Tracheal Cartilage Regeneration Research Articles

Pluronic F127 hydrogel-loaded extracellular vesicles from adipose-derived mesenchymal stem cells promote tracheal cartilage regeneration via SCNN1B delivery

Extracellular vesicles (EVs) derived from adipose-derived mesenchymal stem cells (AMSC-EVs) have been highlighted as a cell-free therapy due to their regenerative capability to enhance tissue and organ regeneration. Herein, we aimed to examine the mechanism of PF127-hydrogel@AMSC-EVs in promoting tracheal cartilage defect repair. Based on bioinformatics methods, SCNN1B was identified as a key gene for the osteogenic differentiation of AMSCs induced by AMSC-EVs. EVs were isolated from rat AMSCs and then loaded onto thermo-sensitive PF-127 hydrogel to develop PF127-hydrogel@AMSC-EVs. It was established that PF127-hydrogel@AMSC-EVs could effectively deliver SCNN1B into AMSCs, where SCNN1B promoted AMSC osteogenic differentiation. The promotive effect was evidenced by enhanced ALP activity, extracellular matrix mineralization, and expression of s-glycosaminoglycan, RUNX2, OCN, collagen II, PERK, and ATF4. Furthermore, the in vivo experiments revealed that PF127-hydrogel@AMSC-SCNN1B-EVs stimulated tracheal cartilage regeneration in rats through PERK/ATF4 signaling axis activation. Therefore, PF127-hydrogel@AMSC-SCNN1B-EVs may be a novel cell-free biomaterial to facilitate tracheal cartilage regeneration and cartilage injury repair.

Temporal control in shell-core structured nanofilm for tracheal cartilage regeneration: synergistic optimization of anti-inflammation and chondrogenesis.

Cartilage tissue engineering offers hope for tracheal cartilage defect repair. Establishing an anti-inflammatory microenvironment stands as a prerequisite for successful tracheal cartilage restoration, especially in immunocompetent animals. Hence, scaffolds inducing an anti-inflammatory response before chondrogenesis are crucial for effectively addressing tracheal cartilage defects. Herein, we develop a shell-core structured PLGA@ICA-GT@KGN nanofilm using poly(lactic-co-glycolic acid) (PLGA) and icariin (ICA, an anti-inflammatory drug) as the shell layer and gelatin (GT) and kartogenin (KGN, a chondrogenic factor) as the core via coaxial electrospinning technology. The resultant PLGA@ICA-GT@KGN nanofilm exhibited a characteristic fibrous structure and demonstrated high biocompatibility. Notably, it showcased sustained release characteristics, releasing ICA within the initial 0 to 15 days and gradually releasing KGN between 11 and 29 days. Subsequent in vitro analysis revealed the potent anti-inflammatory capabilities of the released ICA from the shell layer, while the KGN released from the core layer effectively induced chondrogenic differentiation of bone marrow stem cells (BMSCs). Following this, the synthesized PLGA@ICA-GT@KGN nanofilms were loaded with BMSCs and stacked layer by layer, adhering to a 'sandwich model' to form a composite sandwich construct. This construct was then utilized to repair circular tracheal defects in a rabbit model. The sequential release of ICA and KGN facilitated by the PLGA@ICA-GT@KGN nanofilm established an anti-inflammatory microenvironment before initiating chondrogenic induction, leading to effective tracheal cartilage restoration. This study underscores the significance of shell-core structured nanofilms in temporally regulating anti-inflammation and chondrogenesis. This approach offers a novel perspective for addressing tracheal cartilage defects, potentially revolutionizing their treatment methodologies.

Global Bibliometric and Visualized Analysis of Tracheal Tissue Engineering Research.

