Optimization of chitosan-gelatin-based 3D-printed scaffolds for tissue engineering and drug delivery applications.

The combination of biocompatible materials and advanced three-dimensional (3D) additive manufacturing technologies holds great potential in the development of finely tuned complex scaffolds with reproducible macro- and micro-structural characteristics for biomedical applications, such as tissue engineering and drug delivery. In this study, biocompatible printable inks based on chitosan, collagen and gelatin were developed and 3D-printed with a pneumatic-based extrusion printer. The printability of various chitosan-gelatin (CS-Gel) hydrogel inks was assessed by evaluating the quality of the printed constructs. The inks required an extrusion pressure of 150 ± 40 MPa with G22 and G25 nozzles for optimal printing. Inks with low chitosan concentrations (<4% w/v) exhibited poor printability, while inks with 4 % w/v chitosan and 1 % w/v gelatin (CG) demonstrated satisfactory extrusion and printing quality. The addition of collagen (0.1 % w/v) to the optimized ink (CGC) did not compromise printability. Post-printing stabilization using KOH produced self-supporting scaffolds with consistent morphological integrity, while weaker bases like NaOH/EtOH and ammonia vapors resulted in lower pore sizes and reduced structural stability. Water evaporation studies showed that neutralized samples had slower evaporation rates due to the strong intermolecular interactions formed during the neutralization process, contributing to a stable crosslinked network. FTIR spectra confirmed the formation of polyelectrolyte complexes in the CS-Gel and CS-Gel-Collagen blends, further enhancing structural stability. Swelling tests indicated that neutralized constructs maintained stability in different pH environments, with KOH-treated samples exhibiting the lowest swelling ratios and the highest structural stability. After optimizing the ink composition, 10 wt% Levofloxacin was loaded in the constructs as a model antibiotic and it's in vitro release rate was quantified. Drug loading was approximately 4 % for both ink compositions GC and CGC. CG Levo released over 80 % of levofloxacin within the first hour, reaching full release in 24 h, indicating inadequate control, while CGK Levo exhibited slower initial release (55 % in 15 min) followed by stabilized release after 4 h, likely due to controlled diffusion from expanded constructs. These findings demonstrate that the developed hydrogel inks and optimized printing parameters can produce scaffolds suitable for tissue engineering applications. Finally, the cell compatibility of the 3D-printed constructs was confirmed with MTT assay on fibroblasts and the antimicrobial activity of the drug-loaded constructs was tested against E. coli and S. aureus, showing an increase of the bacteria free zone from 8 ± 0.4 mm of the control against E. coli up to 16.4 ± 0.37 mm in the presence of the KOH-treated CG Levo printed construct.

Knockout Genes in Bowel Anastomoses: A Systematic Review of Literature Outcomes.

BACKGROUND: The intestinal wound healing process is a complex event of three overlapping phases: exudative, proliferative, and remodeling. Although some mechanisms have been extensively described, the intestinal healing process is still not fully understood. There are some similarities but also some differences compared to other tissues. The aim of this systematic review was to summarize all studies with knockout (KO) experimental models in bowel anastomoses, underline any recent knowledge, and clarify further the cellular and molecular mechanisms of the intestinal healing process. A systematic review protocol was performed. MATERIALS AND METHODS: Medline, EMBASE, and Scopus were comprehensively searched. RESULTS: a total of eight studies were included. The silenced genes included interleukin-10, the four-and-one-half LIM domain-containing protein 2 (FHL2), cyclooxygenase-2 (COX-2), annexin A1 (ANXA-1), thrombin-activatable fibrinolysis inhibitor (TAFI), and heparin-binding epidermal growth factor (HB-EGF) gene. Surgically, an end-to-end bowel anastomosis was performed in the majority of the studies. Increased inflammatory cell infiltration in the anastomotic site was found in IL-10-, annexin-A1-, and TAFI-deficient mice compared to controls. COX-1 deficiency showed decreased angiogenesis at the anastomotic site. Administration of prostaglandin E2 in COX-2-deficient mice partially improved anastomotic leak rates, while treatment of ANXA1 KO mice with Ac2-26 nanoparticles reduced colitis activity and increased weight recovery following surgery. CONCLUSIONS: our findings provide new insights into improving intestinal wound healing by amplifying the aforementioned genes using appropriate gene therapies. Further research is required to clarify further the cellular and micromolecular mechanisms of intestinal healing.

