3D-printed PLA/Gel hybrid in liver tissue engineering: Effects of architecture on biological functions.

The liver is one of the vital organs in the body, and the gold standard of treatment for liver function impairment is liver transplantation, which poses many challenges. The specific three-dimensional (3D) structure of liver, which significantly impacts the growth and function of its cells, has made biofabrication with the 3D printing of scaffolds suitable for this approach. In this study, to investigate the effect of scaffold geometry on the performance of HepG2 cells, poly-lactic acid (PLA) polymer was used as the input of the fused deposition modeling (FDM) 3D-printing machine. Samples with simple square and bioinspired hexagonal cross-sectional designs were printed. One percent and 2% of gelatin coating were applied to the 3D printed PLA to improve the wettability and surface properties of the scaffold. Scanning electron microscopy pictures were used to analyze the structural properties of PLA-Gel hybrid scaffolds, energy dispersive spectroscopy to investigate the presence of gelatin, water contact angle measurement for wettability, and weight loss for degradation. In vitro tests were performed by culturing HepG2 cells on the scaffold to evaluate the cell adhesion, viability, cytotoxicity, and specific liver functions. Then, high-precision scaffolds were printed and the presence of gelatin was detected. Also, the effect of geometry on cell function was confirmed in viability, adhesion, and functional tests. The albumin and urea production of the Hexagonal PLA scaffold was about 1.22 ± 0.02-fold higher than the square design in 3 days. This study will hopefully advance our understanding of liver tissue engineering toward a promising perspective for liver regeneration.

Antimicrobial peptide-loaded decellularized placental sponge as an excellent antibacterial skin substitute against XDR clinical isolates.

Post-wound infections have remained a serious threat to society and healthcare worldwide. Attempts are still being made to develop an ideal antibacterial wound dressing with high wound-healing potential and strong antibacterial activity against extensively drug-resistant bacteria (XDR). In this study, a biological-based sponge was made from decellularized human placenta (DPS) and then loaded with different concentrations (0, 16 µg/mL, 32 µg/mL, 64 µg/mL) of an antimicrobial peptide (AMP, CM11) to optimize an ideal antibacterial wound dressing. The decellularization of DPS was confirmed by histological evaluations and DNA content assay. The DPS loaded with different contents of antimicrobial peptides (AMPs) showed uniform morphology under a scanning electron microscope (SEM) and cytobiocompatibility for human adipose tissue-derived mesenchymal stem cells. Antibacterial assays indicated that the DPS/AMPs had antibacterial behavior against both standard strain and XDR Acinetobacter baumannii in a dose-dependent manner, as DPS loaded with 64 µg/mL showed the highest bacterial growth inhibition zone and elimination of bacteria under SEM than DPS alone and DPS loaded with 16 µg/mL and 32 µg/mL AMP concentrations. The subcutaneous implantation of all constructs in the animal model demonstrated no sign of acute immune system reaction and graft rejection, indicating in vivo biocompatibility of the scaffolds. Our findings suggest the DPS loaded with 64 µg/mL as an excellent antibacterial skin substitute, and now promises to proceed with pre-clinical and clinical investigations.

Coculture of adipose-derived mesenchymal stem cells/macrophages on decellularized placental sponge promotes differentiation into the osteogenic lineage.

BACKGROUND: Several factors like three-dimensional microstructure, growth factors, cytokines, cell-cell communication, and coculture with functional cells can affect the stem cells behavior and differentiation. The purpose of this study was to investigate the potential of decellularized placental sponge as adipose-derived mesenchymal stem cells (AD-MSCs) and macrophage coculture systems, and guiding the osteogenic differentiation of stem cells. METHODS: The decellularized placental sponge (DPS) was fabricated, and its mechanical characteristics were evaluated using degradation assay, swelling rate, and pore size determination. Its structure was also investigated using hematoxylin and eosin staining and scanning electron microscopy. Mouse peritoneal macrophages and AD-MSCs were isolated and characterized. The differentiation potential of AD-MSCs co-cultured with macrophages was evaluated by RT-qPCR of osteogenic genes on the surface of DPS. The in vivo biocompatibility of DPS was determined by subcutaneous implantation of scaffold and histological evaluations of the implanted site. RESULTS: The DPS had 67% porosity with an average pore size of 238 µm. The in vitro degradation assay showed around 25% weight loss during 30 days in PBS. The swelling rate was around 50% during 72 h. The coculture of AD-MSCs/macrophages on the DPS showed a significant upregulation of four differentiation osteogenic lineage genes in AD-MSCs on days 14 and 21 and a significantly higher mineralization rate than the groups without DPS. Subcutaneous implantation of DPS showed in vivo biocompatibility of scaffold during 28 days follow-up. CONCLUSIONS: Our findings suggest the decellularized placental sponge as an excellent bone substitute providing a naturally derived matrix substrate with biostructure close to the natural bone that guided differentiation of stem cells toward bone cells and a promising coculture substrate for crosstalk of macrophage and mesenchymal stem cells in vitro.

