Biomaterial surface energy-driven ligand assembly strongly regulates stem cell mechanosensitivity and fate on very soft substrates.

Although mechanisms of cell-material interaction and cellular mechanotransduction are increasingly understood, the mechanical insensitivity of mesenchymal cells to certain soft amorphous biomaterial substrates has remained largely unexplained. We reveal that surface energy-driven supramolecular ligand assembly can regulate mesenchymal stem cell (MSC) sensing of substrate mechanical compliance and subsequent cell fate. Human MSCs were cultured on collagen-coated hydrophobic polydimethylsiloxane (PDMS) and hydrophilic polyethylene-oxide-PDMS (PEO-PDMS) of a range of stiffnesses. Although cell contractility was similarly diminished on soft substrates of both types, cell spreading and osteogenic differentiation occurred only on soft PDMS and not hydrophilic PEO-PDMS (elastic modulus <1 kPa). Substrate surface energy yields distinct ligand topologies with accordingly distinct profiles of recruited transmembrane cell receptors and related focal adhesion signaling. These differences did not differentially regulate Rho-associated kinase activity, but nonetheless regulated both cell spreading and downstream differentiation.

Diabetes-induced morphological, biomechanical, and compositional changes in ocular basement membranes.

The current study investigates the structural and compositional changes of ocular basement membranes (BMs) during long-term diabetes. By comparing retinal vascular BMs and the inner limiting membrane (ILM) from diabetic and non-diabetic human eyes by light and transmission electron microscopy (TEM), a massive, diabetes-related increase in the thickness of these BMs was detected. The increase in ILM thickness was confirmed by atomic force microscopy (AFM) on native ILM flat-mount preparations. AFM also detected a diabetes-induced increase in ILM stiffness. The changes in BM morphology and biophysical properties were accompanied by partial changes in the biochemical composition as shown by immunocytochemistry and western blots: agrin, fibronectin and tenascin underwent relative increases in concentration in diabetic BMs as compared to non-diabetic BMs. Fibronectin and tenascin were particularly high in the BMs of outlining microvascular aneurisms. The present data showed that retinal vascular BMs and the ILM undergo morphological, biomechanical and compositional changes during long-term diabetes. The increase in BM thickness not only resulted from an up-regulation of the standard BM proteins, but also from the expression of diabetes-specific extracellular matrix proteins that are not normally found in retinal BMs.

Material properties of the internal limiting membrane and their significance in chromovitrectomy.

Intraoperative visualization of the internal limiting membrane (ILM), the choice of a point of vantage for lifting an initial flap, the precision with which the ILM is grasped, adhesion between the forceps and the ILM, thickness, stiffness and elasticity of the ILM as well as monitoring of the completeness of ILM removal are all important factors for safety and efficacy of a chromovitrectomy intervention. The understanding of the underlying physical features of the ILM, such as contrast behavior and bioanatomical and biomechanical properties represent, thus, useful prerequisites for successful macular surgery. New analytical tools, such as atomic force microscopy and chromaticity analysis, allow new insights into ILM material characteristics, permitting a systematic approach to refinement of surgical technique. .

The nanomechanical properties of rat fibroblasts are modulated by interfering with the vimentin intermediate filament system.

The contribution of the intermediate filament (IF) network to the mechanical response of cells has so far received little attention, possibly because the assembly and regulation of IFs are not as well understood as that of the actin cytoskeleton or of microtubules. The mechanical role of IFs has been mostly inferred from measurements performed on individual filaments or gels in vitro. In this study we employ atomic force microscopy (AFM) to examine the contribution of vimentin IFs to the nanomechanical properties of living cells under native conditions. To specifically target and modulate the vimentin network, Rat-2 fibroblasts were transfected with GFP-desmin variants. Cells expressing desmin variants were identified by the fluorescence microscopy extension of the AFM instrument. This allowed us to directly compare the nanomechanical response of transfected and untransfected cells at high spatial resolution by means of AFM. Depending on the variant desmin, transfectants were either softer or stiffer than untransfected fibroblasts. Expression of the non-filament forming GFP-DesL345P mutant led to a collapse of the endogenous vimentin network in the perinuclear region that was accompanied by localized stiffening. Correlative confocal microscopy indicates that the expression of desmin variants specifically targets the endogenous vimentin IF network without major rearrangements of other cytoskeletal components. By measuring functional changes caused by IF rearrangements in intact cells, we show that IFs play a crucial role in mechanical behavior not only at large deformations but also in the nanomechanical response of individual cells.

