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. 2019 Aug 19;16(6):605–614. doi: 10.1007/s13770-019-00210-1

Evaluation of Polycaprolactone-Associated Human Nasal Chondrocytes as a Therapeutic Agent for Cartilage Repair

Do Hyun Kim 1,#, Mi Hyun Lim 1,#, Jung Ho Jeun 1,2, Sun Hwa Park 1, WeonSun Lee 2, Sang Hi Park 2, Mi Yeon Kwon 2, Se Hwan Hwang 3,✉,#, Sung Won Kim 1,✉,#
PMCID: PMC6879705  PMID: 31824823

Abstract

Background:

In this study, we manufactured a complex of human nasal septal cartilage (hNC) with polycaprolactone (PCL) for transplantation into cartilaginous skeletal defects and evaluated their characteristics.

Methods:

Nasal septum tissue was obtained from five patients aged ≥ 20 years who were undergoing septoplasty. hNCs were isolated and subcultured for three passages in vitro. To formulate the cell–PCL complex, we used type I collagen as an adhesive between chondrocyte and PCL. Immunofluorescence staining, cell viability and growth in the hNC–PCL complex, and mycoplasma contamination were assessed.

Results:

hNCs in PCL showed viability ≥ 70% and remained at these levels for 9 h of incubation at 4 °C. Immunostaining of the hNC–PCL complex also showed high expression levels of chondrocyte-specific protein, COL2A1, SOX9, and aggrecan during 24 h of clinically applicable conditions.

Conclusion:

The hNC–PCL complex may be a valuable therapeutic agent for implantation into injured cartilage tissue, and can be used clinically to repair cartilaginous skeletal defects. From a clinical perspective, it is important to set the short duration of the implantation process to achieve effective functional implantation.

Keywords: Chondrocyte, Collagen, Human nasal septum, Tissue engineering, Cartilage

Introduction

In a clinical setting, structural defects of the cartilaginous portion due to trauma, previous surgical resection, or congenital anomalies are frequently encountered, and can require tissue regeneration for reconstruction [1]. However, the poor capacity for self-repair among cartilaginous defects has promoted the use of autologous tissues as implants that can be formed into the correct anatomical shape [1]. Among various cartilage sources, such as articular cartilage, costal cartilage, and auricular cartilage, chondrocytes from human nasal septal cartilage (hNCs) are an excellent source of chondrocytes for cartilage tissue regeneration. Septal surgery, such as septoplasty, is among the most common otorhinolaryngological procedure performed worldwide to improve nasal obstruction symptoms. During this procedure, septal cartilage is discarded as surgical waste. Therefore, it is easier and less dangerous to obtain hNCs than chondrocytes from articular cartilage, because the collection procedures are minimally invasive [2, 3]. Several studies have recently indicated that nasal chondrocytes have great potential for tissue engineering and/or regenerative medicine applications. Due to their minimally invasive collection procedures, and to the sustainability of their chondrogenic properties after extensive culture expansion [4–6], hNCs are of major clinical interest for the development of cell-based strategies to treat cartilage defects [7].

In the field of tissue engineering, biodegradable scaffolds have been used as supporting tools for cell proliferation and/or appropriate matrix formation (i.e., protein synthesis) to fill tissue defects with cells [8, 9]. For this purpose, the scaffold must be able to withstand physiological loading until sufficient tissue regeneration occurs. Moreover, the material must be sufficiently porous to allow for effective nutrient transport. Finally, it should be biocompatible and degrade as the tissue matrix is produced, leaving only nontoxic degradation products. To meet these requirements, polycaprolactone (PCL) has been developed as a biocompatible and biodegradable synthetic polymer with appropriate mechanical strength and durability. Considering the ease of its production due to desirable porosity and mechanical properties, and its approval by the US Food and Drug Administration, this polymer may be a promising platform for the production of implants that can be manipulated physically, chemically, and biologically to achieve degradation kinetics tailored to a specific anatomical site. We previously demonstrated the biocompatibility and effectiveness of PCL implants for nasal reconstruction in an experimental rabbit model, and in human clinical trials [10, 11]. Several previous trials have reported that the seeding of chondrocytes into PCL scaffolds efficiently maintained their differentiated phenotype and allowed for synthesis of cartilage-specific extracellular matrix (ECM) proteins [12–14]. Therefore, previous in vivo trials have been conducted using hNC–PCL complexes for cartilage repair [15, 16]. However, from a clinical perspective, it is important to investigate the proper time limit for implantation, because hNCs can deteriorate when introduced into unfamiliar conditions such as the collagen and PCL scaffold space. Therefore, we tried to find the effective functional implantation point of hNC–PCL complexes through this study.

