Discovery of novel 1H-benzo[d]imidazole-4,7-dione based transglutaminase 2 inhibitors as p53 stabilizing anticancer agents in renal cell carcinoma.

Overexpression of transglutaminase 2 (TGase 2; TG2) has been implicated in the progression of renal cell carcinoma (RCC) through the inactivation of p53 by forming a protein complex. Because most p53 in RCC has no mutations, apoptosis can be increased by inhibiting the binding between TG2 and p53 to increase the stability of p53. In the present study, a novel TG2 inhibitor was discovered by investigating the structure of 1H-benzo[d]imidazole-4,7-dione as a simpler chemotype based on the amino-1,4-benzoquinone moiety of streptonigrin, a previously reported inhibitor. Through structure-activity relationship (SAR) studies, compound 8j (MD102) was discovered as a potent TG2 inhibitor with an IC50 value of 0.35 µM, p53 stabilization effect and anticancer effects in the ACHN and Caki-1 RCC cell lines with sulforhodamine B (SRB) GI50 values of 2.15 µM and 1.98 µM, respectively. The binding property of compound 8j (MD102) with TG2 was confirmed to be reversible in a competitive enzyme assay, and the binding interaction was expected to be formed at the ß-sandwich domain, a p53 binding site, in the SPR binding assay with mutant proteins. The mode of binding of compound 8j (MD102) to the ß-sandwich domain of TG2 was analyzed by molecular docking using the crystal structure of the active conformation of human TG2. Compound 8j (MD102) induced a decrease in the downstream signaling of p-AKT and p-mTOR through the stabilization of p53 by TG2 inhibition, resulting in tumor cell apoptosis. In a xenograft animal model using ACHN cancer cells, oral administration and intraperitoneal injection of compound 8j (MD102) showed an inhibitory effect on tumor growth, confirming increased levels of p53 and decreased levels of Ki-67 in tumor tissues through immunohistochemical (IHC) tissue staining. These results indicated that the inhibition of TG2 by compound 8j (MD102) could enhance p53 stabilization, thereby ultimately showing anticancer effects in RCC. Compound 8j (MD102), a novel TG2 inhibitor, can be further applied for the development of an anticancer candidate drug targeting RCC.

Recognition of Fine-Grained Walking Patterns Using a Smartwatch with Deep Attentive Neural Networks.

Generally, people do various things while walking. For example, people frequently walk while looking at their smartphones. Sometimes we walk differently than usual; for example, when walking on ice or snow, we tend to waddle. Understanding walking patterns could provide users with contextual information tailored to the current situation. To formulate this as a machine-learning problem, we defined 18 different everyday walking styles. Noting that walking strategies significantly affect the spatiotemporal features of hand motions, e.g., the speed and intensity of the swinging arm, we propose a smartwatch-based wearable system that can recognize these predefined walking styles. We developed a wearable system, suitable for use with a commercial smartwatch, that can capture hand motions in the form of multivariate timeseries (MTS) signals. Then, we employed a set of machine learning algorithms, including feature-based and recent deep learning algorithms, to learn the MTS data in a supervised fashion. Experimental results demonstrated that, with recent deep learning algorithms, the proposed approach successfully recognized a variety of walking patterns, using the smartwatch measurements. We analyzed the results with recent attention-based recurrent neural networks to understand the relative contributions of the MTS signals in the classification process.

The effect of local anaesthetics on synoviocytes: a possible indirect mechanism of chondrolysis.

