abstract: Anti-retroviral drugs and AIDS prevention programs have helped to decrease the rate of new HIV-1 infections in some communities, however, a prophylactic vaccine is still needed to control the epidemic world-wide. Despite over two decades of research, a vaccine against HIV-1 remains elusive, although recent clinical trials have shown promising results. Recent successes have focused on highly conserved, mucosally-targeted antigens within HIV-1 such as the membrane proximal external region (MPER) of the envelope protein, gp41. MPER has been shown to play critical roles in the viral mucosal transmission, though this peptide is not immunogenic on its own. Gag is a structural protein configuring the enveloped virus particles, and has been suggested to constitute a target of the cellular immunity potentially controlling the viral load. It was hypothesized that HIV-1 enveloped virus-like particles (VLPs) consisting of Gag and a deconstructed form of gp41 comprising the MPER, transmembrane, and cytoplasmic domains (dgp41) could be expressed in plants. Plant-optimized HIV-1 genes were constructed and expressed in Nicotiana benthamiana by stable transformation, or transiently using a tobacco mosaic virus-based expression system or a combination of both. Results of biophysical, biochemical and electron microscopy characterization demonstrated that plant cells could support not only the formation of HIV-1 Gag VLPs, but also the accumulation of VLPs that incorporated dgp41. These particles were purified and utilized in mice immunization experiments. Prime-boost strategies combining systemic and mucosal priming with systemic boosting using two different vaccine candidates (VLPs and CTB-MPR - a fusion of MPER and the B-subunit of cholera toxin) were administered to BALB/c mice. Serum antibody responses against both the Gag and gp41 antigens could be elicited in mice systemically primed with VLPs and these responses could be recalled following systemic boosting with VLPs. In addition, mucosal priming with VLPs allowed for a robust boosting response against Gag and gp41 when boosted with either candidate. Functional assays of these antibodies are in progress to test the antibodies' effectiveness in neutralizing and preventing mucosal transmission of HIV-1. This immunogenicity of plant-based Gag/dgp41 VLPs represents an important milestone on the road towards a broadly-efficacious and inexpensive subunit vaccine against HIV-1. / Dissertation/Thesis / Ph.D. Molecular and Cellular Biology 2011

Human immunodeficiency virus (HIV) is globally spread virus without available cure. Since its life-long presence, virus is carefully monitored as well as patient's immunological status. Replicative fitness of the virus is one of important aspects which can be taken into account, when monitoring HIV. Here, we are measuring HIV replicative fitness of gag recombinant viruses and comparing the results with replicative fitness of primary isolates. Further, we are comparing our findings of replicative fitness change over time with disease progression in the patient. We found that gag can be major contributor to overall fitness, although not in all cases. Additionally, we observed a correlation of replicative fitness development and slope of patient's CD4+ T cells. Moreover, this relation was even more noticeable in patients with slow disease progression or in carriers of protective alleles. In summary, our results extend the understanding of replicative fitness and its role in disease progression; and pave the way to use the recombinant HIV for replicative fitness measurement in clinical practice. Keywords: HIV, replicative fitness, recombinant virus, HIV disease progression, gag

Suite à un accident, les lésions du ligament croisé antérieur sont de plus en plus fréquentes devenant un problème de santé publique. En réponse à la demande de solutions alternatives aux traitements chirurgicaux actuels, nous avons développé un renfort ligamentaire en biomatériaux dégradables. Pour répondre à l'exigence de la régénération ligamentaire, ce dernier doit être suffisamment résistant pour répondre aux contraintes physiologiques du genou, et doit se dégrader tout en permettant la régénération d'un nouveau ligament. Nous avons synthétisé de nouveaux copolymères bloc à base de PLA et de poloxamère ou poloxamine qui ont été filés puis tricotés pour concevoir un textile tubulaire. Ce textile tubulaire possède des caractéristiques mécaniques intéressantes pour le remplacement d'un ligament. En parallèle, a été développée une matrice poreuse à trois dimensions en collagène et glycoaminoglycanes. Cette matrice permet de favoriser l'adhésion et la prolifération cellulaire. Le renfort tricoté associé à la matrice de collagène a été implanté in vivo durant 3 mois. A 3 mois, parfaitement intégrée, la structure composite permet la formation d'un néo-tissu tout en perdant progressivement ses propriétés mécaniques. / Following accidents, anterior cruciate ligament (ACL) damages are increasingly becoming a common public health problem. To comply with the demand for alternatives to current surgical treatments, we have developed ligament reinforcement with degradable biomaterials. To meet the requirement of ligament regeneration, ligament reinforcement must be strong enough to support knee physiological strains, and must degrade while allowing the new ligament regeneration. Novel block copolymers PLA- and poloxamer or poloxamine based have been synthesized which were then spun for designing a tubular knitted fabric. The tubular fabric has interesting mechanical characteristics for ligament replacement. In parallel, a collagen and glycosaminoglycans porous three-dimensional matrix has been developed. This matrix is able to promote cell adhesion and proliferation. The knitted reinforcement associated with the collagen matrix has been implanted in vivo for 3 months. Fully integrated, the composite structure allows néo-tissue formation while gradually losing its mechanical properties after 3 months.

