Enzyme-responsive nanospheres target senescent cells for diabetic wound healing by employing chemodynamic therapy.

Evidence indicates that prolonged low-level inflammation and elevated-glucose-induced oxidative stress in diabetic wounds can accelerate senescence. The accumulation of senescent cells, in turn, inhibits cellular proliferation and migration, aggravating the inflammatory response and oxidative stress, ultimately impeding wound healing. In this study, we exploited the heightened lysosomal ß-galactosidase activity detected in senescent cells to develop an innovative drug delivery system by encapsulating Fe3O4 with galactose-modified poly (lactic-co-glycolic acid) (PLGA) (F@GP). We found that F@GP can selectively release Fe3O4 into senescent cells, inducing ferroptosis via the Fenton reaction in the presence of elevated intracellular H2O2 levels. This showed that F@GP administration can serve as a chemodynamic therapy to eliminate senescent cells and promote cell proliferation. Furthermore, the F@GP drug delivery system gradually released iron ions into the diabetic wound tissues, enhancing the attenuation of cellular senescence, stimulating cell proliferation, promoting re-epithelialization, and accelerating the healing of diabetic wounds in mice. Our groundbreaking approach unveiled the specific targeting of senescence by F@GP, demonstrating its profound effect on promoting the healing of diabetic wounds. This discovery underscores the therapeutic potential of F@GP in effectively addressing challenging cases of wound repair. STATEMENT OF SIGNIFICANCE: The development of galactose-modified PLGA nanoparticles loaded with Fe3O4 (F@GP) represents a significant therapeutic approach for the treatment of diabetic wounds. These nanoparticles exhibit remarkable potential in selectively targeting senescent cells, which accumulate in diabetic wound tissue, through an enzyme-responsive mechanism. By employing chemodynamic therapy, F@GP nanoparticles effectively eliminate senescent cells by releasing iron ions that mediate the Fenton reaction. This targeted approach holds great promise for promoting diabetic wound healing by selectively eliminating senescent cells, which play a crucial role in impairing the wound healing process. The innovative utilization of F@GP nanoparticles as a therapeutic intervention offers a novel and potentially transformative strategy for addressing the challenges associated with diabetic wound healing.

Defective NCOA4-dependent ferroptosis in senescent fibroblasts retards diabetic wound healing.

Cellular senescence describes a state of permanent proliferative arrest in cells. Studies have demonstrated that diabetes promotes the pathological accumulation of senescent cells, which in turn impairs cell movement and proliferation. Historically, senescence has been perceived to be a detrimental consequence of chronic wound healing. However, the underlying mechanism that causes senescent cells to remain in diabetic wounds is yet to be elucidated. Ferroptosis and ferritinophagy observed in diabetes are due to iron metabolism disorders, which are directly associated with the initiation and progression of diabetes. Herein, we reveal that senescent fibroblasts in diabetic wounds are resistant to ferroptosis and that impaired ferritinophagy may be a contributing cause. Further, the expression of NCOA4, a key factor that influences ferritinophagy, is decreased in both diabetic wound tissue and high glucose-induced senescent fibroblasts. Moreover, NCOA4 overexpression could render senescent fibroblasts more vulnerable to ferroptosis. A faster wound healing process was also linked to the induction of ferroptosis. Thus, resistance to ferroptosis impedes the removal of senescent fibroblasts; promoting ferritinophagy could reverse this process, which may have significant implications for the management of diabetic wounds.

Acceleration of Wound Healing through Amorphous Calcium Carbonate, Stabilized with High-Energy Polyphosphate.

