Introducing monitoring and automation in cartilage tissue engineering, toward controlled clinical translation

The clinical application of tissue engineered products requires to be tightly connected with the possibility to control the process, assess graft quality and define suitable release criteria for implantation. The aim of this work is to establish techniques to standardize and control the in vitro engineering of cartilage grafts. The work is organized in three sub-projects: first a method to predict cell proliferation capacity was studied, then an in line technique to monitor the draft during in vitro culture was developed and, finally, a culture system for the reproducible production of engineered cartilage was designed and validated. Real-time measurements of human chondrocyte heat production during in vitro proliferation. Isothermal microcalorimetry (IMC) is an on-line, non-destructive and high resolution technique. In this project we aimed to verify the possibility to apply IMC to monitor the metabolic activity of primary human articular chondrocytes (HAC) during their in vitro proliferation. Indeed, currently, many clinically available cell therapy products for the repair of cartilage lesions involve a process of in vitro cell expansion. Establishing a model system able to predict the efficiency of this lengthy, labor-intensive, and challenging to standardize step could have a great potential impact on the manufacturing process. In this study an optimized experimental set up was first established, to reproducible acquire heat flow data; then it was demonstrated that the HAC proliferation within the IMC-based model was similar to proliferation under standard culture conditions, verifying its relevance for simulating the typical cell culture application. Finally, based on the results from 12 independent donors, the possible predictive potential of this technique was assessed. Online monitoring of oxygen as a non-destructive method to quantify cells in engineered 3D tissue constructs. This project aimed at assessing a technique to monitor graft quality during production and/or at release. A quantitative method to monitor the cells number in a 3D construct, based on the on-line measurement of the oxygen consumption in a perfusion based bioreactor system was developed. Oxygen levels dissolved in the medium were monitored on line, by two chemo-optic flow-through micro-oxygen sensors connected at the inlet and the outlet of the bioreactor scaffold chamber. A destructive DNA assay served to quantify the number of cells at the end of the culture. Thus the oxygen consumption per cell could be calculated as the oxygen drop across the perfused constructs at the end of the culture period and the number of cells quantified by DNA. The method developed would allow to non-invasively monitoring in real time the number of chondrocytes on the scaffold. Bioreactor based engineering of large-scale human cartilage grafts for joint resurfacing. The aim of this project was to upscale the size of engineered human cartilage grafts. The main aim of this project consisted in the design and prototyping of a direct perfusion bioreactor system, based on fluidodynamic models (realized in collaboration with the Institute for Bioengineering of Catalonia, Spain), able to guarantee homogeneous seeding and culture conditions trough the entire scaffold surface. The system was then validated and the capability to reproducibly support the process of tissue development was tested by histological, biochemical and biomechanical assays. Within the same project the automation of the designed scaled up bioreactor system, thought as a stand alone system, was proposed. A prototype was realized in collaboration with Applikon Biotechnology BV, The Netherlands. The developed system allows to achieve within a closed environment both cell seeding and culture, controlling some important environmental parameters (e.g. temperature, CO2 and O2 tension), coordinating the medium flow and tracking culture development. The system represents a relevant step toward process automation in tissue engineering and, as previously discussed, enhancing the automation level is an important requirement in order to move towards standardized protocols of graft generation for the clinical practice. These techniques will be critical towards a controlled and standardized procedure for clinical implementation of tissue engineering products and will provide the basis for controlled in vitro studies on cartilage development. Indeed the resulting methods have already been integrated in a streamlined, controlled, bioreactor based protocol for the in vitro production of up scaled engineered cartilage drafts. Moreover the techniques described will serve as the foundation for a recently approved Collaborative Project funded by the European Union, having the goal to produce cartilage tissue grafts. In order to reach this goal the research based technologies and processes described in this dissertation will be adapted for GMP compliance and conformance to regulatory guidelines for the production of engineered tissues for clinical use, which will be tested in a clinical trial

Chancen und Limitationen früher gesundheitsökonomischer Evaluation zur Unterstützung der Translation medizinischer Innovationen aus dem Bereich der regenerativen Medizin

