External Mechanical Stability Regulates Hematoma Vascularization in Bone Healing Rather than Endothelial YAP/TAZ Mechanotransduction.

Bone fracture healing is regulated by mechanobiological cues. Both, extracellular matrix (ECM) deposition and microvascular assembly determine the dynamics of the regenerative processes. Mechanical instability as by inter-fragmentary shear or compression is known to influence early ECM formation and wound healing. However, it remains unclear how these external cues shape subsequent ECM and microvascular network assembly. As transcriptional coactivators, the mechanotransducers yes-associated protein 1 (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) translate physical cues into downstream signaling events, yet their role in sprouting angiogenesis into the hematoma after injury is unknown. Using bone healing as model system for scar-free regeneration, the role of endothelial YAP/TAZ in combination with tuning the extrinsic mechanical stability via fracture fixation is investigated. Extrinsically imposed shear across the gap delayed hematoma remodeling and shaped the morphology of early collagen fiber orientations and microvascular networks, suggesting that enhanced shear increased the nutrient exchange in the hematoma. In contrast, endothelial YAP/TAZ deletion has little impact on the overall vascularization of the fracture gap, yet slightly increases the collagen fiber deposition under semi-rigid fixation. Together, these data provide novel insights into the respective roles of endothelial YAP/TAZ and extrinsic mechanical cues in orchestrating the process of bone regeneration.

Sex- and age-related differences in the inflammatory properties of cardiac fibroblasts: impact on the cardiosplenic axis and cardiac fibrosis.

Background: Age and sex are prominent risk factors for heart failure and determinants of structural and functional changes of the heart. Cardiac fibroblasts (cFB) are beyond their task as extracellular matrix-producing cells further recognized as inflammation-supporting cells. The present study aimed to evaluate the impact of sex and age on the inflammatory potential of cFB and its impact on the cardiosplenic axis and cardiac fibrosis. Materials: Left ventricles (LV) of 3- and 12-months old male and female C57BL/6J mice were harvested for immunohistochemistry, immunofluorescence and cFB outgrowth culture and the spleen for flow cytometry. LV-derived cFB and respective supernatants were characterized. Results: LV-derived cFB from 3-months old male mice exhibited a higher inflammatory capacity, as indicated by a higher gene expression of CC-chemokine ligand (CCL) 2, and CCL7 compared to cFB derived from 3-months old female mice. The resulting higher CCL2/chemokine C-X3-C motif ligand (Cx3CL1) and CCL7/Cx3CL1 protein ratio in cell culture supernatants of 3-months old male vs. female cFB was reflected by a higher migration of Ly6Chigh monocytes towards supernatant from 3-months old male vs. female cFB. In vivo a lower ratio of splenic pro-inflammatory Ly6Chigh to anti-inflammatory Ly6Clow monocytes was found in 3-months old male vs. female mice, suggesting a higher attraction of Ly6Chigh compared to Ly6Clow monocytes towards the heart in male vs. female mice. In agreement, the percentage of pro-inflammatory CD68+ CD206- macrophages was higher in the LV of male vs. female mice at this age, whereas the percentage of anti-inflammatory CD68+ CD206+ macrophages was higher in the LV of 3-months old female mice compared to age-matched male animals. In parallel, the percentage of splenic TGF-ß+ cells was higher in both 3- and 12-months old female vs. male mice, as further reflected by the higher pro-fibrotic potential of female vs. male splenocytes at both ages. In addition, female mice displayed a higher total LV collagen content compared to age-matched male mice, whereby collagen content of female cFB was higher compared to male cFB at the age of 12-months. Conclusion: Age- and sex-dependent differences in cardiac fibrosis and inflammation are related to age- and sex-dependent variations in the inflammatory properties of cardiac fibroblasts.

The decisive early phase of bone regeneration.