Amorphous calcium carbonate (ACC), precipitated in the presence of inorganic polyphosphate (polyP), has shown promise as a material for bone regeneration due to its morphogenetic and metabolic energy (ATP)-delivering properties. The latter activity of the polyP-stabilized ACC ("ACC∙PP") particles is associated with the enzymatic degradation of polyP, resulting in the transformation of ACC into crystalline polymorphs. In a novel approach, stimulated by these results, it was examined whether "ACC∙PP" also promotes the healing of skin injuries, especially chronic wounds. In in vitro experiments, "ACC∙PP" significantly stimulated the migration of endothelial cells, both in tube formation and scratch assays (by 2- to 3-fold). Support came from ex vivo experiments showing increased cell outgrowth in human skin explants. The transformation of ACC into insoluble calcite was suppressed by protein/serum being present in wound fluid. The results were confirmed in vivo in studies on normal (C57BL/6) and diabetic (db/db) mice. Topical administration of "ACC∙PP" significantly accelerated the rate of re-epithelialization, particularly in delayed healing wounds in diabetic mice (day 7: 1.5-fold; and day 13: 1.9-fold), in parallel with increased formation/maturation of granulation tissue. The results suggest that administration of "ACC∙PP" opens a new strategy to improve ATP-dependent wound healing, particularly in chronic wounds.

Application of collagen-chondroitin sulfate scaffolds with different pore sizes combined with acidic fibroblast growth factor in repairing full thickness skin defects in nude mice.

Wound healing of skin defects is complex. For the treatment of large and deep wounds, it is a good alternative to accept artificial dermis grafting at the first stage surgery, and autologous split-thickness skin grafting 2-3 weeks later at the second stage surgery. In addition, the effectiveness of numerous cytokines such as fibroblast growth factor (FGF) on wounds healing has been widely researched. The traditional view is that direct external application orin vivoinjection of exogenous FGFs may not achieve the desired therapeutic effect as the effective concentration cannot be maintained for a long time. Therefore, some researchers have tried to integrate various cytokines into skin substitutes for combined application. However, we believe that considering the current situation, it is still difficult to achieve mass production of these types of artificial dermis. Here, we manufactured a collagen-chondroitin sulfate scaffold material by imitating the marketed artificial dermis materials. Then, we combined it with recombinant human acidic FGF in a single full dose during the first-stage artificial dermis transplantation, which is simple and completely feasible but always controversial in the current clinical work, to explore whether this combinatorial therapy could serve as an efficient way wound healing in the Balb/c-nu mice full-thickness skin defect model.

The effect of carbon nanotubes on osteogenic functions of adipose-derived mesenchymal stem cells in vitro and bone formation in vivo compared with that of nano-hydroxyapatite and the possible mechanism.

It has been well recognized that the development and use of artificial materials with high osteogenic ability is one of the most promising means to replace bone grafting that has exhibited various negative effects. The biomimetic features and unique physiochemical properties of nanomaterials play important roles in stimulating cellular functions and guiding tissue regeneration. But efficacy degree of some nanomaterials to promote specific tissue formation is still not clear. We hereby comparatively studied the osteogenic ability of our treated multi-walled carbon nanotubes (MCNTs) and the main inorganic mineral component of natural bone, nano-hydroxyapatite (nHA) in the same system, and tried to tell the related mechanism. In vitro culture of human adipose-derived mesenchymal stem cells (HASCs) on the MCNTs and nHA demonstrated that although there was no significant difference in the cell adhesion amount between on the MCNTs and nHA, the cell attachment strength and proliferation on the MCNTs were better. Most importantly, the MCNTs could induce osteogenic differentiation of the HASCs better than the nHA, the possible mechanism of which was found to be that the MCNTs could activate Notch involved signaling pathways by concentrating more proteins, including specific bone-inducing ones. Moreover, the MCNTs could induce ectopic bone formation in vivo while the nHA could not, which might be because MCNTs could stimulate inducible cells in tissues to form inductive bone better than nHA by concentrating more proteins including specific bone-inducing ones secreted from M2 macrophages. Therefore, MCNTs might be more effective materials for accelerating bone formation even than nHA.

A physiologically active interpenetrating collagen network that supports growth and migration of epidermal keratinocytes: zinc-polyP nanoparticles integrated into compressed collagen.

