Advances in the Development of Auricular Cartilage Bioimplants

Abstract

Conditions such as congenital abnormalities, cancer, infections, and trauma can severely impact the integrity of the auricular cartilage, resulting in the need for a replacement structure. Current implants, carved from the patient’s rib, involve multiple surgeries and carry risks of adverse events such as contamination, rejection, and reabsorption. Tissue engineering aims to develop lifelong auricular bioimplants using different methods, different cell types, growth factors and maintenance media formulations, and scaffolding materials compatible with the host. This review aims to examine the progress in auricular bioengineering, focusing on improvements derived from in vivo models and clinical trials, as well as the author’s suggestions to enhance the methods. For this scope review, 30 articles were retrieved through Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, plus 6 manually selected articles. The methods reported in the articles were categorized into four levels according to the development phases: source of cells, cell media supplementation, scaffold, or scaffold-free methods, and experimental in vivo or clinical approaches. Many methods have demonstrated potential for the development of bioimplants; four clinical trials reported a structure like the external ear that could be maintained after overcoming post-transplant inflammation. However, several challenges must be solved, such as obtaining a structure that accurately replicates the shape and size of the patient’s healthy contralateral auricle and improvements to avoid immunological rejection and resorption of the bioimplant.

Impact Statement

This review highlights recent advances and challenges in developing bioimplants for auricular cartilage: (1) the use of mesenchymal stem cells in coculture with auricular cartilage chondrocytes to promote the development of elastic cartilage while maintaining an adequate cell mass; (2) medium supplementation with growth factors and nutrients to promote chondrogenesis and to avoid dedifferentiation; (3) improvements in materials for scaffold assembly or the use of scaffold-free methods to form stratified tissues without exogenous material to prevent immune rejection; and (4) the importance of grafting bioimplants into immunocompetent hosts for proper cartilage regeneration, long-term safety, and shape stability.

Keywords

auricular cartilage scaffold chondrocytes tissue engineering

Introduction

The ear is divided into the external, middle, and internal structures, which are responsible for hearing, balance, and orientation. The function of the outer ear (pinna) is to capture, amplify, and conduct sound waves into the ear channel.¹ It acquires its functional shape through the pinna, an elastic cartilage with multiple semirigid textured concavities, covered and supported by soft tissues such as the perichondrium, adipose tissue, and the skin. It also has variations in size and concavities.² In addition, the pinna is projected symmetrically outside the skull and, in balance with the facial landmarks, contributing to facial aesthetics.^3,4

Microtia is a relatively common malformation of the pinna, with a global prevalence of 8–4.2/10,000 births.^5,6 This malformation can be unilateral or bilateral and is classified by levels of pinna development from a smaller ear to absence (anotia).⁷ This condition affects the craniofacial aesthetic of patients and impacts their psychosocial development and learning.⁸ Other conditions that impact the integrity of the pinna are malignant neoplasms, local traumas, pinna infections, and facial burns.^9–14 The aesthetic of the ears attracts attention on a first impression in the social context, it plays an important role in identity, and has psychological and functional relevance. The patients are seen and treated differently due to the stigma associated with their physical abnormalities.^15,16 The first attempt to reconstruct the pinna dates to 3000 B.C. Since then, techniques have evolved to use autologous or cadaveric rib or synthetic implants that are manually molded by the surgeon.^17–19 However, these implants require multiple surgeries and are prone to adverse outcomes such as tissue calcification, extrusion, fracture, or even bacterial contamination.^20–24 Tissue engineering and regenerative medicine aim to create functional, aesthetic, biocompatible, and lifelong bioimplants. Pinna implants are constructed with cells (seeded or recruited in situ), chondrogenic development factors, and scaffold or scaffold-free methods. The challenges are to get enough cells to populate the scaffolds, to promote the development of cartilage extracellular matrix (ECM), to control postsurgical inflammation and possible immune rejection, and to allow bioimplant vascularization.²⁵ Cartilage is not vascularized; it obtains nutrients through the permeation of fluids from neighboring blood vessels. Therefore, local vascularization is expected to enhance the nutrition and oxygenation of the adjacent tissues such as the skin covering the transplanted bioimplant, preventing its absorption or degradation.²⁶

This review aims to compile the results of auricular cartilage bioimplants that have been developed in preclinical and clinical trials, summarizing key information for the development of bioimplants, as well as the factors contributing to chondrogenesis, chondrocyte phenotype maintenance, ECM synthesis, and prevention of ossification. A brief review of in vivo and clinical trials is also added.

