Endocultivation is a pioneering technique used to grow individually shaped bone replacements within the latissimus dorsi muscle of patients with severe skeletal defects. The goal is to use the patient as his own bioreactor to prevent the common problems of engineering tissue in vitro. Replacement scaffolds are designed prior to surgery using CAD to enable a perfect fit. The site of cultivation is a surgically created pouch in the lateral latissimus dorsi which allows for neovascularization, courtesy of the thoracodorsal artery and vein, thereby allowing subsequent free-flap transfer into the desired recipient region. This independent vascular supply makes it possible to grow constructs of the size of a mandible, or in the future, possibly even a complex organ.

The continuous presence of recombinant human bone morphogenetic protein 2 (rhBMP-2) inside a scaffold may be crucial to the outcome in bone tissue engineering. This study investigated whether the release of the growth factor rhBMP-2 via different continuous application schemes influences histomorphological aspects of the hard and soft tissues induced. Three-dimensionally printed hydroxyapatite scaffolds were implanted into one latissimus dorsi muscle of 42 female Lewis rats. Simultaneously implanted mini-osmotic pumps were used to provide a continuous application of rhBMP-2 over 1, 2, or 4 weeks (total dose 200μg). A reference group received rhBMP-2 at the time of implantation only, and a control group received only block implantation. Bone density and histological examinations were performed after 8 weeks. No significant difference in bone density was found between the groups; however, the blood vessel count differed significantly between the groups receiving continuous treatments and both the control group and simultaneous rhBMP-2 treatment group (P<0.0001). Soft tissue types were distributed differently among the study groups. RhBMP-2 application via mini-osmotic pumps is as suitable for inducing bone formation as a single application at the time of implantation. The time interval over which rhBMP-2 was administered had no impact on the amount of new bone formation, probably due to the study duration and low local concentrations of growth factor.

PURPOSE:Molecular markers are only occasionally used in diagnostics of oral squamous cell carcinoma (OSCC), even though they could influence decision making in individually designed cancer therapies. We analyzed the predictive value of the markers HPV, p16, and HMGA2 and the TNM classification in regard to survival and recurrence rates.MATERIAL AND METHODS:A total of 91 OSCC cases were included in this study, with a follow up of up to 131 months. HPV-DNA was present in 7 carcinomas. p16 was detected by immunohistochemical staining in 14 samples. HMGA2 expression was determined by real-time quantitative polymerase chain reaction (qRT-PCR). Overexpression of HMGA2 was found to vary between 32-fold and 32,000-fold compared to nondysplastic tissue.RESULTS:Cox regression analysis showed that age, sex, smoking status, use of alcohol, human papillomavirus (HPV), and tumor size had no significant effect on overall and progression-free survival. HMGA2 and N-status showed significant effects on overall (HMGA2: p = 0.049; N1: p = 0.019; N2: p = 0.02) and disease-free survival (HMGA2: p = 0.057; N1: p = 0.198; N2: p = 0.02). P16 appeared to be borderline significant but the χ(2) indicated that p16 and N were correlated.CONCLUSION:Our results suggest that HMGA2 expression may have the potential to allow a more precise prognosis on survival in patients with OSCC.

The timing of application of recombinant human bone morphogenic protein-2 (rhBMP-2) may be important in determining the final outcome of engineered bone tissue. This study investigates the impact of repetitive rhBMP-2 application on hard and soft tissue morphology in endocultivation. A 3D-printed scaffold was implanted into a pouch in the latissimus dorsi muscle in 40 Lewis rats. RhBMP-2 was injected at defined time points and animals received a total of 200 μg each. Control groups received either rhBMP-2 simultaneously with scaffold implantation, or solely a scaffold with no rhBMP-2. Fluorescence markers were injected after operation. CT-scans and histological examination were performed after 8 weeks. Multiple comparisons revealed significant differences of bone density between the groups who received delayed injections at two separate time points in time compared to those who had simultaneous rhBMP-2 application (p = 0.0038; p = 0.0063) and the control group (p = 0.017, p = 0.0284). The blood vessel count was significantly higher in groups with repetitive injections compared with both control groups. Two soft tissue types were identified and found to have different distributions in the different study groups. Fluorescence labeling showed active new bone formation after 4-5 weeks in all groups where rhBMP-2 was administered. Multiple repetitive injections were more effective than simultaneous application regarding bone density indicating time-dependent effects of rhBMP-2. Bone formation processes were detectable several weeks after rhBMP-2 application indicating long-term effects.

