In Vitro Assessment of Bacterial Adhesion and Biofilm Formation on Novel Bioactive, Biodegradable Electrospun Fiber Meshes Intended to Support Tendon Rupture Repair.

The surgical repair of a ruptured tendon faces two major problems: specifically increased fibrous adhesion to the surrounding tissue and inferior mechanical properties of the scar tissue compared to the native tissue. Bacterial attachment to implant materials is an additional problem as it might lead to severe infections and impaired recovery. To counteract adhesion formation, two novel implant materials were fabricated by electrospinning, namely, a random fiber mesh containing hyaluronic acid (HA) and poly(ethylene oxide) (PEO) in a ratio of 1:1 (HA/PEO 1:1) and 1:4 (HA/PEO 1:4), respectively. Electrospun DegraPol (DP) treated with silver nanoparticles (DP-Ag) was developed to counteract the bacterial attachment. The three novel materials were compared to the previously described DP and DP with incorporated insulin-like growth factor-1 (DP-IGF-1), two implant materials that were also designed to improve tendon repair. To test whether the materials are prone to bacterial adhesion and biofilm formation, we assessed 10 strains of Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Enterococcus faecalis, known for causing nosocomial infections. Fiber diameter, pore size, and water contact angle, reflecting different degrees of hydrophobicity, were used to characterize all materials. Generally, we observed higher biofilm formation on the more hydrophobic DP as compared to the more hydrophilic DP-IGF-1 and a trend toward reduced biofilm formation for DP treated with silver nanoparticles. For the two HA/PEO implants, a similar biofilm formation was observed. All tested materials were highly prone to bacterial adherence and biofilm formation, pointing toward the need of further material development, including the optimized incorporation of antibacterial agents such as silver nanoparticles or antibiotics.

Insulin-like growth factor-1 (IGF-1) poly (lactic-co-glycolic acid) (PLGA) microparticles - development, characterisation, and in vitro assessment of bioactivity for cardiac applications.

Aim: The aim of this study was to evaluate the formulation of a synthetic IGF-1 (pIGF-1) in PLGA microparticles (MP). Methods: Poly (lactic-co-glycolic acid) (PLGA) MPs loaded with pIGF-1 were prepared, characterised and evaluated using double emulsion solvent evaporation method. Results: Spherical MPs showed an average particle size of 2 µm, encapsulation efficiency (EE) of 67% and 50% degradation over 15 days. With a view to enhancing retention in the myocardium, the MP formulation was encapsulated in a cross-linked hyaluronic acid hydrogel. pIGF-1 released from MPs and from MPs suspended in hyaluronic acid hydrogel remained bioactive, determined by a significant increase in cellular proliferation of c-kit+ cells. Conclusion: This formulation has potential for loco-regional delivery to damaged myocardium to promote the survival of cardiomyocytes.

A novel in vitro model for the assessment of postnatal myonuclear accretion.

BACKGROUND: Due to the post-mitotic nature of myonuclei, postnatal myogenesis is essential for skeletal muscle growth, repair, and regeneration. This process is facilitated by satellite cells through proliferation, differentiation, and subsequent fusion with a pre-existing muscle fiber (i.e., myonuclear accretion). Current knowledge of myogenesis is primarily based on the in vitro formation of syncytia from myoblasts, which represents aspects of developmental myogenesis, but may incompletely portray postnatal myogenesis. Therefore, we aimed to develop an in vitro model that better reflects postnatal myogenesis, to study the cell intrinsic and extrinsic processes and signaling involved in the regulation of postnatal myogenesis. METHODS: Proliferating C2C12 myoblasts were trypsinized and co-cultured for 3 days with 5 days differentiated C2C12 myotubes. Postnatal myonuclear accretion was visually assessed by live cell time-lapse imaging and cell tracing by cell labeling with Vybrant® DiD and DiO. Furthermore, a Cre/LoxP-based cell system was developed to semi-quantitatively assess in vitro postnatal myonuclear accretion by the conditional expression of luciferase upon myoblast-myotube fusion. Luciferase activity was assessed luminometrically and corrected for total protein content. RESULTS: Live cell time-lapse imaging, staining-based cell tracing, and recombination-dependent luciferase activity, showed the occurrence of postnatal myonuclear accretion in vitro. Treatment of co-cultures with the myogenic factor IGF-I (p < 0.001) and the cytokines IL-13 (p < 0.05) and IL-4 (p < 0.001) increased postnatal myonuclear accretion, while the myogenic inhibitors cytochalasin D (p < 0.001), myostatin (p < 0.05), and TNFα (p < 0.001) decreased postnatal myonuclear accretion. Furthermore, postnatal myonuclear accretion was increased upon recovery from electrical pulse stimulation-induced fiber damage (p < 0.001) and LY29004-induced atrophy (p < 0.001). Moreover, cell type-specific siRNA-mediated knockdown of myomaker in myoblasts (p < 0.001), but not in myotubes, decreased postnatal myonuclear accretion. CONCLUSIONS: We developed a physiologically relevant, sensitive, high-throughput cell system for semi-quantitative assessment of in vitro postnatal myonuclear accretion, which can be used to mimic physiological myogenesis triggers, and can distinguish the cell type-specific roles of signals and responses in the regulation of postnatal myogenesis. As such, this method is suitable for both basal and translational research on the regulation of postnatal myogenesis, and will improve our understanding of muscle pathologies that result from impaired satellite cell number or function.

