3D culture model of fibroblast-mediated collagen creep to identify abnormal cell behaviour.

Native collagen gels are important biomimetic cell support scaffolds, and a plastic compression process can now be used to rapidly remove fluid to any required collagen density, producing strong 3D tissue-like models. This study aimed to measure the mechanical creep properties of such scaffolds and to quantify any enhanced creep occurring in the presence of cells (cell-mediated creep). The test rig developed applies constant creep tension during culture and measures real-time extension due to cell action. This was used to model extracellular matrix creep, implicated in the transversalis fascia (TF) in inguinal hernia. Experiments showed that at an applied tension equivalent to 15% break strength, cell-mediated creep over 24-h culture periods was identified at creep rates of 0.46 and 0.38%/h for normal TF and human dermal fibroblasts, respectively. However, hernia TF fibroblasts produced negligible cell-mediated creep levels under the same conditions. Raising the cell culture temperature from 4 to 37 °C was used to demonstrate live cell dependence of this creep. This represents the first in vitro demonstration of TF cell-mediated collagen creep and to our knowledge the first demonstration of a functional, hernia-related cell abnormality.

Investigating the mechanical shear-plane between core and sheath elements of peripheral nerves.

The mechanical architecture of rat sciatic nerve has been described as a central core surrounded by a sheath, although the way in which these structures contribute to the overall mechanical properties of the nerve is unknown. We have studied the retraction responses of the core and sheath following transection, together with their tensile properties and the interface between them. Nerves were harvested and maintained at their in situ tension and then either transected entirely, through the sheath only, or through an exposed section of the core. The retraction of each component was measured within 5 min and again after 45 min. Post mortem loss of retraction was tested 0 min or 60 min after excision. For fresh nerves, immediate retraction was 12.68% (whole nerve), 5.35% (sheath) and 4% (core), with a total retraction of 15%, 7.21% and 5.26% respectively. For stored nerves, immediate retraction was 5.33% (whole nerve) and 5.87% (sheath), with an extension of 0.78% for core, and a total retraction of 6.71% and 7.87% and an extension of 1.74%, respectively. Tensile extension and pullout force profiles were obtained for the sheath, the core and the interface between them. These showed a consistent hierarchy of break strengths that would, under increasing load, result in failure of the interface, then the core and finally the sheath. These data reflect the contributions of material tension and fluid swelling pressure to total retraction, and the involvement of an energy-dependent process that runs down rapidly post mortem. This study increases our understanding of the composite nature of peripheral nerve tissue architecture and quantifies the material properties of the distinct elements that contribute to overall mechanical function.

Peripheral nerves in the rat exhibit localized heterogeneity of tensile properties during limb movement.

Peripheral nerves in the limbs stretch to accommodate changes in length during normal movement. The aim of this study was to determine how stretch is distributed along the nerve relative to local variations in mechanical properties. Deformation (strain) in joint and non-joint regions of rat median and sciatic nerves was measured in situ during limb movement using optical image analysis. In each nerve the strain was significantly greater in the joint rather than the non-joint regions (2-fold in the median nerve, 5- to 10-fold in the sciatic). In addition, this difference in strain was conserved in the median nerve ex vivo, demonstrating an in-built longitudinal heterogeneity of mechanical properties. Tensile testing of isolated samples of joint and non-joint regions of both nerves showed that joint regions were less stiff (more compliant) than their non-joint counterparts with joint: non-joint stiffness ratios of 0.5 +/- 0.07 in the median nerve, and 0.8 +/- 0.02 in the sciatic. However, no structural differences identified at the light microscope level in fascicular/non-fascicular tissue architecture between these two nerve regions could explain the observed tensile heterogeneity. This identification of localized functional heterogeneity in tensile properties is particularly important in understanding normal dynamic nerve physiology, provides clues to why peripheral nerve repair outcomes are variable, and suggests potential novel therapeutic targets.

Engineering and cell attachment properties of human fibronectin-fibrinogen scaffolds for use in tissue engineered blood vessels.

Tissue engineered constructs reported to date have been prepared primarily from poly(glycolic) acid or collagen scaffolds onto which cells are grown and matured. In this paper we report experimental data to demonstrate the use of a natural, human protein, as a tubular scaffold for vascular grafting. Using a manual and a scalable dip-coating technique we prepared fibronectin-based tubes up to 12 cm in length and up to 3 mm in diameter. The tubes were flexible and their mechanical properties, measured in terms of tensile strength and burst pressure as a function of humidity, demonstrated their suitability as scaffolds for use in vascular grafting, e.g. coronary artery by pass grafting. In vitro tests involved the attachment of endothelial cells pumped under laminar flow conditions through the tube lumen and the adherence of smooth muscle cells on the outer surface of the tubes. These tests, carried out in multiwells, showed that the scaffolds had excellent cell attachment and guidance characteristics.

