The ongoing battle against multi-resistant strains: in-vitro inhibition of hospital-acquired MRSA, VRE, Pseudomonas, ESBL E. coli and Klebsiella species in the presence of plant-derived antiseptic oils.

The fight against hospital-acquired infections involving antibiotic-resistant microorganisms has become of critical concern to surgeons worldwide. In addition to the development of new effective antibiotic chemotherapy, exploration of 'forgotten' topical antibacterial agents from the pre-antibiotic era has recently gained new attention. We report the promising efficacy of plant-derived antiseptic oils used in traditional aboriginal and south-east Asian treatments such as Lemongrass, Eucalyptus and Tea Tree Oil in the inhibition of clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), multi-resistant Pseudomonas aeruginosa, ESBL-producing Escherichia coli and Klebsiella pneumoniae in the in-vitro setting. Large consistent zones of inhibition were observed for all three plant-derived oils tested in an agar diffusion test. The commonly used antibacterial agents chlorhexidine 0.1%, and ethanol (70%), and standard olive oil consistently demonstrated notably lower or no efficacy in regard to growth inhibition of strains. Notably, Lemongrass oil proved to be particularly active against gram-positive bacteria, while Tea Tree oil showed superior inhibition of gram-negative microorganisms. As proven in vitro, plant-derived antiseptic oils may represent a promising and affordable topical agent to support surgical treatment against multi-resistant and hospital-acquired infections.

Comparison of in vitro biocompatibility of NanoBone(®) and BioOss(®) for human osteoblasts.

INTRODUCTION: Scaffolds for bone tissue engineering seeded with the patient's own cells might be used as a preferable method to repair bone defects in the future. With the emerging new technologies of nanostructure design, new synthetic biomaterials are appearing on the market. Such scaffolds must be tested in vitro for their biocompatibility before clinical application. However, the choice between a natural or a synthetic biomaterial might be challenging for the doctor and the patient. In this study, we compared the biocompatibility of a synthetic bone substitute, NanoBone(®) , to the widely used natural bovine bone replacement material BioOss(®) . MATERIAL AND METHODS: The in vitro behaviour of human osteoblasts on both materials was investigated. Cell performance was determined using scanning electron microscopy (SEM), cell vitality staining and four biocompatibility tests (LDH, MTT, WST, BrdU). RESULTS: We found that both materials showed low cytotoxicity and good biocompatibility. The MTT proliferation test was superior for Nanobone(®) . DISCUSSION: Both scaffolds caused only little damage to human osteoblasts and justify their clinical application. However, NanoBone(®) was able to support and promote proliferation of human osteoblasts slightly better than BioOss(®) in our chosen test set-up. The results may guide doctors and patients when being challenged with the choice between a natural or a synthetic biomaterial. Further experiments are necessary to determine the comparison of biocompatibility in vivo.

Ceramic scaffolds produced by computer-assisted 3D printing and sintering: characterization and biocompatibility investigations.

Hydroxyapatite (HAP) and tricalcium phosphate (TCP) are two very common ceramic materials for bone replacement. However, in general HAP and TCP scaffolds are not tailored to the exact dimensions of the defect site and are mainly used as granules or beads. Some scaffolds are available as ordinary blocks, but cannot be customized for individual perfect fit. Using computer-assisted 3D printing, an emerging rapid prototyping technique, individual three-dimensional ceramic scaffolds can be built up from TCP or HAP powder layer by layer with subsequent sintering. These scaffolds have precise dimensions and highly defined and regular internal characteristics such as pore size. External shape and internal characteristics such as pore size can be fabricated using Computer Assisted Design (CAD) based on individual patient data. Thus, these scaffolds could be designed as perfect fit replacements to reconstruct the patient's skeleton. Before their use as bone replacement materials in vivo, in vitro testing of these scaffolds is necessary. In this study, the behavior of human osteoblasts on HAP and TCP scaffolds was investigated. The commonly used bone replacement material BioOss(R) served as control. Biocompatibility was assessed by scanning electron microscopy (SEM), fluorescence microscopy after staining for cell vitality with fluorescin diacetate (FDA) and propidium iodide (PI) and the MTT, LDH, and WST biocompatibility tests. Both versions were colonised by human osteoblasts, however more cells were seen on HAP scaffolds than TCP scaffolds. Cell vitality staining and MTT, LDH, and WST tests showed superior biocompatibility of HAP scaffolds to BioOss, while BioOss was more compatible than TCP. Further experiments are necessary to determine biocompatibility in vivo. Future modifications of 3D printed scaffolds offer advantageous features for Tissue Engineering. The integration of channels could allow for vascular and nerve ingrowth into the scaffold. Also the complex shapes of convex and concave articulating joint surfaces maybe realized with these rapid prototyping techniques.