Murine leukemia virus (MLV), classified as a gammaretrovirus, has been studied extensively to enhance our understanding of the biology and replication of retroviral infection. Typically referred to as a simple retrovirus, its usefulness as a model is highlighted owing to its minimal genome. The genetic material for MLV was thought to only code the basic and essential defining features of a retrovirus. Through the understanding developed from the use of simple retroviruses, the clinical and research communities were immeasurably more prepared to combat the more complex and decidedly infamous human immunodeficiency virus (HIV). Interestingly, a scenario of convergent evolution has directed MLV to encode an accessory protein, termed Glycosylated Gag (gGag), that shares functionality reminiscent of several HIV proteins. Herein, I present a dissection of a novel function of this enigmatic protein, paired with an improved understanding of the biology of MLV that was revealed by the development of small particle flow cytometry performed on viruses, also known as flow virometry. Initially, we elucidated that gGag is responsible for the resistance of MLV towards the restriction factor murine APOBEC3 (mA3). I showed that even endogenous mA3 from primary cells exhibited an enhanced enzymatic activity towards MLV with mutant gGag proteins which have lost glycosylation sites. In our following study, I illustrated that these mutants displayed a reduced viral core stability, the severity of which was correlated directly with susceptibility to mA3. These results are in line with the hypothesis that viral core stability and APOBEC3-susceptibility are directly linked. Furthermore, I showed for the first time that unprocessed gGag was associated with viral particles released from producer cells in the orientation of a type I membrane protein, with the structural regions directed within the viral core. This may be the direct evidence of how gGag improves capsid stability, a mechanism which is still unresolved. On the flip side, gGag as a type II membrane protein was observed exclusively on virus-like particles devoid of detectable envelope glycoprotein (Env). This marks a potential new function for gGag in the context of infection. Given the ubiquitous necessity of an optimized core stability for any virus, combined with the overlapping function of gGag with HIV accessory proteins, continuation of this work represents an as of yet clinically unexplored avenue for the development of HIV therapeutics. At the same time, in order to characterize individual viral particles, I played an instrumental role in developing the technique of flow virometry within our core facility. I illustrated that the Env of MLV does not significantly accumulate on extracellular vesicles (EVs) and acts as an effective marker for viral particles. With this evidence in hand, the enumeration of MLV virions was made possible. By correlating this information with an absolute viral genome determination, I was able to estimate the packaging efficiency for MLV in a quantitative manner. This information suggests that roughly 80-85% of MLV particles are missing their essential genetic information. These findings may implicate the disease progression of MLV infection may be enhanced by the use of defective-interfering particles, a theory that has been suggested for HIV. This work highlighted the fact that flow virometry is uniquely capable to discriminate viral particles from other cell-derived membraned vesicles in a highly sensitive manner. Overall, my work has unveiled new complexities of a simple retrovirus, while laying the groundwork towards both diagnostics and therapeutics for the ongoing battle with HIV.

