Versatile polymer microspheres for injection therapy: aspects of fluoroscopic traceability and biofunctionalization.

Synthesis and characterization of a series of novel microspheres featuring (i) radiopacity (i.e., clear fluoroscopic traceability) and (ii) an outer surface exposing aldehyde groups are reported. The aldehydes allowed us to tether proteins onto the particles' surface under mild conditions, under which the protein conformation and, hence, structural motifs for biorecognition are preserved. Essential monomer building blocks were (i) 4-iodobenzoyl-2-oxo-ethylmethacrylate (4-IEMA) for radiopacity and (ii) propenal for surface tethering of proteins. The particles demonstrated good X-ray visibility and cytocompatibility. Procedures to couple proteins onto the surface were optimized using fluorescent bovine serum albumin (FITC-BSA) or collagen (FITC-collagen). Furthermore, radiopaque microparticles with unlabeled bovine collagen type I were produced. The presence of immobilized collagen was verified with narrow-scan X-ray photoelectron spectroscopy. Fibroblasts readily adhere to and grow on the collagen-modified surfaces, whereas this was much less the case for the unmodified controls. The results led us to suggest that immobilized nondenatured collagen may transform filler particles from passive space-occupying objects to particles that cross-talk with surrounding tissues.

New intrinsically radiopaque hydrophilic microspheres for embolization: synthesis and characterization.

Polymeric particles currently used for embolization procedures have the disadvantage that they are radiolucent, that is, invisible on X-ray images, and consequently the interventional radiologist has to resort to angiography to (indirectly) monitor the fate of the particles. Here, we introduce intrinsically radiopaque hydrophilic microspheres. Since these microspheres can directly be visualized on X-ray images, using these microspheres for embolization purposes will allow superprecise location of the embolic material, both during and after the procedure. The microspheres, which are prepared by suspension polymerization, are based on the radiopaque monomer 2-[4-iodobenzoyl]-oxo-ethylmethacrylate and hydroxyethylmethacrylate (HEMA) and/or 1-vinyl-2-pyrrolidinone (NVP) as hydrophilic component. It has been shown that for clinically relevant X-ray visibility the spheres should contain at least 20 wt % iodine. At this iodine content, copolymerization with HEMA results in spheres that hardly imbibe water (EQ = 1.08). When HEMA is replaced by NVP, the volume swelling ratio can be significantly increased (to 1.33).

Effect of Self-Assembly of Oxalamide Based Organic Compounds on Melt Behavior, Nucleation, and Crystallization of Isotactic Polypropylene.

We report on the effect of an aliphatic oxalamide based nucleating agent (OXA3,6) on the melt and crystallization behavior of isotactic polypropylene (iPP) under defined shear conditions. Through polarized optical microscopy, we demonstrate that OXA3,6 self-assembles from the iPP melt into rhombic crystals whereas their size and distribution proved highly dependent on the employed cooling rates. The presence of 0.5 wt % of OXA3,6 in iPP results in a significant suppression in iPP melt viscosity, which could not be explained via molecular modeling. A possible cause for the drop in viscosity in the presence of OXA3,6 is attributed to the interaction (absorption) of high molecular weight iPP chains with the nucleating agent, thereby suppressing their contribution to the viscoelastic response of the melt. This proposed mechanism for the suppression in melt viscosity appears similar to that encountered by the homogeneous distribution of nanoparticles such as CNTs, graphene, and silica. Shear experiments, performed using a slit flow device combined with small-angle X-ray diffraction measurements, indicate that crystallization is significantly enhanced in the presence of OXA3,6 at relatively low shear rates despite its lowered sensitivity to shear. This enhancement in crystallization is attributed to the shear alignment of the rhombic OXA3,6 crystals that provide surface for iPP kebab growth upon cooling. Overall, the suppression in melt viscosity in combination with enhanced nucleation efficiency at low as well as high shear rates makes this self-assembling oxalamide based nucleating agent a promising candidate for fast processing.

New acrylic microspheres for arterial embolization: combining radiopacity for precise localization with immobilized thrombin to trigger local blood coagulation.

Particles currently used in arterial embolization therapy have several disadvantages, most importantly their radiolucency. This means the radiologist cannot precisely asses the fate of embolization particles. Microspheres that combine two additional features have been designed. By incorporating an iodine containing monomer, radiopaque microspheres were obtained that display good visibility under standard X-ray conditions. Incorporation of methacrylic acid makes the surface of the spheres suitable for surface functionalization. Here, thrombin was covalently attached to the surface of the radiopaque microspheres. By induction of a thrombus, improved anchoring of the embolization spheres in the blood vessel can be obtained. The immobilized thrombin induced a biphasic response of the blood namely: (1) fast deposition of fibrin on the surface resulting in sphere aggregation and (2) additional thrombin generation in the surrounding blood and a subsequent local thrombus formation. These microspheres with both intrinsic X-ray visibility and a biofunctionalized surface can potentially improve embolization therapies.

