Interact CardioVasc Thorac Surg 2009;8:27-30. doi:10.1510/icvts.2008.179804
© 2009 European Association of Cardio-Thoracic Surgery
Work in progress report - Thoracic general |
Intra-scaffold continuous medium flow combines chondrocyte seeding and culture systems for tissue engineered trachea construction
Qiang Tana,b,*,
Sven Hillingera,
Clemens A. van Blitterswijkc and
Walter Wedera
a Clinic of Thoracic Surgery, University Hospital Zurich, Raemistrasse 100, CH-8091, Zurich, Switzerland
b Shanghai Lung Tumour Clinical Medical Centre, Shanghai Chest Hospital, Shanghai, China
c Department of Tissue Regeneration, University of Twente, The Netherlands
Received 12 March 2008;
received in revised form 28 May 2008;
accepted 29 May 2008
 This work was supported by Swiss National Foundation (NFP 116807).
*Corresponding author. Tel.: +41 44 255 34 16; fax: +41 44 255 88 05.
E-mail address: tqiang{at}hotmail.com (Q. Tan).
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Abstract
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In this study we tested the possibility of seeding chondrocytes into poly (ethylene glycol)-terephthalate–poly (butylene terephthalate) PEOT/PBT scaffold through an intra-scaffold medium flow and the impact of this continuous medium flow on subsequent chondrocyte-scaffold culture. Eight cubic PEOT/PBT co-polymers (1 cm3) were assigned into two groups. In the semi-dynamic seeding group a continuous medium flow was created inside the scaffolds by a pump system. Around six million chondrocytes were harvested each day, suspended in 1 ml medium and delivered onto the scaffold through the perfusion for a sequential five days. Traditional chondrocytes directly seeding and static culture method was performed as control. Scanning electron microscopy (SEM) and histology assessments were performed to evaluate the distribution of chondrocytes inside the scaffolds and MTT test was chosen to check cell vitality. SEM pictures and histology slices from the perfusion group showed a better three-dimensional cell growth and extensive cell distribution inside the scaffolds; while in the control group chondrocytes only dispersedly formed a monolayer on the surface of scaffolds. Accordingly, MTT results from the perfusion group were much higher than those from control group (0.123 vs. 0.067, P<0.01). Continuous medium perfusion inside PEOT/PBT scaffold effectively combines chondrocyte seeding and culture systems for the reconstruction of tissue engineered trachea.
Key Words: Tissue engineering; Trachea replacement; Bioreactor; Cell seeding; Cell culture
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1. Introduction
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Tissue engineering provides a promising approach to tissue and organ substitution [1]. It typically contains two separated stages: in vitro cell-scaffold construct culture stage and in vivo prosthesis–recipient integration stage. Given the absence of an inner vasculature that can be directly connected to the recipient's circulation system, the tissue engineered prosthesis depends on the in-growth of a capillary net from surrounding host tissues to guarantee nutrient supply [2, 3]. In that case the optimal union between the two stages often needs to be properly defined. Regarding tissue engineered trachea construction the dilemma is: on the one hand, a partially developed cell-scaffold construct facilitates the revascularization process while providing inadequate mechanical strength; on the other hand, long-term cultured mature tissue engineered trachea prosthesis might be strong enough to prevent collapse during inspiration while hindering blood vessel in-growth after implantation [4]. The revascularization process proved to be tooslow to support the seeded cells located in the central part of large size tissue engineered implants.
Such being the background, during our research on tissue engineered trachea we pioneered a novel in vivo bioreactor concept defined as the integration of an intra-scaffold medium flow supported by an extracorporeal portable pump system for in situ tissue engineered substitute regeneration. Previous studies have demonstrated that medium perfusion can increase cell content and matrix synthesis in three-dimensional cell-scaffold construct culture systems [5–7]. Various growth factors and relevant cells could be supplemented to the perfusate and delivered continuously to the implanted tissue engineered substitute. Combining the forementioned two stages in tissue engineered prosthesis reconstruction, the in-vivo bioreactor design solves the dilemma by taking advantage of the recipient's intrinsic regenerative capacity for the reconstruction of tissue engineered prosthesis.
