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.

Scale-up of nature's tissue weaving algorithms to engineer advanced functional materials.

We are literally the stuff from which our tissue fabrics and their fibers are woven and spun. The arrangement of collagen, elastin and other structural proteins in space and time embodies our tissues and organs with amazing resilience and multifunctional smart properties. For example, the periosteum, a soft tissue sleeve that envelops all nonarticular bony surfaces of the body, comprises an inherently "smart" material that gives hard bones added strength under high impact loads. Yet a paucity of scalable bottom-up approaches stymies the harnessing of smart tissues' biological, mechanical and organizational detail to create advanced functional materials. Here, a novel approach is established to scale up the multidimensional fiber patterns of natural soft tissue weaves for rapid prototyping of advanced functional materials. First second harmonic generation and two-photon excitation microscopy is used to map the microscopic three-dimensional (3D) alignment, composition and distribution of the collagen and elastin fibers of periosteum, the soft tissue sheath bounding all nonarticular bone surfaces in our bodies. Then, using engineering rendering software to scale up this natural tissue fabric, as well as multidimensional weaving algorithms, macroscopic tissue prototypes are created using a computer-controlled jacquard loom. The capacity to prototype scaled up architectures of natural fabrics provides a new avenue to create advanced functional materials.

Periosteum mechanobiology and mechanistic insights for regenerative medicine.

Periosteum is a smart mechanobiological material that serves as a habitat and delivery vehicle for stem cells as well as biological factors that modulate tissue genesis and healing. Periosteum's remarkable regenerative capacity has been harnessed clinically for over two hundred years. Scientific studies over the past decade have begun to decipher the mechanobiology of periosteum, which has a significant role in its regenerative capacity. This integrative review outlines recent mechanobiological insights that are key to modulating and translating periosteum and its resident stem cells in a regenerative medicine context.

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.

Translating Periosteum's Regenerative Power: Insights From Quantitative Analysis of Tissue Genesis With a Periosteum Substitute Implant.

: An abundance of surgical studies during the past 2 centuries provide empirical evidence of periosteum's regenerative power for reconstructing tissues as diverse as trachea and bone. This study aimed to develop quantitative, efficacy-based measures, thereby providing translational guidelines for the use of periosteum to harness the body's own healing potential and generate target tissues. The current study quantitatively and qualitatively demonstrated tissue generation modulated by a periosteum substitute membrane that replicates the structural constituents of native periosteum (elastin, collagen, progenitor cells) and its barrier, extracellular, and cellular properties. It shows the potentiation of the periosteum's regenerative capacity through the progenitor cells that inhabit the tissue, biological factors intrinsic to the extracellular matrix of periosteum, and mechanobiological factors related to implant design and implementation. In contrast to the direct intramembranous bone generated in defects surrounded by patent periosteum in situ, tissue generation in bone defects bounded by the periosteum substitute implant occurred primarily via endochondral mechanisms whereby cartilage was first generated and then converted to bone. In addition, in defects treated with the periosteum substitute, tissue generation was highest along the major centroidal axis, which is most resistant to prevailing bending loads. Taken together, these data indicate the possibility of designing modular periosteum substitute implants that can be tuned for vectorial and spatiotemporal delivery of biological agents and facilitation of target tissue genesis for diverse surgical scenarios and regenerative medicine approaches. It also underscores the potential to develop physical therapy protocols to maximize tissue genesis via the implant's mechanoactive properties. SIGNIFICANCE: In the past 2 centuries, the periosteum, a niche for stem cells and super-smart biological material, has been used empirically in surgery to repair tissues as diverse as trachea and bone. In the past 25 years, the number of articles indexed in PubMed for the keywords "periosteum and tissue engineering" and "periosteum and regenerative medicine" has burgeoned. Yet the biggest limitation to the prescriptive use of periosteum is lack of easy access, giving impetus to the development of periosteum substitutes. Recent studies have opened up the possibility to bank periosteal tissues (e.g., from the femoral neck during routine resection for implantation of hip replacements). This study used an interdisciplinary, quantitative approach to assess tissue genesis in modular periosteum substitute implants, with the aim to provide translational strategies for regenerative medicine and tissue engineering.

