Phenotype Transition Of Macropahges In Constructing Vascularized Engineered Adipose Tissue

BackgroundIn maxillofacial and plastic surgery, tumor removal, trauma, infection, congenital atrophy may result in loss of subcutaneous fat layer and sequent large soft tissue defects. Besides impairing physiological function, soft tissue defects also cause aesthetic problems and negative psychological feelings. Therefore, tremendous demand for transplantable and ideally, biologically functional adipose tissue grafts exists in the field of maxillofacial and plastic surgery. Current clinical strategies for soft tissue augmentation include the use of autologous fat tissue and synthetic surrogates. Due to varying degrees of graft resorption over time, considerable donor site morbidity, complicated surgical procedure and suboptimal biocompatibility of the implants, optimal strategy to engineer adipose tissue is being sought in the field of reconstructive surgery. With the development of tissue engineering techniques, promising progress has been made in restoring large soft tissue defects using engineered adipose tissue. As adipose tissue is highly vascularized and possesses a high metabolic activity, the engineered adipose tissue needs a rapid and sufficient vascularization. Vascularization is a critical bottleneck for tissue engineered large volume adipose tissue. A major reason for the problem is insufficient understanding of vascularization. Till now, most studies focused on the seeded cells, such as ASCs/preadipocytes, EPCs/ECs, growth factors and scaffold which could promote the proliferation, differentiation of seeded cells. Other important cell types in the process of angiogenesis and adipogenesis, such as macrophages, are paid less attention to. Recent work has shed light on the diverse nature of macrophages. It is now recognized that macrophages do more than simply protect the host from foreign invaders. Beside their role in innate immunity, macrophages act as regulators of development, tissue homoeostasis, remodeling, repair and tissue regeneration. Recently, macrophages were found involved in the process of angiogenesis and adipogenesis in the tissue engineering model. However, it has been showed that macrophages are a highly heterogeneous population of cells. They demonstrate dynamic and plastic phenotypes, and secret diverse cytokines in response to local microenvironmental signals. However, no studies focus the macrophage phenotype in the adipose tissue regeneration in the field of adipose tissue engineering up to now.In conclusion, our study is aimed to construct vascularized engineered adipose tissue using a tissue engineering model. On the basis of the model, we investigate the distribution and phenotype switch of macrophages in the process of angiogenesis and adipogenesis, as well as the role of phenotype regulation of macrophages on adipose tissue regeneration. The study provides a novel insight into the mechanism of adipose tissue regeneration, and the basis for the development of optimal strategy for adipose tissue engineering.ObjectsTo construct a rat adipose tissue engineering model using tissue engineering chamber and in-vivo prefabricated pedicles. To explore the proper procedure for constructing vascularized adipose tissue based on different modified patterns. To clarify the distribution and phenotype switch of macrophages, as well as the expression of various pro-inflammatory cytokines and anti-inflammatory cytokines. To explore the effect of phenotype regulation of macrophages on vascularization and adipose tissue regeneration in the tissue engineering chamber.Methods1. Animal model of vascularized adipose tissue engineeringA rat tissue engineering model using a silicon chamber enclosing the superficial epigastric pedicle in a Matrigel based environment supplemented with FGF-2 was constructed. The difference of adipose tissue regeneration potential is compared among the three groups, including axial pedicles sealed chamber with bone wax(group A), no axial pedicles sealed chamber with adipose pad(group B) and axial pedicles sealed chamber with adipose pad(group C).2. Transition of macrophage phenotype in the process of adipose tissue regenerationThe adipose tissue regeneration model was used the same as in experiment one. The angiogenesis and adipogenesis were evaluated at different harvest time points. The quantity and phenotype transition of macrophages were assessed by immnohistochemistry staining and double immunofluorescence staining. Enzyme-linked immunosorbent assay was used to evaluate the expression of various pro-inflammatory and anti-inflammatory cytokines.3. The effect of initial depletion of macrophages on vascularization and adipose tissue regenerationWith the liposome-mediated macrophage “suicide” approach, the effect of initial depletion of macrophages in the chamber on vascularization and adipose tissue regeneration based on the tissue engineering chamber model. The density of vascular endothelial cells, neutrophils and macrophages, as well as the phenotype of macrophages was evaluated by immnohistochemistry staining and double immunofluorescence staining. Enzyme-linked immunosorbent assay was used to determine the effect