Suitability of Ex Vivo-Expanded Microtic Perichondrocytes for Auricular Reconstruction.

Tissue engineering (TE) techniques offer solutions for tissue regeneration but require large quantities of cells. For microtia patients, TE methods represent a unique opportunity for therapies with low donor-site morbidity and reliance on the surgeon's individual expertise. Microtia-derived chondrocytes and perichondrocytes are considered a valuable cell source for autologous reconstruction of the pinna. The aim of this study was to investigate the suitability of perichondrocytes from microtia patients for autologous reconstruction in comparison to healthy perichondrocytes and microtia chondrocytes. Perichondrocytes were isolated via two different methods: explant culture and enzymatic digestion. The isolated cells were analyzed in vitro for their chondrogenic cell properties. We examined migration activity, colony-forming ability, expression of mesenchymal stem cell markers, and gene expression profile. We found that microtic perichondrocytes exhibit similar chondrogenic properties compared to chondrocytes in vitro. We investigated the behavior in three-dimensional cell cultures (spheroids and scaffold-based 3D cell cultures) and assessed the expression of cartilage-specific proteins via immunohistochemistry, e.g., collagen II, which was detected in all samples. Our results show that perichondrocytes from microtia patients are comparable to healthy perichondrocytes and chondrocytes in terms of chondrogenic cell properties and could therefore be a promising cell source for auricular reconstruction.

FLight Biofabrication Supports Maturation of Articular Cartilage with Anisotropic Properties.

Tissue engineering approaches that recapitulate cartilage biomechanical properties are emerging as promising methods to restore the function of injured or degenerated tissue. However, despite significant progress in this research area, the generation of engineered cartilage constructs akin to native counterparts still represents an unmet challenge. In particular, the inability to accurately reproduce cartilage zonal architecture with different collagen fibril orientations is a significant limitation. The arrangement of the extracellular matrix (ECM) plays a fundamental role in determining the mechanical and biological functions of the tissue. In this study, it is shown that a novel light-based approach, Filamented Light (FLight) biofabrication, can be used to generate highly porous, 3D cell-instructive anisotropic constructs that lead to directional collagen deposition. Using a photoclick-based photoresin optimized for cartilage tissue engineering, a significantly improved maturation of the cartilaginous tissues with zonal architecture and remarkable native-like mechanical properties is demonstrated.

Combining bioengineered human skin with bioprinted cartilage for ear reconstruction.

Microtia is a congenital disorder that manifests as a malformation of the external ear leading to psychosocial problems in affected children. Here, we present a tissue-engineered treatment approach based on a bioprinted autologous auricular cartilage construct (EarCartilage) combined with a bioengineered human pigmented and prevascularized dermo-epidermal skin substitute (EarSkin) tested in immunocompromised rats. We confirmed that human-engineered blood capillaries of EarSkin connected to the recipient's vasculature within 1 week, enabling rapid blood perfusion and epidermal maturation. Bioengineered EarSkin displayed a stratified epidermis containing mature keratinocytes and melanocytes. The latter resided within the basal layer of the epidermis and efficiently restored the skin color. Further, in vivo tests demonstrated favorable mechanical stability of EarCartilage along with enhanced extracellular matrix deposition. In conclusion, EarCartilage combined with EarSkin represents a novel approach for the treatment of microtia with the potential to circumvent existing limitations and improve the aesthetic outcome of microtia reconstruction.

Engineering Inflammation-Resistant Cartilage: Bridging Gene Therapy and Tissue Engineering.

Articular cartilage defects caused by traumatic injury rarely heal spontaneously and predispose into post-traumatic osteoarthritis. In the current autologous cell-based treatments the regenerative process is often hampered by the poor regenerative capacity of adult cells and the inflammatory state of the injured joint. The lack of ideal treatment options for cartilage injuries motivated the authors to tissue engineer a cartilage tissue which would be more resistant to inflammation. A clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 knockout of TGF-ß-activated kinase 1 (TAK1) gene in polydactyly chondrocytes provides multivalent protection against the signals that activate the pro-inflammatory and catabolic NF-κB pathway. The TAK1-KO chondrocytes encapsulate into a hyaluronan hydrogel deposit copious cartilage extracellular matrix proteins and facilitate integration onto native cartilage, even under proinflammatory conditions. Furthermore, when implanted in vivo, compared to WT fewer pro-inflammatory M1 macrophages invade the cartilage, likely due to the lower levels of cytokines secreted by the TAK1-KO polydactyly chondrocytes. The engineered cartilage thus represents a new paradigm-shift for the creation of more potent and functional tissues for use in regenerative medicine.