PURPOSE: While the effect of local anaesthetics on chondrocyte viability is widely documented, the effect of these medications on synoviocytes is largely unknown. The purpose of this study was to understand the effect of 0.5 % bupivacaine and 0.5 % bupivacaine with epinephrine on synoviocyte viability, cytokine and growth factor release, and breakdown product formation. METHODS: Rabbit fibroblast-like synoviocyte (Type B) cultures were perfused with 0.5 % bupivacaine or 0.5 % bupivacaine with epinephrine (1:200,000) for 24 h. Cell viability was evaluated using a two-colour fluorescence assay. The supernatant was analysed using multiplex inflammatory and matrix metalloproteinase assays. RESULTS: Synoviocytes treated for 24 h with 0.5 % bupivacaine with epinephrine demonstrated a significant decrease in viability (31.3 ± 19.4 % cell death) when compared with synoviocytes cultured in control media (3.8 ± 1.3 % cell death, p = 0.000) and those cultured in 0.5 % bupivacaine alone (12.6 ± 11.1 % cell death, p = 0.003). No significant decrease in cell viability was observed in synoviocytes treated with 0.5 % bupivacaine compared to those in control media (12.6 ± 11.1 % vs 3.8 ± 1.3 % cell death, p = 0.194). Significantly greater amounts of MMP-1 (47.0 ± 9.2 pg/ml) and MMP-3 (250.0 ± 68.8 pg/ml) were observed in 0.5 % bupivacaine cultures compared with controls (14.3 ± 14.3, p = 0.023 and 72.0 ± 84.9, p = 0.045, respectively). CONCLUSIONS: 0.5 % bupivacaine with epinephrine caused a significant increase in cell death of the synoviocytes, while 0.5 % bupivacaine alone produced cell injury and a significant release of matrix metalloproteinases, which may also lead to indirect injury of the surrounding chondrocytes. These results may help explain the onset of chondrolysis observed in patients who have been treated with intra-articular local anaesthetics.

Fine-Grained Motion Recognition in At-Home Fitness Monitoring with Smartwatch: A Comparative Analysis of Explainable Deep Neural Networks.

The squat is a multi-joint exercise widely used for everyday at-home fitness. Focusing on the fine-grained classification of squat motions, we propose a smartwatch-based wearable system that can recognize subtle motion differences. For data collection, 52 participants were asked to perform one correct squat and five incorrect squats with three different arm postures (straight arm, crossed arm, and hands on waist). We utilized deep neural network-based models and adopted a conventional machine learning method (random forest) as a baseline. Experimental results revealed that the bidirectional GRU/LSTMs with an attention mechanism and the arm posture of hands on waist achieved the best test accuracy (F1-score) of 0.854 (0.856). High-dimensional embeddings in the latent space learned by attention-based models exhibit more clustered distributions than those by other DNN models, indicating that attention-based models learned features from the complex multivariate time-series motion signals more efficiently. To understand the underlying decision-making process of the machine-learning system, we analyzed the result of attention-based RNN models. The bidirectional GRU/LSTMs show a consistent pattern of attention for defined squat classes, but these models weigh the attention to the different kinematic events of the squat motion (e.g., descending and ascending). However, there was no significant difference found in classification performance.

Deep Learning Model Based on You Only Look Once Algorithm for Detection and Visualization of Fracture Areas in Three-Dimensional Skeletal Images.

Utilizing "You only look once" (YOLO) v4 AI offers valuable support in fracture detection and diagnostic decision-making. The purpose of this study was to help doctors to detect and diagnose fractures more accurately and intuitively, with fewer errors. The data accepted into the backbone are diversified through CSPDarkNet-53. Feature maps are extracted using Spatial Pyramid Pooling and a Path Aggregation Network in the neck part. The head part aggregates and generates the final output. All bounding boxes by the YOLO v4 are mapped onto the 3D reconstructed bone images after being resized to match the same region as shown in the 2D CT images. The YOLO v4-based AI model was evaluated through precision-recall (PR) curves and the intersection over union (IoU). Our proposed system facilitated an intuitive display of the fractured area through a distinctive red mask overlaid on the 3D reconstructed bone images. The high average precision values (>0.60) were reported as 0.71 and 0.81 from the PR curves of the tibia and elbow, respectively. The IoU values were calculated as 0.6327 (tibia) and 0.6638 (elbow). When utilized by orthopedic surgeons in real clinical scenarios, this AI-powered 3D diagnosis support system could enable a quick and accurate trauma diagnosis.

The chondrotoxicity of single-dose corticosteroids.

