Co-delivery of the embedded growth factor-loaded microspheres and adult stem cells in a hydrogel matrix was studied for its potential as a cell-based therapeutic strategy for cartilage regeneration in partial thickness chondral defects. A photopolymerizable N-methacrylate glycol chitosan (MGC) was employed to form an in situ gel that was embedded with two formulations of growth factor-loaded microspheres and human adipose-derived stem cells (ASC). The polymeric microspheres were used as a delivery vehicle for the controlled release of growth factors to stimulate differentiation of the ASC towards the chondrocyte lineage. The microspheres were made of amphiphilic low molecular weight (Mn < 10,000 Da) poly(1,3-trimethylene carbonate-co--caprolactone)-b-poly(ethylene glycol)-b-poly(1,3-trimethylene carbonate-co--caprolactone) (P(TMC-CL)2-PEG)). This triblock copolymer is solid below 100C, but liquid with a low degree of crystallinity at physiological temperature and degrades slowly, and so acidic degradation products do not accumulate locally. Bone morphogenetic protein-6 (BMP-6) and transforming growth factor-3 (TGF-3) were delivered at 5 ng/day with initial bursts of 14.3 and 23.6%, respectively. Both growth factors were highly bioactive when released, retaining greater than 95% bioactivity for 33 days as measured by cell-based assays. To improve ASC viability within the MGC vehicle, an RGD-containing ligand was grafted to the MGC backbone. Prior to chondrogenic induction within the MGC gel, ASC viability was assessed and greater than 90% of ASC were viable in the gel grafted with cell-adhesive RGD peptides as compared to that in non-RGD grafted gels. For ASC chondrogenesis induced by the sustained release of BMP-6 and TGF-3 in MGC gels, the ASC cellularity and glycosaminosglycan production were similar for 28 days. The ratio of collagen type II to I per cell (normalized to deoxyribonucleic acid content) in the microsphere delivery group was significantly higher than that of non-induced ASC or with soluble growth factor administration in the culture media, and increased with time. Thus, the co-delivery of growth factor-loaded microspheres and ASC in MGC gels successfully induced ASC chondrogenesis and is a promising strategy for cartilage repair. / Thesis (Ph.D, Chemical Engineering) -- Queen's University, 2011-06-07 19:32:50.94

The delivery of growth factors has been attempted for a number of different therapies. The approach of delivering therapeutic growth factors in a safe and efficient manner is difficult and certain criteria should be met. These criteria include: binding the appropriate growth factors, maintaining their bioactivity, and delivering these proteins with controllable release kinetics for an extended period of time. These criteria encompass a set of guidelines that hope to mimic in vivo biological events such as neovascularization. The central goal of this thesis is to meet these criteria by introducing a novel delivery strategy for growth factors using a biocompatible polycation and heparin. It was hypothesized that a polycation could interact with heparin to form a complex with the potential to deliver bioactive growth factors with an adaptable release. This hypothesis was tested by examining the release kinetics of bFGF from the complex and investigating whether the released bFGF maintained its bioactivity. The [polycation:heparin:bFGF] complex was formed by mixing the components in water, resulting in a precipitate. This precipitate was able to deliver bFGF with controllable release kinetics and the bioactivity of the released bFGF was higher than bolus bFGF and comparable to heparin stabilized bFGF. This system is expected to have the ability to bind and deliver numerous heparin-binding growth factors. In conclusion, the delivery system developed in this research provides a novel mechanism for controlled release of growth factors. This delivery strategy has met the criteria listed earlier and this research has laid the foundation for a successful delivery vehicle. Further, a biocompatible polycation was synthesized, which is a critical component of the delivery system. This polycation exhibited in vitro and in vivo biocompatibility that was orders of magnitude higher than existing polycations and has the potential to be very useful in a variety of biomedical applications. This design principle is also expected to serve as a platform for the synthesis of other biocompatible polycations.