Early breaking strength of repaired flexor tendon treated with 5-fluorouracil.

This study investigated the effect of a single intraoperative application of 5-fluorouracil, which may diminish peritendinous adhesion formation, on the tensile strength of repaired digital flexor tendons after 7, 14 and 21 days of healing. Twenty-seven deep flexor tendons from 14 rabbits were exposed to 5-fluorouracil (50 mg/ml) for 5 minutes immediately after repair whereas matched control tendons were exposed to normal saline. Tensile testing at 7, 14 and 21 days revealed no significant differences in the gap or ultimate strengths of the 5-fluorouracil treated and control tendons.

Wet extrusion of fibronectin-fibrinogen cables for application in tissue engineering.

A method for the wet extrusion of human plasma-derived fibronectin-fibrinogen cables is described. Solutions of fibronectin and fibrinogen with and without sodium alginate and carboxymethylcellulose (CMC) are tested. The rheological properties of the protein solutions changed from Newtonian to shear thinning non-Newtonian in the presence of small quantities of these additives, the apparent viscosity increased, and the extrusion properties of the protein solutions improved. Cables were prepared using a capillary with a diameter of 1 mm and overall length of 18 mm. Cable diameter was reduced to about 0.5 mm by drawing using a series of rollers. Cables prepared with sodium alginate were found to have suitable properties, and those made with CMC were sticky and difficult to handle. Solutions containing no sodium alginate required a minimum total protein concentration of about 70 mg/mL for extrusion. Extruded cables were prepared with solutions containing 140 mg/mL total protein with 12.9 mg/mL alginate (high protein), and 46 mg/mL total protein with 47.6 mg/mL of sodium alginate (high alginate). The mechanical strength of the extruded cables was within the range suitable for application in tissue engineering. Extrusion of the protein solutions into cables was achieved in a coagulation bath. Cables with a mechanical strength of approximately 30 N/mm(2), suitable for wound repair and nerve regeneration applications, were prepared with a coagulation bath containing 0.25 M HCl, 2% CaCl(2) at a pH of <0.9. These cables also had a large average elongation at break of 52%, and showed an increase in cable length after breakage (permanent set) of 20%, demonstrating the potential for drawing the cables down to a fine diameter.

A new core suture technique for flexor tendon repair: biomechanical analysis of tensile strength and gap formation.

The purpose of this study was to test in vitro a new flexor tendon suture technique that has been developed to withstand the in vivo forces of active tendon motion. This new core suture technique involves locking loops in the form of a cross stitch. The new technique was tested for ultimate tensile strength and gap formation in cadaver human flexor tendons on a tensile testing machine. The new technique proved significantly stronger than the modified Kessler core suture.

Aortic root characteristics of human pulmonary autografts.

BACKGROUND: After pulmonary autograft replacement of the aortic valve and root, the pulmonary artery (PA) wall is subjected to higher pressures. Concern exists that this may lead to structural and functional changes in the implanted autograft and subsequent aortic root dilatation and neoaortic regurgitation. We therefore assessed root dimensions and neoaortic regurgitation, morphological structure, and mechanical behavior in patients who underwent the Ross operation. METHODS AND RESULTS: Seventy-four patients who were randomized to undergo aortic valve replacement with an aortic homograft or a pulmonary autograft were followed up echocardiographically for up to 4 years and had their aortic root dimensions measured at the level of the annulus, sinuses, and sinotubular junction. In a separate series of 18 patients who underwent pulmonary autograft surgery and 8 normal organ donors, samples from the PA and aorta were analyzed for medial wall thickness, distribution of the staining of collagen and elastin, and elastin fragmentation. Finally, stress-strain curves were obtained from samples of the PA and aorta from 9 patients who underwent pulmonary autograft surgery and from 1 patient in whom a 4-month-old autograft was explanted. No patient in either group had aortic dilatation at any level of >20% or more than mild aortic regurgitation at up to 4 years of follow-up. The aortic media was thicker in both autografts and normal donors (P:<0.01), and there was a trend for the PA media to be thicker in the autograft group. Elastic fiber in all aortas showed little or no variation, whereas in the PA, there was considerable variation in fragmentation. Patients with higher preoperative PA pressures tended to have lower fragmentation scores (chi(2) P:<0.01). The lower stiffness modulus, higher stiffness modulus, and maximum tensile strength of the aorta was 34% to 38% higher than that of the PA (P:<0.01); however, the 4-month-old autograft appeared to show adaptation in mechanical behavior. CONCLUSIONS: In our series of patients, there was no significant progressive dilatation of the aortic root. We demonstrated differences in the anatomic structure and mechanical behavior of the PA in vitro and highlighted histological and mechanical modes of adaptation.

