Characterizing the inherent activity of urinary bladder matrix for adhesion, migration, and activation of fibroblasts as compared with collagen-based synthetic scaffold.

The mechanism of action underlying the intriguing prominent bioactivity of urinary bladder matrix (UBM) for in situ tissue regeneration of soft tissue defects remains to be elucidated. It is speculated that the activity of UBM for cell adhesion, migration, and activation is inherent. The bioactivity of UBM for in situ tissue regeneration and its relation with the structure and intact soluble components of UBM were investigated in comparison to a collagen-based scaffold, PELNAC (PEL). We isolated the soluble component of the two materials with urea buffer, and evaluated the respective effect of these soluble components on the in vitro adhesion and migration of L929 fibroblasts. The spatiotemporal pattern of endogenous-cell ingrowth into the scaffolds and cell activation were investigated using a model of murine subcutaneous implantation. UBM is more capable of promoting the adhesion, migration, and proliferation of fibroblasts than PEL in a serum-independent manner. In vivo, as compared with PEL, UBM exhibits significantly enhanced activity for fast endogenous cell ingrowth and produces a more prominent pro-regenerative and pro-remodeling microenvironment by inducing the expression of TGF-ß1, VEGF, MMP-9, and murine type I collagen. Overall, our results suggest the prominent bioactivity of UBM for in situ tissue regeneration is inherent.

Mesenchymal stromal cells pretreated with proinflammatory cytokines enhance skin wound healing via IL-6-dependent M2 polarization.

BACKGROUND: Numerous studies have shown that mesenchymal stromal cells (MSCs) promote cutaneous wound healing via paracrine signaling. Our previous study found that the secretome of MSCs was significantly amplified by treatment with IFN-γ and TNF-α (IT). It has been known that macrophages are involved in the initiation and termination of inflammation, secretion of growth factors, phagocytosis, cell proliferation, and collagen deposition in wound, which is the key factor during wound healing. In this study, we aim to test whether the supernatant of MSCs pretreated with IT (S-IT MSCs) possesses a more pronounced effect on improving wound healing and describe the interplay between S-IT MSCs and macrophages as well as the potential mechanism in skin wound healing. METHODS: In the present study, we used a unique supernatant of MSCs from human umbilical cord-derived MSCs (UC-MSCs) pretreated with IT, designated S-IT MSCs, subcutaneously injected into a mice total skin excision. We evaluated the effect of S-IT MSCs on the speed and quality of wound repair via IT MSCs-derived IL-6-dependent M2 polarization in vivo by hematoxylin-eosin staining (H&E), immunohistochemistry (IHC), immunofluorescence (IF), Masson's trichrome staining, Sirius red staining, quantitative real-time PCR (qPCR). In addition, the effect of S-IT MSCs on the polarization of macrophages toward M2 phenotype and the potential mechanism of it were also investigated in vitro by flow cytometry (FCM), enzyme-linked immunosorbent assay (ELISA), tube formation assay, and western blot analysis. RESULTS: Compared with control supernatant (S-MSCs), our H&E and IF results showed that S-IT MSCs were more effectively in promoting macrophages convert to the M2 phenotype and enhancing phagocytosis of M2 macrophages. Meanwhile, the results of tube formation assay, IHC, Masson's trichrome staining, Sirius red staining showed that the abilities of M2 phenotype to promote vascularization and collagen deposition were significantly enhanced by S-IT MSCs-treated, thereby accelerating higher quality wound healing. Further, our ELISA, FCM, qPCR and western blot results showed that IL-6 was highly enriched in S-IT MSCs and acted as a key regulator to induce macrophages convert to the M2 phenotype through IL-6-dependent signaling pathways, ultimately achieving the above function of promoting wound repair. CONCLUSIONS: These findings provide the first evidence that the S-IT MSCs is more capable of eliciting M2 polarization of macrophages via IL-6-dependent signaling pathways and accelerating wound healing, which may represent a new strategy for optimizing the therapeutic effect of MSCs on wound healing.

The conditioned medium from mesenchymal stromal cells pretreated with proinflammatory cytokines promote fibroblasts migration and activation.

