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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 1  |  Issue : 1  |  Page : 19-24

Development of tracheal reconstruction methods from scaffold engineering to injectable matrix


1 Mycobacteriology Research Center (MRC), National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran
2 Mycobacteriology Research Center (MRC), National Research Institute of Tuberculosis and Lung Disease (NRITLD); Department of Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Date of Web Publication24-Jul-2017

Correspondence Address:
Jalaledin Ghanavi
Mycobacteriology Research Center (MRC), National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical, Tehran
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bbrj.bbrj_55_17

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  Abstract 

Backgrand: For patients with long segment of tracheal stenosis, resection and replacement is necessary. Tracheal reconstruction associated with complications such as stenosis, insufficiency of blood supply and surgical complication. Methods: In this study, we prepared collagen-chitosan scaffold and sheep's decellularized trachea plus culture medium with nanocup contain growth factors for chondrocyte and fibroblast-epithelial cell culture in rotary bioreactor. After attachment of cells, engineered trachea put into the omentum. Peritoneal stem cell interact with the epithelial and chondrocytes with attendance of growth factors released from nanocup. The bioengineered trachea with omental pedicle transposed from behind the sternum and transplanted in the position of resected trachea. In third experiment, we introduce the in situ tracheal repair technique with injectable matrix for reconstruction of long-segmental stenosis of trachea in a 29-year-old woman. Results: Severe tortuosity in the first experiment and mild stenosis was seen in the second experiment. In third experiment, normal shape in tracheal diameter was seen at injection sites. The interior portion of the trachea at virtual computed tomography (CT) scan and bronchoscopy were similarity to normal after four month fallowing up of injection. Conclusions: Severe tortuosity, mild stenosis in whole length of trachea in this study and disadvantage of trachea transplantation include open surgery, fatal consequences of anastomosis leakage, and rupture besides large mediastinal vessels; hence, we decided to introduce the novel in situ tracheal repair technique. Injectable bioresorbable scaffolds may be used as a temporary scaffolding for transplanted cells and thereby allow the cells to secrete extracellular matrix of their own to enable, in the long term, a complete and natural tissue replacement.

Keywords: Injectable matrix, scaffold engineering, tracheal


How to cite this article:
Ghanavi J, Farnia P, Bahrami A, Jabbari HR, Velayati AA. Development of tracheal reconstruction methods from scaffold engineering to injectable matrix. Biomed Biotechnol Res J 2017;1:19-24

How to cite this URL:
Ghanavi J, Farnia P, Bahrami A, Jabbari HR, Velayati AA. Development of tracheal reconstruction methods from scaffold engineering to injectable matrix. Biomed Biotechnol Res J [serial online] 2017 [cited 2017 Sep 22];1:19-24. Available from: http://www.bmbtrj.org/text.asp?2017/1/1/19/211416


  Introduction Top


Typically, tracheal stenosis has congenital (e.g., hypoplasia), acquired (e.g., postintubation trauma), and cancerous etiologies. Tracheal resection and reanastomosis is usually a successful surgical procedure in the majority of patients.[1] This surgical option is applicable in patients whose affected length exceeds to 50% of the trachea in adults and 30% in children.[2] However, stenosis that affects tracheal length longer than 4.5 cm is more likely to develop postoperative complications. Trachea's function is not limited to carrying air to the lungs and secretions out of them; rather, the specific shape of tracheal cartilage keeps the trachea from collapse in inspiration and bulging of membranous section, which protects alveoli from coughing and sneezing pressure. The trachea is an ideal organ to explore the clinical potential of tissue engineering because severe large airway diseases have been poorly managed by conventional treatments. These unique characteristics make trachea irreplaceable with other organs. As a result, the repair of long-segment tracheal stenosis has remained a surgical challenge.

The application of fascia, which allows for the growth of connective tissue and tracheal epithelialization, has been the first step in tissue engineering for tracheal defect repair.[3] Therefore, the actual tracheal tissue engineering began with decellularization of small bowel, the use of stem of bone marrows – derived progenitor cells, and the application of vascular pedicle samples and costal chondrocyte.[4] Replacement and repair of trachea using stents made of natural and synthetic polymers have been associated with such complications as infection, displacement, vascular stent erosion, and granulation.[5] The use of cadaver,[6] aortic allograft,[5] and tracheal acellular has also ended up in complications in a long term.[7]

In this study, we made scaffolds for tissue engineering consisting of natural polymer or decellularized trachea that have capability of cell attachment and integration with native organ and introduced the in situ procedure for organ regeneration and repair.

