ABSTRACT
The distribution of photo-crosslinkable moieties onto a protein backbone can affect a biomaterial's crosslinking behavior, and therefore also its mechanical and biological properties. A profound insight in this respect is essential for biomaterials exploited in tissue engineering and regenerative medicine. In the present work, photo-crosslinkable moieties have been introduced on the primary amine groups of: (i) a recombinant collagen peptide (RCPhC1) with a known amino acid (AA) sequence, and (ii) bovine skin collagen (COL BS) with an unknown AA sequence. The degree of substitution (DS) was quantified with two conventional techniques: an ortho-phthalic dialdehyde (OPA) assay and 1H NMR spectroscopy. However, neither of both provides information on the exact type and location of the modified AAs. Therefore, for the first time, proteomic analysis was evaluated herein as a tool to identify functionalized AAs as well as the exact position of photo-crosslinkable moieties along the AA sequence, thereby enabling an in-depth, unprecedented characterization of functionalized photo-crosslinkable biopolymers. Moreover, our strategy enabled to visualize the spatial distribution of the modifications within the overall structure of the protein. Proteomics has proven to provide unprecedented insight in the distribution of photo-crosslinkable moieties along the protein backbone, undoubtedly contributing to superior functional biomaterial design to serve regenerative medicine.
ABSTRACT
The placenta is a transient organ, essential for development and survival of the unborn fetus. It interfaces the body of the pregnant woman with the unborn child and secures transport of endogenous and exogenous substances. Maternal and fetal blood are thereby separated at any time, by the so-called placental barrier. Current in vitro approaches fail to model this multifaceted structure, therefore research in the field of placental biology is particularly challenging. The present study aimed at establishing a novel model, simulating placental transport and its implications on development, in a versatile but reproducible way. The basal membrane was replicated using a gelatin-based material, closely mimicking the composition and properties of the natural extracellular matrix. The microstructure was produced by using a high-resolution 3D printing method - the two-photon polymerization (2PP). In order to structure gelatin by 2PP, its primary amines and carboxylic acids are modified with methacrylamides and methacrylates (GelMOD-AEMA), respectively. High-resolution structures in the range of a few micrometers were produced within the intersection of a customized microfluidic device, separating the x-shaped chamber into two isolated cell culture compartments. Human umbilical-vein endothelial cells (HUVEC) seeded on one side of this membrane simulate the fetal compartment while human choriocarcinoma cells, isolated from placental tissue (BeWo B30) mimic the maternal syncytium. This barrier model in combination with native flow profiles can be used to mimic the microenvironment of the placenta, investigating different pharmaceutical, clinical and biological scenarios. As proof-of-principle, this bioengineered placental barrier was used for the investigation of transcellular transport processes. While high molecular weight substances did not permeate, smaller molecules in the size of glucose were able to diffuse through the barrier in a time-depended manner. We envision to apply this bioengineered placental barrier for pathophysiological research, where altered nutrient transport is associated with health risks for the fetus.
ABSTRACT
In the present work, porous gelatin scaffolds were prepared by cryogenic treatment of a chemically cross-linked gelatin hydrogel, followed by removal of the ice crystals formed through lyophilization. This technique often leads to porous gels with a less porous skin. A simple method has been developed to solve this problem. The present study demonstrates that the hydrogel pore size decreased with an increasing gelatin concentration and with an increasing cooling rate of the gelatin hydrogel. Variation of the cryogenic parameters applied also enabled us to develop scaffolds with different pore morphologies (spherical versus transversal channel-like pores). In our opinion, this is the first paper in which temperature gradients during controlled cryogenic treatment were applied to induce a pore size gradient in gelatin hydrogels. With a newly designed cryo-unit, temperature gradients of 10 and 30 degrees C were implemented during the freezing step, resulting in scaffolds with average pore diameters of, respectively, +/-116 and +/-330 microm. In both cases, the porosity and pore size decreased gradually through the scaffolds. Pore size and structure analysis of the matrices was accomplished through a combination of microcomputed tomography using different software packages (microCTanalySIS and Octopus), scanning electron microscopy analysis, and helium pycnometry.
Subject(s)
Freezing , Hydrogels/chemistry , Cross-Linking Reagents , Freeze Drying , Gelatin , Porosity , TemperatureABSTRACT
We report on the feasibility of applying porous gelatin hydrogels, prepared by a novel and controlled cryogenic treatment, as cell-interactive scaffolds for tissue engineering applications. Despite the large number of publications on gelatin as a biomaterial, a detailed study of screening a limited number of gelatin scaffolds for their interaction with a panel of human cells has, to the best of our knowledge, not yet been published. In the present work, we have evaluated two types of porous gelatin scaffolds that differ in their pore geometry and pore size. Type I hydrogels contained top-to-bottom transverse channels (i.e. cones) with a decreasing diameter from the top (330 microm) to the bottom (20-30 microm). Type II hydrogels contained spherical pores with a diameter of 135 microm. Both types of scaffolds were evaluated by confocal laser scanning microscopy in terms of adhesion, spreading, and proliferation of human cells (endothelial, epithelial, fibroblast, glial, and osteoblast) by visualizing cells using calcein-acetoxy methyl ester as a vital stain. The results indicated that cells attached, spread, and proliferated on both types of hydrogels. In addition, the scaffolds developed can be used for the long-term culturing of human cells.