The distinguished property of the physiological polymer, inorganic polyphosphate (polyP), is to act as a bio-intelligent material which releases stimulus-dependent metabolic energy to accelerate wound healing. This characteristic is based on the bio-imitating feature of polyP to be converted, upon exposure to peptide-containing body fluids, from stable amorphous nanoparticles to a physiologically active and energy-delivering coacervate phase. This property of polyP has been utilized to fabricate a wound mat consisting of compressed collagen supplemented with amorphous polyP particles, formed from the inorganic polyanion with an over-stoichiometric ratio of zinc ions. The proliferation and the migration of human skin keratinocytes in those matrices were investigated. If the cells were embedded into the mat they respond with a significantly higher motility when zinc-polyP particles are present. Interestingly, only keratinocytes that were grown in a polyP environment developed well-structured microvilli, reflecting an increased biological activity. The data show that Zn-polyP particles incorporated into wound mats are a potent cell growth and cell migration-stimulating inorganic bio-material.

Magnetic nanoparticles applied in targeted therapy and magnetic resonance imaging: crucial preparation parameters, indispensable pre-treatments, updated research advancements and future perspectives.

Magnetic nanoparticles (MNPs) have attracted much attention in cancer treatment as carriers for drug delivery and imaging contrast agents due to their distinctive performances based on their magnetic properties and nanoscale structure. In this review, we aim to comprehensively dissect how the applications of MNPs in targeted therapy and magnetic resonance imaging are achieved and their specificities by focusing on the following aspects: (1) several important preparation parameters (pH, temperature, ratio of the reactive substances, etc.) that have crucial effects on the properties of MNPs, (2) indispensable treatments to improve the biocompatibility, stability, and targeting ability of MNPs and prolong their circulation time for biomedical applications, (3) the mechanism for MNPs to deliver and release medicine to the desired sites and be applied in magnetic hyperthermia as well as related updated research advancements, (4) comparatively promising research directions of MNPs in magnetic resonance imaging, and (5) perspectives in the further optimization of their preparations, pre-treatments and applications in cancer diagnosis and therapy.

Epithelial differentiation of human adipose-derived stem cells (hASCs) undergoing three-dimensional (3D) cultivation with collagen sponge scaffold (CSS) via an indirect co-culture strategy.

BACKGROUND: Three-dimensional (3D) cultivation with biomaterials was proposed to facilitate stem cell epithelial differentiation for wound healing. However, whether human adipose-derived stem cells (hASCs) on collagen sponge scaffold (CSS) better differentiate to keratinocytes remains unclear. METHODS: 3D cultivation with CSS on hASC epidermal differentiation co-cultured with HaCaT cells at air-liquid interface (ALI) was compared with two-dimensional (2D) form and cultivation without "co-culture" or "ALI." Cellular morphology, cell adhesion, and growth condition were evaluated, followed by the protein and gene expression of keratin 14 (K14, keratinocyte specific marker). RESULTS: Typical cobblestone morphology of keratinocytes was remarkably observed in co-cultured hASCs at ALI, but those seeded on the CSS exhibited more keratinocyte-like cells under an invert microscope and scanning electron microscope. Desired cell adhesion and proliferation were confirmed in 3D differentiation groups by rhodamine-labeled phalloidin staining, consistent with H&E staining. Compared with those cultured in 2D culture system or without "ALI," immunofluorescence staining and gene expression analysis revealed hASCs co-cultured over CSS expressed K14 at higher levels at day 15. CONCLUSIONS: CSS is positive to promote epithelial differentiation of hASCs, which will foster a deeper understanding of artificial dermis in skin wound healing and regeneration.

Preparation and characterization of a gallium-loaded antimicrobial artificial dermal scaffold.