Methods

We carried out a scoping review on “tissue engineering” and “regenerative medicine” for developing human auricular cartilage. Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were used.²⁷

Initial search

The searches were carried out in July 2024 in PubMed by defining publications with a range of six years (2018–2024) of having been published. The following terms were tested: “[(auricular reconstruction) AND (microtia)]” obtaining 213 results, “(((auricular reconstruction) OR (external ear reconstruction)) AND (human))” obtaining 1,102 results and “(((auricle) OR (cartilage xenograft)) AND (human))” obtaining 4,003 results. The last option of terms was selected for an initial list of reports. Additionally, six articles from Scholar Google were manually added after using the search words “auricular reconstruction” and “external ear reconstruction” to explore options that were not systematically included in PubMed. Subsequently, the articles were selected by reading the title and the abstract, and by following the inclusion criteria:

Empirical research and case studies.

Research that developed scaffolds with or without cells.

Research that developed scaffolds-free methods with cells.

Research that used auricular cartilage cells.

Research that used mesenchymal, pluripotent, and stromal cells.

Auricular bioimplants tested in in vivo trials.

Publications between 2018 and 2024.

The exclusion criteria were:

Systematic reviews, meta-analyses, books, or manuals.

Abstracts of conferences, seminars, or congresses.

Articles that develop a different topic in the field of in vitro development of auricular cartilage with in vivo trials.

Research on bioimplants developed from rib or surgical materials.

Investigations without in vivo trials.

In total, 68 articles were considered adequate. After reading the manuscripts, verifying that they met the inclusion criteria, and excluding duplicate articles, 38 articles were discarded since they presented one or more exclusion criteria. We worked with 30 systematically acquired articles plus 6 manually selected articles. This article summarizes the main topics in the field of auricle bioengineering according to the following subjects: source of cells, media and supplements, scaffolds, and experimental in vivo approaches.

Results

Generalities

According to the methods for bioimplant development, the reports were categorized into four steps (I–IV) considering the following technical specifications: (I) cell resource, (II) cellular medium supplementation for cell differentiation or phenotype maintenance, (III) structural support, and (IV) trials (Fig. 1).

FIG. 1.

Bioimplant development stage. I. Cell resource: mesenchymal stem cells (MSCs), chondrocytes, or cocultures of both cell types. II. Cell media supplementation. This can be modified for chondrocyte differentiation when using MSCs or phenotype maintenance when using chondrocytes. III. Structural support of the bioimplant. This may be accomplished by using a scaffold (manual shape or by molds, decellularization, hydrogels, or 3D bioprinting), or by using scaffold-free methods (microsheets or microcultures). IV. Testing bioimplant quality and safety through preclinical and clinical trials.²⁸ 3D, three-dimensional.

I. Cell resource

Seven types of cells and their combinations in coculture have successfully differentiated and produced cartilage scaffolds (Fig. 2). The first cellular resource option is auricular cartilage chondrocytes (ACCs). The cartilage is obtained from otoplasty or microtia reconstruction procedures.¹⁹ ACCs are isolated using enzymatic methods to establish a primary cell culture. ACCs maintain their phenotype until passage 5 and proliferate within the scaffold while synthesizing a significant amount of elastic cartilage ECM.²⁸ However, disturbances in the expression of the Ras Homolog Family Member A (RHOA) gene can occur, leading to decreased cell migration and ECM quality. Transfecting chondrocytes with a vector encoding RHOA resolves this issue.²⁹ Patients with microtia often have cartilage remnants on the affected side.³⁰ Remnants are usually removed and can be used to obtain microtia auricular chondrocytes (mACs), which behave similarly to ACCs under specific conditions.³¹ Nevertheless, the phenotype of mACs can be affected in passage 2, resulting in the development of fibrotic tissue.³² A limitation for ACCs and mACs is the difficulty in achieving optimal cell mass for the cellularization of the bioimplant.²⁵ Another source of patient cells is costal chondrocytes (CCs) isolated from rib cartilage. These cells also exhibit behavior such as ACCs and can develop elastic cartilage ECM. It is essential to consider the patient’s age when utilizing patient-derived cells, as cartilage undergoes age-related modifications.^33,34 The procedure to isolate auricular and CCs requires surgery, which may lead to additional comorbidities for the patient.^22,25 The adipose tissue has been proposed as a patient cell resource because of its abundance and availability through minimally invasive procedures. Using enzymatic techniques, it is possible to isolate adipose-derived stem cells (ASCs), which encompass a variety of progenitor and stem cells, as mesenchymal stem cells (MSCs), multipotent cells with the ability to trigger chondrogenesis when exposed to growth factors. They can be cocultured with chondrocytes.^35,36 Coculturing ACCs with MSCs enhances both cell differentiation and cell mass.³⁷ Microtia auricular adipose-derived mesenchymal stem cells (maMSCs), isolated from the adipose tissue surrounding the remnants in microtia patients, represent another progenitor cell resource that can be differentiated alongside CCs.^25,38 Autologous human induced pluripotent stem cells (iPSCs) also represent a potential alternative due to their increased proliferative capacity and pluripotency.³⁹

FIG. 2.

