Molecular and cellular mechanisms of collagen degradation in the foreign body reaction

Ye, Qingsong (2013) Molecular and cellular mechanisms of collagen degradation in the foreign body reaction. PhD thesis, University of Groningen.

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The biopolymer collagen is often used as a scaffolding biomaterial in tissue engineering. Like any other biomaterial, the implantation of collagen scaffolds induces an inflammatory local tissue reaction, known as foreign body reaction. As introduced in Chapter 1, the interaction of the scaffold with the local tissue generally is species-, biomaterial-, and implant site-dependent. The foreign body Reaction (FBR) against collagen scaffolds involves a complex cascade of spatiotemporally regulated and interconnected processes that include cellular immigration, activation of inflammatory cells, phagocytosis as well as proteolytic activities, which result in the final resolution and degradation of the implanted scaffold. Degradation of collagen scaffold is an important part of the FBR. Ideally the degradation rate should match the speed of tissue regeneration. There are two major pathways for collagen degradation: (a) phagocytosis of collagen bundles by surrounding inflammatory cells, i.e. macrophages and giant cells (the fused macrophages); and (b) enzymatic degradation by secretion enzymes, e.g. matrix metalloproteinase (MMPs). Although several cellular and molecular factors have been shown to be involved in the degradation of collagen degradation, the exact mechanisms are largely unknown. Therefore, a series of experiments was carried out to investigate this topic.

In Chapter 2 we studied the nature and regulation of MMP-based proteolytic degradation of cross-linked collagen scaffolds in subcutaneous and epicardial implantation sites. Collagen disks were quickly degraded epicardially, whereas degradation was attenuated when the disks were implanted subcutaneously. Our data showed that collagenases and gelatinases were present, and that collagenase MMP-13 and gelatinase MMP-2 were predominantly in their active forms at both sites. In contrast, the major MMP inhibitor TIMP-1, was solely observed in subcutaneous implants, which provides an explanation for why MMP-13 and MMP-2 are not able to degrade the collagen scaffold at the subcutaneous implantation site. Remarkably, the potent TIMP-1 inducer interleukin 10 (IL-10) was also mainly detected and co-located with the giant cells subcutaneously. Taken together, we surmise that IL-10, through regulation of the ratio of MMPs and TIMP-1, suppresses the degradation of implanted collagen scaffold in the FBR. This finding indicates that degradation of collagen scaffolds can be regulated via the MMP network by modulating the micro-environment, e.g. by modulating the expression levels of TIMP-1 and/or IL-10.

In Chapter 3, we investigated the fate of the dermal sheep collagen disks at a single implant site, namely subcutaneously, but the disks were different with respect chemical crosslinkers (glutaraldehyde = GDSC, hexamethylenediisocyanate = HDSC). GDSC was almost completely degraded after 28 days, whereas HDSC remained intact. Major differences were seen in the presence of neutrophils inside the two disks: in GDSC neutrophils were present throughout the FBR and massive infiltration of neutrophils was seen at day 2 and day 21, which coincided with high levels of IFN-γ and the phagocytosis of collagen bundles by macrophages. On the other hand, only minor infiltration was seen at day 2 in HDSC and no phagocytosis was observed. Degradation of GDSC occurred through collagenolytic activity and phagocytosis by macrophages. IL-13 was only observed in HDSC, and this resulted in the formation of giant cells in HDSC. In agreement with Chapter 2, the giant cells produced IL-10, that promoted TIMP-1 expression and subsequent inhibition of collagenolytic activity. We conclude (1) that the function of macrophages in mice is largely influenced by differences in micro-environment and that the nature of the micro-environments was induced by the chemical features of the implanted collagen scaffold itself, and that (2) the presence/absence of neutrophils play a major role in the shaping of this micro-environment (high IFN-γ and low IL-10 levels in GDSC, low IFN-γ and high IL-10 levels in HDSC). This study provides insight in how the scaffold itself can regulate its own micro-environment.

