Microtia is a congenital dysplasia of the auricle, which is usually clinically manifested as auricular malformation, often combined with atresia of the external auditory canal and deformity of the middle ear, in layman's terms, that is, smaller than the contralateral ear, and lacks normal ear morphology, it can be called microtia. Microtia can be divided into non-surgical and surgical.
According to the market observation of 3D Science Valley, the medical community has used 3D printing technology to manufacture the implants required for external ear reconstruction surgery in the program of surgical microtia. For instance, 3DBIO Medical, a clinical-stage regenerative medicine company, and the Microtia Congenital Ear Malformation Institute in 2022 performed human ear reconstruction using the Aurinovo implant, an investigational, patient-matched, 3D bioprinted bioprinted ear implant.
EngineeringForLife has recently introduced a novel auricular biomanufacturing method that combines 3D printed molds with advanced casting strategies. Root reported that due to the complex and hierarchical structure of the anatomical tissue, it is a challenge to establish multi-tissue auricular transplantation to ** microtia. To this end, Marcy Zenobi-Wong and his team at ETH Zurich proposed a method that utilizes elution agaroseMoldsHeterogeneous, multilayered, and human-scale tissue transplants are performedA new casting technology for 3D biomanufacturing。In this issueValley. ColumnThe technique will be shared briefly.
These molds are generated by casting agarose into custom-made 3D printed containers, called meta-molds, optimized to facilitate the hydrogel casting process based on geometric and topological constraints. Casting yields high resolution (50 m) and allows further hydrogel layers to be cast on the graft. The multilayered auricular structure is fabricated on a cartilage core consisting of a hyaluronic acid-alginate dual network and an adjacent gelatin-based dermal layer. Bonding between adjacent layers is achieved through orthogonal physical and enzymatic cross-linking of residual functional groups between layers. The material composition and culture time were optimized for each layer of cartilage and prevascularized dermal tissue. To demonstrate the scalability of this technique in the biofabrication of human-sized grafts, double-layered human-sized ears were cast. In general, this new casting technologyIt provides a promising method for the fabrication of complex tissue grafts, overcoming the limitations of other traditional biomanufacturing methods.
The research was published in Advanced Healthcare Materials on October 28, 2023 under the title "Biofabrication of Heterogeneous, Multi-Layered, and Human-Scale Tissue Transplants Using Eluting Mold Casting".
Figure 1 Overview of the experiment.
The study introduces a novel casting technique for the biofabrication of non-uniform bilayer structures, including a vascular-free cartilage core and a pre-vascularized dermis. The proposed technique is based on generating a multi-part agarose mold based on a meta-mold 3D printed using DLP technology. Agarose molds were prepared using a low concentration of agarose (3% W V) and preloaded with calcium chloride (CaCl2) and hydrogen peroxide (H2O2) for ionic crosslinking of alginate and covalent crosslinking of tyramine-modified hyaluronic acid and gelatin-hydrogel, respectively. The cellular hydrogel precursor solution is then cast into a continuous agarose mold to precisely generate multilayer hydrogels. To demonstrate the scalability and potential of this technique, a double-layered, human-sized ear transplant was fabricated in this study (Figure 1). The core structure of auricular cartilage is created by encapsulating human auricular cartilage cells in a hydrogel made of high molecular weight hyaluronic acid tyramide (HA-TYR) and alginate (ALG). A simulated dermal layer is formed by encapsulating Huvecs and primary human dermal fibroblasts in gelatin tyramine (gel-tyr) hydrogels. This bioorthogonal strategy maintains the vascular-free nature of hyaline cartilage while also supporting the development of a microcapillary network adjacent to the dermal tissue layer. The possibility of producing patient-specific mold designs and the multi-layered and scalable approach of this technology make elution molds an attractive solution for the manufacture of human-sized tissues and organs with a wide range of cell densities.
Fig.2 Mold making.
Once the optimal removal direction is found, a set of plastic molds called meta-molds is generated (Figure 2A), in which a molten agarose polymer solution is injected and cooled to room temperature to create an agarose mold (Figure 2B). Next, the agarose mold is extracted from the meta-mold and assembled (Figure 2C). Use a syringe to inject the cell-filled hydrogel precursor solution into the assembled mold (Figure 2D). Depending on the mold design, the two molds can be easily separated to visualize the cast implant (Figure 2e). Subsequent layers can be added to the implant in the same way (Figure 2F-L): repeat this process for the new layer, starting at the top, with the separation direction remaining the same. To cast the second part of this layer, remove the bottom mold, replace it with a new one, and repeat the process (Figure 2F-L).
