Creating High-Resolution Multiscale Maps of Human Tissue Using Multi-beam SEM.

Multi-beam scanning electron microscopy (mSEM) enables high-throughput, nano-resolution imaging of macroscopic tissue samples, providing an unprecedented means for structure-function characterization of biological tissues and their cellular inhabitants, seamlessly across multiple length scales. Here we describe computational methods to reconstruct and navigate a multitude of high-resolution mSEM images of the human hip. We calculated cross-correlation shift vectors between overlapping images and used a mass-spring-damper model for optimal global registration. We utilized the Google Maps API to create an interactive map and provide open access to our reconstructed mSEM datasets to both the public and scientific communities via our website www.mechbio.org. The nano- to macro-scale map reveals the tissue's biological and material constituents. Living inhabitants of the hip bone (e.g. osteocytes) are visible in their local extracellular matrix milieu (comprising collagen and mineral) and embedded in bone's structural tissue architecture, i.e. the osteonal structures in which layers of mineralized tissue are organized in lamellae around a central blood vessel. Multi-beam SEM and our presented methodology enable an unprecedented, comprehensive understanding of health and disease from the molecular to organ length scale.

Mechanistic, mathematical model to predict the dynamics of tissue genesis in bone defects via mechanical feedback and mediation of biochemical factors.

The link between mechanics and biology in the generation and the adaptation of bone has been well studied in context of skeletal development and fracture healing. Yet, the prediction of tissue genesis within - and the spatiotemporal healing of - postnatal defects, necessitates a quantitative evaluation of mechano-biological interactions using experimental and clinical parameters. To address this current gap in knowledge, this study aims to develop a mechanistic mathematical model of tissue genesis using bone morphogenetic protein (BMP) to represent of a class of factors that may coordinate bone healing. Specifically, we developed a mechanistic, mathematical model to predict the dynamics of tissue genesis by periosteal progenitor cells within a long bone defect surrounded by periosteum and stabilized via an intramedullary nail. The emergent material properties and mechanical environment associated with nascent tissue genesis influence the strain stimulus sensed by progenitor cells within the periosteum. Using a mechanical finite element model, periosteal surface strains are predicted as a function of emergent, nascent tissue properties. Strains are then input to a mechanistic mathematical model, where mechanical regulation of BMP-2 production mediates rates of cellular proliferation, differentiation and tissue production, to predict healing outcomes. A parametric approach enables the spatial and temporal prediction of endochondral tissue regeneration, assessed as areas of cartilage and mineralized bone, as functions of radial distance from the periosteum and time. Comparing model results to histological outcomes from two previous studies of periosteum-mediated bone regeneration in a common ovine model, it was shown that mechanistic models incorporating mechanical feedback successfully predict patterns (spatial) and trends (temporal) of bone tissue regeneration. The novel model framework presented here integrates a mechanistic feedback system based on the mechanosensitivity of periosteal progenitor cells, which allows for modeling and prediction of tissue regeneration on multiple length and time scales. Through combination of computational, physical and engineering science approaches, the model platform provides a means to test new hypotheses in silico and to elucidate conditions conducive to endogenous tissue genesis. Next generation models will serve to unravel intrinsic differences in bone genesis by endochondral and intramembranous mechanisms.

Periosteal thickness and cellularity in mid-diaphyseal cross-sections from human femora and tibiae of aged donors.

Due to lack of access in healthy patients, the structural properties underlying the inherent regenerative power and advanced material properties of the human periosteum are not well understood. Periosteum comprises a cellular cambium layer directly apposing the outer surface of bone and an outer fibrous layer encompassed by the surrounding soft tissues. As a first step to elucidating the structural and cellular characteristics of periosteum in human bone, the current study aims to measure cambium and fibrous layer thickness as well as cambium cellularity in human femora and tibiae of aged donors. The major and minor centroidal axes (CA) serve as automated reference points in cross-sections of cadaveric mid-diaphyseal femora and tibiae. Based on the results of this study, within a given individual, the cambium layer of the major CA of the tibia is significantly thicker and more cellular than the respective layer of the femur. These significant intraindividual differences do not translate to significant interindividual differences. Further, mid-diaphyseal periosteal measures including cambium and fibrous layer thickness and cellularity do not correlate significantly with age or body mass. Finally, qualitative observations of periosteum in amputated and contralateral or proximal long bones of the lower extremity show stark changes in layer organization, thickness, and cellularity. In a translational context, these novel data, though inherently limited by availability and accessibility of human mid-diaphyseal periosteum tissue, provide important reference values for the use of periosteum in the context of facilitated healing and regeneration of tissue.

