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An aging population leads to increasing demand for sustained quality of life with the aid of novel implants. Patients expect fast healing and few complications after surgery. Increased biofunctionality and antimicrobial behavior of implants, in combination with supportive stem cell therapy, can meet these expectations. Recent research in the field of bone implants and the implementation of autologous mesenchymal stem cells in the treatment of bone defects is outlined and evaluated in this review. The article highlights several advantages, limitations and advances for metal-, ceramic- and polymer-based implants and discusses the future need for high-throughput screening systems used in the evaluation of novel developed materials and stem cell therapies. Automated cell culture systems, microarray assays or microfluidic devices are required to efficiently analyze the increasing number of new materials and stem cell-assisted therapies. Approaches described in the literature to improve biocompatibility, biofunctionality and stem cell differentiation efficiencies of implants range from the design of drug-laden nanoparticles to chemical modification and the selection of materials that mimic the natural tissue. Combining suitable implants with mesenchymal stem cell treatment promises to shorten healing time and increase treatment success. Most research studies focus on creating antibacterial materials or modifying implants with antibacterial coatings in order to address the increasing number of complications after surgeries that are mostly caused by bacterial infections. Moreover, treatment of multiresistant pathogens will pose even bigger challenges in hospitals in the future, according to the World Health Organization (WHO). These antibacterial materials will help to reduce infections after surgery and the number of antibiotic treatments that contribute to the emergence of new multiresistant pathogens, whilst the antibacterial implants will help reduce the amount of antibiotics used in clinical treatment.

Summary: Biologic mesh is preferred over synthetic mesh for complex and contaminated abdominal wall repairs; however, there are very little data on the risks and complications associated with its use. We report the case of a 67-year-old man with failed synthetic mesh repair for recurrent ventral hernia, who subsequently required an abdominal wall reconstruction (AWR), including the intraperitoneal sublay of noncrosslinked biologic mesh. His postoperative course was complicated with catastrophic sepsis and sustained hemodynamic instability, responding only to mesh explantation. The biologic mesh was subsequently noted to be histologically infected with invasive Candida albicans . Although noncrosslinked biologic mesh is a valuable adjunct to AWR, it is not infection-resistant. Although it is rare, infection of any foreign tissue, including biologic mesh, can occur in the setting of complex ventral abdominal wall repairs. Clinicians should be watchful for such infections in complex repairs as they may require biologic mesh explantation for clinical recovery.

In this study a phosphate-free glass-ceramic porous scaffold was synthesized by a three-step methodology involving slurry preparation, induction of porosity by surfactant-assisted foaming following by freeze-drying and sintering. This inorganic scaffold was characterized by X-ray diffraction, scanning electron microscope (SEM), degradation and bioactivity. Thermal treatment at 750 °C showed two new crystalline phases, combeite and nepheline, into the glassy matrix responsible for its properties. The cell response of the scaffold was also evaluated for using as a bone graft substitute. A commercial Biphasic Calcium Phosphate, BCP, scaffold was assessed in parallel as reference material. Microstructures obtained by SEM showed the presence of macro, meso and microporosity. The glass-ceramic scaffold possesses an interconnected porosity around 31% with a crack-pore system that promote the protein adsorption and cell attachment. Glass-ceramic scaffold with high concentration of calcium ions shows an antimicrobial behavior against Escherichia coli after 24 h of contact. Nepheline phase present in the glass-ceramic structure is responsible for its high mechanical properties being around 87 MPa. Glass-ceramic scaffold promotes greater protein adsorption and therefore the attachment, spreading and osteodifferentiation of Adipose Derived Stem Cells than BCP scaffold. A higher calcification was induced by glass-ceramic scaffold compared to reference BCP material.