The human immunodeficiency virus type-1 matrix (HIV-1 MA) is best described as a multi-functional, structural protein. However, the multitude of functional activities ascribed to this viral component is not nearly as interesting as are its seemingly paradoxical and opposing roles during the viral life cycle. At the time of virus infection, HIV-1 MA remains associated with the reverse transcription complex, in which viral nucleic acids are synthesized, and facilitates its translocation to the host cell nucleus (Bukrinsky, Sharova et al. 1992; Bukrinsky, Sharova et al. 1993). This activity of MA has been proposed to form the basis for the infection of non-dividing cells (Bukrinsky, Haggerty et al. 1993). An interaction between the C-terminally phosphorylated form of MA and HIV-1 integrase, an integral component of the complex, was initially proposed to mediate this association (Gallay, Swingler et al. 1995; Gallay, Swingler et al. 1995). However, conditions which promote dissociation of integrase from the reverse transcription complex do not reduce MA association (Miller, Farnet et al. 1997). The possibility of a direct interaction between MA and the viral genome is discussed in Chapter III. The nucleophilic nature of HIV-1 MA is paradoxical with its reported activity in targeting the viral precursor proteins to the cytoplasmic membrane (Krausslich and Welker 1996), during the particle production phase of the viral life cycle. Furthermore, MA when expressed in the absence of other viral proteins exhibits a cytoplasmic localization (Fouchier, Meyer et al. 1997); a result which does not support a nuclear translocation role for this protein. The work presented here resolves this seemingly controversial issue. We demonstrate that MA exhibits a strong nuclear export activity. This newly discovered activity is designed to effectively counteract the protein's innate nucleophilic nature, thus maintaining a cytoplasmic localization. The nuclear export function of MA is sensitive to changes within the conformation of the protein as C- and N-terminal deletions, as well as point mutations in the protein, abolish the activity. Furthermore, the export activity is mediated by the Crm1 NES receptor (Fornerod, Ohno et al. 1997; Fukuda, Asano et al. 1997; Ossareh-Nazari, Bachelerie et al. 1997) despite the lack of a leucine-rich export signal within the matrix coding region. Therefore, the interaction between matrix protein and Crm1 is most likely to be mediated by another, perhaps cellular, protein. Any changes in matrix structure may lead to the disruption of this protein-protein interaction. We discuss a model implicating a phosphorylation event in the inactivation of this nuclear export signal. An even more fascinating issue regards the role of this nuclear export activity, during the viral life cycle, and is detailed in Chapter II. In short, mutations in MA which impair its nuclear export activity result in nuclear accumulation of the precursor Gag polyprotein (Pr55) and the nucleocapsid-associated viral genomic RNA. As a result, non-infectious virions deficient in genomic viral RNA are produced. Therefore, drugs designed to block this export activity can undermine the carefully orchestrated course of events during HIV replication and can shut down the growth of the virus.

Strahlentherapie im Beckenbereich, wie bei einer Tumortherapie häufig nötig, führt zur Mitbestrahlung der Harnblase. Dieses Normalgewebe zeigt hierbei eine akute und eine späte Strahlenreaktion. Während die akute Strahlenreaktion vollständig reversibel ist, ist die späte Reaktion irreversibel und progradient. Die Strahlenreaktion wurde mittels intravesikaler Zystometrie über das Blasenvolumen ermittelt. Getestet wurden Heparin und PPS als Glykosaminoglykane. Im Rahmen der Arbeit konnte gezeigt werden, dass die akute Strahlenreaktion durch intravesikale Applikation von Glykosaminoglykanen beeinflusst werden kann, und zwar im Sinne einer Reduktion der Reaktion. Die späte Reaktion selbst dagegen nicht. Dafür konnte aber gezeigt werden, dass eine starke konsekutive Komponente der späten Reaktion vorhanden ist. Damit beeinflusst das Maß der akuten Strahlenreaktion die Schwere der späten Reaktion. Eine Verringerung der Reaktionshäufigkeit in der akuten Phase führt zu einer Reduktion der Reaktionshäufigkeit in der Spätphase. / Radiation therapy in the pelvic region leads to a radiation of the bladder too. This normal tissue shows an acute and a late reaction. Whereas the acute one is completely reversible the chronic one is irreversible an progradient. The radiation reaction was measured as a reduction of the volume of the bladder by intravesical cystotonometry. Heparin and PPS were tried as GAG’s. In these examinations could be shown that it is possible to influence the acute reaction by intravesical application of GAG’s . In contrast the chronic phase an influence was impossible. Otherwise it could be proved that there is a high consecutive component in the chronic reaction. That means that a reduction in the frequency of the acute reaction leads to a reduction of the frequency of the chronic reaction.

Since 2021, some state legislators have passed laws that limit what public institutions can teach about discrimination. The number of these laws, also called gag orders, is increasing despite a national outcry against racism, homophobia and transphobia, and other forms of discrimination. Many nursing and other professional healthcare organizations have recognized and published statements decrying racism in healthcare and calling for an increased focus on health disparities and advancing health equity. Similarly, national research institutions and private grant funders are funding health disparities research. Nursing and other faculty in higher education, however, are being gagged by laws and executive orders which prevent them from teaching and conducting research about historic and contemporary health disparities. This commentary seeks to highlight the immediate and long-term impact of academic gag orders and to encourage action in opposition of such legislation. Supported by professional codes of ethics and discipline-specific education, we present concrete activities readers can use to address gag order legislation and in doing so, protect patient and community health outcomes.

