Biodegradable Honeycomb-Mimic Scaffolds Consisting of Nanofibrous Walls.

The scaffold is a porous three-dimensional (3D) material that supports cell growth and tissue regeneration. Such 3D structures should be generated with simple techniques and nontoxic ingredients to mimic bio-environment and facilitate tissue regeneration. In this work, simple but powerful techniques are demonstrated for the fabrication of lamellar and honeycomb-mimic scaffolds with poly(L-lactic acid). The honeycomb-mimic scaffolds with tunable pore size ranging from 70 to 160 µm are fabricated by crystal needle-guided thermally induced phase separation in a directional freezing apparatus. The compressive modulus of the honeycomb-mimic scaffold is ≈4 times higher than that of scaffold with randomly oriented pore structure. The fabricated honeycomb-mimic scaffold exhibits a hierarchical structure from nanofibers to micro-/macro-tubular structures. Pre-osteoblast MC3T3-E1 cells cultured on the honeycomb-mimic nanofibrous scaffolds exhibit an enhanced osteoblastic phenotype, with elevated expression levels of osteogenic marker genes, than those on either porous lamellar scaffolds or porous scaffolds with randomly oriented pores. The advanced techniques for the fabrication of the honeycomb-mimic structure may potentially be used for a wide variety of advanced functional materials.

Poly(N-isopropylacrylamide) based smart nanofibrous scaffolds for use as on-demand delivery systems for oral and dental tissue regeneration.

Stimuli-responsive domains capable of releasing loaded molecules, "on-demand," have garnered increasing attention due to their enhanced delivery, precision targeting, and decreased adverse effects. The development of an on-demand delivery system that can be easily triggered by dental clinicians might have major roles in dental and oral tissue engineering. A series of random graft poly(NIPAm-co-HEMA-Lactate) copolymers were synthesized using 95:5, 85:5, 60:40, and 40:60 ratios of thermosensitive NIPAm and HEMA-poly lactate respectively then electrospun to produce nanofibrous scaffolds loaded with bovine serum albumin (BSA). Cumulative BSA release was assessed at 25C and 37°C. To appraise the use of scaffolds as on-demand delivery systems, they were subjected to thermal changes in the form cooling and warming cycles during which BSA release was monitored. To confirm the triggered releasing ability of the synthesized scaffolds, the copolymer made with 60% NIPAm was selected, based on the results of the release tests, and loaded with bone morphogenetic protein-2 (BMP-2). The loaded scaffolds were placed with mesenchymal-like stem cells (iMSCs) derived from induced pluripotent stem cells (iPSCs), and subjected to temperature alterations. Then, the osteogenic differentiation of iMSCs, which might have resulted from the released protein, was evaluated after 10 days by analyzing runt-related transcription factor 2 (RUNX-2) osteogenic gene expression by the cells using real-time quantitative polymerase chain reaction (qRT-PCR). BSA release profiles showed a burst release at the beginning followed by a more linear pattern at 25°C, and a much slower release at 37°C. The release also decreased when the PNIPAm content decreased in the scaffolds. Thermal triggering led to a step-like release pattern in which the highest release was reported 30 min through the warming cycles. The iMSCs cultivated with scaffolds loaded with BMP-2 and exposed to temperature alteration showed significantly higher RUNX-2 gene expression than cells in the other experimental groups. The synthesized scaffolds are thermo-responsive and could be triggered to deliver biological biomolecules to be used in oral and dental tissue engineering. Thermal stimuli could be simulated by dental clinicians using simple means of cold therapy, for example, cold packs in intraoral accessible sites for specified times.

Synthetic peptides activating discoidin domain receptor 2 and collagen-binding integrins cooperate to stimulate osteoblast differentiation of skeletal progenitor cells.