Micro- and nanomechanical analysis of articular cartilage by indentation-type atomic force microscopy: validation with a gel-microfiber composite.

As documented previously, articular cartilage exhibits a scale-dependent dynamic stiffness when probed by indentation-type atomic force microscopy (IT-AFM). In this study, a micrometer-size spherical tip revealed an unimodal stiffness distribution (which we refer to as microstiffness), whereas probing articular cartilage with a nanometer-size pyramidal tip resulted in a bimodal nanostiffness distribution. We concluded that indentation of the cartilage's soft proteoglycan (PG) gel gave rise to the lower nanostiffness peak, whereas deformation of its collagen fibrils yielded the higher nanostiffness peak. To test our hypothesis, we produced a gel-microfiber composite consisting of a chondroitin sulfate-containing agarose gel and a fibrillar poly(ethylene glycol)-terephthalate/poly(butylene)-terephthalate block copolymer. In striking analogy to articular cartilage, the microstiffness distribution of the synthetic composite was unimodal, whereas its nanostiffness exhibited a bimodal distribution. Also, similar to the case with cartilage, addition of the negatively charged chondroitin sulfate rendered the gel-microfiber composite's water content responsive to salt. When the ionic strength of the surrounding buffer solution increased from 0.15 to 2 M NaCl, the cartilage's microstiffness increased by 21%, whereas that of the synthetic biomaterial went up by 31%. When the nanostiffness was measured after the ionic strength was raised by the same amount, the cartilage's lower peak increased by 28%, whereas that of the synthetic biomaterial went up by 34%. Of interest, the higher peak values remained unchanged for both materials. Taken together, these results demonstrate that the nanoscale lower peak is a measure of the soft PG gel, and the nanoscale higher peak measures collagen fibril stiffness. In contrast, the micrometer-scale measurements fail to resolve separate stiffness values for the PG and collagen fibril moieties. Therefore, we propose to use nanostiffness as a new biomarker to analyze structure-function relationships in normal, diseased, and engineered cartilage.

Superior Rim Stability of the Lens Capsule Following Manual Over Femtosecond Laser Capsulotomy.

PURPOSE: Cataract surgery requires the removal of a circular segment of the anterior lens capsule (LC) by manual or femtosecond laser (FL) capsulotomy. Tears in the remaining anterior LC may compromise surgical outcome. We investigated whether biophysical differences in the rim properties of the LC remaining in the patient after manual or FL capsulotomy (FLC) lead to different risks with regard to anterior tear formation. METHODS: Lens capsule samples obtained by either continuous curvilinear capsulorhexis (CCC) or FLC were investigated by light microscopy, laser scanning confocal microscopy, and scanning electron microscopy; atomic force microscopy (AFM) was used to test the biomechanical properties of the LC. The mechanical stability of the LC following either of the two capsulotomy techniques was simulated by using finite-element modeling. RESULTS: Continuous curvilinear capsulorhexis produced wedge-shaped, uniform rims, while FLC resulted in nearly perpendicular, frayed rims with numerous notches. The LC is composed of two sublayers: a stiff epithelial layer that is abundant with laminin and a softer anterior chamber layer that is predominantly made from collagen IV. Computer models show that stress is uniformly distributed over the entire rim after CCC, while focal high stress concentrations are observed in the frayed profiles of LC after FLC, making the latter procedure more prone to anterior tear formation. CONCLUSIONS: Finite-element modeling based on three-dimensional AFM maps indicated that CCC leads to a capsulotomy rim with higher stress resistance, leading to a lower propensity for anterior radial tears than FLC.

Supramolecular Organization of Collagen Fibrils in Healthy and Osteoarthritic Human Knee and Hip Joint Cartilage.

Cartilage matrix is a composite of discrete, but interacting suprastructures, i.e. cartilage fibers with microfibrillar or network-like aggregates and penetrating extrafibrillar proteoglycan matrix. The biomechanical function of the proteoglycan matrix and the collagen fibers are to absorb compressive and tensional loads, respectively. Here, we are focusing on the suprastructural organization of collagen fibrils and the degradation process of their hierarchical organized fiber architecture studied at high resolution at the authentic location within cartilage. We present electron micrographs of the collagenous cores of such fibers obtained by an improved protocol for scanning electron microscopy (SEM). Articular cartilages are permeated by small prototypic fibrils with a homogeneous diameter of 18 ± 5 nm that can align in their D-periodic pattern and merge into larger fibers by lateral association. Interestingly, these fibers have tissue-specific organizations in cartilage. They are twisted ropes in superficial regions of knee joints or assemble into parallel aligned cable-like structures in deeper regions of knee joint- or throughout hip joints articular cartilage. These novel observations contribute to an improved understanding of collagen fiber biogenesis, function, and homeostasis in hyaline cartilage.