PURPOSE: Corticosteroids are commonly injected into the joint space. However, studies have not examined the chondrotoxicity of one-time injection doses. The purpose of this study is to evaluate the effect of dexamethasone sodium phosphate (Decadron), methylprednisolone acetate (Depo-Medrol), betamethasone sodium phosphate and betamethasone acetate (Celestone Soluspan), and triamcinolone acetonide (Kenalog) on human chondrocyte viability in vitro. METHODS: Single-injection doses of each of the corticosteroids were separately delivered to human chondrocytes for their respective average duration of action and compared to controls using a bioreactor containing a continuous infusion pump constructed to mimic joint fluid metabolism. A 14-day time-controlled trial was also performed. A live/dead reduced biohazard viability/cytotoxicity assay was used to quantify chondrocyte viability. RESULTS: Over their average duration of action, betamethasone sodium phosphate/acetate solution and triamcinolone acetonide caused significant decreases in chondrocyte viability compared to control media (19.8 ± 2.9% vs. 5.2 ± 2.1%, P = 0.0025 and 10.2 ± 1.3% vs. 4.8 ± 0.9%, P = 0.0049, respectively). In the 14-day trial, only betamethasone sodium phosphate/acetate solution caused a significant decrease in chondrocyte viability compared to control media (21.5% vs. 4.6%, P < 0.001). CONCLUSIONS: A single-injection dose of betamethasone sodium phosphate and betamethasone acetate solution illustrated consistent and significant chondrotoxicity using a physiologically relevant in vitro model and should be used with caution. Given the observed chondrotoxicity of triamcinolone acetonide in a single trial, there may be some evidence that this medication is chondrotoxic. However, at 14 days, betamethasone sodium phosphate and betamethasone acetate was the only condition that caused significant cell death.

The effect of local anesthetic and corticosteroid combinations on chondrocyte viability.

PURPOSE: Local anesthetic and corticosteroid combination injections are often used in clinical practice, however research investigating the chondrotoxic properties of these combinations is minimal. The goal of this study was to evaluate the effect of single injection doses of 1% lidocaine or 0.25% bupivacaine in combination with single injection doses of dexamethasone sodium phosphate (Decadron), methylprednisolone acetate (Depo-Medrol), betamethasone sodium phosphate and betamethasone acetate (Celestone Soluspan), or triamcinolone acetonide (Kenalog) on human chondrocyte viability. METHODS: All treatment conditions were delivered to human chondrocytes in vitro for the medication's respective average duration of action using a bioreactor containing a continuous infusion pump constructed to mimic joint fluid metabolism. A two-color fluorescence assay was used to evaluate cell viability. A mixed-effects regression model was used to evaluate the mean differences in cell viability between treatment groups. RESULTS: At 14 days, a single injection dose of 1% lidocaine or 0.25% bupivacaine in combination with betamethasone sodium phosphate and betamethasone acetate solution illustrated significant chondrotoxicity when compared with the local anesthetics alone (P < 0.01). Methylprednisolone acetate and Triamcinolone acetonide both showed significant evidence of chondrotoxicity (P = 0.013; P = 0.016, respectively) when used in combination with 1% lidocaine compared with lidocaine alone, but showed no significant chondrotoxicity in combination with 0.25% bupivacaine (P's = n.s.). CONCLUSIONS: Clinicians should use caution when injecting 1% lidocaine or 0.25% bupivacaine in conjunction with betamethasone sodium phosphate and betamethasone acetate solution due to its pronounced chondrotoxic effect in this study. 1% lidocaine used in combination with methylprednisolone acetate or triamcinolone acetonide also led to significant chondrotoxicity.

Bone tissue engineering with premineralized silk scaffolds.

Silk fibroin biomaterials are being explored as novel protein-based systems for cell and tissue culture. In the present study, biomimetic growth of calcium phosphate on porous silk fibroin polymeric scaffolds was explored to generate organic/inorganic composites as scaffolds for bone tissue engineering. Aqueous-derived silk fibroin scaffolds were prepared with the addition of polyaspartic acid during processing, followed by the controlled deposition of calcium phosphate by exposure to CaCl(2) and Na(2)HPO(4). These mineralized protein-composite scaffolds were subsequently seeded with human bone marrow stem cells (hMSC) and cultured in vitro for 6 weeks under osteogenic conditions with or without BMP-2. The extent of osteoconductivity was assessed by cell numbers, alkaline phosphatase and calcium deposition, along with immunohistochemistry for bone-related outcomes. The results suggest increased osteoconductive outcomes with an increase in initial content of apatite and BMP-2 in the silk fibroin porous scaffolds. The premineralization of these highly porous silk fibroin protein scaffolds provided enhanced outcomes for the bone tissue engineering.