Chapter 4 is a follow-up study to gain further insight into the role of molecular mediators on the micro-environment and consequently on phagocytosis in the foreign body reaction. As shown in Chapter 3, subcutaneously implanted disks of HDSC or GDSC in mice show large differences in degradation rate. Phagocytosis of collagen by macrophages occurred only in GDSC, although comparable numbers of macrophages are seen in HDSC and GDSC. This study aimed to discover the underlined molecular mechanisms of the phagocytosis. Our data showed that Endo180 was expressed in GDSC only, which correlated with the expression of IFN-γ. Endo180 is co-localized with MMP-14 on F4/80 positive cells (murine macrophages), which is likely responsible for the phagocytosis in GDSC. In vitro, IFN-γ induced the expression Endo180 and MMP-14 in murine macrophages cultured on collagen type I (although too high levels of IFN-γ dampened the expression of Endo180 and MMP-14). Moreover, the expression of Endo180 and MMP-14 induced by IFN-γ can be inhibited through IL-10. The differences in micro-environment between GDSC and HDSC (high IFN-γ and low IL-10 levels in GDSC, low IFN-γ and high IL-10 levels in HDSC) provide an explanation why phagocytosis of collagen by macrophages is only seen in GDSC. In summary, we show for the first time that the IFN-γ dependent co-expression of Endo180 and MMP-14 on macrophages coincides with collagen phagocytosis, thus providing evidence that the mechanism of collagen phagocytosis operating in the foreign body reaction by macrophages is comparable with the mechanism of intracellular collagen degradation by fibroblasts seen under physiological tissue repair conditions. Based on these results, regulation of phagocytosis of collagen by macrophage may be feasible through regulation of the levels of IFN-γ or IL-10 (counterpart of IFN-γ). Previous chapters (Chapter 2-4) investigated the molecular and cellular basis for the degradation of collagen scaffolds modified with chemical crosslinkers, yet little is known about the degradation mechanisms of non-crosslinked collagen scaffolds in vivo. In Chapter 5, non-crosslinked dermal sheep collagen (NDSC) and non-crosslinked gelatin (denatured collagen) disks were implanted subcutaneously in mice. Gelatin disks degraded quickly, due to the efficient formation of large giant cells as well as the presence of MMP-13; the inhibitor TIMP-1 was absent. The DDR-2 receptor was not expressed in the gelatin disks. Endo180 and MMP-14 were expressed, but mostly no co-expression was seen. In contrast, NDSC disks showed a very low degradation rate, despite the presence of high numbers of macrophages and the influx of neutrophils. This was attributed to the presence of the matrix metalloproteinase inhibitor TIMP-1. The limited degradation that occurred was mainly in the later stages of the foreign body reaction, and could be attributed to (1) phagocytosis by macrophages due to a co-expression of Endo180 and MMP-14 on these cells (intracellular degradation) and (2) the presence of MMP-13 due to an upregulation of the expression of the DDR-2 receptor (extracellular degradation). We conclude that the physical state of collagen (native or denatured) had a dramatic outcome on the degradation rate and provoked a completely different foreign body reaction.

With regard to the degradation of collagen scaffolds, so far all studies (Chapter 2-5) focussed on the role of matrix metalloproteinases. Yet nothing is known on cathepsin K, a strong collagenolytic enzyme, in the involvement of the FBR. The study in Chapter 6 reports for the first time that cathepsin K and TRAP, two enzymes that are highly expressed in osteoclasts, are present in the FBR towards non-crosslinked collagen (NDSC), glutaraldehyde cross-linked collagen (GDSC) and hexamethylenediisocyanate cross-linked collagen (HDSC), but not in the FBR against denatured collagen (gelatine). Cathepsin K is not involved in the fast degradation of gelatin due to its absence. In the FBR towards NDSC the main cells showing cathepsin K positivity are the multinucleated cells, whereas in the FBR towards GDSC the main cells showing cathepsin K positivity are the mononucleated cells. The giant cells in the FBR towards gelatin and NDSC show different phenotypic properties, as they are cathepsin K negative and cathepsin K positive, respectively. More investigations are needed to gain further insight into the role of cathesin K and giant cell heterogeneity in the foreign body reaction against the collagen scaffolds.