In this proof-of-principle study, we studied the hypothesis of delivering chondrocytes into poly (ethylene glycol)-terephthalate–poly (butylene terephthalate) PEOT/PBT scaffold through an intra-scaffold medium flow and also assessed the effects of this continuous medium flow on subsequent cell culture process.
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2. Materials and methods
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2.1. Scaffolds
2.1.1. Poly (ethylene oxide terephthalate)–poly (butylene terephthalate) PEOT/PBT
Cubic PEOT/PBT co-polymers (1 cm3) were obtained from IsoTis S.A. (Bilthoven, The Netherlands) with a composition denoted as 300/55/45, where 300 represents the PEG MW (g/mol), 55 and 45 represent the wt% of the PEOT and PBT blocks, respectively. These three-dimensional scaffolds were fabricated with a Bioplotter device (Envisiontec GmbH, Germany), which is an XYZ plotting device as described by Landers et al. [8]. Cubic scaffolds designed by computer aided design (CAD) software (Rhinoceros®) were characterised by the fibre diameter (through the nozzle diameter), the spacing between fibres and the layer thickness. These parameter values were set in the software from a literature survey known to favour chondryocytes in-growth [9]. Scanning electron microscopy (SEM) was used to characterise scaffolds with pores in the XYZ plane of approximately 400 µmx400 µmx145 µm and a total porosity of approximately 71%.
2.1.2. Isolation and culture of chondrocytes
Rat chondrocytes were harvested from Lewis rat xiphoids as previously described by our group [10]. Cells up to the fifth passage were collected for use.
2.2. Chondrocytes seeding and culture
2.2.1. Direct chondrocytes seeding and static culture method
Four out of the eight cubic PEOT/PBT co-polymers (1 cm3) were randomly selected and performed direct cell seeding followed with static culture. Five flasks of chondrocytes (around thirty million chondrocytes) were harvested, re-suspended in 1 ml F12 medium and seeded directly onto each scaffold in a Petri dish. The cubic PEOT/PBT scaffolds were then wrapped with xenogeneic (porcine) acellular dermis matrix ADM (Jiangshu Qidong Institute of Medical Supplies, Jiangshu, China) preventing cell suspension leakage. The cells were allowed to attach to the scaffold for 2 h before adding 50 ml Ham's F12 culture medium. The cell-scaffold constructs were cultured in an incubator at 37 °C with 5% CO2 for five days with the medium changes once at day three. During harvest, each sample was evenly cut into three pieces and sent for SEM, histology assessment and MTT, respectively.
2.2.2. Semi-dynamic chondrocytes seeding and culture method
The perfusion system was established as described in our former paper with minor modifications [11]. Briefly, two needles were inserted into the scaffold and connected to two peristaltic pumps (IPC high precision multi-channel dispenser, ISMATEC, Zurich, Switzerland) respectively through Tygon Long Flex Life (LFL) pump tubes. One inlet pump delivered the perfusate into the construct while the other outlet pump sucked the waste medium out. Cell suspension, with 6x106 chondrocytes mixed in 1 cc Ham's F12 medium, was delivered into PEOT/PBT scaffold wrapped with ADM at a volumetric flow speed of 2 ml/h. Once the cell suspension reached the scaffold, the pump system was switched off for 2 h facilitating cell adhesion. After that the cell-scaffold structures were perfused continuously with Ham's F12 medium at a volumetric flow speed of 2 ml/h. This seeding process was repeated daily for five days with totally thirty million cells seeded through the perfusion system before harvest. All four samples were protected in a modified centrifuge tube against contamination and kept in the incubator at 37 °C with 5% CO2. During harvest, each sample was evenly cut into three pieces and sent for SEM, histology and MTT assessment, respectively.
