Inflammation is mediated by blood monocyte-derived or tissue-resident macrophages recruited to the site of tissue injury, be it from acute mechanical forces such as in trauma or surgery, infectious agents, or chronic underlying pathology. In later stages and after resolution of acute inflammation, stromal cells are being recruited, which may lead to fibrosis, scar formation, and organ dysfunction. Currently available therapeutic options to address soft tissue injury include watchful waiting, temporizing measures, such as topical antibiotics, growth factors, or anti-inflammatory applications, or surgical debridement. However, none of these methods are an ideal means to heal the injured tissue, and thus new strategies are being developed to not only replace wounded skin or connective tissues but also to regenerate the native tissue lost.27
One can anticipate that this task will require multi-modality therapy to mediate both the inflammatory resolution and tissue reparative phases of wound healing.
Bioengineered scaffolds could provide a microenvironment that allows for nutrient diffusion as well as biochemical, physical, and cellular stimuli that guides proliferation, differentiation, and migration of implanted or tissue resident cells. While many of these scaffolds are clinically used without cells for soft tissue augmentation or repair, such as collagen or HA, there is increasing interest in combining these ECM-based scaffold materials with cells for in vivo tissue engineering strategies. Biomaterials, such as MSC-hydrogel constructs, hold great promise in tissue engineering, but before the initiation of clinical trials, further studies are needed to investigate their potential interactions with human immune cells. This study demonstrates an in vitro model for investigating the immune response of human peripheral blood monocyte-derived macrophages when cocultured with human MSCs seeded in a hydrogel scaffold. Such studies are crucial in developing more effective biomaterial and cell-based therapies.
There are several factors associated with a material scaffold that can influence cell differentiation or function, including chemical composition (hydrophobicity, chemical stimuli, or added pendant chains), scaffold geometry (2D vs. 3D), and spatial relationship (cell concentration and porosity).28–30
In a similar manner, MSCs have been shown to modulate immune cell function through a variety of paracrine and juxtacrine effects, though these interactions are not completely understood. Over the past two decades there have been an increasing number of injectable fillers based on chemically modified biomaterials to use for soft tissue wound repair.1
The next generation of sECM-based materials, such as the Carbylan–GSX investigated in this study, is of potential interest because it allows for an injectable cell-seeded construct that is capable of cross-linking in situ
for local delivery in wound healing and other tissue engineering strategies.31–33
In this study, we developed a novel coculture system to investigate the potential immunomodulatory and anti-inflammatory properties of such cell–hydrogel constructs. In particular, we determined the phenotype of macrophages cultured with BM- and AT-MSCs with and without an sECM scaffold. We found that the immunophenotypes of macrophages were different among the four different culture conditions, indicating an effect of both the sECM scaffold as well as MSC influence. CD14+/CD16+ monocytes are a subpopulation of circulating monocytes in peripheral blood in normal steady state; however, there is a drastic increase in CD14+/CD16+ cells in both acute and chronic inflammatory conditions.34,35
Such pro-inflammatory monocytes are responsible for the release of tumor necrosis factor-alpha, interleukin (IL)-1, and IL-12, among other cytokines involved in acute phase inflammation as well as frustrated phagocytosis and foreign body response.36,37
An additional marker of inflammation is HLA-DR expression on monocytes facilitating antigen presentation to T lymphocytes and a robust pro-inflammatory response.36,37
Monocyte expression of HLA-DR is used as an assessment of immune status and it is thought that a decrease in circulating HLA-DR+ monocytes is a gauge of a compensatory anti-inflammatory response in systemic illness.37
Alternatively, increased expression of the surface marker CD206 is an indicator of an anti-inflammatory phenotype.16,38,39
Previous work with our novel HA-based sECM scaffold established in vitro
and in vivo
biocompatibility with MSCs.24,31
As CD14+ monocytes differentiated in the presence of our MSC-hydrogel constructs in vitro
, there were no signs of toxicity to the monocytes in culture or induction of a pro-inflammatory phenotype. In fact, the immunophenotype of CD14+ macrophages at the end of the 7-day coculture could be characterized as an anti-inflammatory profile, based on low CD16, high CD206, and low HLA-DR expression. Macrophages cultured on Carbylan–GSX with MSCs expressed lower CD16 and HLA-DR with higher expression of CD206, indicating the least inflammatory profile overall. A unique aspect of this work is the allogeneic nature of the cells in that the macrophages were derived from different donors than the MSCs and that all six MSC lines used were isolated from different donors. Others have shown that while biologic scaffolds can induce an anti-inflammatory macrophage expression, adding a differentiated cellular component will induce a proinflammatory or M1 phenotype in vivo
It is often difficult to predict how the in vitro outcomes will manifest into physiological responses, particularly in wound healing and biomaterial interactions. Translational, in vitro work such as this offers a controlled and manipulatable study environment to investigate the interactions between allogeneic cells, a biomaterial scaffold, and the specific cell type key in regulating these interactions. The statistically significant differences in macrophage surface marker expression among the treatment groups may contribute to understanding the undoubtedly complex mechanisms associated with MSCs in wound healing. Whether or not these differences will translate into physiological outcomes such as improved tissue regeneration or construct “take” can be hypothesized based on this study and warrants further investigation. This work communicates several interesting findings, including (1) the development of a coculture technique using an sECM scaffold to encapsulate MSCs in 3D during monocyte-derived macrophage development; (2) a direct comparison of BM and AT-derived MSCs as well as with a well-studied VF fibroblast cell line sharing similar functional characteristics to MSCs; (3) evidence that our sECM hydrogel scaffold induces an anti-inflammatory surface marker expression independent of 3D cell encapsulation; and (4) evidence supporting the benefit of an MSC-sECM construct for engineering strategies to address both tissue inflammation and repair. In vitro and in vivo work such as this is necessary in accelerating the translation of cell-based therapies from basic science to relevant, well-designed clinical trials.