Bone has a remarkable endogenous regenerative capacity that enables scarless healing and restoration of its prior mechanical function, even under challenging conditions such as advanced age and metabolic or immunological degenerative diseases. However - despite much progress - a high number of bone injuries still heal with unsatisfactory outcomes. The mechanisms leading to impaired healing are heterogeneous, and involve exuberant and non-resolving immune reactions or overstrained mechanical conditions that affect the delicate regulation of the early initiation of scar-free healing. Every healing process begins phylogenetically with an inflammatory reaction, but its spatial and temporal intensity must be tightly controlled. Dysregulation of this inflammatory cascade directly affects the subsequent healing phases and hinders the healing progression. This Review discusses the complex processes underlying bone regeneration, focusing on the early healing phase and its highly dynamic environment, where vibrant changes in cellular and tissue composition alter the mechanical environment and thus affect the signalling pathways that orchestrate the healing process. Essential to scar-free healing is the interplay of various dynamic cascades that control timely resolution of local inflammation and tissue self-organization, while also providing sufficient local stability to initiate endogenous restoration. Various immunotherapy and mechanobiology-based therapy options are under investigation for promoting bone regeneration.

Dissecting the influence of cellular senescence on cell mechanics and extracellular matrix formation in vitro.

Tissue formation and healing both require cell proliferation and migration, but also extracellular matrix production and tensioning. In addition to restricting proliferation of damaged cells, increasing evidence suggests that cellular senescence also has distinct modulatory effects during wound healing and fibrosis. Yet, a direct role of senescent cells during tissue formation beyond paracrine signaling remains unknown. We here report how individual modules of the senescence program differentially influence cell mechanics and ECM expression with relevance for tissue formation. We compared DNA damage-mediated and DNA damage-independent senescence which was achieved through over-expression of either p16Ink4a or p21Cip1 cyclin-dependent kinase inhibitors in primary human skin fibroblasts. Cellular senescence modulated focal adhesion size and composition. All senescent cells exhibited increased single cell forces which led to an increase in tissue stiffness and contraction in an in vitro 3D tissue formation model selectively for p16 and p21-overexpressing cells. The mechanical component was complemented by an altered expression profile of ECM-related genes including collagens, lysyl oxidases, and MMPs. We found that particularly the lack of collagen and lysyl oxidase expression in the case of DNA damage-mediated senescence foiled their intrinsic mechanical potential. These observations highlight the active mechanical role of cellular senescence during tissue formation as well as the need to synthesize a functional ECM network capable of transferring and storing cellular forces.

Characterizing cell recruitment into isotropic and anisotropic biomaterials by quantification of spatial density gradients in vitro.

The success of cell-free in situ tissue engineering approaches depends on an appropriate recruitment of autologous cells from neighboring tissues. This identifies cellular migration as a critical parameter for the pre-clinical characterization of biomaterials. Here, we present a new method to quantify both the extent and the spatial anisotropy of cell migration in vitro. For this purpose, a cell spheroid is used as a cell source to provide a high number of cells for cellular invasion and, at the same time, to guarantee a controlled and spatially localized contact to the material. Therefore, current limitations of assays based on 2D cell sources can be overcome. We tested the method on three biomaterials that are in clinical use for soft tissue augmentation in maxilla-facial surgery and a substrate used for 3D in vitro cell culture. The selected biomaterials were all collagen-derived, but differed in their internal architecture. The analysis of cellular isodensity profiles within the biomaterials allowed the identification of the extent and the preferential directions of migration, as well as their relation to the biomaterials and their specific pore morphologies. The higher cell density within the biomaterials resulting from the here-introduced cell spheroid assay compared to established 2D cell layer assays suggests a better representation of the in vivo situation. Consequently, the presented method is proposed to advance the pre-clinical evaluation of cell recruitment into biomaterials, possibly leading to an improved prediction of the regeneration outcome.

The Degradation of Synthetic Polymeric Scaffolds With Strut-like Architecture Influences the Mechanics-dependent Repair Process of an Osteochondral Defect in Silico.

