The alveolar-capillary interface is the key functional element of gas exchange in the human lung, and disruptions to this interface can lead to significant medical complications. However, it is currently challenging to adequately model this interface in vitro, as it requires not only the co-culture of human alveolar epithelial and endothelial cells but mainly the preparation of a biocompatible scaffold that mimics the basement membrane. This scaffold should support cell seeding from both sides, and maintain optimal cell adhesion, growth, and differentiation conditions. Our study investigates the use of polycaprolactone (PCL) nanofibers as a versatile substrate for such cell cultures, aiming to model the alveolar-capillary interface more accurately. We optimized nanofiber production parameters, utilized polyamide mesh UHELON as a mechanical support for scaffold handling, and created 3D-printed inserts for specialized co-cultures. Our findings confirm that PCL nanofibrous scaffolds are manageable and support the co-culture of diverse cell types, effectively enabling cell attachment, proliferation, and differentiation. Our research establishes a proof-of-concept model for the alveolar-capillary interface, offering significant potential for enhancing cell-based testing and advancing tissue-engineering applications that require specific nanofibrous matrices.
BACKGROUND: Chronic lymphocytic leukemia (CLL) is a common adult leukemia characterized by the accumulation of neoplastic mature B cells in blood, bone marrow, lymph nodes, and spleen. The disease biology remains unresolved in many aspects, including the processes underlying the disease progression and relapses. However, studying CLL in vitro poses a considerable challenge due to its complexity and dependency on the microenvironment. Several approaches are utilized to overcome this issue, such as co-culture of CLL cells with other cell types, supplementing culture media with growth factors, or setting up a three-dimensional (3D) culture. Previous studies have shown that 3D cultures, compared to conventional ones, can lead to enhanced cell survival and altered gene expression. 3D cultures can also give valuable information while testing treatment response in vitro since they mimic the cell spatial organization more accurately than conventional culture. METHODS: In our study, we investigated the behavior of CLL cells in two types of material: (i) solid porous collagen scaffolds and (ii) gel composed of carboxymethyl cellulose and polyethylene glycol (CMC-PEG). We studied CLL cells' distribution, morphology, and viability in these materials by a transmitted-light and confocal microscopy. We also measured the metabolic activity of cultured cells. Additionally, the expression levels of MYC, VCAM1, MCL1, CXCR4, and CCL4 genes in CLL cells were studied by qPCR to observe whether our novel culture approaches lead to increased adhesion, lower apoptotic rates, or activation of cell signaling in relation to the enhanced contact with co-cultured cells. RESULTS: Both materials were biocompatible, translucent, and permeable, as assessed by metabolic assays, cell staining, and microscopy. While collagen scaffolds featured easy manipulation, washability, transferability, and biodegradability, CMC-PEG was advantageous for its easy preparation process and low variability in the number of accommodated cells. Both materials promoted cell-to-cell and cell-to-matrix interactions due to the scaffold structure and generation of cell aggregates. The metabolic activity of CLL cells cultured in CMC-PEG gel was similar to or higher than in conventional culture. Compared to the conventional culture, there was (i) a lower expression of VCAM1 in both materials, (ii) a higher expression of CCL4 in collagen scaffolds, and (iii) a lower expression of CXCR4 and MCL1 (transcript variant 2) in collagen scaffolds, while it was higher in a CMC-PEG gel. Hence, culture in the material can suppress the expression of a pro-apoptotic gene (MCL1 in collagen scaffolds) or replicate certain gene expression patterns attributed to CLL cells in lymphoid organs (low CXCR4, high CCL4 in collagen scaffolds) or blood (high CXCR4 in CMC-PEG).
