Wei Fu

electrospun gelatin/Pcl and collagen/Plcl scaffolds for vascular tissue engineering

Electrospun hybrid nanofibers prepared using combinations of natural and synthetic polymers have been widely investigated in cardiovascular tissue engineering. In this study, electrospun gelatin/polycaprolactone (PCL) and collagen/poly(l-lactic acid-co-ε-caprolactone) (PLCL) scaffolds were successfully produced. Scanning electron micrographs showed that fibers of both membranes were smooth and homogeneous. Water contact angle measurements further demonstrated that both scaffolds were hydrophilic. To determine cell attachment and migration on the scaffolds, both hybrid scaffolds were seeded with human umbilical arterial smooth muscle cells. Scanning electron micrographs and MTT assays showed that the cells grew and proliferated well on both hybrid scaffolds. Gross observation of the transplanted scaffolds revealed that the engineered collagen/PLCL scaffolds were smoother and brighter than the gelatin/PCL scaffolds. Hematoxylin and eosin staining showed that the engineered blood vessels constructed by collagen/PLCL electrospun membranes formed relatively homogenous vessel-like tissues. Interestingly, Young’s modulus for the engineered collagen/PLCL scaffolds was greater than for the gelatin/PCL scaffolds. Together, these results indicate that nanofibrous collagen/PLCL membranes with favorable mechanical and biological properties may be a desirable scaffold for vascular tissue engineering.

Electrospun gelatin/polycaprolactone nanofibrous membranes combined with a coculture of bone marrow stromal cells and chondrocytes for cartilage engineering.

Electrospinning has recently received considerable attention, showing notable potential as a novel method of scaffold fabrication for cartilage engineering. The aim of this study was to use a coculture strategy of chondrocytes combined with electrospun gelatin/polycaprolactone (GT/PCL) membranes, instead of pure chondrocytes, to evaluate the formation of cartilaginous tissue. We prepared the GT/PCL membranes, seeded bone marrow stromal cell (BMSC)/chondrocyte cocultures (75% BMSCs and 25% chondrocytes) in a sandwich model in vitro, and then implanted the constructs subcutaneously into nude mice for 12 weeks. Gross observation, histological and immunohistological evaluation, glycosaminoglycan analyses, Young’s modulus measurement, and immunofluorescence staining were performed postimplantation. We found that the coculture group formed mature cartilage-like tissue, with no statistically significant difference from the chondrocyte group, and labeled BMSCs could differentiate into chondrocyte-like cells under the chondrogenic niche of chondrocytes. This entire strategy indicates that GT/PCL membranes are also a suitable scaffold for stem cell-based cartilage engineering and may provide a potentially clinically feasible approach for cartilage repairs.

Electrospun collagen–poly(L-lactic acid-co-ε-caprolactone) membranes for cartilage tissue engineering

AIMnTo study the feasibility of electrospun collagen-poly(L-lactic acid-co-ε-caprolactone) (collagen-PLCL) membranes for cartilage tissue engineering.nnnMATERIALS & METHODSnCharacteristics and mechanical properties of collagen-PLCL membranes were analyzed. The cell affinity of collagen-PLCL membranes with chondrocytes was also assessed. Then, the cell-scaffold constructs were engineered with collagen-PLCL membranes seeded chondrocytes by a sandwich model. After culture for 1 week in vitro, the constructs were implanted subcutaneously into nude mice for 4, 8 and 12 weeks, followed by evaluation of the quality of neocartilage.nnnRESULTSnCollagen-PLCL membranes exhibited excellent balanced properties without cytotoxicity. With the extension of implantation time in vivo, the constructs revealed more cartilage-like tissue especially at 8 and 12 weeks. The Youngs modulus of the constructs also significantly increased and neared that of native cartilage at 12 weeks postimplantation.nnnCONCLUSIONnWe suggest that collagen-PLCL membranes facilitate the formation of cartilage and thus may represent a promising scaffold for cartilage tissue engineering.