Medial meniscal displacement and strain in three dimensions under compressive loads: MR assessment.

PURPOSE: To investigate the 3D displacement and the local strain of the medial meniscus and its attachments under compressive loading. MATERIALS AND METHODS: Magnetic resonance imaging (MRI) scans of six porcine knee joints were performed under unloaded and loaded conditions (100% and 200% body weight [BW]). Volumes were registered to obtain a 3D displacement field of the medial meniscus and its attachments, which were divided into five anatomic compartments. Finally, displacements of the center of mass of each compartment and the local strain were analyzed. RESULTS: The meniscus and its attachments significantly displaced by up to 2.6 ± 1.2 mm (P < 0.01) under knee joint loads of 200% BW. An increase of 0.9 mm in the distance between posterior and anterior horn (P < 0.001) was observed. The meniscus and its attachment showed an average radial stretch of 0.6%, an average circumferential stretch of 0.9%, and an average axial compression of 11.6% at 200% BW. CONCLUSION: High-resolution MRI was successfully combined with image registration to investigate the displacement and strain of the meniscus and its attachments under compression. The results of this study contribute to the basic understanding of meniscal movement which may impact the design of meniscal implants and the validation of finite element models in the future.

A spike in circulating cytokines TNF-α and TGF-ß alters barrier function between vascular and musculoskeletal tissues.

Molecular transport between the circulatory and musculoskeletal systems regulates articular joint physiology in health and disease. Osteoarthritis (OA) is a degenerative joint disease linked to systemic and local inflammation. Inflammatory events involve cytokines, which are secreted by cells of the immune system and modulate molecular transport across tissue interfaces (referred to as tight junction [TJ] barrier function). In a previous study from our group, OA knee joint tissues were shown to exhibit size separation of different sized molecules delivered as a single bolus to the heart (Ngo et al. in Sci. Rep. 8:10254, 2018). Here, in a follow up study of parallel design, we test the hypothesis that two common cytokines, with multifaceted roles in the etiology of osteoarthritis as well as immune state in general, modulate the barrier function properties of joint tissue interfaces. Specifically, we probe the effect of an acute cytokine increase (spike) on molecular transport within tissues and across tissue interfaces of the circulatory and musculoskeletal systems. A single bolus of fluorescent-tagged 70 kDa dextran, was delivered intracardially, either alone, or with either the pro-inflammatory cytokine TNF-α or the anti-inflammatory cytokine TGF-ß, to skeletally mature (11 to 13-month-old) guinea pigs (Dunkin-Hartley, a spontaneous OA animal model). After five minutes' circulation, whole knee joints were serial sectioned and fluorescent block face cryo-imaged at near-single-cell resolution. The 70 kDa fluorescent-tagged tracer is analogous in size to albumin, the most prevalent blood transporter protein, and quantification of tracer fluorescence intensity gave a measure of tracer concentration. Within five minutes, a spike (acute doubling) in circulating cytokines TNF-α or TGF-ß significantly disrupted barrier function between the circulatory and musculoskeletal systems, with barrier function essentially abrogated in the TNF-α group. In the entire volume of the joint (including all tissue compartments and the bounding musculature), tracer concentration was significantly decreased in the TGF-ß- and TNF-α- compared to the control-group. These studies implicate inflammatory cytokines as gatekeepers for molecular passage within and between tissue compartments of our joints and may open new means to delay the onset and mitigate the progression of degenerative joint diseases such as OA, using pharmaceutical and/or physical measures.

Biomechanical, biophysical and biochemical modulators of cytoskeletal remodelling and emergent stem cell lineage commitment.

