A self-gelling alginate/chitosan based powder containing bioactive nanoparticles for non-compressible bleeding control and promoting wound healing.

The challenge remains in developing hemostatic dressings that can fulfill both hemostatic and repair functions to meet clinical demands worldwide. Herein, the biodegradable powders composed of benzeneboronic acid-modified sodium alginate/catechol-modified quaternized chitosan hydrogel (SBQCC) networks and bioactive cerium oxide nanoparticles (CNPs), were prepared for hemostasis and promoting wound healing. The SBQCC/CNPs powders had good self-gelation ability, water absorption ratio, tissue adhesiveness and biocompatibility. The SBQCC/CNPs powders could not only rapidly absorb a large amount of blood to concentrate coagulation factors when applied on bleeding wounds, but also formed an adhesive hydrogel physical barrier to control bleeding in situ. Meanwhile, the aggregation and activation of red blood cells and platelets induced by the SBQCC/CNPs powders can initiate the forming of internal blood clot with fibrin to further enhance the hemostatic effect. The SBQCC/CNPs powders demonstrated excellent hemostatic performance in non-compressible rat tail vein bleeding and rabbit liver bleeding models. In addition, SBQCC/CNPs powder-derived hydrogels had antibacterial activity and multiple biological activities, including antioxidant, anti-inflammatory and promoting angiogenesis for accelerating wound healing. Therefore, the SBQCC/CNPs powders can accelerate wound healing while achieving effective hemostasis, which will be a promising hemostatic dressing.

Benzeneboronic-alginate/quaternized chitosan-catechol powder with rapid self-gelation, wet adhesion, biodegradation and antibacterial activity for non-compressible hemorrhage control.

Although hemostatic powders have excellent adaptability for irregular and inaccessible wounds, their hemostasis for continuous bleeding or bleeding wounds of non-compressible organs remains a critical challenge. Herein, a series of benzeneboronic acid-modified sodium alginate/catechol-modified quaternized chitosan (SA-BA/QCS-C, SBQCC) powders is developed by borate ester crosslinking for non-compressible hemorrhage control. SBQCC powders possess remarkable tissue adhesion, rapid self-gelation, good cytocompatibility and antibacterial activity against S. aureus and E. coil. The blood coagulation assays show that SBQCC powders display excellent blood clotting ability due to the synergistic effect of SA-BA and QCS-C. The SBQCC2 powder with the SA-BA to QCS-C mass ratio of 5 to 3 has the greatest effect on the blood-clotting rate. Upon depositing SBQCC2 powder to bleeding wounds of rabbit liver, the powder can absorb a large amount of blood and form a stable hydrogel physical barrier at the bleeding wounds in situ to achieve non-pressing rapid hemostasis. The SBQCC2 powder also has good biocompatibility and can be degraded in vivo. Altogether, the SBQCC powders can be a promising candidate for rapid hemostasis, and these findings may provide a new perspective for improving the hemostatic efficiency of the hemostatic powder in biomedical fields.

An antibacterial biomimetic adhesive with strong adhesion in both dry and underwater situations.

Adhesives have attracted extensive attention in biomedical applications in recent years. However, the development of adhesives with strong adhesion in both dry and underwater conditions and antibacterial properties is still a challenge. Herein, a biomimetic adhesive (DP@TA/Gel) was developed based on the adhesion mechanism of mussel in water, from adhesion and solidification to avoiding excessive oxidization processes. DP@TA/Gel exhibited rapid strong nonspecific adhesiveness to diverse materials including wood (485 kPa) metal (507 kPa), plastic (74 kPa), and even fresh biological tissue (39 kPa) in dry conditions. Specially, owing to its biomimetic design, DP@TA/Gel could imitate the mussel adhesion mechanism underwater, endowing it with robust (38 kPa), highly repeatable (at least 15 times) and long-term (at least 120 h) stable adhesion even in underwater conditions. Remarkably, DP@TA/Gel also exhibited high adhesiveness in various water environments, including seawater, and a wide range of pH (3-11) and NaCl concentration (0.9-10%) solutions without any stimulus. In addition, DP@TA/Gel showed excellent biocompatibility and antibacterial properties. Thus, the DP@TA/Gel adhesive has appealing potential biomedical applications such as sutureless wound closure and as a tissue adhesive.