Materials and methods

Preparation of PCL scaffold

A scaffold made up of PCL (PURAC®, Amsterdam, Netherlands) was fabricated using an in-house 3D printing system [17]. Briefly, the granule-typed PCL was fed into a steel syringe equipped in the head part of 3D printing system and melted at 120 °C. The thermally molten PCL was dispensed through a heated nozzle, and the stranded PCL was stacked in multiple layers. The nozzle size was 500 µm and the distance between strands was 500 µm [10]. The overall size of the scaffold was 8 × 8 × 0.8 mm with round edges, and its pore percentage was 50%.

Cell isolation and expansion

hNCs were isolated as described previously [18]. All studies utilizing nasal septal chondrocytes were conducted following written approval (HC13TISI0038) from the Institutional Review Board of the Catholic Medical Center Clinical Research Coordinating Center, and after obtaining written informed consent from the donors. Nasal septum tissue was obtained from five patients aged > 20 years who were undergoing septoplasty. The isolation procedure was performed as reported previously. Briefly, tissues were cut into 1-mm3 pieces and enzymatically digested with 0.2% protease solution (Gibco, Grand Island, NY, USA) for 60 min followed by incubation in 0.3% collagenase (Sigma, St. Louis, MO, USA) for 12 h at 37 °C. The isolated cells were seeded in a 75-mm2 cell culture flask (NUNC, Roskilde, Denmark) and cultured in low-glucose Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL, Waltham, MA, USA) with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) at 37 °C in a 5% CO2 incubator. Chondrocytes were subcultured at a 1:3 ratio using a 0.05% trypsin/EDTA solution (Gibco, Grand Island, NY, USA) for three passages.

Flow cytometry of human nasal septal chondrocytes

Flow cytometry was used to detect expression of chondrocytes surface markers CD9, CD44, CD73, CD90, CD105, and CD166 [19, 20]. CD14, CD19, CD34, and HLA-DR were also conducted for checking hematopoietic markers (Fig. 1). hNCs at passage 3 were placed in a test tube (BD Biosciences, Bedford, MA, USA) at 1 × 105 cells/mL and washed three times with wash buffer (PBS and 3% FBS). The cells were then incubated with monoclonal antibodies against CD markers for 40 min under saturating concentrations. After 3 washes in buffer, cells were centrifuged (1200 rpm, 5 min), resuspended in ice cold PBS, and incubated with FITC- or PE-labeled secondary antibodies for 30 min in the dark at 40 °C. Cell fluorescence was evaluated by flow cytometry with a FACS Calibur instrument (BD Biosciences, Bedford, MA, USA) and data were analyzed using Cell Quest software (BD Biosciences, Bedford, MA, USA).

Fig. 1.

Fig. 1

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Surface marker assay of hNCs with flow cytometry. Chondrogenic surface markers (CD9, CD44, CD73, CD90, CD105, and CD166) showed positive on flow cytometry. On the other hand, the expression of cell surface markers (CD14, CD19, CD34, and HLA-DR) related to hematopoietic cells was negative

Proliferation assay of human nasal septal-derived progenitors

We assessed hNC proliferation using the Cell Counting Kit (CCK-8) (Dojindo Laboratories, Kumamoto, Japan) following the manufacturer’s instructions. Culture medium was removed, and 100 μL fresh medium containing 10 μL CCK-8 was added to each well. The cells were then incubated at 37 °C for 4 h. Cell proliferation was monitored for 2 days. These experiments were performed at least three times on individual donor pools.

Preparation of hNCs–PCL (polycaprolactone) complex

To formulate a cell–PCL complex, we used type I collagen as an adhesive for chondrocyte and PCL. We purchased type I collagen (UBIOSIS, Seongnam, Korea) at a concentration of 30 mg/mL. hNCs were detached from the tissue culture plate with 0.25% trypsin solution, and a cell suspension was prepared. The cell suspension was mixed with the collagen solution at a 2:1 ratio for a final concentration of 20 mg/mL. The total cell concentration was 106 cells/mL. After sampling the ice-cold cell–collagen mixture by pipette, the mixture was applied to the PCL mesh (5 mm × 5 mm × 0.8 mm) using a spatula. The hNC–PCL complex was then kept at 4 °C for further use (Fig. 2).