Artificial dermal scaffolds, which are made of natural or synthetic materials, can improve new blood vessel formation, cell migration and cell proliferation after being implanted into wounds, and they degrade slowly, playing an important role in dermal reconstruction and scar inhibition, finally achieving the goal of wound healing and functional reconstruction. Although these scaffolds have been widely used in clinical applications, biomaterial-associated infection is a deficiency or even a life-threatening problem that must be addressed, as it greatly affects the survival of the scaffolds. The gallium ion (Ga3+) is a novel metallic antimicrobial whose broad-spectrum antimicrobial properties against most bacteria encountered in burn wound infections have been confirmed, and it has been proposed as a promising candidate to prevent implant-associated infections. In this study, a gallium-loaded antimicrobial artificial dermal scaffold was successfully prepared by gallium ions and a collagen solution. The characterization results showed a porous structure with pore sizes ranging from 50 to 150 µm and a large porosity value of 97.4%. The enzymatic degradation rate in vitro was 19 and 28% after 12 and 24 h, respectively. In vitro antimicrobial testing revealed that the 1 h antibacterial rate against Staphylococcus aureus and Pseudomonas aeruginosa was close to 90%, which indicated its great antimicrobial activity. The results of the cytological evaluation showed slight effect on cell proliferation, with a relative growth rate (RGR) value of 80% and great cytocompatibility with cultured cells according to laser scanning confocal microscopy (LSCM) and scanning electron microscope (SEM). Furthermore, the successful prevention of wound infections in SD rats was confirmed with an in vivo antimicrobial evaluation, and the artificial dermal scaffolds also demonstrated great biocompatibility. This gallium-loaded antimicrobial artificial dermal scaffold exerted excellent antimicrobial activity and great biosafety, warranting further research for future clinical applications.

Three-Dimensionally Printed Silk-Sericin-Based Hydrogel Scaffold: A Promising Visualized Dressing Material for Real-Time Monitoring of Wounds.

A wound dressing which can be convenient for real-time monitoring of wounds is particularly attractive and user-friendly. In this study, a nature-originated silk-sericin-based (SS-based) transparent hydrogel scaffold was prepared and evaluated for the visualization of wound care. The scaffold was fabricated from a hybrid interpenetrating-network (IPN) hydrogel composed of SS and methacrylic-anhydride-modified gelatin (GelMA) by 3D printing. The scaffold transformed into a highly transparent hydrogel upon swelling in PBS, and thus, anything underneath could be easily read. The scaffold had a high degree of swelling and presented a regularly macroporous structure with pores around 400 µm × 400 µm, which can help maintain the moist and apinoid environment for wound healing. Meanwhile, the scaffolds were conducive to adhesion and proliferation of L929 cells. A coculture of HaCaT and HSF cells on the scaffold showed centralized proliferation of the two cells in distributed layers, respectively, denoting a promising comfortable environment for re-epithelialization. Moreover, in vivo studies demonstrated that the scaffold showed no excessive inflammatory reaction. In short, this work presented an SS-based transparent hydrogel scaffold with steerable physical properties and excellent biocompatibility through 3D printing, pioneering promising applications in the visualization of wound care and drug delivery.

Immune response of bovine sourced cross-linked collagen sponge for hemostasis.

A comprehensive immunogenicity scheme is proposed to examine immune response of bovine sourced hemostasis collagen sponge to establish foundation for further researches and decrease the incidence of adverse reaction in clinical trials. Compared with negative control group without any implant, spleen and lymph nodes morphology show no apparent swelling in mice with different doses of collagen sponge implants. Immune cells population, especially lymph nodes cells population, is practically coincident with organs. However, splenic cells display slight proliferation in early phase following collagen sponge implantation. Splenic cells apoptosis also demonstrates no significant difference among all groups. T lymphocytes subsets, CD4/CD8 cells ratio, in spleen and lymph nodes are practically normal. Splenic cells Ki67 + proportions do not exhibit significant difference between collagen sponge groups and negative control group. Humoral response is determined by detection of IgG and IgM concentration in serum, not exhibiting remarkable increase with collagen sponge implantation, compared to the drastic increase in positive control group with bovine tendon implantation. Local analysis around implants by hematoxylin-eosin staining discovers slight cell infiltration around collagen sponge. Tumour necrosis factor-α immunostaining indicates slight inflammation in early phase following collagen sponge implantation, but interferon-γ immunostaining is negligible even in positive control group. Collagen sponge, especially in high dose, may have evoked benign immune response in BALB/c mice, but this response is transient. The present evaluation scheme for immune response is integrated and comprehensive, suitable for various biomaterials.

