Development Of Tissue-engineered Constructs Research Articles

Modification of Biodegradable Polymer Nanofibers for Cartilage Tissue Engineering Applications: A Review

Tissue engineering is the use of a combination of cells, engineering and materials methods, and suitable biochemical and physiochemical factors to improve or replace biological functions at the injured site. The designing of a biomaterial that can mimic the three-dimensional tissues in vivo is still challenging. Biodegradable polymers are used for the development of tissue engineering constructs in the form of sponges, films, and macroporous scaffolds, which do not influence cell fate processes such as cell differentiation, migration, and proliferation. Biodegradable polymer nanofibers fabricated by electrospinning have gathered great attention in tissue engineering applications. The electrospun materials have a nanofibrous morphology that is closest to the natural extracellular matrix (ECM). The electrospun material is composed of three-dimensional networks of nanosized fibrous materials that mimic an extracellular matrix such as collagen, elastin, and keratin. These polymers fabricated in the form of fibers in nanosize cause a more favorable microenvironment for cells. We prepared the PLGA/PPG (polylactic-co-glycolic acid/polypropylene glycol) nanofibers by electrospinning technique. PLGA (85:15)/PLGA (75:25) nanofibers are hydrophobic, which can be minimized by the addition of PPG to give better hydrophilicity for cell adhesion for tissue engineering constructs. The morphology of the electrospun fibers of the composite of PLGA and PPG was observed using scanning electron microscopy (SEM). The results proved that the small amount of polypropylene glycol polymer to the polylactic-co-glycolic acid (PLGA 85:15) drastically improves the hydrophilicity of the electrospun nanofibers. The addition of a small amount of hydrophilic polymer to the biodegradable hydrophobic polymer increases the hydrophilic property and can be used for nanofiber-based tissue engineering constructs. In addition, the biomimetic approach for tissue engineering scaffolds for cartilage repair has been discussed.

Mechanical characterization of soft biomaterials: which time and spatial scale to choose?

The mechanical properties of soft gels hold significant relevance in biomedicine and biomaterial design, including the development of tissue engineering constructs and bioequivalents. It is important to adequately characterize the gel's mechanical properties since they play a role both in the overall structural properties of the construct and the physiological responses of cells. The question remains which approach for the mechanical characterization is most suitable for specific biomaterials. Our investigation is centered on the comparison of three types of gels and four distinct mechanical testing techniques: shear rheology, compression, microindentation, and nanoindentation by atomic force microscopy. While analyzing an elastic homogeneous synthetic hydrogel (a polyacrylamide gel), we observed close mechanical results across the different testing techniques. However, our findings revealed more distinct outcomes when assessing a highly viscoelastic gel (Ecoflex) and a heterogeneous biopolymer hydrogel (enzymatically crosslinked gelatin). To ensure precise data interpretation, we introduced correction factors to account for the boundary conditions inherent in many of the testing methods. The results of this study underscore the critical significance of considering both the temporal and spatial scales in mechanical measurements of biomaterials. Furthermore, they encourage the employment of a combination of diverse testing techniques, particularly in the characterization of heterogeneous viscoelastic materials such as biological samples. The obtained results will contribute to the refinement of mechanical testing protocols and advance the development of soft gels for tissue engineering.

Development of a testing machine for biaxial testing of soft tissue and biomaterials

Knowledge of the mechanical properties of soft tissues and biomaterials is the keystone for predictive modeling in cardio- and endovascular surgery, traumatology and orthopedics, neurosurgery, and the development of tissue-engineering constructs. Needed mechanical properties can be only explored via experimental tests. Therefore, mechanical experiments are forming the foundation of medical decision support systems, which are impossible without adequate mathematical models of the processes involved. Sirius University of Science and Technology has launched the project "Experimental research and mathematical modeling of the mechanical behavior of biomaterials". The goal of the project is to create an infrastructure for experimental study of the mechanics of soft tissues and biomaterials. At this stage, a machine for biaxial testing of soft tissues and biomaterials has been developed and put into trial operation. The developed machine takes into account the features of working with soft tissues: small sample size, maintaining certain environmental conditions to preserve the sample, various types of sample gripping to minimize edge effects and no slippage, non-contact methods for assessing displacement and deformation fields, variability in sample loading protocols. The result of the test is a data set that includes a test report and synchronized output readings of strain gauge, temperature, optoelectronic sensors with high-resolution images. Uniaxial verification tests of acrylic tapes VHB4910 were carried out by the developed machine. The results were compared with data obtained on a commercial dynamic mechanical analyzer RSA-G2 (TA Instruments) for similar materials under the same test conditions. A series of biaxial cyclic experiments were carried out for the pericardium of cattle, the cardiovascular patch Ecofolon PS04/PS01 (JSC NPK Ecoflon, St. Petersburg, Russia), and the GoreTex vascular graft (W.L. Gore and Associates, USA).