Nonmulberry Silk Fibroin Scaffold Shows Superior Osteoconductivity Than Mulberry Silk Fibroin in Calvarial Bone Regeneration.

Recent years have witnessed the advancement of silk biomaterials in bone tissue engineering, although clinical application of the same is still in its infancy. In this study, the potential of pure nonmulberry Antheraea mylitta (Am) fibroin scaffold, without preloading with bone precursor cells, to repair calvarial bone defect in a rat model is explored and compared with its mulberry counterpart Bombyx mori (Bm) silk fibroin. After 3 months of implantation, Am scaffold culminates in a completely ossified regeneration with a progressive increase in mineralization at the implanted site. On the other hand, the Bm scaffold fails to repair the damaged bone, presumably due to its low osteoconductivity and early degradation. The deposition of bone matrix on scaffolds is evaluated by scanning electron and atomic force microscopy. These results are corroborated by in vitro studies of enzymatic degradation, colony formation, and secondary conformational features of the scaffold materials. The greater biocompatibility and mineralization in pure nonmulberry fibroin scaffolds warrants the use of these scaffolds as an "ideal bone graft" biomaterial for effective repair of critical size defects.

The role of 3D structure and protein conformation on the innate and adaptive immune responses to silk-based biomaterials.

We have investigated monocyte and T cell responsiveness to silk based biomaterials of different physico-chemical characteristics. Here we report that untransformed CD14+ human monocytes respond to overnight exposure to silk fibroin-based biomaterials in tridimensional form by IL-1ß and IL-6, but not IL-10 gene expression and protein production. In contrast, fibroin based materials in bidimensional form are unable to stimulate monocyte responsiveness. The elicitation of these effects critically requires contact between biomaterials and responding cells, is not sustained and becomes undetectable in longer term cultures. We also observed that NF-κß and p38 MAP kinase play key roles in monocyte activation by silk-based biomaterials. On the other hand, fibroin based materials, irrespective of their physico-chemical characteristics appeared to be unable to induce the activation of peripheral blood T cells from healthy donors, as evaluated by the expression of activation markers and IFN-γ gene.

The bi-functional organization of human basement membranes.

The current basement membrane (BM) model proposes a single-layered extracellular matrix (ECM) sheet that is predominantly composed of laminins, collagen IVs and proteoglycans. The present data show that BM proteins and their domains are asymmetrically organized providing human BMs with side-specific properties: A) isolated human BMs roll up in a side-specific pattern, with the epithelial side facing outward and the stromal side inward. The rolling is independent of the curvature of the tissue from which the BMs were isolated. B) The epithelial side of BMs is twice as stiff as the stromal side, and C) epithelial cells adhere to the epithelial side of BMs only. Side-selective cell adhesion was also confirmed for BMs from mice and from chick embryos. We propose that the bi-functional organization of BMs is an inherent property of BMs and helps build the basic tissue architecture of metazoans with alternating epithelial and connective tissue layers.

Nanoscale topographic and biomechanical studies of the human internal limiting membrane.

PURPOSE: The purpose of this article was to create a nanometer scale topographic and biomechanical profile of the human internal limiting membrane (ILM) under native conditions. METHODS: ILMs from the posterior pole of postmortem human eyes were prepared as flat mounts and investigated by atomic force microscopy (AFM) under physiological conditions. Structural analysis was complemented by transmission electron microscopy. RESULTS: Average thickness of the fully hydrated, native ILMs was 3488 ± 460 nm. Thickness variations from 100 nm to 4326 nm characterized the fovea, which displayed a craterlike morphology. Outside the fovea, thickness distribution was uniform. Although mean ILM thicknesses were similar, standard deviation was higher on the retinal than on the vitreal side, indicating greater roughness. Average ILM stiffness was more than fivefold higher on the retinal than on the vitreal side (227 vs. 44 kPa). CONCLUSIONS: A detailed topographical and nanomechanical profile of native human ILM was generated using AFM. Thickness values were significantly higher than in previous studies because of the preservation of native conditions. Both thickness and stiffness showed marked variations around the fovea but were relatively uniform outside the foveal area. Interestingly, the foveal ILM displayed a craterlike morphological appearance with four distinct layers separated by comparatively steep thickness increments. ILM stiffness was considerably higher on the retinal than on the vitreal side. AFM opens new possibilities for investigating native basement membranes under physiological and pathological conditions. Transmission electron microscopy revealed higher extracellular matrix protein density on the retinal than on the vitreal side.

