Hyperelastic, shape-memorable, and ultra-cell-adhesive degradable polycaprolactone-polyurethane copolymer for tissue regeneration.

Novel polycaprolactone-based polyurethane (PCL-PU) copolymers with hyperelasticity, shape-memory, and ultra-cell-adhesion properties are reported as clinically applicable tissue-regenerative biomaterials. New isosorbide derivatives (propoxylated or ethoxylated ones) were developed to improve mechanical properties by enhanced reactivity in copolymer synthesis compared to the original isosorbide. Optimized PCL-PU with propoxylated isosorbide exhibited notable mechanical performance (50 MPa tensile strength and 1150% elongation with hyperelasticity under cyclic load). The shape-memory effect was also revealed in different forms (film, thread, and 3D scaffold) with 40%-80% recovery in tension or compression mode after plastic deformation. The ultra-cell-adhesive property was proven in various cell types which were reasoned to involve the heat shock protein-mediated integrin (α5 and αV) activation, as analyzed by RNA sequencing and inhibition tests. After the tissue regenerative potential (muscle and bone) was confirmed by the myogenic and osteogenic responses in vitro, biodegradability, compatible in vivo tissue response, and healing capacity were investigated with in vivo shape-memorable behavior. The currently exploited PCL-PU, with its multifunctional (hyperelastic, shape-memorable, ultra-cell-adhesive, and degradable) nature and biocompatibility, is considered a potential tissue-regenerative biomaterial, especially for minimally invasive surgery that requires small incisions to approach large defects with excellent regeneration capacity.

Chemically-induced osteogenic cells for bone tissue engineering and disease modeling.

Cell reprogramming can satisfy the demands of obtaining specific cell types for applications such as tissue regeneration and disease modeling. Here we report the reprogramming of human fibroblasts to produce chemically-induced osteogenic cells (ciOG), and explore the potential uses of ciOG in bone repair and disease treatment. A chemical cocktail of RepSox, forskolin, and phenamil was used for osteogenic induction of fibroblasts by activation of RUNX2 expression. Following a maturation, the cells differentiated toward an osteoblast phenotype that produced mineralized nodules. Bulk and single-cell RNA sequencing identified a distinct ciOG population. ciOG formed mineralized tissue in an ectopic site of immunodeficiency mice, unlike the original fibroblasts. Osteogenic reprogramming was modulated under engineered culture substrates. When generated on a nanofiber substrate ciOG accelerated bone matrix formation in a calvarial defect, indicating that the engineered biomaterial promotes the osteogenic capacity of ciOG in vivo. Furthermore, the ciOG platform recapitulated the genetic bone diseases Proteus syndrome and osteogenesis imperfecta, allowing candidate drug testing. The reprogramming of human fibroblasts into osteogenic cells with a chemical cocktail thus provides a source of specialized cells for use in bone tissue engineering and disease modeling.

Diabetic bone regeneration with nanoceria-tailored scaffolds by recapitulating cellular microenvironment: Activating integrin/TGF-ß co-signaling of MSCs while relieving oxidative stress.

Regenerating defective bone in patients with diabetes mellitus remains a significant challenge due to high blood glucose level and oxidative stress. Here we aim to tackle this issue by means of a drug- and cell-free scaffolding approach. We found the nanoceria decorated on various types of scaffolds (fibrous or 3D-printed one; named nCe-scaffold) could render a therapeutic surface that can recapitulate the microenvironment: modulating oxidative stress while offering a nanotopological cue to regenerating cells. Mesenchymal stem cells (MSCs) recognized the nanoscale (tens of nm) topology of nCe-scaffolds, presenting highly upregulated curvature-sensing membrane protein, integrin set, and adhesion-related molecules. Osteogenic differentiation and mineralization were further significantly enhanced by the nCe-scaffolds. Of note, the stimulated osteogenic potential was identified to be through integrin-mediated TGF-ß co-signaling activation. Such MSC-regulatory effects were proven in vivo by the accelerated bone formation in rat calvarium defect model. The nCe-scaffolds further exhibited profound enzymatic and catalytic potential, leading to effectively scavenging reactive oxygen species in vivo. When implanted in diabetic calvarium defect, nCe-scaffolds significantly enhanced early bone regeneration. We consider the currently-exploited nCe-scaffolds can be a promising drug- and cell-free therapeutic means to treat defective tissues like bone in diabetic conditions.