Fig. 2.

Fig. 2

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Manufacture of the human nasal septal cartilage–polycaprolactone (hNC–PCL) complex. Chondrocytes and type I collagen were prepared on ice to maintain a temperature of 4 °C and the gel form of collagen (A, B). Chondrocytes were blended with collagen using a pipette on ice (C, D). The cell–collagen mixture was extracted using a pipette on ice and applied to the PCL mesh (E, F). Scale bar: 5mm

Scanning electron microscope analysis of the hNCs–PCL complex

The morphology of the 3D-printed PCL mesh was confirmed using a field emission scanning electron microscope (FE-SEM, S-4700, HITACHI Co., Tokyo, Japan) with an acceleration voltage of 10 kV. The mesh was coated for 3 min with platinum using a sputter coater.

Immunofluorescence staining

Cell-collagen mix was resuspended in HBSS containing 10% FBS (5 × 105/mL) and 100 μL of each cell suspension was added to a chamber slide (5 × 104 cells). It was spinned at 800 rpm for 3 min and was removed from chamber slide carefully. Air dry was performed prior to staining. For cell marker immunolabeling, hNCs and the hNC–collagen complex were incubated with 5% goat serum and then double stained. First, cells were incubated with antibody against COL2A1, SOX9, or aggrecan overnight at room temperature, washed three times with 0.01 M phosphate-buffered saline (PBS) containing 3% bovine serum albumin (BSA), and incubated with Alexa Fluor 488-conjugated secondary antibody (1:1000 dilution) for 1 h at room temperature. The cells were then stained with the nuclear stain DAPI. Stained cells were mounted using mounting media (Vectashield; Vector Labs, Burlingame, CA, USA) and observed under a Confocal microscope (LSM510 Meta; Carl Zeiss MeditecAG, Jena, Germany).

Cell viability and growth in hNCs–PCL complex

To evaluate hNC–PCL complex cell viability in the serum-free condition, hNC–PCL was placed in the tube at room temperature (28 °C) or 4 °C for 24 h. hNC–PCL was then treated with 0.2% collagenase I for 5 min in a 5% CO2 incubator at 37 °C. Cell viability was determined using the CCK-8 (Dojindo Laboratories, Kumamoto, Japan).

Detection of mycoplasma contamination

Mycoplasma contamination was evaluated in the cultured hNCs and hNC–PCL by polymerase chain reaction (PCR) analysis. To evaluate mycoplasma contamination at the gene level, PCR was processed using the mycoplasma PCR Detection Kit (TaKaRa, Otsu, Japan) following the manufacturer’s instructions. Briefly, after 1, 9 and 24 h of culture, 1 mL of culture media was collected and centrifuged at 3000 rpm for 5 min. The supernatant was then transferred to a microcentrifuge tube and centrifuged at 12,000 rpm for 10 min. The mycoplasma pellet was collected, suspended with 50 μL dH2O, and then boiled at 98 °C for 10 min. We transferred 50 μL supernatant into new PCR tubes.

We performed two step nested PCR reactions. For 1st PCR reaction, 5 μL of template and 45 μL master mix contains outer primers. 1st PCR reactions were amplified under the following conditions: 1 cycle of pre-denaturation at 94 °C for 30 s, 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 2 min, and extension at 72 °C for 1 min. Next, 2nd PCR was performed by adding 1 μL of 1st PCR template to 45 μL master mix contains inner primers. 2nd PCR amplifications were conducted under the following conditions: 1 cycle of pre-denaturation at 94 °C for 30 s, 30 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 2 min, and extension at 72 °C for 1 min. Finally, 10 μL 2nd PCR products were electrophoresed on 1% agarose gel (Bio-Rad, Hercules, CA, USA) containing 0.4 μg/mL ethidium bromide (Sigma, St. Louis, MO, USA) and detected by ChemiDoc Image Lab (Bio-Rad, Hercules, CA, USA).

