Cartilage 3D bioprinting for rhinoplasty using adipose‐derived stem cells as seed cells: Review and recent advances

Abstract Nasal deformities due to various causes affect the aesthetics and use of the nose, in which case rhinoplasty is necessary. However, the lack of cartilage for grafting has been a major problem and tissue engineering seems to be a promising solution. 3D bioprinting has become one of the most advanced tissue engineering methods. To construct ideal cartilage, bio‐ink, seed cells, growth factors and other methods to promote chondrogenesis should be considered and weighed carefully. With continuous progress in the field, bio‐ink choices are becoming increasingly abundant, from a single hydrogel to a combination of hydrogels with various characteristics, and more 3D bioprinting methods are also emerging. Adipose‐derived stem cells (ADSCs) have become one of the most popular seed cells in cartilage 3D bioprinting, owing to their abundance, excellent proliferative potential, minimal morbidity during harvest and lack of ethical considerations limitations. In addition, the co‐culture of ADSCs and chondrocytes is commonly used to achieve better chondrogenesis. To promote chondrogenic differentiation of ADSCs and construct ideal highly bionic tissue‐engineered cartilage, researchers have used a variety of methods, including adding appropriate growth factors, applying biomechanical stimuli and reducing oxygen tension. According to the process and sequence of cartilage 3D bioprinting, this review summarizes and discusses the selection of hydrogel and seed cells (centered on ADSCs), the design of printing, and methods for inducing the chondrogenesis of ADSCs.

promote chondrogenesis should be considered and weighed carefully. With continuous progress in the field, bio-ink choices are becoming increasingly abundant, from a single hydrogel to a combination of hydrogels with various characteristics, and more 3D bioprinting methods are also emerging. Adipose-derived stem cells (ADSCs) have become one of the most popular seed cells in cartilage 3D bioprinting, owing to their abundance, excellent proliferative potential, minimal morbidity during harvest and lack of ethical considerations limitations. In addition, the coculture of ADSCs and chondrocytes is commonly used to achieve better chondrogenesis. To promote chondrogenic differentiation of ADSCs and construct ideal highly bionic tissue-engineered cartilage, researchers have used a variety of methods, including adding appropriate growth factors, applying biomechanical stimuli and reducing oxygen tension. According to the process and sequence of cartilage 3D bioprinting, this review summarizes and discusses the selection of hydrogel and seed cells (centered on ADSCs), the design of printing, and methods for inducing the chondrogenesis of ADSCs.

