Xeno-Free In Vitro Cultivation and Osteogenic Differentiation of hAD-MSCs on Resorbable 3D Printed RESOMER®.

The development of alloplastic resorbable materials can revolutionize the field of implantation technology in regenerative medicine. Additional opportunities to colonize the three-dimensionally (3D) printed constructs with the patient's own cells prior to implantation can improve the regeneration process but requires optimization of cultivation protocols. Human platelet lysate (hPL) has already proven to be a suitable replacement for fetal calf serum (FCS) in 2D and 3D cell cultures. In this study, we investigated the in vitro biocompatibility of the printed RESOMER® Filament LG D1.75 materials as well as the osteogenic differentiation of human mesenchymal stem cells (hMSCs) cultivated on 3D printed constructs under the influence of different medium supplements (FCS, human serum (HS) and hPL). Additionally, the in vitro degradation of the material was studied over six months. We demonstrated that LG D1.75 is biocompatible and has no in vitro cytotoxic effects on hMSCs. Furthermore, hMSCs grown on the constructs could be differentiated into osteoblasts, especially supported by supplementation with hPL. Over six months under physiological in vitro conditions, a distinct degradation was observed, which, however, had no influence on the biocompatibility of the material. Thus, the overall suitability of the material LG D1.75 to produce 3D printed, resorbable bone implants and the promising use of hPL in the xeno-free cultivation of human MSCs on such implants for autologous transplantation have been demonstrated.

Comparison of different three dimensional-printed resorbable materials: In vitro biocompatibility, In vitro degradation rate, and cell differentiation support.

Biodegradable materials play a crucial role in both material and medical sciences and are frequently used as a primary commodity for implants generation. Due to their material inherent properties, they are supposed to be entirely resorbed by the patients' body after fulfilling their task as a scaffold. This makes a second intervention (e.g. for implant removal) redundant and significantly enhances a patient's post-operative life quality. At the moment, materials for resorbable and biodegradable implants (e.g. polylactic acid or poly-caprolactone polymers) are still intensively studied. They are able to provide mandatory demands such as mechanical strength and attributes needed for high-quality implants. Implants, however, not only need to be made of adequate material, but must also to be personalized in order to meet the customers' needs. Combining three dimensional-printing and high-resolution imaging technologies a new age of implant production comes into sight. Three dimensional images (e.g. magnetic resonance imaging or computed tomography) of tissue defects can be utilized as digital blueprints for personalized implants. Modern additive manufacturing devices are able to use a variety of materials to fabricate custom parts within short periods of time. The combination of high-quality resorbable materials and personalized three dimensional-printing for the custom application will provide the patients with the best suitable and sustainable implants. In this study, we evaluated and compared four resorbable and three dimensional printable materials for their in vitro biocompatibility, in vitro rate of degradation, cell adherence and behavior on these materials as well as support of osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells. The tests were conducted with model constructs of 1 cm2 surface area fabricated with fused deposition modeling three dimensional-printing technology.

Tracking dipeptides at work-uptake and intracellular fate in CHO culture.

Market demands for monoclonal antibodies (mAbs) are steadily increasing worldwide. As a result, production processes using Chinese hamster ovary cells (CHO) are in the focus of ongoing intensification studies for maximizing cell-specific and volumetric productivities. This includes the optimization of animal-derived component free (ADCF) cultivation media as part of good cell culture practice. Dipeptides are known to improve CHO culture performance. However, little or even conflicting assumptions exist about their putative import and functionality inside the cells. A set of well-known performance boosters and new dipeptide prospects was evaluated. The present study revealed that dipeptides are indeed imported in the cells, where they are decomposed to the amino acids building blocks. Subsequently, they are metabolized or, unexpectedly, secreted to the medium. Monoclonal antibody production boosting additives like L-alanine-L-glutamine (AQ) or glycyl-L-glutamine (GQ) can be assigned to fast or slow dipeptide uptake, respectively, thus pinpointing to the need to study dipeptide kinetics and to adjust their feeding individually for optimizing mAb production.

Amino acids in human and animal nutrition.

