A Description of Exercise Programs Implemented During a Firefighter Training Academy.

OBJECTIVE: The aim of the study is to thoroughly describe exercise programs that were performed during a firefighter training academy, including their effects on body composition and physical fitness. METHODS: Fourteen recruits performed exercise programs during a 24-week training academy and several components of their body composition and physical fitness were measured. Moreover, their heart rate (HR) and session rating of perceived exertion (sRPE) were recorded during and after every training session, respectively. RESULTS: Fat-free mass, subcutaneous fat in the abdomen, cardiovascular fitness, flexibility, lower-body power, and muscular endurance significantly improved during the study ( P ≤ 0.05). The highest HR and sRPE responses were observed during aerobic training (AT) sessions. The average HR responses relative to maximum HR were similar during AT, high-intensity circuit training, multimodal high-intensity interval training, and high-intensity functional training; however, the sRPEs during high-intensity circuit training and multimodal high-intensity interval training were significantly lower than AT. CONCLUSIONS: Several effective exercise programs can be implemented during firefighter training academies.

Angiosperm to Gymnosperm host-plant switch entails shifts in microbiota of the Welwitschia bug, Probergrothius angolensis (Distant, 1902).

The adaptation of herbivorous insects to new host plants is key to their evolutionary success in diverse environments. Many insects are associated with mutualistic gut bacteria that contribute to the host's nutrition and can thereby facilitate dietary switching in polyphagous insects. However, how gut microbial communities differ between populations of the same species that feed on different host plants remains poorly understood. Most species of Pyrrhocoridae (Hemiptera: Heteroptera) are specialist seed-feeders on plants in the family Malvaceae, although populations of one species, Probergrothius angolensis, have switched to the very distantly related Welwitschia mirabilis plant in the Namib Desert. We first compared the development and survival of laboratory populations of Pr. angolensis with two other pyrrhocorids on seeds of Welwitschia and found only Pr. angolensis was capable of successfully completing its development. We then collected Pr. angolensis in Namibia from Malvaceae and Welwitschia host plants, respectively, to assess their bacterial and fungal community profiles using high-throughput amplicon sequencing. Comparison with long-term laboratory-reared insects indicated stable associations of Pr. angolensis with core bacteria (Commensalibacter, Enterococcus, Bartonella and Klebsiella), but not with fungi or yeasts. Phylogenetic analyses of core bacteria revealed relationships to other insect-associated bacteria, but also found new taxa indicating potential host-specialized nutritional roles. Importantly, the microbial community profiles of bugs feeding on Welwitschia versus Malvaceae revealed stark and consistent differences in the relative abundance of core bacterial taxa that correlate with the host-plant switch; we were able to reproduce this result through feeding experiments. Thus, a dynamic gut microbiota may provide a means for insect adaptation to new host plants in new environments when food plants are extremely divergent.

An engineered GFP fluorescent bacterial biosensor for detecting and quantifying silver and copper ions.

Presented here are two engineered bacterial biosensors for detecting and quantifying silver and copper ions. The biosensors contain a silver/copper resistance operon and a Green Fluorescent Protein gene that is strictly regulated through silver activated promoter regions normally found on a silver resistance gene (sil operon). The two biosensors efficiently detected silver and copper concentrations of 40 µM-300 µM and 20 µM-600 µM respectively. A strong correlation (R2 = 0.90 or above) between silver/copper and GFP signal makes it possible to quantify the ions using a linear regression. At room temperature incubation, the GFP signal of the biosensors in Ag+ saturated after 13 h. However, a detectable GFP signal was seen in 4 h.

Transmission of mutualistic bacteria in social and gregarious insects.

