Running Deck Flashcards
Why Alginate beads instead of alginate bolus
All the cells in the middle of a large block would be killed - To minimize diffusion limitations… More surface area to volume ratio
- ALSO Encapsulation in general prevents unwanted cell migration
How can you edit the amount of cross-linking of alginate?
Charge the molarity of the cross-linking reagent (Calcium chloride)
What is the ideal location in an animal model for a biocompatibility study
Subcutaneous - Easy to get to, minimally invasive
Don’t have to account for immune response of the surgery itself
Eliminate variables that you would get at a more invasive site
Isolate the immune response to be caused by the injection and not by the surgery itself
What situation would you want to use a t-test vs. ANOVA
T-test for 2 things, ANOVA for more since p-values can compound and make multiple t-tests inaccurate for a set of many things
The effect of the micropattern on cell growth:
With the pattern, there is less space for the cells to grow and proliferate
What substrate parameters can be modified to control cell differentiation?
Stiffness - Depending on stiffness, different amounts of growth factors were observed
Size - Lots of room (SURFACE AREA), more proliferation
Available functional groups - Cells interact differently with different functional groups
What was the purpose of using a hydrogel in tissue engineering for this study?
Similar rate of degradation and other properties with the surrounding tissues and cell types, in terms of mechanical and chemical properties
How did macromer lengths affect sol-gel temperatures?
Longer lengths decreased sol-gel temperature
How did changing modulus affect cellular growth and differentiation
Closer you get to the modulus of the tissue, the most significant differentiation occurs
Most significant MYOGENIC DIFFERENTIATION occurred at 20 kPa, modulus of tissue is 17 kPa
Describe a method to use poragen leaching that ensures a continuous interconnected pore structure and reduces the likelihood of leaving behind residual poragen
Sintering the Poragen so that it forms interconnected pores and dissolves all the way
What is the rationale behind bimodal distribution of pore sizes?
Small pore sizes favor oxygen and nutrient transport
Large size support vasculature and tissue growth
Benefits of bioglass composite scaffold?
High compression modulus High Tg Fastest degradation rate Low inflammation Effectively repaired bone defect
Why controlled release of growth factors?
Can target proliferation and differentiation of cells at different stages of development or cell growth
What electrospinning parameters can you change to control release kinetics from nanofibers?
Voltage difference, viscosity of solution, rate of motion of the target, injection rate with which the solution is injected, all to modulate the nanofiber diameter - which effectively changes the exposed surface area of the nanofibers
Difference between storage and loss modulus of a hydrogel?
Storage modulus measures elastic deformation of hydrogel
Loss modulus measures viscous portion of hydrogel
On a time sweep from rheological data, (Time on x axis, stress on y axis) when does gelation occur?
Gelation occurs at the crossover point between the storage modulus and loss modulus
What is secondary crosslinking within hydrogels?
When they crosslink after the printing to increase mechanical properties
What’s the importance of having a Ca/P ratio below 1.67? What type of Ca/P does it indicate?
Make it more easily resorbable, it is called “Calcium deficient Hydroxyapatite”
What is the relationship between porosity and elastic modulus?
More pores = lower modulus. Less pores = higher modulus
Does anatomical location of mesenchymal stem cell isolation matter?
Yes, they differentiate differently based on where in the body they are from
Primary vs. Cell Lines
Primary = Expensive, but accurately simulate in vivo
– Primary are heterogeneous, first plating, may have other cells in addition to desired cell - Used for Gene therapy
Cell lines, cheaper - may mutate and are used for in vitro
testing conditions where many conditions are tested, more proof of concept
When to use embryonic or induced progenitor stem cell?
Since they self-renew, use for a patient w/ autoimmune neurodegenerative disease as the embryonic stem cells can regenerate
Mesenchymal stem cells in what use?
Artificial hip in order to promote osteoinductivity along the pores of the replacement hip
For testing the stemness of cells, what is added to each type?
Fat - Oil Red O staining to TEST for adipogenesis
Type II collagen used for testing for cartilage
Alkaline Phosphatase for testing for osteogenesis
How to separate cell types, 2 ways
by density- centrifugation
by fluorescent tags and electrostatic charging - then run through electric field to separate different cell types
How to modify a solid material to change properties?
Drill holes in it!
Different combinations of Polylactide and Polyglycolide
Get properties between the two, you can control the ratio
How to make bone cement porous
Mix degradable polymer into bone cement like poragens to make it porous
Harm of too much or too little decellularization
Too much = ECM harmed
Too little = Inflammatory response from remaining cells
How to remove cells (decellularize)
Physically = shake them, freeze thaw
Enzymatic = Trypsin, endonucleases
Chemical - Alkaline, acid
Influence of fluid flow and dynamic loading on cell
Cell experience of pull shear or stretch can influence how it grows and proliferates
Forces and cells
Cells have forces applied onto them, but they also APPLY FORCES to their partners
ex. Change the stiffness of areas of a scaffold to create varied effects when adding cells to the scaffold
How can bone growth be stimulated?
