Improving stability of human three dimensional skin equivalents using plasma surface treatment.

In developing three-dimensional (3D) human skin equivalents (HSEs), preventing dermis and epidermis layer distortion due to the contraction of hydrogels by fibroblasts is a challenging issue. Previously, a fabrication method of HSEs was tested using a modified solid scaffold or a hydrogel matrix in combination with the natural polymer coated onto the tissue culture surface, but the obtained HSEs exhibited skin layer contraction and loss of the skin integrity and barrier functions. In this study, we investigated the method of HSE fabrication that enhances the stability of the skin model by using surface plasma treatment. The results showed that plasma treatment of the tissue culture surface prevented dermal layer shrinkage of HSEs, in contrast to the HSE fabrication using fibronectin coating. The HSEs from plasma-treated surface showed significantly higher transepithelial electrical resistance compared to the fibronectin-coated model. They also expressed markers of epidermal differentiation (keratin 10, keratin 14 and loricrin), epidermal tight junctions (claudin 1 and zonula occludens-1), and extracellular matrix proteins (collagen IV), and exhibited morphological characteristics of the primary human skins. Taken together, the use of plasma surface treatment significantly improves the stability of 3D HSEs with well-defined dermis and epidermis layers and enhanced skin integrity and the barrier functions.

Anomalous mechanics of Zn2+-modified fibrin networks.

Fibrin is the main component of blood clots. The mechanical properties of fibrin are therefore of critical importance in successful hemostasis. One of the divalent cations released by platelets during hemostasis is Zn2+; however, its effect on the network structure of fibrin gels and on the resultant mechanical properties remains poorly understood. Here, by combining mechanical measurements with three-dimensional confocal microscopy imaging, we show that Zn2+ can tune the fibrin network structure and alter its mechanical properties. In the presence of Zn2+, fibrin protofibrils form large bundles that cause a coarsening of the fibrin network due to an increase in fiber diameter and reduction of the total fiber length. We further show that the protofibrils in these bundles are loosely coupled to one another, which results in a decrease of the elastic modulus with increasing Zn2+ concentrations. We explore the elastic properties of these networks at both low and high stress: At low stress, the elasticity originates from pulling the thermal slack out of the network, and this is consistent with the thermal bending of the fibers. By contrast, at high stress, the elasticity exhibits a common master curve consistent with the stretching of individual protofibrils. These results show that the mechanics of a fibrin network are closely correlated with its microscopic structure and inform our understanding of the structure and physical mechanisms leading to defective or excessive clot stiffness.

Construction of enzyme digested holes on hydrogel surface inspired by cell migration processes.

The construction of in vitro capillary network models for drug testing and toxicity evaluation has become a major challenge in the field of tissue engineering. Previously, we discovered a novel phenomenon of hole formation by endothelial cell migration on the surface of fibrin gels. Interestingly, the hole characteristics, such as depth and number, were strongly influenced by the gel stiffness, but the details of hole formation are not to be clarified. In this study, we tried to understand the effect of hydrogel stiffness on the hole formation by dropping collagenase solution onto the surface of the hydrogels because the endothelial cell migration was made possible by the metalloproteinases' digestion. We found that smaller hole structures were formed on stiffer fibrin gels, but larger ones were formed on softer fibrin gels after the hydrogel digestion of the collagenase. This is consistent with our previous results in experiments on hole structures formed by endothelial cells. Furthermore, deep and small hole structures were successfully obtained by optimizing the volume of collagenase solution and incubation time. This unique approach inspired by endothelial cell hole formation may provide new methods of fabricating hydrogels with opening hole structures.

Fibrin gel enhanced trilayer structure in cell-cultured constructs.