Human dermal fibroblasts (HDFs) play important roles in all stages of wound healing. However, in nonhealing wounds, fibroblasts are prone to aging, resulting in insufficient migration, proliferation and secretion functions. Recent studies have suggested that mesenchymal stromal cells (MSCs) are conducive to wound healing and cell growth through paracrine cytokine signaling. In our studies, we found that conditioned medium of MSCs pretreated with IFN-γ and TNF-α (IT MSC-CM) has abundant growth factors associated with wound repair. Our in vitro results showed that the effects of IT MSC-CM on promoting cell migration, proliferation and activation in HDFs were better than those of conditioned medium from mesenchymal stromal cells (MSC-CM). Moreover, we embedded a scaffold material containing IT MSC-CM and reconfirmed that cell migration and activation were superior to that in the presence of MSC-CM in vivo. Generally, PDGF-BB is perceived as a promoter of the migration and proliferation of HDFs. Moreover, a high level of PDGF-BB in IT MSC-CM was detected, according to which we guess that the effect on HDFs may be mediated by the upregulation of PDGF-BB. These studies all showed the potential of IT MSC-CM to promote rapid and effective wound healing.

Injectable silk nanofiber hydrogels as stem cell carriers to accelerate wound healing.

Stem cells have potential utility in wound therapy, however the benefits are often limited due to cell injury from shear stress during injection and poor retention at the wound site. Here, shear-thinning silk nanofiber hydrogels were used to load bone marrow derived mesenchymal stem cells (BMSCs) and inject into wound sites to optimize cell retention and accelerate wound healing. The BMSCs in the silk nanofiber hydrogels maintained stemness better than the cells cultured on plates, and the expression of wound healing-related genes was significantly higher in the hydrogels with higher silk concentrations (2 wt%). The silk nanofibers physically prevented migration of BMSCs from the deposition site in the wound bed. In addition to faster wound healing, these BMSC-loaded hydrogels mediated angiogenesis and inflammation and improved collagen deposition and hair follicle regeneration in vivo in rats. Considering that these silk nanofiber hydrogels were successfully used here as carriers for stem cells to accelerate wound healing, further study for skin regeneration may be warranted.

Microskin-Inspired Injectable MSC-Laden Hydrogels for Scarless Wound Healing with Hair Follicles.

Scarless skin regeneration with functional tissue remains a challenge for full-thickness wounds. Here, mesenchymal stem cell (MSC)-laden hydrogels are developed for scarless wound healing with hair follicles. Microgels composed of aligned silk nanofibers are used to load MSCs to modulate the paracrine. MSC-laden microgels are dispersed into injectable silk nanofiber hydrogels, forming composites biomaterials containing the cells. The injectable hydrogels protect and stabilize the MSCs in the wounds. The synergistic action of silk-based composite hydrogels and MSCs stimulated angiogenesis and M1-M2 phenotype switching of macrophages, provides a suitable niche for functional recovery of wounds. Compared to skin defects treated with MSC-free hydrogels, the defects treated with the MSC-laden composite hydrogels heal faster and form scarless tissues with hair follicles. Wound healing can be further improved by adjusting the ratio of silk nanofibers and particles and the loaded MSCs, suggesting tunability of the system. To the best of current knowledge, this is the first time scarless skin regeneration with hair follicles based on silk material systems is reported. The improved wound healing capacity of the systems suggests future in vivo studies to compare to other biomaterial systems related to clinical goals in skin regeneration in the absence of scarring.

Injectable hydrogel systems with multiple biophysical and biochemical cues for bone regeneration.

Bone regeneration is a complex process in which angiogenesis and osteogenesis are crucial. Introducing multiple angiogenic and osteogenic cues simultaneously into a single system and tuning these cues to optimize the niche remains a challenge for bone tissue engineering. Herein, based on our injectable biomimetic hydrogels composed of silk nanofibers (SNF) and hydroxyapatite nanoparticles (HA), deferoxamine (DFO) and bone morphogenetic protein-2 (BMP-2) were loaded on SNF and HA to introduce more angiogenic and osteogenic cues. The angiogenesis and osteogenesis capacity of injectable hydrogels could be regulated by tuning the delivery of DFO and BMP-2 independently, resulting in vascularization and bone regeneration in cranial defects. The angiogenesis and osteogenesis outcomes accelerated the regeneration of vascularized bones toward similar composition and structure to natural bones. Therefore, the multiple biophysical and chemical cues provided by the nanofibrous structures, organic-inorganic compositions, and chemical and biochemical angiogenic and osteogenic inducing cues suggest the potential for clinical applicability of these hydrogels in bone tissue engineering.