In vivo degradation of three-dimensional silk fibroin scaffolds.

Three-dimensional porous scaffolds prepared from regenerated silk fibroin using either an all-aqueous process or a process involving an organic solvent, hexafluoroisopropanol (HFIP), have shown promise in cell culture and tissue engineering applications. However, their biocompatibility and in vivo degradation have not been fully established. The present study was conducted to systematically investigate how processing method (aqueous vs. organic solvent) and processing variables (silk fibroin concentration and pore size) affect the short-term (up to 2 months) and long-term (up to 1 year) in vivo behavior of the protein scaffolds in both nude and Lewis rats. The samples were analyzed by histology for scaffold morphological changes and tissue ingrowth, and by real-time RT-PCR and immunohistochemistry for immune responses. Throughout the period of implantation, all scaffolds were well tolerated by the host animals and immune responses to the implants were mild. Most scaffolds prepared from the all-aqueous process degraded to completion between 2 and 6 months, while those prepared from organic solvent (hexafluoroisopropanol (HFIP)) process persisted beyond 1 year. Due to widespread cellular invasion throughout the scaffold, the degradation of aqueous-derived scaffolds appears to be more homogeneous than that of HFIP-derived scaffolds. In general and especially for the HFIP-derived scaffolds, a higher original silk fibroin concentration (e.g. 17%) and smaller pore size (e.g. 100-200microm) resulted in lower levels of tissue ingrowth and slower degradation. These results demonstrate that the in vivo behavior of the three-dimensional silk fibroin scaffolds is related to the morphological and structural features that resulted from different scaffold preparation processes. The insights gained in this study can serve as a guide for processing scenarios to match desired morphological and structural features and degradation time with tissue-specific applications.

Non-invasive characterization of structure and morphology of silk fibroin biomaterials using non-linear microscopy.

Designing biomaterial scaffolds remains a major challenge in tissue engineering. Key to this challenge is improved understanding of the relationships between the scaffold properties and its degradation kinetics, as well as the cell interactions and the promotion of new matrix deposition. Here we present the use of non-linear spectroscopic imaging as a non-invasive method to characterize not only morphological, but also structural aspects of silkworm silk fibroin-based biomaterials, relying entirely on endogenous optical contrast. We demonstrate that two photon excited fluorescence and second harmonic generation are sensitive to the hydration, overall beta sheet content and molecular orientation of the sample. Thus, the functional content and high resolution afforded by these non-invasive approaches offer promise for identifying important connections between biomaterial design and functional engineered tissue development. The strategies described also have broader implications for understanding and tracking the remodeling of degradable biomaterials under dynamic conditions both in vitro and in vivo.

Bone regeneration on macroporous aqueous-derived silk 3-D scaffolds.

Spinner flask culture under osteogenic conditions was used to study osteogenic outcomes from human bone marrow-derived mesenchymal stem cells (hMSCs) seeded on aqueous-derived porous silk scaffolds. Of particular novelty was the use of larger sized scaffolds (15 mm diameter, 5 mm thick) and large pore sizes ( approximately 900-1 000 micron diameter). Cultures were maintained for 84 d in the spinner flasks and compared to static controls under otherwise similar conditions. The spinner flask cultures demonstrated enhanced cell proliferation compared to static cultures and the improved fluid flow promoted significantly improved osteogenic related outcomes based on elevated alkaline phosphatase (ALP) activity and the deposition of mineralized matrix. The expression of osteogenic differentiation associated markers based on real time PCR also demonstrated increased responses under the dynamic spinner flask culture conditions. Histological analysis showed organized bone-like structures in the constructs cultured in the spinner flasks after 56 d of culture. These structures stained intensely with von Kossa. The combination of improved transport due to spinner flask culture and the use of macroporous 3D aqueous-derived silk scaffolds with large pore sizes resulted in enhanced outcomes related to bone tissue engineering, even with the use of large sized scaffolds in the study. These results suggest the importance of the structure of the silk biomaterial substrate (water vs. solvent based preparation) and large pore sizes in improved bone-like outcomes during dynamic cultivation.