Current clinical treatments of osteochondral defects in articulating joints are frequently not successful in restoring articular surfaces. Novel scaffold-based tissue engineering strategies may help to improve current treatment options and foster a true regeneration of articulating structures. A frequently desired property of scaffolds is their ability to degrade over time and allow a full restoration of tissue and function. However, it remains largely unknown how scaffold degradation influences the mechanical stability of the tissue in a defect region and, in turn, the regenerative process. Such differing goals-supporting regeneration by degrading its own structure-can hardly be analyzed for tissue engineered constructs in clinical trials and in vivo preclinical experiments. Using an in silico analysis, we investigated the degradation-induced modifications in material and architectural properties of a scaffold with strut-like architecture over the healing course and their influence on the mechanics-dependent tissue formation in osteochondral defects. The repair outcome greatly varied depending on the degradation modality, i.e. surface erosion or bulk degradation with and without autocatalysis, and of the degradation speed, i.e. faster, equal or slower than the expected repair time. Bulk degradation with autocatalysis, independently of degradation speed, caused the mechanical failure of the scaffold prior to osteochondral defect repair and was thereby deemed inappropriate for further application. On the other hand, scaffolds with strut-like architecture degrading by both surface erosion and bulk degradation with slow degradation speed resulted in comparably good repair outcomes, thereby indicating such degradation modalities as favorable for the application in osteochondral defects.

Inner strut morphology is the key parameter in producing highly porous and mechanically stable poly(ε-caprolactone) scaffolds via selective laser sintering.

Selective laser sintering (SLS) is an established method to produce dimensionally accurate scaffolds for tissue engineering (TE) applications, especially in bone. In this context, the FDA-approved, biodegradable polymer poly(ε-caprolactone) (PCL) has been suggested as a suitable scaffold material. However, PCL scaffold mechanical stability - an attribute of particular importance in the field of bone TE - was not considered as a primary target for SLS process parameters optimization so far. Here, we investigated the influence of SLS process parameters on the sintered scaffolds with the aim of producing highly porous (>70% porosity) PCL scaffolds with sub-mm geometrical features for bone TE. Specifically, we studied the influence of laser power, beam compensation and laser beam diameter on the dimensional accuracy and mechanical stiffness of the produced PCL scaffolds. We found that the ratio between the diameter of the molten cross-section within scaffold struts and the outer strut diameter (including partially sintered particles) depended on the SLS process parameters. By maximizing this ratio, the mechanical stability could be optimized. The comparison with in silico predictions of scaffold mechanical stiffness revealed that the diameter of the molten cross-section within struts and not the strut diameter controlled the mechanical behaviour of the scaffold. These observations should be considered when evaluating the quality of the sintering process based on dimensional accuracy, especially for features <1 mm. Based on these findings, we suggested an approach to evaluate the sintering outcome and to define SLS process parameters that enable the production of highly porous scaffolds that are both dimensionally accurate and mechanically stable. Moreover, the cytocompatibility of PCL scaffolds was evaluated by elution tests with primary human mesenchymal stromal cells. No evidence of cytotoxicity was found in any of the investigated scaffolds, confirming the suitability of SLS as production technique of PCL scaffolds for bone TE over a wide range of SLS process parameters.

Scaffold-Dependent Mechanical and Architectural Cues Guide Osteochondral Defect Healing in silico.

Osteochondral defects in joints require surgical intervention to relieve pain and restore function. However, no current treatment enables a complete reconstitution of the articular surface. It is known that both mechanical and biological factors play a key role on osteochondral defect healing, however the underlying principles and how they can be used in the design of treatment strategies remain largely unknown. To unravel the underlying principles of mechanobiology in osteochondral defect healing, i.e., how mechanical stimuli can guide biological tissue formation, we employed a computational approach investigating the scaffold-associated mechanical and architectural properties that would enable a guided defect healing. A previous computer model of the knee joint was further developed to simulate healing of an empty osteochondral defect. Then, scaffolds were implanted in the defect and their architectures and material properties were systematically varied to identify their relevance in osteochondral defect healing. Scaffold mechanical and architectural properties were capable of influencing osteochondral defect healing. Specifically, scaffold material elastic modulus values in the range of cancellous bone (low GPa range) and a scaffold architecture that provided stability, i.e., resistance against displacement, in both the main loading direction and perpendicular to it supported the repair process. The here presented model, despite its simplifications, is regarded as a powerful tool to screen for promising properties of novel scaffold candidates fostering osteochondral defect regeneration prior to their implementation in vivo.

