Scaffold-based non-viral CRISPR delivery platform for efficient and prolonged gene activation to accelerate tissue regeneration.

Clustered regularly interspaced short palindromic repeat activation (CRISPRa) technology has emerged as a precise genome editing tool for activating endogenous transgene expression. While it holds promise for precise cell modification, its translation into tissue engineering has been hampered by biosafety concerns and suboptimal delivery methods. To address these challenges, we have developed a CRISPRa non-viral gene delivery platform by immobilizing non-viral CRISPRa complexes into a biocompatible hydrogel/nanofiber (Gel/NF) composite scaffold. The Gel/NF scaffold facilitates the controlled and sustained release of CRISPRa complexes and also promotes cell recruitment to the scaffold for efficient and localized transfection. As a proof of concept, we employed this CRISPRa delivery platform to activate the vascular endothelial growth factor (VEGF) gene in a rat model with full-thickness skin defects. Our results demonstrate sustained upregulation of VEGF expression even at 21 days post-implantation, resulting in enhanced angiogenesis and improved skin regeneration. These findings underscore the potential of the Gel/NF scaffold-based CRISPRa delivery platform as an efficient and durable strategy for gene activation, offering promising prospects for tissue regeneration. STATEMENT OF SIGNIFICANCE: Translation of clustered regularly interspaced short palindromic repeat activation (CRISPRa) therapy to tissue engineering is limited by biosafety concerns and unsatisfactory delivery strategy. To solve this issue, we have developed a CRISPRa non-viral gene delivery platform by immobilizing non-viral CRISPRa complexes into a biocompatible hydrogel/nanofiber (Gel/NF) composite scaffold. This scaffold enables controlled and sustained release of CRISPRa and can induce cell recruitment for localized transfection. As a proof of concept, we activated vascular endothelial growth factor (VEGF) in a rat model with full-thickness skin defects, leading to sustained upregulation of VEGF expression, enhanced angiogenesis and improved skin regeneration in vivo. These findings demonstrate the potential of this platform for gene activation, thereby offering promising prospects for tissue regeneration.

Simultaneous enhancement of strength and ductility via microband formation and nanotwinning in an L12-strengthened alloy.

Metallic alloys with high strength and large ductility are required for extreme structural applications. However, the achievement of ultrahigh strength often results in a substantially decreased ductility. Here, we report a strategy to achieve the strength-ductility synergy by tailoring the alloy composition to control the local stacking fault energy (SFE) of the face-centered-cubic (fcc) matrix in an L12-strengthened superlattice alloy. As a proof of concept, based on the thermodynamic calculations, we developed a non-equiatomic CoCrNi2(Al0.2Nb0.2) alloy using phase separation to create a near-equiatomic low SFE disordered CoCrNi medium-entropy alloy matrix with in situ formed high-content coherent Ni3(Al, Nb)-type ordered nanoprecipitates (∼ 12 nm). The alloy achieves a high tensile strength up to 1.6 GPa and a uniform ductility of 33%. The low SFE of the fcc matrix promotes the formation of nanotwins and parallel microbands during plastic deformation which could remarkably enhance the strain hardening capacity. This work provides a strategy for developing ultrahigh-strength alloys with large uniform ductility.

Aptamer Functionalized Hypoxia-potentiating Agent and Hypoxia-inducible Factor Inhibitor Combined with Hypoxia-activated Prodrug for Enhanced Tumor Therapy.