Expansion of pluripotent stem cells in defined media devoid of animal-derived feeder cells to generate multilayered three-dimensional (3D) bulk preparations or spheroids, rather than two-dimensional (2D) monolayers, is advantageous for many regenerative, biological or disease-modelling studies. Here we show that electrospun polymer matrices comprised of nanofibres that mimic the architecture of the natural fibrous extracellular matrix allow for feeder-free expansion of pluripotent human induced pluripotent stem cells (IPSCs) and human embryonic stem cells (HESCs) into multilayered 3D 'patty-like' spheroid structures in defined xeno-free culture medium. The observation that IPSCs and HESCs readily revert to 2D growth in the absence of the synthetic nanofibre membranes suggests that this 3D expansion behaviour is mediated by the physical microenvironment and artificial niche provided by the nanofibres only. Importantly, we could show that such 3D growth as patties maintained the pluripotency of cells as long as they were kept on nanofibres. The generation of complex multilayered 3D structures consisting of only pluripotent cells on biodegradable nanofibre matrices of the desired shape and size will enable both industrial-scale expansion and intricate organ-tissue engineering applications with human pluripotent stem cells, where simultaneous coupling of differentiation pathways of all germ layers from one stem cell source may be required for organ formation. Copyright (C) 2014 John Wiley & Sons, Ltd.

Musculoskeletal defects attributable to trauma or infection or as a result of oncologic surgery present a common challenge in reconstructive maxillofacial surgery. The autologous vascularized bone graft still represents the gold standard for salvaging these situations. Preoperative virtual planning offers great potential and provides assistance in reconstructive surgery. Nevertheless, the applicability of autologous bone transfer might be limited within the medically compromised patient or because of the complexity of the defect and the required size of the graft to be harvested. The development of alternative methods are urgently needed in the field of regenerative medicine to enable the regeneration of the original tissue. Since the first demonstration of de novo bone formation by regenerative strategies and the application of bone growth factors some decades ago, further progress has been achieved by tissue engineering, gene transfer, and stem cell application concepts. This review summarizes recent approaches and current developments in regenerative medicine.

Background: The aim of the study was to examine the in vitro antibacterial activity of different oils in comparison to antiseptics against oral microorganisms.Methods: The antimicrobial effect of tea tree oil (TTO), eucalyptus oil (EO), lemon grass oil (LGO), and a eucalyptus-based oil mixture (MXT) were tested in comparison to chlorhexidine digluconate (CHX), povidone-iodine (BTA), and octenidine dihydrochloride (OCT). Oral bacterial strains and candida species using the agar diffusion test were used for the antimicrobial study.Results: All tested oils showed antimicrobial potency against the tested biological indicators. In comparison of all tested substances the largest effective zones were measured for LGO, followed from MXT and CHX. TTO and EO were less effective against the tested micro-organisms followed from BTA.Conclusions: The results of this study show that some essential oils have better antimicrobial properties than standard oral antiseptics. In a follow-up step, the ideal concentrations, the composition of essential oils, and the mode of application will be evaluated. The antibacterial efficacy of essential oils might be promising for use in clinical and oral hygiene applications. The cost reduction and availability particularly in rural areas with easy access to the originating plants might be advantageous factors to be considered.

