Cell and matrix morphology in articular cartilage from adult human knee and ankle joints suggests depth-associated adaptations to biomechanical and anatomical roles

OBJECTIVE Marked differences exist between human knee and ankle joints regarding risks and progression of osteoarthritis (OA). Pathomechanisms of degenerative joint disease may therefore differ in these joints, due to differences in tissue structure and function. Focussing on structural issues which are design goals for tissue engineering, we compared cell and matrix morphologies in different anatomical sites of adult human knee and ankle joints. METHODS Osteochondral explants were acquired from knee and ankle joints of deceased persons aged 20 to 40 years and analyzed for cell, matrix and tissue morphology using confocal and electron microscopy and unbiased stereological methods. Variations associated with joint (knee versus ankle) and biomechanical role (convex versus concave articular surfaces) were identified by 2-way analysis of variance and post-hoc analysis. RESULTS Knee cartilage exhibited higher cell densities in the superficial zone than ankle cartilage. In the transitional zone, higher cell densities were observed in association with convex versus concave articular surfaces, without significant differences between knee and ankle cartilage. Highly uniform cell and matrix morphologies were evident throughout the radial zone in the knee and ankle, regardless of tissue biomechanical role. Throughout the knee and ankle cartilage sampled, chondron density was remarkably constant at approximately 4.2×10(6) chondrons/cm(3). CONCLUSION Variation of cartilage cell and matrix morphologies with changing joint and biomechanical environments suggests that tissue structural adaptations are performed primarily by the superficial and transitional zones. Data may aid the development of site-specific cartilage tissue engineering, and help identify conditions where OA is likely to occur.

Cell therapy for intervertebral disc repair: advancing cell therapy from bench to clinics

Intervertebral disc (IVD) degeneration is a major cause of pain and disability; yet therapeutic options are limited and treatment often remains unsatisfactory. In recent years, research activities have intensified in tissue engineering and regenerative medicine, and pre-clinical studies have demonstrated encouraging results. Nonetheless, the translation of new biological therapies into clinical practice faces substantial barriers. During the symposium "Where Science meets Clinics", sponsored by the AO Foundation and held in Davos, Switzerland, from September 5-7, 2013, hurdles for translation were outlined, and ways to overcome them were discussed. With respect to cell therapy for IVD repair, it is obvious that regenerative treatment is indicated at early stages of disc degeneration, before structural changes have occurred. It is envisaged that in the near future, screening techniques and non-invasive imaging methods will be available to detect early degenerative changes. The promises of cell therapy include a sustained effect on matrix synthesis, inflammation control, and prevention of angio- and neuro-genesis. Discogenic pain, originating from "black discs" or annular injury, prevention of adjacent segment disease, and prevention of post-discectomy syndrome were identified as prospective indications for cell therapy. Before such therapy can safely and effectively be introduced into clinics, the identification of the patient population and proper standardisation of diagnostic parameters and outcome measurements are indispensable. Furthermore, open questions regarding the optimal cell type and delivery method need to be resolved in order to overcome the safety concerns implied with certain procedures. Finally, appropriate large animal models and well-designed clinical studies will be required, particularly addressing safety aspects.

An educational review of cartilage repair: precepts & practice - myths & misconceptions - progress & prospects.

OBJECTIVE The repair of cartilaginous lesions within synovial joints is still an unresolved and weighty clinical problem. Although research activity in this area has been indefatigably sustained, no significant progress has been made during the past decade. The aim of this educational review is to heighten the awareness amongst students and scientists of the basic issues that must be tackled and resolved before we can hope to escape from the whirlpool of stagnation into which we have fallen: cartilage repair redivivus! DESIGN Articular-cartilage lesions may be induced traumatically (e.g., by sports injuries and occupational accidents) or pathologically during the course of a degenerative disease (e.g., osteoarthritis). This review addresses the biological basis of cartilage repair and surveys current trends in treatment strategies, focussing on those that are most widely adopted by orthopaedic surgeons [viz., abrasive chondroplasty, microfracturing/microdrilling, osteochondral grafting and autologous-chondrocyte implantation (ACI)]. Also described are current research activities in the field of cartilage-tissue engineering, which, as a therapeutic principle, holds more promise for success than any other experimental approach. RESULTS AND CONCLUSIONS Tissue engineering aims to reconstitute a tissue both structurally and functionally. This process can be conducted entirely in vitro, initially in vitro and then in vivo (in situ), or entirely in vivo. Three key constituents usually form the building blocks of such an approach: a matrix scaffold, cells, and signalling molecules. Of the proposed approaches, none have yet advanced beyond the phase of experimental development to the level of clinical induction. The hurdles that need to be surmounted for ultimate success are discussed.