| INTRODUCTION
Trauma, burn, tumour, surgery, or congenital malformation of nasal cartilage may compromise a nasal deformity or lead to nasal airway dysfunction, affecting the aesthetics and utility of the nose. 1 Rhinoplasty usually requires trimming of nasal cartilage and implantation of grafts. Therefore, finding the most suitable and compatible cartilage graft material is essential. Ideally, the engineered cartilage graft should have properties to meet the clinical needs of the recipient site, including being able to withstand the external forces in the nasal reconstructive environment, biocompatible, and biologically active to support and promote tissue integration and healing. Besides, it should be readily available or easy to access without contributing to donor site morbidity. Moreover, from a surgical point of view, the graft material needs to be big enough in the case of intraoperative trimming to meet the needs of patients. Chong Zhang and Guanhuier Wang contributed equally to this work.
Currently, commonly used graft materials include autologous cartilage, allografts, and synthetic materials. Autologous cartilage, especially nasal septum cartilage, is an ideal cartilage source. However, the quantity of autologous cartilage is limited, let alone that harvesting auricular cartilage and costal cartilage will inevitably produce additional incisions, which could result in donor site complications.
Widely sourced materials such as allografts and synthetic materials have been used as alternatives to autologous cartilage grafts. Nevertheless, allografts are more susceptible to resorption and infection, while the use of synthetic materials may lead to devastating complications. With further advances in tissue engineering technology, it will be possible in the future to address the issues related to the source of grafts in rhinoplasty by constructing ideal tissueengineered cartilage. Seed cells, scaffold materials, and growth factors were researched and improved to achieve optimization of engineered cartilage. 2 Techniques of constructing scaffolds, including 3D bioprinting (such as inkjet bioprinting, laser-assisted bioprinting, extrusion-based bioprinting, acoustic bioprinting, stereolithography bioprinting, and magnetic bioprinting), direct moulding methods (such as cell sheet stacking, lithography, and injection moulding), and a group of methods for constructing porous scaffolds (such as electrospinning, phase separation, freeze-drying, and self-assembly), are also important to elevating cartilage performance. [3][4][5][6] Nowadays, 3D bioprinting, as a form of cell-laden bottom-up additive manufacturing, has become one of the most promising and advanced tissue engineering methods.
Adipose tissue is widely distributed in various parts of the human body. Adipose-derived stem cells (ADSCs) are undifferentiated mesenchymal stem cells (MSCs) in adult adipose tissue with osteogenic, chondrogenic, and myogenic differentiation potential. The ADSCs play an important role in tissue engineering for advantages such as abundant sources and easy acquisition. In addition, exosomes secreted by ADSCs can promote cartilage regeneration, cell migration, and differentiation as a biological additive. For 3D bioprinting ideal highly bionic tissue-engineered cartilage, it is crucial to successfully induce seed cells such as ADSCs to differentiate into chondrocytes (CCs) and secrete cartilage extracellular matrix (ECM). Researchers have attempted to achieve optimal cartilage by simulating the natural physicochemical environment in vivo, such as adding appropriate growth factors, applying biomechanical stimuli, and reducing oxygen tension.
In this review, we summarize and discuss the selection of hydrogel and seed cells (centered on ADSCs), the design of printing, and the methods for inducing the chondrogenesis of ADSCs, according to the process and sequence of cartilage 3D bioprinting ( Figure 1).
We first introduce the current 3D bioprinting methods and reviewed the commonly used hydrogel scaffold materials and their progress.
Then we mainly introduce the application of ADSCs in tissue engineering cartilage: It can be used as seed cells laden in bio-ink and as the source of extracellular vesicles (EVs) to induce chondrogenesis of cells. Other methods of promoting chondrogenic differentiation of ADSCs were also described. Finally, we summarized and prospected the future directions of 3D bioprinting tissue-engineered cartilage.
2 | 3D BIOPRINTING WITH ADSCs IN HYDROGELS 2.1 | The hydrogel-based 3D bioprinting 3D bioprinting is a form of bottom-up additive manufacturing and tissue engineering technology. Before the printing process begins, an individualized model of the patient is constructed. With the help of medical imaging techniques, usually computerized tomography scan and magnetic resonance imaging, we can reconstruct the patient's nasal shape and use it as a basis to design the model according to the patient's needs, and then import the designed model into the 3D bioprinter. After that, bio-ink containing seed cells, growth factors, and other additives is printed layer by layer to form the construct. And, after a specific in vitro culture, the personalized target tissue is finally constructed. 3D bioprinting technology can be roughly divided into three types: droplet, extrusion, and photocuring bioprinting, which are respectively suitable for bio-ink materials with different curing mechanisms. According to the needs of experimental design, the material of bio-ink is selected according to their biological, biophysical, and biochemical properties. The corresponding 3D bioprinting technique is then selected to construct the scaffold. Several excellent reviews have compared and discussed their advantages and disadvantages, which would not be repeated here. 7,8 Each step of 3D bioprinting will determine the quality of the final construct and the bio-ink based on the hydrogel is the critical core. Hydrogels, a 3D network of molecules composed of hydrophilic polymer chains, can be designed into any shape, size, or form and absorb up to a thousand times their dry weight in a water-rich environment. 9,10 With the development of 3D bioprinting technology, the hydrogel-based system has become a prime candidate as the carrier for cells for a variety of tissue engineering applications.
The ideal bio-ink should have good biocompatibility, biodegradability, and sufficient mechanical properties to support nasal morphology and mimic the natural ECM. In addition, the effect of the hydrogel material and its crosslinking process on the seed cells encapsulated needs to be considered. Since mechanical properties and biocompatibility are often challenging to achieve simultaneously, bio-ink of a single formulation is rarely used at present, and mixed bio-ink has gradually become the mainstream.