3D-Printed Chitosan-Based Hydrogels Loaded with Levofloxacin for Tissue Engineering Applications.

Herein, we demonstrate the feasibility of a three-dimensional printed chitosan (CS)-poly(vinyl alcohol) (PVA)-gelatin (Gel) hydrogel incorporating the antimicrobial drug levofloxacin (LEV) as a potential tissue engineering scaffold. Hydrogels were prepared by physically cross-linking the polymers, and the printability of the prepared hydrogels was determined. The hydrogel with 3% w/v of CS, 3% w/v of PVA, and 2% w/v of Gel presented the best printability, producing smooth and uniform scaffolds. The integrity of 3D-printed scaffolds was improved via a neutralization process since after testing three different neutralized agents, i.e., NH3 vapors, EtOH/NaOH, and KOH solutions. It was proved that the CS/PVA/Gel hydrogel was formed by hydrogen bonds and remained amorphous in the 3D-printed structures. Drug loading studies confirmed the successful incorporation of LEV, and its in vitro release continued for 48 h. The cytotoxicity/cytocompatibility tests showed that all prepared scaffolds were cytocompatible.

Stem Cell Therapies for Epidermolysis Bullosa Treatment.

Epidermolysis bullosa (EB) includes a group of rare skin diseases characterized by skin fragility with bullous formation in the skin, in response to minor mechanical injury, as well as varying degrees of involvement of the mucous membranes of the internal organs. EB is classified into simplex, junctional, dystrophic and mixed. The impact of the disease on patients is both physical and psychological, with the result that their quality of life is constantly affected. Unfortunately, there are still no approved treatments available to confront the disease, and treatment focuses on improving the symptoms with topical treatments to avoid complications and other infections. Stem cells are undifferentiated cells capable of producing, maintaining and replacing terminally differentiated cells and tissues. Stem cells can be isolated from embryonic or adult tissues, including skin, but are also produced by genetic reprogramming of differentiated cells. Preclinical and clinical research has recently greatly improved stem cell therapy, making it a promising treatment option for various diseases in which current medical treatments fail to cure, prevent progression, or alleviate symptoms. So far, stem cells from different sources, mainly hematopoietic and mesenchymal, autologous or heterologous have been used for the treatment of the most severe forms of the disease each one of them with some beneficial effects. However, the mechanisms through which stem cells exert their beneficial role are still unknown or incompletely understood and most importantly further research is required to evaluate the effectiveness and safety of these treatments. The transplantation of skin grafts to patients produced by gene-corrected autologous epidermal stem cells has been proved to be rather successful for the treatment of skin lesions in the long term in a limited number of patients. Nevertheless, these treatments do not address the internal epithelia-related complications manifested in patients with more severe forms.

Poly(Glycerol Succinate) as Coating Material for 1393 Bioactive Glass Porous Scaffolds for Tissue Engineering Applications.