Engineering a large vascularized bone graft is a much greater challenge than engineering small bone tissues. Although this is essentially feasible through an osteogenic factor-based in vivo bioreactor technique, the ossification needs improving. This study was aimed to investigate the possibility and efficacy of ectopic cultivation of sizeable bone grafts with large angiogenic and osteogenic factor-loaded natural bovine bone mineral (NBBM) scaffolds. For this purpose, six groups of sizeable composite scaffolds were constructed, consisting of a titanium mesh cage of NBBM or a mixture of NBBM/autogenous bone particles (AB), which were preloaded with 660 μg recombinant human bone morphogenetic protein-7 (rhBMP-7) and/or 4 μg recombinant human vascular endothelial growth factor165 (rhVEGF165). The scaffolds were implanted in bilateral latissimus dorsi muscles in eight pigs to construct in vivo bioreactors. Sequential fluorescence labeling was then applied to trace bone formation at the early stage. The implants were retrieved 12 weeks later. The undecalcified sections were observed in turn under the fluorescence microscope and light microscope to investigate early stage osteogenesis and histology. Moreover, new bone density (BD) was measured with histomorphometry. Compared with rhBMP-7-delivered NBBM scaffolds, rhVEGF165/rhBMP-7-delivered NBBM scaffolds were with more intense intra-scaffold osteogenesis at the early stage and the ultimate sizeable bone grafts of microstructurally more lamellae and trabeculae, and quantitatively higher BD (31.93% vs. 22.37%, p<0.01). This study demonstrated that as for the endocultivation of a large bone graft with bioactive factor-based in vivo bioreactor technique, dual delivery of rhVEGF165/rhBMP-7 has synergic effects on improving early stage bone formation and subsequently bone quality and quantity of the bone grafts.

There are relatively few nanotechnologies that can produce nanocomposite scaffolds for cell growth. Electrospinning has emerged as the foremost method of producing nanofibrous biomimetic scaffolds for tissue engineering applications. In this study diamond nanoparticles were integrated into a polymer solution to develop a nanocomposite scaffold containing poly(lactide-co-glycolide) (PLGA) loaded with diamond nanoparticles. To investigate the effect of adding diamond nanoparticles to PLGA scaffolds, primary human mesenchymal stem cells (hMSCs) were seeded on the scaffolds. The cytocompatibility results showed that addition of diamond nanoparticles did not impinge upon cell proliferation, nor was there a cytotoxic cellular response after 9 days in culture. Scanning electron microscopy, transmission electron microscopy, atomic force microscopy and confocal microscopy enabled qualitative characterization of the fibres and revealed cell morphology and number. Furthermore, surface roughness was measured to evaluate diamond nanoparticle modifications, and no significant difference was found between the diamond nanocomposite and pure polymer scaffolds. On the other hand, bright spots on phase images performed by atomic force microscopy suggested a higher hardness at certain points on fibers of the PLGA-nanodiamond composites, which was supported by nanoindentation measurements. This study shows that PLGA nanofibers can be reinforced with nanodiamond without adversely affecting cell behaviour, and thus it sets the foundation for future application of these scaffolds in bone tissue engineering.

PURPOSE:Artificial materials such as dental implants are at risk of bacterial contamination in the oral cavity. Human beta defensins (HBDs), small cationic antimicrobial peptides that exert a broad-spectrum antibacterial function at epithelial surfaces and within some mesenchymal tissues, could probably help to reduce such contamination. HBDs also have protective immunomodulatory effects and have been reported to promote bone remodeling. The aim of this study, therefore, was to investigate the influence of recombinant HBD-2 on the proliferation and survival of cells in culture.MATERIALS AND METHODS:Human mesenchymal stem cells (hMSCs), human osteoblasts, human keratinocytes (control), and the HeLa cancer cell line (control) were incubated with recombinant HBD-2 (1, 5, 10, or 20 μg/mL). Cell proliferation and cytotoxicity were evaluated via a water-soluble tetrazolium salt (WST-1) and lactate dehydrogenase assays, respectively.RESULTS:HBD-2 was not toxic in any tested concentration to hMSCs, osteoblasts, keratinocytes, or HeLa cells. Furthermore, proliferation of hMSCs and osteoblasts increased after treatment with HBD-2 at all tested concentrations, and keratinocyte proliferation increased when treated at 20 μg/mL. In contrast, HeLa cancer cells were not affected by HBD-2 as tested.CONCLUSIONS:HBD-2 is not only biocompatible but also promotes proliferation of hMSCs, osteoblasts, and keratinocytes in culture. Further investigation of HBD-2 functional surface coating of artificial materials is recommended.

