Empfohlen
Weitere ähnliche Inhalte
Andere mochten auch
Ähnlich wie Christopher McKay - Rensselaer Polytechnic Institute - Doctoral Defense Presentation
Ähnlich wie Christopher McKay - Rensselaer Polytechnic Institute - Doctoral Defense Presentation (20)
Kürzlich hochgeladen
Kürzlich hochgeladen (20)
Christopher McKay - Rensselaer Polytechnic Institute - Doctoral Defense Presentation
- 1. Hydrogels for Acute Spinal Cord Injury: Physical/Chemical Material Characterization and Assessment of Astrocytic Response Christopher A. McKay Doctoral Thesis Defense April 3rd, 2014
- 2. Societal Impact of Spinal Cord Injury • Lifetime cost at 25 years2: • Incomplete Motor Function (AIS D)- $1,517,806 • High Tetraplegia (C1-C4) - $4,543,182 • Treatment and rehabilitation costs surpass $9.7 billion in the United States alone3 • Less than 1% of patients exhibit complete functional recovery2 1 Christopher and Dana Reeve Foundation 2006, 1 2 NSCISC Study 2012,3 Ackery et al 2004 2
- 3. Societal Impact of Central Nervous System Injury •Clinical Treatments: • Spinal Decompression and Fixation2 • Methylprednisilone3 • Clinical Trials • 50+ ongoing trials4 There is a significant need for an efficacious therapy to improve patient outcome following SCI 1 Christopher and Dana Reeve Foundation 2006, 1 2 Carlson et al 2003, 3Bracken et al 1997, 4 Unite 2 Fight Paralysis 2014 3
- 4. There are three distinctly defined periods of spinal cord injury Acute •Timeframe: 0-48 hours following initial trauma1 • Largely characterized by macroscopic, systemic damage2 •For severe trauma: • Blood vessel hemorrhage • Disruption of blood brain barrier • Infiltration of blood-borne material SCI is a Dynamic Process: Acute Phase 3 Ronsyn et al 2008 3 2 Tator 19981 Liverman 2005 4
- 5. Acute Sub-Acute Time frame: 48 hours – 2 weeks following injury1 Changes in the composition of extracellular environment 2,3 Significant inflammatory response1 Induction of secondary injury cascades2,3 These changes lead to the formation of reactive astrocytes SCI is a Dynamic Process: Sub-Acute Phase 1 Liverman 2005 2 Oyinbo 2011 3 Wingrave et al 2003 4 4 Sofroniew 2009 5
- 6. Postsynaptic Cascade • Activation of glutamatergic receptors • Increase in intracellular Ca2+ • Activation of calpain and mitochondria failure • Activation of caspase Acute Sub-Acute Presynaptic Cascade • Damage to presynaptic neuronal membrane • Accumulation of glutamate in the synaptic cleft Mechanism of Calcium Related Secondary Neuronal Death 1 Syntichaki and Tavernarakis 2003 1 Neuronal apoptosis via calcium dependent glutamate excitotoxicity 6
- 7. Acute Sub-Acute Chronic 1 Liverman 2005 Time frame: 2 weeks following injury – indefinitely1 Characterized by the formation of a glial scar2,3 • Physical and chemical barrier to neuronal regeneration • Key components: activated astrocytes and chondroitin sulfate proteoglycans (CSPGs) 2 Egn et al 1987 3 Berry et al 1983 4 4 Sofroniew 2009 SCI is a Dynamic Process: Chronic Phase 7
- 8. SCI is a Dynamic Process: Review and Experimental Approach Acute Sub-Acute Chronic Solution: Prevent early formation of inhibitory cues within the spinal cord lesion Physical Trauma Structural Damage Calcium Related Neuronal Death Reactive Astrocyte Formation Glial Scar Formation Neuronal Regeneration Failure 8
- 9. Overall Thesis Goal The overall goal of this research is: • The development and characterization of an injectable composite hydrogel system that responds to physiological levels of Ca2+ • Assessment of the astrocytic response to hydrogels of varying composition 9
- 10. Specific Aim 1 Development and Characterization of the Physical, Chemical and Mechanical Behavior of Injectable, Calcium Sensitive Alginate/Chitosan Hydrogels Specific Aim 1 – Hypothesis Injectable alginate/chitosan hydrogels can be fabricated to mimic the elastic modulus of native CNS tissue and respond to change in external Ca2+ concentration while exhibiting tunable physical and mechanical properties 10
- 11. • Spinal cord injuries vary greatly in shape and size •Contusion injuries are the most common type of injury •Hydrogels are injectable – conform to the lesion geometry 1 Silver and Miller 2004 Why use a Hydrogel System? 1 11
- 12. • Homogenous Crosslinking • Easily Injectable • Mimic native CNS mechanical properties • Utilize excess extracellular Ca2+ for in-situ gelation Hydrogel Design Criteria 12
- 13. Specific Aim 1 - Rationale Alginate •Biocompatible, low cytotoxicity •Crosslinks with Ca2+ ions •Negatively charged Chitosan •Positively charged – enhances cellular adhesion Genipin • Natural crosslinking agent • Well suited for spinal cord environments 1 Wee and Gombotz 1998 2 Braccini and Perez 2001 3 Li et al 2007 4 Rowley et al 1999 5 Zuidema et al 2011 6 Moura et al 2011 7 Yamazaki et al 2005 8 Yamazaki et al 2004 9 Koo et al 2006 13
- 14. Specific Aim 1 - Rationale Chitosan/Genipin • Genipin reacts with positive amine groups on chitosan chains2 Genipin/Genipin •Polymerization of genipin molecules between chitosan chain3 Chitosan/Alginate • Oppositely charged – form polyelectrolyte complexes4,5 Alginate/Ca2+ • Ionic bond formation between Ca2+ ions and guluronic acid residues of alginate6,7 1 Chen et al 2006 1 2 Touyama et al 2011 3 Mu et al 2013 4 Sankalia et al 2007 5 Tapia et al 2004 6 Braccini and Perez 2001 7 Li et al 2007 3 14
- 15. Specific Aim 1 - Results 2% Alginate 5% Alginate 5% Alginate 0.5% Chitosan Can alginate hydrogels be used to facilitate in-situ hydrogel formation? • Non-homogenous behavior • Insufficient elastic modulus with CSF Ca2+ 15
- 16. Parameters •Room temperature 37°C Results •Determines rate of hydrogel formation •Fully gelled: <10% change from final modulus •Linear increase is due to evaporation Gelation Point Evaporation Parameters •Equilibrated at 37°C for gelation time Results •Determines linear-viscoelastic (LVE) limit •Hydrogels exhibit deformation above LVE •High strain below the LVE limit provides the best output signal Linear- viscoelastic limit Viable strain values Rheological Characterization Protocol 16