A novel and simplified method of culture of human blood-derived early endothelial progenitor cells for the treatment of ischemic vascular disease.

Endothelial progenitor cells (EPCs) consist of two different subpopulations named early (eEPCs) and late EPCs (lEPCs) that are derived from CD14(+) and CD14(-) circulating cells, respectively. These cells are regularly cultured over fibronectin-coated surfaces in endothelial basal medium (EBM)-2 supplemented with insulin-like growth factor (IGF-1), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF). We have developed a new and simplified method for culturing human EPCs obtained from peripheral blood and tested their ability to preserve cardiac function following infarction. We first demonstrated that eEPCs derived from human peripheral blood mononuclear cells (PBMCs) and cultured in EBM-2 medium supplemented with autologous serum (10%) over fibronectin-coated surfaces (10 µg/ml) in the presence of IGF-1 (50 ng/ml) only, have a secretome similar to eEPCs cultured under regular conditions with IGF-1, VEGF, EGF, and FGF. Our data also indicate that IGF-1 modulates PBMC secretome in a dose-dependent manner. In another series of experiments, we showed that PBMCs cultured in suspension in bags (S-PBMCs) in basal medium supplemented with fibronectin and IGF-1 secrete significant amounts of stem cell factor (SCF, 31.3 ± 3.1 pg/ml)), hepatocyte growth factor (HGF, 438.6 ± 41.4 pg/ml), soluble tumor necrosis factor receptor 1 (sTNFR1, 127.1 ± 9.9 pg/ml), VEGF (139.3 ± 9.6 pg/ml), and IGF-1 (147.2 ± 46.1 pg/ml) but very low levels of TNF-α (13.4 ± 2.5 pg/ml). S-PBMCs injected intravenously into NOD SCID mice migrated to the injured myocardium, reduced cardiac fibrosis, enhanced angiogenesis, and preserved cardiac function after myocardial infarction (MI) in a manner similar to eEPCs cultured under standard conditions. In conclusion, we show in this study a refined and optimized method for culturing eEPCs. Our data indicate that S-PBMCs are composed of several cell populations including eEPCs and that they secrete high amounts of antiapoptotic, anti-inflammatory, and proangiogenic factors capable of preserving cardiac function following MI.

Assessment of growth factor treatment on fibrochondrocyte and chondrocyte co-cultures for TMJ fibrocartilage engineering.

Treatments for patients suffering from severe temporomandibular joint (TMJ) dysfunction are limited, motivating the development of strategies for tissue regeneration. In this study, co-cultures of fibrochondrocytes (FCs) and articular chondrocytes (ACs) were seeded in agarose wells, and supplemented with growth factors, to engineer tissue with biomechanical properties and extracellular matrix composition similar to native TMJ fibrocartilage. In the first phase, growth factors were applied alone and in combination, in the presence or absence of serum, while in the second phase, the best overall treatment was applied at intermittent dosing. Continuous treatment of AC/FC co-cultures with TGF-ß1 in serum-free medium resulted in constructs with glycosaminoglycan/wet weight ratios (12.2%), instantaneous compressive moduli (790 kPa), relaxed compressive moduli (120 kPa) and Young's moduli (1.87 MPa) that overlap with native TMJ disc values. Among co-culture groups, TGF-ß1 treatment increased collagen deposition ∼20%, compressive stiffness ∼130% and Young's modulus ∼170% relative to controls without growth factor. Serum supplementation, though generally detrimental to functional properties, was identified as a powerful mediator of FC construct morphology. Finally, both intermittent and continuous TGF-ß1 treatment showed positive effects, though continuous treatment resulted in greater enhancement of construct functional properties. This work proposes a strategy for regeneration of TMJ fibrocartilage and its future application will be realized through translation of these findings to clinically viable cell sources.