Quantification and statistical analysis

Statistical analyses were conducted using SAS software (ver. 9.4; SAS Institute Inc., Cary, NC, USA). All data are expressed as means ± standard deviation (SD) or standard error of the mean (SEM) from at least three independent experiments. Multiple groups were compared by analysis of variance (ANOVA) with Tukey’s post hoc test to define statistically significant differences among groups. Differences between two samples were detected using Student’s t test. Statistical significance was determined at a level of p < 0.05.

Results

Phenotypic characterization of hNC

The immunophenotypic features of hNC were assessed to identify the expression of CD9, CD14, CD19, CD34, CD44, CD73, CD90, CD105, CD166, and HLA-DR markers. The expression of Chondrogenic associated markers (CD9, CD44, CD73, CD90, CD105, and CD166) was positive. The expression of cell surface markers (CD14, CD19, CD34, and HLA-DR) related to hematopoietic cells was negative.

Proliferation and extracellular matrix (ECM) protein expression in cultured hNCs

After isolation from nasal cartilage, nasal chondrocytes were expanded in a two-dimensional (2D) monolayer culture. After three passages of in vitro culture, the pattern of cell proliferation was monitored for 48 h. After 48 h, the density of cells via absorbance at 450 nm was approximately double that at the initial stage of cultivation (0 h: 1.21 ± 0.09, 24 h: 2.06 ± 0.20, 48 h: 2.67 ± 0.41; Fig. 3A; all p < 0.0001). After three passages of in vitro culture, the expression of ECM components in cultured hNCs was examined by immunostaining. The results indicate that hNCs have adequate chondrogenic potency, suggesting that they are suitable for further experiments to evaluate the hNC–PCL complex for clinical application in cartilage regeneration (Fig. 3). Intracellular levels of COL2A1 and SOX9 were clearly visualized in hNCs compared to the control (Fig. 3B).

Fig. 3.

Fig. 3

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Proliferation activity and extracellular matrix (ECM) protein expression of hNCs in two-dimensional (2D) culture. A Proliferation patterns showed a rapid growth rate, and B Immunofluorescence Imaging. Aggrecan, COL2A1 and SOX9 of hNCs are immunostained in green; nuclei are counterstained with DAPI (blue). Scale bar: 20 μm

Surface morphology analysis of hNCs–PCL using scanning electron microscope

The line width and pore size of the PCL mesh were both 500 μm. hNC–collagen was well attached to PCL mesh structure (Fig. 4).

Fig. 4.

Fig. 4

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hNC–PCL complex analyzed by scanning electron microscopy (SEM). A PCL structure, and B cell morphologies on the surface of scaffold using SEM

Cell viability and ECM protein expression in hNCs–PCL under serum-free conditions

Cell viability and ECM protein expression of hNCs in the hNC–PCL complex were analyzed to evaluate the potential of hNC–PCL for clinical application under serum-free conditions. hNC and hNC–PCL cell viability decreased over time. A significant difference in cell viability was detected between hNCs alone and hNC–PCL complex cells at 9 h under serum-free conditions (hNCs: 85.54 ± 8.25% vs. hNC–PCL: 73.07 ± 9.90% after 9 h serum-free; p = 0.01, Fig. 5). Viability curves generally showed that cell viability was > 70% by 9 h.

Fig. 5.

Fig. 5

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Cell viability in hNC–PCL cells under serum-free conditions. The viability of hNCs and hNC–PCL cells decreased over time. A Viability curves showed that viability was generally > 70% within 9 h in the serum-free condition. *p < 0.05. B Live & dead stain according to the time. Scale bar: 50 μm

The expression of hNC–PCL ECM protein was analyzed by immunostaining and reverse-transcription (RT)-PCR using chondrocyte-specific antibodies under serum-free conditions. Immunofluorescence staining for COL2A1, SOX9, and aggrecan showed that most hNCs expressed these proteins immediately after mixture (1 h), and this level was not elevated at 9 h or 24 h after mixture (Fig. 6). hNCs and hNC–PCL complex cells consistently showed increased expression of mRNAs encoding COL2A1 over time. In contrast, SOX9 gene expression showed an opposite pattern of decreasing expression in two groups. However, a comparison by quantitative PCR indicated that hNC–PCL cells showed a similar expression pattern of COL2A1 and SOX9 during 24 h of cultivation (Fig. 7). Overall, hNCs showed consistent chondrogenic differentiation potentials, regardless of the conditions of the hNC–PCL complex.