| Hydrogels for cartilage tissue engineering and their general properties
The materials of bio-ink for cartilage 3D bioprinting can be divided into natural and synthetic ones ( Figure 2). The natural materials are mainly cartilage matrix components such as hyaluronic acid (HA), collagen, chondroitin sulphate (CS), decellularized ECM (dECM), and so forth. Besides, other proteins and polysaccharides with similar properties are also widely used, such as silk fibroin (SF), agarose, gellan, gelatin, alginate, chitosan, and pectin. While synthetic materials mainly include polycaprolactone (PCL), polylactic acid-glycolic acid copolymer, polyurethane, polylactic acid, polyacrylic acid (PAA), and polyethylene glycol (PEG). Bio-ink is often printed in liquid or gel form and later cured. To balance the printability, mechanical properties, and biocompatibility of the bio-ink, it is essential to have the proper forming and curing method. In general, the method is often determined by the properties of the bio-ink itself.
The common crosslinking mechanisms of hydrogels include physical crosslinking, chemical crosslinking, and enzymatic crosslinking.
Physical crosslinking allows effective forming without any exogenous factors and minimizes chemical toxicity. Physical entanglement of the polymer chains occurs during the gelation of thermally driven hydrogels. This happens with natural polymers like gelatin and synthetic polymers like PAA. 11 Molecular self-assembly is a commonly used strategy for protein-based hydrogels such as collagen through weak noncovalent bonds like ionic bonds, hydrogen bonds, hydrophobic interactions, and van der Waals force. 12 In addition, physical crosslinking can also be based on chelation or electrostatic interaction. 11 Natural ionic polysaccharides such as alginate and pectin can gel in the presence of divalent cations such as calcium ions. 13 Chemical crosslinking is based on covalent bonding between polymer chains, including condensation reactions, radical polymerization, and aldehyde complementation. 10 When compared with physical crosslinking, the covalent bonding chemical crosslinking is more stable and tunable, substantially improving the spatiotemporal precision and flexibility during the printing-curing process. 11 Photo-initiator (PI) is often added to modify the functional groups of free radical polymerization to obtain photocrosslinking properties of hydrogels. Photocrosslinked hydrogels, such as methacrylated gelatin (GelMA) and F I G U R E 1 Cartilage 3D bioprinting for rhinoplasty using adipose-derived stem cells (ADSCs) as seed cells: When fabricating tissueengineered cartilage, the hydrogel-based bio-ink containing seed cells and additives is printed layer by layer to form the targeted construct, combined with other artificial materials or alone, hierarchical or homogeneous. ADSCs stand out from the candidates, especially those from thigh adipose tissue. When mixed with chondrocytes in appropriate proportions (1:3-3:1), more satisfactory cartilage regeneration results can be achieved. Besides, other methods of promoting the chondrogenesis of ADSCs are also imposed. ADSC-EV, ADSC-derived extracellular vesicle; BMP, bone morphogenetic protein; BMSCs, bone marrow-derived MSCs; CCs, chondrocytes; CPCs, chondroprogenitor cells; dECM, decellularized extracellular matrix; ESCs, embryonic stem cells; FGF, fibroblast growth factor; IGF, insulin-like growth factor; iPSCs, induced pluripotent stem cells; MSCs, mesenchymal stem cells; PB-MSCs, peripheral blood-derived MSCs; PDGF, platelet-derived growth factor; PRP, platelet-rich plasma; SMSCs, synovium-derived MSCs; TGF-β, transforming growth factor β; UC-MSCs, umbilical cord blood-derived MSCs.
PEG dimethacrylate (PEGDMA), with crosslinking rates and degrees easy to control, are widely used in 3D bioprinting of cartilage tissue.
Cartilage tissue consists mainly of CCs and ECM, which consist of proteoglycans and collagen fibrils (type II) to form a solid structure. In addition, more CS is observed in the matrix around the cartilage lacu-  Nowadays, natural hydrogels are mainly used for the 3D bioprinting of cartilage tissue. Natural hydrogels commonly have good biocompatibility, and their mechanical properties are lower than that of natural cartilage. Synthetic materials, however, can provide high mechanical strength. Therefore, in the preparation of bio-ink, a mixture is often used to complement their strengths and weaknesses, or other additives are added to natural bio-inks to improve their mechanical properties. Researchers have continuously explored and modified hydrogels and improved bio-ink formulations in recent years. Current studies in the field mainly focus on mixtures of bio-ink with "good biocompatibility" (such as dECM, HA) and "photocrosslinking properties" F I G U R E 2 Natural and synthetic materials for cartilage 3D bioprinting: The natural materials for cartilage 3D bioprinting are mainly matrix components or proteins and polysaccharides with similar properties. Besides, some synthetic materials with high mechanical properties are also widely used. The methacrylated photocrosslinked materials are marked with a sign in the shape of the sun (yellow) on their right side. cdECMMA, methacrylated cartilage-derived dECM; CSMA, methacrylated chondroitin sulfate; dECM, decellularized ECM; ECM, extracellular matrix; GelMA, methacrylated gelatin; HAMA, methacrylated hyaluronic acid; PAA, polyacrylic acid; PCL, polycaprolactone; PEG, polyethylene glycol; PEGDMA, polyethylene glycol dimethacrylate; PLGA, polylactic acid-glycolic acid copolymer; PU, polyurethane; PVA, polyvinyl alcohol; SFMA, methacrylated silk fibroin.
T A B L E 1 Summary of cartilage 3D bioprinting studies in recent years, in which various hybrid hydrogel formulations and seed cells were used.

