Comparison of the biological properties between 3D-printed and decellularized tracheal grafts.

This study sought to characterize the differences between the 3D-printed and decellularized tracheal grafts, providing the basis for the synthesis of the more reasonable and effective tissue-engineered trachea. We compared the biomechanical properties and biocompatibility of the 3D-printed tracheal graft and decellularized tracheal graft in vitro and evaluated the biocompatibility, immune rejection and inflammation of the two materials through in vivo implantation experiments. Compared with the decellularized tracheal graft, the 3D-printed tracheal graft was associated with obviously higher biomechanical properties. The results demonstrated enhanced growth of BMSCs in the decellularized tracheal graft compared to the 3D-printed one when co-culture with two tracheal graft groups. Moreover, the CCK-8 assay demonstrated significant cell proliferation on the decellularized tracheal graft. Serum IgG and IgM measured in vivo by implantation testing indicated that the 3D-Printed tracheal graft exhibited the most significant inflammatory response. HE staining indicated that the inflammatory response in the 3D-printed tracheal graft consisted mainly of eosinophils, while little inflammatory cell infiltrates were observed in the decellularized tracheal graft. CD68 immunohistochemical analysis indicated that the infiltration of macrophages was not significant in both tracheal grafts. Our findings suggest that the biomechanical properties of the 3D-printed tracheal grafts are better than the decellularized tracheal grafts. Nonetheless, the decellularized tracheal graft exhibited better biocompatibility than the 3D-printed tracheal graft.

Exosomes from 3T3-J2 promote expansion of tracheal basal cells to facilitate rapid epithelization of 3D-printed double-layer tissue engineered trachea.

Functional epithelization plays a pivotal role in maintaining long-term lumen patency of tissue-engineered trachea (TET). Due to the slow migration of autologous epithelium, spontaneous epithelization process of transplanted TET is always tardive. Seeding tracheal basal cells (TBCs) on TET before transplantation might be favorable for accelerating epithelization, but rapid expansion of TBCs in vitro is still relatively intractable. In this study, we proposed a promising expansion strategy which enables the TBCs to proliferate rapidly in vitro. TBCs were isolated from the autologous tracheal mucosae of rabbit, and co-cultured with exosomes derived from 3T3-J2 cells. After co-culture with exosomal component, TBCs could vigorously proliferate in vitro and retained their multi-potency. It was in stark contrast to that the single-cultured TBCs could only be expand to passage 2 in about 30 days, moreover, the most majority of single-cultured cells entered late apoptotic stage. On the other hand, a bionic tubular double-layer scaffold with good mechanical property and bio-compatibility was designed and fabricated by 3D printing technology. Then TET with bi-lineage cell-type was constructed in vitro by implanting autologous chondrocytes on the outer-layer of scaffold, and TBCs on the inner-layer, respectively. And then TET was pre-vascularized in vivo, and pedicled transplanted to restore long-segmental defect in recipient rabbits. It was found that the chondrocytes and TBCs seeded on double-layer scaffolds developed well as expected. And almost complete coverage with ciliated epitheliums was observed on the lumen surface of TET 2-week after operation, in comparison with that the epithelization of TET without pre-seeding of TBCs accomplished nearly 2-month after operation. In conclusion, the promising expansion strategy of TBCs together with 3D-printed double-layer scaffolds facilitate the rapid epithelization process of transplanted TET, which might be of vital significance for clinical and translational medicine.

Photocrosslinked natural hydrogel composed of hyaluronic acid and gelatin enhances cartilage regeneration of decellularized trachea matrix.

