Microsurgical Transplantation of Pedicled Muscles in an Isolation Chamber-A Novel Approach to Engineering Muscle Constructs via Perfusion-Decellularization.

Decellularized whole muscle constructs represent an ideal scaffold for muscle tissue engineering means as they retain the network and proteins of the extracellular matrix of skeletal muscle tissue. The presence of a vascular pedicle enables a more efficient perfusion-based decellularization protocol and allows for subsequent recellularization and transplantation of the muscle construct in vivo. The goal of this study was to create a baseline for transplantation of decellularized whole muscle constructs by establishing an animal model for investigating a complete native muscle isolated on its pedicle in terms of vascularization and functionality. The left medial gastrocnemius muscles of 5 male Lewis rats were prepared and raised from their beds for in situ muscle stimulation. The stimulation protocol included twitches, tetanic stimulation, fatigue testing, and stretching of the muscles. Peak force, maximum rate of contraction and relaxation, time to maximum contraction and relaxation, and maximum contraction and relaxation rate were determined. Afterwards, muscles were explanted and transplanted heterotopically in syngeneic rats in an isolation chamber by microvascular anastomosis. After 2 weeks, transplanted gastrocnemius muscles were exposed and stimulated again followed by intravascular perfusion with a contrast agent for µCT analysis. Muscle constructs were then paraffin embedded for immunohistological staining. Peak twitch and tetanic force values all decreased significantly after muscle transplantation while fatigue index and passive stretch properties did not differ between the two groups. Vascular analysis revealed retained perfused vessels most of which were in a smaller radius range of up to 20 µm and 45 µm. In this study, a novel rat model of heterotopic microvascular muscle transplantation in an isolation chamber was established. With the assessment of in situ muscle contraction properties as well as vessel distribution after 2 weeks of transplantation, this model serves as a base for future studies including the transplantation of perfusion-decellularized muscle constructs.

Porous Alumina Ceramics with Multimodal Pore Size Distributions.

Pore networks with multimodal pore size distributions combining advantages from isotropic and anisotropic shaped pores of different sizes are highly attractive to optimize the physical properties of porous ceramics. Multimodal porous Al2O3 ceramics were manufactured using pyrolyzed cellulose fibers (l = 150 µm, d = 8 µm) and two types of isotropic phenolic resin spheres (d = 30 and 300 µm) as sacrificial templates. The sacrificial templates were homogeneously distributed in the Al2O3 matrix, compacted by uniaxial pressing and extracted by a burnout and sintering process up to 1700 °C in air. The amount of sacrificial templates was varied up to a volume content of 67 Vol% to form pore networks with porosities of 0-60 Vol%. The mechanical and thermal properties were measured by 4-point-bending and laser flash analysis (LFA) resulting in bending strengths of 173 MPa to 14 MPa and heat conductivities of 22.5 Wm-1K-1 to 4.6 Wm-1K-1. Based on µCT-measurements, the representative volume-of-interest (VOI) of the samples digital twin was determined for further analysis. The interconnectivity, tortuosity, permeability, the local and global stress distribution as well as strut and cell size distribution were evaluated on the digital twin's VOI. Based on the experimental and simulation results, the samples pore network can be tailored by changing the fiber to sphere ratio and the overall sacrificial template volume. The presence pore formers significantly influenced the mechanical and thermal properties, resulting in higher strengths for samples containing fibrous templates and lower heat conductivities for samples containing spherical templates.

Human Umbilical Vein Endothelial Cell Support Bone Formation of Adipose-Derived Stem Cell-Loaded and 3D-Printed Osteogenic Matrices in the Arteriovenous Loop Model.

Introduction: For the regeneration of large volume tissue defects, the interaction between angiogenesis and osteogenesis is a crucial prerequisite. The surgically induced angiogenesis by means of an arteriovenous loop (AVL), is a powerful methodology to enhance vascularization of osteogenic matrices. Moreover, the AVL increases oxygen and nutrition supply, thereby supporting cell survival as well as tissue formation. Adipose-derived stem cells (ADSCs) are interesting cell sources because of their simple isolation, expansion, and their osteogenic potential. This study targets to investigate the coimplantation of human ADSCs after osteogenic differentiation and human umbilical vein endothelial cells (HUVECs), embedded in a vascularized osteogenic matrix of hydroxyapatite (HAp) ceramic for bone tissue engineering. Materials and Methods: An osteogenic matrix consisting of HAp granules and fibrin has been vascularized by means of an AVL. Trials in experimental groups of four settings were performed. Control experiments without any cells (A) and three cell-loaded groups using HUVECs (B), ADSCs (C), as well as the combination of ADSCs and HUVECs (D) were performed. The scaffolds were implanted in a porous titanium chamber, fixed subcutaneously in the hind leg of immunodeficient Rowett Nude rats and explanted after 6 weeks. Results: In all groups, the osteogenic matrix was strongly vascularized. Moreover, remodeling processes and bone formation in the cell-containing groups with more bone in the coimplantation group were proved successful. Conclusion: Vascularization and bone formation of osteogenic matrices consisting of ADSCs and HUVECs in the rat AVL model could be demonstrated successfully for the first time. Hence, the coimplantation of differentiated ADSCs with HUVECs may therefore be considered as a promising approach for bone tissue engineering.

Injection Molding of 3-3 Hydroxyapatite Composites.

