Cynthia G. Brown-Maupin

Effects of Muscle Contraction on the Load-Strain Properties of Frog Aponeurosis and Tendon

The mechanical properties of the frog semitendinosus (ST) tendon and aponeurosis were measured during passive tensile loading to a force equal to ST maximum tetanic tension and during active isometric muscle contraction. During active contraction, both the tendon and aponeurosis regions initially strained at rates exceeding 400%/s while near the end of the muscle contraction, strain rates were nearly zero. At this point, the strain in the tendon region was equal to that observed during slow passive loading to the same tension level. However, for the aponeurosis, even near the zero strain rate, strain at the end of the active contraction was significantly below that observed during slow passive loading (p < 0.001). Specifically, when aponeurosis strain rate was almost zero, aponeurosis strain was 13.8 ± 3% (means ± SEM, n = 10), which was significantly below that measured during passive loading (23.7 ± 5%) suggesting that active contraction actually altered aponeurosis material properties. These data demonstrate that, while the tendon and aponeurosis regions have different passive biomechanical properties and both demonstrate viscosity typical of other connective tissues, the aponeurosis region of the frog ST actually changed its intrinsic properties during muscle contraction. Thus, extrapolation of biomechanical data obtained at nonphysiological strain rates or under conditions where the muscle-tendon junction has been interrupted should be made with caution.

A101–A107 Skin Substitutes and Wound Healing/Medical Applications

Skin Substitutes and Wound Healing Medical Applications

A201–A209 Gene Therapy and Growth Factors in TE/Basic Sciences/Developmental Biology and TE/Microsystem Technology in TE

Introduction: Cell-based implants for Tissue Engineering are dependent on specific bioactive factors. Transforming Growth Factor-̌ 1 (TGF-̌ 1) has be shown to promote chondrogenesis and may be useful for promoting cartilage repair when expressed from a transgene in a cell-based implant. Retroviral vectors are one of the preferred modes for transgene transduction into mesenchymal cells, however, successful transduction is dependent on active proliferating cells. This study was designed to optimize the TGFˇ1-retroviral transduction efficiency of bone marrow-derived progenitor cells and to study the effects of transgene TGFˇ1 production on chondrogenesis. Methods: Bone marrow-derived mesenchymal progenitor cells, which were obtained from human bone marrow aspirates, were cultured in DMEMC 10% FBS without (group A) or with addition of Epidermal Growth Factor(EGF) (10 ng/ml; group B) to optimize the proliferation rate. After 5 days cell counts were obtained with a MTT proliferation assay. Cells of group A and B were transduced after 24 or 72 hours in culture with retroviral vector DFG-TGF-̌1-Zeo (Polybrene: 8μg/ml; centrifugation with 1700 rpm for 1 hour). Transduced cells were selected with three selection doses of Zeocin after 4 days in culture. The amount of transduced cells was determined with a MTT proliferation assay. The TGFˇ1 protein-production was measured preand post-selection with a TGFˇ1 specific ELISA. Transduced cells of group A and B were assayed for their chondrogenic potential in anin vitro chondrogenesis model [1,2] and compared to the equivalent non-transduced controls. To assay the effect of the transgene expression, cells were incubated with/without exogenous TGF-̌1 protein. Results: An increased proliferation of bone marrow-derived mesenchymal cells could be achieved with exogenous addition of EGF (group B) (in 4 days cell count 40% above control (group A)). Without addition of EGF (group A) the different time points of transduction (transduction after 24 or 72 hours of preculture) showed no significant difference in transduction rate (approximately 10%). However, with addition of EGF (group B) douple as much cells could be transduced after 72 hours of preculture compared to 24 hours of preculture. After preculture for 72 hours the transduction rate was 20%. Furthermore, with EGF addition (group B; 100 pg/1.000 cells) a 100-fold increase of TGF-̌1 protein production could be achieved in comparison to the control (group A; 1 pg/1.000 Zellen). This ratio could also be detected after selection with Zeocin. Transduced cells, which were cultured in DMEM C 10% FBS (group A), underwentin vitro chondrogenesis with and without exogenous addition of TGFˇ1, indicating that the transgene was functional and that the cells were producing enough TGFˇ1 to promote differentiation. Cells under EGF-influence (group B) showed with and without exogenous addition of TGFˇ1 no chondrogenesis. Even in non-transduced controls, which were cultured in EGF-containing medium, no chondrogenic differentiation could be detected. Discussion: TGF-̌ 1 transduced mesenchymal progenitor cells can produce a sufficient amount of active TGFˇ1 protein, to undergo chondrogenesis. EGF-containing medium enhances cell proliferation, retroviral transduction efficiency and TGFˇ1 protein production per cell compared with DMEM and 10% FBS alone. However, mesenchymal progenitor cells seems to loose chondrogenic differentiation potential when cultured in EGF-containing medium. This study shows the potential of gene therapy to optimize cell-based implants for Tissue Engineering. Furthermore the study shows, that methods, to optimize cell-proliferation and transduction, could have also negative effects on cell differentiation.