The development of tracheal tissue engineering (TTE) has seen a rapid growth in recent years. The purpose of this study was to investigate the global status, trends, and hotspots of TTE research based on bibliometrics and visualization analysis. Publications related to TTE were retrieved and included in the Web of Science Core Collection. VOSviewer and CiteSpace were used to generate knowledge maps. Six hundred fifty-five publications were identified, and the quantity of the annual publications worldwide was on the increase. International collaboration is a widespread reality. The United States led the world in the field of trachea tissue engineering, whereas University College London was the institution with the greatest contribution. In addition, Biomaterials had a great influence in this field, attracting the largest number of papers. Moreover, the topics of TTE research largely concentrated on the biomechanical scaffold preparation, the vascularization and epithelialization of scaffold, the tracheal cartilage regeneration, and the tissue-engineered tracheal transplantation. And the research on the application of decellularization and 3D printing for the construction of a tissue-engineered trachea was likely to receive more widespread attention in the future. Impact statement In recent years, tracheal tissue engineering (TTE) has experienced rapid growth. In this study, we investigated the worldwide status and trends of TTE research, and revealed the countries, institutions, journals, and authors that had made significant contributions to the field of TTE. Moreover, the possible research hotspots in the future were predicted. According to our research, researchers can gain a better understanding of the trends in this field, and stay informed of the most current research by tracking key journals, institutions, and authors.

3D-bioprinted cell-laden hydrogel with anti-inflammatory and anti-bacterial activities for tracheal cartilage regeneration and restoration

Despite the notable advances in tissue-engineered tracheal cartilage (TETC), there remain several challenges that need to be addressed, such as uneven cell distribution for cartilage formation, customized C-shaped tracheal morphology, local inflammatory reactions, and infections. To overcome these challenges, this study proposed the addition of icariin (ICA) and chitosan (CS) into a gelatin methacryloyl (GelMA) hydrogel to develop a new ICA/CS/GelMA hydrogel with anti-inflammatory and anti-bacterial properties, and three-dimensional (3D)-bioprinting feasibility. The aim of this study was to construct a TETC, a customized C-shaped cartilage structure, with uniform chondrocyte distribution as well as anti-inflammatory and anti-bacterial functions. Our results confirmed that ICA/CS/GelMA hydrogel provides desirable rheological properties, suitable printability, favorable biocompatibility, and simulated microenvironments for chondrogenesis. Moreover, the addition of ICA stimulated chondrocyte proliferation, extracellular matrix synthesis, and anti-inflammatory ability, while the encapsulation of CS enhanced the hydrogels’ anti-bacterial ability. All these led to the formation of an enhanced TETC after submuscular implantation and an elevated survival rate of experimental rabbits after orthotopic tracheal transplantation. This study provides a reliable cell-laden hydrogel with anti-inflammatory and anti-bacterial activities, suitable printability, and significant advancements in in vivo cartilage regeneration and in situ tracheal cartilage restoration.

Curcumin loaded polycaprolactone scaffold capable of anti-inflammation to enhance tracheal cartilage regeneration

An inflammatory reaction is one of the primary reasons for deteriorating tracheal cartilage regeneration in vivo, hindering tracheal defect restoration with prominent fibrosis and granulation hyperplasia. Therefore, effective anti-inflammatory therapeutic strategies may considerably promote tracheal cartilage regeneration and tracheal defect restoration. We prepared a porous ring-shaped Cur/PCL scaffold by loading curcumin (Cur), an anti-inflammatory compound, into polycaprolactone (PCL) using the supercritical CO2 foaming method. Our results confirmed that the Cur/PCL possessed similar porosity with the PCL alone scaffold. The Cur loaded within the Cur/PCL scaffold exhibited sustained release kinetics, stability and a favorable anti-inflammation property in vitro. Moreover, The Cur/PCL scaffold displayed gratifying biocompatibility and supported ring-shaped tracheal cartilage regeneration. In addition, the engineered ring-shaped tracheal cartilage in the Cur/PCL group showed a potent anti-inflammatory effect on maintaining the cartilaginous phenotype and enhanced tracheal cartilage regeneration when subcutaneously implanted into an autologous rabbit. Furthermore, the engineered cartilage in the Cur/PCL group exerted a favorable anti-inflammatory capacity to significantly suppress fibrogenesis and granulation hyperplasia, enhancing tracheal cartilage regeneration and elevating survival rate of experimental rabbits after orthotopic transplantation. This study provides an effective strategy to prepare an anti-inflammatory scaffold for enhancing cartilage regeneration in vivo and repairing tracheal cartilage defects.