Triple-negative breast cancer (TNBC) is the most lethal subtype of breast cancer. Hypoxia-activated prodrugs (HAPs) have shown promise as potential therapeutic agents for TNBC. While increasing hypoxia levels may promote the HAP activation, it raises concerns regarding HIF1α-dependent drug resistance. It is desirable to develop a targeted approach that enhances tumor hypoxia for HAP activation without promoting HIF1α-dependent drug resistance in TNBC treatment. Herein, we proposed a multi-responsive carrier-free self-assembled nanomedicine named AQ4N@CA4T1ASO. This nanomedicine first targeted tumors by the TNBC-targeting aptamers (T1), and then disassembled in the reductive and acidic conditions within tumors. The released Combretastatin 4 (CA4) could exacerbate hypoxia, thereby promoting the conversion of inactive Banoxantrone (AQ4N) to its active form, AQ4. Simultaneously, the released antisense oligonucleotide (ASO) could attenuate hypoxia-induced HIF1α mRNA expression, thereby sensitizing the tumor to chemotherapy. Overall, this smart nanomedicine represents a profound targeted therapy strategy, combining "hypoxia-potentiating, hypoxia-activated, chemo-sensitization" approaches for TNBC treatment. In vivo study demonstrated significant suppression of tumor growth, highlighting the promising potential of this nanomedicine for future clinical translation.

Sclerostin inhibition in rare bone diseases: Molecular understanding and therapeutic perspectives.

Sclerostin emerges as a novel target for bone anabolic therapy in bone diseases. Osteogenesis imperfecta (OI) and X-linked hypophosphatemia (XLH) are rare bone diseases in which therapeutic potential of sclerostin inhibition cannot be ignored. In OI, genetic/pharmacologic sclerostin inhibition promoted bone formation of mice, but responses varied by genotype and age. Serum sclerostin levels were higher in young OI-I patients, while lower in adult OI-I/III/IV. It's worth investigating whether therapeutic response of OI to sclerostin inhibition could be clinically predicted by genotype and age. In XLH, preclinical/clinical data suggested factors other than identified FGF23 contributing to XLH. Higher levels of circulating sclerostin were detected in XLH. Sclerostin inhibition promoted bone formation in Hyp mice, while restored phosphate homeostasis in age-/gender-dependent manner. The role of sclerostin in regulating phosphate metabolism deserves investigation. Sclerostin/FGF23 levels of XLH patients with/without response to FGF23-antibody warrants study to develop precise sclerostin/FGF23 inhibition strategy or synergistic/additive strategy. Notably, OI patients were associated with cardiovascular abnormalities, so were XLH patients receiving conventional therapy. Targeting sclerostin loop3 promoted bone formation without cardiovascular risks. Further, blockade of sclerostin loop3-LRP4 interaction while preserving sclerostin loop2-ApoER2 interaction could be a potential precise sclerostin inhibition strategy for OI and XLH with cardiovascular safety. The Translational Potential of this Article. Preclinical data on the molecular understanding of sclerostin inhibition in OI and therapeutic efficacy in mouse models of different genotypes, as well as clinical data on serum sclerostin levels in patients with different phenotypes of OI, were reviewed and discussed. Translationally, it would facilitate to develop clinical prediction strategies (e.g. based on genotype and age, not just phenotype) for OI patients responsive to sclerostin inhibition. Both preclinical and clinical data suggested sclerostin as another factor contributing to XLH, in addition to the identified FGF23. The molecular understanding and therapeutic effects of sclerostin inhibition on both promoting bone anabolism and improving phosphate homostasis in Hyp mice were reviewed and discussed. Translationaly, it would facilitate the development of precise sclerostin/FGF23 inhibition strategy or synergistic/additive strategy for the treatment of XLH. Cardiovascular risk could not be ruled out during sclerostin inhibition treatment, especially for OI and XLH patients with cardiovascular diseases history and cardiovascular abnormalities. Studies on the role of sclerostin in inhiting bone formation and protecting cardiovascular system were reviewed and discussed. Translationaly, blockade of sclerostin loop3-LRP4 interaction while preserving sclerostin loop2-ApoER2 interaction could be a potential precise sclerostin inhibition strategy for OI and XLH with cardiovascular safety.

Fabrication of Porous Tantalum with Low Elastic Modulus and Tunable Pore Size for Bone Repair.