Current aspects and future prospects of total anorectal reconstruction - a critical and comprehensive review of the literature

PURPOSE: Many rectal cancer patients undergo abdominoperineal excision worldwide every year. Various procedures to restore perineal (pseudo-) continence, referred to as total anorectal reconstruction, have been proposed. The best technique, however, has not yet been defined. In this study, the different reconstruction techniques with regard to morbidity, functional outcome and quality of life were analysed. Technical and timing issues (i.e. whether the definitive procedure should be performed synchronously or be delayed), oncological safety, economical aspects as well as possible future improvements are further discussed. METHODS: A MEDLINE and EMBASE search was conducted to identify the pertinent multilingual literature between 1989 and 2013. All publications meeting the defined inclusion/exclusion criteria were eligible for analysis. RESULTS: Dynamic graciloplasty, artificial bowel sphincter, circular smooth muscle cuff or gluteoplasty result in median resting and squeezing neo-anal pressures that equate to the measurements found in incontinent patients. However, quality of life was generally stated to be good by patients who had undergone the procedures, despite imperfect continence, faecal evacuation problems and a considerable associated morbidity. Many patients developed an alternative perception for the urge to defecate that decisively improved functional outcome. Theoretical calculations suggested cost-effectiveness of total anorectal reconstruction compared well to life with a permanent colostomy. CONCLUSIONS: Many patients would be highly motivated to have their abdominal replaced by a functional perineal colostomy. Given the considerable morbidity and questionable functional outcome of current reconstruction technique improvements are required. Tissue engineering might be an option to design an anatomically and physiologically matured, and customised continence organ.

Primary human lung pericytes support and stabilize in-vitro perfusable microvessels.

The formation of blood vessels is a complex tissue-specific process that plays a pivotal role during developmental processes, in wound healing, cancer progression, fibrosis and other pathologies. To study vasculogenesis and vascular remodeling in the context of the lung, we developed an in-vitro microvascular model that closely mimics the human lung microvasculature in terms of 3D architecture, accessibility, functionality and cell types. Human pericytes from the distal airway were isolated and characterized using flow cytometry. To assess their role in the generation of normal microvessels, lung pericytes were mixed in fibrin gel and seeded into well-defined microcompartments together with primary endothelial cells (HUVEC). Patent microvessels covering an area of 3.1 mm2 formed within 3-5 days and were stable for up to 14 days. Soluble signals from the lung pericytes were necessary to establish perfusability, and pericytes migrated towards endothelial microvessels. Cell-cell communication in the form of adherens and tight junctions, as well as secretion of basement membrane was confirmed using transmission electron microscopy and immunocytochemistry on chip. Direct co-culture of pericytes with endothelial cells decreased the microvascular permeability by one order of magnitude from 17.8∙10-6 cm/s to 2.0∙10-6 cm/s and led to vessels with significantly smaller and less variable diameter. Upon phenylephrine administration, vasoconstriction was observed in microvessels lined with pericytes but not in endothelial microvessels only. Perfusable microvessels were also generated with human lung microvascular endothelial cells and lung pericytes. Human lung pericytes were thus shown to have a prominent influence on microvascular morphology, permeability, vasoconstriction and long-term stability in an in-vitro microvascular system. This biomimetic platform opens new possibilities to test functions and interactions of patient-derived cells in a physiologically relevant microvascular setting.

Development and characterization of a scaffold-free 3D spheroid model of iPSC-derived human cardiomyocytes

Purpose: Cardiomyocytes are terminally differentiated cells in the adult heart and ischemia and cardiotoxic compounds can lead to cell death and irreversible decline of cardiac function. As testing platforms, isolated organs and primary cells from rodents have been the standard in research and toxicology, but there is a need for better models that more faithfully recapitulate native human biology. Hence, a new in vitro model comprising the advantages of 3D cell culture and the availability of induced pluripotent stem cells (iPSC) from human origin was developed and characterized. Methods: Human cardiomyocytes (CMs) derived from induced pluripotent stem cells (iPSCs) were studied in standard 2D culture and as cardiac microtissues (MTs) formed in hanging drops. 2D cultures were examined using immunofluorescence microscopy and Western blotting while the cardiac MTs were subjected to immunofluorescence, contractility, and pharmacological investigations. Results: iPSC-derived CMs in 2D culture showed well-formed myofibrils, cell-cell contacts positive for connexin-43, and other typical cardiac proteins. The cells reacted to pro-hypertrophic growth factors with a substantial increase in myofibrils and sarcomeric proteins. In hanging drop cultures, iPSC-derived cardiomyocytes formed spheroidal MTs within 4 days showing a homogeneous tissue structure with well-developed myofibrils extending throughout the whole spheroid without a necrotic core. MTs showed spontaneous contractions for more than 4 weeks that were recorded by optical motion tracking, sensitive to temperature, and responsive to electrical pacing. Contractile pharmacology was tested with several agents known to modulate cardiac rate and viability. Calcium-transients underlay the contractile activity and were also responsive to electrical stimulation, caffeine-induced Ca2+-release, extracellular calcium levels. Conclusions: 3D culture using iPSC-derived human cardiomyocytes provides an organoid human-based cellular platform that is free of necrosis and recapitulates vital cardiac functionality, thereby providing new and promising relevant model for the evaluation and development of new therapies and detection of cardiotoxicity.