BACKGROUND: Aliphatic polyesters are widely used for biomedical, pharmaceutical and environmental applications due to their high biodegradability and cost-effective production. Recently, star and hyperbranched polyesters based on glycerol and ω-carboxy fatty diacids have gained considerable interest. Succinic acid and bio-based diacids similar to glycerol are regarded as safe materials according to the US Food and Drug Administration (FDA). Bioactive glass scaffolds utilized in bone tissue engineering are relatively brittle materials. However, their mechanical properties can be improved by using polymer coatings that can further control their degradation rate, tailor their biocompatibility and enhance their performance. The purpose of this study is to explore a new biopolyester poly(glycerol succinate) (PGSuc) reinforced with mesoporous bioactive nanoparticles (MSNs) as a novel coating material to produce hybrid scaffolds for bone tissue engineering. METHODS: Bioactive glass scaffolds were coated with neat PGSuc, PGSuc loaded with dexamethasone sodium phosphate (DexSP) and PGSuc loaded with DexSP-laden MSNs. The physicochemical, mechanical and biological properties of the scaffolds were also evaluated. RESULTS: Preliminary data are provided showing that polymer coatings with and without MSNs improved the physicochemical properties of the 1393 bioactive glass scaffolds and increased the ALP activity and alizarin red staining, suggesting osteogenic differentiation potential when cultured with adipose-derived mesenchymal stem cells. CONCLUSIONS: PGSuc with incorporated MSNs coated onto 1393 bioactive glass scaffolds could be promising candidates in bone tissue engineering applications.

Altered Distribution and Expression of Syndecan-1 and -4 as an Additional Hallmark in Psoriasis.

Syndecans act as independent co-receptors to exert biological activities and their altered function is associated with many pathophysiological conditions. Here, syndecan-1 and -4 were examined in lesional skin of patients with psoriasis. Immunohistochemical staining confirmed altered syndecan-1 distribution and revealed absence of syndecan-4 expression in the epidermis. Fibronectin (FN)-known to influence inflammation and keratinocyte hyperproliferation via α5ß1 integrin in psoriasis-was also decreased. Syndecan-1 and -4 expression was analyzed in freshly isolated lesional psoriatic human keratinocytes (PHK) characterized based on their proliferation and differentiation properties. mRNA levels of syndecan-1 were similar between healthy and PHK, while syndecan-4 was significantly decreased. Cell growth and release of the pro-inflammatory Tumor Necrosis Factor-alpha (TNFα) were selectively and significantly induced in PHKs plated on FN. Results from co-culture of healthy keratinocytes and psoriatic fibroblasts led to the speculation that at least one factor released by fibroblasts down-regulate syndecan-1 expression in PHK plated on FN. To assay if biological treatments for psoriasis target keratinocyte proliferation, gelatin-based patches enriched with inteleukin (IL)-17α or TNFα blockers were prepared and tested using a full-thickness healthy epidermal model (Phenion®). Immunohistochemistry analysis showed that both blockers impacted the localisation of syndecan-1 within the refined epidermis. These results provide evidence that syndecans expression are modified in psoriasis, suggesting that they may represent markers of interest in this pathology.

Benefit of coupling heparin to crosslinked collagen I/III scaffolds for human dermal fibroblast subpopulations' tissue growth.

Currently, there is a lack of models representing the skin dermal heterogeneity for relevant research and skin engineering applications. This is the first study reporting production of dermal equivalents reproducing features of papillary and reticular dermal compartments. Inspired from our current knowledge on the architecture and composition differences between the papillary and reticular dermis, we evaluated different collagen-based porous materials to serve as scaffolds for the three-dimensional expansion of freshly isolated papillary and/or reticular fibroblasts. The scaffolds, composed of either collagen I or collagen I and III mixtures, were prepared by lyophilization. Pore size and hydrolytic stability were controlled by crosslinking with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) or EDC/NHS with covalently bound heparin. The evaluation of the resultant "papillary" and "reticular" dermal equivalents was based on the analysis of characteristic features of each dermal compartment, such as cell density and deposition of newly synthetized extracellular matrix components in histological sections. Crosslinking supported cell growth during dermal tissue formation independent on the fibroblast subpopulation. The presence of collagen III seemed to have some positive but non-specific effect only on the maintenance of the mechanical strength of the scaffolds during dermal formation. Histological analyses demonstrated a significant and specific effect of heparin on generating dermal equivalents reproducing the respective higher papillary than reticular cell densities and supporting distinct extracellular matrix components deposition (three to five times more carbohydrate material deposited by papillary fibroblasts in all scaffolds containing heparin, while higher collagen production was observed only in the presence of heparin).