Background: Xenografts are an attractive alternative to traditional bone grafts because of the large supply from donors with predictable morphology and biology as well as minimal risk of human disease transmission. Clinical series involving xenograft bone transplantation, most commonly from bovine sources, have reported poor results with frequent graft rejection and failure to integrate with host tissue. Failures have been attributed to residual alpha-Gal epitope in the xenograft which humans produce natural antibody against. To the authors' knowledge, there is currently no xenograft-derived bone graft substitute that has been adopted by orthopedic surgeons for routine clinical use., Methods: In the current study, a bone scaffold intended to serve as a bone graft substitute was derived from porcine cancellous bone using a tissue decellularization and chemical oxidation protocol. In vitro cytocompatibility, pathogen clearance, and alpha-Gal quantification tests were used to assess the safety of the bone scaffold intended for human use., Results: In vitro studies showed the scaffold was free of processing chemicals and biocompatible with mouse and human cell lines. When bacterial and viral pathogens were purposefully added to porcine donor tissue, processing successfully removed these pathogens to comply with sterility assurance levels established by allograft tissue providers. Critically, 98.5% of the alpha-Gal epitope was removed from donor tissue after decellularization as shown by ELISA inhibition assay and immunohistochemical staining., Conclusions: The current investigation supports the biologic safety of bone scaffolds derived from porcine donors using a decellularization protocol that meets current sterility assurance standards. The majority of the highly immunogenic xenograft carbohydrate was removed from donor tissue, and these findings support further in vivo investigation of xenograft-derived bone tissue for orthopedic clinical application., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Expanded polytetrafluoroethylene (ePTFE), an ideal bioimplant material, is commonly used in surgical repair to treat soft tissue defects and deformities. However, the main disadvantage of ePTFE is that its distinctive porous ultrastructure is prone to bacterial adhesion that gives rise to infection and chronic inflammation, resulting in functional failure. Herein, a potentially promising approach to ePTFE autologous vascularization (AV-ePTFE) in vivo was established and developed to enhance the material's antibacterial properties., Methods: Hematoxylin and eosin (H&E) staining and visual observation were performed to validate the intensity of the inflammatory response and related histological changes in surgical wounds after AV-ePTFE implantation. In addition, the antibacterial activities of AV-ePTFE were assessed by an in vitro bacterial adhesion assay and scanning electron microscope observation., Results: The optimal time point of AV-ePTFE was 12 weeks after implantation. AV-ePTFE relieved inflammation based on an inflammation grading evaluation and expedited wound healing. Furthermore, AV-ePTFE effectively reduced the number of bacterial adhesions, inhibited bacterial biofilm formation, and prevented the occurrence of infection., Conclusions: We conclude that autologous vascularization is an effective method to improve the antibacterial adhesion properties and biocompatibility of ePTFE after implantation and that it may have a significant effect on clinical application of future porous biomaterials., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Over the past few decades, in vitro cell culture systems have greatly expanded our understanding of host-pathogen interactions. However, studies using these models have been limited by the fact that they lack the complexity of the human body. Therefore, recent efforts that allow tissue architecture to be mimicked during in vitro culture have included the development of methods and technology that incorporate tissue structure, cellular composition, and efficient long-term culture. These advances have opened the door for the study of pathogens that previously could not be cultured and for the study of pathophysiological properties of infection that could not be easily elucidated using traditional culture models. Here we discuss the latest studies using organoids and engineering technology that have been developed and applied to the study of host-pathogen interactions in mucosal tissues.

