Elsevier

Biomaterials

Volume 30, Issue 25, September 2009, Pages 4117-4126
Biomaterials

A scaffold-bioreactor system for a tissue-engineered trachea

https://doi.org/10.1016/j.biomaterials.2009.04.028Get rights and content

Abstract

A scaffold-bioreactor system was developed for growing tissue-engineered trachea and the effect of fluid flow on producing trachea-like neotissue was investigated. Chondrocytes were seeded in the poly(ɛ-caprolactone)-type II collagen scaffold and grown in the bioreactor operated under continuous flow at a rotational speed from 5 to 20 rpm. Flow analysis showed that the maximal and minimal shear stress in the bioreactor was 0.189–0.752 dyne/cm2 and 30.3 × 10−5–104 × 10−5 dyne/cm2, respectively. After 4 and 8 weeks, the constructs were harvested from the bioreactor and analyzed. The application of rotation increased cell proliferation, GAG and collagen content in the constructs. Especially at 15 rpm, a two-fold increase in cell number, 170% increase in GAG, and 240% increase in collagen were found compared to static culture at 8 weeks. H&E staining showed the formation of neocartilage and the alignment of chondrocytes along the flow direction. The constructs grown under 15 rpm was selected for implantation into tracheal defects of rabbits. The mean survival of six animals was 52 days. The re-epithelialization of respiratory epithelium from the anastomotic sites was observed, with granulation tissue overgrowth. This successful initial step would allow us to make further improvement in applying tissue-engineering techniques to regenerate tracheas for practical use.

Introduction

Tracheal tissue is composed by chondrocytes, respiratory epithelial cells, collagen matrix, and blood vessels. The tracheal tube is reinforced by “C” shape cartilage that prevents collapse during inspiration. The surface of trachea is lined with functional ciliated respiratory epithelia. Researches in tracheal tissue engineering are mainly focused on two fields of interests: the tracheal cartilage reconstruction [1], [2], and the lumen respiratory epithelialization [3], [4]. One of the current challenges is to combine two different tissues, i.e. cartilage and epithelia, in tracheal tissue engineering. Kojima et al. made a non-woven poly(glycolic acid) fiber wrapped up silicone tube, with the inner and outer surface of the tube seeded with epithelial cells and chondrocytes, respectively. After implantation into the subcutaneous space of nude mice, the matrix components of the graft showed similarity to those of native tracheal tissue [5]. However, the graft collapsed only 7 days after implantation to sheep cervical trachea [6]. In a recent clinical study by Macchiarini et al., autologous epithelial cells and mesenchymal stem cells derived chondrocytes were seeded to human donor trachea and cultured in a rotational bioreactor. The graft provided the recipient a functional airway in the left bronchus at 4 months [7]. Despite using autologous tissue or tracheal allograft, each construct seems to have its own limitation. For example, the patients receiving donor trachea might require immunosuppressive therapy. Another important concern is the mechanical properties of tissue-engineering trachea, which are still far from satisfactory. To overcome the limitation of mechanical properties in tissue-engineering trachea, a poly(ɛ-caprolactone) (PCL)–collagen tracheal scaffold has been developed in our lab [8]. We showed that this composite scaffold could sustain mechanical strength. Besides, the scaffold was highly porous with type II collagen sponge filled in the multi-ring structure which provided a proper environment for the proliferation of chondrocytes.

Bioreactors are important tools for culturing long-segment tracheal tissue for clinical applications. As mentioned, in a recent clinical study human donor trachea seeded with autologous cells was cultured in a self-designed rotational bioreactor [7]. However, this study and other studies have not focused on hydrodynamic environment in tracheal tissue engineering. Dynamic culture systems can offer several important advantages compared with the static ones. For instance, mass transfer can be enhanced by convective fluid flow. In addition, physiologically relevant physical signals provided by a dynamic system, such as shear stress, compression, pressure, and stretch, can have influences on tissue development. Several studies have demonstrated that shear stress regulates the metabolism of ECM in tissue-engineered cartilage [9], [10], [11], [12]. Since chondrocytes in vivo typically reside in a mechanically dynamic environment, the dynamic environment generated by a bioreactor is believed to provide an opportunity for reconstruction of larger tracheal constructs. A wide variety of bioreactor types have been developed for tissue engineering, such as spinner flasks [13], rotating wall vessels [14], concentric cylinders [15], [16], [17], and the perfusion system [18]. Each of these bioreactors provides a different flow stream and shear stress. Cartilage tissue grown in rotational bioreactors has been reported to be functionally more superior to that in static or in spinner flasks [19]. This might be due to the turbulent flow and higher shear stress created by the spinner flasks compared to the rotational bioreactor, which has predominately laminar flow conditions and lower shear stress.

In the current study, a rotational bioreactor was developed for the uniform production of tissue-engineering trachea. Constructs with the optimal growth condition were then implanted into rabbit cervical tracheal defects. Postoperative evaluation was conducted using flexible bronchoscopy and histological analyses. We expected that by combining tracheal tissue engineering and telescopic anastomosis technology, the lumen surface of the constructs could be epithelialized at an early stage of the healing process after implantation.

Section snippets

Preparation of the tracheal scaffold

The tracheal composite scaffold (a grooved PCL stent in combination with ring-like type II collagen sponge in a tubular design) was fabricated as previously described [8]. Briefly, the stent part of the tracheal scaffold was prepared from 11% (w/v) 1,4-dioxane solution of PCL (Mn = 80 kDa, Aldrich, Milwaukee, WI, USA). The polymer solution was poured into a mold with special groove design and subsequently frozen at a temperature −20 °C for 8 h and then freeze-dried for 48 h to form the porous

Flow simulation

The rotational bioreactor was operated under continuous flow at a rotational speed of 5, 10, 15 or 20 rpm. The medium (∼10 ml) was approximately half full of the glass chamber during the culture period. Based on the simulation, the fluid flow at the construct surface was laminar. The maximal shear stresses were between 0.189 and 0.752 dyne/cm2 and minimal shear stresses were between 30.3 × 10−5 and 104 × 10−5 dyne/cm2 at different rotational speeds (Table 1). Fig. 3A shows vertical cross-sections of

Discussion

Bioreactors play an important role in tissue engineering, because the fluid flow in bioreactors can enhance nutrient mass transfer and stimulate cell proliferation and matrix secretion [25]. Although different bioreactors were designed for applications to cartilage [26], bone [27], [28], skin [29], or blood vessel [30] tissue engineering, those for trachea tissue engineering have not been developed in detail. By moving cells between liquid (medium) and gaseous (air) phases, bioreactors provide

Conclusion

In this study, we demonstrated a bioreactor that could be applied to grow tissue-engineered trachea. The flow generated in the dynamic environment of the bioreactor could stimulate chondrocyte proliferation and matrix secretion. Furthermore, chondrocytes were aligned with the flow direction, and the morphology was similar to that observed in native tracheal cartilage. In animal experiments, tissue-engineered tracheal constructs could resist collapse without deformation during the implantation

Acknowledgements

This work was supported by the National Health Research Institutes, and the National Science Council, Taiwan.

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