Mimicking Bone Extracellular Matrix: From BMP-2-Derived Sequences to Osteogenic-Multifunctional Coatings.

Cell-material interactions are regulated by mimicking bone extracellular matrix on the surface of biomaterials. In this regard, reproducing the extracellular conditions that promote integrin and growth factor (GF) signaling is a major goal to trigger bone regeneration. Thus, the use of synthetic osteogenic domains derived from bone morphogenetic protein 2 (BMP-2) is gaining increasing attention, as this strategy is devoid of the clinical risks associated with this molecule. In this work, the wrist and knuckle epitopes of BMP-2 are screened to identify peptides with potential osteogenic properties. The most active sequences (the DWIVA motif and its cyclic version) are combined with the cell adhesive RGD peptide (linear and cyclic variants), to produce tailor-made biomimetic peptides presenting the bioactive cues in a chemically and geometrically defined manner. Such multifunctional peptides are next used to functionalize titanium surfaces. Biological characterization with mesenchymal stem cells demonstrates the ability of the biointerfaces to synergistically enhance cell adhesion and osteogenic differentiation. Furthermore, in vivo studies in rat calvarial defects prove the capacity of the biomimetic coatings to improve new bone formation and reduce fibrous tissue thickness. These results highlight the potential of mimicking integrin-GF signaling with synthetic peptides, without the need for exogenous GFs.

Protein-reactive nanofibrils decorated with cartilage-derived decellularized extracellular matrix for osteochondral defects.

Cartilage defect is difficult to heal due to its avascular properties. Implantation of mesenchymal stem cell is one of the most promising approach for regenerating cartilage defects. Here we prepared polymeric nanofibrils decorated with cartilage-derived decellularized extracellular matrix (dECM) as a chondroinductive scaffold material for cartilage repair. To fabricate nanofibrils, eletrospun PCL nanofibers were fragmented by subsequent mechanical and chemical process. The nanofibrils were surface-modified with poly(glycidyl methacrylate) (PGMA@NF) via surface-initiated atom transfer radical polymerization (SI-ATRP). The epoxy groups of PGMA@NF were subsequently reacted with dECM prepared from bovine articular cartilage. Therefore, the cartilage-dECM-decorated nanofibrils structurally and biochemically mimic cartilage-specific microenvironment. Once adipose-derived stem cells (ADSCs) were self-assembled with the cartilage-dECM-decorated nanofibrils by cell-directed association, they exhibited differentiation hallmarks of chondrogenesis without additional biologic additives. ADSCs in the nanofibril composites significantly increased expression of chondrogenic gene markers in comparison to those in pellet culture. Furthermore, ADSC-laden nanofibril composites filled the osteochondral defects compactly due to their clay-like texture. Thus, the ADSC-laden nanofibril composites supported the long-term regeneration of 12 weeks without matrix loss during joint movement. The defects treated with the ADSC-laden PGMA@NF significantly facilitated reconstruction of their cartilage and subchondral bone ECM matrices compared to those with ADSC-laden nanofibrils, non-specifically adsorbing cartilage-dECM without surface decoration of PGMA.

Label-Free Fluorescent Mesoporous Bioglass for Drug Delivery, Optical Triple-Mode Imaging, and Photothermal/Photodynamic Synergistic Cancer Therapy.

