Philipp Fisch

Osteochondral defects remain a major clinical challenge, frequently progressing to osteoarthritis and ultimately requiring joint replacement. Despite advances in regenerative strategies, current approaches often fail to restore the structural and functional complexity of the osteochondral unit, resulting in poor integration and limited long-term performance. Here, we developed autologous osteochondral grafts combining mechanically mature nasal chondrocyte-derived cartilage with a 3D-printed strontium–hardystonite–gahnite trabecular bone scaffold, which was tested in an osteochondral defect model in sheep. Nasal chondrocytes were encapsulated in a hyaluronan transglutaminase-alginate hydrogel generating hyaline-like cartilage with physiologically relevant compressive properties (1.0 ± 0.2 MPa) after 6 weeks of in vitro maturation. µCT scans of the osteochondral unit were used to design biomimetic trabecular bone scaffolds. Controlled infiltration of the hydrogel into the trabecular pores and subsequent cartilage maturation created a mechanical interlock between cartilage and bone layer, reaching an interfacial shear strength of 201 ± 42 kPa. The translational potential of this approach was evaluated in a proof-of-concept ovine study. The engineered cartilage remained attached to the bone scaffold for three months following implantation, but osteochondral repair was limited by implant sinking, likely caused by inadequate implant fixation in the defect, resulting in micromotion, synovial fluid infiltration, osteolysis and subsequent bone cyst formation. Together, this work demonstrates the feasibility of engineering mechanically mature autologous osteochondral grafts while identifying subchondral bone integration as the primary bottleneck limiting successful osteochondral regeneration and future clinical translation.

A persistent challenge in cartilage tissue engineering is identifying cell sources capable of regenerating cartilage with native-like properties. Variability in chondrogenic potential among human- and animal-derived cells complicates graft development and disease modeling. Here we benchmarked ten chondrogenic cell sources under standardized culture conditions using hyaluronan-alginate biomaterials and implementing a functionality index combining histological and mechanical metrics referenced to native tissue. Mesenchymal stem cells and fetal chondroprogenitors demonstrated superior regenerative capacity, achieving 103% and 95% of native cartilage functionality, respectively. In contrast, adult articular chondrocytes from healthy and osteoarthritic donors produced fibrocartilaginous tissue with functionality scores below 50%. These findings provide a quantitative framework to guide cell source selection and optimize cartilage graft standardization for clinical applications and physiologically relevant disease models, addressing regulatory demands for product characterization.

Functional elastic cartilage remains an unmet challenge in regenerative medicine, limiting reconstructive options for children born with microtia, the congenital malformation of the auricle. The current gold standard treatment relies on harvesting rib cartilage, an invasive procedure associated with donor site morbidity, impaired aesthetics, and unnaturally stiff ears due to the use of hyaline cartilage. Here, we engineered primary human auricular chondrocytes to generate functional elastic cartilage with near‐native tissue properties. This was achieved through the synergistic integration of growth factor‐controlled chondrogenesis, stress‐relaxing biomaterials that promote elastic cartilage formation, and a bioreactor platform enabling homogeneous tissue maturation. The engineered grafts exhibited a uniform extracellular matrix composition with elastin, glycosaminoglycans, and collagen II comparable to native auricular cartilage, while lacking the fibrocartilage marker collagen I. The resulting tissue achieved a compressive modulus of 1.1 ± 0.03 MPa, matching native cartilage (1.0 ± 0.1 MPa), and maintained its structural integrity and elasticity in vivo for 6 weeks in a subcutaneous rat model. These grafts represent the closest replication of native human elastic cartilage achieved ex vivo to date, establishing a foundation for clinically viable, functional auricular grafts.

