Tracheal Regeneration Research Articles

Engineering the extracellular matrix for clinical applications: Endoderm, mesoderm, and ectoderm

Tissue engineering is rapidly progressing from a research-based discipline to clinical applications. Emerging technologies could be utilized to develop therapeutics for a wide range of diseases, but many are contingent on a cell scaffold that can produce proper tissue ultrastructure. The extracellular matrix, which a cell scaffold simulates, is not merely a foundation for tissue growth but a dynamic participant in cellular crosstalk and organ homeostasis. Cells change their growth rates, recruitment, and differentiation in response to the composition, modulus, and patterning of the substrate on which they reside. Cell scaffolds can regulate these factors through precision design, functionalization, and application. The ideal therapy would utilize highly specialized cell scaffolds to best mimic the tissue of interest. This paper discusses advantages and challenges of optimized cell scaffold design in the endoderm, mesoderm, and ectoderm for clinical applications in tracheal transplant, cardiac regeneration, and skin grafts, respectively.

EFFECTS OF DIFFERENT ANTIOXIDANT ENZYMES ON THE TRACHEAL EPITHELIUM REGENERATION AFTER CHEMICAL BURN

The aim of the study was to investigate a role of different antioxidant enzymes for tracheal epithelium regeneration after chemical burn. Methods. The study was conducted in a rat model of chemical burn of the upper airways caused by inhaled hydrochloric acid. Results. According to results of histological examination, to the 2nd day after the exposure approximately 70 % of the epithelial surface remained injured, mostly due to death of the ciliated cells. The degree of the damage was unchanged to the 4th day after the exposure. Visible regeneration of the tracheal epithelium began to the 7th day. The death of the tracheal epithelial cells was generally due to necrosis though cell apoptosis also occurred. The expression of all antioxidant enzymes was greatly decreased during the 1st day after the exposure followed by its growth to maximum to the 7th day and normal level to the 15th day after the burn. Exposition of superoxide dismutase to the trachea resulted in a significant epithelium destruction. On contrary, peroxiredoxin 6 and a chimeric protein containing peroxiredoxin / superoxide dismutase significantly protected the tracheal epithelium. Conclusion. Peroxiredoxins and their derivates could be used as highly efficient therapeutic agents with potent antioxidant action in patients with burn injury of the upper airways.

RETRACTED: Airway Transplantation

No definitive solution has been discovered for replacing long segments or the entire trachea in humans. Most of this challenge stems from the specific function and mechanics that are almost impossible to replicate except in the setting of an allotransplantation, which requires lifelong immunosuppressive medication. Recently, tissue engineering provided significant evidence concerning the next promising therapeutic alternative for tracheal replacement. Underlying mechanism and pathways of cell-surface interactions, cell migration, and differentiation are essential to understand the complexity of tracheal tissue regeneration. Tracheal replacement remains challenging but initial steps toward an ideal therapeutic concept have been made.

Tracheal cartilage regeneration by progenitor cells derived from the perichondrium

The regenerative potential of adult tracheal cartilage is very limited which makes repair of this cartilage extremely challenging. However, the perichondrium has a large quantity of progenitor cells that differentiate directly into chondrocytes. Bone morphogenetic protein (BMP) stimulates chondrogenesis by inducing the differentiation of progenitor cells into chondrocytes which makes it a potential therapeutic agent for cartilage regeneration. In this present study, we investigated the mesenchymal stem cell characterization of progenitor cells from the perichondrial tissue of tracheal cartilage and the efficiency of the perichondrium for tracheal cartilage regeneration by evaluating the effect of BMP on cartilage regeneration in perichondrial progenitor cells. Progenitor cells were isolated from the fibrous perichodrial tissue of tracheal cartilage and cultured for phenotypic characterization of mesenchymal stem cells. Next, a 0.3–0.5 mm gap was made in the mid-ventral portion of the tracheal cartilages. In the control group (n=5), the resulting gap was left untreated (without perichondrial replacement). In the perichondrial group (n=15), the gap was replaced by fibrous perichondrial tissue. We euthanized all mice in each group (n=5) at 2, 4, and 20 weeks after the perichondrial replacement and examined the replacement site microscopically. The immunohistochemistry for BMP-2 was performed in progenitor cells from perichondrial tissue. The primary cultured cells from fibrous perichondrial tissue were positive for CD29, CD90, and CD105, but negative for CD45. The progenitor cells were capable of multipotent differentiation towards adipogenic and osteogenic lineages. The control group showed no regenerated cartilage 2 weeks after injury. The experimental group demonstrated proliferation of progenitor cells between both stumps at 2 weeks and regenerated hyaline cartilage was observed at 4 and 20 weeks. Regenerated cartilage was observed as separated islets between the ends of the host cartilage stumps. The gaps were nearly filled with regenerated cartilage at 20 weeks. BMP-2 was expressed in both fibrous and cabial perichondria chondrogenesis. The progenitor cells from perichondrial tissue had mesenchymal stem cell-like features and might play a crucial role in tracheal cartilage regeneration and repair. Expression of BMP-2 may induce formation of structural cartilage in a tracheal injury model.

