In Vitro Enzymatic Degradation of Tissue Grafts ... - ACS Publications

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In Vitro Enzymatic Degradation of Tissue Grafts and Collagen Biomaterials by Matrix Metalloproteinases: Improving the Collagenase Assay A.L. Helling,†,‡ E.K. Tsekoura,†,‡ M. Biggs,‡ Y. Bayon,§ A. Pandit,‡ and D.I. Zeugolis*,†,‡ †

Regenerative, Modular & Developmental Engineering Laboratory (REMODEL), Biomedical Sciences Building, ‡Science Foundation Ireland (SFI) Centre for Research in Medical Devices (CÚ RAM), Biomedical Sciences Building, National University of Ireland Galway (NUI Galway), Galway, Ireland § Sofradim Production, A Medtronic Company, Trévoux, France ABSTRACT: Matrix metalloproteinase-1 and -8 are active during the wound healing and remodelling processes, degrading native extracellular matrix and implantable devices. However, traditional in vitro assays utilize primarily matrix metalloproteinase-1 to mimic the in vivo degradation microenvironment. Herein, we assessed the influence of various concentrations of matrix metalloproteinase- 1 and 8 (50, 100, and 200 U/mL) as a function of pH (5.5 and 7.4) and time (3, 6, 9, 12, and 24 h) on the degradation profile of three tissue grafts (chemically cross-linked Permacol, nonchemically crosslinked Permacol and nonchemically cross-linked Strattice) and a collagen biomaterial (nonchemically cross-linked collagen sponge). Chemically cross-linked and nonchemically cross-linked Permacol samples exhibited the highest resistance to enzymatic degradation, while nonchemically cross-linked collagen sponges exhibited the least resistance to enzymatic degradation. Qualitative and quantitative degradation analysis of all samples revealed a similar degradation profile over time, independently of the matrix metalloproteinase used and its respective concentration and pH. These data indicate that matrix metalloproteinase-1 and matrix metalloproteinase-8 exhibit similar degradation profile in vitro, suggesting that matrix metalloproteinase-8 should be used for collagenase assay. KEYWORDS: tissue grafts, collagen biomaterials, collagen cross-linking, in vitro enzymatic degradation, matrix metalloproteinases, collagenase assay

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INTRODUCTION During the remodelling process, extracellular matrix (ECM) is subjected to enzymatic degradation by the action of matrix metalloproteinases (MMPs), a family of zinc-containing endopeptidases.1−4 MMPs degrade ECM molecules, non-ECM molecules, and cell surface molecules, playing that way a pivotal role in diverse physiologic (e.g., development, morphogenesis, wound repair, remodelling) and pathophysiological (e.g., scarring, osteoarthritis, metastasis) processes.5−8 MMP-1, MMP-2, MMP-8, MMP-13, and MMP-14 can cleave native, not denatured, interstitial collagens at a specific helical locus; for example, MMP-1, MMP-8, and MMP-13 degrade collagen types I, II, and III into one-quarter and three-quarter fragments.9−13 MMP-114,15 and MMP-816,17 are secreted by fibroblasts and neutrophils, respectively, and are commonly present during wound healing and remodelling processes.18 MMP-13 is exclusively present in the skeleton during embryonic development (e.g., bone and cartilage cells).19−23 MMP-2 and MMP-14 are frequently associated with diseases, such as Dupuytren’s24−26 and cancer progression.27−30 Tissue grafts and collagen biomaterials are commonly employed as implantable devices.31−40 In vitro enzymatic degradation studies © XXXX American Chemical Society

permit the investigation of the extent of cross-linking and the mechanism of degradation (e.g., kinetics, unfolding profile of collagen chains, preferential interaction of MMPs to collagen chains) of collagen-based devices under defined conditions (e.g., concentration, pH, time).41−49 It is worth pointing out that in vitro degradation assays fail to a large extent to imitate the complex in vivo tissue context, where numerous cell populations and secreted enzymes will interrogate the implantable device.50−53 There are also several important limitations associated with in vitro enzymatic assays, commonly arising from a lack of standardization in the environmental conditions between similar studies54 or even the utilization of appropriate enzymes. For example, in vitro enzymatic degradation of collagenbased devices is frequently assessed with MMP-1.55−58 However, this is not physiologically accurate, as MMP-1 preferentially cleaves collagen type III;59−61 MMP-8 is always the predominant collagenase present in normal healing Special Issue: Tissue Engineering Received: December 30, 2015 Accepted: April 11, 2016

