Antimicrobial effect of emulsion-encapsulated isoeugenol against biofilms of food pathogens and spoilage bacteria.

Food-related biofilms can cause food-borne illnesses and spoilage, both of which are problems on a global level. Essential oils are compounds derived from plant material that have a potential to be used in natural food preservation in the future since they are natural antimicrobials. Bacterial biofilms are particularly resilient towards biocides, and preservatives that effectively eradicate biofilms are therefore needed. In this study, we test the antibacterial properties of emulsion-encapsulated and unencapsulated isoeugenol against biofilms of Lis. monocytogenes, S. aureus, P. fluorescens and Leu. mesenteroides in tryptic soy broth and carrot juice. We show that emulsion encapsulation enhances the antimicrobial properties of isoeugenol against biofilms in media but not in carrot juice. Some of the isoeugenol emulsions were coated with chitosan, and treatment of biofilms with these emulsions disrupted the biofilm structure. Furthermore, we show that addition of the surfactant Tween 80, which is commonly used to disperse oils in food, hampers the antibacterial properties of isoeugenol. This finding highlights that common food additives, such as surfactants, may have an adverse effect on the antibacterial activity of preservatives. Isoeugenol is a promising candidate as a future food preservative because it works almost equally well against planktonic bacteria and biofilms. Emulsion encapsulation has potential benefits for the efficacy of isoeugenol, but the effect of encapsulation depends on the properties of food matrix in which isoeugenol is to be applied.

Effects of Tween 80 on Growth and Biofilm Formation in Laboratory Media.

Tween 80 is a widely used non-ionic emulsifier that is added to cosmetics, pharmaceuticals, and foods. Because of its widespread use we need to understand how it affects bacteria on our skin, in our gut, and in food products. The aim of this study is to investigate how Tween 80 affects the growth and antimicrobial susceptibility of Staphylococcus aureus, Listeria monocytogenes, and Pseudomonas fluorescens, which are common causes of spoilage and foodborne illnesses. Addition of 0.1% Tween 80 to laboratory growth media increased the growth rate of planktonic S. aureus batch cultures, and it also increased the total biomass when S. aureus was grown as biofilms. In contrast, Tween 80 had no effect on batch cultures of L. monocytogenes, it slowed the growth rate of P. fluorescens, and it led to formation of less biofilm by both L. monocytogenes and P. fluorescens. Furthermore, Tween 80 lowered the antibacterial efficacy of two hydrophobic antimicrobials: rifampicin and the essential oil isoeugenol. Our findings underline the importance of documenting indirect effects of emulsifiers when studying the efficacy of hydrophobic antimicrobials that are dispersed in solution by emulsification, or when antimicrobials are applied in food matrixes that include emulsifiers. Furthermore, the species-specific effects on microbial growth suggests that Tween 80 in cosmetics and food products could affect the composition of skin and gut microbiota, and the effect of emulsifiers on the human microbiome should therefore be explored to uncover potential health effects.

Enhancing the antibacterial efficacy of isoeugenol by emulsion encapsulation.

Food spoilage and foodborne illnesses are two global challenges for food manufacturers. Essential oils are natural antibacterials that could have a potential for use in food preservation. Unfortunately high concentrations are needed to obtain the desired antibacterial effect, and this limits their use in food due to their adverse organoleptic properties. Encapsulation could make essential oils more effective by concentrating them in the aqueous phase of the food matrix where the bacteria are present. Here we tested encapsulation of the essential oil isoeugenol in spray-dried emulsions as a means of making isoeugenol a more effective antibacterial for use in food preservation. We used ß-lactoglobulin and n-OSA starch as emulsifiers, and some emulsions were coated with positively charged chitosan to promote the contact with bacteria through electrostatic interactions. The antibacterial efficacy was quantified as the minimal bactericidal concentration in growth media, milk and carrot juice. The emulsion encapsulation system developed in this study provided high loading capacities, and encapsulation enhanced the efficacy of isoeugenol against Gram-positive and -negative bacteria in media and carrot juice but not in milk. Chitosan-coating did not enhance the efficacy further, possibly due to the aggregation of the chitosan-coated emulsions. The encapsulation system is easy to upscale and should be applicable for encapsulation of similar essential oils. Therefore, we believe it has potential to be used for natural food preservation.

