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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 (Ca 3 ZrSi 2 O 9 ) 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.

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.)

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.

A facile and flexible approach for the integration of biomimetically branched microvasculature within bulk hydrogels is presented. For this, sacrificial scaffolds of thermoresponsive poly(2-cyclopropyl-2-oxazoline) (PcycloPrOx) are created using melt electrowriting (MEW) in an optimized and predictable way and subsequently placed into a customized bioreactor system, which is then filled with a hydrogel precursor solution. The aqueous environment above the lower critical solution temperature (LCST) of PcycloPrOx at 25 °C swells the polymer without dissolving it, resulting in fusion of filaments that are deposited onto each other (print-and-fuse approach). Accordingly, an adequate printing pathway design results in generating physiological-like branchings and channel volumes that approximate Murray's law in the geometrical ratio between parent and daughter vessels. After gel formation, a temperature decrease below the LCST produces interconnected microchannels with distinct inlet and outlet regions. Initial placement of the sacrificial scaffolds in the bioreactors in a pre-defined manner directly yields perfusable structures via leakage-free fluid connections in a reproducible one-step procedure. Using this approach, rapid formation of a tight and biologically functional endothelial layer, as assessed not only through fluorescent dye diffusion, but also by tumor necrosis factor alpha (TNF-α) stimulation, is obtained within three days., (© 2022 The Authors. Advanced Materials published by Wiley-VCH GmbH.)

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 development of suitable synthetic scaffolds for use as human tendon grafts to repair tendon ruptures remains a significant engineering challenge. Previous synthetic tendon grafts have demonstrated suboptimal tissue ingrowth and synovitis due to wear particles from fiber-to-fiber abrasion. In this study, we present a novel fiber-reinforced hydrogel (FRH) that mimics the hierarchical structure of the native human tendon for synthetic tendon graft material. Ultrahigh molecular weight polyethylene (UHMWPE) fibers were impregnated with either biosynthetic polyvinyl alcohol/gelatin hydrogel (FRH-PG) or with polyvinyl alcohol/gelatin + strontium-hardystonite (Sr-Ca 2 ZnSi 2 O 7 , Sr-HT) composite hydrogel (FRH-PGS). The scaffolds were fabricated and assessed to evaluate their suitability for tendon graft applications. The microstructure of both FRH-PG and FRH-PGS showed successful impregnation of the hydrogel component, and the tendon scaffolds exhibited equilibrium water content of ∼70 wt %, similar to the values reported for native human tendon, compared to ∼50 wt % water content retained in unmodified UHMWPE fibers. The tensile strength of FRH-PG and FRH-PGS (77.0-81.8 MPa) matched the range of human Achilles' tendon tensile strengths reported in the literature. In vitro culture of rat tendon stem cells showed cell and tissue infiltration into both FRH-PG and FRH-PGS after 2 weeks, and the presence of Sr-HT ceramic particles influenced the expression of tenogenic markers. On the other hand, FRH-PG supported the proliferation of murine C2C12 myoblasts, whereas FRH-PGS seemingly did not support it under static culture conditions. In vivo implantation of FRH-PG and FRH-PGS scaffolds into full-thickness rat patellar tendon defects showed good collagenous tissue ingrowth into these scaffolds after 6 weeks. This study demonstrates the potential viability for our FRH-PG and FRH-PGS scaffolds to be used for off-the-shelf biosynthetic tendon graft material.

Achieving adequate healing in large or load-bearing bone defects is highly challenging even with surgical intervention. The clinical standard of repairing bone defects using autografts or allografts has many drawbacks. A bioactive ceramic scaffold, strontium-hardystonite-gahnite or "Sr-HT-Gahnite" (a multi-component, calcium silicate-based ceramic) is developed, which when 3D-printed combines high strength with outstanding bone regeneration ability. In this study, the performance of purely synthetic, 3D-printed Sr-HT-Gahnite scaffolds is assessed in repairing large and load-bearing bone defects. The scaffolds are implanted into critical-sized segmental defects in sheep tibia for 3 and 12 months, with bone autografts used for comparison. The scaffolds induce substantial bone formation and defect bridging after 12 months, as indicated by X-ray, micro-computed tomography, and histological and biomechanical analyses. Detailed analysis of the bone-scaffold interface using focused ion beam scanning electron microscopy and multiphoton microscopy shows scaffold degradation and maturation of the newly formed bone. In silico modeling of strain energy distribution in the scaffolds reveal the importance of surgical fixation and mechanical loading on long-term bone regeneration. The clinical application of 3D-printed Sr-HT-Gahnite scaffolds as a synthetic bone substitute can potentially improve the repair of challenging bone defects and overcome the limitations of bone graft transplantation., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Synthetic cell carriers (A) represent common scaffold structures for the development of cell-based in vitro models of the human intestine but due to their low porosity or unwanted molecular adhesion effects, synthetic carriers can negatively affect cell function. Alternative scaffolds such as natural extracellular matrices (ECMs) (B) were shown to overcome some of the common drawbacks. However, their fabrication is time-consuming, less well standardized and not entirely conform to the 3R principle (replacement, reduction, refinement). Nowadays, biopolymers such as bacterial nanocellulose (BNC) (C) represent interesting scaffold materials for innovative tissue engineering concepts, as they can be generated in a faster and more standardized process workflow without the need for animal material. In this study, we demonstrate the BNC as suitable carrier for the development of Caco-2-based in vitro models of the human intestine. The BNC-based models exhibit organ-specific properties comprising typical cellular morphologies, characteristic protein expression profiles, representative ultrastructural features and the formation of a tight epithelial barrier. The proof of in vivo -like transport activities further validates the high quality of the BNC-based Caco-2 models. In summary, this illustrates the BNC as alternative bioscaffold of non-animal origin to develop functional organ models in vitro. Unlabelled Image • Comparison of synthetic, tissue-derived and biotechnologically produced scaffolds for tissue engineering applications • Reproducible and morphologically relevant results were obtained for bacterial nanocellulose (BNC)-based scaffolds • BNC as biocompatible as well as biomimetically relevant hydrogel-material shows a high production- and cost- efficiency [ABSTRACT FROM AUTHOR]