Long-Term Chondrocyte Retention in Partially Decellularized Tracheal Grafts

Abstract

Objective.

Decellularized tracheal grafts possess the biological cues necessary for tissue regeneration. However, conventional decellularization approaches to target the removal of all cell populations including chondrocytes lead to a loss of mechanical support. We have created a partially decellularized tracheal graft (PDTG) that preserves donor chondrocytes and the mechanical properties of the trachea. In this study, we measured PDTG chondrocyte retention with a murine microsurgical model.

Study Design.

Murine in vivo time-point study.

Setting.

Research Institute affiliated with Tertiary Pediatric Hospital.

Methods.

PDTG was created using a sodium dodecyl sulfate protocol. Partially decellularized and syngeneic grafts were orthotopically implanted into female C57BL/6J mice. Grafts were recovered at 1, 3, and 6 months postimplant. Pre- and postimplant grafts were processed and analyzed via quantitative immunofluorescence. Chondrocytes (SOX9+, DAPI+) present in the host and graft cartilage was evaluated using ImageJ.

Results.

Partial decellularization resulted in the maintenance of gross tracheal architecture with the removal of epithelial and submucosal structures on histology. All grafts demonstrated SOX9+ chondrocytes throughout the study time points. Chondrocytes in PDTG were lower at 6 months compared to preimplant and syngeneic controls.

Conclusion.

PDTG retained donor graft chondrocytes at all time points. However, PDTG exhibits a reduction in chondrocytes at 6 months. The impact of these histologic changes on cartilage extracellular matrix regeneration and repair remains unclear.

Pathologies resulting from tracheal defects are potentially life-threatening. A majority of tracheal lesions can be resected and primary anastomosis safely performed. However, long-segmental tracheal defects, defined as >30% overall length in children, cannot be repaired with direct anastomosis and tracheal replacement becomes necessary.^1–5 Unfortunately, the ideal tracheal replacement has yet to be identified. Autologous tissues have been associated with donor site morbidity and allogenic tissues have led to immune rejection.^6,7 Therefore, the focus has shifted to engineering tracheal grafts capable of renewal and repair.

Our initial work on tissue-engineered tracheal grafts demonstrated that in contrast to synthetic, decellularized biologic scaffolds can support the regeneration of an epithelium identical to the native trachea. Decellularized tracheal grafts offer the advantage of being nonimmunogenic while preserving native tissue structures, which contribute to the biological cues necessary for clinical tissue regeneration. However, human clinical cases show that failed cartilage regeneration remains an important limiting factor in the clinical adoption of grafts that are completely decellularized.^8,9

Conventional decellularization approaches target the removal of all cell populations including chondrocytes, which leads to a loss of cellular and mechanical support.¹⁰ Chondrocytes and the extracellular matrix (ECM) have been shown to influence surrounding cell behavior, including the regulation of cell proliferation, self-renewal or differentiation, migration, and cell death.^6,7 Given the immunopriviledged nature of cartilage, contemporary approaches have examined partial decellularization techniques that target the removal of immunogenic cell types while preserving the underlying cartilage.^10,11 With these concepts in mind, we created partially decellularized tracheal grafts (PDTG) and demonstrated that they are capable of regenerating tracheal neotissue in vivo.^12,13 While we have extensively characterized host-derived neotissue, the durability of donor-derived tissue (cartilage) in vivo remains unclear. In this study, we qualitatively and quantitatively assess PDTG chondrocytes in vivo in a time-point study using a murine microsurgical model for orthotopic tracheal replacement.

Methods

Animal Care and Ethics Statement

The animal care protocol (AR15-00090) was reviewed and approved by the Institutional Animal Care and Use Committee of the Abigail Wexner Research Institute at Nationwide Children’s Hospital. All animals were treated and cared for according to the regulations outlined in the Animal Welfare Act as per the standards published by the Public Health Service, National Institutes of Health in the Care and Use of Laboratory Animals (2011), US Department of Agriculture.

