The feasibility of human embryonic stem cell (hESC)-derived cartilage for tracheoplasty

Abstract

Introduction

Airway reconstruction with autologous costal cartilage often results in long-term complications. On the other hand, implant-type regenerated cartilage using chondrocytes and scaffolds is associated with better biocompatibility and outcomes. Nevertheless, obtaining a substantial amount of chondrocytes remains a challenge. Allogenic cartilage from human embryonic stem cells (hESCs) can be used as an alternative graft for tracheal reconstruction. The aim of this study was to determine the regenerative potential of hESCs-derived cartilage tissue.

Methods

The clinical grade hESC line sSEES-2 was cultured in chondrocyte differentiation media for eight weeks. Maturation potential was estimated by subcutaneous transplantation into immunodeficient mice and the mechanical properties were measured with a tactile sensor. For tracheoplasty studies, the matured cartilage constructs were implanted into tracheotomized sites in athymic rats, and were evaluated 1- and 3-months post-implantation through endoscopy and histological analysis.

Results

Small cartilage tissues – or “cartilage islets” – were successfully obtained by culturing the SEES2 cells in specialized media. The thickness and strength (Young's modulus) of the grafted cartilage increased one month after implantation, indicating maturation. Furthermore, the cartilage graft successfully restored artificial defects in rat trachea with no structural damage or air leakage. The tracheal mucosa recovered one month after the implantation, and airway patency was maintained for three months. Histological analysis of the tracheal tissues revealed epithelial regeneration, and direct integration between the cartilage graft and native cartilage without the formation of granulation tissue.

Conclusions

Cartilage tissue derived from hESCs successfully maintained airway structure and biocompatibility in a rat model for three months, which supports the application of engineered cartilage for clinical airway reconstruction.

Graphical abstract

Highlights

• •Human embryonic stem cells (hESCs) differentiated into chondrocytes in vitro.
• •Chondrocytes derived from hESCs produced functional cartilage to maintain airway.
• •Cartilage derived from hESCs can be used for reconstructing airways.

1. Introduction

Congenital subglottic stenosis is a severe congenital disease characterized by a narrow subglottic space directly below the vocal cords, which causes respiratory distress immediately after birth. While autologous costal cartilage grafts can achieve life-long laryngotracheal reconstruction [1], the latter is often accompanied by the atrophy and disappearance of the costal cartilage, formation of granulation tissue, and/or ossification, eventually resulting in airway stenosis [2,3]. Thus, costal cartilage is not an ideal material for airway restoration, and more biocompatible grafts are desirable alternatives. Regenerated cartilage derived from autologous chondrocytes has been effective in reconstructing the airway in animal models [4]. A comparative study of costal cartilage patches and implant-type regenerated cartilage in a rabbit model demonstrated neocartilage formation at the interface between native tracheal cartilage and the implant-type regenerated cartilage. In contrast, the junction with costal cartilage was characterized by the presence of granulation tissue. Cartilage generated through autologous approaches offers the advantage of excellent biocompatibility for airway reconstruction. We had designed a clinical study to evaluate airway reconstruction using autologous implant-type regenerative cartilage as a less invasive alternative to autologous costal cartilage patch reconstruction (UMIN000032362). However, the study was ultimately discontinued for reasons related to the company's internal circumstances. Autologous regenerative cartilage is challenging to commercialize due to limited donor tissue availability, donor site injury [5], individual variations in donor cell quality [6], and a protracted [6] and costly manufacturing process [5,6]. The paucity of efficient sources of chondrocytes is a major obstacle to the clinical translation of regenerative cartilage.

Cartilage has limited capacity for self-regeneration and is classified as an immune-privileged tissue, which reduces host immune responses to foreign antigens [7], [8]. Allogenic regenerative cartilage can be implanted in the nose, ears, and airway. Seiiku embryonic stem cells (SEES-1-7, and X), a clinical grade human embryonic stem cell (hESC) line, has been established at the National Center for Child Health and Development (NCCHD) in Japan [9]. Furthermore, researchers at NCCHD have successfully developed engineered cartilage using hESCs-derived embryoid bodies in an adherent culture system [10]. However, the phenotypic characteristics of hESCs-derived chondrocytes, including matrix production, biocompatibility and lifespan, in the airway have not been determined so far. In this study, we evaluated the regenerative ability and biocompatibility of the matrix generated by hESCs-derived chondrocytes in a rat model of tracheotomy to determine its feasibility as an alternative graft material for tracheal reconstruction.

