Bacterial reservoir in deeper skin is a potential source for surgical site and biomaterial-associated infections.

BACKGROUND: The origin of surgical site and biomaterial-associated infection is still elusive. Micro-organisms contaminating the wound may come from the air in the operating theatre, the surgical team or the skin of the patient. The skin of patients is disinfected prior to surgery, but bacteria deeper in the skin (e.g. in sweat glands or sebaceous glands) may not be reached. METHODS: A preliminary cohort study was performed to study the origin of surgical site and biomaterial-associated infection between May 2020 and February 2021. In order to investigate whether cutaneous microbiota colonize the wound when released from the skin upon cutting, aerobic and anaerobic bacteria were isolated, quantified and identified from the skin of 99 patients undergoing trauma surgery, before and after skin disinfection, from knife blades and from the wound directly after the first cut. RESULTS: Ninety-nine percent of the patients were culture-positive before disinfection with chlorhexidine. Of these, 40% were still culture-positive after disinfection. Of these, 54% had a positive culture of the wound after cutting the skin. Twenty percent of the patients with a negative culture after disinfection had a positive wound culture after cutting the skin. Staphylococcus epidermidis and Cutibacterium acnes were the most commonly cultured bacterial species. In 9% of cases, more than 100 bacterial colonies were cultured from the wound; this may cause biomaterial-associated infection. CONCLUSION: Bacteria residing in the skin and not eradicated by disinfection may enter the surgical wound upon cutting, resulting in contamination which may cause biomaterial-associated infection.

Current osteomyelitis mouse models, a systematic review.

Osteomyelitis is an inflammatory bone disease caused by an infecting microorganism leading to a gradual bone loss. Due to the difficulty in studying osteomyelitis directly in patients, animal models allow researchers to investigate the pathogenesis of the infection and the development of novel prophylactic, anti-inflammatory and antimicrobial treatment strategies. This review is specifically focused on the in vivo mouse osteomyelitis studies available in literature. Thus, a systematic search on Web of Science and PubMed was conducted using the query "(infection) AND (mice OR mouse OR murine) AND (model OR models) AND (arthroplasty OR fracture OR (internal fixator) OR (internal fixation OR prosthesis OR implant OR osteomyelitis)". After critical assessment of the studies according to the inclusion and exclusion criteria, 135 studies were included in the detailed analysis. Based on the model characteristics, the studies were classified into five subject groups: haematogenous osteomyelitis, post-traumatic osteomyelitis, bone-implant-related infection, peri-prosthetic joint infection, fracture-related infection. In addition, the characteristics of the mice used, such as inbred strain, age or gender, the characteristics of the pathogens used, the inoculation methods, the type of anaesthesia and analgesia used during surgery and the procedures for evaluating the pathogenicity of the infecting micro-organism were described. Overall, the mouse is an excellent first step in vivo model to study the pathogenesis, inflammation and healing process of osteomyelitis and to evaluate novel prophylaxis and treatment strategies.

A murine Staphylococcus aureus fracture-related infection model characterised by fracture non-union, staphylococcal abscess communities and myeloid-derived suppressor cells.

A fracture-related infection (FRI) is a serious complication that can occur after surgical fixation of bone fractures. Affected patients may encounter delayed healing and functional limitations. Although it is well established that Staphylococcus aureus (S. aureus) is the main causative pathogen of an FRI, the pathophysiology of an S. aureus-induced FRI is not well characterised over time. Therefore, an experimental study in mice comparing S. aureus-inoculated and non-inoculated groups was performed that particularly focused on staphylococcal abscess communities (SACs) and host cellular response. C57Bl/6N female mice received a double osteotomy of the femur, which was stabilised using a titanium 6-hole MouseFix locking plate and four screws. Animals were either S. aureus-inoculated or non-inoculated and euthanised between 1 and 28 d post-surgery. Histopathological evaluation showed normal bone healing for non-inoculated mice, whereas inoculated mice had no fracture consolidation and severe osteolysis. Within the bone marrow of inoculated mice, SACs were observed from 7 d, which increased in size and number over time. A fibrin pseudocapsule enclosed the SACs, which were surrounded by many Ly6G+ neutrophils with some Ly6C+ monocytes and F4/80+ macrophages, the majority of which were viable. The abscesses were encapsulated by fibrin(ogen), collagen and myofibroblasts, with regulatory T cells and M2 macrophages at the periphery. Only bone marrow monocytes and neutrophils of inoculated mice displayed functional suppression of T cells, indicative of myeloid-derived suppressor cells. The present study revealed that an FRI in mice is persistent over time and associated with osteolysis, SAC formation and an immunosuppressive environment.