Organ-to-Cell-Scale Health Assessment Using Geographical Information System Approaches with Multibeam Scanning Electron Microscopy.

This study combines novel multibeam electron microscopy with a geographical information system approach to create a first, seamless, navigable anatomic map of the human hip and its cellular inhabitants. Using spatial information acquired by localizing relevant map landmarks (e.g. cells, blood vessels), network modeling will enable disease epidemiology studies in populations of cells inhabiting tissues and organs.

Engineering and commercialization of human-device interfaces, from bone to brain.

Cutting edge developments in engineering of tissues, implants and devices allow for guidance and control of specific physiological structure-function relationships. Yet the engineering of functionally appropriate human-device interfaces represents an intractable challenge in the field. This leading opinion review outlines a set of current approaches as well as hurdles to design of interfaces that modulate transfer of information, i.a. forces, electrical potentials, chemical gradients and haptotactic paths, between endogenous and engineered body parts or tissues. The compendium is designed to bridge across currently separated disciplines by highlighting specific commonalities between seemingly disparate systems, e.g. musculoskeletal and nervous systems. We focus on specific examples from our own laboratories, demonstrating that the seemingly disparate musculoskeletal and nervous systems share common paradigms which can be harnessed to inspire innovative interface design solutions. Functional barrier interfaces that control molecular and biophysical traffic between tissue compartments of joints are addressed in an example of the knee. Furthermore, we describe the engineering of gradients for interfaces between endogenous and engineered tissues as well as between electrodes that physically and electrochemically couple the nervous and musculoskeletal systems. Finally, to promote translation of newly developed technologies into products, protocols, and treatments that benefit the patients who need them most, regulatory and technical challenges and opportunities are addressed on hand from an example of an implant cum delivery device that can be used to heal soft and hard tissues, from brain to bone.

Prospective randomised evaluation of a collagen/thrombin and autologous platelet haemostatic agent during cementless total hip arthroplasty.

BACKGROUND: Total hip arthroplasty (THA) can be associated with substantial peri-operative blood loss which can negatively influence a patient's clinical outcome. Few haemostatic agents have been tested in THA. The aim of this study was to determine whether the use of a collagen/thrombin/ autologous platelet haemostatic agent would result in a significant decrease of blood transfusions for patients undergoing primary THA. MATERIALS AND METHODS: THA patients meeting inclusion/exclusion criteria (n=109) were enrolled in this prospective, double-blind trial and randomised to a treatment arm (standard haemostatic methods plus haemostatic agent) or control arm (standard haemostatic methods only). The primary outcome was transfusion. Secondary outcome measures included peri-operative narcotic usage and post-operative haemoglobin levels, pain scores, function, and general health quality of life. RESULTS: Transfusions were required by 5/60 (8.3%) patients in the treatment group and 7/49 (14.3%) in the control group (p=0.33). The mean number of units transfused was not significantly different between the treatment group (2.2±1.3) and the control group (1.6±0.5) (p=0.36). Haemoglobin values on post-operative days 1, 2, and 3 were significantly higher in the treatment group (p=0.002, 0.04, and 0.02, respectively). Hip Disability and Osteoarthritis Outcome Score and Short Form-12 scores were not different between the two groups. DISCUSSION: In relatively healthy patients undergoing primary cementless THA there was no significant difference in number of transfusions or number of units transfused. It is unlikely that we will routinely use the investigated haemostatic agent to reduce blood loss in a healthy patient undergoing THA. The product may have some benefit in patients who refuse blood transfusions, have minimal ability to increase blood volume, are undergoing total joint revision, or have markedly low pre-operative haemoglobin levels, but this needs to be demonstrated.

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.

Arthritic periosteal tissue from joint replacement surgery: a novel, autologous source of stem cells.