Co-culture-inducible bacteriocin production in lactic acid bacteria.

It is common knowledge that microorganisms have capabilities, like the production of antimicrobial compounds, which do not normally appear in ideal laboratory conditions. Common antimicrobial discovery techniques require the isolation of monocultures and their individual screening against target microorganisms. One strategy to achieve expression of otherwise hidden antimicrobials is induction by co-cultures. In the area of bacteriocin-producing lactic acid bacteria, there has been some research focusing into the characteristics of co-culture-inducible bacteriocin production and particularly the molecular mechanism(s) of such interactions. No clear relationship has been seen between bacteriocin-inducing and bacteriocin-producing microorganisms. The three-component regulatory system seems to be playing a central role in the induction, but inducing compounds have not been identified or characterized. However, the presence of the universal messenger molecule autoinducer-2 has been associated in some cases with the co-culture-inducible bacteriocin phenotype and it may play the role in the additional regulation of the three-component regulatory system. Understanding the mechanisms of induction would facilitate the development of strategies for screening and development of co-culture bacteriocin-producing systems and novel products as well as the perseverance of such systems in food and down to the intestinal tract, possibly conferring a probiotic effect on the host.

Binary combination of epsilon-poly-L-lysine and isoeugenol affect progression of spoilage microbiota in fresh turkey meat, and delay onset of spoilage in Pseudomonas putida challenged meat.

Proliferation of microbial population on fresh poultry meat over time elicits spoilage when reaching unacceptable levels, during which process slime production, microorganism colony formation, negative organoleptic impact and meat structure change are observed. Spoilage organisms in raw meat, especially Gram-negative bacteria can be difficult to combat due to their cell wall composition. In this study, the natural antimicrobial agents ε-poly-L-lysine (ε-PL) and isoeugenol were tested individually and in combinations for their activities against a selection of Gram-negative strains in vitro. All combinations resulted in additive interactions between ε-PL and isoeugenol towards the bacteria tested. The killing efficiency of different ratios of the two antimicrobial agents was further evaluated in vitro against Pseudomonas putida. Subsequently, the most efficient ratio was applied to a raw turkey meat model system which was incubated for 96 h at spoilage temperature. Half of the samples were challenged with P. putida, and the bacterial load and microbial community composition was followed over time. CFU counts revealed that the antimicrobial blend was able to lower the amount of viable Pseudomonas spp. by one log compared to untreated samples of challenged turkey meat, while the single compounds had no effect on the population. However, the compounds had no effect on Pseudomonas spp. CFU in unchallenged meat. Next-generation sequencing offered culture-independent insight into population diversity and changes in microbial composition of the meat during spoilage and in response to antimicrobial treatment. Spoilage of unchallenged turkey meat resulted in decreasing species diversity over time, regardless of whether the samples received antimicrobial treatment. The microbiota composition of untreated unchallenged meat progressed from a Pseudomonas spp. to a Pseudomonas spp., Photobacterium spp., and Brochothrix thermosphacta dominated food matrix on the expense of low abundance species. We observed a similar shift among the dominant species in meat treated with ε-PL or the antimicrobial blend, but the samples differed markedly in the composition of less abundant species. In contrast, the overall species diversity was constant during incubation of turkey meat challenged with P. putida although the microbiota composition did change over time. Untreated or ε-PL treated samples progressed from a Pseudomonas spp. to a Pseudomonas spp. and Enterobacteriaceae dominated food matrix, while treatment with the antimicrobial blend resulted in increased relative abundance of Hafnia spp., Enterococcaceae, and Photobacterium spp. We conclude that the blend delayed the onset of spoilage of challenged meat, and that all antimicrobial treatments of unchallenged or challenged meat affect the progression of the microbial community composition. Our study confirms that the antimicrobial effects observed in vitro can be extrapolated to a food matrix such as turkey meat. However, it also underlines the consequence of species-to-species variation in susceptibility to antimicrobials, namely that the microbial community change while the CFU remains the same. Addition of antimicrobials may thus prevent the growth of some microorganisms, allowing others to proliferate in their place.