Effects of sterilization on the mechanical properties of poly(methyl methacrylate) based personalized medical devices.

BACKGROUND: Nowadays, personalized medical devices are frequently used for patients. Due to the manufacturing procedure sterilization is required. How different sterilization methods affect the mechanical behavior of these devices is largely unknown. MATERIALS AND METHODS: Three poly(methyl methacrylate) (PMMA) based materials (Vertex Self-Curing, Palacos R+G, and NextDent C&B MFH) were sterilized with different sterilization methods: ethylene oxide, hydrogen peroxide gas plasma, autoclavation, and γ-irradiation. Mechanical properties were determined by testing the flexural strength, flexural modulus, fracture toughness, and impact strength. RESULTS: The flexural strength of all materials was significantly higher after γ-irradiation compared to the control and other sterilization methods, as tested in a wet environment. NextDent C&B MFH showed the highest flexural and impact strength, Palacos R+G showed the highest maximum stress intensity factor and total fracture work. CONCLUSION: Autoclave sterilization is not suitable for the sterilization of PMMA-based materials. Ethylene oxide, hydrogen peroxide gas plasma, and γ-irradiation appear to be suitable techniques to sterilize PMMA-based personalized medical devices.

Poly(trimethylene carbonate) and poly(D,L-lactic acid) modify human dendritic cell responses to staphylococci but do not affect Th1 and Th2 cell development.

Biomaterial-associated infections (BAIs) are frequent complications in the use of medical devices (biomaterials) correlated with considerable patient discomfort and high treatment costs. The presence of a biomaterial in the host causes derangement of local immune responses increasing susceptibility to infection. Dendritic cells (DCs) have an important role in directing the nature of immune responses by activating and controlling CD4+ T helper (Th) cell responses. To assess the immunomodulatory effect of the combined presence of biomaterials and Staphylococcus aureus (S. aureus) or Staphylococcus epidermidis (S. epidermidis), DC-mediated T cell proliferation and Th1/Th2 cell development were measured using an in vitro human cell system. Poly(trimethylene carbonate) (PTMC) and poly(D,L-lactic acid) (PDLLA) modified the production of the DC pro-inflammatory cytokines TNF-α, IL-6 and IL-23 in response to S. aureus and S. epidermidis. However, this modified cytokine production did not cause differences in Th1/Th2 cell polarisation, showing a Th1 cell predominance. In the absence of staphylococci, neither of the biomaterials induced DC-mediated T cell proliferation or Th1/Th2 cell polarisation. Moreover, either in the absence or presence of the biomaterials, S. aureus was a more potent inducer of DC cytokine secretion, T cell proliferation and Th1 cell development than S. epidermidis. In conclusion, although PTMC and PDLLA modulated DC cytokine responses to staphylococci, this did not alter the resulting Th cell development. This result suggested that, in this human cell model, Th1/Th2 cell responses were mainly determined by the species of bacteria and that PTMC or PDLLA did not detectably influence these responses.

The prohibitin-repressive interaction with E2F1 is rapidly inhibited by androgen signalling in prostate cancer cells.