Biomechanical characterization of tissues in Dupuytren's disease.

The aim of this study was to characterize mechanical properties of tissues of Dupuytren's disease and to attempt to identify changes due to cellular activity. Tensile tests confirmed the heterogeneity of Dupuytren's disease tissue with distinct stress-strain curves for the three tissue types normally present, namely, cord, transition zone and nodule. The tensile strength for cord tissue was nearly twice that of nodule tissue, but the latter was nearly twice as stretchable as cord. In contrast, the transition tissue had the tensile strength of cord with the stretchability of nodule. It was found that tensile loading stimulated a cellular response as demonstrated by an increase in the creep strain rate of the tissue at 37 degrees C compared with that at 4 degrees C using Dupuytren's tissue in an in vitro culture test. The creep strain rate for nodule at 37 degrees C was more than seven times that for cord at a nominal creep stress of 0.75 MPa.

Mechanical stress in vitro induces increased expression of MMPs 2 and 9 in excised Dupuytren's disease tissue.

We have previously shown that the ability to mechanically extend Dupuytren's contractures in vivo by the Continuous Elongation Technique before surgery resulted in increased metalloproteinase activity. However, under these conditions it was not possible to show whether the response was proportional to the mechanical stimulus or was inflammatory cell mediated. Using an in vitro system of controlled extensions in which inflammatory involvement is absent, we have now shown that there is a clear correlation between the load applied to the tissue and the release of matrix metalloproteinase-2. The subsequent degradation of the collagen results in a loss of mechanical strength reported in the preceding paper.

Seasonal variation and sex differences of circulating macrophages, immunoglobulins and lymphocytes in healthy school children.

Subpopulations of T and B lymphocytes and levels of serum immunoglobulins G, A, M, E and subclasses G1, G2 and G3 were studied in 45 healthy school children aged 8-16 years during four seasons of the year. There were significant increases in CD4+ T helper cells, total T lymphocytes and CD4+/CD8+ (helper/cytotoxic) T-cell ratio during the spring season. While the levels of CD8+ T cells and total B lymphocytes remained statistically unchanged during all four seasons, the levels of natural (HNK-1) killer cells and macrophages increased significantly during the autumn and summer seasons respectively. The levels of immunoglobulins G, A, M and E remained statistically unchanged during all four seasons. Girls had higher levels of CD4+ T cells and a higher CD4+/CD8+ T-cell ratio than boys. Girls also had slightly higher levels of immunoglobulin G and M. These observations suggest that seasonal variations of some immunological parameters occur in healthy children. This may be an adaptive response to variable climatic and other environmental factors. These natural variations due to seasonal changes should be taken into account when immunological tests are used in clinical investigations.

Prevalence and clinical picture of IDDM in Nigerian Igbo schoolchildren.

OBJECTIVE: To evaluate the prevalence and clinical picture of IDDM in Nigerian Igbo schoolchildren born and living in continental Africa. RESEARCH DESIGN AND METHODS: In three school districts (Ezza, Ishielu, Ohaukwu), 77,862 schoolchildren aged 5-17 yr answered our questionnaires on age, sex, known disease, drugs being taken, family history, and diabetic symptoms like polyuria, polydipsia, polyphagia, and weight loss. Positive respondents were given glucosuric tests, and glucosuric subjects had hyperglycemic tests. Diagnosis of IDDM was established in hyperglycemic patients by referred hospital clinicians based on insulin requirements. RESULTS: Twelve new cases of IDDM were found in addition to 14 previously diagnosed cases, giving a CPR of 0.33/1000. Ishielu had a CPR of 0.46/1000 compared with 0.25 (P less than 0.01) for Ohaukwu. Boys had a CPR of 0.38/1000 compared with 0.25/1000 (P less than 0.06) for girls. Boy-to-girl prevalence ratio was approximately 3:1. CONCLUSIONS: The relatively high prevalence of IDDM in this poor African population, despite potential deaths caused by minimal medical attention, may be because of long-term protein malnutrition and endemic childhood infections, which have been implicated in the etiology of IDDM in similar malnourished populations.