Nanomaterials combined with phototherapy and multimodal imaging are promising for cancer theranostics. Our aim is to develop fluorescent mesoporous bioglass nanoparticles (fBGn) based on carbon dots (CD) with delivery, triple-mode imaging, and photothermal (PTT) properties for cancer theranostics. A direct and label-free approach was used to prepare multicolor fluorescent fBGn with 3-aminopropyl triethoxysilane as the surface-functionalizing agent. The calcination at 400 °C provided fBGn with high fluorescence intensity originating from the CD. In particular, a triple-mode emission [fluorescence imaging, two-photon (TP), and Raman imaging] was observed which depended on CD nature and surface properties such as surface oxidation edge state, amorphous region, nitrogen passivation of surface state, and crystalline region. The fBGn also exhibited phototherapeutic properties such as photodynamic (PDT) and PTT effects. The antitumor effect of the combined PDT/PTT therapy was significantly higher than that of individual (PDT or PTT) therapy. The fBGn, due to the mesoporous structure, the anticancer drug doxorubicin could be loaded and released in a pH-dependent way to show chemotherapy effects on cancer cells. The in vivo imaging and biocompatibility of fBGn were also demonstrated in a nude mouse model. The fBGn, with the combined capacity of anticancer delivery, triple-mode imaging, and PTT/PDT therapy, are considered to be potentially useful for cancer theranostics.

Angiogenesis-promoted bone repair with silicate-shelled hydrogel fiber scaffolds.

Promoting angiogenesis is a key strategy for stimulating the repair of damaged tissues, including bone. Among other proangiogenic factors, ions have recently been considered a potent element that can be incorporated into biomaterials and then released at therapeutic doses. Silicate-based biomaterials have been reported to induce neovascularization through vascular endothelial growth factor signaling pathway, potentiating acceleration of bone regeneration. Here, we designed a silicate-shelled hydrogel fiber scaffold with a hard/soft layered structure to investigate the possibility of silicate coating on biopolymer for enhancing biological properties. An alginate hydrogel was injected to form a fiber scaffold with shape-tunability that was then coated with a thin silicate layer with various sol-gel compositions. The silicate/alginate scaffold could release calcium and silicate ions, and in particular, silicate ion release was highly sustainable for over one week at therapeutically relevant levels. The ionic release was highly effective in stimulating the mRNA expression of angiogenic markers (VEGF, KDR, eNOS, bFGF, and HIF1-α) in endothelial cells (HUVECs). Moreover, the in vitro tubular networking of cells was significantly enhanced (1.5 times). In vivo implantation in subcutaneous tissue revealed more pronounced blood vessel formation around the silicate-shelled scaffolds than around silicate-free scaffolds. The presence of a silicate shell was also shown to accelerate acellular mineral (hydroxyapatite) formation. The cellular osteogenesis potential of the silicate/alginate scaffold was further proven by the enhanced expression of osteogenic genes (Col1a1, ALP and OCN). When implanted in a rat calvarium defect, the silicate-shelled scaffold demonstrated significantly improved bone formation (2-3 times higher in bone volume and density) with a concurrent sign of proangiogenesis. This work highlights that the surface-layering of silicate composition is an effective approach for improving the bone regeneration capacity of polymeric hydrogel scaffolds by stimulating ion-induced angiogenesis and providing bone bioactivity to the surface.

Anti-inflammatory actions of folate-functionalized bioactive ion-releasing nanoparticles imply drug-free nanotherapy of inflamed tissues.