Prohibitin (PHB) is a tumour suppressor molecule with pleiotropic activities across several cellular compartments including mitochondria, cell membrane and the nucleus. PHB and the steroid-activated androgen receptor (AR) have an interplay where AR downregulates PHB, and PHB represses AR. Additionally, their cellular locations and chromatin interactions are in dynamic opposition. We investigated the mechanisms of cell cycle inhibition by PHB and how this is modulated by AR in prostate cancer. Using a prostate cancer cell line overexpressing PHB, we analysed the gene expression changes associated with PHB-mediated cell cycle arrest. Over 1000 gene expression changes were found to be significant and gene ontology analysis confirmed PHB-mediated repression of genes essential for DNA replication and synthesis, for example, MCMs and TK1, via an E2F1 regulated pathway-agreeing with its G1/S cell cycle arrest activity. PHB is known to inhibit E2F1-mediated transcription, and the PHB:E2F1 interaction was seen in LNCaP nuclear extracts, which was then reduced by androgen treatment. Upon two-dimensional western blot analysis, the PHB protein itself showed androgen-mediated charge differentiation (only in AR-positive cells), indicating a potential dephosphorylation event. Kinexus phosphoprotein array analysis indicated that Src kinase was the main interacting intracellular signalling hub in androgen-treated LNCaP cells, and that Src inhibition could reduce this AR-mediated charge differentiation. PHB charge change may be associated with rapid dissociation from chromatin and E2F1, allowing the cell cycle to proceed. The AR and androgens may deactivate the repressive functions of PHB upon E2F1 leading to cell cycle progression, and indicates a role for AR in DNA replication licensing.

A chlorhexidine-releasing epoxy-based coating on titanium implants prevents Staphylococcus aureus experimental biomaterial-associated infection.

Prevention of biomaterial-associated infections (BAI) remains a challenging problem, in particular due to the increased risk of resistance development with the current antibiotic-based strategies. Metallic orthopaedic devices, such as non-cemented implants, are often inserted under high mechanical stress. These non-cemented implants cannot be protected by e.g. antibioticreleasing bone cement or other antimicrobial approaches, such as the use of bioactive glass. Therefore, in order to avoid abrasion during implantation procedures, we developed an antimicrobial coating with great mechanical stability for orthopaedic implants, to prevent Staphylococcus aureus BAI. We incorporated 5 and 10 wt % chlorhexidine in a novel mechanically stable epoxy-based coating, designated CHX5 and CHX10, respectively. The coatings displayed potent bactericidal activity in vitro against S. aureus, with over 80 % of the release (19 µg/cm2 for CHX5 and 41 µg/cm2 for CHX10) occurring within the first 24 h. In mice, the CHX10 coating significantly reduced the number of CFU (colony forming units), both on the implants and in the peri-implant tissues, 1 d after S. aureus challenge. The CHX10-coated implants were well-tolerated by the animals, with no signs of toxicity observed by histological analysis. Moreover, the coating significantly reduced the frequency of culture-positive tissues 1 d, and of culture-positive implants 1 and 4 d after challenge. In summary, the chlorhexidine-releasing mechanically stable epoxy-based CHX10 coating prevented implant colonisation and S. aureus BAI in mice and has good prospects for clinical development.

Fabrication of oxide layer on zirconium by micro-arc oxidation: Structural and antimicrobial characteristics.

The aim of this study was to cover the surfaces of zirconium (Zr) with an antimicrobial layer for biomedical applications. For this purpose, the micro-arc oxidation (MAO) process was employed in a sodium silicate and sodium hydroxide containing base electrolyte with and without addition of silver acetate (AgC2H3O2). In general, synthesized MAO layers were composed of zirconium oxide (ZrO2) and zircon (ZrSiO4). Addition of AgC2H3O2 into the base electrolyte caused homogenous precipitation of silver-containing particles in the MAO layer, which exhibited excellent antibacterial efficiency against methicillin-resistant Staphylococcus aureus (MRSA) as compared to the untreated and MAO-treated Zr.

Prevention of Staphylococcus aureus biomaterial-associated infections using a polymer-lipid coating containing the antimicrobial peptide OP-145.

