| Budget Amount *help |
¥3,650,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥150,000)
Fiscal Year 2007: ¥650,000 (Direct Cost: ¥500,000、Indirect Cost: ¥150,000)
Fiscal Year 2006: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2005: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
This research project is aimed at constructing, based on collagen gel scaffold, engineered cardiac tissue (ECT) that will possess the contractile characteristics of in vivo myocardium. Through 3 years' studies, we obtained the following results and achievements.
1. By comparing the mRNA and protein expression of some vital transcriptional factors and constitutive proteins of intercalated disc in cultured cardiomyocytes with those in myocardium in vivo, we showed that the transcriptional factors, SRF and myocardin, and the constitutive proteins, N-cadherin and connexin43, were at lower expression levels in cultured cardiomyocytes than in the tissues in vivo. It suggested that the attenuation of these molecules may impede the further differentiation of the cardiomyocytes under culture condition and that the formation of intercalated disc of engineered cardiac tissue may be in defect. The weak contractile force of ECT is also considered to mostly owe to the above lower expressions. Based o
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n these findings, gene transfer technology was employed to enforce the expression of N-cadherin in cultured cells to enhance the interaction between adjacent cardiomyocytes.
2. Permeability of the collagen gel sold was increased by the addition of heparin and albumin. With such modified collagen gel, nutrient provision and metabolic waste excretion are improved 4 kinds of additives, heparin, albumin, fibronectin, and fibrinogen were added to collagen gels, respectively, and the permeability of glucose through the gels were measured. The results showed that heparin and albumin were able to increase the permeablity by about two folds.
3. A series of experiments were conducted to carefully search for the optimal conditions to each step of the ECT creation procedure. It came out that the following two treatments were necessary besides prior published protocol: I) the addition of cysteine into culture medium would help to diminish the residual effect of collagenase after cell harvest from natural heart tissue and in gel making process, ii) to prevent the embedded cardiomyocytes from depositing at and attaching to the bottom of culture dishes, the gels should be inverted once just after gelation.
4. A novel electro-tensile bioreactor was developed to promote the continuation of cardiomyocytes differentiation and the maturation of 3D tissue under culture. Differing from the former one developed by us, this new type imposed tensile stress by resisting, with an ingenious way, the contraction of the ECT which is induced by electrical pulse stimulus, rather than directly stretching the ECT. Therefore, the electrical stimulus, contraction strain and contraction stress were orderly occurred in the ECT the harmony of electrical stimulus and the tensile stress was reached by means of the inherent electro-contraction mechanism.
5. We invented an algorithm based on the theory of elastic string vibration to calculate the contractile force in the ECT versus to time, strain, and strain rate, respectively. By analyzing the contraction displacement of the ECT in ring shape, this method provided an effective approach for revealing the characteristics of ECT contraction and can detect quite small contractile force to 1 μN
6. Through the above innovations and improvements, the contractile force exerted by the ECT was increased by 16 folds, and it presented the characteristics that the maximal contractile force preceded the maximal contraction strain but corresponding to maximal strain rate, which is quite similar to the characteristics of natural heart muscle contraction. Less
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