- 17. Gelation Point Evaporation Parameters •Equilibrated at 37°C for gelation time Results •Determines linear-viscoelastic (LVE) limit •Hydrogels exhibit deformation above LVE •High strain below the LVE limit provides the best output signal Linear- viscoelastic limit Viable strain values Rheological Characterization Protocol 17
- 18. Parameters • Strain is chosen based on position of LVE • Equilibrated at 37°C for gelation time Results • Optimal value: low-frequency plateau Parameters • Strain is chosen based on position of LVE • Frequency is chosen from low-frequency plateau • Equilibrated at 37°C for gelation time Results • Determines true ultimate elastic modulus Low- Frequency Plateau Ultimate Elastic Modulus Evaporation Rheological Characterization Protocol 18
- 19. Hydrogel Fabrication Protocol Two Distinct Steps Chitosan/Genipin Crosslinking Alginate/CaCl2 Crosslinking Improving Homogeneity: Novel Fabrication Method
- 20. Hydrogel Fabrication Protocol 6 mL Two Distinct Steps Chitosan/Genipin Crosslinking •Chitosan dissolved in 0.4% acetic acid Alginate/CaCl2 Crosslinking Improving Homogeneity: Novel Fabrication Method
- 21. ~6.8 mL Hydrogel Fabrication Protocol Two Distinct Steps Chitosan/Genipin Crosslinking •Chitosan dissolved in 0.4% acetic acid Alginate/CaCl2 Crosslinking •Neutralized with 0.5M NaOH Improving Homogeneity: Novel Fabrication Method
- 22. Hydrogel Fabrication Protocol ~8.8 mL Two Distinct Steps Chitosan/Genipin Crosslinking •Chitosan dissolved in 0.4% acetic acid Alginate/CaCl2 Crosslinking •Neutralized with 0.5M NaOH •Addition of Genipin Improving Homogeneity: Novel Fabrication Method
- 23. 10 mL Hydrogel Fabrication Protocol Two Distinct Steps Chitosan/Genipin Crosslinking •Chitosan dissolved in 0.4% acetic acid Alginate/CaCl2 Crosslinking •Neutralized with 0.5M NaOH •Addition of Genipin •Brought to final volume with 0.85% NaCl Improving Homogeneity: Novel Fabrication Method
- 24. 10 mL Hydrogel Fabrication Protocol Two Distinct Steps Chitosan/Genipin Crosslinking •Chitosan dissolved in 0.4% acetic acid Alginate/CaCl2 Crosslinking •Neutralized with 0.5M NaOH •Addition of Genipin •Brought to final volume with 0.85% NaCl Improving Homogeneity: Novel Fabrication Method •Incubated at 37°C for 24 hours
- 25. 10 mL Hydrogel Fabrication Protocol Two Distinct Steps Chitosan/Genipin Crosslinking •Chitosan dissolved in 0.4% acetic acid Alginate/CaCl2 Crosslinking •Neutralized with 0.5M NaOH •Addition of Genipin •Brought to final volume with 0.85% NaCl Improving Homogeneity: Novel Fabrication Method •Incubated at 37°C for 24 hours •Alginate dissolved in 0.85% NaCl 5 mL
- 26. 15 mL0 mL Hydrogel Fabrication Protocol Two Distinct Steps Chitosan/Genipin Crosslinking •Chitosan dissolved in 0.4% acetic acid Alginate/CaCl2 Crosslinking •Neutralized with 0.5M NaOH •Addition of Genipin •Brought to final volume with 0.85% NaCl Improving Homogeneity: Novel Fabrication Method •Incubated at 37°C for 24 hours •Alginate dissolved in 0.85% NaCl •Chitosan/Genipin solution is added
- 27. 20 mL0 mL Hydrogel Fabrication Protocol Two Distinct Steps Chitosan/Genipin Crosslinking •Chitosan dissolved in 0.4% acetic acid Alginate/CaCl2 Crosslinking •Neutralized with 0.5M NaOH •Addition of Genipin •Brought to final volume with 0.85% NaCl Improving Homogeneity: Novel Fabrication Method •Incubated at 37°C for 24 hours •Alginate dissolved in 0.85% NaCl •Chitosan/Genipin solution is added •CaCl2 added to solution •Complete solution mixed for 30 minutes
- 28. 0 mL Hydrogel Fabrication Protocol Two Distinct Steps Chitosan/Genipin Crosslinking •Chitosan dissolved in 0.4% acetic acid Alginate/CaCl2 Crosslinking •Neutralized with 0.5M NaOH •Addition of Genipin •Brought to final volume with 0.85% NaCl Improving Homogeneity: Novel Fabrication Method •Incubated at 37°C for 24 hours •Alginate dissolved in 0.85% NaCl •Chitosan/Genipin solution is added •CaCl2 added to solution •Complete solution mixed for 30 minutes 20 mL •Centrifuge for 2 minutes at 2,000 rcf
- 29. 0 mL Hydrogel Fabrication Protocol Two Distinct Steps Chitosan/Genipin Crosslinking •Chitosan dissolved in 0.4% acetic acid Alginate/CaCl2 Crosslinking •Neutralized with 0.5M NaOH •Addition of Genipin •Brought to final volume with 0.85% NaCl Improving Homogeneity: Novel Fabrication Method •Incubated at 37°C for 24 hours •Alginate dissolved in 0.85% NaCl •Chitosan/Genipin solution is added •CaCl2 added to solution •Complete solution mixed for 30 minutes 20 mL •Centrifuge for 2 minutes at 2,000 rcf
- 30. 0 mL Hydrogel Fabrication Protocol Two Distinct Steps Chitosan/Genipin Crosslinking •Chitosan dissolved in 0.4% acetic acid Alginate/CaCl2 Crosslinking •Neutralized with 0.5M NaOH •Addition of Genipin •Brought to final volume with 0.85% NaCl Improving Homogeneity: Novel Fabrication Method •Incubated at 37°C for 24 hours •Alginate dissolved in 0.85% NaCl •Chitosan/Genipin solution is added •CaCl2 added to solution •Complete solution mixed for 30 minutes •Centrifuge for 2 minutes at 2,000 rcf •Excess liquid aspirated and gel used as needed ~2-5 mL of Usable Gel
- 31. Specific Aim 1 - Results 0.25% w/v alginate Sensitivity to changes in Ca2+ concentration • Alginate hydrogels demonstrate sensitivity to differences in calcium concentration as low as 1 mM • Fabrication process may allow for in-situ crosslinking at CSF Ca2+ levels * 31
- 32. Formulation of alginate chitosan hydrogel blends • Ca2+ is varied to control elastic modulus • Hydrogels approximate elastic modulus of spinal cord tissue (300-1000 Pa) Specific Aim 1 - Results • Composition varied to control crosslinking behavior 32
- 33. Mechanical response of alginate/chitosan hydrogels to in-situ conditions Specific Aim 1 - Results In-situ gelation model • 500 μL of hydrogel is injected into a chamber slide • 200 μL neurobasal media is added to chamber slide and replaced daily (1.4 or 6 mM Ca2+) • Hydrogel is incubated at 37°C • Rheological assessment is performed after 2 or 5 days A5/C0 G0/Ca22 Low Ca2+ (1.4 mM) A5/C25 G01/Ca20 Low Ca2+ (1.4 mM) A5/C25 G01/Ca20 High Ca2+ (6 mM) 33
- 34. Mechanical response of alginate/chitosan hydrogels to in-situ conditions Specific Aim 1 - Results A B A5/C0 G0/Ca22 A5/C25 G01/Ca20 In-situ gelation model • 500 μL of hydrogel is injected into a chamber slide • 200 μL neurobasal media is added to chamber slide and replaced daily (1.4 or 6 mM Ca2+) • Hydrogel is incubated at 37°C • Rheological assessment is performed after 2 or 5 days 34