Symbiotic microbes can confer a range of benefits to social, sub-social, and gregarious insects that include contributions to nutrition, digestion, and defense. Transmission of beneficial symbionts to the next generation in these insects sometimes occurs transovarially as in many solitary insects, but primarily through social contact such as coprophagy in gregarious taxa, and trophallaxis in eusocial insects. While these behaviors benefit reliable transmission of multi-microbial assemblages, they may also come at the cost of inviting the spread of parasites and pathogens. Nonetheless, the overall benefit of social symbiont transmission may be one of several important factors that reinforce the evolution of social behaviors and insect eusociality.

Comparative Study of Optical Coherence Tomography Angiography and Phase-Resolved Doppler Optical Coherence Tomography for Measurement of Retinal Blood Vessels Caliber.

PURPOSE: To compare the accuracy of Doppler optical coherence tomography (DOCT) and OCT angiography (OCTA) for measuring retinal blood vessel caliber at different flow rates. METHODS: A research-grade 1060-nm OCT system with 3.5-µm axial resolution in retinal tissue and 92,000 A scan/s image acquisition rate was used in this study. DOCT and OCTA measurements were acquired both from a flow phantom and in vivo from retinal blood vessels in six male Brown Norway rats. The total retinal blood flow (TRBF) was modified from baseline to 70% and 20% of baseline by reducing the ocular perfusion pressure (OPP). The retinal blood vessel caliber (RBVC) was measured from OCTA and DOCT images. The caliber measurements were conducted by two separate graders using a custom MATLAB-based image processing algorithm. RESULTS: The RBVC measured with OCTA and DOCT for normal blood flow rates were not significantly different (56.69 ± 12.17 and 57.17 ± 9.46 µm, P = 0.27, respectively). However, significant differences were detected when TRBF was reduced to 70% (55.69 ± 11.56 vs. 50.62 ± 8.85 µm, P < 0.01) and 20% (50.29 ± 9.29 vs. 44.88 ± 7.13 µm, P < 0.01) of baseline. CONCLUSIONS: Reduced TRBF resulted in inaccuracy of the RBVC measurements with DOCT in both the phantom and animal study. This result suggests that OCTA is a more accurate tool for RBVC evaluation when applied to retinal diseases associated with reduced TRBF, such as glaucoma and diabetic retinopathy. TRANSLATIONAL RELEVANCE: Results from this study are directly applicable to clinical studies of retinal blood flow measured with OCTA and DOCT.

The cotton stainer's gut microbiota suppresses infection of a cotransmitted trypanosomatid parasite.

The evolutionary and ecological success of many insects is attributed to mutualistic partnerships with bacteria that confer hosts with novel traits including food digestion, nutrient supplementation, detoxification of harmful compounds and defence against natural enemies. Dysdercus fasciatus firebugs (Hemiptera: Pyrrhocoridae), commonly known as cotton stainers, possess a simple but distinctive gut bacterial community including B vitamin-supplementing Coriobacteriaceae symbionts. In addition, their guts are often infested with the intestinal trypanosomatid parasite Leptomonas pyrrhocoris (Kinetoplastida: Trypanosomatidae). In this study, using experimental bioassays and fluorescence in situ hybridization (FISH), we report on the protective role of the D. fasciatus gut bacteria against L. pyrrhocoris. We artificially infected 2nd instars of dysbiotic and symbiotic insects with a parasite culture and measured parasite titres, developmental time and survival rates. Our results show that L. pyrrhocoris infection increases developmental time and slightly modifies the quantitative composition of the gut microbiota. More importantly, we found significantly higher parasite titres and a tendency towards lower survival rates in parasite-infected dysbiotic insects compared to symbiotic controls, indicating that the gut bacteria successfully interfere with the establishment or proliferation of L. pyrrhocoris. The colonization of symbiotic bacteria on the peritrophic matrix along the gut wall, as revealed by FISH, likely acts as a barrier blocking parasite attachment or entry into the hemolymph. Our findings show that in addition to being nutritionally important, D. fasciatus' gut bacteria complement the host's immune system in preventing parasite invasions and that a stable gut microbial community is integral for the host's health.

Multi-modal defences in aphids offer redundant protection and increased costs likely impeding a protective mutualism.