Bone tissue engineering put emphasis on fabrication three-dimensional biodegradable porous scaffolds that supporting bone regeneration and functional bone tissue formation. In the present work, we prepared novel 3D tripolymeric scaffolds of nanochitosan (NCS)/silk fibroin (SF)/hyaluronic acid (HA) ternary blends and demonstrating the synergistic effect of scaffolds and its use in tissue engineering applications. The physico-chemical characterization of the prepared scaffold was evaluated by FTIR, XRD and SEM studies. The FT-IR and XRD results confirmed the interfacial bonding interaction existing between polymers. SEM images showed good interconnected porous structure with rough surface morphology. The in vitro cytocompatibility tests carried out with osteoblast cells by the MTT assay demonstrated that the blended scaffold favors the early adhesion, growth and proliferation of preosteoblast MC3T3-E1 cells. The alizarin red assay indicated that the prepared scaffold can promote the osteogenic differentiation and facilitate the calcium mineralization of MC3T3-E1 cells. The alkaline phosphatase assay confirmed that the NCS/SF/HA scaffold provide conducive environment for osteoblast proliferation and mineral deposition. The bactericidal action of NCS/SF/HA scaffold reveals that the prepared sample has the potential to kill the microorganisms to a greater extent. Hence the overall findings concluded that the NCS/SF/HA scaffolds have better applications in tissue engineering., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Materials can be engineered to deliver specific biological cues that control stem cell growth and differentiation. However, current materials are still limited for stem cell engineering as stem cells are regulated by a complex biological milieu that requires spatiotemporal control. Here a new approach of using materials that incorporate designed bacteria as units that can be engineered to control human mesenchymal stem cells (hMSCs), in a highly dynamic-temporal manner, is presented. Engineered Lactococcus lactis spontaneously colonizes a variety of material surfaces (e.g., polymers, metals, and ceramics) and is able to maintain growth and induce differentiation of hMSCs in 2D/3D surfaces and hydrogels. Controlled, dynamic, expression of fibronectin fragments supports stem cell growth, whereas inducible-temporal regulation of secreted bone morphogenetic protein-2 drives osteogenesis in an on-demand manner. This approach enables stem cell technologies using material systems that host symbiotic interactions between eukaryotic and prokaryotic cells., (© 2018 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Living organisms constantly maintain their structural and biochemical integrity by the critical means of response, healing, and regeneration. Inanimate objects, on the other hand, are axiomatically considered incapable of responding to damage and healing it, leading to the profound negative environmental impact of their continuous manufacturing and trashing. Objects with such biological properties would be a significant step towards sustainable technology. In this work we present a feasible strategy for driving regeneration in fabric by means of integration with a bacterial biofilm to obtain a symbiotic-like hybrid - the fabric provides structural framework to the biofilm and supports its growth, whereas the biofilm responds to mechanical tear by synthesizing a silk protein engineered to self-assemble upon secretion from the cells. We propose the term crossbiosis to describe this and other hybrid systems combining organism and object. Our strategy could be implemented in other systems and drive sensing of integrity and response by regeneration in other materials as well.

Background: Recent data suggest an increased risk for infection when acellular dermal matrix is used in breast reconstruction. This may be because some acellular dermal matrices are actually not terminally sterilized but are instead "aseptically processed." This study evaluates aseptic and sterile matrices for evidence of bacterial contamination and whether or not terminal sterilization affects matrix collagen architecture and stem cell ingrowth., Methods: Five separate samples of 14 different matrices were analyzed by fluorescent in situ hybridization using a bacterial DNA probe to detect bacterial DNA on the matrices. Separate samples were incubated for bacteria, acid-fast bacilli, and fungi for 2 to 6 weeks to detect living organisms. The impact of terminal sterilization on the collagen network and stem cell ingrowth on the matrices was then assessed., Results: Traces of bacterial DNA were encountered on all matrices, with more bacteria in the aseptic group compared with the sterile group (3.4 versus 1.6; p = 0.003). The number of positive cultures was the same between groups (3.8 percent). Electron microscopy demonstrated decreased collagen organization in the sterile group. Stem cell seeding on the matrices displayed a wide variation of cellular ingrowth between matrices, with no difference between aseptic and sterile groups (p = 0.2)., Conclusions: Although there was more evidence of prior bacterial contamination on aseptically processed matrices compared with sterile matrices; clinical cultures did not differ between groups. Terminal sterilization does not appear to affect stem cell ingrowth but may come at the cost of damaging the collagen network., Clinical Question/level of Evidence: Therapeutic, V.

It is not known to what extent residual infection may interfere with the success of pulp regeneration procedures. The aim of this study was to determine, radiographically and histologically, the effect of residual bacteria on the outcome of pulp regeneration mediated by a tissue-engineered construct as compared with traditional revascularization. Periapical lesions were induced in 24 canine teeth of 6 ferrets. After disinfection with 1.25% NaOCl and triple antibiotic paste, ferret dental pulp stem cells, encapsulated in a hydrogel scaffold, were injected into half the experimental teeth. The other half were treated with the traditional revascularization protocol with a blood clot scaffold. After 3 mo, block sections of the canine teeth were imaged radiographically and processed for histologic and histobacteriologic analyses. Associations between variables of interest were evaluated through mixed effects regression models. There were no significant differences between the 2 experimental groups in radiographic root development ( P > 0.05). There was a significant association between the presence of persistent periapical radiolucency and root wall thickness ( P = 0.02). There was also no significant difference in histologic findings between the 2 experimental groups ( P > 0.05). The presence of residual bacteria was significantly associated with lack of radiographic growth ( P < 0.001). The amount of dentin-associated mineralized tissue formed in teeth with residual bacteria was significantly less than in teeth with no residual bacteria ( P < 0.001). Residual bacteria have a critical negative effect on the outcome of regenerative endodontic procedures.

