Depth-dependent biomechanical and biochemical properties of fetal, newborn, and tissue-engineered articular cartilage

Adult articular cartilage has depth-dependent mechanical and biochemical properties which contribute to zone-specific functions. The compressive moduli of immature cartilage and tissue-engineered cartilage are known to be lower than those of adult cartilage. The objective of this study was to determine if such tissues exhibit depth-dependent compressive properties, and how these depth-varying properties were correlated with cell and matrix composition of the tissue. The compressive moduli of fetal and newborn bovine articular cartilage increased with depth (p < 0.05) by a factor of 4-5 from the top 0.1 mm (28 +/- 13 kPa, 141 +/- 10 kPa, respectively) to 1 mm deep into the tissue. Likewise, the glycosaminoglycan and collagen content increased with depth (both p < 0.001), and correlated with the modulus (both p < 0.01). In contrast, tissue-engineered cartilage formed by either layering or mixing cells from the superficial and middle zone of articular cartilage exhibited similarly soft regions at both construct surfaces, as exemplified by large equilibrium strains. The properties of immature cartilage may provide a template for developing tissue-engineered cartilage which aims to repair cartilage defects by recapitulating the natural development and growth processes. These results suggest that while depth-dependent properties may be important to engineer into cartilage constructs, issues other than cell heterogeneity must be addressed to generate such tissues. (c) 2005 Elsevier Ltd. All rights reserved.

A software tool for simulating spatial separation and kinetics in biological membranes

Self-segregation and compartimentalisation are observed experimentally to occur spontaneously on live membranes as well as reconstructed model membranes. It is believed that many of these processes are caused or supported by anomalous diffusive behaviours of biomolecules on membranes due to the complex and heterogeneous nature of these environments. These phenomena are on the one hand of great interest in biology, since they may be an important way for biological systems to selectively localize receptors, regulate signaling or modulate kinetics; and on the other, they provide an inspiration for engineering designs that mimick natural systems. We present an interactive software package we are developing for the purpose of simulating such processes numerically using a fundamental Monte Carlo approach. This program includes the ability to simulate kinetics and mass transport in the presence of either mobile or immobile obstacles and other relevant structures such as liquid-ordered lipid microdomains. We also present preliminary simulation results regarding the selective spatial localization and chemical kinetics modulating power of immobile obstacles on the membrane, obtained using the program.

Evaluation of silk sericin as a biomaterial: In vitro growth of human corneal limbal epithelial cells on Bombyx mori sericin membranes

Sericin and fibroin are the two major proteins in the silk fibre produced by the domesticated silkworm, Bombyx mori. Fibroin has been extensively investigated as a biomaterial. We have previously shown that fibroin can function successfully as a substratum for growing cells of the eye. Sericin has been so far neglected as a biomaterial because of suspected allergenic activity. However, this misconception has now been dispelled, and sericin’s biocompatibility is currently indisputable. Aiming at promoting sericin as a possible substratum for the growth of corneal cells in order to make tissue-engineered constructs for the restoration of the ocular surface, in this study we investigated the attachment and growth in vitro of human corneal limbal epithelial cells (HLECs) on sericin-based membranes. Sericin was isolated and regenerated from the silkworm cocoons by an aqueous procedure, manufactured into membranes, and characterized (mechanical properties, structural analysis, contact angles). Primary cell cultures from two donors were established in serum-supplemented media in the presence of murine feeder cells. Membranes made of sericin and fibroin-sericin blends were assessed in vitro as substrata for HLECs in a serum-free medium, in a cell attachment assay and in a 3-day cell growth experiment. While the mechanical characteristics of sericin were found to be inferior to those of fibroin, its ability to enhance the attachment of HLECs was significantly superior to fibroin, as revealed by the PicoGreen® assay. Evidence was also obtained that cells can grow and differentiate on these substrata.

