Development of a 3D-printed synthetic tracheal scaffold for partial tracheal wall reconstruction: an experimental animal study

Abstract

Background

Tracheal reconstruction remains a significant clinical challenge due to the difficulty of maintaining airway patency and promoting epithelial regeneration following tracheal injury or resection. Recent advances in tissue engineering have explored 3D-printed scaffolds as potential solutions; however, limitations remain regarding scaffold biocompatibility, mechanical stability, and integration with host tissue. This study evaluated the feasibility of a non-biodegradable polyurethane–polyvinyl chloride (PU-PVC) composite scaffold for partial tracheal wall reconstruction in a rabbit model.

Methods

Nine New Zealand white rabbits underwent anterior tracheal wall reconstruction using a customized half-pipe PU-PVC scaffold. Scaffold mechanical properties were characterized before implantation. Animals were monitored for 30 days, after which bronchoscopic evaluation was performed to assess luminal patency. Histological analysis was conducted to evaluate epithelialization and inflammatory response. Explanted scaffolds were also assessed for structural integrity and mechanical stability.

Results

The 30-day survival rate was 88.9% (8/9; 95% CI 51.8%–99.7%), with one death occurring on postoperative day 14. Bronchoscopic examination demonstrated complete luminal patency in all surviving animals, with no evidence of stenosis, obstruction, or mucus plugging. Histological findings demonstrated variable epithelialization, with 50% (4/8) of rabbits showing favorable epithelial coverage accompanied by mild inflammation, while the remaining animals exhibited partial or minimal epithelialization associated with greater inflammatory responses. Post-explant evaluation demonstrated preservation of scaffold structure and mechanical properties, with only minor changes in tensile strength and porosity after implantation.

Conclusion

In this preliminary single-cohort feasibility study, the PU-PVC composite scaffold-maintained airway patency throughout the 30-day observation period and demonstrated acceptable short-term mechanical stability following partial tracheal wall reconstruction in a rabbit model. Although epithelialization outcomes were variable and no comparator group was included, the findings support further preclinical investigation of this scaffold platform; however, long-term safety, durability, and biological integration remain to be established. Future studies incorporating appropriate control groups, longer follow-up periods, quantitative assessments of airway patency, and bioactive modifications such as cell seeding or growth factors are warranted to enhance tissue regeneration and better define the scaffold’s translational potential.

Highlights

Innovative 3D-printed PU/PVC scaffold for long-segment tracheal reconstruction.

Maintained luminal patency with high survival rate in a preclinical rabbit model.

Comprehensive assessment including mechanical testing, bronchoscopy, and histology.

Use of locally fabricated, cost-effective materials for potential translational application.

Introduction

Tracheal reconstruction is one of the greatest challenges in thoracic surgery when direct end-to-end anastomosis is impossible or after this procedure has failed. The main indications for tracheal reconstruction include malignant tumors (squamous cell carcinoma, adenoid cystic carcinoma), tracheoesophageal fistula, trauma, unsuccessful surgical results for benign diseases and congenital stenosis [1].

Tracheal defects pose a significant clinical challenge due to the lack of effective substitutes for surgical reconstruction. These defects can arise from benign conditions, such as congenital malformations, trauma, infections, or malignant diseases. While surgical resection with end-to-end anastomosis is the current gold standard treatment, it is only feasible when the excised segment does not exceed half the tracheal length in adults or one-third in children [2]. But even when technically feasible, primary anastomosis is fraught with significant challenges that can compromise long-term outcomes. The trachea’s segmental blood supply renders the anastomotic site highly susceptible to ischemia and dehiscence. Furthermore, the inherent rigidity and limited mobility of the trachea impose severe constraints on the maximum length of resection without creating excessive tension at the suture line. Post-operatively, the healing process itself often becomes a source of complication; chronic inflammation and the formation of granulation tissue in response to sutures can lead to restenosis, while the disruption of the mucociliary epithelium results in impaired mucus clearance and recurrent respiratory infections. These persistent limitations underscore the critical need for a reliable alternative for tracheal reconstruction that can circumvent the pitfalls of conventional anastomosis.” Some of the traditional approaches for tracheal defects, including pericardial patches, autografts, and slide tracheoplasty, often fall short or are impractical [3, 4]. Alternatives like stents or T-tubes are associated with complications and reduced quality of life. Other options, such as autografts, allografts, or prosthetics, face limitations like inflammation, infection, and mismatched physiological properties [5]. Given these challenges, an ideal tracheal substitute must closely replicate the native trachea’s complex anatomy and function.

The trachea’s complex anatomy requires a scaffold that replicates its mechanical and biological functions. The adult human trachea averages 11.8 cm in length (range, 10–13 cm) and features cartilaginous rings for structural support and a pseudo -stratified columnar epithelium for mucociliary clearance. A successful scaffold must maintain luminal patency, resist collapse, and support epithelialization while being biocompatible [6].

