Modification of fibrocyte responses to diminish biomaterial-mediated fibrotic tissue responses

摘要

Introduction: Implant-associated fibrotic tissue responses remain a major challenge in the design of medical devices. However, there is no effective treatment to reduce fibrotic tissue reaction without compromising normal wound healing responses. Recent evidence suggests that circulating fibroblast-like cells, termed fibrocytes, are responsible for the extent of these reactions, presenting an alternative model of repair. These cells are highly migratory and have been shown to be multi-potent, differentiating to different specialized cells, such as myofibroblasts and adipocytes. In the wound environment, fibrocytes differentiate into myofibroblasts which lead to excessive collagen production and scar formation. We assume that the extent of fibrotic tissue reactions may be reduced if fibrocytes are directly differentiated to adipocytes. To test the hypothesis, experiments were carried out to investigate the influence of localized fibrocyte-to-adipocyte differentiation on fibrocyte responses and fibrotic tissue formation surrounding biomaterial implants, respectively. Materials and Methods: Fibrocytes were isolated from the spleen of mice. For in vitro fibrocyte adipogenesis differentiation studies, fibrocytes were seeded on glass cover slips in a 24 well plate and then incubated with DMEM media (as control) or StemPro Adipogenesis Differentiation media, as per the manufacturer's instruction, to stimulate adipogenic differentiation. After cell culture for 14 days, adipocyte differentiation was confirmed through Oil Red O staining while other samples were stained with Sinus Red to identify collagen. In the model of directed fibrocyte differentiations, mini-osmotic pumps (Alzet Model 1002) were used to achieve sustained delivery of DMEM media (as control) or a concentrated adipogenic cocktail (StemPro, Invitrogen) at a constant rate of 0.25 ng/h to scaffold implants for 14 days. At the end of the study, scaffold and surrounding tissue were isolated for histological analyses. Results and Discussion: To investigate the potential of fibrocyte differentiation to mitigate fibrosis, we first confirmed that the Incubation of adipogenic media is capable of prompting differentiation. It is estimated that 2-week treatment leads to ～65-70% of adipogenic differentiation of fibrocytes based on Oil Red O staining. As expected, the increase in lipid accumulation is mirrored by a 3X decrease in the amount of collagen production in fibrocyte-derived adipocyte culture. Subsequent studies were carried out to investigate the effect of adipogenic differentiation on fibrotic tissue responses in vivo. As expected, we found that adipogenic stimulation can significantly alter fibrotic responses (Figure 1). Specifically, we found that adipogenic treatment significantly increased (2.8x) adipocyte accumulations in the implant-surrounding tissue. On the other hand, localized release of adipogenic agent led to 2.3X reduction in collagen production. Further analyses revealed a phenomenon that indicated reciprocal Inhibition of the adipocyte and myofibroblast differentiation pathways of fibrocytes. Conclusion: Understanding and manipulating fibrocyte reactions is becoming increasingly important as a means to mitigate fibrotic reactions to medical implants. Our study shows, for the first time, that directed differentiation of fibrocytes may be a viable strategy to reduce implant-associated fibrotic tissue formation and improving the function of medical implants.