In vitro dose-dependent effects of matrix metalloproteinases on ECM hydrogel biodegradation

Abstract

Matrix metalloproteinases (MMPs) cause proteolysis of extracellular matrix (ECM) in tissues affected by stroke. However, little is known about how MMPs degrade ECM hydrogels implanted into stroke cavities to regenerate lost tissue. To establish a structure-function relationship between different doses of individual MMPs and isolate their effects in a controlled setting, an in vitro degradation assay quantified retained urinary bladder matrix (UBM) hydrogel mass as a measure of degradation across time. A rheological characterization indicated that lower ECM concentrations (<4 mg/mL) did not cure completely at 37 °C and had a high fraction of mobile proteins that were easily washed-out. Hydrolysis by dH₂O caused a steady 2 % daily decrease in hydrogel mass over 14 days. An acceleration of degradation to 6 % occurred with phosphate buffered saline and artificial cerebrospinal fluid. MMPs induced a dose-dependent increase and within 14 days almost completely (>95 %) degraded the hydrogel. MMP-9 exerted the most significant biodegradation, compared to MMP-3 and -2. To model the in vivo exposure of hydrogel to MMPs, mixtures of MMP-2, -3, and -9, present in the cavity at 14-, 28-, or 90-days post-stroke, revealed that 14- and 28-days mixtures achieved an equivalent biodegradation, but a 90-days mixture exhibited a slower degradation. These results revealed that hydrolysis, in addition to proteolysis, exerts a major influence on the degradation of hydrogels. Understanding the mechanisms of ECM hydrogel biodegradation is essential to determine the therapeutic window for bioscaffold implantation after a stroke, and they are also key to determine optimal degradation kinetics to support tissue regeneration. STATEMENT OF SIGNIFICANCE: After implantation into a stroke cavity, extracellular matrix (ECM) hydrogel promotes tissue regeneration through the degradation of the bioscaffold. However, the process of degradation of an ECM hydrogel remains poorly understood. We here demonstrated in vitro under highly controlled conditions that hydrogel degradation is very dependent on its protein concentration. Lower protein concentration hydrogels were weaker in rheological measurements and particularly susceptible to hydrolysis. The proteolytic degradation of tissue ECM after a stroke is caused by matrix metalloproteinases (MMPs). A dose-dependent MMP-driven biodegradation of ECM hydrogel exceeded the effects of hydrolysis. These results highlight the importance of in vitro testing of putative causes of degradation to gain a better understanding of how these factors affect in vivo biodegradation.

Keywords: Biodegradation; Bioscaffold; Extracellular matrix; Hydrogel; Matrix metalloproteinase; Rheology.

Figure 1.. Experimental set-up.

A. To determine the mass of a hydrogel and how this is retained during hydrolysis and biodegradation, transwell inserts were weighed without a hydrogel to gain a baseline measure. Pre-gel ECM protein concentrations were placed in a transwell insert and cured for 60 minutes prior to weighing in at day 0. Hydrogels were immersed in a supernatant consisting of dH₂O, phosphate buffered saline (PBS) or artificial cerebral spinal fluid (aCSF) to investigate the impact of hydrolysis over 14 days. To determine retained mass, transwell inserts with remaining hydrogels were removed and weighed daily before application of new supernatant. Discarded supernatant was collected and stored at −80 °C. To investigate the biodegradation of hydrogels, supernatant (PBS) was supplemented with matrix metalloproteinases (MMP) −2, −3 and −9 at different dosages.

Figure 2.. Rheology of ECM hydrogels.

A. Pre-gel viscosity of different concentrations of ECM proteins were determined to provide a baseline measure of the preparations’ rheological properties. B. Degree of gelation was measured to determine which fraction of ECM proteins cross-linked versus those that are remained mobile and could easily be washed off. C. A time sweep of the storage (shown here) and loss modulus was obtained to contrast the stiffness and elasticity of different ECM concentrations forming a hydrogel. Points show all individual measurements with a fitted curve. D. 50% time to maximum gelation was measured to determine how long it would take for each concentration to complete the process of gelation. E. The storage modulus (G’) indicated a concentration-dependent linear increase in hydrogel stiffness. F. As with the storage modulus, the loss (G”) modulus was highly dependent on ECM protein concentration. (*p<0.05; ** p<0.01; *** p<0.001)

Figure 3.. Hydrolysis assay.