Skeletal progenitor: collagen interactions are critical for bone development and regeneration. Both collagen-binding integrins and discoidin domain receptors (DDR1 and DDR2) function as collagen receptors in bone. Each receptor is activated by a distinct collagen sequence; GFOGER for integrins and GVMGFO for DDRs. Specific triple helical peptides containing each of these binding domains were evaluated for ability to stimulate DDR2 and integrin signaling and osteoblast differentiation. GVMGFO peptide stimulated DDR2 Y740 phosphorylation and osteoblast differentiation as measured by induction of osteoblast marker mRNAs and mineralization without affecting integrin activity. In contrast, GFOGER peptide stimulated focal adhesion kinase (FAK) Y397 phosphorylation, an early measure of integrin activation, and to a lesser extent osteoblast differentiation without affecting DDR2-P. Significantly, the combination of both peptides cooperatively enhanced both DDR2 and FAK signaling and osteoblast differentiation, a response that was blocked in Ddr2-deficient cells. These studies suggest that the development of scaffolds containing DDR and integrin-activating peptides may provide a new route for promoting bone regeneration. STATEMENT OF SIGNIFICANCE: A method for stimulating osteoblast differentiation of skeletal progenitor cells is described that uses culture surfaces coated with a collagen-derived triple-helical peptide to selectively activate discoidin domain receptors. When this peptide is combined with an integrin-activating peptide, synergistic stimulation of differentiation is seen. This approach of combining collagen-derived peptides to stimulate the two main collagen receptors in bone (DDR2 and collagen-binding integrins) provides a route for developing a new class of tissue engineering scaffolds for bone regeneration.

Recent progress in polymeric gene vectors: Delivery mechanisms, molecular designs, and applications.

Gene therapy and gene delivery have drawn extensive attention in recent years especially when the COVID-19 mRNA vaccines were developed to prevent severe symptoms caused by the corona virus. Delivering genes, such as DNA and RNA into cells, is the crucial step for successful gene therapy and remains a bottleneck. To address this issue, vehicles (vectors) that can load and deliver genes into cells are developed, including viral and non-viral vectors. Although viral gene vectors have considerable transfection efficiency and lipid-based gene vectors become popular since the application of COVID-19 vaccines, their potential issues including immunologic and biological safety concerns limited their applications. Alternatively, polymeric gene vectors are safer, cheaper, and more versatile compared to viral and lipid-based vectors. In recent years, various polymeric gene vectors with well-designed molecules were developed, achieving either high transfection efficiency or showing advantages in certain applications. In this review, we summarize the recent progress in polymeric gene vectors including the transfection mechanisms, molecular designs, and biomedical applications. Commercially available polymeric gene vectors/reagents are also introduced. Researchers in this field have never stopped seeking safe and efficient polymeric gene vectors via rational molecular designs and biomedical evaluations. The achievements in recent years have significantly accelerated the progress of polymeric gene vectors toward clinical applications.

Immunomodulatory fibrous hyaluronic acid-Fmoc-diphenylalanine-based hydrogel induces bone regeneration.

AIM: To investigate the potential of an ultrashort aromatic peptide hydrogelator integrated with hyaluronic acid (HA) to serve as a scaffold for bone regeneration. MATERIALS AND METHODS: Fluorenylmethyloxycarbonyl-diphenylalanine (FmocFF)/HA hydrogel was prepared and characterized using microscopy and rheology. Osteogenic differentiation of MC3T3-E1 preosteoblasts was investigated using Alizarin red, alkaline phosphatase and calcium deposition assays. In vivo, 5-mm-diameter calvarial critical-sized defects were prepared in 20 Sprague-Dawley rats and filled with either FmocFF/HA hydrogel, deproteinized bovine bone mineral, FmocFF/Alginate hydrogel or left unfilled. Eight weeks after implantation, histology and micro-computed tomography analyses were performed. Immunohistochemistry was performed in six rats to assess the hydrogel's immunomodulatory effect. RESULTS: A nanofibrous FmocFF/HA hydrogel with a high storage modulus of 46 KPa was prepared. It supported osteogenic differentiation of MC3T3-E1 preosteoblasts and facilitated calcium deposition. In vivo, the hydrogel implantation resulted in approximately 93% bone restoration. It induced bone deposition not only around the margins, but also generated bony islets along the defect. Elongated M2 macrophages lining at the periosteum-hydrogel interface were observed 1 week after implantation. After 3 weeks, these macrophages were dispersed through the regenerating tissue surrounding the newly formed bone. CONCLUSIONS: FmocFF/HA hydrogel can serve as a cell-free, biomimetic, immunomodulatory scaffold for bone regeneration.