Isoeugenol has a non-disruptive detergent-like mechanism of action.

Isoeugenol is an essential oil constituent of nutmeg, clove, and cinnamon. Despite isoeugenol's promising antimicrobial activity, no studies have yet investigated its mode of antibacterial action at the molecular level. The aim of this study is to clarify isoeugenol's antibacterial mode of action using the Gram-negative and Gram-positive model organisms Escherichia coli and Listeria innocua, respectively. We determined the antimicrobial activity of isoeugenol against the model organisms, and examined how isoeugenol affects cell morphology, cell membrane permeabilization, and how isoeugenol interacts with phospholipid membranes using vesicle and supported lipid bilayer models. Isoeugenol demonstrated a bactericidal activity against E. coli and L. innocua that did not affect cell morphology, although the cell membrane was permeabilized. We hypothesized that the cell membrane was the primary site of action, and studied this interaction in further detail using purified membrane model systems. Isoeugenol's permeabilization of calcein-encapsulated vesicles was concentration dependent, and isoeugenol's interaction with giant unilamellar vesicles indicated increased membrane fluidity and a non-disruptive permeabilization mechanism. This contradicted membrane fluidity measurements on supported lipid bilayers (SLBs), which indicated decreased membrane fluidity. However, further investigations demonstrated that the interaction between isoeugenol and bilayers was reversible, and caused membranes to display heterogeneous topography, an increased mass, and a higher degree of hydration. In conclusion, we propose that isoeugenol interacts with membranes in a reversible non-disruptive detergent-like manner, which causes membrane destabilization. Furthermore, we argue that isoeugenol increases membrane fluidity. Our work contributes to the understanding of how essential oil constituents interact with cell components.

The antimicrobial mechanism of action of epsilon-poly-l-lysine.

Epsilon-poly-l-lysine (ε-PL) is a natural antimicrobial cationic peptide which is generally regarded as safe (GRAS) as a food preservative. Although its antimicrobial activity is well documented, its mechanism of action is only vaguely described. The aim of this study was to clarify ε-PL's mechanism of action using Escherichia coli and Listeria innocua as model organisms. We examined ε-PL's effect on cell morphology and membrane integrity and used an array of E. coli deletion mutants to study how specific outer membrane components affected the action of ε-PL. We furthermore studied its interaction with lipid bilayers using membrane models. In vitro cell studies indicated that divalent cations and the heptose I and II phosphate groups in the lipopolysaccharide layer of E. coli are critical for ε-PL's binding efficiency. ε-PL removed the lipopolysaccharide layer and affected cell morphology of E. coli, while L. innocua underwent minor morphological changes. Propidium iodide staining showed that ε-PL permeabilized the cytoplasmic membrane in both species, indicating the membrane as the site of attack. We compared the interaction with neutral or negatively charged membrane systems and showed that the interaction with ε-PL relied on negative charges on the membrane. Suspended membrane vesicles were disrupted by ε-PL, and a detergent-like disruption of E. coli membrane was confirmed by atomic force microscopy imaging of supported lipid bilayers. We hypothesize that ε-PL destabilizes membranes in a carpet-like mechanism by interacting with negatively charged phospholipid head groups, which displace divalent cations and enforce a negative curvature folding on membranes that leads to formation of vesicles/micelles.

Essential oils in food preservation: mode of action, synergies, and interactions with food matrix components.