Proportion of cells utilized in the development of cartilage ECM. Auricular cartilage chondrocytes (ACCs) are the primary choice, accounting for 30% of all cell resources used. Healthy pinnae cells are obtained from donor patients or animals. Microtia auricular chondrocytes (mACs) were utilized in 19% of the reports. These cells are isolated from chondral remnants of patients with microtia. Recent studies have shown that their behavior may differ from mature chondrocytes derived from healthy ears. Acellular scaffolds were recellularized in situ, in 16% of the reports. Once the scaffold is grafted, its biochemical composition triggers signaling for recruiting host cells. Cocultures combining mesenchymal stem cells (MSCs) with chondrocytes from different sources were employed in 12% of the studies. Chondrocytes facilitate MSCs differentiation, and MSCs provide increased cell mass. Other types of cells, such as fibroblasts, were used in 9% of the cases to verify biocompatibility with the scaffold materials. MSCs isolated from adipose tissue were used in 5% of reports. These are differentiated by growth factors to trigger chondrogenesis and to maintain this phenotype. Microtia auricular adipose-derived mesenchymal stem cells (maMSCs) were used in 3% of the reports. These cells are isolated from the adipose tissue surrounding auricular cartilage and contain progenitor cells. Costal chondrocytes (CCs) derived from rib cartilage, along with progenitor cells such as induced pluripotent stem cells (iPSCs) and adipose-derived stromal cells (ASCs) differentiated into chondrocytes, were also reported in 3% of studies. ECM, extracellular matrix.

II. Cellular medium supplementation

The cellular medium and its supplementation are formulated according to the nutritional requirements for chondrogenesis and cell expansion. In general, chondrocyte culture media requires insulin-transferrin-selenium (ITS) with fetal bovine serum, autologous patient’s serum, or xeno-free culture media. ITS promotes the proliferation of auricular chondrocytes and increases the quality and quantity of cartilage ECM components.⁴⁰ Another essential supplement is ascorbic acid, which enhances insulin signaling by facilitating glucose uptake, participates as a cofactor for collagen synthesis, and promotes the expression of pro-osteogenic genes.^41–43 For stem cell differentiation, culture media must contain growth factors or compounds that promote chondrogenesis. A member of the transforming growth factor beta (TGFbeta) superfamily, TGF-β1, activates the TGF-β receptor II (TGFRII) and leads the expression of SRY-Box Transcription Factor 9 (SOX9), type II collagen (COL2A1), and aggrecan, which are essential for cartilage development.⁴⁴ TGF-β3 is another superfamily member that triggers the expression of chondrogenic genes by activating the small mother against decapentaplegic (SMAD) receptors pathway (Smad2/Smad3).⁴⁵ On the other hand, genipin is a natural compound that triggers chondrogenesis in MSCs without the presence of growth factors, it promotes the expression of COL2A1 and aggrecan.⁴⁶ Mature chondrocytes need phenotype maintenance supplementation to avoid dedifferentiation or senescence. Auricular chondrocyte maintenance requires a basic fibroblast growth factor (bFGF) to prevent dedifferentiation.²⁵ A useful biomarker of auricular chondrocyte dedifferentiation is the decreased secretion of glial fibrillary acidic protein and its periodic measurement is recommended through the cellular passages.⁴⁷