The pea aphid, Acyrthosiphon pisum, maintains extreme variation in resistance to its most common parasitoid wasp enemy, Aphidius ervi, which is sourced from two known mechanisms: protective bacterial symbionts, most commonly Hamiltonella defensa, or endogenously encoded defences. We have recently found that individual aphids may employ each defence individually, occasionally both defences together, or neither. In field populations, Hamiltonella-infected aphids are found at low to moderate frequencies and while less is known about the frequency of resistant genotypes, they show up less often than susceptible genotypes in field collections. To better understand these patterns, we sought to compare the strengths and costs of both types of defence, individually and together, in order to elucidate the selective pressures that maintain multi-modal defence mechanisms or that may favour one over the other. We experimentally infected five aphid genotypes (two lowly and three highly resistant), each with two symbiont strains, Hamiltonella-APSE8 (moderate protection) and Hamiltonella-APSE3 (high protection). This resulted in three sublines per genotype: uninfected, +APSE8 and +APSE3. Each of the 15 total sublines was first subjected to a parasitism assay to determine its resistance phenotype and in a second experiment, a subset was chosen to compare fitness (fecundity and survivorship) in the presence and absence of parasitism. In susceptible aphid genotypes, parasitized sublines infected with Hamiltonella generally showed increased protection with direct fitness benefits, but clear infection costs to fitness in the absence of parasitism. In resistant genotypes, Hamiltonella infection rarely conferred additional protection, often further reduced fecundity and survivorship when enemy challenged, and resulted in constitutive fitness costs in the absence of parasitism. We also identified strong aphid genotype × symbiont-strain interactions, such that the best defensive strategy against parasitoids varied for each aphid genotype; one performed best with no protective symbionts, the others with particular strains of Hamiltonella. This surprising variability in outcomes helps explain why Hamiltonella infection frequencies are often intermediate and do not strongly track parasitism frequencies in field populations. We also find that variation in endogenous traits, such as resistance, among host genotypes may offer redundancy and generally limit the invasion potential of mutualistic microbes in insects.

Specificity of Multi-Modal Aphid Defenses against Two Rival Parasitoids.

Insects are often attacked by multiple natural enemies, imposing dynamic selective pressures for the development and maintenance of enemy-specific resistance. Pea aphids (Acyrthosiphon pisum) have emerged as models for the study of variation in resistance against natural enemies, including parasitoid wasps. Internal defenses against their most common parasitoid wasp, Aphidius ervi, are sourced through two known mechanisms- 1) endogenously encoded resistance or 2) infection with the heritable bacterial symbiont, Hamiltonella defensa. Levels of resistance can range from nearly 0-100% against A. ervi but varies based on aphid genotype and the strain of toxin-encoding bacteriophage (called APSE) carried by Hamiltonella. Previously, other parasitoid wasps were found to commonly attack this host, but North American introductions of A. ervi have apparently displaced all other parasitoids except Praon pequodorum, a related aphidiine braconid wasp, which is still found attacking this host in natural populations. To explain P. pequodorum's persistence, multiple studies have compared direct competition between both wasps, but have not examined specificity of host defenses as an indirectly mediating factor. Using an array of experimental aphid lines, we first examined whether aphid defenses varied in effectiveness toward either wasp species. Expectedly, both types of aphid defenses were effective against A. ervi, but unexpectedly, were completely ineffective against P. pequodorum. Further examination showed that P. pequodorum wasps suffered no consistent fitness costs from developing in even highly 'resistant' aphids. Comparison of both wasps' egg-larval development revealed that P. pequodorum's eggs have thicker chorions and hatch two days later than A. ervi's, likely explaining their differing abilities to overcome aphid defenses. Overall, our results indicate that aphids resistant to A. ervi may serve as reservoirs for P. pequodorum, hence contributing to its persistence in field populations. We find that specificity of host defenses and defensive symbiont infections, may have important roles in influencing enemy compositions by indirectly mediating the interactions and abundance of rival natural enemies.