2. Material and methods

2.1. Induction of cartilage differentiation from SEES-2 cells

Engineered cartilage was aseptically harvested from an in vitro culture system [10]. Briefly, hESCs (SEES-2) were cultured on dishes coated with iMatrix-511 silk (892021, Nippi, Incorporated, Tokyo, Japan) in StemFit medium (RCAK02 N, Ajinomoto Healthy Supply Co., Inc., Tokyo, Japan). The medium was changed daily. The ESCs were detached with a 1:1 mixture of 0.5 mM EDTA and TrypLE™ Select (A1217701, Thermo Fisher Scientific, MA, USA) for 5 min, and passaged every week at the density of 550 cells/cm². To produce embryoid bodies, the ESCs were dissociated and plated in ultra-low attachment 96-well plates (7007, Corning, Arz, USA) with XF32 medium containing various supplements. The embryoid bodies (672 in total) were collected 4 days later and plated onto collagen PS (301–84621, nippi)-coated dishes in XF32 medium. The medium was changed every 3 days using an automated medium exchanger integrated with the incubator (CellKeeper (Model SCALE-120ME), RORZE Lifescience Inc. Ibaraki, Japan). The embryoid bodies fused into sheets within a few days, and the "cell sheets" were easily detachable after 60 days.

2.2. Maturation of SEES-2-derived cartilage

Six-weeks-old male immunodeficient NOG (NOD.Cg-PrkdcscidIl2rgtm1Sug/ShiJic) mice were procured from Charles River Laboratories, Inc., MA, USA (protocol No.A2003-002-C20-M01). The mice were anesthetized with 3 % isoflurane gas (099–06571, Fujifilm Wako Pure Chemicals Co., Ltd., Tokyo, Japan). The cartilage harvested from cell sheets was transplanted into the subcutaneous space of mice under sterile conditions. Four weeks later, the mice were euthanized humanely and the cartilage tissues were removed.

2.3. Evaluation of mechanical properties

The mechanical properties of the engineered cartilage were tested using a Venustron tactile sensor (Axiom Co., Ltd., Fukushima, Japan). Young's modulus was calculated by compression strength and frequency decrease using Venus 42 software (Axiom Software, Valhalla, NY, USA) based on a previous report [11]. These parameters were measured three times for three samples at each time point, following in vitro culture and after four weeks of subcutaneous implantation using the Venustron tactile sensor.

2.4. Tracheoplasty with matured SEES-2-derived cartilage

Six-weeks-old male F344/NSlc rats (Japan SLC, Inc., Shizuoka, Japan) were used for tracheoplasty studies. All experimental procedures were approved by the Animal Care Committee of Tokyo University (protocol No. Medical-P21-045). Following anesthetization with isoflurane (Maruishi Pharmaceutical Co. Ltd., Osaka, Japan), a midline cervical incision was made and the trachea was exposed. An 18-gauge plastic cannula was inserted into the trachea for ventilation using an artificial respirator (Shinanoseisakusho Co. Ltd., Tokyo, Japan), and a tracheotomy measuring 2 × 3 mm was performed as an extension of the initial incision for cannulating the plastic tube. Engineered cartilage constructs measuring 2 × 3 mm were implanted into the tracheotomy site, and secured using 8-0 monofilament sutures (Prolene, Ethicon, Somerville, NJ). After reconstructing the airway, air leakage was assessed by filling the wound site with saline.

2.5. Morphological and histological analyses

The morphology of the reconstructed site was assessed 1- or 3-months post implantation by tracheoscopy using an ultra-thin endoscope (TESALA, AVS Co. Ltd., Tokyo, Japan) as described previously [12]. Following endoscopy, the cartilage specimens were harvested, fixed overnight in 4 % paraformaldehyde at 4 °C, embedded in paraffin, and cut into thin sections. Deparaffinization, dehydration, and permeabilization were performed using standard techniques. Hematoxylin-eosin (HE) staining was performed using Carrazzi's hematoxylin solution (30022, Muto Pure Chemicals Co., Ltd., Tokyo, Japan) and eosin Y (32053, Muto Pure Chemicals Co., Ltd.). The sections were then stained with 0.1 % Toluidine blue solution (pH 7.0) for 3 min, rinsed, dehydrated in isopropyl alcohol, and cleared in xylene.