of initial depletion of macrophages on the pro-inflammatory or anti-inflammatory cytokine microenvironment.ResultsAll chambers displayed vascular patency at 6 weeks. The average volume and weight of constructs in group C(axial vessel pedicles sealed chamber with adipose tissue pad) were the largest among the three groups. A large amount of mature adipose tissue surrounded the pedicle was observed in the group. Relatively less neo-adipose tissue was generated in group B(no vessel pedicles sealed chamber with adipose tissue pad). Hardly adipose tissue was observed. The tissue of the constructs was predominantly fibrous connective tissue.Histology analysis showed that obvious circulation infiltration of inflammatory cells and endothelial cells into the Matrigel at 3 days. Moreover, a large number of red blood cells were present in the surrounding matrix, giving a hemorrhagic appearance. At 7 days after implantation, some of the Matrigel was degraded and replaced by newly formed connective tissue and capillaries. At 14 days, the Matrigel was mostly degraded and gradually replaced by newly generated connective tissue and microvessels. However, obvious adipose cells were hardly seen at this time. At 42 days, the Matrigel was completely absorbed, and the constructs mostly contained mature adipose tissue, surrounded by a connective tissue capsule. Macrophages were obvious in the chamber constructs 3 days after implantation, peaked at day 7, and significantly decreased thereafter. At day 3, macrophages were predominantly M1 macrophages(CCR7+), and there were few M2 macrophages(CD206+). At day 7, the percentage of M2 macrophages significantly increased and remained stable at day 14. At 42 days, macrophages became predominant macrophage population. The ratio of M2/M1 significantly increased during the adipose regeneration process. Enzyme-linked immunosorbent assay demonstrated transition of cytokines from pro-inflammatory to anti-inflammatory, which was consistent with the transition of macrophage phenotype from M1 to M2.Macrophage density in the Clodronate group is significantly less the density in its respective control groups, while the density recovered to comparable level to control groups. In terms of macrophage phenotype, the percentage of M1 is significantly more in the Clodronate group than that in the other two groups, while the percentage of M2, as well as the ratio of M2/M1, is remarkably less in the Clodronate group than in other two groups. Significantly more neutrophil were recruited to the clodronate group compared with the other two groups at 4, 14 and 42 days. Enzyme-linked immunosorbent assay showed the expression of pro-inflammatory cytokines, such as IL-1β, TNF-α and IL-6 was significantly more in Clodronate group than in the other two groups. In contrast, the expression of anti-inflammatory cytokines, such as IL-4, IL-10 and TGF-β, was significantly less in Clodronate group than in the other two groups.ConclusionVascularized engineered adipose tissue could be constructed using tissue engineering chamber and in-vivo prefabricated pedicles. Enough physical space provided by the tissue engineering chamber to accommodate tissue growth, sufficient blood supply provided from the axial vessel pedicles and the contact of scaffold with autologous adipose tissue, create proper microenvironment beneficial for angiogenesis and adipogenesis and promote adipose tissue regeneration. Macrophages are involved in the whole process of angiogenesis and adipose tissue regeneration in the tissue engineering chamber. Macrophages infiltrating the chamber undergo a pro-inflammatory to anti-inflammatory transition, while microenvironment in the chamber undergoes the switch from pro-inflammatory state to anti-inflammatory state. Using selective depletion of macrophages based on liposome mediated macrophage “suicide” approach, we found that macrophages play an essential role in both angiogenesis and adipogenesis in the tissue engineering chamber. Clodronate-containing liposomes can efficiently eliminate the macrophages in the chamber at early stage. However the depletion effects will disappear after two weeks. Initial depletion of macrophages leads to significantly delayed vascularization, long-lasting neutrophil infiltration and continuous inflammatory states in the chamber. Initial depletion of macrophages also causes dysfunction of macrophage M1-to M2 transiting. The results indicate that proper regulation of macrophage phenotype, rather than the quantity of macrophages, determines the final outcome of adipose tissue regeneration in the tissue engineering chamber.Our findings provide a novel insight into the role of macrophage in adipose-tissue regeneration and the basis for the development of biomaterials in adipose tissue engineering. The property of a scaffold which is beneficial to the M1-to-M2 transition of macrophages must be considered during scaffold design. Moreover, exogenous cytokines or seed cells could be delivered to induce the transition of macrophage phenotype and local microenvironment from pro-inflammatory state to anti-inflammatory state. The approach based on macrophages could be an innovative vascularization strategy for adipose tissue engineering.