Hepatocyte growth on polycapronolactone and 2-hydroxyethylmethacrylate nanofiber sheets enhanced by bone marrow-derived mesenchymal stromal cells.

BACKGROUND/AIMS: The development of hepatocyte-based Bioartificial Liver Assist Devices, intended for the therapy of chronic and fulminant liver failure, is one of the important tasks in the area of tissue engineering. New advances in the development of semipermeable non-woven nanofiber biomaterials and the co-cultivation of bone marrow mesenchymal stromal cells (BMSC) and hepatocytes could be utilized in order to maintain hepatocyte cultures in these devices. METHODOLOGY: We have compared rat hepatocyte growth on nanofiber biomaterials from different polymers, 2-hydroxyethylmethacrylate (HEMA) and ethoxyethylmethacrylate (EOEMA) copolymers, polyurethane (PUR), chitosan and polycapronolactone (PCL) spun from different solvent mixtures. RESULTS: In all cases the adhesion of hepatocytes to nanofibers was significantly better/stronger than to unstructured polymer surfaces; coating the nanofibers with collagen did not increase cell adhesion. We found the best hepatocyte adhesion on HEMA/EOEMA copolymer nanofibers and PCL nanofibers spun from a mixture of ethylacetate and dimethyl sulphoxide. Using a migration assay, we observed the migration of BMSC towards hepatocytes; hepatocytes cocultivated with BMSC excreted lower amounts of stress enzymes. CONCLUSIONS: The results demonstrate that nonwoven nanofiber layers, particularly those containing BMSC, are a suitable biocompatible support for functional hepatocyte cultures and that they can be used in a laboratory bioreactor or potentially in clinical setting.

A simple drug anchoring microfiber scaffold for chondrocyte seeding and proliferation.

The structural properties of microfiber meshes made from poly(2-hydroxyethyl methacrylate) (PHEMA) were found to significantly depend on the chemical composition and subsequent cross-linking and nebulization processes. PHEMA microfibres showed promise as scaffolds for chondrocyte seeding and proliferation. Moreover, the peak liposome adhesion to PHEMA microfiber scaffolds observed in our study resulted in the development of a simple drug anchoring system. Attached foetal bovine serum-loaded liposomes significantly improved both chondrocyte adhesion and proliferation. In conclusion, fibrous scaffolds from PHEMA are promising materials for tissue engineering and, in combination with liposomes, can serve as a simple drug delivery tool.

Surface modification of hydrogels based on poly(2-hydroxyethyl methacrylate) with extracellular matrix proteins.

Infrared attenuated total reflection spectroscopy was used for in situ observation of the deposition of collagen I on poly(2-hydroxyethyl methacrylate-co-methacrylic acid, 2.9%) hydrogels and subsequent attachment of laminin or fibronectin on the collagen surface. While there was no adsorption of collagen dissolved in an acid solution on the hydrogel surface, it deposited on the surface at pH 6.5. The collagen layers with attached laminin or fibronectin were stable on hydrogel surface in physiological solution. The modification with collagen and particularly with collagen and laminin or fibronectin allowed the adhesion and growth of mesenchymal stromal cells and astrocytes on the hydrogel surface.

Biomimetic modification of dual porosity poly(2-hydroxyethyl methacrylate) hydrogel scaffolds-porosity and stem cell growth evaluation.