Stem cell-based tissue engineering with silk biomaterials.

Silks are naturally occurring polymers that have been used clinically as sutures for centuries. When naturally extruded from insects or worms, silk is composed of a filament core protein, termed fibroin, and a glue-like coating consisting of sericin proteins. In recent years, silk fibroin has been increasingly studied for new biomedical applications due to the biocompatibility, slow degradability and remarkable mechanical properties of the material. In addition, the ability to now control molecular structure and morphology through versatile processability and surface modification options have expanded the utility for this protein in a range of biomaterial and tissue-engineering applications. Silk fibroin in various formats (films, fibers, nets, meshes, membranes, yarns, and sponges) has been shown to support stem cell adhesion, proliferation, and differentiation in vitro and promote tissue repair in vivo. In particular, stem cell-based tissue engineering using 3D silk fibroin scaffolds has expanded the use of silk-based biomaterials as promising scaffolds for engineering a range of skeletal tissues like bone, ligament, and cartilage, as well as connective tissues like skin. To date fibroin from Bombyx mori silkworm has been the dominant source for silk-based biomaterials studied. However, silk fibroins from spiders and those formed via genetic engineering or the modification of native silk fibroin sequence chemistries are beginning to provide new options to further expand the utility of silk fibroin-based materials for medical applications.

Cartilage tissue engineering with silk scaffolds and human articular chondrocytes.

Adult cartilage tissue has poor capability of self-repair, especially in case of severe cartilage damage due to trauma or age-related degeneration. Autologous cell-based tissue engineering using three-dimensional (3-D) porous scaffolds has provided an option for the repair of full thickness defects in adult cartilage tissue. Mesenchymal stem cells (MSCs) and chondrocytes are the two major cell sources for cartilage tissue engineering. Silk fibroin as a naturally occurring degradable fibrous protein with unique mechanical properties, excellent biocompatibility and process-ability has demonstrated strong potential for skeletal tissue engineering. The present study combined adult human chondrocytes (hCHs) with aqueous-derived porous silk fibroin scaffolds for in vitro cartilage tissue engineering. The results were compared with a previous study using the same scaffolds but using MSCs to generate the cartilage tissue outcomes. Culture-expanded hCHs attached to, proliferated and re-differentiated in the scaffolds in a serum-free, chemically defined medium containing TGF-beta1, based on cell morphology, levels of cartilage-related gene transcripts, and the presence of a cartilage-specific ECM. Cell density was critical for the redifferentiation of culture-expanded hCHs in the 3-D aqueous-derived silk fibroin scaffolds. The level of cartilage-related transcripts (AGC, Col-II, Sox 9 and Col-II/Col-I ratio) and the deposition of cartilage-specific ECM were significantly upregulated in constructs initiated with higher seeding density. The hCH-based constructs were significantly different than those formed from MSC-based constructs with respect to cell morphology, zonal structure and initial seeding density needed to successfully generate engineered cartilage-like tissue. These results suggest fundamental differences between stem cell-based (MSC) and primary cell-based (hCH) tissue engineering, as well as the importance of suitable scaffold features, in the optimization of cartilage-related outcomes in vitro. The present work diversifies cell sources in combination with silk fibroin-based tissue engineering applications. Together with our previous studies, the present results show great promise for engineered 3-D silk fibroin scaffolds in autologous cell-based skeletal tissue engineering.

Electrospun silk-BMP-2 scaffolds for bone tissue engineering.