Antimicrobial Peptide immunity protects human nasal and auricular cartilage against infection.

BACKGROUND: Despite being impervious to surveillance by the adaptive immune system because of its lack of vascularity, infection of the nasal and auricular cartilage after surgery such as rhinoplasty or otoplasty is rare. Why is this so? Our goal was to determine whether the expression of antimicrobial peptides provides a previously unrecognized nonepithelial layer of innate immune defense within the nasal and auricular cartilage. MATERIALS AND METHODS: Seven samples of nasal septum cartilage and 2 biopsies from auricular cartilage grafts were harvested during rhinoplasty and otoplasty procedures. Ten cadaveric samples of auricular and 9 samples of nasal cartilage were also obtained. Immunohistochemical staining was directed against the human beta-defensin antimicrobial peptides (hBD) 1, 2, and 3. A semiquantitative analysis was performed to measure immunoreactivity. RESULTS: All 3 human beta-defensins were detected along the perichondral line and within the cartilage matrix in the nasal and auricular samples. Areas with positive immunohistochemical staining were also detected within chondrocyte cytoplasm. CONCLUSIONS: We provide the first evidence of antimicrobial peptide expression (hBD-1, -2 and -3) within the perichondrium and cartilage matrix layers of the nasal and auricular cartilage. This previously unrecognized innate immune function of perichondrocytes and chondrocytes may explain the resistance of the nasal and auricular cartilage to infection after surgical procedures despite the absence of a vascular system.

Endocultivation: does delayed application of BMP improve intramuscular heterotopic bone formation?

INTRODUCTION: The time point of Bone morphogenetic protein (BMP) delivery on matrices in vivo may play an important role. Delayed application could be advantageous as this would allow soft tissue (ST) ingrowth and vascularisation of scaffolds prior to BMP-loading. The aim of this study was to compare the application of BMP injected simultaneously during matrix implantation with delayed application four weeks after matrix implantation for endocultivation in a rat model. MATERIAL AND METHODS: Bovine hydroxyapatite blocks were placed in pouches in the Musculus latissimus dorsi in 6 Lewis rats unilaterally to allow for soft tissue ingrowth. Four weeks later, a second block was inserted on the contralateral side of each rat. At that time point, 100microg rhBMP-2 in 2ml sodium chloride was injected on both sides to induce bone formation. For eight weeks, bone regeneration was monitored by computed tomography (CT) and fluorescent labelling. RESULTS: The simultaneous and delayed BMP application groups were significantly different (p=0.01). Slightly lower bone densities were seen for the delayed BMP application with a mean of 588 Hounsfield Units (HU) (standard deviation (SD) 30HU). Simultaneous BMP application revealed slightly higher densities with a mean of 633HU (SD 30HU). The largest differences were observed when comparing bone density directly after implantation or at the end of the observation period (p<0.0001). CONCLUSION: Bone density was slightly lower in the case of delayed application of BMP-2. The increase of bone density after application of BMP-2 was similar for both groups. Thus, delayed application of BMP had no advantageous effect in this particular study design. Further studies are needed to explore if varying delays, different material designs or special BMP application devices may alter these results.

Endocultivation: 3D printed customized porous scaffolds for heterotopic bone induction.