Repair of long segmental trachea defects is always a great challenge in the clinic. The key to solving this problem is to develop an ideal trachea substitute with biological function. Using of a decellularized trachea matrix based on laser micropore technique (LDTM) demonstrated the possibility of preparing ideal trachea substitutes with tubular shape and satisfactory cartilage regeneration for tissue-engineered trachea regeneration. However, as a result of the very low cell adhesion of LDTM, an overly high concentration of seeding cell is required, which greatly restricts its clinical translation. To address this issue, the current study proposed a novel strategy using a photocrosslinked natural hydrogel (PNH) carrier to enhance cell retention efficiency and improve tracheal cartilage regeneration. Our results demonstrated that PNH underwent a rapid liquid-solid phase conversion under ultraviolet light. Moreover, the photo-generated aldehyde groups in PNH could rapidly react with inherent amino groups on LDTM surfaces to form imine bonds, which efficiently immobilized the cell-PNH composite to the surfaces of LDTM and/or maintained the composite in the LDTM micropores. Therefore, PNH significantly enhanced cell-seeding efficiency and achieved both stable cell retention and homogenous cell distribution throughout the LDTM. Moreover, PNH exhibited excellent biocompatibility and low cytotoxicity, and provided a natural three-dimensional biomimetic microenvironment to efficiently promote chondrocyte survival and proliferation, extracellular matrix production, and cartilage regeneration. Most importantly, at a relatively low cell-seeding concentration, homogeneous tubular cartilage was successfully regenerated with an accurate tracheal shape, sufficient mechanical strength, good elasticity, typical lacuna structure, and cartilage-specific extracellular matrix deposition. Our findings establish a versatile and efficient cell-seeding strategy for regeneration of various tissue and provide a satisfactory trachea substitute for repair and functional reconstruction of long segmental tracheal defects.

Prevascularized Tracheal Scaffolds Using the Platysma Flap for Enhanced Tracheal Regeneration.

OBJECTIVES: One of the greatest hurdles in tracheal tissue engineering is insufficient vascularization, which leads to delayed mucosal regeneration, inflammation, and restenosis. This study investigated whether a prevascularized segmental tracheal substitute using platysma can enhance tracheal mucosal regeneration. METHODS: Three-dimensional (3D) printed scaffolds with (group M) or without (group S) Matrigel coating were implanted under the feeding vessels of the platysma in New Zealand White rabbits (n = 3) to induce vascularization. After 1 or 2 weeks, tracheal defects were created and vascularized scaffolds with feeders of the platysma were transplanted as rotational flaps. As controls, scaffolds with or without Matrigel coating was transplanted into a tracheal defect without prevascularization. Airway patency and epithelization were examined using a rigid bronchoscope every 2 weeks. Surviving animals were euthanized at 24 weeks, and microcomputed tomography and histological evaluation were performed. RESULTS: Animals with 2 weeks of prevascularization showed longer survival than animals with 0 or 1 weeks of prevascularization regardless of the Matrigel coating. Wider airway patency was observed in group M than group S. Group M showed migration of epithelium over the scaffold from 4 weeks after transplantation and complete coverage with epithelium at 12 weeks, whereas group S showed migration of the epithelium from 14 weeks and incomplete coverage with epithelium even at 24 weeks. CONCLUSION: This two-step method, utilizing the platysma as an in vivo bioreactor, may be a promising approach to achieve long-term survival and enhanced luminal patency. Matrigel coating on the scaffold had a synergistic effect on epithelial regeneration. LEVEL OF EVIDENCE: NA Laryngoscope, 131:1732-1740, 2021.

Deconstructing tissue engineered trachea: Assessing the role of synthetic scaffolds, segmental replacement and cell seeding on graft performance.

The ideal construct for tracheal replacement remains elusive in the management of long segment airway defects. Tissue engineered tracheal grafts (TETG) have been limited by the development of graft stenosis or collapse, infection, or lack of an epithelial lining. We applied a mouse model of orthotopic airway surgery to assess the impact of three critical barriers encountered in clinical applications: the scaffold, the extent of intervention, and the impact of cell seeding and characterized their impact on graft performance. First, synthetic tracheal scaffolds electrospun from polyethylene terephthalate / polyurethane (PET/PU) were orthotopically implanted in anterior tracheal defects of C57BL/6 mice. Scaffolds demonstrated complete coverage with ciliated respiratory epithelium by 2 weeks. Epithelial migration was accompanied by macrophage infiltration which persisted at long term (>6 weeks) time points. We then assessed the impact of segmental tracheal implantation using syngeneic trachea as a surrogate for the ideal tracheal replacement. Graft recovery involved local upregulation of epithelial progenitor populations and there was no evidence of graft stenosis or necrosis. Implantation of electrospun synthetic tracheal scaffold for segmental replacement resulted in respiratory distress and required euthanasia at an early time point. There was limited epithelial coverage of the scaffold with and without seeded bone marrow-derived mononuclear cells (BM-MNCs). We conclude that synthetic scaffolds support re-epithelialization in orthotopic patch implantation, syngeneic graft integration occurs with focal repair mechanisms, however epithelialization in segmental synthetic scaffolds is limited and is not influenced by cell seeding. STATEMENT OF SIGNIFICANCE: The life-threatening nature of long-segment tracheal defects has led to clinical use of tissue engineered tracheal grafts in the last decade for cases of compassionate use. However, the ideal tracheal reconstruction using tissue-engineered tracheal grafts (TETG) has not been clarified. We addressed the core challenges in tissue engineered tracheal replacement (re-epithelialization and graft patency) by defining the role of cell seeding with autologous bone marrow-derived mononuclear cells, the mechanism of respiratory epithelialization and proliferation, and the role of the inflammatory immune response in regeneration. This research will facilitate comprehensive understanding of cellular regeneration and neotissue formation on TETG, which will permit targeted therapies for accelerating re-epithelialization and attenuating stenosis in tissue engineered airway replacement.

