Continuous Low-Intensity Ultrasound Promotes Native-to-Native Cartilage Integration.

Failure of the host/graft interface to integrate impedes the success of cartilage repair protocols. Continuous low-intensity ultrasound (cLIUS) at the resonant frequency of 5 MHz is proposed as a treatment modality for promoting native-to-native cartilage integration in vitro. Cylindrical incisions (4 mm) simulating chondral discontinuity were made in bovine cartilage and osteochondral explants, and maintained under cLIUS stimulation (14 kPa [5 MHz, 2.5 Vpp], 20 min, four times/day) for 28 days. Incised cartilage and osteochondral explants were categorized into three study groups; Group I: cLIUS was applied immediately upon incision; Group II: cLIUS was applied after 14 days following incision; Group-III: after 14 days following incision, explants were treated with 0.1% hyaluronidase and 30 U/mL collagenase VII. As a separate study group, incised osteochondral explants were treated immediately with cLIUS at a nonresonant frequency of 2 MHz (14 kPa [2 MHz, 6 Vpp], 20 min, four times/day). Cellular migration was analyzed by scratch assays, and by visualizing migrating cells into the hydrogel core of cartilage/hydrogel constructs. Explants under cLIUS (5 MHz) displayed higher percent apposition along with gap closures when compared with untreated controls and explants treated with cLIUS at 2 MHz. cLIUS (5 MHz)-treated explants were immunopositive for type II collagen. The strength of native-to-native cartilage integration was higher (p = 0.005) in cLIUS-treated cartilage explants at 0.19 ± 0.08 MPa as compared with 0.05 ± 0.03 MPa in untreated controls. Enhanced cartilage phenotype coupled with increased cellular migration were noted under cLIUS (5 MHz), alluding to the observed integration between cartilage interfaces. Collectively, cLIUS at cell resonant frequency promoted integrative cartilage repair, therefore, has the potential to improve cartilage repair outcomes. Impact Statement Lack of integration between the host and graft cartilage interfaces impedes the success of cartilage repair techniques. Continuous low-intensity ultrasound (cLIUS) is documented to induce chondrogenesis and chondrocyte phenotype. However, integrative cartilage repair under cLIUS has not been evaluated. Our results demonstrated integration between cartilage interfaces, increased percent apposition, increased strength of integration, and maintenance of cartilage phenotype under cLIUS (5 MHz). Integrative repair under cLIUS (5 MHz) stemmed from enhanced migration of cells and increased expression of cartilage-specific genes, namely SOX9 and COL2A1. Thus, cLIUS has the potential to improve the outcomes of grafting protocols for cartilage repair.

Continuous low-intensity ultrasound attenuates IL-6 and TNFα-induced catabolic effects and repairs chondral fissures in bovine osteochondral explants.

BACKGROUND: Cartilage repair outcomes are compromised in a pro-inflammatory environment; therefore, the mitigation of pro-inflammatory responses is beneficial. Treatment with continuous low-intensity ultrasound (cLIUS) at the resonant frequency of 5 MHz is proposed for the repair of chondral fissures under pro-inflammatory conditions. METHODS: Bovine osteochondral explants, concentrically incised to create chondral fissures, were maintained under cLIUS (14 kPa (5 MHz, 2.5 Vpp), 20 min, 4 times/day) for a period of 28 days in the presence or absence of cytokines, interleukin-6 (IL-6) or tumor necrosis factor (TNF)α. Outcome assessments included histological and immunohistochemical staining of the explants; and the expression of catabolic and anabolic genes by qRT-PCR in bovine chondrocytes. Cell migration was assessed by scratch assays, and by visualizing migrating cells into the hydrogel core of cartilage-hydrogel constructs. RESULTS: Both in the presence and absence of cytokines, higher percent apposition along with closure of fissures were noted in cLIUS-stimulated explants as compared to non-cLIUS-stimulated explants on day 14. On day 28, the percent apposition was not significantly different between unstimulated and cLIUS-stimulated explants exposed to cytokines. As compared to non-cLIUS-stimulated controls, on day 28, cLIUS preserved the distribution of proteoglycans and collagen II in explants despite exposure to cytokines. cLIUS enhanced the cell migration irrespective of cytokine treatment. IL-6 or TNFα-induced increases in MMP13 and ADAMTS4 gene expression was rescued by cLIUS stimulation in chondrocytes. Under cLIUS, TNFα-induced increase in NF-κB expression was suppressed, and the expression of collagen II and TIMP1 genes were upregulated. CONCLUSION: cLIUS repaired chondral fissures, and elicited pro-anabolic and anti-catabolic effects, thus demonstrating the potential of cLIUS in improving cartilage repair outcomes.