The development of rapid, chemoselective covalent bond-forming reactions enables the assembly of hydrogel scaffolds suitable for applications in cellular encapsulation. We previously reported that amide-forming ligations between potassium acyltrifluoroborates (KATs) and hydroxylamines produce robust hydrogels that have excellent cytocompatibility, but the requirement for somewhat acidic conditions for efficient hydrogel assemblies limited their application to robust cell types. To overcome this constraint, we have recently found that quinolinium acyltrifluoroborates (QATs) serve as highly efficient reaction partners for amide-forming reactions at neutral pH. In this article, we document the construction of poly(ethylene glycol) (PEG)-derived hydrogels by efficient cross-linking of QAT-functionalized macromers with a partner hydroxylamine-functionalized macromer. Gelation occurs at physiological pH in under 2 min, offering a rapid and facile approach to the immobilization of delicate stromal cells. The cytocompatibility of the cross-linking was demonstrated by in situ gelation in the presence of human mesenchymal stem cells and sustained cell viability for 7 days. Facile incorporation of a cyclic cell adhesion peptide, simply by including the reaction partner in the gelation reactions, illustrated that the desired components can be introduced into the gels without further elaboration.

Two‐photon polymerization (2PP) has garnered increasing attention for engineering hydrogels with tailored architectures and controlled cellular responses. However, current 2PP strategies typically rely on (meth)acrylated proteins and inefficient chain‐growth crosslinking mechanisms. Although thiol‐ene photo‐click reactions can enhance 2PP efficiency, commercial water‐soluble thiol crosslinkers (e.g., DTT—dithiothreitol) tend to form intramolecular loops and introduce structural defects due to their short molecular length. As a result, high polymer concentrations (often up to 20%–50%) are required to achieve satisfactory print fidelity. Here, we develop a series of water‐soluble, polyvinyl alcohol macromolecular thiol (PVASH, bearing 10–35 thiol groups) for fast high‐fidelity hydrogel microfabrication via 2PP. A two‐step synthesis yields PVASH with tunable degrees of substitution and excellent water‐solubility. Compared to DTT and polyethylene glycol di‐thiol, PVASH‐based hydrogels exhibit reduced swelling, enhanced mechanical properties, and significantly improved printing fidelity. Notably, several complex hydrogel structures are fabricated at laser power as low as 20 mW and high scanning speeds of up to 400 mm s ⁻¹ , achieving sub‐micron feature size at 3% polymer concentration. After biofunctionalization with RGD motifs, the micro‐scaffolds support cell infiltration, adhesion, proliferation, and osteogenic differentiation. Altogether, this work reports a new strategy for 2PP microfabrication of cell‐interactive hydrogel structures with unprecedented printing efficiency and precision.

Patients born with microtia, the congenital malformation of the external ear, face substantial psychosocial strain. Current reconstruction relies on harvesting rib cartilage, an invasive procedure associated with donor site morbidity and unnaturally stiff ears due to the use of hyaline cartilage. Tissue engineered auricles could overcome these drawbacks by providing patient-specific elastic cartilage without the need for rib harvest. Yet, key challenges such as fibrocartilage formation, inhomogeneous extracellular matrix formation and mechanical inferiority during ex vivo maturation remain, often leading to graft deformation and degradation in vivo . To address this gap, we integrated approaches maintaining the chondrogenic potential with growth factors, promoting elastic cartilage formation through stress-relaxing materials, and achieving homogeneous maturation by culturing grafts on an elevated bioreactor platform that enables uniform nutrient diffusion, using primary human auricular chondrocytes. Together these approaches resulted in the maturation of bioprinted auricular grafts that closely resemble native human auricular cartilage, demonstrated by the uniform distribution of elastin, glycosaminoglycans, and collagen II, and lack of collagen I. RNA sequencing revealed gene expression patterns consistent with the transition from fibrocartilage towards elastic cartilage. On a functional level, grafts achieved a compressive modulus of 1.1±0.03 MPa, matching that of native human auricular cartilage (1.0±0.1 MPa) and maintained their structural integrity for 6 weeks in a subcutaneous rat model, where they transitioned towards mature elastic fibers. These grafts represent the closest approximation of native elastic cartilage achieved ex vivo to date, bringing the field closer to a clinically viable, long-term therapy for children affected by microtia. One-sentence summary: Bioprinted auricular grafts develop native-like elastic cartilage and advance towards a durable therapy for children with microtia.