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DOI: 10.1021/acsbiomaterials.5b00563 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

Enzymatic Degradation. Enzymatic degradation of ∼0.05 mg chemically cross-linked Permacol, nonchemically cross-linked Permacol, nonchemically cross-linked Strattice and nonchemically crosslinked collagen sponges was carried out in phosphate buffered saline at 5.5 and 7.4 pH, using 50, 100, and 200 U/ml MMP-1 (Clostridium histolyticum, Sigma-Aldrich, C0130) or 50, 100, and 200 U/ml MMP-8 (Clostridium histolyticum, Invitrogen, 17101015) for 3, 6, 9, 12, and 24 h, under static conditions. Given that previous studies have demonstrated a temperature dependent MMP activity,73−75 this experimentation was conducted at physiological temperature (37 °C). Qualitative Determination of Enzymatic Degradation. Enzymatic degradation was initially assessed visually. Selective samples were assessed using environmental scanning electron microscopy (ESEM, Hitachi S2600N, Japan) at low vacuum (2−6 Torr). Further, selective samples were stained with Picro Sirius Red as follows: All samples were fixed in formalin, dehydrated through a series of ascending alcohol concentrations, embedded in paraffin, using a tissue processor (Leica Asp300) and sectioned (4 μm) using a microtome (Leica EG 1150C, Germany). The samples were then deparaffinised in xylene and in descending alcohol concentrations and stained in Weigert’s hematoxylin for 8 min, 0.2% phosphomolybdic acid for 2 min, and then for 1 h in a Picro-Sirius Red solution. Finally, the samples were washed in acidified water and in a series of ascending alcohol concentrations. Quantitative Determination of Enzymatic Degradation. Sample weight was recorder prior to enzymatic digestion. At a given digestion time-point (3, 6, 9, 12, and 24 h), the supernatant was removed and samples were freeze-dried again. The weight of the freeze-dried material was then recorded. Weight loss, as a function of enzymatic degradation, was assessed using the following formula: W = [(Wo − Wt)/Wo]100, where Wo represents the initial mass of the sample, Wt represents the mass after enzymatic degradation, and W represents mass loss percentage. Statistical Analysis. One-way analysis of variance (ANOVA) was performed using Minitab 16 (USA), after confirmation of the assumptions of parametric statistics. Statistical significance was set at the p < 0.05. Post hoc Tukey’s analysis was employed to identify significant differences.

wounds;62 and MMP-8 degrades collagen type I more efficiently than MMP-1.63−67 To assess whether the widely used collagenase assay for collagen-based devices should be conducted with MMP-1 or MMP-8, herein, the enzymatic degradation of Permacol (chemically cross-linked with hexamethylene diisocyanate), nonchemically cross-linked Permacol, nonchemically crosslinked Strattice, and nonchemically cross-linked collagen sponge was assessed as a function of MMP-1 and MMP-8 concentration (50, 100, and 200 U/mL), pH (5.5 and 7.4), and exposure time (3 h, 6 h, 9 h, 12 and 24 h).