Load-induced osteogenic differentiation of mesenchymal stromal cells is caused by mechano-regulated autocrine signaling.

Mechanical boundary conditions critically influence the bone healing process. In this context, previous in vitro studies have demonstrated that cyclic mechanical compression alters migration and triggers osteogenesis of mesenchymal stromal cells (MSC), both processes being relevant to healing. However, it remains unclear whether this mechanosensitivity is a direct consequence of cyclic compression, an indirect effect of altered supply or a specific modulation of autocrine bone morphogenetic protein (BMP) signaling. Here, we investigate the influence of cyclic mechanical compression (ε = 5% and 10%, f = 1 Hz) on human bone marrow MSC (hBMSC) migration and osteogenic differentiation in a 3D biomaterial scaffold, an in vitro system mimicking the mechanical environment of the early bone healing phase. The open-porous architecture of the scaffold ensured sufficient supply even without cyclic compression, minimizing load-associated supply alterations. Furthermore, a large culture medium volume in relation to the cell number diminished autocrine signaling. Migration of hBMSCs was significantly downregulated under cyclic compression. Surprisingly, a decrease in migration was not associated with increased osteogenic differentiation of hBMSCs, as the expression of RUNX2 and osteocalcin decreased. In contrast, BMP2 expression was significantly upregulated. Enabling autocrine stimulation by increasing the cell-to-medium ratio in the bioreactor finally resulted in a significant upregulation of RUNX2 in response to cyclic compression, which could be reversed by rhNoggin treatment. The results indicate that osteogenesis is promoted by cyclic compression when cells condition their environment with BMP. Our findings highlight the importance of mutual interactions between mechanical forces and BMP signaling in controlling osteogenic differentiation.

Collagen Fibrils Mechanically Contribute to Tissue Contraction in an In Vitro Wound Healing Scenario.

Wound contraction is an ancient survival mechanism of vertebrates that results from tensile forces supporting wound closure. So far, tissue tension was attributed to cellular forces produced by tissue-resident (myo-)fibroblasts alone. However, difficulties in explaining pathological deviations from a successful healing path motivate the exploration of additional modulatory factors. Here, it is shown in a biomaterial-based in vitro wound healing model that the storage of tensile forces in the extracellular matrix has a significant, so-far neglected contribution to macroscopic tissue tension. In situ monitoring of tissue forces together with second harmonic imaging reveal that the appearance of collagen fibrils correlates with tissue contraction, indicating a mechanical contribution of tensioned collagen fibrils in the contraction process. As the re-establishment of tissue tension is key to successful wound healing, the findings are expected to advance the understanding of tissue healing but also underlying principles of misregulation and impaired functionality in scars and tissue contractures.

Cell-Perceived Substrate Curvature Dynamically Coordinates the Direction, Speed, and Persistence of Stromal Cell Migration.

Adherent cells residing within tissues or biomaterials are presented with 3D geometrical cues from their environment, often in the form of local surface curvatures. While there is growing evidence that cellular decision-making is influenced by substrate curvature, the effect of physiologically relevant, cell-scale anisotropic curvatures remains poorly understood. This study systematically explores the migration behavior of human bone marrow stromal cells (hBMSCs) on a library of anisotropic curved structures. Analysis of cell trajectories reveals that, on convex cylindrical structures, hBMSC migration speed and persistence are strongly governed by the cellular orientation on the curved structure, while migration on concave cylindrical structures is characterized by fast but non-aligned and non-persistent migration. Concurrent presentation of concave and convex substrates on toroidal structures induces migration in the direction where hBMSCs can most effectively avoid cell bending. These distinct migration behaviors are found to be universally explained by the cell-perceived substrate curvature, which on anisotropic curved structures is dependent on both the temporally varying cell orientation and the 3D cellular morphology. This work demonstrates that cell migration is dynamically guided by the perceived curvature of the underlying substrate, providing an important biomaterial design parameter for instructing cell migration in tissue engineering and regenerative medicine.