Porous tantalum (Ta) is a potential bone substitute due to its excellent biocompatibility and desirable mechanical properties. In this work, a series of porous Ta materials with interconnected micropores and varying pore sizes from 23 to 210 µm were fabricated using spark plasma sintering. The porous structure was formed by thermal decomposition of ammonium bicarbonate powder premixed in the Ta powder. The pore size and porosity were controlled by the categorized particle size of ammonium bicarbonate. The porous Ta has elastic moduli in the range of 2.1-3.2 GPa and compressive yield strength in the range of 23-34 MPa, which are close to those of human bone. In vitro, as-fabricated porous Ta demonstrates excellent biocompatibility by supporting adhesion and proliferation of preosteoblasts. In vivo studies also validate its bone repair capability after implantation in a rat femur defect model. The study demonstrates a facile strategy to fabricate porous Ta with controllable pore size for bone repair.

The roles of hepatokine and osteokine in liver-bone crosstalk: Advance in basic and clinical aspects.

Both the liver and bone are important secretory organs in the endocrine system. By secreting organ factors (hepatokines), the liver regulates the activity of other organs. Similarly, bone-derived factors, osteokines, are created during bone metabolism and act in an endocrine manner. Generally, the dysregulation of hepatokines is frequently accompanied by changes in bone mass, and osteokines can also disrupt liver metabolism. The crosstalk between the liver and bone, particularly the function and mechanism of hepatokines and osteokines, has increasingly gained notoriety as a topic of interest in recent years. Here, based on preclinical and clinical evidence, we summarize the potential roles of hepatokines and osteokines in liver-bone interaction, discuss the current shortcomings and contradictions, and make recommendations for future research.

Controlled pVEGF delivery via a gene-activated matrix comprised of a peptide-modified non-viral vector and a nanofibrous scaffold for skin wound healing.

Regulating cell function and tissue formation by combining gene delivery with functional scaffolds to create gene-activated matrices (GAMs) is a promising strategy for tissue engineering. However, fabrication of GAMs with low cytotoxicity, high transfection efficiency, and long-term gene delivery properties remains a challenge. In this study, a non-viral DNA delivery nanocomplex was developed by modifying poly (D, L-lactic-co-glycolic acid)/polyethylenimine (PLGA/PEI) nanoparticles with the cell-penetrating peptide KALA. Subsequently, the nanocomplex carrying plasmid DNA encoding vascular endothelial growth factor (pVEGF) was immobilized onto a polydopamine-coated electrospun alginate nanofibrous scaffold, resulting in a GAM for enhanced skin wound healing. The nanocomplex exhibited much lower cytotoxicity and comparable or even higher transfection efficiency compared with PEI. The GAM enabled sustained gene release and long-tern transgene expression of VEGF in vitro. In an excisional full-thickness skin wound rat model, the GAM could accelerate wound closure, promote complete re-epithelization, reduce inflammatory response, and enhance neovascularization, ultimately enhancing skin wound healing. The current GAM comprising a low-toxic gene delivery nanocomplex and a biocompatible 3D nanofibrous scaffold demonstrates great potential for mediating long-term cell functions and may become a powerful tool for gene delivery in tissue engineering. STATEMENT OF SIGNIFICANCE: Gene delivery is a promising strategy in promoting tissue regeneration as an effective alternative to growth factor delivery, but the study on three-dimensional gene-activated scaffolds remains in its infancy. Herein, a biodegradable nanofibrous gene-activated matrix integrating non-viral nanoparticle vector was designed and evaluated both in vitro and in vivo. The results show that the nanoparticle vector provided high transfection efficiency with minimal cytotoxicity. After surface immobilization of the nanocomplexes carrying plasmid DNA encoding vascular endothelial growth factor (pVEGF), the nanofibrous scaffold enabled sustained DNA release and long-term transgene expression in vitro. In a rat full-thickness skin wound model, the scaffold could accelerate wound healing. This innovative gene-activated matrix can be a promising candidate for tissue regeneration.

Spatiotemporal Delivery of pBMP2 and pVEGF by a Core-Sheath Structured Fiber-Hydrogel Gene-Activated Matrix Loaded with Peptide-Modified Nanoparticles for Critical-Sized Bone Defect Repair.