Collagen/chitosan based two-compartment and bi-functional dermal scaffolds for skin regeneration.

Inspired from the sophisticated bilayer structures of natural dermis, here, we reported collagen/chitosan based two-compartment and bi-functional dermal scaffolds. Two functions refer to mediating rapid angiogenesis based on recombinant human vascular endothelial growth factor (rhVEGF) and antibacterial from gentamicin, which were encapsulated in PLGA microspheres. The gentamicin and rhVEGF encapsulated PLGA microspheres were further combined with collagen/chitosan mixtures in low (lower layer) and high (upper layer) concentrations, and molded to generate the two-compartment and bi-functional scaffolds. Based on morphology and pore structure analyses, it was found that the scaffold has a distinct double layered porous and connective structure with PLGA microspheres encapsulated. Statistical analysis indicated that the pores in the upper layer and in the lower layer have great variations in diameter, indicative of a two-compartment structure. The release profiles of gentamicin and rhVEGF exceeded 28 and 49 days, respectively. In vitro culture of mouse fibroblasts showed that the scaffold can facilitate cell adhesion and proliferation. Moreover, the scaffold can obviously inhibit proliferation of Staphylococcus aureus and Serratia marcescens, exhibiting its unique antibacterial effect. The two-compartment and bi-functional dermal scaffolds can be a promising candidate for skin regeneration.

Effect of stabilizers on bioactivity of peptide-24 in PLGA microspheres.

In the present study, Poly (D,L-lactide-co-glycolide) microspheres (PLGA MSs) were prepared for delivering a novel oligopeptide derived from rhBMP-2 (Peptide-24). Hydroxypropyl-ß-cyclodextrin (HP-ß-CD) and Bovine serum albumin (BSA) were used as stabilizers for retaining bioactivity of the oligopeptide. The morphology, diameter, drugloading rates and encapsulation rates of the PLGA MSs were detected and compared. The PLGA MSs were incubated for 3 and 30 days respectively to obtain the release supernatant containing Peptide-24. The structure and bioactivity of released Peptide-24 from PLGA MSs were evaluated through physicochemical detections and cell culture. The structure integrity of the Peptide-24 was confirmed by Far-UV circular dichroism and matrix-assisted laser desorption/ionization time-of-flight Mass Spectrometer (MALDI-TOF-MS) analysis. The interaction between PLGA matrix and loaded Peptide- 24 was verified through Raman. The results showed that the diameter of PLGA MSs was from 8.62 to 15.34 µm, the loading rate was 0.7-0.8%, and the encapsulation rate was 69.3-85.3%. The released Peptide-24 from PLGA MSs was proved to retain original bioactivity by the cellular activity and alkaline phosphatase (ALP) test. HP-ß-CD is a kind of excellent stabilizer for retaining the bioactivity of Peptide-24 in PLGA MSs.

Repair of rat calvarial bone defects by controlled release of rhBMP-2 from an injectable bone regeneration composite.

The objective of the present study was to enhance the regeneration ability of an injectable bone regeneration composite (IBRC) by the controlled release of recombinant human bone morphogenetic protein-2 (rhBMP-2). The IBRC comprised nano-hydroxyapatite/collagen (nHAC) particles in an alginate hydrogel carrier. First, bovine serum albumin (BSA) as a model protein was released from IBRC to evaluate its release rules. The results suggested that IBRC is a good controlled release carrier for BSA in the range 5-75 µg/ml. In the in vitro study the rhBMP-2 released from IBRC was determined by an enzyme-linked immunosorbent assay specific for rhBMP-2. The bioactivity of the released rhBMP-2 was evaluated through differentiated function of marrow mesenchymal stem cells (MSCs), as measured by alkaline phosphatase activity. The results of an in vitro study confirmed that rhBMP-2 released continuously for 21 days, and its bioactivity was well preserved during this period. The bone formation ability was assessed using a rat calvarial defect model of critical size. Micro-computed tomography (micro-CT) and histological analysis demonstrated that the IBRC had good bone formation ability, which was promoted through rhBMP-2 released from IBRC/rhBMP-2. In vitro and in vivo studies suggested that the present system is a potential bone critical defect repair material for clinical applications.

