Kickstarting BMP-2 induced Bone Tissue Engineering

Summary Growth factor-based therapies have been shown to facilitate bone tissue engineering. Bone morphogenetic protein-2 (BMP-2) is a growth factor extensively used in the clinic for the purpose of stimulating bone growth. Although preclinical studies showed impressive results and low side effect rates, the high doses needed for effective bone formation in humans are associated with various serious side effects. To prolong its site specific pharmacological actions, biomaterials are used for local delivery. However, the optimal delivery rate and biomaterial conditions are still unknown. This thesis focusses on identifying and optimizing parameters that influence BMP-2 release and corresponding bone formation. Adjustments of the growth factor release rate often requires changes to the biomaterial characteristics, which may influence bone formation. To solve this complex puzzle, a biomaterial was developed capable of modifying BMP-2 release without changing the biomaterial characteristics as well as modifying the biomaterial characteristics without changing the BMP-2 release profiles. Hereby this thesis demonstrated that the local pharmacological actions of BMP-2 are highly dependent on three main parameters, i.e. BMP-2 pharmacokinetics, biomaterial chemistry and application site. Whereas BMP-2 release profiles influenced bone formation when applied under the skin, no effect was seen when applied in a bone defect. Based on this thesis, we have to conclude that “one size fits all” will not apply for the optimal release profile in BMP-2 based bone tissue engineering. Most likely, biomaterial characteristics and release profiles need to be tailored to meet the requirements of the clinical application

Kickstarting BMP-2 induced Bone Tissue Engineering

Summary Growth factor-based therapies have been shown to facilitate bone tissue engineering. Bone morphogenetic protein-2 (BMP-2) is a growth factor extensively used in the clinic for the purpose of stimulating bone growth. Although preclinical studies showed impressive results and low side effect rates, the high doses needed for effective bone formation in humans are associated with various serious side effects. To prolong its site specific pharmacological actions, biomaterials are used for local delivery. However, the optimal delivery rate and biomaterial conditions are still unknown. This thesis focusses on identifying and optimizing parameters that influence BMP-2 release and corresponding bone formation. Adjustments of the growth factor release rate often requires changes to the biomaterial characteristics, which may influence bone formation. To solve this complex puzzle, a biomaterial was developed capable of modifying BMP-2 release without changing the biomaterial characteristics as well as modifying the biomaterial characteristics without changing the BMP-2 release profiles. Hereby this thesis demonstrated that the local pharmacological actions of BMP-2 are highly dependent on three main parameters, i.e. BMP-2 pharmacokinetics, biomaterial chemistry and application site. Whereas BMP-2 release profiles influenced bone formation when applied under the skin, no effect was seen when applied in a bone defect. Based on this thesis, we have to conclude that “one size fits all” will not apply for the optimal release profile in BMP-2 based bone tissue engineering. Most likely, biomaterial characteristics and release profiles need to be tailored to meet the requirements of the clinical application

In Vitro and In Vivo Correlation of BMP-2 Release Profiles from Complex Delivery Vehicles

Local sustained delivery of bioactive molecules from biomaterials is a promising strategy to enhance bone regeneration. To optimize delivery vehicles for bone formation, the design characteristics are tailored with consequential effect on BMP-2 release and bone regeneration. Complying with the 3R principles, the growth factor release is often investigated in vitro using several buffers to mimic the in vivo physiological environment. However, this remains an unmet need. Therefore, this study investigates the correlation between the in vitro and in vivo (IVIVC) BMP-2 release from complex delivery vehicles in several commonly used in vitro buffers: cell culture model, phosphate buffered saline, and a strong desorption buffer. The results from this study showed that the release environment affected the BMP-2 release profiles, creating distinct relationships between release versus time and differences in extent of release. According to the guidance set by the U.S. Food and Drug Administration (FDA), in vitro- in vivo correlation resulted in level A internal predictability for individual composites. Since the IVIVC was influenced by the BMP-2 loading method and composite surface chemistry, the external predictive value of the IVIVCs was limited. These results show that the IVIVCs can be used for predicting the release of an individual composite. However, the models cannot be used for predicting in vivo release for different composite formulations since they lack external predictability. Potential confounding effects of drug type, delivery vehicle formulations and application site should be added to the equation to develop one single IVIVC applicable for complex delivery vehicles. Altogether, these results imply that more sophisticated in vitro systems should be used in bone regeneration to accurately discriminate and predict in vivo BMP-2 release from different complex delivery vehicles