Dentin Sialoprotein/Phosphophoryn (DSP/PP) as Bio-Inductive Materials for Direct Pulp Capping.

Conventional direct pulp capping, such as calcium hydroxide (Ca(OH)2) or silicate products, usually induces an inflammatory reaction to provoke pulp regeneration. Phosphophoryn (PP) and dentin sialoprotein (DSP), the two most abundant non-collagenous proteins in the dentin matrix, are responsible for dentin mineralization, pulp cell migration, and differentiation. Here we examined the PP and combined DSP/PP as bio-inductive pulp capping materials by in vitro and in vivo tests. Firstly, the effects of the PP dose on pulp cell migration and matrix protein expression were examined by an agarose bead test. Secondly, the role of recombinant DSP (recDSP) and recDSP/PP on stimulating DSP-PP transcript expression was examined by RT-PCR. DSPP mRNA was also knocked down by RNA interference (RNAi) to examine their functions on dentin matrix mineralization. Finally, we used ferret animal models to test PP and recDSP/PP acting as capping agents on in vivo pulp responses and reparative dentin formation. The result showed that intermediate-dose PP was the most effective to enhance cell migration and differentiation. RecDSP/PP strongly enhanced the DSP-PP transcript expression, while inhibition of DSPP mRNA expression by siRNAs partially or completely affected dental pulp cell mineralization. The in vivo results showed that intermediate-dose PP and recDSP/PP proteins induced less pulp inflammation and promoted reparative dentin formation. Contrarily, conventional calcium hydroxide induced severe pulp inflammation. With these findings, DSP and PP could serve as capping agents for pulp capping therapy.

Thixotropic Red Microalgae Sulfated Polysaccharide-Peptide Composite Hydrogels as Scaffolds for Tissue Engineering.

Sulfated polysaccharides of red marine microalgae have recently gained much attention for biomedical applications due to their anti-inflammatory and antioxidant properties. However, their low mechanical properties limit their use in tissue engineering. Herein, to enhance the mechanical properties of the sulfated polysaccharide produced by the red marine microalga, Porphyridium sp. (PS), it was integrated with the fluorenylmethoxycarbonyl diphenylalanine (FmocFF) peptide hydrogelator. Transparent, stable hydrogels were formed when mixing the two components at a 1:1 ratio in three different concentrations. Electron microscopy showed that all hydrogels exhibited a nanofibrous structure, mimicking the extracellular matrix. Furthermore, the hydrogels were injectable, and tunable mechanical properties were obtained by changing the hydrogel concentration. The composite hydrogels allowed the sustained release of curcumin which was controlled by the change in the hydrogel concentration. Finally, the hydrogels supported MC3T3-E1 preosteoblasts viability and calcium deposition. The synergy between the sulfated polysaccharide, with its unique bioactivities, and FmocFF peptide, with its structural and mechanical properties, bears a promising potential for developing novel tunable scaffolds for tissue engineering that may allow cell differentiation into various lineages.

A library of Rhodamine6G-based pH-sensitive fluorescent probes with versatile in vivo and in vitro applications.

Acidic pH is critical to the function of the gastrointestinal system, bone-resorbing osteoclasts, and the endolysosomal compartment of nearly every cell in the body. Non-invasive, real-time fluorescence imaging of acidic microenvironments represents a powerful tool for understanding normal cellular biology, defining mechanisms of disease, and monitoring for therapeutic response. While commercially available pH-sensitive fluorescent probes exist, several limitations hinder their widespread use and potential for biologic application. To address this need, we developed a novel library of pH-sensitive probes based on the highly photostable and water-soluble fluorescent molecule, Rhodamine 6G. We demonstrate versatility in terms of both pH sensitivity (i.e., pK a) and chemical functionality, allowing conjugation to small molecules, proteins, nanoparticles, and regenerative biomaterial scaffold matrices. Furthermore, we show preserved pH-sensitive fluorescence following a variety of forms of covalent functionalization and demonstrate three potential applications, both in vitro and in vivo, for intracellular and extracellular pH sensing. Finally, we develop a computation approach for predicting the pH sensitivity of R6G derivatives, which could be used to expand our library and generate probes with novel properties.