Silk fibroin fiber scaffolds containing bone morphogenetic protein 2 (BMP-2) and/or nanoparticles of hydroxyapatite (nHAP) prepared via electrospinning were used for in vitro bone formation from human bone marrow-derived mesenchymal stem cells (hMSCs). BMP-2 survived the aqueous-based electrospinnig process in bioactive form. hMSCs were cultured for up to 31 days under static conditions in osteogenic media on the scaffolds (silk/PEO/BMP-2, silk/PEO/nHAP, silk/PEO/nHAP/BMP-2) and controls (silk/PEO, silk/PEO extracted). Electrospun silk fibroin-based scaffolds supported hMSC growth and differentiation toward osteogenic outcomes. The scaffolds with the co-processed BMP-2 supported higher calcium deposition and enhanced transcript levels of bone-specific markers than in the controls, indicating that these nanofibrous electrospun silk scaffolds were an efficient delivery system for BMP-2. X-ray diffraction (XRD) analysis revealed that the apatite formed on the silk fibroin/BMP-2 scaffolds had higher crystallinity than on the silk fibroin scaffold controls. In addition, nHAP particles were incorporated into the electrospun fibrous scaffolds during processing and improved bone formation. The coexistence of BMP-2 and nHAP in the electrospun silk fibroin fibers resulted in the highest calcium deposition and upregulation of BMP-2 transcript levels when compared with the other systems. The results suggest that electrospun silk-fibroin-based scaffolds are potential candidates for bone tissue engineering. Furthermore, the mild aqueous process required to spin the fibers offers an important option for delivery of labile cytokines and other components into the system.

In vivo degradation profile of porcine cartilage-derived extracellular matrix powder scaffolds using a non-invasive fluorescence imaging method.

We present a non-invasive fluorescence method for imaging of scaffold degradation in vivo by quantifying the degradation of porcine cartilage-derived extracellular matrix powder (PCP).Three-dimensional porous scaffolds should be biocompatible and bioresorbable, with a controllable degradation and resorption rate to match tissue growth. However, in vivo scaffold degradation and tissue ingrowth processes are not yet fully understood. Unfortunately, current analysis methods require animal sacrifice and scaffold destruction for the quantification of scaffold degradation and cannot monitor the situation in real time. In this study, Cy3, a fluorescent dye, was used for visualizing PCP and a real-time degradation profile was obtained quantitatively by a non-invasive method using an imaging system in which the reduction in fluorescence intensity depended on PCP scaffold degradation. Real-time PCP scaffold degradation was confirmed through changes in the volume and morphology of the scaffold using micro-computed tomography and microscopy. Our results suggest that extracellular matrix degradation was induced by collagen degradation because of the binding between Cy3 and collagen. This non-invasive real-time monitoring system for scaffold degradation will increase our understanding of in vivo matrix and/or scaffold degradation.

Influence of macroporous protein scaffolds on bone tissue engineering from bone marrow stem cells.

The aim of this study was to investigate the effect of three-dimensional silk fibroin scaffold preparation methods (aqueous and solvent) on osteogenic responses by human bone marrow stem cells (hMSCs). Macroporous 3D protein scaffolds with similar sized pores of 900+/-50 microm were prepared either by an organic solvent process (hexafluoro-2-propanol, HFIP) or an aqueous process. hMSCs were expanded, seeded on the scaffolds, and cultured up to 28 days under static conditions in osteogenic media. hMSCs seeded onto the water-based silk scaffolds showed a significant increase in cell numbers (p<0.01) vs. the HFIP-prepared silk scaffolds. Significantly higher (p<0.01) alkaline phosphatase (ALPase) activity and calcium deposition were apparent after 28 days of culture in the water-based silk scaffolds when compared to the HFIP-derived silk scaffolds. Transcript levels for collagen type I (Col I), ALP, and osteopontin (OP) increased (p<0.05) in the water-based silk scaffolds in comparison to the HFIP-derived materials. At early stages of culture, increased expression of OP and collagen type II (Col II) were also observed in both scaffolds. Expression of Col II, MMP 13, Col I, and OP proteins increased in the water-based silk scaffolds in comparison to the HFIP-derived scaffolds while bone sialoprotein (BSP) proteins increased in the HFIP-derived silk scaffolds in comparison to the water-based scaffolds after 28 days of culture. Histological analysis showed the development of bone-like trabeculae with cuboid cells in an extracellular matrix (ECM) in the water-based silk scaffolds with more organization than in the HFIP-derived material after 28 days of culture. Alcian blue staining demonstrated the presence of proteoglycan in the ECM formed in the water-based scaffolds but not in the HFIP-prepared silk scaffolds. The results suggest that macroporous 3D aqueous-derived silk fibroin scaffolds provide improved bone-related outcomes in comparison to the HFIP-derived systems. These data illustrate the importance of materials processing on biological outcomes, as the same protein, silk fibroin, was used in both preparations.