The aim of this study was to evaluate the ability of computer assisted designed (CAD) synthetic hydroxyapatite and tricalciumphosphate blocks to serve as precise scaffolds for intramuscular bone induction in a rat model. A central channel to allow for vessel pedicle or nerve integration was added. Natural bovine hydroxyapatite blocks served as controls to evaluate and compare biocompatibility of the new matrices. Individually designed 3D-printed rounded and porous hydroxyapatite (HA) and tricalcium phosphate (TCP) blocks were placed in pouches in the Musculus latissimus dorsi in 12 Lewis rats bilaterally. Bovine hydroxyapatite blocks with and without a central channel served as controls. Simultaneously, 200 microg rhBMP-2 in 1 ml sodium chloride was injected on both sides. For 8 weeks, bone generation was monitored by computer tomography and fluorescence labeling. The increase rates of bone density in CT examinations were higher in the HA groups (184-220 HU 8 weeks after implantation) compared to the TCP group (18 HU; p<0.0001). Microradiography and fluorescence microscopy 8 weeks after implantation showed new bone formation for all materials tested. For all scaffolds, toluidine staining revealed vital bone directly on the scaffold materials but also in the gaps between. It can be concluded from our data that the specially shaped hydroxyapatite and tricalcium phosphate blocks tested against the bovine hydroxyapatite blocks showed good biocompatibility and osteoinductivity in vivo. Further studies should explore if the stability of the individually designed blocks is sufficient to cultivate larger replacements without an external matrix for support.

Porous polymer/hydroxyapatite scaffolds: characterization and biocompatibility investigations.

Poly-lactic-glycolic acid (PLGA) has been widely used as a scaffold material for bone tissue engineering applications. 3D sponge-like porous scaffolds have previously been generated through a solvent casting and salt leaching technique. In this study, polymer-ceramic composite scaffolds were created by immersing PLGA scaffolds in simulated body fluid, leading to the formation of a hydroxyapatite (HAP) coating. The presence of a HAP layer was confirmed using scanning electron microscopy, energy dispersive X-ray spectroscopy and Fourier transform infrared spectroscopy in attenuated total reflection mode. HAP-coated PLGA scaffolds were tested for their biocompatibility in vitro using human osteoblast cell cultures. Biocompatibility was assessed by standard tests for cell proliferation (MTT, WST), as well as fluorescence microscopy after standard cell vitality staining procedures. It was shown that PLGA-HAP composites support osteoblast growth and vitality, paving the way for applications as bone tissue engineering scaffolds.

The battle against multi-resistant strains: Renaissance of antimicrobial essential oils as a promising force to fight hospital-acquired infections.

Hospital-acquired infections and antibiotic-resistant bacteria continue to be major health concerns worldwide. Particularly problematic is methicillin-resistant Staphylococcus aureus (MRSA) and its ability to cause severe soft tissue, bone or implant infections. First used by the Australian Aborigines, Tea tree oil and Eucalyptus oil (and several other essential oils) have each demonstrated promising efficacy against several bacteria and have been used clinically against multi-resistant strains. Several common and hospital-acquired bacterial and yeast isolates (6 Staphylococcus strains including MRSA, 4 Streptococcus strains and 3 Candida strains including Candida krusei) were tested for their susceptibility for Eucalyptus, Tea tree, Thyme white, Lavender, Lemon, Lemongrass, Cinnamon, Grapefruit, Clove Bud, Sandalwood, Peppermint, Kunzea and Sage oil with the agar diffusion test. Olive oil, Paraffin oil, Ethanol (70%), Povidone iodine, Chlorhexidine and hydrogen peroxide (H(2)O(2)) served as controls. Large prevailing effective zones of inhibition were observed for Thyme white, Lemon, Lemongrass and Cinnamon oil. The other oils also showed considerable efficacy. Remarkably, almost all tested oils demonstrated efficacy against hospital-acquired isolates and reference strains, whereas Olive and Paraffin oil from the control group produced no inhibition. As proven in vitro, essential oils represent a cheap and effective antiseptic topical treatment option even for antibiotic-resistant strains as MRSA and antimycotic-resistant Candida species.

Rapid prototyping: porous titanium alloy scaffolds produced by selective laser melting for bone tissue engineering.