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MATERIALS AND METHODS

Materials. Three porcine dermis tissue grafts were assessed in this work: hexamethylene diisocyanate (HMDI) cross-linked and gamma irradiated Permacol (REF: 5110−150, LOT: 12B06−6, Covidien, France), gamma irradiated nonchemically cross-linked noncommercial variant of Permacol (Preparation: 12 × 028PS, provided by Covidien, France) and E-beam treated nonchemically cross-linked Strattice (REF: 0816001 EU, LOT: S11052−264, Life Cell, USA). Nonchemically cross-linked collagen (pepsin extracted and purified from porcine dermis, Covidien, France) sponges were also used as example of collagen-based biomaterial for selective experimentation. All chemicals and reagents were purchased from Sigma-Aldrich (Ireland), unless otherwise stated. Physical Characterization. The surface morphology of wet tissue grafts was assessed using stereomicroscopy (Nikon SMZ 800, Japan). The thickness of the tissue grafts was measured using digital callipers (Scienceware, Digi-Max, Sigma-Aldrich, Ireland). The superficial density was calculated as mass (g) per unit area (1 cm × 1 cm). To assess the level of hydration, tissue grafts were hydrated in phosphate buffered saline and were subsequently incubated at 103 ± 2 °C overnight. The moisture content (% M) was calculated according to the formula: % M = [(Ww − Wd)/Ww]100, where Ww represents the initial wet mass of the sample and Wd represents the final dry mass. The wet to dry ratio was calculated according to the following formula: wet to dry ratio = Ww/Wd. The denaturation temperature of tissue grafts was assessed using differential scanning calorimetry (Shimadzu DSC-60, Japan), as has been described previously.68 Heat was applied in hermetically sealed aluminum pans at atmospheric pressure at a rate of 5 °C/min from 20 to 90 °C. All samples had been hydrated in PBS for 24 h. Collagen Content Determination. Collagen content was determined using hydroxyproline assay.69,70 Briefly, samples were lyophilized and subsequently hydrolyzed in concentrated HCl for 16 h at 100 °C. The samples were then diluted by 50× in distilled H2O, vortexed and diluted in isopropanol (2:1 ratio of isopropanol to distilled H2O). Chloramine T reagent, citrate buffer, distilled H2O, Ehrlich’s reagent, perchloric acid, and isopropanol were then added, the solution was homogenized and incubated at 70 °C for 10 min. The absorbance was determined at 555 nm. The amount of hydroxyproline was calculated using a standard curve prepared with hydroxyproline standards. Lipid Content Determination. Lipid content was determined using the Folch method.71 Briefly, samples were homogenized in chloroform/methanol (2 to 1 ratio) and agitated for 15 min in an orbital shaker at room temperature. The homogenate was filtrated to recover the liquid phase and the solution was washed. After vortexing for 60 s, the mixture was centrifuged at 2000 rpm to separate the fraction rich in lipids. Removal of the methanol phase was carried out using siphoning and the lower chloroform phase, containing lipids, was subsequently evaporated under vacuum in a rotary evaporator. Carbohydrate Content Determination. Carbohydrate content was determined using the Anthrone method.72 Briefly, samples were hydrolyzed with 2.5 N HCl for 3 h at 95 °C, neutralized with Na2CO3 and centrifuged at 10 000 rpm for 8 min. The supernatant was collected and suspended in 100 mL of ice cold 95% H2SO4 containing Anthrone reagent. The mixture was heated for 8 min at 95 °C and cooled rapidly to 0 °C. The absorbance was measured at 630 nm and compared with that of a standard glucose sample.

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RESULTS Physical Characterization. Morphological analysis (Figure 1) revealed similar surface morphology for all tissue grafts and the presence of hair follicles. Thickness analysis (Table 1) revealed that chemically cross-linked Permacol was the thickest of all tissue grafts (p < 0.001), while nonchemically cross-linked Permacol was the thinnest (p < 0.001). With respect to superficial density (Table 1), chemically cross-linked Permacol exhibited the highest superficial density (p < 0.001), whereas no difference was observed between nonchemically cross-linked Permacol and nonchemically cross-linked Strattice (p > 0.05). Moisture content analysis (Table 1) revealed that nonchemically cross-linked Strattice exhibited significantly higher (p < 0.001) moisture content than Permacol (cross-linked with HMDI) and nonchemically cross-linked Permacol, while no significant difference (p > 0.05) was observed between chemically crosslinked Permacol and nonchemically cross-linked Permacol. Differential scanning calorimetry analysis (Table 1) revealed that nonchemically cross-linked Permacol exhibited significantly lower (p < 0.001) denaturation temperature than chemically cross-linked Permacol and nonchemically cross-linked Strattice, whereas no significant difference (p > 0.05) was observed between chemically cross-linked Permacol and nonchemically cross-linked Strattice. Quantitative Determination of Collagen, Lipid, and Carbohydrates Content. Quantitative compositional analysis (Table 2) revealed that nonchemically cross-linked Permacol exhibited significantly lower (p < 0.001) collagen and lipids B


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Figure 1. (A−C) Stereomicroscopy and (D−F) scanning electron microscopy analysis of: (A, D) chemically cross-linked Permacol, (B, E) nonchemically cross-linked Permacol and (C, F) nonchemically cross-linked Strattice. Stereomicroscopy scale bar: 200 mm.