Electric field-driven building blocks for introducing multiple gradients to hydrogels.

Gradient biomaterials are considered as preferable matrices for tissue engineering due to better simulation of native tissues. The introduction of gradient cues usually needs special equipment and complex process but is only effective to limited biomaterials. Incorporation of multiple gradients in the hydrogels remains challenges. Here, beta-sheet rich silk nanofibers (BSNF) were used as building blocks to introduce multiple gradients into different hydrogel systems through the joint action of crosslinking and electric field. The blocks migrated to the anode along the electric field and gradually stagnated due to the solution-hydrogel transition of the systems, finally achieving gradient distribution of the blocks in the formed hydrogels. The gradient distribution of the blocks could be tuned easily through changing different factors such as solution viscosity, which resulted in highly tunable gradient of mechanical cues. The blocks were also aligned under the electric field, endowing orientation gradient simultaneously. Different cargos could be loaded on the blocks and form gradient cues through the same crosslinking-electric field strategy. The building blocks could be introduced to various hydrogels such as Gelatin and NIPAM, indicating the universality. Complex niches with multiple gradient cues could be achieved through the strategy. Silk-based hydrogels with suitable mechanical gradients were fabricated to control the osteogenesis and chondrogenesis. Chondrogenic-osteogenic gradient transition was obtained, which stimulated the ectopic osteochondral tissue regeneration in vivo. The versatility and highly controllability of the strategy as well as multifunction of the building blocks reveal the applicability in complex tissue engineering and various interfacial tissues.

Natural Nanofiber Shuttles for Transporting Hydrophobic Cargo into Aqueous Solutions.

Hydrophobic biomolecules realize their functions in vivo in aqueous environments, often through a delicate balance of amphiphilicity and chaperones. Introducing exogenous hydrophobic biomolecules into in vivo aqueous systems is a challenge in drug delivery and regenerative medicine, where labile linkers, carriers, and fusions or chimeric molecules are often designed to facilitate such aqueous interfaces. Here, we utilize naturally derived silk nanofiber shuttles with the capacity to transport hydrophobic cargos directly into aqueous solutions. These nanofibers disperse in organic solvents and in aqueous solutions because of their inherent amphiphilicity, with enriched hydrophobicity and strategically interspersed negatively charged groups. Hydrophobic molecules loaded on these shuttles in organic solvent-water systems separated from the solvent after centrifugation. These concentrated hydrophobic molecule-loaded nanofibers could then be dispersed into aqueous solution directly without modification. These shuttle systems were effective for different hydrophobic molecules such as drugs, vitamins, and dyes. Improved biological stability and functions of hydrophobic cargos after loading on these nanofibers suggest potential applications in drug delivery, cosmetology, medical diagnosis, and related health fields, with a relatively facile process.

Water-insoluble amorphous silk fibroin scaffolds from aqueous solutions.

Regenerated silk fibroin (RSF) is emerging as promising biomaterial for regeneration, drug delivery and optical devices, with continued demand for mild, all-aqueous processes to control microstructure and the performance. Here, temperature control of assembly kinetics was introduced to prepare the water-insoluble scaffolds from neutral aqueous solutions of RSF protein. Higher temperatures were used to accelerate the assembly rate of the silk fibroin protein chains in aqueous solution and during the lyophilization process, resulting in water-insoluble scaffold formation. The scaffolds were mainly composed of amorphous states of the silk fibroin chains, endowing softer mechanical properties. These scaffolds also showed nanofibrous structures, improved cell proliferation in vitro and enhanced neovascularization and tissue regeneration in vivo than previously reported silk fibroin scaffolds. These results suggest utility of silk scaffolds in soft tissue regeneration.

Amorphous Silk Fibroin Nanofiber Hydrogels with Enhanced Mechanical Properties.