Automated Expansion of Primary Human T Cells in Scalable and Cell-Friendly Hydrogel Microtubes for Adoptive Immunotherapy.

Adoptive immunotherapy is a highly effective strategy for treating many human cancers, such as melanoma, cervical cancer, lymphoma, and leukemia. Here, a novel cell culture technology is reported for expanding primary human T cells for adoptive immunotherapy. T cells are suspended and cultured in microscale alginate hydrogel tubes (AlgTubes) that are suspended in the cell culture medium in a culture vessel. The hydrogel tubes protect cells from hydrodynamic stresses and confine the cell mass less than 400 µm (in radial diameter) to ensure efficient mass transport, creating a cell-friendly microenvironment for growing T cells. This system is simple, scalable, highly efficient, defined, cost-effective, and compatible with current good manufacturing practices. Under optimized culture conditions, the AlgTubes enable culturing T cells with high cell viability, low DNA damage, high growth rate (≈320-fold expansion over 14 days), high purity (≈98% CD3+), and high yield (≈3.2 × 108 cells mL-1 hydrogel). All offer considerable advantages compared to current T cell culturing approaches. This new culture technology can significantly reduce the culture volume, time, and cost, while increasing the production.

Scalable Culturing of Primary Human Glioblastoma Tumor-Initiating Cells with a Cell-Friendly Culture System.

Glioblastoma is the most aggressive and deadly brain cancer. There is growing interest to develop drugs that specifically target to glioblastoma tumor-initiating cells (TICs). However, the cost-effective production of large numbers of high quality glioblastoma TICs for drug discovery with current cell culturing technologies remains very challenging. Here, we report a new method that cultures glioblastoma TICs in microscale alginate hydrogel tubes (or AlgTubes). The AlgTubes allowed long-term culturing (~50 days, 10 passages) of glioblastoma TICs with high growth rate (~700-fold expansion/14 days), high cell viability and high volumetric yield (~3.0 × 108 cells/mL) without losing the stem cell properties, all offered large advancements over current culturing methods. This method can be applied for the scalable production of glioblastoma TICs at affordable cost for drug discovery.

Theoretically proposed optimal frequency for ultrasound induced cartilage restoration.

BACKGROUND: Matching the frequency of the driving force to that of the system's natural frequency of vibration results in greater amplitude response. Thus we hypothesize that applying ultrasound at the chondrocyte's resonant frequency will result in greater deformation than applying similar ultrasound power at a frequency outside of the resonant bandwidth. Based on this resonant hypothesis, our group previously confirmed theoretically and experimentally that ultrasound stimulation of suspended chondrocytes at resonance (5 MHz) maximized gene expression of load inducible genes. However, this study was based on suspended chondrocytes. The resonant frequency of a chondrocyte does not only depend on the cell mass and intracellular stiffness, but also on the mechanical properties of the surrounding medium. An in vivo chondrocyte's environment differs whether it be a blood clot (following microfracture), a hydrogel or the pericellular and extracellular matrices of the natural cartilage. All have distinct structures and compositions leading to different resonant frequencies. In this study, we present two theoretical models, the first model to understand the effects of the resonant frequency on the cellular deformation and the second to identify the optimal frequency range for clinical applications of ultrasound to enhance cartilage restoration. RESULTS: We showed that applying low-intensity ultrasound at the resonant frequency induced deformation equivalent to that experimentally calculated in previous studies at higher intensities and a 1 MHz frequency. Additionally, the resonant frequency of an in vivo chondrocyte in healthy conditions, osteoarthritic conditions, embedded in a blood clot and embedded in fibrin ranges from 3.5 - 4.8 MHz. CONCLUSION: The main finding of this study is the theoretically proposed optimal frequency for clinical applications of therapeutic ultrasound induced cartilage restoration is 3.5 - 4.8 MHz (the resonant frequencies of in vivo chondrocytes). Application of ultrasound in this frequency range will maximize desired bioeffects.

