Reconstruction of connective tissue from fibrin-based dermal equivalent transplanted to animals with experimental wounds

Wound healing in rats transplanted with dermal equivalent (DE) based on fibrin with dermal fibroblasts has been examined in this work. Histological studies of biopsy samples from dermis newly formed in the process of the model wound recovery in laboratory animals have shown the positive influence of DE on wound healing. It was found that the area of collagen fibers, number of precapillaries, capillaries and post-capillaries in granulation tissue were significantly increased in animals with transplanted fibrin-based DE compared to the rats of the control group indicating more intensive repair.     

Water transport and IIF parameters for a connective tissue equivalent

Understanding the biophysical processes that govern freezing injury of a tissue equivalent (TE) is an important step in characterizing and improving the cryopreservation of these systems. TEs were formed by entrapping human dermal fibroblasts (HDFs) in collagen or in fibrin gels. Freezing studies were conducted using a Linkam cryostage fitted to an optical microscope allowing observation of the TEs cooled under controlled rates between 5 and 130 degrees C/min. Typically, freezing of cellular systems results in two biophysical processes that are both dependent on the cooling rate: dehydration and/or intracellular ice formation (IIF). Both these processes can potentially be destructive to cells. In this study, the biophysics of freezing cells in collagen and fibrin TEs have been quantified and compared to freezing cells in suspension. Experimental data were fitted in numerical models to extract parameters that governed water permeability, E(Lp) and L(pg), and intracellular ice nucleation, omega(o) and kappa(o). Results indicate that major differences exist between freezing HDFs in suspension and in a tissue equivalent. During freezing, 55% of the HDFs in suspension formed IIF as compared to 100% of HDFs forming IIF in collagen and fibrin TE at a cooling rate of 130 degrees C/min. Also, both the water permeability and the IIF parameters were determined to be higher for HDFs in TEs as compared to cell suspensions. Between the TEs, HDFs in fibrin TE exhibited higher values for the biophysical parameters as compared to HDFs in collagen TE. The observed biophysics seems to indicate that cell-cell and cell-matrix interactions play a major role in ice propagation in TEs.     

Acceptance of a connective tissue equivalent for grafting and capsuloligamentary reconstruction

A strip of a connective tissue equivalent prepared by using the patient's fibroblasts cultivated in vitro and calf skin collagen, was used for capsuloligamentary reconstruction of the knee. The study of the humoral and cell-mediated immune responses indicated that there was no cell-mediated immune reaction and only a transient humoral reaction 2 months after implantation. This first assay in human surgery gave a good functional result, and an immunological response was no longer observed 5 months after grafting.     

Organization of connective tissue patterns by dermal fibroblasts in the regenerating axolotl limb

A set of tendons, aponeurotic sheets and retinaculae, which transduce muscle action from proximal limb levels to flexion and extension of the digits, is found in limbs of many vertebrates. This set of structures, here termed the digit tendon complex, is described for the axolotl forelimb. We show that the complex forms autonomously in muscleless axolotl limb regenerates produced from a cuff of unirradiated dermis surrounding an irradiated limb stump, and persists for up to a year after amputation. The pattern of other connective tissue structures, including the skeleton, is also normal. Fibroblast condensations that may represent sets of these cells normally associated with muscles in the extensor and flexor compartments of the carpal region also form in muscleless limbs. The results are discussed in terms of the importance of the dermis in pattern regulation, selforganization of connective tissues in general and autonomous development of the digit tendon complex in particular.     

Influence of dermal equivalent maturation on the development of a cultured skin equivalent.

Histologic and immunofluorescence methods were used to analyse the presence of fibronectin, chondroitin-4-sulphate and chondroitin-6-sulphate, type III and IV collagens, laminin, and keratins to assess the maturation level of cultured dermal and skin equivalents. In a first phase, fibroblasts in monolayer culture were compared with dermal equivalents in which fibroblasts are embedded in a type I collagen gel. Different fluorescent patterns were observed depending on the culture system used. A sequential appearance of macromolecules was noticed in dermal equivalents. Fibronectin was first detected after 4 days of culture, whereas chondroitin-4-sulphate and chondroitin-6-sulphate and type III collagen were present after 7 days. In contrast, all three macromolecules were detected at 24 h of culture in fibroblastic monolayer cultures. In a second phase, the quality of our skin equivalents was evaluated according to the seeding time of epidermal cells upon dermal equivalents (1, 4, or 7 days). A satisfactory stratification was obtained when keratinocytes were seeded after 4 and 7 days of dermal equivalent culture. Laminin and fibronectin were detected at the dermo-epidermal junction, but type IV collagen was absent. Various keratins, as detected by the AE1, AE2, and AE3 antibodies, were present in the epidermal layer. Following keratinocyte confluence, a change in the organization pattern of type III collagen in the dermal fraction of the skin equivalent was also noticed. Our comparative results show that seeding of epidermal cells on a more mature dermal equivalent leads to improved differentiation status of the epidermal layer.     