Silk fibroin (SF) hydrogels have been engineered as universal substrates for various tissue regenerations and drug delivery. Although different physical and chemical crosslinking strategies are developed to form SF hydrogels with suitable performances, a significant gap remains to match specific requirements of various tissues. Here, amorphous SF nanofibers with more tyrosine residues outside the surfaces are used to replace traditional SF. Under the same crosslinking conditions, the use of amorphous SF nanofibers results in tougher properties, four times higher stiffness than that from traditional SF solutions. Unlike previous SF hydrogels, the SF nanofiber hydrogels show high tunability in wide modulus range of 0.6-160 kPa under low SF concentrations (below 5 wt%), showing improved mechanical match with various soft tissues. Better stability and cytocompatibility are also achieved, further confirming the superiority of the hydrogels as the tissue substrates. Therefore, a feasible strategy is developed to optimize the performances of SF hydrogel via tuning the nano-structural state in aqueous solutions, which will enrich SF-based hydrogel family in future.

Injectable Silk-Vaterite Composite Hydrogels with Tunable Sustained Drug Release Capacity.

Improving the efficiency of chemotherapy remains a key challenge in drug delivery. Many drug carriers have been designed to achieve multifunctional factors as part of their performance, including controlled release, dispersibility in aqueous environments, and targeting to cancer sites. However, it is difficult to optimize multiple properties simultaneously for a single carrier system. Here, synergistic carriers composed of vaterite microspheres and silk nanofiber hydrogels were developed to improve the dispersibility of vaterite spheres and the control of drug delivery without compromising the injectability or sensitivity to pH. The vaterite microspheres were dispersed homogeneously and remained stable in the silk nanofiber hydrogels. Doxorubicin (DOX) was effectively loaded on the vaterite spheres and silk nanofibers, forming synergistic silk-vaterite hydrogel delivery systems. The sustained delivery of DOX was tuned and controlled by vaterite stability and the DOX content loaded on the spheres and nanofibers. The cytotoxicity was regulated via the controlled delivery of DOX, suggesting the possibility of optimizing chemotherapeutic strategies. These silk-vaterite delivery hydrogels suggest a useful strategy for designing novel delivery systems for improved delivery and therapeutic benefits.

BN nanospheres functionalized with mesoporous silica for enhancing CpG oligodeoxynucleotide-mediated cancer immunotherapy.

CpG oligodeoxynucleotides (CpG ODNs) possess strong immunostimulatory activity, which hold great promise in cancer immunotherapy. However, their therapeutic efficacy is largely limited due to nuclease degradation and poor cellular internalization. Efficiently delivering CpG ODNs into target cells is crucial to improve their therapeutic efficacy. Boron nitride nanospheres (BNNS) possess advantage as carriers for CpG ODNs. However, their poor aqueous dispersity and low CpG ODN loading capacity became a big obstacle for further applications. Herein, we develop amino group grafted, mesoporous silica (MS)-functionalized BNNS as novel nanovectors for CpG ODN delivery. Modification of BNNS with MS significantly improved the dispersity of BNNS and CpG ODN loading. BNNS@MS-NH2 exhibited no cytotoxicity and enhanced the delivery of CpG ODNs into macrophages. BNNS@MS-NH2/CpG ODN complexes triggered enhanced immunostimulation and induced higher amounts of cytokines. Most importantly, BNNS@MS-NH2/CpG ODN complexes induced bifurcated cytokines, which simultaneously simulated the secretion of IL-6, TNF-α and IFN-α. In contrast, CpG ODN and BNNS/CpG ODN complexes could not. The result of the Transwell plate assay suggested that BNNS@MS-NH2/CpG ODN complexes were more effective in inhibiting cancer cell growth. Taken together, our findings provide a promising strategy for enhancing CpG ODN-mediated cancer immunotherapy.

Extracellular Matrix Component Shelled Nanoparticles as Dual Enzyme-Responsive Drug Delivery Vehicles for Cancer Therapy.