Bioengineering of fibroblast-conditioned polycaprolactone/gelatin electrospun scaffold for skin tissue engineering.

BACKGROUND: Synthetic tissue engineering scaffolds has poor biocompatiblity with very low angiogenic properties. Conditioning the scaffolds with functional groups, coating with biological components, especially extracellular matrix (ECM), is an excellent strategy for improving their biomechanical and biological properties. METHODS: In the current study, a composite of polycaprolactone and gelatin (PCL/Gel) was electrospun in the ratio of 70/30 and surface modified with 1% gelatin-coating (G-PCL/Gel) or plasma treatment (P-PCL/Gel). The surface modification was determined by SEM and ATR-FTIR spectroscopy, respectively. The scaffolds were cultured with fibroblast 3T3, then decellularized during freeze-thawing process to fabricate a fibroblast ECM-conditioned PCL/Gel scaffold (FC-PCL/Gel). The swelling and degaradtion as well as in vitro and in vivo biocompatibility and angiogenic properties of the scaffolds were evaluated. RESULTS: The structure of the surface-modified G-PCL/Gel and P-PCL/Gel were unique and not changed compared with the PCL/Gel scaffolds. ATR-FTIR analysis admitted the formation of oxygen-containing groups, hydroxyl and carboxyl, on the surface of the P-PCL/Gel scaffold. The SEM micrographs and DAPI staining confirmed the cell attachment and the ECM deposition on the platform and successful removal of the cells after decellularization. P-PCL/Gel showed better cell attachment, ECM secretion and deposition after decellularization compared with G-PCL/Gel. The FC-PCL/Gel was considered as an optimized scaffold for further assays in this study. The FC-PCL/Gel showed increased hydrophilic behavior and cytobiocompatibility compared with P-PCL/Gel. The ECM on the FC-PCL/Gel scaffold showed a gradual degradation during 30 days of degradation time, as a small amount of ECM remained over the FC-PCL/Gel scaffold at day 30. The FC-PCL/Gel showed significant biocompatibility and improved angiogenic property compared with P-PCL/Gel when subcutaneously implanted in a mouse animal model for 7 and 28 days. CONCLUSIONS: Our findings suggest FC-PCL/Gel as an excellent biomimetic construct with high angiogenic properties. This bioengineered construct can serve as a possible application in our future pre-clinical and clinical studies for skin regeneration.

Pretreatment of Mesenchymal Stem Cells With Leishmania major Soluble Antigens Induce Anti-Inflammatory Properties in Mouse Peritoneal Macrophages.

Recent studies have demonstrated the influential role of microbial stimulus in characteristics and immunomodulatory effects of mesenchymal stem cells (MSCs). Due to the migration of MSCs to infection site, it is of importance to understand the interaction of microbial ligands with MSCs in order to clarify the positive or negative role of MSCs in the control of infection. In this research, we assess leishmanial soluble antigen (LSA)-primed MSCs on macrophage immune responses to lipopolysaccharide (LPS). For this purpose, the effects of both conditioned media (CM) and cell-cell contact of LSA primed MSCs were determined on macrophage responses to LPS. According to the obtained results, MSC-treated macrophages demonstrated an alternatively activated macrophages with higher levels of interleukin-10 (IL-10) and transforming growth factor-alpha (TNF-α) and lower levels of IL-6 and nitric oxide (NO) production as compared to the controls. In addition, phagocytosis of apoptotic thymocytes was induced in MSC-treated macrophages. In conclusion, it seems that MSCs trigger an anti-inflammatory phenotype in macrophages at Leishmania infected sites in order to enhance the induction of immune regulatory cells and clearance of apoptotic cells. J. Cell. Biochem. 118: 2764-2779, 2017. © 2017 Wiley Periodicals, Inc.

Decellularized Placental Sponge: A Platform for Coculture of Mesenchymal Stem Cells/Macrophages to Assess an M2 Phenotype and Osteogenic Differentiation In Vitro and In Vivo.

