d-amino Acid Inhibits Biofilm but not New Bone Formation in an Ovine Model

Background: Infectious complications of musculoskeletal trauma are an important factor contributing to patient morbidity. Biofilm-dispersive bone grafts augmented with d-amino acids (d-AAs) prevent biofilm formation in vitro and in vivo, but the effects of d-AAs on osteocompatibility and new bone formation have not been investigated. Questions/purposes: We asked: (1) Do d-AAs hinder osteoblast and osteoclast differentiation in vitro? (2) Does local delivery of d-AAs from low-viscosity bone grafts inhibit new bone formation in a large-animal model? Methods: Methicillin-sensitive Staphylococcus aureus and methicillin-resistant S aureus clinical isolates, mouse bone marrow stromal cells, and osteoclast precursor cells were treated with an equal mass (1:1:1) mixture of d-Pro:d-Met:d-Phe. The effects of the d-AA dose on biofilm inhibition (n = 4), biofilm dispersion (n = 4), and bone marrow stromal cell proliferation (n = 3) were quantitatively measured by crystal violet staining. Osteoblast differentiation was quantitatively assessed by alkaline phosphatase staining, von Kossa staining, and quantitative reverse transcription for the osteogenic factors a1Col1 and Ocn (n = 3). Osteoclast differentiation was quantitatively measured by tartrate-resistant acid phosphatase staining (n = 3). Bone grafts augmented with 0 or 200 mmol/L d-AAs were injected in ovine femoral condyle defects in four sheep. New bone formation was evaluated by μCT and histology 4 months later. An a priori power analysis indicated that a sample size of four would detect a 7.5% difference of bone volume/total volume between groups assuming a mean and SD of 30% and 5%, respectively, with a power of 80% and an alpha level of 0.05 using a two-tailed t-test between the means of two independent samples. Results: Bone marrow stromal cell proliferation, osteoblast differentiation, and osteoclast differentiation were inhibited at d-AAs concentrations of 27 mmol/L or greater in a dose-responsive manner in vitro (p < 0.05). In methicillin-sensitive and methicillin-resistant S aureus clinical isolates, d-AAs inhibited biofilm formation at concentrations of 13.5 mmol/L or greater in vitro (p < 0.05). Local delivery of d-AAs from low-viscosity grafts did not inhibit new bone formation in a large-animal model pilot study (0 mmol/L d-AAs: bone volume/total volume = 26.9% ± 4.1%; 200 mmol/L d-AAs: bone volume/total volume = 28.3% ± 15.4%; mean difference with 95% CI = −1.4; p = 0.13). Conclusions: d-AAs inhibit biofilm formation, bone marrow stromal cell proliferation, osteoblast differentiation, and osteoclast differentiation in vitro in a dose-responsive manner. Local delivery of d-AAs from bone grafts did not inhibit new bone formation in vivo at clinically relevant doses. Clinical Relevance: Local delivery of d-AAs is an effective antibiofilm strategy that does not appear to inhibit bone repair. Longitudinal studies investigating bacterial burden, bone formation, and bone remodeling in contaminated defects as a function of d-AA dose are required to further support the use of d-AAs in the clinical management of infected open fractures.