Functional Assessment of Human Articular Cartilage Using Second Harmonic Generation (SHG) Imaging: A Feasibility Study.

Many arthroscopic tools developed for knee joint assessment are contact-based, which is challenging for in vivo application in narrow joint spaces. Second harmonic generation (SHG) laser imaging is a non-invasive and non-contact method, thus presenting an attractive alternative. However, the association between SHG-based measures and cartilage quality has not been established systematically. Here, we investigated the feasibility of using image-based measures derived from SHG microscopy for objective evaluation of cartilage quality as assessed by mechanical testing. Human tibial plateaus harvested from nine patients were used. Cartilage mechanical properties were determined using indentation stiffness (Einst) and streaming potential-based quantitative parameters (QP). The correspondence of the cartilage electromechanical properties (Einst and QP) and the image-based measures derived from SHG imaging, tissue thickness and cell viability were evaluated using correlation and logistic regression analyses. The SHG-related parameters included the newly developed volumetric fraction of organised collagenous network (Φcol) and the coefficient of variation of the SHG intensity (CVSHG). We found that Φcol correlated strongly with Einst and QP (ρ = 0.97 and - 0.89, respectively). CVSHG also correlated, albeit weakly, with QP and Einst, (|ρ| = 0.52-0.58). Einst and Φcol were the most sensitive predictors of cartilage quality whereas CVSHG only showed moderate sensitivity. Cell viability and tissue thickness, often used as measures of cartilage health, predicted the cartilage quality poorly. We present a simple, objective, yet effective image-based approach for assessment of cartilage quality. Φcol correlated strongly with electromechanical properties of cartilage and could fuel the continuous development of SHG-based arthroscopy.

Supercritical Fluid-Based Decellularization Technologies for Regenerative Medicine Applications.

Supercritical fluid-based extraction technologies are currently being increasingly utilized in high purity extract products for food industries. In recent years, supercritical fluid-based extraction technology is transformed in biomaterials process fields to be further utilized for tissue engineering and other biomedical applications. In particular, supercritical fluid-based decellularization protocols have great advantage over the conventional decellularization as it may allow preservation of extracellular matrix components and structures. In this review, the latest technological development utilizing the supercritical fluid-based decellularization for regenerative medicine is introduced.

Decellularized matrices for tumor cell modeling.

Collagen is the main component of the extracellular matrix and it plays a key role in tumor progression. Commercial collagen solutions are derived from animals, such as rat-tail and bovine or porcine skin. Their cost is quite high and the product is stable only at low temperature, with the disadvantage of a short expiring date. Most importantly, lot-to-lot variability can occur and the reconstituted collagen gels differ significantly from native tissues in terms of both structure and stiffness. In this chapter, we describe a straightforward method to use native, collagen rich skin samples derived from by-products of the tanning industry. The protocol proposed preserves the microstructure of the ovine skin collagen network, offering structurally competent and more relevant model to investigate cell behavior in vitro. Other advantages of the proposed procedure consist in the cost-effectiveness of the process and an increased level of reproducibility. The decellularized ovine skin samples support the adhesion and growth of different cancer cell lines (pancreatic, breast and melanoma cells). The proposed decellularized skin scaffolds are meant as future low-cost competitors for conventional porous scaffold derived by biomaterials, since they offer a biomimetic environment for the cells.

Quantitative Assessment of Tendon Hierarchical Structure by Combined Second Harmonic Generation and Immunofluorescence Microscopy.