The in situ tracheal repair technique is an injecting matrix used for regeneration and repair of long segment of tracheal stenosis. The injectable matrix consists of natural polymer with stem cell niche and cup-shaped nanoparticles filled with growth factor. The injectable matrix injects to create the three-dimensional (3D) matrix system or network in segment of tracheal stenosis after stenting and granulation tissue formation (if needed). The injected area of the trachea as a whole matrix allows the migration of a plurality stem cells or tissue-specific progenitor cells to the 3D matrix system or network; cells in the presence of thein vivo shear stress depend on airflow in inspiration, expiration, airway secretion, and swallowing caused organ maturation.


  Subjects and Methods Top


The first experiment

The chitosan/collagen composite was used to prepare scaffolds. The chitosan solution (75%–85% deacetylated; Sigma-Aldrich) prepared with 0.75 g chitosan dissolved in 50 mL of acetic acid 1% w/v (Merck), mixed with collagen type II (concentration 0.5% w/v) at a 3:1 v/v rate. The produced gel was molded into the shape of tracheal scaffold, using chemical crosslink with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, lyophilization, and gamma irradiation method [Figure 1] and [Figure 2].
Figure 1: The chitosan/collagen scaffolds on porous polytetrafluoroethylene tube

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Figure 2: Transmission electron microscopy image of matrix comprising collagen, chitosan, and nanocup

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A specific rotary reactor with peristaltic pump designed for continues circulation and cell culture. Culture medium circulated with peristaltic pump (3 ml/min) from inside of lumen space to outside. Dulbecco's Modified Eagle Medium (DMEM) medium with nanocup containing transforming growth factor-beta (TGFB) and basic fibroblast growth factor (bFGF) mid-distribution contains 10 ng/ml bFGF and TGFB 10 ng/ml [Figure 3]. To fabricate the tracheal cartilage, the extracellular matrix (ECM) containing chondrocytes obtained from sheep nasal cartilage was used.[8] The culture media was changed every 3 days.
Figure 3: Rotary bioreactor for cell deposition and induced mechanical stress

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After 7 days, the fibroblast cell, along with the nasal epithelial cell of the sheep, was co-cultured in DMEM and Ham's nutrient mixture F12 (3:1, v/v) circulated with peristaltic pump (1ml/min) from inside of lumen space to outside. The culture medium with nanocup containing EGE and bFGF mid-distribution bFGF 10 ng/ml and EGE 10 ng/ml for 24 h [Figure 4].
Figure 4: Transmission electron microscopy image of a nanocup with/or without growth factor

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The second experiment

In the second step to repair sheep trachea, we acellularized it using enzymatic method.

The sheep trachea was removed through aseptic method and immediately rinsed with phosphate-buffered saline (PBS) containing penicillin-streptomycin and amphotericin B. Then, mucus and blood remains were completely removed. The trachea remained in sterile distilled water in a fridge at 4°C for 48 h and was exchanged every 24 h. Then, it was kept in a sodium deoxycholate solution (1%) at 4°C in shaker at 100 rpm for 2 days. The solution was replaced every 24 h. The trachea kept in continual circulation with sterile distilled water at 4°C for 24 h. It was then immersed in a DNase1 solution (2000 U/ml), along with 0.5 mol NaCl at 4°C in shaker at 50 rpm for 24 h. The sample was then rinsed with distilled water at 4°C for three times. After this stage, the trachea in chloroform at 0.001 bar for 3 h and was kept in chloroform at 4°C for 24 h [Figure 5].
Figure 5: Extracellular matrix of hyaline cartilage of decellularized trachea with empty lacuna

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The sample was removed from the chloroform and freeze-drying at the 0.001 Torr vacuum. The trachea was rinsed with PBS. Chondrocyte, epithelial, and fibroblast cells achieved at the same manner describe in the first experiment.

Transplantation

Four sheep operated for each of experiments. After inducing general anesthesia with ketamine hydrochloride and xylazine and continuing with inhalation anesthesia, laparotomy was carried out. The engineered trachea obtained from both experiments was removed from the rotary reactor and was placed in the omental wrap and fixed with nylon 4/0 [Figure 6]. After 20 days, the samples implanted intrathoracic transposition at the site of tracheal resection (6 cm defect).
Figure 6: Bioartificial trachea twisted with omentum

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The third experiment

The in situ tracheal repair technique promotes angiogenesis, selection of appropriate cell sources, reproducible differentiation of the selected cell type or types along organ-specific lineages and enhances supports three-dimensional (3D) production of tissues in living system with normal shear stress and function. Due to complexity of trachea and involvement of more than one cell type, an understanding of the factors involved in the differentiation potential of the selected cell source atin vitro is invaluable.

