Ligament-derived matrix stimulates a ligamentous phenotype in human adipose-derived stem cells.

Human adipose stem cells (hASCs) can differentiate into a variety of phenotypes. Native extracellular matrix (e.g., demineralized bone matrix or small intestinal submucosa) can influence the growth and differentiation of stem cells. The hypothesis of this study was that a novel ligament-derived matrix (LDM) would enhance expression of a ligamentous phenotype in hASCs compared to collagen gel alone. LDM prepared using phosphate-buffered saline or 0.1% peracetic acid was mixed with collagen gel (COL) and was evaluated for its ability to induce proliferation, differentiation, and extracellular matrix synthesis in hASCs over 28 days in culture at different seeding densities (0, 0.25 x 10(6), 1 x 10(6), or 2 x 10(6) hASC/mL). Biochemical and gene expression data were analyzed using analysis of variance. Fisher's least significant difference test was used to determine differences between treatments following analysis of variance. hASCs in either LDM or COL demonstrated changes in gene expression consistent with ligament development. hASCs cultured with LDM demonstrated more dsDNA content, sulfated-glycosaminoglycan accumulation, and type I and III collagen synthesis, and released more sulfated-glycosaminoglycan and collagen into the medium compared to hASCs in COL (p

Injectable laminin-functionalized hydrogel for nucleus pulposus regeneration.

Cell delivery to the pathological intervertebral disc (IVD) has significant therapeutic potential for enhancing IVD regeneration. The development of injectable biomaterials that retain delivered cells, promote cell survival, and maintain or promote an NP cell phenotype in vivo remains a significant challenge. Previous studies have demonstrated NP cell - laminin interactions in the nucleus pulposus (NP) region of the IVD that promote cell attachment and biosynthesis. These findings suggest that incorporating laminin ligands into carriers for cell delivery may be beneficial for promoting NP cell survival and phenotype. Here, an injectable, laminin-111 functionalized poly(ethylene glycol) (PEG-LM111) hydrogel was developed as a biomaterial carrier for cell delivery to the IVD. We evaluated the mechanical properties of the PEG-LM111 hydrogel, and its ability to retain delivered cells in the IVD space. Gelation occurred in approximately 20 min without an initiator, with dynamic shear moduli in the range of 0.9-1.4 kPa. Primary NP cell retention in cultured IVD explants was significantly higher over 14 days when cells were delivered within a PEG-LM111 carrier, as compared to cells in liquid suspension. Together, these results suggest this injectable laminin-functionalized biomaterial may be an easy to use carrier for delivering cells to the IVD.

Improving the lens design and performance of a contemporary electromagnetic shock wave lithotripter.

The efficiency of shock wave lithotripsy (SWL), a noninvasive first-line therapy for millions of nephrolithiasis patients, has not improved substantially in the past two decades, especially in regard to stone clearance. Here, we report a new acoustic lens design for a contemporary electromagnetic (EM) shock wave lithotripter, based on recently acquired knowledge of the key lithotripter field characteristics that correlate with efficient and safe SWL. The new lens design addresses concomitantly three fundamental drawbacks in EM lithotripters, namely, narrow focal width, nonidealized pulse profile, and significant misalignment in acoustic focus and cavitation activities with the target stone at high output settings. Key design features and performance of the new lens were evaluated using model calculations and experimental measurements against the original lens under comparable acoustic pulse energy (E+) of 40 mJ. The -6-dB focal width of the new lens was enhanced from 7.4 to 11 mm at this energy level, and peak pressure (41 MPa) and maximum cavitation activity were both realigned to be within 5 mm of the lithotripter focus. Stone comminution produced by the new lens was either statistically improved or similar to that of the original lens under various in vitro test conditions and was significantly improved in vivo in a swine model (89% vs. 54%, P = 0.01), and tissue injury was minimal using a clinical treatment protocol. The general principle and associated techniques described in this work can be applied to design improvement of all EM lithotripters.

Transcription factors MYOCD, SRF, Mesp1 and SMARCD3 enhance the cardio-inducing effect of GATA4, TBX5, and MEF2C during direct cellular reprogramming.

