Jonsson School Undergraduate Research Scholars Award The ... · on top of these collagen fibrils,...
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Relative Location of Scan (100μm) The Development of a Methodology to Fabricate Collagen Patches of Known Dimensions to Analyze Keratocyte Behavior Jacob A. Awkal 1 , Alex M. Najjar 1 , Kevin H. Lam 1 , W. Matthew Petroll 2 , David W. Schmidtke 1 1 Biomedical Engineering, The University of Texas at Dallas, Department of Bioengineering , 2 University of Texas Southwestern Medical Center, Department of Surgery & Ophthalmology Abstract Methods Conclusion References Seeded Keratocytes 1. Tuyer-Mai M. Phan, et. al. (1989). Role of Fibronectin and Fibrinogen in Healing of Corneal Epithelial Scrape Wounds. Association for Research in Vision and Ophthalmology. 2. William D. Staatz, et.al. (1990). The α 2 β 1 . Integrin Cell Surface Collagen Receptor Binds to the α 1 (I)-CB3 Peptide of Collagen. J Biol Chem 3. Kivanany, P.B.; Grose, K.C.; Petroll, W.M. Temporal and spatial analysis of stromal cell and extracellular matrix patterning following lamellar keratectomy. Exp. Eye Res. 2016, 153, 56–64. “The Cornea.” Hybrid Cornea, www.hybridcornea.org/aboutcornea.htm. Figure 1. depicts the layers of the cornea, the relative thickness of each layer are, Epithelial ~50 um, Bowman’s ~5 um, Stroma ~500 um, Descemet’s membrane ~10 um, and Endothelial ~5 um. Response of Keratocytes to Aligned Collagen Fibers To determine the response of keratocytes to aligned collagen fibers, we plated rabbit corneal keratocytes with 10,000 cells/mL and 50ng/mL PDGF (BB-subunit monomer), serum-free on top of the aligned fibrillar collagen patch substrates and characterized their response by optical and fluorescence microscopy. Figure B shows a Differential Interface Contrast (DIC) image of the cells and portrays that the cells align in the direction of the collagen fibers. Similarly, figure F shows a fluorescent image of the actin cytoskeleton of keratocytes seeded on the collagen fibers. Contact & Acknowledgments For contact: Jacob Adam Awkal, [email protected] Acknowledgment: I would like to thank Kevin H. Lam and Tarik Z. Shihabeddin for their encouragement, insight, and enthusiasm during this project. Finally, thank you Dr. Schmidtke for allowing me to pursue a research initiative with the Jonsson School Research Scholars Program in the Schmidtke Lab. Abnormal corneal wound healing is a significant medical problem that has been widely addressed in the bioengineering community. It is estimated by the Federal Occupational Health that there are ~ 2.5 million eye injuries in the United States each year, and about 40,000 corneal transplants occur annually, according to the National Eye Institute. Following surgery or traumatic injury, corneal wound healing can lead to impairment of ocular function via scarring and corneal fibrosis. This fibrosis is caused by the activation of corneal keratocytes from their native quiescent state to an activated my fibroblastic phenotype. This transformation is tied to the release of platelet-derived growth factor (PDGF) upon damage of the basement membrane of the corneal epithelium. Thus, activated by PDGF, the keratocytes begin to secrete extracellular matrix (ECM) proteins and exert elongation forces to help repair the wound. Consequently, however, these behaviors can lead to a disordered tissue microstructure within the corneal stroma, which causes corneal hazing or a reduction in the transparency of the cornea. Although it is well known that the response of keratocytes in corneal wound healing is regulated by both soluble biochemical cues and insoluble biophysical cues (e.g. topography, ECM composition, elasticity) present in the local microenvironment, there are still significant gaps in our knowledge of the factors and mechanism(s) that lead to stromal fibrosis and corneal haze instead of normal wound repair. Currently, there are no efficient technique that can recreate the orthogonal collagen patterning of the stroma, in the form of a patch in vitro. After laser eye surgery or a keratectomy, there are areas of collagen that have been severed and acellular zones. By creating collagen patches, the biomimicry of wound injury can be modeled for cellular responses due to the release of PDGF. When a section of