The overarching aim of this study is to assess the feasibility of using periosteal tissue from the femoral neck of arthritic hip joints, usually discarded in the normal course of hip replacement surgery, as an autologous source of stem cells. In addition, the study aims to characterize intrinsic differences between periosteum-derived cell (PDC) populations, isolated via either enzymatic digestion or a migration assay, including their proliferative capacity, surface marker expression, and multipotency, relative to commercially available human bone marrow-derived stromal cells (BMSCs) cultured under identical conditions. Commercial BMSCs and PDCs were characterized in vitro, using a growth assay, flow cytometry, as well as assay of Oil Red O, alizarin red, and Safranin O/Fast Green staining after respective culture in adipo-, osteo-, and chondrogenic media. Based on these outcome measures, PDCs exhibited proliferation rate, morphology, surface receptor expression, and multipotency similar to those of BMSCs. No significant correlation was observed between outcome measures and donor age or diagnosis (osteoarthritis [OA] and rheumatoid arthritis [RA], respectively), a profound finding given recent rheumatological studies indicating that OA and RA share not only common biomarkers and molecular mechanisms but also common pathophysiology, ultimately resulting in the need for joint replacement. Furthermore, PDCs isolated via enzymatic digestion and migration assay showed subtle differences in surface marker expression but otherwise no significant differences in proliferation or multipotency; the observed differences in surface marker expression may indicate potential effects of isolation method on the population of cells isolated and/or the behavior of the respective isolated cell populations. This study demonstrates, for the first time to our knowledge, the feasibility of using arthritic tissue resected during hip replacement as a source of autologous stem cells. In sum, periosteum tissue that is resected with the femoral neck in replacing the hip represents an unprecedented and, to date, unstudied source of stem cells from OA and RA patients. Follow-up studies will determine the degree to which this new, autologous source of stem cells can be banked for future use.

Lithotripsy stimulates new bone formation and mitigates loss of bone due to disuse in aged rats.

BACKGROUND: Normal "wear and tear" of bones during weight bearing activity creates microdamage that triggers bone to heal itself. OBJECTIVE: A controlled laboratory study was carried out to determine the effect of lithotripsy on bone apposition and resorption in osteopenic, hind-limb suspended, aged rats compared to age-matched controls allowed normal weight bearing (cage) activity. METHODS: First, we tested the feasibility of using a clinical lithotripsy device, designed for treatment of kidney stones, to create microdamage in bone. In a second step, we tested the hypothesis that microdamage induced through lithotripsy treatment increases bone apposition in aged rats. In a third step, we exposed osteopenic, aged rats to lithotripsy to evaluate the effectiveness of lithotripsy in counteracting bone loss due to simulated disuse. RESULTS: Both in aged, weight bearing as well as aged, osteopenic rats, we showed that lithotripsy effectively increases the area of bone apposition along the periosteal and endosteal surfaces. While new bone apposition concentrates in areas of lithotripsy treatment in aged bone of weight bearing rats, new bone apposition extends beyond the immediate treatment site (to the contralateral limb) of osteopenic animals. Furthermore, bone resorption decreases in osteopenic (hindlimb suspended) and aged rat femora treated with lithotripsy, compared to baseline and hindlimb suspended controls. This decrease in resorption is not observed in the contralateral limb of osteopenic animals. CONCLUSION: Taken as a whole, lithotripsy may offer a viable treatment method for disuse osteopenia or osteoporosis, particularly for aging individuals or for those who are limited in their weight bearing activities.

Periosteum, bone's "smart" bounding membrane, exhibits direction-dependent permeability.

The periosteum serves as bone's bounding membrane, exhibits hallmarks of semipermeable epithelial barrier membranes, and contains mechanically sensitive progenitor cells capable of generating bone. The current paucity of data regarding the periosteum's permeability and bidirectional transport properties provided the impetus for the current study. In ovine femur and tibia samples, the periosteum's hydraulic permeability coefficient, k, was calculated using Darcy's Law and a custom-designed permeability tester to apply controlled, volumetric flow of phosphate-buffered saline through periosteum samples. Based on these data, ovine periosteum demonstrates mechanically responsive and directionally dependent (anisotropic) permeability properties. At baseline flow rates comparable to interstitial fluid flow (0.5 µL/s), permeability is low and does not exhibit anisotropy. In contrast, at high flow rates comparable to those prevailing during traumatic injury, femoral periosteum exhibits an order of magnitude higher permeability compared to baseline flow rates. In addition, at high flow rates permeability exhibits significant directional dependence, with permeability higher in the bone to muscle direction than vice versa. Furthermore, compared to periosteum in which the intrinsic tension (pre-stress) is maintained, free relaxation of the tibial periosteum after resection significantly increases its permeability in both flow directions. Hence, the structure and mechanical stress state of periosteum influences its role as bone's bounding membrane. During periods of homeostasis, periosteum may serve as a barrier membrane on the outer surface of bone, allowing for equal albeit low quiescent molecular communication between tissue compartments including bone and muscle. In contrast, increases in pressure and baseline flow rates within the periosteum resulting from injury, trauma, and/or disease may result in a significant increase in periosteum permeability and consequently in increased molecular communication between tissue compartments. Elucidation of the periosteum's permeability properties is key to understanding periosteal mechanobiology in bone health and healing, as well as to elucidate periosteum structure and function as a smart biomaterial that allows bidirectional and mechanically responsive fluid transport.