Frequency sensitive mechanism in low-intensity ultrasound enhanced bioeffects.

This study presents two novel theoretical models to elucidate frequency sensitive nuclear mechanisms in low-intensity ultrasound enhanced bioeffects. In contrast to the typical 1.5 MHz pulsed ultrasound regime, our group previously experimentally confirmed that ultrasound stimulation of anchored chondrocytes at resonant frequency maximized gene expression of load inducible genes which are regulatory markers for cellular response to external stimuli. However, ERK phosphorylation displayed no frequency dependency, suggesting that the biochemical mechanisms involved in enhanced gene expression is downstream of ERK phosphorylation. To elucidate such underlying mechanisms, this study presents a theoretical model of an anchored cell, representing an in vitro chondrocyte, in an ultrasound field. The model results showed that the mechanical energy storage is maximized at the chondrocyte's resonant frequency and the energy density in the nucleus is almost twice as high as in the cytoplasm. Next, a mechanochemical model was developed to link the mechanical stimulation of ultrasound and the increased mechanical energy density in the nucleus to the downstream targets of the ERK pathway. This study showed for the first time that ultrasound stimulation induces frequency dependent gene expression as a result of altered rates of transcription factors binding to chromatin.

Theoretical evaluation of the acoustic field in an ultrasonic bioreactor.

Ultrasound-assisted bioreactors that provide mechanical conditioning to cells have broad applicability in tissue engineering, but biological experiments with ultrasound are very sensitive to environmental conditions. A mathematical model was developed to complement experimental measurements, as well as to describe ultrasonic fields existing in regions where measurements are impossible, specifically, within microporous tissue engineering scaffolds. The model uniquely combines Biot theory to predict the ultrasonic field in the scaffold with an electromechanical transducer model to couple the mechanical stimulation experienced by cells to the external electrical input. In the specific example examined here, cells immobilized on scaffolds are subjected to different forms of ultrasonic stimulation due to the formation of standing wave fields and vertical high-pressure bands. The model confirms the sensitivity of the supplied acoustic power to the liquid level in sonobioreactors and identifies the input electrical impedance as a method of detecting resonance effects.

Enhanced depth-independent chondrocyte proliferation and phenotype maintenance in an ultrasound bioreactor and an assessment of ultrasound dampening in the scaffold.

Chondrocyte-seeded scaffolds were cultured in an ultrasound (US)-assisted bioreactor, which supplied the cells with acoustic energy around resonance frequencies (~5.0 MHz). Polyurethane-polycarbonate (BM), chitosan (CS) and chitosan-n-butanol (CSB) based scaffolds with varying porosities were chosen and the following US regimen was employed: 15 kPa and 60 kPa, 5 min per application and 6 applications per day for 21 days. Non-stimulated scaffolds served as control. For BM scaffolds, US stimulation significantly impacted cell proliferation and depth-independent cell population density compared to controls. The highest COL2A1/COL1A1 ratios and ACAN mRNA were noted on US-treated BM scaffolds compared to controls. A similar trend was noted on US-treated cell-seeded CS and CSB scaffolds, though COL2A1/COL1A1 ratios were significantly lower compared to BM scaffolds. Expression of Sox-9 was also elevated under US and paralleled the COL2A1/COL1A1 ratio. As an original contribution, a simplified mathematical model based on Biot theory was developed to understand the propagation of the incident US wave through the scaffolds and the model analysis was connected to cellular responses. Scaffold architecture influenced the distribution of US field, with the US field being the least attenuated in BM scaffolds, thus coupling more mechanical energy into cells, and leading to increased cellular activity.

Mathematical modeling of pathogen trajectory in a patient care environment.