Selective laser melting (SLM), a method used in the nuclear, space, and racing industries, allows the creation of customized titanium alloy scaffolds with highly defined external shape and internal structure using rapid prototyping as supporting external structures within which bone tissue can grow. Human osteoblasts were cultured on SLM-produced Ti6Al4V mesh scaffolds to demonstrate biocompatibility using scanning electron microscopy (SEM), fluorescence microscopy after cell vitality staining, and common biocompatibility tests (lactate dihydrogenase (LDH), 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), 5-bromo-2-deoxyuridine (BrdU), and water soluble tetrazolium (WST)). Cell occlusion of pores of different widths (0.45-1.2 mm) was evaluated. Scaffolds were tested for resistance to compressive force. SEM investigations showed osteoblasts with well-spread morphology and multiple contact points. Cell vitality staining and biocompatibility tests confirmed osteoblast vitality and proliferation on the scaffolds. Pore overgrowth increased during 6 weeks' culture at pore widths of 0.45 and 0.5 mm, and in the course of 3 weeks for pore widths of 0.55, 0.6, and 0.7 mm. No pore occlusion was observed on pores of width 0.9-1.2 mm. Porosity and maximum compressive load at failure increased and decreased with increasing pore width, respectively. In summary, the scaffolds are biocompatible, and pore width influences pore overgrowth, resistance to compressive force, and porosity.

Man as living bioreactor: fate of an exogenously prepared customized tissue-engineered mandible.

In 2004, we reported a novel method of repairing a human mandible by in vivo tissue engineering. The patient served as his own bioreactor as the exogenously prepared customized mandible replacement was grown inside his latissimus dorsi muscle prior to transplantation to repair the existing defect. Our technique was developed through extensive experience with an animal model. We describe our and the patient's experiences with this procedure. We give details to the benefits and limitations of this technique as it stands and outline issues that should be addressed in future human clinical trials.

The mechanical integrity of in vivo engineered heterotopic bone.

Recent advances in tissue engineering have aroused interest in growth of heterotopic bone for the repair of skeletal defects. This study demonstrates an in vivo method in minipigs of engineering individual human-sized mandible replacements of heterotopic bone with a mechanical integrity similar to natural bone. Ten individualized mandible replacement scaffolds were created using computer-aided design (CAD) techniques. Five had a resorbable external scaffold made of polylactite mesh (test group 1) and five had had a non-resorbable external scaffold of titanium mesh (test group 2). The mesh scaffolds were loaded each with five BioOss blocks serving as internal scaffolds and 3.5 mg recombinant human Bone Morphogenetic Protein-7. The loaded mesh scaffolds were implanted into the latissimus dorsi muscles of five infant minipigs. After 6 weeks the mandible replacements were harvested. Core biopsy cylinders were taken from the replacements of both test groups and from the natural pig mandibles (control 1). Also, core biopsies from plain BioOss Blocks were gained (control 2). The core biopsy cylinders were loaded axially into a compression test device to evaluate the mechanical compression resistance. Additional specimen underwent histological examination. Both test groups resulted in successful bone induction with degrees of compression resistance [Test 1: 1.62 MPa (SD+/-0.73); Test 2: 1.51 MPa (SD+/-0.56)] statistically insignificant when compared to natural porcine mandibular bone [1.75 MPa (SD+/-0.69)]. This differed significantly from the much lower compression resistance seen in the unadulterated BioOss [0.92 MPa (SD+/-0.04)]. Following this, the in vivo engineered bone has a similar mechanical compression stability as natural bone.

Minimal invasive surgery for hip replacement: a new technique using the NILNAV hip system.

BACKGROUND: Minimal invasive surgical techniques are used for cruciate ligament reconstructions, unicondylar knee replacements and, more recently, for fixation of fractures. This is a report of the first instrumented technique for hip replacement using a 5-cm incision without the need for a navigation system or X-rays. METHODS: It uses the C.F.P stem (LINK) but is universal. It includes jigs for the osteotomy of the neck, a right-angled reamer and spacers (lollipops) to orientate the acetabular cup to the femoral stem. A case series of 14 patients using this new hip replacement technique (called NILNAV Hip System) is reported. RESULTS: The procedure was successfully performed on all seven patients, with reduced postoperative pain and stiffness, and increased quality of life and functional status. All patients were discharged on postoperative Days 1 and 2 with minimal pain and blood loss. CONCLUSIONS: This new minimal access total hip replacement technique was successfully performed on seven patients. There are several advantages of using this system compared with the more traditional techniques. Such a technique should help reduce morbidity and mortality rates for those patients undergoing a total hip replacement.