Essential oils are aromatic and volatile liquids extracted from plants. The chemicals in essential oils are secondary metabolites, which play an important role in plant defense as they often possess antimicrobial properties. The interest in essential oils and their application in food preservation has been amplified in recent years by an increasingly negative consumer perception of synthetic preservatives. Furthermore, food-borne diseases are a growing public health problem worldwide, calling for more effective preservation strategies. The antibacterial properties of essential oils and their constituents have been documented extensively. Pioneering work has also elucidated the mode of action of a few essential oil constituents, but detailed knowledge about most of the compounds' mode of action is still lacking. This knowledge is particularly important to predict their effect on different microorganisms, how they interact with food matrix components, and how they work in combination with other antimicrobial compounds. The main obstacle for using essential oil constituents as food preservatives is that they are most often not potent enough as single components, and they cause negative organoleptic effects when added in sufficient amounts to provide an antimicrobial effect. Exploiting synergies between several compounds has been suggested as a solution to this problem. However, little is known about which interactions lead to synergistic, additive, or antagonistic effects. Such knowledge could contribute to design of new and more potent antimicrobial blends, and to understand the interplay between the constituents of crude essential oils. The purpose of this review is to provide an overview of current knowledge about the antibacterial properties and antibacterial mode of action of essential oils and their constituents, and to identify research avenues that can facilitate implementation of essential oils as natural preservatives in foods.

Antimicrobial mechanism of monocaprylate.

Monoglyceride esters of fatty acids occur naturally and encompass a broad spectrum of antimicrobial activity. Monocaprylate is generally regarded as safe (GRAS) and can function both as an emulsifier and as a preservative in food. However, knowledge about its mode of action is lacking. The aim of this study was therefore to elucidate the mechanism behind monocaprylate's antimicrobial effect. The cause of cell death in Escherichia coli, Staphylococcus xylosus, and Zygosaccharomyces bailii was investigated by examining monocaprylate's effect on cell structure, membrane integrity, and its interaction with model membranes. Changes in cell structure were visible by atomic force microscopy (AFM), and propidium iodide staining showed membrane disruption, indicating the membrane as a site of action. This indication was confirmed by measuring calcein leakage from membrane vesicles exposed to monocaprylate. AFM imaging of supported lipid bilayers visualized the integration of monocaprylate into the liquid disordered, and not the solid ordered, phase of the membrane. The integration of monocaprylate was confirmed by quartz crystal microbalance measurements, showing an abrupt increase in mass and hydration of the membrane after exposure to monocaprylate above a threshold concentration. We hypothesize that monocaprylate destabilizes membranes by increasing membrane fluidity and the number of phase boundary defects. The sensitivity of cells to monocaprylate will therefore depend on the lipid composition, fluidity, and curvature of the membrane.

Flow perfusion culture of human mesenchymal stem cells on coralline hydroxyapatite scaffolds with various pore sizes.

Bone grafts are widely used in orthopaedic reconstructive surgery, but harvesting of autologous grafts is limited due to donor site complications. Bone tissue engineering is a possible alternative source for substitutes, and to date, mainly small scaffold sizes have been evaluated. The aim of this study was to obtain a clinically relevant substitute size using a direct perfusion culture system. Human bone marrowderived mesenchymal stem cells were seeded on coralline hydroxyapatite scaffolds with 200 µm or 500 µm pores, and resulting constructs were cultured in a perfusion bioreactor or in static culture for up to 21 days and analysed for cell distribution and osteogenic differentiation using histological stainings, alkaline phosphatase activity assay, and real-time RT-PCR on bone markers. We found that the number of cells was higher during static culture at most time points and that the final number of cells was higher in 500 µm constructs as compared with 200 µm constructs. Alkaline phosphatase enzyme activity assays and real time RT-PCR on seven osteogenic markers showed that differentiation occurred primarily and earlier in statically cultured constructs with 200 µm pores compared with 500 µm ones. Adhesion and proliferation of the cells was seen on both scaffold sizes, but the vitality and morphology of cells changed unfavorably during perfusion culture. In contrast to previous studies using spinner flask that show increased cellularity and osteogenic properties of cells when cultured dynamically, the perfusion culture in our study did not enhance the osteogenic properties of cell/scaffold constructs. The statically cultured constructs showed increasing cell numbers and abundant osteogenic differentiation probably because of weak initial cell adhesion due to the surface morphology of scaffolds. Our conclusion is that the specific scaffold surface microstructure and culturing system flow dynamics has a great impact on cell distribution and proliferation and on osteogenic differentiation, and the data presented warrant careful selection of in vitro culture settings to meet the specific requirements of the scaffolds and cells, especially when natural biomaterials with varying morphology are used.

Effect of dynamic 3-D culture on proliferation, distribution, and osteogenic differentiation of human mesenchymal stem cells.

