Molecular responses of human dermal fibroblasts to dual cues: contact guidance and mechanical load.

Fibroblast contraction in wound healing involves the interaction of several cell types, cytokines, and extracellular matrix molecules. We have previously developed fibroblast alignment models using precise uniaxial mechanical loads in 3D culture and using contact guidance on fibronectin strands. Our aim here was to use contact guidance to place fibroblasts in their potentially most sensitive configuration, i.e., perpendicular to the axis of loading, to present cells with conflicting guidance cues. Gene expression at the mRNA level of cells recovered from different zones of the 3D collagen gel (with distinct orientation) was determined by quantitative RT-PCR for the matrix proteases MMP1, 2, and 3, and inhibitors TIMP1 and 2. Our results show a 2-, 4-, and 3-fold increase in MMP1, 2, and 3, respectively, in the non-aligned strain zone, relative to the aligned strain zone. These results suggest that cells unable to align to applied loads remodel their matrix far more rapidly than orientated cells. Where fibroblasts were held in an alignment perpendicular to the applied load by contact guidance, the fall in MMP mRNA expression was largely abolished, indicating that these cells remained in a mechano-activated state. The protease inhibitors TIMP1 and 2 were poorly mechano-responsive, further suggesting that changes in MMP expression result in functional matrix remodelling. These results indicate how mechanical loading in tissues may influence matrix remodelling, particularly under conflicting guidance cues.

Effect of precise mechanical loading on fibroblast populated collagen lattices: morphological changes.

The contraction of a collagen lattice by resident fibroblasts causes strains to be developed within that lattice. These strains can be increased or decreased by altering the aspect ratio (ratio of length/width/thickness) of the fibroblast populated collagen lattice, as the cross-sectional area resisting the strain is changed and by the application of an external load. The fibroblasts align themselves with the direction of the maximum principle strain; in effect, these cells are "hiding" from the perceived strain. The direction of the maximum principle strain can be predetermined by the use of a computational finite element analysis. Using the tensioning-Culture Force Monitor to apply pre-determined loading patterns of known repeatable magnitudes, as calculated by the finite element analysis, we have succeeded in aligning fibroblasts into a deliberate predicted orientation. This study has shown that the resident fibroblast population will respond to changes in strain resulting from the most subtle of mechanical loads. This may be an important mechanism in development and repair of connective tissue.

Clinical heterogeneity of silent-gene b-thalassemia among Indians.

This paper describes six Indian families in which silent-gene beta-thalassemia was encountered by us in the past 2 years. This stresses the importance of globin-chain synthesis in the diagnosis of beta-thalassemia. Of these six cases, five were diagnosed retrospectively when they had a homozygous beta-thalassemia offspring. The beta/alpha synthetic ratios ranged from 0.45 to 0.60. Four belong to group-I silent beta-thalassemia and two cases to group II. The severity of thalassemia major in the offspring of these silent carriers cannot be predicted.

Characterization of G6PD Rohini--a new class III Indian variant.

A new Indian G6PD variant was detected in a 15 yr old Maratha male during a population screening programme in high school children in Bombay (India). The propositus and two family members having the same variant were apparently healthy. This enzyme variant has a mild erythrocyte G6PD deficiency and a slow electrophoretic mobility. It is characterized by a high Michaelis-Menton constant for G6P, a bimodal curve for pH optima and a slight decrease in the thermostability. The rate of utilization of substrate analogue is similar to that of normal. These observations suggest identification of a new class III variant, designated as G6PD Rohini.