III. Structural support: Scaffold and scaffold-free methods

Scaffolds may be developed from organic polymers, protein fibers, reconstituted materials from animal tissues, decellularized tissue, ceramics, or a mix of these materials.⁴⁸ Scaffolding offers biologically mimetic-shaped support for cell development and allows reinforcement of delicate regions in the ear structure to avoid deformations in vivo.^38,49 In addition, some biological fibers sustain cell adhesion and trigger signaling pathways to promote cell differentiation, such as the RGD (Arg-Gly-Asp) peptide contained in collagen fibers, which interacts with integrins to regulate cellular processes.⁵⁰ On the other side, assembled scaffolds may not have the porosity necessary for cell movement and vascularization.⁵¹ Some scaffold materials are prone to degradation by proteolytic enzymes or trigger reactive oxygen species production. They also may shrink or distort over time.^33,52 Scaffold materials may cause immune rejection, inflammation, fibrosis, or tissue ulcerations.^53,54 Therefore, scaffold-free methods use only cellular material. By avoiding scaffold materials, these risks are reduced, improving the biocompatibility and security of the bioimplants. However, only small cartilage structures have been successfully developed using scaffold-free methods, whereas scaffolding techniques allow for the creation of larger constructs. The limitations of scaffold-free methods highlight the need for innovative strategies that can effectively support cell growth and organization, enabling the engineering of larger and more functional cartilage bioimplants that can better mimic the properties of native cartilage. In addition, sufficient cell mass is required.^55–57 Both scaffolding and scaffold-free methods report the same cellular behavior: when cells are in a monolayer, they exhibit a fibroblastic morphology, which transforms into a spheroidal shape when transferred to a three-dimensional (3D) culture. Taking advantage of the benefits of both, combinations of these methods have been developed, as chondrocyte ECM microsheets developed by scaffold-free methods and embedded in a 3D bioprinted scaffold, or decellularized and grinded cartilage used to develop hydrogel scaffolds.^58,59 However, for classification purposes, only the final method used to shape the scaffold will be classified (Figs. 3 and 4).

FIG. 3.

Classification of Methods for Developing Bioimplants. Scaffolding methods are utilized in 83% of the reports, employing exogenous extracellular matrix (ECM) as support for bioimplant assembly. In contrast, scaffold-free methods are reported in 17% of the studies. These methods allow cells to autonomously synthesize and assemble their own ECM for structural support.

FIG. 4.

Classification of scaffolding and scaffold-free methods. Scaffolding methods: 3D bioprinting scaffolds are reported in 33% of the studies, utilizing hydrogels with rheological properties that facilitate both, the printing and gelling of complex three-dimensional structures. Manual shaping and casting techniques account for 31% of the reports, providing an easier approach to shaping scaffolds according to specific requirements. Decellularized scaffolds, which comprise 14% of the studies, involve xenotransplantation of cartilage that has undergone a washing process to remove cells while preserving ECM proteins. These scaffolds can recruit cells in situ upon grafting. Hydrogels composed of biomaterials and cells are featured in 5% of the reports, highlighting their versatility to match the physicochemical properties of cartilage. Scaffold-free methods: microsheets are reported in 14% of the studies. They consist of multilayer cell culture structures where the ECM is entirely synthesized by the cells themselves. Additionally, micro 3D spheroids, used in 3% of reports, mimic the embryonic development of auricular hillocks, offering insights into cellular interactions without the need for scaffolding materials.

Scaffolding methods

The scaffold materials must mimic the cartilage’s mechanical properties, such as elasticity and shape-retention stability.⁶⁰ As well as biological properties allowing attachment, proliferation, and permeability.⁶¹ Scaffolds can be developed from manual methods such as assembling the scaffold biomaterials into manufactured molds to fully 3D printed bioimplants (Fig. 4). Progress has been made in understanding the scaffold biomaterials properties and their contribution to the development of bioimplants (Table 1).

Table 1.

The Main Findings from Preclinical and Clinical Trials on Biomaterials and Scaffolding Techniques Are Summarized

