Design and Characterization of an Intraoperatively Loaded Protein Delivery Device for the Treatment of Open Tibial Fractures
Authors:   , Yu Aaron , Wheatley Margaret A. 
Published: July 2021

The FDA approved INFUSE Bone Graft is the standard of care for the therapeutic delivery of BMP-2 in open tibial fractures stabilized via intramedullary nailing. Though the device is considered clinically effective, several serious and potentially preventable side effects induced by the Bone Graft's initial burst release characteristics, such as massive swelling, cystic bone growth, and ectopic bone growth have negatively impacted the lives of countless patients. Based on analysis of recent studies involving BMP-2 delivery technologies, it was determined that a lower dose, sustained release of BMP-2, averaging 1.37 to 25.7 [mu]g per day over at the duration of at least 2-4 weeks, absent a burst release, would dramatically decrease the chances of adverse side effects while still providing the therapeutic benefits of BMP-2. Several new technologies have been developed that provide similar release profiles to the abovementioned, and they have shown much promise in several in vivo and in vitro studies. However, the majority of those technologies rely on pre-encapsulation of the BMP-2 and harsh polymer processing techniques that facilitate denaturation of the protein. Intraoperative loading circumvents this shortcoming, since the BMP-2 is stored in its stable, lyophilized form until needed. Over the course of this design project, a novel, biodegradable protein delivery device was developed in an attempt to combine the advantages of intraoperative loading with a more clinically relevant delivery profile for BMP-2. The proposed design consisted of a two component system comprising a Polycaprolactone : PEG 400 composite outer pouch with an internal, fibrous sodium alginate sponge. The system was designed for intraoperative loading using a technique similar to the INFUSE Bone Graft, where a reconstituted model protein solution (bovine serum albumin) is injected into the PCL: PEG 400 pouch containing an alginate sponge. After a brief 5 minute wait, a 10 % w/v CaCl2 solution is injected into the pouch, causing crosslinking of the swelled alginate and encapsulation of the protein. The pouch is then tightly sealed with a solvent bonding approach using ethyl acetate. This pouch design allows for successful loading with reconstituted protein solution within 20 minutes. The release characteristics of the device, as well as alternative preparation methods and their effect on the pouch release rate were investigated through dissolution testing in simulated physiological conditions (PBS, pH 7.4 & 37°C) and the Bradford-Coomassie assay. Several pouch formulations were assessed, and three were capable of providing sustained release of BSA in the desired 1.37-25.7 [mu]g range over 14 days, with no burst release. It was determined that modifying the ratio of PCL to PEG 400 would cause a predictable change in the release profile of the pouches, in that the higher the PCL : PEG 400 ratio, the slower the release. In addition to the protein delivery aspects, the material degradation and surface morphologies of the pouch were monitored over the desired dissolution period to understand this novel technology to the degree that future modifications to the design could be conducted if necessary. The polymer degradation rate of the pouch was assessed with inherent viscosity testing and GPC. The results of these tests show a minimal decrease in IV, Mn, and Mw over 16.4 days, suggesting that no substantial degradation occurs in the pouch material during the delivery period. SEM imaging of the pouch material over the delivery period combined with the previous studies suggest that when the material is placed in aqueous media, the PEG 400 will dissolve out rapidly. A mass change experiment was attempted to quantify the amount of PEG 400 lost over time once placed in aqueous media, which showed that 90.4 % of the PEG 400 was lost within 1 minute, 98.3 % within 1 hour, and ~100% of the total PEG 400 content within 5 hours of submersion. The removal of PEG 400 from the pouch left behind a porous PCL material exhibiting distributed pores approximately 2-5 [mu]m in diameter. This network acted as a rate controlling membrane, limiting mass transport into and out of the pouch, allowing for sustained release of the encapsulated protein from the sodium alginate via diffusion, while preventing a burst release. A preliminary BSA specific competitive ELISA was conducted to determine if the model protein could be encapsulated and released from the delivery device over a two week period without damage to its binding sites specific to the ELISA. Successful binding would therefore suggest that the overall tertiary structure of the BSA was uncompromised. The results of the ELISA showed consistent, sustained release from three different pouch formulations, thereby verifying that a complex protein could be successfully loaded into the device and delivered in a controlled, predictable manner, with no adverse effects on the protein structure. Bioactivity testing with a BMP-2 specific ELISA and the Alkaline Phosphatase assay (ALP) with C2C12 cells was planned, but due to time and cost constraints, the test was postponed. Nevertheless, the previous results have been quite encouraging, and low bioactivity from BMP-2 released from the device was not expected.