HSP70-A key regulator in chondrocyte homeostasis under naturally coupled hydrostatic pressure-thermal stimuli.

OBJECTIVE: During physical activities, chondrocytes experience coupled stimulation of hydrostatic pressure (HP) and a transient increase in temperature (T), with the latter varying within a physiological range from 32.5 °C to 38.7 °C. Previous short-term in vitro studies have demonstrated that the combined hydrostatic pressure-thermal (HP-T) stimuli more significantly enhance chondroinduction and chondroprotection of chondrocytes than isolated applications. Interestingly, this combined benefit is associated with a corresponding increase in HSP70 levels when HP and T are combined. The current study therefore explored the indispensable role of HSP70 in mediating the combined effects of HP-T stimuli on chondrocytes. DESIGN: In this mid-long-term study of in vitro engineered cartilage constructs, we assessed chondrocyte responses to HP-T stimuli using customized bioreactor in standard and HSP70-inhibited cultures. RESULTS: Surprisingly, under HSP70-inhibited conditions, the usually beneficial HP-T stimuli, especially its thermal component, exerted detrimental effects on chondrocyte homeostasis, showing a distinct and unfavorable shift in gene and protein expression patterns compared to non-HSP70-inhibited settings. Such effects were corroborated through mechanical testing and confirmed using a secondary cell source. A proteomic-based mechanistic analysis revealed a disruption in the balance between biosynthesis and fundamental cellular structural components in HSP70-inhibited conditions under HP-T stimuli. CONCLUSIONS: Our results highlight the critical role of sufficient HSP70 induction in mediating the beneficial effects of coupled HP-T stimulation on chondrocytes. These findings help pave the way for new therapeutic approaches to enhance physiotherapy outcomes and potentially shed light on the elusive mechanisms underlying the onset of cartilage degeneration, a long-standing enigma in orthopedics.

Applying support-vector machine learning algorithms toward predicting host-guest interactions with cucurbit[7]uril.

Machine learning is a valuable tool in the development of chemical technologies but its applications into supramolecular chemistry have been limited. Here, the utility of kernel-based support vector machine learning using density functional theory calculations as training data is evaluated when used to predict equilibrium binding coefficients of small molecules with cucurbit[7]uril (CB[7]). We find that utilising SVMs may confer some predictive ability. This algorithm was then used to predict the binding of drugs TAK-580 and selumetinib. The algorithm did predict strong binding for TAK-580 and poor binding for selumetinib, and these results were experimentally validated. It was discovered that the larger homologue cucurbit[8]uril (CB[8]) is partial to selumetinib, suggesting an opportunity for tunable release by introducing different concentrations of CB[7] or CB[8] into a hydrogel depot. We qualitatively demonstrated that these drugs may have utility in combination against gliomas. Finally, mass transfer simulations show CB[7] can independently tune the release of TAK-580 without affecting selumetinib. This work gives specific evidence that a machine learning approach to recognition of small molecules by macrocycles has merit and reinforces the view that machine learning may prove valuable in the development of drug delivery systems and supramolecular chemistry more broadly.

Cucurbit[8]uril-Derived Graphene Hydrogels.

The scalable production of uniformly distributed graphene (GR)-based composite materials remains a sizable challenge. While GR-polymer nanocomposites can be manufactured at a large scale, processing limitations result in poor control over the homogeneity of hydrophobic GR sheets in the matrices. Such processes often result in difficulties controlling stability and avoiding aggregation, therefore eliminating benefits that might have otherwise arisen from the nanoscopic dimensions of GR. Here, we report an exfoliated and stabilized GR dispersion in water. Cucurbit[8]uril (CB[8])-mediated host-guest chemistry was used to obtain supramolecular hydrogels consisting of uniformly distributed GR and guest-functionalized macromolecules. The obtained GR hydrogels show superior bioelectrical properties over identical systems produced without CB[8]. Utilizing such supramolecular interactions with biologically derived macromolecules is a promising approach to stabilize graphene in water and avoid oxidative chemistry.

Protein-mediated gelation and nano-scale assembly of unfunctionalized hyaluronic acid and chondroitin sulfate.