Researchers have done their best in the last decades to find the best way to reconstruct the trachea. Each of the options had its limitations. Autologous autografts have been clinically applied for small airway defects; however, it is not applicable for large tracheal defects. Cadaveric allografts have also been used, but it has the limitation of a shortage of donors. There are also a high risk of rejection and a need for lifelong immunosuppression. It also may have a high rate of major complications and uncertainty regarding its long-term effects. A modified form of allograft, which is performed by decellularizing the cadaveric trachea, has solved some of these drawbacks, but the processing is more complicated and time-consuming [2].

Recent advances in tissue engineering and additive manufacturing have accelerated the development of patient-specific tracheal substitutes [7–9]. Over the past several years, numerous studies have investigated 3D-printed biodegradable scaffolds composed of materials such as polycaprolactone (PCL), polylactic acid (PLA), and their composites [10, 11]. These scaffolds can be fabricated with customizable architectures and pore structures that promote cellular infiltration and tissue regeneration [12, 13]. In parallel, biomimetic strategies incorporating extracellular matrix components, stem cells, epithelial cells, chondrocytes, and growth factors have been explored to enhance epithelialization and improve functional integration with host tissue [14, 15]. Despite encouraging preclinical results, many biodegradable scaffolds remain susceptible to complications related to insufficient mechanical strength, uncontrolled degradation, granulation tissue formation, airway stenosis, and incomplete mucosal regeneration [16, 17]. Consequently, maintaining a balance between structural stability and biological integration remains a major challenge in tracheal tissue engineering.

Given these limitations, alternative scaffold designs with enhanced mechanical durability continue to be investigated. In the present study, we evaluated a non-biodegradable polyurethane–polyvinyl chloride (PU-PVC) composite scaffold fabricated using 3D-printing technology. Unlike biodegradable constructs that gradually lose mechanical integrity during degradation, this scaffold was designed to provide structural support during the healing process. The present study aimed to generate preliminary feasibility data regarding its short-term in vivo performance. The objective of this preliminary feasibility study was to assess the short-term mechanical performance, airway patency, and histological response of the scaffold in a rabbit model of partial tracheal wall reconstruction.

Synthetic scaffolds have also been being explored, such as those made from polycaprolactone (PCL) or polycaprolactone-polyurethane (PCL-PU), to overcome these challenges [10, 18–20]. PU, known for its flexibility and biocompatibility, is widely used in tissue engineering, while polyvinyl chloride (PVC) offers rigidity and durability [21–23]. This study aimed to develop and evaluate a synthetic PU-PVC scaffold for partial tracheal wall reconstruction in a rabbit model. Such a synthetic scaffold must resist luminal collapse yet be flexible, have a sufficient degree of porosity to permit vascular and cellular ingrowth, but also be liquid and airtight and must facilitate the growth of functional airway epithelium to avoid infection and aid in mucociliary clearance. Finally, the scaffold must also be biocompatible to avoid implant rejection [24].

Materials and methods

Materials and scaffold fabrication

The scaffolds were fabricated from a non-biodegradable polyurethane (PU) and polyvinyl chloride (PVC) composite. Polyurethane was selected because of its flexibility, elasticity, and mechanical strength, while PVC was incorporated to enhance structural stability and durability. The combination was intended to provide sufficient flexibility while maintaining resistance to airway collapse.

The scaffolds were designed as cylindrical tubes with an inner diameter of 8 mm and a length of 15 mm to match the anatomical dimensions of the rabbit trachea. Each scaffold was longitudinally sectioned to create a half-pipe configuration corresponding to approximately one-half of the tracheal circumference (Fig. 1). The resulting scaffold geometry was designed to match the dimensions of the anterior tracheal wall defect. To facilitate implantation, each scaffold was longitudinally sectioned to create a half-pipe patch suitable for anterior tracheal wall reconstruction. The scaffold dimensions (8 mm inner diameter and 15 mm length) were selected to approximate the dimensions of the adult New Zealand white rabbit trachea. A standardized anterior tracheal wall defect measuring 15 mm in longitudinal length and extending across the anterior half of the tracheal circumference was designed to create a partial tracheal wall reconstruction model involving approximately four tracheal rings while maintaining airway continuity and animal survival.

Fig. 1.

Schematic representation of the tracheal defect and scaffold implantation geometry. (A) Native rabbit trachea shown in cross-section and longitudinal view. (B) Creation of a standardized anterior tracheal wall defect measuring 15 mm in length and extending across approximately one-half of the tracheal circumference (180°). (C) Dimensionally matched half-pipe PU-PVC scaffold (8-mm internal diameter, 15-mm length). (D) Reconstruction of the anterior tracheal wall by suturing the scaffold to the defect margins using interrupted 5 − 0 Prolene sutures

3D Printing process and sterilization

The scaffolds were fabricated from a polyurethane (PU) and polyvinyl chloride (PVC) composite at a PU/PVC ratio of 70:30 (w/w). PU and PVC were dissolved in a 1:1 dimethylformamide (DMF)/tetrahydrofuran (THF) solvent system at a polymer concentration of 18–22 wt%. The mixture was magnetically stirred for 12 h at 40 °C until a homogeneous solution was obtained.