Preparation and the hemostatic property study of porous gelatin microspheres both in vitro and in vivo.

Rapid hemostasis is crucial to saving the lives of traumatic patients in emergency medical treatment. To improve the hemostatic performance of the gelatin microspheres (GMs) in vivo and in vitro, in the study, porous gelatin microspheres (PGMs) were prepared through water-in-oil emulsion method combined with vacuum freeze-drying after cross-linking with glutaraldehyde and prefreeze in liquid nitrogen. Owing to the porous structure and rough surface, the obtained PGMs were effective to induce red blood cells aggregation and promote fibrin generation, which led to improved hemostatic potential than GMs in blood coagulation time, whole blood clotting rate, and the hemostatic efficiency of rabbit liver wound and ear vein cut models tests. The potent hemostatic effect of the PGMs could be attributed to the synergistic effects of the physiological blood coagulation activated by negatively charged surface and physical barriers reinforced by fibrin. Moreover, PGMs with high water absorption rate and porous structure showed better hemostatic performance than commercial hemostatic powder (chitosan hemostasis power and Yunnan Baiyao) in vitro and in vivo. Cytotoxicity tests with bone marrow mesenchymal stem cells of murine showed that PGMs with excellent cytocompatibility were conducive to cell proliferation. Therefore, it could be concluded that PGMs were more suitable for rapid hemostasis than GMs and had a great potential for hemostatic applications.

A Spray-Filming Self-Healing Hydrogel Fabricated from Modified Sodium Alginate and Gelatin as a Bacterial Barrier.

Self-healing hydrogels as wound dressings still face challenges in infection prevention, especially in the dressing of mass wounds, due to their inflexibility and the slow formation of the protective film on the wound. Therefore, designing a spray-filming (rapid-forming) hydrogel that can serve as a bacterial barrier is of particular significance in the development of wound dressings. Here, a self-healing hydrogel based on adipic acid dihydrazide-modified gelatin (Gel-ADH) and monoaldehyde-modified sodium alginate(SA-mCHO) is prepared. Using dynamic, Schiff base bonds, the hydrogels exhibit excellent self-healing properties. Moreover, the gelation time of SA-mCHO/Gel-ADH (SG) hydrogels is shortened to 2-21 s, resulting in rapid filming by spraying the two precursor solutions. In addition, the rapid spray-filming ability might offer sufficient flexibility and rapidity for dealing with mass and irregular wounds. Notably, the bacterial barrier experiments show that the SG hydrogel films could form an effective barrier to Staphylococcus aureus and Candida albicans for 12 h. Therefore, SG hydrogels could be used in wound dressings and they show great promise in applications associated with mass and irregular traumas.

Injectable negatively charged gelatin microsphere-based gels as hemostatic agents for intracavitary and deep wound bleeding in surgery.

Gelatin, as natural macromolecular material, has been used in biomedical fields widely. In this study, various injectable gelatins A, B, and their compound AB microsphere-based gels (A-GMGs, B-GMGs and AB-GMGs) were prepared through water-in-oil emulsion method for hemostasis, and the effects of blood coagulation in vitro and surgical hemostasis (a deep liver wound model) in vivo were evaluated. Furthermore, the influences of gelatin sorts, the size of microsphere, zeta potential (ZP) and viscoelastic properties on hemostasis were also assessed. Results showed that the gelatin microspheres (GMs) exhibited smooth surface, good sphericity and the particle size of a rough normal distribution. GMs carried negative charges and their electronegativity was stronger than that of gelatin A (GA) and gelatin B (GB) raw materials. Rheological analysis showed that a decreasing particle size of the microspheres led to stronger gel strength, and solid-like gels were exhibited under low stress conditions and liquid-like gels were exhibited under high stress conditions. The blood clotting time of B-GMGs was within 60 s, which exhibited a significantly higher blood clotting effect compared with control groups. The hemostasis assay in vivo showed that the gels had better hemostatic effect on a deep liver wound bleeding model compared with control groups, especially B-GMGs. However, in vivo and vitro hemostatic experiments, particle size of GMs had no obvious influence on the hemostatic effect of the gels. In addition, the CCK-8 assay of bone marrow mesenchymal stem cells of murine (mMSCs) indicated non-cytotoxicity of GMs for cells. These results demonstrated that the gelatin microsphere-based gels (GMGs) had potential to be an effective hemostatic material for intracavitary and deep wound bleeding in surgery.