The scarcity of current antibiotic-based strategies to prevent biomaterial-associated infections (BAI) and their risk of resistance development prompted us to develop a novel antimicrobial implant-coating to prevent Staphylococcus aureus-induced BAI. We incorporated the antimicrobial peptide OP-145 into a Polymer-Lipid Encapsulation MatriX (PLEX)-coating to obtain high peptide levels for prolonged periods at the implant-tissue interphase. We first confirmed that OP-145 was highly effective in killing S. aureus and inhibiting biofilm formation in vitro. OP-145 injected along S. aureus-inoculated implants in mice significantly reduced the number of culture-positive implants. OP-145 was released from the PLEX coating in a controlled zero-order kinetic rate after an initial 55%-burst release and displayed bactericidal activity in vitro. In a rabbit intramedullary nail-related infection model, 67% of rabbits with PLEX-OP-145-coated nails had culture-negative nails after 28days compared to 29% of rabbits with uncoated nails. In rabbits with PLEX-OP-145-coated nails, bone and soft tissue samples were culture-negative in 67% and 80%, respectively, whereas all bone samples and 71% of the soft tissue samples of rabbits with uncoated nails were infected. Together, PLEX-OP-145 coatings, of which both compounds have already been found safe in man, can prevent implant colonization and S. aureus-induced BAIs.

Real-time quantification of matrix metalloproteinase and integrin αvß3 expression during biomaterial-associated infection in a murine model.

Biomaterial implants and devices increase the risk of microbial infections due to the biofilm mode of growth of infecting bacteria on implant materials, in which bacteria are protected against antibiotic treatment and the local immune system. Matrix-metalloproteinases (MMPs) and cell surface integrin receptors facilitate transmigration of inflammatory cells toward infected or inflamed tissue. This study investigates the relationship between MMP- and integrin-expression and the clearance of infecting Staphylococcus aureus around implanted biomaterials in a murine model.MMP- and integrin αvß3-expression were monitored in mice, with and without subcutaneously implanted biomaterial samples, in the absence and presence of bioluminescent S. aureus Xen36. Staphylococcal persistence was imaged longitudinally over time using bioluminescence imaging. The activatable MMPSense®680 and integrin-targeted IntegriSense®750 probes were injected on different days after implantation and their signal intensity and localisation monitored using fluorescence imaging. After sacrifice 7 or 16 days post-implantation, staphylococci from biomaterial samples and surrounding tissues were cultured on agar-plates and presence of host inflammatory cells was histologically evaluated.MMP- and integrin-expression were equally enhanced in presence of staphylococci or biomaterials up to 7 days post-implantation, but their localisation along the biomaterial samples differed. Bacterial clearance from tissue was higher in the absence of biomaterials. It is of clinical relevance that MMP- and integrin-expression were enhanced in presence of both staphylococci and biomaterials, although the immune system in the presence of biomaterials remained hampered in eradicating bacteria during the first 7 days post-implantation.

In vitro cytotoxicity evaluation of porous TiO2-Ag antibacterial coatings for human fetal osteoblasts.

Implant-associated infections (IAIs) may be prevented by providing antibacterial properties to the implant surface prior to implantation. Using a plasma electrolytic oxidation (PEO) technique, we produced porous TiO2 coatings bearing various concentrations of Ag nanoparticles (Ag NPs) (designated as 0 Ag, 0.3 Ag and 3.0 Ag) on a Ti-6Al-7Nb biomedical alloy. This study investigates the cytotoxicity of these coatings using a human osteoblastic cell line (SV-HFO) and evaluates their bactericidal activity against methicillin-resistant Staphylococcus aureus (MRSA). The release of Ag and the total amount of Ag in the coatings were determined using a graphite furnace atomic absorption spectrometry technique (GF-AAS) and flame-AAS, respectively. Cytotoxicity was evaluated using the AlamarBlue assay coupled with the scanning electron microscopy (SEM) observation of seeded cells and by fluorescence microscopy examination of the actin cytoskeleton and nuclei after 48 h of incubation. Antibacterial activity was assessed quantitatively using a direct contact assay. AlamarBlue viability assay, SEM and fluorescence microscopy observation of the SV-HFO cells showed no toxicity for 0 Ag and 0.3 Ag specimens, after 2, 5 and 7 days of culture, while 3.0 Ag surfaces appeared to be extremely cytotoxic. All Ag-bearing surfaces had good antibacterial activity, whereas Ag-free coatings showed an increase in bacterial numbers. Our results show that the 0.3 Ag coatings offer conditions for optimum cell growth next to antibacterial properties, which makes them extremely useful for the development of new antibacterial dental and orthopedic implants.