Ultrasound or EM waves
Pros/Cons of injectable cell based therapies
Pros = minimal invasive, reduce risk + healing time, Cons = low cell survival and retention rate
Types of stem cells
totipotent can be any tissue pluripotent = any of 3 germ layers Multipotent = multiple tissue types mesenchymal can be bone, fat, or muscle iPS = Can be reversed and then redifferentiated
How to induce differentiation
Addition of growth factors, chemical stimuli
Co-culture with another cell type
Physical manipulation - Culture beyond confluence or modulate fluid flow over cells
Polyethylene importance
Mech properties in the range of bone, depending on molecular weight
Hydrogel importance
Drug delivery, drug eluted when matrix swells
Natural material - good biocompatibility
Ceramics importance
Bioactive glass (providing Ca or P ions into solution), good osteoinductivity - results in strong cohesive strength between implant and bone
Requirements for scaffold design
Tissue independent: Biocompatible, tunable properties (mechanical, structural, topographical, degradable)
Tissue Dependent: Mechanical, degradation time, pore structure, overall shape and structure, surface functionality
Photolithography
Coated surface resists imprinting by beam - results in textured surface - Cells can grow
Relevance of microfluidics
Capillary analysis and design - mimicking smallest level of fluid flow to understand shear forces on relevant cells
Nanofibers use
to replicate ECM to give cells a familiar environment
Random vs. aligned nanofibers
Aligned for tendons and ligaments
Random for collagen
Sterilizing decellularized ECM
Can change mech props based on dose and method
and change bioactivity - ethylene oxide induces immune response
What is move favorable, a slow sustained release of BMP-2 or a rapid diffusion over a period of a few hours? Why?
Days because the lower concentration prevents negative side effects
Is it bad for molecules to display brownian motion during delivery? How can we avoid this?
Brownian will carry anywhere, may not target tissue
Localized drug delivery would help
Could bottom up delivery of BMP-2 be used in other types of biomaterials/structures? Do you think this method would be more advantageous?
Stiffness of surface affects cell behavior
Could be either, different reasons why
Why is a protective sheath or shell beneficial for a protein delivery system such as the one described here?
Increases time of viability
Molecule can diffuse farther
What follow-up studies would be recommended to further develop this system towards the clinical applications we previously discussed?
In vitro, multiple cell types
In vivo
Attempting to carry/release other types of molecules
Figure 5 - release kinetics. Could the fact that there is a dramatic pH change be used to their advantage?
Yes, pH in saliva very different than pH in stomach where it is very acidic
Could engineer it to pass through stomach and release in GI tract
Issues with high dosages of BMP-2 and what are 2 ways they can be circumvented
Issues: Adiopogenesis, inflammation, cyst like bone formation, ectopic bone formation
Solutions: Upregulation of Phenamil, use less BMP 2 bc there are only picograms in the body
What is the function of a knockdown?
To create a negative control in the design
Why is PRP beneficial for creating vascular networks?
PRP gel contains the growth factors needed so additional ones are not required
What needs to be considered when developing PRP gels for in vivo study?
Degradation, cell viability, stiffness (stimulate bone = make cells stiffer), site implantation
Piezoelectricity
Ability of certain materials to generate electric charge in response to mechanical stress or vice versa - based on crystal lattice
Static vs. Pulsed EM Field
Static = rare earth magnet, static field, strong health applications Pulsed = dynamic, pulsed
Clinical bone repair
Surgical = bone grafts Non-invasive = Ultrasound, EM stimulation
Electrostimulation
Low level electric current to tissue –> alteration in intracellular calcium –> Increased activated calmodulin –> increase proliferation of bone
LIPUS
Low Intensity Pulsed Ultrasound - stimulates bone and causes it to reform and repair,
It can be modulated to deform hydrogels more or less depending on force provided by LIPUS
Capacitive coupling
Electrodes placed on either side of fracture, creates alternating current
Inductive coupling
EM Field around fracture, low frequency (LIPUS) or combined (CEMF)
PMA
Pre market approval - safe AND effective, 1% of new devices
510k
premarket clearance - establish that a device is substantially equivalent to a previously cleared device, (previous device = “Predicate”)
Class 1, 2, 3 devices
1: little to no risk (bandages)
2: higher risk, (needles, monitors, implanted devices)
3: Clinical trials, high risk
Hydrogels have ____ bonds with ___ interactions
covalent, many interactions
Poragen leaching results in products that are always interconnected T/F
False, not always interconnected
Polylactide true facts
Tunable mech properties depending on final form of scaffold
Relatively hydrophobic compared to polyglycolide
Degrades over longer period of time than polyglycolide (6 months to 2 years)
