Release of antimicrobial peptide Dhvar-5 from polymethylmethacrylate beads.

Osteomyelitis is still a major cause of morbidity and remains a difficult complication to treat in orthopaedic surgery. The treatment of choice is a combination of systemic and local antibiotics. The insertion of gentamicin-loaded polymethylmethacrylate (PMMA) beads into the bone results in high local concentrations of gentamicin and low systemic concentrations. However, the effectiveness of this treatment is being hampered by the emergence of antimicrobial resistance. New antimicrobial agents are therefore needed. One new class of promising antibiotics is antimicrobial peptides (AMP). Derived from natural human peptides, these have a low tendency to induce antimicrobial resistance. Dhvar-5 is an antimicrobial peptide based on histatin-5, which is found in human saliva and consists of 14 amino acids. It has demonstrated bactericidal activity in vitro. In order to develop a new local treatment using Dhvar-5 for osteomyelitis, we investigated its release from PMMA beads and its antimicrobial activity against a clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA) before and after release from PMMA beads. Specific amounts of Dhvar-5 were incorporated into PMMA mini beads, containing 120, 600 and 1200 microg of Dhvar-5, respectively. Dhvar-5 was released from the beads in all three groups. Total release from the 120 microg beads was 9 microg per bead after 7 days. However, the release per bead in the 600 and 1200 microg beads was far more, respectively, 416 and 1091 microg over a 28 day period. After release, the Dhvar-5 also retained its antimicrobial activity against MRSA. On the basis of these data we conclude that the amount of Dhvar-5 release from PMMA beads is not proportionate to the amount incorporated; instead, it demonstrated an exponential relationship to the amount of total peptide released. Furthermore, the released peptide remained biologically active against a clinical isolate of MRSA.

Degradable bisphosphonate-alkaline phosphatase-complexed hydroxyapatite implants in vitro.

Degradable hydroxyapatite (HA) implants complexed with the resorption inhibiting agent bisphosphonate (PCP) and the mineralizing agent alkaline phosphatase (ALP) can theoretically maintain alveolar bone mass directly after extraction of teeth. The present in vitro study investigated the surface properties of PCP-ALP-complexed HA implants in relation to the requirements of implant behavior and action. Adsorbed PCP (pH 3.49) resulted in a flattening and broadening of the phosphate peaks and the formation of carbonate peaks in the HA pattern of the implant indicating a chemical alteration of the HA surface. Adsorption of ALP onto PCP-altered HA surfaces was 26% lower than onto HA implant blank surfaces. PCP-ALP-complexed HA implants released the PCP and ALP steadily and continuously over observation periods of, respectively, 75 and 14 days. During these observation periods, the ceramic grains of the HA implant became smaller and intergrain boundaries became broader. These morphologic characteristics suggested preconditioning of the HA implant surface for future bonding and degradation in vivo. Individual grains were no longer bonded to other grains and detached from the implant which had become rounded in shape. From in vitro mice experiments we found that PCP concentrations between 10(-4) and 10(-3) M resulted in 45Ca-release from the bone HA. Our calculations showed, however, that only a total concentration of 1.4 x 10(-4) M PCP was gradually released over the whole observation period. In another experiment, it appeared that a PCP concentration in solution < 10(-3) M did not reduce ALP activity. It is concluded that release of PCP by the PCP-ALP-complexed implants is maintained at levels in the range to impair osteoclast bone resorption but not high enough to block osteoblast activity. The amount of ALP released can lead to induction of bone formation onto implant surfaces. pH-induced alterations in the microstructure and chemistry of the HA surface allow for controlled degradation of the HA implants in vitro. A PCP-ALP-complexed HA implant acting as temporary scaffolding for alveolar bone growth enhancement, mineralization, and maintenance seems to be a reasonable concept for preservation of the edentulous alveolus.

Dual X-ray absorptiometry for alveolar bone: precision of peri-implant mineral measurements ex vivo.

The precision of measurements of minor mineral changes in alveolar bone mineral content (ABMC) and alveolar bone mineral density (ABMD) on implant surfaces was determined in small regions (0.03 +/- 0.005 cm2) using dual X-ray absorptiometry (DXA). Dog hemimandibles with alveolar processes containing 17 implants were studied ex vivo. The precision was expressed as the coefficient of variation in percent (c.v. %). The ultra-high resolution protocol was applied to the mesial, distal and apical subregions of each implant. The line spacing was 0.0254 cm and the point resolution was 0.0127 cm. The mean c.v. (%) +/- s.d. for the ABMC in the mesial, distal and apical regions were 0.42 +/- 17, 0.47 +/- 0.21 and 0.48 +/- 0.18, respectively. For the ABMD these values were 0.42 +/- 0.16, 0.47 +/- 0.19 and 0.48 +/- 0.16. For each region approximately 68% of the 17 c.v. values were distributed within 1 s.d. from the mean c.v. These results indicate that measurements are highly reproducible (better than 0.48%) and that there are no differences in precision between several peri-implant regions. Changes as small as 0.85% in ABMC and ABMD in small areas adjacent to implant surfaces are measurable with a confidence level of 95%. Therefore the DXA technique will be expedient for our research evaluating the efficacy of the ceramic hydroxyapatite implant releasing agents affecting or inducing alveolar bone- and root cementum-like materials on its surface.

Dual-energy X-ray absorptiometry for histologic bone sections.

In fundamental osteoporosis research precise and accurate assessment of the mineral quantity in histological bone sections is of particular importance when studying the local effects of implants releasing bone modulating agents. A potentially useful technique to estimate the bone mineral density (BMD) is dual-energy X-ray absorptiometry (DXA). A highly collimated (0.13 mm) Hologic 2000 with a line spacing and point resolution of 0.13 mm was used. The mineral content was measured in regions of 3.1 mm(2). A ceramic hydroxyapatite (CHA) phantom was developed as a reference standard. The phantom was made of a single-phase hydroxyapatite starting powder by compressing and sintering at 1000 degrees Celsius. The true density was 3.14 + or - 0.001 g/cm(3). The calcium/phosphorus ratio was close to the theoretical one of 1.67. The mean precision error expressed as the coefficient of variation (CV) of the mineral density (MD) measurements of the phantoms with thicknesses of 1, 2, and 3 mm was 0.2%. Embedded undecalcified alveolar bone sections of dogs (0.0015-1 mm in thickness) were scanned simultaneously with a phantom 1 mm in thickness. The precision error (CV) of the BMD measurements calculated by DXA for sections > or = 0.1 mm and with a BMD > or = 0.14 g/cm(2) was 0.81%. There was a linear relationship between the BMD calculated by DXA and the estimated BMD in the histological bone sections by means of the true density of the phantom. It is concluded that DXA using a standard CHA phantom is a precise and accurate method to measure MD changes as small as 1% in histological bone areas of 3.1 mm(2) provided that the loss or gain in BMD is > or = 0.14 g/cm(2).