Feasibility of tracheal reconstruction using silicone-stented aortic allografts

Abstract

OBJECTIVES

Tracheal reconstruction post-extensive resection remains an unresolved challenge in thoracic surgery. This study evaluates the use of aortic allografts (AAs) for tracheal replacement and reconstruction in a rat model, aiming to elucidate the underlying mechanisms of tracheal regeneration.

METHODS

AAs from female rats were employed for tracheal reconstruction in 36 male rats, with the replacement exceeding half of the tracheal length. To avert collapse, silicone stents were inserted into the AA lumens. No immunosuppressive therapy was administered. The rats were euthanized biweekly, and the AAs were examined for neovascularization, cartilage formation, respiratory epithelial ingrowth, submucosal gland regeneration and the presence of the Sex-determining region of Y-chromosome (SRY) gene.

RESULTS

All procedures were successfully completed without severe complications. The AA segments were effectively integrated into the tracheal framework, with seamless distinction at suture lines. Histological analysis indicated an initial inflammatory response, followed by the development of squamous and mucociliary epithelia, new cartilage ring formation and gland regeneration. In situ hybridization identified the presence of the SRY gene in newly formed cartilage rings, confirming that regeneration was driven by recipient cells.

CONCLUSIONS

This study demonstrates the feasibility of AAs transforming into functional tracheal conduits, replicating the main structural and functional characteristics of the native trachea. The findings indicate that this approach offers a novel pathway for tissue regeneration and holds potential for treating extensive tracheal injuries.

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INTRODUCTION

Tracheal reconstruction, particularly of the proximal airway involving the trachea and main carina, has transitioned from a high-risk endeavour to a technically intricate yet increasingly feasible surgical intervention. This transformation, initially considered insurmountable prior to the 1960s, owes much to pioneering efforts and groundbreaking studies in both human cadavers and animal models. These foundational studies illuminated the nuances of segmental blood supply to the trachea and established the safe resection length, often up to 5 cm, followed by primary reanastomosis. The contributions of surgical luminaries like Grillo, Pearson and Perelman have been pivotal in the development of methodologies for safe resection under these complex conditions, particularly for diseases rarely encountered in surgical practice [1]. Advancements in radiological imaging and anaesthetic care have further augmented the safety and efficacy of these procedures. The implementation of standardized intraoperative airway management techniques, eliminating the reliance on cardiopulmonary bypass, has significantly reduced the associated surgical risks. Despite this progress, reconstructing the trachea, especially for extensive lesions, remains a formidable challenge, representing one of the most intricate tasks in thoracic surgery [2].

The dilemma of effectively treating patients requiring extensive central airway resections has led to the exploration of a spectrum of tracheal replacement strategies. These include the utilization of autogenous tissues, nonviable materials, foreign materials, transplantation and tissue engineering approaches. However, a majority of these strategies have not yielded successful clinical outcomes, often failing to replicate the complex structural and functional dynamics of the native trachea [3]. The approach of tracheal allotransplantation, while conceptually promising, is fraught with limitations such as the need for ABO compatibility, extended preparation times and the imperative for immunosuppression. These constraints are particularly problematic in oncological patients, rendering the approach less viable [4, 5].

In previous investigations, we explored the use of living autologous aortic conduits in rabbit models to circumvent immune responses. This approach, while innovative, led to the inflammatory destruction of the aortic tissue, followed by its transformation into a structure resembling the trachea’s mucociliary epithelium and cartilage [6]. While this method demonstrated potential, it posed significant clinical risks due to the extraction of an aortic segment from patients. Consequently, our focus shifted to aortic allografts (AAs), with the aim to evaluate their capability for tracheal regeneration without the risks associated with autologous tissue extraction. This approach, however, raised new questions about the potential immunologic implications and the ability to replicate the regenerative phenomena observed in autologous tissues within an allogenic context.

To address these challenges, we formulated a hypothesis exploring the feasibility of tracheal regeneration using AAs. We selected a rat model for its suitability in terms of technical simplicity and relevance to the study. This model was instrumental in evaluating the efficacy of AAs as a tracheal substitute, aiming to elucidate the complex mechanisms underlying tracheal regeneration and to potentially establish AAs as a viable option for tracheal reconstruction in cases where extensive resection is necessary.