The Interaction of BMP2-Induced Defect Healing in Rat and Fixator Stiffness Modulates Matrix Alignment and Contraction.

Successful fracture healing requires a tight interplay between mechanical and biological cues. In vitro studies illustrated that mechanical loading modulates bone morphogenetic protein (BMP) signaling. However, in the early phases of large bone defect regeneration in vivo, the underlying mechanisms leading to this mechanosensation remained unknown. We investigated the interaction of BMP2 stimulation and mechanical boundary conditions in a rat critical-sized femoral defect model (5 mm) stabilized with three distinctly different external fixator stiffness. Defects were treated with 5 µg rhBMP2 loaded on an absorbable collagen sponge. Early matrix alignment was monitored by second-harmonic generation imaging. Bony bridging of defects and successive healing was monitored by histology at day 7 and day 14 as well as in vivo microCT at days 10, 21, and 42 post-operation. Femora harvested at day 42 were characterized mechanically assessing torsional load to failure ex vivo. At tissue level, differences between groups were visible at day 14 with manifest bone formation in the microCT. Histologically, we observed prolonged chondrogenesis upon flexible fixation, whereas osteogenesis started earlier after rigid and semirigid fixation. At later time points, there was a boost of bone tissue formation upon flexible fixation, whereas other groups already displayed signs of tissue maturation. Based on gene expression profiling, we analyzed the mechanobiological interplay. Already at day 3, these analyses revealed differences in expression pattern, specifically of genes involved in extracellular matrix formation. Gene regulation correlating with fixator stiffness was pronounced at day 7 comprising genes related to immunological processes and cellular contraction. The influence of loading on matrix contraction was further investigated and confirmed in a 3D bioreactor. Taken together, we demonstrate an early onset of mechanical conditions influencing BMP2-induced defect healing and shed light on gene regulatory networks associated with extracellular matrix organization and contraction that seemed to directly impact healing outcomes. © 2018 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

Mesoscale substrate curvature overrules nanoscale contact guidance to direct bone marrow stromal cell migration.

The intrinsic architecture of biological tissues and of implanted biomaterials provides cells with large-scale geometrical cues. To understand how cells are able to sense and respond to complex structural environments, a deeper insight into the cellular response to multi-scale and conflicting geometrical cues is needed. In this study, we subjected human bone marrow stromal cells (hBMSCs) to mesoscale cylindrical surfaces (diameter 250-5000 µm) and nanoscale collagen fibrils (diameter 100-200 nm) that were aligned perpendicular to the cylinder axis. On flat surfaces and at low substrate curvatures (cylinder diameter d > 1000 µm), cell alignment and migration were governed by the nanoscale collagen fibrils, consistent with the contact guidance effect. With increasing surface curvature (decreasing cylinder diameter, d < 1000 µm), cells increasingly aligned and migrated along the cylinder axis, i.e. the direction of zero curvature. An increase in phosphorylated myosin light chain levels was observed with increasing substrate curvature, suggesting a link between substrate-induced cell bending and the F-actin-myosin machinery. Taken together, this work demonstrates that geometrical cues of up to 10× cell size can play a dominant role in directing hBMSC alignment and migration and that the effect of nanoscale contact guidance can even be overruled by mesoscale curvature guidance.

Mechanobiologically optimized 3D titanium-mesh scaffolds enhance bone regeneration in critical segmental defects in sheep.

Three-dimensional (3D) titanium-mesh scaffolds offer many advantages over autologous bone grafting for the regeneration of challenging large segmental bone defects. Our study supports the hypothesis that endogenous bone defect regeneration can be promoted by mechanobiologically optimized Ti-mesh scaffolds. Using finite element techniques, two mechanically distinct Ti-mesh scaffolds were designed in a honeycomb-like configuration to minimize stress shielding while ensuring resistance against mechanical failure. Scaffold stiffness was altered through small changes in the strut diameter only. Honeycombs were aligned to form three differently oriented channels (axial, perpendicular, and tilted) to guide the bone regeneration process. The soft scaffold (0.84 GPa stiffness) and a 3.5-fold stiffer scaffold (2.88 GPa) were tested in a critical size bone defect model in vivo in sheep. To verify that local scaffold stiffness could enhance healing, defects were stabilized with either a common locking compression plate that allowed dynamic loading of the 4-cm defect or a rigid custom-made plate that mechanically shielded the defect. Lower stress shielding led to earlier defect bridging, increased endochondral bone formation, and advanced bony regeneration of the critical size defect. This study demonstrates that mechanobiological optimization of 3D additive manufactured Ti-mesh scaffolds can enhance bone regeneration in a translational large animal study.