Employing the cyclophosphate to accelerate the degradation of nano-hydroxyapatite/poly(amino acid) (n-HA/PAA) composite materials.

Owing to the good degradability and biocompatibility of polyphosphoesters (PPEs), the aim of the current study was to investigate a novel degradable composite of nano-hydroxyapatite/poly(amino acid) (n-HA/PAA) with cyclophosphate (CPE) via in situ melting polymerization to improve the degradation of n-HA/PAA. The structure of each composite was characterized via Fourier transform infrared spectroscopy, X-ray diffraction, and X-ray photoelectron spectroscopy. The degradation properties were studied in terms of the weight loss and pH in a phosphate-buffered saline (PBS) solution, while the surface morphology was examined using a scanning electron microscope-energy dispersive spectrometer (SEM-EDS) after soaking the surface in simulated body fluid (SBF). The cell proliferation, cell adhesion, and alkaline phosphatase (ALP) activity were used for the analysis of cytocompatibility. The weight loss results showed that the n-HA/PAA composite was 9.98 wt%, weighed after soaking in the PBS solution for 12 weeks, whereas the nano-hydroxyapatite/polyphosphoester-amino acid (n-HA/PPE-AA) composite was 46.94 wt%. The pH of the composites was in a suitable range between 6.64 to 7.06 and finally stabilized at 7.39. The SEM and EDS results revealed the formation of an apatite-like layer on the surface of the n-HA/PPE-AA composites after soaking in SBF for one week. The cell counting Kit 8 (CCK-8) assay of the cell culture in the leaching liquid of the n-HA/PPE-AA composites exhibited non-cytotoxicity and high-proliferation, and the cell adhesion showed the well spreading and normal phenotype extension of the cells on the n-HA/PPE-AA composites surface. Concurrently, the co-culture results of the composites and cells confirmed that the n-HA/PPE-AA composites exhibited a higher ALP activity. In summary, the results demonstrated that the n-HA/PPE-AA composites had a controllable degradation property, good bioactivity, and cytocompatibility.

Mechanics, degradability, bioactivity, in vitro, and in vivo biocompatibility evaluation of poly(amino acid)/hydroxyapatite/calcium sulfate composite for potential load-bearing bone repair.

A ternary composite of poly(amino acid), hydroxyapatite, and calcium sulfate (PAA/HA/CS) was prepared using in situ melting polycondensation method and evaluated in terms of mechanical strengths, in vitro degradability, bioactivity, as well as in vitro and in vivo biocompatibility. The results showed that the ternary composite exhibited a compressive strength of 147 MPa, a bending strength of 121 MPa, a tensile strength of 122 MPa, and a tensile modulus of 4.6 GPa. After immersion in simulated body fluid, the compressive strength of the composite decreased from 147 to 98 MPa for six weeks and the bending strength decreased from 121 to 75 MPa for eight weeks, and both of them kept stable in the following soaking period. The composite could be slowly degraded with 7.27 wt% loss of initial weight after soaking in phosphate buffered solution for three weeks when started to keep stable weight in the following days. The composite was soaked in simulated body fluid solution and the hydroxyapatite layer, as flower-like granules, formed on the surface of the composite samples, showing good bioactivity. Moreover, it was found that the composite could promote proliferation of MG-63 cells, and the cells with normal phenotype extended and spread well on the composite surface. The implantation of the composite into the ulna of sheep confirmed that the composite was biocompatible and osteoconductive in vivo, and offered the PAA/HA/CS composite promising material for load-bearing bone substitutes for clinical application.

In vivo biocompatibility of new nano-calcium-deficient hydroxyapatite/poly-amino acid complex biomaterials.

