Tomáš Suchý

Collagen hydrogels serve as biomimetic scaffolds that closely resemble the natural extracellular matrix, thus providing an ideal 3D biocompatible environment for cells. However, based on our previous experience, not all collagen isolates are capable of gelling, which appears to depend on the type, origin, species, age and sex of the source animal and the collagen isolation method applied. We therefore decided to evaluate porcine collagen-rich materials isolated from two different porcine genotypes applying two different specific isolation methods, and to analyse other main components, i.e., lipids and glycosaminoglycans, as well as amino acid composition and structural and morphological properties. While all the collagen isolates obtained were subjected to the gelling process, only one of them successfully gelled. In addition, the gelling ability of this isolate was confirmed repeatedly on collagens that were isolated from other pigs of the same porcine genotype. The results revealed that the gelling process proceeds via cooperation between the composition and the structure of the collagen isolate. With respect to the composition, one of the most important factors in terms of the success of the gelation process of collagen isolates concerns elevated glycosaminoglycan contents. The structural factors that characterise collagen isolates, i.e., cross-links (immature and mature) and their mutual ratio, as well as the presence of telopeptides, strongly impact the progress of the gelling process and the resulting character of the hydrogel structure. All these factors are influenced by the isolation procedure.

Collagen nanofibrous materials have become integral to tissue engineering due to their exceptional properties and biocompatibility. Dehydrothermal crosslinking (DHT) enhances stability and maintains structural integrity without the formation of toxic residues. The study involved the crosslinking of electrospun collagen, applying DHT with access to air and under vacuum conditions. Various DHT exposure times of up to 72 h were applied to examine the time dependance of the DHT process. The DHT crosslinked collagen was subsequently chemically crosslinked using carbodiimides. The material crosslinked in this way evinced elevated Young’s modulus values and ultimate tensile strength values, a lower swelling rate and lower shrinkage ratio during crosslinking, and a higher degree of resistance to degradation than the material crosslinked solely with DHT or carbodiimides. It was shown that the crosslinking mechanism using DHT occupies different binding sites than those using chemical crosslinking. Access to air for 12 h or less did not exert a significant impact on the material properties compared to DHT under vacuum conditions. However, concerning longer exposure times, it was determined that access to air results in the deterioration of the properties of the material and that reactions take place that occupy the free bonding sites, which subsequently reduces the effectiveness of chemical crosslinking using carbodiimides.

Diamond-like carbon (DLC) layers are known for their high corrosion and wear resistance, low friction, and high biocompatibility. However, it is often necessary to dope DLC layers with additional chemical elements to strengthen their adhesion to the substrate. Ti-DLC layers (doped with 0.4, 2.1, 3.7, 6.6, and 12.8 at.% of Ti) were prepared by dual pulsed laser deposition, and pure DLC, glass, and polystyrene (PS) were used as controls. In vitro cell–material interactions were investigated with an emphasis on cell adhesion, proliferation, and osteogenic differentiation. We observed slightly increasing roughness and contact angle and decreasing surface free energy on Ti-DLC layers with increasing Ti content. Three-week biological experiments were performed using adipose tissue-derived stem cells (ADSCs) and bone marrow mesenchymal stem cells (bmMSCs) in vitro. The cell proliferation activity was similar or slightly higher on the Ti-doped materials than on glass and PS. Osteogenic cell differentiation on all materials was proved by collagen and osteocalcin production, ALP activity, and Ca deposition. The bmMSCs exhibited greater initial proliferation potential and an earlier onset of osteogenic differentiation than the ADSCs. The ADSCs showed a slightly higher formation of focal adhesions, higher metabolic activity, and Ca deposition with increasing Ti content.

Diamond-like carbon (DLC) layers are known for their high corrosion and wear resistance, low friction, and high biocompatibility. However, it is often necessary to dope DLC layers with additional chemical elements to strengthen their adhesion to the substrate. Ti-DLC layers (doped with 0.4, 2.1, 3.7, 6.6, 12.8 at.% of Ti) were prepared by dual pulsed laser deposition, and pure DLC, glass, and polystyrene (PS) were used as controls. In vitro cell-material interactions were investigated with an emphasis on cell adhesion, proliferation, and osteogenic differentiation. We observed slightly increasing roughness and contact angle and decreasing surface free energy on Ti-DLC layers with increasing Ti content. Three-week biological experiments were performed using adipose tissue-derived stem cells (ADSCs) and bone marrow mesenchymal stem cells (bmM-SCs) in vitro. The cell proliferation activity was similar or slightly higher on the Ti-doped materials than on glass and PS. Osteogenic cell differentiation on all materials was proved by collagen and osteocalcin production, ALP activity, and Ca deposition. The bmMSCs exhibited greater initial proliferation potential and an earlier onset of osteogenic differentiation than the ADSCs. The ADSCs showed a slightly higher formation of focal adhesions, higher metabolic activity, and Ca deposition with increasing Ti content.

The physical properties and structure of collagen treated with high-pressure technologies have not yet been investigated in detail. The main goal of this work was to determine whether this modern gentle technology significantly changes the properties of collagen. High pressure in the range of 0–400 MPa was used, and the rheological, mechanical, thermal, and structural properties of collagen were measured. The rheological properties measured in the area of linear viscoelasticity do not statistically significantly change due to the influence of pressure or the duration of pressure exposure. In addition, the mechanical properties measured by compression between two plates are not statistically significantly influenced by pressure value or pressure hold time. The thermal properties T_on and ∆H measured by differential calorimetry depend on pressure value and pressure hold time. Results from amino acids and FTIR analyses show that exposure of collagenous gels to high pressure (400 MPa), regardless of applied time (5 and 10 min), caused only minor changes in the primary and secondary structure and preserved collagenous polymeric integrity. SEM analysis did not show changes in collagen fibril ordering orientation over longer distances after applying 400 MPa of pressure for 10 min.

Scaffolds made of degradable polymers, such as collagen, polyesters or polysaccharides, are promising matrices for fabrication of bioartificial vascular grafts or patches. In this study, collagen isolated from porcine skin was processed into a gel, reinforced with collagen particles and with incorporated adipose tissue-derived stem cells (ASCs). The cell-material constructs were then incubated in a DMEM medium with 2% of FS (DMEM_part), with added polyvinylalcohol nanofibers (PVA_part sample), and for ASCs differentiation towards smooth muscle cells (SMCs), the medium was supplemented either with human platelet lysate released from PVA nanofibers (PVA_PL_part) or with TGF-β1 + BMP-4 (TGF + BMP_part). The constructs were further endothelialised with human umbilical vein endothelial cells (ECs). The immunofluorescence staining of alpha-actin and calponin, and von Willebrand factor, was performed. The proteins involved in cell differentiation, the extracellular matrix (ECM) proteins, and ECM remodelling proteins were evaluated by mass spectrometry on day 12 of culture. Mechanical properties of the gels with ASCs were measured via an unconfined compression test on day 5. Gels evinced limited planar shrinkage, but it was higher in endothelialised TGF + BMP_part gel. Both PVA_PL_part samples and TGF + BMP_part samples supported ASC growth and differentiation towards SMCs, but only PVA_PL_part supported homogeneous endothelialisation. Young modulus of elasticity increased in all samples compared to day 0, and PVA_PL_part gel evinced a slightly higher ratio of elastic energy. The results suggest that PVA_PL_part collagen construct has the highest potential to remodel into a functional vascular wall.