Transplanted retinal pigment epithelium (RPE) cells hold promise for treatment of age-related macular degeneration (AMD) and Stargardt disease (SD), but it is conceivable that the degenerated host Bruch's membrane (BM) as a natural substrate for RPE might not optimally support transplanted cell survival with correct cellular organization. We fabricated novel ultrathin three-dimensional (3-D) nanofibrous membranes from collagen type I and poly(lactic-co-glycolic acid) (PLGA) by an advanced clinical-grade needle-free electrospinning process. The nanofibrillar 3-D networks closely mimicked the fibrillar architecture of the native inner collagenous layer of human BM. Human RPE cells grown on our nanofibrous membranes bore a striking resemblance to native human RPE. They exhibited a correctly orientated monolayer with a polygonal cell shape and abundant sheet-like microvilli on their apical surfaces. RPE cells built tight junctions and expressed RPE65 protein. Flat 2-D PLGA film and cover glass as controls delivered inferior RPE layers. Our nanofibrous membranes may imitate the natural BM to such extent that they allow for the engineering of an in vivo-like human RPE monolayer that maintains the natural biofunctional characteristics. Such ultrathin membranes may provide a promising vehicle for a functional RPE cell monolayer implantation in the subretinal space in patients with AMD or SD.

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.

AIM Biomaterials that mimic the nanofibrous architecture of the natural extracellular matrix (ECM) are in the focus for stem cell hosting or delivery in tissue engineering of multilayered soft tissues such as skin, mucosa, or retina. Synthetic nanofibers for such ECM are usually produced by single-syringe electrospinning with only one needle-jet at very low production rates of 0.005-0.008 g·min⁻¹. The aim of this study was to utilize a novel industrial needle-free multijet electrospinning device with the potential for mass production of nanofibrous ECM (NF-ECM) exhibiting a controlled three-dimensional (3D) morphology for large-scale applications such as large area skin regeneration in patients with burns. METHODS The novel NanoSpider™ NS200, an industrial apparatus originally designed for electrospinning of nanofibrous textile meshes, was used to fabricate 3D NF-ECMs of the following synthetic and natural biopolymers: collagen, gelatin, poly(caprolactone) (PCL), and poly(L-lactide-co-glycolide) (PLGA). Different concentrations of Gelatin polymer solution were electrospun under varying processing conditions, namely speed of spinning electrode rotation (u) and electric field intensity (E) by altering applied voltage (v) or the distance between electrodes (h) to achieve homogeneous desirable 3D morphology. Nanofiber diameters were assessed by scanning electron microscopy (SEM). Biocompatibility was tested by WST-1 (water-soluble tetrazolium salt) proliferation assay of seeded human mesenchymal stem cells (HMSCs). Biological performance of HMSCs on 3D PLGA NF-ECM was compared to two-dimensional (2D) PLGA film controls via SEM and confocal microscopy. Western blotting addressed the expression of surface adhesion proteins; focal adhesion kinase (FAK), phosphorylated FAK (pY397), α-tubulin, paxillin, vinculin. and integrin subunits; α5, αv, and β1 proteins. RESULTS Large-scale mass production of NF-ECM membranes with a highly homogenous nanofiber morphology and 3D architecture could be produced with an extremely high production rate of 0.394±0.013 g·min⁻¹·m⁻¹ when compared to standard procedures. This was achieved by electrospinning a 20% (wt)/v gelatin solution, in an electric field intensity of 0.381 kV·mm⁻¹. The nanofibers possessed diameters of around 180±40 nm with 28% deviation. HSMCs proliferation was significantly improved on NF-ECMs derived from collagen, gelatin, and PLGA when compared to PCL or flat coverglass controls (p<0.01). PLGA NF-ECM in 3D nanofibrous architecture possessed significantly superior biocompatibility when compared to flat 2D PLGA film (p<0.05). Furthermore, on 3D PLGA NF-ECMs, HSMCs expressed a higher amount of α-tubulin and paxillin compared to the HMSCs cultured on a 2D PLGA film (p<0.05). HMSCs exhibited a complex multifaceted morphology on all NF-ECMs, where cells appeared to be integrated into the 3D NF-ECMs niches with complex cell filopodia extending into to all directions. In contrast, HMSCs on flat 2D films of the same materials or on coverglass displayed a simple flattened, monolayered structure. CONCLUSION Needle-free multijet electrospinning can be used to mass produce artificial ECMs with intrinsic biocompatibility and desirable integration of stem cells for large-scale applications.