Table 1. Analysis of Chemically Cross-Linked Permacol. Nonchemically Cross-Linked Permacol, and Nonchemically CrossLinked Stratticea sample

thickness (mm)

superficial density (g/cm2)

moisture (%)

denaturation temperature (°C)

chemically cross-linked Permacol nonchemically cross-linked Permacol nonchemically cross-linked Strattice

1.58 ± 0.07 1.29 ± 0.04 1.41 ± 0.04

0.165 ± 0.007 0.137 ± 0.004 0.131 ± 0.002

69.84 ± 3.55 70.60 ± 2.73 78.12 ± 0.97b

59.94 ± 0.16 56.61 ± 0.17b 59.21 ± 0.21

b

b

a

Chemically cross-linked permacol revealed that chemically cross-linked permacol was the thickest of all tissue grafts (p < 0.001. N = 5). Chemically cross-linked Permacol exhibited the highest superficial density (p < 0.001; N = 5). Nonchemically cross-linked Strattice exhibited the highest moisture content (p < 0.001; N = 5). Nonchemically cross-linked Permacol demonstrated the lowest denaturation temperature (p < 0.001; N = 3). b Indicates significant difference (p < 0.001).

sponges within 3 h, even at lowest, 50 U/mL, concentration (Figure 2). At pH 7.4, nonchemically cross-linked collagen sponges withheld MMP-1 degradation for 6 h, independently of the concentration used (50, 100, and 200 U/mL), whereas complete degradation was observed after 9 h for all MMP-1 concentrations (Figure 2). Visual inspection of the enzymatic degradation (50, 100, and 200 U/mL MMP-8, at pH 5.5 and 7.4, and 3, 6, 9, 12, and 24 h) revealed that nonchemically cross-linked Strattice was completely degraded after 24 h at pH 5.5 and pH 7.4 at 200 U/ml (Figure 3). Chemically cross-linked Permacol and nonchemically cross-linked Permacol were able to withheld MMP-8 degradation at both pHs, even at 200 U/mL and ever after 24 h, albeit the nonchemically cross-linked Permacol to a lesser extent than chemically cross-linked Permacol (Figure 3). At pH 5.5, MMP-8 completed degraded nonchemically cross-linked collagen sponges within 3 h, even at lowest, 50 U/mL, concentration (Figure 3). At pH 7.4, nonchemically cross-linked collagen sponges withheld MMP-8 degradation for 6 h, independently of the concentration used (50, 100, and 200 U/mL), whereas complete degradation was observed after 9 h for all MMP-8 concentrations (Figure 3). ESEM analysis of tissue grafts made apparent that the degradation was initiated and progressed at the hair follicles, whereas Picro Sirius Red staining revealed that even after 3 h of exposure to MMPs, the cohesiveness of tissue grafts was reduced (Figure 4). Quantitative Determination of MMP-1 and MMP-8 Degradation. None of the various treatments (50, 100, and 200 U/mL MMP-1 and MMP-8, at pH 5.5 and 7.4 and 3, 6, 9, 12,

Table 2. Quantitative Compositional Analysis of Chemically Cross-Linked Permacol, Nonchemically Cross-Linked Permacol, and Nonchemically Cross-Linked Strattice sample chemically cross-linked permacol nonchemically cross-linked permacol nonchemically cross-linked strattice a

collagen lipid carbohydrate content (%) content (%) content (%) 86.1 81.4a 90.1

1.0 3.0a ∼0.0

∼0.0 ∼0.0 ∼0.0

Indicates significant difference (p < 0.001) (% of dry weight).