Die Erkenntnisse medizinischer Forschung haben maßgeblich dazu beigetragen die öffentliche Gesundheit in den Industrienationen im letzten Jahrhundert signifikant zu verbessern. Hierfür ist es gelungen, grundlegende Ergebnisse aus dem Forschungslabor in Anwendungen zur tatsächlichen Verbesserung der öffentlichen Gesundheit zu „übersetzen“. Ein Prozess, der unter dem Terminus „Translation“ in die wissenschaftliche Literatur eingegangen ist. Die regenerative Medizin ist ein relativ neues medizinisches Forschungsfeld, welches untersucht, inwieweit die Heilung von Krankheiten durch die Wiederherstellung der Funktion von Zellen, Geweben oder Organen erreicht werden kann. Bisher haben sich nicht alle Erwartungen, die in regenerativmedizinische Ansätze gesetzt worden sind, erfüllt, da die Translation für viele Entdeckungen nicht erfolgreich abgeschlossen werden konnte. Ein Grund dafür wird in der mangelnden Berücksichtigung der Anforderungen nationaler Gesundheitssysteme hinsichtlich der Wirtschaftlichkeit neuer Technologien durch die Entdecker gesehen. Auf Grund knapper Ressourcen im deutschen Gesundheitswesen gewinnen gesundheitsökonomische Analysen neuer medizinischer Technologien bei der Translation zunehmend an Bedeutung. Für Arzneimittel beispielsweise ist Evidenz über die Wirtschaftlichkeit, nach dem Nachweis der Qualität, Wirksamkeit und Sicherheit, die vierte regulatorische Hürde auf dem Weg zur Erstattung durch die Kostenträger. Die gesundheitsökonomische Evaluation hat sich dabei als das Mittel der Wahl zur Erfassung von Kosten und Nutzen etabliert, da sie einen systematischen Vergleich der ökonomischen und medizinischen Auswirkungen verschiedener Therapieoptionen erlaubt. Zur erfolgreichen Verbreitung neuer Therapien im Gesundheitssystem wäre es für deren Entdecker nützlich, möglichst frühzeitig bei ihren Entscheidungen auf gesundheitsökonomische Daten zurückgreifen zu können. Sie erlauben eine erste Einschätzung der Erstattungswahrscheinlichkeit und somit des kommerziellen Potenzials. Außerdem können durch die Daten gewonnene Erkenntnisse noch kostengünstig bei der Produktentwicklung berücksichtigt werden. Klinische und ökonomische Daten stehen in frühen Phasen der Technologieentwicklung jedoch meist nicht in ausreichendem Umfang zur Verfügung. Ein wichtiges Werkzeug vergleichender gesundheitsökonomischer Evaluationen ist daher die entscheidungsanalytische Modellierung, welche es ermöglicht, ein komplexes System realitätsnah darzustellen und, auf Grundlage der besten, verfügbaren Evidenz, die Auswirkungen verschiedener Handlungsalternativen auf dieses System abzuschätzen. Ziel der vorliegenden Arbeit ist es, die frühzeitige Nutzbarkeit gesundheitsökonomischer Modelle zur Unterstützung der Translation medizinischer Innovationen aus dem Gebiet der regenerativen Medizin empirisch zu erforschen. Hierfür werden Fallstudien aus zwei Indikationsgebieten hinzugezogen, für die jeweils ein entscheidungsanalytisches Kosten-Nutzwert-Modell programmiert wird. Im ersten Aufsatz werden die generelle Machbarkeit sowie Chancen und Limitationen der Modellierung im Kontext einer Innovation zur Behandlung von Knorpelschäden des Knies untersucht. Im zweiten Aufsatz werden diese Erkenntnisse auf ein Fallbeispiel aus dem Bereich der Behandlung von Komplikationen in Folge der Prostatektomie angewendet. Eine frühe Modellierung erwies sich im Fall der ausgewählten Innovationen als machbar. Es konnten für beide Fallstudien auf Grundlage der Modelle Schlussfolgerungen für die weitere Produktentwicklung gezogen werden, beispielsweise durch die Identifikation von Patientengruppen, die in besonderem Maße von der Innovation profitieren. Den Limitationen der Modellierung aufgrund der Ergebnisunsicherheit des Modells im ersten Fallbeispiel, konnte im zweiten Fallbeispiel teilweise durch eine genauere Erfassung dieser Unsicherheit entgegengewirkt werden

Modulation of the in vitro mechanical and chemical environment for the optimization of tissue-engineered articular cartilage