Periodontal and peri-implant wound healing following laser therapy

Laser irradiation has numerous favorable characteristics, such as ablation or vaporization, hemostasis, biostimulation (photobiomodulation) and microbial inhibition and destruction, which induce various beneficial therapeutic effects and biological responses. Therefore, the use of lasers is considered effective and suitable for treating a variety of inflammatory and infectious oral conditions. The CO2 , neodymium-doped yttrium-aluminium-garnet (Nd:YAG) and diode lasers have mainly been used for periodontal soft-tissue management. With development of the erbium-doped yttrium-aluminium-garnet (Er:YAG) and erbium, chromium-doped yttrium-scandium-gallium-garnet (Er,Cr:YSGG) lasers, which can be applied not only on soft tissues but also on dental hard tissues, the application of lasers dramatically expanded from periodontal soft-tissue management to hard-tissue treatment. Currently, various periodontal tissues (such as gingiva, tooth roots and bone tissue), as well as titanium implant surfaces, can be treated with lasers, and a variety of dental laser systems are being employed for the management of periodontal and peri-implant diseases. In periodontics, mechanical therapy has conventionally been the mainstream of treatment; however, complete bacterial eradication and/or optimal wound healing may not be necessarily achieved with conventional mechanical therapy alone. Consequently, in addition to chemotherapy consisting of antibiotics and anti-inflammatory agents, phototherapy using lasers and light-emitting diodes has been gradually integrated with mechanical therapy to enhance subsequent wound healing by achieving thorough debridement, decontamination and tissue stimulation. With increasing evidence of benefits, therapies with low- and high-level lasers play an important role in wound healing/tissue regeneration in the treatment of periodontal and peri-implant diseases. This article discusses the outcomes of laser therapy in soft-tissue management, periodontal nonsurgical and surgical treatment, osseous surgery and peri-implant treatment, focusing on postoperative wound healing of periodontal and peri-implant tissues, based on scientific evidence from currently available basic and clinical studies, as well as on case reports.

A novel organ-slice culturing system to simulate meniscal repair: Proof of concept using a synovium-based pool of meniscoprogenitor cells.

Meniscal injuries can occur secondary to trauma or be instigated by the changes in knee-joint function that are associated with aging, osteo- and rheumatoid arthritis, disturbances in gait and obesity. Sixty per cent of persons over 50 years of age manifest signs of meniscal pathology. The surgical and arthroscopic measures that are currently implemented to treat meniscal deficiencies bring only transient relief from pain and effect but a temporary improvement in joint function. Although tissue-engineering-based approaches to meniscal repair are now being pursued, an appropriate in-vitro model has not been conceived. The aim of this study was to develop an organ-slice culturing system to simulate the repair of human meniscal lesions in vitro. The model consists of a ring of bovine meniscus enclosing a chamber that represents the defect and reproduces its sequestered physiological microenvironment. The defect, which is closed with a porous membrane, is filled with fragments of synovial tissue, as a source of meniscoprogenitor cells, and a fibrin-embedded, calcium-phosphate-entrapped depot of the meniscogenic agents BMP-2 and TGF-ß1. After culturing for 2 to 6 weeks, the constructs were evaluated histochemically and histomorphometrically, as well as immunohistochemically for the apoptotic marker caspase 3 and collagen types I and II. Under the defined conditions, the fragments of synovium underwent differentiation into meniscal tissue, which bonded with the parent meniscal wall. Both the parent and the neoformed meniscal tissue survived the duration of the culturing period without significant cell losses. The concept on which the in-vitro system is based was thus validated. This article is protected by copyright. All rights reserved.

