Polyglycerol sebacate, a soft substrate for tissue engineering Chris Highley April 3, 2007 Prof. Matyjaszewski Polymer

1. Poly(glycerol sebacate), a soft substrate for tissue engineeringChris HighleyApril 3, 2007Prof. MatyjaszewskiPolymer Chemistry

2. The body is a mechanically dynamic environment Deformation of implants must be sustainable without irritation to surrounding tissue Three classes of biodegradable elastomers reported: Hydrogels Elastin-like peptides Polyhydroxyalkanoates (PHAs) Research shows that substrate stiffness influences contractility, motility, and spreading of cells. Image shows the �interplay of physical and biochemical signals in the feedback of matrix stiffness on contractility and cell signalling� People suspect stiffness of surroundings have effects on the paths cells take in differentiation--for example in organogenesis during development; there are implications for disease and regeneration as well. Kris Dahl, a prof in BME is looking at how the mechanics within an hESC affect its fate. Clearly internal factors are tied to external.Research shows that substrate stiffness influences contractility, motility, and spreading of cells. Image shows the �interplay of physical and biochemical signals in the feedback of matrix stiffness on contractility and cell signalling� People suspect stiffness of surroundings have effects on the paths cells take in differentiation--for example in organogenesis during development; there are implications for disease and regeneration as well. Kris Dahl, a prof in BME is looking at how the mechanics within an hESC affect its fate. Clearly internal factors are tied to external.

3. Hydrogels But hydrogels lack toughness� CMC = Carboxymethyl cellulose MC = Methyl cellulose HPMC = Hydroxypropylmethyl cellulose Cellulose is plant derived--humans cannot digest this (but does not mean it�s not biocompatible) Crosslinking when hydrazide and aldehyde groups form a hydrazone bond Degradation mechanism: enzymatic hydrolysis (hyaluronidase catalyzes this) Crosslinking has an effect on mechanical properties--crosslinker type and density (Lee et al.): looked at alginate and compared covalent crosslinking using EDC (and adipic dihydrazide or PEG or lysine) and ionic crosslinking using Ca2+ HA-DEX synthesis Step 1: Prepare dex-suc ester by reacting dexamethasone with succinic anhydride and 4-dimethylaminopyridine in anhydrous acetone Step 2: Prepare NHS-dex-suc ester by reacting dex-suc ester with N-hydroxysuccinimide and dicyclohexyl carbodiimide in acetone Step 3: React NHS-dex-suc in dimethylformamide with HA-A in sodium bicarbonate HA-A synthesis: HA reacted with large molar excess of adipic dihydrazide in presence of 1-ethyl-3-carbodiimide (EDC) and 1-hydroxybenzotriazole (HOBt) and purified HA-B: Add sodium periodate, terminate reaction with ethylene glycolCMC = Carboxymethyl cellulose MC = Methyl cellulose HPMC = Hydroxypropylmethyl cellulose Cellulose is plant derived--humans cannot digest this (but does not mean it�s not biocompatible) Crosslinking when hydrazide and aldehyde groups form a hydrazone bond Degradation mechanism: enzymatic hydrolysis (hyaluronidase catalyzes this) Crosslinking has an effect on mechanical properties--crosslinker type and density (Lee et al.): looked at alginate and compared covalent crosslinking using EDC (and adipic dihydrazide or PEG or lysine) and ionic crosslinking using Ca2+ HA-DEX synthesis Step 1: Prepare dex-suc ester by reacting dexamethasone with succinic anhydride and 4-dimethylaminopyridine in anhydrous acetone Step 2: Prepare NHS-dex-suc ester by reacting dex-suc ester with N-hydroxysuccinimide and dicyclohexyl carbodiimide in acetone Step 3: React NHS-dex-suc in dimethylformamide with HA-A in sodium bicarbonate HA-A synthesis: HA reacted with large molar excess of adipic dihydrazide in presence of 1-ethyl-3-carbodiimide (EDC) and 1-hydroxybenzotriazole (HOBt) and purified HA-B: Add sodium periodate, terminate reaction with ethylene glycol