Implantation site-dependent differences for tracheal regeneration with induced pluripotent stem cells (iPS cells)

Conclusion: The histological findings and quantitative measurements demonstrated that there were differences in teratoma formation according to the site of implantation. Elucidating the mechanisms of the teratoma formation caused by the site of implantation moves the field another step closer to clinical use of induced pluripotent stem (iPS) cells for tracheal regeneration. Objectives: Our previous study demonstrated the potential for iPS cells to be used as a new cell source for tracheal regeneration. However, teratoma formation remains a major problem. Implantation site-dependent differences in teratoma formation have been reported. In this study, the teratoma-forming propensity after implantation into tracheal defects and abdominal subcutaneous tissue was examined histologically and quantitatively. Methods: Mouse iPS cells were cultured in artificial material under various conditions. After cultivation in vitro, artificial materials with cultured iPS cells were then implanted into cervical tissue around tracheal defects and into abdominal subcutaneous tissue in nude rats. Teratoma formation was evaluated histologically and quantitatively with measurement of maximum diameter (MD). Results: Teratoma was observed in 10 of 11 rats with cervical tissue around tracheal defects and in 3 of 11 rats with abdominal subcutaneous tissue implants. The average MD was 5.36 mm in the trachea and 0.97 mm in the abdomen.

Evaluation of the Use of Induced Pluripotent Stem Cells (iPSCs) for the Regeneration of Tracheal Cartilage

The treatment of laryngotracheal stenosis remains a challenge as treatment often requires multistaged procedures, and successful decannulation sometimes fails after a series of operations. Induced pluripotent stem cells (iPSCs) were generated in 2006. These cells are capable of unlimited symmetrical self-renewal, thus providing an unlimited cell source for tissue-engineering applications. We have previously reported tracheal wall regeneration using a three-dimensional (3D) scaffold containing iPSCs. However, the efficiency of differentiation into cartilage was low. In addition, it could not be proven that the cartilage tissues were in fact derived from the implanted iPSCs. The purpose of this study was to evaluate and improve the use of iPSCs for the regeneration of tracheal cartilage. iPSCs were cultured in vitro in a 3D scaffold in chondrocyte differentiation medium. After cultivation, differentiation into chondrocytes was examined. The ratio of undifferentiated cells was analyzed by flow cytometry. The 3D scaffolds were implanted into tracheal defects, as an injury site, in 24 nude rats. Differentiation into chondrocytes in vitro was confirmed histologically, phenotypically, and genetically. Flow cytometric analysis demonstrated that the population of undifferentiated cells was decreased. Cartilage tissue was observed in the regenerated tracheal wall in 6 of 11 rats implanted with induced iPSCs, but in none of 13 rats implanted with the control and noninduced iPSCs. The expression of cartilage-specific protein was also demonstrated in vivo in 3D scaffolds containing iPSCs. The presence of the GFP gene derived from iPSCs was confirmed in samples of cartilage tissue by the combination of laser microdissection (LMD) and polymerase chain reaction (PCR) techniques. Our study demonstrated that iPSCs have the potential to differentiate into chondrogenic cells in vitro. Cartilage tissue was regenerated in vivo. Our results suggest that iPSCs could be a new cell source for the regeneration of tracheal cartilage.