Histological evaluation of healing tendons is primarily focused on monitoring restoration of longitudinal collagen alignment, although the elastic property of energy-storing flexor tendons is largely attributed to interfascicular sliding facilitated by the interfascicular matrix (IFM). The objectives of this study were to explore the utility of second harmonic generation (SHG) imaging to objectively assess cross-sectional tendon fascicle architecture, to combine SHG microscopy with elastin immunofluorescence to assess the ultrastructure of collagen and elastin in longitudinal and transverse sections, and lastly, to quantify changes in IFM elastin and fascicle collagen alignment of normal and collagenase-injured flexor tendons. Paraffin-embedded transverse and longitudinal histological sections (10-µm thickness) derived from normal and collagenase-injured (6- and 16-week time-points) equine superficial digital flexor tendons were de-paraffinized, treated with Tris EDTA at 80°C for epitope retrieval, and incubated with mouse monoclonal anti-elastin antibody (1:100 dilution) overnight. Anti-mouse IgG Alexa Flour 546 secondary antibody was applied, and sections were mounted with ProLong Gold reagent with 4',6-diamidino-2-phenylindole (DAPI). Nuclei (DAPI) and elastin (Alexa Fluor 546) signals were captured by using standard confocal imaging with 405 and 543 nm excitation wavelengths, respectively. The SHG signal was captured by using a tunable Ti:Sapphire laser tuned to 950 nm to visualize type I collagen. Quantitative measurements of fascicle cross-sectional area (CSA), IFM thickness in transverse SHG-DAPI merged z-stacks, fascicle/IFM elastin area fraction (%), and elastin-collagen alignment in longitudinal SHG-elastin merged z-stacks were conducted by using ImageJ software. Using this methodology, fascicle CSA, IFM thickness, and IFM elastin area fraction (%) at 6 weeks (∼2.25-fold; ∼2.8-fold; 60% decrease; p < 0.001) and 16 weeks (∼2-fold; ∼1.5-fold; 70% decrease; p < 0.001) after collagenase injection, respectively, were found to be significantly different from normal tendon. IFM elastin and fascicle collagen alignment characterized via fast Fourier transform (FFT) frequency plots at 16 weeks demonstrated that collagen re-alignment was more advanced than that of elastin. The integration of SHG-derived quantitative measurements in transverse and longitudinal tendon sections supports comprehensive assessment of tendon structure. Our findings demonstrate the importance of including IFM and non-collagenous proteins in tendon histological evaluations, tasks that can be effectively carried out by using SHG and immunofluorescence microscopy. Impact statement This work demonstrated that second harmonic generation microscopy in conjunction with elastin immunofluorescence provided a comprehensive assessment of multiscale structural re-organization in healing tendon than when restricted to longitudinal collagen fiber alignment alone. Utilizing this approach for tendon histomorphometry is ideal not only to improve our understanding of hierarchical structural changes that occur after tendon injury and during remodeling but also to monitor the efficacy of therapeutic approaches.

Scalable and cost-effective generation of osteogenic micro-tissues through the incorporation of inorganic microparticles within mesenchymal stem cell spheroids.

Mesenchymal stem cells (MSCs) are considered primary candidates for treating complex bone defects in cell-based therapy and tissue engineering. Compared with monolayer cultures, spheroid cultures of MSCs (mesenspheres) are favorable due to their increased potential for differentiation, extracellular matrix (ECM) synthesis, paracrine activity, and in vivo engraftment. Here, we present a strategy for the incorporation of microparticles for the fabrication of osteogenic micro-tissues from mesenspheres in a cost-effective and scalable manner. A facile method was developed to synthesize mineral microparticles with cell-sized spherical shape, biphasic calcium phosphate composition (hydroxyapatite and ß-tricalcium phosphate), and a microporous structure. Calcium phosphate microparticles (CMPs) were incorporated within the mesenspheres through mixing with the single cells during cell aggregation. Interestingly, the osteogenic genes were upregulated significantly (collagen type 1 (Col 1) 30-fold, osteopontin (OPN) 10-fold, and osteocalcin (OCN) 3-fold) after 14 days of culture with the incorporated CMPs, while no significant upregulation was observed with the incorporation of gelatin microparticles. The porous structure of the CMPs was exploited for loading and sustained release of an angiogenic small molecule. Dimethyloxaloylglycine (DMOG) was loaded efficiently onto the CMPs (loading efficiency: 65.32 ± 6%) and showed a sustained release profile over 12 days. Upon incorporation of the DMOG-loaded CMPs (DCMPs) within the mesenspheres, a similar osteogenic differentiation and an upregulation in angiogenic genes (VEGF 5-fold and kinase insert domain (KDR) 2-fold) were observed after 14 days of culture. These trends were also observed in immunostaining analysis. To evaluate scalable production of the osteogenic micro-tissues, the incorporation of microparticles was performed during cell aggregation in a spinner flask. The DCMPs were efficiently incorporated and directed the mesenspheres toward osteogenesis and angiogenesis. Finally, the DCMP mesenspheres were loaded within a three-dimensional printed cell trapper and transplanted into a critical-sized defect in a rat model. Computed tomography and histological analysis showed significant bone formation with blood vessel reconstruction after 8 weeks in this group. Taken together, we provide a scalable and cost-effective approach for fabrication of osteogenic micro-tissues, as building blocks of macro-tissues, that can address the large amounts of cells required for cell-based therapies.

