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Background The efficiency of transdermal drug delivery may be increased by pretreating the skin with microneedles, but distinct effects of microneedles and the microneedle-enhanced delivery of vasoactive drugs on the skin microvasculature are still not well investigated. Materials and methods In eight healthy human subjects, we measured the microvascular response to microneedle-induced microtraumas in the skin microvasculature using polarized light spectroscopy imaging (Tissue Viability imaging, TiVi). The microvascular response was assessed for up to 48 hours for three microneedle sizes (300 µm, 500 µm, and 750 µm) and for different pressures and application times. Results In our results, microneedle application increased the local red blood cell (RBC) concentration for up to 24 hours dependent on the needle lengths, applied time, and force. Conclusion Optimization of microneedles size, pressure, and application time should be taken into account for future protocols for drug delivery and experimental provocations.

The natural extracellular matrix (ECM) provides the optimal environment for cells. Many enzymatic or non-enzymatic based strategies to extract ECM proteins from tissues were published over the past years. However, every single isolation strategy reported so far is associated with specific bottlenecks. In this study, frequently used strategies to isolate ECM from human placenta or adipose tissue using Tris-, serum-, or pepsin-based buffers were compared. The resulting ECM proteins were biochemically characterized by analysis of cellular remnants using Hoechst DNA staining, glycosaminoglycan (GAG) content by dimethylmethylene blue, visualization of protein bands using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis combined with amino acid quantification, and assessment of the proangiogenic profile using an angiogenesis array. Tris-NaCl-extracted ECM proteins showed a high heterogenic degree of extracted proteins, bioactive growth factors, and GAGs, but no collagen-I. Active serum-extracted ECM showed significant lower DNA remnants when compared with the Tris-NaCl isolation strategy. Pepsin-extracted ECM was rich in collagen-I and low amounts of remaining bioactive growth factors. This strategy was most effective to reduce DNA amounts when compared with the other isolation strategies. Pepsin-extracted ECM from both tissues easily gelled at 37°C, whereas the other extracted ECM strategies did not gel at 37°C (Tris-NaCl: liquid; serum: sponge). All relevant characteristics (DNA residues, ECM diversity and bioactivity, shape) of the extracted ECM proteins highly depend on its isolation strategy and could still be optimized. Impact statement The natural human extracellular matrix (ECM) is the ideal cell niche. Various strategies were reported to isolate human ECM components from various sources. In this article, we compared frequently used methods and compared their characteristics (DNA remnants, glycosaminoglycan content, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, amino acid quantification, angiogenesis array, and gel formation). We conclude that more research is still necessary to optimize current isolation approaches for

Laminins are major components of all basement membranes surrounding nerve or vascular tissues. In particular laminin-111, the prototype of the family, facilitates a large spectrum of fundamental cellular responses in all eukaryotic cells. Laminin-111 is a biomaterial frequently used in research, however it is primarily isolated from non-human origin or produced with time-intensive recombinant techniques at low yield.Here, we describe an effective method for isolating laminin-111 from human placenta, a clinical waste material, for various tissue engineering applications. By extraction with Tris-NaCl buffer combined with non-protein-denaturation ammonium sulfate precipitation and rapid tangential flow filtration steps, we could effectively isolate native laminin-111 within only 4 days. The resulting material was biochemically characterized using a combination of dot blot, SDS-PAGE, Western blot and HPLC-based amino acid analysis. Cytocompatibility studies demonstrated that the isolated laminin-111 promotes rapid and efficient adhesion of primary Schwann cells. In addition, the bioactivity of the isolated laminin-111 was demonstrated by (a) using the material as a substrate for outgrowth of NG 108-15 neuronal cell lines and (b) promoting the formation of interconnected vascular networks by GFP-expressing human umbilical vein endothelial cells.In summary, the isolation procedure of laminin-111 as described here from human placenta tissue, fulfills many demands for various tissue engineering and regenerative medicine approaches and therefore may represent a human alternative to various classically used xenogenic standard materials.