Structure and biocompatibility of an injectable bone regeneration composite.

With the development of minimally invasive techniques, injectable materials have become one of the major hotspots in the biomaterial field. We have developed an injectable bone regeneration composite (IBRC) using calcium alginate hydrogel as matrix to carry nano-hydroxyapatite/collagen particles. In this work, we evaluated the homogeneity of IBRC by dry/wet weight ratio test. The results showed that the structural homogeneity was determined by controlling the molar ratios of trisodium phosphate to calcium sulfate rather than alginate concentration in the studied ranges. Pore sizes of wet IBRC samples were characterized by thermoporometry. The pore properties of dried IBRC were tested by mercury porosimetry. Average pore size and porosity of dried IBRC declined with increasing alginate concentration. In contrast, surprisingly, pore size of wet homogeneous IBRC increased with increasing alginate concentration. Meanwhile, the swelling ratio did not increase with varying alginate concentration, but the swelling degree increased with increasing alginate concentration. In vitro cell culture showed that IBRC had no obvious cytotoxic effect on the rat bone mesenchymal stem cells. The morphology and viability of cells were also related to MR value. IBRC had good histocompatibility with a mild short-term inflammatory response in rat dorsum muscle. In addition, the excellent ability of IBRC to promote bone healing was confirmed by 5-mm-diameter cranial defects using histological analysis and bone mineral density measurement.

Self-assembly model, hepatocytes attachment and inflammatory response for silk fibroin/chitosan scaffolds.

Silk fibroin is an attractive natural fibrous protein for biomedical application due to its good biocompatibility and high tensile strength. Silk fibroin is apt to form a sheet-like structure during the freeze-drying process, which is not suitable for the scaffold of tissue engineering. In our former study, the adding of chitosan promoted the self-assembly of silk fibroin/chitosan (SFCS) into a three-dimensional (3D) homogeneous porous structure. In this study, a model of the self-assembly is proposed; furthermore, hepatocytes attachment and inflammatory response for the SFCS scaffold were examined. The rigid chain of chitosan may be used as a template for beta-sheet formation of silk fibroin, and this may break the sheet structure of the silk fibroin scaffold and promote the formation of a 3D porous structure of the SFCS scaffold. Compared with the polylactic glycolic acid scaffold, the SFCS scaffold further facilitates the attachment of hepatocytes. To investigate the inflammatory response, SFCS scaffolds were implanted into the greater omentum of rats. From the results of implantation, we could demonstrate in vivo that the implantation of SFCS scaffolds resulted in only slight inflammation. Keeping the good histocompatibility and combining the advantages of both fibroin and chitosan, the SFCS scaffold could be a prominent candidate for soft tissue engineering, for example, in the liver.

Silk fibroin/chitosan scaffold: preparation, characterization, and culture with HepG2 cell.

Tissue engineering requires the development of three-dimensional water-stable scaffolds. In this study, silk fibroin/chitosan (SFCS) scaffold was successfully prepared by freeze-drying method. The scaffold is water-stable, only swelling to a limited extent depending on its composition. Fourier Transform Infrared (FTIR) spectra and X-Ray diffraction curves confirmed the different structure of SFCS scaffolds from both chitosan and silk fibroin. The homogeneous porous structure, together with nano-scale compatibility of the two naturally derived polymers, gives rise to the controllable mechanical properties of SFCS scaffolds. By varying the composition, both the compressive modulus and compressive strength of SFCS scaffolds can be controlled. The porosity of SFCS scaffolds is above 95% when the total concentration of silk fibroin and chitosan is below 6 wt%. The pore sizes of the SFCS scaffolds range from 100 microm to 150 microm, which can be regulated by changing the total concentration. MTT assay showed that SFCS scaffolds can promote the proliferation of HepG2 cells (human hepatoma cell line) significantly. All these results make SFCS scaffold a suitable candidate for tissue engineering.