Seed cells
The cell-laden hydrogels Sparkles Reference Articular CCs GelMA, PEGDMA, and gelatin Integrated cartilage-mimetic tissue with a biosensing platform, and developed an odourperceptive nose-like hybrid 14 HAMA, pHPMA-lac-PEG, and PCL Limited fibrocartilage formation, generated complex 3D constructs with mechanical stiffness 15 GelMA and gellan gum Increased the construct stiffness, supported chondrogenesis 16 pHPMA-lac-PEG, CSMA, or HAMA Increased the storage modulus of polymer mixtures, decreased the degradation kinetics in crosslinked hydrogels 17 GelMA and HAMA Opened up the perspective of hybrid and zonal stratification bio-ink to generate more complex cartilage structures 18 Alginate and sub-micron PLA Higher Young's modulus and cell viability 19 HA and alginate or PLA Good printability, degradation, mechanical properties, retained more than 85% cell viability 20 GelMA, HAMA, and CSMA Enhanced cartilage formation, promoted matrix distribution, improved mechanical properties 21 Auricular chondrocytes PAOXA, alginate, and NFC Broader tuning potential of mechanical properties increased cytocompatibility 22 cdECMMA Printed in ear shape, adequate mechanical properties and structural integrity 23 NSCs NFC and alginate Increased shape fidelity, printing resolution, and storage modulus correlating 24 Gelatin and alginate-di-aldehyde Good cell viability, increased expression of cartilage-specific markers, not degrading within 28 days 25 BMSCs SF and dECM Good mechanical strength, released chondrogenic growth factors, enhanced chondrogenesis of BMSCs 26 Alginate-GelMA interpenetrating network and alginate sulfate Improved mechanical properties, continuously released TGF-β3 with high-affinity binding, promoting chondrogenesis and suppressing hypertrophy of BMSCs 27 Acrylated peptides (RGD) and PEGDMA Enhanced the osteogenic and chondrogenic differentiation of BMSCs with minimal printhead clogging 28 PEGDMA and GelMA Bionic compressive modulus and promoted differentiation 29 Alginate, GelMA, CS, amino ethyl methacrylate, and HAMA Favoured the differentiation of BMSCs toward hypertrophic cartilage, supported the hypothesis that too crosslinked matrix does not allow for proper macromolecular diffusivity and hinders cartilage development 30 Fibrin-HA and HA-Tyramine Supported cell migration and enhanced cartilage regeneration 31 Alginate, GelMA, and β-tricalcium phosphate Regulated the differentiation of BMSCs to form osteochondral tissue calcification zone 32 ADSCs Hydroxybutyl chitosan and oxidized CS Good injectability and controllable shape 33 iPSCs Gelatin and PU Convenient printing processes, tunable mechanical properties, and degradation rates 34 (Continues) (such as, methacrylated hyaluronic acid [HAMA], GelMA, PEGDMA) or "good mechanical properties "(such as silk protein, synthetic materials; Table 1).

| The shift in 3D bioprinting ideas
As bioprinting materials have continued to progress over the years, some researchers have shifted their attention from traditional 3D bioprinting to more diverse hybrid printing. In some studies, 3D scaffolds with excellent mechanical properties are printed with synthetic materials and filled with cell-laden hydrogels later ( Figure 3). The combination of two tissue-engineered cartilage methods provides another direction to optimize engineered cartilage. In this way, synthetic materials represented by PCL have played a more critical role in 3D bioprinting with their outstanding mechanical properties (Table 2). Besides, Wu et al. 37 used pre-differentiated ADSCs to form cartilage scaffold-free tissue strands and constructed zonally stratified cartilage, in which collagen fibres are aligned with designated orientation in each zone imitating the anatomical regions and matrix orientation of native articular cartilage and with satisfying compression modulus at the same time.