Oriented lamellar silk fibrous scaffolds to drive cartilage matrix orientation: towards annulus fibrosus tissue engineering.

A novel design of silk-based scaffold is developed using a custom-made winding machine, with fiber alignment resembling the anatomical criss-cross lamellar fibrous orientation features of the annulus fibrosus of the intervertebral disc. Crosslinking of silk fibroin fibers with chondroitin sulphate (CS) was introduced to impart superior biological functionality. The scaffolds, with or without CS, instructed alignment of expanded human chondrocytes and of the deposited extracellular matrix while supporting their chondrogenic redifferentiation. The presence of CS crosslinking could not induce statistically significant changes in the measured collagen or glycosaminoglycan content, but resulted in an increased construct stiffness. By offering the combined effect of cell/matrix alignment and chondrogenic support, the silk fibroin scaffolds developed with precise fiber orientation in lamellar form represent a suitable substrate for tissue engineering of the annulus fibrosus part of the intervertebral disc.

The nanomechanical signature of breast cancer.

Cancer initiation and progression follow complex molecular and structural changes in the extracellular matrix and cellular architecture of living tissue. However, it remains poorly understood how the transformation from health to malignancy alters the mechanical properties of cells within the tumour microenvironment. Here, we show using an indentation-type atomic force microscope (IT-AFM) that unadulterated human breast biopsies display distinct stiffness profiles. Correlative stiffness maps obtained on normal and benign tissues show uniform stiffness profiles that are characterized by a single distinct peak. In contrast, malignant tissues have a broad distribution resulting from tissue heterogeneity, with a prominent low-stiffness peak representative of cancer cells. Similar findings are seen in specific stages of breast cancer in MMTV-PyMT transgenic mice. Further evidence obtained from the lungs of mice with late-stage tumours shows that migration and metastatic spreading is correlated to the low stiffness of hypoxia-associated cancer cells. Overall, nanomechanical profiling by IT-AFM provides quantitative indicators in the clinical diagnostics of breast cancer with translational significance.

Imaging articular cartilage tissue using atomic force microscopy (AFM).

Cartilage is a complex avascular tissue composed of cells ("chondrocytes") embedded in an extracellular matrix (ECM) consisting of 70%-80% water. The primary components of the ECM are negatively charged aggrecans and collagen II fibrils, which possess a characteristic, ordered three-dimensional structure. The components interact to ensure that the cartilage is able to absorb shock and can function to protect the bone ends. Atomic force microscopy (AFM) can be used to examine structure-function relationships of cartilage at both micrometer and nanometer scales. When imaged at the micrometer scale with microspheres, only the ECM and chondrocytes can be distinguished. Correspondingly, mechanical testing of cartilage at the micrometer scale results in unimodal distribution of the stiffness because the bulk elastic property of the ECM is probed. In contrast, bare AFM tips are able to reveal the molecular components of the ECM at the nanometer scale. Mechanical testing at the nanometer scale reveals a bimodal distribution of the stiffness and reflects the distinct stiffness of the collagen network and the proteoglycan moiety. In this protocol, the corresponding AFM image and force map are used to reveal the distinct morphology of the collagen fibers and proteoglycan gel. Although, in principle, these experiments can be performed using any AFM, an AFM with tube scanners that have manual screws for tilting the sample is preferable because cartilage has macroscopically rough surface features. By manually tilting the probe over the sample, an optimal angle for tip approach can be achieved.

Imaging fibroblast cells using atomic force microscopy (AFM).

Atomic force microscopy (AFM) can be used to visualize the three major cytoskeletal components that contribute to the mechanical properties of the cell. These are actin microfilaments, intermediate filaments, and microtubules. In this protocol, rat embryonic fibroblasts expressing actin tagged with green fluorescent protein (GFP) are used to demonstrate this procedure.

Imaging collagen II using atomic force microscopy (AFM).