3D printing technology and tissue engineering technology in tracheal replacement: Application and hotspot research / 中国组织工程研究

BACKGROUND: Functional tracheal reconstruction remains a surgical challenge due to the lack of satisfactory tracheal substitutes. OBJECTIVE: To review the research hotspot, clinical application, and main obstacles of tissue-engineered trachea METHODS: A computer-based search of PubMed, Medline, and WanFang databases was performed to retrieve relevant articles published from 2004 to 2019 with the search terms “3D printing, tissue-engineered trachea, trachea reconstruction, tracheal replacement” in English and Chinese. A total of 47 literatures were included in the final analysis. RESULTS AND CONCLUSION: At present, the methods of tracheal reconstruction mainly include artificial tracheal transplantation, allotransplantation, autologous tissue transplantation and tissue-engineered tracheal transplantation. Artificial trachea transplants often fail due to rupture, infection and narrowing of the trachea. Allotransplantation requires long-term immunosuppressive therapy, and death is often caused by necrosis and infection because of insufficient angiogenesis after transplantation. Autogenous tissue has limited ability to replicate the structure and function of the trachea and also has surgical trauma. Tissue-engineered trachea can simulate the biological structure and function similar to natural trachea by selecting suitable scaffold materials and implanting seed cells evenly in the scaffold. It seems to be an ideal tracheal substitute. An intact tracheal scaffold was prepared with biodegradable material using 3D printing technology combined with tissue engineering technology and then implanted into the tissue-engineered trachea cultured with mesenchymal stem cells. This provides a new approach to long-segment tracheal defect reconstruction.

Long-segmental tracheal reconstruction in rabbits with pedicled Tissue-engineered trachea based on a 3D-printed scaffold.

Long-segmental tracheal defects constitute an intractable clinical problem, due to the lack of satisfactory tracheal substitutes for surgical reconstruction. Tissue engineered artificial substitutes could represent a promising approach to tackle this challenge. In our current study, tissue-engineered trachea, based on a 3D-printed poly (l-lactic acid) (PLLA) scaffold with similar morphology to the native trachea of rabbits, was used for segmental tracheal reconstruction. The 3D-printed scaffolds were seeded with chondrocytes obtained from autologous auricula, dynamically pre-cultured in vitro for 2 weeks, and pre-vascularized in vivo for another 2 weeks to generate an integrated segmental trachea organoid unit. Then, segmental tracheal defects in rabbits were restored by transplanting the engineered tracheal substitute with pedicled muscular flaps. We found that the combination of in vitro pre-culture and in vivo pre-vascularization successfully generated a segmental tracheal substitute with bionic structure and mechanical properties similar to the native trachea of rabbits. Moreover, the stable blood supply provided by the pedicled muscular flaps facilitated the survival of chondrocytes and accelerated epithelialization, thereby improving the survival rate. The segmental trachea substitute engineered by a 3D-printed scaffold, in vitro pre-culture, and in vivo pre-vascularization enhanced survival in an early stage post-operation, presenting a promising approach for surgical reconstruction of long segmental tracheal defects. STATEMENT OF SIGNIFICANCE: We found that the combination of in vitro pre-culture and in vivo pre-vascularization successfully generated a segmental tracheal substitute with bionic structure and mechanical properties similar to the native trachea of rabbits. Moreover, the stable blood supply provided by the pedicled muscular flaps facilitated the survival of chondrocytes and accelerated epithelialization, thereby improving the survival rate of the rabbits. The segmental trachea substitute engineered by a 3D-printed scaffold, in vitro pre-culture, and in vivo pre-vascularization enhanced survival in an early stage post-operation, presenting a promising approach for surgical reconstruction of long segmental tracheal defects.