Accumulation of human EGF in nectar of transformed plants of Nicotiana langsdorffii x N. sanderae and transfer to honey by bees.

Honey has been used successfully in wound healing for thousands of years. The peptide hormone human epidermal growth factor (hEGF) is also known to have a beneficial effect in various wound healing processes via mechanisms that differ from those for honey. In this study, we show that hEGF can be incorporated into honey via nectar. Plants of Nicotiana langsdorffii x N. sanderae were transformed with the gene for hEGF, equipped with a nectary-targeted promoter and a signal sequence for secretion to nectar. These plants accumulated hEGF in the nectar. The maximum hEGF concentration recorded with ELISA in these plants is 2.5 ng·ml⁻¹. There is a significant linear relationship (P<0.001) between hEGF concentration and induction of hEGF-receptor phosphorylation. Since the flower morphology of these plants did not allow production of honey from their nectar, we used feeding solutions, spiked with synthetic hEGF, to study transfer of this peptide into honey through bee activity. Transfer of hEGF from a feeding solution to honey by bees occurred with retention of the hEGF concentration and the capacity to induce hEGF-receptor phosphorylation. These observations indicate that plants can function as a production platform for honey containing biologically active peptides, which may enhance wound healing and other biological processes.

Medical-grade honey enriched with antimicrobial peptides has enhanced activity against antibiotic-resistant pathogens.

Honey has potent activity against both antibiotic-sensitive and -resistant bacteria, and is an interesting agent for topical antimicrobial application to wounds. As honey is diluted by wound exudate, rapid bactericidal activity up to high dilution is a prerequisite for its successful application. We investigated the kinetics of the killing of antibiotic-resistant bacteria by RS honey, the source for the production of Revamil® medical-grade honey, and we aimed to enhance the rapid bactericidal activity of RS honey by enrichment with its endogenous compounds or the addition of antimicrobial peptides (AMPs). RS honey killed antibiotic-resistant isolates of Pseudomonas aeruginosa, Staphylococcus epidermidis, Enterococcus faecium, and Burkholderia cepacia within 2 h, but lacked such rapid activity against methicillin-resistant S. aureus (MRSA) and extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli. It was not feasible to enhance the rapid activity of RS honey by enrichment with endogenous compounds, but RS honey enriched with 75 µM of the synthetic peptide Bactericidal Peptide 2 (BP2) showed rapid bactericidal activity against all species tested, including MRSA and ESBL E. coli, at up to 10-20-fold dilution. RS honey enriched with BP2 rapidly killed all bacteria tested and had a broader spectrum of bactericidal activity than either BP2 or honey alone.

Microscopic detection of viable Staphylococcus epidermidis in peri-implant tissue in experimental biomaterial-associated infection, identified by bromodeoxyuridine incorporation.

Infection of biomedical devices is characterized by biofilm formation and colonization of surrounding tissue by the causative pathogens. To investigate whether bacteria detected microscopically in tissue surrounding infected devices were viable, we used bromodeoxyuridine (BrdU), a nucleotide analogue that is incorporated into bacterial DNA and can be detected with antibodies. Infected human tissue was obtained postmortem from patients with intravascular devices, and mouse biopsy specimens were obtained from mice with experimental biomaterial infection. In vitro experiments showed that Staphylococcus epidermidis incorporated BrdU, as judged from staining of the bacteria with anti-BrdU antibodies. After incubation of bacteria with BrdU and subsequent staining of microscopic sections with anti-BrdU antibodies, bacteria could be clearly visualized in the tissue surrounding intravascular devices of deceased patients. With this staining technique, relapse of infection could be visualized in mice challenged with a low dose of S. epidermidis and treated with dexamethasone between 14 and 21 days after challenge to suppress immunity. This confirms and extends our previous findings that pericatheter tissue is a reservoir for bacteria in biomaterial-associated infection. The pathogenesis of the infection and temporo-spatial distribution of viable, dividing bacteria can now be studied at the microscopic level by immunolabeling with BrdU and BrdU antibodies.