Tracheal Graft Preparation

PDTG and syngeneic grafts were previously prepared from harvested tracheas from 6- to 8-week-old female C57BL/6J mice, as previously described.^14,15 Approximately 5 mm of the proximal trachea was removed from donor mice. Syngeneic and PDTG grafts were implanted freshly. PDTG was prepared as previously described.^11,12 Briefly, sodium dodecyl sulfate was utilized for shorter durations and with a lower rotation speed to help limit protein alteration. The grafts were then washed with Triton-X to remove the residual DNA. To examine total chondrocyte loss over the course of the study, preimplantation controls of native tracheal grafts and PDTG were directly processed for histology following explantation and production, respectively.

Tracheal Graft Implantation

Native tracheal grafts and PDTG were implanted into syngeneic female C57BL/6J mice (6 to 8 weeks old) following established protocols.^14,15 Recipient mice were anesthetized and sedated, followed by an aseptic midline incision from the sternum to the hyoid bone. The overlying strap musculature was divided, and the surrounding soft tissue was dissected from the trachea circumferentially, paying careful attention to avoid disruption of the recurrent laryngeal nerves or esophagus. A 5 mm full circumferential segment of the trachea was removed via transverse proximal and distal cuts. The distal end of the tracheal segment was secured to the sternal notch to create a temporary tracheostomy site. The graft was implanted in the proximal native airway with a 9–0 sterile nylon suture. The tracheostomy site was released with resection of a segment of the native trachea followed by distal anastomosis. Animals were carefully observed following recovery and euthanized if humane endpoint criteria were met (respiratory distress, loss of >20% preimplantation weight). PDTG and syngeneic graft animals were euthanized using a ketamine-xylazine cocktail at study endpoints of 1, 3, and 6 months. Only animals that survived to the planned endpoint were included for analysis. Tracheal grafts were harvested, and tissues were formalin-fixed, embedded, and sectioned. We performed chondrocyte retention analysis on PDTG (not implanted) (n = 3) and fresh, native trachea (n = 3), which acted as preimplantation controls. Postimplantation, animals from PDTG were analyzed for chondrocyte retention at 1 month (n = 4), 3 months (n = 4), and 6 months (n = 4). Finally, syngeneic grafts acted as surgical controls and were similarly analyzed at 1 month (n = 6), 3 months (n = 3), and 6 months (n = 3).

Immunofluorescent staining for chondrocyte-specific Sox9 and nuclei-specific DAPI was performed on the native trachea (n = 3) and PDTG (n = 3) before implantation. Chondrocytes (Sox9+) were measured in grafts at 1 (n = 4), 3 (n = 4), and 6 months (n = 4). As a surgical control, chondrocytes in syngeneic grafts were quantified at 1 (n = 6), 3 (n = 3), and 6 (n = 3) months (Figure 2).

Figure 2.

Tracheal cartilage of syngeneic and partially decellularized tracheal grafts in vivo. (A) Longitudinal section of chondrocytes (Sox9+, green) yellow arrows, select Sox9+/DAPI+ cells. (B) Representative images of cartilage at each time point (red *, chondrocyte). PDTG, partially decellularized tracheal graft.

Histology

Native trachea and grafts were fixed in a 10% neutral-buffered formalin solution followed by paraffin embedding. Specimens were longitudinally sectioned at 6 to 8 μm thickness. Representative sections were decalcified and stained with hematoxylin and eosin (H&E), Masson’s Trichrome, and Alcian Blue, respectively (Sigma-Aldrich). For immunofluorescence, sections were deparaffinized with xylene washes, rehydrated through graded alcohols, and equilibrated with 1X phosphate-buffered saline (PBS) solution. Sections were blocked for 30 minutes in a blocking buffer composed of 5% bovine serum albumin and 0.1% Triton X-100 detergent in PBS. SOX9 primary antibody (EMD Millipore) was diluted to a working concentration of 1:400 using a blocking buffer. A primary antibody cocktail was added to sections and incubated overnight at 4°C. Slides were washed for 10 minutes in 1X PBS in a dim room, followed by the addition of secondary antibody solution (1:500 dilution of goat anti-rabbit immunoglobulin G with Alexa Fluor 488 [Invitrogen] in blocking buffer). The secondary antibody was incubated in the dark for 45 minutes at room temperature, followed by a single wash with 1X PBS. Slides were dipped 5 times in distilled water, blotted to remove excess water, then mounted using Vectashield mounting medium with DAPI counterstain (Vector Laboratories). Images were captured using digital microscopy (Zen Blue Edition; Zeiss) for analysis and archiving. Chondrocytes present in host and graft cartilage were evaluated and manually quantified using ImageJ (NIH).

