About 3D Printability of Thermoplastic Collagen for Biomedical Applications.

With more than 1.5 million total knee and hip implants placed each year, there is an urgent need for a drug delivery system that can effectively support the repair of bone infections. Scaffolds made of natural biopolymers are widely used for this purpose due to their biocompatibility, biodegradability, and suitable mechanical properties. However, the poor processability is a bottleneck, as highly customizable scaffolds are desired. The aim of the present research is to develop a scaffold made of thermoplastic collagen (TC) using 3D printing technology. The viscosity of the material was measured using a rheometer. A 3D bioplotter was used to fabricate the scaffolds out of TC. The mechanical properties of the TC scaffolds were performed using tension/compression testing on a Zwick/Roell universal testing machine. TC shows better compressibility with increasing temperature and a decrease in dynamic viscosity (η), storage modulus (G'), and loss modulus (G″). The compressive strength of the TC scaffolds was between 3-10 MPa, depending on the geometry (cylinder or cuboid, with different infills). We have demonstrated for the first time that TC can be used to fabricate porous scaffolds by 3D printing in various geometries.

Additive Manufacturing with Thermoplastic Collagen.

Thermoplastic collagen is a partially denatured collagen powder which can be processed by thermoplastic methods such as extrusion and injection molding, but was hitherto not adapted for the use in additive manufacturing (AM) techniques. This paper describes the first successful application of collagen/water/glycerol mixtures in an AM process using a BioScaffolder 3.2 from GeSiM mbH. Strands of molten collagen were deposited onto a building platform forming differently shaped objects. The collagen melt was characterized rheologically and optimal processing conditions were established. The technique includes the use of supporting structures of PLA/wood composite for samples with complex geometry as well as post-processing steps such as the removal of the supporting structure and manual surface smoothing. The manufactured objects are characterized concerning water solubility, swelling behavior and compressibility. Possible applications are in the non-medical sector and include collagen-based pet food or customized organ models for medical training.

Synthesis and structure-activity relationship of beta-defensins, multi-functional peptides of the immune system.

beta-defensins are a large family of multiple disulfide-bonded peptides occurring in mammals and birds. They play an important role in the innate immune system, directly killing microbial organisms. Recent research has demonstrated that beta-defensins are important for other biological functions beyond antimicrobial effects, including inhibition of viral infection, interaction with Toll-like receptors, chemotactic effects, and sperm function. The corresponding broad spectrum of activities makes this peptide class an important subject and tool in immunologic research. In this review, we summarize the current status of the routes to obtain synthetic beta-defensins, their major structural properties and structure-activity relationship.

Structure-activity relation of human beta-defensin 3: influence of disulfide bonds and cysteine substitution on antimicrobial activity and cytotoxicity.

Human beta-defensins form a group of cysteine-rich antimicrobial peptides which have been found in epithelial tissue and, more recently, in the male genital tract. They play a role in the defense against microbial pathogens in innate immunity and display additional chemotactic functions in the adaptive immune system. An important characteristic of antimicrobial peptides is that they also exhibit toxic potential on eukaryotic cells. Very little is known about the structure dependence of antimicrobial and cytotoxic effects. We investigated human beta-defensin 3 (hBD-3), a potent broad-spectrum antimicrobial effector peptide, regarding the influence of structural parameters on the antimicrobial and cytotoxic activity. We have established a structure-activity relation of the hBD-3 using synthetic derivatives differing in length, charge, disulfide connectivity, and overall hydrophobicity. The antimicrobial activity of the peptides was compared to the cyctotoxic effects on monocytic THP-1 cells and the hemolytic activity on human erythrocytes. We found that it is not important for antimicrobial and cytotoxic activity whether and how cysteine residues are arranged to form disulfide bonds. Substitution of half-cystinyl residues by tryptophan resulted in increased activities, while other substitutions did not change activity. Correlation of activities with the structural changes demonstrates that the activity on eukaryotic cells appears to depend strongly on the overall hydrophobicity. In contrast, the antimicrobial potency of hBD-3 peptides is determined by the distribution of positively charged amino acid residues and hydrophobic side chains. The results facilitate the understanding of beta-defensin interaction with different cell types and guide the design of antimicrobially active peptides.

Engineering disulfide bonds of the novel human beta-defensins hBD-27 and hBD-28: differences in disulfide formation and biological activity among human beta-defensins.

