Enhanced Osteosarcoma Immunotherapy via CaCO3 Nanoparticles: Remodeling Tumor Acidic and Immune Microenvironment for Photodynamic Therapy.

Osteosarcoma (OS) is a "cold" tumor enriched in noninflammatory M2 phenotype tumor-associated macrophages (TAMs), which limits the efficacy of immunotherapy. The acidic tumor microenvironment (TME), generated by factors such as excess hydrogen (H+) ions and high lactate levels, activates immunosuppressive cells, further promoting a suppressive tumor immune microenvironment (TIME). Therefore, a multitarget synergistic combination strategy that neutralizes the acidic TME and reprograms TAMs can be beneficial for OS therapy. Here, a calcium carbonate (CaCO3)/polydopamine (PDA)-based nanosystem (A-NPs@(SHK+Ce6)) is developed. CaCO3 nanoparticles are used to neutralize H+ ions and alleviate the suppressive TIME, and the loaded SHK not only synergizes with photodynamic therapy (PDT) but also inhibits lactate production, further reversing the acidic TME and repolarizing TAMs to consequently lead to enhanced PDT-induced tumor suppression and comprehensive beneficial effects on antitumor immune responses. Importantly, A-NPs@(SHK+Ce6), in combination with programmed cell death protein 1 (PD-1) checkpoint blockade, shows a remarkable ability to eliminate distant tumors and promote long-term immune memory function to protect against rechallenged tumors. This work presents a novel multiple-component combination strategy that coregulates the acidic TME and TAM polarization to reprogram the TIME.

Bone-inspired stress-gaining elastomer enabled by dynamic molecular locking.

The limited capacity of typical materials to resist stress loading, which affects their mechanical performance, is one of the most formidable challenges in materials science. Here, we propose a bone-inspired stress-gaining concept of converting typically destructive stress into a favorable factor to substantially enhance the mechanical properties of elastomers. The concept was realized by a molecular design of dynamic poly(oxime-urethanes) network with mesophase domains. During external loading, the mesophase domains in the condensed state were aligned into more ordered domains, and the dynamic oxime-urethane bonds served as the dynamic molecular locks disassociating and reorganizing to facilitate and fix the mesophase domains. Consequently, the tensile modulus and strength were enhanced by 1744 and 49.3 times after four cycles of mechanical training, respectively. This study creates a molecular concept with stress-gaining properties induced by repeated mechanical stress loading and will inspire a series of innovative materials for diverse applications.

Pro-vasculogenic Fibers by PDA-Mediated Surface Functionalization Using Cell-Free Fat Extract (CEFFE).

Formation of adequate vascular network within engineered three-dimensional (3D) tissue substitutes postimplantation remains a major challenge for the success of biomaterials-based tissue regeneration. To better mimic the in vivo angiogenic and vasculogenic processes, nowadays increasing attention is given to the strategy of functionalizing biomaterial scaffolds with multiple bioactive agents. Aimed at engineering electrospun biomimicking fibers with pro-vasculogenic capability, this study was proposed to functionalize electrospun fibers of polycaprolactone/gelatin (PCL/GT) by cell-free fat extract (CEFFE or FE), a newly emerging natural "cocktail" of cytokines and growth factors extracted from human adipose tissue. This was achieved by having the electrospun PCL/GT fiber surface coated with polydopamine (PDA) followed by PDA-mediated immobilization of FE to generate the pro-vasculogenic fibers of FE-PDA@PCL/GT. It was found that the PDA-coated fibrous mat of PCL/GT exhibited a high FE-loading efficiency (∼90%) and enabled the FE to be released in a highly sustained manner. The engineered FE-PDA@PCL/GT fibers possess improved cytocompatibility, as evidenced by the enhanced cellular proliferation, migration, and RNA and protein expressions (e.g., CD31, vWF, VE-cadherin) in the human umbilical vein endothelial cells (huvECs) used. Most importantly, the FE-PDA@PCL/GT fibrous scaffolds were found to enormously stimulate tube formation in vitro, microvascular development in the in ovo chick chorioallantoic membrane (CAM) assay, and vascularization of 3D construct in a rat subcutaneous embedding model. This study highlights the potential of currently engineered pro-vasculogenic fibers as a versatile platform for engineering vascularized biomaterial constructs for functional tissue regeneration.