Scaffolding techniques	Material	Cells	In vivo trial	Main findings	References
Manual shape and molds	PGA	Human ACCs, mACs, and CCs. Rabbit ACCs and MSCs	SCID hairless congenic mice and immunocompetent rabbits Humans	The PGA scaffold can be grafted without the need for in vitro maturation time, if exposure to degradation products is controlled to avoid triggering an immunological reaction. The mechanical properties of the biomaterial provide a suitable environment for chondrocytes. After 1 month of grafting, the biomaterial shows neocartilage in its structure with mechanical properties like those of native cartilage.	^32,53
	PLA	Human iPSCs and ACCs	Nude mice Humans	PLA has a three-dimensional shape memory that preserves the reliefs present in the ear shape. Its slow degradation and absorption avoid severe inflammation and consequent ossification.	^39,53,63
	Bacterial cellulose membrane	—	Wistar rats	The scaffold mimics the resistance and elasticity of elastic cartilage. Does not suffer degradation or rejection by the body. Allows the development of a vascularization network on its periphery.	⁶⁰
	Poly-L-lactic acid (PLLA)	Human ACCs	Nude mice	PLLA acts as a physical barrier to inflammation and ossification factors. It allows the development of perichondrium on its surface.	⁹⁷
Decellularization	Human ear	Human aortic endothelial cells	Wistar rats	Significantly reduced the presence of total native DNA, proinflammatory cytokines, and immunogenicity. While the native cartilage structure was preserved, there was a marked decrease in GAGs concentration, which may impact the elasticity of the final tissue.	⁷⁰
Decellularization	Goat auricular cartilage	—	Humans mice	Treated cartilage allows interspecies tissue transplantation and cell recruitment in situ without major clinical complications.	^71,72
Hydrogel	HPCH	Goat ACCs	Athymic mice	It can be used as an injectable cellularized scaffold because it gels at body temperature. Maintains its original shape when neocartilage is generated.	⁷⁸
3D bioprinting	PU	MSCs derived from tonsils	Mice C57BL/6	The biomaterial has optimal porosity and allows neovascularization. Its physical properties allow it to maintain its original shape when grafted.	⁵¹
	PU	Human dermal fibroblasts	BALB/c Mice	It can be modified using argon to improves cell attachment on its surface without deformation or toxicity.	⁸⁷
	PCL	Human ACCs	Athymic rats	Porosity of 200 µm in scaffolds allows cell migration and proliferation inside.	³⁸
		Human ACCs	Athymic rats	Pretreatment with ethanol in polymers as scaffold material improves cell attachment.	⁸⁹
		—	Athymic rats	Cell recruitment. Scaffolds with precise anatomical shape. Permeable structure that promotes angiogenesis. The stiffness should be modified at the points of greatest skin tension to avoid ulcerations.	⁵²
		Porcine ACCs	Athymic rats	It has a great rate of degradation, and its degradation product does not trigger serious immunological reactions. Previous cellularization of the PCL reduces the appearance of ulcers because there is less friction between the material and the skin.	⁵⁴
		Porcine ACCs	RNU nude rat	Allows random and spherical porosity that promotes chondrogenesis.	⁸⁸
	Bioink based on goat ear cartilage	Cellularization by recruitment of chondrocytes in situ	Wistar chicken and rat embryo	Cartilage with alkaline digestion treatment suppresses its immunogenicity.	⁵⁸
	HDPE	Mouse Fibroblasts L929 cells	Sprague-Dawley (SD) rat	A pretreatment with polydopamine in the scaffold improves cell attachment, migration, and has antibacterial effect that avoids infections when transplanted.	⁹³

Some biomaterials have not been tested on nonimmunosuppressed animals or humans; therefore, the results and contributions may vary when biomaterials interact with the immune system.

ACCs, auricular cartilage chondrocytes; CCs, costal chondrocytes; HPCH, hydroxypropyl chitin; mACs, microtia auricular chondrocytes; MSCs, mesenchymal stem cells; PCL, polycaprolactone; PGA, polyglycolic acid; PLA, polylactic acid; PU, polyurethane.

a. Manual shape and casts

Some biomaterials are easier to shape through manual methods and casting. They have low initial viscosity, which enhances their ability to fill casts and adapt to desired geometries when a crosslinker is added. Fibrin network, with fibrinogen and thrombin, is a moldable biomaterial that can reach a solid phase with hardness comparable to that of cartilage.⁶² However, it is prone to degradation and morphological changes when tested in vivo due to the material’s nature and softness.⁶³ Fibrin is highly degradable by fibrinolysis due to its role in the coagulation cascade. Its affinity for cells can be enhanced by adding other materials, improving its physicochemical features and long-term stability.^64,65

The polylactic acid (PLA) has a thermo-responsive shape-memory effect that allows it to maintain the shape of complex three-dimensional structures as the pinna. Also, a combination of filler biomaterials can enhance the physicochemical properties of the constructs, such as a scaffold made with polycaprolactone (PCL), polyglycolic acid (PGA), and PLA.^53,63 On the other hand, the degradation products of PGA may trigger a host immune response.^66,67 To address this issue, it is crucial to incubate the scaffold with cells before grafted. This preincubation enables the cells to degrade the surface of the exposed PGA and to produce sufficient autologous ECM instead, which significantly reduces the material’s exposure to the immune system.^53,67 Despite the encouraging long-term results in cartilage development within living systems, there is a major challenge in preventing the invasion of host connective tissue into the bioimplant during the healing process, observed with the presence of type I collagen in the construct.³⁹