Scaffolds were manufactured using an extrusion-based fused deposition modeling (FDM) 3D printer (MobioTech, Iran). The printing temperature was maintained at 180 °C (processing range 175–185 °C) using a 200-µm nozzle. Printing was performed at a speed of 10 mm/s with an extrusion flow rate of 1.2 mm³/s. The layer thickness was set at 200 μm and filament spacing at 400–500 μm using a 0°/90° grid lattice infill pattern with an infill density of 50%.

The resulting scaffolds exhibited an average pore size of 250–400 μm and a porosity of approximately 60 ± 5%. Printing was performed with a bed temperature of 40–50 °C under ambient conditions (20–25 °C; relative humidity 40–50%).

Following fabrication, scaffolds were dried under vacuum at 40 °C for 24 h and subsequently cured at 50 °C for 12 h to facilitate solvent removal and scaffold stabilization prior to sterilization and implantation (Fig. 2).

Fig. 2.

Workflow for 3D-Printed Tracheal Scaffold Preparation and Transplantation in Rabbits. This figure illustrates the complete workflow of tracheal scaffold fabrication and transplantation. Filament preparation shows PVC and PU combined using hot melt extrusion to produce the material for printing. The trachea design panel demonstrates the digital CAD modeling of the scaffold, specifying its shape and dimensions. Printing the trachea panel depicts the 3D printing process of the scaffold prior to implantation. The printed trachea image shows the final scaffold (8 mm diameter × 15 mm length) ready for transplant. Finally, the artificial trachea transplant panel shows placement of the scaffold in the rabbit’s tracheal defect, demonstrating the surgical implantation step

Mechanical testing and porosity analysis

The tensile properties of the PU-PVC scaffold material were evaluated using a universal testing machine (Instron 5960) according to ASTM Standard D638. Scaffold samples (n = 5) were cut into dog-bone shapes and stretched at a constant rate of (5 mm/min) until failure. The ultimate tensile strength (UTS), elastic modulus (Young’s modulus), and elongation at break were calculated from the resulting stress-strain curves.

The porosity of the 3D-printed scaffolds was determined using the liquid displacement. Briefly, a known volume (V1) of scaffold was immersed in a graduated cylinder containing a known volume (V2) of ethanol. The total volume after the scaffold was fully saturated and the air bubbles were removed was recorded as V3. The porosity (%) was calculated using the formula:

Animals and experimental groups

Nine female, 3-month-old New Zealand white rabbits, weighing between 2.5 and 3 kg, were utilized in this study. The animals were obtained from the Experimental Medicine Center of SUMS and housed in the university’s specialized post-operative care unit. All rabbits were assigned to a single experimental group and underwent the identical surgical procedure of anterior tracheal wall reconstruction using the 3D-printed scaffold. A control or sham group was not included in this preliminary feasibility study. All experimental procedures were approved by the Animal Care and Experiment Committee of Shiraz University School of Medicine under license ID IR.SUMS.AEC.1401.016.

Surgical procedure of tracheal reconstruction

General anesthesia was induced via intramuscular injection of ketamine (25 mg/kg) and xylazine (5 mg/kg). A single prophylactic dose of ampicillin was administered 15 min prior to the incision. Oxygen was supplied via a mask throughout the procedure. Following a midline neck incision, the skin, subcutaneous tissue, and strap muscles were dissected to expose the tracheal rings. A 15-mm-long anterior tracheal wall defect encompassing approximately four tracheal rings and extending across the anterior half of the tracheal circumference was created approximately 1 cm distal to the thyroid cartilage. The dimensions of the defect were matched to those of the implanted half-pipe scaffold (8-mm internal diameter, 15-mm length). The half-pipe scaffold patch was sutured to the edges of the tracheal defect using interrupted 5 − 0 Prolene sutures (Fig. 3).

Fig. 3.

Surgical Procedure for Tracheal Reconstruction in Rabbits. This figure shows the key steps of tracheal reconstruction using the PU-PVC scaffold in rabbits. Panel A shows the anesthetized rabbit prepared with an endotracheal tube. Panel B shows the trachea exposed and the scaffold positioned at the defect. Panel C illustrates the scaffold being sutured for stability. Panel D shows the completion of suturing, securing the scaffold in place

Postoperative care and monitoring

After the surgery, the rabbits were monitored for 2 h before being transferred to the recovery unit. Postoperative analgesia (meloxicam) and antibiotic (ampicillin) were administered for 3 consecutive days. The general condition and respiratory status of the animals were assessed daily.

Endpoint evaluation and histological analysis

After a 30-day period, the surviving rabbits underwent bronchoscopy examination using a 6-mm probe to assess luminal patency. Subsequently, the animals were euthanized in a CO2 chamber. The reconstructed tracheal segments were carefully excised, fixed in 10% formalin, and processed for histological analysis. The samples were embedded in paraffin, sectioned into 10-µm slices, and stained with Hematoxylin and Eosin (H&E). A qualified pathologist, blinded to the study groups, examined the sections under a light microscope to evaluate parameters of epithelialization, inflammation, and tissue integration.

Epithelialization was assessed semi-quantitatively using a three-grade system: Grade 1 (favorable), epithelial coverage of more than 50% of the reconstructed luminal surface; Grade 2 (unfavorable), epithelial coverage of less than 50%; and Grade 3 (scant), minimal or absent epithelial coverage. Inflammatory response was evaluated semi-quantitatively based on the density and distribution of inflammatory cell infiltrates and categorized as mild, moderate, or severe.