Utilization of an Acellular Cartilage Matrix‐Based Photocrosslinking Hydrogel for Tracheal Cartilage Regeneration and Circumferential Tracheal Repair

AbstractAcellular cartilage matrix (ACM)‐based hydrogels hold promise for use in the context of tracheal cartilage regeneration owing to the ease with which they can be molded into specific shapes and their intrinsic cartilage‐specific microenvironmental properties. In this report, an acrylic anhydride‐modified ACM (Ac‐matrix) hydrogel is fabricated for utilization in the context of tracheal cartilage regeneration and circumferential tracheal repair. Hydrogels are successfully prepared with a range of Ac‐matrix concentrations (4%, 6%, and 8%) and found that mechanical strength increases with rising Ac‐matrix concentrations whereas swelling and degradation ratio values decline. These Ac‐matrix hydrogels exhibit satisfactory cytocompatibility when loaded with chondrocytes. Chondrocyte‐loaded 6% hydrogels are able to support cartilage formation both in vitro and in vivo across a range of chondrocyte densities (5, 10, 20, and 40 million chondrocytes per mL), with increasing density being conducive to more homogenous cartilage formation. A homogeneously tubular tracheal cartilage construct is additionally generated and supports circumferential tracheal reconstruction in a rabbit tracheal lesion model system, with a biological structure and function consistent with those of the native trachea. These results suggest that this Ac‐matrix hydrogel platform is well‐suited to use in the context of tracheal cartilage regeneration, thus offering the potential to support circumferential tracheal reconstruction.

Curcumin Loaded Polycaprolactone Scaffold Capable of Anti-Inflammation to Enhance Tracheal Cartilage Regeneration

Curcumin Loaded Polycaprolactone Scaffold Capable of Anti-Inflammation to Enhance Tracheal Cartilage Regeneration

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.

Nanofibrillar Decellularized Wharton's Jelly Matrix for Segmental Tracheal Repair

AbstractWharton's jelly (WJ) is considered a potential scaffold in tissue‐engineered trachea for its similar composition and function to cartilage tissue. However, the feasibility of using WJ to construct engineered neocartilage tissue has not been reported, let alone tubular tracheal cartilage regeneration and segmental tracheal lesion repair. Here, electrospun nanofibrous membranes composed of three different decellularized WJ matrix (DWJM)/poly(ε‐caprolactone) (PCL) ratios (8:2, 5:5, and 2:8) are fabricated. The results demonstrate improved degradation speed, absorption, and cell adhesion capacity but weakened mechanical properties with increased DWJM content, but satisfactory homogeneous cartilage regeneration is only achieved in the DWJM/PCL (8:2) group after 12 weeks in vivo culture. Furthermore, homogeneous, 3D, tubular, trachea‐shaped cartilage is constructed with a controllable lumen diameter and wall thickness based on the 2D nanofibrous membrane using a modified sandwich model, in which the chondrocyte‐membrane construct is rolled around a silicon tube. Most importantly, by combining the above schemes with previously established vascularization and epithelialization techniques, chondrification, vascularization, and epithelialization are achieved simultaneously thus realizing long‐term (6 months) circumferential tracheal lesion repair in a rabbit model with a biological structure and function similar to that of native trachea, representing a promising approach for the clinical application of tracheal tissue engineering.

