title: Bone Morphogenetic Protein-2 Adsorption onto Poly-ɛ-caprolactone Better Preserves Bioactivity In Vitro and Produces More Bone In Vivo than Conjugation Under Clinically Relevant Loading Scenarios.
abstract: BACKGROUND
  One strategy to reconstruct large bone defects is to prefabricate a vascularized flap by implanting a biomaterial scaffold with associated biologics into the latissimus dorsi and then transplanting this construct to the defect site after a maturation period. This strategy, similar to all clinically and regulatory feasible biologic approaches to surgical reconstruction, requires the ability to quickly (<1 h within an operating room) and efficiently bind biologics to scaffolds. It also requires the ability to localize biologic delivery. In this study, we investigated the efficacy of binding bone morphogenetic protein-2 (BMP2) to poly-ɛ-caprolactone (PCL) using adsorption and conjugation as a function of time.
  
  
  METHODS
  BMP2 was adsorbed (Ads) or conjugated (Conj) to PCL scaffolds with the same three-dimensional printed architecture while altering exposure time (0.5, 1, 5, and 16 h), temperature (4°C, 23°C), and BMP2 concentration (1.4, 5, 20, and 65 μg/mL). The in vitro release was quantified, and C2C12 cell alkaline phosphatase (ALP) expression was used to confirm bioactivity. Scaffolds with either 65 or 20 μg/mL Ads or Conj BMP2 for 1 h at 23°C were implanted subcutaneously in mice to evaluate in vivo bone regeneration. Micro-computed tomography, compression testing, and histology were performed to characterize bone regeneration.
  
  
  RESULTS
  After 1 h exposure to 65 μg/mL BMP2 at 23°C, Conj and Ads resulted in 12.83 ± 1.78 and 10.78 ± 1.49 μg BMP2 attached, respectively. Adsorption resulted in a positive ALP response and had a small burst release; whereas conjugation provided a sustained release with negligible ALP production, indicating that the conjugated BMP2 may not be bioavailable. Adsorbed 65 μg/mL BMP2 solution resulted in the greatest regenerated bone volume (15.0 ± 3.0 mm³), elastic modulus (20.1 ± 3.0 MPa), and %bone ingrowth in the scaffold interior (17.2% ± 5.4%) when compared with conjugation.
  
  
  CONCLUSIONS
  Adsorption may be optimal for the clinical application of prefabricating bone flaps due to BMP2 binding in a short exposure time, retained BMP2 bioactivity, and bone growth adhering to scaffold geometry and into pores with healthy marrow development.
authors.affiliation: Department of Biomedical Engineering, University of Michigan , Ann Arbor, Michigan.
authors.forename: Janki J
authors.initials: JJ
authors.lastname: Patel
authors.forename: Colleen L
authors.initials: CL
authors.lastname: Flanagan
authors.forename: Scott J
authors.initials: SJ
authors.lastname: Hollister
mesh_terms: D000327:Adsorption; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D055396:Bone Morphogenetic Protein 2 / Q000008:administration & dosage / Q000737:chemistry*; D001861:Bone Regeneration; D001842:Bone and Bones / Q000166:cytology / Q000378:metabolism*; D002478:Cells, Cultured; D019245:Compressive Strength; D006801:Humans; D007700:Kinetics; D051379:Mice; D011091:Polyesters / Q000737:chemistry*; D016062:Porosity; D013314:Stress, Mechanical; D013696:Temperature; D054457:Tissue Scaffolds / Q000737:chemistry; D055114:X-Ray Microtomography
chemical_list: C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D011091:Polyesters; C016240:polycaprolactone; D000469:Alkaline Phosphatase