Shape-memory responses compared between random and aligned electrospun fibrous mats.

Significant progress has been made in the design of smart fibers toward achieving improved efficacy in tissue regeneration. While electrospun fibers can be engineered with shape memory capability, both the fiber structure and applied shape-programming parameters are the determinants of final performance in applications. Herein, we report a comparison study on the shape memory responses compared between electrospun random and aligned fibers by varying the programming temperature T prog and the deforming strain ε deform . A PLLA-PHBV (6:4 mass ratio) polymer blend was first electrospun into random and aligned fibrous mat forms; thereafter, the effects of applying specific T prog (37°C and 46°C) and ε deform (30%, 50%, and 100%) on the morphological change, shape recovery efficiency, and switching temperature T sw of the two types of fibrous structures were examined under stress-free condition, while the maximum recovery stress σ max was determined under constrained recovery condition. It was identified that the applied T prog had less impact on fiber morphology, but increasing ε deform gave rise to attenuation in fiber diameters and bettering in fiber orientation, especially for random fibers. The efficiency of shape recovery was found to correlate with both the applied T prog and ε deform , with the aligned fibers exhibiting relatively higher recovery ability than the random counterpart. Moreover, T sw was found to be close to T prog , thereby revealing a temperature memory effect in the PLLA-PHBV fibers, with the aligned fibers showing more proximity, while the σ max generated was ε deform -dependent and 2.1-3.4 folds stronger for the aligned one in comparison with the random counterpart. Overall, the aligned fibers generally demonstrated better shape memory properties, which can be attributed to the macroscopic structural orderliness and increased molecular orientation and crystallinity imparted during the shape-programming process. Finally, the feasibility of using the shape memory effect to enable a mechanoactive fibrous substrate for regulating osteogenic differentiation of stem cells was demonstrated with the use of aligned fibers.

Formation of low-density electrospun fibrous network integrated mesenchymal stem cell sheet.

Cell sheets combined with electrospun fibrous mats represent an attractive approach for the repair and regeneration of injured tissues. However, the conventional dense electrospun mats as supportive substrates in forming "cell sheet on fiber mat" complexes suffer from problems of limiting the cellular function and eliciting a host response upon implantation. To give full play to the role of electrospun biomimicking fibers in forming quality cell sheets, this study proposed to develop a cell-fiber integrated sheet (CFIS) featuring a spatially homogeneous distribution of cells within the fiber structure by using a low-density fibrous network for cell sheet formation. A low-density electrospun polycaprolactone (PCL) fibrous network at a density of 103.8 ± 16.3 µg cm-2 was produced by controlling the fiber deposition for a short period of 1 min and subsequently transferred onto polydimethylsiloxane rings for facilitating cell sheet formation, in which rat bone marrow-derived mesenchymal cells were used. Using a dense electrospun PCL fibrous mat (481.5 ± 7.5 µg cm-2) as the control, it was found that cells on the low-density fibrous network (L-G) exhibited improved capacities in spreading, proliferation, stemness maintenance and matrix-remodeling during the process of CFIS formation. Structurally, the CFIS constructs revealed strong integration between the cells and the fibrous network, thus providing excellent cohesion and physical integrity to enable strengthening of the formed cell sheet. By contrast, the cell sheet formed on the dense fibrous mat (D-G) showed a two-layer (biphasic) structure due to the limitation of cellular invasion. Moreover, such engineered CFIS was identified with enhanced immunomodulatory effects by promoting LPS-stimulated macrophages towards an M2 phenotype in vitro. Our results suggest that the CFIS may be used as a native tissue equivalent "cell sheet" for improving the efficacy of the tissue engineering approach for the repair and regeneration of impaired tissues.

Engineering a Mechanoactive Fibrous Substrate with Enhanced Efficiency in Regulating Stem Cell Tenodifferentiation.