Porous decellularized trachea scaffold prepared by a laser micropore technique

Rapid development of tissue engineering technology provides new methods for tracheal cartilage regeneration. However, the current lack of an ideal scaffold makes engineering of trachea cartilage tissue into a three-dimensional (3-D) tubular structure a great challenge. Although a decellularized trachea matrix (DTM) has become a recognized scaffold for trachea cartilage regeneration, it is difficult for cells to detach from or penetrate the matrix because of its non-porous structure. To tackle these problems, a laser micropore technique (LMT) was applied in the current study to enhance trachea sample porosity, and facilitate decellularizing treatment and cell ingrowth. Furthermore, after optimizing LMT and decellularizing treatment parameters, LMT-treated DTM (LDTM) retained its natural tubular structure with only minor extracellular matrix damage. Moreover, compared with DTM, the current study showed that LDTM significantly improved the adherence rate of cells with perfect cell biocompatibility. Moreover, the optimal implantation cell density for chondrogenesis with LDTM was determined to be 1 × 108 cells/ml. Collectively, the results suggest that the novel LDTM is an ideal scaffold for trachea tissue engineering.

Enhanced chondrogenic differentiation of human mesenchymal stems cells on citric acid-modified chitosan hydrogel for tracheal cartilage regeneration applications.

Congenital tracheal stenosis in infants and children is a worldwide clinical problem. Tissue engineering is a promising method for correcting long segmental tracheal defects. Nonetheless, the lack of desirable scaffolds always limits the development and applications of tissue engineering in clinical practice. In this study, a citric-acid-functionalized chitosan (CC) hydrogel was fabricated by a freeze–thaw method. Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) confirmed that citric acid was successfully attached to the chitosan hydrogel. Scanning electron microscopy (SEM) images and compression tests showed that the CC hydrogel had an interconnected porous structure and better wet mechanical properties. Using morphological and proliferation analyses, cell biocompatibility of the CC hydrogel was shown by culturing human mesenchymal stem cells (hMSCs) on it. Specific expression of cartilage-related markers was analyzed by real-time polymerase chain reaction and western blotting. The expression of chondrocytic markers was strongly upregulated in the culture on the CC hydrogel. Hematoxylin and eosin staining revealed that the cells had the characteristic shape of chondrocytes and clustered into the CC hydrogel. Both Alcian blue staining and a sulfated glycosaminoglycan (sGAG) assay indicated that the CC hydrogel promoted the expression of glycosaminoglycans (GAGs). In a nutshell, these results suggested that the CC hydrogel enhanced chondrogenic differentiation of hMSCs. Thus, the newly developed CC hydrogel may be a promising tissue-engineered scaffold for tracheal cartilage regeneration.

Evaluation of the potential of kartogenin encapsulated poly(L-lactic acid-co-caprolactone)/collagen nanofibers for tracheal cartilage regeneration.

Tracheal stenosis is one of major challenging issues in clinical medicine because of the poor intrinsic ability of tracheal cartilage for repair. Tissue engineering provides an alternative method for the treatment of tracheal defects by generating replacement tracheal structures. In this study, we fabricated coaxial electrospun fibers using poly(L-lactic acid-co-caprolactone) and collagen solution as shell fluid and kartogenin solution as core fluid. Scanning electron microscope and transmission electron microscope images demonstrated that nanofibers had uniform and smooth structure. The kartogenin released from the scaffolds in a sustained and stable manner for about 2 months. The bioactivity of released kartogenin was evaluated by its effect on maintain the synthesis of type II collagen and glycosaminoglycans by chondrocytes. The proliferation and morphology analyses of mesenchymal stems cells derived from bone marrow of rabbits indicated the good biocompatibility of the fabricated nanofibrous scaffold. Meanwhile, the chondrogenic differentiation of bone marrow mesenchymal stem cells cultured on core-shell nanofibrous scaffold was evaluated by real-time polymerase chain reaction. The results suggested that the core-shell nanofibrous scaffold with kartogenin could promote the chondrogenic differentiation ability of bone marrow mesenchymal stem cells. Overall, the core-shell nanofibrous scaffold could be an effective delivery system for kartogenin and served as a promising tissue engineered scaffold for tracheal cartilage regeneration.