No description available.

Depuis sa découverte il y a plus de 30 ans, le Virus de l’Immunodéficience Humaine est à l’origine d’une importante mortalité dans le monde. De par la difficulté de tester l’efficacité de formulations thérapeutiques et/ou vaccinales directement chez l’homme, des études d’infections modèles du VIH, comme celle du Virus de l’Immunodéficience Féline (FIV), ont été entreprises ces dernières années. Au-delà de son intérêt vétérinaire, l’étude du FIV représente un avantage important pour trouver un moyen de contrôler les infections par les lentivirus tel que le VIH. Elle peut permettre de développer et surtout de tester l’efficacité des vaccins et/ou thérapies spécifiques chez le chat, dont le SIDA mime les symptômes et les modifications hématologiques rencontrés chez l’homme. Ce manuscrit s’est intéressé à l’étude structurale de deux familles de protéines virales de ces virus, les protéines lentivirales précoces (protéine Tat du VIH) et tardives (domaines Capside CA et Matrice MA de Gag du FIV). L’étude structurale de ces protéines et leur compréhension fonctionnelle au sein de l’hôte pourront à l’avenir ouvrir de nouvelles voies thérapeutiques et/ou vaccinales contre les lentivirus, palliant ainsi les problèmes existants de résistances virales / Since its discovery 30 years ago, the Human Immunodeficiency Virus is the cause of an important mortality worldwide. Because of the difficulty to test the efficiency of therapeutical and/or vaccinal formulations directly in humans, studies of models of HIV infections, such as the Feline Immunodeficiency Virus (FIV), have been performed in recent years. In addition to its veterinary interest, the study of FIV is an important issue to find a way to control infections by lentiviruses such as HIV. It can help to develop and test the efficiency of specific therapies and/or vaccines for cats, where AIDS mimics the symptoms and hematologic changes observed in humans. This manuscript describes the structural study of two types of viral proteins of these viruses, early lentiviral proteins (HIV Tat protein) and late lentiviral proteins (CA capsid and MA Matrix domains of FIV Gag). The structural study of these proteins and their functional understanding into the host will open new therapeutic and/or vaccine strategies against these lentiviruses in the future, in order to overcome the existing problems of viral resistance

Synthetic biomaterials are constantly being developed and play central roles in contemporary strategies in regenerative medicine and tissue engineering as artificial extracellular microenvironments. Such scaffolds provide 2D- and 3D-support for interaction with cells and thus convey spatial and temporal control over their function and multicellular processes, such as differentiation and morphogenesis. A model fibrillar system with tunable viscoelastic properties, comprised of 2 native ECM components like collagen type I and the GAG heparin, is presented here. Although the individual components comply with the adhesive, mechanical and bioinductive requirements for artificial reconstituted ECMs, their interaction and structural characterization remains an intriguing conundrum. The aim of the work was to analyze and structurally characterize a xenogeneic in vitro cell culture scaffold reconstituted from two native ECM components, collagen type I and the highly negatively charged glycosaminoglycan heparin. Utilizing a broad spectrum of structural analysis it could be shown that pepsin-solubilized collagen type I fibrils, reconstituted in vitro in the presence of heparin, exhibit an unusually thick and straight shape, with a non-linear dependence in size distribution, width-to-length ratio, and morphology over a wide range of GAG concentrations. The experiments imply a pronounced impact of the nucleation phase on the cofibril morphology as a result of the strong electrostatic interaction of heparin with atelocollagen. Heparin is assumed to stabilize the collagen-GAG complexes and to enhance their parallel accretion during cofibrillogenesis, furthermore corroborated by the heparin quantitation data showing the GAG to be intercalated as a linker molecule with a specific binding site inside the cofibrils. In addition, the exerted morphogenic effect of the GAG, appears to be influenced by factors as degree of sulfation, charge, and