- MeSH
- buněčné kultury metody MeSH
- chronická lymfatická leukemie * patologie metabolismus MeSH
- gely chemie MeSH
- kolagen * chemie farmakologie MeSH
- lidé MeSH
- polyethylenglykoly * chemie MeSH
- receptory CXCR4 metabolismus MeSH
- sodná sůl karboxymethylcelulosy * chemie farmakologie MeSH
- techniky 3D buněčné kultury metody MeSH
- tkáňové podpůrné struktury * chemie MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
This study develops and characterizes novel biodegradable soft hydrogels with dual porosity based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers cross-linked by hydrolytically degradable linkers. The structure and properties of the hydrogels are designed as scaffolds for tissue engineering and they are tested in vitro with model mesenchymal stem cells (rMSCs). Detailed morphological characterization confirms dual porosity suitable for cell growth and nutrient transport. The dual porosity of hydrogels slightly improves rMSCs proliferation compared to the hydrogel with uniform pores. In addition, the laminin coating supports the adhesion of rMSCs to the hydrogel surface. However, hydrogels modified by heptapeptide RGDSGGY significantly stimulate cell adhesion and growth. Moreover, the RGDS-modified hydrogels also affect the topology of proliferating rMSCs, ranging from single-cell to multicellular clusters. The 3D reconstruction of the hydrogels with cells obtained by laser scanning confocal microscopy (LSCM) confirms cell penetration into the inner structure of the hydrogel and its corresponding microstructure. The prepared biodegradable oligopeptide-modified hydrogels with dual porosity are suitable candidates for further in vivo evaluation in soft tissue regeneration.
The aim of this study was to develop multifunctional magnetic poly(ε-caprolactone) (PCL) mats with antibacterial properties for bone tissue engineering and osteosarcoma prevention. To provide good dispersion of magnetic iron oxide nanoparticles (IONs), they were first grafted with PCL using a novel three-step approach. Then, a series of PCL-based mats containing a fixed amount of ION@PCL particles and an increasing content of ascorbic acid (AA) was prepared by electrospinning. AA is known for increasing osteoblast activity and suppressing osteosarcoma cells. Composites were characterized in terms of morphology, mechanical properties, hydrolytic stability, antibacterial performance, and biocompatibility. AA affected both the fiber diameter and the mechanical properties of the nanocomposites. All produced mats were nontoxic to rat bone marrow-derived mesenchymal cells; however, a composite with 5 wt.% of AA suppressed the initial proliferation of SAOS-2 osteoblast-like cells. Moreover, AA improved antibacterial properties against Staphylococcus aureus and Escherichia coli compared to PCL. Overall, these magnetic composites, reported for the very first time, can be used as scaffolds for both tissue regeneration and osteosarcoma prevention.
- MeSH
- antibakteriální látky chemie farmakologie MeSH
- Escherichia coli účinky léků MeSH
- kosti a kostní tkáň MeSH
- krysa rodu rattus MeSH
- kyselina askorbová * chemie farmakologie MeSH
- lidé MeSH
- magnetické nanočástice chemie MeSH
- nádorové buněčné linie MeSH
- nanokompozity chemie MeSH
- osteoblasty metabolismus cytologie MeSH
- osteosarkom patologie MeSH
- polyestery * chemie MeSH
- Staphylococcus aureus * účinky léků růst a vývoj MeSH
- testování materiálů MeSH
- tkáňové inženýrství * MeSH
- tkáňové podpůrné struktury chemie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Herein, the recent advances in the development of resorbable polymeric-based biomaterials, their geometrical forms, resorption mechanisms, and their capabilities in various biomedical applications are critically reviewed. A comprehensive discussion of the engineering approaches for the fabrication of polymeric resorbable scaffolds for tissue engineering, drug delivery, surgical, cardiological, aesthetical, dental and cardiovascular applications, are also explained. Furthermore, to understand the internal structures of resorbable scaffolds, representative studies of their evaluation by medical imaging techniques, e.g., cardiac computer tomography, are succinctly highlighted. This approach provides crucial clinical insights which help to improve the materials' suitable and viable characteristics for them to meet the highly restrictive medical requirements. Finally, the aspects of the legal regulations and the associated challenges in translating research into desirable clinical and marketable materials of polymeric-based formulations, are presented.
- MeSH
- biokompatibilní materiály chemie MeSH
- lékové transportní systémy * metody MeSH
- lidé MeSH
- polymery * chemie MeSH
- tkáňové inženýrství * metody MeSH
- tkáňové podpůrné struktury chemie MeSH
- vstřebatelné implantáty MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Previously, a new biodegradable poly(ester urethane urea) was synthesized based on polycaprolactone-diol and fish gelatin (PU-Gel). In this work, the potential of this new material for neural tissue engineering is evaluated. Membranes with randomly oriented fibers and with aligned fibers are produced using electrospinning and characterized regarding their mechanical behavior under both dry and wet conditions. Wet samples exhibit a lower Young's modulus than dry ones and aligned membranes are stiffer and more brittle than those randomly oriented. Cyclic tensile tests are conducted and high values for recovery ratio and resilience are obtained. Both membranes exhibited a hydrophobic surface, measured by the water contact angle (WCA). Human mesenchymal stem cells from umbilical cord tissue (UC-MSCs) and human neural stem cells (NSCs) are seeded on both types of membranes, which support their adhesion and proliferation. Cells stained for the cytoskeleton and nucleus in membranes with aligned fibers display an elongated morphology following the alignment direction. As the culture time increased, higher cell viability is obtained on randomfibers for UC-MSCs while no differences are observed for NSCs. The membranes support neuronal differentiation of NSCs, as evidenced by markers for a neuronal filament protein (NF70) and for a microtubule-associated protein (MAP2).