MATERIALS AND METHODS

Tracheal replacement with fresh AAs was conducted in male Wistar rats, aged 10–12 weeks. To prevent confusion with previous repair procedures involving conduit contraction and remaining tracheal extension, we performed 8–10 mm tracheal resections instead of the 5 mm used in our preliminary experiments. To facilitate the detection of recipient-derived cells within the grafts, we utilized aortic segments from female rats and transplanted them into male recipients. In our study, we utilized polymerase chain reaction (PCR) technology to specifically target and detect the SRY gene, a sex-determining region on the Y chromosome. This gene’s presence in the newly formed cartilage tissue within the AAs implanted into male recipients, originally harvested from female donors, served as definitive evidence for the participation of recipient-derived cells in the regeneration process. This methodological choice was critical for our investigation, as it provided a clear, molecular-level distinction between donor and recipient cell contributions, enabling us to accurately trace the origin of regenerative cells within the grafts. For the silicone stent, we employed thin-walled PVC tubes, which were critical in maintaining the aortic grafts’ structural integrity. Detailed visualization of these stents and their application is provided in Fig. 2A. The animals received care following China Regulations and Institutional Ethical Committee Guidelines for animal research, and were raised in our institution at the Department of Experimental Research, The First Hospital of Jilin University (No. 2023-0639).

Harvest of the aortic allografts

Eighteen female rats weighing 200–220 g were subjected to left thoracotomy under general anaesthesia to obtain a 3-cm descending thoracic aortic segment. The AAs were divided into 2 1.5-cm segments, which were subsequently placed in phosphate buffer saline solution before being transplanted within 60 min of harvesting. Matching of blood or tissue compatibility between recipient and donor was not attempted.

Anaesthesia, surgery and postoperative management

Recipient rats were fasted for 8 h before being intramuscularly injected with ketamine (1 mg/100g) at 30 min prior to induction. After induction with intravenous propofol (1%; 80 mg/100g) and endotracheal intubation, ventilation was carried out using a Siemens 900 C ventilator (Siemens, Solna, Sweden). In the meanwhile, 60% oxygen inhalation and 2–2.5% isoflurane were applied for maintenance anaesthesia until the surgery was completed. The animals were placed in a supine position and a median incision was made. Then, tracheal artery was ligated, followed by excision of six distal tracheal rings away from 3 to 5 tracheal rings. Freshly collected AAs with silicone stents were then anastomosed with the recipient tracheal ends on the caudal side. To initiate this procedure, 1 row of interrupted 8–0 non-absorbable sutures were placed on posterior membranous and later on anterolateral cartilaginous wall. Afterwards, four 8–0 non-absorbable sutures were put at 0, 3, 6 and 9 points of circular rings (Fig. 1). No immunosuppressive therapy was given. And benzathine penicillin (400 000 U) was administered in every animal through intramuscular injection. Our surgical protocols were approved by Ethics and Animal Use Committee of First Hospital of Jilin University.

Follows-up and evaluation

Pulse oximetry was measured during the whole operation. Daily clinical examinations were conducted until the 7th postoperative day, followed by weekly assessments thereafter. Data on respiration, weight and overall condition were collected. To thoroughly evaluate the regeneration process over time, animals were scheduled for euthanasia at revised intervals of 2 weeks, 1 month, 2 months, 3 months and 6 months postoperation (n = 6 for each time point). This adjustment was based on our observations from preliminary experiments, where significant airway structure regeneration was evident by the 3-month mark. Animals showing respiratory distress (or equivalent) signs prior to the scheduled euthanasia date were given euthanasia for systematic autopsy.

Histological examinations

After sacrifice, the full-length trachea and adjacent tissue were subjected to en bloc resection for macroscopic and microscopic analyses. Upon removal of the tracheal stent and evaluation of the graft macroscopically, post-mortem samples were immediately placed in a 4% paraformaldehyde solution for preservation. The samples were then subjected to paraffin embedding, sectioned at 3-μm thickness and stained with haematoxylin–eosin or toluidine blue before observation under a light microscope.