Introduction History of Trauma and Orthopaedic Surgery Clinical History and Examination Techniques Research Methods, Epidemiology and Statistics Musculoskeletal Structure, Function, and Healing Surgical Anatomy and Embryology of the Musculoskeletal System Bone Physiology and Repair Soft Tissue Physiology and Healing Central and Peripheral Nervous System Peri, Intra and Post Operative Care The Physiology of Aging Principles of Orthopaedic Pharmacology Basic Science Molecular and Cell Biology, Immunology and Genetics Musculoskeletal Imaging Nuclear Medicine Biomechanics and Biomaterials Metabolic Bone Disease Developmental and Inflammatory Conditions Kinesiology Infection Trauma ATLS and Polytrauma Major Incident Management The Closed Treatment of Common Fractures Principles of Operative Fracture Fixation, (AO) Shoulder Girdle and Humerus Fractures Elbow and Forearm Wrist and Hand Fractures Pelvic and Acetabular Fractures Hip and Proximal Femur Femoral Fractures Knee and Tibia Ankle Fractures Foot Fractures Spinal Fractures Pediatric Fractures and Dislocations Management of Infections and Non-Unions. (Complications) Paediatric Orthopaedic Surgery Clinical Assessment, Investigations and Screening Bone Dysplasias Chromosomal Disorders Metabolic Diseases Infections Neuromuscular Affections Paediatric Spine Upper Extremity Lower Extremity Adolescent Disorders Syndromes Sports Medicine Exercise Physiology, Epidemiology and Special Considerations Pathophysiology of Cartilage, Meniscus, Tendon and Ligament Essential Arthroscopic Skills and Concepts Shoulder Elbow, Wrist and Hand Pelvis, Hip and Thigh The Knee Leg, Foot and Ankle Musculoskeletal Oncology Pathophysiology, Molecular and Cell Biology of Tumors Clinical Evaluation, Principles of Biopsy and Staging Principles of Chemotherapy and Radiotherapy Bone Tumours Soft Tissue Tumours Metastatic Tumours Tumour Reconstruction Surgery Amputations Prosthetics and Orthotics The Spine History, Physical Examination and Investigations Pathophysiology of Back Pain Spinal Biomechanics Cervical Spine Thoracic Spine Lumbosacral Spine Scoliosis Spinal Instrumentation Complications and Management Thereof Non-Fusion Technologies Adult Reconstruction Surgery Arthritis AVN / Osteonecrosis Principles of Arthroplasty / Methods of Fixation Alternatives to Arthroplasty (Osteotomies) Upper Limb Primary THA Revision THA Primary TKA UKA Revision TKA Bone Conserving Options Ankle Arthroplasty Wear Strategies in Managing Infections Periprosthetic Fractures Other Complications Hand and Upper Limb Finger tip Injuries Tendon Injuries Nerve and Vessel Injuries Dupuytrens Tendonitis and Triggering Osteoarthritis Rheumatoid Arthritis Arthrodesis The Thumb Replantation and Microsurgery Nerve Compression Syndromes Foot and Ankle Ligament and Tendon Injuries Hallux Valgus Lesser Toe Deformities Pes Planus and Pes Cavus The Diabetic Foot Neurologic Disorders The Rheumatoid Foot Nerve Compression Syndromes New technologies and best clinical practice Minimal Invasive Surgery (MIS) Computer Aided Orthopaedic Surgery (CAOS) MIS Meets CAOS Tissue Engineering Genomics and Proteomics Endocultivation Bone Antibacterial Peptides Selected Ethical Issues Occupation Related Illness and Compensation