Surgical membranes as directional delivery devices to generate tissue: testing in an ovine critical sized defect model.

PURPOSE: Pluripotent cells residing in the periosteum, a bi-layered membrane enveloping all bones, exhibit a remarkable regenerative capacity to fill in critical sized defects of the ovine femur within two weeks of treatment. Harnessing the regenerative power of the periosteum appears to be limited only by the amount of healthy periosteum available. Here we use a substitute periosteum, a delivery device cum implant, to test the hypothesis that directional delivery of endogenous periosteal factors enhances bone defect healing. METHODS: Newly adapted surgical protocols were used to create critical sized, middiaphyseal femur defects in four groups of five skeletally mature Swiss alpine sheep. Each group was treated using a periosteum substitute for the controlled addition of periosteal factors including the presence of collagen in the periosteum (Group 1), periosteum derived cells (Group 2), and autogenic periosteal strips (Group 3). Control group animals were treated with an isotropic elastomer membrane alone. We hypothesized that periosteal substitute membranes incorporating the most periosteal factors would show superior defect infilling compared to substitute membranes integrating fewer factors (i.e. Group 3>Group 2>Group 1>Control). RESULTS: Based on micro-computed tomography data, bone defects enveloped by substitute periosteum enabling directional delivery of periosteal factors exhibit superior bony bridging compared to those sheathed with isotropic membrane controls (Group 3>Group 2>Group 1, Control). Quantitative histological analysis shows significantly increased de novo tissue generation with delivery of periosteal factors, compared to the substitute periosteum containing a collagen membrane alone (Group 1) as well as compared to the isotropic control membrane. Greatest tissue generation and maximal defect bridging was observed when autologous periosteal transplant strips were included in the periosteum substitute. CONCLUSION: Periosteum-derived cells as well as other factors intrinsic to periosteum play a key role for infilling of critical sized defects.

Multiscale mechanobiology of de novo bone generation, remodeling and adaptation of autograft in a common ovine femur model.

The link between mechanics and biology in the generation and the adaptation of bone has been studied for more than a century in the context of skeletal development and fracture healing. However, the interplay between mechanics and biology in de novo generation of bone in postnatal defects as well as healing of morcellized bone graft or massive cortical bone autografts is less well understood. To address this, here we integrate insights from our previously published studies describing the mechanobiology on both de novo bone generation and graft healing in a common ovine femoral defect model. Studying these effects in a common experimental model provides a unique opportunity to elucidate factors conducive to harnessing the regenerative power of the periosteum, and ultimately, to provide mechanistic insights into the multiscale mechanobiology of bone generation, remodeling and adaptation. Taken together, the studies indicate that, as long as adequate, directional transport of cells and molecules can be insured (e.g. with periosteum in situ or a delivery device), biological factors intrinsic to the periosteum suffice to bridge critical sized bone defects, even in the absence of a patent blood supply. Furthermore, mechanical stimuli are crucial for the success of periosteal bone generation and bone graft healing. Interestingly, areas of highest periosteal strain around defects correlate with greatest amounts albeit not greatest mineralization of newly generated bone. This may indicate a role for convection enhanced transport of cells and molecules in modulation of tissue generation by pluripotent cells that ingress into the defect center, away from the periosteum and toward the surface of the intramedullary nail that fills the medullary cavity. These insights bring us much closer to understanding the mechanobiological environment and stimuli that stimulate the proliferation and differentiation of periosteum-derived progenitor cells and ultimately drive the generation of new bone tissue. Furthermore, these insights provide a foundation to create virtual predictive computational models of bone mechanophysiology, to develop cell seeding protocols for scale up and manufacture of engineered tissues, to optimize surgical procedures, and to develop post-surgical therapies with the ultimate goal of achieving the best possible healing outcomes for treatment and/or reconstruction of postnatal bone defects.