Purpose . Manuka honey (MH) is an antibacterial agent specific to the islands of New Zealand containing both hydrogen peroxide and a Unique Manuka Factor (UMF). Although the antibacterial properties of MH have been studied, the effect of varying UMF of MH incorporated into tissue engineered scaffolds have not. Therefore, this study was designed to compare silk fibroin cryogels and electrospun scaffolds incorporated with a 5% MH concentration of various UMF. Methods . Characteristics such as porosity, bacterial clearance and adhesion, and cytotoxicity were compared. Results . Pore diameters for all cryogels were between 51 and 60  µ m, while electrospun scaffolds were 10  µ m. Cryogels of varying UMF displayed clearance of approximately 0.16 cm for E. coli and S. aureus . In comparison, the electrospun scaffolds clearance ranged between 0.5 and 1 cm. A glucose release of 0.5 mg/mL was observed for the first 24 hours by all scaffolds, regardless of UMF. With respect to cytotoxicity, neither scaffold caused the cell number to drop below 20,000. Conclusions . Overall, when comparing the effects of the various UMF within the two scaffolds, no significant differences were observed. This suggests that the fabricated scaffolds in this study displayed similar bacterial effects regardless of the UMF value., Competing Interests: The authors declare that they have no competing interests.

The use of electrospun nanofibers for tissue engineering and regenerative medicine applications is a growing trend as they provide improved support for cell proliferation and survival due, in part, to their morphology mimicking that of the extracellular matrix. Sterilization is a critical step in the fabrication process of implantable biomaterial scaffolds for clinical use, but many of the existing methods used to date can negatively affect scaffold properties and performance. Poly(lactic-co-glycolic acid) (PLGA) has been widely used as a biodegradable polymer for 3D scaffolds and can be significantly affected by current sterilization techniques. The aim of this study was to investigate pulsed ozone gas as an alternative method for sterilizing PLGA nanofibers. The morphology, mechanical properties, physicochemical properties, and response of cells to PLGA nanofiber scaffolds were assessed following different degrees of ozone gas sterilization. This treatment killed Geobacillus stearothermophilus spores, the most common biological indicator used for validation of sterilization processes. In addition, the method preserved all of the characteristics of nonsterilized PLGA nanofibers at all degrees of sterilization tested. These findings suggest that ozone gas can be applied as an alternative method for sterilizing electrospun PLGA nanofiber scaffolds without detrimental effects.

Porous polyurethane (PU) structures raise increasing interest as scaffolds in tissue engineering applications. Understanding the effects of sterilization on their properties is mandatory to assess their potential use in the clinical practice. The aim of this work is the evaluation of the effects of two innovative sterilization techniques (i.e. plasma, Sterrad(®) system, and ozone) on the morphological, chemico-physical and mechanical properties of a PU foam synthesized by gas foaming, using water as expanding agent. In addition, possible toxic effects of the sterilization were evaluated by in vitro cytotoxicity tests. Plasma sterilization did not affect the morphological and mechanical properties of the PU foam, but caused at some extent degradative phenomena, as detected by infrared spectroscopy. Ozone sterilization had a major effect on foam morphology, causing the formation of new small pores, and stronger degradation and oxidation on the structure of the material. These modifications affected the mechanical properties of the sterilized PU foam too. Even though, no cytotoxic effects were observed after both plasma and ozone sterilization, as confirmed by the good values of cell viability assessed by Alamar Blue assay. The results here obtained can help in understanding the effects of sterilization procedures on porous polymeric scaffolds, and how the scaffold morphology, in particular porosity, can influence the effects of sterilization, and viceversa.