In Vitro and In Vivo Correlation of BMP-2 Release Profiles from Complex Delivery Vehicles

Local sustained delivery of bioactive molecules from biomaterials is a promising strategy to enhance bone regeneration. To optimize delivery vehicles for bone formation, the design characteristics are tailored with consequential effect on BMP-2 release and bone regeneration. Complying with the 3R principles, the growth factor release is often investigated in vitro using several buffers to mimic the in vivo physiological environment. However, this remains an unmet need. Therefore, this study investigates the correlation between the in vitro and in vivo (IVIVC) BMP-2 release from complex delivery vehicles in several commonly used in vitro buffers: cell culture model, phosphate buffered saline, and a strong desorption buffer. The results from this study showed that the release environment affected the BMP-2 release profiles, creating distinct relationships between release versus time and differences in extent of release. According to the guidance set by the U.S. Food and Drug Administration (FDA), in vitro- in vivo correlation resulted in level A internal predictability for individual composites. Since the IVIVC was influenced by the BMP-2 loading method and composite surface chemistry, the external predictive value of the IVIVCs was limited. These results show that the IVIVCs can be used for predicting the release of an individual composite. However, the models cannot be used for predicting in vivo release for different composite formulations since they lack external predictability. Potential confounding effects of drug type, delivery vehicle formulations and application site should be added to the equation to develop one single IVIVC applicable for complex delivery vehicles. Altogether, these results imply that more sophisticated in vitro systems should be used in bone regeneration to accurately discriminate and predict in vivo BMP-2 release from different complex delivery vehicles

Effect of biomaterial electrical charge on in vivo bone formation

Biomaterials that act as both protein delivery vehicle and scaffold, can improve the safety and efficacy of bone morphogenetic protein-2 (BMP-2) for clinical applications. However, the optimal scaffold characteristics are not known. The osteoinductive and osteoconductive capacity of a fixed electrically charged surface is thus far unexplored. Therefore, in this study we aim to investigate the effect of different electrical states on BMP-2 induced bone formation in oligo[(polyethylene glycol) fumarate] (OPF) hydrogels. Neutral, negatively or positively charged scaffolds were fabricated using unmodified OPF (neutral charge), sodium methacrylate (SMA) crosslinked OPF (negative charge), or [2-(methacryloyloxy) ethyl] trimethylammonium chloride (MAE) crosslinked OPF (positive charge), respectively. To allow investigation of surface charge for different BMP-2 release rates, three BMP-2 release profiles were generated by protein encapsulation into poly(lactic-co-glycolic acid) (PLGA) microspheres and/or adsorption on the OPF composite. Release of radiolabeled 125I-BMP-2 was analyzed in vitro and in vivo and bone formation was assessed after 9 weeks of subcutaneous implantation in rats. Negatively charged OPF generated significantly more bone formation compared to neutral and positively charged OPF. This effect was seen for all three loading methods and subsequent BMP-2 release profiles. Along with charge modifications, a more sustained release of BMP-2 improved bone formation in OPF composites. Overall, this study clearly shows that negative charge enhances bone formation compared to neutral and positive charge in OPF composites