Post-implantation scaffold retrieval and mechanical evaluation

Following euthanasia, the reconstructed tracheal segments were carefully excised en bloc. Surrounding soft tissues were gently dissected under direct visualization to expose the implanted scaffold. Adherent tissue was removed using microsurgical instruments while preserving the structural integrity of the scaffold. The retrieved scaffolds were rinsed with sterile saline to remove residual biological debris. Because the dimensions and geometry of the explanted half-pipe scaffolds did not permit preparation of standard ASTM D638 dog-bone specimens, post-implantation mechanical testing was performed using a modified protocol designed to evaluate relative changes in scaffold properties after implantation. The explanted scaffolds were subjected to tensile and compression testing to assess structural integrity and compare their mechanical performance with pre-implantation measurements.

Post-implantation tensile and compressive testing were performed using the same universal testing machine and testing parameters described for the pre-implantation scaffolds. Mechanical properties, including ultimate tensile strength, elastic modulus, elongation at break, and compressive strength, were recorded and compared descriptively with baseline values. Scaffold porosity and density were also reassessed after explantation using the same methods applied before implantation.

Statistical analysis

Given the exploratory nature of this preliminary feasibility study and the absence of a control or sham group, analyses were limited to descriptive statistics. Continuous variables, including scaffold mechanical properties, were summarized as mean ± standard deviation (SD). Categorical outcomes, including luminal patency and epithelialization grades, were reported as frequencies and percentages.

No formal hypothesis testing or between-group statistical comparisons were performed. Data visualization was performed using SigmaPlot (version 10; Systat Software, Richmond, CA, USA) and R software (version 4.6.0; R Core Team, 2026, Windows x86_64).

Results

Survival and postoperative observations

In this research, a total of nine female New Zealand white rabbits, aged 3 months and weighing between 2.5 and 3 kg, were utilized as the experimental animal model. The 30-day survival rate was found to be 88.9% (8/9 animals; 95% Clopper–Pearson confidence interval: 51.8%–99.7%). A single rabbit succumbed on postoperative day 14, displaying symptoms of subcutaneous emphysema in the neck three days prior to death. A post-mortem examination revealed subcutaneous emphysema and purulent discharge surrounding the reconstructed trachea. However, because microbiological cultures, histopathological evaluation, and formal assessment of airway integrity were not performed, the precise cause of death could not be determined. Potential contributing factors include infection, anastomotic or suture-line failure, or a combination of these processes. The remaining eight rabbits were successfully monitored until the end of the study period.

Mechanical characterization of the scaffold

The PU/PVC composite scaffold exhibited excellent mechanical properties prior to implantation, including high tensile strength (25 ± 3 MPa), flexibility (Elongation at break: 350 ± 50%), pore size ranging from 250 to 400 μm and appropriate compressive strength (800 ± 100 kPa) to withstand respiratory pressures. After 30 days of implantation, the scaffold retained most of its mechanical integrity, with a slight reduction in tensile strength (23 ± 4 MPa) and elasticity, which can be attributed to tissue integration and biological remodelling. The porosity decreased from 60 ± 5% before implantation to 55 ± 8% after explantation, which may reflect cellular infiltration and extracellular matrix deposition. These results confirm the suitability of the scaffold for tracheal reconstruction applications. The key results are comprehensively summarized in (Table 1).

Table 1.

Mechanical and physical properties of the PU-PVC scaffold before and after 30 days of implantation. This table summarizes the mechanical and physical properties of the PU-PVC scaffold before implantation and after 30 days in vivo. It includes tensile strength, elasticity (elongation at break), compressive strength, density, porosity, and hardness

Mechanical Property	Before Implantation	After 30-Day Implantation	Test Standard
Tensile Strength (MPa)	25 ± 3	23 ± 4	ASTM D638
Young’s Modulus (MPa)	150 ± 20	145 ± 25	ASTM D638
Elongation at Break (%)	350 ± 50	320 ± 60	ASTM D638
Compressive Strength (kPa)	800 ± 100	750 ± 120	ASTM D695
Density (g/cm³)	1.20 ± 0.05	1.21 ± 0.07	ASTM D792
Porosity (%)	60 ± 5	55 ± 8	Liquid displacement
Hardness (Shore A)	85 ± 5	82 ± 6	ASTM D2240

Summary of Results: The scaffold maintained high tensile strength and compressive strength after 30 days with only slight reductions. Elasticity remained high, density was stable, and porosity slightly decreased, indicating tissue integration. Overall, mechanical integrity was preserved

Terminology and Units:

- MPa: Megapascal - unit of tensile stress

- kPa: Kilopascal - unit of compressive stress

- %: Percentage - used for elongation at break and porosity

- Shore A: Hardness measured on Shore A scale

- N/A: Not Applicable - data not available

- Grade 1 / 2 / 3: Categorization for luminal patency or epithelialization

- Favorable / Unfavorable / Scant: Degree of epithelial coverage

- Mild / Moderate / Severe: Degree of inflammation observed

Prior to implantation, the scaffolds demonstrated mechanical characteristics highly suitable for tracheal reconstruction. The high ultimate tensile strength (25 ± 3 MPa) and compressive strength (800 ± 100 kPa) indicated a robust structure capable of resisting the mechanical stresses of the tracheal environment, including coughing and respiratory pressures. Concurrently, the very high elongation at break (350 ± 50%) revealed superior elasticity and flexibility, which are critical for accommodating the dynamic movements of the trachea without fracturing. The elastic modulus (150 ± 20 MPa) suggested a material with a balanced stiffness, designed to provide support without causing irritation through a mismatch in compliance with the native tissue.