Mycoplasma pneumoniae P1 type 1- and type 2-specific sequences within the P1 cytadhesin gene of individual strains.

Mycoplasma pneumoniae strains traditionally are divided into two types, based on sequence variation in the P1 gene. Recently, however, we have identified 8 P1 subtypes by restriction fragment length polymorphism analysis. In the present study the P1 gene sequences of three P1 type 1 and two P1 type 2 M. pneumoniae strains were analyzed. A new P1 gene sequence in a type 1 strain with partial similarity to a recently reported variable region in the P1 gene of an M. pneumoniae type 2 strain (T. Kenri, R. Taniguchi, Y. Sasaki, N. Okazaki, M. Narita, K. Izumikawa, M. Umetsu, and T.Sasaki, Infect. Immun. 67:4557-4562, 1999) was identified. In addition, the P1 gene of the type 1 strain contained another region with nucleotide polymorphisms identical to a stretch in the P1 gene of one of our type 2 strains. These findings indicate that recombination between sequences specific for P1 type 1 and type 2 had occurred and that P1 type 1 and type 2 hybrid sequences can be present within the P1 gene of an individual strain. Identical or nearly identical variable P1 gene sequences were present in several repetitive regions outside the P1 gene locus in the genome of M. pneumoniae strain M129, implying recombination as a mechanism for generation of the P1 gene variation. Additionally, in the P1 gene sequences of four of the five strains studied, single-nucleotide polymorphisms different from the previously reported P1 type 1 and 2 characteristic sequences were identified. The polymorphic sites are candidate targets for genotyping of M. pneumoniae by direct sequencing of amplicons from clinical specimens.

Platelet microbicidal activity is an important defense factor against viridans streptococcal endocarditis.

To study the role of platelet microbicidal activity in host defense against infective endocarditis (IE) due to viridans streptococci (VS), the susceptibility to platelet releasate of blood and oral VS isolates from patients with and without IE was compared. The influence of neutralization of platelet microbicidal activity was studied in 2 experimental IE models. Resistance to platelet releasate was more prevalent among VS from blood of patients with IE than from blood of bacteremic patients without IE and among oral VS isolates. Serum from rabbits vaccinated with human platelet sonicate supernatants neutralized human and rabbit platelet-released microbicidal activity and had antibodies recognizing microbicidal proteins thrombocidin-1 and -2 and other human platelet proteins. In the 2 rabbit IE models, vaccination increased the susceptibility to experimental IE due to platelet releasate-susceptible VS. Thus, platelet-released microbicidal activity is an important host defense factor against IE due to VS.

The effect of pseudomonas exotoxin A on cytokine production in whole blood exposed to Pseudomonas aeruginosa.

To determine the effect of Pseudomonas aeruginosa exotoxin A (P-ExA) on cytokine production, we studied cytokine release induced by heat-killed P. aeruginosa (HKPA) in human whole blood in the presence or absence of P-ExA. P-ExA (0.01-1 microgram ml(-1)) caused a dose-dependent decrease in HKPA-induced production of tumor necrosis factor alpha (TNF), interleukin (IL-) 10, IL-6 and IL-8 (all P<0.05). P-ExA-induced inhibition of IL-10, IL-6 and IL-8 release was not dependent on reduced TNF concentrations, since the relative attenuation of the production of these cytokines was similar in the presence or absence of a neutralizing anti-TNF antibody. The effect of P-ExA on cytokine production may offer a disadvantage to the host with respect to clearance of the infection.