b. Decellularized scaffolds

Decellularized scaffolds imply a meticulous process to remove cellular material while preserving the essential physicochemical properties of the ECM components of the tissue.⁶⁸ Decellularization can be performed by physical techniques, chemical agents, enzymes, or a combination of them.⁶⁹ The whole human pinna can be decellularized through a solvent perfusion method that preserves native growth factors and the vascularization network.⁷⁰ It is important to remark that auricle procuration is challenging in many countries due to cosmetic, ethical, and legal concerns. The decellularization process involves a combination of mechanical disintegration, washing cycles, and gamma irradiation.⁷¹ Washing cycles with sodium deoxycholate or dimethyl sulfoxide preserve the cartilage porosity, mechanical stability, and flexibility.⁷² During the decellularization is important to include genipin for supporting cell reattaching to the scaffold.^46,73 However, detergents for decellularization, remanent cell debris, and molecules such as galactosyl-alpha-(1,3)-galactose (α-Gal) can trigger immune-inflammatory responses in the host.⁵⁹ Another remaining challenge is the elimination of DNA residues. Although it is proposed to use a DNase solution, its efficiency depends on the tissue size, as larger tissues continue to present DNA even after 10 days of treatment.⁷⁴ Decellularized goat auricular cartilage has been used as a safe xenogeneic decellularized scaffold.⁷¹

c. Hydrogels

Hydrogels for bioimplants can simulate the physicochemical and mechanical characteristics of soft and elastic tissues.^75,76 Hydrogel scaffolds must be biocompatible, nontoxic, biodegradable, deformable, and must allow cell proliferation.⁷⁷ Hydroxypropyl chitin (HPCH), a thermosensitive hydrogel, solidifies in less than 18 s at body temperature, achieving an elasticity comparable to auricular cartilage tissue. HPCH is an ideal scaffold for encapsulating cells and can even be injected into a living system to generate cartilage bioimplants in situ.⁷⁸ In contrast, certain hydrogels, such as poly (ethylene glycol) (PEG), cannot be utilized as the unique scaffold material. This limitation arises from PEG’s reticulated chain’s nonadhesive properties, which inhibit cell attachment and cell-scaffold interactions.⁷⁹ Despite the wide use of popular hydrogel materials for in vitro tissue engineering, such as matrigel and gelatin-methacrylate (GelMA), they are restricted for use in clinical bioimplants due to safety limitations.^80–82

d. 3D bioprinting scaffolds

3D bioprinting allows greater parameter control to obtain scaffolds with the patient’s pinna shape.⁸³ The injection system supports the homogeneous extrusion of the bioink to print cellularized or acellularized scaffolds. The bioink consists of gels or hydrogels of natural or synthetic polymers that can incorporate cells and other biological materials such as growth factors and supplements.^84–86 Bioprinting enhances the manipulability of synthetic polymers, such as polyurethane (PU) and PCL, while also allowing for precise regulation of their porosity, making them more conducive to cell-friendly environments.^51,87–90 Among the disadvantages of these scaffold materials, PU may induce granulomatous reactions in in vivo protocols. PU chemical modifications are required to improve biocompatibility and to reduce adverse reactions in living systems.⁹¹ Regarding PCL, this material is slowly reabsorbed, compromising the implant structural support, and causing deformations.⁹² High-density polyethylene (HDPE) is the simplest polymer but unsuitable for tissue engineering applications. However, it can be used as scaffold when is pretreated with polydopamine for enhancing cell attachment and migration on its surface.⁹³ An important technical disadvantage of bioprinting using cells is the mechanical stress and long exposure time during the extrusion and polymerization of the bioink.⁹⁴ To solve this, it is recommended to analyze cell viability following bioprinting. This analysis provides critical insights that can guide the adjustment of the mechanical parameters of the bioprinter, ensuring optimal conditions for cell survival and functionality.⁹⁵

Scaffold-free methods

Scaffold-free methods are emerging in the development of cartilage bioimplants. These methods rely on the capacity of cells to create tissues without the need for exogenous scaffold.⁹⁶ Therefore, scaffold-free methods use only tissue-specific ECM synthesized by cells. By avoiding scaffold biomaterials, the immune response to the grafted bioimplant is decreased, improving the biocompatibility and the security of the construct (Table 2).^57,98

Table 2.