This work evaluated the effects of UV irradiation, plasma radiation, steam and 70% ethanol treatments on the sterilization and integrity of auto-crosslinked hyaluronic acid (HA-ACP) scaffolds structured in microparticles and sponges aiming in vivo applications for regenerative medicine of bone tissue. The integrity of the microparticles was characterized by rheological behavior, while for the sponges, it was characterized by scanning electron microscopy, Fourier transform infrared spectroscopy and differential scanning calorimetry. The effectiveness of the sterilization treatment was verified by the number of microorganism colonies in the samples after the treatments. In conclusion, plasma radiation was the best treatment for the sponges, while steam sterilization in the autoclave at 126°C (1.5 kgf/cm2) for 5 min was the best treatment for the microparticles.

Cell culture techniques are essential for studying host-pathogen interactions. In addition to the broad range of single cell type-based two-dimensional cell culture models, an enormous amount of coculture systems, combining two or more different cell types, has been developed. These systems enable microscopic visualization and molecular analyses of bacterial adherence and internalization mechanisms and also provide a suitable setup for various biochemical, immunological, and pharmacological applications. The implementation of natural or synthetical scaffolds elevated the model complexity to the level of three-dimensional cell culture. Additionally, several transwell-based cell culture techniques are applied to study bacterial interaction with physiological tissue barriers. For keeping highly differentiated phenotype of eukaryotic cells in ex vivo culture conditions, different kinds of microgravity-simulating rotary-wall vessel systems are employed. Furthermore, the implementation of microfluidic pumps enables constant nutrient and gas exchange during cell cultivation and allows the investigation of long-term infection processes. The highest level of cell culture complexity is reached by engineered and explanted tissues which currently pave the way for a more comprehensive view on microbial pathogenicity mechanisms., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Various compositions of synthetic calcium phosphates (CaP) have been proposed and their use has considerably increased over the past decades. Besides differences in physico-chemical properties, resorption and osseointegration, artificial CaP bone graft might differ in their resistance against biofilm formation. We investigated standardised cylinders of 5 different CaP bone grafts (cyclOS, chronOS (both β-TCP (tricalcium phosphate)), dicalcium phosphate (DCP), calcium-deficient hydroxyapatite (CDHA) and α-TCP). Various physico-chemical characterisations e.g., geometrical density, porosity, and specific surface area were investigated. Biofilm formation was carried out in tryptic soy broth (TSB) and human serum (SE) using Staphylococcus aureus (ATCC 29213) and S. epidermidis RP62A (ATCC 35984). The amount of biofilm was analysed by an established protocol using sonication and microcalorimetry. Physico-chemical characterisation showed marked differences concerning macro- and micropore size, specific surface area and porosity accessible to bacteria between the 5 scaffolds. Biofilm formation was found on all scaffolds and was comparable for α-TCP, chronOS, CDHA and DCP at corresponding time points when the scaffolds were incubated with the same germ and/or growth media, but much lower for cyclOS. This is peculiar because cyclOS had an intermediate porosity, mean pore size, specific surface area, and porosity accessible to bacteria. Our results suggest that biofilm formation is not influenced by a single physico-chemical parameter alone but is a multi-step process influenced by several factors in parallel. Transfer from in vitro data to clinical situations is difficult; thus, advocating the use of cyclOS scaffolds over the four other CaP bone grafts in clinical situations with a high risk of infection cannot be clearly supported based on our data.

Biomimetic in vitro intestinal models are becoming useful tools for studying host-microbial interactions. In the past, these models have typically been limited to simple cultures on 2-D scaffolds or Transwell inserts, but it is widely understood that epithelial cells cultured in 3-D environments exhibit different phenotypes that are more reflective of native tissue, and that different microbial species will preferentially adhere to select locations along the intestinal villi. We used a synthetic 3-D tissue scaffold with villous features that could support the coculture of epithelial cell types with select bacterial populations. Our end goal was to establish microbial niches along the crypt-villus axis in order to mimic the natural microenvironment of the small intestine, which could potentially provide new insights into microbe-induced intestinal disorders, as well as enabling targeted probiotic therapies. We recreated the surface topography of the small intestine by fabricating a biodegradable and biocompatible villous scaffold using poly lactic-glycolic acid to enable the culture of Caco-2 with differentiation along the crypt-villus axis in a similar manner to native intestines. This was then used as a platform to mimic the adhesion and invasion profiles of both Salmonella and Pseudomonas, and assess the therapeutic potential of Lactobacillus and commensal Escherichia coli in a 3-D setting. We found that, in a 3-D environment, Lactobacillus is more successful at displacing pathogens, whereas Nissle is more effective at inhibiting pathogen adhesion.