Human beta-defensins comprise a large number of peptides that play a functional role in the innate and adaptive immune system. Recently, clusters of new beta-defensin genes with predominant expression in testicular tissue have been discovered on different chromosomes by bioinformatics. beta-Defensins share a common pattern of three disulfides that are essential for their biological effects. Here we report for the first time the chemical synthesis of the new fully disulfide-bonded beta-defensins hBD-27 and hBD-28, and compare the results with synthetic procedures to obtain the known hBD-2 and hBD-3. While hBD-27 was readily converted into a product with the desired disulfide pattern by oxidative folding, hBD-28 required a selective protective group strategy to introduce the three disulfide bonds. The established synthetic processes were applied to the synthesis of hBD-2, which, like hBD-27, was accessible by oxidative folding, whereas hBD-3 required a selective strategy comparable to hBD-28. Experimental work demonstrated that trityl, acetamidomethyl, and t-butyl are superior to other protection strategies. However, the suitable pairwise arrangement of the protective groups can be different, as shown here for hBD-3 and hBD-28. Determination of the minimum inhibitory concentration against different bacteria revealed that hBD-27, in contrast to other beta-defensins tested, has virtually no antimicrobial activity. Compared to the other peptides tested, hBD-27 showed almost no cytotoxic activity, measured by hemoglobin release of erythrocytes. This might be due to the low positive net charge, which is significantly higher for hBD-2, hBD-3, and hBD-28.

Stability and cleavage conditions of (2-furyl)-L-alanine-containing peptides.

The furyl group of (2-furyl)-L-alanine-containing peptides obtained from Fmoc solid-phase synthesis is partially degraded to several by-products during the final TFA-mediated deprotection in the presence of cation scavengers such as ethanedithiol and propanedithiol. The major by-product corresponds to a bis-dithioacetale formed after acidic hydrolysis of the furyl group. We examined several cleavage conditions and found that cleavage cocktails containing water and triisopropylsilane or 3,6-dioxa-1,8-octanedithiol (DODT) in trifluoroacetic acid are sufficient to minimize the side reaction.

Isolation and biochemical characterization of LEAP-2, a novel blood peptide expressed in the liver.

The human genome contains numerous genes whose protein products are unknown in terms of structure, interaction partner, expression, and function. To unravel the function of these orphan genes, it is of particular value to isolate native forms of protein and peptide products derived from these genes. From human blood ultrafiltrate, we characterized a novel gene-encoded, cysteine-rich, and cationic peptide that we termed liver-expressed antimicrobial peptide 2 (LEAP-2). We identified several circulating forms of LEAP-2 differing in their amino-terminal length, all containing a core structure with two disulfide bonds formed by cysteine residues in relative 1-3 and 2-4 positions. Molecular cloning of the cDNA showed that LEAP-2 is synthesized as a 77-residue precursor, which is predominantly expressed in the liver and highly conserved among mammals. This makes it a unique peptide that does not exhibit similarity with any known human peptide regarding its primary structure, disulfide motif, and expression. Analysis of the LEAP-2 gene resulted in the identification of an alternative promoter and at least four different splicing variants, with the two dominating transcripts being tissue-specifically expressed. The largest native LEAP-2 form of 40 amino acid residues is generated from the precursor at a putative cleavage site for a furin-like endoprotease. In contrast to smaller LEAP-2 variants, this peptide exhibited dose-dependent antimicrobial activity against selected microbial model organisms. LEAP-2 shares some characteristic properties with classic peptide hormones and it is expected that the isolation of this novel peptide will help to unravel its physiological role.

Hemofiltrate CC chemokine 1[9-74] causes effective internalization of CCR5 and is a potent inhibitor of R5-tropic human immunodeficiency virus type 1 strains in primary T cells and macrophages.

Proteolytic processing of the abundant plasmatic human CC chemokine 1 (HCC-1) generates a truncated form, HCC-1[9-74], which is a potent agonist of CCR1, CCR3, and CCR5; promotes calcium influx and chemotaxis of T lymphoblasts, monocytes, and eosinophils; and inhibits infection by CCR5-tropic human immunodeficiency virus type 1 (HIV-1) isolates. In the present study we demonstrate that HCC-1[9-74] interacts with the second external loop of CCR5 and inhibits replication of CCR5-tropic HIV-1 strains in both primary T cells and monocyte-derived macrophages. Low concentrations of the chemokine, however, frequently enhanced the replication of CCR5-tropic HIV-1 isolates but not the replication of X4-tropic HIV-1 isolates. Only HCC-1[9-74] and HCC-1[10-74], but not other HCC-1 length variants, displayed potent anti-HIV-1 activities. Fluorescence-activated cell sorter analysis revealed that HCC-1[9-74] caused up to 75% down-regulation of CCR5 cell surface expression, whereas RANTES (regulated on activation, normal T-cell expressed and secreted) achieved a reduction of only about 40%. Studies performed with green fluorescent protein-tagged CCR5 confirmed that both HCC-1[9-74] and RANTES, but not full-length HCC-1, mediated specific internalization of the CCR5 HIV-1 entry cofactor. Our results demonstrate that the interaction with HCC-1[9-74] causes effective intracellular sequestration of CCR5, but they also indicate that the effect of HCC-1[9-74] on viral replication is subject to marked cell donor- and HIV-1 isolate-dependent variations.