Preliminary Evaluation of 3D Printed Chitosan/Pectin Constructs for Biomedical Applications.

In the present study, chitosan (CS) and pectin (PEC) were utilized for the preparation of 3D printable inks through pneumatic extrusion for biomedical applications. CS is a polysaccharide with beneficial properties; however, its printing behavior is not satisfying, rendering the addition of a thickening agent necessary, i.e., PEC. The influence of PEC in the prepared inks was assessed through rheological measurements, altering the viscosity of the inks to be suitable for 3D printing. 3D printing conditions were optimized and the effect of different drying procedures, along with the presence or absence of a gelating agent on the CS-PEC printed scaffolds were assessed. The mean pore size along with the average filament diameter were measured through SEM micrographs. Interactions among the characteristic groups of the two polymers were evident through FTIR spectra. Swelling and hydrolysis measurements confirmed the influence of gelation and drying procedure on the subsequent behavior of the scaffolds. Ascribed to the beneficial pore size and swelling behavior, fibroblasts were able to survive upon exposure to the ungelated scaffolds.

Preparation and Evaluation of Collagen-Based Patches as Curcumin Carriers.

Patients with psoriasis are dissatisfied with the standard pharmacological treatments, whether systemic or topical, with many of them showing interest in complementary and alternative medicine. Curcumin (Cur), a natural polyphenol derived from turmeric, has recently gained attention for skin-related diseases because of its proven anti-inflammatory action. However, topical treatment with Cur would be inadequate because of its hydrophobicity, instability, and low bioavailability. In addition, hyperkeratosis and lack of moisture in psoriatic skin result in low penetration that would prevent actives from permeating the stratum corneum. In this work, a polymer-based formulation of Cur for the topical treatment of psoriasis is reported. To improve the physicochemical stability of Cur, it was first encapsulated in chitosan nanoparticles. The Cur-loaded nanoparticles were incorporated in a hydrophilic, biocompatible collagen-based patch. The nanoparticle-containing porous collagen patches were then chemically cross-linked. Morphology, chemical interactions, swelling ratio, enzymatic hydrolysis, and Cur release from the patches were evaluated. All patches showed excellent swelling ratio, up to ~1500%, and after cross-linking, the pore size decreased, and their hydrolysis rates decelerated. The in vitro release of Cur was sustained with an initial burst release, reaching 55% after 24 h. Cur within the scaffolds imparted a proliferation inhibitory effect on psoriatic human keratinocytes in vitro.

A novel mechanism in wound healing: Laminin 332 drives MMP9/14 activity by recruiting syndecan-1 and CD44.

Re-epithelialization describes the resurfacing of a skin wound with new epithelium. In response to various stimuli including that of growth factors, cytokines and extracellular matrix (ECM), wound edge epidermal keratinocytes undergo cytoskeleton rearrangements compatible with their motile behavior and develop protrusive adhesion contacts. Matrix metalloproteinases (MMP) expression is crucial for proper cell movement and ECM remodeling; however, their deposition mechanism is unknown in keratinocytes. Here, we show that similar to cytokine IL-1ß, the precursor laminin 332 pro-migratory fragment G45 induces expression of the MMP-9 pro-enzyme, which together with MMP-14, further exerts its proteolytic activity within epithelial podosomes. This event strictly depends on the expression of the proteoglycan receptor syndecan-1 that was found in a ring surrounding the podosome core, co-localised with CD44. Our findings uncover that by directly recruiting both syndecan-1 and CD44, the laminin-332 G45 domain plays a major role in regulating mechanisms underlying keratinocyte / ECM remodeling during wound repair.

Chitosan dressings containing inorganic additives and levofloxacin as potential wound care products with enhanced hemostatic properties.