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<p>Collagen crosslinking with a polypropyleneimine octaamine dendrimers, via carbodiimide chemistry, was further exploited to demonstrate the ability of this technology for various tissue engineering strategies, including tissue engineered corneal equivalents (TECE) and blood-contacting biomaterials. In addition, modification with extracellular matrix components and other biomimetic molecules may enhance tissue-host interactions for greater <em>in vivo </em>compatibility.</p> <p>First, the efficacy of the dendrimer crosslinking technology was further validated with commercially available collagen-based materials, from bovine or human sources (Chapter 4: Paper 1), as determined via transmittance, water uptake, differential scanning calorimetry, collagenase stability and <em>in vitro </em>cell compatibility. Despite gel formation, the matrix integrity was compromised with collagen-based materials manufactured under acidic conditions and purified via freeze-drying.</p> <p>To continue the theme of dendrimer crosslinked collagen gels as TECE materials, growth factor incorporation was investigated with epidermal growth factor (EGF) and heparin-binding EGF (HB-EGF), as a method for improving device epithelialization and subsequent host integration. However, given the short half lives of these growth factors, an effective growth factor delivery system is necessary to protect growth factor bioactivity. As heparan sulphate proteoglycans sequester and release heparin-binding growth factors <em>in vivo</em>, the use of heparinized dendrimer crosslinked collagen (CHG) gels for HB-EGF delivery would provide prolonged, controlled delivery, while maintaining growth factor effectiveness (Chapter 5: Paper 2). HB-EGF release was prolonged and capable of inducing human cornea epithelial cell (HCEC) proliferation. Thus, HB-EGF delivery from CHG gels could aid in TECE device retention through enhanced device-host integration via epithelialization.</p> <p>Alternatively, tethering EGF or HB-EGF to dendrimer crosslinked collagen (CG) gels could also supply growth factor stimulation in a manner that maintains bioactivity, while stimulating growth factor receptors continually with minute concentrations (Chapter 6: Paper 3). Growth factor uptake and bioactivity was assessed with radiolabeled growth factor and through <em>in vitro </em>epithelial cell culture, respectively. Surface-modification of CG gels with growth factors demonstrated greater bioactivity, compared to growth factor bulk-modification of CG gels.</p> <p>Finally, dendrimer crosslinked collagen gels, with pre-activated heparin (PH gels) were investigated as a tissue engineered blood-contacting biomaterial (Chapter 7: Paper 4), as we hypothesized that biomaterial induced coagulation is not only influenced by an anticoagulant surface, but also by the underlying material and that improved blood-biomaterial interactions may be achieved by utilizing a natural polymer that emulates biomimetic properties. Pre-activation of heparin was utilized to increase heparin gel content, while antithrombotic properties were evaluated via antithrombin and fibrinogen adsorption and plasma recalcification times. PH gels had increased heparinization, but extensive crosslinking compromised antithrombin-heparin interactions, compared to CHG gels. CHG gels demonstrated improved antithrombotic properties and further evaluation of these gels for blood-contacting applications is warranted.</p> / Doctor of Philosophy (PhD)