- 35. Specific Aim 1 - Results Degradation Assay • 500 μL of hydrogel is injected into a 24 well plate • 200 μL of aCSF was added to each well and changed daily (1.4 mM Ca2+) • Hydrogel removed from well and weighed at designated time points 35
- 36. Specific Aim 1 - Results Hydrogel Electrical Charge • Ninhydrin Assay •Ninhydrin binds to primary amines on chitosan •Absorbance correlates to free amine concentration •Free amine groups are related to hydrogel charge Alginate Chitosan Genipin Positive Charge 36
- 37. • Overall similar structure for all hydrogel blends • Pores size appears concentration dependent •Hydrogel appearance is composition dependent – related to genipin concentration Specific Aim 1 - Results A25/C0 G0/Ca22 A5/C0 G0/Ca22 A25/C125 G1/Ca23 A25/C25 G05/Ca18 A5/C125 G1/Ca24 A5/C25 G01/Ca20 Hydrogel Appearance and Internal Structure 37
- 38. Specific Aim 1 - Conclusions • Alginate/chitosan hydrogels can be fabricated to mimic the elastic modulus of native CNS tissue • Hydrogels demonstrate sensitivity to mM changes in Ca2+ concentration • Changes in mechanical behavior are observed following incubation in Ca2+ containing media, in a concentration dependent manner – indicative of change in crosslinking behavior • Degradation, electrical charge and porosity are tunable by altering hydrogel composition Specific Aim 1 – Hypothesis Injectable alginate/chitosan hydrogels can be fabricated to mimic the elastic modulus of native CNS tissue and respond to change in external Ca2+ concentration while exhibiting tunable physical and mechanical properties 38
- 39. Specific Aim 2 Characterization of Astrocyte Attachment and Activation in Response to Interaction with Alginate/Chitosan Hydrogels Specific Aim 2 – Hypothesis Astrocytes will exhibit greater attachment to hydrogels that demonstrate a higher positive charge while exhibiting no significant increase in reactivity relative to astrocytes cultured on poly-D-lysine coated glass 39
- 40. 1 Sofroniew and Vinters 2010 1 1 22 Allen and Barres 2009 Specific Aim 2 - Rationale 2 Astrocytes are Active Participants in the CNS Environment 40
- 41. Specific Aim 2 - Rationale 1Powell et al 1997 2Liesi and Silver 1988 3Tom et al 2004 4Beck et al 2008 5Gris et al 2007 6Christopherson et al 2005 7Hurtado et al 2011 8 Deng et al 2011 Astrocytes Can Direct Neurite Outgrowth • ECM molecule production1-3 • Inhibitory: CSPGs, versican, keratin sulfate • Beneficial: Laminin, Fibronectin • Signaling molecules • Axon growth inhibition: ERG-14, TGFβ5, SOX95 • Synapse formation: Thrombospondins6 7 8 Neurons follow migrating astrocytes into biomaterials within the lesion site GFAP/SMI-31 41
- 42. Hydrogel Composition Influences Astrocyte Attachment Behavior Specific Aim 2 - Results A5/C0/G0/Ca22 A5/C125/G1/Ca24 A5/C25/G01/Ca22 Mag – 10X Scale bar – 300 μm Calcein-AM Hoechst 33342 Attachment Assay • 500 μL of hydrogel was injected into a chamber slide well • 200 μL of astrocyte media was added • Stained with Calcein-AM and Hoechst 33342 after two days 42
- 43. Hydrogel Composition Influences Astrocyte Attachment Behavior Specific Aim 2 - Results • Astrocyte attachment is composition dependent • Non-homogenous attachment – Clustering behavior is observed A A B CC A B B A A A A 43
- 44. Hydrogel Composition Influences Astrocyte Activation Specific Aim 2 - Results Astrocyte Activation Assay • 500 μL of hydrogel injected into chamber slide well • Astrocytes cultured for 2 days in astrocyte media. • Protein is isolated and expression was quantified via Western blot 44
- 45. Specific Aim 2 - Conclusions • Astrocyte Attachment is Composition Dependent • Astrocytes Exhibit Differential Attachment Behavior Across Hydrogel Surface • Hydrogel Composition Significantly Influences Astrocyte Activation Specific Aim 2 – Hypothesis Astrocytes will exhibit greater attachment to hydrogels that demonstrate a higher positive charge while exhibiting no significant increase in reactivity relative to astrocytes cultured on poly-D-lysine coated glass 45
- 46. Specific Aim 3 Assessment of the Mechanism of Astrocyte Attachment to Alginate/Chitosan Hydrogels and the Influence of Activation on Astrocyte Adhesion Specific Aim 3 – Hypothesis Differential astrocyte adhesion that is observed on hydrogels with different compositions is a consequence of altered astrocyte behavior and the transition of astrocytes to a more reactive state 46
- 47. Contradictory Attachment Behavior is Observed Specific Aim 3 - Rationale Attachment is not correlated with an increased positive charge 47
- 48. Specific Aim 3 - Rationale Possible Mechanisms Influencing Attachment • Increased astrocyte activation • Increased production of various ECM molecules • Significant morphological changes hypertrophy and branching • Increased astrocyte proliferation – Proliferation is correlated with astrocyte reactive state • Hydrogel crosslinking behavior 48
- 49. Specific Aim 3 - Results Artificial Astrocyte Activation • Astrocytes cultured on poly-D-lysine coated well plates or hydrogels for two days • Media was DMEM or DMEM with transforming growth factor β1 (TGF-β1) Influence of Increased Astrocyte Activation on Astrocyte Attachment 49
- 50. Specific Aim 3 - Results Influence of Hydrogel Components on Astrocyte Proliferation Astrocyte Proliferation Assay • Astrocytes were cultured on poly- D-lysine coated well plates • 24 hour attachment period • Following 24 hours, addition of media containing hydrogel components • After 24 hours, astrocytes stained for Ki-67 and Hoechst 33342 50
- 51. Specific Aim 3 - Results Influence of Hydrogel Components on Astrocyte Attachment Astrocyte Attachment Assay • Astrocytes were cultured on poly- D-lysine coated well plates • Media containing hydrogel components added simultaneously with astrocytes • After 48 hours, media was removed and cells were stained with Calcein-AM and Hoechst 33342 51
- 52. Specific Aim 3 - Results Influence of Hydrogel Components on Astrocyte Morphology 0.5% Alginate GNP 0.1 24 mM Ca2+ Poly-D-lysine coated glass control GFAP/Hoechst 33342 0.125% Chitosan/0.1% Genipin 0.25% Chitosan/0.01% Genipin 52
