The results of the present study confirm the intuitive concept that blood-derived mononuclear macrophages play a necessary and important role in the early in vivo degradation process of ECM scaffolds and autologous tissue grafts, and that chemical crosslinking of the ECM by compounds such as CDI effectively inhibits the degradation process. These findings are important in the context of regenerative medicine and biologic scaffolds because the proposed mechanisms of the constructive remodeling response typically require scaffold degradation to realize the optimal beneficial effects. Because the presence of mononuclear macrophages is usually associated with inflammatory processes and the expected downstream consequences of tissue necrosis and scar tissue formation, the findings of the present study emphasize the wisdom of evaluating the role of macrophages with an open mind at least with regard to ECM scaffold remodeling.
ECM scaffolds such as SIS-ECM and urinary bladder matrix (UBM-ECM) have been shown to degrade rapidly in vivo
, typically within 8–10 weeks after implantation,21,22
and yet have been associated with long-term functional tissue remodeling in many anatomic locations.21,37,38
Degradation-dependent events such as the release of constituent growth factors, the production and release of matricryptic peptides, and the recruitment of host progenitor cells and stem cells contribute to the well-documented dynamic histomorphologic changes that characterize the host response to such scaffold materials during this early postoperative period.3,4,6,9,12,39–43
It is logical to speculate, based upon the results of the present study, that blood-derived mononuclear macrophages are at least important, if not essential, mediators of these degradation-dependent events. It is therefore reasonable to conclude that the degradation of an ECM scaffold is beneficial and necessary to tissue remodeling efforts. Such an interpretation is in stark contrast to the conclusion that a macrophage-rich tissue response after ECM scaffold implantation necessarily represents an adverse host–patient reaction.44
The clinical utility of ECM scaffolds depends upon the ability of the scaffold material to provide adequate mechanical support to the injured or missing tissue being repaired. The degradation rate of nonchemically crosslinked ECM appears to be compatible with the rate of new host tissue deposition, such that the scaffold retains sufficient strength to serve as a functional replacement until the host can deposit new host tissue.45–48
However, there is a measurable loss of strength that occurs before transfer of mechanical function to the new host tissue. A common approach for increasing the strength of biologic scaffolds composed of ECM or components of ECM (e.g., purified type I collagen) has been the use of chemical crosslinking agents such as CDI,24,32–34
While chemical crosslinking does maintain the mechanical strength of the scaffold, it also necessarily alters the kinetics of degradation and has a deleterious effect on the biologic activity of the degradation products. The present study showed that CDI effectively inhibited ECM scaffold degradation regardless of the presence of macrophages. This finding was not unexpected and was consistent with recent studies that show a markedly altered, proinflammatory host tissue response to ECM scaffolds that have been chemically crosslinked.23
In clinical applications, noncrosslinked ECM scaffolds are the only grafts that are indicated for contaminated wounds because of the antibacterial effects associated with their degradation.53
Taken together, the beneficial biologic effects of ECM scaffold degradation and the loss of scaffold mechanical strength associated with scaffold degradation would suggest that a balance of these seemingly mutually exclusive events is necessary to realize functional tissue reconstruction.
It has recently been shown that macrophage phenotype is an important determinant of tissue remodeling in the context of regenerative medicine.18
The M2 macrophage phenotype is considered to be an immunomodulatory and tissue remodeling phenotype. In contrast, proinflammatory macrophages, designated as an M1 phenotype, are associated with chronic inflammation and a foreign body reaction.54
Although not evaluated in the present study, the macrophage phenotype profile was characterized in a study that used the same animal model and showed the predominant macrophage phenotype to ECM scaffolds that are not chemically crosslinked to be M2 from 1 week postimplantation to at least 4 months postimplantation.18
In contrast, the predominant macrophage phenotype to CDI crosslinked ECM was M1 during the same time period. Therefore, it is important to consider not only the simple presence of macrophages at the site of scaffold remodeling but also the phenotype of this important cell population when attempting to predict downstream remodeling outcomes.
Although not directly investigated in this study, the depletion of macrophages would logically alter the temporal and spatial response of other cell types toward an implanted scaffold material, including ECM scaffolds. The acute host tissue response to an implanted foreign material, as all of these materials represent, is typically characterized by the presence of neutrophils. After 3–5 days, the neutrophil population decreases and the mononuclear macrophage population becomes the predominant cell response. This balance of neutrophil departure and mononuclear macrophage arrival was disrupted by the depletion of macrophages in the present study. Neutrophils were still the prominent feature of the host cell response 2 weeks after implantation in the animals that were implanted with the 14C-SIS scaffold and that received clodronate-containing liposomes. The present results show that neutrophils do indeed participate in ECM scaffold remodeling, but do not appear to be major determinants of early scaffold degradation based upon the quantitative 14C measurements. It is logical that at least a certain degree of neutrophil-mediated scaffold degradation should occur, but the ability of neutrophils alone to provide for all of the biologic effects that are associated with ECM scaffold degradation remains to be investigated.
The present study is limited by the fact that the results were confined to the early stages of scaffold remodeling, that is, 2 weeks. The marked depletion of mononuclear macrophages for longer periods of time places the host at great risk for sepsis and makes such studies difficult. Further, repeated administration of clodronate was performed to ensure that the results were not confounded by newly formed circulating macrophages later in the study period. It should also be noted that the present study is limited to a single type of biologic scaffold material, specifically the SIS-ECM, but the learned principles may be applicable to all biologic scaffold materials.
In summary, macrophages play an important and central role in biologic scaffold degradation and remodeling. Their presence within and surrounding scaffold materials after in vivo implantation should not only be expected but also perhaps be recognized as a favorable course of events and may predict a constructive remodeling outcome.