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To control the release of nerve growth factor (NGF) in the injured peripheral nerve, NGF-loaded chitosan/PLGA composite microspheres ionically cross-linked by tripolyphosphate (TPP/Chitosan/PLGA-NGF) were prepared. The encapsulation efficiency of NGF ranged from 83.4 ± 1.5 % to 72.1 ± 1.6 % with TPP concentrations from 1 % to 10 %. Zeta potential and FT-IR analyses together with confocal microscopy demonstrated that multiple NGF-loaded PLGA microspheres were embedded in chitosan matrix, the mean size of TPP/Chitosan/PLGA-NGF microspheres ranged from 40.2 ± 3.4 to 49.3 ± 3.1 μm. The increase of TPP concentration improved the network stability and decreased the swelling ratio, resulting in the decreased NGF release from 67.7 ± 1.2 % to 45.7 ± 0.8 % in 49 days. The sustained release of NGF could promote PC12 cells differentiation and neurite growth in vitro. Moreover, in comparison with NGF solution without microencapsulation, TPP/Chitosan/PLGA-NGF microspheres enhanced sciatic nerve regeneration and prevented gastrocnemius muscle atrophy in rats. These results demonstrate the feasibility of using TPP/Chitosan/PLGA-NGF microspheres for neural tissue repair.

Insulin-like growth factor (IGF-1) has encouraged researchers to investigate its various potential therapeutic uses such as in the treatment of osteoporosis and repair of articular cartilage. The purpose of this study was to develop core-shell double-layer structural microspheres for the long-term delivery of rhIGF-1 for the prevention of bone loss and articular degeneration. rhIGF-1-loaded poly(lactic acid)/hydroxyapatite@Eudragit (PLA/HA@Eu) double-layer microspheres with a core-shell structure were formulated using poly(lactic acid) and hydroxyapatite by an oil-in-water-in-oil (w/o/w) technique. The in vitro release profiles of rhIGF-1 and the surface morphology and cytocompatibility of microspheres were investigated. In vitro release of rhIGF-1 from PLA/HA@Eu microspheres was maintained over 180 days. In vivo experiments were performed on male C57BL/6 mice with two different ages: adult mice (three months old) and old mice (ten months old). PLA/HA@Eu microspheres containing 1 mg rhIGF-1 were subcutaneously implanted into the back of mice. The rhIGF-1 amounts in the sera, livers and bones of mice were monitored for 6 months. The in vivo rhIGF-1 release patterns were similar to those observed from in vitro release. By analyzing the effects of rhIGF-1-loaded microspheres on bone mass in adult mice and cartilage in old mice via micro-CT and histological measurements, we observed beneficial trends. These results suggest that PLA/HA@Eu double-layer microspheres providing long-term delivery of rhIGF-1 were effective in systemic preventive therapy of bone loss in adult mice and delaying the progressive degeneration of articular cartilage in old mice. The long-term delivery strategy demonstrated herein may be a promising therapeutic approach for delaying osteoporosis and articular degeneration.

In offshore gas well drilling and production, methane hydrate may block the tubing, resulting in the stoppage of gas production. Conventional methods such as injection of thermal hydrate inhibitors, thermal insulating or heating, gas dehydration and reducing pressure are time-consuming and expensive, and sometimes, they are not realistic in production conditions. New methods are needed to lower the cost of gas hydrate prevention and to overcome these limitations. The thermal effect of cavitation was applied to the prevention of gas hydrate in this study. The thermal impact of cavitation, supposed to heat the fluids and prevent the formation of gas hydrate, was evaluated. Numerical simulation was performed to study the thermal performance of cavitation. Furthermore, experimental studies of the influence of initial temperature, flow rate, fluid volume and fluid viscosity on the thermal effect of cavitation were performed, and the results were analysed.