Across complex, multi-time and -length scale biological systems, redundancy confers robustness and resilience, enabling adaptation and increasing survival under dynamic environmental conditions; this review addresses ubiquitous effects of cytoskeletal remodelling, triggered by biomechanical, biophysical and biochemical cues, on stem cell mechanoadaptation and emergent lineage commitment. The cytoskeleton provides an adaptive structural scaffold to the cell, regulating the emergence of stem cell structure-function relationships during tissue neogenesis, both in prenatal development as well as postnatal healing. Identification and mapping of the mechanical cues conducive to cytoskeletal remodelling and cell adaptation may help to establish environmental contexts that can be used prospectively as translational design specifications to target tissue neogenesis for regenerative medicine. In this review, we summarize findings on cytoskeletal remodelling in the context of tissue neogenesis during early development and postnatal healing, and its relevance in guiding lineage commitment for targeted tissue regeneration. We highlight how cytoskeleton-targeting chemical agents modulate stem cell differentiation and govern responses to mechanical cues in stem cells' emerging form and function. We further review methods for spatiotemporal visualization and measurement of cytoskeletal remodelling, as well as its effects on the mechanical properties of cells, as a function of adaptation. Research in these areas may facilitate translation of stem cells' own healing potential and improve the design of materials, therapies, and devices for regenerative medicine.

Connectomics of Bone to Brain-Probing Physical Renderings of Cellular Experience.

"Brainless" cells, the living constituents inhabiting all biological materials, exhibit remarkably smart, i.e., stimuli-responsive and adaptive, behavior. The emergent spatial and temporal patterns of adaptation, observed as changes in cellular connectivity and tissue remodeling by cells, underpin neuroplasticity, muscle memory, immunological imprinting, and sentience itself, in diverse physiological systems from brain to bone. Connectomics addresses the direct connectivity of cells and cells' adaptation to dynamic environments through manufacture of extracellular matrix, forming tissues and architectures comprising interacting organs and systems of organisms. There is imperative to understand the physical renderings of cellular experience throughout life, from the time of emergence, to growth, adaptation and aging-associated degeneration of tissues. Here we address this need through development of technological approaches that incorporate cross length scale (nm to m) structural data, acquired via multibeam scanning electron microscopy, with machine learning and information transfer using network modeling approaches. This pilot case study uses cutting edge imaging methods for nano- to meso-scale study of cellular inhabitants within human hip tissue resected during the normal course of hip replacement surgery. We discuss the technical approach and workflow and identify the resulting opportunities as well as pitfalls to avoid, delineating a path for cellular connectomics studies in diverse tissue/organ environments and their interactions within organisms and across species. Finally, we discuss the implications of the outlined approach for neuromechanics and the control of physical behavior and neuromuscular training.

Osteoarthritis: New Strategies for Transport and Drug Delivery Across Length Scales.

Osteoarthritis (OA) is the fourth leading cause of disability in adults. Yet, few viable pharmaceutical options exist for pain abatement and joint restoration, aside from joint replacement at late and irreversible stages of the disease. From the first onset of OA, as joint pain increases, individuals with arthritis increasingly reach for drug delivery solutions, from taking oral glycosaminoglycans (GAGs) bought over the counter from retail stores (e.g., Costco) to getting injections of viscous, GAG-containing synovial fluid supplement in the doctor's office. Little is known regarding the efficacy of delivery mode and/or treatment by such disease-modulating agents. This Review addresses the interplay of mechanics and biology on drug delivery to affected joints, which has profound implications for molecular transport in joint health and (patho)physiology. Multiscale systems biology approaches lend themselves to understand the relationship between the cell and joint health in OA and other joint (patho)physiologies. This Review first describes OA-related structural and functional changes in the context of the multilength scale anatomy of articular joints. It then summarizes and categorizes, by size and charge, published molecular transport studies, considering changes in permeability induced through inflammatory pathways. Finally, pharmacological interventions for OA are outlined in the context of molecular weights and modes of drug delivery. Taken together, the current state-of-the-art points to a need for new drug delivery strategies that harness systems-based interactions underpinning molecular transport and maintenance of joint structure and function at multiple length scales from molecular agents to cells, tissues, and tissue compartments which together make up articular joints. Cutting edge and cross-length and -time scale imaging represents a key discovery enabling technology in this process.