Main Findings from Preclinical and Clinical Trials in Scaffold-Free Technique

Scaffold-free technique	Material	Cells	In vivo trial	Main findings	References
ECM microsheets	ECM of chondrocytes	Rabbit ACCs	Nude mice	Microsheets promote cell attachment because of native ECM properties.	⁶³
		Human ACCs, CCs		Microsheets and supplementation with bFGF promote a physicochemical and mechanical microenvironment for phenotype maintenance and cell proliferation.	²⁵
				A tissue without post-transplant deformations was obtained, however, only one cubic centimeter of cartilage developed.	⁵⁶
				Microsheets mature to cartilage-like tissue in 2 weeks in vitro incubation with appropriate culture conditions.	¹⁰¹
		Human mACs		miR-193b-3p promotes resistant ECM synthesis without enzymatic degradation in vivo.	¹⁰²
		Human mACs	Humans	The microsheets can be incubated in the patient’s abdominal adipose tissue to mature and form native neocartilage. The neocartilage bioimplant can be sculpted into an ear structure to be used clinically.	^100,109
Microcultures	—	Atrial perichondrium chondroprogenitor cells derived from the ear with microtia	NOD/Scid mice	Mimicking the morphological conditions of embryonic chondrogenic induction triggers gene expression of craniofacial cartilage development. The microspheres fuse to form a single neocartilage tissue.	¹⁰⁴

These methods simulate the development of cartilage using native material synthesized from chondrocytes.

ECM, extracellular matrix.

a. ECM microsheets

ECM microsheet techniques take advantage of the capability of cells to develop a native stratified tissue for assembling a bioimplant without exogenous biomaterials.⁹⁹ Microsheets are developed as a multilayer culture that fuses to create an injectable gel of chondrocytes and ECM.¹⁰⁰ When these constructs are grafted, chondrocytes maintain their ability to synthesize cartilage during the interaction with the host microenvironment.⁹² However, bioimplant shrinking has been reported.^56,101 This may be corrected with genetically modified chondrocytes to avoid enzymatic degradation of the ECM during the post-transplant inflammatory response. These chondrocytes are transfected with miRNAs for inhibiting the expression of matrix metalloproteinases.¹⁰² In this method, the third cell passage should be utilized at the latest to obtain a larger cell population. However, a limitation is the content of sulfated glycosaminoglycans (GAG) and type II collagen in the microsheets, as chondrocytes tend to synthesize smaller amounts of these biomaterials with each subsequent passage.¹⁰³

b. Micro 3D spheroids

Chondrogenesis in chondroprogenitor cells can be triggered in vitro by mimicking the morphology of the pinna hillocks, structures where the chondroprogenitor cells of auricular perichondria develop in their native state during the embryonic phase.² When cells are seeded in U-bottom microwelled plates, they develop spheroids, such as the auricular hillocks, and they express chondrogenic genes.¹⁰⁴ This protocol enables the fabrication of small structures with a diameter of 4.3 mm. However, larger and more complex structures, such as the pinna, have not yet been developed. Additionally, the efficient differentiation of various types of progenitor cells through this protocol could be limited, as chondroprogenitor cells exhibit higher expression levels of type II collagen and aggrecan compared to others.^105,106

Preclinical and clinical trials

Preclinical trials are necessary to confirm the efficacy and safety of bioimplants in a living system.¹⁰⁷ They are performed in small animals to validate the compatibility of new materials and cartilage maturation, or in large animals to confirm the preservation and lifespan of the ear structure.¹⁰⁸ Usually, bioimplants are grafted in the abdomen, dorsum, or near the ribs and ears, depending on the tested species.^32,71,109

a. In vivo experimental approaches

The grafted auricle bioimplant must retain the cartilage structure and function. In addition, it may support the growth of surrounding skin and must facilitate tissue revascularization.¹¹⁰ Furthermore, the bioimplant constructs are designed to be gradually reabsorbed.¹¹¹ During the grafting, it is important to prevent the skin from becoming excessively tight, and inflammation must be minimized to avoid postsurgical ulcerations and deformations generated by the physiological interactions between the covering skin and the bioimplant. Cellularized scaffolds generate an interface that isolates the skin from the scaffold material which may prevent these adverse outcomes. To avoid deformities, the areas of the scaffold that are most exposed to friction in the host may be reinforced during the 3D design and assembling of the pinna. During the 3D design, some pinna areas must be reinforced and cellularized in vitro before the bioimplant grafting to prevent post-transplant ulcerations and deformations (Fig. 5).^38,52,54

FIG. 5.