concentration. Further detailed structural analysis of the PSC-heparin gels using TEM and SFM showed a hierarchy involving 3 different structural levels and banding patterns in the system: asymmetric segment longspacing (SLS) fibrils and symmetric segments with an average periodicity (AP) of 250 - 260 nm, symmetric fibrous longspacing (FLS IV) nanofibrils with AP of 165 nm, and cofibrils exhibiting an asymmetric D-periodicity of 67 nm with a striking resemblance to the native collagen type I banding pattern. The intercalation of the high negatively charged heparin in the cofibrils was suggested as the main trigger for the hierarchical formation of the polymorphic structures. We also proposed a model explaining the unexpected presence of a symmetric and asymmetric form in the system and the principles governing the symmetric or asymmetric fate of the molecules. The last section of the experiments showed that the presence of telopeptides and heparin both had significant effects on the structural and mechanical characteristics of in vitro reconstituted fibrillar collagen type I. The implemented structural analysis showed that the presence of telopeptides in acid soluble collagen (ASC) impeded the reconstitution of D-periodic collagen fibrils in the presence of heparin, leaving behind only a symmetric polymorphic form with a repeating unit of 165 nm (FLS IV). Further x-ray diffraction analysis of both telopeptide-free and telopeptide-intact collagen fibrils showed that the absence of the flanking non-helical termini in pepsin-solubilized collagen (PSC) resulted in a less compact packing of triple helices of atelocollagen with an increase of interhelical distance from 1.0 to 1.2 nm in dried samples. The looser packing of the triple helices was accompanied by a decrease in bending stiffness of the collagen fibrils, which demonstrated that the intercalated heparin cannot compensate for the depletion of telopeptides. Based on morphological, structural and mechanical differences between ASC and PSC-heparin fibrils reported here, we endorsed the idea that heparin acts as an intrafibrillar cross-linker which competed for binding sites at places along the atelocollagen helix that are occupied in vivo by telopeptides in the fibrillar collagen type I. The performed studies are of particular interest for understanding and gaining control over a rather versatile and already exploited xenogeneic cell culture system. The reconstituted cofibrils with their unusual morphology and GAG intercalation – a phenomenon not reported in vivo – are expected to exhibit interesting biochemical behavior as a biomaterial for ECM scaffolds. Varying the experimental conditions, extent of telopeptide removal, and heparin concentration provides powerful means to control the kinetics, structure, dimensions, as well as mechanical properties of the system which is particularly important for predicting a certain cell behavior towards the newly developed matrix. The GAG intercalation could be interesting for studies with required long-term 'release upon demand' of the GAG, as well as native binding and stabilization of growth factors, cytokines, chemokines, thus providing a secondary tool to control cell signaling and fate, and later on tissue morphogenesis. / Synthetische Biomaterialien werden stetig weiterentwickelt und spielen als künstliche Mikroumgebungen eine zentrale Rolle in den modernen Strategien der regenerativen Medizin und des Tissue Engineerings. Solche sogenannten Scaffolds liefern eine 2D- und 3D-Struktur zur Interaktion mit Zellen und üben somit eine räumliche und zeitliche Kontrolle auf ihre Funktion und multizelluläre Prozesse aus, wie die Differenzierung und Morphogenese. Obwohl häufig die adhäsiven, mechanischen und bioinduzierenden Eigenschaften von Einzelkomponenten aus natürlichen Bestandteilen der extrazellulären Matrix (ECM) rekonstituierten Trägerstrukturen bekannt sind, bleiben die funktionalen und strukturellen Auswirkungen in Mehrkomponentensystemen eine faszinierende Fragestellung. Das Ziel der Arbeit war die Analyse und die strukturelle Charakterisierung einer xenogenen in vitro Zellkultur-Trägerstruktur, die aus den zwei nativen ECM Komponenten Kollagen Typ I und das stark negativ geladene Glykosaminoglykan (GAG) Heparin rekonstituiert wurde. Unter Nutzung eines breiten Spektrums von Methoden