Biomimetic approach to tissue engineering

The overall goal of tissue engineering is to create functional tissue grafts that can regenerate or replace our defective or worn out tissues and organs. Examples of grafts that are now in pre-clinical studies or clinical use include engineered skin, cartilage, bone, blood vessels, skeletal muscle, bladder, trachea, and myocardium. Engineered tissues are also finding applications as platforms for pharmacological and physiological studies in vitro. To fully mobilize the cell's biological potential, a new generation of tissue engineering systems is now being developed to more closely recapitulate the native developmental milieu, and mimic the physiologic mechanisms of transport and signaling. We discuss the interactions between regenerative biology and engineering, in the context of (i) creation of functional tissue grafts for regenerative medicine (where biological input is critical), and (ii) studies of stem cells, development and disease (where engineered tissues can serve as advanced 3D models).     

Leech responses to tissue transplantation

The aim of the present work is to describe histologically, histochemically and immunocytochemically, the sequence of events that lead to first and second set rejection of allo- or xenograft in leeches. Graft responses of leeches are comparable and are described following specific steps: inflammatory phase, rejection phase and granulation tissue formation (including re-epithelialisation, angiogenesis and fibroplasia).The responses to first and second graft in first set graft rejection as well as to the first transplant in second set graft experiments are identical and in the time span of a week all grafts are destroyed and disappear. In the second set graft rejection experiments the responses against the second transplant are markedly accelerated. The second graft shows massive structural alterations and it is rapidly rejected, within 3-4 days.Our results permit to highlight that in leeches there is a specific responsiveness of immune system similar to those described in highly divergent phyla.     

Dystocia due to soft tissue

In dystocia caused by abnormal conditions of the soft parts, the etiologic changes may be either in the genital tissues or in adjacent soft structures. Broadly, the conditions causing the difficulty may be grouped as follows: (1) anomalies or congenital modifications; (2) tumors; (3) modifications due to age, accident or surgical operations; (4) modification of the expulsive forces; (5) abnormalities of the products of conception. Often in such circumstances cesarean section is necessary. Sometimes when tumor is present it can be removed before it interferes with delivery, but decision to excise the growth must be guided by such factors as the location of the lesion and the stage of gestation. This would determine to what extent the maintenance of pregnancy would be jeopardized by surgical intervention before term.     

Sensitivity of tissue differentiation and bone healing predictions to tissue properties

Computational models are employed as tools to investigate possible mechano-regulation pathways for tissue differentiation and bone healing. However, current models do not account for the uncertainty in input parameters, and often include assumptions about parameter values that are not yet established. The aim was to clarify the importance of the assumed tissue material properties in a computational model of tissue differentiation during bone healing. An established mechano-biological model was employed together with a statistical approach. The model included an adaptive 2D finite element model of a fractured long bone. Four outcome criteria were quantified: (1) ability to predict sequential healing events, (2) amount of bone formation at specific time points, (3) total time until healing, and (4) mechanical stability at specific time points. Statistical analysis based on fractional factorial designs first involved a screening experiment to identify the most significant tissue material properties. These seven properties were studied further with response surface methodology in a three-level Box–Behnken design. Generally, the sequential events were not significantly influenced by any properties, whereas rate-dependent outcome criteria and mechanical stability were significantly influenced by Young's modulus and permeability. Poisson's ratio and porosity had minor effects. The amount of bone formation at early, mid and late phases of healing, the time until complete healing and the mechanical stability were all mostly dependent on three material properties; permeability of granulation tissue, Young's modulus of cartilage and permeability of immature bone. The consistency between effects of the most influential parameters was high. To increase accuracy and predictive capacity of computational models of bone healing, the most influential tissue mechanical properties should be accurately quantified.     