collagen is severed (i.e. Photorefractive Keratectomy (PRK) or Laster In Situ Keratomileusis (LASIK)). In this study, we answer the following question: using microfluidic devices can we develop a technique to fabricate orthogonal collagen patterned patches in vitro? Step 1: Functionalize glass coverslip with hydrophobic coating Step 2: Treat PDMS microfluidic device with Oxygen Plasma and place on aquasil coated coverslip Step 3: Attach PDMS to syringe pump and on hotplate (37°C) and infuse chilled collagen (7.5μL/min for 225μL) Hypothesis • By placing an orthogonal poly-dimethyl siloxane (PDMS) microfluidic device on a fabricated collagen line, the underlying collagen layer will be ripped off where it meets the stamp, producing a collagen area of known dimension. • During normal wound healing in vivo, keratocytes are exposed to various stimuli that can have additive, antagonistic, synergistic, or potentiating effects on the keratocyte response. Step 4: Attach PDMS to syringe pump and on hotplate (37°C) and infuse chilled collagen (7.5μL/min for 225μL) Collagen Area • We have developed a novel method for creating patches of aligned collagen fibrils of well-defined widths (50-1500 um) that can serve as biomimicry of wound injury. • We have demonstrated that when keratocytes are cultured on top of these collagen fibrils, there was co-alignment between the keratocytes and the collagen fibrils. • These substrates can be used to further understand how topography regulates the adhesion and migration of corneal keratocytes during wound healing. Atomic Force Microscopy Discussion Figure 4. Depicts Atomic Force Microscopy (AFM) images of an aligned collagen patch formed with a constant shear rate of 150 s -1 of the green location. (c) Diagonal top view of the 3D topography of the edge of the collagen patch to emphasize the periodicity of the fibers. (d) Top view of the 3D topography of the edge of the collagen patch to show fiber density. Figure 2. Schematic of the procedure for fabricating aligned collagen patches via microfluidic patterning. Developed using Paint-3D & Photoshop. Figure 5. Differential Interference Contrast (DIC) and fluorescent images of aligned collagen fibers formed with their respective orientation index and alignment. Future Goals • Use TGF-β 1 to investigate its contractile forces and stress-fibers to further understand corneal wound healing. • Analyze the interaction between stromal and epithelial wound healing as related to corneal wound healing. • Investigate fibronectin as it relates to fibroblast adhesion to fibrin substrates and collagen matrices, as it pertains to corneal wound healing. Figure 3. Differential Interference Contrast (DIC) images of (a) and (b) aligned collagen fibers, and the respective patch generated (1500μm x 1500μm) from removal of orthogonal microfluidic device formed under microfluidic patterning. Orientation Index Glass Area Orientation Index Collagen Area Orientation Index Patch Area Orientation Index Glass Area Orientation Index Collagen Area Orientation Index Glass Area Keratocytes on Collagen (Middle) Keratocytes on Glass Keratocytes on Collagen (Middle) Jonsson School Undergraduate Research Scholars Award Keratocytes on Collagen (Middle) Keratocytes on Glass Keratocytes on Glass Orientation Index for Collagen Patch Orientation Index for Collagen Patch Patches Generated (1500μm x 1500μm & 1500μm x 50μm) Patch Generated (1500μm x 1500μm) Orientation Index for Collagen Patch Collagen Area 50um 50μm 1500μm I ∅ , cell alignment as a function of cell radial angle, is quantified using Fourier Transform algorithms. The degree of cell alignment is calculated using an Orientation Index (OI) in the form of the equation: OI = 2 < cos 2 > −1 ∗ 100% where, cos 2 = 1000 ∗ ∫ −90 90 I ∅ cos 2 ∅− d∅ ∫ −90 90 I ∅ d∅ With I ∅ centered at θ =0° and evaluated over the direction of flow during deposition along the aligned collagen fibers at θ∈(-90°, 90°). For cells completely aligned with collagen, the OI is equal to 100%; however, cells perpendicular to the collagen fibers have an OI of -100%. 20μm (a) (b) (c) Figure 4. depicts collagen patches formed from 50μm-1500μm (d) 20μm 20μm 20μm 20μm 20μm 20μm Glass Collagen
Transcript of Jonsson School Undergraduate Research Scholars Award The ... · on top of these collagen fibrils,...