Tubular open-porous ß-tricalcium phosphate polycaprolactone scaffolds as guiding structure for segmental bone defect regeneration in a novel sheep model.

Large segmental bone defect reconstruction with sufficient functional restoration is one of the most demanding challenges in orthopaedic surgery. Available regenerative treatment options, as the vascularized bone graft transfer, the Masquelet technique or the Ilizarov distraction osteogenesis, are associated with specific indications and distinct limitations. As an alternative, a hollow cylindrical ceramic-polymer composite scaffold (ß-tricalcium phosphate and poly-lactid co-ε- caprolactone), facilitating a strong surface guiding effect for tissue ingrowth (group 1; n = 6) was investigated here. In combination with an additional autologous, cancellous bone graft filling, the scaffold's ability to work as an open-porous membrane to improve the defect healing process was analysed (group 2; n = 6). A novel model of a critical size (40 mm) tibia osteotomy defect stabilized with an external hybrid-ring fixator, was established in sheep. Segmental defect regeneration and tissue organization in relation to the scaffold were analysed radiologically, (immune-) histologically, and with second-harmonic generation imaging 12 weeks after surgery. The scaffold's tubular shape and open-porous structure controlled the collagen fibre orientation within the bone defect and guided the following mineralization process along the scaffold surface. In combination with the osteoinductive stimulus, a unilateral bony bridging of the critically sized defect was achieved in one third of the animals. The external hybrid-ring fixator was appropriate for large segmental defect stabilization in sheep.

T Lymphocytes Influence the Mineralization Process of Bone.

Bone is a unique organ able to regenerate itself after injuries. This regeneration requires the local interplay between different biological systems such as inflammation and matrix formation. Structural reconstitution is initiated by an inflammatory response orchestrated by the host immune system. However, the individual role of T cells and B cells in regeneration and their relationship to bone tissue reconstitution remain unknown. Comparing bone and fracture healing in animals with and without mature T and B cells revealed the essential role of these immune cells in determining the tissue mineralization and thus the bone quality. Bone without mature T and B cells is stiffer when compared to wild-type bone thus lacking the elasticity that helps to absorb forces, thus preventing fractures. In-depth analysis showed dysregulations in collagen deposition and osteoblast distribution upon lack of mature T and B cells. These changes in matrix deposition have been correlated with T cells rather than B cells within this study. This work presents, for the first time, a direct link between immune cells and matrix formation during bone healing after fracture. It illustrates specifically the role of T cells in the collagen organization process and the lack thereof in the absence of T cells.

Surface Curvature Differentially Regulates Stem Cell Migration and Differentiation via Altered Attachment Morphology and Nuclear Deformation.

Signals from the microenvironment around a cell are known to influence cell behavior. Material properties, such as biochemical composition and substrate stiffness, are today accepted as significant regulators of stem cell fate. The knowledge of how cell behavior is influenced by 3D geometric cues is, however, strongly limited despite its potential relevance for the understanding of tissue regenerative processes and the design of biomaterials. Here, the role of surface curvature on the migratory and differentiation behavior of human mesenchymal stem cells (hMSCs) has been investigated on 3D surfaces with well-defined geometric features produced by stereolithography. Time lapse microscopy reveals a significant increase of cell migration speed on concave spherical compared to convex spherical structures and flat surfaces resulting from an upward-lift of the cell body due to cytoskeletal forces. On convex surfaces, cytoskeletal forces lead to substantial nuclear deformation, increase lamin-A levels and promote osteogenic differentiation. The findings of this study demonstrate a so far missing link between 3D surface curvature and hMSC behavior. This will not only help to better understand the role of extracellular matrix architecture in health and disease but also give new insights in how 3D geometries can be used as a cell-instructive material parameter in the field of biomaterial-guided tissue regeneration.

