Ultrasound triggered topical delivery of Bmp7 mRNA for white fat browning induction via engineered smart exosomes.

BACKGROUND: Efficient and topical delivery of drugs is essential for maximized efficacy and minimized toxicity. In this study, we aimed to design an exosome-based drug delivery platform endowed with the ability of escaping from phagocytosis at non-target organs and controllably releasing drugs at targeted location. RESULTS: The swtichable stealth coat CP05-TK-mPEG was synthesized and anchored onto exosomes through the interaction between peptide CP05 and exosomal surface marker CD63. Chlorin e6 (Ce6) was loaded into exosomes by direct incubation. Controllable removal of PEG could be achieved by breaking thioketal (TK) through reactive oxygen species (ROS), which was produced by Ce6 under ultrasound irradiation. The whole platform was called SmartExo. The stealth effects were analyzed in RAW264.7 cells and C57BL/6 mice via tracing the exosomes. To confirm the efficacy of the engineered smart exosomes, Bone morphogenetic protein 7 (Bmp7) mRNA was encapsulated into exosomes by transfection of overexpressing plasmid, followed by stealth coating, with the exosomes designated as SmartExo@Bmp7. Therapeutic advantages of SmartExo@Bmp7 were proved by targeted delivering Bmp7 mRNA to omental adipose tissue (OAT) of obese C57BL/6 mice for browning induction. SmartExo platform was successfully constructed without changing the basic characteristics of exosomes. The engineered exosomes effectively escaped from the phagocytosis by RAW264.7 and non-target organs. In addition, the SmartExo could be uptaken locally on-demand by ultrasound mediated removal of the stealth coat. Compared with control exosomes, SmartExo@Bmp7 effectively delivered Bmp7 mRNA into OAT upon ultrasound irradiation, and induced OAT browning, as evidenced by the histology of OAT and increased expression of uncoupling protein 1 (Ucp1). CONCLUSIONS: The proposed SmartExo-based delivery platform, which minimizes side effects and maximizing drug efficacy, offers a novel safe and efficient approach for targeted drug delivery. As a proof, the SmartExo@Bmp7 induced local white adipose tissue browning, and it would be a promising strategy for anti-obesity therapy.

An easy long-acting BMP7 release system based on biopolymer nanoparticles for inducing osteogenic differentiation of adipose mesenchymal stem cells.

In contrast to the early acting bone morphogenetic protein 2, bone morphogenetic protein 7 (BMP7) plays a decisive role mainly in the late stages of bone formation. To overcome deactivation and degradation of expensive BMP7, we designed a novel long-acting BMP7 release system based on poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB) nanoparticles to enable the induction of osteogenic differentiation in human adipose mesenchymal stem cells (ADSCs). In order to improve the encapsulation efficiency of BMP7 and avoid damage by organic solvents, BMP7 was modified and protected using the biosurfactant soybean lecithin. In an in vitro test, BMP7-soybean lecithin-P34HB nanoparticles (BMP7-SPNPs) showed a short initial burst of BMP7 release during the first 24h, followed by a steady increase to a cumulative 80% release in 20days. Compared with the rapid release of control P34HB nanoparticles without soybean phospholipids loaded with BMP7 without soybean lecithin, BMP7-SPNPs significantly reduced the initial burst of BMP7 release and stabilized the content of BMP7 to allow long-term osteogenic differentiation during the late phase of bone development. Human ADSCs treated with BMP7-SPNPs showed higher alkaline phosphatase activity and higher expression levels of genetic markers of osteogenic differentiation compared with the control group. Thus, the results indicate that BMP7-SPNPs can be used as a rapid and long-acting BMP7 delivery system for osteogenic differentiation.

Intra-articular delivery of synovium-resident mesenchymal stem cells via BMP-7-loaded fibrous PLGA scaffolds for cartilage repair.

Delivery of synovium-resident mesenchymal stem cells (synMSCs) to cartilage defect site might provide a novel therapeutic modality for treatment of articular cartilage diseases. However, low isolation efficiency of synMSCs limits their therapeutic application. Niche-preserving non-enzymatic isolation of synMSCs was firstly attempted by employing micro-organ culture system based on recapitulating tissue-specific homeostasis ex vivo. The isolated synMSCs retained superior long-term growth competency, proliferation and chondrogenic potential to bone marrow-derived MSCs (BMSCs). It was noted that synMSCs demonstrated 9-fold increase in cartilaginous micro-tissue formation and 13-fold increase in sulfated proteoglycans deposition compared to BMSCs. For delivery of synMSCs, fibrous PLGA scaffolds were specifically designed for full-thickness osteochondral defects in rabbits. The scaffolds provided effective micro-environment for growth and host-integration of synMSCs. Combined delivery of synMSCs with bone morphogenetic proteins-7 (BMP-7) was designed to achieve synergistic therapeutic efficacy. BMP-7-loaded PLGA nanoparticles electrosprayed onto the scaffolds released BMP-7 over 2 weeks to conform with its aimed role in stimulating early stage endochondral ossification. Scaffold-supported combined administration of synMSCs with BMP-7 resulted in high proteoglycan and collagen type II induction and thick hyaline cartilage formation. Intra-articular co-delivery of synMSCs with BMP-7 via fibrous PLGA scaffolds may be a promising therapeutic modality for articular cartilage repair.