The macroporous synthetic poly(2-hydroxyethyl methacrylate) (pHEMA) hydrogels as 3D cellular scaffolds with specific internal morphology, so called dual pore size, were designed and studied. The morphological microstructure of hydrogels was characterized in the gel swollen state and the susceptibility of gels for stem cells was evaluated. The effect of specific chemical groups covalently bound in the hydrogel network by copolymerization on cell adhesion and growth, followed by effect of laminin coating were investigated. The evaluated gels contained either carboxyl groups of the methacrylic acid or quaternary ammonium groups brought by polymerizable ammonium salt or their combinations. The morphology of swollen gel was visualized using the laser scanning confocal microscopy. All hydrogels had very similar porous structures - their matrices contained large pores (up to 102 µm) surrounded with gel walls with small pores (100 µm). The total pore volume in hydrogels swollen in buffer solution ranged between 69 and 86 vol%. Prior to the seeding of the mouse embryonal stem cells, the gels were coated with laminin. The hydrogel with quaternary ammonium groups (with or without laminin) stimulated the cell growth the most. The laminin coating lead to a significant and quaternary ammonium groups. The gel chemical modification influenced also the topology of cell coverage that ranged from individual cell clusters to well dispersed multi cellular structures. Findings in this study point out the laser scanning confocal microscopy as an irreplaceable method for a precise and quick assessment of the hydrogel morphology. In addition, these findings help to optimize the chemical composition of the hydrogel scaffold through the combination of chemical and biological factors leading to intensive cell attachment and proliferation.

The Effect of iPS-Derived Neural Progenitors Seeded on Laminin-Coated pHEMA-MOETACl Hydrogel with Dual Porosity in a Rat Model of Chronic Spinal Cord Injury.

Spinal cord injury (SCI), is a devastating condition leading to the loss of locomotor and sensory function below the injured segment. Despite some progress in acute SCI treatment using stem cells and biomaterials, chronic SCI remains to be addressed. We have assessed the use of laminin-coated hydrogel with dual porosity, seeded with induced pluripotent stem cell-derived neural progenitors (iPSC-NPs), in a rat model of chronic SCI. iPSC-NPs cultured for 3 weeks in hydrogel in vitro were positive for nestin, glial fibrillary acidic protein (GFAP) and microtubule-associated protein 2 (MAP2). These cell-polymer constructs were implanted into a balloon compression lesion, 5 weeks after lesion induction. Animals were behaviorally tested, and spinal cord tissue was immunohistochemically analyzed 28 weeks after SCI. The implanted iPSC-NPs survived in the scaffold for the entire experimental period. Host axons, astrocytes and blood vessels grew into the implant and an increased sprouting of host TH+ fibers was observed in the lesion vicinity. The implantation of iPSC-NP-LHM cell-polymer construct into the chronic SCI led to the integration of material into the injured spinal cord, reduced cavitation and supported the iPSC-NPs survival, but did not result in a statistically significant improvement of locomotor recovery.

Acute and delayed implantation of positively charged 2-hydroxyethyl methacrylate scaffolds in spinal cord injury in the rat.

OBJECT: Hydrogels are nontoxic, chemically inert synthetic polymers with a high water content and large surface area that provide mechanical support for cells and axons when implanted into spinal cord tissue. METHODS: Macroporous hydrogels based on 2-hydroxyethyl methacrylate (HEMA) were prepared by radical copolymerization of monomers in the presence of fractionated NaCl particles. Male Wistar rats underwent complete spinal cord transection at the T-9 level. To bridge the lesion, positively charged HEMA hydrogels were implanted either immediately or 1 week after spinal cord transection; control animals were left untreated. Histological evaluation was performed 3 months after spinal cord transection to measure the volume of the pseudocyst cavities and the ingrowth of tissue elements into the hydrogels. RESULTS: The hydrogel implants adhered well to the spinal cord tissue. Histological evaluation showed ingrowth of connective tissue elements, blood vessels, neurofilaments, and Schwann cells into the hydrogels. Morphometric analysis of lesions showed a statistically significant reduction in pseudocyst volume in the treated animals compared with controls and in the delayed treatment group compared with the immediate treatment group (p < 0.001 and p < 0.05, respectively). CONCLUSIONS: Positively charged HEMA hydrogels can bridge a posttraumatic spinal cord cavity and provide a scaffold for the ingrowth of regenerating axons. The results indicate that delayed implantation can be more effective than immediate reconstructive surgery.

Surface morphology of contact lenses probed with microscopy techniques.