An in vivo and in vitro assessment of autophagy-related gene expression in muscle of rainbow trout (Oncorhynchus mykiss).

In mammals, new evidence has demonstrated the important role of the autophagic/lysosomal pathway in regulating muscle mass and identified the transcription factor FoxO3 as a key factor of the control of this proteolytic system by inducing several autophagy-related genes. In contrast, the mechanisms responsible for the regulation of autophagy have not been investigated in teleosts, known to exhibit different muscle growth dynamics. The present work aimed to characterize both in vivo and in vitro the transcriptional regulation of several major genes involved in autophagy (LC3B, gabarapl1, atg12l, atg4b) in the white skeletal muscle of rainbow trout. We found that fasting fish for 14days or serum depletion of trout myocytes strongly induces the expression of all studied genes. Our in vitro study on trout myocytes indicated that IGF1 induces FoxO3 phosphorylation but has a low or no effect on autophagy-related gene expression, suggesting a moderate role for this transcription factor on the autophagic/lysosomal pathway in this species. Data reported here show for the first time in a lower vertebrate, the existence and the regulation of several major genes involved in the autophagy, opening a new area of research on the molecular bases of muscle protein degradation in teleosts.

Systematic assessment of growth factor treatment on biochemical and biomechanical properties of engineered articular cartilage constructs.

OBJECTIVE: To determine the effects of bone morphogenetic protein-2 (BMP-2), insulin-like growth factor (IGF-I), and transforming growth factor-beta1 (TGF-beta1) on the biochemical and biomechanical properties of engineered articular cartilage constructs under serum-free conditions. METHODS: A scaffoldless approach for tissue engineering, the self-assembly process, was employed. The study consisted of two phases. In the first phase, the effects of BMP-2, IGF-I, and TGF-beta1, at two concentrations and two dosage frequencies each were assessed on construct biochemical and biomechanical properties. In phase II, the effects of growth factor combination treatments were determined. Compressive and tensile mechanical properties, glycosaminoglycan (GAG) and collagen content, histology for GAG and collagen, and immunohistochemistry (IHC) for collagen types I and II were assessed. RESULTS: In phase I, BMP-2 and IGF-I treatment resulted in significant, >1-fold increases in aggregate modulus, accompanied by increases in GAG production. Additionally, TGF-beta1 treatment resulted in significant, approximately 1-fold increases in both aggregate modulus and tensile modulus, with corresponding increases in GAG and collagen content. In phase II, combined treatment with BMP-2 and IGF-I increased aggregate modulus and GAG content further than either growth factor alone, while TGF-beta1 treatment alone remained the only treatment to also enhance tensile properties and collagen content. DISCUSSION: This study determined systematically the effects of multiple growth factor treatments under serum-free conditions, and is the first to demonstrate significant increases in both compressive and tensile biomechanical properties as a result of growth factor treatment. These findings are exciting as coupling growth factor application with the self-assembly process resulted in tissue engineered constructs with functional properties approaching native cartilage values.

Growth and growth factor production by human nasal septal chondrocytes in serum-free media.

BACKGROUND: Tissue-engineered human cartilage offers vast possibilities as a source of graft implant material for reconstructive surgery. Serum-supplemented growth media is successful in supporting chondrocyte proliferation in vitro. Serum, however, contains exogenous growth factors that hamper the identification and quantification of growth factors autogenously produced by chondrocytes. We explore the possibility of using a commercially available serum-free medium UltraCULTURE as an alternative to modified Webber's medium (MWM), the standard media used in chondrocyte cell culture. METHODS: Human nasal septal chondrocytes were grown in UltraCULTURE containing various concentrations of basic fibroblast growth factor (bFGF; 0, 1, 10, and 100 ng/mL) with or without insulin-like growth factor and compared with chondrocytes grown in MWM. Growth curves and transforming growth factor (TGF) beta 1 production were analyzed. RESULTS: We found no differences in the ability to sustain cell viability in culture between the two base media types. We also found no statistically significant differences in TGF-beta 1 production by chondrocytes grown in either system. Finally, there were no statistically significant differences in chondrocyte proliferation between cultures supplemented with bFGF at 10 and 100 ng/mL. CONCLUSION: UltraCULTURE media is a cost-effective, serum-free alternative to standard media with compatible growth characteristics. It offers specific advantages over standard serum-containing media for the precise measurement of autogenous growth factor production by cultured chondrocytes. Furthermore, UltraCULTURE's serum-free environment would be ideal for safely producing tissue-engineered cartilage grafts.