In vitro biocompatibility and biomechanics study of novel, Microscopy Aided Designed and ManufacturEd (MADAME) materials emulating natural tissue weaves and their intrinsic gradients.

This study conducted biomechanical and biocompatibility tests of textiles and textile composites, created using recursive logic to emulate the properties of natural tissue weaves and their intrinsic mechanical stiffness gradients. Two sets of samples were created, first to test feasibility on textile samples designed as periosteum substitutes with elastane fibers mimicking periosteum's endogenous elastin and nylon fibers substituting for collagen, and then on composites comprising other combinations of suture materials before and after sterilization. In the first part, the bulk tensile mechanical stiffness of elastane-nylon textiles were tuned through respective fiber composition and orientation, i.e., aligned with and orthogonal to loading direction. Cell culture biocompatibility studies revealed no significant differences in proliferation rates of embryonic murine stem cells seeded on textiles compared to collagen membrane controls. Until the 15th day of culture, cells were rarely observed in direct contact with the elastane fibers, similar to previous observations with elastomeric sheets used in periosteum substitute implants. In the second part of the study textile samples were created from FDA-approved medical sutures comprising silk, expanded polytetrafluoroethylene, and polybutester. Biocompatibility and mechanical stiffness were assessed as a function of sterilization/disinfection mode (steam, ethylene oxide, and serial disinfection with ethanol). Cell proliferation rates did not differ significantly from controls, except for silk-suture containing textiles, which showed bacterial contamination and no viable cells after 15 days' culture for all sterilization methods. Sterilization had mixed (mostly not significant) effects on textile stiffness, except for the case of polybutester suture-based textiles that showed a significant increase in stiffness with ethylene oxide sterilization. In general, all textile combinations exhibited significantly higher stiffness than periosteum. Textiles comprising medical sutures of different stiffnesses arranged in engineered patterns offer a novel means to achieve mechanical gradients in medical device materials, emulating those of nature's own.

Electron Microscopy Sample Preparation Protocol Enabling Nano-to-mesoscopic Mapping of Cellular Connectomes and Their Habitats in Human Tissues and Organs.

Multibeam scanning electron microscopy (multiSEM) provides a technical platform for seamless nano-to-mesoscale mapping of cells in human tissues and organs, which is a major new initiative of the U.S. National Institutes of Health. Such cross-length-scale imaging is expected to provide unprecedented understanding of relationships between cellular health and tissue-organ as well as organismal-scale health outcomes. For example, understanding relationships between loss in cell viability and cell network connectivity enables identification of emergent behaviors and prediction of degenerative disease onset, in organs as diverse as bone and brain, at early timepoints, providing a basis for future treatments and prevention. Developed for rapid throughput imaging of minute defects on semiconductor wafers, multiSEM has recently been adapted for imaging of human organs, their constituent tissues, and their respective cellular inhabitants. Through integration of geospatial approaches, statistical and network modelling, advances in computing and the management of immense datasets, as well as recent developments in machine learning that enable the automation of big data analyses, multiSEM and other cross- cutting imaging technologies have the potential to exert a profound impact on elucidation of disease mechanisms, translating to improvements in human health. Here we provide a protocol for acquisition and preparation of sample specimen sizes of diagnostic relevance for human anatomy and physiology. We discuss challenges and opportunities to integrate this approach with multibeam scanning electron microscopy workflows as well as multiple imaging modalities for mapping of organ and tissue structure and function.

Mechanomics Approaches to Understand Cell Behavior in Context of Tissue Neogenesis, During Prenatal Development and Postnatal Healing.