Electrospun-aligned fibers in ultrathin fineness have previously demonstrated a limited capacity in driving stem cells to differentiate into tendon-like cells. In view of the tendon's mechanoactive nature, endowing such aligned fibrous structure with mechanoactivity to exert in situ mechanical stimulus by itself, namely, without any forces externally applied, is likely to potentiate its efficiency of tenogenic induction. To test this hypothesis, in this study, a shape-memory-capable poly(l-lactide-co-caprolactone) (PLCL) copolymer was electrospun into aligned fibrous form followed by a "stretching-recovery" shape-programming procedure to impart shape memory capability. Thereafter, in the absence of tenogenic supplements, human adipose-derived stem cells (ADSCs) were cultured on the programmed fibrous substrates for a duration of 7 days, and the effects of constrained recovery resultant stress-stiffening on cell morphology, proliferation, and tenogenic differentiation were examined. The results indicate that the in situ enacted mechanical stimulus due to shape memory effect (SME) did not have adverse influence on cell viability and proliferation, but significantly promoted cellular elongation along the direction of fiber alignment. Moreover, it revealed that tendon-specific protein markers such as tenomodulin (TNMD) and tenascin-C (TNC) and gene expression of scleraxis (SCX), TNMD, TNC, and collagen I (COL I) were significantly upregulated on the mechanoactive fibrous substrate with higher recovery stress compared to the counterparts. Mechanistically, the Rho/ROCK signaling pathway was identified to be involved in the substrate self-actuation-induced enhancement in tenodifferentiation. Together, these results suggest that constrained shape recovery stress may be employed as an innovative loading modality to regulate the stem cell tenodifferentiation by presenting the fibrous substrate with an aligned tendon-like topographical cue and an additional mechanoactivity. This newly demonstrated paradigm in modulating stem cell tenodifferentiation may improve the efficacy of tendon tissue engineering strategy for tendon healing and regeneration.

Facile preparation of a controlled-release tubular scaffold for blood vessel implantation.

Salvianic acid-loaded mesoporous silica nanoparticles into gelatin/polyurethane bilayered small-diameter tubular scaffold were prepared by thermally induced phase separation (TIPS) and electrospinning. Mesoporous silica nanoparticles (MSNs) were selected as carriers to load salvianic acid (SAL). The SAL-loaded MSNs (SAL@MSNs) with an optimized SAL loading efficiency of 10% was initially dispersed in gelatin solution and under a vacuum freeze-drying process as an inner layer of vascular scaffolds. Then, poly(ester-urethane)urea (C-PEEUU) nanofibers were electrospun outside the SAL@MSNs/Gelatin vascular scaffold to strengthen the spongy matrix. The loaded SAL within the MSNs/Gelatin/C-PEEUU bilayered small-diameter tubular scaffold showed a sustained release profile and good mechanical properties. In addition, the drug-loaded composite scaffold showed no unfavorable effects on the adhesion and proliferation of endothelial cells. Moreover, no intimal hyperplasia and acute thrombosis was observed in the short-term implantation in rabbit's carotid artery. We believe the SAL@MSNs/Gelatin/C-PEEUU bilayered vascular scaffolds have promise for vascular tissue engineering applications.

The fabrication of 3D surface scaffold of collagen/poly (L-lactide-co-caprolactone) with dynamic liquid system and its application in urinary incontinence treatment as a tissue engineered sub-urethral sling: In vitro and in vivo study.

AIMS: To fabricate a novel nanoyarn biomaterial via a dynamic liquid electrospinning system, and to simultaneously evaluate whether nanoyarn is capable of being applied as a urinary sling for future clinical transfer. METHODS: Nanoyarn was cultured with adipose-derived stem cells (ADSCs). Cell morphology and function were observed on nanoyarn. Female rats that underwent vagina dilatation (VD) and bilateral ovarian resection (BOR) were used as the urinary incontinence model. After 2 weeks, the cells-sling was fixed to the suburethra. A commercial sling that tension-free vaginal tape-obturator (TVT-O) was used as a control. The urodynamic test for leak point pressure (LPP) and histological tests were used to evaluate the sling's performance in vivo. RESULTS: The nanoyarn possessed beneficial properties and the actin filament from ADSCs, which is very similar to muscle. Rats that underwent VD and BOR maintained a low LPP, whereas the LPP in rats with VD alone recovered to normal levels within 2 weeks. LPP in the nanoyarn group gradually decreased on the three urodynamic tests post-suburethral surgery, however, the cell-laden nanoyarn maintained LPP at normal levels for 8 weeks; the TVT-O group showed a significant increase in LPP at 8 weeks. Cell-laden nanoyarn was infiltrated with more cells, collagen, and vessels than the controls. CONCLUSIONS: The nanoyarn showed sufficient efficacy to maintain LPP in urinary incontinence rat model. In addition, it improved cell infiltration, collagen and muscle development compared to TVT-O. Thus, the combination of ADSCs and a nanoyarn scaffold could be a promising tissue-engineered sling for the treatment of urinary incontinence.