Generating 3D bone cell networks in vitro that mimic the dynamic process during early bone formation remains challenging. Here, we report a synthetic biodegradable microporous hydrogel for efficient formation of 3D networks from human primary cells, analysis of cell-secreted extracellular matrix (ECM) and microfluidic integration. Using polymerization-induced phase separation, we demonstrate dynamic in situ formation of microporosity (5–20 µm) within matrix metalloproteinase-degradable polyethylene glycol hydrogels in the presence of living cells. Pore formation is triggered by thiol-Michael-addition crosslinking of a viscous precursor solution supplemented with hyaluronic acid and dextran. The resulting microporous architecture can be fine-tuned by adjusting the concentration and molecular weight of dextran. After encapsulation in microporous hydrogels, human mesenchymal stromal cells and osteoblasts spread rapidly and form 3D networks within 24 hours. We demonstrate that matrix degradability controls cell-matrix remodeling, osteogenic differentiation, and deposition of ECM proteins such as collagen. Finally, we report microfluidic integration and proof-of-concept osteogenic differentiation of 3D cell networks under perfusion on chip. Altogether, this work introduces a synthetic microporous hydrogel to efficiently differentiate 3D human bone cell networks, facilitating future in vitro studies on early bone development.

The gold standard of auricular reconstruction involves manual graft assembly from autologous costal cartilage. The intervention may require multiple surgical procedures and lead to donor‐site morbidity, while the outcome is highly dependent on individual surgical skills. A tissue engineering approach provides the means to produce cartilage grafts of a defined shape from autologous chondrocytes. The use of autologous cells minimizes the risk of host immune response; however, factors such as biomaterial compatibility and in vitro maturation of the tissue‐engineered (TE) cartilage may influence the engraftment and shape‐stability of TE implants. Here, this work tests the biocompatibility of bioprinted autologous cartilage constructs in a rabbit model. The TE cartilage is produced by embedding autologous auricular chondrocytes into hyaluronan transglutaminase (HATG) based bioink, previously shown to support chondrogenesis in human auricular chondrocytes in vitro and in immunocompromised xenotransplantation models in vivo. A drastic softening and loss of cartilage markers, such as sulfated glycosaminoglycans (GAGs) and collagen type II are observed. Furthermore, fibrous encapsulation and partial degradation of the transplanted constructs are indicative of a strong host immune response to the autologous TE cartilage. The current study thus illustrates the crucial importance of immunocompetent autologous animal models for the evaluation of TE cartilage function and compatibility.

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.

The creation of multi‐tissue auricular transplants for the treatment of microtia is a challenge due to the complex and layered structure of this anatomical tissue. A novel casting technique for the 3D biofabrication of heterogeneous, multi‐layered, and human‐scale tissue transplants using eluting agarose molds is presented. The molds are generated by casting agarose into custom 3D‐printed containers, termed metamolds, optimized to facilitate the hydrogel casting process based on geometric and topological constraints. Casting yields high resolution (50 µm) and allows for subsequent casting of further hydrogel layers on the transplant. Multi‐layered auricular constructs are fabricated on a cartilage core consisting of a hyaluronic acid‐alginate double network and an adjacent gelatin‐based dermal layer. Bonding between adjacent layers is achieved by orthogonal physical and enzymatic crosslinking of residual functional groups between each layer. Material composition and culture duration are optimized for each layer allowing for maturation into cartilaginous and pre‐vascularized dermal tissues. To demonstrate the scalability of this technique for the biofabrication of human‐sized transplants, bi‐layered human‐sized ears are cast. Overall, this novel casting technique offers a promising approach for the fabrication of complex tissue grafts, overcoming the limitations of other traditional biofabrication methods.