content than chemically cross-linked Permacol and nonchemically cross-linked Strattice, whereas no significant difference (p > 0.05) was observed between chemically cross-linked Permacol and nonchemically cross-linked Strattice. No significant difference (p > 0.05) was observed in carbohydrates content between all samples. Qualitative Determination of MMP-1 and MMP-8 Degradation. Visual inspection of the enzymatic degradation (50, 100, and 200 U/mL MMP-1, at pH 5.5 and 7.4 and 3, 6, 9, 12, and 24 h) revealed that nonchemically cross-linked Strattice was completely degraded after 24 h at pH 5.5 and 200 U/mL MMP-1 and after 24 h at pH 7.4 at 100 and 200 U/mL MMP-1 (Figure 2). Chemically cross-linked Permacol and nonchemically cross-linked Permacol were able to withheld MMP-1 degradation at both pHs, even at 200 U/mL and ever after 24 h, albeit the nonchemically cross-linked Permacol to a lesser extent than chemically cross-linked Permacol (Figure 2). At pH 5.5, MMP-1 completed degraded nonchemically cross-linked collagen C


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Figure 2. Qualitative MMP-1 degradation analysis of (A) chemically cross-linked Permacol, (B) nonchemically cross-linked Permacol,(C) nonchemically cross-linked Strattice, and (D) nonchemically cross-linked collagen sponge, as a function of MMP-1 concentration, pH, and MMP-1 exposure time.

Figure 3. Qualitative MMP-8 degradation analysis of (A) chemically cross-linked Permacol, (B) nonchemically cross-linked Permacol, (C) nonchemically cross-linked Strattice, and (D) nonchemically cross-linked collagen sponge, as a function of MMP-8 concentration, pH, and MMP-8 exposure time.

and 24 h) was able to degrade more than 30% dry weight of the chemically cross-linked Permacol (Figure 5). With respect to nonchemically cross-linked Permacol (Figure 6) and nonchemically

cross-linked Strattice (Figure 7), an increase in degradation as a function of time, at a given concentration of MMP-1 or MMP-8, was observed and after 24 h, more than 60% dry weight of the D


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Figure 4. Microscopy analysis revealed that degradation was initiated and progressed at the hair follicles (top row; hair follicles are recognized with arrows), whereas even 3 h of incubation with matrix metalloproteinases is enough to interrupt the cohesive structure of tissue grafts (bottom row).

Figure 5. Quantitative analysis (dry weight loss) of chemically cross-linked Permacol as a function of MMP-1 and MMP-8 concentration (50, 100, and 200 U/mL), pH (5.5 and 7.4), and exposure time (3, 6, 9, 12, and 24 h) to respective MMP.

biodegradation rate at a given microenvironment.53,76−85 Herein, we assessed the influence of various MMP-1 and MMP-8 concentrations (50, 100, and 200 U/mL), as a function of pH (5.5 and 7.4) and exposure time (3, 6, 9, 12, and 24 h) to the respective MMP solution, on the enzymatic degradation of Permacol (cross-linked with HMDI), nonchemically crosslinked Permacol, nonchemically cross-linked Strattice and nonchemically cross-linked collagen sponge. Although differences were observed in the structural (stereomicroscopy), biophysical (thickness, superficial density, moisture content, and denaturation temperature) and compositional (collagen, lipids and carbohydrates) properties of the tissues grafts, in general, their features were not out of proportion different, given the biological origin of the materials. For example, significant regional differences in porcine dermis and epidermis

samples had been degraded, independently of the pH, MMP used and its concentration. At pH 5.5, MMP-1 and MMP-8 degraded over 80% dry weight of nonchemically cross-linked collagen sponges within 3 h, even at the lowest, 50 U/ml, concentration (Figure 8). At pH 7.4, MMP-1 degraded over 80% dry weight of nonchemically cross-linked collagen sponges within 3 h, even at the lowest, 50 U/ml, concentration (Figure 8), whereas MMP-8 required 6 h to degrade 80% dry weight of the nonchemically cross-linked collagen sponges, independently of the MMP-8 concentration (Figure 8).