Biomechanical assessment of tissue retrieved after in vivo cartilage defect repair: tensile modulus of repair tissue and integration with host cartilage.

Failure to restore the mechanical properties of tissue at the repair site and its interface with host cartilage is a common problem in tissue engineering procedures to repair cartilage defects. Quantitative in vitro studies have helped elucidate mechanisms underlying processes leading to functional biomechanical changes. However, biomechanical assessment of tissue retrieved from in vivo studies of cartilage defect repair has been limited to compressive tests. Analysis of integration following in vivo repair has relied on qualitative histological methods. The objectives of this study were to develop a quantitative biomechanical method to assess (1) the tensile modulus of repair tissue and (2) its integration in vivo, as well as determine whether supplementation of transplanted chondrocytes with IGF-I affected these mechanical properties. Osteochondral blocks were obtained from a previous 8 month study on the effects of IGF-I on chondrocyte transplantation in the equine model. Tapered test specimens were prepared from osteochondral blocks containing the repair/native tissue interface and adjacently located blocks of intact native tissue. Specimens were then tested in uniaxial tension. The tensile modulus of repair tissue averaged 0.65 MPa, compared to the average of 5.2 MPa measured in intact control samples. Integration strength averaged 1.2 MPa, nearly half the failure strength of intact cartilage samples, 2.7 MPa. IGF-I treatment had no detectable effects on these mechanical properties. This represents the first quantitative biomechanical investigation of the tensile properties of repair tissue and its integration strength in an in vivo joint defect environment.

Culture of bovine preantral follicles in a serum-free system: markers for assessment of growth and development.

Satisfactory development of bovine follicles in vitro remains elusive. This study used a serum-free system to evaluate the effects of insulin-like growth factor-1 (IGF-1) on bovine preantral follicles in culture and to identify the activity of gelatinase matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) in vitro to assess their potential as markers of development. Preantral follicles were cultured for 6 days in serum-free medium containing insulin and IGF-1 (10 ng/ml). No difference was observed in follicular growth, health, or antrum formation between IGF-1-treated follicles and controls. However, IGF-1 had a negative effect (P < 0.01) on oocyte size and granulosa cell proliferation. When MMP-9 was secreted, the probability of follicles having healthy granulosa or theca cells at the end of the culture period was 0.85 and 0.60, respectively. If TIMP-1 was released, the probability of follicles having healthy somatic cells was 0.79. When TIMP-2 was detected, the probability of granulosa and theca cell health was 0. 78 and 0.67, respectively. These results demonstrate no positive effects of IGF-1 on bovine follicles in this system. Furthermore, MMP-9 and TIMPs are related to follicular health and, therefore, can be used as markers of follicular development.

Assessment of growth factor effects on post-thaw development of cryopreserved mouse morulae to the blastocyst stage.

The objective of this study was to assess the influence of specific factors on post-thaw development of mouse cryopreserved morulae. Thawed morulae (n = 206) were randomly distributed between 10 treatment groups: medium alone control (CT), Vero (VR) cells, leukaemia inhibitory factor (1 ng/ml), interleukin-6 (1 ng/ml), transforming growth factor (TGF) alpha (2 ng/ml), epidermal growth factor (EGF) (4 ng/ml), platelet-derived growth factor (1 ng/ml), insulin-like growth factor (IGF)-I (30 ng/ml), IGF-II (1 ng/ml) and TGFbeta (2 ng/ml). At 4, 8, 20, 30 and 48 h, a digitized image of each thawed embryo was captured and stored for later analysis. The following parameters were examined: blastocoel formation, blastocyst expansion, zona thickness and hatching. At termination of the experiment, cell number per embryo was determined by bisbenzimide staining. When contrasted to the medium alone control, co-culture consistently accelerated the development of frozen-thawed morulae to the hatched blastocyst stage, allowing embryos to recover rapidly from any damage sustained during the cryopreservation process. While no single growth factor/cytokine was able to completely mimic the results achieved with co-culture, all of the growth factors impacted positively on at least one of the morphological parameters studied. Cell proliferation was significantly stimulated by just 48 h exposure to growth factors, either through co-culture or by direct media supplementation. Co-culture again yielded the best results with a mean cell count of 217 +/- 76 cells per blastocyst as compared with 131 +/- 36 in control medium alone. Amongst the factors tested, IGF-I, IGF-II and EGF had the greatest impact, with mean cell counts of 172 +/- 50, 168 +/- 50 and 179 +/- 55 respectively. Whereas only 5% of CT embryos developed to blastocysts with > 200 cells, 51% of thawed embryos placed on co-culture monolayers and 25-32% of embryos cultured with IGF-I, IGF-II or EGF had > 200 cells. This study for the first time systematically describes the effect of culture regimen and growth factor additives on the post-thaw development of cryopreserved embryos.