Efficient cell seeding and subsequent support from a substrate ensure optimal cell growth and neotissue development during tissue engineering, including heart valve tissue engineering. Fibrin gel as a cell carrier may provide high cell seeding efficiency and adhesion property, improved cellular interaction, and structural support to enhance cellular growth in trilayer polycaprolactone (PCL) substrates that mimic the structure of native heart valve leaflets. This cell carrier gel coupled with a trilayer PCL substrate may enable the production of native-like cell-cultured leaflet constructs suitable for heart valve tissue engineering. In this study, we seeded valvular interstitial cells onto trilayer PCL substrates with fibrin gel as a cell carrier and cultured them for 1 month in vitro to determine if this gel can improve cell proliferation and production of extracellular matrix within the trilayer cell-cultured constructs. We observed that the fibrin gel enhanced cellular proliferation, their vimentin expression, and collagen and glycosaminoglycan production, leading to improved structure and mechanical properties of the developing PCL cell-cultured constructs. Fibrin gel as a cell carrier significantly improved the orientations of the cells and their produced tissue materials within trilayer PCL substrates that mimic the structure of native heart valve leaflets and, thus, may be highly beneficial for developing functional tissue-engineered leaflet constructs.

In situ sprayed NIR-responsive, analgesic black phosphorus-based gel for diabetic ulcer treatment.

The treatment of diabetic ulcer (DU) remains a major clinical challenge due to the complex wound-healing milieu that features chronic wounds, impaired angiogenesis, persistent pain, bacterial infection, and exacerbated inflammation. A strategy that effectively targets all these issues has proven elusive. Herein, we use a smart black phosphorus (BP)-based gel with the characteristics of rapid formation and near-infrared light (NIR) responsiveness to address these problems. The in situ sprayed BP-based gel could act as 1) a temporary, biomimetic "skin" to temporarily shield the tissue from the external environment and accelerate chronic wound healing by promoting the proliferation of endothelial cells, vascularization, and angiogenesis and 2) a drug "reservoir" to store therapeutic BP and pain-relieving lidocaine hydrochloride (Lid). Within several minutes of NIR laser irradiation, the BP-based gel generates local heat to accelerate microcirculatory blood flow, mediate the release of loaded Lid for "on-demand" pain relief, eliminate bacteria, and reduce inflammation. Therefore, our study not only introduces a concept of in situ sprayed, NIR-responsive pain relief gel targeting the challenging wound-healing milieu in diabetes but also provides a proof-of-concept application of BP-based materials in DU treatment.

Biocompatible exosome-modified fibrin gel accelerates the recovery of spinal cord injury by VGF-mediated oligodendrogenesis.

Exosomes show potential for treating patients with spinal cord injury (SCI) in clinical practice, but the underlying repair mechanisms remain poorly understood, and biological scaffolds available for clinical transplantation of exosomes have yet to be explored. In the present study, we demonstrated the novel function of Gel-Exo (exosomes encapsulated in fibrin gel) in promoting behavioural and electrophysiological performance in mice with SCI, and the upregulated neural marker expression in the lesion site suggested enhanced neurogenesis by Gel-Exo. According to the RNA-seq results, Vgf (nerve growth factor inducible) was the key regulator through which Gel-Exo accelerated recovery from SCI. VGF is related to myelination and oligodendrocyte development according to previous reports. Furthermore, we found that VGF was abundant in exosomes, and Gel-Exo-treated mice with high VGF expression indeed showed increased oligodendrogenesis. VGF was also shown to promote oligodendrogenesis both in vitro and in vivo, and lentivirus-mediated VGF overexpression in the lesion site showed reparative effects equal to those of Gel-Exo treatment in vivo. These results suggest that Gel-Exo can thus be used as a biocompatible material for SCI repair, in which VGF-mediated oligodendrogenesis is the vital mechanism for functional recovery.

Optimization of Fibrin Scaffolds to Study Friction in Cultured Mesothelial Cells.

To study the friction of cell monolayers avoiding damage due to stress concentration, cells can be cultured on fibrin gels, which have a structure and viscoelasticity similar to that of the extracellular matrix. In the present research, we studied different gel compositions and surface coatings in order to identify the best conditions to measure friction in vitro. We examined the adhesion and growth behavior of mesothelial cell line MET-5A on fibrin gels with different fibrinogen concentrations (15, 20, and 25 mg/mL) and with different adhesion coatings (5 µg/mL fibronectin, 10 µg/mL fibronectin, or 10 µg/mL fibronectin + 10 µg/mL collagen). We also investigated whether different substrates influenced the coefficient of friction and the ability of cells to stick to the gel during sliding. Finally, we studied the degradation rates of gels with and without cells. All substrates tested provided a suitable environment for the adherence and proliferation of mesothelial cells, and friction measurements did not cause significant cell damage or detachment. However, in gels with a lower fibrinogen concentration, cell viability was higher and cell detachment after friction measurement was lower. Fibrinolysis was negligible in all the substrates tested.

