Influence of Absorbable Calcium Sulfate-Based Bone Substitute Materials on Human Haemostasis-In Vitro Biological Behavior of Antibiotic Loaded Implants.

Calcium sulfate (CS) formulations are frequently implanted as antibiotically impregnated bone substitutes in orthopedic and trauma surgery to prevent or treat bone infections. Calcium ions have been discussed as candidates to accelerate blood coagulation. The goal of this study is to evaluate substance-specific influences of CS formulations on blood coagulation. Specific ELISAs were conducted to determine markers of activated blood coagulation after incubation of human blood with CS beads. Additionally, wettability with freshly drawn human blood was measured. Three different types of CS bone substitute beads were compared (CS dihydrate with tripalmitin, containing Gentamicin (Herafill®-G: Group A) or Vancomycin (CaSO4-V: Group B); and a CS hemihydrate with Tobramycin (Osteoset®: Group C)). Examinations were performed by ELISA assays for F1+2, FXIIa and C3a. Our results prove that none of the CS preparations accelerated single specific assays for activated coagulation markers. This allows the conclusion that neither Herafill®-G (CaSO4-G) nor CaSO4-V alter haemostasis negatively. Blood samples incubated with Osteoset® display an elevated F1+2-activity. The addition of tripalmitin in Herafill®-G shifts the original into a significantly hydrophobic formulation. This was additionally proven by contact angle examination of the three substances with freshly drawn human blood, showing that acceleration of plasmatic coagulation is hindered by lipids and induced by surface effects caused by presence of rapidly soluble calcium ions in the Osteoset® preparation.

Correction: Viable adhered Staphylococcus aureus highly reduced on novel antimicrobial sutures using chlorhexidine and octenidine to avoid surgical site infection (SSI).

[This corrects the article DOI: 10.1371/journal.pone.0190912.].

Viable adhered Staphylococcus aureus highly reduced on novel antimicrobial sutures using chlorhexidine and octenidine to avoid surgical site infection (SSI).

BACKGROUND: Surgical sutures can promote migration of bacteria and thus start infections. Antiseptic coating of sutures may inhibit proliferation of adhered bacteria and avoid such complications. OBJECTIVES: This study investigated the inhibition of viable adhering bacteria on novel antimicrobially coated surgical sutures using chlorhexidine or octenidine, a critical factor for proliferation at the onset of local infections. The medical need, a rapid eradication of bacteria in wounds, can be fulfilled by a high antimicrobial efficacy during the first days after wound closure. METHODS: As a pretesting on antibacterial efficacy against relevant bacterial pathogens a zone of inhibition assay was conducted with middle ranged concentrated suture coatings (22 µg/cm). For further investigation of adhering bacteria in detail the most clinically relevant Staphylococcus aureus (ATCC®49230™) was used. Absorbable braided sutures were coated with chlorhexidine-laurate, chlorhexidine-palmitate, octenidine-laurate, and octenidine-palmitate. Each coating type resulted in 11, 22, or 33 µg/cm drug content on sutures. Scanning electron microscopy (SEM) was performed once to inspect the coating quality and twice to investigate if bacteria have colonized on sutures. Adhesion experiments were assessed by exposing coated sutures to S. aureus suspensions for 3 h at 37°C. Subsequently, sutures were sonicated and the number of viable bacteria released from the suture surface was determined. Furthermore, the number of viable planktonic bacteria was measured in suspensions containing antimicrobial sutures. Commercially available sutures without drugs (Vicryl®, PGA Resorba®, and Gunze PGA), as well as triclosan-containing Vicryl® Plus were used as control groups. RESULTS: Zone of inhibition assay documented a multispecies efficacy of novel coated sutures against tested bacterial strains, comparable to most relevant S. aureus over 48 hours. SEM pictures demonstrated uniform layers on coated sutures with higher roughness for palmitate coatings and sustaining integrity of coated sutures. Adherent S. aureus were found via SEM on all types of investigated sutures. The novel antimicrobial sutures showed significantly less viable adhered S. aureus bacteria (up to 6.1 log) compared to Vicryl® Plus (0.5 log). Within 11 µg/cm drug-containing sutures, octenidine-palmitate (OL11) showed the highest number of viable adhered S. aureus (0.5 log), similar to Vicryl® Plus. Chlorhexidine-laurate (CL11) showed the lowest number of S. aureus on sutures (1.7 log), a 1.2 log greater reduction. In addition, planktonic S. aureus in suspensions were highly inhibited by CL11 (0.9 log) represents a 0.6 log greater reduction compared to Vicryl® Plus (0.3 log). CONCLUSIONS: Novel antimicrobial sutures can potentially limit surgical site infections caused by multiple pathogenic bacterial species. Therefore, a potential inhibition of multispecies biofilm formation is assumed. In detail tested with S. aureus, the chlorhexidine-laurate coating (CL11) best meets the medical requirements for a fast bacterial eradication. This suture coating shows the lowest survival rate of adhering as well as planktonic bacteria, a high drug release during the first-clinically most relevant- 48 hours, as well as biocompatibility. Thus, CL11 coatings should be recommended for prophylactic antimicrobial sutures as an optimal surgical supplement to reduce wound infections. However, animal and clinical investigations are important to prove safety and efficacy for future applications.