The present study is bringing a comparison of surface morphology for various types of contact lenses. A novel method--scanning electron microscopy under aqueous conditions (cryo-SEM)--was tested for visualization of lenses at magnifications up to 2000x. For imaging lens surface on nanometre scale, we employed atomic force microscopy (AFM) in aqueous media. Various materials of lenses, based on silicone hydrogels or conventional hydrogels, were investigated. Total, 10 types of contact lenses from five manufacturers were selected and probed. We found that different methods of lens manufacture (lathe-cutting, cast-moulding, and spin casting) led to different values of surface roughness. In the swollen state, roughness values of lens surfaces lie between 4 and 140 nm. Lenses manufactured by lathe-cutting exhibit notable higher values, so that they could be easily distinguished from others. In cast-moulded lenses, the surface roughness decreased with increasing water content. Moreover, additional treatments of lenses introduced unique structural motifs onto surface. For instance, porous structure was found on lens surface finalized with plasma oxidation.

New perfluoroalkylated amphiphilic methacrylates bearing sulfinyl group as monomers for biomedical applications: water content and oxygen permeability of their copolymers with DEGMA.

Perfluoroalkylated methacrylates 7a-c bearing sulfinyl group within a straight-chain ester group, i.e. CH(2)=C(CH(3))CO(2)CH(2)CH(2)S(O)-CH(2)CH(2)CF(2)(CF(2)CF(2))(n)CF(3) (n=1-3) were prepared by two alternative synthetic sequences from 2-[(polyfluoroalkyl)sulfanyl]ethanols HOCH(2)CH(2)SCH(2)CH(2)CF(2)(CF(2)CF(2))(n)CF(3) (n=1-3) in overall yields of 88-91%. Copolymers of 7a-c with diethylene glycol methacrylate (DEGMA) prepared in bulk under radical conditions display high transparency, increased water content and good oxygen permeability properties, which are advantageous for their application in ophthalmology and as prosthetic materials.

Ice-templated hydrogels based on chitosan with tailored porous morphology.

Preparation and morphological characterization of some novel hydrogels based on chitosan (CS) with porous structure tailored by ice-templating and porogen leaching are presented in the paper. Poly(methylmethacrylate) (PMMA), as fractionated particles, has been used as polymer porogen. The influence of the mesh of the fractionated PMMA particles, the weight ratio between CS and fractionated PMMA particles, and the speed of the crystallization, on the internal morphology of the hydrogels have been deeply investigated. The morphology of the obtained hydrogels was observed by scanning electron microscopy (SEM). As a function of the synthesis conditions, hydrogels with a heterogeneous morphology consisting of randomly and evenly distributed polyhedral pores, or with an oriented structure, which has microchanneled structures arranged along the freezing direction, were generated.

Treating spinal cord injury in rats with a combination of human fetal neural stem cells and hydrogels modified with serotonin.

Currently, there is no effective strategy for the treatment of spinal cord injury (SCI). A combination of biomaterials and stem cell therapy seems to be a promising approach to increase regenerative potential after SCI. We evaluated the use of a cellpolymer construct based on a combination of the conditionally immortalized spinal progenitor cell line SPC-01_GFP3, derived from human fetal spinal cord tissue, with a serotonin-modified poly(2-hydroxyethyl methacrylate) hydrogel (pHEMA-5HT). We compared the effect of treatment with a pHEMA-5HT hydrogel seeded with SPC-01_GFP3 cells, treatment with a pHEMA-5HT only and no treatment on functional outcome and tissue reconstruction in hemisected rats. Prior to transplantation the cell-polymer construct displayed a high potential to support the growth, proliferation and differentiation of SPC-01 cells in vitro. One month after surgery, combined hydrogel-cell treatment reduced astrogliosis and tissue atrophy and increased axonal and blood vessel ingrowth into the implant; however, two months later only the ingrowth of blood vessels remained increased. SPC-01_GFP3 cells survived well in vivo and expressed advanced markers of neuronal differentiation. However, a majority of the transplanted cells migrated out of the lesion and only rarely remained in the hydrogel. No differences among the groups in motor or sensory recovery were observed. Despite the support of the hydrogel as a cell carrier in vitro, and good results in vivo one month postsurgery, there was only a small effect on long term recovery, mainly due to the limited ability of the hydrogels to support the in vivo growth and differentiation of cells within the implant. Further modifications will be necessary to achieve stable long term improvement in functional outcome.