Effective communication between immune and bone-forming cells is crucial for the successful healing of bone defects. This study aimed to assess the potential of a decellularized placental sponge (DPS) as a coculture system for inducing M1/M2 polarization in macrophages and promoting osteogenic differentiation in adipose-derived mesenchymal stem cells (AD-MSCs), both in vitro and in vivo. We prepared the DPS and conducted a comprehensive characterization of its biomechanical properties, antibacterial activity, and biocompatibility. In vitro, we examined the influence of the DPS on the polarization of macrophages cocultured with AD-MSCs through nitric oxide assays, cytokine assays, phagocytosis tests, and real-time polymerase chain reaction (PCR). For in vivo assessment, we utilized micro-CT imaging, histological evaluations, and real-time PCR to determine the impact of the DPS seeded with Wharton's jelly mesenchymal stem cells (WJ-MSCs) on bone regeneration in a calvarial bone defect model. The coculture of AD-MSCs and macrophages on the DPS led to increased production of IL-10, upregulation of CD206, Arg1, and YM1 gene expression, and enhanced phagocytic capacity for apoptotic thymocytes. Concurrently, it reduced the secretion of TNF-α and nitric oxide (NO), downregulated the expression of CD86, NOS2, and IRF5 genes, and decreased macrophage phagocytosis of yeast. These results indicated polarization of macrophages toward an M2-like phenotype. In vivo, the presence of the DPS resulted in enhanced bone formation at the defect site. Immunostaining demonstrated that both the DPS and DPS + WJ-MSC constructs induced macrophage polarization toward an M2 phenotype, as compared to the control defect. In conclusion, this immunomodulatory effect, coupled with its biocompatibility and biomechanical properties resembling natural bone, positions the DPS as an attractive candidate for further exploration in the field of bone tissue engineering and regenerative medicine.

Decellularized Placental Sponge Seeded with Human Mesenchymal Stem Cells Improves Deep Skin Wound Healing in the Animal Model.

Skin injuries lead to a large burden of morbidity. Although numerous clinical and scientific strategies have been investigated to repair injured skin, optimal regeneration therapy still poses a considerable obstacle. To address this challenge, decellularized extracellular matrix-based scaffolds recellularized with stem cells offer significant advancements in skin regeneration and wound healing. Herein, a decellularized human placental sponge (DPS) was fabricated using the decellularization and freeze-drying technique and then recellularized with human adipose-derived mesenchymal cells (MSCs). The biological and biomechanical properties and skin full-thickness wound healing capacity of the stem cells-DPS constructs were investigated in vitro and in vivo. The DPS exhibited a uniform 3D microstructure with an interconnected pore network, 89.21% porosity, a low degradation rate, and good mechanical properties. The DPS and MSCs-DPS constructs were implanted in skin full-thickness wound models in mice. An accelerated wound healing was observed in the wounds implanted with the MSCs-DPS construct when compared to DPS and control (wounds with no treatment) during 7 and 21 days postimplantation follow-up. In the MSCs-DPS group, the wound was completely re-epithelialized, the epidermis layer was properly organized, and the dermis and epidermis' bilayer structures were restored after 7 days. Our findings suggest that DPS is an excellent carrier for MSC culture and delivery to skin wounds and now promises to proceed with clinical evaluations.

Chitosan-Placental ECM Composite Thermos-Responsive Hydrogel as a Biomimetic Wound Dressing with Angiogenic Property.

Attempts are being made to develop an ideal wound dressing with excellent biomechanical and biological properties. Here, a thermos-responsive hydrogel is fabricated using chitosan (CTS) with various concentrations (1%, 2.5%, and 5% w/v) of solubilized placental extracellular matrix (ECM) and 20% ß-glycerophosphate to optimize a smart wound dressing hydrogel with improved biological behavior. The thermo-responsive CTS (TCTS) alone or loaded with ECMs (ECM-TCTS) demonstrate uniform morphology using SEM. TCTS and ECM1%-TCTS and ECM2.5%-TCTS show a gelation time of 5 min at 37 °C, while no gel formation is observed at 4 and 25 °C. ECM5%-TCTS forms gel at both 25 and 37 °C. The degradation and swelling ratios increase as the ECM content of the hydrogel increase. All the constructs show excellent biocompatibility in vitro and in vivo, however, the hydrogels with a higher concentration of ECM demonstrate better cell adhesion for fibroblast cells and induce expression of angiogenic factors (VEGF and VEGFR) from HUVEC. Only the ECM5%-TCTS has antibacterial activity against Acinetobacter baumannii ATCC 19606. The data obtained from the current study suggest the ECM2.5%-TCTS as an optimized smart biomimetic wound dressing with improved angiogenic properties now promises to proceed with pre-clinical and clinical investigations.