Novel high efficient coatings for anti-microbial surgical sutures using chlorhexidine in fatty acid slow-release carrier systems.

Sutures can cause challenging surgical site infections, due to capillary effects resulting in bacteria permeating wounds. Anti-microbial sutures may avoid these complications by inhibiting bacterial pathogens. Recently, first triclosan-resistances were reported and therefore alternative substances are becoming clinically relevant. As triclosan alternative chlorhexidine, the "gold standard" in oral antiseptics was used. The aim of the study was to optimize novel slow release chlorhexidine coatings based on fatty acids in surgical sutures, to reach a high anti-microbial efficacy and simultaneously high biocompatibility. Sutures were coated with chlorhexidine laurate and chlorhexidine palmitate solutions leading to 11, 22 or 33 µg/cm drug concentration per length. Drug release profiles were determined in aqueous elutions. Antibacterial efficacy against Staphylococcus aureus was assessed in agar diffusion tests. Biocompatibility was evaluated via established cytotoxicity assay (WST-1). A commercially triclosan-containing suture (Vicryl Plus), was used as anti-microbial reference. All coated sutures fulfilled European Pharmacopoeia required tensile strength and proved continuous slow drug release over 96 hours without complete wash out of the coated drug. High anti-microbial efficacy for up to 5 days was observed. Regarding biocompatibility, sutures using 11 µg/cm drug content displayed acceptable cytotoxic levels according to ISO 10993-5. The highest potential for human application were shown by the 11 µg/cm chlorhexidine coated sutures with palmitic acid. These novel coated sutures might be alternatives to already established anti-microbial sutures such as Vicryl Plus in case of triclosan-resistance. Chlorhexidine is already an established oral antiseptic, safety and efficacy should be proven for clinical applications in anti-microbial sutures.

Local application of BMP-2 specific plasmids in fibrin glue does not promote implant fixation.

BACKGROUND: BMP-2 is known to accelerate fracture healing and might also enhance osseointegration and implant fixation. Application of recombinant BMP-2 has a time-limited effect. Therefore, a gene transfer approach with a steady production of BMP-2 appears to be attractive. The aim of this study was to examine the effect of locally applied BMP-2 plasmids on the bone-implant integration in a non-weight bearing rabbit tibia model using a comparatively new non-viral copolymer-protected gene vector (COPROG). METHODS: Sixty rabbits were divided into 4 groups. All of them received nailing of both tibiae. The verum group had the nails inserted with the COPROG vector and BMP-2 plasmids using fibrin glue as a carrier. Controls were a group with fibrin glue only and a blank group. After 28 and 56 days, these three groups were sacrificed and one tibia was randomly chosen for biomechanical testing, while the other tibia underwent histomorphometrical examination. In a fourth group, a reporter-gene was incorporated in the fibrin glue instead of the BMP-2 formula to prove that transfection was successful. RESULTS: Implant fixation strength was significantly lower after 28 and 56 days in the verum group. Histomorphometry supported the findings after 28 days, showing less bone-implant contact.In the fourth group, successful transfection could be confirmed by detection of the reporter-gene in 20 of 22 tibiae. But, also systemic reporter-gene expression was found in heterotopic locations, showing an undesired spreading of the locally applied gene formula. CONCLUSION: Our results underline the transfecting capability of this vector and support the idea that BMP-2 might diminish osseointegration. Further studies are necessary to specify the exact mechanisms and the systemic effects.