2.2.3. Scanning electron microscopy (SEM)
Samples for SEM analysis were fixed in 2% phosphate-buffered glutaraldehyde solution, then dehydrated with a graded isopropanol series and air dried. Before analysis, the dried samples were mounted on aluminium supports and sputter-coated with gold. The percent of the scaffold pores attached with chondrocytes were counted on both the top (exterior) side and the cutting (interior) side.
2.2.4. Histology
The PEOT/PBT samples are fixed in 4% buffered formalin for at least 18 h and afterwards dehydrated using graded ethanol solutions and xylene. Thereafter the probes are paraffin-embedded and sliced. The 3 µm thick slices are mounted on a slide and stained with hematoxylin and eosin (HE-staining).
2.3. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay
The MTT tests were performed according to the method previously described [10]. MTT assay determines viable cell numbers and is based on the mitochondrial conversion of the tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). For each sample, we obtained three MTT results.
2.4. Statistical analysis
The percent of chondrocyte-attached pores both inside and outside the scaffold were compared between the two groups. The probability values were assessed using the  2-test, and P<0.01 was considered significant. The MTT data from the two groups were expressed as mean±S.D. and statistic analysis by unpaired t-test at a given P-value of <0.05, which was considered as statistically significant.
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3. Results
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3.1. Scanning electron microscopy (SEM)
The percent of scaffold pores attached with chondrocytes from both the exterior and interior faces of each sample are listed in Table 1. Generally, scaffolds from the perfusion group were more evenly distributed with cells densely packed. In one sample, the exterior surface was covered with multi-layer three-dimensional growth chondrocytes (Fig. 1a). The cells also formed some dense cell clusters inside the scaffold (Fig. 1i–l). In contrast, samples from the static seeding group showed some cell clusters near the top surface (Fig. 1e–h) and there was almost no three-dimensional cell growth inside the scaffold (m–p). The cells only form a mono-layer on the surface of the scaffold in some areas (Fig. 1). The statistic analysis between the two groups regarding the chondrocyte-attached pores showed significant difference from both the exterior surface (P<0.003) and the interior surface (P<0.001).
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Table 1 The chondrocyte-attached pores percent of the PEOT/PBT samples from perfusion seeding and static seeding group
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Fig. 1. SEM picture of the chondrocytes distribution inside the PEOT/PBT scaffolds. (a–d) The exterior surface of the PEOT/PBT scaffolds from the perfusion seeding group, the chondrocytes formed 3-dimensional cell cluster in 22–100% of the scaffold pores. (e–h) The exterior surface of the PEOT/PBT scaffolds from the static culture group, in most cases the chondrocytes only formed a mono-layer on the surface of the scaffold instead of covering the pores. (i–l) The interior cutting surface of the scaffolds from the perfusion group, chondrocytes migrated deep into and showed 3-dimensional cell growth inside the scaffold. (m–p) The interior cutting surface of the scaffolds from the static culture group, no chondrocytes were found inside the scaffolds.
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3.2. Histology
On the edges and in the pores of the scaffold from the semi-dynamic seeding group a moderate amount of spindle-shaped cells are visible (Fig. 2a). In some areas the spindle-shaped cells form thin bundles or small round clusters. The cells show an elongated, activated nucleus with one good visible basophilic nucleolus.
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Fig. 2. Histology results, HE staining x100. (a) In the perfusion group, the chondrocytes not only formed one monolayer on the surface of the scaffold but also tried to cover the pores of the scaffold. (b) No chondrocytes were found in most of the histology slices of the static culture group.
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On histology from the static group sample, we failed to find any cells in the slides (Fig. 2b).
3.3. MTT
The MTT test results showed significant difference (P<0.01) between the semi-dynamic seeding group and directly seeding static culture group (0.123 vs. 0.067) (Fig. 3).
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Fig. 3. MTT results showed significant difference between the perfusion group (0.123±0.04) and the static culture group (0.067±0.013).