| The shift in the designs for 3D bioprinting constructs
In addition to the diversification in the modalities of 3D bioprinting, the requirements for the constructs are also changing ( Figure 4). Conventional 3D bioprinting constructs are often homogeneous cartilagelike tissue. However, the layered structure of cartilage tissue plays an important role in its physiology. Histologically, the nasal cartilage is divided into the superficial zone and the deep zone, composing a sandwich-like three-layer structure. When coming close to the deeper zone, cells gradually become larger, more rounded, and less frequent. 46 Besides, the oxygen tension and nutrient availability of the tissue and the CCs population with hypertrophic and ossification markers such as Runt-related transcription factor 2 (RUNX2) and collagen type X (COL X) are increasing. 47  In the study of Sun et al., 45     times more than that in marrow. 55 Overall, ADSCs offer practical advantages as seed cells for cartilage regeneration.

| Where to access the adipose tissue?
In plastic surgery, if the patient also plans to undergo liposuction, ADSCs can be extracted directly from the adipose tissue obtained during the surgery. If not, it is necessary to find the most suitable site to harvest ADSCs.
Adipose tissue can be classified by morphology into white, brown, and beige subsets. 56   Although few studies have been done, the mechanical sorting method is more direct and easier to use than the immunophenotypic sorting method, which broadens the methods of sorting ADSCs.

| A mix of seed cells: co-culturing with the chondrocytes
Despite all the advantages of ADSCs as seed cells mentioned earlier, the application of MSCs alone as seed cells could result in unstable and hypertrophic cartilage tissue with a high tendency to ossification. 75 (Table 3).
Taken together, there is no co-identification in the ratio of ADSCs to CCs. Nevertheless, what is clear is that the induction effect of CCs is dose-dependent: the higher the proportion of CCs, the more GAG was produced. 86 On balance, a ratio from 1:3 to 3:1 of ADSCs to CCs may be a relatively practical and conservative choice ( Figure 5). However, the most precise ratio needs to be further investigated.

| CHONDROGENIC CULTURE OF ADSCs
For the construction of ideal highly bionic tissue-engineered cartilage, it is not enough to consider the selection and design of seed cells and bio-ink. It is also crucial to successfully induce seed cells such as ADSCs to differentiate into CCs and secrete cartilage ECM.
Researchers have attempted to achieve optimal cartilage by simulating the natural physicochemical environment in vivo, such as adding appropriate growth factors, applying biomechanical stimuli, and reducing oxygen tension ( Figure 6).  F I G U R E 6 Strategies for inducing the chondrogenic differentiation of adipose-derived stem cells (ADSCs). ADSC-EV, ADSC-derived extracellular vesicle; BMP, bone morphogenetic protein; FGF, fibroblast growth factor; IGF, insulin-like growth factor; PDGF, platelet-derived growth factor; PRP, platelet-rich plasma; TGF-β, transforming growth factor β.

| Biochemical additives for chondrogenic differentiation
In addition, there are also other attractive biochemical factors beneficial to MSCs differentiation and cartilage regeneration (Table 4).

| Natural combinations of biofertilizers
In addition to adding a variety of growth factors, the growth and chondrogenic differentiation of ADSCs can also be promoted by directly adding "natural combinations of growth factors" such as platelet-rich plasma (PRP) and ADSC-derived EVs (ADSC-EVs).
PRP is a part of whole blood with a high concentration of platelets. In addition to the platelets, PRP also contains a large number of cytokines, such as TGF-β, IGF-1, epidermal growth factor (EGF), PDGF, and so forth. 115,116 As described in the previous part, these cytokines play an important role in supporting the growth and differentiation of MSCs. 117  and PI3K-AKT signalling pathways. 125 However, apparently few differences were found between ADSC-EVs and BMSC-EVs in the study of Gorgun et al., 126 and both of them could possess immunomodulatory, pro-regenerative, pro-angiogenic, and anti-apoptotic properties.
In the study of Xue et al., 127  In addition to bioreactors that provide the usual mechanical stimulation described above, low amplitude high frequency vibration loading, as a model of mechanical stimulation, has been demonstrated to promote chondrogenic differentiation of bone marrow stem cells with the involvement of the β-catenin signalling pathway. 137 Besides, electromagnetic field (EMF) and ultrasonic stimulation are also used in promoting the chondrogenesis of MSCs. The EMF can be divided into static and pulsed magnetic fields according to the changing pattern of the magnetic field over time. 129