Human corneal epithelial equivalents constructed on Bombyx mori silk fibroin membranes

Membranes prepared from a protein, fibroin, isolated from domesticated silkworm (Bombyx mori) silk, support the cultivation of human limbal epithelial (HLE) cells and thus display significant potential as biomaterials for ocular surface reconstruction. We presently extend this promising avenue of research by directly comparing the attachment, morphology and phenotype of primary HLE cell cultures grown on fibroin to that observed on donor amniotic membrane (AM), the current clinical standard substrate for HLE transplantation. Fibroin membranes measuring 6.3 ± 0.5 μm (mean ± sd) in thickness and permeable to FITC dextran of a molecular weight up to 70 kDa, were used. Attachment of HLE cells to fibroin was similar to that supported by tissue culture plastic but approximately 6-fold less than that observed on AM. Nevertheless, epithelia constructed from HLE on fibroin maintained evidence of corneal phenotype (K3/K12 expression) and displayed a comparable number and distribution of ΔNp63+ progenitor cells to that seen in cultures grown on AM. These results support the suitability of membranes constructed from Bombyx mori silk fibroin as substrata for HLE cultivation and encourage progression to studies of efficacy in preclinical models.

Antenatal ureaplasma infection impairs development of the fetal ovine gut in an IL-1-dependent manner

Ureaplasma infection of the amniotic cavity is associated with adverse postnatal intestinal outcomes. We tested whether interleukin-1 (IL-1) signaling underlies intestinal pathology following ureaplasma exposure in fetal sheep. Pregnant ewes received intra-amniotic injections of ureaplasma or culture media for controls at 3, 7, and 14 d before preterm delivery at 124 d gestation (term 150 d). Intra-amniotic injections of recombinant human interleukin IL-1 receptor antagonist (rhIL-1ra) or saline for controls  were given 3 h before and every 2 d after Ureaplasma injection. Ureaplasma exposure caused fetal gut inflammation within 7 d with damaged villus epithelium and gut barrier loss. Proliferation, differentiation, and maturation of enterocytes were significantly reduced after 7 d of ureaplasma exposure, leading to severe villus atrophy at 14 d. Inflammation, impaired development and villus atrophy of the fetal gut was largely prevented by intra-uterine rhIL-1ra treatment. These data form the basis for a clinical understanding of the role of ureaplasma in postnatal intestinal pathologies.

Parents’ experience of time distortion following diagnosis of a serious or lethal fetal anomaly

PURPOSE: To explore the experience of couples who continued pregnancy following a diagnosis of serious or lethal fetal anomaly. STUDY DESIGN: Thirty-one male and female participants were recruited from a high-risk maternal–fetal medicine clinic in Washington State. Data were collected using in-depth interviews during pregnancy and after the birth of their baby. Transcribed interviews were thematically analyzed through the phenomenological lens of Merleau-Ponty. FINDINGS: Participants described how time became reconfigured and reconstituted as they tried to compress a lifetime of love for their future child into a limited period. Participants’ concepts of time became distorted and were related to their perceptual lived experience rather than the schedule-filled,regimented, linear clock time that governed the health professionals. CONCLUSION: Living in distorted time may be a mechanism parents use to cope with overwhelming and disorienting feelings when their unborn baby is diagnosed with a fetal anomaly.

Ureaplasma parvum multiple banded antigen (MBA) size variation - association with fetal inflammation in a sheep model (Published abstract)

Background: Ureaplasma species are the most prevalent isolates from women who deliver preterm. The MBA, a surface exposed lipoprotein, is a key virulence factor of ureaplasmas. We investigated MBA variation after chronic and acute intra-amniotic (IA) ureaplasma infections. Method: U. parvum serovar 3 (2x104 colony-forming-units) was injected IA into pregnant ewes at: 55 days gestation (d, term = 145d) (n=8); 117d (n=8) and 121d (n=8). Fetuses were delivered surgically (124d) and ureaplasmas cultured from amniotic fluid (AF), chorioamnion, fetal lung (FL) and umbilical cord were tested by western blot and PCR assays to demonstrate MBA and mba gene variation respectively. Tissue sections were sectioned and stained by haemotoxylin and eosin and inflammatory cell counts and pathology were reported (blinded to outcome). Results: Numerous MBA/mba variants were generated in vivo after chronic exposure to ureaplasma infection but after acute infection no variants (3d) or very few variants (7d) were generated. Identical MBA variants were detected within the AF and FL but different ureaplasma variants were detected within chorioamnion specimens. The severity of inflammation within chronically infected tissues varied between animals ranging from no inflammation to severe inflammation with/without fibrosis. Chorioamnion, FL and cord from the same animal demonstrated the same degree of inflammation. Conclusions: MBA/mba variation in vivo occurred after the initiation of the host immune response and we propose that ureaplasmas vary the MBA antigen to evade the host immune response. In some animals there was no inflammation despite colonisation with high numbers of ureaplasmas.