Tracheal regeneration: Evidence of bone marrow mesenchymal stem cell involvement

Recent advances in airway transplantation have shown the ability of exvivo or invivo tracheal regeneration with bioengineered conduits or biological substitutes, respectively. Previously, we established a process of invivo-guided tracheal regeneration using vascular allografts as a biological scaffold. We theorized that tracheal healing was the consequence of a mixed phenomenon associating tracheal contraction and regeneration. The aim of the present study was to determine the role that bone marrow stem cells play in that regenerative process. Three groups of 12 rabbits underwent a gender-mismatched aortic graft transplantation after tracheal resection. The first group received no cells (control group), the second group had previously received autologous green fluorescent protein-labeled mesenchymal stem cell transplantation, and the third group received 3 labeled mesenchymal stem cell injections on postoperative days 0, 10, and 21. The clinical results were impaired by stent complications (obstruction or migration), but no anastomotic leakage, dehiscence, or stenosis was observed. The rabbits were killed, and the trachea was excised for analysis at 1 to 18 months after tracheal replacement. In all 3 groups, microscopic examination showed an integrated aortic graft lined by metaplastic epithelium. By 12 months, immature cartilage was detected among disorganized elastic fibers. Positive SRY gene detection served as evidence for engraftment of cells derived from the male recipient. EF-green fluorescent protein detection showed bone marrow-derived mesenchymal stem cell involvement. The results of the present study imply a role for bone marrow stem cells in tracheal regeneration after aortic allografting. Studies are necessary to identify the local and systemic factors stimulating that regenerative process.

Regeneration of Tracheal Epithelium Using a Collagen Vitrigel—Sponge Scaffold Containing Basic Fibroblast Growth Factor

Our group has had good results in tracheal mucosal regeneration using a collagen vitrigel-sponge scaffold in an animal model. In this study, the effectiveness of this scaffold with the application of basic fibroblast growth factor (b-FGF) was investigated. A collagen vitrigel-sponge scaffold was fabricated with simultaneous addition of b-FGF. Three types of collagen vitrigel-sponge scaffolds were made: no b-FGF, 10 ng of b-FGF, and 100 ng of b-FGF. At 3, 5, 7, and 14 days after implantation in rats, the tracheas were removed and histologically evaluated. The regeneration of mucosal epithelium and the subepithelial layer was evaluated. Mucosal epithelium, including pseudostratified epithelium and ciliated cells, regenerated earlier in the scaffolds when b-FGF was applied than when b-FGF was not applied. Regeneration of the subepithelial layer, infiltration of inflammatory cells and fibroblasts, and angiogenesis were promoted earlier in the scaffolds with b-FGF application. Our technique for tracheal reconstruction using collagen vitrigel-sponge scaffolds with b-FGF application affords a feasible approach for accelerating the regeneration of the intraluminal surface and subepithelial layer of tracheal tissue.

Collagen vitrigel scaffold for regenerative medicine of the trachea: Experimental study and quantitative evaluation

Conclusion: Our technique for tracheal regeneration using a collagen vitrigel scaffold affords a feasible approach for accelerating epithelial regeneration on the intraluminal surface of the host tracheal defect. Objectives: The purpose of the study was to quantitatively evaluate the effectiveness of a collagen vitrigel scaffold for the regeneration of the tracheal epithelium. Methods: The collagen vitrigel scaffold was developed by conjugating a collagen vitrigel membrane to a collagen sponge to promote both epithelial cell growth and mesenchymal cell infiltration. The collagen vitrigel scaffold was implanted into tracheal defects in rats as the vitrigel model, and a conventional collagen sponge was implanted as a control model. The structure of the regenerated tissue was observed and thickness of the epithelium was quantitatively evaluated by histological examination. Results: Histological findings showed the surface of the collagen vitrigel scaffold to be flat in comparison with that of the conventional collagen sponge. At 7, 14, and 28 days post-implantation, the average thickness of the regenerated epithelial layer in the vitrigel model group was greater than that in the control group.

The Future of Tissue-Engineered Organs for Transplant? It's Here!