Bone morphogenetic protein-7 incorporated polycaprolactone scaffold has a great potential to improve survival and proliferation rate of the human embryonic kidney cells.

Renal failures treatment has been faced with several problems during the last decades. Kidney tissue engineering has been created many hopes to improve treatment procedures with scaffold fabrication that can modulate kidney cells/stem cells migration to the lesion site and increase the survival of these cells at that site with imitating the role of the kidney extracellular matrix. In this study, bone morphogenetic protein-7 (BMP7) as a vital factor for kidney development and regeneration was incorporated in the polycaprolactone (PCL) nanofibers and after morphological, mechanical, and biocompatible characterization, proliferation, and survival of the human embryonic kidney cells (HEK) were investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), flow cytometry, and gene expression while cultured on scaffolds. Mechanical properties of the PCL nanofibers modulated after combining with BMP7 and hydration degree, protein adsorption and cell adhesion were enhanced in PCL-BMP7 compared to the pure PCL. Proliferation rate and growth increased significantly in HEK cells cultured on PCL-BMP7 when compared with that of PCL and tissue culture plate, whereas these data were also confirmed via significant decrease in apoptotic genes expression level in HEK cell cultured on PCL-BMP7. According to the results, PCL-BMP7 demonstrated positive effects on the survival and proliferation rate of the kidney cells and showed has also a great potential to use as a bioimplant for kidney tissue engineering applications.

Mechanisms of Bone Morphogenetic Protein-7 Protective Effects Against Cold Ischemia-Induced Renal Injury in Rats.

Deceased donor kidneys are exposed to cold ischemic insult which makes them particularly susceptible to the effects of cold ischemic injury during hypothermic preservation resulting in high rates of delayed graft function. Bone morphogenetic protein-7 (BMP-7) is a valuable reagent in the field of tissue regeneration and preservation under ischemic conditions. Following these insights, we investigated the effect of recombinant human BMP-7 (rhBMP-7) on graft preservation during cold ischemia. The study was conducted on an experimental model of kidney cold ischemia in rats. Kidneys were perfused with University of Wisconsin (UW) saline solution, rhBMP-7, or rhBMP-7 + UW, and exposed to cold ischemia for 6, 12, and 24 hours. In tubular epithelial cells of kidneys perfused with rhBMP-7 and rhBMP-7+UW solution, the expression of BMP-7 and E-cadherin was observed after 24 hours of cold ischemia. In kidneys not perfused with rhBMP-7, high expression of transforming growth factor-ß and α-smooth muscle actin was found. Also, in kidneys perfused with rhBMP-7 solution, statistically higher levels of Smad1, Smad5, and Smad8 messenger RNA expressions were proven. BMP-7 maintains the morphology of kidney tissue better than UW solution during 24 hours of cold ischemia. BMP-7 prevents epithelial to mesenchymal transformation and consequently maintains epithelial phenotype of tubular cells.

Effect of dual delivery of antibiotics (vancomycin and cefazolin) and BMP-7 from chitosan microparticles on Staphylococcus epidermidis and pre-osteoblasts in vitro.

The main aims of this manuscript are to: i) determine the effect of commonly used antibiotics to treat osteoarticular infections on osteoblast viability, ii) study the dual release of the growth factor (BMP-7) and antibiotics (vancomycin and cefazolin) from chitosan microparticles iii) demonstrate the bioactivity of the antibiotics released in vitro on Staphylococcus epidermidis. The novelty of this work is dual delivery of growth factor and antibiotic from the chitosan microparticles in a controlled manner without affecting their bioactivity. Cefazolin and vancomycin have different therapeutic concentrations for their action in vivo and therefore, two different concentrations of the drugs were used. Osteoblast cytotoxicity test concluded that cefazolin concentrations of 50 and 100µg/ml were found to have positive influence on osteoblast proliferation. A significant increase in osteoblast proliferation was observed in the presence of cefazolin and BMP-7 in comparison with BMP-7 alone group; indicating cefazolin might play a role in osteoblast proliferation. On the other hand, vancomycin concentration of 1000µg/ml was found to significantly reduce (p<0.01) osteoblast proliferation in comparison with controls. The microbial study indicated that cefazolin at a minimum concentration of 21.5µg/ml could inhibit ~85% growth of S. epidermidis, whereas vancomycin at a concentration of 30µg/ml was found to inhibit ~80% bacterial growth.

Effect of scaffold architecture and BMP-2/BMP-7 delivery on in vitro bone regeneration.