After fixation and decalcification, the samples were embedded in gelatine. After fixation and decalcification, the samples were embedded in gelatin and stored at –80°C. Ten-micrometre sections were used for fluorescent immunohistochemistry staining. Whole-mount staining was carried out by immersing the entire tissue in 4% paraformaldehyde at room temperature for 60 min. To retrieve antigens, a sodium citrate buffer was used with gentle agitation. For blocking and permeabilization, the tissue was submerged in serum and 10% Triton X-100 for 24 h. Sections were incubated with primary antibody at 4°C overnight and with secondary antibodies at room temperature in the dark for 75 min. Cell nuclei were counterstained with 4',6-diamidino-2-phenylindole (Sigma) for 30 min in the dark. Sections were mounted and dried at 4°C overnight. The tissue was then sandwiched between 2 slides and imaged using confocal microscopy.

Scanning electron microscopy and transmission electron microscopy examination

For scanning electron microscopy examination, the harvested specimens were fixed in 2.5% glutaraldehyde in 0.1 mol/l cacodylate buffer, pH 7.4 for 24 h. They were then postfixed with 2% osmium tetroxide in the same buffer for 2 h. The specimens were dehydrated using graded alcohol and critical point dried over CO₂. To make them conductive, they underwent progressive osmium impregnation in a vacuum evaporator. Finally, they were coated with gold–palladium and examined in a Phillips 505 microscope (XL29, 25 kV, Kassel, Germany).

For transmission electron microscopy examination, the harvested specimens were fixed overnight in a mixture of cold 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH = 7.2) and 2% paraformaldehyde in 0.1 M phosphate or cacodylate buffer (pH = 7.2). They were then embedded with epoxy resin. The samples mixed with epoxy resin were loaded into capsules and polymerized at 38°C for 12 h and 60°C for 48 h. Thin sections were sliced on an ultramicrotome (RMC MT-XL; RMC Products, Tucson, AZ, USA) and collected on a copper grid. Appropriate areas for thin sectioning were cut at 65 nm and stained with saturated 4% uranyl acetate and 4% lead citrate before examination on a transmission electron microscope (JEM-1400; JEOL, Tokyo, Japan) at 80 kV. Two pathologists who were blind to the study read all the results simultaneously.

In situ hybridization

To investigate the newly formed tissue, in particular cartilage, we performed in situ hybridization at 8 weeks post-transplantation. The aortic tissue from female rats was transplanted into male rats to analyse the presence of the Y chromosome through PCR detection of the Sex-determining region of Y-chromosome (SRY) gene, as previously described [6]. Tracheal tissues were collected from both male and female control rats, as well as from male rats 8 weeks following transplantation of female AAs. Cartilage rings in control rats and newly formed cartilage rings in grafts were collected for DNA detection. Total DNA extraction was carried out using the classic chloroform/phenol approach following the specific instructions. The cartilages were then incubated with proteinase K at 56°C overnight.

SRY gene was amplified in every sample on the Perkin-Elmer instrument (Perkin-Elmer, Norwalk, CT). Primers for insulin-like growth factor 1 gene, which was the tracer for DNA isolation and amplification, were used as positive control. The PCR system was 50 l in volume, consisting of respective primers (29 pmol), 10* Taq polymerase buffer, magnesium chloride (0.75 mmol/l), dNTPs (200 mol/l), Taq polymerase (2.5 U) and extracted DNA (5 l, 50 ng/l). The obtained PCR products included 114 and 116 base pairs for SRY and IGF, respectively. Thermal cycling conditions were shown below, 10-min denaturation under 95°C; 30 s under 95°C, 30 s under 53°C, 10 s under 72°C for 40 cycles and 10-min incubation under 72°C. When negative results were obtained for SRY gene amplification, we examined whether insulin-like growth factor 1 gene was correctly amplified. Positive PCR results suggested that the process was efficient, confirming the negative result in detecting SRY gene.

RESULTS

Clinical outcomes and postoperative assessment

In this study, AAs were harvested from 18 female donor rats and transplanted into 36 male recipient Wistar rats. The postoperative period was marked by stable recovery in most of the animals (refer to Fig. 2A–C for detailed observation). Notably, only 1 rat succumbed to anastomotic dehiscence within 48 h post-surgery. This isolated incident did not reflect any systemic flaws in the surgical procedure, as subsequent post-mortem evaluations showed no structural abnormalities or complications attributable to the tracheal reconstruction itself. The strategic implementation of non-absorbable sutures played a pivotal role in securing the silicone stents within the grafts. This approach effectively prevented graft collapse (malacia), ensuring long-term stability of the tracheal substitute. Nevertheless, it is important to note that this technique carried a minor risk of partial stent displacement, particularly if the sutures excessively penetrated or weakened the native tracheal cartilage. Throughout the extensive six-month follow-up period, clinical observations remained consistently positive. There were no signs of complications typically associated with tracheal reconstructions, such as anastomotic leakage, luminal stenosis or graft dehiscence. These findings affirm the structural integrity and functional adequacy of the AAs as tracheal replacements.