Análisis de micromovilidad en vástagos femorales mediante ensayo dinámico de oscilaciones libres

La artroplastia de cadera se considera uno de los mayores avances quirúrgicos de la Medicina. La aplicación de esta técnica de Traumatología se ha incrementado notablemente en los últimos anos, a causa principalmente del progresivo incremento de la esperanza de vida. En efecto, con la edad aumentan los problemas de artrosis y osteoporosis, enfermedades típicas de las articulaciones y de los huesos que requieren en muchos casos la sustitución protésica total o parcial de la articulación. El buen comportamiento funcional de una prótesis depende en gran medida de la estabilidad primaria, es decir, el correcto anclaje de la prótesis en el momento de su implantación. Las prótesis no cementadas basan su éxito a largo plazo en la osteointegración que tiene lugar entre el material protésico y el tejido óseo, y para lograrla es imprescindible conseguir unas buenas condiciones de estabilidad primaria. El aflojamiento aséptico es la principal causa de fallo de artroplastia total de cadera. Este es un fenómeno en el que, debido a complejas interacciones de factores mecánicos y biológicos, se producen movimientos relativos que comprometen la funcionalidad del implante. La minimización de los correspondientes danos depende en gran medida de la detección precoz del aflojamiento. Para lograr la detección temprana del aflojamiento aséptico del vástago femoral se han ensayado diferentes técnicas, tanto in vivo como in vitro: análisis numéricos y técnicas experimentales basadas en sensores de movimientos provocados por cargas transmitidas natural o artificialmente, tales como impactos o vibraciones de distintas frecuencias. Los montajes y procedimientos aplicados son heterogéneos y, en muchas ocasiones, complejos y costosos, no existiendo acuerdo sobre una técnica simple y eficaz de aplicación general. Asimismo, en la normativa vigente que regula las condiciones que debe cumplir una prótesis previamente a su comercialización, no hay ningún apartado referido específicamente a la evaluación de la bondad del diseño del vástago femoral con respecto a la estabilidad primaria. El objetivo de esta tesis es desarrollar una metodología para el análisis, in vitro, de la estabilidad de un vástago femoral implantado, a fin de poder evaluar las técnicas de implantación y los diferentes diseños de prótesis previamente a su oferta en el mercado. Además se plantea como requisito fundamental que el método desarrollado sea sencillo, reversible, repetible, no destructivo, con control riguroso de parámetros (condiciones de contorno de cargas y desplazamientos) y con un sistema de registro e interpretación de resultados rápido, fiable y asequible. Como paso previo, se ha realizado un análisis cualitativo del problema de contacto en la interfaz hueso-vástago aplicando una técnica optomecánica del campo continuo (fotoelasticidad). Para ello se han fabricado tres modelos en 2D del conjunto hueso-vástago, simulando tres tipos de contactos en la interfaz: contacto sin adherencia y con holgura, contacto sin adherencia y sin holgura, y contacto con adherencia y homogéneo. Aplicando la misma carga a cada modelo, y empleando la técnica de congelación de tensiones, se han visualizado los correspondientes estados tensionales, siendo estos más severos en el modelo de unión sin adherencia, como cabía esperar. En todo caso, los resultados son ilustrativos de la complejidad del problema de contacto y confirman la conveniencia y necesidad de la vía experimental para el estudio del problema. Seguidamente se ha planteado un ensayo dinámico de oscilaciones libres con instrumentación de sensores resistivos tipo galga extensométrica. Las muestras de ensayo han sido huesos fémur en todas sus posibles variantes: modelos simplificados, hueso sintético normalizado y hueso de cadáver, seco y fresco. Se ha diseñado un sistema de empotramiento del extremo distal de la muestra (fémur) con control riguroso de las condiciones de anclaje. La oscilación libre de la muestra se ha obtenido mediante la liberación instantánea de una carga estética determinada y aplicada previamente, bien con una maquina de ensayo o bien por gravedad. Cada muestra se ha instrumentado con galgas extensométricas convencionales cuya señal se ha registrado con un equipo dinámico comercial. Se ha aplicado un procedimiento de tratamiento de señal para acotar, filtrar y presentar las respuestas de los sensores en el dominio del tiempo y de la frecuencia. La interpretación de resultados es de tipo comparativo: se aplica el ensayo a una muestra de fémur Intacto que se toma de referencia, y a continuación se repite el ensayo sobre la misma muestra con una prótesis implantada; la comparación de resultados permite establecer conclusiones inmediatas sobre los efectos de la implantación de la prótesis. La implantación ha sido realizada por un cirujano traumatólogo utilizando las mismas técnicas e instrumental empleadas en el quirófano durante la práctica clínica real, y se ha trabajado con tres vástagos femorales comerciales. Con los resultados en el dominio del tiempo y de la frecuencia de las distintas aplicaciones se han establecido conclusiones sobre los siguientes aspectos: Viabilidad de los distintos tipos de muestras sintéticas: modelos simplificados y fémur sintético normalizado. Repetibilidad, linealidad y reversibilidad del ensayo. Congruencia de resultados con los valores teóricos deducidos de la teoría de oscilaciones libres de barras. Efectos de la implantación de tallos femorales en la amplitud de las oscilaciones, amortiguamiento y frecuencias de oscilación. Detección de armónicos asociados a la micromovilidad. La