Mechanomics represents the natural progression of knowledge at the intersection of mechanics and biology with the aim to codify the role of mechanical environment on biological adaptation. Compared to the mapping of the human genome, the challenge of mapping the mechanome remains unsolved. Solving this grand challenge will require both top down and bottom up R&D approaches using experimental and computational tools to visualize and measure adaptation as it occurs. Akin to a mechanical test of a smart material that changes its mechanical properties and local environment under load, stem cells adapt their shape, cytoskeletal architecture, intrinsic mechanical properties, as well as their own niche, through cytoskeletal adaptation as well as up- and down-regulation of structural proteins that modulate their mechanical milieux. Recent advances in live cell imaging allow for unprecedented study and measurements of displacements, shape and volume changes in stem cells, reconfiguring of cytoskeletal machinery (nucleus, cytoskeleton), in response to controlled mechanical forces and stresses applied at cellular boundaries. Coupled with multiphysics computational and virtual power theoretical approaches, these novel experimental approaches enable mechanical testing of stem cells, multicellular templates, and tissues inhabited by stem cells, while the stem cells themselves evolve over time. The novel approach is paving the way to decipher mechanisms of structural and functional adaptation of stem cells in response to controlled mechanical cues. This mini-review outlines integrated approaches and methodologies implemented to date in a series of studies carried out by our consortium. The consortium's body of work is described in context of current roadblocks in the field and innovative, breakthrough solutions and is designed to encourage discourse and cross disciplinary collaboration in the scientific community.

Mechanical modulation of osteochondroprogenitor cell fate.

Mesenchymal cells are natural tissue builders. They exhibit an extraordinary capacity to metamorphize into differentiated cells, using extrinsic spatial and temporal inputs and intrinsic algorithms, as well as to build and adapt their own habitat. In addition to providing a habitat for osteoprogenitor cells, tissues of the skeletal system provide mechanical support and protection for the multiple organs of vertebrate organisms. This review examines the role of mechanics on determination of cell fate during pre-, peri- and postnatal development of the skeleton as well as during tissue genesis and repair in postnatal life. The role of cell mechanics is examined and brought into context of intrinsic cues during mesenchymal condensation. Remarkable new insights regarding structure function relationships in mesenchymal stem cells, and their influence on determination of cell fate are integrated in the context of de novo tissue generation and postnatal repair. Key differences in the formation of osteogenic and chondrogenic condensations are discussed in relation to direct intramembranous and indirect endochondral ossification. New approaches are discussed to elucidate and exploit extrinsic cues to generate tissues in the laboratory and in the clinic.

Idealization of pericellular fluid space geometry and dimension results in a profound underprediction of nano-microscale stresses imparted by fluid drag on osteocytes.

To date, no published study has examined quantitatively the effect of geometric and dimensional idealization on prediction of the mechanical signals imparted by fluid drag to cell surfaces. We hypothesize that this idealization affects the magnitude and range of imparted forces predicted to occur at a subcellular level. Hence, we used computational fluid dynamics to predict magnitudes and spatial variation of fluid velocity and pressure, as well as shear stress, on the cell surface in two- and three-dimensional models of actual and idealized pericellular canalicular geometries. Furthermore, variation in actual pericellular space dimensions was analyzed statistically based on high-resolution transmitted electron micrographs (TEM). Accounting for the naturally occurring protrusions of the pericellular space delineating lamina limitans resulted in predictions of localized stress spikes on the cell surface, up to five times those predicted using idealized geometries. Predictions accounting for actual pericellular geometries approached those required to trigger cell activity in in vitro models. Furthermore, statistical analysis of TEM-based dimensions showed significant variation in the width of the canalicular space as well as the diameter of the cell process, both of which decrease with increasing distance from the cell body. For the first time to our knowledge, this study shows the influence of physiologic geometry per se on the nano-scale flow regimes in bone, and the profound influence of physiologic geometry on force magnitudes and variations imparted locally to cells through load-induced fluid flow.

Knee Joint Tissues Effectively Separate Mixed Sized Molecules Delivered in a Single Bolus to the Heart.