Two-phase electrospinning to incorporate growth factors loaded chitosan nanoparticles into electrospun fibrous scaffolds for bioactivity retention and cartilage regeneration.

Growth factor is an essential ingredient to regulate mesenchymal stem cells (MSCs) chondrogenic differentiation in cartilage tissue engineering. However, non-osteochondral specification, short plasma half time and bioactivity loss restrict growth factor's application. Thus, novel chondrogenic growth factors, specifically target osteochondral lineage cells, that can be sustained release and bioactivity protected to exert functions continually and effectively have attracted increasing researchers' interest. To achieve these goals, chitosan nanoparticles and electrospun fiber scaffolds were used as dual release system to sustain release Nel-like molecule-1 (Nell-1) growth factor and protect bioactivity, then the effect and mechanism of Nell-1 on inducing human bone MSCs (hBMSCs) differentiate toward chondrocytes were investigated. For release and bioactivity protection study, preloading Nell-1 into chitosan nanoparticles significantly extended the release time, increased the released Nell-1's bioactivity than directly incorporating Nell-1 into the scaffolds. Furthermore, Nell-1 specifically promotes hBMSCs in vitro chondrogenic differentiation by increasing expression of chondrogenic related genes and proteins. These findings suggest the potential utility of Nell-1 incorporated dual release scaffold for cartilage tissue engineering.

Development of Dynamic Liquid and Conjugated Electrospun Poly(L-lactide-co-caprolactone)/Collagen Nanoyarns for Regulating Vascular Smooth Muscle Cells Growth.

Simulating the modeling of smooth muscle layer in the vascular structure makes a great difference for vascular tissue regeneration. A functional tissue engineered vascular media shall promote the aligned organization and three-dimensional penetration of smooth muscle cells (SMCs) into the scaffold. To this aim, dynamic liquid and conjugated nanoyarns based on poly(L-lactide-co-caprolactone) (P(LLA-CL)) and collagen (COL) with a weight ratio at 3:1 were fabricated by electrospinning methods, with random and aligned nanofibers as control groups. The Fourier transform infrared spectroscopy and X-ray diffraction analyses confirmed the preservation of P(LLA-CL)/COL components and structure. Scanning electron microscope (SEM) results indicated a significant increase of yarn diameters at 19.27 ± 6.16 µm (dynamic liquid) and 10.24 ± 3.09 µm (conjugated), and both of the nanoyarns had improved mechanical tensile properties than the random nanofibers. Compared with random and aligned nanofibers, the nanoyarns presented significant higher porosity and larger pore diameter, leading to a decrease of water contact angle and a promotion of SMCs proliferation and migration. Better SMCs orientation was observed on the conjugated nanoyarns, while superior SMCs penetration was achieved on the dynamic liquid nanoyarns, owing to the differences in yarns microstructure. Herein, this study demonstrated that the aligned and porous P(LLA-CL)/COL nanoyarns fabricated by dynamic liquid and conjugated electrospinning were beneficial to regulating vascular SMCs outgrowth, which had important implications for functional reconstruction of vascular media.

Electrospun nanoyarn seeded with myoblasts induced from placental stem cells for the application of stress urinary incontinence sling: An in vitro study.

