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

Abstract

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:DMet: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 lCT 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). InThe institution of one or more of the authors (SAG, FE, JCW) has received, during the study period, funding from the Orthopaedic Extremity Trauma Research Program (SAG and JCW), the National Institute of Arthritis and Musculoskeletal Diseases (SAG, JCW, and FE), Medtronic, Inc (SAG); Oak Ridge Institute (AJH); and the National Institutes of Health (SAG). One of the authors certifies that he (SAG), or a member of his or her immediate family, has or may receive payments or benefits, during the study period, an amount less than USD 10,000, from Medtronic Spine and Biologics (Memphis, TN, USA).

Remodeling of injectable, low‐viscosity polymer/ceramic bone grafts in a sheep femoral defect model

“…Bone volume/total volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) within the regions of interest were computed using SCANCO's Medical microCT systems software as described previously . A host bone control was run using the same parameters and analyzed for comparison to the graft groups . Additional scans were run on LV/A and LV/CM composites (prepared as above) and packed ceramic particles (to replicate group C) to provide initial morphometric parameter values for all test groups.…”

“…Residual matrix particles (A or CM), residual PUR, and newly formed bone were quantified for each sample (two data points per slide) in an area of interest (6 × 14.6 mm) in the center of each defect using color analysis on Metamorph software (Version 7.0.1, Waltham, MA). The area of interest was divided into 6 regions measuring 1.83 mm wide and corresponding to the volumes of interest measured by µCT as described previously …”

“…15,18 A host bone control was run using the same parameters and analyzed for comparison to the graft groups. 30 Additional scans were run on LV/A and LV/CM composites (prepared as above) and packed ceramic particles (to replicate group C) to provide initial morphometric parameter values for all test groups.…”

Bacterial d -amino acids suppress sinonasal innate immunity through sweet taste receptors in solitary chemosensory cells

“…D-amino acids may stimulate biofilm disassembly in B. subtilis (36) and inhibit biofilm formation of Pseudomonas aeruginosa (37, 38), Staphylococcus aureus (39) (40), and Staphylococcus epidermidis (41) as well as cause dispersal or detachment of cells from single-species and multi-species biofilms containing P. aeruginosa (42), S. epidermidis (41), or Staphylococcus aureus (43, 44). D-amino acids may inhibit adhesion of bacterial cells to one another (45, 46).…”

Eradication of Enterococcus faecalis Biofilms on Human Dentin

“…The biocompatibility of D -amino acids is especially promising as they were non-cytotoxic to human osteoblasts at concentrations less than 50 mmol/L, 25 times more than the required concentration for biofilm inhibition (Harmata et al, 2015) and were non-toxic when orally administrated (Tsume et al, 2014). Inducing dispersal by a sub-toxic concentrations of an anti-biofilm agent is of high interest (Kolodkin-Gal et al, 2010; Romero and Kolter, 2011; Bucher et al, 2015, 2016), as it is expected to reduce the selective pressure for the success of resistant mutants.…”