Noninvasive ultrasound imaging for assessment of intact microstructure of extracellular matrix in tissue engineering.

Development of artificial tissues or organs is one of the actual tasks in regenerative medicine that requires observation and evaluation of intact volume microstructure of tissue engineering products at all stages of their formation, from native donor tissues and decellularized scaffolds to recipient cell migration in the matrix. Unfortunately in practice, methods of vital noninvasive imaging of volume microstructure in matrixes are absent. In this work, we propose a new approach based on high-frequency acoustic microscopy for noninvasive evaluation and visualization of volume microstructure in tissue engineering products. The results present the ultrasound characterization of native rat diaphragms and lungs and their decellularized scaffolds. Verification of the method for visualization of tissue formation in the matrix volume was described in the model samples of diaphragm scaffolds with stepwise collagenization. Results demonstrate acoustic microscopic sensitivity to cell content concentration, variation in local density, and orientation of protein fibers in the volume, micron air inclusions, and other inhomogeneities of matrixes.

In Vivo Assessment of Decellularized Porcine Myocardial Slice as an Acellular Cardiac Patch.

Acellular cardiac patches made of various biomaterials have shown to improve heart function after myocardial infarction (MI). Extracellular matrix scaffold derived from a decellularized tissue has unique advantages to serve as an acellular cardiac patch due to its biomimetic nature. In this study, we examined the therapeutic outcomes of using a decellularized porcine myocardium slice (dPMS) as an acellular patch in a rat acute MI model. dPMSs with two different thicknesses (300 and 600 µm) were patched to the infarcted area of the rat myocardium, and their effects on cardiac function and host interactions were assessed. We found that the implanted dPMS firmly attached to host myocardium after implantation and prevented thinning of the left ventricular (LV) wall after an MI. A large number of host cells were identified to infiltrate into the implanted dPMS, and a significant number of vessel structures was observed in the dPMS and infarcted area. We detected a significantly higher density of M2 macrophages in the groups treated with dPMSs as compared to the MI group. Contraction of the LV wall and cardiac functional parameters (left ventricular ejection fraction and fractional shortening) was significantly improved in the treatment groups (300 and 600 µm dPMS) 4 weeks after surgery. Our results proved the therapeutic benefits of using dPMS as an acellular cardiac patch for the treatment of acute myocardial infarction.

Rapid 3D bioprinting of decellularized extracellular matrix with regionally varied mechanical properties and biomimetic microarchitecture.

Hepatocellular carcinoma (HCC), as the fifth most common malignant cancer, develops and progresses mostly in a cirrhotic liver where stiff nodules are separated by fibrous bands. Scaffolds that can provide a 3D cirrhotic mechanical environment with complex native composition and biomimetic architecture are necessary for the development of better predictive tissue models. Here, we developed photocrosslinkable liver decellularized extracellular matrix (dECM) and a rapid light-based 3D bioprinting process to pattern liver dECM with tailorable mechanical properties to serve as a platform for HCC progression study. 3D bioprinted liver dECM scaffolds were able to stably recapitulate the clinically relevant mechanical properties of cirrhotic liver tissue. When encapsulated in dECM scaffolds with cirrhotic stiffness, HepG2 cells demonstrated reduced growth along with an upregulation of invasion markers compared to healthy controls. Moreover, an engineered cancer tissue platform possessing tissue-scale organization and distinct regional stiffness enabled the visualization of HepG2 stromal invasion from the nodule with cirrhotic stiffness. This work demonstrates a significant advancement in rapid 3D patterning of complex ECM biomaterials with biomimetic architecture and tunable mechanical properties for in vitro disease modeling.

Label-Free Assessment of Collagenase Digestion on Bovine Pericardium Properties by Fluorescence Lifetime Imaging.