Evaluation of the potential of rhTGF- β3 encapsulated P(LLA-CL)/collagen nanofibers for tracheal cartilage regeneration using mesenchymal stems cells derived from Wharton's jelly of human umbilical cord

Tracheal injuries are one of major challenging issues in clinical medicine because of the poor intrinsic ability of tracheal cartilage for repair. Tissue engineering provides an alternative method for the treatment of tracheal defects by generating replacement tracheal structures. In this study, core-shell nanofibrous scaffold was fabricated to encapsulate bovine serum albumin & rhTGF-β3 (recombinant human transforming growth factor-β3) into the core of the nanofibers for tracheal cartilage regeneration. Characterization of the core-shell nanofibrous scaffold was carried out by scanning electron microscope (SEM), transmission electron microscope (TEM), laser scanning confocal microscopy (LSCM), and tensile mechanical test. The rhTGF-β3 released from the scaffolds in a sustained and stable manner for about 2months. The bioactivity of released rhTGF-β3 was evaluated by its effect on the synthesis of type II collagen (COL2) and glycosaminoglycans (GAGs) by chondrocytes. The results suggested that its bioactivity was retained during release process. The proliferation and morphology analyses of mesenchymal stems cells derived from Wharton's jelly of human umbilical cord (WMSCs) indicated the good biocompatibility of the fabricated nanofibrous scaffold. Meanwhile, the chondrogenic differentiation of WMSCs cultured on core-shell nanofibrous scaffold was evaluated by real-time qPCR and histological staining. The results suggested that the core-shell nanofibrous scaffold with rhTGF-β3 could promote the chondrogenic differentiation ability of WMSCs. Therefore, WMSCs could be a promising seed cells in the construction of tissue-engineered tracheal cartilage. Overall, the core-shell nanofibrous scaffold could be an effective delivery system for rhTGF-β3 and served as a promising tissue engineered scaffold for tracheal cartilage regeneration.

A novel tissue-engineered trachea with a mechanical behavior similar to native trachea

A novel tissue-engineered trachea was developed with appropriate mechanical behavior and substantial regeneration of tracheal cartilage. We designed hollow bellows scaffold as a framework of a tissue-engineered trachea and demonstrated a reliable method for three-dimensional (3D) printing of monolithic bellows scaffold. We also functionalized gelatin sponge to allow sustained release of TGF-β1 for stimulating tracheal cartilage regeneration and confirmed that functionalized gelatin sponge induces cartilaginous tissue formation in vitro. A tissue-engineered trachea was then created by assembling chondrocytes-seeded functionalized gelatin sponges into the grooves of bellows scaffold and it showed very similar mechanical behavior to that of native trachea along with substantial regeneration of tracheal cartilage in vivo. The tissue-engineered trachea developed here represents a novel concept of tracheal substitute with appropriate mechanical behavior similar to native trachea for use in reconstruction of tracheal stenosis.

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.

Bioengineered trachea using autologous chondrocytes for regeneration of tracheal cartilage in a rabbit model

In this study, a bioengineered trachea composed of autologous chondrocytes was developed, and its effect on cartilaginous regeneration was evaluated by implantation into tracheal defects in rabbits. Prospective controlled trial in an animal model. The tracheal prosthesis used in this study was composed of polypropylene (the frame) and collagen sponge (the scaffold). Chondrocytes were harvested from the costal cartilage of rabbits and seeded into the tracheal prosthesis. The bioengineered trachea, consisting of the tracheal prosthesis with chondrocytes, was implanted into surgically created tracheal defects of rabbits in the bioengineered group, and a tracheal prosthesis without chondrocytes was implanted in the control group. After implantation, the presence of regenerated cartilage was observed in the bioengineered trachea but not in the tracheal prosthesis without chondrocytes. It was confirmed in this study that the implanted chondrocytes proliferated in an appropriate portion of the tracheal defect and that the partially resected tracheal cartilage was repaired with regenerated cartilaginous tissue into a ring-shaped form as a whole. These results demonstrate the feasibility of cartilaginous regeneration using a bioengineered trachea with autologous chondrocytes.

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.