- 53. Proposed Hydrogel Crosslinking and Attachment Mechanism Specific Aim 3 - Conclusions Alginate Only 0.125% Chitosan / 0.1% Genipin 0.25% Chitosan / 0.01% Genipin High genipin/genipin Low alginate/chitosan Low genipin/genipin High alginate/chitosan 53
- 54. • Increasing astrocyte activation does not influence astrocyte attachment • Differential attachment is not a consequence of astrocyte proliferation • Hydrogel components influence astrocyte morphology and attachment in a concentration dependent manner Specific Aim 3 - Conclusions Specific Aim 3 – Hypothesis Differential astrocyte adhesion that is observed on hydrogels with different compositions is a consequence of altered astrocyte behavior and the transition of astrocytes to a more reactive state 54
- 55. Thesis Summary • Developed an injectable, calcium sensitive hydrogel material for use in acute SCI • Demonstrates increased crosslinking in an in situ gelation model • Variable degradation rate, porosity and charge • Promotes the attachment and activation of astrocytes in a composition dependent manner • Activation without proliferation mild activation? • Attachment is likely controlled by dominant forms of crosslinking within hydrogels • Varying hydrogel composition can promote/inhibit attachment Overall, the work in this thesis provide insight into the potential use of a novel calcium sensing biomaterial which may prove beneficial in providing an environment conducive to neuronal regeneration in a combinatorial treatment for acute spinal cord injury 55
- 56. Future Directions • Modifying alginate polymer composition to increase sensitivity to physiological Ca2+ concentrations • What is the role of ECM production? Do astrocyte produce significant inhibitory ECM molecules? • Influence on neuronal behavior? Do hydrogels have a significant influence on neuronal excitoxicity in response to increased Ca2+ in media? 56
- 57. Acknowledgements • Thesis Advisor • Dr. Ryan Gilbert • Committee Members • Dr. Deanna Thompson • Dr. Guohao Dai • Dr. Pankaj Karande • Gilbert Lab Graduate Students • Dr. Christopher Rivet • Jonathan Zuidema • Nicholas Schaub • Christopher Johnson • Dr. Deanna Thompson Lab • Dr. Linxia Zhang • Dr. Abby Koppes • Courtney Dumont • Chris Bertucci • Kathryn Kearns This work was funded by support from the National Institutes of Health, National Insititute of Neurological Disorders and Stroke R21NS62392 and NSF CAREER Award 1150125 to R. Gilbert. • Dr. Lee Ligon Lab • Dr. Lee Ligon • Joshua McLane • Undergraduate Research Students • Rebecca Pomrenke • Elise DeSimone • Nicholas Zaccor • Greg Desmond • High School Researchers • Addison Haxo • Austin Kim • David Frey – Scanning Electron Microscopy • Cindy, Dennis and Sarah McKay • Jacqueline Zaccor 57
Hinweis der Redaktion
- There are over a million people living in the United States with spinal cord injury with approximately 40,000 new cases occurring every year. There is a large financial cost of living with spinal cord injury over the course of a patients lifetime. Depending on the severity of the injury, total lifetime costs can total close to 5 million dollars. Within the United States alone, the cost of treatment and rehabilitation for all patients with spinal cord injuries nearly surpasses 10 billion dollars. Furthermore, if you enter the hospital with a spinal cord injury, there is less than a 1% chance that you will leave the hospital with the same amount of function as before your injury.
- Clinically, treatment options are rather limited and are mostly focused on the decompression and fixation of the spinal column to provide some stability to the area. Or with treatment with the anti-inflammatory steroid methylprednisilone. However, the use of methylprednisilone has recent come under debate as to its efficacy as a treatment for acute spinal cord injury and it is not a proven standard of care. According to the group Unite 2 Fight paralysis, there are over 50 clinical trials that are ongoing for various types of spinal cord injury. While a number of these have shown promise, 8 of them are in phase 3 trials, none have been thus far been demonstrated to be a complete cure for SCI. Thus, there is a significant need for an efficacious treatment therapy that is able to improve patient outcome following spinal cord injury.
- Physiologically, spinal cord injury is generally characterized by three distinct time periods, the first of which is termed the acute phase. The acute phase is generally defined as the first 48 hours following the initial traumatic event and is characterized by large scale changes to the spinal cord tissue at a macroscopic level. Trauma causes destruction of tissue structure and large-scale neuronal death and dieback, as well as destruction of near by blood vessels harming the vascular structure of the spinal cord. If the damage is severe enough, blood vessels may hemorrhage causing damage to the blood brain barrier and allowing the migration of blood-borne materials into the spinal cord environment
- Including shifts in the concentrations of electrolytes such as K, H, Na and Ca as well as accumulation of growth factors such as glutamate within the spinal cord lesion. There is a significant inflammatory response that is dependent on the severity of the injury, with inflammatory cells such as microglia and macrophages being recruited to the lesion site and releasing cytokines into the local environment. Also, there is the induction of a number of secondary injury cascades including free radical production, lipid peroxidation, calcium related neuronal apoptosis and ischemia leading to oxygen and glucose deprivation. Together, a number of these factors induce a condition called reactive astrogliosis, which is the transition of into a more reactive state when they exhibit significant changes in morphology and protein expression. In this image on the right we have astrocytes in healthy tissue that are stained for the intermediate filament glial fibrillary acidic protein. On the right are astrocyte that are slightly reactive and it can be seen that the exhibit significant morphological changes such as increased branching and a hypertrophic cell body.