Enhanced therapeutic index of liposomal doxorubicin Myocet locally delivered by fibrin gels in immunodeficient mice bearing human neuroblastoma.

Caelyx and Myocet are clinically used liposomal forms of doxorubicin (Dox). To explore ways to improve their therapeutic index, we have studied their activity in vitro and in vivo when locally delivered by fibrin gels (FBGs). In vivo local toxic and anti-tumour activities of loaded FBGs were assessed in two immunodeficient mouse orthotopic human neuroblastoma (NB) models after application in the visceral space above the adrenal gland, either still tumour-bearing or after tumour removal. In parallel, in vitro assays were used to mimic the in vivo overlaying of FBGs on the tumour surface. FBGs were prepared with different concentrations of fibrinogen (FG) and clotted in the presence of Ca2+ and thrombin. The in vitro assays showed that FBGs loaded with Myocet possess a cytotoxic activity against NB cell lines generally greater than those loaded with free Dox or Caelyx. In vivo FBGs loaded with Myocet showed lower general and local toxicities as compared to gels loaded with Caelyx or free Dox, and also to free Dox administered i.v. (all treatments with Dox at 2.5 mg/Kg). The anti-tumour activity, evaluated in the two mouse orthotopic NB models of adjuvant and neo-adjuvant therapy, resulted in a better performance of FBGs loaded with Myocet compared to the other local (FBGs loaded with Caelyx or free Dox) or systemic (free Dox) treatments (administered at 2.5 and 5 mg/Kg Dox). Specifically, the application of FBGs at 40 mg/mL in the adjuvant model caused 92 % tumour volume reduction, while by the neo-adjuvant application of FBGs at 22 mg/mL a re-growing tumour volume reduction of 89 % was obtained. Taken together, our in vitro and in vivo results indicate a significantly higher activity for the FBGs loaded with Myocet. In particular, the lower toicity coupled with the higher anti-tumour activity on both the local treatment modalities strongly suggest a better therapeutic index when Myocet is administered through FBGs. Therefore, FBGs loaded with Myocet may be considered as a possible new tool for the loco-regional treatment of NB or even other tumour histotypes treatable by loco-regional chemotherapy.

Effect of Hypochlorite- and Peroxide-Induced Oxidation of Fibrinogen on the Fibrin Structure.

Using the methods of dynamic and elastic light scattering and confocal laser scanning microscopy, the damage in the spatial fibrin structure during peroxide- and hypochlorite-induced oxidation of fibrinogen was studied. Peroxide had a weak effect on the structural organization of fibrin, whereas hypochlorite caused the formation of abnormal fibrin with reduced individual fiber diameter and decreased porosity. Measurements of the size distributions of the native and oxidized fibrinogen revealed a decrease in the hydrodynamic size of the oxidized fibrinogen molecule with an increase in the concentration of oxidizers. These results indicate that the hydrophobicity of fibrinogen surface increased and its colloidal stability decreased. The possible role of oxidative sites in the assembly of structurally abnormal fibrin is analyzed.

Blood Clot Phenotyping by Rheometry: Platelets and Fibrinogen Chemistry Affect Stress-Softening and -Stiffening at Large Oscillation Amplitude.

(1) Background: Together with treatment protocols, viscoelastic tests are widely used for patient care. Measuring at broader ranges of deformation than currently done will add information on a clot's mechanical phenotype because fibrin networks follow different stretching regimes, and blood flow compels clots into a dynamic non-linear response. (2) Methods: To characterize the influence of platelets on the network level, a stress amplitude sweep test (LAOStress) was applied to clots from native plasma with five platelet concentrations. Five species were used to validate the protocol (human, cow, pig, rat, horse). By Lissajous plots the oscillation cycle for each stress level was analyzed. (3) Results: Cyclic stress loading generates a characteristic strain response that scales with the platelet quantity at low stress, and that is independent from the platelet count at high shear stress. This general behavior is valid in the animal models except cow. Here, the specific fibrinogen chemistry induces a stiffer network and a variant high stress response. (4) Conclusions: The protocol provides several thresholds to connect the softening and stiffening behavior of clots with the applied shear stress. This points to the reversible part of deformation, and thus opens a new route to describe a blood clot's phenotype.