The extracellular matrix architecture of bovine pericardium (BP) has distinct biochemical and biomechanical properties that make it a useful biomaterial in the field of regenerative medicine. Collagen represents the dominant structural protein of BP and is therefore intimately associated with the properties of this biomaterial. Enzymatic degradation of collagen molecules is critical for extracellular matrix turnover, remodeling and ultimately tissue regeneration. We present a quantitative, label-free and non-destructive method for monitoring changes in biochemical and biomechanical properties of BP during tissue degradation, based on multi-spectral fluorescence lifetime imaging (ms-FLIm). Strong correlations of fluorescence intensity ratio and average fluorescence lifetime were identified with collagen content, Young's Modulus and Ultimate tensile strength during collagenase degradation, indicating the potential of optically monitoring collagen degradation using ms-FLIm. The obtained results demonstrate the value of ms-FLIm to assess the quality of biomaterials in situ for applications in regenerative medicine.

Assessment of extracellular matrix-related biomarkers in patients with lower extremity artery disease.

BACKGROUND: The prevalence of lower extremity artery disease (LEAD) is high (20%-25%) in the population older than 65 years, but patients are seldom identified until the disease is advanced. Circulating markers of disease activity might provide patients with a key opportunity for timely treatment. We tested the hypothesis that measuring blood-specific fragments generated during degradation of the extracellular matrix (ECM) could provide further insight into the pathophysiologic mechanism of arterial remodeling. METHODS: The protein profile of diseased arteries from patients undergoing infrainguinal limb revascularization was assessed by a liquid chromatography and tandem mass spectrometry, nontargeted proteomic approach. The information retrieved was the basis for measurement of neoepitope fragments of ECM proteins in the blood of 195 consecutive patients with LEAD by specific enzyme-linked immunosorbent assays. RESULTS: Histologic and proteomic analyses confirmed the structural disorganization of affected arteries. Fourteen of 81 proteins were identified as differentially expressed in diseased arteries with respect to healthy tissues. Most of them were related to ECM components, and the difference in expression was used in multivariate analyses to establish that severe arterial lesions in LEAD patients have a specific proteome. Analysis of neoepitope fragments in blood revealed that fragments of versican and collagen type IV, alone or in combination, segregated patients with mild to moderate symptoms (intermittent claudication, Fontaine I-II) from those with severe LEAD (critical limb ischemia, Fontaine III-IV). CONCLUSIONS: We propose noninvasive candidate biomarkers with the ability to be clinically useful across the LEAD spectrum.

Matching material and cellular timescales maximizes cell spreading on viscoelastic substrates.

Recent evidence has shown that, in addition to rigidity, the viscous response of the extracellular matrix (ECM) significantly affects the behavior and function of cells. However, the mechanism behind such mechanosensitivity toward viscoelasticity remains unclear. In this study, we systematically examined the dynamics of motor clutches (i.e., focal adhesions) formed between the cell and a viscoelastic substrate using analytical methods and direct Monte Carlo simulation. Interestingly, we observe that, for low ECM rigidity, maximum cell spreading is achieved at an optimal level of viscosity in which the substrate relaxation time falls between the timescale for clutch binding and its characteristic binding lifetime. That is, viscosity serves to stiffen soft substrates on a timescale faster than the clutch off-rate, which enhances cell-ECM adhesion and cell spreading. On the other hand, for substrates that are stiff, our model predicts that viscosity will not influence cell spreading, since the bound clutches are saturated by the elevated stiffness. The model was tested and validated using experimental measurements on three different material systems and explained the different observed effects of viscosity on each substrate. By capturing the mechanism by which substrate viscoelasticity affects cell spreading across a wide range of material parameters, our analytical model provides a useful tool for designing biomaterials that optimize cellular adhesion and mechanosensing.

A Cost-Effective Culture System for the In Vitro Assembly, Maturation, and Stimulation of Advanced Multilayered Multiculture Tubular Tissue Models.