Growth inhibition of Staphylococcus aureus induced by low-frequency electric and electromagnetic fields.

Magnetic field therapy is an established technique in the treatment of pseudarthrosis. In cases of osteomylitis, palliation is also observed. This study focuses on the impact of different electric and electromagnetic fields on the growth of Staphylococcus aureus by in vitro technologies. Cultures of Staphylococcus aureus in fluid and gel-like medium were exposed to a low-frequency electromagnetic field, an electromagnetic field combined with an additional electric field, a sinusoidal electric field and a static electric field. In gel-like medium no significant difference between colony-forming units of exposed samples and non-exposed references was detected. In contrast, Staphylococcus aureus concentrations in fluid medium could clearly be reduced under the influence of the four different applied fields within 24 h of experiment. The strongest effects were observed for the direct current electric field which could decrease CFU/ml of 37%, and the low-frequency electromagnetic field with additional induced electric alternating field with a decrease of Staphylococci concentration by 36%. The effects of the electromagnetic treatment on Staphylococci within fluid medium are significantly higher than in gel-like medium. The application of low-frequency electromagnetic fields corroborates clinical situations of bone infections during magnetic field therapy.

Safety and effectiveness of extracorporeal shockwave therapy: results of a rabbit model of chronic osteomyelitis.

Extracorporeal shockwave therapy (ESWT) is applied successfully in various orthopedic disorders. Since shockwaves have demonstrated significant bactericidal effectiveness in vitro, safety and effectiveness of ESWT in vivo were evaluated in a rabbit model of osteomyelitis. Chronic osteomyelitis was induced by injecting sodium morrhuate and Staphylococcus aureus into the proximal tibia of 12 New Zealand white rabbits. Four and five wk after the initial operation, soft focused ESWT was applied twice to the infected limbs. Clinical parameters and laboratory values were followed and blood samples were taken for culture before and 30 min after ESWT. Following sacrifice after 8 wk, lungs, spleen and kidneys were studied histologically for signs of sepsis and secondary infection. Tibial osteomyelitis was assessed clinically, and by radiologic, microbiologic and histologic procedures. Signs of bacterial spreading were not detectable after ESWT, neither in blood cultures nor in histologic analyses of representative organs. Temperature, body weight, C-reactive protein and white blood cell levels also remained unchanged after ESWT. Of particular interest, histologic scores of osteomyelitis were significantly decreased in the ESWT-group compared to the untreated control (p = 0.019). However, S. aureus was still detectable in tissue samples of all animals. This is the first study investigating the effects of ESWT applied to infected target areas. ESWT of infected bone did neither induce bacterial spreading nor worsening of infection, and the results suggest the reported treatment protocol of ESWT to be beneficial in the treatment of chronic bone infections.

A fibrin glue composition as carrier for nucleic acid vectors.