metodología se ha demostrado apta para ser incorporada a la normativa de prótesis, es de aplicación universal y abre vías para el análisis de la detección y caracterización de la micromovilidad de una prótesis frente a las cargas de servicio. ABSTRACT Total hip arthroplasty is considered as one of the greatest surgical advances in medicine. The application of this technique on Traumatology has increased significantly in recent years, mainly due to the progressive increase in life expectancy. In fact, advanced age increases osteoarthritis and osteoporosis problems, which are typical diseases of joints and bones, and in many cases require full or partial prosthetic replacement on the joint. Right functional behavior of prosthesis is highly dependent on the primary stability; this means it depends on the correct anchoring of the prosthesis at the time of implantation. Uncemented prosthesis base their long-term success on the quality of osseointegration that takes place between the prosthetic material and bone tissue, and to achieve this good primary stability conditions is mandatory. Aseptic loosening is the main cause of failure in total hip arthroplasty. This is a phenomenon in which relative movements occur, due to complex interactions of mechanical and biological factors, and these micromovements put the implant functionality at risk. To minimize possible damage, it greatly depends on the early detection of loosening. For this purpose, various techniques have been tested both in vivo and in vitro: numerical analysis and experimental techniques based on sensors for movements caused by naturally or artificially transmitted loads, such as impacts or vibrations at different frequencies. The assemblies and methods applied are heterogeneous and, in many cases, they are complex and expensive, with no agreement on the use of a simple and effective technique for general purposes. Likewise, in current regulations for governing the conditions to be fulfilled by the prosthesis before going to market, there is no specific section related to the evaluation of the femoral stem design in relation to primary stability. The aim of this thesis is to develop a in vitro methodology for analyzing the stability of an implanted femoral stem, in order to assess the implantation techniques and the different prosthesis designs prior to its offer in the market. We also propose as a fundamental requirement that the developed testing method should be simple, reversible, repeatable, non-destructive, with close monitoring of parameters (boundary conditions of loads and displacements) and with the availability of a register system to record and interpret results in a fast, reliable and affordable manner. As a preliminary step, we have performed a qualitative analysis of the contact problems in the bone-stem interface, through the application of a continuous field optomechanical technique (photoelasticity). For this proposal three 2D models of bone–stem set, has been built simulating three interface contact types: loosened an unbounded contact, unbounded and fixed contact, and bounded homogeneous contact. By means of applying the same load to each model, and using the stress freezing technique, it has displayed the corresponding stress states, being more severe as expected, in the unbounded union model. In any case, the results clearly show the complexity of the interface contact problem, and they confirm the need for experimental studies about this problem. Afterward a free oscillation dynamic test has been done using resistive strain gauge sensors. Test samples have been femur bones in all possible variants: simplified models, standardized synthetic bone, and dry and cool cadaveric bones. An embedding system at the distal end of the sample with strong control of the anchoring conditions has been designed. The free oscillation of the sample has been obtained by the instantaneous release of a static load, which was previously determined and applied to the sample through a testing machine or using the gravity force. Each sample was equipped with conventional strain gauges whose signal is registered with a marketed dynamic equipment. Then, it has applied a signal processing procedure to delimit, filter and present the time and frequency response signals from the sensors. Results are interpreted by comparing different trials: the test is applied to an intact femur sample which is taken as a reference, and then this test is repeated over the same sample with an implanted prosthesis. From comparison between results, immediate conclusions about the effects of the implantation of the prosthesis can be obtained. It must be said that the implementation has been made by an expert orthopedic surgeon using the same techniques and instruments as those used in clinical surgery. He has worked with three commercial femoral stems. From the results obtained in the time and frequency domains for the different applications the following conclusions have been established: Feasibility of the different types of synthetic samples: simplified models and standardized synthetic femur. Repeatability, linearity and reversibility of the testing method. Consistency of results with theoretical values deduced from the bars free oscillations theory. Effects of introduction of femoral stems in the amplitude, damping and frequencies of oscillations Detection of micromobility associated harmonics. This methodology has been proved suitable to be included in the standardization process of arthroplasty prosthesis, it is universally applicable and it allows establishing new methods for the analysis, detection and characterization of prosthesis micromobility due to functional loads.