Major and minor centroidal axes serve as objective, automatable reference points to test mechanobiological hypotheses using histomorphometry.

Recent studies show that minor and major centroidal axes (CA) of long bone cross sections provide valuable predictions of prevailing loading patterns in age and treatment matched cohorts of animals. Furthermore, using CA, we recently showed that the degree of mineralization and area of woven bone laid down in the first two weeks after creation of a critical sized bone defect relate inversely and correlate significantly to loading patterns. Here, we aim to determine how closely independent measures of the spatial distribution of bone apposition determined using the major and minor CA as reference points correlate to those using anatomically defined axes as reference points. In histological sections from the previous study, we found no statistically significant difference between the anatomical and centroidal axes with respect to the centroid location or axis rotation, but there is a significant albeit small difference in the average distance between centroids. Outcome measures calculated in areas of bone defined by 15°, 30°, 45°, 60°, or 90° sectors when using the CA differ less than 5% from those calculated using anatomical axes as reference points. Hence, the major and minor CA provide objective reference points for comparison of mechanobiological outcome measures between animals in matched cohorts. Calculation of major and minor CA is automated, which reduces the potential for observer bias. A major advantage of using the major and minor CA as reference points is that it allows for direct relation of outcome measures to loading patterns in age and treatment matched cohorts, ultimately providing a tool to test mechanobiological hypotheses on histological cross sections of bone.

Net change in periosteal strain during stance shift loading after surgery correlates to rapid de novo bone generation in critically sized defects.

In an ovine femur model, proliferative woven bone fills critically sized defects enveloped by periosteum within 2 weeks of treatment with the one-stage bone-transport surgery. We hypothesize that mechanical loading modulates this process. Using high-definition optical strain measurements we determined prevailing periosteal strains for normal and surgically treated ovine femora subjected ex vivo to compressive loads simulating in vivo stance shifting (n = 3 per group, normal vs. treated). We determined spatial distribution of calcein green, a label for bone apposition in first the 2 weeks after surgery, in 15°, 30°, and 45° sectors of histological cross sections through the middle of the defect zone (n = 6 bones, three to four sections per bone). Finally, we correlated early bone formation to either the maximal periosteal strain or the net change in maximal periosteal strain. We found that treatment with the one-stage bone-transport surgery profoundly changes the mechanical environment of cells within the periosteum during stance shift loading. The pattern of early bone formation is repeatable within and between animals and relates significantly to the actual strain magnitude prevailing in the periosteum during stance shift loading. Interestingly, early bone apposition after the surgery correlates well to the maximal net change in strain (above circa 2000-3000 µÎµ, in tension or compression) rather than strain magnitude per se, providing further evidence that changes in cell shape may drive mechanoadaptation by progenitor cells. These important insights regarding mechanobiological factors that enhance rapid bone generation in critically sized defects can be translated to the tissue and organ scale, providing a basis for the development of best practices for clinical implementation and the definition of movement protocols to enhance the regenerative effect.

Effects of mechanical loading patterns, bone graft, and proximity to periosteum on bone defect healing.