Tissue-engineered trachea from a 3D-printed scaffold enhances whole-segment tracheal repair in a goat model.

Traditional treatment therapies for tracheal stenosis often cause severe post-operative complications. To solve the current difficulties, novel and more suitable long-term treatments are needed. A whole-segment tissue-engineered trachea (TET) representing the native goat trachea was 3D printed using a poly(caprolactone) (PCL) scaffold engineered with autologous auricular cartilage cells. The TET underwent mechanical analysis followed by in vivo implantations in order to evaluate the clinical feasibility and potential. The 3D-printed scaffolds were successfully cellularized, as observed by scanning electron microscopy. Mechanical force compression studies revealed that both PCL scaffolds and TETs have a more robust compressive strength than does the native trachea. In vivo implantation of TETs in the experimental group resulted in significantly higher mean post-operative survival times, 65.00 ± 24.01 days (n = 5), when compared with the control group, which received autologous trachea grafts, 17.60 ± 3.51 days (n = 5). Although tracheal narrowing was confirmed by bronchoscopy and computed tomography examination in the experimental group, tissue necrosis was only observed in the control group. Furthermore, an encouraging epithelial-like tissue formation was observed in the TETs after transplantation. This large animal study provides potential preclinical evidence around the employment of an orthotopic transplantation of a whole 3D-printed TET.

Mouse Model of Tracheal Replacement With Electrospun Nanofiber Scaffolds.

OBJECTIVES: The clinical experience with tissue-engineered tracheal grafts (TETGs) has been fraught with graft stenosis and delayed epithelialization. A mouse model of orthotopic replacement that recapitulates the clinical findings would facilitate the study of the cellular and molecular mechanisms underlying graft stenosis. METHODS: Electrospun nanofiber tracheal scaffolds were created using nonresorbable (polyethylene terephthalate + polyurethane) and co-electrospun resorbable (polylactide-co-caprolactone/polyglycolic acid) polymers (n = 10/group). Biomechanical testing was performed to compare load displacement of nanofiber scaffolds to native mouse tracheas. Mice underwent orthotopic tracheal replacement with syngeneic grafts (n = 5) and nonresorbable (n = 10) and resorbable (n = 10) scaffolds. Tissue at the anastomosis was evaluated using hematoxylin and eosin (H&E), K5+ basal cells were evaluated with the help of immunofluorescence testing, and cellular infiltration of the scaffold was quantified. Micro computed tomography was performed to assess graft patency and correlate radiographic and histologic findings with respiratory symptoms. RESULTS: Synthetic scaffolds were supraphysiologic in compression tests compared to native mouse trachea ( P < .0001). Nonresorbable scaffolds were stiffer than resorbable scaffolds ( P = .0004). Eighty percent of syngeneic recipients survived to the study endpoint of 60 days postoperatively. Mean survival with nonresorbable scaffolds was 11.40 ± 7.31 days and 6.70 ± 3.95 days with resorbable scaffolds ( P = .095). Stenosis manifested with tissue overgrowth in nonresorbable scaffolds and malacia in resorbable scaffolds. Quantification of scaffold cellular infiltration correlated with length of survival in resorbable scaffolds (R2 = 0.95, P = .0051). Micro computed tomography demonstrated the development of graft stenosis at the distal anastomosis on day 5 and progressed until euthanasia was performed on day 11. CONCLUSION: Graft stenosis seen in orthotopic tracheal replacement with synthetic tracheal scaffolds can be modeled in mice. The wide array of lineage tracing and transgenic mouse models available will permit future investigation of the cellular and molecular mechanisms underlying TETG stenosis.

[The biocompatibility and immunogenicity study of decellularized tracheal matrix].