Ex vivo engineering of autologous bone tissue as an alternative to bone grafting is a major clinical need. In the present study, we evaluated the effect of 3-D dynamic spinner flask culture on the proliferation, distribution, and differentiation of human mesenchymal stem cells (MSCs). Immortalized human MSCs were cultured on porous 75:25 PLGA scaffolds for up to 3 weeks. Dynamically cultured cell/scaffold constructs demonstrated a 20% increase in DNA content (21 days), enhanced ALP specific activity (7 days and 21 days), a more than tenfold higher Ca2+ content (21 days), and significantly increased transcript levels of early osteogenesis markers (e.g., COL1A1, BMP2, RUNX-2) as compared with static culture. Despite the formation of a dense superficial cell layer, markedly increased cell ingrowth was observed by fluorescence microscopy on day 21. Furthermore, increased extracellular matrix deposition was visualized by scanning electron microscopy after 1 and 3 weeks of dynamic culture. The observed increased ingrowth and osteogenic differentiation of 3-D dynamically cultured human MSCs can be explained by generation of fluid shear stress and enhanced mass transport to the interior of the scaffold mimicking the native microenvironment of bone cells. This study provides evidence for the effectiveness of dynamic culture of human MSCs during the initial phase of ex vivo osteogenesis.

Flow perfusion culture of human mesenchymal stem cells on silicate-substituted tricalcium phosphate scaffolds.

Autologous bone grafts are currently the gold standard for treatment of large bone defects, but their availability is limited due to donor site morbidity. Different substitutes have been suggested to replace these grafts, and this study presents a bone tissue engineered alternative using silicate-substituted tricalcium phosphate (Si-TCP) scaffolds seeded with human bone marrow-derived mesenchymal stem cells (hMSC). The cells were seeded onto the scaffolds and cultured either statically or in a perfusion bioreactor for up to 21 days and assessed for osteogenic differentiation by alkaline phosphatase activity assays and by quantitative real-time RT-PCR on bone markers. During culture, cells from the flow cultured constructs demonstrated improved proliferation and osteogenic differentiation verified by a more pronounced expression of several bone markers, e.g. alkaline phosphatase, osteopontin, Runx2, bone sialoprotein II, and bone morphogenetic protein 2. Cells and matrix were distributed homogeneously throughout the entire scaffold in flow culture, whereas only a peripheral layer was obtained after static culture. A viable and homogenous ex vivo bone construct with superior osteogenic properties was produced in dynamic culture and may provide a replacement for autologous grafts.

Multilineage differentiation of porcine bone marrow stromal cells associated with specific gene expression pattern.

There are increasing reports regarding differentiation of bone marrow stromal cells (BMSC) from human and various species of animals including pigs. The phenotype and function of BMSC along a mesenchymal lineage differentiation are well characterized by specific transcription factors and marker genes. However, it is not fully clear whether multilineage differentiation (osteogenesis, chondrogenesis, and adipogenesis) of BMSC is associated with a specific gene expression pattern. In the present study, we investigated the gene expression pattern of representative transcription factors and marker genes along those three mesenchymal lineages during a particular lineage differentiation of porcine BMSC by means of real-time PCR measurement. In an osteogenic medium, the mRNA levels of cbfa1, osterix, alkaline phosphatase, type 1 collagen, osteonectin, bone sialoprotein, and osteocalcin were induced stepwise. Meanwhile, sox9, specific to chondrogenic differentiation, was inhibited but not PPARgamma2 specific to adipogenic differentiation. In an adipogenic medium, adipogenic differentiation was confirmed by upregulation of PPARgamma2 and aP2 and downregulation of osteogenic genes and sox9. Chondrogenic differentiation was induced in cell pellet culture by expression of sox9, type 2 collagen, and aggrecan. Cbfa1 and PPARgamma2 were inhibited in chondrogenic medium. These results indicate that the differentiation potential of BMSC to a particular mesenchymal lineage relies upon specific gene expression pattern, namely upregulation of genes specific for this lineage and suppression of other lineage differentiation.