The osteoinductive effect of controlled BMP-2 release is location-dependent

The main challenge in BMP-2 based application lies in finding strategies that prolong its effective period, as it has a short biological half-life. Several BMP-2 release profiles have shown to enhance bone formation at various application sites. However, it remains to be determined which BMP-2 release profile best augments bone formation and whether this effect is location-dependent. Therefore, the aim of this study was to investigate the effect of BMP-2 release from oligo[(polyethylene glycol) fumarate] bis(2-(methacryloyloxy)ethyl) phosphate (OPF-BP) composites on the osteoinductive efficacy at ectopic versus orthotopic application. By varying the BMP-2 loading method, three different OPF-BP composites were created with varied release profiles. The composites were compared to unloaded OPF-BP as negative control, and to the clinically used Infuse® absorbable collagen sponge (ACS) as positive control. Bone formation was assessed by micro-computed tomography after 9 weeks of subcutaneous implantation and 3, 6, and 9 weeks of orthotopic implantation in rats (n=48). Whereas a BMP-2 burst release of >49% generated significantly more bone compared to sustained release (burst release <30%) at the subcutaneous implantation site, differential release did not affect bone formation at the orthotopic site. Furthermore, all BMP-2 containing OPF-BP composites showed significantly more bone formation compared to ACS in the orthotopic implantation site. In conclusion, this study clearly shows that the osteoinductive effect of different BMP-2 release profiles is location dependent. Additionally, more bone formation in OPF-BP compared to ACS at both application sites emphasizes the role of biomaterials as a scaffold to achieve proper bone tissue formation

Effect of biomaterial electrical charge on in vivo bone formation

Biomaterials that act as both protein delivery vehicle and scaffold, can improve the safety and efficacy of bone morphogenetic protein-2 (BMP-2) for clinical applications. However, the optimal scaffold characteristics are not known. The osteoinductive and osteoconductive capacity of a fixed electrically charged surface is thus far unexplored. Therefore, in this study we aim to investigate the effect of different electrical states on BMP-2 induced bone formation in oligo[(polyethylene glycol) fumarate] (OPF) hydrogels. Neutral, negatively or positively charged scaffolds were fabricated using unmodified OPF (neutral charge), sodium methacrylate (SMA) crosslinked OPF (negative charge), or [2-(methacryloyloxy) ethyl] trimethylammonium chloride (MAE) crosslinked OPF (positive charge), respectively. To allow investigation of surface charge for different BMP-2 release rates, three BMP-2 release profiles were generated by protein encapsulation into poly(lactic-co-glycolic acid) (PLGA) microspheres and/or adsorption on the OPF composite. Release of radiolabeled 125I-BMP-2 was analyzed in vitro and in vivo and bone formation was assessed after 9 weeks of subcutaneous implantation in rats. Negatively charged OPF generated significantly more bone formation compared to neutral and positively charged OPF. This effect was seen for all three loading methods and subsequent BMP-2 release profiles. Along with charge modifications, a more sustained release of BMP-2 improved bone formation in OPF composites. Overall, this study clearly shows that negative charge enhances bone formation compared to neutral and positive charge in OPF composites

The osteoinductive effect of controlled BMP-2 release is location-dependent

The main challenge in BMP-2 based application lies in finding strategies that prolong its effective period, as it has a short biological half-life. Several BMP-2 release profiles have shown to enhance bone formation at various application sites. However, it remains to be determined which BMP-2 release profile best augments bone formation and whether this effect is location-dependent. Therefore, the aim of this study was to investigate the effect of BMP-2 release from oligo[(polyethylene glycol) fumarate] bis(2-(methacryloyloxy)ethyl) phosphate (OPF-BP) composites on the osteoinductive efficacy at ectopic versus orthotopic application. By varying the BMP-2 loading method, three different OPF-BP composites were created with varied release profiles. The composites were compared to unloaded OPF-BP as negative control, and to the clinically used Infuse® absorbable collagen sponge (ACS) as positive control. Bone formation was assessed by micro-computed tomography after 9 weeks of subcutaneous implantation and 3, 6, and 9 weeks of orthotopic implantation in rats (n=48). Whereas a BMP-2 burst release of >49% generated significantly more bone compared to sustained release (burst release <30%) at the subcutaneous implantation site, differential release did not affect bone formation at the orthotopic site. Furthermore, all BMP-2 containing OPF-BP composites showed significantly more bone formation compared to ACS in the orthotopic implantation site. In conclusion, this study clearly shows that the osteoinductive effect of different BMP-2 release profiles is location dependent. Additionally, more bone formation in OPF-BP compared to ACS at both application sites emphasizes the role of biomaterials as a scaffold to achieve proper bone tissue formation