Synthesis and characterization of a new vertebroplasty cement based on gold-containing PMMA microspheres.

There are a number of drawbacks to incorporating large concentrations of barium sulfate (BaSO4) as the radiopacifier in PMMA-based bone cements for percutaneous vertebroplasty. These include adverse effects on injectability, viscosity profile, setting time, mechanical properties of the cement and bone resorption. We have synthesized a novel cement that is designed to address some of these drawbacks. Its powder includes PMMA microspheres in which gold particles are embedded and its monomer is the same as that used in commercial cements for vertebroplasty. In comparison to one such commercial cement brand, VertaPlex™, the new cement has longer doughing time, longer injection time, higher compressive strength, higher compressive modulus, and is superior in terms of cytotoxicity. For augmentation of fractured fresh-frozen cadaveric vertebral bodies (T6-L5) using simulated vertebroplasty, results for compressive strength and compressive stiffness of the construct and the percentage of the volume of the vertebral body filled by the cement were comparable for the two cements although the radiopacity of the new cement was significantly lower than that for VertaPlex™. The present results indicate that the new cement warrants further study.

Development of new injectable bulking agents: biocompatibility of radiopaque polymeric microspheres studied in a mouse model.

Radiopaque polymeric microspheres have a potential as new bulking agents for treatment of stress urinary incontinence (SUI). The advantage over existing bulking agents lies in their X-ray visibility in situ; other polymeric bulking agents (e.g., PTFE or silicone rubbers) are practically radiolucent (i.e., incapable of absorbing X-radiation). Radiopacity is useful in practice because of the high spatial accuracy of X-ray imaging. For instance, X-ray fluoroscopy can be used to assess possible migration of the bulking agent over time or to provide guidance in cases in which a second injection of a bulking agent is necessary (repeated treatment of SUI). Biocompatibility of injected radiopaque microspheres was investigated in vivo by using the mouse as a model. Microspheres were injected subcutaneously (9 animals) or intramuscularly (9 animals), and follow-up was 8 days or 3 months. X-ray fluoroscopy gave clear images of the microspheres as an ensemble, and it was found that no migration occurred during 3 months. Histopathology confirmed that all microspheres stayed close to the site of the injection. The microspheres appeared to be well tolerated; only a few giant cells, manifesting a mild inflammatory reaction, were encountered. At 3 months, capillary blood vessels were observed throughout the microsphere beds, and macrophages and fibroblast cells were seen in between the microspheres. This is encouraging with respect to the intended application, although it must be acknowledged that the data refer merely to a mouse model. Further experiments with larger, more representative models (rabbit and goat) are in progress.

Design, synthesis, imaging, and biomechanics of a softness-gradient hydrogel nucleus pulposus prosthesis in a canine lumbar spine model.

A hydrogel nucleus pulposus prosthesis (NPP) was designed to swell in situ, have intrinsic radiopacity, and restore intervertebral disc height and biomechanical functionality. These features were examined using an ex vivo canine lumbar model. Nine NPPs were implanted in five spines and their visibility was assessed on radiography, computed tomography (CT), and magnetic resonance imaging (MRI). The NPPs were visible on all imaging modalities and 8/9 NPPs stayed intact and in situ. Six other NPPs were tested biomechanically in six canine lumbar spines. Removal of the nucleus pulposus (nuclectomy) caused significant changes in biomechanical parameters. After implantation and swelling of the NPP, values were not significantly different from the native state for range of motion (ROM) of flexion-extension (FE) and lateral bending (LB), the neutral zone (NZ) of all motion directions, and the NZ stiffness (NZS) of FE. Biomechanical restoration by the NPP compared with the nuclectomized state was significant for the ROM of FE and axial rotation, the NZ of FE and LB, and the NZS of FE and LB. Disc height was significantly restored and 6/6 NPPs stayed intact and in situ. In conclusion, the NPPs swell in situ, have intrinsic radiopacity and restored disc height and aforementioned biomechanical properties.

A highly radiopaque vertebroplasty cement using tetraiodinated o-carborane additive.

Bone cements for vertebroplasty must have a much better radiocontrast level than cements for knee or hip arthroplasty. This is generally accomplished by adding a relatively large portion of BaSO(4), although this affects the physical-mechanical and biological properties of the cement. This prompted us to develop an alternative radiopaque cement, on the basis of unique highly radiopaque methacrylic microspheres. These contain iodine in two modalities: (i) covalently linked to the methacrylic polymer, and (ii) as constituent of the stable tetraiodocarborane 8,9,10,12-I(4)-1,2-closo-C(2)B(10)H(8). The total iodine content in these particles exceeded 30% by mass. These radiopaque microspheres as well as the cement made thereof were characterized extensively, e.g., by scanning electron microscopy, X-ray contrast measurements, X-ray photoelectron spectroscopy, measurements of compressive strength, infrared spectroscopy, and solid state (11)B{(1)H} NMR spectroscopy. Furthermore, the new cement was subjected to several biocompatibility tests in vitro. The results show that the new bone cement fulfills all physico-chemical criteria for use in vertebroplasty. Further data on the cement's biocompatibility (in vitro), as well as on the handling parameters and doughviscosity, indicate that this material has a potential to become an alternative to vertebroplasty cements with a high BaSO(4) content. The new cement provides two significant advantages: (i) controlled viscosity in the dough phase, which facilitates precise injection during the vertebroplasty procedure; (ii) excellent structural stability, which precludes leaching of contrast post-implantation.

