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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.

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]