Temporal and spatial distribution of macrophage phenotype markers in the foreign body response to glutaraldehyde-crosslinked gelatin hydrogels

Abstract

In recent years, macrophage phenotype has emerged as an important determinant of the success or failure of implanted polymeric biomaterials. However, it is not well understood how changes in biomaterials properties affect the foreign body response or macrophage behavior. Because failed attempts at biomaterial degradation by macrophages have been linked to frustrated phagocytosis, a defining feature of the foreign body response, we hypothesized that increased hydrogel crosslinking density (and decreased degradability) would exacerbate the foreign body response. Gelatin hydrogels were crosslinked with glutaraldehyde (0.05%, 0.1%, and 0.3%) and implanted subcutaneously in C57BL/6 mice over the course of 3 weeks. Histological and immunohistochemical analysis was used to characterize fibrous capsule formation and the temporal and spatial distribution of macrophage phenotype markers. Interestingly, changes in hydrogel crosslinking did not affect the thickness of the fibrous capsule surrounding the hydrogels, expression of the pan-macrophage marker F480, expression of three macrophage phenotype markers (iNOS, Arg1, CD163), or expression of the myofibroblast marker aSMA. With respect to temporal changes, the level of expression of the M1 marker (iNOS) remained relatively constant throughout the study, while the M2 markers Arg1 and CD163 increased over time. Expression of these M2 markers was highly correlated with fibrous capsule thickness. Differences in spatial distribution of staining also were noted, with the strongest staining for iNOS at the hydrogel surface and increasing expression of the myofibroblast marker aSMA toward the outer edge of the fibrous capsule. These results confirm previous reports that macrophages in the foreign body response exhibit characteristics of both M1 and M2 phenotypes. Understanding the effects (or lack of effects) of biomaterial properties on the foreign body response and macrophage phenotype may aid in the rational design of biomaterials to integrate with surrounding tissue.

Humanized mouse model for assessing the human immune response to xenogeneic and allogeneic decellularized biomaterials

“…Each material was injected subcutaneously, a common delivery route for ISO standard biocompatibility tests, and at least eight different injections (n=8−16) were analyzed histologically three days and one week post injection. These time points were selected for assessing the early and mid-phase immune response since previous studies have shown macrophage response and polarization during this timeframe to be representative of long term biocompatibility and tissue repair [27–29]. Longer time points were not investigated since these biomaterials degrade completely by 2–3 weeks in vivo .…”

“…Cellular infiltration was observed to be unevenly distributed in the injected material, particularly towards the center in the Hu-mice samples (Figure 2A). To distinguish cells clustered at the material border versus cells that infiltrated deeper into the biomaterial, cell density was quantified as two measures: cells throughout the whole biomaterial and cells in only the core region as defined as 200 μm inward from the biomaterial border [29]. …”

Macrophage-based therapeutic strategies in regenerative medicine

“…The same is true for M2-like macrophages, which have been associated with both tissue regeneration [5, 110] and fibrosis [35, 36]. With respect to biomaterial-mediated tissue repair, both M1- and M2-like macrophages have been separately associated with either vascular integration or fibrous encapsulation [6, 7, 90, 111, 112]. We propose that these apparently conflicting reports are related to the timing of macrophage activation, with any aberrations from the natural M1-to-M2 sequence resulting in impaired healing, as discussed throughout this review.…”

“…A properly regulated inflammatory response is important for efficient healing, not only for preventing infection and clearing necrotic tissue but also for regulating tissue formation and remodeling. Indeed, the pharmacologic depletion of immune cells causes impaired tissue healing and regeneration in experimental models of injury [4, 5], and dysregulation of immune cell activation results in encapsulation of biomaterials in fibrous tissue in the foreign body response [6–8]. …”

Drug delivery strategies to control macrophages for tissue repair and regeneration

“…However, perhaps because of their role in promoting cell proliferation and collagen deposition, M2a macrophages are also associated with fibrous encapsulation of biomaterials. 16,57 Indeed, delivery of the M2a-promoting factor IL4 exacerbates the foreign body response. 39 For more information on the contribution of M1 and M2a macrophages to tissue repair and regenerative medicine strategies, the reader is referred to Spiller et al 58 and Nassiri et al 59 Another activation state of macrophages that is emerging as a major contributor to tissue repair and regeneration is the so-called M2c phenotype, which are stimulated with IL10 or glucocorticoids.…”