The manufacturing of ideal implants requires fabrication processes enabling an adjustment of the shape, porosity and pore sizes to the patient-specific defect. To meet these criteria novel porous hydroxyapatite (HAp) implants were manufactured by combining ceramic injection molding (CIM) with sacrificial templating. Varied amounts (Φ = 0-40 Vol%) of spherical pore formers with a size of 20 µm were added to a HAp-feedstock to generate well-defined porosities of 11.2-45.2 Vol% after thermal debinding and sintering. At pore former contents Φ ≥ 30 Vol% interconnected pore networks were formed. The investigated Young's modulus and flexural strength decreased with increasing pore former content from 97.3 to 29.1 GPa and 69.0 to 13.0 MPa, agreeing well with a fitted power-law approach. Additionally, interpenetrating HAp/polymer composites were manufactured by infiltrating and afterwards curing of an urethane dimethacrylate-based (UDMA) monomer solution into the porous HAp ceramic preforms. The obtained stiffness (32-46 GPa) and Vickers hardness (1.2-2.1 GPa) of the HAp/UDMA composites were comparable to natural dentin, enamel and other polymer infiltrated ceramic network (PICN) materials. The combination of CIM and sacrificial templating facilitates a near-net shape manufacturing of complex shaped bone and dental implants, whose properties can be directly tailored by the amount, shape and size of the pore formers.

Hierarchical Surface Texturing of Hydroxyapatite Ceramics: Influence on the Adhesive Bonding Strength of Polymeric Polycaprolactone.

The tailored manipulation of ceramic surfaces gained recent interest to optimize the performance and lifetime of composite materials used as implants. In this work, a hierarchical surface texturing of hydroxyapatite (HAp) ceramics was developed to improve the poor adhesive bonding strength in hydroxyapatite and polycaprolactone (HAp/PCL) composites. Four different types of periodic surface morphologies (grooves, cylindric pits, linear waves and Gaussian hills) were realized by a ceramic micro-transfer molding technique in the submillimeter range. A subsequent surface roughening and functionalization on a micron to nanometer scale was obtained by two different etchings with hydrochloric and tartaric acid. An ensuing silane coupling with 3-aminopropyltriethoxysilane (APTES) enhanced the chemical adhesion between the HAp surface and PCL on the nanometer scale by the formation of dipole-dipole interactions and covalent bonds. The adhesive bonding strengths of the individual and combined surface texturings were investigated by performing single-lap compressive shear tests. All individual texturing types (macro, micro and nano) showed significantly improved HAp/PCL interface strengths compared to the non-textured HAp reference, based on an enhanced mechanical, physical and chemical adhesion. The independent effect mechanisms allow the deliberately hierarchical combination of all texturing types without negative influences. The hierarchical surface-textured HAp showed a 6.5 times higher adhesive bonding strength (7.7 ± 1.5 MPa) than the non-textured reference, proving that surface texturing is an attractive method to optimize the component adhesion in composites for potential medical implants.

Modular Lattice Constructs for Biological Joint Resurfacing.

IMPACT STATEMENT: The repair of large articular cartilage lesions is still a major challenge. In particular, the fixation of the grafts to the subchondral bone plate represents an unresolved problem. In this work, we present a completely novel concept based on a modular lattice, combining building blocks of different ceramic materials, anchoring pins and space for cell-loaded hydrogels or other scaffold materials. This concept targets not only circumscribed cartilage defects but also large osteoarthritic lesions. It spans the bridge between cell therapy and artificial joint arthroplasty, and thus is of significant medical and socioeconomic impact.

Modular ceramic scaffolds for individual implants.

Ideal artificial bone grafts aim for multiscale porosity, high mechanical strength and ensure rapid vascularization for bone ingrowth. In this work modular ceramic arteriovenous loops (AV-loops) with a hierarchical porosity approach were designed and manufactured to meet these criteria and to exceed the poor mechanical strength of monolithic scaffolds. Bioactive building blocks (ß-TCP, HAp, BCP) with dimensions of 1.5-3.0 mm were prepared by injection molding and assembled to complex AV-loop scaffolds using a customized automated assembly technology (pick and place). The building blocks were bonded with a biocompatible adhesive. Single building blocks are characterized by a compressive strength of 112.4-134.5 MPa with a residual sintering porosity of 32.2-41.5%, matching the strength of cortical bone of 100-230 MPa. The compressive strength of the modular assemblies varied between 22.3 and 47.6 MPa primary depending on the building block arrangement. The achieved compressive strengths are superior to current monolithic AV-scaffolds and sufficient for the implantation as non-load-bearing AV-loop scaffolds in isolation chambers. The modular AV-loop scaffolds provide a hierarchical interconnected pore network (P = 58.8%) combining small macropores of 4.1-4.3 µm size for possible enhanced protein absorption and large gradient macropores of 200-1700 µm size for optimum vascularization and complete bone ingrowth. The modular building block approach allows to design patient individualized scaffolds with complex hierarchical pore networks. The pore volume, size and geometry as well as the biological response can effectively be tuned by changing the dimensions, shape and placing gap of the bioactive building blocks. STATEMENT OF SIGNIFICANCE: Gold standard of bone replacement in case of surgery or cancer is still own bone material usually taken from the hip/arm or leg in second surgery with poor mechanical properties and limited amount. To avoid a second surgery and provide mechanical strong scaffold structures for fast patient regeneration a novel modular building block approach is used. This allows complex scaffold geometry with a hierarchical interconnection porosity for blood vessel ingrowth. The pore volume, size and geometry as well as the biological response can effectively be tuned by changing the dimensions, shape and placing gap of the bioactive building blocks.