Following the 30-day implantation period, the explanted scaffolds were re-evaluated. A slight yet observable change in mechanical properties was recorded, indicating an interaction between the scaffold and the biological environment. The ultimate tensile strength saw a minimal reduction to 23 ± 4 MPa, while the elastic modulus decreased to 145 ± 25 MPa. This marginal decline suggests a slight softening of the material, potentially due to water absorption or initial superficial degradation, which did not, however, compromise the scaffold’s structural integrity or primary function. Notably, the scaffold largely retained its exceptional flexibility, with the elongation at break measured at 320 ± 60%. Similarly, the compressive strength experienced a slight decrease to 750 ± 120 kPa.

Critically, the density showed no significant change (1.21 ± 0.07 g/cm³) providing strong evidence that no substantial swelling, mass loss or structural comprise occurred. This stability is paramount for ensuring the scaffold s predictable performance and long –term safety in vivo.

Sterilization procedure

Following fabrication, the scaffolds were sterilized using a combined protocol of autoclaving and ethylene oxide (EtO) gas sterilization prior to implantation. Autoclaving was applied as an initial sterilization step, followed by EtO gas sterilization to ensure adequate sterilization of heat-sensitive components and to achieve comprehensive microbial elimination. After EtO processing, the scaffolds were aerated according to the manufacturer’s recommendations before implantation. The potential effects of the combined sterilization methods on scaffold mechanical properties, surface characteristics, residual sterilant levels, and biological performance were not independently evaluated and therefore represent a limitation of the present study.

Bronchoscopic examination and luminal patency

Luminal patency was the first parameter assessed, graded as follows:

• Grade 1: 100% patent.

• Grade 2: More than 50% patent.

• Grade 3: Less than 50% patent.

No tracheal stenosis or obstruction within the tracheal lumen in any rabbits was observed. Furthermore, no mucous plugs or purulent discharge were observed in the trachea, suggesting that the airways remained clear and patent throughout the study period consistent with 100% or Grade 1 patency (Fig. 4).

Fig. 4.

Bronchoscopic Evaluation of Tracheal Lumen. The bronchoscopic image demonstrates the tracheal lumen is patent, showing no evidence of stenosis or obstruction, confirming maintained airway following scaffold implantation and indicating successful initial airway reconstruction

Histologic examination

The second variable assessed was epithelialization, which was categorized into three grades mentioned earlier. Gross and microscopic examination revealed the following distribution of epithelialization (Fig. 5). 50% of rabbits (4/8) showed favorable epithelialization with low inflammation (Grade 1). 25% of rabbits (2/8) exhibited unfavorable epithelialization with moderate inflammation (Grade 2). 25% of rabbits (2/8) showed scant epithelialization with severe inflammation (Grade 3).

Fig. 5.

Histologic Evaluation of Tracheal Wall After Scaffold Implantation. Panel A shows a normal tracheal cross-section with intact ciliary epithelium (arrowhead) and C-shaped cartilage (black arrow), representing healthy tissue architecture. Panel B illustrates the scaffold (white arrow) covered by regenerated ciliary epithelium (arrowhead) along with mild to moderate inflammation (star), indicating partial tissue healing and integration of the scaffold. Panel C demonstrates the scaffold (white arrow) without epithelial coverage and with moderate to severe inflammation (star), reflecting incomplete epithelial regeneration and potential challenges in scaffold integration. This figure provides a comprehensive view of the histologic outcomes of scaffold implantation, highlighting differences between normal tissue and varying degrees of regenerative success and inflammatory response

Inflammation and tissue integration

In the cases of favorable epithelialization, there was mild to moderate inflammation, suggesting that the new tissue was integrating well with the native trachea and the scaffold. In contrast, the rabbits with unfavorable epithelialization and scant epithelialization exhibited moderate to severe inflammation, likely indicating an incomplete healing process and possible challenges with scaffold integration. The degree of inflammation may have been influenced by factors such as infection control measures and the scaffold’s ability to support tissue growth and epithelialization.

Summary of key findings

Survival Rate: 88.9% at 30 days.

Bronchoscopic Findings: No stenosis, obstruction, mucous plugs, or pus in any of the reconstructed tracheas.

Epithelialization: 50% favorable epithelialization (more than 50% coverage).

25% moderate epithelialization (less than 50% coverage).

25% scant epithelialization (minimal or no coverage).

Inflammation: Varying degrees of inflammation observed, with lower inflammation associated with favorable epithelialization. The key results summarized in.