Taken together, we can conclude the following regarding the degradation of collagen disks in the foreign body reaction:

1) Enzymatic degradation of collagen scaffolds Generally, collagen is degraded through the interplay between collagenases and gelatinases. MMPs are produced and secreted as inactive precursors (proMMP) by inflammatory cells such as macrophages. These cells also produce the membrane-bound MMP-14. Together these MMPs degrade collagen and gelatin to smaller products. This process is inhibited through binding of e.g. TIMP-1 to MMPs. This thesis (Chapter 2) has deepened the understanding of the molecular mechanisms and regulation of the MMP-based proteolytic network in the degradation of collagen disks during the FBR. In the absence of TIMP-1, i.e. in epicardial implants, both collagenases (MMP-13) and gelatinases (MMP-2 and 9) are present as active enzymes and act sequentially to fully degrade the implanted disk. Subcutaneously, however, the high expression of TIMP-1 prevents the collagenolytic activity of the various collagenases.

Besides MMPs, so far hardly any attention has been given towards other proteinases, e.g. cathepsin K, in the foreign body reaction directed against implanted materials. Cathepsin K, a strong collagenolytic enzyme, was reported to be present in giant cells in the foreign body reaction against wear debris derived from the gliding surfaces of prostheses used in joint replacements. Yet nothing is known on cathepsin K in the involvement of the FBR towards collagen scaffolds. In this thesis (Chapter 6), we report for the first time the presence of cathepsin K in the FBR against collagen scaffolds in mice subcutaneously. Interestingly, cathepsin K was not present in the gelatin (denatured collagen) scaffolds. Though it is insufficient to draw any conclusion regarding the precise roles of cathepsin K in the FBR at this stage, our pilot data show that it is necessary to further explore the involvement of cathepsin K in the degradation of collagen scaffolds.

2) Phagocytosis of collagen scaffolds Apart from enzymatic degradation, clearance of collagen scaffolds can be alternatively achieved through phagocytosis, an intracellular process of collagen degradation. Accumulating knowledge has been gained about the mechanisms of collagen phagocytosis by fibroblasts under physiological circumstance, yet vitrually nothing is known whether the inflammatory cells phagocytose fibrillar collagen in the FBR against collagen implants. This thesis for the first time shows that phagocytic cells, such as macrophages and giant cells, play a crucial role in the breakdown of collagen sacaffolds through phagocytosis.

Collagen phagocytosis by macrophages was observed in GDSC scaffolds at day 2 and 21 (Chapter 3), as well as in NDSC at day 21 and 28 (Chapter 5). Further study (Chapter 4) has shown that the collagen phagocytosis correlated with the interplay between the membrane-located mediators Endo180 and MMP-14. This appears to be logical that the collagen phagocytotic process by macrophage is realized through the ability of macrophages to cleave collagen fibriles near the cell membrane by MMP-14 followed by the lysosomal delivery of cleaved collagen. However, in HDSC there was a different type of macrophage present, which did not express Endo180 and therefore no phogocytotic activity was associated with this type of macrophages.

Moreover, collagen phagocytosis by giant cells was also seen in gelatin scaffolds and NDSC (Chapter 5 and 6). Interestingly, the presence of giant cells in these two biomaterials exhibited totally different characteristics. The giant cells in gelatin showed more nuclei and greater surface area than the giant cells in NDSC. Endo180 and MMP-14 were expressed in both scaffolds; however, their distribution patterns differed. Endo180 and MMP-14 co-localized in NDSC, suggesting that the collagen phagocytosis by giant cells followed a comparable way as it has been observed in fibroblasts. In contrast, Endo180 and MMP-14 were not co-localized in gelatin; indicating that collagen phagocytosis by giant cells in gelatin may follow a different way. Further study is required to understand this mechanism. Furthermore, the giant cells in NDSC were positive for cathepsin K and TRAP enzymes, whilst both enzymes were negative in giant cells in gelatin scaffolds. Together, our data clearly showed two distinct sub-sets of giant cells in terms of size, morphology and biological behaviors, during the FBR against non-crosslinked collagen and gelatin scaffolds.