The first-in-man bioengineered artificial tracheobronchial transplant consisting of a synthetic scaffold and autologous bone marrow-derived mesenchymal cells was recently reported. Extensive evaluation of the patient before and after transplant provides some insight into the potential mechanisms of stem cell mobilization and tracheal tissue regeneration.

Acceptable Warm Ischemia Time of Tracheal Grafts From Non–heart-beating Donors in Rats

Objectives Warm ischemia (WI)-induced airway complications are common in clinical lung transplantation. However, the acceptable WI time of tracheal grafts from non–heart-beating donors (NHBDs) is unknown. The purpose of this study was to determine the acceptable WI time by observing tracheal epithelial regeneration among NHBD. Method Forty-eight rats were randomly divided into four groups (each with 12 rats): WI-0 minutes (group A), WI-30 minutes (group B), WI-45 minutes (group C), and WI-60 minutes (group D). In each group, the tracheas from 6 rats were imbedded in the greater omentum of 6 other rats. Fourteen days later, the transplanted trachea was obtained from the recipient to evaluate epithelial thickness and regeneration. Six tracheas were obtained from living donors as a control group. Results There were no significant differences in tracheal transplantation time (mean, 17.66 ± 1.21 minutes). There were no significant differences in epithelial thickness and regeneration between the controls and groups A, B, and C ( P < .05). Group D showed no normal epithelial structure of the trachea only with monolayer cells. Conclusions The time limits of tolerance to WI of tracheal grafts from NHBDs may be 45 minutes.

Bone Marrow-Derived Mesenchymal Stem Cells Enhance Cryopreserved Trachea Allograft Epithelium Regeneration and Vascular Endothelial Growth Factor Expression

Epithelium regeneration and revascularization of tracheal implants are challenging issues to be solved in tracheal transplantation research. Bone marrow-derived mesenchymal stem cells (BMSCs) can migrate to the damaged tissue and promote functional restoration. Here, we applied intravenous transplantation of BMSCs combined with a cryopreserved allograft to investigate the role of BMSCs in enhancing implant survival, tracheal epithelium regeneration and revascularization. After transplantation with cryopreserved allografts, PKH-26 labeled 3 to 5 passage BMSCs were injected into recipient rats through the tail vein. Rats in the control groups were injected with a comparable amount of phosphate-buffered saline. We observed the histology of the tracheal allograft and measured vascular endothelial growth factor (VEGF) protein levels in the epithelium to evaluate the effect of BMSCs on epithelium regeneration and revascularization. Histologic observation of the rats from the BMSCs injection groups showed that the tracheal lumen was covered by pseudostriated ciliated columnar epithelium. The cartilage structure was intact. There were no signs of denaturation or necrosis. PKH-26 labeled BMSCs migrated to the implant site and exhibited red fluorescence, with the brightest red fluorescence at the anastomotic site. VEGF protein levels in the allograft epithelium of the BMSCs injection group were higher than the levels in the phosphate-buffered saline injection group. Our results indicate that given systemic administration, BMSCs may enhance epithelium regeneration and revascularization by upregulating VEGF expression.

Development of Nanofiber Coated Indomethacin—Eluting Stent for Tracheal Regeneration

In the case of tracheal rupture or stenosis, most effective way is to insert a commercially available metal stent. However, the implantation often causes a fever or a pain on the contact surface between trachea and the stent. And also the metal stent should be removed after a certain time implantation. Thus, we developed a functional tracheal drug eluting stent consisting of indomethacin, a nonsteroidal anti-inflammatory drug (NSAID), loaded nanofibers on a bare metal stent. To control the drug release kinetics and enhancement of mucosal regeneration, gelatin and PLCL were coated layer by layer on a metal stent by an electrospinning method. Indomethacin was loaded in the gelatin layer by soaking and drying method (0.1, 0.5, and 1 wt% in ethanol for 10 min). The morphology of functional drug eluting tracheal stent was characterized by scanning electron microscope (SEM). And mechanical properties of the constructs such as air leak pressure, ultimate tensile stress, and modulus were calculated and evaluated. Drug release was performed by a high performance liquid chromatography (HPLC). Stably coated gelatin and poly(L-lactide- co-epsilon-caprolactone) (PLCL) nanofibers were observed by SEM. Bi-layered nanofibers-coated stent showed enough mechanical properties as a tracheal stent, which confirmed by a custom-designed air leak mechanical test. For indomethacin loading on a stent, stent was immersed in a series of drug solutions (different concentrations) for 10 min. At the result of HPLC, total amounts of indomethacin on a stent were approximately 77, 323, and 670 ug/stent, respectively. Time dependent drug release kinetics of the tracheal stent showed a sustained release profile regardless of indomethacin content. Thus, functionally designed nanofiber coated tracheal stent with anti-inflammatory drug may be useful for tracheal regeneration.