Stimuli-responsive drug delivery systems with reduced side effects offer promising prospects for cancer therapy. In this study, we developed an enzyme-responsive nanomedicine system based on extracellular matrix components (ECM) shelled mesoporous silica nanoparticles. The covalently conjugated ECM biomacromolecules, hyaluronic acid and collagen I, can not only enhance the biocompatibility of the particles and avoid early drug leakage, but also allow selective biodegradation triggered by hyaluronidase (HAase) and Matrix metalloproteinases 2 (MMP-2), which are overexpressed enzymes in some tumor tissues. The in vitro cytotoxicity test confirmed favorable biocompatibility of the as-prepared nanomedicine system. Moreover, this system showed distinguishing controlled release efficiency toward cancer cells induced by different levels of HAase and MMP-2. The in vivo antitumor test demonstrated the excellent efficiency of our system for tumor targeting drug delivery and tumor growth inhibition. Therefore, this dual enzyme-responsive drug delivery system provided an efficient platform for cancer therapy.

Effect of heparan sulfate mimetics from Escherichia coli K5 polysaccharide on SDF-1/CXCL12-induced endothelial progenitor cells in vitro.

In tumorigenesis, CXCL12 level increases sharply because of tumor tissue hypoxia. As CXCR4 cells, endothelial progenitor cells (EPCs) are mobilized to tumor bed through the CXCL12/CXCR4 axis and are involved in tumor angiogenesis. In this process, either glycosaminoglycan (GAG) or heparan sulfate (HS) carried by membrane proteoglycans is implicated. Exogenous soluble HS mimetics can act as a competitive inhibitor of membranous HS, thereby preventing the formation of a normal signal axis. In this work, the effect of HS mimetics on the CXCL12-induced EPCs in vitro was investigated. HS mimetics, named as K5PSs, were obtained from sulfated Escherichia coli K5 polysaccharide/heparosan, and EPCs were collected from rat bone marrow. Results showed that CXCL12 could promote EPCs viability. This promotion might be related to its regulation of cell cycle and anti-apoptosis activity; it also could promote EPCs migration and secretion of pro-angiogenesis factors. All its functions were obtained by activation of MAPK/ERK pathway, FAK pathway, and PI3k/AKT pathway. However, its effect on EPCs was attenuated by K5PSs, and the existence of sulfate groups both at 2-O-position and N-position in K5PSs is essential to inhibit its effect on EPCs. This work suggested that K5PSs could be applied in anti-tumor treatment through inhibiting tumor angiogenesis.

Silk Biomaterials with Vascularization Capacity.

Functional vascularization is critical for the clinical regeneration of complex tissues such as kidney, liver or bone. The immobilization or delivery of growth factors has been explored to improve vascularization capacity of tissue engineered constructs, however, the use of growth factors has inherent problems such as the loss of signaling capability and the risk of complications such as immunological responses and cancer. Here, a new method of preparing water-insoluble silk protein scaffolds with vascularization capacity using an all aqueous process is reported. Acid was added temporally to tune the self-assembly of silk in lyophilization process, resulting in water insoluble scaffold formation directly. These biomaterials are mainly noncrystalline, offering improved cell proliferation than previously reported silk materials. These systems also have appropriate softer mechanical property that could provide physical cues to promote cell differentiation into endothelial cells, and enhance neovascularization and tissue ingrowth in vivo without the addition of growth factors. Therefore, silk-based degradable scaffolds represent an exciting biomaterial option, with vascularization capacity for soft tissue engineering and regenerative medicine.

Direct Formation of Silk Nanoparticles for Drug Delivery.

Silk is useful as a drug carrier because of its biocompatibility, tunable degradation, and capacity in maintaining the function of drugs. However, further refinements are still required for silk-based nanoparticles to optimize applications as anticancer drug delivery systems. Here, a novel strategy was developed to prepare silk nanoparticles with improved performance. Unlike previous preparation methods that first obtain silk solutions and then induce nanoparticle formation through different treatments, here silk nanoparticles were directly prepared after a modified dissolution process. The nanoparticles had amorphous structure and homogeneous morphology, as well as improved dispersion in water and PBS solutions and improved pH-dependent drug release behavior when compared with the traditionally prepared silk nanoparticles. These improvements resulted in better uptake of the nanoparticles into cancer cells and higher cytotoxicity against cancer cells. These properties, when combined with the simpler and milder preparation process, indicate potential utility for anticancer drug delivery.

c9, t11- conjugated linoleic acid induces HCC cell apoptosis and correlation with PPAR-γ signaling pathway.