The clinical translation of bioactive scaffolds for the treatment of large segmental bone defects remains a grand challenge. The gene-activated matrix (GAM) combining gene therapy and tissue engineering scaffold offers a promising strategy for the restoration of structure and function of damaged or dysfunctional tissues. Herein, a gene-activated biomimetic composite scaffold consisting of an electrospun poly(ε-caprolactone) fiber sheath and an alginate hydrogel core which carried plasmid DNA encoding bone morphogenetic protein 2 (pBMP2) and vascular endothelial growth factor (pVEGF), respectively, is developed. A peptide-modified polymeric nanocarrier with low cytotoxicity and high efficiency serves as the nonviral DNA delivery vector. The obtained GAM allows spatiotemporal release of pVEGF and pBMP2 and promotes osteogenic differentiation of preosteoblasts in vitro. In vivo evaluation using a critical-sized segmental femoral defect model in rats shows that the dual gene delivery system can significantly accelerate bone healing by activating angiogenesis and osteogenesis. These findings demonstrate the effectiveness of the developed dual gene-activated core-sheath structured fiber-hydrogel composite scaffold for critical-sized bone defect regeneration and the potential of cell-free scaffold-based gene therapy for tissue engineering.

High-Strength, Biomimetic Functional Chitosan-Based Hydrogels for Full-Thickness Osteochondral Defect Repair.

Fabrication of a hydrogel scaffold for full-thickness osteochondral defect repair remains a grand challenge. Developing layered and multiphasic hydrogels to mimic the intrinsic hierarchical structure of the osteochondral unit is a promising strategy. Chitosan-based hydrogels are widely applied for biomedical applications. However, insufficient mechanical strength and lack of biological cues to restore damaged cartilage and subchondral tissue significantly hinder their application in osteochondral tissue engineering. In this study, a strong and tough, osteochondral-mimicking functional chitosan-based hydrogel (bilayer-gel) with an in situ mineralized, osteoconductive lower layer and a basic fibroblast growth factor (bFGF)-incorporated, chondrogenic inducing upper layer was developed. The obtained bilayer-gel showed a depth-dependent gradient pore structure and composition. The strong double crosslinked hydrogel network and the homogeneous deposition of hydroxyapatite nanoparticles (HAp) at the lower layer provided a compressive strength of up to 2.5 MPa and a compressive strain of up to 40%. In vitro study showed that the bilayer-gel facilitates both chondrogenic differentiation in the upper layer and osteogenic differentiation in the lower layer. In vivo implantation revealed that the bilayer-gel could simultaneously promote hyaline cartilage and subchondral bone formation, thus resulting in an improved osteochondral reconstruction outcome. The present bilayer-gel thus shows great potential for full-thickness osteochondral defect repair.

Targeting loop3 of sclerostin preserves its cardiovascular protective action and promotes bone formation.

Sclerostin negatively regulates bone formation by antagonizing Wnt signalling. An antibody targeting sclerostin for the treatment of postmenopausal osteoporosis was approved by the U.S. Food and Drug Administration, with a boxed warning for cardiovascular risk. Here we demonstrate that sclerostin participates in protecting cardiovascular system and inhibiting bone formation via different loops. Loop3 deficiency by genetic truncation could maintain sclerostin's protective effect on the cardiovascular system while attenuating its inhibitory effect on bone formation. We identify an aptamer, named aptscl56, which specifically targets sclerostin loop3 and use a modified aptscl56 version, called Apc001PE, as specific in vivo pharmacologic tool to validate the above effect of loop3. Apc001PE has no effect on aortic aneurysm and atherosclerotic development in ApoE-/- mice and hSOSTki.ApoE-/- mice with angiotensin II infusion. Apc001PE can promote bone formation in hSOSTki mice and ovariectomy-induced osteoporotic rats. In summary, sclerostin loop3 cannot participate in protecting the cardiovascular system, but participates in inhibiting bone formation.

