(M-482) Microparticle Fabrication for Scaffolds with Sustained Delivery of Bioactive Factors that Promote Vascular Regeneration

Introduction::

Every year the number of people that find themselves on an organ transplant waiting list increases by 4%. (Howard, 2007). The current treatments rely on autologous and allogenic transplants. While this strategy works well, some major drawbacks include transplant rejection and limited donor tissue. An alternative treatment would be tissue engineered scaffolds and grafts; however, a major limitation preventing clinical translation of tissue engineered products is insufficient vascularization. Therapeutic angiogenesis is being explored as a method to promote vascular sprouting and maturation (Brudno et.al., 2013).  Engineered scaffolds can either be pre-vascularized in vitro, or they can be functionalized to release therapeutic agents that promote rapid vascular ingrowth. To ensure vascular functionality, biochemical signaling cues that encourage vascular stabilization/maturation must be continually delivered to the newly formed vasculature. As such, pro-angiogenic scaffolds should incorporate a drug delivery method that can achieve sustained release kinetics, one such method is the inclusion of microparticles (< 100 μm in diameter) with tunable crosslinking densities The aim of this research is to fabricate Chondroitin Sulfate Methacrylate (CSMA) microparticles with tunable release kinetics, to sustain the delivery of growth factors in a scaffold to promote the rapid formation of mature blood vessels. As such, we hypothesize that higher concentration CSMA microparticles will result in a more sustained release profile.

Materials and Methods:: Chondroitin sulfate (CS) from bovine trachea and glycidyl methacrylate were reacted for 24 hours followed by precipitation, dialysis, and lyophilization to make chondroitin sulfate methacrylate (CSMA). Dry CSMA was dissolved in DI water to create 10, 15, and 20% solutions. Bovine serum albumin (BSA) and lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate (LAP) photoinitiator were added to the CSMA to make final concentrations of 30mg/mL and 2% respectively. Fifty μL of the CSMA mixture were added to a cylindrical mold and crosslinked with a 405 nm blue light for 3 minutes. After gelation the gels were loaded into a 28G insulin syringe and sheared through the needle. The microparticles were then suspended in phosphate buffered saline (PBS) at a concentration of 75 mg of microparticles per mL. A protein release analysis was performed by incubating the BSA loaded microparticles at 37C for 6 days. Supernatant was collected every 24 hours and stored at -20℃. Protein quantification was performed using a bicinchoninic acid (BCA) assay according to the manufacturer instructions. To analyze the particle size distribution, the microparticles were stored at room temperature for 24 hours and then stained with 1% methylene blue. Brightfield microscopy and subsequent ImageJ analysis was used to quantify the feret diameters.

Results, Conclusions, and Discussions::

Quantitative morphometric analyses of microparticle images show a variety of particle sizes (Figure 1). The 10, 15, and 20% microparticles had average feret diameters of ­28.31< ![if !msEquation] >< ![if !vml] >< ![endif] >< ![endif] > 5.56 μm, 10.94< ![if !msEquation] >< ![if !vml] >< ![endif] >< ![endif] > 6.12μm, and 19.77< ![if !msEquation] >< ![if !vml] >< ![endif] >< ![endif] > 2.35 μm respectively. Average feret diameters are between 5μm and 50 μm. The 10% microparticles may exhibit larger feret diameters because of lower crosslinking density and increased swelling. The total protein released from the 10, 15, and 20% microparticles was 18.525%, 17.24%, and 4.80% respectively. 10% CSMA microparticles showed the most protein release while 20% CSMA microparticles showed the least protein release (Figure 2). The 10% microparticles exhibited similar release profiles to the 15% microparticles. The 20% microparticles exhibited the slowest release kinetics of the three experimental conditions, likely because of increased crosslinking densities.

Overall, the 10 and 15% concentration CSMA microparticles showed larger sustained therapeutic payload delivery than the 20% microparticles. Additionally, 15% microparticles showed a relatively smaller swelling. As such these microparticles are a promising candidate for the sustained release of maturation factors during vascular regeneration. Future work will focus on producing more uniform microparticle sizes as well as loading the particles with specific growth factors such as Ang-1 and PDGF-BB and evaluating the degree of vascular maturation with and without microparticles present.

Acknowledgements (Optional): : Funding for this project was provided by NSF REU grant EEC 2150076.

References (Optional): :

Howard, David, H. 2007. "Producing Organ Donors." Journal of Economic Perspectives, 21 (3): 25-36.

DOI: 10.1257/jep.21.3.25 

Brudno, Y., Ennett-Shepard, A.B., Chen, R.R, Aizenberg, M., & Monney, D.J. (2013). Enhancing microvascular formation and vessel maturation through temporal control over multiple pro-angiogenic and pro-maturation factors. In Biomaterials (Vol. 34. Issue 36, pp 9201-9209). Elsevier BV. https://doi.org/10.1016/j.biomaterials.2013.08.007.