Kartogenin preconditioning commits mesenchymal stem cells to a precartilaginous stage with enhanced chondrogenic potential by modulating JNK and ß-catenin-related pathways.

Cartilage engineering strategies using mesenchymal stem cells (MSCs) could provide preferable solutions to resolve long-segment tracheal defects. However, the drawbacks of widely used chondrogenic protocols containing TGF-ß3, such as inefficiency and unstable cellular phenotype, are problematic. In our research, to optimize the chondrogenic differentiation of human umbilical cord MSCs (hUCMSCs), kartogenin (KGN) preconditioning was performed prior to TGF-ß3 induction. hUCMSCs were preconditioned with 1 µM of KGN for 3 d, sequentially pelleted, and incubated with TGF-ß3 for 28 d. Then, the expression of chondrogenesis- and ossification-related genes was evaluated by immunohistochemistry and RT-PCR. The underlying mechanism governing the beneficial effects of KGN preconditioning was explored by phosphorylated kinase screening and validated in vitro and in vivo using JNK inhibitor (SP600125) and ß-catenin activator (SKL2001). After KGN preconditioning, expression of fibroblast growth factor receptor 3, a marker of precartilaginous stem cells, was up-regulated in hUCMSCs. Furthermore, the KGN-preconditioned hUCMSCs efficiently differentiated into chondrocytes with elevated chondrogenic gene ( SOX9, aggrecan, and collagen II) expression and reduced expression of ossific genes (collagen X and MMP13) compared with hUCMSCs treated with TGF-ß3 only. Phosphokinase screening indicated that the beneficial effects of KGN preconditioning are directly related to an up-regulation of JNK phosphorylation and a suppression of ß-catenin levels. Blocking and activating tests revealed that the prochondrogenic effects of KGN preconditioning was achieved mainly by activating the JNK/Runt-related transcription factor (RUNX)1 pathway, and antiossific effects were imparted by suppressing the ß-catenin/RUNX2 pathway. Eventually, tracheal patches, based on KGN-preconditioned hUCMSCs and TGF-ß3 encapsulated electrospun poly( l-lactic acid-co-ε-caprolactone)/collagen nanofilms, were successfully used for restoring tracheal defects in rabbit models. In summary, KGN preconditioning likely improves the chondrogenic differentiation of hUCMSCs by committing them to a precartilaginous stage with enhanced JNK phosphorylation and suppressed ß-catenin. This novel protocol consisting of KGN preconditioning and subsequent TGF-ß3 induction might be preferable for cartilage engineering strategies using MSCs.-Jing, H., Zhang, X., Gao, M., Luo, K., Fu, W., Yin, M., Wang, W., Zhu, Z., Zheng, J., He, X. Kartogenin preconditioning commits mesenchymal stem cells to a precartilaginous stage with enhanced chondrogenic potential by modulating JNK and ß-catenin-related pathways.

Rare Copy Number Variations Might Not be Involved in the Molecular Pathogenesis of PA-IVS in an Unselected Chinese Cohort.

Congenital heart defect (CHD) is one of the most common birth defects in China, while pulmonary atresia with intact ventricular septum (PA-IVS) is the life-threatening form of CHD. Numerous previous studies revealed that rare copy number variants (CNVs) play important roles in CHD, but little is known about the prevalence and role of rare CNVs in PA-IVS. In this study, we conducted a genome-wide scanning of rare CNVs in an unselected cohort consisted of 54 Chinese patients with PA-IVS and 20 patients with pulmonary atresia with ventricular septal defect (PA-VSD). CNVs were identified in 6/20 PA-VSD patients (30%), and three of these CNVs (15%) were considered potentially pathogenic. Two pathogenic CNVs occurred at a known CHD locus (22q11.2) and one likely pathogenic deletion located at 13q12.12. However, no rare CNVs were detected in patients with PA-IVS. Potentially pathogenic CNVs were more enriched in PA-VSD patients than in PA-IVS patients (p = 0.018). No rare CNVs were detected in patients with PA-IVS in our study. PA/IVS might be different from PA/VSD in terms of genetics as well as anatomy.

Advances in molecular genetics for pulmonary atresia.

Genetic and environmental factors may be similar in certain CHD. It has been widely accepted that it is the cumulative effect of these risk factors that results in disease. Pulmonary atresia is a rare type of complex cyanotic CHD with a poor prognosis. Understanding the molecular mechanism of pulmonary atresia is essential for future diagnosis, prevention, and therapeutic approaches. In this article, we reviewed several related copy number variants and related genetic mutations, which were identified in patients with pulmonary atresia, including pulmonary atresia with ventricular septal defect and pulmonary atresia with intact ventricular septum.

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.

Restoring tracheal defects in a rabbit model with tissue engineered patches based on TGF-ß3-encapsulating electrospun poly(l-lactic acid-co-ε-caprolactone)/collagen scaffolds.

Long segment tracheal stenosis often has a poor prognosis due to the limited availability of materials for tracheal reconstruction. Tissue engineered tracheal patches based on electrospun scaffolds and stem cells present ideal solutions to this medical challenge. However, the established engineering process is inefficient and time-consuming. In our research, to optimize the engineering process, core-shell nanofilms encapsulating TGF-ß3 were fabricated as scaffolds for tracheal patches. The morphological and mechanical characteristics, degradation and biocompatibility of poly(l-lactic acid-co-ε-caprolactone)/collagen (PLCL/collagen) scaffolds with different compositions (PLCL:collagen 75:25, 50:50 and 25:75, respectively) were comparatively evaluated to determine the preferable compositional ratio. Then the chondrogenesis-inducing potential is investigated, and tracheal patches based on electrospun scaffolds and bone marrow mesenchymal stem cells (BMSCs) were constructed to restore tracheal defects in rabbit models. The results indicated that core-shell scaffolds with a PLCL/collagen proportion of 75:25 were eligible for tracheal patches. The stable and sustained release of TGF-ß3 from scaffolds could efficiently promote the chondrogenic differentiation of BMSCs and shorten the incubation time. Tracheal integrity was well maintained for 2 months after restoration; meanwhile, re-epithelialization also achieved. In conclusion, TGF-ß3-encapsulating core-shell electrospun scaffolds with a PLCL/collagen proportion of 75:25 could be used to optimize engineering process of tracheal patches.

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.

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

Long segmental repair of trachea stenosis is an intractable condition in the clinic. The reconstruction of an artificial substitute by tissue engineering is a promising approach to solve this unmet clinical need. 3D printing technology provides an infinite possibility for engineering a trachea. Here, we 3D printed a biodegradable reticular polycaprolactone (PCL) scaffold with similar morphology to the whole segment of rabbits' native trachea. The 3D-printed scaffold was suspended in culture with chondrocytes for 2 (Group I) or 4 (Group II) weeks, respectively. This in vitro suspension produced a more successful reconstruction of a tissue-engineered trachea (TET), which enhanced the overall support function of the replaced tracheal segment. After implantation of the chondrocyte-treated scaffold into the subcutaneous tissue of nude mice, the TET presented properties of mature cartilage tissue. To further evaluate the feasibility of repairing whole segment tracheal defects, replacement surgery of rabbits' native trachea by TET was performed. Following postoperative care, mean survival time in Group I was 14.38 ± 5.42 days, and in Group II was 22.58 ± 16.10 days, with the longest survival time being 10 weeks in Group II. In conclusion, we demonstrate the feasibility of repairing whole segment tracheal defects with 3D printed TET.