Inflammation prevailing conditions delay healing processes of damaged tissues, leading to a functional impairment. Although anti-inflammatory drugs are clinically available, they often cause unwanted side effects thus being considered suboptimal. Here we report drug-free synthetic nanoparticles that target and internalize pro-inflammatory cells and release ions, ultimately demonstrating profound anti-inflammatory functions. We introduce folate-functionalized bioactive glass nanoparticle BGN(F) that can bind to pro-inflammatory cells to endocytose and release ions. The folate-conjugation significantly enhanced the nanoparticle internalization to LPS-induced pro-inflammatory cells. The direct treatment of BGN(F) at proper doses (80-160 µg/mL) substantially down-regulated pro-inflammatory molecules, including TNF-α, IL-6, iNOS and COX-2, at both gene and protein levels. The phosphorylation of intracellular signaling molecules involved in the inflammatory events, such as p38 MAPK, ERK (1/2), SAPK/JANK, IκBα, and NF-κB, were significantly suppressed by the BGN(F) treatment. Furthermore, BGN(F) was potential to switch the macrophage polarization from M1 to M2. The released ions, not the physical interactions, of nanoparticles were observed to contribute in major part to the anti-inflammatory actions of BGN(F). The BGN(F), when locally administered to a Notexin-induced myoinjury tissue in mice, significantly down-regulated IL-6 and TNF-α, switched the macrophage phenotype from M1 to M2, and accelerated tissue healing. The current findings that demonstrate profound anti-inflammatory actions of BGN(F) in vitro and in vivo support their uses as novel drug-free nanotherapeutic platform for the treatment of inflamed tissues.

Intracellular co-delivery of Sr ion and phenamil drug through mesoporous bioglass nanocarriers synergizes BMP signaling and tissue mineralization.

Inducing differentiation and maturation of resident multipotent stem cells (MSCs) is an important strategy to regenerate hard tissues in mal-calcification conditions. Here we explore a co-delivery approach of therapeutic molecules comprised of ion and drug through a mesoporous bioglass nanoparticle (MBN) for this purpose. Recently, MBN has offered unique potential as a nanocarrier for hard tissues, in terms of high mesoporosity, bone bioactivity (and possibly degradability), tunable delivery of biomolecules, and ionic modification. Herein Sr ion is structurally doped to MBN while drug Phenamil is externally loaded as a small molecule activator of BMP signaling, for the stimulation of osteo/odontogenesis and mineralization of human MSCs derived from dental pulp. The Sr-doped MBN (85Si:10Ca:5Sr) sol-gel processed presents a high mesoporosity with a pore size of ∼6nm. In particular, Sr ion is released slowly at a daily rate of ∼3ppm per mg nanoparticles for up to 7days, a level therapeutically effective for cellular stimulation. The Sr-MBN is internalized to most MSCs via an ATP dependent macropinocytosis within hours, increasing the intracellular levels of Sr, Ca and Si ions. Phenamil is loaded maximally ∼30% into Sr-MBN and then released slowly for up to 7days. The co-delivered molecules (Sr ion and Phenamil drug) have profound effects on the differentiation and maturation of cells, i.e., significantly enhancing expression of osteo/odontogenic genes, alkaline phosphatase activity, and mineralization of cells. Of note, the stimulation is a result of a synergism of Sr and Phenamil, through a Trb3-dependent BMP signaling pathway. This biological synergism is further evidenced in vivo in a mal-calcification condition involving an extracted tooth implantation in dorsal subcutaneous tissues of rats. Six weeks post operation evidences the osseous-dentinal hard tissue formation, which is significantly stimulated by the Sr/Phenamil delivery, based on histomorphometric and micro-computed tomographic analyses. The bioactive nanoparticles releasing both Sr ion and Phenamil drug are considered to be a promising therapeutic nanocarrier platform for hard tissue regeneration. Furthermore, this novel ion/drug co-delivery concept through nanoparticles can be extensively used for other tissues that require different therapeutic treatment. STATEMENT OF SIGNIFICANCE: This study reports a novel design concept in inorganic nanoparticle delivery system for hard tissues - the co-delivery of therapeutic molecules comprised of ion (Sr) and drug (Phenamil) through a unique nanoparticle of mesoporous bioactive glass (MBN). The physico-chemical and biological properties of MBN enabled an effective loading of both therapeutic molecules and a subsequently sustained/controlled release. The co-delivered Sr and Phenamil demonstrated significant stimulation of adult stem cell differentiation in vitro and osseous/dentinal regeneration in vivo, through BMP signaling pathways. We consider the current combination of Sr ion with Phenamil is suited for the osteo/odontogenesis of stem cells for hard tissue regeneration, and further, this ion/drug co-delivery concept can extend the applications to other areas that require specific cellular and tissue functions.