A 29-year-old female was having 8.5 cm long tracheal destruction and stenosis which occurred 10 years back after trauma in a car accident.

She was treated with argon plasma coagulation laser during these years. Computed tomography (CT), radiologic, and bronchoscopic findings showed the stenosis started from 2 cm below the vocal cord to 2.5 cm until to tracheal bifurcation, and only 2–3 mm from tracheal lumen was opened [Figure 7].
Figure 7: Bronchoscopic interior view of tracheal stenosis in a 29-year-old woman with cervical trauma

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After preparation for bronchoscopy, under general anesthesia, fiber-optic bronchoscopy was passed through the upper part of the trachea to detect exact location of stenosis. Rigid bronchoscopy number 4 through 6.5 was inserted orderly for dilatation, and then, holmium laser ablation was performed for the stenosis lumen.

Matrix composed of chitosan/collagen and cup-shaped nanoparticles. The chitosan solution (75%–85% deacetylated) prepared with 0.25 g chitosan dissolved in 50 mL of acetic acid 0.25% w/v, mixed with collagen type II (concentration 0.2% w/v) at a 3:1 v/v rate in sterile condition. Nanocups for chondrocyte matrix in mid-distribution contains 10 ng/ml of TGFB and bFGF, respectively. Similarly, nanocups for fibroblast/epithelial cell in mid- distribution contains 10 ng/ml EGE and bFGF.

A first-stage operation was done with chondrocyte matrix injection by needle bronchoscopy for tracheal cartilage reconstruction. Amount of injectable matrix was 0.1 ml and distance between locations of injection was 1 mm apart. Injection depth was 2–3 mm and configuration of injections was C-shape (anatomic shape of cartilages of the trachea). The second-stage operation was done with fibroblast/epithelial matrix injection for tracheal epithelial reconstruction by injection needle of bronchoscopy after 3 days. Amount of injectable matrix was 0.1 ml and locations of injection were 1 mm apart. Injection depth was 1–2 mm and configuration of injections was circle shape. In this site, procedures are complete.


  Results Top


The first and second experimental results were compared. In the first experiment, severe tortuosity occurred in two trachea; it caused by heterogeneous placement of chondrocyte in matrix. In the second experiment, all of the trachea following placed in the omental wrap had mild stenosis in whole length, and after postimplantation, the ventilation care was very difficult and shortness of breath improved slightly over time.

Human study in the third experiment shows normal shape in injection sites and interior portion of the trachea at virtual CT scan after the 1st and 3rd month of injection. At the 1st month of follow-up, virtual CT showed that the injection site of trachea was almost the same as the native trachea [Figure 8] and [Figure 9].
Figure 8: Interior view of tracheal virtual computed tomography scan after 1 month of injection

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Figure 9: Interior view of tracheal virtual computed tomography scan after 3 months of injection

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Bronchoscopy demonstrated that the injected sites were normal and the inner side of trachea was smooth with normal color. Diameter of the injection site of trachea was equal to patient's own trachea. An interior view of tracheal bronchoscopy after 4 months of injection of the injectable matrix can be seen that the injection sites have completely normal shape. At the 4th month follow-up, everything was normal [Figure 10].
Figure 10: Interior view of the tracheal bronchoscopy after 4 months of injection

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  Discussion Top


Long segment of tracheal stenosis is life-threatening because irreplaceable and the vascular pedicle anastomosis is still impossible. Tissue engineering ingredient consists of the cells (mesenchymal or stem cell), materials (natural or synthetic matrix), and biochemical factors. The efficacy of scaffolds for cell seeding is the important factor in tissue engineering.

A nonimmunogenic scaffold with suitable mechanical characteristics would be the ideal tracheal graft. Therefore, tissue engineering is the next most promising technology to potentially offer such a solution.[9]

In the first experiment, we utilize collagen-chitosan spongy tubular scaffold for seeding of chondrocyte and epithelial cell and made the bioartificial trachea.