Biomimetic approach to cardiac tissue engineering

Here, we review an approach to tissue engineering of functional myocardium that is biomimetic in nature, as it involves the use of culture systems designed to recapitulate some aspects of the actual in vivo environment. To mimic the capillary network, subpopulations of neonatal rat heart cells were cultured on a highly porous elastomer scaffold with a parallel array of channels perfused with culture medium. To mimic oxygen supply by haemoglobin, the culture medium was supplemented with a perfluorocarbon (PFC) emulsion. Constructs cultivated in the presence of PFC contained higher amounts of DNA and cardiac markers and had significantly better contractile properties than control constructs cultured without PFC. To induce synchronous contractions of cultured constructs, electrical signals mimicking those in native heart were applied. Over only 8 days of cultivation, electrical stimulation induced cell alignment and coupling, markedly increased the amplitude of synchronous construct contractions and resulted in a remarkable level of ultrastructural organization. The biomimetic approach is discussed in the overall context of cardiac tissue engineering, and the possibility to engineer functional human cardiac grafts based on human stem cells.     

New approaches to insect tissue culture

Conclusion Current methods of insect cell culture have produced a limited variety of cell types in an ever expanding list of insect cell lines. In developing midgut epithelial cell lines, we found that traditional methods in insect cell culture failed to provide healthy cells from mature tissues. Examination of mammalian cell culture literature for this particular cell type provided the insight required to successfully develop a cell-specific line (Baines et al., 1994). The potential applications for cell-specific lines from insects are numerous. This paper is a compilation of ideas that will hopefully enable other researchers to develop additional cell-specific lines.     

Circulating autoantibodies to mammalian tissue kallikreins

Autoantibodies to tissue kallikrein (EC 3.4.21.35) were discovered in normal human, rat, mouse, and guinea pig sera. Three independent methods--binding of iodolabeled antigen, enzyme-linked immunosorbent assay (ELISA), and immunoblotting--were used to demonstrate these kallikrein autoantibodies. Autoantibodies from rat and human sera were purified, using rat and human tissue kallikrein-affinity chromatography, respectively. Purified rat kallikrein autoantibody bound 50% of 125I-labeled rat urinary kallikrein upon incubation of antibody at 2.5 X 10(-10) M. The subtypes of rat and human kallikrein autoantibodies were determined by an ELISA, using antisera to immunoglobulin subclasses. In both species, autoantibody was predominantly IgG (approximately 80%) and some IgM (approximately 20%). Purified autoantibodies from rat and human sera were separated on sodium deodecyl sulfate-polyacrylamide gels, and their subunits were identified by Western blot analyses, using anti-rat and anti-human IgG antibodies, respectively. When primary cultures of mouse spleen cells were incubated for 1 to 5 days with lipopolysaccharide (1 to 5 micrograms/ml), the anti-kallikrein antibodies in the media increased up to seven-fold. We have demonstrated circulating autoantibodies that recognize and bind both autologous and heterologous kallikrein; however, their significance to the function of the tissue kallikrein-kinin system in normal and disease states remains to be explored.     

Regulatory mechanisms to control tissue alpha-tocopherol

To test the hypothesis that hepatic regulation of alpha-tocopherol metabolism would be sufficient to prevent overaccumulation of alpha-tocopherol in extrahepatic tissues and that administration of high doses of alpha-tocopherol would up-regulate extrahepatic xenobiotic pathways, rats received daily subcutaneous injections of either vehicle or 0.5, 1, 2, or 10 mg alpha-tocopherol/100 g body wt for 9 days. Liver alpha-tocopherol increased 15-fold in rats given 10 mg alpha-tocopherol/100 g body wt (mg/100 g) compared with controls. Hepatic alpha-tocopherol metabolites increased with increasing alpha-tocopherol doses, reaching 40-fold in rats given the highest dose. In rats injected with 10 mg/100 g, lung and duodenum alpha-tocopherol concentrations increased 3-fold, whereas alpha-tocopherol concentrations of other extrahepatic tissues increased 2-fold or less. With the exception of muscle, daily administration of less than 2 mg/100 g failed to increase alpha-tocopherol concentrations in extrahepatic tissues. Lung cytochrome P450 3A and 1A levels were unchanged by administration of alpha-tocopherol at any dose. In contrast, lung P-glycoprotein (MDR1) levels increased dose dependently and expression of this xenobiotic transport protein was correlated with lung alpha-tocopherol concentrations (R(2)=0.88, p<0.05). Increased lung MDR1 may provide protection from exposure to environmental toxins by increasing alveolar space alpha-tocopherol.