In vitro cartilage tissue engineering with 3D porous aqueous-derived silk scaffolds and mesenchymal stem cells.

Adult cartilage tissue has limited self-repair capacity, especially in the case of severe damages caused by developmental abnormalities, trauma, or aging-related degeneration like osteoarthritis. Adult mesenchymal stem cells (MSCs) have the potential to differentiate into cells of different lineages including bone, cartilage, and fat. In vitro cartilage tissue engineering using autologous MSCs and three-dimensional (3-D) porous scaffolds has the potential for the successful repair of severe cartilage damage. Ideally, scaffolds designed for cartilage tissue engineering should have optimal structural and mechanical properties, excellent biocompatibility, controlled degradation rate, and good handling characteristics. In the present work, a novel, highly porous silk scaffold was developed by an aqueous process according to these criteria and subsequently combined with MSCs for in vitro cartilage tissue engineering. Chondrogenesis of MSCs in the silk scaffold was evident by real-time RT-PCR analysis for cartilage-specific ECM gene markers, histological and immunohistochemical evaluations of cartilage-specific ECM components. Dexamethasone and TGF-beta3 were essential for the survival, proliferation and chondrogenesis of MSCs in the silk scaffolds. The attachment, proliferation, and differentiation of MSCs in the silk scaffold showed unique characteristics. After 3 weeks of cultivation, the spatial cell arrangement and the collagen type-II distribution in the MSCs-silk scaffold constructs resembles those in native articular cartilage tissue, suggesting promise for these novel 3-D degradable silk-based scaffolds in MSC-based cartilage repair. Further in vivo evaluation is necessary to fully recognize the clinical relevance of these observations.

Three-dimensional aqueous-derived biomaterial scaffolds from silk fibroin.

A new all-aqueous process is described to form three-dimensional porous silk fibroin matrices with control of structural and morphological features. The result of this process are scaffolds with controllable porosity and pore sizes that fully degrade in the presence of proteases, unlike prior methods to generate silk-based biomaterials that required the use of organic solvent treatments to impart control of structure and stability in aqueous environments, with low rates of proteolytic hydrolysis. A mechanism is proposed for this novel process that imparts physical stability via hydrophobic interactions. Adjusting the concentration of silk fibroin in water, and the particle size of granular NaCl used in the process, leads to the control of morphological and functional properties of the scaffolds. The aqueous-derived scaffolds had highly homogeneous and interconnected pores with pore sizes ranging from 470 to 940 microm, depending on the mode of preparation. The scaffolds had porosities >90% and compressive strength and modulus up to 320 +/- 10 and 3330 +/- 500 KPa, respectively, when formed from 10% aqueous solutions of fibroin. The scaffolds fully degraded upon exposure to protease during 21 days, unlike the scaffolds prepared from organic solvent processing. These new silk-based three-dimensional matrices provide useful properties as biomaterial matrices due to the all-aqueous mode of preparation, control of pore size, connectivity of pores, degradability and useful mechanical features. Importantly, this process offers an entirely new window of materials properties when compared with traditional silk fibroin-based materials.

Characterization of subpopulated articular chondrocytes separated by Percoll density gradient.

The aim of this study was to develop a method for fractionation of articular chondrocytes from the entire thickness of the tissue. Isolated chondrocytes from rabbit articular cartilage fractionated by centrifugation in a discontinuous Percoll gradient resulted in four cell fractions with two differing properties. The lowest-density fraction consisted mainly of large cells with small nuclei proliferated actively, maintained the chondrocytic phenotype, and secreted larger amounts of proteoglycan. In contrast, the highest-density fraction consisted of small cells with large nuclei proliferated slowly, did not express the chondrocytic phenotype, and produced larger amounts of interleukin 1-induced nitric oxide. Comparing our results with other previous reports, we find that fraction 1 cells are likely originated from the deep layer of the articular cartilage, whereas fraction 4 cells are tentatively categorized as chondrocytes from the superficial layer of cartilage. Centrifugal fractionation of articular chondrocytes via Percoll density gradient permits clear separation of these heterogeneous cells into different phenotypic populations and allows distinguishing of cells from the different layers of articular cartilage. This simple novel method will provide ready separation of articular chondrocytes for the investigation of the pathogenesis of articular cartilage.