Optimal structural and physical properties of aerogels for promoting robust neurite extension in vitro.

Cell behaviour is influenced by external factors including the physical properties of the substrate such as its surface topography and stiffness. Recent studies have demonstrated the potential of aerogels as biomaterials and specifically as neural scaffolds. The 3-D structure inherent to aerogels offers an advantage over other biocompatible substrates which lack the dimensionality needed to mimic the in vivo topography of tissues. Here, we used a variety of aerogel types to correlate the extension of neurites by neuronal cells with surface roughness ranging from 0 to 3 µm and stiffness 10 kPa-4 MPa. This investigation reveals that the optimal surface features for neurite extension are a surface roughness of 0.5 µm and a Young's modulus between 1 and 3.5 MPa. The significance of these findings to optimize materials for nerve repair is discussed.

Noninvasive Detection, Tracking, and Characterization of Aerogel Implants Using Diagnostic Ultrasound.

Medical implants are routinely tracked and monitored using different techniques, such as MRI, X-ray, and ultrasound. Due to the need for ionizing radiation, the two former methods pose a significant risk to tissue. Ultrasound imaging, however, is non-invasive and presents no known risk to human tissue. Aerogels are an emerging material with great potential in biomedical implants. While qualitative observation of ultrasound images by experts can already provide a lot of information about the implants and the surrounding structures, this paper describes the development and study of two simple B-Mode image analysis techniques based on attenuation measurements and echogenicity comparisons, which can further enhance the study of the biological tissues and implants, especially of different types of biocompatible aerogels.

Safety and efficacy assessment of aerogels for biomedical applications.

The unique physicochemical properties of aerogels have made them an attractive class of materials for biomedical applications such as drug delivery, regenerative medicine, and wound healing. Their low density, high porosity, and ability to regulate the pore structure makes aerogels ideal nano/micro-structures for loading of drugs and active biomolecules. As a result of this, the number of in vitro and in vivo studies on the therapeutic efficacy of these porous materials has increased substantially in recent years and continues to be an area of great interest. However, data about their in vivo performance and safety is limited. Studies have shown that polymer-based, silica-based and some hybrid aerogels are generally regarded as safe but given that studies on the acute, subacute, and chronic toxicity for the majority of aerogel types is missing, more work is still needed. This review presents a comprehensive summary of different biomedical applications of aerogels proposed to date as well as new and innovative applications of aerogels in other areas such as decontamination. We have also reviewed their biological effect on cells and living organisms with a focus on therapeutic efficacy and overall safety (in vivo and in vitro).

Enhanced neurite outgrowth on electrically conductive carbon aerogel substrates in the presence of an external electric field.

Previous works from our laboratory have firmly established that aerogels are a suitable substrate to elicit accelerated neurite extension. On non-conducting aerogels, in the presence of an externally-applied DC bias, neurons extended neurites which were preferentially aligned towards the anode. In this investigation, we sought to determine whether electrically-conductive carbon aerogels elicited a more robust alignment of neurites toward the anode than non-conductive aerogels due to the capacity of conductive aerogels to sustain a current, thereby providing a direct interface between neurons and the external electrical stimulus. To determine if this was the case, we plated PC12 neuronal cells on electrically conductive carbon aerolges derived from acetic acid-catalized resorcinol formaldehyde aerogels (ARF-CA) and subjected them to an external electric field. The voltages applied at the electrodes of the custom-built electro-stimulation chamber were 0 V, 15 V, and 30 V. For each voltage, the directionality and length of the neurites extended by PC12 cells were determined and compared to those observed when PC12 cells were plated on non-conductive aerogels subjected to the same voltage. The results show that the directionality of neurite extension was similar between conductive and non-conductive aerogels. A higher neurite length difference was observed on conductive aerogels with increasing voltage, 43% and 106% for 0-15 V and 0-30 V respectively, compared to non-conductive aerogels, 12% and 20%. These findings indicate that conductive carbon aerogels have a greater potential as scaffolds for nerve regeneration than non-conductive ones.

Nerve Response to Superelastic Shape Memory Polyurethane Aerogels.

