Quantification of Cell Subpopulations, Fractions of Dead Cells and Debris in Cell Suspensions by Laser Diffractometry

Laser diffractometry was employed for size analysis in liver cell and blood cell suspensions to assess its suitability for characterizing cell populations. The method proved sensitive to detect subpopulations in liver cells (bimodal or trimodal distributions) and to quantify their volume fractions. Cell debris and aggregates of cells could also be quantified, dead cell populations recognized by their shift in the mean cell diameter. Laser diffractometry is therefore suitable for determining the quality of cell isolations (e.g. by liver perfusion) or for following alterations in cell populations during culture of cells in suspension. Analysis of human blood allowed differenciations to be made between thrombocytes and other blood cells. No peak separation was obtained for the populations of erythrocytes and granulocytes due to their similarity in size. Monocytes could not be detected due to their extremely low number in the blood indicating the limit of the metho

Effects of Bone Morphogenic Proteins on Engineered Cartilage

A report describes experiments on the effects of bone morphogenic proteins (BMPs) on engineered cartilage grown in vitro. In the experiments, bovine calf articular chondrocytes were seeded onto biodegradable polyglycolic acid scaffolds and cultured in, variously, a control medium or a medium supplemented with BMP-2, BMP-12, or BMP-13 in various concentrations. Under all conditions investigated, cell-polymer constructs cultivated for 4 weeks macroscopically and histologically resembled native cartilage. At a concentration of 100 ng/mL, BMP-2, BMP-12, or BMP-13 caused (1) total masses of the constructs to exceed those of the controls by 121, 80, or 62 percent, respectively; (2) weight percentages of glycosaminoglycans in the constructs to increase by 27, 18, or 15, respectively; and (3) total collagen contents of the constructs to decrease to 63, 89, or 83 percent of the control values, respectively. BMP-2, but not BMP-12 or BMP-13, promoted chondrocyte hypertrophy. These observations were interpreted as suggesting that the three BMPs increase the growth rates and modulate the compositions of engineered cartilage. It was also concluded that in vitro engineered cartilage is a suitable system for studying effects of BMPs on chondrogenesis in a well-defined environment

Biomechanical Analysis of the Efficacy of Locking Plates during Cyclic Loading in Metacarpal Fractures

Purpose. To analyse the biomechanical characteristics of locking plates under cyclic loading compared to a nonlocking plate in a diaphyseal metacarpal fracture. Methods. Oblique diaphyseal shaft fractures in porcine metacarpal bones were created in a biomechanical fracture model. An anatomical reduction and stabilization with a nonlocking and a comparable locking plate in mono- or bicortical screw fixation followed. Under cyclic loading, the displacement, and in subsequent load-to-failure tests, the maximum load and stiffness were measured. Results. For the monocortical screw fixation of the locking plate, a similar displacement, maximum load, and stiffness could be demonstrated compared to the bicortical screw fixation of the nonlocking plate. Conclusions. Locking plates in monocortical configuration may function as a useful alternative to the currently common treatment with bicortical fixations. Thereby, irritation of the flexor tendons would be avoided without compromising the stability, thus enabling the necessary early functional rehabilitation

Bonding of articular cartilage using a combination of biochemical degradation and surface cross-linking

After trauma, articular cartilage often does not heal due to incomplete bonding of the fractured surfaces. In this study we investigated the ability of chemical cross-linkers to facilitate bonding of articular cartilage, either alone or in combination with a pre-treatment with surface-degrading agents. Articular cartilage blocks were harvested from the femoropatellar groove of bovine calves. Two cartilage blocks, either after pre-treatment or without, were assembled in a custom-designed chamber in partial apposition and subjected to cross-linking treatment. Subsequently, bonding of cartilage was measured as adhesive strength, that is, the maximum force at rupture of bonded cartilage blocks divided by the overlap area. In a first approach, bonding was investigated after treatment with cross-linking reagents only, employing glutaraldehyde, 1-ethyl-3-diaminopropyl-carbodiimide (EDC)/N-hydroxysuccinimide (NHS), genipin, or transglutaminase. Experiments were conducted with or without compression of the opposing surfaces. Compression during cross-linking strongly enhanced bonding, especially when applying EDC/NHS and glutaraldehyde. Therefore, all further experiments were performed under compressive conditions. Combinations of each of the four cross-linking agents with the degrading pre-treatments, pepsin, trypsin, and guanidine, led to distinct improvements in bonding compared to the use of cross-linkers alone. The highest values of adhesive strength were achieved employing combinations of pepsin or guanidine with EDC/NHS, and guanidine with glutaraldehyde. The release of extracellular matrix components, that is, glycosaminoglycans and total collagen, from cartilage blocks after pre-treatment was measured, but could not be directly correlated to the determined adhesive strength. Cytotoxicity was determined for all substances employed, that is, surface degrading agents and cross-linkers, using the resazurin assay. Taking the favourable cell vitality after treatment with pepsin and EDC/NHS and the cytotoxic effects of guanidine and glutaraldehyde into account, the combination of pepsin and EDC/NHS appeared to be the most advantageous treatment in this study. In conclusion, bonding of articular cartilage blocks was achieved by chemical fixation of their surface components using cross-linking reagents. Application of compressive forces and prior modulation of surface structures enhanced cartilage bonding significantly. Enzymatic treatment in combination with cross-linkers may represent a promising addition to current techniques for articular cartilage repair

