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The cultivation of cells in 3D systems is commonly regarded to be more physiological than in 2D as it comes much closer to the natural situation in tissues in many different aspects. However, 3D cell culture is much more complex. Cells within the pores of a printed 3D scaffold face a special situation concerning cell-material interaction and cell adhesion, cell proliferation, and supply of medium and oxygen into the core of the scaffolds. Biological assays (for cell proliferation, viability, and activity) have been validated primarily for 2D cell cultures and need to be adapted for 3D cultures. Likewise, in imaging, a number of points need to be taken into account in order to get a clear picture of the cells in 3D scaffolds, preferably with the method of multiphoton microscopy. Here, we describe a method for pretreatment and cell seeding of porous inorganic composite scaffolds (α-TCP/HA) for bone tissue engineering and for cultivation of the cell-scaffold constructs. The analytical methods described are the cell proliferation assay and the ALP activity assay. A step-by-step protocol is provided here that safely tackles typical difficulties that arise with this 3D cell-scaffold setting. In addition, MPM imaging of cells is described both with and without labeling. The combination of biochemical assays and imaging provides valuable insights into the possibilities of analysis with this 3D cell-scaffold system., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: Decellularizing solid organs is a promising top-down process to produce acellular bio-scaffolds for 'de novo' regrowth or application as tissue 'patches' that compensate, e.g., large volumetric muscle loss in reconstructive surgery. Therefore, generating standardized acellular muscle scaffolds marks a pressing area of need. Although animal muscle decellularization protocols were established, those are mostly manually performed and lack defined bioreactor environments and metrologies to assess decellularization quality in real-time. To close this gap, we engineered an automated bioreactor system to provide chemical decellularization solutions to immersed whole rat gastrocnemius medialis muscle through perfusion of the main feeding arteries., Results: Perfusion control is adjustable according to decellularization quality feedback. This was assessed both from (i) ex situ assessment of sarcomeres/nuclei through multiphoton fluorescence and label-free Second Harmonic Generation microscopy and DNA quantification, along with (ii) in situ within the bioreactor environment assessment of the sample's passive mechanical elasticity., Conclusion: We find DNA and sarcomere-free constructs after 72 h of 0.1% SDS perfusion-decellularization. Furthermore, passive elasticity can be implemented as additional online decellularization quality measure, noting a threefold elasticity decrease in acellular constructs., Significance: Our MyoBio represents a novel and useful automated bioreactor environment for standardized and controlled generation of acellular whole muscle scaffolds as a valuable source for regenerative medicine.

In situ tissue engineering is an emerging field aiming at the generation of ready-to-use three-dimensional tissues. One solution to supply a proper vascularization of larger tissues to provide oxygen and nutrients is the arteriovenous loop (AVL) model. However, for this model, suitable scaffold materials are needed that are biocompatible/non-immunogenic, slowly degradable, and allow vascularization. Here, we investigate the suitability of the known gelatin methacryloyl (GelMA)-based hydrogel for in-situ tissue engineering utilizing the AVL model. Rat AVLs are embedded by two layers of GelMA hydrogel in an inert PTFE chamber and implanted in the groin. Constructs were explanted after 2 or 4 weeks and analyzed. For this purpose, gross morphological, histological, and multiphoton microscopic analysis were performed. Immune response was analyzed based on anti-CD68 and anti-CD163 staining of immune cells. The occurrence of matrix degradation was assayed by anti-MMP3 staining. Vascularization was analyzed by anti- α -smooth muscle actin staining, multiphoton microscopy, as well as expression analysis of 53 angiogenesis-related proteins utilizing a proteome profiler angiogenesis array kit. Here we show that GelMA hydrogels are stable for at least 4 weeks in the rat AVL model. Furthermore, our data indicate that GelMA hydrogels are biocompatible. Finally, we provide evidence that GelMA hydrogels in the AVL model allow connective tissue formation, as well as vascularization, introducing multiphoton microscopy as a new methodology to visualize neovessel formation originating from the AVL. GelMA is a suitable material for in situ and in vivo TE in the AVL model., (Creative Commons Attribution license.)

The goal of bone tissue engineering is to build artificial bone tissue with properties that closely resemble human bone and thereby support the optimal integration of the constructs (biografts) into the body. The development of tissues in 3D scaffolds includes several complex steps that need to be optimized and monitored. In particular, cell–material interaction during seeding, cell proliferation and cell differentiation within the scaffold pores play a key role. In this work, we seeded two types of 3D-printed scaffolds with pre-osteoblastic MC3T3-E1 cells, proliferated and differentiated the cells, before testing and adapting different assays and imaging methods to monitor these processes. Alpha-TCP/HA (α-TCP with low calcium hydroxyapatite) and baghdadite (Ca3ZrSi2O9) scaffolds were used, which had comparable porosity (~50%) and pore sizes (~300–400 µm). Cell adhesion to both scaffolds showed ~95% seeding efficiency. Cell proliferation tests provided characteristic progression curves over time and increased values for α-TCP/HA. Transmitted light imaging displayed a homogeneous population of scaffold pores and allowed us to track their opening state for the supply of the inner scaffold regions by diffusion. Fluorescence labeling enabled us to image the arrangement and morphology of the cells within the pores. During three weeks of osteogenesis, ALP activity increased sharply in both scaffolds, but was again markedly increased in α-TCP/HA scaffolds. Multiphoton SHG and autofluorescence imaging were used to investigate the distribution, morphology, and arrangement of cells; collagen-I fiber networks; and hydroxyapatite crystals. The collagen-I networks became denser and more structured during osteogenic differentiation and appeared comparable in both scaffolds. However, imaging of the HA crystals showed a different morphology between the two scaffolds and appeared to arrange in the α-TCP/HA scaffolds along collagen-I fibers. ALP activity and SHG imaging indicated a pronounced osteo-inductive effect of baghdadite. This study describes a series of methods, in particular multiphoton imaging and complementary biochemical assays, to validly measure and track the development of bone tissue in 3D scaffolds. The results contribute to the understanding of cell colonization, growth, and differentiation, emphasizing the importance of optimal media supply of the inner scaffold regions. [ABSTRACT FROM AUTHOR]