Isolation and characterization of a Sca-1 + /CD31 progenitor cell lineage derived from mouse heart tissue

BackgroundMyocardial infarction remains the leading cause of mortality in developed countries despite recent advances in its prevention and treatment. Regenerative therapies based on resident cardiac progenitor cells (CPCs) are a promising alternative to conventional treatments. However, CPCs resident in the heart are quite rare. It is unclear how these CPCs can be isolated and cultured efficiently and what the effects of long-term culture in vitro are on their ‘stemness’ and differentiation potential, but this is critical knowledge for CPCs’ clinical application.ResultsHere, we isolated stem cell antigen-1 positive cells from postnatal mouse heart by magnetic active cell sorting using an iron-labeled anti-mouse Sca-1 antibody, and cultured them long-term in vitro. We tested stemness marker expression and the proliferation ability of long-term cultured Sca-1+ cells at early, middle and late passages. Furthermore, we determined the differentiation potential of these three passages into cardiac cell lineages (cardiomyocytes, smooth muscle and endothelial cells) after induction in vitro. The expression of myocardial, smooth muscle and endothelial cell-specific genes and surface markers were analyzed by RT-PCR and IF staining. We also investigated the oncogenicity of the three passages by subcutaneously injecting cells in nude mice. Overall, heart-derived Sca-1+ cells showed CPC characteristics: long-term propagation ability in vitro, non-tumorigenic in vivo, persistent expression of stemness and cardiac-specific markers, and multipotent differentiation into cardiac cell lineages.ConclusionsOur research may bring new insights to myocardium regeneration, for which even a small number of biopsy-derived CPCs could be enriched and propagated long term in vitro to obtain sufficient seed cells for cell injection or cardiac tissue engineering.

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 14.38u2009±u20095.42 days, and in Group II was 22.58u2009±u200916.10 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.

Re-epithelialization: a key element in tracheal tissue engineering

Trachea-tissue engineering is a thriving new field in regenerative medicine that is reaching maturity and yielding numerous promising results. In view of the crucial role that the epithelium plays in the trachea, re-epithelialization of tracheal substitutes has gradually emerged as the focus of studies in tissue-engineered trachea. Recent progress in our understanding of stem cell biology, growth factor interactions and transplantation immunobiology offer the prospect of optimization of a tissue-engineered tracheal epithelium. In addition, advances in cell culture technology and successful applications of clinical transplantation are opening up new avenues for the construction of a tissue-engineered tracheal epithelium. Therefore, this review summarizes current advances, unresolved obstacles and future directions in the reconstruction of a tissue-engineered tracheal epithelium.

Cell sources for trachea tissue engineering: past, present and future.

Trachea tissue engineering has been one of the most promising approaches to providing a potential clinical application for the treatment of long-segment tracheal stenosis. The sources of the cells are particularly important as the primary factor for tissue engineering. The use of appropriate cells seeded onto scaffolds holds huge promise as a means of engineering the trachea. Furthermore, appropriate cells would accelerate the regeneration of the tissue even without scaffolds. Besides autologous mature cells, various stem cells, including bone marrow-derived mesenchymal stem cells, adipose tissue-derived stem cells, umbilical cord blood-derived mesenchymal stem cells, amniotic fluid stem cells, embryonic stem cells and induced pluripotent stem cells, have received extensive attention in the field of trachea tissue engineering. Therefore, this article reviews the progress on different cell sources for engineering tracheal cartilage and epithelium, which can lead to a better selection and strategy for engineering the trachea.

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.

Restoring tracheal defects in a rabbit model with tissue engineered patches based on TGF-β3-encapsulating electrospun poly(l-lactic acid-co-ε-caprolactone)/collagen scaffolds

Abstract 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-β3 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-β3 from scaffolds could efficiently promote the chondrogenic differentiation of BMSCs and shorten the incubation time. Tracheal integrity was well maintained for 2u2009months after restoration; meanwhile, re-epithelialization also achieved. In conclusion, TGF-β3-encapsulating core–shell electrospun scaffolds with a PLCL/collagen proportion of 75:25 could be used to optimize engineering process of tracheal patches.