PURPOSE: Gene delivery from biomaterials has become an important tool in tissue engineering. The purpose of this study was to generate a gene vector-doted fibrin glue as a versatile injectable implant to be used in gene therapy supported tissue regeneration. METHODS: Copolymer-protected polyethylenimine(PEI)-DNA vectors (COPROGs), naked DNA and PEI-DNA were formulated with the fibrinogen component of the fibrin glue TISSUCOL and lyophilized. Clotting parameters upon rehydration and thrombin addition were measured, vector release from fibrin clots was determined. Structural characterizations were carried out by electron microscopy. Reporter and growth factor gene delivery to primary keratinocytes and chondrocytes in vitro was examined. Finally,chondrocyte colonized clots were tested for their potency in cartilage regeneration in a osteochondral defect model. RESULTS: The optimized glue is based on the fibrinogen component of TISSUCOL, a fibrin glue widely used in the clinics, co-lyophilized with copolymer-protected polyethylenimine(PEI)- DNA vectors (COPROGs). This material, when rehydrated, forms vector-containing clots in situ upon thrombin addition and is suitable to mediate growth factor gene delivery to primary keratinocytes and primary chondrocytes admixed before clotting. Unprotected PEI-DNA in the same setup was comparatively unsuitable for clot formation while naked DNA was ineffective in transfection. Naked DNA was released rapidly from fibrin clots (>70% within the first seven days) in contrast to COPROGs which remained tightly immobilized over extended periods of time (0.29% release per day). Electron microscopy of chondrocytecolonized COPROG-clots revealed avid endocytotic vector uptake. In situ BMP-2 gene transfection and subsequent expression in chondrocytes grown in COPROG clots resulted in the upregulation of alkaline phosphatase expression and increased extracellular matrix formation in vitro. COPROG-fibrinogen preparations with admixed autologous chondrocytes when clotted in situ in osteochondral defects in the patellar grooves of rabbit femura gave rise to luciferase reporter gene expression detectable for two weeks (n=3 animals per group). However, no significant improvement in cartilage formation in osteochondral defects filled with autologous chondrocytes in BMP-2-COPROG clots was achieved in comparison to controls (n=8 animals per group). CONCLUSIONS: COPROGs co-lyophilized with fibrinogen are a simple basis for an injectable fibrin gluebased gene-activated matrix. The preparation can be used is complete analogy to fibrin glue preparations that are used in the clinics. However, further improvements in transgene expression levels and persistence are required to yield cartilage regeneration in the osteochondral defect model chosen in this study.

Effective inhibition of experimental metastasis and prolongation of survival in mice by a potent factor Xa-specific synthetic serine protease inhibitor with weak anticoagulant activity.

Clinical and experimental evidence suggests that the blood coagulation system is involved in the dissemination of malignant tumors. Consequently, anticoagulant agents have been tested as metastasis suppressors in experimental models. Recently, we have found a close correlation between factor Xa (FXa)-specificity of a series of synthetic serine protease inhibitors and their anti-metastatic potential in a murine T-cell lymphoma metastasis model. Interference of such inhibitors with blood-coagulation may represent a major experimental and clinical obstacle. Here, we test anti-metastatic effects of a recently developed, highly specific 3-amidinophenylalanine-type FXa inhibitor, WX-FX4, with weaker anticoagulant activity when compared to well-established FXa inhibitors, such as DX-9065a, as measured by the activated partial thromboplastin time, prothrombin time, prothrombinase complex activity, and coagulation time. Treatment of mice with WX-FX4 (1.5 mg/kg twice daily) led to significant reduction of experimental liver metastasis of a syngeneic T-cell lymphoma in DBA/2 mice (> 90%), and of experimental lung metastasis of a human fibrosarcoma in CD1 nu/nu mice (> 60%). Due to its relatively low anticoagulant activity, daily treatment over 100 days was possible, leading to significant survival benefits without inducing bleeding anomalities. FXa-inhibitors with highly efficient anti-metastatic potential without coagulation-related side effects may represent important new tools as anticancer agents.

Biomechanical and allergological characteristics of a biodegradable poly(D,L-lactic acid) coating for orthopaedic implants.

