A tissue engineered medical device is typically comprised of a matrix that is populated with multiple layers of cells. The matrix provides a structure that the cells can grow on and in order for the cells to grow, they must be exposed to a nutrient media. Additionally, inputting mechanical stresses and strains into the cells stimulates faster growth, orientation and enhanced material properties. For example, mechanical strain applied to fibroblasts seeded on a collagen matrix, induces fibroblast elongation and alignment of the cells. Mechanical strain also promotes smooth muscle cell proliferation.
At the beginning of the stimulation process, there are few cells on the construct and they are not aligned. Consequently, the tissue construct response is "weak" or highly damped. Figure I depicts what the mechanical response of the construct might look like at this early stage. Over time as the stimulation continues, the cells proliferate, differentiate and align making the tissue construct stronger. Figure II shows what a more developed construct response might look like. It's important to note that the specimen response in both figures is viscoelastic meaning it exhibits an elastic stiffness and viscous damping component. To measure and characterize these components, the SMA GrowthApp acquires the load and displacement data and then processes it to determine the dynamic stiffness and loss stiffness for the construct. If the cross-sectional information and length of the construct are known, these can also be entered into the App to calculate stress and strain to determine the storage and loss modulus. The above process is often referred to as Dynamic Materials Characterization (DMC) or Dynamic Mechanical Analysis (DMA). In addition to determining how the construct growth is progressing, the dynamic material properties are also important in predicting the in-vivo performance of the matured tissue construct (also called a bioprosthesis) at various loads and frequencies. For example, the load cycle imparted by a normal footstep to an ACL tendon, knee joint or spinal joint is a complex waveform. This waveform can be made up of loading components that range from DC to 20 Hz. It is important in tissue engineered medical device design to ensure that its response closely matches the native tissue. For example, a spinal disk medical device that is too stiff will place undue loading on the surrounding vertebral disks. Likewise, a spinal disk medical device that is too soft will end up absorbing too much motion which may lead to early failure of the device. It is also advantageous if the medical device stiffness matches the native tissue response across all frequencies. For example, a spinal disk medical device needs to work just as well during jogging (higher frequency and load components) as it does when the patient is sitting (mostly static loading).
The Stiffness Measurement & Analysis (SMA) GrowthApp enables the system to make real-time measurements of the sample stiffness during culture. Described in U.S. Patent 7,410,792 B2 Instrumented bioreactor with material property measurement capability and process-based adjustment for conditioning tissue engineered medical products, the application measures the mechanical response of the tissue construct as it is being stimulated and conditioned. The stiffness measurements can then be used as guidelines for bench marking progress during culturing or for modifying the stimulation profile. Based upon the measured response, the bioreactor system is able to alter the stimulation profile. This technology will enable researchers to automatically culture the bioprosthesis from initial seeding to an implantation-ready state with minimal operator intervention.
The SMA GrowthApp has four plots and one panel which display the acquired stress, strain, modulus, pressure and volume data (lumen construct only). The panel on the right side of the screen displays the numerical value of the dynamic and storage moduli, the phase angle between stress and strain, the corresponding loss coefficient as well as the initial diameter measurement and scan number. The following is a short description of each plot.
The blue line of the top left plot displays the applied stress with its axis being on the left. The red line displays the strain with its axis being on the right.
The value of the average measured dynamic modulus is displayed for each scan as a green dot on the top right plot. The plot is refreshed every 100 scans.
The bottom left plot displays the normalized stress vs. strain curve. The center blue dot is the origin of the axis while the second blue dot indicates the location on the curve used for the storage modulus calculation. The storage modulus is considered to be the slope of a line from the origin to the maximum strain point on the elliptical hysteresis loop.
The bottom right plot displays the pressure-volume curve. The volume is considered to be the product of the effective cross sectional area of the stimulator diaphragm multiplied by its displacement.
While the SMA GrowthApp is useful in determining the tissue construct or bioprosthesis response across varying loading and frequencies, it also sets the foundation for taking the growth stimulation a very important step further. Once the researcher has determined the key property milestones that should be exhibited at various stages throughout the tissue construct growth cycle, these milestones could be used to trigger different conditioning sequences. Using the Figure I example above again, the tissue construct initially exhibits a soft response. The conditioning sequence for this stage of the tissue construct development would most likely be a low frequency sinewave of small volume or pressure amplitude. As the construct grows and develops strength, the SMA GrowthApp continually measures the tissue construct material response. Once the tissue construct properties indicate that it has progressed along the growth cycle (ie: increased dynamic modulus), a higher force/frequency conditioning sequence could then be initiated automatically. This process could then be repeated (ie: higher tissue construct strength means higher load conditioning) until the tissue construct reaches maturity. Future versions of the SMA GrowthApp will incorporate this adaptation capability. While this discussion focuses only on desired material properties such as storage and loss stiffness (or modulus), other properties such as the chemistry of the nutrient conditioning media could also be used. These additional parameters would necessitate adding sensors to the system that are capable of measuring these conditions (ie: O2, N2, PH, etc).
A concept created and patented by Tissue Growth Technologies, the intelligent Stiffness Measurement and Analysis of the SMA GrowthApp sets the stage for automated tissue engineered medical device development for years to come.