Fig. 6.

Fig. 6

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ECM protein expression in the hNC–PCL complex under serum-free conditions. ECM protein expression in hNC–PCL cells was analyzed by immunostaining under serum-free conditions. Immunofluorescence staining for COL2A1, SOX9, and aggrecan showed that most hNCs expressed these proteins during the follow-up period. Scale bar: 50 μm

Fig. 7.

Fig. 7

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ECM gene expression in hNC–PCL cells under serum-free conditions. Expression patterns of mRNAs encoding COL2A1 consistently increased in hNCs and hNC–PCL cells over time, whereas SOX9 expression decreased over time in two groups

Mycoplasma detection of hNCs–collagen in culture

Mycoplasma detection is necessary for the therapeutic application of hNC–collagen for cartilage regeneration. To evaluate mycoplasma contamination of hNC–collagen at the gene level, PCR analysis was performed using a mycoplasma PCR detection kit with primers designed to react specifically with the highly conserved coding region of the mycoplasma genome. Cultured hNCs and a hNC–collagen mixture were tested for mycoplasma contamination and all PCR products showed one band in all tested samples. This ca. 700-bp band was an internal DNA band, which confirms the results of the PCR reaction (Fig. 8), further indicating that collagen does not affect mycoplasma contamination in hNCs. The clinical relevance of this result is underlined by the potential for clinical application of an hNC–collagen mixture in patients with cartilage defects.

Fig. 8.

Fig. 8

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Mycoplasma polymerase chain reaction (PCR) analysis of cultured hNCs and the hNC–PCL complex. PCR analysis of samples from collagen, 2D-cultured hNCs, and hNCs encapsulated in collagen (hNC–collagen) cultured for 24 h

Discussion

Tissue engineering techniques that combine scaffolding with autologous chondrocytes have been developed to address various clinical problems [14, 21, 22]. Scaffolding provides a 3D biodegradable structure for in vitro growth of living cells and their subsequent implantation [23]. Based on these principles, various scaffolds have been introduced as carriers for chondrocyte implantation in clinical practice. Among these, chondrocyte/PCL-scaffold implants have been reported to achieve effective cartilage regeneration, allowing chondrocytes to proliferate for transfer to host cartilage tissues [12–14].

Previous studies have reported that hNCs proliferated approximately four times faster than human articular chondrocytes in monolayer culture, and had markedly higher chondrogenic capacity in in vitro-engineered constructs [24]. These results were consistent regardless of the age of the donor [25]. The large tissue yield and consequently large cell yield from donors with a wide age range (15–60 years) have been reported as a potential source of chondrocytes for large-volume tissue-engineered implants [6]. Considering these favorable findings, hNCs could be a good cell source for in vitro engineering of autologous cartilage grafts [5, 24, 25]. However, studies examining the combination of hNCs and PCL are rare in the context of craniofacial defects or degenerative disease. For clinical application, the effectiveness and quality of septal chondrocytes combined with PCL must be evaluated.

Cartilage volume is composed of 1% chondrocyte and 99% ECM. ECM controls the mechanical, compressive, and load-bearing properties of cartilage, whereas chondrocytes synthesize and maintain ECM [26]. Aggrecan and COL2A1 are the main components of cartilage ECM, and SOX9 plays a key role in chondrogenesis [27]. In the current study, we measured chondrogenic potential via immunostaining and Western blot analysis using ECM markers. Cultured hNCs exhibited higher expression levels of COL2A1 and SOX9, indicating that hNCs may be a valuable candidate for cartilage engineering.