- MeSH
- biokompatibilní materiály chemie farmakologie MeSH
- buněčná adheze účinky léků MeSH
- buněčná diferenciace účinky léků MeSH
- kultivované buňky MeSH
- lidé MeSH
- mezenchymální kmenové buňky * cytologie účinky léků metabolismus MeSH
- nervové kmenové buňky * cytologie účinky léků metabolismus MeSH
- pevnost v tahu MeSH
- polyestery * chemie farmakologie MeSH
- polyurethany * chemie farmakologie MeSH
- proliferace buněk účinky léků MeSH
- testování materiálů MeSH
- tkáňové inženýrství * metody MeSH
- tkáňové podpůrné struktury chemie MeSH
- viabilita buněk účinky léků MeSH
- želatina * chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- MeSH
- fyziologická neovaskularizace MeSH
- intravitální mikroskopie metody MeSH
- klinické praxe MeSH
- lidé MeSH
- modely nemocí na zvířatech MeSH
- myši MeSH
- regenerativní endodoncie * metody výchova MeSH
- tkáňové podpůrné struktury MeSH
- transplantace kmenových buněk metody MeSH
- zubní kořen chirurgie MeSH
- zubní zárodek MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
Lately, the need for three-dimensional (3D) cell culture has been recognized in order to closely mimic the organization of native tissues. Thus, 3D scaffolds started to be employed to facilitate the 3D cell organization and enable the artificial tissue formation for the emerging tissue engineering applications. 3D scaffolds can be prepared by various techniques, each with certain advantages and disadvantages. Decellularization is an easy method based on removal of cells from native tissue sample, yielding extracellular matrix (ECM) scaffold with preserved architecture and bioactivity. This chapter provides a detailed protocol for decellularization of pig lung and also some basic assays for evaluation of its effectivity, such as determination of DNA content and histological verification of the selected ECM components. Such decellularized scaffold can subsequently be used for various tissue engineering applications, for example, for recellularization with cells of interest, for natural ECM hydrogel preparation, or as a bioink for 3D bioprinting.
- MeSH
- extracelulární matrix MeSH
- hydrogely MeSH
- plíce * MeSH
- prasata MeSH
- tkáňové inženýrství * metody MeSH
- tkáňové podpůrné struktury * MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: The purpose of dermal substitutes is to mimic the basic properties of the extracellular matrix of human skin. The application of dermal substitutes to the defect reduces the formation of hypertrophic scars and improves the scar quality. This study aims to develop an original dermal substitute enriched with stable fibroblast growth factor 2 (FGF2-STAB®) and test it in an animal model. METHODS: Dermal substitutes based on collagen/chitosan scaffolds or collagen/chitosan scaffolds with nanofibrous layer were prepared and enriched with FGF2-STAB® at concentrations of 0, 0.1, 1.0, and 10.0 μg ‧ cm-2. The performance of these dermal substitutes was tested in vivo on artificially formed skin defects in female swine. The outcomes were evaluated using cutometry at 3 and 6 months. In addition, visual appearance was assessed based on photos of the scars at 1-month, 3-month and 6-month follow-ups using Yeong scale and Visual Analog Scale. RESULTS: The dermal substitute was fully integrated into all defects and all wounds healed successfully. FGF2-STAB®-enriched matrices yielded better results in cutometry compared to scaffolds without FGF2. Visual evaluation at 1, 3, and 6 months follow-ups detected no significant differences among groups. The FGF2-STAB® effectiveness in improving the elasticity of scar tissues was confirmed in the swine model. This effect was independently observed in the scaffolds with nanofibres as well as in the scaffolds without nanofibres. CONCLUSION: The formation of scars with the best elasticity was exhibited by addition 1.0 μg ‧ cm-2of FGF2-STAB® into the scaffolds, although it had no significant effect on visual appearance at longer follow-ups. This study creates the basis for further translational studies of the developed product and its progression into the clinical phase of the research.