Therapeutic aptamer targeting sclerostin loop3 for promoting bone formation without increasing cardiovascular risk in osteogenesis imperfecta mice.

Rationale: Sclerostin inhibition demonstrated bone anabolic potential in osteogenesis imperfecta (OI) mice, whereas humanized therapeutic sclerostin antibody romosozumab for postmenopausal osteoporosis imposed clinically severe cardiac ischemic events. Therefore, it is desirable to develop the next generation sclerostin inhibitors to promote bone formation without increasing cardiovascular risk for OI. Methods and Results: Our data showed that sclerostin suppressed inflammatory responses, prevented aortic aneurysm (AA) and atherosclerosis progression in hSOSTki.Col1a2+/G610C.ApoE-/- mice. Either loop2&3 deficiency or inhibition attenuated sclerostin's suppressive effects on expression of inflammatory cytokines and chemokines in vitro, whilst loop3 deficiency maintained the protective effect of sclerostin on cardiovascular system both in vitro and in vivo. Moreover, loop3 was critical for sclerostin's antagonistic effect on bone formation in Col1a2+/G610C mice. Accordingly, a sclerostin loop3-specific aptamer aptscl56 was identified by our lab. It could recognize both recombinant sclerostin and sclerostin in the serum of OI patients via targeting loop3. PEG40k conjugated aptscl56 (Apc001PE) demonstrated to promote bone formation, increase bone mass and improve bone microarchitecture integrity in Col1a2+/G610C mice via targeting loop3, while did not show influence in inflammatory response, AA and atherosclerosis progression in Col1a2+/G610C.ApoE-/- mice with Angiotensin II infusion. Further, Apc001PE had no influence in the protective effect of sclerostin on cardiovascular system in hSOSTki.Col1a2+/G610C.ApoE-/- mice, while it inhibited the antagonistic effect of sclerostin on bone formation in hSOSTki.Col1a2+/G610C mice via targeting loop3. Apc001PE was non-toxic to healthy rodents, even at ultrahigh dose. Apc001PE for OI was granted orphan drug designation by US-FDA in 2019 (DRU-2019-6966). Conclusion: Sclerostin loop3-specific aptamer Apc001PE promoted bone formation without increasing cardiovascular risk in OI mice.

The Synergy of Topographical Micropatterning and Ta|TaCu Bilayered Thin Film on Titanium Implants Enables Dual-Functions of Enhanced Osteogenesis and Anti-Infection.

Poor osteogenesis and implant-associated infection are the two leading causes of failure for dental and orthopedic implants. Surface design with enhanced osteogenesis often fails in antibacterial activity, or vice versa. Herein, a surface design strategy, which overcomes this trade-off via the synergistic effects of topographical micropatterning and a bilayered nanostructured metallic thin film is presented. A specific microgrooved pattern is fabricated on the titanium surface, followed by sequential deposition of a nanostructured copper (Cu)-containing tantalum (Ta) (TaCu) layer and a pure Ta cap layer. The microgrooved patterns coupled with the nanorough Ta cap layer shows strong contact guidance to preosteoblasts and significantly enhances the osteogenic differentiation in vitro, while the controlled local sustained release of Cu ions is responsible for high antibacterial activity. Importantly, rat calvarial defect models in vivo further confirm that the synergy of microgrooved patterns and the Ta|TaCu bilayered thin film on titanium surface could effectively promote bone regeneration. The present effective and versatile surface design strategy provides significant insight into intelligent surface engineering that can control biological response at the site of healing in dental and orthopedic implants.

Exosomal transfer of osteoclast-derived miRNAs to chondrocytes contributes to osteoarthritis progression.