Raised IgG and IgM in "epidemic" IDDM suggest that infections are responsible for the seasonality of type I diabetes.

Serum levels of immunoglobulins G, A and M were quantitatively measured at diagnosis and at regular intervals for four years in 92 type I (insulin-dependent) diabetes patients. The patients were 0.8-15.99 years of age at diagnosis. Thirty-six of them got diabetes during periods of high incidence (the "epidemic" group) and 56 of them were diagnosed during periods of seemingly low incidence (the non-"epidemic" patients). Fifty percent (18/36) of the "epidemic" group had infections less than two months preceding diagnosis as compared to 29% (16/56) (p less than 0.01) of the non-"epidemic" patients. At diagnosis immunoglobulins G, and M in the "epidemic" group were 11.28 +/- 2.0 and 1.97 +/- 0.77 as compared to 9.9 +/- 2.3 (p less than 0.01) and 1.31 +/- 0.58 (p less than 0.001) respectively in the non-"epidemic" patients. The same highly significant differences were observed when mean values of IgG and IgM were compared between children who had infections less than two months before diagnosis and those without preceding infections. Except for slightly higher (p less than 0.12) total (OKT3) T-lymphocytes and higher (p less than 0.03) B-lymphocytes at diagnosis in the "epidemic" group, there were no significant differences in quantitative T- and B-lymphocyte subpopulations between various groups. 6.3% (2/32) of the patients who had preceding infections had HLA-DR3/non-DR4 genotypes as compared to 30.6% (15/49); p less than 0.001, of those patients without preceding infections.(ABSTRACT TRUNCATED AT 250 WORDS)

Seasonality of type 1 (insulin-dependent) diabetes mellitus: values of C-peptide, insulin antibodies and haemoglobin A1c show evidence of a more rapid loss of insulin secretion in epidemic patients.

According to month of diagnosis, 165 children who developed Type 1 (insulin-dependent) diabetes mellitus at the age of 0-16.2 years (mean +/- SD, 7.6 +/- 4.1 years) could be divided into 69 patients diagnosed during peak seasons (epidemic cases) and 96 patients diagnosed during months of low incidence (non-epidemic cases). Seasonality of onset of symptoms and of diagnosis was observed in both sexes in all age groups. The patients diagnosed during peak seasons had shorter duration of symptoms (13.2 +/- 8.1 days) as compared to 22.9 +/- 10.3 days; p less than 0.001 in the patients diagnosed during months of low incidence. At diagnosis, 88.4% (61/69) of the epidemic group had ketonuria as compared to 71.9% (69/96); p less than 0.06 in the non-epidemic patients. The values of C-peptide, insulin antibodies, haemoglobin A1c and HLA-DR phenotype frequencies in the 69 epidemic patients were compared with those of the 96 non-epidemic patients. In the epidemic patients, the C-peptide values of 0.11 +/- 0.05 mmol/l at diagnosis had increased to 0.12 +/- 0.05 mmol/l at one month and 0.13 +/- 0.06 mmol/l at 3 months. These values were significantly lower (p less than 0.001) than in the non-epidemic patients at the same time points: 0.17 +/- 0.08 nmol/l; p less than 0.001, 0.23 +/- 0.11 nmol/l; p less than 0.001, and 0.22 +/- 0.10 nmol/l.(ABSTRACT TRUNCATED AT 250 WORDS)

Synovial fluid circulation in the hip joint.

Early in our study of the role of incongruency in the hip (Ref. 1) we observed by x-ray screening that, under load, a drop of barium sulphate placed between the articular surfaces spread out between them and a substantial portion was extruded from the intercartilage space into the fossa; all the barium was drawn back as load was reduced. This observation and others (Ref. 2) which demonstrated the location, size and behaviour of intercartilage space with movement and load, prompted exploration of the hypothesis that the space due to joint incongruency serves as a pump to transport synovial fluid in the articular cavity in aid of nutrition and lubrication. This had already been suggested by others (Ref. 3); but none have closely studied the hypothesis. The following is an expansion of this idea, which leads to the concept of a systematic circulation of fluid through the hip joint.