Interleukin-1 receptor type I gene-deficient mice are less susceptible to Staphylococcus epidermidis biomaterial-associated infection than are wild-type mice.

Elevated concentrations of interleukin-1 (IL-1) were found in tissue surrounding biomaterials infected with Staphylococcus epidermidis. To determine the role of IL-1 in biomaterial-associated infection (BAI), IL-1 receptor type I-deficient (IL-1R(-/-)) and wild-type mice received subcutaneous implants of silicon elastomer (SE) or polyvinylpyrrolidone-grafted SE (SEpvp), combined with an injection of 10(6) CFU of S. epidermidis or sterile saline. Neither mouse strain was susceptible to BAI around SE. IL-1R(-/-) mice with SEpvp implants had a no abscess formation and a reduced susceptibility to persistent S. epidermidis infection. The normal foreign body response, characterized by giant-cell formation and encapsulation, was delayed around SEpvp in wild-type mice but not in IL-1R(-/-) mice. This coincided with enhanced local IL-4 production in IL-1R(-/-) mice. These data suggest that inhibition of local IL-1 activity may be beneficial for the outcome of BAI.

In vitro and in vivo evaluation of gelatin-chondroitin sulphate hydrogels for controlled release of antibacterial proteins.

Chemically cross-linked gelatin-chondroitin sulphate (ChS) hydrogels, impregnated in Dacron, were evaluated as drug delivery systems for antibacterial proteins. The gelatin-chondroitin sulphate gels, plain or impregnated in Dacron, were cross-linked with a water-soluble carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The release of lysozyme and recombinant thrombocidin (rTC-1), an antibacterial protein derived from human blood platelets, from the gelatin-ChS gels in Dacron in phosphate-buffered saline at 37 degrees C was determined, and compared to the release from gelatin gels in Dacron and plain gelatin-ChS gels. The incorporation of chondroitin sulphate into gelatin gels, caused a marked increase in lysozyme loading capacity, and a slower release rate. The relative release profiles for rTC-1 and lysozyme were equal for cross-linked gelatin as well as for cross-linked gelatin-ChS gels. Furthermore, rTC-1 showed no loss of antibacterial activity after 1 week of release. The lysozyme concentration profiles in the samples and in the surrounding medium as a function of time were calculated using mathematical solutions for Ficks second law of diffusion for a semi-infinite composite medium, which is a schematic representation of a slab in a surrounding medium. The biocompatibility and degradation of the Dacron matrices impregnated with gelatin-ChS gels was studied after implantation in subcutaneous pockets in rats. Chemically cross-linked gelatin-Ch5 gels showed a mild tissue reaction, and almost complete degradation within 18 weeks of implantation.

Thrombocidins, microbicidal proteins from human blood platelets, are C-terminal deletion products of CXC chemokines.

Antibacterial proteins are components of the innate immune system found in many organisms and produced by a variety of cell types. Human blood platelets contain a number of antibacterial proteins in their alpha-granules that are released upon thrombin activation. The present study was designed to purify these proteins obtained from human platelets and to characterize them chemically and biologically. Two antibacterial proteins were purified from platelet granules in a two-step protocol using cation exchange chromatography and continuous acid urea polyacrylamide gel electrophoresis and were designated thrombocidin (TC)-1 and TC-2. Characterization of these proteins using mass spectrometry and N-terminal sequencing revealed that TC-1 and TC-2 are variants of the CXC chemokines neutrophil-activating peptide-2 and connective tissue-activating peptide-III, respectively. TC-1 and TC-2 differ from these chemokines by a C-terminal truncation of 2 amino acids. Both TCs, but not neutrophil-activating peptide-2 and connective tissue-activating peptide-III, were bactericidal for Bacillus subtilis, Escherichia coli, Staphylococcus aureus, and Lactococcus lactis and fungicidal for Cryptococcus neoformans. Killing of B. subtilis by either TC appeared to be very rapid. Because TCs were unable to dissipate the membrane potential of L. lactis, the mechanism of TC-mediated killing most probably does not involve pore formation.