Lysozyme concentration in the loose connective tissue of animals with different species sensitivity to brucellosis]

Lysozyme content was determined in the subcutaneous cellular tissue of guinea pigs and albino rats differing by species resistance to brucella infection. Experiments were conducted on intact animals and those inoculated subcutaneously with live Br. abortus 19-BA vaccine. Connective tissue was taken from the site of the vaccine administration and from the contralateral side (control). Observations showed connective tissue of the animals highly sensitive to brucella infection to be exceedingly poor in this enzyme; as to connective tissue of albino rats with low sensitivity--it contained high amount of this enzyme. Lysozyme content increased considerably in the animals belonging to both species in the inflammatory focus developing at the site of the vaccine inoculation.     

Heterophile antibodies to the antigens of myocardial interstitial connective tissue and erythrocytes in animals of different species

Reactions of heterophilic antibodies with antigens of the interstitial connective tissue (ICT) of the myocardium of different animals were studied and compared. The reactions were identical on bovine, pig, sheep and rabbit myocardium. The heterophilic antigen of the ICT of bovine myocardium was also found on bovine, rabbit and rat red cells and was absent on sheep red blood cells. It is suggested that heterophilic antigen of the ICT of the bovine myocardium is a novel heterophilic antigen, the specificity of which is linked with D-galactose and which differs from antigens of other heterophilic systems.     

The use of human fibroblasts grown on microcarriers for the formation of the connective tissue equivalent

Live equivalents of tissues, specifically those produced on the basis of fibroblasts and collagen gel, are widely used for repair of organ and tissues defects. In clinical practice, it is more convenient to use the fibroblasts grown on microcarriers or such a connective tissue equivalent when the fibroblasts on microcarriers are embedded in collagen gel. We studied the properties of a connective tissue equivalent produced by embedding the fibroblasts grown on microcarriers in collagen gel for its prospective use in clinical practice. According to our results, the optimal time of use of the live tissue equivalent amounts to three--four days after embedding of fibroblasts on microcarriers in gel. At that time, contraction only begins, which facilitates manipulations with the gel.     

Bone and soft connective tissue alterations result from loss of fibrillin-2 expression

Fibrillins 1, 2 and 3 make up a family of genes that encode large, cysteine-rich extracellular matrix glycoproteins found in connective tissues, lung, blood vessels and other extensible tissues. Fibrillins 1 and 2 have both overlapping as well as separate distributions in human embryonic and adult tissues. Fibrillin-containing microfibrils are known to modulate morphogenetic events by proper targeting of growth factors to the extracellular matrix. Mutation of the fibrillin-2 gene causes a genetic disorder, congenital contractural arachnodactyly (CCA), that results in flexion contractures. Previously, we have shown a distinct fibrillin-2 distribution in the pericellular matrix of interior tenocytes and later demonstrated a unique fibrillin-2 containing structure that runs along the tendon cell arrays in the canine flexor tendon. We hypothesized that loss of these fibrillin-2 containing structures might affect normal tendon development. To test our hypothesis, connective tissues from mice null for fibrillin-2 gene expression were studied. Murine flexor digitorum longus tendons were evaluated for total collagen content, and the intermolecular collagen cross-links hydroxylysyl and lysyl pyridinoline. The results show decreased collagen cross-links in fibrillin-2 null mice, however total collagen content remained the same when compared to wild type. Bone morphology was studied using micro computed tomography (CT). Fibrillin-2 null mice display a focal area of decreased bone length in the extremities as compared to wild type mice. Together, these results demonstrate a role for fibrillin-2 in bone and soft connective tissue morphological and biochemical processes.     

Collagen-polysaccharide gel as a model of intercellular substance of the connective tissue]

Gel samples forming at 37 degrees C in the solutions containing tropocollagen and various polysaccharides were examined by electron microscopy. Contracting gel clots formed in the solutions containing chondroitin sulfate, proteoglycine from the tracheal cartilage, gum arabic. Electron microscopy showed such clots to be permeated with collagen fibrillae with transverse striations and a period of 640 A. An association between the density of the forming gel and the nature of the polysaccharide component is discussed. Gel forming in the solutions containing tropocollagen and various polysaccharides is regarded as a model of the connective tissue intercellular substance.