Factors limiting the spread of the protective symbiont Hamiltonella defensa in Aphis craccivora Aphids.

Many insects are associated with heritable symbionts that mediate ecological interactions, including host protection against natural enemies. The cowpea aphid, Aphis craccivora, is a polyphagous pest that harbors Hamiltonella defensa, which defends against parasitic wasps. Despite this protective benefit, this symbiont occurs only at intermediate frequencies in field populations. To identify factors constraining H. defensa invasion in Ap. craccivora, we estimated symbiont transmission rates, performed fitness assays, and measured infection dynamics in population cages to evaluate effects of infection. Similar to results with the pea aphid, Acyrthosiphon pisum, we found no consistent costs to infection using component fitness assays, but we did identify clear costs to infection in population cages when no enemies were present. Maternal transmission rates of H. defensa in Ap. craccivora were high (ca. 99%) but not perfect. Transmission failures and infection costs likely limit the spread of protective H. defensa in Ap. craccivora. We also characterized several parameters of H. defensa infection potentially relevant to the protective phenotype. We confirmed the presence of H. defensa in aphid hemolymph, where it potentially interacts with endoparasites, and performed real-time quantitative PCR (qPCR) to estimate symbiont and phage abundance during aphid development. We also examined strain variation of H. defensa and its bacteriophage at multiple loci, and despite our lines being collected in different regions of North America, they were infected with a nearly identical strains of H. defensa and APSE4 phage. The limited strain diversity observed for these defensive elements may result in relatively static protection profile for this defensive symbiosis.

Aphid-encoded variability in susceptibility to a parasitoid.

BACKGROUND: Many animals exhibit variation in resistance to specific natural enemies. Such variation may be encoded in their genomes or derived from infection with protective symbionts. The pea aphid, Acyrthosiphon pisum, for example, exhibits tremendous variation in susceptibility to a common natural enemy, the parasitic wasp Aphidius ervi. Pea aphids are often infected with the heritable bacterial symbiont, Hamiltonella defensa, which confers partial to complete resistance against this parasitoid depending on bacterial strain and associated bacteriophages. That previous studies found that pea aphids without H. defensa (or other symbionts) were generally susceptible to parasitism, together with observations of a limited encapsulation response, suggested that pea aphids largely rely on infection with H. defensa for protection against parasitoids. However, the limited number of uninfected clones previously examined, and our recent report of two symbiont-free resistant clones, led us to explicitly examine aphid-encoded variability in resistance to parasitoids. RESULTS: After rigorous screening for known and unknown symbionts, and microsatellite genotyping to confirm clonal identity, we conducted parasitism assays using fifteen clonal pea aphid lines. We recovered significant variability in aphid-encoded resistance, with variation levels comparable to that contributed by H. defensa. Because resistance can be costly, we also measured aphid longevity and cumulative fecundity of the most and least resistant aphid lines under permissive conditions, but found no trade-offs between higher resistance and these fitness parameters. CONCLUSIONS: These results indicate that pea aphid resistance to A. ervi is more complex than previously appreciated, and that aphids employ multiple tactics to aid in their defense. While we did not detect a tradeoff, these may become apparent under stressful conditions or when resistant and susceptible aphids are in direct competition. Understanding sources and amounts of variation in resistance to natural enemies is necessary to understand the ecological and evolutionary dynamics of antagonistic interactions, such as the potential for coevolution, but also for the successful management of pest populations through biological control.

Microablation of collagen-based substrates for soft tissue engineering.