- Of particular importance to this research is the mechanism of calcium related secondary neuronal death, which I am going to briefly describe for you Here we have a schematic of a synaptic cleft with pre synaptic and post synaptic neurons as well as an astrocyte process that is surrounding the synaptic cleft. Activation of glutamatergic receptors, in particular the NMDA calcium transoprters, increases intracellular calcium. This leads to a depolarization of the post synaptic neuron and the opening of voltage gated calcium channels. Due to the large calcium concentration gradient between the extracelllular and intracellular fluid, this promotes a significant increase in the intracellular calcium concentration.
- Astrocytes here are stained for the intermediate filament glial fibrillary acidic protein, a structural protein that is frequently used as an astrocyte marker. While all astrocytes do not necessarily express GFAP when healthy, the expression of GFAP is necessary for the formation of the glial scar and its expression is significantly upregulated following spinal cord injury. For this reason, the amount of GFAP that is expressed by astrocytes is often used as a method to determine how reactive astrocytes are and how capable they are of forming glial scar tissue.
- Solution: Development of a biomaterial system that is able to reduce the inhibitory nature of the spinal cord lesion during the acute and sub acute phase of spinal cord injury and promote the formation of an environment that is more conducive to restoration of function following spinal cord injury.
- There are two overall goals of this thesis: The development and characterization of an injectable composite hydrogel system that responds to physiological levels of Ca2+ in an attempt to promote in situ hydrogel formation and inhibit calcium related secondary injury Assessment of the astrocytic response to these hydrogels to examine their capacity for promoting or inhibiting scar formation To explore these goals, three specific aims were proposed, the first of which is:
- So why would we try to utilize a hydrogel system for spinal cord injury over that of other biomaterial systems? SCI vary in size and shape. On the left is a complete transection of the spinal cord, the middle is a blunt contusive injury and the right is a stab wound. In the clinical setting, complete transections of the spinal cord are relatively rare. Contusive injuries are by far the most common type of injury. They generally have irregular, non-uniform geometries and every type of injury is different. Hydrogels are perfect for this type of injury as they can be easily injected into lesion site and will conform to the geometry of the lesion, as opposed to other biomaterial treatments that may need to be fabricated in specific shapes.
- When trying to determine the best way to design this hydrogel material, there were a number of criteria that we wanted our material to exhibit. We wanted out material to exhibit homogenous crosslinking behavior so that portions of the spinal cord lesion would experience the material identically. Additionally, we wanted the material to be easily injectable. We wanted our material to mimic the mechanical properties of native CNS tissue. Neurons exhibit increased neurite branching and extension and astrocyte exhibit less reactivity on substrates that approximate the mechanical properties of their native tissue. Lastly, we wanted a hydrogel material that could respond to and utilize the increased extracellular Ca2+ present after SCI as a means for in situ gelation and possibly reduce the potential for calcium related secondary injury.
- - lack of inflammatory response in SCI models1 – potential for in-situ gelation using cerebrospinal fluid2,3 – poor cellular adhesion4 via electrostatic interactions5 To help control the phsical properties of our hydrogels, the natural crosslinking agent genipin was added to our hydrogel system. A number of hydrogel properties can be varied by controlling the chitosan/genipin crosslinking ratio. Genipin is expecially well suited for the spinal cord environment as it is non-cytotoxic at low concentrations, has been shown to induce neurite outgrowth and is anti-inflammatory.
- There are four possible types of crosslinking within a hydrogel that contains these materials. Chitosan/genipin crosslinking occurs between genipin and the amine groups on chitosan chains and the degree of crosslinking contributes to the overall hydrogel charge. Genipin/genpin crosslinking occurs through polymerization of genipin molecules and contributes to the interchain spacing and entanglement between chitosan chains. Chitosan/alginate crosslinking occurs through electrostatic interactions between negatively charged alginate and positively charged chitosan, leading to polyelecrolyte complex formation. Excess polyelectrolyte formation can make the material difficult to inject. Alginate/Ca2+ binding occurs though ionic bond formation between Ca2+ ions and guluronic acid residues of alginate. The degree of alginate/Ca2+ crosslinking contributes to the elastic modulus of the material.
- In order to test the potential for in situ gelation of alginate hydogels, solutions of alginate or alginate/chitosan were directly injected either an artificial CSF solution containing 1.4 mM CaCl2 or a 10 mM CaCl2 solution. Alginate solutions injected into aCSF exhibited little hydrogel formation. The images shown here are hydrogels after 2% aginate, 5% alginate or 5% algiante/0.5% chitosan were injected into a solution of 10 mM CaCl2. Due to the nearly instantaneous way that alginate and Ca2+ form crosslinks, these hydrogels are very non-homogenous. To assess the mechanical behavior of these gels, strain sweeps were performed. The graph shown here provides the elastic modulus for 5% alginate hydrogels injected in to either 10 mM or aCSF as a function of strain. \ These results demonstrate that the Ca2+ concentration present within aCSF is insufficient to provide an elastic modulus comparable to native spinal cord tissue (300-1000 Pa). These results indicate that this method is unlikely to form a homogenous and injectable material that able to sufficient interact with Ca ions in situ. Thus, I developed a novel hydrogel fabrication procudure that I am going to discuss briefly. Backwards graph labels
- In order to properly characterize the mechanical properties of our hydrogel materials, 4 rheological tests are performed in a specific order. I’m going to go over this rheological procedure briefly. First is a gelation time test. The hydrogel is fabricated at room temperature and then placed on the rheometer that has been heated to 37C and a time sweep is run at a constant strain and frequency. This graph depicts the results of a typical gelation time test, with elastic modulus on the y-axis and time on the x-axis. Hydrogels were considered equlibrated and fully gelled when the elastic modulus reaches a point that is less than ten percent that at the end of the test. Due to the nature of the test, the linear increase observed is due to evaporation of liquid in the hdyrogel. The next test was a strain sweep. This graph shows a typical strain sweep result, with elastic modulus on the y axis and strain on the x axis. Prior to running this test, the hydrogel is equilibrated on the rheometer for its respective gelation time, as previously determined. This test allows us to determine the linear viscoelastic limit of out material. This is the point at which a further increase in strain will cause permenant material deformation. It also allows us to determine viable strain values for further testing. The optimal choice is a strain value near, but not above the linear viscoelastic limit. These strain sweeps are important as the position of the linear viscoelastic limit provides important information on how strongly crosslinked a hydrogel material is.