Bone defects and fracture non-unions remain a substantial challenge for clinicians due to a high occurrence of delayed union or non-union requiring surgical intervention. The current grafting procedures used to treat these injuries have many limitations and further long-term complications associated with them. This has resulted in research efforts to identify graft substitution therapies that are able to repair and replace tissue function. Many of these tissue engineered products include the use of growth factors to induce cell differentiation, migration, proliferation, and/or matrix production. However, current growth factor delivery methods are limited by poor retention of growth factors upon implantation resulting in low bioactivity. These limiting factors lead to the use of high doses and frequent injections, putting the patients at risk for adverse effects. The goal of this work was to develop and evaluate the efficacy of BMP-2 delivery systems to improve bone regeneration. We examined two approaches for delivery of BMP-2 in this work. First, we evaluated the use of a self-assembling lipid microtube system for the sustained delivery of BMP-2. We determined that sustained delivery of BMP-2 from the lipid microtube system was able to enhance osteogenic differentiation compared to empty microtubes, however did not demonstrate a significant advantage compared to a bolus BMP-2 dose in vitro. Second, we developed and assessed the functionality of an affinity-based system to sequester BMP-2 at the implant site and retain bioactivity by incorporating heparin within a collagen matrix. Incorporation of heparin in the collagen matrix improved BMP-2 retention and bioactivity, thus enhancing cell-mediated mineralized matrix deposition in vitro. Lastly, the affinity-based BMP-2 delivery system was evaluated in a challenging in vivo bone repair model. Delivery of pre-bound BMP-2 and heparin in a collagen matrix resulted in new bone formation with mechanical properties not significantly different to those of intact bone. Whereas delivery of BMP-2 in collagen or collagen/heparin matrices had similar volumes of regenerated mineralized tissue but resulted in mechanical properties significantly less than intact bone properties. The work presented in this thesis aimed to address parameters currently preventing optimal performance of protein therapies including stability, duration of exposure, and localization at the treatment site. We were able to demonstrate that sustained delivery of BMP-2 from lipid microtubes was able to induce osteogenic differentiation, although this sustained delivery approach was not significantly advantageous over a bolus dose. Additionally, we demonstrated that the affinity-based system was able to improve BMP-2 retention within the scaffold and in vitro activity. However, in vivo implantation of this system demonstrated that only delivery of pre-complexed BMP-2 and heparin resulted in regeneration of bone with mechanical properties not significantly different from intact bone. These results indicate that delivery of BMP-2 and heparin may be an advantageous strategy for clinically challenging bone defects.

The aim of this thesis is to determine the feasibility of an injectable delivery system based on 5-ethylene ketal ε-caprolactone for localized delivery of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) for treating critical limb ischemia. HGF and VEGF were chosen because of their ability to simultaneously stimulate the proliferation and migration of endothelial cells, to initiate the formation of blood vessels and the recruitment of pericytes to stabilize the blood vessels. Homopolymer of 5-ethylene ketal ε-caprolactone and its copolymer with D,L-Lactide were synthesized by ring opening polymerization using hydrophobic initiator (octan-1-ol) or an hydrophilic initiator (MPEG), and stannous octanoate as a co-initiator/catalyst. The resulting polymers were amorphous and viscous liquids at room temperature. The viscosity, biodegradation rate, and release rate were varied by copolymerizing with D,L-lactide and/or initiating with MPEG or octan-1-ol. In vitro, the polymers degraded with surface erosion characterized by a nearly linear mass loss with time with no significant change in number average molecular weight and glass transition temperature. The ratio of EKC to DLLA in the copolymer remained the same throughout the degradation studies. A similar degradation mechanism was observed in vivo when the copolymer initiated with octan-1-ol was implanted subcutaneously in rats. In vivo, the polymer exhibited a moderate chronic inflammatory response, characterized by the presence of neutrophils, macrophages, fibroblasts and fibrous capsule formation. The inflammatory response decreased with time but was still on going after 18 weeks of subcutaneous implantation. Protein release from the polymer was transported by convection through the hydrated polymer region, at a rate determined by the osmotic pressure generated and the hydraulic conductivity of the polymer. Highly bioactive VEGF and HGF were released in a sustained manner, without burst effect for over 41 days when delivered simultaneously, using the osmotic release mechanism. VEGF was released at the rate of 36 ± 7 ng/day for 41 days, while HGF was released at the rate of 16 ± 2 ng/day for 70 days. Factors that influenced release of proteins were their solubility in the concentrated trehalose solution and hydraulic permeability of the polymer. This delivery system can serve as a potential vehicle for controlled release of VEGF and HGF for treating critical limb ischemia or the controlled release of other proteins for other clinical applications. / Thesis (Ph.D, Chemical Engineering) -- Queen's University, 2012-05-23 10:18:48.307

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