We have previously shown the suitability of aerogels as scaffolds for neuronal cells. Here, we report on the use of superelastic shape memory polyurethane aerogels (SSMPA). SSMPA have a distinctly different stiffness than previously reported aerogels. The soft and deformable nature of SSMPA allowed for radial compression of the aerogel induced by a custom designed apparatus. This radial compression changed the pore diameter and surface roughness (Sa) of SSMPA, while maintaining similar stiffness. Two varieties of SSMPA were used, Mix-14 and Mix-18, with distinctly different pore diameters and Sa. Radial compression led to a decreased pore diameter, which, in turn, decreased the Sa. The use of custom designed apparatus and two types of SSMPA allowed us to examine the influence of stiffness, pore size, and Sa on the extension of processes (neurites) by PC12 neuronal cells. PC12 cells plated on SSMPA with a higher degree of radial compression extended fewer neurites per cell when compared to other groups. However, the average length of the neurites was significantly longer when compared to the unrestricted group and to those extended by cells plated on SSMPA with less radial compression. These results demonstrate that SSMPA with 1.9 µm pore diameter, 1.17 µm Sa, and 203 kPa stiffness provides the optimum combination of physical parameters for nerve regeneration.

Growing Neural PC-12 Cell on Crosslinked Silica Aerogels Increases Neurite Extension in the Presence of an Electric Field.

Externally applied electrical stimulation (ES) has been shown to enhance the nerve regeneration process and to influence the directionality of neurite outgrowth. In addition, the physical and chemical properties of the substrate used for nerve-cell regeneration is critical in fostering regeneration. Previously, we have shown that polyurea-crosslinked silica aerogels (PCSA) exert a positive influence on the extension of neurites by PC-12 cells, a cell-line model widely used to study neurite extension and electrical excitability. In this work, we have examined how an externally applied electric field (EF) influences the extension of neurites in PC-12 cells grown on two substrates: collagen-coated dishes versus collagen-coated crosslinked silica aerogels. The externally applied direct current (DC) bias was applied in vitro using a custom-designed chamber containing polydimethysiloxane (PDMS) embedded copper electrodes to create an electric field across the substrate for the cultured PC-12 cells. Results suggest orientation preference towards the anode, and, on average, longer neurites in the presence of the applied DC bias than with 0 V DC bias. In addition, neurite length was increased in cells grown on silica-crosslinked aerogel when compared to cells grown on regular petri-dishes. These results further support the notion that PCSA is a promising material for nerve regeneration.

Investigation of surface topography and stiffness on adhesion and neurites extension of PC12 cells on crosslinked silica aerogel substrates.

Fundamental understanding and characterization of neural response to substrate topography is essential in the development of next generation biomaterials for nerve repair. Aerogels are a new class of materials with great potential as a biomaterial. In this work, we examine the extension of neurites by PC12 cells plated on matrigel-coated and collagen-coated mesoporous aerogel surfaces. We have successfully established the methodology for adhesion and growth of PC12 cells on polyurea crosslinked silica aerogels. Additionally, we have quantified neurite behaviors and compared their response on aerogel substrates with their behavior on tissue culture (TC) plastic, and polydimethylsiloxane (PDMS). We found that, on average, PC12 cells extend longer neurites on crosslinked silica aerogels than on tissue culture plastic, and, that the average number of neurites per cluster is lower on aerogels than on tissue culture plastic. Aerogels are an attractive candidate for future development of smart neural implants and the work presented here creates a platform for future work with this class of materials as a substrate for bioelectronic interfacing.

Novel technique for repair of severed peripheral nerves in rats using polyurea crosslinked silica aerogel scaffold.

PURPOSE/AIM: To design, synthesize, and test in vivo an aerogel-based top-open peripheral nerve scaffold to simultaneously support and guide multiple completely severed peripheral nerves in a rat model. Also, to explore options for immobilizing severed nerves on the aerogel material without the use of sutures resulting in reduced surgical time. MATERIALS AND METHOD: A novel material and approach was developed for the reattachment of severed peripheral nerves. Nerve confinement and alignment in this case relies on the surface properties of a lightweight, highly porous, polyurea crosslinked silica aerogel scaffold. The distal and proximal ends of completely transected nerve terminals were positioned inside prefabricated "top-open" corrugated channels that cradled approximately two thirds of the circumference of the nerve trunk and connectivity of the severed nerves was evaluated using sciatic function index (SFI) technique for five months post-surgery on 10 female Sprague-Dawley rats then compared with the gold standard for peripheral nerve repair. The interaction of nerves with the surface of the scaffold was investigated also. RESULTS AND CONCLUSION: Multichannel aerogel-based nerve support scaffold showed similar SFI recovery trend as the case suture repair technique. Usage of an adhesion-promoting coating reduced the friction between the nerve and the scaffold leading to slippage and lack of attachment between nerve and surface. The aerogel scaffold used in this study did not collapse under pressure during the incubation period and allowed for a rapid and non-invasive peripheral nerve repair approach without the demands of microsurgery on both time and surgical expertise. This technique may allow for simultaneous repair and reconnection of multiple severed nerves particularly relevant to nerve branching sites.