B601–B626 Cartilage Repair/Skeletal Tissue/Stem Cells

In the process of tissue-engineering controlled ex-vivo expansion of autologous cells is still a major limiting factor. Current practice employs serum mitogens to stimulate proliferation. Besides concerns about low virus diseases the variation in serum batches results in hardly predictable outcomes in cellular proliferation and differentiation. The decision to proliferate and enter the cell cycle is made in G1 at the restriction point and at the molecular level requires the activation of certain cell cycle reglators. These include the CyclinDependent Kinases (CDK’s) their catalytic subunits, the cyclins and their inhibitors, the CDK-Inhibitors (CKI’s). In order to gain a better understanding about the molecular regulation of human chondrocyte proliferation we analyzed the expression profiles of the G1 cell-cycle regulators of the CDK’s, the cyclins and the CKI’s (INK, CIP/KIP) and the Retinoblastoma proteinfamily. Comparisons were made between native human cartilage and different stages of cultivated human chondrocytes including proliferating, quiescent and senescent stages by immunostaining and Ribonuclease Protection Assay (RPA). Human chondrocytes were isolated and cultivated in monolayer culturesin vitro. The proliferation rates were observed to decrease with increasing passage numbers until finally-after 8–12 passagesthe cells stop to proliferate and reach replicative senescence. Like other cultivated primary cells chondrocytes are dependent on serum mitogens to proliferate. Deprived of these serum mitogens they complete mitosis, undergo cell cycle arrest and become quiescent. Quiesecnee was induced by serum withdrawal in passage two. The expression patterns differ between native human cartilage, proliferating, quiescent and senescent human chondrocytes, particularly an upregulation of CKI’s in quiescent and senescent states. This provides a molecular basis for a targeted intervention. By the addition of antisense oligonucleotides directed against particular CKI’s chondrocytes can be cultivated under reduced serum mitogen dependence. This could allow serum free cultures of chondrocytes beyond the limitations of quiescence and replicative senescence which has an important implication for ex-vivoexpansion in tissueengineering. This work was supported by the “Deutsches Zentrum f ̈ r Biomaterialien und Organersatz (BMOZ). Stuttgart-Tubingen”.

A601–A607 Cartilage Repair/Skeketal Tissue/Stem Cells in TE

For tissue-engineering concepts with cartilage cells PGA-scaffolds serve in many studies as attaching constituents for cells and matrix. The cells are seeded on the scaffold material and start proliferation and distribution over the materials inner surface. The matrix proteins are layed down in the pore spaces. The aim of this study was to determine, if there is any positive effect of the polymer material on cartilage cells other than giving attaching facility and forming the developing matrix. Pig chondrocytes obtained from adult animals were used from knee and hipjoint. Cells were isolated in digestion chamber and proliferated in monolayer culture with addition of EGF and bFGF on agarose coated cellculture dishes. After several passages of expansion we brought the cells in high density conditions to stimulate chondrogenesis adding TGFˇ and IGF-I. For this study the cells were seeded after amplification under high density conditions on PGA-fleece for chondrogensis, leaving out growth factors TGF-̌ and IGF-I. Control experiments were carried out with the same type of cells under high density conditions without PGA-scaffolds and without growth factors. After one week under high density conditions the production of matrix protein stopped and dissolution of the matrix began. Two days later the DNA content fell by apoptotic processes: the neocartilage pellets started to dissolve. The cells seeded on PGA did not show a matrix dissolution and extensive programmed celldeath, instead the matrix volume increased with cellular expression. This positive effect on chondrogenesis is comparable with the results under influence of both growth factors TGF-̌and IGF-I. In 21 days of chondrogenesis mechanically resistant cartilage pellets develop. The collagen II content of the matrix is still lower than in native cartilage, but modification of the Polymer-fleece might lead to better results in future experiments. With this paper we demonstrate relevant positive effects on chondrogensis for PGA-fleece, which have not been found by other groups as their concepts included both procedures, proliferation and chondrogenesis on this Biomaterial. Expanding the cells separately before starting the chondrogenetic process makes PGA play a positive role in neo-cartilage formation.