Tracheal Replacement with Fresh and Cryopreserved Aortic Allograft in Adult Dog

Many reports have described tracheal replacement using aortic allografts, with varying results and minimal understanding of the mechanism of tracheal regeneration. The present study attempted tracheal regeneration in adult dogs using fresh aortic allografts (FAA) and cryopreserved aortic allografts (CAA). Twelve adult beagles underwent tracheal resection and were transplanted with FAA (n = 5) or CAA (n = 7). Animals were followed-up with serial radiography and magnetic resonance imaging, and were euthanized at predetermined times up to 16 mo post-surgery. There were no procedural deaths, but two animals died due to stent migration. Stent migration occurred in seven of the 12 animals. Evidence of regeneration of tracheal epithelium was observed in the surviving animals, with the transformation of squamous metaplasia to mucociliary epithelium being time-dependent. Islet of cartilage were observed in animals after 6 mo, but ring-like cartilage structures were absent, even after 16 mo. During autopsy, axial graft contractions up to 68% were observed. Serial radiographs show that most of the contraction occurred within 1 mo. The results of the MRI showed that the graft area was strongly enhanced for up to 2 mo, but was clearly reduced after 3 mo. Tracheal replacement in adult dogs using FAA or CAA is feasible. However, immaturity of the neotracheal cartilage did not allow the tissue to function as native tracheal tissue. Prolonged stenting should be considered in adult if the procedure is to be clinically contemplated.

Re: Moving towards in situ tracheal regeneration: the bionic tissue engineered transplantation approach J. Cell. Mol. Med. Vol. 14, No. 7, 2010, pp. 1877–1889

Re: Moving towards <i>in situ</i> tracheal regeneration: the bionic tissue engineered transplantation approach <i>J. Cell. Mol. Med.</i> Vol. 14, No. 7, 2010, pp. 1877–1889

Implantation site dependent differences for tracheal regeneration with iPS cells

Maximum diameter of teratoma formation in tracheal area Maximum diameter of teratoma formation in abdominal area Implantation site dependent differences for tracheal regeneration with iPS cells Mitsuyoshi Imaizumi, MD1; Yukio Nomoto, MD1; Takashi Sugino, MD2; Yuka Sato, PhD3; Koichi Omori, MD1 Department of 1Otolaryngology, 2Basic Pathology and 3Microbiology, Fukushima Medical University School of Medicine, Fukushima, Japan

Variation of Safety for Tracheal Regeneration with iPS Cells

METHOD: In this retrospective case series performed at a tertiary care center, four patients treated for papillary thyroid cancer between September 2009 and February 2010 were found to have unusual sites of metastases. We report on this series and perform a review of the current literature regarding typical sites of spread. RESULTS: Metastatic papillary thyroid cancer was found in the following locations: 1) within the sternocleidomastoid muscle, 2) occipital skin, skull, dura, and within the internal jugular vein, 3) retropharynx, and 4) in the right atrium. Review of current literature demonstrated the most common locations of regional metastases are level VI (77%), followed by level III (69%), level IV (66%), and level II (46%) of the deep cervical chain. CONCLUSION: Our patients novel sites of metastatic disease have not been depicted in previous reports. In our series all patients with unusual metastatic disease had a more aggressive course, more aggressive histology, less iodine avidity, and had worse outcomes. Although this disease most often spreads to the deep cervical chain, atypical sites of distant metastases should always be considered in patients presenting with a new mass who have known history of papillary thyroid cancer.