Evaluation of the Feasibility of Endothelial Colony-Forming Cells to Develop Tissue-Engineered Vascular Grafts Based on the Gene Expression Profile Analysis.

In the experiment, we used the endothelial colony-forming cells (ECFC) obtained from the peripheral blood of patients who underwent percutaneous coronary intervention. The cells were isolated on a Histopaque 1077 density gradient (Sigma-Aldrich, USA), and then cultured in EGM-2MV culture medium (Lonza, Switzerland). A commercial culture of primary human coronary artery endothelial cells (HCAEC) was used as a control. The cells were unfrozen and cultured according to the manufacturer's recommendations in MesoEndo Cell Growth Medium (Cell Applications, USA).The experiment was carried out in specialized μ-Luer plates in the perfusion system (IBIDI, Germany), which provided a continuous unidirectional flow of the culture medium with a shear stress of 5 dyn/cm2. Control plates were cultured under standard conditions for a similar period of time. Total RNA was isolated from cell samples. The expression of the genes NOTCH4, NRP2, PLAT, PLAU, NOTCH1, FLT1, COL4A2, CD34, SERPINE1, HEY2, MKI67, KLF4, LYVE1, FLT4 was assessed using a quantitative real-time polymerase chain reaction. The expression of the genes was calculated by the ΔCt method and expressed on a logarithmic (log10) scale as a fold change relating to the control samples. In mature endothelial cells HCAEC when exposed to a laminar flow, only the transcription factor KLF4 and venous differentiation NRP2 marker values increased significantly. ECFC showed statistically significant growth in KLF4, NRP2, CD34, and LYVE1, as well as PLAU expression decrease. In addition, we observed the overexpression of FLT4, LYVE1, NOTCH4, and NRP2 in ECFC in relation to HCAEC and HEY2 hypoexpression. CD34 overexpression characteristic of progenitor cells was also found. An increase in COL4A2 expression associated with type IV collagen synthesis was a characteristic feature of ECFC. The gene expression profile of endothelial colony-forming cells is quite close to that of primary endothelial cells of the human coronary artery, and thus, the cells obtained from patients' peripheral blood can be used to develop personalized tissue-engineered constructs.

Angiogenic Potential of VEGF Mimetic Peptides for the Biofunctionalization of Collagen/Hydroxyapatite Composites.

Currently, the focus on bioinspired concepts for the development of tissue engineering constructs is increasing. For this purpose, the combination of collagen (Coll) and hydroxyapatite (HA) comes closest to the natural composition of the bone. In order to confer angiogenic properties to the scaffold material, vascular endothelial growth factor (VEGF) is frequently used. In the present study, we used a VEGF mimetic peptide (QK) and a modified QK-peptide with a poly-glutamic acid tag (E7-QK) to enhance binding to HA, and analyzed in detail binding efficiency and angiogenic properties. We detected a significantly higher binding efficiency of E7-QK peptides to hydroxyapatite particles compared to the unmodified QK-peptide. Tube formation assays revealed similar angiogenic functions of E7-QK peptide (1µM) as induced by the entire VEGF protein. Analyses of gene expression of angiogenic factors and their receptors (FLT-1, KDR, HGF, MET, IL-8, HIF-1α, MMP-1, IGFBP-1, IGFBP-2, VCAM-1, and ANGPT-1) showed higher expression levels in HUVECs cultured in the presence of 1 µM E7-QK and VEGF compared to those detected in the negative control group without any angiogenic stimuli. In contrast, the expression of the anti-angiogenic gene TIMP-1 showed lower mRNA levels in HUVECs cultured with E7-QK and VEGF. Sprouting assays with HUVEC spheroids within Coll/HA/E7-QK scaffolds showed significantly longer sprouts compared to those induced within Coll/HA/QK or Coll/HA scaffolds. Our results demonstrate a significantly better functionality of the E7-QK peptide, electrostatically bound to hydroxyapatite particles compared to that of unmodified QK peptide. We conclude that the used E7-QK peptide represents an excellently suited biomolecule for the generation of collagen/hydroxyapatite composites with angiogenic properties.