Scaffold Pore Curvature Influences ΜSC Fate through Differential Cellular Organization and YAP/TAZ Activity.

Tissue engineering aims to repair, restore, and/or replace tissues in the human body as an alternative to grafts and prostheses. Biomaterial scaffolds can be utilized to provide a three-dimensional microenvironment to facilitate tissue regeneration. Previously, we reported that scaffold pore size influences vascularization and extracellular matrix composition both in vivo and in vitro, to ultimately influence tissue phenotype for regenerating cranial suture and bone tissues, which have markedly different tissue properties despite similar multipotent stem cell populations. To rationally design biomaterials for specific cell and tissue fate specification, it is critical to understand the molecular processes governed by cell-biomaterial interactions, which guide cell fate specification. Building on our previous work, in this report we investigated the hypothesis that scaffold pore curvature, the direct consequence of pore size, modulates the differentiation trajectory of mesenchymal stem cells (MSCs) through alterations in the cytoskeleton. First, we demonstrated that sufficiently small pores facilitate cell clustering in subcutaneous explants cultured in vivo, which we previously reported to demonstrate stem tissue phenotype both in vivo and in vitro. Based on this observation, we cultured cell-scaffold constructs in vitro to assess early time point interactions between cells and the matrix as a function of pore size. We demonstrate that principle curvature directly influences nuclear aspect and cell aggregation in vitro. Scaffold pores with a sufficiently low degree of principle curvature enables cell differentiation; pharmacologic inhibition of actin cytoskeleton polymerization in these scaffolds decreased differentiation, indicating a critical role of the cytoskeleton in transducing cues from the scaffold pore microenvironment to the cell nucleus. We fabricated a macropore model, which allows for three-dimensional confocal imaging and demonstrates that a higher principle curvature facilitates cell aggregation and the formation of a potentially protective niche within scaffold macropores which prevents MSC differentiation and retains their stemness. Sufficiently high principle curvature upregulates yes-associated protein (YAP) phosphorylation while decreased principle curvature downregulates YAP phosphorylation and increases YAP nuclear translocation with subsequent transcriptional activation towards an osteogenic differentiation fate. Finally, we demonstrate that the inhibition of the YAP/TAZ pathway causes a defect in differentiation, while YAP/TAZ activation causes premature differentiation in a curvature-dependent way when modulated by verteporfin (VP) and 1-oleyl-lysophosphatidic acid (LPA), respectively, confirming the critical role of biomaterials-mediated YAP/TAZ signaling in cell differentiation and fate specification. Our data support that the principle curvature of scaffold macropores is a critical design criterion which guides the differentiation trajectory of mesenchymal stem cells' scaffolds. Biomaterial-mediated regulation of YAP/TAZ may significantly contribute to influencing the regenerative outcomes of biomaterials-based tissue engineering strategies through their specific pore design.

Retraction: A highly antibacterial polymeric hybrid micelle with efficiently targeted anticancer siRNA delivery and anti-infection in vitro/in vivo.

Retraction of 'A highly antibacterial polymeric hybrid micelle with efficiently targeted anticancer siRNA delivery and anti-infection in vitro/in vivo' by Li Zhou et al., Nanoscale, 2018, 10, 17304-17317, DOI: 10.1039/C8NR03001D.

Delivery of CRISPR/Cas9 Plasmid DNA by Hyperbranched Polymeric Nanoparticles Enables Efficient Gene Editing.

