Three-dimensional printing of a patient-specific engineered nasal cartilage for augmentative rhinoplasty

Abstract

Autologous cartilages or synthetic nasal implants have been utilized in augmentative rhinoplasty to reconstruct the nasal shape for therapeutic and cosmetic purposes. Autologous cartilage is considered to be an ideal graft, but has drawbacks, such as limited cartilage source, requirements of additional surgery for obtaining autologous cartilage, and donor site morbidity. In contrast, synthetic nasal implants are abundantly available but have low biocompatibility than the autologous cartilages. Moreover, the currently used nasal cartilage grafts involve additional reshaping processes, by meticulous manual carving during surgery to fit the diverse nose shape of each patient. The final shapes of the manually tailored implants are highly dependent on the surgeons' proficiency and often result in patient dissatisfaction and even undesired separation of the implant. This study describes a new process of rhinoplasty, which integrates three-dimensional printing and tissue engineering approaches. We established a serial procedure based on computer-aided design to generate a three-dimensional model of customized nasal implant, and the model was fabricated through three-dimensional printing. An engineered nasal cartilage implant was generated by injecting cartilage-derived hydrogel containing human adipose-derived stem cells into the implant containing the octahedral interior architecture. We observed remarkable expression levels of chondrogenic markers from the human adipose-derived stem cells grown in the engineered nasal cartilage with the cartilage-derived hydrogel. In addition, the engineered nasal cartilage, which was implanted into mouse subcutaneous region, exhibited maintenance of the exquisite shape and structure, and striking formation of the cartilaginous tissues for 12 weeks. We expect that the developed process, which combines computer-aided design, three-dimensional printing, and tissue-derived hydrogel, would be beneficial in generating implants of other types of tissue.

Keywords: Three-dimensional bioprinting; computer aided design and manufacturing; decellularization; rhinoplasty; tissue engineering.

Figure 1.

Computer-aided design and 3D printing of a patient-customized nasal implant. (a) The process of generating the custom design of the nasal implant model. The difference between the preoperative and postoperative nose geometrical shapes was calculated. A 3D solid model was then generated according to the geometric difference. Finally, an octahedral pattern architecture was designed in the nasal implant model, and a cover mold model was designed based on the nasal implant model. (b) Schematic elucidating the principle of fabricating a 3D construct by the pMSTL system. (c) Photographs of the fabricated PCL nasal implant and OrmoComp cover mold with the patient-specific design (scale bars = 5 mm).

Figure 2.

Process of cell-laden hydrogel injection technique and cell distribution in the scaffold. (a) Assembling cover molds and scaffolds for cell-laden hydrogel injection. (b) A schematic of the cell-laden hydrogel pre-gel injection procedure. (c) Calcein AM staining of each scaffold after cell seeding and injecting (scale bar = 200 μm, left). Quantification of cell distribution in each scaffold (n = 3 per experimental group, ****p < 0.0001, right).

Figure 3.

Cell viability and hypoxia in engineered nasal cartilage. (a) Assembled nasal implant and cover mold (left); hydrogel-injected nasal implant (right). (b) LIVE/DEAD staining of engineered nasal cartilage at days 4, 10, and 14. Star symbols indicate PCL region, green indicates live cells, and red indicates dead cells in the hydrogel region (scale bar = 200 μm). (c) Pimonidazole staining of engineered nasal cartilage at day 14: green indicates hypoxia, and blue indicates nucleus (DAPI; scale bar = 200 μm).

Figure 4.

Chondrogenic differentiation in engineered cartilage in vitro. (a) Changes in mRNA expression levels and (b) GAG amount normalized to DNA amount, and the production of (c) aggrecan and (d) type-2 collagen in the hASCs injected with alginate hydrogel or cartilage-derived hydrogel (n = 4 per experimental group for real-time qPCR; n = 3 per experimental group for biochemical assay; ***p < 0.001; ****p < 0.0001; scale bar = 200 μm).

Figure 5.

Subcutaneous implantation of the engineered nasal cartilage: (a) schematic and photograph of the construct implanted in a dorsal subcutaneous region (scale bar = 10 mm) and (b) gross image of the retrieved implant after 12 weeks post-implantation. The ruler is graduated in mm.

Figure 6.

The histological analysis of the implanted engineered nasal cartilages. Representative images obtained after H&E and collagen II staining of the implants retrieved at (a) 6 and (b) 12 weeks (scale bars = 200 μm). The red dotted line and the star symbols indicate the PCL region.