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DISCUSSION The physicochemical properties (e.g., composition, architecture, density) and the processing (e.g., decellularisation, sterilization, cross-linking) of a medical device significantly influence its E


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Figure 6. Quantitative analysis (dry weight loss) of nonchemically cross-linked Permacol as a function of MMP-1 and MMP-8 concentration (50, 100, and 200 U/mL), pH (5.5 and 7.4), and exposure time (3, 6, 9, 12, and 24 h) to respective MMP.

Figure 7. Quantitative analysis (dry weight loss) of nonchemically cross-linked Strattice as a function of MMP-1 and MMP-8 concentration (50, 100, and 200 U/mL), pH (5.5 and 7.4), and exposure time (3, 6, 9, 12, and 24 h) to respective MMP.

thickness and collagen content have been documented,86 which can explain differences between the tested tissue grafts. All tissue grafts had moisture content between 70 and 78%. Moisture content is a major determinant of the physicomechanical

properties of collagen scaffolds and various cross-linking methods are utilized to tailor swelling/mechanical properties to the tissue to be replaced.87−90 All tissue grafts had collagen content between 81 and 90% dry weight, which is in agreement F


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Figure 8. Quantitative analysis (dry weight loss) of nonchemically cross-linked collagen sponge as a function of MMP-1 and MMP-8 concentration (50, 100, and 200 U/mL), pH (5.5 and 7.4) and exposure time (3, 6, 9, 12, and 24 h) to respective MMP.

nonchemically cross-linked Strattice can be attributed to the age and gender of the animal, the location from where the grafts were obtained and the processing, which have also been shown to influence the properties of collagen-based devices.52,70,84,86,123−131 It is worth pointing out that resistance to enzymatic (collagenase) degradation is only indicative of the extent of cross-linking and should not be used alone as a means to assess the efficacy and safety of the device. Indeed, nonchemically cross-linked Strattice has been used successfully for numerous clinical indications, including omphalocele,132 breast reconstruction,133 and hernia,134 as indicative examples. Qualitative and quantitative degradation analysis of chemically cross-linked (HMDI) Permacol, nonchemically cross-linked Permacol, nonchemically cross-linked Strattice, and nonchemically cross-linked collagen sponge samples revealed a similar degradation profile over time (3, 6, 9, 12, and 24 h), independently of the MMP (1 and 8) concentration (50, 100, and 200 U/mL), and pH (5.5 and 7.4). This surprising behavior of MMP-1 may be attributed to its bacterial (clostridium) origin. Previous studies have shown that clostridium collagenase (DFB Pharmaceuticals, USA; 1 mg/mL; type not specified) was able to degrade human collagen types I, III, IV, V, but not collagen type VI.135 Nonetheless, given that MMP-8 is more economical than MMP-1 and that MMP-8 has higher specificity to collagen type I than MMP-1, it may be more reasonable to utilize 50 U/ml MMP-8 for customarily in vitro degradation assays, unless a specific property of MMP-1 is required. In the present study, we ventured to rationally improve the customarily used collagenase assay using MMP-8, as opposed to MMP-1, for collagen-based devices that are primarily composed of collagen type I. However, to create in vivo surrogate degradation assays numerous parameters should be considered, including: origin of the enzyme(s) (e.g., human or bacterial);

with the expected collagen content of dermis and previous publications.91−94 Lipids were less than 3% dry weight, which is not surprising, given that a delipidation process takes place to produce tissue grafts out of porcine dermis and high lipid content negatively impacts on cell attachment and proliferation and reduces biocompatibility.95,96 Among the materials assessed herein, the chemically crosslinked with HMDI Permacol exhibited the highest resistance to enzymatic degradation, whereas nonchemically cross-linked collagen sponges exhibited the least resistance to enzymatic degradation. With respect to HMDI cross-linked Permacol, this is not surprising, given that HMDI is an effective collagen crosslinker, with biophysical and biological properties superior to those of glutaraldehyde cross-linked collagen-based materials,97−103 enabling HMDI cross-linked Permacol to be an effective implantable device for numerous soft tissue repair clinical targets, including complex abdominal wall reconstruction,104 chest wall reconstruction,105 and complex vein wounds,106 as indicative examples. Nonetheless, attention should be paid when a cross-linking method is employed, as extensive cross-linking/ high resistance to collagenase degradation may be associated with elevated foreign body response.53,107 The nonchemically cross-linked collagen sponges exhibited the least resistance to enzymatic degradation, which can be attributed to the loose packing density, as compared to tissue grafts, of collagen-based biomaterials. For example, previous studies have demonstrated that reconstituted forms of collagen exhibit weaker second harmonic generation signals than native tissues.108−110 Similarly, collagen fibers,111,112 due to the closer packing of their constituent molecules, exhibit higher denaturation temperature and resistance to degradation than collagen hydrogels,49,113−115 films116−119 and sponges.120−122 The differences between the nonchemically cross-linked Permacol and G