Gene editing nucleases such as CRISPR/Cas9 have enabled efficient and precise gene editing in vitro and hold promise of eventually achieving in vivo gene editing based therapy. However, a major challenge for their use is the lack of a safe and effective virus-free system to deliver gene editing nuclease elements. Polymers are a promising class of delivery vehicle due to their higher safety compared to currently used viral vectors, but polymers suffer from lower transfection efficiency. Polymeric vectors have been used for small nucleotide delivery but have yet to be used successfully with plasmid DNA (pDNA), which is often several hundred times larger than small nucleotides, presenting an engineering challenge. To address this, we extended our previously reported hyperbranched polymer (HP) delivery system for pDNA delivery by synthesizing several variants of HPs: HP-800, HP-1.8K, HP-10K, HP-25K. We demonstrate that all HPs have low toxicity in various cultured cells, with HP-25K being the most efficient at packaging and delivering pDNA. Importantly, HP-25K mediated delivery of CRISPR/Cas9 pDNA resulted in higher gene-editing rates than all other HPs and Lipofectamine at several clinically significant loci in different cell types. Consistently, HP-25K also led to more robust base editing when delivering the CRISPR base editor "BE4-max" pDNA to cells compared with Lipofectamine. The present work demonstrates that HP nanoparticles represent a promising class of vehicle for the non-viral delivery of pDNA towards the clinical application of gene-editing therapy.

Biomimetic tubular scaffold with heparin conjugation for rapid degradation in in situ regeneration of a small diameter neoartery.

To address the clinical need for readily available small diameter vascular grafts, biomimetic tubular scaffolds were developed for rapid in situ blood vessel regeneration. The tubular scaffolds were designed to have an inner layer that is porous, interconnected, and with a nanofibrous architecture, which provided an excellent microenvironment for host cell invasion and proliferation. Through the synthesis of poly(spirolactic-co-lactic acid) (PSLA), a highly functional polymer with a norbornene substituting a methyl group in poly(l-lactic acid) (PLLA), we were able to covalently attach biomolecules onto the polymer backbone via thiol-ene click chemistry to impart desirable functionalities to the tubular scaffolds. Specifically, heparin was conjugated on the scaffolds in order to prevent thrombosis when implanted in situ. By controlling the amount of covalently attached heparin we were able to modulate the physical properties of the tubular scaffold, resulting in tunable wettability and degradation rate while retaining the porous and nanofibrous morphology. The scaffolds were successfully tested as rat abdominal aortic replacements. Patency and viability were confirmed through dynamic ultrasound and histological analysis of the regenerated tissue. The harvested tissue showed excellent vascular cellular infiltration, proliferation, and migration with laminar cellular arrangement. Furthermore, we achieved both complete reendothelialization of the vessel lumen and native-like media extracellular matrix. No signs of aneurysm or hyperplasia were observed after 3 months of vessel replacement. Taken together, we have developed an effective vascular graft able to generate small diameter blood vessels that can function in a rat model.

Macropore design of tissue engineering scaffolds regulates mesenchymal stem cell differentiation fate.

Craniosynostosis is a debilitating birth defect characterized by the premature fusion of cranial bones resulting from premature loss of stem cells located in suture tissue between growing bones. Mesenchymal stromal cells in long bone and the cranial suture are known to be multipotent cell sources in the appendicular skeleton and cranium, respectively. We are developing biomaterial constructs to maintain stemness of the cranial suture cell population towards an ultimate goal of diminishing craniosynostosis patient morbidity. Recent evidence suggests that physical features of synthetic tissue engineering scaffolds modulate cell and tissue fate. In this study, macroporous tissue engineering scaffolds with well-controlled spherical pores were fabricated by a sugar porogen template method. Cell-scaffold constructs were implanted subcutaneously in mice for up to eight weeks then assayed for mineralization, vascularization, extracellular matrix composition, and gene expression. Pore size differentially regulates cell fate, where sufficiently large pores provide an osteogenic niche adequate for bone formation, while sufficiently small pores (<125 µm in diameter) maintain stemness and prevent differentiation. Cell-scaffold constructs cultured in vitro followed the same pore size-controlled differentiation fate. We therefore attribute the differential cell and tissue fate to scaffold pore geometry. Scaffold pore size regulates mesenchymal cell fate, providing a novel design motif to control tissue regenerative processes and develop mesenchymal stem cell niches in vivo and in vitro through biophysical features.

Scaffolds with controlled release of pro-mineralization exosomes to promote craniofacial bone healing without cell transplantation.