The development of tubular engineered tissues is a challenging research area aiming to provide tissue substitutes but also in vitro models to test drugs, medical devices, and even to study physiological and pathological processes. In this work, the design, fabrication, and validation of an original cost-effective tubular multilayered-tissue culture system (TMCS) are reported. By exploiting cellularized collagen gel as scaffold, a simple moulding technique and an endothelialization step on a rotating system, TMCS allowed to easily prepare in 48 h, trilayered arterial wall models with finely organized cellular composition and to mature them for 2 weeks without any need of manipulation. Multilayered constructs incorporating different combinations of vascular cells are compared in terms of cell organization and viscoelastic mechanical properties demonstrating that cells always progressively aligned parallel to the longitudinal direction. Also, fibroblast compacted less the collagen matrix and appeared crucial in term of maturation/deposition of elastic extracellular matrix. Preliminary studies under shear stress stimulation upon connection with a flow bioreactor are successfully conducted without damaging the endothelial monolayer. Altogether, the TMCS herein developed, thanks to its versatility and multiple functionalities, holds great promise for vascular tissue engineering applications, but also for other tubular tissues such as trachea or oesophagus.

Diffusion tensor optical coherence tomography.

In situ measurements of diffusive particle transport provide insight into tissue architecture, drug delivery, and cellular function. Analogous to diffusion-tensor magnetic resonance imaging (DT-MRI), where the anisotropic diffusion of water molecules is mapped on the millimeter scale to elucidate the fibrous structure of tissue, here we propose diffusion-tensor optical coherence tomography (DT-OCT) for measuring directional diffusivity and flow of optically scattering particles within tissue. Because DT-OCT is sensitive to the sub-resolution motion of Brownian particles as they are constrained by tissue macromolecules, it has the potential to quantify nanoporous anisotropic tissue structure at micrometer resolution as relevant to extracellular matrices, neurons, and capillaries. Here we derive the principles of DT-OCT, relating the detected optical signal from a minimum of six probe beams with the six unique diffusion tensor and three flow vector components. The optimal geometry of the probe beams is determined given a finite numerical aperture, and a high-speed hardware implementation is proposed. Finally, Monte Carlo simulations are employed to assess the ability of the proposed DT-OCT system to quantify anisotropic diffusion of nanoparticles in a collagen matrix, an extracellular constituent that is known to become highly aligned during tumor development.

Linear array of multi-substrate tracts for simultaneous assessment of cell adhesion, migration, and differentiation.

Cell migration, which is central to a wide variety of life processes, involves integration of the extracellular matrix (ECM) with the internal cytoskeleton and motor proteins via receptors spanning the plasma membrane. Cell migration can be induced by a variety of signals, including gradients of external soluble molecules, differences in ECM composition, or electrical gradients. Current in vitro methods to study cell migration only test one substrate at a time. Here, we present a method for assessing cell adhesion, migration, and differentiation in up to 20 different test conditions simultaneously, using only minute amounts of target substrate. Our system, which we call the linear array of multi-substrate cell migration assay (LAMA), has two configurations for direct comparison of one or two cell types in response to an array of ECM constituents under the same culture conditions. This culture model utilizes only nanogram amounts of test substrates and a minimal number of cells, which maximizes the use of limited and expensive test reagents. Moreover, LAMA can also be used for high-throughput screening of potential pharmaceuticals that target ECM-dependent cell behavior and differentiation.

Use of an Ovine Collagen Dressing with Intact Extracellular Matrix to Improve Wound Closure Times and Reduce Expenditures in a US Military Veteran Hospital Outpatient Wound Center.

A novel, comprehensive decision-making and treatment algorithm was established within a US government-run military veteran hospital in an attempt to standardize the process of outpatient wound care and streamline costs. All patients were systematically evaluated and treated using the comprehensive algorithm over a span of nine months. After three months of adherence to the algorithm, the algorithm was modified to include ovine-based collagen extracellular matrix (CECM) dressings as a first-line conventional treatment strategy for all appropriate wounds. The purpose of this retrospective analysis was to evaluate the hospital's change in cellular and/or tissue-based graft usage and cost, as well as wound healing outcomes following modification of the wound care standardization algorithm. Data from the first quarter (Q1; three months) of protocol implementation were compared to the subsequent two quarters (six months), during which time the first-line dressing modification of the protocol was implemented. Results showed that between quarters 1 and 3, the percentage of wounds healed increased by 95.5% (24/64 to 80/109), and the average time to heal each wound decreased by 22.6% (78.8 days to 61.0 days). Cellular and/or tissue-based graft unit usage decreased by 59.7% (144 units to 58 units), and expenditures on cellular and/or tissue-based grafts decreased by 66.0% ($212,893 to $72,412). Results of this analysis displayed a trend toward decreased expenditures, faster healing times, and a greater number of healed wounds following modification of an evidence-based algorithm to incorporate CECM dressings as a first-line treatment strategy in managing chronic wounds.