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(4) Shingleton, W.; Hodges, D.; Brick, P.; Cawston, T. Collagenase: A key enzyme in collagen turnover. Biochem. Cell Biol. 1996, 74, 759−775. (5) Armstrong, D.; Jude, E. The role of matrix metalloproteinases in wound healing. Journal of the American Podiatric Medical Association 2002, 92, 12−18. (6) Woessner, J. Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J. 1991, 5, 2145−2154. (7) Streuli, C. Extracellular matrix remodelling and cellular differentiation. Curr. Opin. Cell Biol. 1999, 11, 634−640. (8) Chen, Q.; Jin, M.; Yang, F.; Zhu, J.; Xiao, Q.; Zhang, L. Matrix metalloproteinases: Inflammatory regulators of cell behaviors in vascular formation and remodeling. Mediators Inflammation 2013, 2013, 928315. (9) Aimes, R.; Quigley, J. Matrix metalloproteinase-2 is an interstitial collagenase. Inhibitor-free enzyme catalyzes the cleavage of collagen fibrils and soluble native type I collagen generating the specific 3/4- and 1/4-length fragments. J. Biol. Chem. 1995, 270, 5872−5876. (10) Seandel, M.; Noack-Kunnmann, K.; Zhu, D.; Aimes, R.; Quigley, J. Growth factor-induced angiogenesis in vivo requires specific cleavage of fibrillar type I collagen. Blood 2001, 97, 2323−2332. (11) Ohuchi, E.; Imai, K.; Fujii, Y.; Sato, H.; Seiki, M.; Okada, Y. Membrane type 1 matrix metalloproteinase digests interstitial collagens and other extracellular matrix macromolecules. J. Biol. Chem. 1997, 272, 2446−2451. (12) Balbín, M.; Fueyo, A.; Knäuper, V.; López, J.; Alvarez, J.; Sánchez, L.; Quesada, V.; Bordallo, J.; Murphy, G.; López-Otín, C. Identification and enzymatic characterization of two diverging murine counterparts of human interstitial collagenase (MMP-1) expressed at sites of embryo implantation. J. Biol. Chem. 2001, 276, 10253−10262. (13) Welgus, H.; Jeffrey, J.; Stricklin, G.; Eisen, A. The gelatinolytic activity of human skin fibroblast collagenase. J. Biol. Chem. 1982, 257, 11534−11539. (14) Pardo, A.; Selman, M. MMP-1: The elder of the family. Int. J. Biochem. Cell Biol. 2005, 37, 283−288. (15) Arakaki, P.; Marques, M.; Santos, M. MMP-1 polymorphism and its relationship to pathological processes. J. Biosci. 2009, 34, 313−320. (16) Gutiérrez-Fernández, A.; Inada, M.; Balbín, M.; Fueyo, A.; Pitiot, A.; Astudillo, A.; Hirose, K.; Hirata, M.; Shapiro, S.; Noël, A.; Werb, Z.; Krane, S.; López-Otín, C.; Puente, X. Increased inflammation delays wound healing in mice deficient in collagenase-2 (MMP-8). FASEB J. 2007, 21, 2580−2591. (17) Balbín, M.; Fueyo, A.; Knäuper, V.; Pendás, A.; López, J.; Jiménez, M.; Murphy, G.; López-Otín, C. Collagenase 2 (MMP-8) expression in murine tissue-remodeling processes. Analysis of its potential role in postpartum involution of the uterus. J. Biol. Chem. 1998, 273, 23959− 23968. (18) Nwomeh, B.; Liang, H.; Diegelmann, R.; Cohen, I.; Yager, D. Dynamics of the matrix metalloproteinases MMP-1 and MMP-8 in acute open human dermal wounds. Wound Repair and Regeneration 1998, 6, 127−134. (19) Mattot, V.; Raes, M.; Henriet, P.; Eeckhout, Y.; Stehelin, D.; Vandenbunder, B.; Desbiens, X. Expression of interstitial collagenase is restricted to skeletal tissue during mouse embryogenesis. J. Cell Sci. 1995, 108, 529−535. (20) Gack, S.; Vallon, R.; Schmidt, J.; Grigoriadis, A.; Tuckermann, J.; Schenkel, J.; Weiher, H.; Wagner, E.; Angel, P. Expression of interstitial collagenase during skeletal development of the mouse is restricted to osteoblast-like cells and hypertrophic chondrocytes. Cell Growth Differentiation 1995, 6, 759−767. (21) Tuckermann, J.; Pittois, K.; Partridge, N.; Merregaert, J.; Angel, P. Collagenase-3 (MMP-13) and integral membrane protein 2a (Itm2a) are marker genes of chondrogenic/osteoblastic cells in bone formation: Sequential temporal, and spatial expression of Itm2a, alkaline phosphatase, MMP-13, and osteocalcin in the mouse. J. Bone Miner. Res. 2000, 15, 1257−1265. (22) Johansson, N.; Saarialho-Kere, U.; Airola, K.; Herva, R.; Nissinen, L.; Westermarck, J.; Vuorio, E.; Heino, J.; Kähäri, V. Collagenase-3 (MMP-13) is expressed by hypertrophic chondrocytes, periosteal cells, and osteoblasts during human fetal bone development. Dev. Dyn. 1997, 208, 387−397.