OBJECTIVE: To evaluate the compatibility of novel nano-calcium-deficient hydroxyapatite/poly-amino acid (n-CDHA/PAA) complex biomaterials with muscle and bone tissue in an in vivo model. METHODS: Thirty-two New Zealand white rabbits were used in this study. Biomaterials were surgically implanted into each rabbit in the back erector spinae and in tibia with induced defect. Polyethylene was implanted into rabbits in the control group and n-CDHA/PAA into those of the experimental group. Animals were examined at four different points in time: 2 weeks, 4 weeks, 12 weeks, and 24 weeks after surgery. They were euthanized after embolization. Back erector spinae muscles with the surgical implants were examined after hematoxylin and eosin (HE) staining at these points in time. Tibia bones with the surgical implants were examined by X-ray and scanning electron microscopy (SEM) at these points in time to evaluate the interface of the bone with the implanted biomaterials. Bone tissues were sectioned and subjected to HE, Masson, and toluidine blue staining. RESULTS: HE staining of back erector spinae muscles at 4 weeks, 12 weeks, and 24 weeks after implantation of either n-CDHA/PAA or polyethylene showed disappearance of inflammation and normal arrangement in the peripheral tissue of implant biomaterials; no abnormal staining was observed. At 2 weeks after implantation, X-ray imaging of bone tissue samples in both experimental and control groups showed that the peripheral tissues of the implanted biomaterials were continuous and lacked bone osteolysis, absorption, necrosis, or osteomyelitis. The connection between implanted biomaterials and bone tissue was tight. The results of HE, Masson, toluidine blue staining and SEM confirmed that the implanted biomaterials were closely connected to the bone defect and that no rejection had taken place. The n-CDHA/PAA biomaterials induced differentiation of a large number of chondrocytes. New bone trabecula began to form at 4 weeks after implanting n-CDHA/PAA biomaterials, and lamellar bone gradually formed at 12 weeks and 24 weeks after implantation. Routine blood and kidney function tests showed no significant changes at 2 weeks and 24 weeks after implantation of both biomaterials. CONCLUSION: n-CDHA/PAA composites showed good compatibility in in vivo model. In this study, n-CDHA/PAA were found to be safe, nontoxic, and biologically active in bone repair.

Mesoporous calcium-silicon xerogels with mesopore size and pore volume influence hMSC behaviors by load and sustained release of rhBMP-2.

Mesoporous calcium-silicon xerogels with a pore size of 15 nm (MCS-15) and pore volume of 1.43 cm(3)/g were synthesized by using 1,3,5-mesitylene (TMB) as the pore-expanding agent. The MCS-15 exhibited good degradability with the weight loss of 50 wt% after soaking in Tris-HCl solution for 56 days, which was higher than the 30 wt% loss shown by mesoporous calcium-silicon xerogels with a pore size of 4 nm (MCS-4). The pore size and pore volume of MCS-15 had significant influences on load and release of recombinant human bone morphogenetic protein-2 (rhBMP-2). The MCS-15 had a higher capacity to encapsulate a large amount of rhBMP-2; it could adsorb 45 mg/g of rhBMP-2 in phosphate-buffered saline after 24 hours, which was more than twice that with MCS-4 (20 mg/g). Moreover, the MCS-15 system exhibited sustained release of rhBMP-2 as compared with MCS-4 system (showing a burst release). The MCS-15/rhBMP-2 system could promote the proliferation and differentiation of human mesenchymal stem cells, showing good cytocompatibility and bioactivity. The results indicated that MCS-15, with larger mesopore size and higher pore volume, might be a promising carrier for loading and sustained release of rhBMP-2, which could be used as bone repair material with built-in osteoinduction function in bone reconstruction.

An HPLC-MS/MS method for simultaneous determination of the active metabolites of febuxostat (67M-1, 67M-2 and 67M-4) in human plasma.