Moving towards in situ tracheal regeneration: the bionic tissue engineered transplantation approach

In June 2008, the world’s first whole tissue-engineered organ – the windpipe – was successfully transplanted into a 31-year-old lady, and about 18 months following surgery she is leading a near normal life without immunosuppression. This outcome has been achieved by employing three groundbreaking technologies of regenerative medicine: (i) a donor trachea first decellularized using a detergent (without denaturing the collagenous matrix), (ii) the two main autologous tracheal cells, namely mesenchymal stem cell derived cartilage-like cells and epithelial respiratory cells and (iii) a specifically designed bioreactor that reseed, before implantation, the in vitro pre-expanded and pre-differentiated autologous cells on the desired surfaces of the decellularized matrix. Given the long-term safety, efficacy and efforts using such a conventional approach and the potential advantages of regenerative implants to make them available for anyone, we have investigated a novel alternative concept how to fully avoid in vitro cell replication, expansion and differentiation, use the human native site as micro-niche, potentiate the human body’s site-specific response by adding boosting, permissive and recruitment impulses in full respect of sociological and regulatory prerequisites. This tissue-engineered approach and ongoing research in airway transplantation is reviewed and presented here.

A tissue-engineered trachea derived from a framed collagen scaffold, gingival fibroblasts and adipose-derived stem cells

In some types of tracheal disease, tracheal resection is required. For patients with tracheal resection, artificial grafts, made from collagen sponge with a spiral polypropylene stent and mesh, have been clinically used by our group. However, epithelial regeneration was confirmed to be slow. In the present study, we investigated the potential of gingival fibroblasts (GFBs) and adipose-derived stem cells (ASCs) as autologous transplanted cells in combination with artificial graft for tracheal epithelial regeneration. In in vitro co-culturing with tracheal epithelial cells, GFBs stimulated epithelial cell differentiation and reconstruction of a pseudostratified epithelium. ASCs stimulated epithelial cell proliferation and reconstruction of a multi-layered epithelium. Subsequently, we prepared three kinds of bioengineered scaffolds from GFBs and/or ASCs and implanted them into rat tracheal defects. The bioengineered scaffolds containing GFBs were covered with tracheal epithelial cells after 1 week, and highly ciliated epithelium was formed after 2 weeks of transplantation. The bioengineered scaffold containing ASCs induced thick epithelium, and then pseudostratified epithelium containing goblet cells was formed. Furthermore, the application of both GFBs and ASCs had synergistic effects on tracheal epithelial regeneration, suggesting that bioengineered scaffolds containing GFBs and ASCs are useful for hastening tracheal epithelial regeneration.

Endogenous and exogenous stem cells: a role in lung repair and use in airway tissue engineering and transplantation

Rapid repair of the denuded alveolar surface after injury is a key to survival. The respiratory tract contains several sources of endogenous adult stem cells residing within the basal layer of the upper airways, within or near pulmonary neuroendocrine cell rests, at the bronchoalveolar junction, and within the alveolar epithelial surface, which contribute to the repair of the airway wall. Bone marrow-derived adult mesenchymal stem cells circulating in blood are also involved in tracheal regeneration. However, an organism is frequently incapable of repairing serious damage and defects of the respiratory tract resulting from acute trauma, lung cancers, and chronic pulmonary and airway diseases. Therefore, replacement of the tracheal tissue should be urgently considered. The shortage of donor trachea remains a major obstacle in tracheal transplantation. However, implementation of tissue engineering and stem cell therapy-based approaches helps to successfully solve this problem. To date, huge progress has been achieved in tracheal bioengineering. Several sources of stem cells have been used for transplantation and airway reconstitution in animal models with experimentally induced tracheal defects. Most tracheal tissue engineering approaches use biodegradable three-dimensional scaffolds, which are important for neotracheal formation by promoting cell attachment, cell redifferentiation, and production of the extracellular matrix. The advances in tracheal bioengineering recently resulted in successful transplantation of the world's first bioengineered trachea. Current trends in tracheal transplantation include the use of autologous cells, development of bioactive cell-free scaffolds capable of supporting activation and differentiation of host stem cells on the site of injury, with a future perspective of using human native sites as micro-niche for potentiation of the human body's site-specific response by sequential adding, boosting, permissive, and recruitment impulses.