Objective: To investigate the biocompatibility and immunogenicity of the tracheal matrix decellularized by sodium perchlorate (NaClO 4). Methods: Bone marrow mesenchymal stem cells (BMSCs) were divided from 2-month-old New Zealand white rabbits. The trachea of 6-month-old New Zealand white rabbits were trimmed to a length of 1.5 cm and randomly divided into control group (group A 1, n=5, just stripped the loose connective tissue outside the trachea) and experimental group (group B 1, n=5, decellularized by improved NaClO 4 immersion method). The cytotoxicity of the scaffold leaching solution was detected by MTT assay, and the major histocompatibility complex (MHC) expression was detected by immunohistochemical method. The 4th generation of BMSCs were seeded onto the scaffold of 2 groups, and the cell activity around the material was observed by inverted microscope after Giemsa staining at 48 hours, while the cells states on the scaffold were observed at 7 and 14 days after culturing by scanning electron microscope. Another 10 6-month-old New Zealand white rabbits were randomly divided into control group (group A 2, n=5) and experimental group (group B 2, n=5), which implanted the native trachea and decellularized tracheal matrix into the subcutaneous sac of the back neck, respectively. The serum immunoglobulin IgM and IgG contents were analysed at 5, 10, 15, 20, 25, and 30 days after operation, and HE staining observation was performed at 30 days after operation. Results: MTT assay showed that the proliferation activity of BMSCs cultured in the leach liquor of group B 1 was well, showing no significant difference when compared with group A 1 and negative control group with pure culture medium ( P>0.05). The immunohistochemical staining showed that the decellularized process could significantly reducing the antigenicity of matrix materials. Giemsa staining showed that BMSCs grew well around the two tracheal matrixs (groups A 1 and B 1) in vitro. Scanning electron microscope observation showed that the cells were attached to the outer wall of the tracheal material in group A 1, which present a flat, round, oval shaped, tightly arranged cells and cluster distribution; and in group B 1, the cells formed a single lamellar sheet cover the outer wall of the tracheal material, whose morphology was similar to that in group A 1, and the growth trend was better. In vivo experimental results showed that the rejection of group B 2 was lower than that of group A 2. The contens of IgM and IgG in group A 2 were significantly higher than those in group B 2 at each time point after operation ( P<0.05). HE staining showed no signs of rejection, macrophagocyte, or lymphocyte infiltration occurred, and the collagen fibers maintained their integrity in group B 2. Conclusion: The decellularized matrix treated by NaClO 4 has a fine biocompatibility, while its immunogenicity decreased, and it is suitable for the scaffold material for constructing of tissue engineered trachea.

Biomechanical properties and cellular biocompatibility of 3D printed tracheal graft.

The goals of our study were to evaluate the biomechanical properties and cellular biocompatibility of 3D printed tracheal graft fabricated by polycaprolactone (PCL). Compared with native tracheal patch, there was a significant increase in maximum stress and elastic modulus for 3DP tracheal graft (p < 0.05). BMSCs were co-cultured under four different conditions to investigate cytotoxicity of the graft: (1) co-cultured with normal culture medium, as blank control; (2) co-cultured with perfluoropropylene, as negative control; (3) co-cultured with 3DP tracheal graft; and (4) co-cultured with polyvinyl chloride, as positive control. Moreover, the results of SRB assay showed that compared with blank and negative control group, there was no significant difference in the cell proliferation of 3DP tracheal graft group for 21 days (p > 0.05). These results revealed that 3DP tracheal graft in our study has favorable cellular biocompatibility and biomechanical properties, and, therefore, will be a promising alternative for tissue-engineered trachea.

Tissue-engineered trachea regeneration using decellularized trachea matrix treated with laser micropore technique.