OBJECTIVE: To fabricate a novel electrospun nanoyarn using dynamic liquid electrospinning technique. The nanoyarn will be seeded with myoblasts differentiated from placental stem cells (PSCs) to evaluate the feasiblity of the cell-scaffold construct as a tissue engineering sling to treat stress urinary incontinence. MATERIAL AND METHODS: PSCs were induced to myoblasts with 5-azacytidine and horse serum. Myoblasts differentiation was confirmed by immunofluorescence and western blot. Western blot was also used to assess the change of extracellular matrix (ECM) expression. A dynamic liquid electrospinning system was used to fabricate a novel nanoyarn scaffold for myoblast seeding. Cell morphology and proliferation on nanoyarn and nanofiber scaffold were compared with scanning electron microscopy (SEM) and MTS assay respectively. Filament actin development was tested with Rhodamine-labeled phalloidin stainning; cell infiltration into scaffolds was observed with H&E stainning. ECM expression was evaluated by a collagen assay, immunofluorescence imaging and real-time PCR. RESULTS: Myoblasts showed increased expression of α-SMA, desmin, and collagen type 1, 3 when compared to PSCs. The nanoyarn possessed higher porosity, larger pore size, and aligned fibers/yarns as compared to nanofiber scaffold. Cell proliferation was significantly improved on nanoyarn scaffold. Cells could infiltrate deeply in the nanoyarn scaffold after 7 days in culture, however, they could only proliferate on the surface of the nanofiber scaffold. The myoblast-nanoyarn constructs seemed to be more like a muscle tissue. The myoblasts spreading on the nanoyarn scaffold were visible with aligned actin filaments in the horizontal view, whereas myoblasts spreading on the nanofiber scaffold were visible with unaligned actin filaments. Nanoyarn myoblasts exhibited higher production and density of type 1, 3 collagen and elastin. CONCLUSIONS: PSCs could be induced into myoblast and expressed elevated myogenic markers and ECM. PSCs are potential cell source for a tissue engineered sling. The novel electrospun nanoyarn scaffold showed potential for use as a sling for treatment of stress urinary incontinence. In vitro studies demonstrated that the nanoyarn scaffold could improve cell proliferation, muscular tissue development, and ECM expression compared to random nanofiber scaffolds. The combination of myoblasts and nanoyarn scaffold could be a promising tissue engineered sling for future in vivo studies.

Application of Wnt Pathway Inhibitor Delivering Scaffold for Inhibiting Fibrosis in Urethra Strictures: In Vitro and in Vivo Study.

OBJECTIVE: To evaluate the mechanical property and biocompatibility of the Wnt pathway inhibitor (ICG-001) delivering collagen/poly(L-lactide-co-caprolactone) (P(LLA-CL)) scaffold for urethroplasty, and also the feasibility of inhibiting the extracellular matrix (ECM) expression in vitro and in vivo. METHODS: ICG-001 (1 mg (2 mM)) was loaded into a (P(LLA-CL)) scaffold with the co-axial electrospinning technique. The characteristics of the mechanical property and drug release fashion of scaffolds were tested with a mechanical testing machine (Instron) and high-performance liquid chromatography (HPLC). Rabbit bladder epithelial cells and the dermal fibroblasts were isolated by enzymatic digestion method. (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay) and scanning electron microscopy (SEM) were used to evaluate the viability and proliferation of the cells on the scaffolds. Fibrolasts treated with TGF-ß1 and ICG-001 released medium from scaffolds were used to evaluate the anti-fibrosis effect through immunofluorescence, real time PCR and western blot. Urethrography and histology were used to evaluate the efficacy of urethral implantation. RESULTS: The scaffold delivering ICG-001 was fabricated, the fiber diameter and mechanical strength of scaffolds with inhibitor were comparable with the non-drug scaffold. The SEM and MTT assay showed no toxic effect of ICG-001 to the proliferation of epithelial cells on the collagen/P(LLA-CL) scaffold with ICG-001. After treatment with culture medium released from the drug-delivering scaffold, the expression of Collagen type 1, 3 and fibronectin of fibroblasts could be inhibited significantly at the mRNA and protein levels. In the results of urethrography, urethral strictures and fistulas were found in the rabbits treated with non-ICG-001 delivering scaffolds, but all the rabbits treated with ICG-001-delivering scaffolds showed wide caliber in urethras. Histology results showed less collagen but more smooth muscle and thicker epithelium in urethras repaired with ICG-001 delivering scaffolds. CONCLUSION: After loading with the Wnt signal pathway inhibitor ICG-001, the Collagen/P(LLA-CL) scaffold could facilitate a decrease in the ECM deposition of fibroblasts. The ICG-001 delivering Collagen/P(LLA-CL) nanofibrous scaffold seeded with epithelial cells has the potential to be a promising substitute material for urethroplasty. Longer follow-up study in larger animals is needed in the future.