Chitosan-gelatin hydrogels were utilized to growth-supportive substrata for culture and growth of substratum for respiratory epithelial cells.[10] Collagen sponge and gel accelerating tracheal epithelial cells were regenerated and replaced with recipient epithelial cells and promote mesenchymal cell infiltration. Furthermore, collagen (type II) induced chondrocyte growth to form cartilage-like tissue.[11],[12],[13]

Tissue culture medium in rotary bioreactor contains nanocups, nasal epithelial and fibroblast cells of the recipient sheep that circulated inside to outside of the lumen, until the cells gets seeded. Coculture of these cells and epithelial–mesenchymal interactions continued in omentum twist and in the presence of peritoneal stem cell to covering inside the lumen. This interaction plays a crucial role in the morphogenesis and differentiation of the epithelium of the trachea.[14]

Bioengineered scaffold coated with tracheal fibroblasts were effective at accelerating regeneration of the tracheal epithelium when implanted into rabbits with tracheal defects.[15] Umbilical cord blood-mesenchymal stem cells seeded onto 3D scaffolds have been successfully engineered to cartilaginous tissuein vitro that is very similar to hyaline cartilage for type II collagen secretion and low elastin expression.[16]

Combination of epithelial stem/progenitor cells and mesenchymal stem cell-derived chondrocytes repaired airway defects.[17] Chondrogenesis induced by TGF-β1 and stimulated by microgravity.[18]

The type II collagen network confers to cartilage its strength against tensile forces.[19] The porous chitosan scaffold environments alone caused proliferation of the chondrocytes with the effect of slower release rate TGF-β1 from microspheres.[20] Mechanical stimulation enhances cartilage formation and development of tissue-engineered cartilage.[18] Chondrocyte proliferation and matrix production are supported by the use of a rotary bioreactor.[21],[22]

After implantation of scaffold that wrapped in omentum, the entrapped growth factors were released slowly in peritoneal cavity which consequently caused the mesenchymal peritoneal stem cell infiltration and transformation. Thereof, the combination of mesenchymal stem cell with chondrocytes and epithelial cells will accelerate the maturation of trachea.

The presence of pluripotent mesenchymal cells raising from the peritoneal mesothelium is endowed with such a degree of plasticity that, if placed in the appropriate microenvironment, they have a remarkable potential to generate other mesenchymal-derived cell lines.[23] Matrix ( first experiment) and natural organ (second experiment) with expose to growth factor released from nanocup define the best environmental conditions to take advantage plasticity and make the peritoneal mesenchymal cells attach and transform.

The fabrication of collagen-chitosan matrix in the first method and use of natural tracheal matrix in the second method and placement of cells in both of matrix containing collagen and a specific the tripeptide Arg-Gly-Asp(RGD) motif as the niche of stem cells, along with their mechanical progression in the placement site, result in cellular evolution.

Tissue engineering requires the use of a cell source to allow for the generation and maintenance of tissue-specific biological functions as well as the use of injectable matrix to support and guide tissue development.

Following these results from two experiments and disadvantage of trachea transplantation include open surgery, fatal consequences of anastomosis leakage, tissue necrosis, and rejection besides large mediastinal vessels, we decided to introduce the novel in situ tracheal repair technique.

A major problem in engineering of any tissue for clinical application is selecting human cell sources with the potential to provide sufficient number of cells for the development of tissue used to repair defects caused by disease or injury beyond the repair capabilities of the human body. Stem cells exist in every organ and tissue.

The niche is a specialized microenvironment housing stem cells.[24] The niche dynamically regulates stem cell behavior, maintaining a balance between quiescence, self-renewal, and differentiation.[25] Recapitulating the native ECM is the key to providing the 3D structure and biological cues for homing and delivery of stem cells for tissue engineering and organ regeneration purposes.[26]

The biophysical signs, regarding the micron sizes of cells, topographical feature, and the pressure on matrix in nano- and pico-newton range (shear stress), depend on airflow in inspiration, expiration, airway secretion, and swallowing caused organ maturation.

Mechanical interactions between cells and the environment significantly affect cell differentiation. These mechanical forces are caused by cell-cell and cell-matrix interactions, which direct differentiation and evolution of a complex tissue pathway.

It is essential to prepare an environment similar to natural ECM in tissue engineering. Natural polymers such as collagen or gelatin resemble most the natural ECM and contain specific signals such as Arg-Gly-Asp (RGD) motifs for cell adhesion and allow cell infiltration providing a biological advantage.[27]

RGD sequence is widely used for binding to integrin. However, RGD peptide contains several peptide motifs with more control over integrin features, cell adhesion, fate, behavior, and evolution.[28],[29]

Advantage of using the injectable matrix described is the ability to create 3D matrix systems.

Injectable bioresorbable scaffolds may be used as a temporary scaffolding for transplanted cells and thereby allow the cells to secrete ECM of their own to enable, in the long term, a complete and natural tissue replacement.[30]

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10]



 

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