Tissue-engineered trachea provides a promising approach for reconstruction of long segmental tracheal defects. However, a lack of ideal biodegradable scaffolds greatly restricts its clinical translation. Decellularized trachea matrix (DTM) is considered a proper scaffold for trachea cartilage regeneration owing to natural tubular structure, cartilage matrix components, and biodegradability. However, cell residual and low porosity of DTM easily result in immunogenicity and incomplete cartilage regeneration. To address these problems, a laser micropore technique (LMT) was applied in the current study to modify trachea sample porosity to facilitate decellular treatment and cell ingrowth. Decellularization processing demonstrated that cells in LMT treated samples were more easily removed compared with untreated native trachea. Furthermore, after optimizing the protocols of LMT and decellular treatments, the LMT-treated DTM (LDTM) could retain their original tubular shape with only mild extracellular matrix damage. After seeding with chondrocytes and culture in vitro for 8 weeks, the cell-LDTM constructs formed tubular cartilage with relatively homogenous cell distribution in both micropores and bilateral surfaces. In vivo results further confirmed that the constructs could form mature tubular cartilage with increased DNA and cartilage matrix contents, as well as enhanced mechanical strength, compared with native trachea. Collectively, these results indicate that LDTM is an ideal scaffold for tubular cartilage regeneration and, thus, provides a promising strategy for functional reconstruction of trachea cartilage. STATEMENT OF SIGNIFICANCE: Lacking ideal biodegradable scaffolds greatly restricts development of tissue-engineered trachea. Decellularized trachea matrix (DTM) is considered a proper scaffold for trachea cartilage regeneration. However, cell residual and low porosity of DTM easily result in immunogenicity and incomplete cartilage regeneration. By laser micropore technique (LMT), the current study efficiently enhanced the porosity and decellularized efficacy of DTM. The LMT-treated DTM basically retained the original tubular shape with mild matrix damage. After chondrocyte seeding followed by in vitro culture and in vivo implantation, the constructs formed mature tubular cartilage with matrix content and mechanical strength similar to native trachea. The current study provides an ideal scaffold and a promising strategy for cartilage regeneration and functional reconstruction of trachea.

Endoscopic management of tissue-engineered tracheal graft stenosis in an ovine model.

OBJECTIVE: To evaluate the safety and efficacy of bronchoscopic interventions in the management of tissue-engineered tracheal graft (TETG) stenosis. STUDY DESIGN: Animal research study. METHODS: TETGs were constructed with seeded autologous bone marrow-derived mononuclear cells on a bioartificial graft. Eight sheep underwent tracheal resection and orthotopic implantation of this construct. Animals were monitored by bronchoscopy and fluoroscopy at 3 weeks, 6 weeks, 3 months, and 4 months. Bronchoscopic interventions, including dilation and stenting, were performed to manage graft stenosis. Postdilation measurements were obtained endoscopically and fluoroscopically. RESULTS: Seven dilations were performed in six animals. At the point of maximal stenosis, the lumen measured 44.6 ± 8.4 mm2 predilation and 50.7 ± 14.1 postdilation by bronchoscopy (P = 0.3517). By fluoroscopic imaging, the airway was 55.9 ± 12.9 mm2 predilation and 65.9 ± 22.4 mm2 postdilation (P = 0.1303). Stents were placed 17 times in six animals. Pre- and poststenting lumen sizes were 62.8 ± 38.8 mm2 and 80.1 ± 54.5 mm2 by bronchoscopy (P = 0.6169) and 77.1 ± 38.9 mm2 and 104 ± 60.7 mm2 by fluoroscopy (P = 0.0825). Mortality after intervention was 67% with dilation and 0% with stenting (P = 0.0004). The average days between bronchoscopy were 8 ± 2 for the dilation group and 26 ± 17 in the stenting group (P = 0.05). One hundred percent of dilations and 29% of stent placements required urgent follow-up bronchoscopy (P = 0.05). CONCLUSION: Dilation has limited efficacy for managing TETG stenosis, whereas stenting has a more lasting clinical effect. LEVEL OF EVIDENCE: NA. Laryngoscope, 127:2219-2224, 2017.

Current Solutions for Long-Segment Tracheal Reconstruction.

This article is a continuation of previous reviews about the appropriate method for long-segment tracheal reconstruction. We attempted to cover the most recent, successful and promising results of the different solutions for reconstruction that are rather innovative and suitable for imminent clinical application. Latest efforts to minimize the limitations associated with each method have been covered as well. In summary, autologous and allogenic tissue reconstruction of the trachea have been successful methods for reconstruction experimentally and clinically. Autologous tissues were best utilized clinically to enhance revascularization, whether as a definitive airway or as an adjunct to allografts or tissue-engineered trachea (TET). Allogenic tissue transplantation is, currently, the most suitable for clinical application, especially after elimination of the need for immunosuppressive therapy with unlimited supply of tissues. Similar results have been reported in many studies that used TET. However, clinical application of this method was limited to use as a salvage treatment in a few studies with promising results. These results still need to be solidified by further clinical and long-term follow-up reports. Combining different methods of reconstruction was often required to establish a physiological rather than an anatomical trachea and have shown superior outcomes.