The role of molecular size selectivity in the onset and progression of osteoarthritis (OA), a degenerative disease of the musculoskeletal system and the most common cause of disability in aging adults, is unknown. Here we delivered a mixture of Texas-red (70 kDa), and Rhodamine-green (10 kDa) tagged, dextrans of neutral charge in a single bolus via heart injection to middle aged (8-10 months) and aged (17-19 months) Dunkin-Hartley Guinea pigs, a natural model for OA. We quantified tracer transport in serial-sectioned, cryofixed block specimens after five minutes' circulation. A remarkable separation of the molecules was observed in serial fluorescent images of whole joint sections. The larger, 70 kDa red tracer was abundant in the marrow cavity albeit less prevalent or absent in the bone, cartilage, meniscus and other tissues of the joint. Tissues of the meniscus, ligament, and tendon exhibited abundant 10 kDa tracer; volumes of tissue containing this molecular tracer were significantly lower in older than in younger animals. Surprisingly, muscle fiber bundles exhibited little fluorescence, while their bounding fasciae fluoresced either red or green. Small caliber channels through the articular cartilage appeared to show a degree of green fluorescence not observed in the surrounding cartilage matrix. This study opens up new avenues for study of musculoskeletal physiology in health and disease as well as new strategies for drug delivery.

Prospective Design, Rapid Prototyping, and Testing of Smart Dressings, Drug Delivery Patches, and Replacement Body Parts Using Microscopy Aided Design and ManufacturE (MADAME).

Natural materials exhibit smart properties including gradients in biophysical properties that engender higher order functions, as well as stimuli-responsive properties which integrate sensor and/or actuator capacities. Elucidation of mechanisms underpinning such smart material properties (i), and translation of that understanding (ii), represent two of the biggest challenges in emulating natural design paradigms for design and manufacture of disruptive materials, parts, and products. Microscopy Aided Design And ManufacturE (MADAME) stands for a computer-aided additive manufacturing platform that incorporates multidimensional (multi-D) printing and computer-controlled weaving. MADAME enables the creation of composite design motifs emulating e.g., patterns of woven protein fibers as well as gradients in different caliber porosities, mechanical, and molecular properties, found in natural tissues, from the skin on bones (periosteum) to tree bark. Insodoing, MADAME provides a means to manufacture a new genre of smart materials, products and replacement body parts that exhibit advantageous properties both under the influence of as well as harnessing dynamic mechanical loads to activate material properties (mechanoactive properties). This Technical Report introduces the MADAME technology platform and its associated machine-based workflow (pipeline), provides basic technical background of the novel technology and its applications, and discusses advantages and disadvantages of the approach in context of current 3 and 4D printing platforms.

Biotechnologies toward Mitigating, Curing, and Ultimately Preventing Edema through Compression Therapy.

For a century-old problem, edema and its treatment have gone remarkably unnoticed by the biomedical community. Given the prevalence of lymphedema and its debilitating repercussions, there is an acute need for both efficacy-based measures and clinical standards to guide compression garment design and therapeutic application. This review outlines the current state of the art in compression treatment and suggests an integrated biomedical engineering approach going forward. Characterizing the pressure gradient profiles of commercial compression sleeves is necessary to better understand the role of compression treatment in the mitigation of swelling. Integration of pressure sensor technologies with advanced materials design and manufacture provides a critical path not only to elucidate the mechanisms of but also to improve on current compression-based therapies and associated therapeutic devices.

Design of tissue engineering scaffolds as delivery devices for mechanical and mechanically modulated signals.