Collagen II is a fibrous protein that assembles from basic tropocollagen subunits to form extracellular supramolecular fiber networks within cartilage tissue. Tropocollagen subunits of ~300 nm in length self-assemble first into pentameric uniform microfibrils, which fuse into bigger collagen fibrils that can range from 10 nm to 500 nm in diameter. The collagen fibrils display a characteristic 67-nm repeat because of the staggering of individual collagen molecules with respect to each other. This protocol demonstrates how to prepare collagen protein samples for analysis by atomic force microscopy (AFM). It also describes the steps for generating AFM images of collagen samples during and after manipulation to analyze collagen self-assembly.

Preparation of microsphere tips for atomic force microscopy (AFM).

This protocol describes the preparation and use of spherical indenters for micrometer-scale imaging and mechanical testing with atomic force microscopy (AFM). A spherical indenter is prepared by gluing a hard borosilicate sphere to a tipless cantilever. For this purpose, a stereomicroscope with a micromanipulator attachment is employed. The spheres are cleaned prior to gluing to remove any contamination from their surfaces. Once they are cleaned, the borosilicate spheres are stored in absolute ethanol in a glass tube.

Atomic force microscopy for biological imaging and mechanical testing across length scales.

Atomic force microscopy (AFM) offers researchers a unique opportunity to visualize, manipulate, and quantitatively assess structural and mechanical aspects of native biological samples with nanometer resolution. An unparalleled advantage of AFM over other high-resolution microscopes is that biological specimens, ranging from tissues to cells to molecules, can be investigated in physiologically relevant aqueous environments. The AFM can be operated at 37°C, which makes it ideal for in situ cell or tissue studies. Combining an optical microscope with an AFM makes it possible to directly correlate structural/nanomechanical changes with optical/fluorescence images. This ability to simultaneously acquire structural and function information is unprecedented in biology. This article introduces the basics of AFM for imaging and investigating the properties of biological samples.

Anabolic and catabolic responses of human articular chondrocytes to varying oxygen percentages.

INTRODUCTION: Oxygen is a critical parameter proposed to modulate the functions of chondrocytes ex-vivo as well as in damaged joints. This article investigates the effect of low (more physiological) oxygen percentage on the biosynthetic and catabolic activity of human articular chondrocytes (HAC) at different phases of in vitro culture. METHODS: HAC expanded in monolayer were cultured in pellets for two weeks (Phase I) or up to an additional two weeks (Phase II). In each Phase, cells were exposed to 19% or 5% oxygen. Resulting tissues and culture media were assessed to determine amounts of produced/released proteoglycans and collagens, metalloproteinases (MMPs), collagen degradation products and collagen fibril organization using biochemical, (immuno)-histochemical, gene expression and scanning electron microscopy analyses. In specific experiments, the hypoxia-inducible factor-1alpha (HIF-1alpha) inhibitor cadmium chloride was supplemented in the culture medium to assess the involvement of this pathway. RESULTS: Independent from the oxygen percentage during expansion, HAC cultured at 5% O(2) (vs 19% O(2)) during Phase I accumulated higher amounts of glycosaminoglycans and type II collagen and expressed reduced levels of MMP-1 and MMP-13 mRNA and protein. Switching to 19% oxygen during Phase II resulted in reduced synthesis of proteoglycan and collagen, increased release of MMPs, accumulation of type II collagen fragments and higher branching of collagen fibrils. In contrast, reducing O(2) during Phase II resulted in increased proteoglycan and type II collagen synthesis and reduced expression and release of MMP-13 mRNA and protein. Supplementation of cadmium chloride during differentiation culture at 5% O(2) drastically reduced the up-regulation of type II collagen and the down-regulation of MMP-1 mRNA. CONCLUSIONS: The application of more physiologic oxygen percentage during specific phases of differentiation culture enhanced the biosynthetic activity and reduced the activity of catabolic enzymes implicated in cartilage breakdown. Modulation of the oxygen percentage during HAC culture may be used to study pathophysiological events occurring in osteoarthritis and to enhance properties of in vitro engineered cartilaginous tissues.

Stretching, unfolding, and deforming protein filaments adsorbed at solid-liquid interfaces using the tip of an atomic-force microscope.

Cells move by actively remodeling a dense network of protein filaments. Here we analyze the force response of various filaments in a simplified experimental setup, where single filaments are moved with an atomic-force microscope (AFM) tip against surface friction, with the AFM operating in the torsional mode. Our experimental findings are well explained within a simple model based on Newtonian mechanics: we observe force plateaus, which are the signature of the sequential stretching of single repeat units, followed ultimately by deformation of the whole polymer shape.