Microcomputed tomography (μCT) is widely used for the study of mineralized tissues, but a similar use for soft tissues is hindered by their low X-ray attenuation. This limitation can be overcome by the recent development of different staining techniques. Staining with Lugol’s solution, a mixture of one part iodine and two parts potassium iodide in water, stands out among these techniques for its low complexity and cost. Currently, Lugol staining is mostly used for anatomical examination of tissues. In the present study, we seek to optimize the quality and reproducibility of the staining for ex vivo visualization of soft tissues in the context of a peripheral nerve regeneration model in the rat. We show that the staining result not only depends on the concentration of the staining solution but also on the amount of stain in relation to the tissue volume and composition, necessitating careful adaptation of the staining protocol to the respective specimen tissue. This optimization can be simplified by a stepwise staining which we show to yield a similar result compared to staining in a single step. Lugol staining solution results in concentration-dependent tissue shrinkage which can be minimized but not eliminated. We compared the shrinkage of tendon, nerve, skeletal muscle, heart, brain, and kidney with six iterations of Lugol staining. 60 ml of 0.3% Lugol’s solution per cm³ of tissue for 24 h yielded good results on the example of a peripheral nerve regeneration model, and we were able to show that the regenerating nerve inside a silk fibroin tube can be visualized in 3D using this staining technique. This information helps in deciding the region of interest for histological imaging and provides a 3D context to histological findings. Correlating both imaging modalities has the potential to improve the understanding of the regenerative process.

Natural extracellular matrix (ECM) derived biomaterials from decellularized allogenic tissues are of increasing interest for tissue engineering since their structure and composition provide a complexity that is not achievable with current manufacturing techniques. The prerequisite to bring allogenic tissue from bench to bedside as a functional biomaterial is the full removal of cells while preserving most of its native characteristics such as structure and composition. The exceptionally dense structure of articular cartilage, however, poses a special challenge for decellularisation, scaffold preparation and reseeding. Therefore, we tested 24 different protocols aiming to remove cells and glycosaminoglycans (GAG) while preserving the collagen backbone and ultrastructure. The resulting matrices were analyzed for cell removal (DNA-quantification, HE-staining), GAG content (DMMB-assay, Alcian blue staining, µCT), collagen integrity (immunohistochemistry, ultrastructure) and biomechanics (compression test). Furthermore, seeding tests were conducted to evaluate cell viability and attachment to the scaffolds. SDS-based protocols yielded satisfactory reduction of DNA content, yet had negative effects on cell viability and attachment. Hydrochloric acid efficiently decellularized the scaffold and pepsin emerged as best option for GAG depletion. Combining these two reagents lead to our final protocol, most efficient in DNA and GAG depletion while preserving the collagen architecture. The compressive modulus decreased in the absence of GAG to approximately 1/3 of native cartilage, which is significantly higher compared to commercially available scaffolds tested as a reference (ranging from 1/25 to 1/100 of native cartilage). Cytocompatibility tests showed that human adipose-derived stromal cells readily adhered to the scaffold. In this study, we established a protocol combining freeze/thaw cycles, osmotic shock and treatment with hydrochloric acid followed by a pepsin digestion step, achieving successful decellularization and GAG depletion within one week resulting in a cytocompatible material with intact collagen structure. The protocol provides a basis for the generation of allogeneic scaffolds, potentially substituting manufactured scaffolds currently used in clinical articular cartilage treatment.

