In vitro culture increases mechanical stability of human tissue engineered cartilage constructs by prevention of microscale scaffold buckling.

Many studies have measured the global compressive properties of tissue engineered (TE) cartilage grown on porous scaffolds. Such scaffolds are known to exhibit strain softening due to local buckling under loading. As matrix is deposited onto these scaffolds, the global compressive properties increase. However the relationship between the amount and distribution of matrix in the scaffold and local buckling is unknown. To address this knowledge gap, we studied how local strain and construct buckling in human TE constructs changes over culture times and GAG content. Confocal elastography techniques and digital image correlation (DIC) were used to measure and record buckling modes and local strains. Receiver operating characteristic (ROC) curves were used to quantify construct buckling. The results from the ROC analysis were placed into Kaplan-Meier survival function curves to establish the probability that any point in a construct buckled. These analysis techniques revealed the presence of buckling at early time points, but bending at later time points. An inverse correlation was observed between the probability of buckling and the total GAG content of each construct. This data suggests that increased GAG content prevents the onset of construct buckling and improves the microscale compressive tissue properties. This increase in GAG deposition leads to enhanced global compressive properties by prevention of microscale buckling.

Correlating rheological properties and printability of collagen bioinks: the effects of riboflavin photocrosslinking and pH.

Collagen has shown promise as a bioink for extrusion-based bioprinting, but further development of new collagen bioink formulations is necessary to improve their printability. Screening these formulations by measuring print accuracy is a costly and time consuming process. We hypothesized that rheological properties of the bioink before, during, and/or after gelation can be used to predict printability. In this study, we investigated the effects of riboflavin photocrosslinking and pH on type I collagen bioink rheology before, during, and after gelation and directly correlated these findings to the printability of each bioink formulation. From the riboflavin crosslinking study, results showed that riboflavin crosslinking increased the storage moduli of collagen bioinks, but the degree of improvement was less pronounced at higher collagen concentrations. Dots printed with collagen bioinks with riboflavin crosslinking exhibited smaller dot footprint areas than those printed with collagen bioinks without riboflavin crosslinking. From the pH study, results showed that gelation kinetics and final gel moduli were highly pH dependent and both exhibited maxima around pH 8. The shape fidelity of printed lines was highest at pH 8-9.5. The effect of riboflavin crosslinking and pH on cell viability was assessed using bovine chondrocytes. Cell viability in collagen gels was found to decrease after blue light activated riboflavin crosslinking but was not affected by pH. Correlations between rheological parameters and printability showed that the modulus associated with the bioink immediately after extrusion and before deposition was the best predictor of bioink printability. These findings will allow for the more rapid screening of collagen bioink formulations.

Mechanical properties and structure-function relationships of human chondrocyte-seeded cartilage constructs after in vitro culture.

Autologous Chondrocyte Implantation (ACI) is a widely recognized method for the repair of focal cartilage defects. Despite the accepted use, problems with this technique still exist, including graft hypertrophy, damage to surrounding tissue by sutures, uneven cell distribution, and delamination. Modified ACI techniques overcome these challenges by seeding autologous chondrocytes onto a 3D scaffold and securing the graft into the defect. Many studies on these tissue engineered grafts have identified the compressive properties, but few have examined frictional and shear properties as suggested by FDA guidance. This study is the first to perform three mechanical tests (compressive, frictional, and shear) on human tissue engineered cartilage. The objective was to understand the complex mechanical behavior, function, and changes that occur with time in these constructs grown in vitro using compression, friction, and shear tests. Safranin-O histology and a DMMB assay both revealed increased sulfated glycosaminoglycan (sGAG) content in the scaffolds with increased maturity. Similarly, immunohistochemistry revealed increased lubricin localization on the construct surface. Confined compression and friction tests both revealed improved properties with increased construct maturity. Compressive properties correlated with the sGAG content, while improved friction coefficients were attributed to increased lubricin localization on the construct surfaces. In contrast, shear properties did not improve with increased culture time. This study suggests the various mechanical and biological properties of tissue engineered cartilage improve at different rates, indicating thorough mechanical evaluation of tissue engineered cartilage is critical to understanding the performance of repaired cartilage. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2298-2306, 2017.

A comparison of three techniques for quantitative carbohydrate analysis used in characterization of therapeutic antibodies.

A comparison of three techniques for quantitative analysis of galactosylation present on immunoglobulins is described. ESIMS, MALDI-TOF MS, and anion-exchange chromatography with fluorescence detection were evaluated in terms of repeatability, limit of quantitation, selectivity, and linearity. A recombinant monoclonal IgG was enzymatically modified in vitro to produce essentially completely galactosylated and degalactosylated forms of the immunoglobulin. Samples of known galactosylation levels were prepared by mixing the modified forms with the native form of the immunoglobulin. Good repeatability and linearity were demonstrated for all three assays (RSDs<1.0%, correlation coefficients>0.99). Differences in selectivity, sensitivity, and other performance aspects of the three techniques are also discussed in this paper.

Rapid quantitative capillary zone electrophoresis method for monitoring the micro-heterogeneity of an intact recombinant glycoprotein.

A simple high-resolution capillary zone electrophoresis (CZE) method capable of rapidly assessing the micro-heterogeneity of a 24 kDa molecular weight glycoprotein, has been developed. Separation is carried out using a bare silica capillary at a pH of 2.5 in a commercially available electrophoresis buffer system composed of triethanolamine and phosphoric acid. Over 30 peaks were detected within a run time of 15 min using a 27 cm capillary and approximately 60 peaks were detected using a 77 cm capillary. Although most of the peaks arise from differences in the oligosaccharide structures present on the one glycosylation site on this molecule, other forms of micro-heterogeneity due to the presence of the nonglycosylated form of this glycoprotein and various types of chemical degradation, e.g., deamidation, are also responsible for the multitude of peaks observed. Although the exact chemical identity of each peak in the resulting electropherogram of this glycoprotein is not known, useful information can be obtained for assessing comparability, stability, and batch consistency. Factors impacting the resolution, precision, accuracy, and robustness of the assay are also discussed along with inherent advantages and limitations associated with measuring the micro-heterogeneity of intact glycoproteins.