Native and solubilized decellularized extracellular matrix: A critical assessment of their potential for improving the expansion of mesenchymal stem cells.

Capturing the promise of mesenchymal stem cell (MSC)-based treatments is currently limited by inefficient production of cells needed for clinical therapies. During conventional ex vivo expansion, a large portion of MSCs lose the properties that make them attractive for use in cell therapies. Decellularized extracellular matrix (dECM) has recently emerged as a promising substrate for the improved expansion of MSCs. MSCs cultured on these surfaces exhibit improved proliferation capacity, maintenance of phenotype, and increased differentiation potential. Additionally, these dECMs can be solubilized and used to coat new cell culture surfaces, imparting key biological properties of the native matrices to other surfaces such as tissue engineering scaffolds. Although this technology is still developing, there is potential for an impact in the fields of MSC biology, biomaterials, tissue engineering, and therapeutics. In this article, we review the role of dECM in MSC expansion by first detailing the decellularization methods that have been used to produce the dECM substrates; discussing the shortcomings of current decellularization methods; describing the improved MSC characteristics obtained when the cells are cultured on these surfaces; and considering the effect of the passage number, age of donor, and dECM preparation method on the quality of the dECM. Finally we describe the critical roadblocks that must be addressed before this technology can fulfil its potential, including elucidating the mechanism by which the dECMs improve the expansion of primary MSCs and the identification of a readily available source of dECM. STATEMENT OF SIGNIFICANCE: Current mesenchymal stem cell (MSC) culture methods result in premature cellular senescence or loss of differentiation potential. This creates a major bottleneck in their clinical application, as prolonged expansion is necessary to achieve clinically relevant numbers of cells. Recently, decellularized extracellular matrix (dECM) produced by primary MSC has emerged as an attractive substrate for the improved expansion of MSC; cells cultured on these surfaces retain their desired stem cell characteristics for prolonged times during culture. This review article describes the inception and development of this dECM-based technology, points out existing challenges that must be addressed, and suggests future directions of research. To our knowledge, this is the first review written on the use of dECM for improved mesenchymal stem cell expansion.

Molecular assessment of collagen denaturation in decellularized tissues using a collagen hybridizing peptide.

Decellularized extracellular matrix (ECM) derived from tissues and organs are emerging as important scaffold materials for regenerative medicine. Many believe that preservation of the native ECM structure during decellularization is highly desirable. However, because effective techniques to assess the structural damage in ECM are lacking, the disruptive effects of a decellularization method and the impact of the associated structural damage upon the scaffold's regenerative capacity are often debated. Using a novel collagen hybridizing peptide (CHP) that specifically binds to unfolded collagen chains, we investigated the molecular denaturation of collagen in the ECM decellularized by four commonly used cell-removing detergents: sodium dodecyl sulfate (SDS), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), sodium deoxycholate (SD), and Triton X-100. Staining of the detergent-treated porcine ligament and urinary bladder matrix with carboxyfluorescein-labeled CHP demonstrated that SDS and Triton X-100 denature the triple helical collagen molecule while CHAPS and SD do not, although second harmonic generation imaging and transmission electron microscopy (TEM) revealed that all four detergents disrupt collagen fibrils. Our findings from the CHP staining were further confirmed by the circular dichroism spectra of intact triple helical collagen molecules in CHAPS and SD solutions, and the TEM images of CHP-conjugated gold nanoparticles binding only to the SDS and Triton X-100 treated collagen fibrils. CHP is a powerful new tool for direct and reliable measurement of denatured collagen molecules in decellularized tissues. It is expected to have wide applications in the development and standardization of the tissue/organ decellularization technology. STATEMENT OF SIGNIFICANCE: Preservation of the native ECM structure in decellularized tissues is highly desirable, since denaturation of ECM molecules (e.g., collagen) during decellularization can strongly influence the cellular response. Unfortunately, conventional techniques (SEM, SHG) are not conducive to identifying denatured collagen molecules in tissues. We demonstrate the first investigation into the molecular denaturation of collagen in decellularized ECM enabled by a novel Collagen Hybridizing Peptide (CHP) that specifically binds to unfolded collagen chains. We show that SDS and Triton X-100 denature collagen molecules while CHAPS and SD cannot. Such detection has been nearly impossible with other existing techniques. The CHP technique will advance our understanding about the effect of the cell-removing process on ECM, and lead to development of the decellularization technology.