- In order to properly characterize the mechanical properties of our hydrogel materials, 4 rheological tests are performed in a specific order. I’m going to go over this rheological procedure briefly. First is a gelation time test. The hydrogel is fabricated at room temperature and then placed on the rheometer that has been heated to 37C and a time sweep is run at a constant strain and frequency. This graph depicts the results of a typical gelation time test, with elastic modulus on the y-axis and time on the x-axis. Hydrogels were considered equlibrated and fully gelled when the elastic modulus reaches a point that is less than ten percent that at the end of the test. Due to the nature of the test, the linear increase observed is due to evaporation of liquid in the hdyrogel. The next test was a strain sweep. This graph shows a typical strain sweep result, with elastic modulus on the y axis and strain on the x axis. Prior to running this test, the hydrogel is equilibrated on the rheometer for its respective gelation time, as previously determined. This test allows us to determine the linear viscoelastic limit of out material. This is the point at which a further increase in strain will cause permenant material deformation. It also allows us to determine viable strain values for further testing. The optimal choice is a strain value near, but not above the linear viscoelastic limit. These strain sweeps are important as the position of the linear viscoelastic limit provides important information on how strongly crosslinked a hydrogel material is.
- The next test is a frequency sweep. This test is performed using the stain derived from our strains sweeps and hydrogels are equilibrated at 37C for the previously determined gelation time prior to testing. The elastic modulus is then measured as a function of the frequency used to strain the material. The optimal value to choose for further testing is within the low frequency plateau as highlighted here. At this point, the elastic modulus is stable and is relatively insensitive to frequency. Next we perform a time sweep using the parameters that were determined with out previous test. Performing a time sweep with these parameters allows us to determine the ultimate elastic modulus of our material, as the use of these parameters ensures that our material is only being elastically deformed and and no changes in the material are being made due to the testing parameters.
- There are two distinct step in the fabrication process for my hydrogel material.
- There are two distinct step in the fabrication process for my hydrogel material.
- There are two distinct step in the fabrication process for my hydrogel material.
- There are two distinct step in the fabrication process for my hydrogel material.
- There are two distinct step in the fabrication process for my hydrogel material.
- There are two distinct step in the fabrication process for my hydrogel material.
- There are two distinct step in the fabrication process for my hydrogel material.
- There are two distinct step in the fabrication process for my hydrogel material.
- There are two distinct step in the fabrication process for my hydrogel material.
- There are two distinct step in the fabrication process for my hydrogel material.
- There are two distinct step in the fabrication process for my hydrogel material.
- There are two distinct step in the fabrication process for my hydrogel material.
- Using this protocol, alginate hydrogels were fabricated using different amounts of CaCl2 and strain sweeps were performed in order to determine how sensitive mechanical behavior was to small changes in Ca during fabrication. 0.5% alginate hydrogels were fabricated with either 21, 22 or 23 mM CaCl2. The y-axis on this graph present the elastic modulus, while the x-axis is the strain applied. The results of this experiment demonstrate that alginate hydrogels fabricated with my procure are very sensitive to differences in Ca concentration as low as 1 mM during fabrication. These results provide evidence that hydrogels fabricated with this procedure may allow for further in situ crosslinking with the 1.4 mM Ca2+ concentration present in CSF.
- Utilizing this information, I fabricated 6 different hydrogel blends with varying concentrations of alginate, chitosan and genipin in order to create hydrogels with different relative amounts of crosslinking. Then by altering the caclium concentration to control alginate/calcium crosslinking, the ultimate elastic modulus of each hydrogel blend was modified so that there was no significant difference between blends and that all of the hydrogels exhibit an elastic modulus that approximates that of native spinal cord tissue. This graph demonstrates the ultimate elastic modulus of all hydrogels fabrciated as described previously. The three graphs on the left are fabricated with 0.5% alginate, while the three on the right are 0.25% alginate. For each group of three, the concentration of chiotsan is increasing from left to right.
- In order to test how these materials might be have within the spinal cord lesion, hydrogels were subjected an in situ gelation model. EXPLAIN MODEL VIA ON SCREEEN TEXT Here we have a hydrogel composed only of alginate and calcium that was subjected to media containing 1.4 mM Ca, the amount in healthy CSF. All incubated hydrogels exhibit a shift in the linear viscoelastic limit and an increased resistance to change at higher elastic modulus values. This indicates that a change in the internal crosslinking structure is occurring in response to exposure to this calcium containing media, similar what was shown earlier during explanation of the rheology protocol. This behavior is also dependent on the composition of the hydrogel. Here is a composite hydrogels where 0.25% chitosan and 0.01% genipin has been added during fabrication. Again, a shift in the linear viscoelastic limit is observed along with an increase in elastic modulus at high strain values indicative of a more strongly crosslinked hydrogel. This behavior is transient, as this resistance decreases from 2 days to 5 days in culture. Furthermore, an increase in the calcium concentration within the media influences the behavior of the hydrogel, preventing the transient decrease in elastic modulus after 2 days in culture.
- The concentration of Ca within media also significantly influences the ultimate elastic modulus of our material within an in situ gelation model. In this graph, we have the ultimate elastic modulus on the y axis, while the x-axis provides information about the length of time in the in situ model and the concentration of ca2+ in the media. Letters above the bars indicate statistically similar groups. Black bars indicate hydrogels that have been exposed to high concnetrations of Ca, while gray bars are hydrogels expose to low Ca concentrations. A significant decrease in ultimate elastic modulus is observed in hydrogels exposed to low levels of Ca over the course of the 5 day test period. However, increasing the concentration of Ca to levels observed following injury prevents this decrease in ultimate elastic modulus over. Again, this behavior is dependent on the composition of the hydrogel. For hydrogels fabricated with high chitosan and low genipin, a significant drop in ultimate elastic modulus is observed after 5 days in our in situ gelation model with low Ca2+, however the drop is significantly less than that observed for hydrogels without chitosan and genipin. Again, increasing the calcium concentration to injury like levels prevents this decrease after 5 days.