A poly(D,L-lactic acid) surface coating (PDLLA) has been developed to optimize interactions at the implant-tissue interface. Mechanical and allergological characteristics were evaluated in the present study to elucidate possible indications and limitations prior to clinical application. Implants of stainless steel and Ti-6Al-4V and Co-Cr-Mo alloys were coated with PDLLA, and mechanical stability was studied during intramedullary implantation into rat and human cadaver bones and during dilation of coronary artery stents. Elongation resistance was examined on AlMgSi alloy specimens. Furthermore, proliferation of peripheral blood mononuclear cells of nickel-allergic donors and controls and interleukin-4 and interferon-gamma levels were measured in the presence of coated/uncoated implants and after stimulation with phytohemagglutinin or NiSO4. PDLLA remained stable on the implants with a minimum of 96% of the original coating mass and tolerated lengthening of at least 8%. Further lengthening was followed by microcracking and cohesive failure within the coating. PDLLA exerted no suppressive effect upon spontaneous and pan-T-cell mitogen inducible T-cell proliferation. Furthermore, specific proliferation to nickel in cells of nickel-allergic blood donors and production of interleukin-4 and IFN-gamma were not influenced by the coating. PDLLA coating proved high mechanical stability on different orthopaedic implants and did not influence in vitro T-cell reactivity towards specific biomaterials.

Triple-coated stents (Hirudin/Iloprost/Paclitaxel): an in vitro approach for characterizing the antiproliferative potential of each individual compound.

BACKGROUND: Hirudin (H)/iloprost (I)/paclitaxel (P)-coated stents represent a multifactorial approach to reducing the proliferative response caused by ballooning and stenting. The study presented compares the net effect of each individual compound of HIP-coated stents with the summed effect of the compounds in the stent coating. METHODS AND RESULTS: For proliferation prescreening studies, human coronary smooth muscle cells were incubated with H (0.005-500 microg/ml), I (0.00001-1 microg/ml), and P (0.0001-10 microg/ml). After 5 days, cell number was studied in a cell analyzer system. Secondly, 8-mm stents were coated with (1) HI, (2) HIP-10 microg/20 microg/40 microg (HIP5%/10%/20%), (3) P-40 microg (P), (4) IP-40 microg (IP), and (5) HP-40 microg (HP). After 5 days, the effect on cell proliferation and cytoskeletal structures was studied. No antiproliferative effect was found after incubation with H; significant inhibition was seen after incubation with I (p<0.05) or lipophilically dissolved P (p<0.001). After 5 days incubation with HIP5%-, HIP10%-, HIP20%-, P20%-, IP20%-, and HP20%-coated stents, cell proliferation was inhibited by 55.5% (p<0.05), 61% (p<0.05), 57.9% (p<0.05), 59.5% (p<0.001), 59.8% (p<0.001), and 63.3% (p<0.001), respectively. HI- and HIP-coated stents caused a severe destruction of the cytoskeletal structures smooth muscle alpha-actin and alpha-tubulin; despite the destruction, vital cells could be identified with positive FDA staining. CONCLUSIONS: Although both lipophilically dissolved P and hydrophilically dissolved I contributed to the antiproliferative effect, no additive effect of the two compounds was detected. In vivo P can be released more easily from the coating material due to the permanent lipophilic contact of the stent struts with the vessel wall. The current study is the first report on a clear and uncomplicated technique to obtain information on the antiproliferative potential of coated stents before large experimental studies are initiated.

Effects of osteopromotive and anti-infective membranes on bone regeneration: an experimental study in rat mandibular defects.

PURPOSE: Reconstructive surgical treatment with osteopromotive membranes has become an integral part of implant dentistry. Nevertheless, there are still instances in which this technique alone is of limited or no benefit. The aim of the present study was to determine whether a combination of titanium membranes coated with transforming growth factor beta1 (TGF-beta1) and insulin-like growth factor I (IGF-I) is of value in the regeneration of so-called critical-size defects in the rat model. An analysis was made of whether or not locally administered antibiotics are deleterious to bone regeneration. MATERIALS AND METHODS: A total of 24 rats were included in the study and were divided into 4 groups, each with 6 animals. Critical-size defects were created bilaterally and covered by titanium membranes coated with (1) polylactide, (2) polylactide and clindamycin, (3) polylactide and growth factors, or (4) polylactide, clindamycin, and growth factors. All 24 contralateral defects were covered by titanium membranes without any substrate (controls). Four weeks after treatment the animals were sacrificed. RESULTS: In groups 3 and 4, most defects showed thin but almost complete bridging of the defects with new bone formation. In particular, clindamycin had no inhibitory effect on the regeneration of bone. Nevertheless, after 28 days, there was no significant difference between the individual groups (including controls) with respect to the total amount of newly formed bone. DISCUSSION AND CONCLUSION: These results support the hypothesis that coating titanium membranes with TGF-beta1/IGF-I leads to almost complete bony bridging of critical-size defects without voluminous carrier materials. Moreover, simultaneous administration of clindamycin seems possible.