The aim of this study was to develop 3-D tissue engineered constructs that mimic the in vivo conditions through a self-contained growth factor delivery system. A set of nanoparticles providing the release of BMP-2 initially followed by the release of BMP-7 were incorporated in poly(ε-caprolactone) scaffolds with different 3-D architectures produced by 3-D plotting and wet spinning. The release patterns were: each growth factor alone, simultaneous, and sequential. The orientation of the fibers did not have a significant effect on the kinetics of release of the model protein BSA; but affected proliferation of bone marrow mesenchymal stem cells. Cell proliferation on random scaffolds was significantly higher compared to the oriented ones. Delivery of BMP-2 alone suppressed MSC proliferation and increased the ALP activity to a higher level than that with BMP-7 delivery. Proliferation rate was suppressed the most by the sequential delivery of the two growth factors from the random scaffold on which the ALP activity was the highest. Results indicated the distinct effect of scaffold architecture and the mode of growth factor delivery on the proliferation and osteogenic differentiation of MSCs, enabling us to design multifunctional scaffolds capable of controlling bone healing.

Hyaluronan promotes the chondrocyte response to BMP-7.

OBJECTIVE: Chondrocytes exhibit specific responses to bone morphogenetic proteins (BMPs) and transforming growth factor-betas (TGF-betas). The bioactivity of these growth factors is regulated by numerous mediators. In our previous study, Smad1 was found to interact with the cytoplasmic domain of the hyaluronan receptor CD44. The purpose of this study was to determine the ability of hyaluronan in the pericellular matrix to modulate the chondrocyte responses to BMP-7 or TGF-beta1. EXPERIMENTAL DESIGN: Nuclear translocation of Smad1, Smad2 and Smad4 was studied in bovine articular chondrocytes in response to BMP-7 and TGF-beta1. The effects of matrix disruption by hyaluronidase treatment and the initiation of matrix repair by the addition of hyaluronan on the nuclear translocation of Smad proteins, Smad1 phosphorylation and luciferase expression by a CD44 reporter construct in response to BMP-7 were also studied. RESULTS: The disruption of the hyaluronan-dependent pericellular matrix of chondrocytes resulted in diminished nuclear translocation of endogenous Smad1 and Smad4 in response to BMP-7; however, the nuclear translocation of Smad2 and Smad4 in these matrix-depleted chondrocytes in response to TGF-beta1 was not diminished. Incubation of the matrix-depleted chondrocytes with exogenous hyaluronan restored Smad1 and Smad4 nuclear translocation and increased pCD44(499)-Luc luciferase expression in response to BMP-7. Both exogenous hyaluronan and matrix re-growth enhanced by hyaluronan synthase-2 (HAS2) transfection restored Smad1 phosphorylation. CONCLUSIONS: Disruption of hyaluronan-CD44 interactions has little effect on the TGF-beta responses; however, re-establishing CD44-hyaluronan ligation promotes a robust cellular response to BMP-7 by articular chondrocytes. Thus, changes in cell-hyaluronan interactions may serve as a mechanism to modulate cellular responsiveness to BMP-7.

Weekly intra-articular injections of bone morphogenetic protein-7 inhibits osteoarthritis progression.

INTRODUCTION: We investigated the ability of a weekly intra-articular injection of bone morphogenetic protein (BMP)-7 to prevent osteoarthritis in rabbits with anterior cruciate ligament transections. METHODS: First, 36 knee joints were randomly divided into four groups: 50, 500, 5,000 ng BMP-7, and control. Knee cartilage was evaluated at 4, 8, and 12 weeks. Then, in order to control for individual differences, 500 ng BMP-7 was injected into one knee and phosphate-buffered saline (PBS) into the other, and the two knees were compared at 4, 8, and 12 weeks (n = 5). For pharmacokinetic analysis, cartilage was harvested at 1 hour and 1, 2, 4, and 7 days after knee injection of 500 ng BMP-7 or PBS (n = 3). RESULTS: Histological scores in the 500 and 5,000 ng BMP-7 groups were significantly better than those in the other groups at 12 weeks. Matched pair analysis demonstrated that both macroscopic and histological scores in the 500 ng BMP-7 group were better than those in the control group. Immunohistochemical analysis revealed higher BMP-7 expression by chondrocytes in the BMP-7 injected knees. Histology of whole knee and quantitative micro computed tomography analysis showed that weekly injections of 500 ng BMP-7 did not induce synovial fibrosis, ectopic bone, or osteophyte formation. As detected by enzyme-linked immunosorbent assay, BMP-7 concentration in the cartilage tissue was still higher in the BMP-7 treated group 7 days after the injection. CONCLUSIONS: Weekly intra-articular injections of BMP-7 inhibited progression of osteoarthritis. Obvious adverse effects were not observed. BMP-7 concentration and expression in cartilage were still higher 7 days after injection.