OBJECTIVE: Minimizing healthcare worker exposure to airborne infectious pathogens is an important infection control practice. This study utilized mathematical modeling to evaluate the trajectories and subsequent concentrations of particles following a simulated release in a patient care room. DESIGN: Observational study. SETTING: Biocontainment unit patient care room at a university-affiliated tertiary care medical center. METHODS: Quantitative mathematical modeling of airflow in a patient care room was achieved using a computational fluid dynamics software package. Models were created on the basis of a release of particles from various locations in the room. Computerized particle trajectories were presented in time-lapse fashion over a blueprint of the room. A series of smoke tests were conducted to visually validate the model. RESULTS: Most particles released from the head of the bed initially rose to the ceiling and then spread across the ceiling and throughout the room. The highest particle concentrations were observed at the head of the bed nearest to the air return vent, and the lowest concentrations were observed at the foot of the bed. CONCLUSIONS: Mathematical modeling provides clinically relevant data on the potential exposure risk in patient care rooms and is applicable in multiple healthcare delivery settings. The information obtained through mathematical modeling could potentially serve as an infection control modality to enhance the protection of healthcare workers.

Mechanotransduction of ultrasound is frequency dependent below the cavitation threshold.

This study provides evidence that low-intensity ultrasound directly affects nuclear processes, and the magnitude of the effect varies with frequency. In particular, we show that the transcriptional induction of first load-inducible genes, which is independent of new protein synthesis, is frequency dependent. Bovine chondrocytes were exposed to low-intensity (below the cavitational threshold) ultrasound at 2, 5 and 8 MHz. Ultrasound elevated the expression of early response genes c-Fos, c-Jun and c-Myc, maximized at 5 MHz. The phosphorylated ERK inhibitor PD98059 abrogated any increase in c-series gene expression, suggesting that signaling occurs via the MAPPK/ERK pathway. However, phosphorylated ERK levels did not change with ultrasound frequency, indicating that processes downstream of ERK phosphorylation (such as nuclear transport and chromatin reorganization) respond to ultrasound with frequency dependence. A quantitative, biphasic mathematical model based on Biot theory predicted that cytoplasmic and nuclear stress is maximized at 5.2 ± 0.8 MHz for a chondrocyte, confirming experimental measurements.

A new rapid method for Clostridium difficile DNA extraction and detection in stool: toward point-of-care diagnostic testing.

We describe a new method for the rapid diagnosis of Clostridium difficile infection, with stool sample preparation and DNA extraction by heat and physical disruption in a single-use lysis microreactor (LMR), followed by a rapid PCR amplification step. All steps can be accomplished in <20 minutes overall. Gel electrophoresis is currently used to detect the amplification product, pending real-time availability with an ultra-rapid thermocycler. Compared with the dual enzyme immunoassay (EIA) screening test (C. diff Quik Chek Complete; Techlab, Blacksburg, VA), the novel LMR/PCR assay showed complete concordance with all glutamate dehydrogenase (GDH) results (GDH(+)/toxin(+), n = 48; GDH(-)/toxin(-), n = 81). All 69 stool samples with discordant EIA results (GDH(+)/toxin(-)) were tested by both the LMR/PCR assay and the loop-mediated isothermal amplification test (LAMP) (Illumigene C. difficile; Meridian Bioscience, Cincinnati, OH). In 64/69 EIA-discordant samples, LAMP and LMR/PCR results matched (both positive in 29 sample and both negative in 35 samples); in the remaining 5 samples, results were discrepant between the LAMP assay (all five negative) and the LMR/PCR assay (all 5 positive). Overall, LMR/PCR testing matched the current algorithm of EIA and/or LAMP reflex testing in 193/198 (97.5%) samples. The present proof-of-concept study suggests that the novel LMR/PCR technique described here may be developed as an inexpensive, rapid, and reliable point-of-care diagnostic test for C. difficile infection and other infectious diseases.

Experimental Validation of a Fundamental Model for PCR Efficiency.