4. Elastin-like peptides Drawbacks: Expensive Immunogenic Endotoxins may be present Synthesis of elastin-like peptides can be done using bacterial fermentation: create DNA plasmid coding for your desired polypeptide, and perform a bacterial transformation. It seems organic chemistry techniques make it difficult to obtain high molecular weight polymers, and elastin yields from bacteria are also high. Also allows complete control of protein structure. In the Macromolecules study, their aim was to develop materials for vascular engineering These polymers not only provide a flexible substrate, but you can also engineer binding sequences into them specific to the cell types you want to grown on them (Welsh and Tirrell). Amine specific cross linker avoided disturbing their binding domain.Synthesis of elastin-like peptides can be done using bacterial fermentation: create DNA plasmid coding for your desired polypeptide, and perform a bacterial transformation. It seems organic chemistry techniques make it difficult to obtain high molecular weight polymers, and elastin yields from bacteria are also high. Also allows complete control of protein structure. In the Macromolecules study, their aim was to develop materials for vascular engineering These polymers not only provide a flexible substrate, but you can also engineer binding sequences into them specific to the cell types you want to grown on them (Welsh and Tirrell). Amine specific cross linker avoided disturbing their binding domain.

5. Polyhydroxyalkanoates Drawback: Reversible deformations are not as large as those seen in PGS PHAs are polyesters that naturally accumulate in bacteria, and they are inherently biodegradable by hydrolysis. They occur as a result of synthesis pathways which are favored when bacteria are grown in media with excess carbon (eg glucose) but limited in an essential nutrient, such as nitrogen or phosphate--the synthesis acts as a carbon reserve and an electron sink. PHB is a thermoplastic which when compolymerized with other PHAs acquires favorable properties (eg easier processing, less stiff, more tough). Image is a transgenic Arabidopsis expressing the PHB pathway in the cytoplasm. Red represents agglomerations of PHA inclusions (result of staining with Nile Blue A--a lipophilic dye). Model plant for genetic and molecular studies of plants and closely related to rapeseed, which would be used to produce PHA on a large scale.PHAs are polyesters that naturally accumulate in bacteria, and they are inherently biodegradable by hydrolysis. They occur as a result of synthesis pathways which are favored when bacteria are grown in media with excess carbon (eg glucose) but limited in an essential nutrient, such as nitrogen or phosphate--the synthesis acts as a carbon reserve and an electron sink. PHB is a thermoplastic which when compolymerized with other PHAs acquires favorable properties (eg easier processing, less stiff, more tough). Image is a transgenic Arabidopsis expressing the PHB pathway in the cytoplasm. Red represents agglomerations of PHA inclusions (result of staining with Nile Blue A--a lipophilic dye). Model plant for genetic and molecular studies of plants and closely related to rapeseed, which would be used to produce PHA on a large scale.

6. PGS qualities Good mechanical properties from covalent crosslinking and H-bonding interactions Rubberlike elasticity can be obtained by building 3-d network of random coils through copolymerization where at least one monomer is trifunctional Meets the following design criteria: Hydrolysis is the degradation mechanism (as opposed to relying on enzymes can result in individual differences) Low crosslinking density (not as brittle) Wanted to have crosslinks be hydrolyzable as well for homogeneous degradation Nontoxic monomers, with one trifunctional Ester bond hydrolysisEster bond hydrolysis

7. Monomers Gycerol: CH2(OH)CH(OH)CH2OH--basic building block of lipids Sebacic acid: (HOOC(CH2)8COOH) Natural metabolic intermediate in ?-oxidation of fatty acids Not too short (short chain, dicarboxylic acids more likely to cyclize) or too long (hydrophobicity and poor mixing with glycerol) Both demonstrated safe in vivo and approved for medical applications. Also, they are cheap.

8. PGS �biorubber� not the first PGS synthesis Nagata et al. synthesized a number of network aliphatic copolyesters The copolyesters synthesized were rigid, totally crosslinked polymers Network copolyesters prepared from gylcerol and sebacic acid with 10-90 mol % of either succinic acid, 1,12-dodecanedicarboxylic acid, 1,18-octadecanedicarboxylic acid, or terephthalic acid Prepared using melt-polycondensation, cast in dimethylformamide solution, and post-polymerized at 230-250C to create polymer network In previous studies had shown that films created from network polyester were degraded by lipase enzymes (they hydrolyze ester bonds) They used 60 mmol dicarboxylic acid mixtures and 40 mmol glycerol and heated under conditions varying slightly by the makeup of the mixture Wide angle X-ray diffractometer gave them figure 2. It shows a distinct peak at ca 20 degrees for all the copolymers, indicating an ordered structure. And the peak angle is not affected by the copolymerization. The paper simply listed their observations of the properties of the various polymers.Network copolyesters prepared from gylcerol and sebacic acid with 10-90 mol % of either succinic acid, 1,12-dodecanedicarboxylic acid, 1,18-octadecanedicarboxylic acid, or terephthalic acid Prepared using melt-polycondensation, cast in dimethylformamide solution, and post-polymerized at 230-250C to create polymer network In previous studies had shown that films created from network polyester were degraded by lipase enzymes (they hydrolyze ester bonds) They used 60 mmol dicarboxylic acid mixtures and 40 mmol glycerol and heated under conditions varying slightly by the makeup of the mixture Wide angle X-ray diffractometer gave them figure 2. It shows a distinct peak at ca 20 degrees for all the copolymers, indicating an ordered structure. And the peak angle is not affected by the copolymerization. The paper simply listed their observations of the properties of the various polymers.