Antibacterial poly(D,L-lactic acid) coating of medical implants using a biodegradable drug delivery technology.

OBJECTIVES: Biomaterial-associated bacterial infections present common and challenging complications with medical implants. The purpose of this study was to determine the antibacterial properties of a low molecular weight biodegradable poly(D,L-lactic acid) coating with integrated antibiotics gentamicin and teicoplanin. METHODS: Coating of Kirschner-wires was carried out by a solvent casting technique under aseptic conditions with and without incorporated antibiotics. Release kinetics of gentamicin and teicoplanin were studied in phosphate-buffered saline. Initial bacterial adhesion of Staphylococcus epidermidis on coated and bare implants was determined by radiolabelling and counts of detached viable organisms. RESULTS: The incorporated antibiotics showed a continuous release over a period of at least 96 h with an initial peak of release in the first 6 h. Attachment of non-viable microorganisms, detected by radiolabelled bacteria, was increased significantly by the polymer coatings (P < 0.05). In contrast, the number of viable bacteria was reduced by the pure polymer (P < 0.01) and further by the polymer-antibiotic combinations (P < 0.05). CONCLUSIONS: Poly(D,L-lactic acid) coating of implants could offer new perspectives in preventing biomaterial-associated infections. Combinations with other drugs to formulate custom-tailored implant surfaces are feasible.

Nonviral vector loaded collagen sponges for sustained gene delivery in vitro and in vivo.

BACKGROUND: Naked DNA and standard vectors have previously been used for gene delivery from implantable carrier matrices with great potential for gene therapeutic assistance of wound healing or tissue engineering. We have previously developed copolymer-protected gene vectors which are inert towards opsonization. Here we examine their potency in carrier-mediated gene delivery in comparison to standard vectors using a vector-loaded collagen sponge model. METHODS: Equine collagen type I sponges were loaded by a lyophilization method with naked DNA, polyethylenimine (PEI)-DNA, DOTAP/cholesterol-DNA and copolymer-protected PEI-DNA. These preparations were characterized in terms of vector-release, cell growth on the matrices and reporter gene expression by cells colonizing the sponges in vitro and in vivo. Subcutaneous implantation of sponges in rats served as an in vivo model. RESULTS: At the chosen low vector dose, the loading efficiency was at least 86%. Naked DNA-loaded collagen matrices lost 77% of the DNA dose in an initial burst in aqueous buffer in vitro. The other preparations examined displayed a sustained vector release. There was no difference in cell growth and invasion of the sponges between vector-loaded and untreated collagen grafts. Reporter gene expression from cells colonizing the sponges in vitro was observed for not more than 7 days with naked DNA, whereas the lipoplex and polyplex preparations yielded long-term expression throughout the experimental period of up to 56 days. The highest expression levels were achieved with the PEI-DNA-PROCOP (protective copolymer) formulation. Upon subcutaneous implantation in rats, no luciferase expression was detected with naked DNA preparations. DOTAP/cholesterol-DNA and PEI-DNA-loaded implants lead to reporter gene expression for at least 3 days, but with poor reproducibility. PEI-DNA-PROCOP collagen matrices yielded consistently the highest reporter gene expression levels for at least 7 days with good reproducibility. CONCLUSIONS: With the preparation method chosen, lipoplex- and polyplex-loaded collagen sponges are superior in mediating sustained gene delivery in vitro and local transfection in vivo as compared to naked DNA-loaded sponges. Protective copolymers are particularly advantageous in promoting the tranfection capacity of polyplex-loaded sponges upon subcutaneous implantation, likely due to their stabilizing and opsonization-inhibiting properties.