Recently a theoretical analysis of PCR efficiency has been published by Booth et al., (2010). The PCR yield is the product of three efficiencies: (i) the annealing efficiency is the fraction of templates that form binary complexes with primers during annealing, (ii)the polymerase binding efficiency is the fraction of binary complexes that bind to polymerase to form ternary complexes and (iii)the elongation efficiency is the fraction of ternary complexes that extend fully. Yield is controlled by the smallest of the three efficiencies and control could shift from one type of efficiency to another over the course of a PCR experiment. Experiments have been designed that are specifically controlled by each one of the efficiencies and the results are consistent with the mathematical model. The experimental data has also been used to quantify six key parameters of the theoretical model. An important application of the fully characterized model is to calculate initial template concentration from real-time PCR data. Given the PCR protocol, the midpoint cycle number (where the template concentration is half that of the final concentration) can be theoretically determined and graphed for a variety of initial DNA concentrations. Real-time results can be used to calculate the midpoint cycle number and consequently the initial DNA concentration, using this graph. The application becomes particularly simple if a conservative PCR protocol is followed where only the annealing efficiency is controlling.

A model of tuberculosis transmission and intervention strategies in an urban residential area.

The model herein aims to explore the dynamics of the spread of tuberculosis (TB) in an informal settlement or township. The population is divided into households of various sizes and also based on commuting status. The model dynamics distinguishes between three distinct social patterns: the exposure of commuters during travel, random diurnal interaction and familial exposure at night. Following the general SLIR models, the population is further segmented into susceptible (S), exposed/latently infected (L), active/infectious (I), and recovered (R) individuals. During the daytime, commuters travel on public transport, while non-commuters randomly interact in the community to mimic chance encounters with infectious persons. At night, each family interacts and sleeps together in the home. The risk of exposure to TB is based on the proximity, duration, and frequency of encounters with infectious persons. The model is applied to a hypothetical population to explore the effects of different intervention strategies including vaccination, wearing of masks during the commute, prophylactic treatment of latent infections and more effective case-finding and treatment. The most important findings of the model are: (1) members of larger families are responsible for more disease transmissions than those from smaller families, (2) daily commutes on public transport provide ideal conditions for transmission of the disease, (3) improved diagnosis and treatment has the greatest impact on the spread of the disease, and (4) detecting TB at the first clinic visit, when patients are still smear negative, is key.

Efficiency of the Polymerase Chain Reaction.

The polymerase chain reaction (PCR) has found wide application in biochemistry and molecular biology such as gene expression studies, mutation detection, forensic analysis and pathogen detection. Increasingly quantitative real time PCR is used to assess copy numbers from overall yield. In this study the yield is analyzed as a function of several processes: (1) thermal damage of the template and polymerase occurs during the denaturing step, (2) competition exists between primers and templates to either anneal or form dsDNA, (3) polymerase binding to annealed products (primer/ssDNA) to form ternary complexes and (4) extension of ternary complexes. Explicit expressions are provided for the efficiency of each process, therefore reaction conditions can be directly linked to the overall yield. Examples are provided where different processes play the yield-limiting role. The analysis will give researchers a unique understanding of the factors that control the reaction and will aid in the interpretation of experimental results.

A model of the complex response of Staphylococcus aureus to methicillin.

It is widely accepted that beta-lactam antimicrobials cause cell death through a mechanism that interferes with cell wall synthesis. Later studies have also revealed that beta-lactams modify the autolysis function (the natural process of self-exfoliation of the cell wall) of cells. The dynamic equilibrium between growth and autolysis is perturbed by the presence of the antimicrobial. Studies with Staphylococcus aureus to determine the minimum inhibitory concentration (MIC) have revealed complex responses to methicillin exposure. The organism exhibits four qualitatively different responses: homogeneous sensitivity, homogeneous resistance, heterogeneous resistance and the so-called 'Eagle-effect'. A mathematical model is presented that links antimicrobial action on the molecular level with the overall response of the cell population to antimicrobial exposure. The cell population is modeled as a probability density function F(x,t) that depends on cell wall thickness x and time t. The function F(x,t) is the solution to a Fokker-Planck equation. The fixed point solutions are perturbed by the antimicrobial load and the advection of F(x,t) depends on the rates of cell wall synthesis, autolysis and the antimicrobial concentration. Solutions of the Fokker-Planck model are presented for all four qualitative responses of S. aureus to methicillin exposure.

