Last Edited : April 26, 2009
DATE CREATED: Dec 27 2008
ACTION PLAN
PROBLEMS IDENTIFIED (PI) / OUTSIDE SKILL REQUIRED (OSR) / RESOLVED (R)
Cardiac arrest starts with scar tissue formation in the heart muscles.
For cardiac tissue engineering, there were many basic researches with cell therapy, tissue engineering, and micropatterning of cardiomyocytes were suggested.cell therapy, ### et. al reported cell injected cardiac tissue regenerated with stem cells and the cardiac function increased. However, there were still challenging problems with cardiac tissue regeneration. In patterning tissue structures with predetermined or guided regeneration of cardiac tissue with textured structures showed better function and natural[Langer-Acordian shape ] functions. Precise control of tissue textures with patterning or microengineering of cells' microenvironments can be advantageous for regeneration of heart muscles and cardiac function recovery and evaluation.
Cell culturing substrate materials have essential functions in tissue engineering. In tissue engineering, the scaffold materials can support initial architectures of final tissue and can have function of cytokine, growth factor and gene delivery. They can support cell adhesion, proliferation and 3D tissue formation. In patterning of cells for higher complexity of functional organs, scaffold materials are also important in chemical and mechanical point of view. In addition to chemical substance delivery, substrate materials can control tissue formation with mechanical aspects. *** et al reported the cell cytoskeleton change with substrate stiffness and ** reported the mechanical microenvironments of cell affect different results with cell types.
Cardiac cells can be patterned in two dimensionally on well defined patterns. Cardiomyocytes aligned along the patterned sufaces and formed myofibers to show the potencial of cell patterning.
In mechanical point of view, the substrate materials were petri dish, glass or silicon wafers. For 3D patterned cardiac tissue regeneration, scaffold material has to support mechanical stability as well as chemical functions.
Recently, it is reported cardiomyocytes cultured on controlled substrate stiffness showed different cytoskeleton and cell shapes. These results implies the cardiac tissue formation is dependent on the mechanical strength of the substrates.
Our hypothesis is that the cardiomyocytes in the low stiffness hydrogel usually cannot make myofibers, but they may form the myofibers on hydrogels with optimal stiffness range.
To prove this hypothesis, we investigated the difference of primary cultured cardiomyocytes on the mechanically different gelatin hydrogels.
And also investigated the relation between myofiber formation and mechanical condition of substrates with three dimensional microchannel.
Mechanical effects of hydrogel mechanical stiffness on cardiomyocytes' morphology and cytoskeletal change were also investigated.
Various stiffness in the hydrogel scaffolds with channel pattern to guide myofiber formation.
PAPER TITLE : Cardiac myofiber formation with controlled hydrogel stiffness
A) BACKGROUND:This study reports the mechanical effect of scaffold stiffness on cardiomyocytes myofiber formation.
Cardiac patch generation will be a good choice for heart failure patients.
Fiber shape cardiac myofiber generation will be efficient way to generate 3D cardiac tissue patch
B) HYPOTHESIS
Cardiac myofiber can be formed in fiber of fibrin or collagen or Matrigel
Cardiac cells cannot secrete MMPs so need space for extension
C) SPECIFIC AIMS
AIM 1 -
Demonstrated myofiber formation of primary cultured cardiomyocytes in fibrin fibers
Measure contractile force under electrical stimulation
AIM 2 -
Apply to 3D cardiac tissue formation with fiber bundle
Introduction
MATERIALS and METHODS (GENERAL EXPERIMENTAL APPROACH)
Primary culture of cardiomyocytes
Dr. Jinseok will prepare
Cardiac cells were prepared by primary culture of neonate rats. The cardiac cell isolation procedures were reported previously[ref]. In brief, neonatal SD rats of day 2 were anastesized with @@ and *** prior to incision, with carefully open the chest and harvested hearts. The hearts were immersed in heparin containing PBS and trimmed small vessels and connective tissues other than heart muscle. Heart tissues were cut to 4~5 pieces and washed with heparin-PBS to remove red blood cells. Tissue pieces were chopped and digested with collagenase type II for 20 minutes, and isolated cells were separated with cell strainer. This cell isolation process were repeated 4~6 times until all the tissue pieces were digested. Collected cells were preplated to petri-dish for 30 minutes, and suspended cells were recollected by centrifuge.
All the animal protocol were reviewed and approved from Animal Experiment Review Board[proof] and approved as protocol number 35742[proof] of Harvard university.
Microfluidic spinning of cardiac cell-incorporated fibers
Gel preparation
Dr. Hojae will prepare
Gelatin hydrogels were prepared by enzymatic crosslinking reaction. Microbial transglutaminase(mTGase) was used as crosslinking enzyme [ref]. Gelatin solution was prepared by adding 1g/mL of gelatin type A from porcine skin (bloom 300, Sigma) to PBS and autoclaved for sterilization. Crosslinking enzyme mTGase was dissolved in PBS with concentration of 20 wt% and filtered with 0.2um syringe filter and stored at -20oC. MTGase stock solution was added to gelatin solution to make final mTGase concentration as 0.2, 1.0, 2.0 %. Mixed solutions were gelated in the 37oC cell culture incubator for 1 h. Mechanical properties were evaluated with texture analyser in Dr. Hojae's University.
Myofiber formation in hydrogel matrix
For cardiomyocyte culture, 50uL of gelatin-mTGase solution was dropped on 18mm diameter glass coverslip and covered with PDMS membrane during gelation. Gelated coverslip-hydrogel samples were seeded with various cell concentration of 10^4, 10^5, 10^6 cardiac cells per sample.
Electrical function evaluation of cardiac cell
Mechanical stiffness of gelatin hydrogel
Electrical signal from cardiac cells
Immunofluorescence of Actin, myosin, intercalating disc
RESULTS AND DISCUSSION
E-1) DISCUSSION - DESIGN PITFALLS AND ALTERNATIVES
F) FIGURES FOR PAPER
Fig. 1 Mechanical properties of gelatin hydrogel.
Fig. 2 Biocompatibility of hydrogels : Cell area and viability on each gel stiffness.[control : petri dish]
Fig. 3 Cardiomyocyte actin filament on different hydrogel stiffness
Fig. 4 Myofiber formation with cell concentration and stiffness [connexin, intercalating disc]
Fig. 5 Beating characteristics of myofibers[# in total cells, beat per min]
Fig. 6 Electric properties of myofibers [action potential]
Fig. 7 Contractile forces of myofibers with fluorescent microparticle tracing
Fig. 8
G) FUTURE DIRECTIONS
Gelatin ; micropatterned scaffold