Gradual conversion of cellular stress patterns into pre-stressed matrix architecture during in vitro tissue growth.

The complex arrangement of the extracellular matrix (ECM) produced by cells during tissue growth, healing and remodelling is fundamental to tissue function. In connective tissues, it is still unclear how both cells and the ECM become and remain organized over length scales much larger than the distance between neighbouring cells. While cytoskeletal forces are essential for assembly and organization of the early ECM, how these processes lead to a highly organized ECM in tissues such as osteoid is not clear. To clarify the role of cellular tension for the development of these ordered fibril architectures, we used an in vitro model system, where pre-osteoblastic cells produced ECM-rich tissue inside channels with millimetre-sized triangular cross sections in ceramic scaffolds. Our results suggest a mechanical handshake between actively contracting cells and ECM fibrils: the build-up of a long-range organization of cells and the ECM enables a gradual conversion of cell-generated tension to pre-straining the ECM fibrils, which reduces the work cells have to generate to keep mature tissue under tension.

A double-layer patch design for local and controlled drug delivery as an intraoperative custom-made implant-coating technology.

BACKGROUND: The specific biological need of patients frequently becomes obvious just in the intraoperative setting. We hypothesized that a double-layer patch approach that allowed rapid attachment to an implant surface would represent a potential solution for technically challenging intraoperative personalized local drug delivery. METHODS: Dexamethasone-loaded poly[(rac-lactide)-co-glycolide] (PLGA) microparticles were embedded within a polyvinyl alcohol (PVA) patch that was attached to metal implant surfaces by in situ polymerization of alkyl-2-cyanoacrylates (CAs). Hydroxyapatite (HA) nanoparticles were also embedded in the PVA patch. RESULTS: Very rapid dexamethasone-release profiles were observed from the PLGA microparticles / PVA patches. The incorporation of HA nanoparticles into the PVA enabled control of CA penetration within the patch, and improved significantly its attachment, while no interference with the drug release was observed. CONCLUSIONS: Double-layered patches with 1 layer for drug delivery and 1 as gluing interface could represent a solution for safe and controlled local drug delivery from implant surfaces or other, even biological, materials. The technology platform presented here opens the opportunity for personalized medicine by allowing local administration of drugs with customized release based on an intraoperative application.

The emergence of extracellular matrix mechanics and cell traction forces as important regulators of cellular self-organization.

Physical cues play a fundamental role in a wide range of biological processes, such as embryogenesis, wound healing, tumour invasion and connective tissue morphogenesis. Although it is well known that during these processes, cells continuously interact with the local extracellular matrix (ECM) through cell traction forces, the role of these mechanical interactions on large scale cellular and matrix organization remains largely unknown. In this study, we use a simple theoretical model to investigate cellular and matrix organization as a result of mechanical feedback signals between cells and the surrounding ECM. The model includes bi-directional coupling through cellular traction forces to deform the ECM and through matrix deformation to trigger cellular migration. In addition, we incorporate the mechanical contribution of matrix fibres and their reorganization by the cells. We show that a group of contractile cells will self-polarize at a large scale, even in homogeneous environments. In addition, our simulations mimic the experimentally observed alignment of cells in the direction of maximum stiffness and the building up of tension as a consequence of cell and fibre reorganization. Moreover, we demonstrate that cellular organization is tightly linked to the mechanical feedback loop between cells and matrix. Cells with a preference for stiff environments have a tendency to form chains, while cells with a tendency for soft environments tend to form clusters. The model presented here illustrates the potential of simple physical cues and their impact on cellular self-organization. It can be used in applications where cell-matrix interactions play a key role, such as in the design of tissue engineering scaffolds and to gain a basic understanding of pattern formation in organogenesis or tissue regeneration.