Fig.3 Simulated 100 mmCaCl2 and 0Finite element analysis of 01% H2O2 diffusion from agarose mold into cast hydrogel.
To estimate the time required for crosslinking of each layer, a finite element analysis (FEA) simulation was set up in COMSOL multiphysics (Figure 3). Figure 3a shows 100 mMcAc2 and 0., respectively01% H2O2 diffusion over time. The simulation estimated that the time for complete diffusion of 100 mCaCl2 into the cast cartilage structure was 30 min, 0The time of 01% H2O2 was 20 min. Figure 3b shows the same simulation of the second layer casting. In this case, only H2O2 is required for crosslinking of the casting material.
Fig.4 In vitro characterization of the cartilage control group.
Figures 4A, B show the chondrogenesis potential of condition 2 with 30 106 cells in 49 days. At all time points, the cells remained more than 93% viable (Figure 4C). Using intensity quantification based on immunohistological staining, semi-quantitative assessment of glycosaminoglycans (GAGs) and collagen deposition in samples can be performed (Figure 4D). Compression testing showed a stable and significantly increased stiffness of the sample during the 49-day incubation period (Figure 4E). RT-qPCR data confirmed that the expression of collagen type I (col1a1) gene was not significantly increased at selected time points (Figure 4F). In contrast, the marker genes of the cartilage extracellular matrix (ECM) type II collagen (COL2A1) and aggrecan (ACAN) showed significant upregulation over time (Figure 4F).
Fig.5 In vitro characterization of vascularized dermal control.
3% and 4The 5% condition allows for rapid development of the vascular network in the 3D matrix (Figure 5A). Unrestricted compression measurements show that the elastic modulus continues to increase with the polymer content, and is 1709 102 pA at 6% gel-tyr (Fig. 5b). There were significant differences in vascularization area and total vessel length between 3% and 6% gel-tyr conditions (Figure 5C). Colocalization of CD90+ fibroblasts and microcapillaries was confirmed using CD90(thy-1) CD31 co-staining (Figure 5D). Long-term build-up cultures lead to the formation of a highly interconnected capillary network (Figure 5E). After 7 days, lumen formation is clearly visible (Figure 5F).
Fig.6. Casting of humanoid multi-layered ears.
To demonstrate the scalability and potential of meta-model technology, one was madeTwo-layer, human-sized ear graft technique。Start at the auricular cartilage core and cast from the inside to the outside (Figure 6A). Cross-linking is initiated by the diffusion of CaCl2 and H2O2 from the agarose mold into the injected polymer solution for 30 minutes (Figure 6A). Between these two steps, cross-link the polymer solution for 15 min by allowing H2O2 to diffuse from the agarose mold (Figure 6A). Finally, the top agarose mold is discarded, while the bottom agarose mold serves as a resting surface for multilayer structures during incubation (Figure 6A). As shown in Figure 6a, good shape retention was observed throughout the 49-day incubation period due to the agarose mold, as the agarose mold supports the overhang of the multilayer ear at the initial time point where its stiffness is low. At the last time point, make a precise incision at the interface of the cartilage and dermis using a scalpel to assess the increase in the compressive modulus of the cartilage core (Figure 6B). At the last time point, 3 regions from different parts of the mature ear structure were assessed using histological analysis (Figure 6C). As shown in Figure 6D, the resulting cartilage quality is the best, with strong staining of GAGS and type II collagen, and absence of type I collagen staining.
In summary, this study adopts a novel biomanufacturing method based on advanced casting strategiesLarge tissue grafts are made using elution molds。Compared to traditional casting methods, this method enables the biofabrication of more complex structures, including different layers composed of different materials and cell types. Since there are no strict rheological requirements, this new biomanufacturing method allows the use of a wide range of biomaterials. Different materials and element types can be combined in a layered manner, independent of the chosen design. This work provided a proof of concept for this technology, using two different hydrogels and multiple cell types to biofabricate human-sized load cell structures. Future work will focus on more intensive characterization of each layer after casting, as well as improving the culture conditions for such large structures to mitigate the effects of the diffusion limit through the agarose mold. This can be achieved using custom plastic grid supports to preserve the structure without introducing a physical barrier.
Article**: Knowing is deep, and doing is far. Based on a global network of manufacturing experts, 3D Science Valley provides the industry with an in-depth view of additive and intelligent manufacturing from a global perspective. For more analysis in the field of additive manufacturing, follow the *** series released by 3D Science Valley.
*Submissions l Send to 2509957133@qqcom