The Structural-Functional Damage of Fibrinogen Oxidized by Hydrogen Peroxide.

The effect of peroxide-induced oxidation of fibrinogen on modification of its primary structure and functional properties was investigated. The oxidation sites were shown to be Met, Trp, and His residues. Using the DLS method, it was found that the oxidative modification of fibrinogen results in the change of microrheological characteristics of fibrin network. The fibrinogen oxidation diminishes its tolerance to plasmin hydrolysis and deteriorates the factor XIIIa ability to stabilize the fibrin gel.

Fibrin gels entrapment of a doxorubicin-containing targeted polycyclodextrin: Evaluation of in vivo antitumor activity in orthotopic models of human neuroblastoma.

In vivo local antitumor activity of fibrin gels (FBGs) loaded with the poly-cyclodextrin oCD-NH2/Dox, compared to free Dox, was evaluated in two mouse orthotopic neuroblastoma (NB) models, after positioning of the releasing devices in the visceral space. FBGs were prepared at the fibrinogen (FG) concentrations of 22 and 40 mg/ml clotted in the presence of 0.81 mM/mg FG Ca2+ and 1.32 U/mg FG thrombin. Our results indicate that FBGs loaded with oCD-NH2/Dox and applied as neoadjuvant loco-regional treatment, show an antitumor activity significantly greater than that displayed by the same FBGs loaded with identical dose of Dox or after free Dox administered intra venous (iv). In particular, FBGs prepared at 40 mg/ml showed a slightly lower antitumor activity, although after their positioning we observed a significant initial reduction of tumor burden lasting for several days after gel implantation. FBGs at 22 mg/ml loaded with oCD-NH2/Dox and applied after tumor removal (adjuvant treatment model) showed a significantly better antitumor activity than the iv administration of free Dox, with 90% tumor regrowth reduction compared to untreated controls. In all cases the weight loss post-treatment was limited after gel application, although in the adjuvant treatment the loss of body weight lasted longer than in the other treatment modality. In accordance with our recent published data on the low local toxic effects of FBGs, the present findings also underline an increase of the therapeutic index of Dox when locally administered through FBGs loaded with the oCD-NH2/Dox complex.

Fibrin Gels Entrapment of a Poly-Cyclodextrin Nanocarrier as a Doxorubicin Delivery System in an Orthotopic Model of Neuroblastoma: Evaluation of In Vitro Activity and In Vivo Toxicity.

PURPOSE: Fibrin gels (FBGs) are potential delivery vehicles for many drugs, and can be easily prepared from purified components. We previously demonstrated their applicability for the release of different doxorubicin (Dox) nanoparticles used clinically or in an experimental stage, such as its inclusion complex with the amino ß-cyclodextrin polymer (oCD-NH2/Dox). Here we extend these studies by in vitro and in vivo evaluations. METHODS: An in vitro cytotoxicity model consisting of an overlay of a neuroblastoma (NB) cell-containing agar layer above a drug-loaded FBG layer was used. Local toxicity in vivo (histology and blood analysis) was studied in a mouse orthotopic NB model (SHSY5YLuc+ cells implanted into the left adrenal gland). RESULTS: In vitro data show that FBGs loaded with oCD-NH2/Dox have a slightly lower cytotoxicity against NB cell lines than those loaded with Dox. Fibrinogen (FG), and Ca2+ concentrations may modify this activity. In vivo data support a lower general and local toxicity for FBGs loaded with oCD-NH2/Dox than those loaded with Dox. CONCLUSION: Our results suggest a possible increase of the therapeutic index of Dox when locally administered through FBGs loaded with oCD-NH2/Dox, opening the possibility of using these releasing systems for the treatment of neuroblastoma.

Utility of platelet rich fibrin gel therapy in nonhealing ulcer secondary to ecthyma gangrenosum.