Amino acids are key components of human and animal nutrition, both as part of a protein-containing diet, and as supplemented individual products. In the last 10 years there has been a marked move away from the extraction of amino acids from natural products, which has been replaced by efficient fermentation processes using nonanimal carbon sources. Today several amino acids are produced in fermentation plants with capacities of more than 100,000 tonnes to serve the requirements of animal feed and human nutrition. The main fermentative amino acids for animal nutrition are L-lysine, L-threonine, and L-tryptophan. DL-Methionine continues to be manufactured for animal feed use principally by chemical synthesis, and a pharmaceutical grade is manufactured by enzymatic resolution. Amino acids play an important role in medical nutrition, particularly in parenteral nutrition, where there are high purity requirements for infusion grade products. Amino acids are also appearing more often in dietary supplements, initially for performance athletes, but increasingly for the general population. As the understanding of the effects of the individual amino acids on the human metabolism is deepened, more specialized product mixtures are being offered to improve athletic performance and for body-building.

Proline availability regulates proline-4-hydroxylase synthesis and substrate uptake in proline-hydroxylating recombinant Escherichia coli.

Microbial physiology plays a crucial role in whole-cell biotransformation, especially for redox reactions that depend on carbon and energy metabolism. In this study, regio- and enantio-selective proline hydroxylation with recombinant Escherichia coli expressing proline-4-hydroxylase (P4H) was investigated with respect to its interconnectivity to microbial physiology and metabolism. P4H production was found to depend on extracellular proline availability and on codon usage. Medium supplementation with proline did not alter p4h mRNA levels, indicating that P4H production depends on the availability of charged prolyl-tRNAs. Increasing the intracellular levels of soluble P4H did not result in an increase in resting cell activities above a certain threshold (depending on growth and assay temperature). Activities up to 5-fold higher were reached with permeabilized cells, confirming that host physiology and not the intracellular level of active P4H determines the achievable whole-cell proline hydroxylation activity. Metabolic flux analysis revealed that tricarboxylic acid cycle fluxes in growing biocatalytically active cells were significantly higher than proline hydroxylation rates. Remarkably, a catalysis-induced reduction of substrate uptake was observed, which correlated with reduced transcription of putA and putP, encoding proline dehydrogenase and the major proline transporter, respectively. These results provide evidence for a strong interference of catalytic activity with the regulation of proline uptake and metabolism. In terms of whole-cell biocatalyst efficiency, proline uptake and competition of P4H with proline catabolism are considered the most critical factors.

Deactivation of formate dehydrogenase (FDH) in solution and at gas-liquid interfaces.

Enzymes, increasingly important in the synthesis of fine chemicals and pharmaceutical intermediates, are often insufficiently stable under reacting conditions. We have investigated the stability, in homogeneous aqueous solution and at gas-liquid interfaces, of formate dehydrogenase (FDH), important for cofactor regeneration, from Candida boidinii and overexpressed in E. coli. When exposed to mechanical stress, residual activity, [E](t)/[E](0), and residual protein were found to scale proportionally with gas-liquid surface area in the bubble column, verifying a surface-driven process, and with time and total throughput in a gear pump, but did not seem to be influenced much by shear in a Couette viscometer. All FDH variants are deactivated by chaotropes but not kosmotropes: the first-order deactivation constant k(d) correlates well with the Jones-Dole coefficient B but not well with the surface tension increment deltasigma of various concentrated ammonium salt solutions. This finding might provide guidance for focusing the search for quantitative theories of Hofmeister effects.

Membrane reactors at Degussa.

The review covers the development of membrane reactor technologies at Degussa for the synthesis of fine chemicals. The operation of fed-batch or continuous biocatalytic processes in the enzyme membrane reactor (EMR) is well established at Degussa. Degussa has experience of running EMRs from laboratory gram scale up to a production scale of several hundreds of tons per year. The transfer of the enzyme membrane reactor from biocatalysis to chemical catalysis in the chemzyme membrane reactor (CMR) is discussed. Various homogeneous catalysts have been investigated in the CMR, and the scope and limitation of this new technique is discussed.