Relative Location of Scan (100μm)
The Development of a Methodology to Fabricate Collagen Patches of Known Dimensions to Analyze Keratocyte Behavior
Jacob A. Awkal1, Alex M. Najjar1, Kevin H. Lam1, W. Matthew Petroll2, David W. Schmidtke11Biomedical Engineering, The University of Texas at Dallas, Department of Bioengineering , 2University of Texas Southwestern Medical Center, Department of Surgery & Ophthalmology
Abstract Methods
Conclusion References
Seeded Keratocytes
1. Tuyer-Mai M. Phan, et. al. (1989). Role of Fibronectin and Fibrinogen in Healing of CornealEpithelial Scrape Wounds. Association for Research in Vision and Ophthalmology.2. William D. Staatz, et.al. (1990). The α2β 1. Integrin Cell Surface Collagen Receptor Binds to theα1 (I)-CB3 Peptide of Collagen. J Biol Chem3. Kivanany, P.B.; Grose, K.C.; Petroll, W.M. Temporal and spatial analysis of stromal cell andextracellular matrix patterning following lamellar keratectomy. Exp. Eye Res. 2016, 153, 56–64.
“The Cornea.” Hybrid Cornea, www.hybridcornea.org/aboutcornea.htm.Figure 1. depicts the layers of the cornea, the relative thickness of each layer are, Epithelial ~50 um, Bowman’s ~5 um, Stroma ~500 um, Descemet’s membrane ~10 um, and Endothelial ~5 um.
Response of Keratocytes to Aligned Collagen FibersTo determine the response of keratocytes to aligned collagen fibers, we plated rabbitcorneal keratocytes with 10,000 cells/mL and 50ng/mL PDGF (BB-subunit monomer),serum-free on top of the aligned fibrillar collagen patch substrates and characterized theirresponse by optical and fluorescence microscopy. Figure B shows a Differential InterfaceContrast (DIC) image of the cells and portrays that the cells align in the direction of thecollagen fibers. Similarly, figure F shows a fluorescent image of the actin cytoskeleton ofkeratocytes seeded on the collagen fibers.
Contact & Acknowledgments For contact: Jacob Adam Awkal, [email protected]: I would like to thank Kevin H. Lam and Tarik Z. Shihabeddin fortheir encouragement, insight, and enthusiasm during this project. Finally, thank youDr. Schmidtke for allowing me to pursue a research initiative with the Jonsson SchoolResearch Scholars Program in the Schmidtke Lab.
Abnormal corneal wound healing is a significant medical problemthat has been widely addressed in the bioengineering community. It isestimated by the Federal Occupational Health that there are ~ 2.5 millioneye injuries in the United States each year, and about 40,000 cornealtransplants occur annually, according to the National Eye Institute.Following surgery or traumatic injury, corneal wound healing can lead toimpairment of ocular function via scarring and corneal fibrosis. This fibrosisis caused by the activation of corneal keratocytes from their nativequiescent state to an activated my fibroblastic phenotype. Thistransformation is tied to the release of platelet-derived growth factor(PDGF) upon damage of the basement membrane of the cornealepithelium. Thus, activated by PDGF, the keratocytes begin to secreteextracellular matrix (ECM) proteins and exert elongation forces to helprepair the wound. Consequently, however, these behaviors can lead to adisordered tissue microstructure within the corneal stroma, which causescorneal hazing or a reduction in the transparency of the cornea. Although itis well known that the response of keratocytes in corneal wound healing isregulated by both soluble biochemical cues and insoluble biophysical cues(e.g. topography, ECM composition, elasticity) present in the localmicroenvironment, there are still significant gaps in our knowledge of thefactors and mechanism(s) that lead to stromal fibrosis and corneal hazeinstead of normal wound repair.Currently, there are no efficient technique that can recreate the orthogonalcollagen patterning of the stroma, in the form of a patch in vitro. After lasereye surgery or a keratectomy, there are areas of collagen that have beensevered and acellular zones. By creating collagen patches, the biomimicryof wound injury can be modeled for cellular responses due to the releaseof PDGF. When a section of collagen is severed (i.e. PhotorefractiveKeratectomy (PRK) or Laster In Situ Keratomileusis (LASIK)).