- Next, to determine how long our hydrogel material might persist within the spinal cord lesion, we performed a degradation assay. READ DEGRADATION ASSAY PROTOCOL FROM TEXT This table provides the results of this degradation assay as a percentage of the amount of gel that was remaining at certain time points. The first three columns are hydrogels with 0.5% alginate and an increasing amount of chitosan, while the next three are hydrogels with 0.25% alginate and an increasing amount of chitosan. Hydrogels without chitosan are completely degraded in approximately 7 days. The addition of chitosan and genipin significantly decreases the degradation rate of the hydrogels. This effect is composition dependent, hydrogels with high chitosan concentrations that form a significant amount of polyelectrolyte complexes show the slowest degradation rate, with one of them having 60% remaining at the end of the 28 day time period.
- Next we performed a ninhydrin assay in order to examine the positive charge character of our hydrogels. The results demonstrate that hydrogel composition has a significant influence on the positive charge character of our material. The y-axis on this figure is the number of free amine groups within each hydrogel blend. The absorbance of our hydrogels was compared to the abosrbance of a glycine standard curve in order to determine the number of free amine groups. These results are just as would have been expected. A decrease in the alginate concentration results in a increase in positive charge by decreasing polyelectrolyte complex formation. Additionally, an increase in chitosan and decrease in genipin results in a higher positive charge by decreasing the amount of chitosan/genipin crosslinking.
- To examine the internal structure of our hydrogel materials, we performed scanning electron microscopy on lyophollized hydrogels. The first row contains pictures and micrographs of hydrogels without chitosan and genipin, while the middle row contains hydrogels with low chitosan and high genipin and the bottom row contains hdyrogels with high chitosan and low genipin. Results demonstrate that there is an overall similar porous structure for all hydrogel blends. However, qualitatively there appears to be an increase in pore size for hydrogels fabricated with low chitosan and high genipin concentations. A further increase in chitosan with a decrease in genipin concentration appears to reduce the pore size back towards that of hdyrogels without chitosan and genipin.
- For years, astrocytes were thought of as only reactive and not active participants in the CNS environment. However, recent research has demonstrated that this is not the case. Astrocytes a number of duties within the healthy CNS including maintaining connection with the blood brain barrier, to control CNS metabolism and influence CNS blood flow through dilation and contraction of vasculature. This figure shows an astrocyte end foot interacting with a CNS blood vessel. Astrocytes also significantly interact with neurons both on nodes of ranvier and synaptic boutons on dendrites and at axonal synapses. This figure show an astrocyte that is extending processes and enveloping a neuron. Astrocytes can interact with up to 100,000 synapses in certain parts of the CNS. In this image, an astrocyte is interacting with the synapse between two neurons. Astrocytes not only help to control the concentration of neurotransmitters within the synaptic cleft, but they also send release their own “gliatransmitters” to influence neuronal behavior.
- Astrocytes also possess the ability to directly influence neurite outgrowth, through the production of a number of ECM molecules. Molcules that are inhibitory to neuronal growth include chondroitin sulfate proteoglycans, and other proteoglycans including versican and keratin sulfate proteoglycans. They also produce ECM molecules that may promote neuronal growth including laminin and fibronectin. The release a number of signalling molecules that can influence astrocyte behavior including the axonal inhibitory molecules ERG-1, TGFb and SOX9. They also release thrombonspondins which have been shown to increase neuronal synapse formation and may help guide neuronal growth. Furthermore, astrocytes have been shown to migrate first into biomaterials within in vivo models of spinal cord injury, with neurons often folllowing and migrating parallel to astrocytes. However, most biomaterials are designed specifically for promoting neuronal growth and how they influence astrocyte behavior is often an afterthought My contention is that promoting positive interaction with astrocytes is just as important as promoting neuronal growth. If a material is placed within the spinal cord lesion and it induces significant astrocyte activation and glial scar production, it does not matter how well the material promotes neuronal regeneration as neurons will not like ever reach the material.
- Thus, we decided to focus on characterize the astrocyte interaction with our biomaterial. The first experiment we ran was an attachment assay in order to examine how well astrocytes attach the the surface of our hydrogels with different compositions. READ ASSAY PROTOCOL. Results show that the hydrogel composition has a significant influence on astrocyte attachment to the hydrogel surface. The image on the left shows astrocytes on hydrogels without chitosan/genipin, in the middle are hydrogels with low chitosan and high genipin and on the right are hydrogels with high chitosan and low genipin. Qualitatively, the greatest amount of astrocyte attachment is observed on hydrogels with low chitosan and high genipin, compared to other hydrogel groups. Additionally, astrocytes appear to spread out across the entirety of the hydrogel surface.
- Matlab was used to quanitify the number of astrocytes attached to each hydrogel type as well as examine clustering behavior on the hydrogel surface. In this graph, the y-axis is the average number of cells per sample. The x-axis is the type of hydrogels. The first three bars are low alginate hydogels, while the second three bars are high alginate hydrogels. Black bars represent hydrogels without chitosan, light gray bars are hydrogels with low chitosan and high genipin and dark grey bars are hydrogels with high chitosan and low genipin. Letters above bars represent groups that are statistically similar. Results demonstrate that attachment to hydrogels is indeed composition dependent. 1st (Red) – The addition of 0.125% chitosan and 0.1% genipin results in increased attachment relative to hydrogels with no chitosan or genipin. 2nd (Blue) – An Increase in alginate concentration from 0.25% to 0.5% when all else is constant results in decreased astrocyte attachment 3rd (Green) – Furthermore, an increase in chitosan to 0.25% and decrease in genipin to 0.01% results in decreased attachment, relative to hydrogels with low chitosan and high genipin as seen by the red bars. 4th (Orange) – Additionally, clustering analysis demonstrated that astrocytes ten to cluster in large groups on hydrogels with high chitosan and low genipin, while astrocytes spread out evenly over the surface of all other hydrogels.
- Next, we examined the reactivity response of astrocytes to culture on the surface of our hydrogels. To do this, we performed an astrocyte activation assay. READ TEXT ON SLIDE. The top of this figure is representative Western blots for GFAP and the control protein alpha tubulin. As described previously, GFAP is an astrocyte marker that is used to assess the reactivity of astrocytes as it is significantly increased following spinal cord injury and its expression is necessary for the formation of the glial scar. The bottom is a quantification of Western blot results. The y axis show the GFAP expression normalized to the expression of the control protein alpha tubulin. The x axis shows the type of surface astrocytes were cultured on. Results demonstrate that GFAP expression is increased on all hydrogels relative to expression on poly-D-lysine coated glass, however only astrocytes cultured on hydrogels with low chitosan and high genipin concentrations show significant. Furthermore, GFAP expression appears to be correlated with astrocyte attachment, providing a possible mechanism for the increased attachment that is observed on some hydrogels.
- 2 - Clustering behavior on hydrogels with higher or no chitosan Astrocyte activation appears to correlate with attachment
- In order to do this, three specific aims were proposed. The first of which is…..
