Jiří Michálek

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 Basso–Beattie-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.

Non-fouling Hydrogels of 2-Hydroxyethyl Methacrylate and Zwitterionic Carboxybetaine (Meth)acrylamides

Five poly(betaine) brushes were prepared, and their resistance to blood plasma fouling was studied. Two carboxybetaines monomers were copolymerized with 2-hydroxyethyl methacrylate (HEMA) to prepare novel hydrogels. By increasing the content of the zwitterionic comonomer, a 4-fold increase in the water content could be achieved while retaining mechanical properties close to the widely used poly(HEMA) hydrogels. All hydrogels showed an unprecedentedly low fouling from blood plasma. Remarkably, by copolymerization with 10 mol % of carboxybetaine acrylamide, hydrogels fully resistant to blood plasma were prepared.

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.

Morphological characterization of nanofibers: methods and application in practice

Biomedical applications such as wound dressing for skin regeneration, stem cell transplantation, or drug delivery require special demands on the three-dimensional porous scaffolds. Besides the biocompatibility and mechanical properties, the morphology is the most important attribute of the scaffold. Specific surface area, volume, and size of the pores have considerable effect on cell adhesion, growth, and proliferation. In the case of incorporated biologically active substances, their release is also influenced by the internal structure of nanofibers. Although many scientific papers are focused on the preparation of nanofibers and evaluation of biological tests, the morphological characterization was described just briefly as service methods. The aim of this paper is to summarize the methods applicable for morphological characterization of nanofibers and supplement it by the results of our research. Needleless electrospinning technique was used to prepare nanofibers from polylactide, poly(e-caprolactone), gelatin, and polyamide. Scanning electron microscopy was used to evaluate the fiber diameters and to reveal eventual artifacts in the nanofibrous structure. Nitrogen adsorption/desorption measurements were employed to measure the specific surface areas. Mercury porosimetry was used to determine total porosities and compare pore size distributions of the prepared samples.

Light absorption properties of the rabbit cornea repeatedly irradiated with UVB rays.

Under normal conditions, the cornea absorbs the majority of UVB (ultraviolet B, 280–320 nm) rays, which is very important for the protection of the inner eye against their damaging effect. Our previous studies have shown that repeated irradiation of the rabbit cornea with UVB rays for 5 days (daily dose of 1.01 J cm−2) caused photokeratitis accompanied by swelling (hydration) of the corneal stroma, thinning of the corneal epithelium and decrease in antioxidants. The purpose of this study was to examine the light absorption properties of such damaged rabbit cornea. Results of both spectrophotometry of the whole corneal buttons and corneal tissue dissolved in sodium hydroxide show that because of above mentioned disturbances, UVB‐irradiated cornea absorbs more light throughout the whole measurable UV–VIS spectral range than the normal cornea. Increased corneal thickness (result of hydration), changes of corneal transparency (the cornea becomes grayish) and some increase in protein content all contribute to the increased light absorption of UVB irradiated corneas. We suggest that the UVB‐irradiated cornea, although damaged and nearly without antioxidants, might actually through its higher UV absorbance protect the inner eye against further damage from UVB rays.

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.

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.

Macroporous 2-hydroxyethyl methacrylate hydrogels of dual porosity for cell cultivation: morphology, swelling, permeability, and mechanical behavior

AbstractMacroporous hydrogels of dual porosity based on cross-linked poly(2-hydroxyethyl methacrylate) (pHEMA) were prepared, and their swelling, mechanical responses, and hydraulic permeability, as well as cell adhesion and proliferation, were examined on gel-based scaffolds. Large pores on the order of tens and hundreds of microns were generated by adding a solid porogen (fractionated particles of sodium chloride) to the polymerization mixture, which was subsequently washed out of the gel. Small pores on the order of a few microns were formed by the reaction-induced phase separation mechanism caused by addition of a diluent (1-dodecanol), which is a poor solvent for pHEMA. Morphological studies using light and electron microscopy techniques revealed that the large pores were embedded in the pHEMA matrix containing small fused spherical pores. Mechanical behavior of the dual-porosity hydrogels characterized by oscillatory shear measurements revealed that despite very high pore volume, the hydrogels were sufficiently stiff and self-supportive. The cell cultivation pilot experiment showed that the morphology (dual porosity) demonstrated marked effects on the promotion of cell adhesion, growth, and proliferation, not only on the gel surface but in the large pores within the gel bulk as well. Graphical AbstractDual porosity hydrogel was formed using washable templates (large pores) and utilizing the phase separation within the hydrogel walls (fine porosity) in one step. The hydrogel structure was visualized by scanning electron microscopy (gel without cells) - the image confirmed dual porosity.

2,4,4,5,7,7,8,8,9,9,9-Undecafluoro-2,5-bis(trifluoromethyl)-3,6-dioxanonyl methacrylate

The title monomer (4) was prepared from the trimer of hexafluoropropene-1,2-oxide, 2,4,4,5,7,7,8,8,9,9,9-undecafluoro-2,5-bis(trifluoromethyl)-3,6-dioxanonanoyl fluoride (1), via methyl ester 2 that was reduced by sodium borohydride to the corresponding alkanol 3, which was finally acylated by methacryloyl chloride.