Electrospun nanofibers represent an ideal matrix for the purpose of skeletal muscle tissue engineering due to their highly aligned structure in the nanoscale, mimicking the extracellular matrix of skeletal muscle. However, they often consist of high-density packed fibers, which might impair vascularization. The integration of polyethylene oxide (PEO) sacrificial fibers, which dissolve in water, enables the creation of less dense structures. This study examines potential benefits of poly-ε-caprolactone-collagen I-PEO-nanoscaffolds (PCP) in terms of neovascularization and distribution of newly formed vessels compared to poly-ε-caprolactone -collagen I-nanoscaffolds (PC) in a modified arteriovenous loop model in the rat. For this purpose, the superficial inferior epigastric artery and vein as well as a motor nerve branch were integrated into a multilayer three-dimensional nanofiber scaffold construct, which was enclosed by an isolation chamber. Numbers and spatial distribution of sprouting vessels as well as macrophages were analyzed via immunohistochemistry after two and four weeks of implantation. After four weeks, aligned PC showed a higher number of newly formed vessels, regardless of the compartments formed in PCP by the removal of sacrificial fibers. Both groups showed cell influx and no difference in macrophage invasion. In this study, a model of combined axial vascularization and neurotization of a PCL-collagen I-nanofiber construct could be established for the first time. These results provide a foundation for the in vivo implantation of cells, taking a major step towards the generation of functional skeletal muscle tissue. [ABSTRACT FROM AUTHOR]

Aims: Desminopathies comprise hereditary myopathies and cardiomyopathies caused by mutations in the intermediate filament protein desmin that lead to severe and often lethal degeneration of striated muscle tissue. Animal and single cell studies hinted that this degeneration process is associated with massive ultrastructural defects correlating with increased susceptibility of the muscle to acute mechanical stress. The underlying mechanism of mechanical susceptibility, and how muscle degeneration develops over time, however, has remained elusive. Methods: Here, we investigated the effect of a desmin mutation on the formation, differentiation, and contractile function of in vitro‐engineered three‐dimensional micro‐tissues grown from muscle stem cells (satellite cells) isolated from heterozygous R349P desmin knock‐in mice. Results: Micro‐tissues grown from desmin‐mutated cells exhibited spontaneous unsynchronised contractions, higher contractile forces in response to electrical stimulation, and faster force recovery compared with tissues grown from wild‐type cells. Within 1 week of culture, the majority of R349P desmin‐mutated tissues disintegrated, whereas wild‐type tissues remained intact over at least three weeks. Moreover, under tetanic stimulation lasting less than 5 s, desmin‐mutated tissues partially or completely ruptured, whereas wild‐type tissues did not display signs of damage. Conclusions: Our results demonstrate that the progressive degeneration of desmin‐mutated micro‐tissues is closely linked to extracellular matrix fibre breakage associated with increased contractile forces and unevenly distributed tensile stress. This suggests that the age‐related degeneration of skeletal and cardiac muscle in patients suffering from desminopathies may be similarly exacerbated by mechanical damage from high‐intensity muscle contractions. We conclude that micro‐tissues may provide a valuable tool for studying the organization of myocytes and the pathogenic mechanisms of myopathies. [ABSTRACT FROM AUTHOR]

Two‐dimensional (2D) cancer models have been the standard for drug development over the past few years, but they frequently do not resemble in vivo properties adequately. 3D models are superior in many aspects and are, therefore, more similar to human pathophysiology. Over the past years, the emerging field of biofabrication has made significant advances, resulting in even more sophisticated 3D models. With this study, a hydrogel is created for biofabrication that is suitable for mimicking the tumor microenvironment in vitro and is further tested as a new vascularized melanoma model in vivo. The alginate/hyaluronic acid/gelatin bioink shows good shape‐fidelity, high cell survival rates, and enables successful cultivation of melanoma cells and adipose‐derived stem cells as well as cell differentiation in vitro. In vivo, in the arteriovenous loop model, it proves to be a unique method to study melanoma progression, tumor vascularization, and ultimately and reliably metastases in an isolated and controlled environment. These results show that this 3D model is very application‐oriented for molecular research and therapy development. [ABSTRACT FROM AUTHOR]