The performance of a hydrogel nucleus pulposus prosthesis in an ex vivo canine model.

A nucleus pulposus prosthesis (NPP) made of the hydrogel N-vinyl-2-pyrrolidinone copolymerized with 2-(4'-iodobenzoyl)-oxo-ethyl methacrylate has recently been developed. The special features of this NPP, i.e. intrinsic radiopacity and its ability to swell in situ to fill the nucleus cavity and restore disc height, were investigated ex vivo in canine spinal specimens. L7-S1 intervertebral discs were isolated from three canine spinal specimens, and the dimensions of the nuclei pulposi were measured. Based on these averaged measurements, the NPP prototype was made and inserted in its dry form (xerogel) into a canine cadaveric spinal segment and allowed to swell overnight at 38 degrees C. The integrity of the NPP and the filling of the nucleus cavity were assessed before and after swelling, using radiography, computed tomography, and magnetic resonance imaging. The ability of the NPP to restore disc height was assessed on radiographs of 10 spinal specimens. Thereafter the NPP was macroscopically assessed in situ by dissection of the spinal specimen. Both on imaging and macroscopically, 9/10 NPPs appeared to have a near perfect fit and disc height was restored in 8/10 spinal segments. The NPP may thus be an acceptable treatment option for low back patients meeting the requirements for NPP treatment.

Radio-opaque and surface-functionalized polymer microparticles: potentially safer biomaterials for different injection therapies.

Injectable polymer particles with a diameter in the range of 30-300 microm find applications as a biomaterial in different clinical fields, such as cosmetic surgery, reconstructive surgery, and urology. However, clinical effects tend to disappear after several months, either due to migration of the particles away from the injection site (caused by weak adherence with the surrounding soft tissues) or due to fibrosis (caused by excessive encapsulation of the particles by fibrous tissue). Little is known about the fate of injected microparticles, due to the fact that they are extremely difficult to trace in a noninvasive manner. Design, synthesis, and characterization of new polymeric microspheres with two additional features that can enhance safety and can help to overcome drawbacks of existing products are reported. First, the new microparticles feature clear radio-opacity (X-ray visibility) as they are prepared on the basis of a reactive methacrylic monomer that contains covalently bound iodine. Model experiments reveal that the level of X-ray contrast is sufficient for clinical monitoring; they can be visualized both during the injection and afterward. The particles feature excellent cytocompatibility in vitro and in vivo. Second, a method is explored to functionalize the surface of the particles, for example, through immobilization of collagen. Other extracellular matrix proteins can also be immobilized, and this provides a mechanism to control anchoring of the particles in soft tissue. The results are briefly discussed in the context of improved biomaterials, contemporary X-ray imaging, and control over biomaterial-soft tissue interactions in vivo.

Injectable polymeric microspheres with X-ray visibility. Preparation, properties, and potential utility as new traceable bulking agents.

The copolymer of methyl methacrylate (MMA) and 2-[2',3',5'-triiodobenzoyl]oxoethyl methacrylate (1), ratio 3:1 (mass:mass), was prepared via a free-radical polymerization in bulk. The copolymer (M(w) = 97.8 kD and M(n) = 41.5 kD) was dissolved in chloroform and subsequently transformed into beads with a diameter in the micrometer range, using a solvent evaporation technique. The resulting microbeads were characterized by different techniques, including NMR spectroscopy, differential scanning calorimetry, gel permeation chromatography, and scanning electron microscopy. The latter technique was used as the basis for statistical analysis of the bead size. Typically, an average diameter of 96 microm and a standard deviation of 21 microm were obtained. The beads were also subjected to some preliminary tests regarding cytotoxicity. The copolymer of MMA and 1 contains covalently bound iodine. Therefore, the material is intrinsically radiopaque, i.e., capable of absorbing X-radiation while no contrast additive is needed. Our interest in these microspheres stems primarily from their possible utility as injectable and afterward traceable (radiopaque) bulking agents, e.g., for use in urology for the treatment of female stress incontinence due to sphincter deficiency. As a first test into this direction, a sample of the microbeads was mixed with ethylene glycol, and the resulting suspension was studied with respect to injectability and radiopacity. The results suggest that the radiopaque microbeads may provide access to improved bulking agents. Further modification of the surface may be necessary in order to suppress the migratory aptitude of the radiopaque polymeric microspheres in vivo.