3D-Printed Reinforcement Scaffolds with Targeted Biodegradation Properties for the Tissue Engineering of Articular Cartilage.

Achieving regeneration of articular cartilage is challenging due to the low healing capacity of the tissue. Appropriate selection of cell source, hydrogel, and scaffold materials are critical to obtain good integration and long-term stability of implants in native tissues. Specifically, biomechanical stability and in vivo integration can be improved if the rate of degradation of the scaffold material matches the stiffening of the sample by extracellular matrix secretion of the encapsulated cells. To this end, a novel 3D-printed lactide copolymer is presented as a reinforcement scaffold for an enzymatically crosslinked hyaluronic acid hydrogel. In this system, the biodegradable properties of the reinforced scaffold are matched to the matrix deposition of articular chondrocytes embedded in the hydrogel. The lactide reinforcement provides stability to the soft hydrogel in the early stages, allowing the composite to be directly implanted in vivo with no need for a preculture period. Compared to pure cellular hydrogels, maturation and matrix secretion remain unaffected by the reinforced scaffold. Furthermore, excellent biocompatibility and production of glycosaminoglycans and collagens are observed at all timepoints. Finally, in vivo subcutaneous implantation in nude mice shows cartilage-like tissue maturation, indicating the possibility for the use of these composite materials in one-step surgical procedures.

Improved accuracy and precision of bioprinting through progressive cavity pump-controlled extrusion.

3D bioprinting has seen a tremendous growth in recent years in a variety of fields such as tissue engineering, drug testing and regenerative medicine, which has led researchers and manufacturers to continuously advance and develop novel bioprinting techniques and materials. Although new bioprinting methods are emerging (e.g. contactless and volumetric bioprinting), micro-extrusion bioprinting remains the most widely used method. Micro-extrusion bioprinting, however, is still largely dependent on the conventional pneumatic extrusion process, which relies heavily on homogenous biomaterial inks and bioinks to maintain a constant material flow rate. Augmenting the functionality of the bioink with the addition of nanoparticles, cells or biopolymers can induce inhomogeneities resulting in uneven material flow during printing and/or clogging of the nozzle, leading to defects in the printed construct. In this work, we evaluated a novel extrusion technique based on a miniaturized progressive cavity pump (PCP) which allows precise control over the volumetric flow rate by positive displacement. We compared the accuracy and precision of this system to the pneumatic extrusion system and tested both systems for their effect on cell viability after extrusion. The PCP achieved a significantly higher accuracy and precision compared to the pneumatic system, while maintaining good viability. These improvements were independent of the bioink composition, printing speed or nozzle size. This study demonstrates the merit of precise extrusion-process control in bioprinting by PCPs and investigates their influence on process-induced cell damage. PCPs are a promising tool for bioprinting and could help provide standardized and validated bioprinted constructs while leaving the researcher more freedom in the design of the bioinks.

Cell-Laden Agarose-Collagen Composite Hydrogels for Mechanotransduction Studies.

The increasing investigation of cellular mechanotransduction mechanisms requires biomaterials combining biofunctionality and suitable mechanical properties. Agarose is a standard biomaterial for cartilage and intervertebral disc mechanobiology studies, but lacks adhesion motifs and the necessary cell-matrix interaction for mechanotransduction. Here, collagen type I was blended at two concentrations (2 and 4.5 mg/mL) with agarose 2% wt/vol. The composite hydrogels were characterized in terms of structural homogeneity, rheological properties and size stability. Nucleus pulposus (NP) cell viability, proliferation, morphology, gene expression, GAG production, adhesion and mechanotransduction ability were further tested. Blended hydrogels presented a homogenous network of the two polymers. While the addition of 4.5 mg/mL collagen significantly decreased the storage modulus and increased the loss modulus of the gels, blended gels containing 2 mg/mL collagen displayed similar mechanical properties to agarose. Hydrogel size was conserved over 21 days for all agarose-based gels. Embedded cells were viable (>80%) and presented reduced proliferation and a round morphology typical of NP cells in vivo. Gene expression of collagen types I and II and aggrecan significantly increased in blended hydrogels from day 1 to 7, further resulting in a significantly superior GAG/DNA ratio compared to agarose gels at day 7. Agarose-collagen hydrogels not only promoted cell adhesion, contrary to agarose gels, but also showed a 5.36-fold higher focal adhesion kinase phosphorylation (pFAK/ß-tubulin) when not compressed, and increased pFAK/FAK values 10 min after compression. Agarose-collagen thus outperforms agarose, mimics native tissues constituted of non-fibrillar matrix and collagens, and allows exploring complex loading in a highly reproducible system.