In vivo ultrasonic detection of polyurea crosslinked silica aerogel implants.

BACKGROUND: Polyurea crosslinked silica aerogels are highly porous, lightweight, and mechanically strong materials with great potential for in vivo applications. Recent in vivo and in vitro studies have demonstrated the biocompatibility of this type of aerogel. The highly porous nature of aerogels allows for exceptional thermal, electric, and acoustic insulating capabilities that can be taken advantage of for non-invasive external imaging techniques. Sound-based detection of implants is a low cost, non-invasive, portable, and rapid technique that is routinely used and readily available in major clinics and hospitals. METHODOLOGY: In this study the first in vivo ultrasound response of polyurea crosslinked silica aerogel implants was investigated by means of a GE Medical Systems LogiQe diagnostic ultrasound machine with a linear array probe. Aerogel samples were inserted subcutaneously and sub-muscularly in a) fresh animal model and b) cadaveric human model for analysis. For comparison, samples of polydimethylsiloxane (PDMS) were also imaged under similar conditions as the aerogel samples. CONCLUSION/SIGNIFICANCE: Polyurea crosslinked silica aerogel (X-Si aerogel) implants were easily identified when inserted in either of the regions in both fresh animal model and cadaveric model. The implant dimensions inferred from the images matched the actual size of the implants and no apparent damage was sustained by the X-Si aerogel implants as a result of the ultrasonic imaging process. The aerogel implants demonstrated hyperechoic behavior and significant posterior shadowing. Results obtained were compared with images acquired from the PDMS implants inserted at the same location.

Histological evaluation of the biocompatibility of polyurea crosslinked silica aerogel implants in a rat model: a pilot study.

BACKGROUND: Aerogels are a versatile group of nanostructured/nanoporous materials with physical and chemical properties that can be adjusted to suit the application of interest. In terms of biomedical applications, aerogels are particularly suitable for implants such as membranes, tissue growth scaffolds, and nerve regeneration and guidance inserts. The mesoporous nature of aerogels can also be used for diffusion based release of drugs that are loaded during the drying stage of the material. From the variety of aerogels polyurea crosslinked silica aerogels have the most potential for future biomedical applications and are explored here. METHODOLOGY: This study assessed the short and long term biocompatibility of polyurea crosslinked silica aerogel implants in a Sprague-Dawley rat model. Implants were inserted at two different locations a) subcutaneously (SC), at the dorsum and b) intramuscularly (IM), between the gluteus maximus and biceps femoris of the left hind extremity. Nearby muscle and other internal organs were evaluated histologically for inflammation, tissue damage, fibrosis and movement (travel) of implant. CONCLUSION/SIGNIFICANCE: In general polyurea crosslinked silica aerogel (PCSA) was well tolerated as a subcutaneous and an intramuscular implant in the Sprague-Dawley rat with a maximum incubation time of twenty months. In some cases a thin fibrous capsule surrounded the aerogel implant and was interpreted as a normal response to foreign material. No noticeable toxicity was found in the tissues surrounding the implants nor in distant organs. Comparison was made with control rats without any implants inserted, and animals with suture material present. No obvious or noticeable changes were sustained by the implants at either location. Careful necropsy and tissue histology showed age-related changes only. An effective sterilization technique for PCSA implants as well as staining and sectioning protocol has been established. These studies further support the notion that silica-based aerogels could be useful as biomaterials.

Effect of surface plasma treatments on the adhesion of Mars JSC 1 simulant dust to RTV 655, RTV 615, and Sylgard 184.