9. The biorubber synthesis Transparent, colorless elastomeric product Product features small number of crosslinks and hydroxyl groups directly attached to the backbone FTIR confirms ester bonds and indicates hydroxyl groups are hydrogen bonded Polycondensation of 0.1 mol of each (glycerol and sebacic acid) at 120C under argon for 24 h before reducing pressure from 1 torr to 40 mtorr over 5 h Kept at 120C and 40 mtorr for 48h KBr pellet of polymer used on Nicolet Magna-IR 550 spectrometer C=O stretch at 1740 confirms ester bonds OH stretch (broad, intense) at 3448 indicates hydroxyl groups are hydrogen bondedPolycondensation of 0.1 mol of each (glycerol and sebacic acid) at 120C under argon for 24 h before reducing pressure from 1 torr to 40 mtorr over 5 h Kept at 120C and 40 mtorr for 48h KBr pellet of polymer used on Nicolet Magna-IR 550 spectrometer C=O stretch at 1740 confirms ester bonds OH stretch (broad, intense) at 3448 indicates hydroxyl groups are hydrogen bonded

10. Product characterization Hydroxyl groups = hydrophilic; water-in-air contact angle: 32.0� In water: insoluble, swells 2.1 ? 0.33% after 24 h Composition of monomers approximately 1:1 (by elemental analysis) Crosslinking density 38.3 ? 3.4 mol/m3, MC = 18,300 ? 1,620 TC = -52.14 and -18.50�C; TM = 5.23 and 37.62�C (from DSC) Contact angle: nearly identical to flat 2.7 nm thick type I collagen (31.9) Differential scanning calorimetery done on Perkin-Elmer DSC calorimeter (no glass transition temp above -80, which was lower limit of instrument) Contact angle measured at room temp using sessile-drop method and VCA2000 video contact angle system to analyze drop profile Element analysis performed by QTI (C13H22O5 is monomer if 1:1; C should be 60.47, found 60.46; H should be 8.53, found 8.36) Cross-linking density (moles of active network chains per unit volume): 38.3+/-3.4 mol/m^3 Molecular mass between crosslinks: from n = E/3RT = p/M (E=Young�s modulus, R=gas const, T=temp, p=density) They have created sheets and foams using melting/solubility in organics: creating NaCl particles and a PTFE mold, curing in vacuum at 120C and 100 mtorr, then salt leaching Curve shape resembles that of ligament and vulcanized rubber--repeated elongation to at least 3x original length; tensile Young�s modulus is ca. 0.282 MPa--indicating soft material (P4HB, is a reportedly elastomeric degradable PHA, much stiffer) YM of PGS is between ligaments (which contain elastin in addition to collagen) and tendon (mostly collagen) Soaking polymer does not alter mechanical propertiesContact angle: nearly identical to flat 2.7 nm thick type I collagen (31.9)

11. Biocompatibility of PGS Growth rate of fibroblasts greater on PGS (MTT assay) Morphology differences in PGS-coated dishes (A) compared to PLGA (B) In vivo results show biocompatibility equal to, or better than, PLGA Another study showed biocompatibility with nerves Open circle (PGS) and open square (PLGA) show inflammatory zones (implants into rats) to be the same. However, thickness of fibrous capsule (closed shapes)--an avascular collagen layer--was very different. This fibrous capsule is important in implants, because it can block mass transfer between an implant and surrounding tissues. Found PGS to completely reabsorbed and undetectable at 60 days; data suggest that mechanical strength decreases linearly with mass loss--important for tissue eng, drug del, and in vivo sensors Sundback et al found biocompatibility with nerves--plated nerves on PGS; also implant PGS next to sciatic nerve in ratsOpen circle (PGS) and open square (PLGA) show inflammatory zones (implants into rats) to be the same. However, thickness of fibrous capsule (closed shapes)--an avascular collagen layer--was very different. This fibrous capsule is important in implants, because it can block mass transfer between an implant and surrounding tissues. Found PGS to completely reabsorbed and undetectable at 60 days; data suggest that mechanical strength decreases linearly with mass loss--important for tissue eng, drug del, and in vivo sensors Sundback et al found biocompatibility with nerves--plated nerves on PGS; also implant PGS next to sciatic nerve in rats