Transient overexpression of defined combinations of master regulator genes can effectively induce cellular reprogramming: the acquisition of an alternative predicted phenotype from a differentiated cell lineage. This can be of particular importance in cardiac regenerative medicine wherein the heart lacks the capacity to heal itself, but simultaneously contains a large pool of fibroblasts. In this study we determined the cardio-inducing capacity of ten transcription factors to actuate cellular reprogramming of mouse embryonic fibroblasts into cardiomyocyte-like cells. Overexpression of transcription factors MYOCD and SRF alone or in conjunction with Mesp1 and SMARCD3 enhanced the basal but necessary cardio-inducing effect of the previously reported GATA4, TBX5, and MEF2C. In particular, combinations of five or seven transcription factors enhanced the activation of cardiac reporter vectors, and induced an upregulation of cardiac-specific genes. Global gene expression analysis also demonstrated a significantly greater cardio-inducing effect when the transcription factors MYOCD and SRF were used. Detection of cross-striated cells was highly dependent on the cell culture conditions and was enhanced by the addition of valproic acid and JAK inhibitor. Although we detected Ca(2+) transient oscillations in the reprogrammed cells, we did not detect significant changes in resting membrane potential or spontaneously contracting cells. This study further elucidates the cardio-inducing effect of the transcriptional networks involved in cardiac cellular reprogramming, contributing to the ongoing rational design of a robust protocol required for cardiac regenerative therapies.

Interstitial engraftment of adipose-derived stem cells into an acellular dermal matrix results in improved inward angiogenesis and tissue incorporation.

Acellular dermal matrices (ADM) are commonly used in reconstructive procedures and rely on host cell invasion to become incorporated into host tissues. We investigated different approaches to adipose-derived stem cells (ASCs) engraftment into ADM to enhance this process. Lewis rat adipose-derived stem cells were isolated and grafted (3.0 × 10(5) cells) to porcine ADM disks (1.5 mm thick × 6 mm diameter) using either passive onlay or interstitial injection seeding techniques. Following incubation, seeding efficiency and seeded cell viability were measured in vitro. In addition, Eighteen Lewis rats underwent subcutaneous placement of ADM disk either as control or seeded with PKH67 labeled ASCs. ADM disks were seeded with ASCs using either onlay or injection techniques. On day 7 and or 14, ADM disks were harvested and analyzed for host cell infiltration. Onlay and injection techniques resulted in unique seeding patterns; however cell seeding efficiency and cell viability were similar. In-vivo studies showed significantly increased host cell infiltration towards the ASCs foci following injection seeding in comparison to control group (p < 0.05). Moreover, regional endothelial cell invasion was significantly greater in ASCs injected grafts in comparison to onlay seeding (p < 0.05). ADM can successfully be engrafted with ASCs. Interstitial engraftment of ASCs into ADM via injection enhances regional infiltration of host cells and angiogenesis, whereas onlay seeding showed relatively broad and superficial cell infiltration. These findings may be applied to improve the incorporation of avascular engineered constructs.

The effect of nitric oxide surface flux on the foreign body response to subcutaneous implants.

Although the release of nitric oxide (NO) from biomaterials has been shown to reduce the foreign body response (FBR), the optimal NO release kinetics and doses remain unknown. Herein, polyurethane-coated wire substrates with varying NO release properties were implanted into porcine subcutaneous tissue for 3, 7, 21 and 42 d. Histological analysis revealed that materials with short NO release durations (i.e., 24 h) were insufficient to reduce the collagen capsule thickness at 3 and 6 weeks, whereas implants with longer release durations (i.e., 3 and 14 d) and greater NO payloads significantly reduced the collagen encapsulation at both 3 and 6 weeks. The acute inflammatory response was mitigated most notably by systems with the longest duration and greatest dose of NO release, supporting the notion that these properties are most critical in circumventing the FBR for subcutaneous biomedical applications (e.g., glucose sensors).

Similar T-cell immune responses induced by group M consensus env immunogens with wild-type or minimum consensus variable regions.