Background: Hyaluronic acid (HA) is a major component of the extracellular matrix (ECM) in the central nervous system and the only purely supramolecular glycosaminoglycan. Much focus has been given to using this high molecular weight polysaccharide for tissue engineering applications. In most studies, the backbone of HA is functionalized with moieties that can facilitate network formation through physical self-assembly, or covalent crosslinking (e.g. photo-catalyzed) at concentrations where the polysaccharide does not gel on its own. However, these crosslinks often utilize functional groups not found in biological tissues. Methods: Oscillatory rheology, dynamic light scattering, and scanning electron microscopy were used to study albumin/HA structures. Dynamic light scattering and transmission electron microscopy were used to study albumin/chondroitin sulfate (CS) structures. UV-vis spectroscopy was used to demonstrate the potential for using protein-polymer blends as an ECM-mimetic model to study transport of small molecules. Results: We examine the intermolecular interactions of two major glycosaminoglycans found in the human brain, HA and the lower molecular weight CS, with the model protein albumin. We report the properties of the resulting micro- and nano materials. Our albumin/HA systems formed gels, and albumin/CS systems formed micro- and nanoparticles. These systems are formed from unfunctionalized polysaccharides, which is an attractive and simple method of forming HA hydrogels and CS nanoparticles. We also summarize the concentrations of HA and CS found in various mammalian brains, which could potentially be useful for biomimetic scaffold development. Conclusions: Simple preparation of commercially available charged biomacromolecules results in interesting materials with structures at the micron and nanometer length-scales. Such materials may have utility in serving as cost-effective models of nervous system electrostatic interactions and as in vitro drug release and model system for ECM transport studies.

A Real-Time Skin Dose Tracking System for Biplane Neuro-Interventional Procedures.

A biplane dose-tracking system (Biplane-DTS) that provides a real-time display of the skin-dose distribution on a 3D-patient graphic during neuro-interventional fluoroscopic procedures was developed. Biplane-DTS calculates patient skin dose using geometry and exposure information for the two gantries of the imaging system acquired from the digital system bus. The dose is calculated for individual points on the patient graphic surface for each exposure pulse and cumulative dose for both x-ray tubes is displayed as color maps on a split screen showing frontal and lateral projections of a 3D-humanoid graphic. Overall peak skin dose (PSD), FOV-PSD and current dose rates for the two gantries are also displayed. Biplane-DTS uses calibration files of mR/mAs for the frontal and lateral tubes measured with and without the table in the beam at the entrance surface of a 20 cm thick PMMA phantom placed 15 cm tube-side of the isocenter. For neuro-imaging, conversion factors are applied as a function of entrance field area to scale the calculated dose to that measured with a Phantom Laboratory head phantom which contains a human skull to account for differences in backscatter between PMMA and the human head. The software incorporates inverse-square correction to each point on the skin and corrects for angulation of the beam through the table. Dose calculated by Biplane DTS and values measured by a 6-cc ionization chamber placed on the head phantom at multiple points agree within a range of -3% to +7% with a standard deviation for all points of less than 3%.

Incorporating Corrections for the Head-Holder and Compensation Filter when Calculating Skin Dose during Fluoroscopically-Guided Interventions.

The skin dose tracking system (DTS) that we developed provides a color-coded illustration of the cumulative skin dose distribution on a 3D graphic of the patient during fluoroscopic procedures for immediate feedback to the interventionist. To improve the accuracy of dose calculation, we now have incorporated two additional important corrections (1) for the holder used to immobilize the head in neuro-interventions and (2) for the built-in compensation filters used for beam equalization. Both devices have been modeled in the DTS software so that beam intensity corrections can be made. The head-holder is modeled as two concentric hemi-cylindrical surfaces such that the path length between those surfaces can be determined for rays to individual points on the skin surface. The head-holder on the imaging system we used was measured to attenuate the primary x-rays by 10 to 20% for normal incidence, and up to 40% at non-normal incidence. In addition, three compensation filters of different shape are built into the collimator apparatus and were measured to have attenuation factors ranging from 58% to 99%, depending on kVp and beam filtration. These filters can translate and rotate in the beam and their motion is tracked by the DTS using the digital signal from the imaging system. When it is determined that a ray to a given point on the skin passes through the compensation filter, the appropriate attenuation correction is applied. These corrections have been successfully incorporated in the DTS software to provide a more accurate determination of skin dose.