Adjusting the chemical and physical properties of hydrogels leads to improved stem cell survival and tissue ingrowth in spinal cord injury reconstruction: a comparative study of four methacrylate hydrogels.

Currently, there is no effective strategy for the treatment of spinal cord injury (SCI). A suitable combination of modern hydrogel materials, modified to effectively bridge the lesion cavity, combined with appropriate stem cell therapy seems to be a promising approach to repair spinal cord damage. We demonstrate the synergic effect of porosity and surface modification of hydrogels on mesenchymal stem cell (MSC) adhesiveness in vitro and their in vivo survival in an experimental model of SCI. MSCs were seeded on four different hydrogels: hydroxypropylmethacrylate-RGD prepared by heterophase separation (HPMA-HS-RGD) and three other hydrogels polymerized in the presence of a solid porogen: HPMA-SP, HPMA-SP-RGD, and hydroxy ethyl methacrylate [2-(methacryloyloxy)ethyl] trimethylammonium chloride (HEMA-MOETACl). Their adhesion capability and cell survival were evaluated at 1, 7, and 14 days after the seeding of MSCs on the hydrogel scaffolds. The cell-polymer scaffolds were then implanted into hemisected rat spinal cord, and MSC survival in vivo and the ingrowth of endogenous tissue elements were evaluated 1 month after implantation. In vitro data demonstrated that HEMA-MOETACl and HPMA-SP-RGD hydrogels were superior in the number of cells attached. In vivo, the highest cell survival was found in the HEMA-MOETACl hydrogels; however, only a small ingrowth of blood vessels and axons was observed. Both HPMA-SP and HPMA-SP-RGD hydrogels showed better survival of MSCs compared with the HPMA-HS-RGD hydrogel. The RGD sequence attached to both types of HPMA hydrogels significantly influenced the number of blood vessels inside the implanted hydrogels. Further, the porous structure of HPMA-SP hydrogels promoted a statistically significant greater ingrowth of axons and less connective tissue elements into the implant. Our results demonstrate that the physical and chemical properties of the HPMA-SP-RGD hydrogel show the best combination for bridging a spinal cord lesion, while the HEMA-MOETACl hydrogel serves as the best carrier of MSCs.

Treatment of ocular surface injuries by limbal and mesenchymal stem cells growing on nanofiber scaffolds.

Stem cell (SC) therapy represents a promising approach to treat a wide variety of injuries, inherited diseases, or acquired SC deficiencies. One of the major problems associated with SC therapy remains the absence of a suitable matrix for SC growth and transfer. We describe here the growth and metabolic characteristics of mouse limbal stem cells (LSCs) and mesenchymal stem cells (MSCs) growing on 3D nanofiber scaffolds fabricated from polyamide 6/12 (PA6/12). The nanofibers were prepared by the original needleless electrospun Nanospider technology, which enables to create nanofibers of defined diameter, porosity, and a basis weight. Copolymer PA6/12 was selected on the basis of the stability of its nanofibers in aqueous solutions, its biocompatibility, and its superior properties as a matrix for the growth of LSCs, MSCs, and corneal epithelial and endothelial cell lines. The morphology, growth properties, and viability of cells grown on PA6/12 nanofibers were comparable with those grown on plastic. LSCs labeled with the fluorescent dye PKH26 and grown on PA6/12 nanofibers were transferred onto the damaged ocular surface, where their seeding and survival were monitored. Cotransfer of LSCs with MSCs, which have immunosuppressive properties, significantly inhibited local inflammatory reactions and supported the healing process. The results thus show that nanofibers prepared from copolymer PA6/12 represent a convenient scaffold for growth of LSCs and MSCs and transfer to treat SC deficiencies and various ocular surface injuries.