In this study, we answer the following question:using microfluidic devices can we develop a technique to fabricateorthogonal collagen patterned patches in vitro?
Step 1: Functionalize glass coverslip with hydrophobic coating
Step 2: Treat PDMS microfluidic device with Oxygen Plasma and place on aquasil coated coverslip
Step 3: Attach PDMS to syringe pump and on hotplate (37°C) and infuse chilled collagen (7.5μL/min for 225μL)
Hypothesis• By placing an orthogonal poly-dimethyl siloxane (PDMS)
microfluidic device on a fabricated collagen line, the underlying collagen layer will be ripped off where it meets the stamp, producing a collagen area of known dimension.
• During normal wound healing in vivo, keratocytes are exposed to various stimuli that can have additive, antagonistic, synergistic, or potentiating effects on the keratocyte response.
Step 4: Attach PDMS to syringe pump and on hotplate (37°C) and infuse chilled collagen (7.5μL/min for 225μL)
Collagen Area
• We have developed a novel method for creating patches ofaligned collagen fibrils of well-defined widths (50-1500 um)that can serve as biomimicry of wound injury.
• We have demonstrated that when keratocytes are culturedon top of these collagen fibrils, there was co-alignmentbetween the keratocytes and the collagen fibrils.
• These substrates can be used to further understand howtopography regulates the adhesion and migration ofcorneal keratocytes during wound healing.
Atomic Force Microscopy
Discussion
Figure 4. Depicts Atomic Force Microscopy (AFM) images of an aligned collagen patch formedwith a constant shear rate of 150 s-1 of the green location. (c) Diagonal top view of the 3Dtopography of the edge of the collagen patch to emphasize the periodicity of the fibers. (d) Topview of the 3D topography of the edge of the collagen patch to show fiber density.
Figure 2. Schematic of the procedure for fabricating aligned collagen patches via microfluidic patterning. Developed using Paint-3D & Photoshop.
Figure 5. Differential Interference Contrast (DIC) and fluorescent images of aligned collagen fibers formed with their respective orientation index and alignment.
Future Goals• Use TGF-β1 to investigate its contractile forces and stress-fibers
to further understand corneal wound healing.
• Analyze the interaction between stromal and epithelial woundhealing as related to corneal wound healing.
• Investigate fibronectin as it relates to fibroblast adhesion to fibrinsubstrates and collagen matrices, as it pertains to corneal woundhealing.
Figure 3. Differential Interference Contrast (DIC) images of (a) and (b) aligned collagen fibers, and the respective patch generated (1500μm x 1500μm) from removal of orthogonal microfluidic device formed under microfluidic patterning.
Orientation Index Glass AreaOrientation Index Collagen Area Orientation Index Patch Area Orientation Index Glass AreaOrientation Index Collagen Area Orientation Index Glass Area
Keratocytes on Collagen (Middle) Keratocytes on GlassKeratocytes on Collagen (Middle)
Jonsson School Undergraduate Research Scholars Award
Keratocytes on Collagen (Middle) Keratocytes on Glass Keratocytes on Glass
Orientation Index for Collagen Patch Orientation Index for Collagen Patch
Patches Generated (1500μm x 1500μm & 1500μm x 50μm)
Patch Generated (1500μm x 1500μm)
Orientation Index for Collagen Patch
Collagen Area
50um 50μm 1500μm
I ∅ , cell alignment as a function of cell radial angle, is quantified using Fourier Transform algorithms. Thedegree of cell alignment is calculated using an Orientation Index (OI) in the form of the equation:
OI 𝜃𝜃 = 2 < cos2 𝜃𝜃 > −1 ∗ 100%where,
cos2 𝜃𝜃 = 1000 ∗∫−9090 I ∅ cos2 ∅ − 𝜃𝜃 d∅
∫−9090 I ∅ d∅
With I ∅ centered at θ = 0° and evaluated over the direction of flow during deposition along the alignedcollagen fibers at θ∈(-90°, 90°).
For cells completely aligned with collagen, the OI is equal to 100%; however, cells perpendicular to the collagenfibers have an OI of -100%.
20μm
(a) (b)
(c)
Figure 4. depicts collagen patches formed from 50μm-1500μm
(d)
20μm20μm20μm20μm20μm20μm
GlassCollagen