The present paper is focused on a study regarding the possibility of obtaining hydroxyapatite-silver nanoparticle coatings on porous polyurethane scaffold. The method applied is based on a combined strategy involving hydroxyapatite biomimetic deposition on polyurethane surface using a Supersaturated Calcification Solution (SCS), combined with silver ions reduction and in-situ crystallization processes on hydroxyapatite-polyurethane surface by sample immersing in AgNO3 solution. The morphology, composition and phase structure of the prepared samples were characterized by scanning electron microscopy coupled with energy dispersive X-ray spectroscopy (SEM-EDX), X-ray diffraction (XRD), UV-Vis spectroscopy and X-ray photoelectron spectroscopy (XPS) measurements. The data obtained show that a layer of hydroxyapatite was deposited on porous polyurethane support and the silver nanoparticles (average size 34.71 nm) were dispersed among and even on the hydroxyapatite crystals. Hydroxyapatite/polyurethane surface acts as a reducer and a stabilizing agent for silver ions. The surface plasmon resonance peak in UV-Vis absorption spectra showed an absorption maximum at 415 nm, indicating formation of silver nanoparticles. The hydroxyapatite-silver polyurethane scaffolds were tested against Staphylococcus aureus and Escherichia coli and the obtained data were indicative of good antibacterial properties of the materials., (© 2013.)

Infectious complications of open fractures continue to be a significant factor contributing to non-osseous union and extremity amputation. The persistence of bacteria within biofilms despite meticulous debridement and antibiotic therapy is believed to be a major cause of chronic infection. Considering the difficulties in treating biofilm-associated infections, the use of biofilm dispersal agents as a therapeutic strategy for the prevention of biofilm-associated infections has gained considerable interest. In this study, we investigated whether local delivery of D-Amino Acids (D-AAs), a biofilm dispersal agent, protects scaffolds from contamination and reduces microbial burden within contaminated rat segmental defects in vivo. In vitro testing on biofilms of clinical isolates of Staphylococcus aureus demonstrated that D-Met, D-Phe, D-Pro, and D-Trp were highly effective at dispersing and preventing biofilm formation individually, and the effect was enhanced for an equimolar mixture of D-AAs. Incorporation of D-AAs into polyurethane scaffolds as a mixture (1:1:1 D-Met:D-Pro:D-Trp) significantly reduced bacterial contamination on the scaffold surface in vitro and within bone when implanted into contaminated femoral segmental defects. Our results underscore the potential of local delivery of d-AAs for reducing bacterial contamination by targeting bacteria within biofilms, which may represent a treatment strategy for improving healing outcomes associated with open fractures., (Published by Elsevier Ltd.)

The hierarchical electrostatic deposition of anionic microgels and a cationic oligopeptide throughout a PCL-chitosan nanofiber scaffold inhibits S. epidermidis colonization of the scaffold interior while promoting osteoblast adhesion, spreading, and proliferation on the scaffold surface., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The ex vivo cultured limbal stem cells over a biocompatible scaffold are used in the management of limbal stem cell deficiency as an ideal replacement for human amniotic membrane (HAM). A novel source of collagen from fish scales (FSC) was used to fabricate the scaffold. In this study, we have evaluated the physicochemical, mechanical, and culture characteristics of FSC and compared with denuded HAM. The cultured corneal cells were characterized by real-time polymerase chain reaction for putative stem cell markers. The swelling ratio, collagenase assay, and microbial resistance of FSC gave better results when compared to those of HAM. The mechanical and physical strengths of FSC were good enough to handle when compared to HAM. Under microscopic observation, epithelial migration was noted at the end of 48 h from limbal explants plated on FSC and on HAM at the end of 72 h. By the end of the 15th day, 90 to 100% confluent growth was seen resembling the morphological features of limbal epithelium. In conclusion, FSCs from a novel renewable biological source were optically clear with sufficient strength, and gave encouraging results in culture studies; the same may be tried as potential candidate for corneal transplantation after in vivo studies., (© 2012, Copyright the Authors. Artificial Organs © 2012, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.)