The goal of this study is to elucidate whether mechanobiological factors, including mechanical loading patterns, presence of bone graft, and proximity to the periosteum, correlate to de novo tissue generation and healing in critical sized long bone defects, which are enveloped by periosteum in situ and are bridged at 16 weeks, in sheep femora. Quantitative histomorphometric measures of defect cross sections show that, along the axis least able to resist bending loads (minor centroidal axis, CA), bone laid down in the first two weeks after surgery exhibits more mineralization albeit less total area compared to bone along the axis most able to resist bending loads (major CA). Similarly, areas of the cross section along the minor CA show a higher degree of perfusion albeit less total area of perfusion compared to bone along the major CA. Furthermore, proximity to the periosteal niche, in conjunction with the presence of bone graft and predominant loading patterns, relates significantly to the radial distribution of early bone apposition and perfusion of bone at 16 weeks after surgery (linear regression with R(2)>0.80). In the absence of graft, early bone density is relatively evenly distributed in the defect zone, as is the intensity of perfused tissue. As measured by a steeper average slope in intensity of fluorochrome (new bone) distribution between the periosteum and the IM nail, the presence of bone graft retards initial bone formation in the defect zone and is associated with less evenly distributed tissue perfusion (steeper slope) persisting 16 weeks after surgery. Finally, although the mean area of bone resorption is not significantly different within or between groups defined by the presence of graft and/or mechanical loading patterns in the defect zone, the mean area of infilling resorption spaces is significantly higher in areas of the defect zone least able to resist bending (minor CA) but is not significantly related to the presence of bone graft. To our knowledge, the use of the major and minor centroidal axes to relate prevailing mechanical loading patterns to area and density of early bone generation in bone defects has not been reported prior to this study and may provide a new means to assess structure-function relationships in de novo bone generation and healing of bone defects.

Role of mechanical loading in healing of massive bone autografts.

We assessed healing of a 3.5 cm autograft transport segment, denuded of periosteum, and docked to the healthy distal femur with an intramedullary nail. We hypothesized that healing relates to proximity to the healthy distal femur and to mechanical loading patterns. Total bone area, area of new bone apposition, and quality of new bone formed in the 2 weeks after surgery, and area and degree of perfusion 16 weeks after surgery were measured as a function of proximity and loading patterns (as defined by the major and minor centroidal axes, CA). At 16 weeks, no significant differences in early bone apposition or perfusion were observed as a function of distance from the healthy distal femur. Qualitatively, bone was well perfused, both vascularly and pericellularly, and highly remodeled. When cross-sections were pooled from distal to proximal through the docking zone and normalized for total bone area, significant differences in the amount of early proliferative woven bone were related to loading patterns. In contrast, no differences in normalized perfusion area were attributable to loading patterns. Furthermore, early bone apposition and perfusion decreased with increasing radial distance from the bone surface toward the intramedullary nail. Finally, no differences were observed in areas of resorption within the docking zone compared to baseline levels measured in the control (in bone removed to create the defect zone at the time of surgery). Interestingly, infilling of resorption spaces within docking zone specimens related significantly to predominant loading patterns, where areas within the major CA exhibited significantly more infilling.

Bone regeneration in long-bone defects: tissue compartmentalisation? In vivo study on bone defects in sheep.

Regeneration of living tissue varies with species, age and type of tissue, and undoubtedly with the biological and mechanical environment of the precise tissue. Autologous cancellous bone grafting is a well-known technique that provides bony regeneration. We investigated the efficiency of autologous bone grafting in a well-vascularised muscle environment, and additionally when isolated from the muscle and connected only to the bony environment. We designed a reproducible animal model producing a stable 3cm middiaphyseal bone and periosteal defect on sheep femurs and created a foreign-body membrane with a temporary poly-methylmethacrylate spacer. The foreign-body membrane had the outer dimension of the removed bone segment. We then ascertained the bony regeneration potential within the bone defect using autologous cancellous bone graft. Regeneration of bone is enhanced considerably by an autologous foreign-body membrane that separates the interfragmentary space from the muscular environment. This effect is independent of the autologous bone graft. The results suggest that bone behaves like a compartment that protects its specific humoral or its cellular environment, or both. Regeneration of bone can be enhanced by compartmentalisation of the bone defect.

Bilateral periprosthetic joint infection caused by Salmonella enterica serotype Enteritidis, and identification of Salmonella sp using molecular techniques.

Salmonella septic arthritis is rare. Our objective was to identify bacterial species from joint fluid using a broad-range real-time PCR and pyrosequencing technique. We describe a case of bilateral Salmonella enterica serotype Enteritidis infection of right and left total knee arthroplasties. DNA was extracted from the joint fluid of the left knee, amplified by PCR, and the amplicons were evaluated by pyrosequencing. The patient was treated with ciprofloxacin, and the polyethylene liners were replaced in both knees. The results of pyrosequencing detected a Salmonella species. To the best of our knowledge, this is the first report describing the detection of Salmonella in joint fluid by universal PCR followed by pyrosequencing.