Statistical Analysis

Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software Inc.). The Shapiro-Wilk test was used to assess the normality of the data. Data with normal distribution were compared using Welch’s t test for data with significantly different variances or using an unpaired t test for data with equal variances. The Mann–Whitney nonparametric test was used for data with nonnormal distribution. Paired t test was used to compare data between graft and host in each animal. Statistical difference was considered significant at p < .05. All experimental data were conveyed as mean ± standard deviation (SD).

Results

Partial Decellularization Results in Removal of Epithelium With Gross Preservation of Cartilaginous Structures

PDTG were compared to syngeneic controls. Partial decellularization resulted in the maintenance of gross tracheal architecture and lumen patency similar to the native trachea. Consistent with previous findings, partial decellularization was confirmed histologically by the absence of epithelial and submucosal structures on H&E, conservation of collagen (Masson’s Trichrome staining), and glycosaminoglycans (Alcian Blue) within the tracheal rings (Figure 1).¹¹ The preservation of donor cartilage included chondrocytes, which were morphologically comparable to the native trachea. Chondrocytes were similarly identifiable in both PDTG and native tissue.

Figure 1.

Histologic characterization of partially decellularized tracheal grafts. Axial sections of H&E, Masson’s Trichrome, and Alcian blue for native trachea (control; top row) and PDTG (bottom row). Cartilage ring in red. Yellow arrows, chondrocytes. H&E, hematoxylin and eosin; PDTG, partially decellularized tracheal graft.

Chondrocytes Were Present at All Time Points in PDTG

For preimplantation controls, immunofluorescent staining for chondrocyte-specific Sox9 and nuclei-specific DAPI was performed on native trachea (n = 3) and PDTG (n = 3). Postimplantation, chondrocytes (Sox9+) were measured in PDTG grafts at 1 month (n = 4), 3 months (n = 4), and 6 months (n = 4). As a surgical control, chondrocytes in syngeneic grafts were quantified at 1 month (n = 6), 3 months (n = 3), and 6 months (n = 3) (Figure 2).

All grafts demonstrated chondrocytes at 1-, 3-, and 6-month time points. Syngeneic grafts at all time points contained a similar number of chondrocytes compared to native controls, with quantification demonstrating an average of 3103 and 2972 Sox9+ cells/mm², respectively. Aggregate PDTG yielded average counts of 2012 Sox9+ cells/mm² preimplantation and 2774 Sox9+ cells/mm² postimplantation. Overall, the number of chondrocytes in PDTG was highly variable, exhibiting larger SD compared to the control, and was particularly notable in our preimplanted grafts (Figure 3). Among implanted grafts, syngeneic and PDTG had a comparable number of chondrocytes when examined against preimplanted controls, with no difference observed across any time point. Chondrocytes in PDTG were lower at 6 months compared to both preimplanted native (p = .0352) and syngeneic (p = .0430) controls. Of note, this difference was not seen within the PDTG group across time points. Furthermore, no difference was found at any time point between syngeneic and native trachea, suggesting that chondrocyte durability is retained following syngeneic transplant.

Figure 3.

Quantification of chondrocytes in vivo. Chondrocyte (Sox9+) quantification of native, syngeneic, and partially decellularized tracheal graft (PDTG). PDTG contains less chondrocytes at 6 months compared to preimplantation PDTG and syngeneic grafts at the same time point (*, p < .05).