Both of the osteogenic factor and the suitable delivery system in bone tissue engineering are essential for bone regeneration. In this study, we manufactured two kinds of composite vehicles for sustained release of rhBMP-2 and its derived synthetic oligopeptide (Peptide-24, abbreviated as P24) for osteogenesis and bone defect repair. The composite vehicle was based on cross-linked chitosan, nano-hydroxyapatite/collagen (nHAC), and poly (lactide-co-glycolide) acid microsphere. The physicochemical properties of the composite vehicles (abbreviated as CS/nHAC/PLGA-MS) were investigated. The rhBMP-2 and P24 release kinetics from the vehicles were examined and the secondary structure of rhBMP-2 and P24 after 28 days' release process was analyzed. In vitro cell proliferation, osteogenic differentiation and rat calvarial defect repair were evaluated. The results proved that the composite vehicle had favorable compressive strength, elastic modulus, the porosity, and the bulk density. The secondary structures of rhBMP-2 and P24 kept stability during microencapsulation and release process. P24 from the vehicle kept a geared-up release and rhBMP-2 from the vehicle kept a three-stage mode release process. The results of in vitro cell detection showed that the composite vehicle had good biocompatibility and osteoinduction. In vivo rat calvarial defect repair demonstrated that both groups of vehicles with rhBMP2 and P24 exhibited satisfied bone defect repair. This research showed that the composite vehicle could exhibit sustained release of osteogenic factors. CS/nHAC/PLGA-MS loading rhBMP-2 or P24 could be a novel and ideal scaffold for bone regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1593-1606, 2017.

We have designed a rapid and ultrasensitive electrochemiluminescent (ECL) competitive immunoassay for the determination of mercury(II) ion. It is based on the use of CdSe quantum dots (QDs), methylmercury-6-mercaptonicotinic acid-ovalbumin as coating antigen and specific monoclonal antibodies (mAbs) against Hg(II). The latter is quite selective for Hg(II). The coating antigen was immobilized on the surface of a gold electrode via reaction between the functional groups of cysteamine and glutaraldehyde. The mercury(II) ions in a sample and the coating antigen compete for binding sites of QD-labeled monoclonal antibody which binds specifically to Hg(II) ions. The ECL of the system decreases with increasing concentration of Hg(II) because less QD-labeled mAbs are present on the surface of the electrode. Under optimal conditions, the decrease of ECL intensity is linearly related to the logarithm of the Hg(II) concentration in the range from 0.02 to 100 ng mL−1, with a detection limit of 6.2 pg mL−1. As far as we know, this is the first report on an ECL immunoassay for Hg(II) based on a specific monoclonal antibody. The favorable results obtained when this method was applied to real samples indicate that this detection scheme can widely enlarge the applicability of detecting heavy metal ions by exploiting the ECL of QDs for immunoassays.

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-β-cyclodextrin (HP-β-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-β-CD is a kind of excellent stabilizer for retaining the bioactivity of Peptide-24 in PLGA MSs.

A kind of PLGA microspheres was prepared with bovine serum albumin (BSA) as the model drug and poly (lactide-co-glycolide) as the matrix. The polylactic acid/hydroxyapatite (PLA/HA) scaffold was manufactured through 3D printing technology. Then the PLGA microspheres were composited in the scaffold. It was also explored about the feasibility of skeletal scaffolds loaded with bone growth factor. The BSA loading PLGA microspheres were prepared by W/O/W method and the scaffolds were prepared by 3D-printing using PLA and HA as raw materials. The composite scaffold was fabricated by adsorbing the microspheres/ethanol suspension into scaffolds under negative pressure. The cell-adhesion ability, hydrophilicity, scaffold morphology, release properties and biocompatibility of the composite scaffold were characterized, respectively. The results show no burst release of BSA from the PLGA microspheres at beginning stage and sustained longer than 35 days. Drug-loading rate of microspheres was 0.64%. PLA/HA scaffold shows enhanced hydrophilicity as well as excellent cell compatibility and cell adhesion property. SEM images show PLGA microspheres were successfully absorbed in PLA/HA scaffold. MTT experiments of the composite scaffold show non cytotoxic and its cell relative proliferation rate is up to 88.37%. These studies show the feasibility of skeletal scaffolds loaded with bone growth factor. Through low-temperature rapid prototyping technology, the long-effective bioactive bone scaffold can be prepared and have a well application prospect.