Noting the abundance and importance of collagen as a biomaterial, we have developed a facile method for the production of a dense fibrillar extracellular matrix mimicking collagen-elastin hybrids with tunable mechanical properties. Through the use of excimer-laser technology, we have optimized conditions for the ablation of collagen lamellae without denaturation of protein, maintenance of fibrillar ultrastructure and preservation of native D-periodicity. Strengths of collagen-elastin hybrids ranged from 0.6 to 13 MPa, elongation at break from 9 to 70% and stiffness from 2.9 to 94 MPa, allowing for the design of a wide variety of tissue specific scaffolds. Further, large (centimeter scale) lamellae can be fabricated and embedded with recombinant elastin to generate collagen-elastin hybrids. Exposed collagen in hybrids act as cell adhesive sites for rat mesenchymal stem cells that conform to ablate waveforms. The ability to modulate these features allows for the generation of a class of biopolymers that can architecturally and physiologically replicate native tissue.

How resident microbes modulate ecologically-important traits of insects.

The microbiota inhabiting insects influence a wide range of ecologically-important traits. In addition to their better-known roles in nutrient provisioning and degrading plant polymers, there is emerging evidence that microorganisms also aid herbivores in countering plant defenses. The latter can be mediated by enzymes that degrade plant allelochemicals or via the modulation of plant signaling pathways. Symbionts are also increasingly recognized to protect insects from attack by a wide range of natural enemies. Underlying mechanisms are poorly understood, but some microbes produce antimicrobials or toxins, while others modulate insect immune responses. Ecologically-relevant symbioses can exhibit dynamic variation in strength and specificity of conferred phenotypes, transfer key traits among unrelated insects, and have effects that extend to interacting players and beyond.

Effects of crosslinking on the mechanical properties, drug release and cytocompatibility of protein polymers.

Recombinant elastin-like protein polymers are increasingly being investigated as component materials of a variety of implantable medical devices. This is chiefly a result of their favorable biological properties and the ability to tailor their physical and mechanical properties. In this report, we explore the potential of modulating the water content, mechanical properties, and drug release profiles of protein films through the selection of different crosslinking schemes and processing strategies. We find that the selection of crosslinking scheme and processing strategy has a significant influence on all aspects of protein polymer films. Significantly, utilization of a confined, fixed volume, as well as vapor-phase crosslinking strategies, decreased protein polymer equilibrium water content. Specifically, as compared to uncrosslinked protein gels, water content was reduced for genipin (15.5%), glutaraldehyde (GTA, 24.5%), GTA vapor crosslinking (31.6%), disulfide (SS, 18.2%) and SS vapor crosslinking (25.5%) (P<0.05). Distinct crosslinking strategies modulated protein polymer stiffness, strain at failure and ultimate tensile strength (UTS). In all cases, vapor-phase crosslinking produced the stiffest films with the highest UTS. Moreover, both confined, fixed volume and vapor-phase approaches influenced drug delivery rates, resulting in decreased initial drug burst and release rates as compared to solution phase crosslinking. Tailored crosslinking strategies provide an important option for modulating the physical, mechanical and drug delivery properties of protein polymers.

Effects of parasitism on aphid nutritional and protective symbioses.

Insects often carry heritable symbionts that negotiate interactions with food plants or natural enemies. All pea aphids, Acyrthosiphon pisum, require infection with the nutritional symbiont Buchnera, and many are also infected with Hamiltonella, which protects against the parasitoid Aphidius ervi. Hamiltonella-based protection requires bacteriophages called APSEs with protection levels varying by strain and associated APSE. Endoparasitoids, including A. ervi, may benefit from protecting the nutritional symbiosis and suppressing the protective one, while the aphid and its heritable symbionts have aligned interests when attacked by the wasp. We investigated the effects of parasitism on the abundance of aphid nutritional and protective symbionts. First, we determined strength of protection associated with multiple symbiont strains and aphid genotypes as these likely impact symbiont responses. Unexpectedly, some A. pisum genotypes cured of facultative symbionts were resistant to parasitism and resistant aphid lines carried Hamiltonella strains that conferred no additional protection. Susceptible aphid clones carried protective strains. qPCR estimates show that parasitism significantly influenced both Buchnera and Hamiltonella titres, with multiple factors contributing to variation. In susceptible lines, parasitism led to increases in Buchnera near the time of larval wasp emergence consistent with parasite manipulation, but effects were variable in resistant lines. Parasitism also resulted in increases in APSE and subsequent decreases in Hamiltonella, and we discuss how this response may relate to the protective phenotype. In summary, we show that parasitism alters the within-host ecology of both nutritional and protective symbioses with effects likely significant for all players in this antagonistic interaction.