For a comprehensive understanding of the grafts’ integration and performance, scheduled euthanasia and systematic evaluations were conducted. These assessments, performed at pre-designated intervals, focused on histological examinations to elucidate the progressive adaptation and transformation of the grafts within the tracheal environment. Notably, all sacrificed animals were in a stable and healthy condition, underscoring the success and reliability of the tracheal reconstruction methodology employed in this study.

Pathological results and dynamic regeneration process

The chronological examination of the AAs post-tracheal reconstruction offers a comprehensive view of the airway regeneration process. Initially, within the 1st month, macroscopic evaluation demonstrated effective revascularization in the grafts, crucial for tissue viability and integration (Fig. 2D). By the 3rd month postoperation, the integration of the graft with the native trachea was remarkably seamless, evidenced by the indistinguishable anastomosis, indicating the graft’s progressive adaptation to the host environment (Fig. 2E). This integration was further advanced by the 6th month, where the grafts displayed structurally and functionally mature cartilaginous rings, akin to those in the native trachea (Fig. 2F).

Histologically, the transformation was marked by a series of regenerative phases. Initially, HE staining at 1 month postoperation identified sporadic squamous epithelial cells in the graft lumen. By the 2nd month, this pattern had progressed to the formation of a respiratory ciliated epithelium along with the emergence of submucosal glands (Fig. 3C). This phase marked the onset of a functional airway lining. By the six-month mark, the grafts had developed a complete respiratory epithelium, closely resembling the native tracheal histology. Toluidine blue staining complemented these findings by illustrating the early appearance of cartilaginous islands at the one-month mark (Fig. 3E). By 6 months, these islands had evolved into well-formed cartilage rings, similar to the native tracheal structure (Fig. 3F). This histological progression was supported by advanced imaging techniques. scanning electron microscopy and transmission electron microscopy analyses confirmed the gradual regeneration of key tracheal components, including epithelial lining, cartilage formation and submucosal gland development (Fig. 4). This meticulous documentation of the regenerative process underscores the potential of AAs as robust scaffolds for airway reconstruction.

In sum, these results paint a detailed picture of the regenerative timeline within AAs, emphasizing their capacity for remodelling into functional airway structures. This transition from an inflammatory response to the development of a fully differentiated tracheal anatomy over 6 months highlights the AAs’ potential in reconstructing complex airway tissues, providing a promising approach for extensive tracheal injuries.

Immunohistochemical and in situ hybridization insights

Initial CD68 immunostaining, indicative of macrophage activity, revealed a minimal presence on the 1st and 3rd postoperative days (Fig. 5Aa and b), signifying the onset of an innate immune response. This activity escalated to a peak at 1 week (Fig. 5Ac), reflecting the critical inflammatory phase necessary for initial wound healing and graft integration, and persisted through the 2nd week (Fig. 5Ad). This pattern of macrophage infiltration is consistent with the early stages of graft acceptance and incorporation, as macrophages play a vital role in both immune response modulation and tissue remodelling.

The gradual increase in CD31 expression, a marker of neovascularization, from nearly undetectable at 1 week to pronounced at 2 weeks (Fig. 5Ba and b), culminated in extensive microvascular formation at 1 month (Fig. 5Bc). By 3 months, revascularization was robust throughout the graft surface and matrix (Fig. 5Bd). This progression underscores the crucial establishment of a new vascular network within the graft, essential for its long-term viability and function. Revascularization is a key component in tissue engineering, facilitating the integration of the graft with the host tissue, as it ensures the supply of essential nutrients and oxygen for cellular activities and tissue maintenance.