In this paper, we document the process and findings of preparing dual poly (lactide-co-glycolide)/chitosan microspheres (PLGA/CS MSs) for osteoinductive oligopeptide derived from BMP-2 (abbreviated as Peptide-24). Through adjusting the amount of Peptide-24, three kinds of PLGA/CS MSs were successfully constructed in twice encapsulations. We studied the morphology, size distribution and loading efficiency of the PLGA/CS MSs. We also focused on the pH change of the environment and the molecular weight of the matrix during the degradation process of PLGA/CS MSs. More specifically, the release of Peptide-24 from three kinds of PLGA/CS MSs was monitored in PBS at 37 °C and pH 7.4. The structural stability of the released Peptide-24 was detected by Far-UV circular dichroism and MALDI-TOF-MS analysis. The mean sizes of the three kinds of PLGA/CS MSs are 47.5, 63.0 and 89.1 μm; and their drug-loading rates are 2.61, 3.21 and 2.21%, respectively. Comparing with Chitosan microspheres (abbreviated as CS MSs), the PLGA/CS MSs have excellent release curves with zero-order kinetics and controllable model. The incubation solution of PLGA/CS MSs avoided producing acid environment as poly (lactide-co-glycolide) microspheres (PLGA MSs) did, which was explained by analyzing the molecular weight of the matrix. The released oligopeptide kept its original structure and relative molecular weight throughout the procedures of encapsulation, storage and release. This indicates its structure stability. Thus, we conclude that dual PLGA/CS MSs is a promising vehicle that is suitable for the delivery of bioactive factors.

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.

In present study, protein loaded poly (lactide-co-glycolide)/chitosan microspheres (PLGA/CS MSs) with spheres-in-sphere structure were prepared in order to weaken the burst release of protein from PLGA microspheres (PLGA MSs) and to buffer acidic micro-milieu. The PLGA MSs and PLGA/CS MSs were characterized in terms of their size distribution, morphology, drug-loading rate, zeta potential and physical–chemical properties. The incubation experiments of PLGA MSs and PLGA/CS MSs were manipulated in PBS solution at pH 7.4, 37 °C to monitor the release of BSA and the vehicles degradation. The release kinetic of BSA was illuminated mainly based on the degradation processes of the matrices. External CS crusts were proved to strikingly improve the release kinetic of the model protein by reducing initial burst release and extending continuous release while acting as a diffusion barrier. Moreover, using PLGA/CS MSs could avoid the decrease of pH value resulted from the acidic products of PLGA MSs because of the effective buffer action of the basic groups in CS. The results demonstrated that the spheres-in-sphere structure is an effective way to control the initial burst release of protein and to overcome the acidic problem of protein-loading PLGA MSs.

It is advantageous to incorporate controlled growth factor delivery into tissue engineering strategies. The purpose of the present study was to develop a novel tissue engineering scaffold with the capability of controlled releasing BMP-2-derived synthetic peptide. Porous nano-hydroxyapatite/collagen/poly(L-lactic acid)/chitosan microspheres (nHAC/PLLA/CMs) composite scaffolds containing different quantities of chitosan microspheres (CMs) were prepared by a thermally induced phase separation method. Dioxane was used as the solvent for PLLA. Introduction of less than 30% of CMs (on PLLA weight basis) did not remarkably affect the morphology and porosity of the nHAC/PLLA/CMs scaffolds. However, as the microspheres contents increased to 50%, the porosity of the composite decreased rapidly. The compressive modulus of the composite scaffolds increased from 15.4 to 25.5 MPa, while the compressive strength increased from 1.42 to 1.63 MPa as the microspheres contents increased from 0% to 50%. The hydrolytic degradation and synthetic peptide release kinetics in vitro were investigated by incubation in phosphate buffered saline solution (pH 7.4). The results indicated that the degradation rate of the scaffolds was increased with the enhancement of CMs dosage. The synthetic peptide was released in a temporally controlled manner, depending on the degradation of both incorporated chitosan microspheres and PLLA matrix. In vitro bioactivity assay revealed that the encapsulated synthetic peptide was biologically active as evidenced by stimulation of rabbit marrow mesenchymal stem cells (MSCs) alkaline phosphatase (ALP) activity. The successful microspheres-scaffold system offers a new delivery method of growth factors and a novel scaffold design for bone regeneration.

