Atorvastatin-loaded hydrogel affects the smooth muscle cells of human veins.

Intimal hyperplasia (IH) is the major cause of stenosis of vein grafts. Drugs such as statins prevent stenosis, but their systemic administration has limited effects. We developed a hyaluronic acid hydrogel matrix, which ensures a controlled release of atorvastatin (ATV) at the site of injury. The release kinetics demonstrated that 100% of ATV was released over 10 hours, independent of the loading concentration of the hydrogel. We investigated the effects of such a delivery on primary vascular smooth muscle cells isolated from human veins. ATV decreased the proliferation, migration, and passage of human smooth muscle cells (HSMCs) across a matrix barrier in a similar dose-dependent (5-10 µM) and time-dependent manner (24-72 hours), whether the drug was directly added to the culture medium or released from the hydrogel. Expression analysis of genes known to be involved in the development of IH demonstrated that the transcripts of both the gap junction protein connexin43 (Cx43) and plasminogen activator inhibitor-1 (PAI-1) were decreased after a 24-48-hour exposure to the hydrogel loaded with ATV, whereas the transcripts of the heme oxygenase (HO-1) and the inhibitor of tissue plasminogen activator were increased. At the protein level, Cx43, PAI-1, and metalloproteinase-9 expression were decreased, whereas HO-1 was upregulated in the presence of ATV. The data demonstrate that ATV released from a hydrogel has effects on HSMCs similar to the drug being freely dissolved in the environment.

Injectable rhBMP-2-loaded chitosan hydrogel composite: osteoinduction at ectopic site and in segmental long bone defect.

Carriers for bone morphogenetic protein-2 (BMP-2) used in clinical practice still suffer from limitations such as insufficient protein retention. In addition, there is a clinical need for injectable carriers. The main objective of this study was to assess bone forming ability of rhBMP-2 combined either with chitosan hydrogel (rhBMP-2/CH) or chitosan hydrogel containing ß-tricalcium phosphate (ß-TCP) (rhBMP-2/CH/TCP). Formulations were first compared in a rat ectopic intramuscular bone formation model, and the optimal formulation was further evaluated in healing of 15-mm critical size defect in the radius of a rabbit. Three weeks after injection ectopically formed bone was analyzed by microcomputerized tomography (micro-CT) and histology. Significantly higher (4.7-fold) mineralized bone formation was observed in the rhBMP-2/CH/TCP group compared to rhBMP-2/CH group. In a pilot study, defect in a rabbit radius treated with rhBMP-2/CH/TCP showed incomplete regeneration at 8 weeks with composite leakage from the defect, indicating the need for formulation refinement when segmental defect repair is foreseen.

The effects of carrier nature and pH on rhBMP-2-induced ectopic bone formation.

Carrier nature and pH are important factors for rhBMP-2 osteoinductive activity. As for formulation pH, rhBMP-2 undergoes conformational changes and aggregates when shifting from acidic to neutral pH conditions. The present work investigates, to our knowledge for the first time, the effect of the carrier pH on rhBMP-2 bioactivity in a rat ectopic bone formation model. In addition, the influence of the carrier nature on rhBMP-2 osteoinductive activity was studied by comparing under identical experimental conditions two biopolymers having a very similar chemical structure but opposite charges. Specifically, rhBMP-2 was incorporated into chitosan (CH) and hyaluronan (HY) hydrogels at two different pH values. Hydrogels (0.2 mL) containing rhBMP-2 (150 µg) were injected into quadriceps muscle of Sprague-Dawley rats. Three weeks after injection, animals were euthanized and explanted specimens were analyzed by microcomputerized tomography (micro-CT) and histology. Bone formation was observed in quadriceps muscle with both rhBMP-2-loaded carriers at both pH values. A trend towards higher mineralized bone formation (1.7-fold for CH, 1.4-fold for HY) was observed for rhBMP-2-loaded hydrogels at low pH (4.8 ± 0.2) compared to high pH (6.2 ± 0.2). Significantly higher (two- to three-fold) mineralized bone formation was observed with rhBMP-2/HY hydrogel compared to rhBMP-2/CH hydrogel, although bone was more mature with the CH hydrogel. These results indicate that both hydrogels are effective carriers for rhBMP-2 and that the carrier nature influences bone formation in terms of volume and quality. This study also provided evidence that the formulation pH is a very important factor that may be critical to design efficient carriers for BMP-2.

Physical instability, aggregation and conformational changes of recombinant human bone morphogenetic protein-2 (rhBMP-2).

The influence of two different pH values on the physical stability of recombinant human bone morphogenetic protein-2 (rhBMP-2) in aqueous solution was evaluated in the present work. RhBMP-2 in solution at pH 4.5 or 6.5 was characterized by intrinsic and extrinsic (Nile Red and 1,8-ANS) fluorescence spectroscopy, 90 degrees light-scattering and transmission electron microscopy (TEM). Compared to the pH 4.5 solution, rhBMP-2 at pH 6.5 had (i) a stronger intrinsic fluorescence intensity, (ii) a longer fluorescence lifetime, (iii) a stronger 90 degrees light-scattering intensity, (iv) a stronger Nile Red fluorescence intensity, (v) a higher Nile Red fluorescence anisotropy, (vi) a lower 1,8-ANS fluorescence intensity, (vii) a higher 1,8-ANS fluorescence anisotropy and (viii) a longer 1,8-ANS fluorescence lifetime. Electron microscopy showed that rhBMP-2 at pH 4.5 contained aggregates of about 100 nm in diameter. More and larger protein aggregates (0.1-2 microm) were observed in solution at pH 6.5. Taken together, these results indicate conformational changes and increased aggregation of rhBMP-2 at pH 6.5 compared to pH 4.5, demonstrating a strong influence of pH on rhBMP-2 physical stability. These observations must be considered when developing a delivery system for rhBMP-2.

Comparison of volatile constituents of Persicaria odorata(Lour.) Soják (Polygonum odoratum Lour.) and Persicaria hydropiper L. Spach (Polygonum hydropiper L.).

Polygonum odoratum Lour. has been reclassified as Persicaria odorata (Lour.) Soják [Wilson, K. L. Polygonum sensu lato (Polygonaceae) in Australia. Telopea 1988, 3, 177-182]; other synonyms currently used are Vietnamese mint or Vietnamese coriander and, in Malaysia, Daun Laksa or Laksa plant. The aerial parts of Laksa plant are highly aromatic, and they contain many organic compounds such as (Z)-3-hexenal, (Z)-3-hexenol, decanal, undecanal, and dodecanal that are typical for green, citrus, orange peel, and coriander odors. In addition to these aldehydes, 3-sulfanyl-hexanal and 3-sulfanyl-hexan-1-ol were discovered for the first time in this herb. The fresh leaves are pungent when they are chewed, although the active compound has never been identified. The pungency of Persicaria hydropiper (L.) Spach (formerly Polygonum hydropiper L., synonym water pepper) is produced by polygodial, a 1,4-dialdehyde derived from drimane terpenoids. We also identified polygodial as the active pungent compound in P. odorata (Lour.) Soják.