BACKGROUND: Dust accumulation on surfaces of critical instruments has been a major concern during lunar and Mars missions. Operation of instruments such as solar panels, chromatic calibration targets, as well as Extra Vehicular Activity (EVA) suits has been severely compromised in the past as a result of dust accumulation and adhesion. Wind storms with wind speeds of up to 70 mph have not been effective in removing significant amounts of the deposited dust. This is indeed an indication of the strength of the adhesion force(s) involved between the dust particles and the surface(s) that they have adhered to. Complications associated with dust accumulation are more severe for non-conducting surfaces and have been the focus of this work. METHODOLOGY: Argon plasma treatment was investigated as a mechanism for lowering dust accumulation on non-conducting polymeric surfaces. Polymers chosen for this study include a popular variety of silicones routinely used for space and terrestrial applications namely RTV 655, RTV 615, and Sylgard 184. Surface properties including wettability, surface potential, and surface charge density were compared before and after plasma treatment and under different storage conditions. Effect of ultraviolet radiation on RTV 655 was also investigated and compared with the effect of Ar plasma treatment. CONCLUSION/SIGNIFICANCE: Gravimetric measurements proved Ar plasma treatment to be an effective method for eliminating dust adhesion to all three polymers after short periods of exposure. No physical damage was detected on any of the polymer surfaces after Ar plasma treatment. The surface potential of all three polymers remained zero up to three months post plasma exposure. Ultraviolet radiation however was not effective in reducing surface and caused damage and significant discoloration to RTV 655. Therefore, Ar plasma treatment can be an effective and non-destructive method for treating insulating polymeric surfaces in order to eliminate dust adhesion and accumulation.

Investigation of polyurea-crosslinked silica aerogels as a neuronal scaffold: a pilot study.

BACKGROUND: Polymer crosslinked aerogels are an attractive class of materials for future implant applications particularly as a biomaterial for the support of nerve growth. The low density and nano-porous structure of this material combined with large surface area, high mechanical strength, and tunable surface properties, make aerogels materials with a high potential in aiding repair of injuries of the peripheral nervous system. however, the interaction of neurons with aerogels remains to be investigated. METHODOLOGY: In this work the attachment and growth of neurons on clear polyurea crosslinked silica aerogels (PCSA) coated with: poly-L-lysine, basement membrane extract (BME), and laminin1 was investigated by means of optical and scanning electron microscopy. After comparing the attachment and growth capability of neurons on these different coatings, laminin1 and BME were chosen for nerve cell attachment and growth on PCSA surfaces. The behavior of neurons on treated petri dish surfaces was used as the control and behavior of neurons on treated PCSA discs was compared against it. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that: 1) untreated PCSA surfaces do not support attachment and growth of nerve cells, 2) a thin application of laminin1 layer onto the PCSA discs adhered well to the PCSA surface while also supporting growth and differentiation of neurons as evidenced by the number of processes extended and b3-tubulin expression, 3) three dimensional porous structure of PCSA remains intact after fixing protocols necessary for preservation of biological samples and 4) laminin1 coating proved to be the most effective method for attaching neurons to the desired regions on PCSA discs. This work provides the basis for potential use of PCSA as a biomaterial scaffold for neural regeneration.

Organization of membrane motor in outer hair cells: an atomic force microscopic study.

Using atomic force microscopy, we imaged the cytosolic surface of the lateral plasma membrane of outer hair cells from guinea pigs' inner ear. We used a "cell-free" preparation, in which a patch of plasma membrane was firmly attached to a substrate and the cytoplasmic face was exposed. The membrane patches contained densely packed particles whose diameter, after correcting for the geometry of the probing tip, was approximately 10 nm. The particles were predominantly aligned unidirectionally with spacing of approximately 36 nm. The density of the particle was approximately 850 microm(-2), which could be an underestimate presumably due to the method of sample preparation. Antibody-labeled specimens showed particles more elevated than unlabeled preparation indicative of primary and secondary antibody complexes. The corrected diameters of these particles labeled with anti-actin were approximately 12 nm while that with antiprestin were approximately 8 nm. The alignment pattern in antiprestin-labeled specimens resembled that of the unlabeled preparation. Specimens labeled with actin antibodies did not show such alignment. We interpret that the particles observed in the unlabeled membranes correspond to the 10-nm particles reported by electron microscopy and that these particles contain prestin, a member of the SLC26 family, which is essential for electromotility.