For immunostaining, the deparaffinized sections were first incubated in histophine (415,211, Nichirei Corporation, Tokyo, Japan) in a microwave oven at a power setting of 700 W for 20 min to unmask the epitopes. After three washes with PBS, the sections were incubated overnight with primary antibodies (412,811, Nichirei Corporation) at 4 °C, and then incubated with 3,3′-diaminobenzidine (4065–1, Muto Pure Chemicals Co., Ltd.) and counterstained with hematoxylin.

2.6. Statistical analysis

The Young's moduli of the cartilage induced in vitro and the cartilage matured in vivo were compared using the two-sided Mann-Whitney U test implemented in Python 3.11 using SciPy (Scientific Python) version 1.11.1. The Mann-Whitney U statistic and two-tailed p-value were calculated, and p-values below 0.05 were considered statistically significant.

3. Results

3.1. Characterization of hESCs-derived cartilage

Cartilage was generated from SEES-2 cells after eight weeks of culture. As shown in Fig. 1a, the cartilage tissues measured less than 1 cm, and their surface was smooth, semi-translucent, and white. Four weeks after in vivo implantation, the cartilage grafts exhibited increased thickness and size (Fig. 1b). Furthermore, the median Young's modulus of the cartilage obtained in vitro was 11 kPa, and increased to 349 kPa at four weeks post-implantation. Thus, in vivo maturation of the cartilage grafts led to a significant increase in Young's modulus, which was indicative of high elasticity and flexibility (Fig. 1c).

Fig. 1.

Engineered cartilage derived from hESCs. (a) Representative image of hESCs-derived cartilage showing smooth, semi-translucent, and white surface, and measuring less than 1 cm in diameter prior to in vitro culture. (b) Representative image of the grafted cartilage four weeks after in vivo implantation showing increased thickness and size. (c) Median Young's moduli of the in vitro engineered cartilage (11 kPa) and in vivo matured cartilage four weeks after implantation (349 kPa; p = 0.015).

3.2. Tracheoplasty with cartilage derived from hESCs

The hESCs-derived cartilage grafts measuring 2 × 3 mm were implanted into tracheotomy sites as a patch material. The engineered cartilage did not sustain any structural damage due to needle or suture penetration, and was resistant to any mechanical alterations during ligation or suturing. In addition, the grafted cartilage matched the texture of native cartilage and effectively prevented any air leakage from the trachea, thereby retaining the original functions of cartilage tissues (Fig. 2). All experimental animals survived until autopsy.

Fig. 2.

Tracheoplasty with cartilage derived from hESCs.

The hESC-derived cartilage was implanted into tracheotomy sites measuring 3 × 2 mm. The engineered cartilage did not sustain any structural damage from needle or suture penetration and showed sufficient resistance to manipulation during ligation or suturing. The texture of the engineered cartilage closely resembled that of native cartilage. Furthermore, no air leakage was observed at the reconstructed site, indicating that the engineered cartilage can form highly functional tissues in vivo.

3.3. The engineered cartilage achieved airway reconstruction and epithelialization within three months post-implantation

The respiratory status of the operated animals remained within normal limits for a period of three months post-implantation, and no abnormalities were observed (Fig. 3a). Endoscopic examination revealed that the reconstructed airway maintained its lumen during inspiration and expiration until three months following cartilage grafting (Fig. 3b). The color of the lumen at the reconstruction site was comparable to that of a normal airway, suggesting that the lumen was covered with mucosa. Furthermore, although some deformation was noted at the site of tracheal reconstruction compared to the normal trachea, no significant stenosis was observed (Fig. 3a and b). Thus, hESCs-derived engineered cartilage was able to support airway structure for up to three months after implantation. In addition, H&E staining of the tracheal lumen showed epithelial regeneration in all specimens, which was also confirmed by intense staining for keratin, indicating that hESCs-derived cartilage was epithelialized after four weeks of tracheoplasty (Fig. 4). Furthermore, the grafted cartilage was present three months after implantation, as indicated by H&E staining of the cartilage matrix (Fig. 5b). Notably, a continuous tissue block was observed at certain regions of the interface between the rat tracheal cartilage and hESCs-derived cartilage, without any evidence of granulation tissue (Fig. 5c).

Fig. 3.

Macroscopic and endoscopic findings three months post-implantation. (a) Macroscopic observation indicated normal respiratory status. (b) Endoscopic examination revealed that the reconstructed airway maintained its lumen until three months.