The collagen phagocytotic activity in GDSC showed strong correlation with the presence of neutrophils (Chapter 3), but there is no evidence that neutrophils are directly involved in phagocytosing the collagen scaffold. The presence of neutrophils apparently induced the expression of pro-inflammatory cytokines including IFN-γ, the latter being known as a major activator for phagocytosis by macrophages. Therefore, we conclude that neutrophils play a crucial regulatory role in the collagen phagocytosis by macrophages through IFN-γ.

3) Regulation of collagen degradation through changes in the molecular mediators in the FBR micro-environment Extensive investigations have been carried out in this thesis (Chapter 2-5) with regard to the roles of cytokines (IL-1β, IL-4, IL-6, IL-10, IL-13, TNF-α, IFN-γ) and chemokines (CCL-2, CCL-3) in the regulation of proteolytic activities and cellular phagocytosis.

We have showed that in the proteolytic activities, the expression of TIMP-1 (a major regulator of MMP activity) depends on the presence of IL-10 (Chapter 2). IL-10 in HDSC was mainly produced by giant cells. One of the prime mediators that drives giant cells formation is IL-13, which was highly expressed in HDSC (Chapter 3). Interestingly, the collagen receptor DDR-2 possesses an impact in up-regulating MMP-13 expression (Chapter 5). These findings provide cues and clues to modulate the enzymatic degradation of collagen scaffold in future therapies in tissue engineering: a) reduce the MMP-based enzymatic degradation of collagen through increasing of the expression levels of IL-10 and/or IL-13; and b) speed up the enzymatic degradation via stimulating the expression of DDR-2.

On the other hand, our data showed that the collagen receptor Endo180 is crucial for the collagen phagocytosis by macrophages (Chapter 3), and that the expression of Endo180 in macrophage specifically relied on the presence of IFN-γ, but not on the other pro-inflammatory factors such as IL-1β and TNF-α. Interestingly, the IFN-γ inducing Endo180 effect can be blocked by IL-10 in a dose-dependent manner (Chapter 4). Therefore, IFN-γ, produced by netrophils, appears to be the key regulator for the collagen phagocytosis by macrophages. However, it might be not true for the collagen phagocytosis by giant cells in the gelatin scaffold, where the neutrophils and IFN-γ were virtually absent (Chapter 5). This suggests that the collagen phagocytosis by giant cells in gelatin is regulated through other unknown pathways, which requires further investigation.

4) Regulation of collagen degradation by changing the nature of chemical cross-linking It is known that the FBR reaction differs between species, biomaterials and implant sites. Our current studies deepened this knowledge and showed that the differences in the FBR were due to the different micro-environment created by the different types of implanted collagen scaffolds. A change in the chemical nature of collagen scaffold, e.g. cross-linking the native coll

Item ID: 28790
Item Type: Thesis (PhD)
Keywords: proefschriften (vorm); metalloprotei͏̈nases; fagocytose; collageen; immuunreacties; weefseltechniek; immunologie (geneeskunde); medische fysica
Additional Information:

This thesis is openly accessible from the link to the University of Groningen's institutional repository above.

Date Deposited: 24 Jun 2015 02:48
FoR Codes: 09 ENGINEERING > 0903 Biomedical Engineering > 090301 Biomaterials @ 40%
09 ENGINEERING > 0903 Biomedical Engineering > 090303 Biomedical Instrumentation @ 20%
11 MEDICAL AND HEALTH SCIENCES > 1199 Other Medical and Health Sciences > 119999 Medical and Health Sciences not elsewhere classified @ 40%
SEO Codes: 92 HEALTH > 9201 Clinical Health (Organs, Diseases and Abnormal Conditions) > 920103 Cardiovascular System and Diseases @ 40%
92 HEALTH > 9201 Clinical Health (Organs, Diseases and Abnormal Conditions) > 920199 Clinical Health (Organs, Diseases and Abnormal Conditions) not elsewhere classified @ 60%
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