Incorporation of fibronectin to enhance cytocompatibility in multilayer elastin-like protein scaffolds for tissue engineering.

Recombinant, elastin-like protein (ELP) polymers are of significant interest for the engineering of compliant, resilient soft tissues due to a wide range of tunable mechanical properties, biostability, and biocompatibility. Here, we enhance endothelial cell (EC) and mesenchymal stem cell compatibility with ELP constructs by addition of fibronectin (Fn) to the surface or bulk of ELP hydrogels. We find that cell adhesion, proliferation, and migration can be modulated by Fn addition. Adsorption of Fn to the hydrogel surface is more efficient than bulk blending. Surface immobilization of Fn by genipin crosslinking leads to stability without loss of bioactivity. Gels of varying mechanical modulus do not alter cell adhesion, proliferation, and migration in the range we investigate. However, more compliant gels promote an EC morphology suggesting tubulogenesis or network formation, whereas stiffer gels promote cobblestone morphology. Multilayer structures consisting of thin ELP sheets reinforced with collagen microfiber are fabricated and laminated through the culture of MSCs at layer interfaces. High cell viability in the resulting three-dimensional constructs suggests the applicability of Fn to the design of strong, resilient artificial blood vessels and other soft tissue replacements.

Effect of bone marrow-derived extracellular matrix on cardiac function after ischemic injury.

Ischemic heart disease is a leading cause of death, with few options to retain ventricular function following myocardial infarction. Hematopoietic-derived progenitor cells contribute to angiogenesis and tissue repair following ischemia reperfusion injury. Motivated by the role of bone marrow extracellular matrix (BM-ECM) in supporting the proliferation and regulation of these cell populations, we investigated BM-ECM injection in myocardial repair. In BM-ECM isolated from porcine sternum, we identified several factors important for myocardial healing, including vascular endothelial growth factor, basic fibroblast growth factor-2, and platelet-derived growth factor-BB. We further determined that BM-ECM serves as an adhesive substrate for endothelial cell proliferation. Bone marrow ECM was injected in a rat model of myocardial infarction, with and without a methylcellulose carrier gel. After one day, reduced infarct area was noted in rats receiving BM-ECM injection. After seven days we observed improved fractional shortening, decreased apoptosis, and significantly lower macrophage counts in the infarct border. Improvements in fractional shortening, sustained through 21 days, as well as decreased fibrotic area, enhanced angiogenesis, and greater c-kit-positive cell presence were associated with BM-ECM injection. Notably, the concentrations of BM-ECM growth factors were 10(3)-10(8) fold lower than typically required to achieve a beneficial effect, as reported in pre-clinical studies that have administered single growth factors alone.

Hydrazone self-crosslinking of multiphase elastin-like block copolymer networks.

Biosynthetic strategies for the production of recombinant elastin-like protein (ELP) triblock copolymers have resulted in elastomeric protein hydrogels, formed through rapid physical crosslinking upon warming of concentrated solutions. However, the strength of physically crosslinked networks can be limited, and options for non-toxic chemical crosslinking of these networks are not optimal. In this report, we modify two recombinant elastin-like proteins with aldehyde and hydrazide functionalities. When combined, these modified recombinant proteins self-crosslink through hydrazone bonding without requiring initiators or producing by-products. Crosslinked materials are evaluated for water content and swelling upon hydration, and subject to tensile and compressive mechanical tests. Hydrazone crosslinking is a viable method for increasing the mechanical strength of elastin-like protein polymers, in a manner that is likely to lend itself to the biocompatible in situ formation of chemically and physically crosslinked ELP hydrogels.