Fig. 4.

Regenerated tracheal epithelium.

Representative immunostaining image showing keratin deposition in the regenerated tracheal epithelium.

Fig. 5.

Histological examination of the implantation site. (a) Normal trachea (b)(c)Cartilage derived from hESCs remained present three months after being implanted into a section of the airway. The cartilage matrix was maintained until three months of implantation. The hESCs-derived and native tracheal cartilage eventually became a continuous block without granulation formation. The clear absence of granulation tissue at the interface is indicative of superior mechanical properties of the engineered cartilage, and the direct integration between the implanted and native cartilages.

4. Discussion

Chondrocytes derived from hESCs are capable of generating adequate functional amounts of cartilage matrix through a specialized adherent culture system and suitable in vivo conditions. In this study, we used engineered cartilage for tracheoplasty in a rat model, and were able to demonstrate excellent biocompatibility for up to three months post-implantation. Furthermore, auto self-regeneration of the mucosal epithelium was confirmed four weeks after implantation. Tracheoplasty using engineered cartilage was maintained until three months as the grafted material formed a continuous interface with the native trachea cartilage.

The median Young's modulus of the engineered cartilage constructs was approximately 350 kPa after in vivo incubation, indicating that the grafts can maintain their mechanical properties within airway constructs. In addition, the flexibility and hardness exhibited by the engineered cartilage during surgery were similar to that of native cartilage. Furthermore, the grafts stably provided mechanical air sealing in the airway reconstruction model. These findings indicate that chondrocytes derived from hESCs can produce cartilage proteins like collagen type 2 and glycosaminoglican in quantities and quality that are sufficient to provide the mechanical strength needed for airway reconstruction. In other words, the quantitative as well as qualitative maturation of the cartilage matrix in vivo reflect the functional competence of the regenerated tissue. This study confirmed that the cartilage grafts exhibited Toluidine Blue staining patterns comparable to native tracheal cartilage, thereby verifying the presence and distribution of proteoglycans within the cartilage matrix. The observed mechanical characteristics of the cartilage graft are attributed to the validated integrity of the cartilage matrix.

The SEES-2 cells have been successfully differentiated into hepatocyte-like cells (HLCs), which were tested in a clinical trial (JMA-ⅡA00412) as a bridging therapy prior to liver transplantation for five infants diagnosed with urea cycle disorder, and resulted in improvements in hyperammonemia and growth [13]. SEES-2, an hESC cell line developed for industrial and clinical applications, was used as the source of engineered cartilage allografts in this study.

The hESCs-derived and native tracheal cartilage formed a cartilage mass without granulation in the airway reconstruction model. Similar outcomes have been observed for autologous engineered cartilage and native tracheal cartilage in a rabbit model. A single reconstructed annular cartilage segment within the trachea may exhibit enhanced mechanical characteristics suitable for airway reconstruction. Engineered cartilage is capable of integrating with native cartilage, even in heterologous transplants, and may obtain nutrients, tropic factors, and oxygen from the surrounding native cartilage in the short-term. In fact, radioimmunoassay has demonstrated the progressive remodeling of matrix tissue within the engrafted cartilage [14]. Furthermore, the native tracheal cartilage may supply chondroblasts, the progenitor cells of chondrocytes, via the junction over time, resulting in the gradual remodeling and self-integration of the implanted cartilage.

SEES-2 cells have been utilized in clinical applications, and have documented safety profiles. In addition, chondrocytes obtained from SEES-2 cells can produce a matrix that supports airway structure. Although further studies using large animal models are warranted, these results indicate the potential feasibility of airway reconstruction using hESCs-derived allogeneic cartilage.

5. Conclusions

Cartilage derived from hESCs successfully reconstructed the airway in a rat model, and maintained airway lumen and biocompatibility for up to three months. In addition, the hESCs-derived chondrocytes were able to produce a functional matrix. Furthermore, the lack of granulation in the interface between native tracheal cartilage and implanted cartilage indicates that engineered cartilage may possess adequate mechanical properties and can undergo remodeling to gradually replace site cartilage. Taken together, engineered cartilage tissue has clinical potential for tissue regeneration and similar applications.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Makoto Komura and Hiroko Komura have been affiliated with the social cooperation program of SheepMedical Co., Ltd. since November 1st, 2022. The other authors have no conflicts of interest to declare.