PLGA-PTMC-Cultured Bone Mesenchymal Stem Cell Scaffold Enhances Cartilage Regeneration in Tissue-Engineered Tracheal Transplantation.

The treatment of long-segment tracheal defect requires the transplantation of effective tracheal substitute, and the tissue-engineered trachea (TET) has been proposed as an ideal tracheal substitute. The major cause of the failure of segmental tracheal defect reconstruction by TET is airway collapse caused by the chondromalacia of TET cartilage. The key to maintain the TET structure is the regeneration of chondrocytes in cartilage, which can secrete plenty of cartilage matrices. To address the problem of the chondromalacia of TET cartilage, this study proposed an improved strategy. We designed a new cell sheet scaffold using the poly(lactic-co-glycolic acid) (PLGA) and poly(trimethylene carbonate) (PTMC) to make a porous membrane for seeding cells, and used the PLGA-PTMC cell-scaffold to pack the decellularized allogeneic trachea to construct a new type of TET. The TET was then implanted in the subcutaneous tissue for vascularization for 2 weeks. Orthotopic transplantation was then performed after implantation. The efficiency of the TET we designed was analyzed by histological examination and biomechanical analyses 4 weeks after surgery. Four weeks after surgery, both the number of chondrocytes and the amount of cartilage matrix were significantly higher than those contained in the traditional stem-cell-based TET. Besides, the coefficient of stiffness of TET was significantly larger than the traditional TET. This study provided a promising approach for the long-term functional reconstruction of long-segment tracheal defect, and the TET we designed had potential application prospects in the field of TET reconstruction.

Cellular biocompatibility and biomechanical properties of 3D printed tracheal graft / 国际生物医学工程杂志

Objective To prepare 3D printed tracheal graft and investigate its cellular biocompatibility and biomechanical properties.Methods Bone marrow was isolated from tibial plateau of young New Zealand white rabbit,and bone mesenchymal stem cells (BMSCs) were obtained by whole bone marrow culture method and adherent purification method.Biomechanical test was performed for 3D printed trachea graft.After co-cultured of 3D printed trachea graft and BMSCs,cell morphology was observed and the proliferation index of the cells on 3D printed trachea graft was quantified using sulforhodamine B (SRB) assay.Results 3D printed trachea graft showed excellent biomechanical properties.Cell morphology was normal and cells grew well after co-culture with 3D printed trachea graft.The SRB assay indicated good proliferation of BMSCs on 3D printed trachea graft.Conclusions 3D printed trachea graft shows favorable cellular biocompatibility and biomechanical properties,and therefore can be used as a scaffold material for tissue-engineered trachea.

Biological characteristics of newborn rabbit tracheal chondrocytes / 中国病理生理杂志

[ ABSTRACT] AIM:To investigate the biological characteristics of newborn rabbit tracheal chondrocytes in vitro. METHODS:Newborn rabbit tracheal chondrocytes were obtained by the method of enzyme digestion, and then cultured in monolayer in vitro.Morphological and growth observations were performed under inverted phase contrast microscope.The ultrastructures of the cells were observed under scanning electron microscope and transmission electron microscope.The bi-ological characteristics of secreted extracellular matrix components were detected by real-time PCR, immunocytochemistry staining and toluidine blue staining.RESULTS: Newborn rabbit tracheal chondrocytes isolated and cultured in vitro showed short triangular or irregular shapes, and adherent growth very well.The ultrastructures of the cells showed pore and abundant cytoplasm and organelles, with a lot of protein secretions in the cells.The chondrocytes expressed the mRNA of collagen I, collagen II and proteoglycans, mainly collagen II and proteoglycans.Immunocytochemistry staining showed col-lagen II and SOX9 positive, and collagen I weakly positive.Toluidine blue staining was also positive.CONCLUSION:Enzyme digestion and monolayer culture are suitable method to obtain newborn rabbit tracheal chondrocytes.These cells, secreting extracellular matrix components, are able to be selected as seed cells for tissue engineering of trachea in vitro, and used to study the therapeutic method for neonatal rabbit tracheal stenosis.