zur strukturellen Analyse konnte gezeigt werden, dass im Beisein von Heparin rekonstituierte Pepsin-gelöste Kollagen Typ I Fibrillen eine ungewöhnlich dicke und gerade Form, mit nichtlinearen Abhängigkeiten der Größenverteilung, des Breite-zu-Länge Verhältnises und der Morphologie für eine Reihe von GAG Konzentrationen, aufweisen. Die Experimente deuten auf eine besondere Wirkung der Nukleierungsphase auf die Kofibrillmorphologie hin, als Folge der starken elektrostatischen Inteaktionen Heparins mit Atelokollagen. Es wird angenommen, dass Heparin die Komplexe aus Kollagen-GAG stabilisiert, die parallele Anlagerung während der Kofibrillogenese verbessert und dass überdies, belegt durch Heparin Quantitätsdaten, als Verbindungsmolekül mit einer spezifischen Anbindungsstelle innerhalb der Kofibrillen eingelagert wird. Darüber hinaus scheint der ausgeübte morphogene Effekt des GAGs Heparins von Faktoren wie Grad der Sulfatierung, Ladung und Konzentration abzuhängen. Weitere detailierte Strukturanalysen der PSC - Heparin Gele mit TEM und SFM zeigten eine Hierarchie mit drei unterschiedlichen strukturellen Ebenen und Bandmustern im System: asymmetrisch segmentierte, weitabständige Fibrillen (SLS) und symmetrische Segmente mit einem AP von 250-260 nm, symmetrische fibrose weitabständige (FLS IV) Nanofibrillen mit einem AP von von 165 nm und Kofibrillen asymmetrischer D-Periodizität von 67 nm, die eine erstaunliche Ähnlichkeit zum natürlichen Kollagen Typ I Bandmuster haben. Die Einlagerung des sehr negativ geladenen Heparins in die Kofibrillen wurde als Hauptauslöser der hierarchischen Formation der polymorphen Strukturen betrachtet. Wir schlugen ebenso ein Model vor, welches sowohl das unerwartete Vorhandensein symmetrischer und asymmetrischer Formen im System als auch die Regeln erklärt, die das symmetrische oder asymmetrische Schicksal der Moleküle steuern. Der letzte Abschnitt der Experimente zeigte, dass die Anwesenheit der Telopeptide und Heparins eine signifikante Wirkung auf die strukturellen und mechanischen Charakteristika der in vitro rekonstituierten Kollagen Typ I Fibrillen hatte. Die durchgeführten Strukturanalysen zeigten außerdem, dass die Anwesenheit der Telopeptide in säurelöslichem Kollagen (ASC) die Rekonstitution D-periodischer Kollagenfibrillen mit Heparin verhinderte, sodass nur symmetrisch polymorphe Formen mit einer Wiederholeinheit von 165 nm möglich waren (FLS IV). Weitere Messungen der Telopeptid-freien und Telopeptid-intakten Kollagenfibrillen mit Röntgendiffraktometrie ergaben, dass die Abwesenheit der nicht-helix-strukturierten Enden in Pepsin-gelöstem Kollagen (PSC) zu einer weniger kompakten Anordnung der Tripelhelices von Atelokollagen führte. Der interhelix Abstand erhöhte sich von 1,0 zu 1,2 nm für getrocknete Proben. Das zeigt, dass die losere Anordnung der Tripelhelices einhergeht mit der Verringerung der Biege-Elastizitäts-module der Kollagenfibrillen,. Basierend auf den hier vorgestellten morphologischen, strukturellen und mechanischen Unterschieden zwischen ASC und PSC-Heparin Fibrillen wird die Idee unterstützt, dass Heparin als intrafibrillärer Vernetzer fungiert und an Bindungsstellen der Helix bindet, welche in vivo bei Kollagen Typ I Fibrillen durch Telopeptide besetzt sind. Die durchgeführten Studien sind von besonderem Interesse für das Verständnis und die Steuerung eines sehr vielseitigen und bereits verwendeten xenogenes Zellkultursystem für das Tissue Engineering. Von den rekonstituierten Kofibrillen mit ihrer ungewöhnlichen Morphologie und GAG Einlagerung - ein in vivo nicht bekanntes Phänomen - erwartet man, dass sie ein intressantes biochemisches Verhalten als Biomaterial für ECM Scaffolds zeigen. Variationen der experimentellen Bedingungen, des Ausmaßes der Telopeptidentfernung und der Heparinkonzentration liefern vielfältige Möglichkeiten um die Kinetik, Struktur, Dimension sowie die mechanischen Eigenschaften des Systems zu kontrollieren. Damit sollte es möglich sein, ein bestimmtes Zellverhalten gegenüber der neu entwickelten Matrix vorherzusagen. Die GAG-Einlagerung bietet interessante Optionen für eine langfristige Freisetzung des GAGs 'on demand', sowie die native Bindung und Stabilisierung von Wachstumsfaktoren, Cytokinen, Chemokinen, womit zusätzlich Zellsignalisierung und -schicksal und später Gewebemorphogenese kontrolliert werden kann.