Osteoarthritis (OA) is a prevalent aging-related joint disease lacking disease-modifying therapies. Here, we identified an upregulation of circulating exosomal osteoclast (OC)-derived microRNAs (OC-miRNAs) during the progression of surgery-induced OA in mice. We found that reducing OC-miRNAs by Cre-mediated excision of the key miRNA-processing enzyme Dicer or blocking the secretion of OC-originated exosomes by short interfering RNA-mediated silencing of Rab27a substantially delayed the progression of surgery-induced OA in mice. Mechanistically, the exosomal transfer of OC-miRNAs to chondrocytes reduced the resistance of cartilage to matrix degeneration, osteochondral angiogenesis and sensory innervation during OA progression by suppressing tissue inhibitor of metalloproteinase-2 (TIMP-2) and TIMP-3. Furthermore, systemic administration of a new OC-targeted exosome inhibitor (OCExoInhib) blunted the progression of surgery-induced OA in mice. We suggest that targeting the exosomal transfer of OC-miRNAs to chondrocytes represents a potential therapeutic avenue to tackle OA progression.

Author Correction: HIF1α inhibition facilitates Leflunomide-AHR-CRP signaling to attenuate bone erosion in CRP-aberrant rheumatoid arthritis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Engineering High-Resolution Micropatterns Directly onto Titanium with Optimized Contact Guidance to Promote Osteogenic Differentiation and Bone Regeneration.

Topographical cues play an important role in directing cell behavior, and thus, extensive research efforts have been devoted to fabrication of surface patterns and exploring the contact guidance effect. However, engineering high-resolution micropatterns directly onto metallic implants remains a grand challenge. Moreover, there still lacks evidence that allows translation of in vitro screening to in vivo tissue response. Herein, we demonstrate a fast, cost-effective, and feasible approach to the precise fabrication of shape- and size-controlled micropatterns on titanium substrates using a combination of photolithography and inductively coupled plasma-based dry etching. A titanium TopoChip containing 34 microgrooved patterns with varying geometry parameters and a flat surface as the control was designed for a high-throughput in vitro study of the contact guidance of osteoblasts. The correlation between the surface pattern dimensions, cell morphological characteristics, proliferation, and osteogenic marker expression was systematically investigated in vitro. Furthermore, the surface with the highest osteogenic potential in vitro along with representative controls was evaluated in rat cranial defect models. The results show that microgrooved pattern parameters have almost no effect on osteoblast proliferation but significantly regulate the cell morphology, orientation, focal adhesion (FA) formation, and osteogenic differentiation in vitro. In particular, a specific groove pattern with a ridge width of 3 µm, groove width of 7 µm, and depth of 2 µm can most effectively align the cells through regulating the distribution of FAs, resulting in an anisotropic actin cytoskeleton, and thereby promoting osteogenic differentiation. In vivo, microcomputed tomography and histological analyses show that the optimized pattern can apparently stimulate new bone formation. This study not only offers a microfabrication method that can be extended to fabricate various shape- and size-controlled micropatterns on titanium alloys but also provides insight into the surface structure design of orthopedic and dental implants for enhanced bone regeneration.

HIF1α inhibition facilitates Leflunomide-AHR-CRP signaling to attenuate bone erosion in CRP-aberrant rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disorder characterized by progressive bone erosion. Leflunomide is originally developed to suppress inflammation via its metabolite A77 1726 to attenuate bone erosion. However, distinctive responsiveness to Leflunomide is observed among RA individuals. Here we show that Leflunomide exerts immunosuppression but limited efficacy in RA individuals distinguished by higher serum C-reactive protein (CRPHigher, CRPH), whereas the others with satisfactory responsiveness to Leflunomide show lower CRP (CRPLower, CRPL). CRP inhibition decreases bone erosion in arthritic rats. Besides the immunomodulation via A77 1726, Leflunomide itself induces AHR-ARNT interaction to inhibit hepatic CRP production and attenuate bone erosion in CRPL arthritic rats. Nevertheless, high CRP in CRPH rats upregulates HIF1α, which competes with AHR for ARNT association and interferes Leflunomide-AHR-CRP signaling. Hepatocyte-specific HIF1α deletion or a HIF1α inhibitor Acriflavine re-activates Leflunomide-AHR-CRP signaling to inhibit bone erosion. This study presents a precision medicine-based therapeutic strategy for RA.