Discussion

Tracheal reconstruction represents a challenging clinical endeavor in restoring the structural and functional integrity of the trachea. Tissue engineering approaches, notably those leveraging 3D printing technology, have emerged as a promising solution, although further validation through animal studies is warranted before clinical translation [25–28]. 3D printing presents innovative possibilities for developing personalized tracheal substitutes that could potentially overcome current treatment limitations. Recent investigations have showcased successful applications of 3D printing in fabricating tissue-engineered tracheas, underscoring its potential for tracheal reconstruction [28, 29]. However, the majority of these studies have focused on repairing partial tracheal defects, with relatively few addressing extensive tracheal defects or large airway defects, which are far more complex. Furthermore, the intricate and specialized procedures involved in creating 3D-printed tracheal scaffolds hinder their widespread clinical adoption, as they require significant technical expertise, equipment, and resources.

Previous studies utilized PCL scaffolds, which were effective but still resulted in significant post-operative complications including due to excessive granulation tissue, lack of re-epithelization, infections, pneumonia, and stenosis [10, 18, 30]. However, in this present study, a synthetic scaffold comprising a composite of PU and PVC was locally fabricated at our university lab for partial tracheal wall reconstruction. The purpose of this study was not to develop a highly engineered biomimetic construct, but rather to evaluate the feasibility of a simple, locally fabricated scaffold platform capable of maintaining airway patency in a partial tracheal wall defect model. The observed short-term in vivo response, mechanical stability, and cost-effectiveness of this scaffold rendered it an appealing choice for initial experiments.

Primary re-epithelialization is particularly important for minimizing the development of complications such as granulation tissue formation, fibrosis, and wound contraction [28, 31–33]. In our study, we observed some mucosal regeneration at the site of implantation, indicating that epithelial cells from the adjacent tracheal mucosa successfully migrated onto the scaffold and began forming a respiratory epithelium. However, given that this scaffold is intended for potential use in humans, the extent of epithelialization observed in this study was not sufficient to meet clinical standards.

Multiple factors may have contributed to the observed low levels of epithelialization in this study. One potential explanation is the relatively brief postoperative observation period of only one month. It is plausible that if the rabbits had been monitored over a more extended timeframe, the epithelialization process may have advanced further, yielding a higher percentage of epithelial coverage. Lengthening the observation period would also facilitate a more comprehensive evaluation of the scaffold’s long-term stability and its integration into the host tissue. Additionally, improvements in infection control protocols might positively influence epithelialization rates. While this study adhered to a standard antibiotic regimen following the surgical procedure, the implementation of more rigorous infection control measures—such as enhanced sterilization techniques in the operating room and prolonged antibiotic administration—could potentially mitigate inflammation and infection. Both of these factors are known to impede epithelial proliferation and tissue regeneration. Variability in epithelialization may also have been influenced by subtle differences in surgical technique, local tissue response, or postoperative healing among individual animals. This observation implies that the surgical precision in scaffold placement may significantly affect the quality of epithelialization. Moreover, it indicates that employing more refined surgical techniques, particularly concerning the removal of the anterior tracheal wall and the accurate placement of the scaffold, may enhance tissue integration and promote epithelial migration.

A pivotal determinant of epithelial cell migration and proliferation is the presence of an appropriate extracellular matrix (ECM). The ECM supplies essential structural support and biochemical signals that facilitate cellular growth, proliferation, and integration within the scaffold. Research has demonstrated that the incorporation of stem cells or growth factors significantly enhances epithelialization, overall tissue integration, wound healing rates, and the quality of skin regeneration [34, 35].

In comparison to similar studies, our investigation reported a relatively lower mortality rate, which can be interpreted as a favorable outcome [10, 18, 24]. This observation may be attributed to the minimally invasive nature of the surgical intervention coupled with high standards of postoperative care, which encompassed effective pain management and vigilant monitoring. The reduced mortality rate is particularly significant, as it suggests that both the scaffold and the surgical approach were well-tolerated in the rabbit model, suggesting that the scaffold and surgical procedure were generally well tolerated in this rabbit model during the 30-day observation period.

One of the most noteworthy findings of this study was the endoscopic assessment, which revealed an absence of stenosis in all reconstructed tracheas. This indicates that the synthetic scaffold effectively maintained tracheal patency and successfully prevented airway narrowing, a critical requirement for effective tracheal reconstruction. Tracheal stenosis is a common complication associated with reconstructive procedures, and the absence of such complications in all test subjects provides promising evidence that the scaffold preserved structural integrity and did not precipitate any obstructive effects as commonly observed in PCL scaffolds [10, 18, 32].

Although all surviving animals demonstrated complete luminal patency at the 30-day evaluation, this finding should be interpreted cautiously. The preservation of airway caliber may have been attributable, at least in part, to the mechanical support provided by the non-degradable PU-PVC scaffold rather than complete physiological tissue repair or functional regeneration of the tracheal wall. An important finding of this study was the apparent discrepancy between the bronchoscopic and histological outcomes. Despite the absence of stenosis, more than half of the evaluated specimens exhibited unfavorable or scant epithelialization accompanied by moderate-to-severe inflammation. These findings suggest that short-term maintenance of airway patency does not necessarily reflect complete biological integration or mucosal regeneration. It is possible that the scaffold’s mechanical properties were sufficient to preserve airway architecture during the observation period despite incomplete epithelial healing.