OBJECTIVE: Cis9, trans11 conjugated linoleic acid (c9, t11-CLA.) is one of the most important isomers of conjugated linoleic acid, which have a strong anti-tumor effects. Based on previous studies, we further explored the molecular mechanism of inducing cells apoptosis in human hepatocellular carcinoma cell line HepG2 and Hep3B. METHODS: Cell Counting Kit 8 (CCK-8) assay was used to investigate the effects of c9, t11-CLA on cell viability and cell proliferation ability; The effects of c9, t11-CLA on cell apoptosis was analyzed by DNA ladder assay, immuno-fluorescence and flow cytometry, respectively. Apoptotic related gene (Bcl-2, Bcl-XL, Bcl-w, Mcl-1, Bax, Bak, Bad, Bid and Bim), PPAR family member (PPAR-α, PPAR-ß and PPAR-γ), and Cox2 mRNA and protein expression were analyzed by RT-PCR and western blotting. ELISA assay was used to detect the content of Caspase-3. RESULTS: Our data were confirmed that c9, t11-CLA could inhibit the HCC cells proliferation ability and decrease the cells viability. RT-PCR and western blotting assay verified that c9, t11-CLA obviously increased the transcription and protein expression levels of PPAR-γ. The synchronism and correlation between PPAR-γ and apoptotic proteins Bcl-2, Bax and Caspase-3 were found with a dose- and time-dependent manner. PPAR-γ inhibitor GW9662 and activator Rosilitazone were further verified that there was cooperative relation between them. CONCLUSION: In our study, we first report that c9, t11-CLA induces apoptosis in HCC cells by activation of PPARγ-Bcl-2-Caspase-3 signal pathway. These results indicated that c9, t11-CLA will be useful for clinic therapy of anti-tumor and as a new regulator of PPAR-γ in the future.

Construction of serum resistant micelles based on heparosan for targeted cancer therapy.

A novel micelle based on heparosan and deoxycholic acid (DOCA) conjugate (HD) as drug carrier was reported here. As the surface was negatively charged, this micelle could resist serum adsorption, showing favorable stability. Moreover, fluorescence observation confirmed that it was able to deliver model hydrophobic drug doxorubicin (DOX) into HeLa cells efficiently. The DOX-loaded micelles showed sustained release behavior at pH 7.4, and accelerated release behavior at pH 5.0 or in the presence of ß-glucuronidase, which over-expressed in tumor cells. In vitro cytotoxicity assay demonstrated that the half-maximal inhibitory concentration (IC50) of DOX-loaded micelles against HeLa cells was much lower than that of COS7 cells, showing significant therapeutic distinction between tumor cells and normal cells. Combining with the good biocompatibility and biodegradability of heparosan, this micelle may be promising in clinical application for targeted drug delivery.

[An experimental study on the in vivo intermingled culture of rat autologous and allogeneic epithelial cells].

OBJECTIVE: To explore a method for in vivo skin reconstruction. METHODS: Thirty Sprague-Dawley (SD) rats were randomly divided into two paired groups, i.e. in vivo culture (A group, 10 pairs) and in vitro grafting (B group, 5 pairs). Skin samples were harvested from the rats of the two groups for the isolation of epithelial cells which were then mixed cultured in vitro in 1:1 ratio. Mixed cellular suspension in A group was harvested 4 days after culture. The mixed cellular sheets were harvested 14 days after culture. The cultured cells and sheets were then transplanted onto total skin loss wounds of donor rats for further cultivation. The wounds in A group were covered with allogeneic full-thickness skin. While the wounds in B group were covered by collagen membrane and gauze. Wound repair was observed and compared between the two groups at 2 - 3 post-operative weeks. RESULTS: Most of the wounds in A group healed after 2 - 3 weeks with smooth surface, and the peithelium connected closely and tightly with the subcutaneous tissue. In the wounds in B group on 5 post-operative day, some of the cellular sheets survived and some fell off. Even the healed wounds in B group would be injured again resulting in protracted small wounds. CONCLUSION: In vivo in situ epithelial culture might be an optional method of skin reconstruction for wound healing.