HPMA-RGD hydrogels seeded with mesenchymal stem cells improve functional outcome in chronic spinal cord injury.

Chronic spinal cord injury (SCI) is characterized by tissue loss and a stable functional deficit. While several experimental therapies have proven to be partly successful for the treatment of acute SCI, treatment of chronic SCI is still challenging. We studied whether we can bridge a chronic spinal cord lesion by implantation of our newly developed hydrogel based on 2-hydroxypropyl methacrylamide, either alone or seeded with mesenchymal stem cells (MSCs), and whether this treatment leads to functional improvement. A balloon-induced compression lesion was performed in adult 2-month-old male Wistar rats. Five weeks after injury, HPMA-RGD hydrogels [N-(2-hydroxypropyl)-methacrylamide with attached amino acid sequences--Arg-Gly-Asp] were implanted into the lesion, either with or without seeded MSCs. Animals with chronic SCI served as controls. The animals were behaviorally tested using the Basso­Beattie-Breshnahan (BBB) (motor) and plantar (sensory) tests once a week for 6 months. Behavioral analysis showed a statistically significant improvement in rats with combined treatment, hydrogel and MSCs, compared with the control group (P < 0.05). Although a tendency toward improvement was found in rats treated with hydrogel only, this was not significant. Subsequently, the animals were sacrificed 6 months after SCI, and the spinal cord lesions evaluated histologically. The combined therapy (hydrogel with MSCs) prevented tissue atrophy (P < 0.05), and the hydrogels were infiltrated with axons myelinated with Schwann cells. Blood vessels and astrocytes also grew inside the implant. MSCs were present in the hydrogels even 5 months after implantation. We conclude that 5 weeks after injury, HPMA-RGD hydrogels seeded with MSCs can successfully bridge a spinal cord cavity and provide a scaffold for tissue regeneration. This treatment leads to functional improvement even in chronic SCI.

Cultivation of human keratinocytes without feeder cells on polymer carriers containing ethoxyethyl methacrylate: in vitro study.

The cell/tissue engineering therapy of extensive or chronic skin wounds is a highly topical task of the contemporary medicine. One of possible therapeutic approaches is grafting of in vitro cultured keratinocytes directly to the wound bed, where the cells colonize the wound, proliferate and improve the re-epithelization process. Because the successful cultivation of keratinocytes needs an application of feeder cells, the exclusion of these cells from the cultivation system is highly required. In this study we show a positive influence of 2-ethoxyethyl methacrylate as a component of cultivation support on growth of keratinocytes without feeder cells. Keratinocytes cultured on these surfaces are able to migrate to the model wound bed in vitro, where they form distinct colonies and have a normal differentiation potential.

Macroporous hydrogels based on 2-hydroxyethyl methacrylate. Part 5: hydrolytically degradable materials.

Macroporous hydrogels based on 2-hydroxyethyl methacrylate, 2-ethoxyethyl methacrylate and N-(2-hydroxypropyl)methacrylamide, methacrylic acid and [2-(methacryloyloxy)ethyl]trimethylammonium chloride crosslinked with N,O-dimethacryloylhydroxylamine were prepared. Hydrogels were degraded in a buffer of pH 7.4. Completely water-soluble polymers were obtained over time periods ranging from 2 to 40 days. The process of degradation was followed gravimetrically and by optical and electron microscopy. In vivo biological tests with hydrogels based on copolymers of 2-ethoxyethyl methacrylate/N-(2-hydroxypropyl)methacrylamide were performed.

Evaluation of biocompatibility of the copolymer of 2-hydroxyethyl methacrylate with 2-(methylsulfanyl)ethyl methacrylate.

This study compares subcutaneous and intracerebral biocompatibilty of two hydrogels: copolymer of 2-hydroxyethyl methacrylate with 2-(methylsulfanyl)ethyl methacrylate and poly(2-hydroxyethyl methacrylate) as reference polymer. The experimental copolymer was more biologically inert than poly(2-hydroxyethyl methacrylate) in both the studied parameters, hence the former material is a suitable candidate for biomedical application.