Effect of hyaluronan on osteogenic differentiation of porcine bone marrow stromal cells in vitro.

Hyaluronan (HA) plays a predominant role in tissue morphogenesis, cell migration, proliferation, and cell differentiation. The aims of the present study were to investigate whether (i) prolonged presence of high concentration (4.0 mg/mL) 800 KDa HA and (ii) pretreatment with HA can modify osteogenic differentiation of pig bone marrow stromal cells (pBMSC). Cell proliferation and mineralization were measured. Expression of differentiation-related genes was evaluated by means of real-time reverse transcription polymerase chain reaction (RT-PCR). HA increased cell proliferation on day 7. HA decreased the basal level of bone-related gene expression and increased the basal level of sox9 marginally during 7-day pretreatment with HA. HA increased calcium deposit on day 21. cbfa1, ALP, and type 1 alpha collagen (Col1) expression was increased when pBMSC were cultivated in osteogenic medium, whereas their expression was decreased in the presence of HA on day 7. On day 14, the addition of HA upregulated cbfa1 and ALP expression compared to osteogenic medium group; there was no significant difference in Col1 expression. At day 21, osteocalcin (OC) expression showed 2.5-fold upregulation over osteogenic medium. These results suggest that exogenous HA stimulates endogenous HA, which together may play a synergetic role in osteogenic differentiation under osteoinducing conditions although gene expression was inhibited at the early stage.

Morphology, proliferation, and osteogenic differentiation of mesenchymal stem cells cultured on titanium, tantalum, and chromium surfaces.

Metallic implants are widely used in orthopedic surgery and dentistry. Durable osseous fixation of an implant requires that osteoprogenitor cells attach and adhere to the implant, proliferate, differentiate into osteoblasts, and produce mineralized matrix. In the present study, we investigated the interactions between human mesenchymal stem cells (MSCs) and smooth surfaces of titanium (Ti), tantalum (Ta), and chromium (Cr). Mean cellular area was quantified using fluorescence microscopy (4 h). Cellular proliferation was assessed by (3)H-thymidine incorporation and methylene blue cell counting assays (4 days). Osteogenic differentiation response was quantified by cell-specific alkaline phosphatase activity (ALP) assay (4 days), expression analysis of bone-related genes (4 days), and mineralization assay (21 days). Undifferentiated and osteogenically stimulated MSCs cultured on the different surfaces showed the same tendencies for proliferation and differentiation. MSCs exposed to Ti surfaces demonstrated enhanced proliferation compared with Ta and Cr surfaces. Cultivation of MSCs on Ta surfaces resulted in significantly increased mean cellular area and cell-specific ALP activity compared with the other surfaces tested. Cells cultured on Cr demonstrated reduced spreading and proliferation. In conclusion, Ta metal, as an alternative for Ti, can be considered as a promising biocompatible material, whereas further studies are needed to fully understand the role of Cr and its alloys in bone implants.

Cobalt-chromium-molybdenum alloy causes metal accumulation and metallothionein up-regulation in rat liver and kidney.

Cobalt-chromium-molybdenum (CoCrMo) metal-on-metal hip prosthesis has had a revival due to their excellent wear properties. However, particulate wear debris and metal ions liberated from the CoCrMo alloys might cause carcinogenicity, hypersensitivity, local and general tissue toxicity, genotoxicity and inflammation-generating qualities. Nine months after implanting small pieces of CoCrMo alloy intramuscularly and intraperitoneally in rats, we analysed the accumulation of metals with a multi-element analysis, and the levels of metallothionein I/II with real-time reverse transcriptase-polymerase chain reaction in liver and kidney. We found that metal ions are liberated from CoCrMo alloys and suggest that they are released by dissolucytosis, a process where macrophages causes the metallic surface to release metal ions. Animals with intramuscular implants accumulated metal in liver and kidney and metallohionein I/II were elevated in liver tissue. The present data do not tell whether kidney and liver are the primary target organs or what possible toxicological effect the different metal ions might have, but they show that metal ions are liberated from CoCrMo alloys that are not subjected to mechanical wear and that they accumulate in liver and kidney tissue. That the liberated metal ions affect the tissues is supported by an up-regulation of the detoxifying/pacifying metalloprotein I/II in the liver.