The evolution of α-tubulin expression, specific to ciliated respiratory epithelium, further highlighted the maturation of the airway lining. The gradual increase from faint positivity at 1 month post-transplantation (Fig. 5Ca) to strong positivity at 6 months (Fig. 5Cc) illustrated the successful regeneration and functional establishment of a ciliated respiratory epithelium within the graft. This development is indicative of the graft’s transformation into a structure closely resembling native tracheal tissue, both morphologically and functionally.

Molecular insights were further enhanced by in situ hybridization for the SRY gene, revealing the involvement of recipient-derived cells in the formation of new cartilage structures within the grafts. This finding, evidenced by the amplification of the SRY gene in male recipients’ AAs at 8 weeks (Fig. 5D), provides compelling evidence of the chimeric nature of the regenerated tissue. It suggests that the graft environment may facilitate the recruitment and differentiation of recipient stem cells, contributing to the formation of cartilage and other tracheal components. This phenomenon aligns with recent advancements in tissue engineering and regenerative medicine, which emphasize the role of the host microenvironment in guiding stem cell differentiation and tissue regeneration.

DISCUSSION

The evolution of tracheal reconstruction techniques, particularly for extensive lesions, has been a complex journey in thoracic surgery. The shift from the restrictive ‘2-cm Belsey rule’, which posited the impossibility of primary reconstruction for resections exceeding 2 cm, to the current state of advanced tracheal surgery, is a testament to the field’s progress [7]. Pioneers like Hermes C. Grillo and others have played a pivotal role in overcoming challenges related to perioperative ventilation and cartilage healing [1]. Today, surgeons can safely resect significant portions of the trachea and apply primary anastomosis, extending the scope of surgery to include complex laryngotracheal and carinal reconstructions. However, extensive tracheal lesions remain a high-risk domain, often relegating patients to limited palliative care options [8, 9].

In this context, our study introduces a novel approach using silicone-stented AAs for tracheal reconstruction, marking a significant advancement in addressing the need for reliable tracheal substitutes. The seamless integration of AAs into the host trachea, replicating both structural and functional aspects of the native tissue, is a breakthrough. This study builds upon the foundational work in tracheal reconstruction using AAs, acknowledging the seminal efforts by Martinod et al. While not the 1st, our research further elucidates the regenerative capacity of AAs, contributing to the evolving methodologies in tracheal reconstruction.

In our experimental procedures, both silk and absorbable sutures were employed for anastomosing the AAs to the recipient trachea. Throughout the follow-up period, preliminary observations suggested no significant difference in the performance of these suture materials regarding the critical outcomes of graft integration and healing. However, it is pertinent to mention that this study was not designed as a randomized controlled trial to systematically compare the efficacy of silk versus absorbable sutures in tracheal reconstruction. As such, our findings regarding suture material should be interpreted with caution, and further research is warranted to conclusively determine the optimal suture choice for this application.

In our observations, the presence of necrotic areas within the AAs was noted, highlighting the critical challenge of maintaining graft viability throughout the transplantation process. This finding prompts a closer examination of factors that may contribute to tissue necrosis, including the timing of graft harvesting and implantation, preservation methods and the host’s immune response. It also underscores the importance of meticulous graft preparation and postoperative monitoring to identify and address signs of compromised graft viability early. Future research should aim to refine these aspects of the transplantation protocol to reduce the incidence of necrosis and improve the outcomes of tracheal reconstruction using AAs.

A critical aspect of our study is the demonstration of the reduced immunogenic response of AAs. This property is particularly advantageous compared to other graft materials that necessitate heavy immunosuppression, a factor that limits their use, especially in cancer patients. The favourable immunological profile of AAs makes them suitable for a broader patient demographic, including those with malignancies where immunosuppressive therapy is contraindicated [10]. When contrasted with other tracheal substitutes like prostheses or decellularized grafts, AAs present several unique benefits. The biological scaffold of AAs naturally aligns with the biomechanical and histological properties of the trachea, a feature not fully replicated in synthetic or decellularized materials. Additionally, AAs support vascularization and integration with host cells without the need for extensive external bioengineering, giving them an edge over other grafting techniques [10, 11].

The regenerative process within AAs, including the development of ciliated epithelium and cartilage formation, indicates a potential role for basal stem cells in airway regeneration. This finding resonates with the growing understanding of stem cell-mediated repair mechanisms in tissue engineering and regenerative medicine, suggesting the graft environment may foster the recruitment and differentiation of recipient stem cells into tracheal tissues [12].