Assessment of in vivo glucose kinetics using stable isotope tracers to determine their alteration in humans during critical illness.

Glucose plays a central role in energy metabolism, and alterations in its utilization have been reported under a variety of physiological and pathological conditions. The extent and direction of its changes provide useful information to promote the understanding of pathophysiology. The regulation of in vivo glucose kinetics is important because it is closely linked to energy production and the control of amino acid and protein metabolism. Although alterations in glucose kinetics have been demonstrated in critically ill patients, the mechanisms responsible are not well understood. The measurement of glucose kinetics in humans using stable isotopic glucose tracers provides a better understanding of the responses to nutritional support in these patients. While tracer methods have been used to quantitatively measure in vivo kinetics in patients with a variety of critical illnesses as well as in normal volunteers during fasting and exercise in European countries and the United States, they have not received the same attention in Japan. Stable isotopic glucose tracers can be safely given to humans since they are themselves naturally occurring substances, accounting for a small percentage of the total, depending on the isotopic species. The intravenous administration of a glucose tracer allows quantitative assessment of in vivo glucose kinetics under a variety of conditions. This method has wide potential for obtaining kinetic data on all aspects of in vivo glucose metabolism, with major advantages for conducting metabolic studies in humans.

Growth hormone or insulin-like growth factor I increases fat oxidation and decreases protein oxidation without altering energy expenditure in parenterally fed rats.

We assessed whether the increased growth in parenterally fed rats treated with growth hormone (GH) or insulin-like growth factor I (IGF-I) or both is associated with alterations in energy expenditure or macronutrient oxidation or both. Surgically stressed male rats (approximately 235 g) were given total parenteral nutrition (TPN) and treated with recombinant human GH (rhGH) (800 micrograms/d), rhIGF-I (800 micrograms/d), rhGH+rhIGF-I (800 micrograms/d of each), or TPN alone for 3 d. Treatment with GH or IGF-I or both resulted in significantly greater body weight gain, nitrogen retention, and serum total IGF-I concentrations compared with TPN alone (P < 0.0001). Assessment of respiratory gas exchange and motor activity for 23 h on day 3 indicated no significant differences between groups in either total or activity-related rates of energy expenditures (kJ/kg0.75). Estimates based on the nitrogen-free respiratory quotient (RQ) revealed fat oxidation to be significantly increased by GH (P < 0.001) and IGF-I (P < 0.03), whereas protein oxidation was significantly reduced (P < 0.0001) by these growth factors. GH and IGF-I combined further enhanced fat oxidation while reducing protein catabolism. Serum insulin concentrations were significantly increased by GH but decreased by IGF-I. GH significantly decreased serum total triiodothyronine concentrations and IGF-I significantly decreased serum corticosterone concentrations. These results suggest that treatment with GH or IGF-I can increase fat oxidation and spare protein for growth without altering energy expenditure in surgically stressed rats maintained with TPN.

IGF-I alters energy expenditure and protein metabolism during parenteral feeding in rats.

In this study, 20 micrograms.kg.-1.h-1 of recombinant human insulin-like growth factor I (IGF-I) was infused into normal healthy rats to examine the effects of IGF-I on glucose, protein, and energy metabolism in either overnight-fasting or parenteral-feeding states. The fed state was maintained by continuous intravenous feeding of a complete diet containing 838 kJ.kg-1.d-1, 2 g N.kg-1.d-1, and no fat. At each nutritional state, one-half of the animals received IGF-I infusion while the other half received saline as control. [14C-1]leucine and [3H-6]glucose were used to determine the effects of IGF-I on protein and glucose kinetics during fed and fasting states. The results showed that 1) infusion of IGF-I at this amount did not alter plasma glucose appearance and only marginally decreased plasma glucose concentrations in both nutritional states; 2) during fasting, IGF-I did not show anabolic effects on protein metabolism either at the whole-body level or in individual tissues. However, during feeding, IGF-I significantly stimulated exogenous nitrogen utilization by the whole body; and 3) IGF-I reduced the thermogenic effect of feeding. The results suggest that parenteral feeding may be an important variable in the response of protein anabolism to IGF-I in vivo.