Consensus HIV-1 genes can decrease the genetic distances between candidate immunogens and field virus strains. To ensure the functionality and optimal presentation of immunologic epitopes, we generated two group-M consensus env genes that contain variable regions either from a wild-type B/C recombinant virus isolate (CON6) or minimal consensus elements (CON-S) in the V1, V2, V4, and V5 regions. C57BL/6 and BALB/c mice were primed twice with CON6, CON-S, and subtype control (92UG37_A and HXB2/Bal_B) DNA and boosted with recombinant vaccinia virus (rVV). Mean antibody titers against 92UG37_A, 89.6_B, 96ZM651_C, CON6, and CON-S Env protein were determined. Both CON6 and CON-S induced higher mean antibody titers against several of the proteins, as compared with the subtype controls. However, no significant differences were found in mean antibody titers in animals immunized with CON6 or CON-S. Cellular immune responses were measured by using five complete Env overlapping peptide sets: subtype A (92UG37_A), subtype B (MN_B, 89.6_B and SF162_B), and subtype C (Chn19_C). The intensity of the induced cellular responses was measured by using pooled Env peptides; T-cell epitopes were identified by using matrix peptide pools and individual peptides. No significant differences in T-cell immune-response intensities were noted between CON6 and CON-S immunized BALB/c and C57BL/6 mice. In BALB/c mice, 10 and eight nonoverlapping T-cell epitopes were identified in CON6 and CON-S, whereas eight epitopes were identified in 92UG37_A and HXB2/BAL_B. In C57BL/6 mice, nine and six nonoverlapping T-cell epitopes were identified after immunization with CON6 and CON-S, respectively, whereas only four and three were identified in 92UG37_A and HXB2/BAL_B, respectively. When combined together from both mouse strains, 18 epitopes were identified. The group M artificial consensus env genes, CON6 and CON-S, were equally immunogenic in breadth and intensity for inducing humoral and cellular immune responses.

Low demographic variability in wild primate populations: fitness impacts of variation, covariation, and serial correlation in vital rates.

In a stochastic environment, long-term fitness can be influenced by variation, covariation, and serial correlation in vital rates (survival and fertility). Yet no study of an animal population has parsed the contributions of these three aspects of variability to long-term fitness. We do so using a unique database that includes complete life-history information for wild-living individuals of seven primate species that have been the subjects of long-term (22-45 years) behavioral studies. Overall, the estimated levels of vital rate variation had only minor effects on long-term fitness, and the effects of vital rate covariation and serial correlation were even weaker. To explore why, we compared estimated variances of adult survival in primates with values for other vertebrates in the literature and found that adult survival is significantly less variable in primates than it is in the other vertebrates. Finally, we tested the prediction that adult survival, because it more strongly influences fitness in a constant environment, will be less variable than newborn survival, and we found only mixed support for the prediction. Our results suggest that wild primates may be buffered against detrimental fitness effects of environmental stochasticity by their highly developed cognitive abilities, social networks, and broad, flexible diets.

Fatty acid composition of wild anthropoid primate milks.

Fatty acids in milk reflect the interplay between species-specific physiological mechanisms and maternal diet. Anthropoid primates (apes, Old and New World monkeys) vary in patterns of growth and development and dietary strategies. Milk fatty acid profiles also are predicted to vary widely. This study investigates milk fatty acid composition of five wild anthropoids (Alouatta palliata, Callithrix jacchus, Gorilla beringei beringei, Leontopithecus rosalia, Macaca sinica) to test the null hypothesis of a generalized anthropoid milk fatty acid composition. Milk from New and Old World monkeys had significantly more 8:0 and 10:0 than milk from apes. The leaf eating species G. b. beringei and A. paliatta had a significantly higher proportion of milk 18:3n-3, a fatty acid found primarily in plant lipids. Mean percent composition of 22:6n-3 was significantly different among monkeys and apes, but was similar to the lowest reported values for human milk. Mountain gorillas were unique among anthropoids in the high proportion of milk 20:4n-6. This seems to be unrelated to requirements of a larger brain and may instead reflect species-specific metabolic processes or an unknown source of this fatty acid in the mountain gorilla diet.

The impact of cropping on wild populations of Saguinus mystax and Saguinus fuscicollis in Peru

A transect census technique was used to estimate the population densities of Saguinus mystax and Saguinus fuscicollis at two sites in Peru. Cropping of these two species had occurred five years before the census at one site and two years before at the other. The populations of S. mystax at both sites had recovered completely from cropping, and the relationship between S. mystax and S. fuscicollis had not been altered at one site and had been reversed in favor of S. mystax at the other. Copyright © 1984 Wiley‐Liss, Inc., A Wiley Company

A coprological survey of parasites of wild mantled howling monkeys, Alouatta palliata palliata.

