Figure S1. Aldehyde modifications. Aldehyde groups were synthesized on HA (a) and DS (b) polysaccharide using sodium periodate (NaIO4) oxidation of diol group. Different reaction conditions were chosen for each polysaccharide due to differences in the amount of diol groups per repeat unit. For HA modification, 1:2 NaIO4:HA for 2 hr was used, while for DS modification, 2:1 NaIO4:HA for 5 hr was used.
Figure S2. Periodate polysaccharide degradation. HA polymers degraded due to non-specific reactions during the oxidation reaction. Starting with a 390 kDa HA, the HA degraded to 55 kDa following 1:2 NaIO4:HA reaction for 2 hr. All oxidation reactions were for 2 hr. Values reported are number-averaged molecular weights (Mn) as determined by gel permeation chromatography.
Figure S3. Peptide synthesis. Peptides synthesized through solid-phase peptide synthesis were analyzed using MALDI-TOF mass spectroscopy to verify mass. A large peak at 1582 Da confirmed successful synthesis of the peptide GCNSGGRMSMPVSNGG-Hyd.
Figure S4. Peptide coupling. The characteristic 1H NMR peak from maleimide protons was used to verify coupling of a thiol containing peptide to a maleimide modified HA. The tert-butylammonium salt of HA (HATBA) was first modified with maleimide groups through a (Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) catalyzed coupling of an amine terminated maleimide to carboxylic acid groups along the HA backbone. Approximately 38% of the repeating disaccharides were modified with a maleimide group. Peptides were then coupled to HA-maleimide through a thiol-maleimide click reaction between the thiol of the cysteine in the peptide sequence and the maleimide on HA-maleimide. Disappearance of the maleimide peak after the coupling reaction confirmed complete conversion of the maleimide groups following reaction with thiols.
Figure S5. Hydrogel rheological characterization. The crosslinking behavior of hydrogels over a range of macromer concentrations (1-5 wt%) was monitored with rheology. Hydrogels with a range of gel points (~0.5 to 5 min) and final storage moduli (~6 to 1500 Pa) were obtained through changes in macromer concentration.
Figure S6. MMP mediated hydrogel degradation. Hydrogels over a range of macromer concentrations (2-5 wt%) were incubated in active MMPs (20 U/mL collagenase, type 4) and mass loss was quantified using an uronic acid assay for HA content (mean±SEM, n=3 gels per formulation). Hydrogels with higher macromer concentrations degraded more slowly than those at lower macromer concentrations in the same amount of active MMP due to the higher number of MMP degradable crosslinks present.
Figure S7. Passive rTIMP-3 release from hydrogels. The release behavior of encapsulated rTIMP-3 was quantified from hydrogels over a range of macromer concentrations (2-5 wt%) using ELISA. Passive rTIMP-3 release was reduced by increasing macromer weight percent from 2 to 3.5 wt%, but no further reduction was observed by increasing the macromer weight percent from 3.5 to 5 wt% (mean±SEM, n=3 gels per formulation).
Figure S8. rMMP-2 activity in vitro. rMMP-2 activity using in vitro fluorogenic substrate assay. Following activation of recombinant proMMP-2 (R&D systems), MMP-2 gradually decreases in protease activity.
Figure S9. Hydrogel reconstruction with ITKsnap. Representative MRI images of myocardium from remote and hydrogel injection regions (scale bars = 10 mm). Pockets of hydrogel were identified within the myocardium due to the increased MRI signal intensity of the hydrogels compared to myocardium (white arrows).
Figure S10. HA hydrogel confirmation. Tissue sections were harvested 14 days post MI, frozen, sectioned and stained with alcian blue to visualize HA hydrogels. (a) Representative photomicrograph of hydrogel within the myocardium. (b) Hydrogel pockets degraded after incubation with hyaluronidase, confirming the presence of the HA based hydrogel. Scale bars = 100 µm.
Figure S11. Myocardial MMP expression 14 days post MI. Myocardial expression of MMP subclasses within the MI region and remote myocardium 14 days following MI induction including the gelatinases (MMP-2 and 9), collagenases (MMP-13) and membrane-type MMPs (MMP-14). Expression of all MMP subclasses was significantly elevated 14 days in the MI region following MI induction. Hydrogel injections significantly inhibited MMP-14 expression compared to the MI only group, while hydrogel/rTIMP-3 injections significantly inhibited MMP-13 expression compared to the MI only and hydrogel injection groups. Only MMP-9 expression was significantly upregulated in the remote myocardium following MI, albeit slightly, while hydrogel injections did not significantly alter MMP expression in the remote myocardium compared to sham (sham: n=5; MI: n=6;MI/hydrogel: n=7; MI/hydrogel/rTIMP-3: n=7; *p<0.05 vs. sham; +p<0.05 vs. MI; #p<0.05 vs. MI/hydrogel).
Figure S12. Biochemical analysis of myocardial tissue. Tissue sections harvested from the MI region 14 days post MI were stained with picrosirius red (PSR) for fibrillar collagen and α-smooth muscle actin (SMA), a marker for vascular smooth muscle cells and myofibroblasts. Representative photomicrographs of PSR and SMA stains from all groups, scale bars = 100µm. SMA quantification revealed significant increases following MI, but no significant differences with hydrogel or hydrogel/rTIMP-3 delivery. (d) PSR quantification revealed a significant increase in collagen content following MI, but no effect of hydrogel or hydrogel/rTIMP-3 injections on collagen content. All values are mean±SEM; pairwise t-test with Bonferroni correction; *p<0.05 vs. sham; sham n=5, MI n=6, MI/hydrogel n=7, MI/hydrogel/rTIMP-3 n=7.
Figure S13. Myocardial fibroblast expression. Myocardial fibroblast phenotype in the MI region was assessed 14 days following MI induction. Expression of a fibronectin variant, extra domain-1 (EDA), was significantly upregulated in all groups. However, expression of a myosin heavy chain isoform (MYH14) was significantly upregulated only with hydrogel/rTIMP-3 injection, indicating the presence of a more mature contractile myofibroblast phenotype (sham: n=5; MI: n=7; MI/hydrogel: n=6; MI/hydrogel/rTIMP-3: n=8; *p<0.05 vs. sham).
Table S1. 28 day serial echocardiography. Left ventricle geometry and function was assessed for 28 days following MI induction. MI induction caused a gradual decline in ejection fraction (EF) over 28 days which was significantly attenuated by hydrogel/rTIMP-3 injections. MI induction caused a gradual dilation of the LV end diastolic volume (LVEDV) which was significantly attenuated by hydrogel/rTIMP-3 injections. MI induction caused progressive thinning of the LV posterior wall thickness at diastole (LVPWThd) which was significantly attenuated by both hydrogel and hydrogel/rTIMP-3 injections at early timepoints, but only hydrogel/rTIMP-3 injections significantly attenuated wall thinning after day 14. MI induction caused a steady increase in pulmonary capillary wedge pressure (PCWP) which was significantly attenuated by hydrogel/rTIMP-3 delivery. (*=p<0.05 vs. Baseline; +=p<0.05 vs. Respective MI Only Value; #= p<0.05 vs. Respective MI/hydrogel Value.)
Treatment Day EF (%) LVEDV (mL) LVPWThd (cm) PCWP (mmHg)
Table S2. PCR table. Primers used for the expression of matrix metalloproteinase (MMP) subclasses and markers of myofibroblast differentiation EDA fibronectin-1 (FN1) and myosin heavy chain 14 (MYH14) in the myocardium.