Articular cartilage is the connective tissue lining the ends of long bones, providing a dynamic surface that bears load while providing a smooth surface for articulation. When damaged, however, this tissue exhibits a poor capacity for repair, lacking the lymphatics and vasculature necessary for remodeling. Osteoarthritis (OA), a growing health and economic burden, is the most common disease afflicting the knee joint. Impacting nearly thirty million Americans and responsible for approximately $90 billion in total annual costs, this disease is characterized by a progressive loss of cartilage accompanied by joint pain and dysfunction. Moreover, while generally considered to be a disease of the elderly (65 years and up), evidence suggests the disease may be traced to joint injuries in young, active individuals, of whom nearly 50% will develop signs of OA within 20 years of the injury. For these reasons, significant research efforts are directed at developing tissue-engineered cartilage as a cell-based approach to articular cartilage repair. Clinical success, however, will depend on the ability of tissue-engineered cartilage to survive and thrive in a milieu of harsh mechanical and chemical agents. To this end, previous work in our laboratory has focused on growing tissues appropriate for repair of focal defects and entire articular surfaces, thereby investigating the role of mechanical and chemical stimuli in tissue development. While we have had success at producing replacement tissues with certain qualities appropriate for clinical function, engineered cartilage capable of withstanding the full range of insults in vivo has yet to be developed. For this reason, and in an effort to address this shortcoming, the work described in this dissertation aims to (1) further characterize and (2) optimize the response of tissue-engineered cartilage to physical loading and the concomitant chemical insult found in the injured or diseased diarthrodial joint, as well as (3) provide a clinically relevant strategy for joint resurfacing. Together, this holistic approach maximizes the chances for in vivo success of tissue-engineered cartilage. Regular joint movement and dynamic loads are important for the maintenance of healthy articular cartilage. Extensive work has been done demonstrating the impact of mechanical load on the composition of the extracellular matrix and the biosynthetic activity of resident chondrocytes in explant cultures as well as in tissue-engineered cartilage. In further characterizing the response of tissue-engineered cartilage to mechanical load, the work in this dissertation demonstrated the impact of displacement-controlled and load-controlled stimulation on the mechanical and biochemical properties of engineered cartilage. Additionally, these studies captured tension-compression nonlinearity in tissue-engineered cartilage, highlighting the role of the proteoglycan-collagen network in the ability to withstand dynamic loads in vivo, and optimized a commercial bioreactor for use with engineered cartilage. The deleterious chemical environment of the diseased joint is also well investigated. It is therefore essential to consider the impact of pro-inflammatory cytokines on the mechanical and biochemical development of tissue-engineered cartilage, as chemical injury is known to promote degradation of extracellular matrix constituents and ultimately failure of the tissue. Combining expertise in interleukin-1\alpha, dexamethasone, and drug delivery systems, a dexamethasone drug delivery system was developed and demonstrated to provide chondroprotection for tissue-engineered cartilage in the presence of supraphysiologic doses of pro-inflammatory cytokines. These results highlight the clinical relevance of this approach and indicate potential success as a therapeutic strategy. Clinical success, however, will not only be determined by the mechanical and biochemical properties of tissue-engineered cartilage. For engineered cartilage to bear loads in vivo, it is necessary to match the natural topology of the articular surface, recapitulating normal contact geometries and load distribution across the joint. To ensure success, a method for fabricating a bilayered engineered construct with biofidelic cartilage and subchondral bone curvatures was developed. This approach aims to create a cell-based cartilage replacement that restores joint congruencies, normalizes load distributions across the joint, and serves as a potential platform for the repair of both focal defects and full joint surfaces. The research described in this dissertation more fully characterizes the benefits of mechanical stimulation, prescribes a method for chondroprotection in vivo, and provides a strategy for creating a cartilage replacement that perfectly matches the native architecture of the knee, thus laying the groundwork for clinical success of tissue-engineered cartilage

EFFECT OF A 12-WEEK HOME-BASED NEUROMUSCULAR ELECTRICAL STIMULATION TREATMENT ON CLINICAL OUTCOMES FOLLOWING ARTICULAR CARTILAGE KNEE SURGERY

Articular cartilage defects in the knee are common, and can result in pain, decreased function and decreased quality of life. Untreated defects are considered to be a risk factor for developing osteoarthritis, a progressive degenerative joint disease with minimal treatment options. To address these issues, various surgical procedures are available to treat articular cartilage defects in the knee. While these procedures overall have positive results, after surgery patients experience large and persistent deficits in quadriceps strength. A contributing factor to this post-surgical weakness is believed to be the extended post-operative non-weight bearing period, with full weight bearing not initiated until approximately 4 – 6 weeks after surgery. During this non-weight bearing period a minimal amount of demand is placed upon the muscle. Subsequently, the quadriceps muscle undergoes a large degree of atrophy with a significant decrease in muscle strength. Muscular strength deficits reduce the knee joint stability, also increasing the risk of osteoarthritis development. Interventions that can be used to facilitate quadriceps strength while protecting the articular cartilage repair are needed. Neuromuscular electrical stimulation (NMES) is an effective post-knee surgery rehabilitation technique to regain quadriceps musculature. In recent years manufactures have been developing knee sleeve garments integrated with NMES allowing for portability of the NMES treatment. The primary aim of this study was to evaluate the effectiveness of a 12-week home-based neuromuscular electrical stimulation treatment on post-surgical clinical outcomes (quadriceps strength, lower extremity function, and patient reported outcomes) after articular cartilage knee surgery. Patients were randomized between a standard of care home-treatment group and a NMES home-treatment group. Patients completed isometric quadriceps strength testing, the Y-balance test, and the Knee Injury and Osteoarthritis Outcome Score (KOOS) before surgery and at 3-months after surgery. The secondary aims of this study were to determine the most effective NMES parameters for post-surgical quadriceps strength; and to develop a framework to identify factors that may influence a patient’s adherence to a prescribed therapy program. From our results we can make several conclusions. First, we found only a small number of studies utilize similar parameters for post-surgical quadriceps strength treatments. The majority of the parameters reported in the literature were highly variable between studies. Second, clinicians can utilize the expanded Health Belief Model to identify situational and personal factors unique to a patient that may impact adherence to a prescribed treatment. Clinicians can then implement the proposed interventional strategies to address the identified situational and personal factors. Finally, there was no difference in quadriceps strength, lower extremity function, or self-reported scores at 3-month between a home-based NMES treatment and a standard of care home-based treatment. Patients’ adherence to the treatment protocols may have been a major factor contributing to these results. Utilizing a model, such as the proposed expanded Health Belief Model, may assist clinicians in improving a patients’ adherence to future prescribed home-treatment programs