Laminins are major components of all basement membranes surrounding nerve or vascular tissues. In particular laminin-111, the prototype of the family, facilitates a large spectrum of fundamental cellular responses in all eukaryotic cells. Laminin-111 is a biomaterial frequently used in research, however it is primarily isolated from non-human origin or produced with time-intensive recombinant techniques at low yield.Here, we describe an effective method for isolating laminin-111 from human placenta, a clinical waste material, for various tissue engineering applications. By extraction with Tris-NaCl buffer combined with non-protein-denaturation ammonium sulfate precipitation and rapid tangential flow filtration steps, we could effectively isolate native laminin-111 within only 4 days. The resulting material was biochemically characterized using a combination of dot blot, SDS-PAGE, Western blot and HPLC-based amino acid analysis. Cytocompatibility studies demonstrated that the isolated laminin-111 promotes rapid and efficient adhesion of primary Schwann cells. In addition, the bioactivity of the isolated laminin-111 was demonstrated by (a) using the material as a substrate for outgrowth of NG 108-15 neuronal cell lines and (b) promoting the formation of interconnected vascular networks by GFP-expressing human umbilical vein endothelial cells.In summary, the isolation procedure of laminin-111 as described here from human placenta tissue, fulfills many demands for various tissue engineering and regenerative medicine approaches and therefore may represent a human alternative to various classically used xenogenic standard materials.

Objective: After iontophoresis of vasoactive drugs into the skin, a decrease in perfusion is commonly observed. We delivered vasoactive drugs by iontophoresis using different delivery protocols to study how these affect this decrease in perfusion as measured using LDF. Methods: We measured skin perfusion during iontophoresis of (ACh), MCh, andNAusing a single pulse or separate pulses at different skin sites, and during repeated delivery of ACh at the same site. Results: Perfusion half-life was 6.1 (5.6-6.6) minutes for ACh and 41 (29-69) minutes for MCh (p less than 0.001). The maximum response with multiple pulses of ACh iontophoresis was lower than with a single pulse, 30 (22-37) PU vs. 43 (36-50) PU, p less than 0.001. Vasoconstriction to NA was more rapid with a single pulse than with multiple pulses. The perfusion half-life of ACh decreased with repeated delivery of ACh at the same site-first 16 (14-18), second 5.9 (5.1-6-9) and third 3.2 (2.9-3.5) minutes, p less than 0.001. Conclusions: The drug delivery protocol affects microvascular responses to iontophoresis, possibly as a result of differences in the dynamics of local drug concentrations. Perfusion half-life may be used as a measure to quantify the rate of perfusion recovery after iontophoresis of vasoactive drugs.

Pepsin-solubilized atelocollagen can be used to form highly complex three-dimensional matrices for a broad spectrum of tissue engineering applications. Moreover, it has a long history as a favorable biomaterial in pharmaceutical and medical industries. So far, the main sources for these approaches are collagens from xenogenic sources. Yet, these nonhuman collagens carry a risk of provoking immune reactions in patients. Here we describe an effective method of isolating atelocollagen type 1/3 (COL1/3) from human placenta. By combining a single pepsin digestion step with tangential flow filtration and further precipitation steps, we could purify COL1/3 within only 4 days of processing. The resulting COL1/3 was biochemically characterized by determining residual DNA content, proving the absence of impurities by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) analysis combined with total amino acid quantification, identifying the isolated collagen types by Western blot analysis, and analyzing the spontaneous formation of fibrous structures on freeze-drying via scanning electron microscopy. Finally, the cytocompatibility of the isolated collagen was demonstrated in two dimensional using primary rat hepatocytes and in three dimensional by a sprouting assay of human umbilical vein endothelial cell. The isolation method described not only fulfills demands for cost-efficient bioengineering using a human waste material but also potentially increases overall safety for patients by use of homologous products.

Schwann cells play a key role in peripheral nerve regeneration. Failure in sufficient formation of Bungner bands due to impaired Schwann cell proliferation has significant effects on the functional outcome after regeneration. Therefore, the growth substrate for Schwann cells should be considered with highest priority in any peripheral nerve tissue engineering approach. Due to its excellent biocompatibility silk fibroin has most recently attracted considerable interest as a biomaterial for use as conduit material in peripheral nerve regeneration. In this study we established a protocol to covalently bind collagen and laminin, which have been isolated from human placenta, to silk fibroin utilizing carbodiimide chemistry. Altered adhesion, viability and proliferation of Schwann cells were evaluated. A cell adhesion assay revealed that the functionalization with both, laminin or collagen, significantly improved Schwann cell adhesion to silk fibroin. Moreover laminin drastically accelerated adhesion. Schwann cell proliferation and viability assessed with BrdU and MTT assay, respectively, were significantly increased in the laminin-functionalized groups. The results suggest beneficial effects of laminin on both, cell adhesion as well as proliferative behaviour of Schwann cells. To conclude, the covalent tailoring of silk fibroin drastically enhances its properties as a cell substratum for Schwann cells, which might help to overcome current hurdles bridging long distance gaps in peripheral nerve injuries with the use of silk-based nerve guidance conduits.