When bone morphogenetic protein (BMP) is delivered to matrices in vivo may affect tissue engineered bone constructs for jaw reconstruction after cancer surgery. This study compared the effects of BMP application at different times after matrix implantation for heterotopic bone induction in a rat model. Hydroxyapatite blocks were implanted unilaterally onto the surface of the latissimus dorsi muscle. A second block was implanted onto the contralateral muscle after 1, 2 or 4 weeks and 200μg rhBMP-2 was injected into the blocks on both sides. Bone formation and density inside the blocks was analysed by CT and histology. 8 weeks after BMP application increases in bone density within the scaffolds were most pronounced in the simultaneous application group (179 HU). Less pronounced increases were observed for the 1 (65 HU), 2 (58 HU) and 4 (31 HU; p<0.0001) week delay group. Homogeneous bone induction started from the central channel of the blocks. Capillaries and larger vessels were seen in all constructs, samples receiving delayed BMP treatment demonstrated significantly greater neovascularization. Delayed application of BMP was less effective for heterotopic bone formation than simultaneous application. A central channel allows homogeneous bone induction directly from the centre of the blocks.

Novel clinical grade electrospinning methods could provide three-dimensional (3D) nanostructured biomaterials comprising of synthetic or natural biopolymer nanofibers. Such advanced materials could potentially mimic the natural extracellular matrix (ECM) accurately and may provide superior niche-like spaces on the subcellular scale for optimal stem-cell attachment and individual cell homing in regenerative therapies. The goal of this study was to design several novel nanofibrous extracellular matrices (NF-ECMs) with a natural mesh-like 3D architecture through a unique needle-free multi-jet electrospinning method in highly controlled manner to comply with good manufacturing practices (GMP) for the production of advanced healthcare materials for regenerative medicine, and to test cellular behavior of human mesenchymal stem cells (HMSCs) on these. Biopolymers manufactured as 3D NF-ECM meshes under clinical grade GMP-like conditions show higher intrinsic cytobiocompatibility with superior cell integration and proliferation if compared to their 2D counterparts or a clinically-approved collagen membrane.

The transplantation of human stem cells seeded on biomaterials holds promise for many clinical applications in cranio-maxillo-facial tissue engineering and regenerative medicine. However, stem cell propagation necessary to produce sufficient cell numbers currently utilizes fetal calf serum (FCS) as a growth supplement which may subsequently transmit animal pathogens. Human platelet lysate (HPL) could potentially be utilized to produce clinical-grade stem cell-loaded biomaterials as an appropriate FCS substitute that is in line with clinically-applicable practice. The goal of this study was to investigate whether HPL can be successfully used to propagate human mesenchymal stem cells (HMSCs) seeded on clinically-approved collagen materials under clinically-applicable conditions using FCS as a control.HMSCs were isolated from bone marrow and cultured in the presence of 10% FCS or 10% HPL. Characterization of HMSCs was performed by flow cytometry and through osteogenic and adipogenic differentiation assays. Proliferative capacity of HMSCs on both matrices was investigated by mitochondrial dehydrogenase assays (WST) and tissue coverage scanning electron microscopy (SEM).The isolated HMSC differentiated into osteogenic and adipogenic cells authenticating the multipotentiality of the HMSCs. WST tests and the SEM images demonstrated that HPL was generally superior to FCS in promoting growth of seeded HMSCs. For all other tests HPL supported HMSCs at least equal to FCS.In conclusion, HPL is an effective growth factor to allow expansion of clinical-grade HMSCs on clinically-approved biomaterials for maxillofacial and oral implantology applications.