Platelet rich fibrin (PRF) gel is a new second generation platelet concentrate, which has been widely used in various dermatological conditions such as nonhealing diabetic foot ulcers, venous ulcers, and trophic ulcers in leprosy. In this case report, we present a patient with nonhealing ulcer secondary to ecthyma gangrenosum, who showed dramatic response with PRF gel. This case suggests a role of PRF gel in nonhealing ulcers of infectious etiology which has not been described in English language literature. Considering previous literature and our case, it may be concluded that PRF gel may be utilized as an effective alternative for nonhealing ulcers of nonneoplastic etiology.

VEGF/BMP-2 loaded three-dimensional model for enhanced angiogenic and odontogenic potential of dental pulp stem cells.

AIM: To investigate the proliferation and differentiation potential of human dental pulp stem cells (DPSCs) in a three-dimensional culture model (TDM) by incorporation of VEGF and BMP-2. METHODOLOGY: TDM was established using fibrin gel (fg) as a soft tissue matrix and demineralized dentine disc (dd) as a hard tissue matrix. DPSCs and vascular endothelial growth factor (VEGF) were encapsulated in fibrin gel (fg-VEGF) and then inserted into bone morphogenetic protein (BMP-2)-coated demineralized dentine discs (dd-BMP-2). DPSCs were incubated for 28 days in various fg/dd combinations in the absence or presence of VEGF and BMP-2. Proliferation and morphology of DPSCs in fibrin gel were analysed using MTT and Live&Dead assays. Release profiles of VEGF and BMP-2 from fibrin gel and dentine discs were quantified using ELISA, and the expressions of angiogenic and odontogenic differentiation markers were determined with RT-qPCR analysis. Data were analysed statistically using Wilcoxon signed rank tests, Kruskal-Wallis tests with Mann-Whitney U tests and Bonferroni adjustment. The level of significance was set at P < 0.05. RESULTS: DPSCs were able to proliferate and showed interconnected cellular elongations in fibrin gel depending on fibrinogen concentration whilst monolayer control group showed typical fibroblast-like cell morphology. Encapsulating of VEGF in fibrin gel and BMP-2 in gelatin that was used to coat dentine discs allowed the controlled releases of growth factors, which induced angiogenic and odontogenic gene expressions by DPSCs. Higher expressions of PECAM as an angiogenic factor, and BSP, DMP-1, OCN and CBFA as odontogenic factors, were observed in TDM as compared to the other fg/dd combinations and the monolayer control group (P < 0.05). CONCLUSIONS: TDM consisting of fibrin gel and dentine matrix allowed cell-cell interactions. TDM was highly effective in delivering both VEGF and BMP-2 that enhanced the angiogenic and odontogenic potential of DPSCs.

Biopolymer gels as a basis of cryoprotective medium for testicular tissue of rats.

Cryopreservation of testis tissue is a promising approach to save fertility in prepubertal boys under going gonadotoxic cancer therapies. The using biopolymers as a basis of cryoprotective medium can be effective for the optimization of cryopreservation protocols of immature testicular tissue. The research purpose was to determine morphological parameters and metabolic activity of seminiferous tubules of immature rat testes under exposure to cryoprotective solution (DMSO) based on collagen or fibrin gels (CG or FG) as one of the first stages of developing the cryopreservation protocol. It was found that 30-min exposure of tissue samples to CG and FG with 0.6 M DMSO did not impair the spermatogenic epithelium and metabolic activity of the cells (MTT test and total lactate dehydrogenase activity). The use of FG at the time of exposure of 45 min did not lead to significant changes in the metabolic activity in contrast to other groups. The findings could be used to substantiate and develop the effective techniques for cryopreservation of immature seminiferous tubules.

Fabrication and Handling of 3D Scaffolds Based on Polymers and Decellularized Tissues.

Polymeric, ceramic and hybrid material-based three-dimensional (3D) scaffold or matrix structures are important for successful tissue engineering. While the number of approaches utilizing the use of cell-based scaffold and matrix structures is constantly growing, it is essential to provide a framework of their typical preparation and evaluation for tissue engineering. This chapter describes the fabrication of 3D scaffolds using two-photon polymerization, decellularization and cell encapsulation methods and easy-to-use protocols allowing assessing the cell morphology, cytotoxicity and viability in these scaffolds.

Towards xeno-free cultures of human limbal stem cells for ocular surface reconstruction.