Despite the progress in the development of hemostatic products, efficient treatment solutions to control hemorrhage upon wounding are still necessary. Chitosan (CS) is a natural hydrogel-forming polysaccharide, easy to modify for specific applications. Inorganic compounds in turn possess documented hemostatic properties. In this study, innovative hemostatic products based on CS, containing the inorganic additives aluminum chloride, aluminum sulfate hydrate or iron(III) sulfate and the antibiotic Levofloxacin were prepared, and their potential use as hemostatic materials was investigated. Structural characteristics, physical state and drug loading/release properties were examined. Strong interactions developed between CS and the additives, the pore size in the resulting products was affected, swelling increased up to 2500% and the stability of the wound dressings improved. The crystallinity of Levofloxacin reduced, and its release was immediate. The materials showed biocompatibility upon contact with cultured keratinocytes, hemocompatibility and hemostatic efficacy in vitro and in vivo.

Isolation of a novel embryonic stem cell cord blood-derived population with in vitro hematopoietic capacity in the presence of Wharton's jelly-derived mesenchymal stromal cells.

BACKGROUND: Recent studies highlight the existence of a population of cord blood (CB)-derived stem cells that bare embryonic features (very small embryonic-like stem cells [VSELs]) as the most primitive CB-stem cell population. In the present study, we present for the first time a novel and high purity isolation method of VSELs with in vitro hematopoietic capacity in the presence of Wharton's jelly-derived mesenchymal stromal cells (WJ-MSCs). METHODS: The experimental procedure includes isolation upon gradually increased centrifugation spins and chemotaxis to Stromal cell-derived factor 1a (SDF-1a). Τhis cell population is characterized with flow cytometry, alkaline phosphatase (ALP) staining and qRT-PCR. The functional role of the isolated VSELs is assayed following co-culture with WJ-MSCs or bone marrow-derived mesenchymal stromal cells (BM-MSCs), whereas the stimulation of the quiescent VSEL population is verified via cell cycle analysis. The in vitro hematopoietic capacity is evaluated in methylcellulose cultures and also through induction of erythroid differentiation. RESULTS: The final isolated subpopulation is characterized as a small-sized CD45/Lineage-/CXCR4+/CD133+/SSEA-4+cell population, positive in ALP staining and overexpressing the Oct3/4, Nanog and Sox-2 transcription factors. Upon the co-culture with MSCs, a stimulation of the quiescent VSEL population is observed. An impressive increase in the co-expression of the CD34+/CD45+ markers is observed following the co-culture with the WJ-MSCs, which is confirmed by the intense clonogenic ability suggesting in vitro differentiation toward all of the hematopoietic cell lineages and successful differentiation toward erythrocytes. DISCUSSION: Conclusively, we propose a novel, rapid and rather simplified isolation method of CB-VSELs, capable of in vitro hematopoiesis.

Perlecan expression influences the keratin 15-positive cell population fate in the epidermis of aging skin.

The epidermis is continuously renewed by stem cell proliferation and differentiation. Basal keratinocytes append the dermal-epidermal junction, a cell surface-associated, extracellular matrix that provides structural support and influences their behaviour. It consists of laminins, type IV collagen, nidogens, and perlecan, which are necessary for tissue organization and structural integrity. Perlecan is a heparan sulfate proteoglycan known to be involved in keratinocyte survival and differentiation. Aging affects the dermal epidermal junction resulting in decreased contact with keratinocytes, thus impacting epidermal renewal and homeostasis. We found that perlecan expression decreased during chronological skin aging. Our in vitro studies revealed reduced perlecan transcript levels in aged keratinocytes. The production of in vitro skin models revealed that aged keratinocytes formed a thin and poorly organized epidermis. Supplementing these models with purified perlecan reversed the phenomenon allowing restoration of a well-differentiated multi-layered epithelium. Perlecan down-regulation in cultured keratinocytes caused depletion of the cell population that expressed keratin 15. This phenomenon depended on the perlecan heparan sulphate moieties, which suggested the involvement of a growth factor. Finally, we found defects in keratin 15 expression in the epidermis of aging skin. This study highlighted a new role for perlecan in maintaining the self-renewal capacity of basal keratinocytes.

How do epidermal matrix metalloproteinases support re-epithelialization during skin healing?