enzyme(s) selection/permutations based on the composition of the device or the cell population at the site of implantation; environmental conditions at the side of implantation (e.g., mechanical loads). We anticipate that advancements in multifactorial approaches would enable in the years to come the development of more physiologically relevant in vitro assays.

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CONCLUSION During wound healing and remodelling, matrix metalloproteinases degrade native extracellular matrix and implantable devices. Although both matrix metalloproteinase-1 and matrix metalloproteinase-8 are responsible for the enzymatic degradation in vivo, traditional in vitro assays, aiming to recapitulate the in vivo degradation in vitro, utilize primarily matrix metalloproteinase-1. Herein, we assessed the influence of various concentrations of matrix metalloproteinase- 1 and 8, as a function of pH and time on the degradation profile of tissue grafts and a collagen biomaterial. Our data indicate that tissue origin/characteristics, processing and cross-linking influence enzymatic degradation in vitro and matrix metalloproteinase- 1 and 8 induce similar degradation profile, suggesting that the customarily used collagenase assay should be utilizing matrix metalloproteinase-8, as opposed to matrix metalloproteinase-1, as a means to interrogate collagen-based devices in vitro. Elucidation of the actions of matrix metalloproteinases in vivo and in vitro would enable rational design of in vitro degradation assays.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +353 (0) 9149 3166. Fax: +353 (0) 9156 3991. Notes

The authors declare the following competing financial interest(s): Y.B. is an employee of Sofradim Production, A Medtronic Company.

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ACKNOWLEDGMENTS This work is supported by the: Covidien Research Project, under Grant 81952 to D.Z.; Health Research Board, Health Research Awards Programme, under Grant HRA_POR/2011/84 to D.Z. M.B. acknowledges Science Foundation Ireland, Starting Investigators Research Programme, under Grant 11/SIRG/ B2135. This publication has also been supported from Science Foundation Ireland and the European Regional Development Fund (Grant 13/RC/2073) to A.P.. The authors acknowledge the Electron Microscopy Unit within the Centre for Microscopy & Imaging and the Histology Core Facility of the National Centre for Biomedical Engineering Science (NCBES) at NUI Galway, which are funded by NUI Galway and the Irish Government’s Programme for Research in Third Level Institutions, Cycles 4 and 5, National Development Plan 2007−2013. Y.B. is an employee of Sofradim Production, A Medtronic Company

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