A. Higher concentrations of ECM hydrogel were less susceptible to hydrolysis by PBS, whereas a 2 mg/mL concentration was rapidly degraded. However, the cumulative protein released into the supernatant was higher for the higher concentrations of ECM protein, potentially reflecting the greater amounts of protein contained within these gels rather than their level of degradation. Note that protein measurements were not normalized to a pre-degradation measurement. B. dH₂O exerted a minor, but consistent hydrolysis of a 4 mg/mL hydrogel, but this was considerably accelerated in the presence of electrolytes, which are present in PBS and aCSF, which more truthfully reflect the liquids present in biological tissues. aCSF exerted a greater release of protein over time compared to PBS and dH₂O, highlighting the potential contribution of hydrolysis to the degradation of ECM hydrogels in the brain.

Figure 4.. MMP assay.

A. Even at its lowest concentration, MMP-2 exerted a biodegradation effect that exceeded the hydrolysis effect by PBS. Higher doses (>1 ng/mL) of MMP-2 accelerated the degradation of a 4 mg/mL ECM hydrogel. A higher (100 ng/mL) dose of MMP-2 increased the protein release from ECM hydrogel compared to a lower (1 ng/mL) dose, which in turn was significantly higher than the protein released through PBS-hydrolysis. B. MMP-3 exhibited a greater separation between lower doses (0.01 and 0.1 ng/mL) and higher doses (≥1 ng/mL), with higher doses achieving an almost complete biodegradation of ECM hydrogel over 14 days. C. MMP-9 followed a similar pattern to MMP-3 with a lower and higher dose separation, including an almost complete biodegradation of the ECM hydrogel by 14 days. The protein release characteristics of these higher doses followed a similar pattern, indicating no increase in biodegradation with doses higher than 1 ng/mL.

Figure 5.. MMP comparison.

To compare the biodegradation efficiency of different MMPs, a direct comparison of these revealed that MMP-3 and −9 were equivalent, but significantly more efficient than MMP-2. In contrast, MMP-2 revealed a higher level of cumulative protein release from the hydrogels, potentially indicating that MMP-2 acts on a different set of ECM proteins.

Figure 6.. Impact of stroke cavity MMP mixture on biodegradation.

To model the proteolysis of multiple MMPs acting together, a mixture of MMP-2, −3 and −9 at dosage levels present within a stroke cavity at different time points was used to assay its effect on biodegradation. During the initial 7 days phase, the 14 and 28days post-stroke MMP mixtures were more effective at biodegrading the ECM hydrogel than the 90 days mixture.

Figure 7.. Contribution of individual MMPs to MMP mixture.

To determine the unique contribution of individual MMPs to the effects of the MMP mixtures, individual MMPs at their post-stroke dosage were compared and then contrasted with their corresponding MMP mixtures. A. The MMP-2 dosage in the 14- and 28-days post-stroke mixture were equivalent and more efficient at degrading ECM hydrogel than the 90 days MMP-2 dosage. At all post-stroke time points, the MMP mixture was more efficient than the individual MMP-2. B. The MMP-3 dosage at 14- and 28-days post-stroke achieved an equivalent level of biodegradation, whereas at 90 days post-stroke no MMP-3 was detected and hence no comparison could be performed. C. The MMP-9 dosage was equivalent across all time points post-stroke. At the 14- and 28-days post-stroke time points, the individual MMP-9 dosage was not as efficient as the MMP mixture, highlighting additional contribution to the biodegradation by MMP-2 and MMP-3. However, at 90-days post-stroke, the biodegradation response of the individual MMP-9 and the MMP mixture were equivalent, suggesting that MMP-2 did not add to the biodegradation observed at this time point.