To develop a novel tissue engineering scaffold with the capability of controlled releasing BMP-2-derived synthetic peptide, porous poly(lactic acid)/chitosan microspheres (PLA/CMs) composites containing different quantities of chitosan microspheres were prepared by a thermally induced phase separation method. FTIR analysis revealed that there were strong hydrogen bond interactions between the PLA and chitosan component. Introduction of less than 30% CMs (on PLA weight basis) did not remarkably affect the morphology and porosity of the PLA/CMs scaffolds. The compressive strength of the composite scaffolds increased from 0.48 to 0.66 MPa, while the compressive modulus increased from 7.29 to 8.23 MPa as the microspheres' contents increased from 0% to 50%. In vitro degradability investigation indicated that the dissolution of chitosan component was preferential than PLA matrix and the inclusion of CMs could neutralize the acidity of PLA degradation products. Compared with the rapid release from CMs, the synthetic peptide was released from PLA/CMs scaffolds in a temporally controlled manner, mainly depending on the degradation of PLA matrix. The promising microspheres based scaffold release system can be used to deliver bioactive factors for a variety of non-loaded bone regeneration and tissue engineering application.

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.

The goal for treating osteoarthritis (OA) is finding ways to decrease joint pain and dysfunction and prevent and slow the cartilage degeneration. Extracorporeal shock-wave therapy (ESWT) has been found to improve motor dysfunction and ameliorate pain with OA in animals. However, few studies have found that it can prevent and slow joint degeneration in vivo. The aim of study was to investigate the effect of ESWT on OA in rabbit.A total of 30 male New Zealand white rabbits were divided into 3 groups: control, OA induced by anterior cruciate ligament transaction (ACLT), and ALCT plus ESWT. The animals were killed at 4 and 8 weeks. Nitric oxide (NO) level was measured in the synovial cavity of knee joints, and cartilage sections were graded macroscopically by a Mankin scoring system. Chondrocyte apoptosis was investigated by flow cytometry and the expression of active caspase 3 by indirect immunohistochemistry.ESWT significantly reduced the NO level in the synovial cavity of knee joints (P0.05) and chondrocyte apoptosis (P0.05) of rabbits with OA. ESWT treatment significantly decreased the severity of cartilage lesions at both times as compared to rabbits with OA alone (P0.05).ESWT reduced the progression of OA in rabbits. This effect may be related to decreased level of NO and is likely mediated by reduced chondrocyte apoptosis. ESWT may be a useful treatment for knee OA.

The effects of the transplanted bone morphogenetic protein-2 (BMP2) -related peptide P24 and rhBMP2 combined with poly(lactic-co-glycolic acid) (PLGA)/chitosan (CS) microspheres were investigated in promoting the repair of rat cranial bone defect. Forty white rats were selected and equally divided into four groups (group A: 1 μg of rhBMP2/PLGA/CS composite; group B: 3 mg of P24/PLGA/CS composite; group C: 0.5 μg of rhBMP2 + 1.5 mg of P24/PLGA/CS composite; group D: blank PLGA/CS material), and rat cranial bone defect models with a diameter of 5 mm were established. The materials were transplanted to the cranial bone defects. The animals were sacrificed on weeks 6 and 12 post-operation. Radiographic examinations (x-ray imaging and 3D CT scanning) and histological evaluations were performed. The repaired areas of cranial bone defects were measured, and the osteogenetic abilities of various materials were compared. Cranial histology, imaging, and repaired area measurements showed that the osteogenetic effects at two time points (weeks 6 and 12) in group C were better than those in groups A and B. The effects in groups A and B were similar. Group D achieved the worst repair effect of cranial bone defects, where a large number of fibrous connective tissues were observed. The PLGA/CS composite microspheres loaded with rhBMP2 and P24 had optimal concrescence and could mutually increase their osteogenesis capability. rhBMP2 + P24/PLGA/CS composite is a novel material for bone defect repair with stable activity to induce bone formation.