Multi-Conformation Monte Carlo: A Method for Introducing Flexibility in Efficient Simulations of Many-Protein Systems.

We present a novel multi-conformation Monte Carlo simulation method that enables the modeling of protein-protein interactions and aggregation in crowded protein solutions. This approach is relevant to a molecular-scale description of realistic biological environments, including the cytoplasm and the extracellular matrix, which are characterized by high concentrations of biomolecular solutes (e.g., 300-400 mg/mL for proteins and nucleic acids in the cytoplasm of Escherichia coli). Simulation of such environments necessitates the inclusion of a large number of protein molecules. Therefore, computationally inexpensive methods, such as rigid-body Brownian dynamics (BD) or Monte Carlo simulations, can be particularly useful. However, as we demonstrate herein, the rigid-body representation typically employed in simulations of many-protein systems gives rise to certain artifacts in protein-protein interactions. Our approach allows us to incorporate molecular flexibility in Monte Carlo simulations at low computational cost, thereby eliminating ambiguities arising from structure selection in rigid-body simulations. We benchmark and validate the methodology using simulations of hen egg white lysozyme in solution, a well-studied system for which extensive experimental data, including osmotic second virial coefficients, small-angle scattering structure factors, and multiple structures determined by X-ray and neutron crystallography and solution NMR, as well as rigid-body BD simulation results, are available for comparison.

3D discrete angiogenesis dynamic model and stochastic simulation for the assessment of blood perfusion coefficient and impact on heat transfer between nanoparticles and malignant tumors.

Early detection of malignant tumors plays a crucial role in the survivability chances of the patient. Therefore, new and innovative tumor detection methods are constantly searched for. Tumor-specific magnetic-core nano-particles can be used with an alternating magnetic field to detect and treat tumors by hyperthermia. For the analysis of the method effectiveness, the bio-heat transfer between the nanoparticles and the tissue must be carefully studied. Heat diffusion in biological tissue is usually analyzed using the Pennes Bio-Heat Equation, where blood perfusion plays an important role. Malignant tumors are known to initiate an angiogenesis process, where endothelial cell migration from neighboring vasculature eventually leads to the formation of a thick blood capillary network around them. This process allows the tumor to receive its extensive nutrition demands and evolve into a more progressive and potentially fatal tumor. In order to assess the effect of angiogenesis on the bio-heat transfer problem, we have developed a discrete stochastic 3D model & simulation of tumor-induced angiogenesis. The model elaborates other angiogenesis models by providing high resolution 3D stochastic simulation, capturing of fine angiogenesis morphological features, effects of dynamic sprout thickness functions, and stochastic parent vessel generator. We show that the angiogenesis realizations produced are well suited for numerical bio-heat transfer analysis. Statistical study on the angiogenesis characteristics was derived using Monte Carlo simulations. According to the statistical analysis, we provide analytical expression for the blood perfusion coefficient in the Pennes equation, as a function of several parameters. This updated form of the Pennes equation could be used for numerical and analytical analyses of the proposed detection and treatment method.

Membrane sorting via the extracellular matrix.

We consider the coupling between a membrane and the extracellular matrix. Computer simulations demonstrate that the latter coupling is able to sort lipids. It is assumed that membranes are elastic manifolds, and that this manifold is disrupted by the extracellular matrix. For a solid-supported membrane with an actin network on top, regions of positive curvature are induced below the actin fibers. A similar mechanism is conceivable by assuming that the proteins which connect the cytoskeleton to the membrane induce local membrane curvature. The regions of non-zero curvature exist irrespective of any phase transition the lipids themselves may undergo. For lipids that prefer certain curvature, the extracellular matrix thus provides a spatial template for the resulting lateral domain structure of the membrane.