Reactive oxygen species (ROS) have recently been recognized as important signal transducers, particularly regulating proliferation and differentiation of cells. Diphenyleneiodonium (DPI) is known as an inhibitor of the nicotinamide adenine dinucleotide phosphate oxidase (NOX) and is also affecting mitochondrial function. The aim of this study was to investigate the effect of DPI on ROS metabolism and mitochondrial function in human amniotic membrane mesenchymal stromal cells (hAMSCs), human bone marrow mesenchymal stromal cells (hBMSCs), hBMSCs induced into osteoblast-like cells, and osteosarcoma cell line MG-63. Our data suggested a combination of a membrane potential sensitive fluorescent dye, tetramethylrhodamine methyl ester (TMRM), and a ROS-sensitive dye, CM-H2DCFDA, combined with a pretreatment with mitochondria-targeted ROS scavenger MitoTEMPO as a good tool to examine effects of DPI. We observed critical differences in ROS metabolism between hAMSCs, hBMSCs, osteoblast-like cells, and MG-63 cells, which were linked to energy metabolism. In cell types using predominantly glycolysis as the energy source, such as hAMSCs, DPI predominantly interacted with NOX, and it was not toxic for the cells. In hBMSCs, the ROS turnover was influenced by NOX activity rather than by the mitochondria. In cells with aerobic metabolism, such as MG 63, the mitochondria became an additional target for DPI, and these cells were prone to the toxic effects of DPI. In summary, our data suggest that undifferentiated cells rather than differentiated parenchymal cells should be considered as potential targets for DPI.

Microcomputed tomography (μCT) is widely used for the study of mineralized tissues but a similar use for soft tissues is hindered by their low X-ray attenuation. This limitation can be overcome by the recent development of different staining techniques. Staining with Lugol’s solution, a mixture of one part iodine and two parts potassium iodide in water, stands out among these techniques for its low complexity and cost. Currently, Lugol staining is mostly used for anatomical examination of tissues. In the present study we seek to optimize the quality and reproducibility of the staining for ex vivo visualization of soft tissues in the context of a peripheral nerve regeneration model in the rat. We show that the staining result not only depends on the concentration of the staining solution, but also on the amount of stain in relation to the tissue volume and composition, necessitating careful adaptation of the staining protocol to the respective specimen tissue. This optimization can be simplified by a stepwise staining which we show to yield a similar result compared to staining in a single step. Lugol staining solution results in concentration dependent tissue shrinkage which can be minimized but not eliminated. We compared the shrinkage of tendon, nerve, skeletal muscle, heart, brain and kidney with six iterations of Lugol staining. 60 ml of 0.3% Lugol’s solution per cm ³ of tissue for 24h yielded good results on the example of a peripheral nerve regeneration model and we were able to show that the regenerating nerve inside a silk fibroin tube can be visualized in 3D using this staining technique. This information helps in deciding the region of interest for histological imaging and provides a 3D context to histological findings. Correlating both imaging modalities has the potential to improve the understanding of the regenerative process.

Decellularized articular cartilage of human origin presents itself as the most homologous filling material for focal cartilage defects. Yet, the full repopulation of the exceptionally dense collagen construct has never been achieved without providing host cells with artificially created migration paths into the matrix. Within this study, we examine the use of a femtosecond laser to engrave fine patterns into human articular cartilage before decellularization and GAG depletion (decell‐deGAG). Scaffolds were tested for decellularization success and mechanical behavior. Seeding tests were performed to assess biocompatibility and examine the performance in a simulated defect environment using an osteochondral plug model in vitro and in vivo in an ectopic nude mouse model. The composition and structure of the newly formed repair tissue and macrophage recruitment were observed via histology. The femtosecond laser was successful in engraving deep, fine structures into the matrix without the thermal damage found with other laser techniques. Engraving was also beneficial for decellularization success. The resulting decell‐deGAG scaffold featured a compressive modulus many times stronger than other biomaterials commonly used for cartilage regeneration and presents a defect filling material that is similar to the tissue it is meant to replace. Moreover, the incisions promoted the repopulation with therapeutically relevant cells. A favorable spatial environment inside the incisions facilitated the formation of repair tissue that mimics hyaline cartilage in composition and collagen orientation. Scaffolds were well‐integrated within simulated defects. Femtosecond laser–engraved cartilage poses an authentic defect filling material with cartilage‐like properties. When used in combination with cell seeding, it promotes the formation of differentiated repair tissue. Thus, the hereby presented biomaterial shows great potential in improving the repair of focal cartilage defects and reducing long‐term graft failures.