| Hypoxic environment
The average partial pressure of oxygen measured at the septum and inferior turbinate was 10.5 ± 10.1 mmHg (1.4% ± 1.3%) and 27.6 ± 12.4 mmHg (3.6% ± 1.6%), respectively. 142 At the same time, the physiological oxygen level within human articular cartilage ranges from 2% to 5% and 7% in the bone marrow. 143 Most traditional tissue culture incubators operate at atmospheric oxygen levels (20%) that are actually nonphysiological and hyperoxygen environments for seed cells.
In the study of Scotti et al., 144 human articular CCs displayed a positive response to low oxygen culture, while human nasal CCs were only slightly affected by oxygen percentage. However, it has a significant influence on MSCs. In particular, increasing evidence has demonstrated that low oxygen tension promotes cell proliferation, 76 enhances chondrogenic differentiation capacity, 145 and increases migration ability 146 but inhibits osteogenic differentiation. 147 The study of Shearier et al. 148 showed that the biochemical components of cartilage in vitro are more similar to natural tissue when cultured under hypoxic conditions. Co-culture with ADSCs and CCs under hypoxia was shown to induce or enhance ADSCs to chondrogenic differentiation successfully. Specifically, chondrogenic marker genes: ACAN, COL II, and SOX9 were remarkably enhanced to express in both ADSCs and CCs after crosstalk under low oxygen tension. 76 By increasing the level of hypoxia-inducible factor 1α (HIF-1α), vascular endothelial growth factor A/B (VEGF-A/B), BMP-2/-4/-6, FGF-2, and IGF-1, hypoxia pretreatment could increase the proliferation and chondrogenic differentiation of ADSCs but decrease the osteogenic differentiation of ADSCs. 76,146 Besides, Yasui et al. 145 found that hypoxic treatment (5% oxygen) of human SMSCs prevented senescence induction, evidenced by 2.70-fold-lower expression levels for P16.

| SUMMARY AND FUTURE OUTLOOKS
Three-dimensional bioprinting technology is developing rapidly, and different improved printing systems and construction methods are emerging. Zhou et al. developed a multi-axis robot-based bioprinting system supporting natural cell function preservation and cardiac tissue fabrication, enabling cell printing on 3D complex-shaped vascular scaffolds from all directions. 149 Lan et al. 150 reported an in vitro nasal cartilage tissue engineering method via the freeform reversible embedding of suspended hydrogel 3D bioprinting. Hwang et al. 151 presented a 3D bioprinting platform on a high-throughput scale, which is capable of rapid, continuous 3D printing of constructs with small sizes (<10 μ) and complex shapes and controlling the mechanical property. Although some of them are not yet used for cartilage tissue engineering, they provide more ideas and are expected to be used in the future to print more complex and nuanced cartilage tissue or complex systems containing cartilage tissue efficiently.
Different hydrogel materials for 3D bioprinting have emerged in recent years, and different combinations of bio-ink have been explored. Current studies in the field mainly focus on mixtures of bioink with "good biocompatibility" (such as dECM, HA) and "photocrosslinking properties" (such as HAMA, GelMA, PEGDMA) or "good mechanical properties "(such as silk protein, synthetic materials).
However, no ink formulation has been proven to have absolute dominance. What is certain is that the main trend is to construct tissueengineered cartilage that is more histologically bionic, which can be achieved with the help of layered design and switching printing modes (bionic bio-ink printing paths and fibre arrangement). Moreover, ADSCs have become one of the most popular seed cells in cartilage 3D bioprinting, owing to their abundance, excellent proliferative potential, minimal morbidity during harvest, and lack of ethical considerations limitations. In addition, the co-culture of ADSCs and CCs is also commonly used to achieve better chondrogenesis. At the same time, the optimal selection of the chondroblast differentiation mode of the printed body has not been determined. It is believed that with the efforts of researchers, a standardized protocol for construct handling and implantation will be produced in the future.