Discussion

PDTG has the potential to serve as a solution for the treatment of long-segment tracheal defects. In a previous study, we demonstrated that cartilage-sparing decellularization protocols improve the mechanical and biochemical properties of tracheal grafts relative to completely decellularized constructs.¹¹ In contrast to partial decellularization, complete decellularization disrupts not only graft mechanical properties but also surrounding cell viability and the ECM.¹⁰ Additionally, the preservation of cartilage, and underlying chondrocytes, may serve more than the maintenance of graft biomechanics. Chondrocytes have been shown to influence surrounding cell migration and are essential to cartilage homeostasis. Thus, their long-term preservation could be important for graft healing and reepithelialization^6,16,17

Our PDTG retained graft-derived chondrocytes at all study time points, demonstrating that chondrocytes not only remain after partial decellularization but are preserved throughout the microsurgical and in vivo tracheal repair processes as well. This is supported by quantifying all surgically implanted grafts, both syngeneic and PDTG, corresponding chondrocyte populations to those observed in native controls across all time points. Similar chondrocyte populations are seen between PDTG and control through the 3-month time point. However, at 6 months, chondrocytes in PDTG were lower than both native and syngeneic controls. Chondrocyte populations in PDTG demonstrated high variance in their chondrocyte population, as evidenced by a larger SD compared to the control (Figure 3). Moreover, PDTG chondrocytes had fewer DAPI-positive nuclei when compared with adjacent host tissue or controls. This contrast was most prominent at later time points (Figure 3).

There are several potential sources for the quantitative changes seen in PDTG chondrocytes. The most relevant include mechanical damage at the time of surgery, delayed perfusion because of limited neovascularization postimplantation, and exposure to decellularization detergent during graft processing. Chondrocyte apoptosis and other forms of cell death have been shown to be induced by mechanical damage in articular cartilage.¹⁸ However, given the delayed effect of chondrocyte loss, exposure to decellularization detergents and limited neovascularization seem the most likely sources of injury to PDTG chondrocytes.

In this murine study, PDTG served as a successful tracheal replacement with the endurance of graft-derived chondrocytes throughout the time points of the study. Previously, we demonstrated that PDTG can support host-derived epithelial, endothelial regeneration, and chondrocyte viability for 28 days.¹¹ In contrast, we proved that PDTG retains graft-derived chondrocytes similar to native and surgical controls at least until 3 months postimplantation. This finding is vitally important for the translation of PDTG to humans, as functional chondrocytes may be critical for long-term graft durability.^6,16,17 Prior studies have demonstrated how exposure to reagents for complete decellularization protocols can injure chondrocytes.^9,19 Moreover, neovascularization has been identified as a leading failure of synthetic tracheal grafts.^20,21 Given the role of the chondrocyte in cartilage homeostasis, improvements in chondrocyte viability are likely beneficial to long-term graft health and could also influence neovascularization. The functional impact of the high chondrocyte variance and quantitative changes seen on histology remain unclear and the role of chondrocyte viability on graft performance and neovascularization remains a priority for our group. This information is significant, as it supports the rational design of tracheal grafts with improved chondrocyte retention and creates a foundation to examine the long-term implications of chondrocyte retention on graft performance.

The study had several limitations. First, the function of chondrocytes, such as collagen II homeostasis, was not assessed in this study. Secondly, while this study confirmed chondrocytes throughout in vivo implantation of our PDTG, it did not address the significance of their presence on graft function. Future studies devoted to assessing changes in Sox9 localization within the chondrocyte can help elucidate cell functionality. Finally, our study had a relatively low N per group, which could influence statistical findings.

Conclusion

PDTGs are a potential solution to segmental tracheal defects. Partial decellularization protocol preserves underlying donor-derived chondrocytes, which remained present at 6 months. Chondrocytes are vital for cartilage homeostasis, so a graft that retains donor-derived chondrocytes could provide an improved environment for graft healing and repair.