Improved-Resolution, Real-Time Skin-Dose Mapping for Interventional Fluoroscopic Procedures.

We have developed a dose-tracking system (DTS) that provides a real-time display of the skin-dose distribution on a 3D patient graphic during fluoroscopic procedures. Radiation dose to individual points on the skin is calculated using exposure and geometry parameters from the digital bus on a Toshiba C-arm unit. To accurately define the distribution of dose, it is necessary to use a high-resolution patient graphic consisting of a large number of elements. In the original DTS version, the patient graphics were obtained from a library of population body scans which consisted of larger-sized triangular elements resulting in poor congruence between the graphic points and the x-ray beam boundary. To improve the resolution without impacting real-time performance, the number of calculations must be reduced and so we created software-designed human models and modified the DTS to read the graphic as a list of vertices of the triangular elements such that common vertices of adjacent triangles are listed once. Dose is calculated for each vertex point once instead of the number of times that a given vertex appears in multiple triangles. By reformatting the graphic file, we were able to subdivide the triangular elements by a factor of 64 times with an increase in the file size of only 1.3 times. This allows a much greater number of smaller triangular elements and improves resolution of the patient graphic without compromising the real-time performance of the DTS and also gives a smoother graphic display for better visualization of the dose distribution.

Updates in the real-time Dose Tracking System (DTS) to improve the accuracy in calculating the radiation dose to the patients skin during fluoroscopic procedures.

We have developed a dose-tracking system (DTS) to manage the risk of deterministic skin effects to the patient during fluoroscopic image-guided interventional cardiac procedures. The DTS calculates the radiation dose to the patient's skin in real-time by acquiring exposure parameters and imaging-system geometry from the digital bus on a Toshiba C-arm unit and displays the cumulative dose values as a color map on a 3D graphic of the patient for immediate feedback to the interventionalist. Several recent updates have been made to the software to improve its function and performance. Whereas the older system needed manual input of pulse rate for dose-rate calculation and used the CPU clock with its potential latency to monitor exposure duration, each x-ray pulse is now individually processed to determine the skin-dose increment and to automatically measure the pulse rate. We also added a correction for the table pad which was found to reduce the beam intensity to the patient for under-table projections by an additional 5-12% over that of the table alone at 80 kVp for the x-ray filters on the Toshiba system. Furthermore, mismatch between the DTS graphic and the patient skin can result in inaccuracies in dose calculation because of inaccurate inverse-square-distance calculation. Therefore, a means for quantitative adjustment of the patient-graphic-model position and a parameterized patient-graphic library have been developed to allow the graphic to more closely match the patient. These changes provide more accurate estimation of the skin-dose which is critical for managing patient radiation risk.

Verification of the performance accuracy of a real-time skin-dose tracking system for interventional fluoroscopic procedures.

A tracking system has been developed to provide real-time feedback of skin dose and dose rate during interventional fluoroscopic procedures. The dose tracking system (DTS) calculates the radiation dose rate to the patient's skin using the exposure technique parameters and exposure geometry obtained from the x-ray imaging system digital network (Toshiba Infinix) and presents the cumulative results in a color mapping on a 3D graphic of the patient. We performed a number of tests to verify the accuracy of the dose representation of this system. These tests included comparison of system-calculated dose-rate values with ionization-chamber (6 cc PTW) measured values with change in kVp, beam filter, field size, source-to-skin distance and beam angulation. To simulate a cardiac catheterization procedure, the ionization chamber was also placed at various positions on an Alderson Rando torso phantom and the dose agreement compared for a range of projection angles with the heart at isocenter. To assess the accuracy of the dose distribution representation, Gafchromic film (XR-RV3, ISP) was exposed with the beam at different locations. The DTS and film distributions were compared and excellent visual agreement was obtained within the cm-sized surface elements used for the patient graphic. The dose (rate) values agreed within about 10% for the range of variables tested. Correction factors could be applied to obtain even closer agreement since the variable values are known in real-time. The DTS provides skin-dose values and dose mapping with sufficient accuracy for use in monitoring diagnostic and interventional x-ray procedures.