Widespread and Early Tracheal Cartilage Regeneration by Synchronous Slow Release of b-FGF and BMP-2

Our previous studies have demonstrated that slow release of basic fibroblast growth factor (b-FGF) or bone morphogenetic protein 2 (BMP-2) induces cartilage regeneration. In the present study, we investigated whether synchronous slow release of b-FGF and BMP-2 would induce more widespread and earlier cartilage regeneration than that induced by each growth factor alone. A 1-cm defect was made in the mid-ventral portion of each of 10 consecutive tracheal rings. In four controls, the defect was left untreated. In the gelatin group (n = 4), empty gelatin sponge was implanted. In the b-FGF + BMP-2 group (n = 5), two gelatin sponges containing 100 microg of b-FGF or BMP-2 solution were implanted. After various periods, we euthanatized the dogs, and examined the implant sites. In the b-FGF + BMP-2 group, regenerated fibrous cartilage connected the host cartilage stumps and completely filled the defect between them at 1, 2, 3, and 12 months. Regenerated cartilage was covered by regenerated perichondrium originating from the host perichondrium, and showed neovascularization in the extracellular matrix. We succeeded in inducing more widespread and earlier cartilage regeneration using synchronous slow release of b-FGF and BMP-2 than that induced by release of each growth factor alone.

Tracheal Cartilage Regeneration and New Bone Formation by Slow Release of Bone Morphogenetic Protein (BMP)-2

We investigated the efficiency of bone morphogenetic protein (BMP)-2 released slowly from gelatin sponge for tracheal cartilage regeneration. A 1-cm gap was made in the mid-ventral portion of each of 10 consecutive tracheal cartilages. In the control group (n = 4), the resulting gap was left untreated. In the gelatin group (n = 4), plain gelatin was implanted in the gap. In the BMP-2 group (n = 4), gelatin containing 100 microg BMP-2 was implanted. We euthanatized all dogs in each group at 1, 3, 6, and 12 months after the implantation, respectively, and then examined the implant site macro- and microscopically. In the BMP-2 group, regenerated fibrous cartilage and newly formed bone were observed at 1 and 12 months. Regenerated cartilage was observed at the ends of the host cartilage stumps, with newly formed bone in the middle portion. The gaps were filled with regenerated cartilage and newly formed bone. At 3 and 6 months, regenerated cartilage, but not newly formed bone, was evident. The regenerated cartilage was covered with perichondrium and showed continuity with the host cartilage. We succeeded in inducing cartilage regeneration and new bone formation in canine trachea by slow release of 100 microg BMP-2 from gelatin.

Tracheal cartilage regeneration by slow release of basic fibroblast growth factor from a gelatin sponge

We investigated whether implantation of a gelatin sponge, releasing basic fibroblast growth factor slowly (b-FGF) into a tracheal cartilage defect, would induce regeneration of autologous tracheal cartilage. We created a 1-cm defect in the midventral portion of each of 10 consecutive cervical tracheal cartilage rings in 12 experimental dogs. In the control group (n = 4), the resulting defects were left untreated. In the gelatin group (n = 4), empty gelatin sponges were implanted in the defects. In the basic fibroblast growth factor group (n = 4), gelatin sponges incorporating 100 microg of b-FGF solution were implanted in the defects. We killed the 4 dogs in each group at 1, 3, 6, and 12 months after implantation, respectively, and examined the implant sites macro- and microscopically. In the control and gelatin groups, no regenerated cartilage was observed in the tracheal cartilage defects, and the width of the gap between the host cartilage stumps had shrunk. In the b-FGF group, regenerated cartilage was observed in all dogs. The proportion of the defect in the host cartilage occupied by regenerated cartilage was 13%, 84%, 75%, and 69% at 1, 3, 6, and 12 months, respectively. The regenerated cartilage was fibrous cartilage covered with perichondrium, which grew from the host perichondrium and showed continuity with the host cartilage stumps. Implantation of a gelatin sponge slowly releasing basic fibroblast growth factor induces tracheal cartilage regeneration, which subsequently fills a large proportion of experimentally created tracheal cartilage defects within 12 months after implantation.