Mesenchymal stem cell ingrowth and differentiation on coralline hydroxyapatite scaffolds.

Culture of osteogenic cells on a porous scaffold could offer a new solution to bone grafting using autologous human mesenchymal stem cells (hMSC) from the patient. We compared coralline hydroxyapatite scaffolds with pore sizes of 200 and 500 microm for expansion and differentiation of hMSCs. We cultivated the hMSC statically or in spinner flasks for 1, 7, 14 and 21 days and found that the 200-microm pore scaffolds exhibited a faster rate of osteogenic differentiation than did the 500-microm pore scaffolds as shown by an alkaline phosphatase activity assay and real-time reverse transcriptase polymerase chain reaction for 10 osteogenic markers. The 500-microm scaffolds had increased proliferation rates and accommodated a higher number of cells (shown by DNA content, scanning electron microscopy and fluorescence microscopy). Thus the porosity of a 3D microporous biomaterial may be used to steer hMSC in a particular direction. We found that dynamic spinner flask cultivation of hMSC/scaffold constructs resulted in increased proliferation, differentiation and distribution of cells in scaffolds. Therefore, spinner flask cultivation is an easy-to-use inexpensive system for cultivating hMSCs on small to intermediate size 3D scaffolds.

Optimizing viral and non-viral gene transfer methods for genetic modification of porcine mesenchymal stem cells.

INTRODUCTION: Mesenchymal stem cells (MSCs) provide an excellent source of pluripotent progenitor cells for tissue-engineering applications due to their proliferation capacity and differentiation potential. Genetic modification of MSCs with genes encoding tissue-specific growth factors and cytokines can induce and maintain lineage-specific differentiation. Due to anatomical and physiological similarities to humans, porcine research models have been proven valuable for the preclinical testing of tissue engineering protocols in large animals. The aim of this study was to evaluate optimized viral and non-viral ex vivo gene delivery systems with respect to gene transfer efficiency, maintenance of transgene expression, and safety issues using primary porcine MSCs as target cells. MATERIALS AND METHODS: MSCs were purified from bone marrow aspirates from the proximal tibiae of four 3-month-old Danish landrace pigs by Ficoll step gradient separation and polystyrene adherence technique. Vectors expressing enhanced green fluorescent protein (eGFP) and human bone morphogenetic protein-2 (BMP-2) were transferred to the cells by different non-viral methods and by use of recombinant adeno-associated virus (rAAV)-mediated and retroviral gene delivery. Each method for gene delivery was optimized. Gene transfer efficiency was compared on the basis of eGFP expression as assessed by fluorescence microscopy and fluorescence-activated flow cytometry. BMP-2 gene expression and osteogenic differentiation were evaluated by realtime quantitative RT-PCR and histochemical detection of alkaline phosphatase activity, respectively. RESULTS: Non-viral gene delivery methods resulted in transient eGFP expression by less than 2% of the cells. Using high titer rAAV-based vector up to 90% of the cells were transiently transduced. The efficiency of rAAV-mediated gene delivery was proportional to the rAAV vector titer applied. Retroviral gene delivery resulted in long-term transgene expression of porcine MSCs. A 26-fold increase in percentage of eGFP expressing cells (1.7%+/-0.2% versus 44.1% +/-5.0%, mean +/-SD) and a 68-fold increase in mean fluorescence intensity (327.4+/-56.6 versus 4.8+/-1.3) was observed by centrifugation of retroviral particles onto the target cell layer. Porcine MSCs that were BMP-2 transduced by optimized retroviral gene delivery demonstrated a significant increase in BMP-2 gene expression and showed increased osteogenic differentiation. Retrovirally transduced porcine MSCs were furthermore tested free of replication-competent viruses. DISCUSSION: The non-viral gene transfer methods applied were significantly less efficient compared to the viral methods tested. However, due to advantages with respect to safety issues and ease of handling, improvement of non-viral gene delivery to primary MSCs deserves further attention. The high efficiency of rAAV-mediated gene delivery observed at high titers can be explained by the ability of rAAV vector to transduce nondividing cells and by its tropism towards porcine MSCs. rAAV-mediated gene delivery resulted in transient transgene expression due to lack of stable AAV genome integration. MLV-mediated retroviral gene delivery can be considered a safe method for long-term transgene expression by porcine MSCs, and is therefore particularly attractive for advanced tissue engineering strategies requiring extended transgene expression.