In the present study, we used two types of xenogeneic materials (i.e., porcine collagen and FBS). Although the use of these xenogeneic materials may confer risks related to unknown antigens or microbes, and high levels of xenogeneic proteins raise the concern of immune reactions in human patients [28, 29], porcine collagen has been used successfully in various cosmetic and reconstructive applications [22]. We used this material as an adhesive for chondrocytes and PCL. FBS has been used in several clinical trials without any adverse side effects [30–32]. However, despite the clinical acceptance of these xenogeneic materials [22], it is highly recommended to wash cells sufficiently after harvest and before formulation to decrease xenogeneic protein levels to acceptable levels [32, 33]. Considering the manufacture and preparation of cell–PCL complexes, chondrocytes in such complexes must endure serum-free conditions for considerable periods of time. We therefore evaluated the chondrogenic potential of hNCs in the hNC–PCL complex during 24 h in the absence of FBS. At all time points, the chondrogenic-specific markers aggrecan, COL2A1, and SOX9 in the collagen complex were detected by immunofluorescence staining. Most hNCs expressed these chondrogenic markers consistently from 1 to 24 h after serum-free cultivation. These results were similar to those reported for articular cartilage cultivated in collagen with FBS [34, 35]. It is important to determine the time limit for achieving effective implantation. In this study, we showed that cell viability differed between hNCs alone and the hNC–PCL complex at 9 h. hNC–PCL complex should be utilized in clinical field within 9 h after serum free cultivation because hNC–collagen complex cell viability significantly dropped by 70% at 9 h cultivation. This result represents vital information for clinical trials of hNC–collagen use in patients. Cultured hNCs or hNC–complex cells were also tested at the gene level for mycoplasma contamination. Mycoplasma screening tests were negative in all specimens, indicating that the isolation and culture of chondrocytes and manufacture of the hNC–PCL complex would be safe for clinical application. These findings support the possibility of clinical application of hNCs with PCL gel for cartilaginous skeletal defects.

In tissue engineering, highly proliferative cells are necessary to produce a sufficient numbers of cells [26]. In our study, cell proliferation was monitored over a period of 2 days. hNCs from three different donors showed very high proliferation and rapid growth. In general, monolayer expansion of human chondrocytes in vitro has become an essential step in the process of tissue engineering [36]. In a previous study, articular chondrocytes were shown to double in number after 1 week of cultivation in a conventional monolayer culture [35, 37]. In the current study, the proliferation of hNCs was much more rapid, consistent with a previous finding that hNCs proliferated approximately four times faster than articular chondrocytes [24]. Considering that it is essential to obtain a sufficient amount of viable chondrocytes for clinical application [38], hNCs have great potential as a source of clinically available chondrocytes in tissue engineering for cartilage defects.

Seed cells have always been the focus of tissue engineering studies. Ideal seed cells should have no or little limitations in terms of availability, capacity for expansion, and functional properties [39]. Based on our results, hNCs appear to have high proliferation and ECM secretion potential in combination with PCL. These favorable findings could open the possibility of septal chondrocyte application for cartilaginous skeletal disease. Furthermore, cartilage has been variously described as non-immunogenic or weakly immunogenic [38], which could render cartilage allografts immunoprivileged [40]. The storage and application of tissue that is normally discarded during surgery may be the foundation for developing custom-made tissue and cell banks for patients who require cell therapy.

In conclusion, the isolation of hNCs and manufacture of hNC–PCL complexes would be safe and effective. hNCs proliferate rapidly and maintain chondrogenic differentiation within the PCL complex. To achieve effectively functioning implants, it is important to perform implantation within 9 h of culture. To develop an effective tissue regeneration method using hNCs, additional studies are needed to identify the various regulation and differentiation mechanisms of these cells in vivo.

Acknowledgements

This research was supported by a grant (18172MFDS185) from Ministry of Food and Drug Safety of Korea 2019. This work was supported by the Korea Health Industry Development Institute funded by the Ministry of Health and Welfare (HI14C3228, HI14C0113), by the Basic Science Research Program through the National Research Foundation of Korea (NRF) grant funded by the Ministry of Education (2017R1D1A1B03027903, 2018R1D1A1B07045421), and by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Ministry of Science & ICT (2018M3A9E8020856). This work was also supported by the Institute of Clinical Medicine Research of Bucheon St. Mary’s Hospital, Research Fund, 2018. The sponsors had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Compliance with ethical standards

Conflict of interest

The authors indicate no potential conflict of interest.

Ethical statement

All studies utilizing nasal septal chondrocytes were conducted following written approval (HC13TISI0038) from the Institutional Review Board of the Catholic Medical Center Clinical Research Coordinating Center, and after obtaining written informed consent from the donors.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Do Hyun Kim and Mi Hyun Lim have contributed equally to this work as first author.

Se Hwan Hwang and Sung Won Kim have contributed equally to this work as corresponding author.

Contributor Information

Se Hwan Hwang, Phone: +82 32 340 7044, Email: yellobird@catholic.ac.kr.

Sung Won Kim, Phone: +82-2-2258-6216, Email: kswent@catholic.ac.kr.

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