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4. Discussion
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Tissue engineering aims to provide off-the-shelf organ substitutes reconstructed by autologous cells and biodegradable scaffolds. To date in the field of tissue engineered trachea we are still far away from this ultimate goal. Abundant knowledge of normal tracheal constituents and structure does not translate smoothly into successful tracheal reconstruction in vitro. Living tissues undergo special regeneration processes that are still, so far, too complex to be mimicked in vitro [12].
To improve our understanding of regeneration mechanisms and to further interfere with these processes, we put forward the in-vivo bioreactor concept which aims to take advantage of the patient's own regenerative capacity and improves it by the supplement of appropriate cells and various kinds of growth factors. The traditionally separated in vitro and in vivo stages in tissue engineering approach are merged simply by a continuous medium flow inside the a vascular tissue-engineered construct.
The aim of this study was to test the possibility of seeding chondrocytes onto PEOT/PBT tissue-engineered tracheal co-polymers by means of intra-scaffold medium flow. During the pilot exams we mixed one flask of chondrocytes into 250 cc medium and tried to seed the cells onto the scaffold through continuous perfusion. Unfortunately, we hardly found any cells attached to the scaffold due to low medium cell concentration and less time for cell adhesion under appropriate perfusion speed which proved to be crucial cell culture. In that case, we chose a semi-dynamic seeding procedure characterised with 1 cc of high cell concentration perfusate accompanied with a 2-h perfusion pause for cell adhesion. With this approach a better three-dimensional cell growth was found compared with the directly cell seeding static culture method. These preliminary results may be the first step for future alternative clinical applications, especially in emergency cases where the patients are not able to wait for the time-consuming in vitro reconstruction of the desired organ grafts. In such a critical situation, scaffolds could be first implanted and continuously perfused without any seeding cells. During the operation tissue could be harvested for later isolation and expansion of appropriate autologous cells in the laboratory. The cells could then be seeded into the implanted scaffolds through daily autologous cell transfusions which should be a physiological and economical way for tissue engineered prosthesis construction.
Besides, cell dedifferentiation after several passages in vitro proliferation remains a challenge in tissue engineering research. The in vivo bioreactor design provides an opportunity to technically circumvent this obstacle by extending the cell seeding protocol covering the whole regeneration process. Taking the reconstruction of tissue-engineered trachea as an example, the autologous chondrocytes within limited generations can be seeded through an in vivo bioreactor into the implanted tissue graft during the whole regeneration period. These cells will contribute to the repair and the remodelling of the impaired tissue according to the signaling given by the local regenerative niche.
In this proof-of-principle study the seeded cells were still unevenly distributed inside the scaffold. This indicates that, to guarantee a sufficient perfusion throughout the whole construct, the inner perfusion system of tissue engineering scaffolds should present a customized design according to the geometry of the defect, the porosity and the size of the scaffolds chosen. The flow rate should also be adjustable depending on the cell concentration used and the stage of tissue regeneration to avoid detrimental shear stress. Various kinds of medium, selected growth factors and different cell types are acquired to form an appropriate perfusate facilitating the regeneration and revascularization processes. In addition, artificial oxygen carriers, such as perfluorocarbon (PFC) emulsion, might be applied to increase the oxygen concentration within the scaffolds [13–15]. All these possibilities deserve further in vitro and in vivo experimentations.
In conclusion, our studies showed that chondrocytes can be delivered to and further proliferate within PEOT/PBT scaffolds which are permeated by a continuous medium flow. This offers the possibility to combine the traditionally separated in vitro and in vivo parts of tissue engineering by a novel in-vivo bioreactor concept.
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Acknowledgements
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The SEM pictures were taken by Mr. Klaus Marquardt, Electronic Microscopy Centre of University Zurich, and are gratefully acknowledged. The histology was performed by Dr. med. Vet. Monika Hilbe, ECVP Institute of Veterinary Pathology, and are gratefully acknowledged.
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Abstract
1. Introduction