Furthermore, because the observation period was limited to 30 days, delayed complications such as progressive granulation tissue formation, chronic inflammation, airway remodeling, and secondary stenosis could not be adequately assessed. Persistent inflammation and limited epithelialization may increase the risk of such complications during longer follow-up periods. Consequently, the absence of stenosis at 30 days should not be interpreted as evidence that late airway narrowing or granulation tissue-induced obstruction would not occur. Therefore, the present findings demonstrate short-term structural feasibility rather than long-term functional success.

Another limitation is that the mechanical properties of the scaffold were not directly compared with those of native rabbit tracheal tissue. Although the scaffold demonstrated adequate structural integrity during the study period, the degree to which its biomechanical behavior approximates that of the native trachea remains to be determined.

Although the scaffold demonstrated adequate mechanical performance and maintained airway patency during the study period, detailed microstructural characterization was beyond the scope of the present feasibility study. Consequently, relationships between scaffold microarchitecture, tissue infiltration, epithelialization, and host response could not be assessed and warrant investigation in future studies.

Conclusion

In this preliminary feasibility study, the PU-PVC composite scaffold-maintained airway patency during the 30-day observation period and preserved its structural integrity following implantation in a rabbit model of partial tracheal wall reconstruction. However, epithelialization was variable, and persistent inflammatory responses were observed in a subset of animals, indicating incomplete biological integration. Although no bronchoscopic evidence of airway stenosis was observed at the study endpoint, the relatively short follow-up period may not have been sufficient to detect delayed complications such as progressive granulation tissue formation, airway remodeling, or secondary stenosis. Therefore, the present findings should be interpreted as evidence of short-term mechanical feasibility and early in vivo performance rather than long-term functional success. Given the short observation period, absence of a control group, and lack of comprehensive biocompatibility testing, further studies incorporating longer follow-up durations, serial airway evaluations, appropriate comparator groups, quantitative airway assessments, and comprehensive biological safety testing are required to determine the scaffold’s long-term performance, safety, and translational potential.

Strengths

The study demonstrates several important strengths. It presents a locally fabricated 3D-printed PU/PVC composite scaffold for partial tracheal reconstruction, addressing a critical challenge in tissue engineering. The scaffold exhibited excellent mechanical stability and maintained airway patency in a preclinical rabbit model. The high survival rate of 88.9% and the absence of tracheal stenosis or obstruction underline the safety and feasibility of the approach. Additionally, the combination of mechanical testing, bronchoscopic evaluation, and histological analysis provided a comprehensive assessment of scaffold performance. The use of locally fabricated, cost-effective materials further enhances the potential for broader application and future translational research.

Limitations

Despite these strengths, the study has several important limitations. First, epithelialization outcomes were variable, with only partial mucosal regeneration observed during the 30-day study period. The relatively short 30-day follow-up period limits assessment of long-term scaffold integration, airway remodeling, durability, and functional outcomes. In particular, late complications such as progressive granulation tissue formation, delayed airway stenosis, chronic foreign-body inflammation, scaffold fatigue or fracture, and potential long-term material-related toxicities could not be evaluated. Consequently, the present study cannot establish the long-term safety, durability, or clinical applicability of the scaffold and should be interpreted as an early feasibility investigation.

Second, this was a preliminary feasibility study conducted in a single experimental cohort without a sham, autograft, or standard-of-care control group. Consequently, although the findings demonstrate that the PU-PVC scaffold was capable of maintaining airway patency during the observation period, direct comparisons with established reconstructive techniques or alternative scaffold materials cannot be made. The absence of a comparator group limits the ability to determine the relative efficacy and safety of the proposed scaffold and restricts the conclusions to observational feasibility outcomes.

Third, the scaffold design did not incorporate advanced bioengineering features such as cell seeding, extracellular matrix components, growth factors, biomimetic surface modifications, or functionalized scaffold architectures. Therefore, the present study should be interpreted as a preliminary feasibility assessment of a simple scaffold platform rather than an evaluation of a fully engineered regenerative tracheal construct. The absence of these biological and structural enhancements may have contributed to the variable epithelialization observed and limited tissue regeneration.

Fourth, bronchoscopic evaluation was performed only at the 30-day endpoint, and quantitative measurements of luminal diameter or cross-sectional area were not obtained. Consequently, the assessment of airway patency was qualitative and limited to short-term observations. Furthermore, because granulation tissue formation and secondary airway stenosis may continue to evolve beyond the first postoperative month, the true long-term incidence of scaffold-related airway narrowing could not be determined. Serial bronchoscopic evaluations and longer follow-up periods would be required to adequately assess late airway remodeling and delayed stenosis. In addition, detailed material characterization, including comprehensive morphological, physicochemical, and surface analyses, was beyond the scope of the present study and should be included in future investigations.

Fifth, histological evaluation was performed by a single experienced pathologist. Consequently, inter-observer agreement and reproducibility could not be assessed. Future studies should incorporate multiple blinded observers and formal assessment of inter-rater reliability.