Background: Bacterial inclusion bodies (IBs), mechanically stable, submicron protein particles of 50-500 nm dramatically favor mammalian cell spread when used for substrate surface decoration. The mechanisms supporting fast colonization of IB-modified surfaces have not yet been identified., Results: This study provides evidence of mechanotransduction-mediated stimulation of mammalian cell proliferation on IB-decorated surfaces, as observed by the enhanced phosphorylation of the signal-regulated protein kinase and by the dramatic emission of filopodia in the presence of IBs. Interestingly, the results also show that IBs are highly bioadhesive materials, and that mammalian cell expansion on IBs is synergistically supported by both enhanced adhesion and mechanical stimulation of cell division., Discussion: The extent in which these events influence cell growth depends on the particular cell line response but it is also determined by the genetic background of the IB-producing bacteria, thus opening exciting possibilities for the fine tailoring of protein nanoparticle features that are relevant in tissue engineering.

Decellularization and cellularization of organs have emerged as disruptive methods in tissue engineering and regenerative medicine. Porous hydrogel scaffolds have widespread applications in tissue engineering, regenerative medicine and drug discovery as viable tissue mimics. However, the existing hydrogel fabrication techniques suffer from limited control over pore interconnectivity, density and size, which leads to inefficient nutrient and oxygen transport to cells embedded in the scaffolds. Here, we demonstrated an innovative approach to develop a new platform for tissue engineered constructs using live bacteria as sacrificial porogens. E.coli were patterned and cultured in an interconnected three-dimensional (3D) hydrogel network. The growing bacteria created interconnected micropores and microchannels. Then, the scafold was decellularized, and bacteria were eliminated from the scaffold through lysing and washing steps. This 3D porous network method combined with bioprinting has the potential to be broadly applicable and compatible with tissue specific applications allowing seeding of stem cells and other cell types.

Pulsed electric field (PEF) treatment has been proposed as a decontamination method for labile matrices used in tissue engineering applications. Through the application of PEF, a non-thermal treatment that causes bacterial inactivation through the irreversible rupture of microbial cell membranes, inactivation is achieved without loss of scaffold structure and function. However, some microorganisms are less susceptible to PEF treatment. This study shows that treatment with PEF and nisin, a food preservative bacteriocin, has a synergistic effect on the inactivation of Staphylococcus epidermidis in collagen gels. Almost complete inactivation of a 10(3) -10(4) CFU/mL S. epidermidis population was achieved when treated with a combination of PEF and 500 IU/mL nisin, with results demonstrating a 3.4 log(10) reduction, compared with 0.66 log(10) reduction with PEF alone. Nisin, at concentrations up to 3000 IU/mL, had no discernable toxicity to mammalian 3T3 cells when added to the culture medium or incorporated into the collagen gels. This combined decontamination method, involving PEF plus nisin, may provide a non-destructive process for inactivation of PEF-resistant bacteria in labile tissue engineering scaffolds., (Copyright © 2010 Wiley Periodicals, Inc.)

In this study, silver-loaded coral hydroxyapatites (SLCHAs) were used as scaffolds for bone tissue engineering. The SLCHAs were prepared by surface adsorption process and ion-exchange reaction between Ca(2+) of coral hydroxyapatite (CHA) and Ag(+) of silver nitrate with different concentrations at room temperature. The properties of the composite SLCHAs were investigated by inductively coupled plasma-atomic emission spectrometry (ICP-AES), scanning electron microscropy (SEM) equipped with backscattered electron detector (BSE), and energy-dispersive X-ray spectrometer (EDS). The SEM images showed that the morphology of the SLCHAs depended on the content of Ag(+), and the silver ions were uniformly distributed on the surface of SLCHAs. The ICP-AES results demonstrated that the silver content of the SLCHAs decreased along with the decrease of the concentration of silver nitrate. The SLCHAs were found effective against Escherichia coli and Staphylococcus aureus by antibacterial test. Mouse embryonic pre-osteoblast cells (MC3T3-E1) were used to test the cytocompatibility of SLCHAs, CHA, and pure coral. Cell morphology and cell proliferation were studied with SEM, laser scanning confocal microscope (LSCM), and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay after 1, 3, and 5 days of culture. The results indicated the cell morphology and proliferation on the scaffolds of Ag(+) (13.6 microg/ml)/CHA and Ag(+) (1.7 microg/ml)/CHA were better than that on Ag(+) (170 microg/ml)/CHA. In addition, adhesion of MC3T3-E1 on the scaffolds showed that the confluent cells showed fusiform shape and arranged tightly on the scaffolds. All the results showed that the antibacterial SLCHAs would have potential clinical application as the scaffolds for bone tissue engineering.