Cell-assembled extracellular matrix (CAM) sheet production: Translation from using human to large animal cells

We have created entirely biological tissue-engineered vascular grafts (TEVGs) using sheets of cell-assembled extracellular matrix (CAM) produced by human fibroblasts in vitro. A large animal TEVG would allow long-term pre-clinical studies in a clinically relevant setting (graft size and allogeneic setting). Therefore, canine, porcine, ovine, and human skin fibroblasts were compared for their ability to form CAM sheets. Serum sourcing greatly influenced CAM production in a species-dependent manner. Ovine cells produced the most homogenous and strongest animal CAM sheets but remained ≈3-fold weaker than human sheets despite variations of serum, ascorbate, insulin, or growth factor supplementations. Key differences in cell growth dynamics, tissue development, and tissue architecture and composition were observed between human and ovine. This study demonstrates critical species-to-species differences in fibroblast behavior and how they pose a challenge when attempting to substitute animal cells for human cells during the development of tissue-engineered constructs that require long-term cultures.

Biofabrication Strategies and Engineered In Vitro Systems for Vascular Mechanobiology.

The vascular system is integral for maintaining organ-specific functions and homeostasis. Dysregulation in vascular architecture and function can lead to various chronic or acute disorders. Investigation of the role of the vascular system in health and disease has been accelerated through the development of tissue-engineered constructs and microphysiological on-chip platforms. These in vitro systems permit studies of biochemical regulation of vascular networks and parenchymal tissue and provide mechanistic insights into the biophysical and hemodynamic forces acting in organ-specific niches. Detailed understanding of these forces and the mechanotransductory pathways involved is necessary to develop preventative and therapeutic strategies targeting the vascular system. This review describes vascular structure and function, the role of hemodynamic forces in maintaining vascular homeostasis, and measurement approaches for cell and tissue level mechanical properties influencing vascular phenomena. State-of-the-art techniques for fabricating in vitro microvascular systems, with varying degrees of biological and engineering complexity, are summarized. Finally, the role of vascular mechanobiology in organ-specific niches and pathophysiological states, and efforts to recapitulate these events using in vitro microphysiological systems, are explored. It is hoped that this review will help readers appreciate the important, but understudied, role of vascular-parenchymal mechanotransduction in health and disease toward developing mechanotherapeutics for treatment strategies.

Development of Porous Hydrogel Scaffolds with Multiple Cues for Liver Tissue Engineering

Development of tissue-engineered construct for hepatic regeneration remains challenging due to the lack of potential milieu to control the trans-differentiation of hepatocytes. In the present study, galactose containing poly(vinyl alcohol) (PVA) and gelatin (8:2 and 9:1) hydrogels impregnated with hepatocyte growth factor (HGF)-loaded nanoparticles (241 ± 56 nm) using freeze/thaw technique was fabricated. The scaffolds with the HGF encapsulation efficiency of 57 ± 2.9% exhibited two-stage release kinetics for 30 days. Hepatogenic potential of the fabricated scaffolds were evaluated by determining the adhesion, viability, and proliferation with functional activity of rat primary hepatocytes for 28 days. Scaffolds of 8:2 ratios of PVA and gelatin maintained spheroidal morphology of hepatocytes and exhibited significantly higher metabolic secretions for 28 days (p < 0.05). Hence, galactose-based PVA-gelatin (8:2) hydrogel scaffold could be a promising alternative substrate to retain the phenotype and functional characteristics of hepatocytes due to the co-existence of bioactive motif and trophic factors. The developed 3D scaffold with growth factors encapsulated nanospheres possesses characteristic features for hepatic regeneration and is proposed to be a candidate material for further invivo evaluation

Microspheres leaching for scaffold porosity control

Scaffold morphology plays a key role in the development of tissue engineering constructs. The control of pore size, shape and interconnection is needed to achieve adequate nutrient transport and cell ingrowth. Several techniques are available for scaffold manufacturing, but none allows easy control of morphology and is, at the same time, applicable to a wide variety of materials. To investigate the possibility of processing a wide range polymers by solvent casting/particulate leaching with accurate control of scaffold morphology, three different porogens (gelatin microspheres, paraffin microspheres and sodium chloride crystals) were used to fabricate scaffolds from commonly employed biodegradable polymers. The outcome of processing was evaluated in terms of scaffold morphology and structure/properties relationships. Highly porous scaffolds were obtained with all porogens and well defined spherical pores resulted from microspheres leaching. Furthermore, scaffolds with spherical pores showed better mechanical performance and lower flow resistance. Cytocompatibility tests performed showed no evidence of processing residuals released from the scaffolds. Solvent casting/microspheres leaching, particularly gelatin microspheres leaching, can be used to process a large number of polymers and enables to tailor scaffold pore size, shape and interconnection, thus providing a powerful tool for material selection and optimization of scaffold morphology.