The tri-frame model.

The tri-frame model gives mathematical expression to the transcription and translation processes, and considers all three reading frames (RFs). RNA polymerases transcribe DNA in single nucleotide increments, but ribosomes translate mRNA in pairings of three (triplets or codons). The set of triplets in the mRNA, starting with the initiation codon (usually AUG) defines the open reading frame (ORF). Since ribosomes do not always translocate three nucleotide positions, two additional RFs are accessible. The -1 RF and the +1 RF are triplet pairings of the mRNA, which are accessed by shifting one nucleotide position in the 5' and 3' directions, respectively. Transcription is modeled as a linear operator that maps the initial codons in all three frames into other codon sets to account for possible transcriptional errors. Translational errors (missense errors) originate from misacylation of tRNAs and misreading of aa-tRNAs by the ribosome. Translation is modeled as a linear mapping from codons into aa-tRNA species, which includes misreading errors. A final transformation from aa-tRNA species into amino acids provides the probability distributions of possible amino acids into which the codons in all three frames could be translated. An important element of the tri-frame model is the ribosomal occupancy probability. It is a vector in R(3) that gives the probability to find the ribosome in the ORF, -1 or +1 RF at each codon position. The sequence of vectors, from the first to the final codon position, gives a history of ribosome frameshifting. The model is powerful: it provides explicit expressions for (1) yield of error-free protein, (2) fraction of prematurely terminated polypeptides, (3) number of transcription errors, (4) number of translation errors and (5) mutations due to frameshifting. The theory is demonstrated for the three genes rpsU, dnaG and rpoD of Escherichia coli, which lie on the same operon, as well as for the prfB gene.

Principles of rapid polymerase chain reactions: mathematical modeling and experimental verification.

Polymerase chain reaction (PCR) is an important diagnostic tool for the amplification of DNA. The PCR process can be treated as a problem in biochemical engineering. This study focuses on the development of a mathematical model of the polymerase chain reaction. The PCR process consists of three steps: denaturation of target DNA, annealing of sequence-specific oligonucleotide primers and the enzyme-catalyzed elongation of the annealed complex (primer:DNA:polymerase). The denaturation step separates the double strands of DNA; this model assumes denaturation is complete. The annealing step describes the formation of a primer-fragment complex followed by the attachment of the polymerase to form a ternary complex. This step is complicated by competitive annealing between primers and incomplete fragments including primer-primer reactions. The elongation step is modeled by a stochastic method. Species that compete during the elongation step are deoxynucleotide triphosphates dCTP, dATP, dTTP, dGTP, dUTP, and pyrophosphate. Thermal deamination of dCTP to form dUTP is included in the model. The probability for a species to arrive at the active site is based on its molar fraction. The number of random insertion events depends on the average processing speed of the polymerase and the elongation time of the simulation. The numerical stochastic experiment is repeated a sufficient number of times to construct a probability density distribution (PDF). The moment of the PDF and the annealing step products provide the product distribution at the end of the elongation step. The overall yield is compared to six experimental values of the yield. In all cases the comparisons are very good.

Solitary waves and supersonic reaction front in metastable solids.

Motivated by an increasing number of remarkable experimental observations on the role of pressure and shear stress in solid reactions, explosions, and detonations, we present a simple one-dimensional model that embodies nonlinear elasticity and dispersion as well as chemical or phase transformation. This generalization of the Toda lattice provides an effective model for the description of the organization during an abrupt transformation in a solid. One of the challenges is to capture both the equilibrium degrees of freedom as well as to quantify the possible role of out-of-equilibrium perturbations. In the Toda lattice, we verify that the particle velocities converge in distribution towards the Maxwell-Boltzmann distribution, thus allowing us to define a bonafide temperature. In addition, the balance between nonlinearity and wave dispersion may create solitary waves that act as energy traps. In the presence of reactive chemistry, we show that the trapping of the released chemical energy in solitary waves that are excited by an initial perturbation provides a positive feedback that enhances the reaction rate and leads to supersonic explosion front propagation. These modes of rupture observed in our model may provide a first-order description of ultrafast reactions of heterogeneous mixtures under mechanical loading.