Development and thorough characterization of the processing steps of an ink for 3D printing for bone tissue engineering.

Achieving reproducibility in the 3D printing of biomaterials requires a robust polymer synthesis method to reduce batch-to-batch variation as well as methods to assure a thorough characterization throughout the manufacturing process. Particularly biomaterial inks containing large solid fractions such as ceramic particles, often required for bone tissue engineering applications, are prone to inhomogeneity originating from inadequate mixing or particle aggregation which can lead to inconsistent printing results. The production of such an ink for bone tissue engineering consisting of gellan gum methacrylate (GG-MA), hyaluronic acid methacrylate and hydroxyapatite (HAp) particles was therefore optimized in terms of GG-MA synthesis and ink preparation process, and the ink's printability was thoroughly characterized to assure homogeneous and reproducible printing results. A new buffer mediated synthesis method for GG-MA resulted in consistent degrees of substitution which allowed the creation of large 5 g batches. We found that both the new synthesis as well as cryomilling of the polymer components of the ink resulted in a decrease in viscosity from 113 kPa·s to 11.3 kPa·s at a shear rate of 0.1 s-1 but increased ink homogeneity. The ink homogeneity was assessed through thermogravimetric analysis and a newly developed extrusion force measurement setup. The ink displayed strong inter-layer adhesion between two printed ink layers as well as between a layer of ink with and a layer without HAp. The large polymer batch production along with the characterization of the ink during the manufacturing process allows ink production in the gram scale and could be used in applications such as the printing of osteochondral grafts.

Cartilage tissue formation through assembly of microgels containing mesenchymal stem cells.

Current clinical approaches to treat articular cartilage degeneration provide only a limited ability to regenerate tissue with long-term durability and functionality. In this application, injectable bulk hydrogels and microgels containing stem cells can provide a suitable environment for tissue regeneration. However insufficient cell-cell interactions, low differentiation efficiency and poor tissue adhesion hinder the formation of high-quality hyaline type cartilage. Here, we have designed a higher order tissue-like structure using injectable cell-laden microgels as the building blocks to achieve human bone marrow-derived mesenchymal stem cell (hBMSC) long-term maintenance and chondrogenesis. We have demonstrated that a 4-arm poly(ethylene glycol)-N-hydroxysuccinimide (NHS) crosslinker induces covalent bonding between the microgel building blocks as well as the surrounding tissue mimic. The crosslinking process assembles the microgels into a 3D construct and preserves the viability and cellular functions of the encapsulated hBMSCs. This assembled microgel construct encourages upregulation of chondrogenic markers in both gene and glycosaminoglycan (GAG) expression levels. In addition, the regenerated tissue in the assembled microgels stained positively with Alcian blue and Safranin O exhibiting unique hyaline-like cartilage features. Furthermore, the immunostaining showed a favourable distribution and significantly higher content of type II collagen in the assembled microgels when compared to both the bulk hydrogel and pellet cultures. Collectively, this tissue adhesive hBMSC-laden microgel construct provides potential clinical opportunities for articular cartilage repair and other applications in regenerative medicine. STATEMENT OF SIGNIFICANCE: A reliable approach to reconstruct durable and fully functional articular cartilage tissue is required for effective clinical therapies. Here, injectable hydrogels together with cell-based therapies offer new treatment strategies in cartilage repair. For effective cartilage regeneration, the injectable hydrogel system needs to be bonded to the surrounding tissue and at the same time needs to be sufficiently stable for prolonged chondrogenesis. In this work, we utilised injectable hBMSC-laden microgels as the building blocks to create an assembled construct via N-hydroxysuccinimide-amine coupling. This crosslinking process also allows for rapid bonding between the assembled microgels and a surrounding tissue mimic. The resultant assembled microgel-construct provides both a physically stable and biologically dynamic environment for hBMSC chondrogenesis, leading to the production of a mature hyaline type cartilage structure.