Fecal samples from 155 mantled howling monkeys (Alouatta palliata palliata) examined at Centro Ecologico La Pacifica, Guanacaste Province, Costa Rica, revealed 75 (48%) had parasitic infections. A sampling of nine howling monkeys from Santa Rosa National Park. Costa Rica indicated only one infected animal (11%). Only three of 19 (16%) spider monkeys (Ateles geoffroyi) also from Santa Rosa were infected. Controrchis biliophilus, Trypanoxyuris minutus, unidentified strongylid eggs and Isospora sp. oocysts were found. Three monkeys from La Pacifica died and were examined for adult helminths. They were infected with Ascaris lumbricoides, C. biliophilus and T. minutus.

Breech delivery and birth‐related behaviors in wild mantled howling monkeys

The breech birth of an infant mantled howling monkey was observed on February 12, 1990. The mother assisted the successful delivery by pulling on the infant's tail and hindleg. No other members of the social group attended the mother or demonstrated any interest in the birth process. Copyright © 1991 Wiley‐Liss, Inc., A Wiley Company

Dental microwear in live, wild-trapped Alouatta palliata from Costa Rica.

One problem with dental microwear analyses of museum material is that investigators can never be sure of the diets of the animals in question. An obvious solution to this problem is to work with live animals. Recent work with laboratory primates has shown that high resolution dental impressions can be obtained from live animals. The purpose of this study was to use similar methods to begin to document rates and patterns of dental microwear for primates in the wild. Thirty-three Alouatta palliata were captured during the wet season at Hacienda La Pacifica near Canas, Costa Rica. Dental impressions were taken and epoxy casts of the teeth were prepared using the methods of Teaford and Oyen (1989a). Scanning electron micrographs were taken of the left mandibular second molars at magnifications of 200x and 500x. Lower magnification images were used to calculate rates of wear, and higher magnification images were used to measure the size and shape of microwear features. Results indicate that, while basic patterns of dental microwear are similar in museum samples and samples of live, wild-trapped animals of the same species, ecological differences between collection locales may lead to significant intraspecific differences in dental microwear. More importantly, rates of microwear provide the first direct evidence of differences in molar use between monkeys and humans.

Parasites of wild howlers (Alouatta spp.)

A literature review of howler parasites provides the basis for an overview of the ecological significance of parasite surveys in primates. Within this framework, we have added insights into the interactions between primate hosts and their parasites from a long-term study in Costa Rica. We collected fecal samples from mantled howlers (Alouatta palliata) over a 9-year period (19861994 inclusive) and analyzed them for parasite eggs, larvae, cysts, and oocysts. We found many misperceptions inherent in the typical methodology of primate parasite surveys and in the reporting of the findings. Our work in Costa Rica suggests that a snapshot effect occurs with most surveys. A static view does not reflect the dynamic and changing ecological interaction between host and parasite. We describe some problems with parasite data analyses that emphasize the need for long-term longitudinal surveys in wild primate groups.

Sensory information and associative cues used in food detection by wild vervet monkeys.

Understanding animals' spatial perception is a critical step toward discerning their cognitive processes. The spatial sense is multimodal and based on both the external world and mental representations of that world. Navigation in each species depends upon its evolutionary history, physiology, and ecological niche. We carried out foraging experiments on wild vervet monkeys (Chlorocebus pygerythrus) at Lake Nabugabo, Uganda, to determine the types of cues used to detect food and whether associative cues could be used to find hidden food. Our first and second set of experiments differentiated between vervets' use of global spatial cues (including the arrangement of feeding platforms within the surrounding vegetation) and/or local layout cues (the position of platforms relative to one another), relative to the use of goal-object cues on each platform. Our third experiment provided an associative cue to the presence of food with global spatial, local layout, and goal-object cues disguised. Vervets located food above chance levels when goal-object cues and associative cues were present, and visual signals were the predominant goal-object cues that they attended to. With similar sample sizes and methods as previous studies on New World monkeys, vervets were not able to locate food using only global spatial cues and local layout cues, unlike all five species of platyrrhines thus far tested. Relative to these platyrrhines, the spatial location of food may need to stay the same for a longer time period before vervets encode this information, and goal-object cues may be more salient for them in small-scale space.