Biomimetic bone regeneration methods which demonstrate both clinical and manufacturing feasibility, as alternatives to autogenic or allogenic bone grafting, remain a challenge to the field of tissue engineering. Here, we report the pro-osteogenic capacity of exosomes derived from human dental pulp stem cells (hDPSCs) to facilitate bone marrow stromal cell (BMSC) differentiation and mineralization. To support their delivery, we engineered a biodegradable polymer delivery platform to improve the encapsulation and the controlled release of exosomes on a tunable time scale from poly(lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) triblock copolymer microspheres. Our delivery platform integrates within three-dimensional tissue engineering scaffolds to enable a straightforward surgical insertion into a mouse calvarial defect. We demonstrate the osteogenic potential of these functional constructs in vitro and in vivo. Controlled release of osteogenic hDPSC-derived exosomes facilitates osteogenic differentiation of BMSCs, leading to mineralization to a degree which is comparable to exogenous administration of the same exosomes in human and mouse BMSCs. By recruiting endogenous cells to the defects and facilitating their differentiation, the controlled release of osteogenic exosomes from a tissue engineering scaffold demonstrates accelerated bone healing in vivo at 8 weeks. Exosomes recapitulate the advantageous properties of mesenchymal stem/progenitor cells, without manufacturing or immunogenic concerns associated with transplantation of exogenous cells. This biomaterial platform enables exosome-mediated bone regeneration in an efficacious and clinically relevant way.

Biodegradable nanofibrous temperature-responsive gelling microspheres for heart regeneration.

Myocardial infarction (heart attack) is the number one killer of heart patients. Existing treatments for heart attack do not address the underlying problem of cardiomyocyte (CM) loss and cannot regenerate the myocardium. Introducing exogenous cardiac cells is required for heart regeneration due to the lack of resident progenitor cells and very limited proliferative potential of adult CMs. Poor retention of transplanted cells is the critical bottleneck of heart regeneration. Here, we report the invention of a poly(l-lactic acid)-b-poly(ethylene glycol)-b-poly(N-Isopropylacrylamide) copolymer and its self-assembly into nanofibrous gelling microspheres (NF-GMS). The NF-GMS undergo thermally responsive transition to form not only a 3D hydrogel after injection in vivo, but also exhibit architectural and structural characteristics mimicking the native extracellular matrix (ECM) of nanofibrous proteins and gelling proteoglycans or polysaccharides. By integrating the ECM-mimicking features, injectable form, and the capability of maintaining 3D geometry after injection, the transplantation of hESC-derived CMs carried by NF-GMS led to a striking 10-fold graft size increase over direct CM injection in an infarcted rat model, which is the highest reported engraftment to date. Furthermore, NF-GMS carried CM transplantation dramatically reduced infarct size, enhanced integration of transplanted CMs, stimulated vascularization in the infarct zone, and led to a substantial recovery of cardiac function. The NF-GMS may also serve as advanced injectable and integrative biomaterials for cell/biomolecule delivery in a variety of biomedical applications.

Nanofibrous spongy microspheres to deliver rabbit mesenchymal stem cells and anti-miR-199a to regenerate nucleus pulposus and prevent calcification.

Lower back pain is mainly caused by intervertebral disc degeneration, in which calcification is frequently involved. Here novel nanofibrous spongy microspheres (NF-SMS) are used to carry rabbit bone marrow mesenchymal stromal cells (MSCs) to regenerate nucleus pulposus tissues. NF-SMS are shown to significantly enhance the MSC seeding, proliferation and differentiation over control microcarriers. Furthermore, a hyperbranched polymer (HP) with negligible cytotoxicity and high microRNA (miRNAs) binding affinity is synthesized. The HP can complex with anti-miR-199a and self-assemble into "double shell" polyplexes which are able to achieve high transfection efficiency into MSCs. A double-emulsion technique is used to encapsulate these polyplexes in biodegradable nanospheres (NS) to enable sustained anti-miR-199 delivery. Our results demonstrate that MSC/HP-anti-miR-199a/NS/NF-SMS constructs can promote the nucleus pulposus (NP) phenotype and resist calcification in vitro and in a subcutaneous environment. Furthermore, injection of MSC/HP-anti-miR-199a/NS/NF-SMS can stay in place, produce functional extracellular matrix, maintain disc height and prevent intervertebral disc (IVD) calcification in a rabbit lumbar degeneration model.