Shaping Synthetic Multicellular and Complex Multimaterial Tissues via Embedded Extrusion-Volumetric Printing of Microgels

In living tissues, cells express their functions following complex signals from their surrounding microenvironment. Capturing both hierarchical architectures at the micro- and macroscale, and anisotropic cell patterning remains a major challenge in bioprinting, and a bottleneck toward creating physiologically-relevant models. Addressing this limitation, a novel technique is introduced, termed Embedded Extrusion-Volumetric Printing (EmVP), converging extrusion-bioprinting and layer-less, ultra-fast volumetric bioprinting, allowing spatially pattern multiple inks/cell types. Light-responsive microgels are developed for the first time as bioresins (µResins) for light-based volumetric bioprinting, providing a microporous environment permissive for cell homing and self-organization. Tuning the mechanical and optical properties of gelatin-based microparticles enables their use as support bath for suspended extrusion printing, in which features containing high cell densities can be easily introduced. µResins can be sculpted within seconds with tomographic light projections into centimeter-scale, granular hydrogel-based, convoluted constructs. Interstitial microvoids enhanced differentiation of multiple stem/progenitor cells (vascular, mesenchymal, neural), otherwise not possible with conventional bulk hydrogels. As proof-of-concept, EmVP is applied to create complex synthetic biology-inspired intercellular communication models, where adipocyte differentiation is regulated by optogenetic-engineered pancreatic cells. Overall, EmVP offers new avenues for producing regenerative grafts with biological functionality, and for developing engineered living systems and (metabolic) disease models

Biofabrication : reappraising the definition of an evolving field

Biofabrication is an evolving research field that has recently received significant attention. In particular, the adoption of Biofabrication concepts within the field of Tissue Engineering and Regenerative Medicine has grown tremendously, and has been accompanied by a growing inconsistency in terminology. This article aims at clarifying the position of Biofabrication as a research field with a special focus on its relation to and application for Tissue Engineering and Regenerative Medicine. Within this context, we propose a refined working definition of Biofabrication, including Bioprinting and Bioassembly as complementary strategies within Biofabrication

Characterization of Colloidal Drug Carriers

Hydrophobic interaction chromatography (HIC) is presented as a suitable method to determine the surface hydrophobicity of colloidal drug carriers. The experimental design is described in great detail. Results obtained with surface-modified polystyrene particles as model carriers and parenteral fat emulsions are discussed as examples. HIC was able to distinguish between particles chemically modified by the introduction of functional groups. Polystyrene particles of various size were surface-modified by adsorption of the block-polymer Poloxamine 908. HIC distinguished between the hydrophobicities of these adsorption layers. The measured surface hydrophobicities of particles and fat emulsions were well in agreement with the in vivo organ distribution data obtained after i. v. injection of the carriers

Colloidal carriers for intravenous drug targeting: plasma protein adsorption patterns on surface-modified latex particles evaluated by two-dimensional polyacrylamide gel electrophoresis

Targeting to specific sites of the body via colloidal carriers is sought in order to reduce drug side effects. The adsorption of plasma proteins on intravenously injected particles is regarded as the key factor in explaining their organ distribution: total bound protein, or, more likely, the presence of specific proteins and their conformation, are expected to influence macrophage uptake. Polystyrene beads, 60 nm in diameter, were used as model carriers; their surface was differentially modified by adsorption of increasingly hydrophilic block copolymers, poloxamers 184, 188 and 407. After incubation in plasma, the patterns of protein adsorption onto coated beads were analyzed by high-resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The behavior of some representative proteins was monitored, including albumin, fibrinogen, IgG, factor B and the apolipoproteins, A-I, A-IV, C-III, E and J. The more hydrophobic the particles, the larger the total amount of bound protein. However, this correlation was not valid for all of the analyzed protein species, which proves that it is insufficient to look only at physicochemical data to predict organ distribution. On the contrary, it is essential to use 2-D PAGE to establish the correlation between adsorbed proteins and carrier behavior in vivo