Porous tantalum trabecular metal scaffolds in combination with a novel marrow processing technique to replace autograft.

INTRODUCTION: Interbody fusion requires a structural member to carry load while the autograft or osteoinductive agent stimulates bone formation. In the present study, we evaluated the potential use of extracted nucleated cells from bone marrow mixed in hyaluronic acid gel as an osteoinductive agent, in comparison to Collagraft loaded with nucleated cells or rhBMP-2 in the porous tantalum ring, in an anterior lumbar interbody fusion (ALIF) in pigs. METHODS: Four 3-month-old female Danish landrace pigs were employed in the current study. Bone marrow was collected by means of aspiration, from the medullary cavity of the proximal tibia. The nucleated cells were isolated with a Ficoll step gradient centrifugation. The cell adhered rate after 24 hours of cultivation and ALP activity in the osteogenic medium following 4 days of cultivation were measured. Cell numbers in the porous tantalum discs were assessed by CyQuant measurement, and fluorescent live/dead cell staining in the porous tantalum discs was performed after periods of 24 hours and 7 days of cultivation. The nucleated cells mixed in hyaluronic acid gel were cultivated on slides for 24 hours, 7 days and 21 days. The live/dead cell staining, ALP staining or osteocalcin staining, were performed. A porous tantalum ring was loaded with nucleated cells in hyaluronic acid gel or packed with Collagraft strips also with nucleated cells or rhBMP-2. Immediately after preparation, one of three implants was inserted into L2-3, L4-5 or L6-7 respectively. The pigs were killed 3 months postoperatively. The lumbar spine specimens were prepared for histological evaluation. RESULTS: The adhered rate, of the nucleated cells, was 2.26+/-0.56%. ALP activity was no different in the osteogenic culture compared to DMEM/10%FBS alone. Cell numbers and live/dead cells showed no difference in the porous tantalum discs. Histological appearance showed that nucleated cells mixed with hyaluronic acid gel, had more mature bone in the central hole of the porous tantalum ring, compared to Collagraft strips with nucleated cells or rhBMP-2. Bone volume fraction did not differ within the three porous tantalum rings; however, more marrow space in the central hole of the porous tantalum ring was present when nucleated cells mixed with hyaluronic acid gel (57.4%) compared to Collagraft strips with rhBMP-2 (29.7%). CONCLUSION: In the current study, we demonstrate that nucleated cells, which were isolated from bone marrow intraoperatively, could be used to replace autograft if nucleated cells mixed with hyaluronic acid gel or with Collagraft strips packed into the porous tantalum ring in the pig ALIF model.

Molecular mechanism of osteochondroprogenitor fate determination during bone formation.

Osteoblasts and chondrocytes, which derive from a common mesenchymal precursor (osteochondroprogenitor), are involved in bone formation and remodeling in vivo. Determination of osteochondroprogenitor fate is under the control of complex hormonal and local factors converging onto a series of temporospatial dependent transcription regulators. Sox9, together with L-Sox5 and Sox6, of the Sox family is required for chondrogenic differentiation commitment, while Runx2/Cbfa 1, a member of runt family and Osterix/Osx, a novel zinc finger-containing transcription factor play a pivotal role in osteoblast differentiation decision and hypertrophic chondrocyte maturation. Recent in vitro and in vivo evidence suggests beta-catenin, a transcriptional activator in the canonical Wnt pathway, can act as a determinant factor for controlling chondrocyte and osteoblast differentiation. Here we focus on several intensively studied transcription factors and Wnt/beta-catenin signal molecules to illustrate the regulatory mechanism in directing commitment between osteoblast and chondrocyte, which will eventually allow us to properly manipulate the mesenchymal progenitor cell differentiation on bone and regeneration of cartilage tissue engineering.