- MeSH
- chitosan * MeSH
- fibroblastový růstový faktor 2 * MeSH
- hojení ran účinky léků MeSH
- jizva hypertrofická MeSH
- kolagen MeSH
- kůže MeSH
- modely nemocí na zvířatech MeSH
- nanovlákna terapeutické užití MeSH
- popálení MeSH
- prasata MeSH
- pružnost * MeSH
- tkáňové podpůrné struktury MeSH
- umělá kůže * MeSH
- viskozita MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
PURPOSE OF THE STUDY: The preclinical study aimed to compare the healing of segmental bone defects treated with biodegradable hyaluronic acid and tricalcium phosphate-based hydrogel with the established autologous spongioplasty. Another aim was to evaluate the hydrogel as a scaffold for osteoinductive growth factor of bone morphogenetic protein-2 (BMP-2) and stem cells. MATERIAL AND METHODS: The study was conducted in an in vivo animal model. A standardized rabbit model of a 15 mm long segmental bone defect of left radius was used. A total of 40 animals were divided into 5 groups of 8 individuals. In the KO- (negative control) group, the created defect was left to heal spontaneously. In the KO+ (positive control) group, the defect was filled with morselized bone autograft prepared from the resected segment. In the study group A, the defect was filled with hydrogel based on hyaluronic acid derivative and tricalcium phosphate. In the study group B, the defect was filled with hydrogel based on hyaluronic acid derivative, tricalcium phosphate and bone marrow aspirate. In the study group C, the defect was filled with hydrogel based on hyaluronic acid derivative, tricalcium phosphate, bone marrow aspirate and BMP-2. Healing was assessed using radiographs at 1, 6, and 12 weeks postoperatively and histology specimens were collected at 16 weeks postoperatively. RESULTS: Altogether 35 rabbits survived (KO- 7, KO+ 7, A 7, B 6, C 8) until the end of the study. As concerns the radiographic assessment, the best results were achieved by the groups KO+ and C, where new bone formation across the entire width of the bone defect was clearly seen at 6 and 12 weeks and the osteotomy line was completely healed too. At 12 weeks, complete bone remodelling was observed in all animals in the group KO+, whereas in the group C, bone remodelling was fully completed in 5 animals and partially completed in 3 animals. In terms of histological assessment, however, the best results were achieved by the group C, where the bone defect was completely remodelled into lamellar bone in 7 specimens, while in 1 specimen it healed with bony callus formation. In the group KO+, the defect was healed in 4 specimens by cartilaginous callus with loci of remodelling into bony callus, in 2 specimens the bony callus was predominant with cartilaginous callus areas, and only one defect was completely remodelled into lamellar bone. DISCUSSION: Compared to autografts that manifest osteogenic, osteoinductive and osteoconductive properties, the biodegradable hyaluronic acid and tricalcium phosphate-based hydrogel has osteoconductive properties only. Thus, it was also tested in our study as a scaffold for bone marrow cells and BMP-2 osteoinductive growth factor. Thanks to its semi-liquid properties, the biodegradable hyaluronic acid and tricalcium phosphate-based hydrogel is a promising material for use in 3D printing. CONCLUSIONS: The preclinical study in an in vivo animal model confirmed the beneficial effect of the biodegradable hyaluronic acid and tricalcium phosphate-based hydrogel on the healing of critical-size segmental bone defects. Better healing of these defects was also confirmed for filling composed of hydrogel and BMP-2 osteoinductive growth factor. The benefit of bone marrow aspirate mixed with hydrogel was not confirmed. KEY WORDS: bone defect, non-union, rabbit, hyaluronic acid, calcium phosphate, stem cells, BMP-2, scaffold, bone healing, spongioplasty.
- MeSH
- fosforečnany vápenaté * farmakologie MeSH
- hydrogely farmakologie MeSH
- kostní morfogenetický protein 2 * MeSH
- králíci MeSH
- kyselina hyaluronová * farmakologie MeSH
- modely nemocí na zvířatech MeSH
- radius chirurgie zranění MeSH
- regenerace kostí účinky léků MeSH
- tkáňové podpůrné struktury * MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- zvířata MeSH
- Publikační typ
- anglický abstrakt MeSH
- časopisecké články MeSH