12. PGS for vessels Engineered blood vessels Favorable immune response reported Platelet adherence also in favor of PGS a is glass, b PGS, c ePTFE, d PLGA Microporous PGS structure can also be created using salt-fusion Vessels: dip-coated glass rods with PGS; porous poly(1,8-octanediol citrate) (POC) outer layer added by placing PGS-coated rod in a Teflon tube mold containing a salt-POC slurry, then post-polymerizing ePTFE (Teflon): expanded polytetrafluoroethylene, a standard for vascular grafts Salt fusion technique: create a mold, fill it with salt, incubate at 37C and 88% humidity, and the salt fuses. You can then cure PGS on this and use the same salt leaching technique to remove the salt, leaving connected pores allowing for mass-transfer and cell-cell communication.Vessels: dip-coated glass rods with PGS; porous poly(1,8-octanediol citrate) (POC) outer layer added by placing PGS-coated rod in a Teflon tube mold containing a salt-POC slurry, then post-polymerizing ePTFE (Teflon): expanded polytetrafluoroethylene, a standard for vascular grafts Salt fusion technique: create a mold, fill it with salt, incubate at 37C and 88% humidity, and the salt fuses. You can then cure PGS on this and use the same salt leaching technique to remove the salt, leaving connected pores allowing for mass-transfer and cell-cell communication.

13. Microfabrication of PGS Photolithography and etching techniques (commonly used in MEMS devices) allow the creation of wafers for patterning. Sucrose was spin coated onto the silicon wafer to create a release layer (additionally smoothing the ridges and grooves). PGS was molded on the surface, cured, and removed by dissolving the sucrose in water. Resulting substrate is flexible and biodegradable. (A) Shows a PGS substrate which was created with this microfabrication technique. Bovine aortic epithelial cells are growing on it. The topology has a 2.5 um period (scale bars are 10 um and 1 um). Inset is showing how the filipodia like to touch the apex of the microsctructures (indicating why at larger periods, less cell alignment was seen). MEMS techniques also used to create a microvasculature, which was endothelialized under flow conditions with part of the lumens reaching confluence within 14 days. This could lead to tissue-engineered microvasculatute with is critical in engineering vital organs. Patterned PGS shown with 500 um scale bar and 200 um (bottom) With cells: 10x and 40x magnifitcationPhotolithography and etching techniques (commonly used in MEMS devices) allow the creation of wafers for patterning. Sucrose was spin coated onto the silicon wafer to create a release layer (additionally smoothing the ridges and grooves). PGS was molded on the surface, cured, and removed by dissolving the sucrose in water. Resulting substrate is flexible and biodegradable. (A) Shows a PGS substrate which was created with this microfabrication technique. Bovine aortic epithelial cells are growing on it. The topology has a 2.5 um period (scale bars are 10 um and 1 um). Inset is showing how the filipodia like to touch the apex of the microsctructures (indicating why at larger periods, less cell alignment was seen). MEMS techniques also used to create a microvasculature, which was endothelialized under flow conditions with part of the lumens reaching confluence within 14 days. This could lead to tissue-engineered microvasculatute with is critical in engineering vital organs. Patterned PGS shown with 500 um scale bar and 200 um (bottom) With cells: 10x and 40x magnifitcation

14. The future of PGS A limitation from a tissue engineering standpoint is cell seeding Also, due to synthesis conditions, loading drugs (certain proteins) may be difficult Surface modifications may alleviate some of this However, degradation is by surface erosion, rather than bulk degradation* Composites of this and another material(s) (such as PLGA or a hydrogel) could prove to be useful tissue engineering platforms Control over surface morphology also may allow for novel composites Not sure if the �softness� of this substrate is perceptible to a cell (in comparison to PLGA) Still appears to involve a lot of the hand-waving/trial and error that seems to be everywhere in tissue engineering: throw some cells onto a new chemistry, watch what happens (either in vitro or in vivo)