New approaches to tissue engineering aim to exploit endogenous strategies such as those occurring in prenatal development and recapitulated during postnatal healing. Defining tissue template specifications to mimic the environment of the condensed mesenchyme during development allows for exploitation of tissue scaffolds as delivery devices for extrinsic cues, including biochemical and mechanical signals, to drive the fate of mesenchymal stem cells seeded within. Although a variety of biochemical signals that modulate stem cell fate have been identified, the mechanical signals conducive to guiding pluripotent cells toward specific lineages are less well characterized. Furthermore, not only is spatial and temporal control of mechanical stimuli to cells challenging, but also tissue template geometries vary with time due to tissue ingrowth and/or scaffold degradation. Hence, a case study was carried out to analyze flow regimes in a testbed scaffold as a first step toward optimizing scaffold architecture. A pressure gradient was applied to produce local (nm-micron) flow fields conducive to migration, adhesion, proliferation, and differentiation of cells seeded within, as well as global flow parameters (micron-mm), including flow velocity and permeability, to enhance directed cell infiltration and augment mass transport. Iterative occlusion of flow channel dimensions was carried out to predict virtually the effect of temporal geometric variation (e.g., due to tissue development and growth) on delivery of local and global mechanical signals. Thereafter, insights from the case study were generalized to present an optimization scheme for future development of scaffolds to be implemented in vitro or in vivo. Although it is likely that manufacture and testing will be required to finalize design specifications, it is expected that the use of the rational design optimization will reduce the number of iterations required to determine final prototype geometries and flow conditions. As the range of mechanical signals conducive to guiding cell fate in situ is further elucidated, these refined design criteria can be integrated into the general optimization rubric, providing a technological platform to exploit nature's endogenous tissue engineering strategies for targeted tissue generation in the lab or the clinic.

Testing of a new one-stage bone-transport surgical procedure exploiting the periosteum for the repair of long-bone defects.

BACKGROUND: A recently proposed one-stage bone-transport surgical procedure exploits the intrinsic osteogenic potential of the periosteum while providing mechanical stability through intramedullary nailing. The objective of this study was to assess the efficacy of this technique to bridge massive long-bone defects in a single stage. METHODS: With use of an ovine femoral model, an in situ periosteal sleeve was elevated circumferentially from healthy diaphyseal bone, which was osteotomized and transported over an intramedullary nail into a 2.54-cm (1-in) critical-sized diaphyseal defect. The defect-bridging and bone-regenerating capacity of the procedure were tested in five groups of seven animals each, which were defined by the absence (Group 1; control) or presence of the periosteal sleeve alone (Group 2), bone graft within the periosteal sleeve (Groups 3 and 5), as well as retention of adherent, vascularized cortical bone chips on the periosteal sleeve with or without bone graft (Groups 4 and 5). The efficacy of the procedure was assessed qualitatively and quantitatively. RESULTS: At sixteen weeks, osseous bridging of the defect was observed in all twenty-eight experimental sheep in which the periosteal sleeve was retained; the defect persisted in the remaining seven control sheep. Among the experimental groups 2 through 5, significant differences were observed in the density of the regenerated bone tissue; the two groups in which vascularized bone chips adhered to the inner surface of the periosteal sleeve (Groups 4 and 5) showed a higher mean bone density in the defect zone (p < 0.02) than did the other groups. In these two groups with the highest bone density, the addition of bone graft was associated with a significantly lower callus density than that observed without bone graft (p < 0.05). The volume of regenerate bone (p < 0.02) was significantly greater in the groups in which the periosteal sleeve was retained than it was in the control group. Among the experimental groups (groups 2 through 5), however, with the numbers studied, no significant differences in the volume of regenerate bone could be attributed to the inclusion of bone graft within the sleeve or to vascularized bone chips remaining adherent to the periosteum. CONCLUSIONS: The novel surgical procedure was shown to be effective in bridging a critical-sized defect in an ovine femoral model. Vascularized bone chips adherent to the inner surface of the periosteal sleeve, without the addition of morselized cancellous bone graft within the sleeve, provide not only a comparable volume of regenerate bone and composite tissue (callus and bone) but also a superior density of regenerate bone compared with that after the addition of bone graft.

Open access to novel dual flow chamber technology for in vitro cell mechanotransduction, toxicity and pharamacokinetic studies.