Hybrid scaffolds for tissue engineering are becoming increasingly complex through incorporation of biologically active biomacromolecules. There is a need to develop a compatible sterilization method that is capable of killing microorganisms, without adversely affecting the labile scaffold biomaterials or biomacromolecular components. Pulsed electric field (PEF) treatment has been successful as a nonthermal microbial inactivation-pasteurization method within the food industry. We have previously demonstrated that PEF treatment inactivates E. coli seeded in collagen gels. Here, we show that PEF treatment does not affect the structural integrity of the collagen molecule or its adsorption to polystyrene and hydroxyapatite surfaces. Moreover, osteoblast cells cultured on PEF-treated collagen, which was coated onto two- and three-dimensional scaffolds, retained their normal morphology, growth rate, and functionality. PEF treatment, therefore, shows great potential to be used as a sterilization method for collagen-based biomaterials.

Aims: To investigate the effectiveness of pulsed electric field (PEF) treatment as a new method for inactivation of micro-organisms in complex biomatrices and to assess this by quantifying the inactivation of Escherichia coli seeded in collagen gels., Methods and Results: PEF was applied to E. coli seeded collagen gels in static (nonflowing) chambers. The influence of electric field strength, pulse number and seeded cell densities were investigated. The highest level of inactivation was obtained at the maximum field strength of 45 kV cm(-1). For low levels of E. coli contamination (10(3) CFU ml(-1)), PEF treatment resulted in no viable E. coli being recovered from the gels. However, PEF treatment of gels containing higher cell densities (>or=10(4) CFU ml(-1)) did not achieve complete inactivation of E. coli., Conclusions: PEF treatment successfully inactivated E. coli seeded in collagen gels by 3 log(10) CFU ml(-1). Complete inactivation was hindered at high cell densities by the tailing effect observed., Significance and Impact of the Study: PEF shows potential as a novel, nondestructive method for decontamination of collagen-based matrices. Further investigation is required to ensure its compatibility with other proteins and therapeutic drugs for tissue engineering and drug delivery applications.

Chitosan is a well sought-after polysaccharide in biomedical applications due to its biocompatibility, biodegradability to non-toxic substances, and ease of fabrication into various configurations. However, alterations in the anti-bacterial properties of chitosan in various forms is not completely understood. The objective of this study was to evaluate the anti-bacterial properties of chitosan matrices in different configurations against two pathogens-Gram-positive Streptococcus mutans and Gram-negative Actinobacillus actinomycetemcomitans. Two-dimensional (2-D) membranes and three-dimensional (3-D) porous scaffolds were synthesized by air drying and controlled-rate freeze drying. Matrices were suspended in bacterial broths with or without lysozyme (enzyme that degrades chitosan). Influences of pore size, blending with Polycaprolactone (PCL, a synthetic polymer), and neutralization process on bacterial proliferation were studied. Transient changes in optical density of the broth, adhesion characteristics, viability, and contact-dependent bacterial activity were assessed. 3-D porous scaffolds were more effective in reducing the proliferation of S. mutans in suspension than 2-D membranes. However, no significant differences were observed on the proliferation of A. actinomycetemcomitans. Presence of lysozyme significantly increased the antibacterial activity of chitosan against A. actinomycetemcomitans. Pore size did not affect the proliferation kinetics of either species, with or without lysozyme. NaOH neutralization of chitosan increased bacterial adhesion whereas ethanol neutralization inhibited adhesion without lowering proliferation. Mat culture tests indicated that chitosan does not allow proliferation on its surface and it loses antibacterial activity upon blending with PCL. Results suggest that the chemical and structural characteristics of chitosan-based matrices can be manipulated to influence the interaction of different bacterial species.