability and difficulty of attachment to the underlying polymer compromise its success. This work describes the hydrogel composites made of pHEMA and graphene-based materials (GBM) that meet the biological and mechanical requirements for a stand-alone BCD. Five GBM differing in thickness, oxidation degree, and lateral size were incorporated in pHEMA, revealing that only oxidized-GBM can reinforce pHEMA. pHEMA/oxidized-GBM composites are cytocompatible and prevent the adhesion of endothelial cells, blood platelets, and bacteria (S. aureus), thus maintaining pHEMA's anti-adhesive properties. As a proof of concept, the thrombogenicity of the tubular prototypes of the best formulation (pHEMA/Graphene oxide (GO)) was evaluated in vivo, using a porcine arteriovenous-shunt model. pHEMA/GO conduits withstand the blood pressure and exhibit negligible adhesion of blood components, revealing better hemocompatibility than ePTFE, a commercial material for vascular access. Our findings reveal pHEMA/GO, a synthetic and off-the-shelf hydrogel, as a preeminent material for the design of blood-contacting devices that prevent thrombosis and bacterial adhesion.

Riboflavin-mediated photooxidation to improve the characteristics of decellularized human arterial small diameter vascular grafts.

Vascular grafts with a diameter of less than 6 mm are made from a variety of materials and techniques to provide alternatives to autologous vascular grafts. Decellularized materials have been proposed as a possible approach to create extracellular matrix (ECM) vascular prostheses as they are naturally derived and inherently support various cell functions. However, these desirable graft characteristics may be limited by alterations of the ECM during the decellularization process leading to decreased biomechanical properties and hemocompatibility. In this study, arteries from the human placenta chorion were decellularized using two distinct detergents (Triton X-100 or SDS), which differently affect ECM ultrastructure. To overcome biomechanical strength loss and collagen fiber exposure after decellularization, riboflavin-mediated UV (RUV) crosslinking was used to uniformly crosslink the collagenous ECM of the grafts. Graft characteristics and biocompatibility with and without RUV crosslinking were studied in vitro and in vivo. RUV-crosslinked ECM grafts showed significantly improved mechanical strength and smoothening of the luminal graft surfaces. Cell seeding using human endothelial cells revealed no cytotoxic effects of the RUV treatment. Short-term aortic implants in rats showed cell migration and differentiation of host cells. Functional graft remodeling was evident in all grafts. Thus, RUV crosslinking is a preferable tool to improve graft characteristics of decellularized matrix conduits.

Acellular vascular matrix grafts from human placenta chorion: Impact of ECM preservation on graft characteristics, protein composition and in vivo performance.

Small diameter vascular grafts from human placenta, decellularized with either Triton X-100 (Triton) or SDS and crosslinked with heparin were constructed and characterized. Graft biochemical properties, residual DNA, and protein composition were evaluated to compare the effect of the two detergents on graft matrix composition and structural alterations. Biocompatibility was tested in vitro by culturing the grafts with primary human macrophages and in vivo by subcutaneous implantation of graft conduits (n = 7 per group) into the flanks of nude rats. Subsequently, graft performance was evaluated using an aortic implantation model in Sprague Dawley rats (one month, n = 14). In situ graft imaging was performed using MRI angiography. Retrieved specimens were analyzed by electromyography, scanning electron microscopy, histology and immunohistochemistry to evaluate cell migration and the degree of functional tissue remodeling. Both decellularization methods resulted in grafts of excellent biocompatibility in vitro and in vivo, with low immunogenic potential. Proteomic data revealed removal of cytoplasmic proteins with relative enrichment of ECM proteins in decelluarized specimens of both groups. Noteworthy, LC-Mass Spectrometry analysis revealed that 16 proteins were exclusively preserved in Triton decellularized specimens in comparison to SDS-treated specimens. Aortic grafts showed high patency rates, no signs of thrombus formation, aneurysms or rupture. Conduits of both groups revealed tissue-specific cell migration indicative of functional remodeling. This study strongly suggests that decellularized allogenic grafts from the human placenta have the potential to be used as vascular replacement materials. Both detergents produced grafts with low residual immunogenicity and appropriate mechanical properties. Observed differences in graft characteristics due to preservation method had no impact on successful in vivo performance in the rodent model.

Mechanisms of vasculogenesis in 3D fibrin matrices mediated by the interaction of adipose-derived stem cells and endothelial cells.

Vascularization of tissue-engineered constructs is essential to provide sufficient nutrient supply and hemostasis after implantation into target sites. Co-cultures of adipose-derived stem cells (ASC) with outgrowth endothelial cells (OEC) in fibrin gels were shown to provide an effective possibility to induce vasculogenesis in vitro. However, the mechanisms of the interaction between these two cell types remain unclear so far. The aim of this study was to evaluate differences of direct and indirect stimulation of ASC-induced vasculogenesis, the influence of ASC on network stabilization and molecular mechanisms involved in vascular structure formation. Endothelial cells (EC) were embedded in fibrin gels either containing non-coated or ASC-coated microcarrier beads as well as ASC alone. Moreover, EC-seeded constructs incubated with ASC-conditioned medium were used in addition to constructs with ASC seeded on top. Vascular network formation was visualized by green fluorescent protein expressing cells or immunostaining for CD31 and quantified. RT-qPCR of cells derived from co-cultures in fibrin was performed to evaluate changes in the expression of EC marker genes during the first week of culture. Moreover, angiogenesis-related protein levels were measured by performing angiogenesis proteome profiler arrays. The results demonstrate that proximity of endothelial cells and ASC is required for network formation and ASC stabilize EC networks by developing pericyte characteristics. We further showed that ASC induce controlled vessel growth by secreting pro-angiogenic and regulatory proteins. This study reveals angiogenic protein profiles involved in EC/ASC interactions in fibrin matrices and confirms the usability of OEC/ASC co-cultures for autologous vascular tissue engineering.