Stenosis or malacia of the trachea can result in difficult breathing, exercise intolerance, sleep apnea, tracheotomy dependence and/or death [1] . When the malacia or stenosis is restricted to the upper trachea or subglottis, surgeons can bypass the narrowing with a tracheotomy. In certain situations, where a short segment of the cartilage rings is either destroyed, patients may be candidates for either surgery to remove the extrinsic compression or tracheal resection [2] . In non-neoplastic cases, intraluminal silicone stenting can provide relief, albeit with the very significant risks of mucous plugging, infection or migration [3] . In neoplastic cases, the affected section must be removed.

Up to 30% of the tracheal length can be excised and the two ends can be sutured together with a tension-free anastomosis. If more than 30% of the trachea is excised, released maneuvers are often required to allow for a tension free closure. To address long-segment stenosis (>50% of the trachea) or revision tracheal resection where an adequate length of trachea is not available, a variety of tracheoplasty techniques have been attempted with varying degrees of success. There is clearly an upper limit to the length of trachea that is possible to safely resect and, thus, a segment of patients for whom an adequate surgical solution is not readily available. In order to address the upper limit of length, one can turn to create a biologic implant utilizing the patient’s own cells, potentially placing the harvested cells within a scaffold. However, harvesting tracheal chondrocytes from human patients is likely not feasible. There are many other less invasive sources of chondrocytes available for use, including nasal and auricular that have been shown to produce cartilage in tissue engineering applications [4] [5] . Additionally, the use of marrow stromal cells in cartilage tissue engineering is a promising development for future clinical translation [6] [7] .

Scaffold-based tracheal reconstruction has been investigated in pre-clinical models. However, few have been translated to clinical applications. A 3D printed external splint made of polycaprolactone has been investigated in 3 pediatric patients to treat tracheobronchomalacia, but will not suffice as a treatment for neoplasia [7] . A bioengineered and trachealbronchial replacement was implanted in a human patient with primary tracheal cancer for whom other reconstructive options were not possible. This graft was fabricated from polyhedral oligomeric silsesquioxane (POSS) covalently bonded to poly-[carbonate-urea] urethane (PCU) (POSS-PCU), and seeded with autologous mononuclear cells. However, the fabrication process and pre-implantation culture technique are exceedingly complex, prohibiting widespread use and may not be necessary [7] [8] .

This report details a method of creating a three-dimensionally (3D) printed and tissue engineered biodegradable tracheal ring that is seeded with chondrocytes as a first step towards addressing this unmet clinical need.

Our results demonstrate the feasibility of 3D printing a CAD design of a tracheal ring that closely approximates normal tissue. The cell proliferation assay demonstrated that cells seeded in the scaffold had an average initial viability of 87.5% and cell number increased over 7 days to approximately twice the initial concentration (Figure 2). The cells grown in the scaffold demonstrated a similar proliferation trend compared to control cells (T-test comparing the proliferation rates; p = 0.42).

Biomechanical testing revealed that the cell-seeded scaffolds (group 3) retained their physical properties throughout the study when compared to non-treated controls (group 1 and 2). Cell seeding didn’t alter the mechanical properties of the scaffolds during the study period. Specifically, no significant differences in the stiffness (Youngs Modulus) were identified over a 21-day period (p = 0.99) (Table 1).

Histology of the cell seeded scaffold 1 day after incubation showed equal distribution of cells throughout the graft (Figure 3). Upon visual inspection, the cells appeared to retain their characteristic round shape and equal distribution throughout the 21-day study period. Numerous islands of Safranin-O positive staining were seen within the scaffold, demonstrating the presence of aggrecan components. The quantity of Safranin-O positive staining increased over the 21-day study period indicating that the chondrocytes retained their biological properties (Figure 4).

Patients with long segment tracheal stenosis or malacia or patients who require revision tracheal resection represent clinical challenges for the airway surgeon. Allogeneic tracheal transplantation can provide an excellent functional result, but requires lifelong immunosuppression and is dependent on the supply of donor tracheas [9] . To

address the issue of immunosuppression, some surgeons have proposed transplanting devitalized tracheal allografts [10] . These grafts are treated by washing away all donor cellular material, rendering them immunologically inert, and mitigating the need for the immune suppression normally required for transplantation [10] . The grafts are then seeded with stem cells from the recipient to produce true biological replacement prosthesis. The allograft itself serves merely as a biological scaffold on which to seed recipient stem cells prior to transplantation [5] . While this is a promising development, it requires extensive time and cost, and can result in biomechanical degradation requiring stenting [9] [11] .

Another promising development is the emergence of 3D printing technology in which digital designs can be virtually modeled and printed into the physical world with precision. If this technology can be adapted into the realm of tracheal reconstruction, 3D printing can allow for individualized constructs with ideal biomechanical properties and physiologic function. Indeed, surgeons have already attempted to address airway malacia by means of external airway splinting using biodegradable 3D printed scaffolds without chondrocytes [3] [12] . Animal models exploring the biology underlying this approach have yielded improved respiration, but premature splint degradation or improper fit can cause a recurrence of tracheomalacia [13] . This failure may be attributed to the lack of cartilaginous in growth to support the trachea as the splint degrades.

The combination of living cells, whose proliferation rates are comparable to native cells [14] [15] , and a 3D modeled patient specific graft may address some of these failures. In this study we demonstrate the ability to fabricate a customized 3D printed biodegradable PLA scaffold containing proliferating chondrocytes. Our data indicate that the cells within the scaffold are viable and proliferate and retain their biological function by synthesizing extracellular matrix. As well, when comparing Young’s modulus of native cartilage tissue to the produced scaffold, has higher values of 0.77 kPa compared to an average of 0.26 kPa of native cartilage [16] . This proof of concept study represents a first step towards producing a 3D printed and tissue engineered long segment tracheal replacement graft for airway surgery.

Other groups have recently studied tracheal replacement grafts with some level of success [15] [17] . However, all these approaches have potential drawbacks, including a complex scaffold fabrication process which prohibits widespread use, use of stem cells with unknown long-term phenotype, and use of growth factors in the incubation phase with unknown neoplastic potential [15] [17] . Our approach involves the creation of a 3D printed CAD design and optimizes that design using commercially available software. The design can then be 3D printed using a readily available device. PLA is chosen to fabricate the scaffold, as it is FDA approved for various uses ranging from bone screws to wrinkle reducing injections. We are then able to seed the scaffold with mature chondrocytes, which survive and retain their biological function, thus avoiding the risks associated with stem cells and/or growth factors.

Although the complex and specialized technology is extremely valuable in solving many biomedical problems, the cost can be prohibitive for many institutions. With our model, we have shown that widely available technology, combined with innovative optimization, can make these solutions accessible to almost any health care facility. In addition to accessibility, our proof of concept model of tissue engineered cartilaginous grafts can be utilized for various medical purposes. 3D printed tissue engineered constructs using a patient’s own cells allow for many new implantable applications far beyond the trachea. Future directions include exploring the use of other polymers with varying degradation times and expanding the application of this technology to address other unmet challenges in otolaryngology, such as auricular reconstruction, facial contouring, and nasal reconstruction.

Todd A. Goldstein,Lee P. Smith,Benjamin D. Smith,Daniel A. Grande,David Zeltsman, (2015) Integrating Biodegradable 3-Dimensional-Printing into Tracheal Reconstruction. Open Journal of Regenerative Medicine,04,27-33. doi: 10.4236/ojrm.2015.43004