Microfabrication and nanotechnology in stent design.

Intravascular stents were first introduced in the 1980s as an adjunct to primary angioplasty for management of early complications, including arterial dissection, or treatment of an inadequate technical outcome due to early elastic recoil of the atherosclerotic lesion. Despite the beneficial effects of stenting, persistent high rates of restenosis motivated the design of drug-eluting stents for delivery of agents to limit the proliferative and other inflammatory responses within the vascular wall that contribute to the development of a restenotic lesion. These strategies have yielded a significant reduction in the incidence of restenosis, but challenges remain, including incomplete repair of the endothelium at the site of vascular wall injury that may be associated with a late risk of thrombosis. A failure of vessel wall healing has been attributed primarily to the use of polymeric stent coatings, but the effects of the eluted drug and other material properties or design features of the stent cannot be excluded. Improvements in stent microfabrication, as well as the introduction of alternative materials may help to address those limitations that inhibit stent performance. This review describes the application of novel microfabrication processes and the evolution of new nanotechnologies that hold significant promise in eliminating existing shortcomings of current stent platforms.

The use of microfiber composites of elastin-like protein matrix reinforced with synthetic collagen in the design of vascular grafts.

Collagen and elastin networks contribute to highly specialized biomechanical responses in numerous tissues and species. Biomechanical properties such as modulus, elasticity, and strength ultimately affect tissue function and durability, as well as local cellular behavior. In the case of vascular bypass grafts, compliance at physiologic pressures is correlated with increased patency due to a reduction in anastomotic intimal hyperplasia. In this report, we combine extracellular matrix (ECM) protein analogues to yield multilamellar vascular grafts comprised of a recombinant elastin-like protein matrix reinforced with synthetic collagen microfibers. Structural analysis revealed that the fabrication scheme permits a range of fiber orientations and volume fractions, leading to tunable mechanical properties. Burst strengths of 239-2760 mm Hg, compliances of 2.8-8.4%/100 mm Hg, and suture retention strengths of 35-192 gf were observed. The design most closely approximating all target criteria displayed a burst strength of 1483 +/- 143 mm Hg, a compliance of 5.1 +/- 0.8%/100 mm Hg, and a suture retention strength of 173 +/- 4 gf. These results indicate that through incorporation of reinforcing collagen microfibers, recombinant elastomeric protein-based biomaterials can play a significant role in load bearing tissue substitutes. We believe that similar composites can be incorporated into tissue engineering schemes that seek to integrate cells within the structure, prior to or after implantation in vivo.

Fibrillogenesis in continuously spun synthetic collagen fiber.

The universal structural role of collagen fiber networks has motivated the development of collagen gels, films, coatings, injectables, and other formulations. However, reported synthetic collagen fiber fabrication schemes have either culminated in short, discontinuous fiber segments at unsuitably low production rates, or have incompletely replicated the internal fibrillar structure that dictates fiber mechanical and biological properties. We report a continuous extrusion system with an off-line phosphate buffer incubation step for the manufacture of synthetic collagen fiber. Fiber with a cross-section of 53+ or - 14 by 21 + or - 3 microm and an ultimate tensile strength of 94 + or - 19 MPa was continuously produced at 60 m/hr from an ultrafiltered monomeric collagen solution. The effect of collagen solution concentration, flow rate, and spinneret size on fiber size was investigated. The fiber was further characterized by microdifferential scanning calorimetry, transmission electron microscopy (TEM), second harmonic generation (SHG) analysis, and in a subcutaneous murine implant model. Calorimetry demonstrated stabilization of the collagen triple helical structure, while TEM and SHG revealed a dense, axially aligned D-periodic fibril structure throughout the fiber cross-section. Implantation of glutaraldehyde crosslinked and noncrosslinked fiber in the subcutaneous tissue of mice demonstrated limited inflammatory response and biodegradation after a 6-week implant period.