Sixth, the effects of the sterilization procedure on scaffold mechanical properties, surface characteristics, residual sterilant levels, and biological performance were not independently assessed. Future studies should evaluate sterilization-induced changes in material properties and potential biological effects using standardized testing methods.

In addition, detailed microstructural characterization of the scaffold was not performed. Specifically, scanning electron microscopy (SEM), micro-computed tomography (micro-CT), and quantitative analyses of pore size distribution, pore interconnectivity, and surface architecture were not conducted before or after implantation. Consequently, the microstructural features of the scaffold and potential structural changes associated with implantation and tissue infiltration could not be comprehensively evaluated. Future studies should incorporate these analyses to optimize scaffold design and better characterize scaffold–tissue interactions.

Finally, comprehensive material safety and biocompatibility evaluations were not performed before animal implantation. Specifically, no in vitro cytotoxicity or cell-compatibility studies, extractable/leachable analyses, ethylene oxide residual testing, or ISO 10,993-compliant biocompatibility assessments were conducted. Therefore, definitive conclusions regarding the long-term biological safety or biocompatibility of the PU-PVC composite cannot be drawn from the present study. Additional in vitro and long-term in vivo investigations are required to evaluate cellular responses, potential material-related toxicities, chronic inflammatory responses, and the overall safety profile of the scaffold.

Future studies should incorporate appropriate control groups, larger sample sizes, substantially longer follow-up periods, serial bronchoscopic and imaging assessments, quantitative airway measurements, comprehensive biocompatibility testing, detailed material characterization, evaluation of long-term mechanical integrity, and advanced bioengineering strategies to more rigorously assess scaffold performance, long-term safety, and translational potential.

Abbreviations

PU

Polyurethane

PVC

Polyvinyl Chloride

PCL

Polycaprolactone

UTS

Ultimate Tensile Strength

ECM

Extracellular Matrix

H&E

Hematoxylin and Eosin

SUMS

Shiraz University of Medical Sciences

SD

Standard Deviation

Author contributions

Ali Tadayon, MD: Conceptualization, Methodology, Writing – Original Draft, Visualization, Supervision, Validation, Project Administration, Literature Review, Final Approval Maryam Bahrani, MD: Investigation, Data Curation, Writing – Original Draft, Final Approval Taha Negahdari, MD: Conceptualization, Methodology, Writing – Original Draft, Writing – Review & Editing, Visualization, Supervision, Validation, Project Administration, Literature Review, Final Approval, Formal Analysis Mahsa Askari Jahromi, PhD: Methodology, Data Curation, Writing – Original Draft, Writing – Review & Editing, Final Approval Shayan Yousufzai, MD: Methodology, Data Curation, Writing – Original Draft, Writing – Review & Editing, Final Approval Sepehr Moradi, MD: Methodology, Data Curation, Writing – Original Draft, Writing – Review & Editing, Final Approval Mobin Haghdel, PhD: Conceptualization, Methodology, Writing – Original Draft, Visualization, Supervision, Validation, Project Administration, Literature Review, Final Approval

Funding

No funding was received for this project.

Data availability

All data generated or analyzed during this study are included in this published article. Further datasets are available from the corresponding author on reasonable request.

Declarations

Ethical approval

All experimental procedures involving animals were approved by the Animal Care and Experiment Committee of Shiraz University of Medical Sciences (License ID: IR.SUMS.AEC.1401.016). All protocols adhered to institutional and international guidelines for the care and use of laboratory animals, including proper anesthesia, perioperative monitoring, and humane endpoints.

Informed consent

Not applicable. This study was conducted entirely on an animal model (New Zealand white rabbits). Instead of human consent, all animal welfare protocols were strictly followed, including anesthesia, postoperative care, and minimization of pain and distress.

Consent for publication

Not applicable. This study did not involve human participants, human data, or human tissue. All experimental procedures were conducted in animals, and therefore consent for publication was not required.

Declaration of the use of generative AI and AI-assisted technologies in the manuscript preparation process

The authors declare that the use of artificial intelligence and AI-assisted technologies in this manuscript was limited strictly to the following tools and purposes: 1. Wordvice.ai (https://wordvice.ai) This AI-assisted language editing tool was used solely for language refinement, paragraph structuring, grammar correction, and proofreading. It did not contribute to scientific interpretation, data analysis, clinical reasoning, or drafting of the core academic content. 2. Rayyan – Intelligent Systematic Review Platform (https://www.rayyan.ai) Rayyan was used exclusively for screening similar articles, screening relevant literature, and assisting in the identification and extraction of relevant textual material for the Introduction and Discussion sections. It was not used for data generation, clinical interpretation, or scientific decision-making. 3. Zotero Reference Management Software (https://www.zotero.org). Zotero was used for citation management and formatting of references. All scientific content, clinical data interpretation, manuscript drafting, and final approval were performed by the authors. All authors reviewed and approved the final manuscript and confirmed the accuracy and integrity of the reported data.

IRCT code:

Not applicable. This research was a preclinical animal study and did not involve any human participants or clinical trial registration.

Competing interests

The authors declare no competing interests.