Epithelialization of normal wounds occurs by an orderly series of events whereby keratinocytes migrate, proliferate, and differentiate to restore the epidermal barrier function. Keratinocyte migration is one of the earliest and crucial events determining the efficiency of the overall wound repair process. In response to various stimuli including that of growth factors, cytokines and the extracellular matrix, activated keratinocytes at the edges of the wound undergo dramatic morphological changes according to their migratory behaviour through development of protrusive adhesion contacts and cytoskeleton rearrangements. These phenotypic changes are accompanied by the upregulated expression of a new set of genes, among which are adhesion receptors and specific matrix degrading enzymes named matrix metalloproteinases (MMPs). The tightly regulated spatial and temporal MMP expression is crucial for proper re-epithelialization. These multi-domain zinc-containing endopeptidases are necessary for the proper completion of multiple features of epidermal regeneration. They play a key role in the migration process by controlling the repeated cycles of keratinocyte attachment and retraction. In the meantime, they process, degrade or remodel the extracellular matrix often producing cleavages in a gain-of-function manner.

Aprotinin reduces oxidative stress induced by pneumoperitoneum in rats.

BACKGROUND: Ischemia-reperfusion injury induced by pneumoperitoneum is a well-studied entity, which increases oxidative stress during laparoscopic operations. The reported anti-inflammatory action of aprotinin was measured in a pneumoperitoneum model in rats for the first time in this study. MATERIALS AND METHODS: A total of 60 male Albino Wistar rats were used in our protocol. Prolonged pneumoperitoneum (4 h) was applied, causing splanchnic ischemia and a period of reperfusion with a duration of 60 or 180 min followed. Several cytokines and markers of oxidative stress were measured in liver, small intestine, and lungs to compare the aprotinin group with the control group. Tissue inflammation was also evaluated and compared between groups using a five-scaled histopathologic score. RESULTS: In aprotinin group values of biochemical markers (tumor necrosis factor α, interleukin 6, endothelin 1, C reactive protein, pro-oxidant-antioxidant balance, and carbonyl proteins) were lower in all tissues studied. Statistical significance was greater in liver and lungs (P < 0.05). Histopathologic examination revealed significant difference between control and aprotinin groups in all tissues examined. Aprotinin groups showed mild to moderate lesions, while in control groups severe to very severe inflammation was present. Aprotinin subgroup with prolonged reperfusion period (180 min) showed milder lesions in all tissues than the rest of the groups. CONCLUSIONS: Aprotinin reduced inflammatory response and oxidative stress induced by pneumoperitoneum in liver, small intestine, and lungs.

Asymmetric dimethyloarginin (ADMA) concentration in the aqueous humor of patients with exfoliation syndrome or exfoliative glaucoma.

BACKGROUND: Asymmetric dimethyloarginin or dimethylarginin (ADMA) is a marker and maker of oxidative stress. It is elevated in several pathological conditions, such as hyperhomocysteinemia and endothelial dysfunction, which have also been reported in patients with exfoliation syndrome (XFS), or exfoliative glaucoma (XFG). We evaluated ADMA levels in the aqueous humor of XFS and XFG patients. METHODS: This study included 48 aqueous samples; 16 from cataract patients with XFS, 16 from cataract patients with XFG and 16 from age-matched cataract control patients. ADMA levels were determined employing a commercial ELISA kit. RESULTS: ADMA concentration was significantly greater in XFG patients (0.398 ± 0.026 µM) compared to either XFS (0.168 ± 0.028 µM; p < 0.0001) or normal cataract controls (0.245 ± 0.025 µM; p = 0.0002). In contrast, no significant difference was detected for ADMA levels in the aqueous of XFS patients as compared to normal controls (p = 0.0477). CONCLUSIONS: Aqueous humor ADMA concentration is significantly elevated in XFG patients supporting the view that oxidative stress plays a key role in XFG pathobiology. The lower level of this marker in XFS suggests that the development of XFG is a prerequisite for ADMA elevation.