Purpose: Osteoporosis is a serious public health threat that costs the US over 19 billion dollars annually, and is the leading cause of the 1.5 million bone fractures that occur every year. Those over age 60, are at the highest risk of developing this deadly disease. Osteoporosis is marked by progressively decreased bone mass attributed to an imbalance of bone growth and resorption that occurs over time; creating fragile bones, prone to fracture. The resultant fractures often lead to significant debilitation, of which healing time is a critical determinant in the risk of death. Most fractures are healed enough to bare weight after 4 weeks, with full healing occurring months to years later. Yet, this time frame for fracture healing continues to result in debilitation and death for many patients. In order to increase fracture healing rate, bone regeneration must occur. Currently, there are agents that can accomplish this, the most efficacious of which are the proteins known as bone morphogenic proteins, or BMPs. These endogenous proteins result in significantly increased bone regeneration by increasing differentiation and activity of cells that create bone, osteoblasts. However, these highly potent proteins can have some very serious, even life-threatening, side effects. Thus, there remains a critical need to find a suitable delivery system for appropriate and safe administration of BMPs. Hydrogels are an attractive candidate for this application because of the wide range of potential compositions and manipulations they can undergo to fulfill delivery-specific needs. For practicability in bone regeneration, a thermoreversible hydrogel platform that necessitates less drug, provides direct and continuous site deposition, and allows single dose administration, is most advantageous. Thus, the highly biocompatible copolymer, Poly(DL-lactide)-b-Poly(ethylene glycol)-b-Poly(DL-lactide), or PLA-PEG-PLA, is uniquely favorable for utility as a delivery platform due to its ability to form a hydrogel at concentrations that match the necessary requirements. This includes the ability to remain liquid at ambient temperature with subsequent gel transition at 37°C, which allows 1) direct site deposition, 2) continuous release of drug, and 3) a single, safe injection by negating typical thermal requirements that could otherwise further injure the fracture site. Therefore, loading PLA-PEG-PLA hydrogels with BMP-2 has the potential to be clinically relevant as a therapy for increasing the rate of bone regeneration, especially in osteoporotic fractures.
Methods: Hydrogel preparation involves dissolving, PLA-PEG-PLA copolymer (MW range 1000-1700 Da per block) at concentrations of 10-30% (w/v) in water. Drug is loaded while in solution after gel formation. Characterization includes phase diagram measurements to assess gelation and the Nano-ZS90 Zetasizer to assess micelle size/distribution and thermoreversibility, at varying temperatures (10-40°C). Gel dissolution assesses stability of hydrogels in PBS over time (37°C, 30 rpm) with daily extraction/replacement of PBS until gel is disintegrated. Drug release is performed analogously, with quantification of BMP-2 or model protein from extracted PBS by ELISA. Extracted PBS is also incubated with pre-osteoblastic cells (MC3T3-E1, a murine cell line) in vitro for testing of cell viability with alamar blue, and efficacy with alkaline phosphotase (ALP) assay and stain.
Results: After successful hydrogel preparation, the highest molecular weight block polymers (10-30% w/v) were chosen for the solution-gel transition at 37°C (Figure 1). Light scattering data showed increasing micelle size/distribution with increasing temperature, confirming thermoreversibility, and a strong peak shift at 37°C, validating the sol-gel transition (Figure 2). Dissolution studies demonstrated a disintegration profile between 16-22 days, dependent on block weight and concentration (n=3-7 per formulation); an appropriate time frame for reduced fracture healing rate. Model drug release showed a favorable trend with stable unloading of protein over a similar time frame. Cell studies with MC3T3 confirm the biocompatibility of the platform and functional efficacy of the released drug from the platform.
Conclusion: Our data confirms the applicability of our drug-loaded platform for use in bone regeneration models. Characterizations of the hydrogel indicate a thermoreversible, injectable gel that transitions to a solid at body temperature, while releasing optimal concentrations of bone regenerative agents over a well-matched, reduced fracture healing time frame. Our data will provide clinically relevant information regarding the use of our BMP-2 loaded hydrogels for bone regeneration.
Moses Oyewumi– Associate Professor, Northeast Ohio Medical University