Isolated limbal epithelial stem cells (LESCs) were cultured with or without a 3T3 murine fibroblast feeder-layer (FL) in 4 different culture media on culture plates or on denuded human amniotic membrane (AM) support and fibrin gel support: (1) control medium supplemented with fetal bovine serum; (2) control medium supplemented with the synthetic serum "XerumFree™ XF205" (XF); (3) CnT-20 medium supplemented with "XerumFree™ XF205" (CnT-XF) and (4) CnT-20 medium supplemented with human AB serum (CnT-AB). The three xenogeneic media were compared to standard condition (control + FL) and parameters assessed included cell morphology, proliferative potential, number of passages, assessment of clonogenic and abortive colonies, life span, ∆Np63α expression and epithelial morphology on AM. During serial cultivation of LESCs, most of the tested xeno-free media supported similar numbers of cell passages, total colony number, cumulative cell doublings (CCD) rates and expression of ∆Np63α compared to control. The conditions cultivated with a FL showed a non-statistically significant higher number of cell passages and CCD rates before senescence when compared to the same conditions cultured without FL. Except for the control medium, only XF medium enabled the growth of cells on AM. The expression of ∆Np63α was comparable in all the cultures grown onto AM, when compared to the controls on fibrin gel. In conclusion, the xeno-free media enabled LESC culture both on plastic and on denuded human AM. Despite the analyses were carried out in a statistically low number of samples and need re-assessment in a larger cohort, our results suggest that the production of a completely xeno-free LESC graft could be beneficial for future clinical applications.

Localized delivery of methylprednisolone sodium succinate with polymeric nanoparticles in experimental injured spinal cord model.

With important social and economic consequences, spinal cord injuries (SCIs) still exist among major health problems. Although many therapeutic agents and methods investigated for the treatment of acute SCI, only high dose methylprednisolone (MP) is being used currently in practice. Due to the serious side effects, high dose systemic MP administration after SCI is a critical issue that is mostly considered controversial. In our study, it is aimed to develop a nanoparticle-gel combined drug delivery system for localization of MP on trauma site and eliminating dose-dependent side effects by lowering the administered dose. For this purpose, methyl prednisolone sodium succinate (MPSS) loaded polycaprolactone based nanoparticles were developed and embedded in an implantable fibrin gel. The effects of MPSS delivery system are evaluated on an acute SCI rat model, by quantification the levels of three inflammatory cytokines (interleukin-1ß, interleukin-6 and caspase-3) and assessment of the damage on ultrastructural level by transmission electron microscopy. Developed NP-gel system showed very similar results with systemic high dose of MPSS. It is believed that developed system may be used as a tool for the safe and effective localized delivery of several other therapeutic molecules on injured spinal cord cases.

Fibrin gels loaded with cisplatin and cisplatin-hyaluronate complexes tested in a subcutaneous human melanoma model.

Fibrin gels are attractive biomaterials for local delivery of a variety of agents, from drugs to proteins. Similarly, polymer-anticancer-drug conjugates and nanoparticles are emerging as potential candidates for cancer treatment. Combining these different approaches, we have studied the efficacy of fibrin gels loaded with cisplatin (DDP) and a complex of DDP with hyaluronate (DDP-HA) for tumor growth inhibition in a melanoma model. Loaded gels prepared at relatively high fibrinogen concentration (22 mg/ml) showed good in vitro antiproliferative activities, prolonged release of the anticancer drug, and a long persistence (10-15 days) in vivo when implanted subcutaneously (sc) in immunodeficient mice. Gels loaded with DDP or DDP-HA containing 1/3 or even 1/6 of their systemic dose (6 mg/kg) and positioned under the tumor mass in mice bearing a sc human SK-Mel-28 tumor showed an antitumor activity better than that of the original parent compound given intraperitoneally (ip). Moreover, in an additional experiment in vivo, fibrin gels loaded with N-trimethyl chitosan-based nanoparticles containing a DDP-HA complex were assayed, resulting in a further 8 % improvement of anticancer activity, with lesser adverse systemic toxic effects. Taken together, these results suggest that the combination of fibrin gels and drugs complexed with suitable macromolecules holds great promise for loco-regional anticancer therapy of melanoma and other surgically removable cancer types.