Areas that undergo greater stress in the grafting stage. The areas that have presented ulceration during grafting recovery because of the skin tension are highlighted in red. In vitro cellularization of the scaffold reduces ulcer development in post-transplant recovery.^52,54 The areas suggested to be reinforced to avoid deformation are highlighted in blue. This reinforcement must be considered during the scaffold design so that it is not degraded by the mechanical pressure of adjacent tissues when grafting or by the immune system response.³⁸

The tissue-engineered human skin graft (EarSkin) is a bioimplant composed of skin cells that cover an imprinted pinna (bioink: poly-N-acetyl glucosamine nanofibrils, factor XIII, thrombin, and ACCs). During grafting, the interaction skin–host panniculus carnosus–cartilage resembled the physicochemical features of the native elastic cartilage in terms of biochemical composition, flexibility, and shape stability.⁹⁵ However, there was uneven diffusion of nutrients through the pinna, resulting in an inconsistent cell population between the edges and the center. The morphology of the pores significantly influences the transport of nutrients, allowing or interrupting permeation.¹¹² Despite the great progress that has been made when testing bioimplants in living systems, most protocols have been conducted in immunosuppressed animals. This creates a gap in understanding the immune response against the bioimplant.¹¹³ Immune cells play a crucial role in the process of bioimplant maturation since grafting enhances immunocompetent animals.³² It has been observed that during inflammation, immune system cells infiltrate the bioimplant site and assist in the development of fibrovascular tissue around the neocartilage.^114,115

b. Clinical trials

Bioimplants have been tested in humans, specifically in children with microtia, some with long-term follow-up. Pinna bioimplants made of mACs and PCL/PGA/PLA have demonstrated biocompatibility and shape maintenance through the years, and elastic neocartilage development at 6 months after grafting. The PCL core wrapped with PGA nonwoven fibers provided structure and allowed cell migration and proliferation into the entire bioimplant, whereas PLA coating minimizes the direct exposure of residual PGA to the immune system.⁵³ The rate of PGA degradation can lead to significant acidosis in the surrounding microenvironment and trigger a severe host response during direct exposure.¹¹⁶ However, the bioimplants were shaped in casts and did not recreate the patient’s contralateral pinna shape details. The appearance of the pinna may still be improved using other more precise techniques such as 3D bioprinting.¹¹⁷ Bioimplants of goat-decellularized auricular xenotransplant demonstrated their security in clinical trials. The decellularization method completely removed cellular debris and preserved the ECM native biomolecules, facilitating in situ recellularization for the reconstruction of auricles and noses. It is essential to highlight that the final shape of these bioimplants was highly dependent on the surgeon’s skills.⁷⁸ On the other hand, bioimplants free of exogenous material were developed through a novel scaffold-free method. Bioimplants were created with a gel embedded with mACs and autologous serum and incubated in the patient’s lower abdomen. After 6 months, a large cartilage structure was obtained that was manually shaped before grafting.¹⁰⁰ Adverse reactions, such as absorption issues and allergic responses, can still be observed.¹⁰⁹ The in vitro maturation time of the cartilage bioimplant is substituted for grafting in patients. The surrounding host adipose tissue provides essential nutrients and support, creating a microenvironment that is necessary for the maturation of the bioimplant.¹¹⁸

In the studies described above, follow-up assessments were conducted over the years. However, none of these studies reported that the bioimplants grew at the same rate as the patient’s facial development. This highlights the need for further research to synchronize bioimplant growth with facial growth or to determine the optimal timing for grafting to prevent deformities during the facial development.^119–121

Conclusions

The development of auricular implants in in vivo models and clinical trials has demonstrated significant progress in the last decade. Among the advances, the following are worth highlighting. Cocultures, particularly with adipose tissue MSCs and ear chondrocytes, improve cell differentiation and the structure of the bioimplant. Additionally, media supplementation with bFGF is key for phenotype maintenance. Cells can be seeded on a scaffold or stimulated to synthesize small tissues using microspheres. Advances in scaffold-free methods are expected to overcome the use of exogenous material. Bioimplants have been successfully implanted in children, yielding satisfactory long-term results. However, further improvements are required to ensure that bioimplants achieve shape and size like that of the healthy contralateral pinna. Finally, further research is required to prevent adverse events that force bioimplant removal, even in the case of bioimplants manufactured with autologous materials.

Authors’ Contributions

L.M.R.-R. and A.R.-M.: Idea and planning of the review, wrote or contributed to the writing of the article, and references review.

Footnotes

Acknowledgments

L.M.R.-R. acknowledges the scholarships granted by Consejo Nacional de Humanidades Ciencias y Tecnologías (No. CVU 933406) and Tecnologico de Monterrey.

Funding Information

This article was supported in part by the grant Consorcio UANL-TEC 2023 number D5998 and the scholarships granted to L.M.R.-R. by Consejo Nacional de Humanidades Ciencias y Tecnologías (No. CVU 933406) and Tecnologico de Monterrey.

Disclosure Statement

No competing financial interests exist.

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