An HPLC-MS/MS method for simultaneously determination of the active metabolites (67M-1, 67M-2 and 67M-4) in human plasma using clopidogrel as the internal standard was developed and validated. The compounds were extracted by protein precipitation using acetonitrile and separated using a C8 column by a gradient elution with the mobile phase consisting of acetonitrile (containing 0.1% formic acid) and 0.1% formic acid. Quantification was performed using multiple reaction monitoring in positive mode with m/z transitions of 333.1-261.0, 333.1-261.0, 347.0-261.0 and 322.2-184.1 for 67M-1, 67M-2, 67M-4 and clopidogrel (Internal Standard), respectively. This method was validated in terms of specificity, linearity, precision, accuracy, and stability. The lower limit of quantification of this method was 0.5 ng/mL and the calibration curve was linear over the concentration range of 0.5-150 ng/mL. The intra- and inter-run precision was less than 11.67% and 8.64%, respectively, with the accuracy between 98.33% and 108.38%. The samples were stable under all the tested conditions. This method has been successfully applied to the pharmacokinetic study of febuxostat in healthy Chinese volunteers following oral administration of 40 mg and 80 mg febuxostat.

Degradability and cytocompatibility of tricalcium phosphate/poly(amino acid) composite as bone tissue implants in orthopaedic surgery.

In this study, a tricalcium phosphate (TCP) and poly (amino acid) copolymer (PAA) biocomposite were fabricated for bone repair and characterized. The results show that the compressive strength of the TCP/PAA composites increased with an increase in the TCP content at TCP contents less than 40 w%. The weight loss of the composite after soaking in phosphate buffered saline for 12 weeks significantly increased with an increase in the TCP content, revealing its good degradability. In addition, the composite maintained adequate mechanical strength during the degradation period because it underwent a surface erosion process. In vitro MG63 cell culture experiments showed that the composite is non-cytotoxic and thus allows cells to adhere, proliferate and differentiate. Osteoid formation was evidenced on the composite surfaces 12 weeks after its implantation into the femoral bone of dogs. Furthermore, the composite combined directly with the host bone tissue without fibrous capsule tissue, and no inflammatory responses were found, showing the good biocompatibility of the composite. It is expected that the composite may be used for the development of bone implants for orthopaedic surgery.

Preparation and properties of BSA-loaded microspheres based on multi-(amino acid) copolymer for protein delivery.

A multi-(amino acid) copolymer (MAC) based on ω-aminocaproic acid, γ-aminobutyric acid, L-alanine, L-lysine, L-glutamate, and hydroxyproline was synthetized, and MAC microspheres encapsulating bovine serum albumin (BSA) were prepared by a double-emulsion solvent extraction method. The experimental results show that various preparation parameters including surfactant ratio of Tween 80 to Span 80, surfactant concentration, benzyl alcohol in the external water phase, and polymer concentration had obvious effects on the particle size, morphology, and encapsulation efficiency of the BSA-loaded microspheres. The sizes of BSA-loaded microspheres ranged from 60.2 µm to 79.7 µm, showing different degrees of porous structure. The encapsulation efficiency of BSA-loaded microspheres also ranged from 38.8% to 50.8%. BSA release from microspheres showed the classic biphasic profile, which was governed by diffusion and polymer erosion. The initial burst release of BSA from microspheres at the first week followed by constant slow release for the next 7 weeks were observed. BSA-loaded microspheres could degrade gradually in phosphate buffered saline buffer with pH value maintained at around 7.1 during 8 weeks incubation, suggesting that microsphere degradation did not cause a dramatic pH drop in phosphate buffered saline buffer because no acidic degradation products were released from the microspheres. Therefore, the MAC microspheres might have great potential as carriers for protein delivery.

Composite scaffolds of nano calcium deficient hydroxyapatite/multi-(amino acid) copolymer for bone tissue regeneration.

In this study, nano calcium deficient hydroxyapatite (n-DA)/multi-(amino acid) copolymer composite scaffolds were prepared by injection molding foaming method using calcium sulphate dihydrate as a foaming agent. The composite scaffolds showed well interconnected macropores with the pore size of ranging from 100 to 600 µm, porosity of 81 % and compressive strength of 12 MPa, and the compressive strength obviously affected by the porosity. The composite scaffolds could be slowly degraded in phosphate buffered solution (PBS), which lost its initial weight of 61 w % after immersion into PBS for 12 weeks, and the porosity significantly affected the degradability of the scaffolds. Moreover, it was found that the composite scaffolds could promote the MG-63 cells growth and proliferation, and enhance its alkaline phosphatase activity. The implantation of the scaffolds into the femoral bone of rabbits confirmed that the composite scaffolds were biocompatibitive, degradable, and osteoconductive in vivo.