Ziyad S Haidar

ABSTRACT Distraction osteogenesis (DO) is a prevalent surgical technique for the correction of congenital orthopaedic deformities and craniofacial developmental conditions. Yet, clinical benefits continue to be limited by a number of complications mainly as a result of the protracted treatment time during which the fixator has to be kept in situ until the newly-formed bone in the distracted zone consolidates (or hardens), thus exacerbating significant medical, psychological and socio-economical problems on patients, their families and caregivers. On the other hand, protein therapy particularly with the use of potent osteoinductive cytokines from the TGF-B superfamily has been hailed as the most promising alternative to conventional bone grafts. Currently, rhBMP-2 and rhBMP-7/OP-1 have been approved for their "restricted" clinical use in long bone healing and spinal fusion. Prospective clinical trials have reported variability in results ranging from full bone bridging to no bone union and to optimize the therapeutical outcome, the incorporated high and unsafe dosages of the growth factors, timing of release and their application systems necessitate further development. Thus far, loading the protein solution into collagen sponges prior to surgical implantation has shown poor retention and rapid clearance of BMPs within a much shorter period than bone healing requires, especially in humans. Also, such carriers do not provide controlled or customizable release and can comprise outcome by foreign body reactions due to their nature, composition and incomplete degradation. Hence, biocompatible delivery systems that release the bioactive load locally and continuously over proper periods of time for the regeneration of native bone using lower and safer drug concentrations are needed. This doctoral dissertation describes the development and evaluation of a novel hybrid nanoparticulate rhOP-1 delivery system demonstrating characteristics suitable for enhancing de novo bone regeneration and accelerating consolidation in DO. The work is divided into two main phases: (1) Formulating biocompatible, biodegradable, monodisperse, physically-stable and cytocompatible cationic core-shell nanoparticles with good protein encapsulation efficiency to provide sustained release over prolonged periods of time. The resulting suspension of nanoparticles also exhibited an extended shelf-life with leeway for drug loading via simple rehydration immediately prior to use thus preventing degradation or loss of the encapsulant. (2) Evaluating the effect of a single injection of the hybrid nanoparticles loaded with low dosages of rhOP-1 on new bone regeneration and consolidation in a rabbit model of long bone distraction osteogenesis. Findings demonstrate the potential of the core-shell nanoparticles in minimizing the therapeutic protein dosage for DO via their localized, release-controlled, osteogenic and naturally-biocompatible polymeric properties without causing any clinical side effects or pre-mature ossification that often requires repeat osteotomies with considerable morbidity. Furthermore, results suggested that the released bioactive load from the delivery system as well as any resulting effects were restricted or confined to the site of administration in the muscle tissue of young rats with no complications from any degradation by-products. Consequently, a novel, safe and promising injectable delivery system for the administration of exogenous growth factors is presented. In a clinical setting, it can be expected to shorten the treatment period of DO and improve the functional outcome in patients via the earlier removal of the fixator. With the continually increasing understanding of morphogens, future studies might exploit the therapeutic potential and cost-effectiveness of the hybrid nanoparticles incorporated with combinations of BMPs, other morphogens and/or biomolecules in pathologies and conditions beyond DO or bone defects and fracture healing. L'ostéogenèse par distraction osseuse (OD) est une technique chirurgicale répandue pour la correction de difformités orthopédiques congénitales et craniofaciales. Pourtant, les avantages cliniques continuent d'être limités par de complications comme à la suite d'un traitement de longue durée pendant lequel le fixateur doit être gardé in situ jusqu'à ce que l'os nouvellement formé dans la zone distraite se consolide; pouvant significativement engendrer voire aggraver certains problèmes médicaux, psychologiques et socio-économiques. D'un autre côté, l'utilisation de puissantes cytokines de la superfamille TGF-B a été considérée comme l'alternative la plus prometteuse à la greffe conventionnelle d'os. Actuellement, BMP-2 et BMP-7/OP-1 ont été approuvés pour leur utilisation clinique "restreinte" dans la longue guérison osseuse et la fusion spinale. Pourtant, certaines études ont rapporté une certaine variabilité dans les résultats,…

A hybrid, localized and release-controlled delivery system for bone growth factors consisting of a liposomal core incorporated into a shell of alternating layer-by-layer self-assembled natural polyelectrolytes has been formulated. Hydrophilic, monodisperse, spherical and stable cationic nanoparticles (< or =350 nm) with an extended shelf-life resulted. Cytocompatibility was previously assayed with MC3T3-E1.4 mouse preosteoblasts showing no adverse effects on cell viability. In this study, the in vivo biocompatibility of unloaded and loaded nanoparticles with osteogenic protein-1 or OP-1 was investigated. Young male Wistar rats were injected intramuscularly and monitored over a period of 10 weeks for signs of inflammation and/or adverse reactions. Blood samples (600 microL/collection) were withdrawn followed by hematological and biochemical analysis. Body weight changes over the treatment period were noted. Major organs were harvested, weighed and examined histologically for any pathological changes. Finally, the injection site was identified and examined immunohistochemically. Overall, all animals showed no obvious toxic health effects, immune responses and/or change in organ functions. This hybrid core-shell nanoparticulate delivery system localizes the effect of the released bioactive load within the site of injection in muscle with no significant tissue distress. Hence, a safe and promising carrier for therapeutic growth factors and possibly other biomolecules is presented.

The effect of an early single injection of biodegradable core-shell nanoparticles (NPs) loaded with various low doses of recombinant human bone morphogenetic protein-7 (rhBMP-7/rhOP-1) on new bone regeneration and consolidation in a rabbit model of tibial distraction osteogenesis (DO) was investigated. The Regenerate bone was examined using soft radiography, densitometry, micro-computed tomography and histomorphometry. Compared to control, higher bone fill scores and a two- to three-fold increase in the quantity of mineralized tissue were prominent in the 1.0 and 5.0 microg OP-1/NPs groups, 3 weeks post-injections (P>0.05). Histologically, the distraction gap was completely ossified and the osteotomy margins poorly demarcated in those groups, one week into the consolidation phase. An up-regulation of various growth factors, ligands, and receptors was observed using immunohistochemistry. This novel hybrid delivery system maintains the bioactivity of the encapsulant, minimizes the therapeutic doses of rhOP-1, and accelerates DO via its localized, release-controlled, osteogenic, and naturally biocompatible polymeric properties.

The present work is focused on the formulation of core-shell nanoparticles via the layer-by-layer (L-b-L) self-assembly technique for delivery of biomacromolecules such as bone growth factors. The drug encapsulation efficiency of liposomes is enhanced with the increased stability of polyelectrolyte systems achieved through the alternate adsorption of several layers of natural polymers: anionic alginate and cationic chitosan on cationic nanosized phospholipid vesicles. The resulting particles were characterized for their size, surface charge, morphology, encapsulation efficiency, loading capacity and release kinetics over an extended period of 30 days. The L-b-L deposition technique succeeded in building a spherical, monodisperse and stable hybrid nanoparticulate protein delivery system with a cumulative size of 383+/-11.5 nm and zeta potential surface charge of 44.61+/-3.31 mV for five bilayered liposomes. The system offers numerous compartments for encapsulation including the aqueous core and within the polyelectrolyte shell demonstrating good entrapment and sustained linear release of a model protein, bovine serum albumin, in vitro. Our results demonstrate that this delivery system features an extended shelf life and can be loaded immediately prior to administration, thus preventing any loss of the protein.