BACKGROUND: A major stumbling block for researchers developing experimental models of mechanotransduction is the control of experimental variables, in particular the transmission of the mechanical forces at the cellular level. A previous evaluation of state of the art commercial perfusion chambers showed that flow regimes, applied to impart a defined mechanical stimulus to cells, are poorly controlled and that data from studies in which different chambers are utilized can not be compared, even if the target stress regimes are comparable. METHODS: This study provides a novel chamber design to provide both physiologically-based flow regimes, improvements in control of experimental variables, as well as ease of use compared to commercial chambers. This novel design achieves controlled stresses through five gasket designs and both single- and dual-flow regimes. RESULTS: The imparted shear stress within the gasket geometry is well controlled. Fifty percent of the entire area of the 10 x 21 mm universal gasket (Gasket I, designed to impart constant magnitude shear stresses in the center of the chamber where outcome measures are taken), is exposed to target stresses. In the 8 mm diameter circular area at the center of the chamber (where outcome measures are made), over 92% of the area is exposed to the target stress (+/- 2.5%). In addition, other gasket geometries provide specific gradients of stress that vary with distance from the chamber inlet. Bench-top testing of the novel chamber prototype shows improvements, in the ease of use as well as in performance, compared to the other commercial chambers. The design of the chamber eliminates flow deviations due to leakage and bubbles and allows actual flow profiles to better conform with those predicted in computational models. CONCLUSION: The novel flow chamber design provides predictable and well defined mechanical forces at the surface of a cell monolayer, showing improvement over previously tested commercial chambers. The predictability of the imparted stress improves both experiment repeatability as well as the accuracy of inter-study comparisons. Carefully controlling the stresses on cells is critical in effectively mimicking in vivo situations. Overall, the improved perfusion flow chamber provides the needed resolution, standardization and in vitro model analogous to in vivo conditions to make the step towards greater use in research and the opportunity to enter the diagnostic and therapeutic market.

Engineering mechanical gradients in next generation biomaterials - Lessons learned from medical textile design.

Nonwoven and textile membranes have been applied both externally and internally to prescribe boundary conditions for medical conditions as diverse as oedema and tissue defects. Incorporation of mechanical gradients in next generation medical membrane design offers great potential to enhance function in a dynamic, physiological context. Yet the gradient properties and resulting mechanical performance of current membranes are not well described. To bridge this knowledge gap, we tested and compared the mechanical properties of bounding membranes used in both external (compression sleeves for oedema, exercise bands) and internal (surgical membranes) physiological contexts. We showed that anisotropic compression garment textiles, isotropic exercise bands and surgical membranes exhibit similar ranges of resistance to tension under physiologic strains. However, their mechanical gradients and resulting stress-strain relationships show differences in work capacity and energy expenditure. Exercise bands' moduli of elasticity and respective thicknesses allow for controlled, incremental increases in loading to facilitate healing as injured tissues return to normal structure and function. In contrast, the gradients intrinsic to compression sleeve design exhibit gaps in the middle range (1-5N) of physiological strains and also inconsistencies along the length of the sleeve, resulting in less than optimal performance of these devices. These current shortcomings in compression textile and garment design may be addressed in the future through implementation of novel approaches. For example, patterns, fibre compositions, and fibre anisotropy can be incorporated into biomaterial design to achieve seamless mechanical gradients in structure and resulting dynamic function, which would be particularly useful in physiological contexts. These concepts can be applied further to biomaterial design to deliver pressure gradients during movement of oedematous limbs (compression garments) and facilitate transport of molecules and cells during tissue genesis within tissue defects (surgical membranes). STATEMENT OF SIGNIFICANCE: External and internal biomaterial membranes prescribe boundary conditions for treatment of medical disorders, from oedema to tissue defects. Studies are needed to guide the design of next generation biomaterials and devices that incorporate gradient engineering approaches, which offer great potential to enhance function in a dynamic and physiological context. Mechanical gradients intrinsic to currently implemented biomaterials such as medical textiles and surgical interface membranes are poorly understood. Here we characterise quantitatively the mechanics of textile and nonwoven biomaterial membranes for external and internal use. The lack of seamless gradients in compression medical textiles contrasts with the graded mechanical effects achieved by elastomeric exercise bands, which are designed to deliver controlled, incremental increases in loading to facilitate healing as injured tissues return to normal structure and function. Engineering textiles with a prescient choice of fibre composition/size, type of knit/weave and inlay fibres, and weave density/anisotropy will enable creation of fabrics that can deliver spatially and temporally controlled mechanical gradients to maintain force balances at tissue boundaries, e.g. to treat oedema or tissue defects.