- As was mentioned previously, astrocyte attachment is not necessarily correlated with the positive charge of the material, as measured by ninhydrin assay. This shows evidence of that. The y axis is the amount of free amine groups in each hydrogel as previously measured by the ninhydrin assay and the x axis is the average number of cells that were attached to each hydrogel sample. The first thing to notice is that the is no linear relationship between the amount of free amine groups and the number of attached cells. A decrease in the alginate concentration within hydrogels results increase in charge and cell attachment, shown by markers with the same color. However, when the chitosan concentration is increased and genipin concentration is increased, an increase in positive charge and a decrease in attachment are observed, as shown by markers of the same shape.
- A number of experiments were explored in order to explore the possible reasons for this contradiction. Increased astrocyte activation could cause altered astrocyte attachment. Astrocytes produce a number of ECM molecules when they are activated including various proteoglycans, laminin and fibronectin that may be inhibitory or promote cellular attachment. Additionally, astrocytes undergo significant morphological changes after activation including increased branching of processes and hypertrophy of the cell body that may influence the ability of the astrocyte to attach. Increased astrocyte proliferation may be responsible for the reason more cells appear on some hydrogel compositions more than others. Astrocyte proliferation is increased during reactive astrogliosis and GFAP expression was positively correlated with astrocyte attachment on our hydrogels. Lastly, the different hydrogel components and their crosslinked forms may be directly influencing the ability of astrocytes to the hydrogel surface.
- The first experiment that we looked at was the influence of increased astrocyte activation on astrocyte attachment to the hydrogel surface. In order to do this, astrocytes were cultured on either poly-D-lysine coated well plates or hydrogels for two days. Media used during this either DMEM or DMEM with transforming growth factor beta 1 or TGF beta 1. TGFb1 was utilized to activated astrocytes as it is strongly correlated with the ability for astrocytes to exhibit a scar forming phenotype and is strongly anti-proliferation. Thus any changes observed will be solely due to astrocyte activation and not proliferation. Astrocytes were first cultured on PDL coated glass and GFAP expression was quantified via Western blot. Astrocytes were exposed to either 0, 10 or 25 ng/mL of TGFb1. The top graph are representative Western blots for GFAP and the control protein alpha actin. The bottom graph is a quantifcation of this blot, with the y-axis being the expression of GFP relative to alpha actin.The results demonstrate that TGFb1 is able to increase the reactivity of astrocytes in a concentration dependent manner. Astrocytes were then culture on hydrogels with either 0 or 25 ng/mL. After two days in culture, astrocytes were stained with Calcein-AM and Hoechst 33342 and co-stained cells were counted. This graph shows the average cells per sample on the y-axis and the type of hydrogel on the x axis. Black bars represent astrocytes subject to only DMEM while grey bars are astrocytes subjected to 25 ng/mL TGFb. The results demonstrate that astrocyte activation with TGFb produces no significant change in astrocyte attachment.
- Next, the influence of different hydrogel components on astrocyte proliferation was assessed. Astrocytes were cultured on poly-D-lysine coated well plates and allowed to attach for 24 hours. Following 24 hours, media was removed and replaced with media containing either alginate, chitosan, genipin, increased calcium or alginate/chitosan crosslinked groups or genipin/chitosan crosslinked groups at the concentrations they are present at within hydrogels. After 24 hours, astrocytes were stained for Hoechst 33342 and the prolifearation Ki-67. Ki-67 is expressed within all phases of the cell cycle besides the resting phase and assesses actively proliferating astrocytes. On this graph, the y-axis represents the ratio of Ki-67 positive cells to the number of Hoechst 33342 positive cells and indicates the number of actively proliferating astrocytes. The x-axis is the type and concentration of hydrogel component astrocytes were exposed to. GO OVER THE GROUPS. The results demonstrate that, although there is a difference in proliferation between some individual component groups, there is no significant difference in proliferation between any component group and astrocytes on glass controls. This indicated that proliferation is unlikely the reason for differential astrocyte attachment to hydrogels.
- In a similar experiment, the influence of hdyrogel components on the ability to attach to PDL coated glass was assessed. This method was chosen as the amine groups on the PDL coating represent a model of how these hydrogel components may influence astrocyte attachment to chitosan on our hydrogels. The y-axis on this graph is the average number of cells that were co-stained for Calcein-AM and Hoechst 33342, while the x-axis is the type of treatment as before. Results demonstrate that all component treatments result in a significant decrease in astrocyte attachment to PDL coated glass, relative to control with only astrocyte media. Of particular note is that components that are known to react with amine groups generally cause the greatest decrease in attachment including alginate, genipin as well as their respective crosslinked groups.
- Furthermore, hydrogel components have a significant influence on astrocyte morphology. The same experiment as previous was repeated, however astrocyte were stained with GFAP and Hoechst 33342 in order to get a better image of astrocyte morphology. The large graph is astrocyte cultured on poly-D-lysine coated glass and these astrocyte look healthy and extend a number of processes out. The addition of alginate to media significantly decreases the number of attached astrocytes. Furthermore, these astrocytes exhibited a very balled up appearance and do not extend out many processes. Similarly, astrocytes treated with 0.125% chitosan and 0.1% genipin exhibit and unhealthy balled up appearance as well. However, when the concentration of chitosan is increase and genipin is increased, astrocytes exhibit a morphology resembling that of the control group. Add scale bar images
- Using all of this data, I have come up with a proposal for how hydrogel crosslinking is influencing astrocyte attachment to the surface of our hydrogels. This first graph is a schematic of crosslinking within hydrogels not containing chitosan or genipin. Alginate/calcium crosslinks can be seen between calcium ions and guluronic acid residues on the alginate chains in an egg box shape, with astrocytes attaching in between these crosslinks. When 0.125% chitosan and 0.1% genipin was added to these hydrogels, a significant increase in astrocyte attachment was observed. The high genipin/genipin crosslinking in these gels creates long polymerized genipin molecules between chitosan chains, increasing the space between chitosan chains and spacing out free amine groups. This also would help to limit the formation of large sections of alginate/chitosan polyelectrolyte complexes. This is possibly the reason that astrocyte attach to the entire surface of these gels. When chitosan is increased and genipin is decreased, the number of free amine groups increases significantly because of less chitosan/genipin crosslinking. This allows for a significant increase in the formation of large groups of algiante/chitosan polyelectrolyte complexes. Due to the neutral charge of these crosslinks, astrocytes would likely avoid these as attachment sites and might we why astrocytes cluster on these hydrogel surfaces.