Our study’s findings on AAs for tracheal reconstruction enhance our comprehension of tissue engineering and regenerative medicine, suggesting a potential shift in therapeutic strategies for complex tissue reconstructions. AAs offer a natural, less invasive and potentially more effective alternative compared to existing methods. Moreover, the study emphasizes the importance of understanding graft material immunogenicity, particularly for oncological patients.

Our study’s findings underscore the potential of silicone-stented AAs in the reconstruction of extensive tracheal injuries. Looking forward, the application in human medicine, especially using cadaveric donors, presents a promising frontier. The viability of harvested AAs from cadaveric donors hinges on timely procurement post-mortem, ideally within a few hours to minimize cellular degradation and maintain structural integrity. Preserving these allografts requires meticulous methods, such as cryopreservation or perfusion with preservation solutions, to ensure their functional and structural preservation until transplantation. Future research is essential to establish the long-term efficacy and safety of AAs in tracheal reconstruction. Expanding to larger animal models and eventually human clinical trials will be crucial in transitioning these laboratory findings into clinical applications. Further exploration into the role of stem cells within the graft environment could unveil novel methods to optimize tissue regeneration, leading to revolutionary therapeutic interventions not only in tracheal surgery but potentially across various fields of regenerative medicine. This could significantly enhance patient outcomes, offering new hope and possibilities in the treatment of complex tracheal injuries and other challenging medical conditions.

Limitations

This study, utilizing a rat model for tracheal regeneration, may have limited applicability to larger animals or humans, highlighting a need for broader research. The lack of advanced bronchoscopic techniques for extended monitoring in rats is another limitation. Additionally, the exact role of recipient mesenchymal stem cells in graft regeneration remains undefined. Further investigation through comprehensive clinical trials is essential to validate the efficacy and safety of this approach for human tracheal reconstruction.

While our study offers promising insights, limitations exist, particularly regarding the direct translation of findings from a rat model to human applications. Future research should focus on the optimization of allograft preservation techniques, the window for post-mortem harvesting that ensures graft viability and the immunological considerations unique to human recipients. Additionally, exploring the ethical and logistical frameworks for utilizing cadaveric donors in tracheal transplantation will be crucial. Investigating these areas will pave the way for clinical trials, bringing us closer to realizing the potential of AAs in addressing the complex challenge of extensive tracheal injuries in humans.

CONCLUSION

This study introduces silicone-stented AAs as a novel approach for tracheal reconstruction in a rat model, representing a significant advancement in thoracic surgery. The successful integration of AAs into the tracheal structure without the need for immunosuppressive therapy demonstrates their potential as viable substitutes for extensive tracheal lesions. Histological findings indicate the ability of AAs to mimic native tracheal tissue, showing regeneration of crucial components such as ciliated epithelium, cartilage rings and submucosal glands. This process is likely facilitated by the recruitment and differentiation of recipient cells within the graft environment, underscoring the role of the host microenvironment in tissue regeneration. The findings from this research offer a promising direction for future studies in regenerative medicine and tissue engineering, potentially leading to innovative treatments for complex tracheal injuries and other challenging medical conditions.

FUNDING

Natural Science Foundation of Jilin Province (YDZJ202301ZYTS456); Education Department of Jilin Province (JJKH20231207KJ); Youth Development Fund of the First Hospital of Jilin University (04046910001).

Conflict of interest: none declared.

DATA AVAILABILITY

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Author contributions

Shixiong Wei: Data curation; Writing—original draft. Yiyuan Zhang: Data curation; Formal analysis; Investigation. Feixiang Luo: Methodology; Software; Supervision; Validation. Kexing Duan: Project administration; Resources; Software; Supervision; Visualization. Mingqian Li: Conceptualization; Formal analysis; Methodology; Writing—review and editing. Guoyue Lv: Conceptualization; Writing—review and editing.

Reviewer information

European Journal of Cardio-Thoracic Surgery thanks Mohamed Rahouma, Luca Voltolini and the other anonymous reviewers for their contribution to the peer review process of this article.

REFERENCES

ABBREVIATIONS

ABBREVIATIONS
 
• AAs

  Aortic allografts

 
• PCR

  Polymerase chain reaction

Author notes