Patients with advanced stages of glaucoma remain challenging to treat, especially when filtering surgeries fail owing to bleb fibrosis. Despite intensive research on bleb fibrosis, most studies have focused on trabeculectomy, and few have involved patients with XEN or Preserflo implants. In this regard, the data presented in this study may contribute to a better understanding of bleb fibrosis after MIBS with the XEN or Preserflo.

In our study, almost all samples demonstrated characteristic fibrotic features such as activated and enlarged fibroblasts in either parallel or irregular orientations in failed blebs after implantation of the XEN or Preserflo. Multimodal imaging with confocal microscopy and OCT has previously demonstrated a significant increase in reflectivity of failed XEN blebs [16], likely attributable to the presence of increased fibrosis, as confirmed in our histopathological analysis. These results align with previous reports on failed trabeculectomy eyes, in which a similar increase in the reflectivity of the bleb and a high density of collagen connective tissue were found [17, 18].

In general, three factors are believed to be important in the development of filtering bleb fibrosis: first, the trauma of the surgery, which initiates the wound healing process and inflammation; second, the new aqueous humor channel, which brings the aqueous humor into direct contact with the wound; and third, the surface of the implanted material.

Regarding postoperative inflammation, the presence of many lymphocytes and neutrophil granulocytes is expected, but in this study, we observed large numbers of macrophages only and no other inflammatory cells. In addition, the fibrotic features of the blebs were not dependent on the time after primary surgery. Taken together, these observations indicated that postoperative inflammation was not the major cause of fibrosis in our patients. Factors related to the aqueous humor might play a more important role by activating subconjunctival macrophages and fibroblasts, leading to bleb fibrosis.

It has long been discussed that the aqueous humor is involved in processes that result in post-trabeculectomy bleb fibrosis and, finally, surgical failure [19, 20]. Cytokines present in the aqueous humor such as TGF-β, PDGF, VEGF, and TNF-α are important factors in postoperative wound healing. Their role in bleb fibrosis has been extensively studied. The concentrations of these cytokines are largely increased in the vitreous humor of patients with glaucoma [21,22,23]. In particular, TGF-β seems to induce the differentiation of fibroblasts to α-SMA-expressing myofibroblasts, which can modulate the extracellular matrix and exhibit contractile properties, contributing to bleb fibrosis and failure. Accordingly, we found sm-actin-positive myofibroblasts in almost all samples. Interestingly, different types of glaucoma seem to have varying concentrations of cytokines in the vitreous [24]. However, our analysis of patients with POAG and uveitic glaucoma showed no differences in any of the histological parameters analyzed. This may be due to the small sample size, but it is also consistent with the finding that trabeculectomy in secondary uveitic glaucoma produces comparable results to those in patients with POAG [25, 26].

The fibrotic response to MIBS has also been previously studied in animals. One study investigated the fibrotic response to the Preserflo implant in rabbits and found polymorphonuclear leukocytes, foreign body giant cells, and many myofibroblasts located mostly in the fibrotic bleb wall 40 days after surgery [27]. In contrast, another study analyzed rabbit eyes 100 days after Preserflo implantation and found no myofibroblasts [28]. The latter finding mostly agrees with that of our study, although a comparison between human and rabbit eyes should be undertaken with caution. We did not observe any giant multinucleated cells in either the XEN- or Preserflo-implanted eyes but noted singular myofibroblasts in 23 of 25 samples and parallel-oriented myofibroblasts in 7 of 25 samples. Similarly, the XEN implant has not been reported to cause pronounced immune reactions in rabbits and dogs [29].

The stent surface is another factor influencing postoperative bleb fibrosis. Accordingly, efforts have been made to use innovative materials to reduce bleb fibrosis. For example, nanofiber-based materials mimic the natural extracellular matrix better than the flat surfaces of XEN implants or the silicon tube of Baerveldt glaucoma implants [30]. A recent study showed that this new material minimized biomaterial-associated bleb fibrosis in rabbits and could extend the lifespan of a filtrating stent [30]. Thus, a combination of surface material modification and longer-term cytokine milieus regulation is likely crucial in ensuring the long-term success of glaucoma implants and in inhibiting bleb fibrosis.

Numerous pre-, intra-, and postoperative interventions have been studied over the past decades in an attempt to reduce the risk of bleb fibrosis and failure [31, 32]. The use of antimetabolites such as MMC and 5-FU is now well established in glaucoma surgery and can improve long-term success, although side effects along with non-cell-specific cytotoxic effects must be acknowledged [33]. Nevertheless, intraoperative administration of antimetabolites can have only a temporary influence on the local environment, whereas prolonged effects might be necessary for long-term success. A previous histological analysis of failed trabeculectomies showed that eyes operated without MMC presented with parallel-oriented fibroblasts, whereas eyes operated with MMC had only few fibroblasts, with neither contractile fibers nor a particular orientation [19]. This finding contrasts with our data that nearly all samples contained numerous fibroblasts, and 60% presented with parallel-oriented fibroblasts, indicating strong bleb fibrosis, even though all patients received MMC during primary surgery. Additionally, the majority of our samples stained positive for singular sm-actin-positive myofibroblasts. In the abovementioned previous histological analysis, no association was found between histological features and time after surgery, agreeing with our findings.

A limitation of our study is that we included patients with different types of glaucoma and varying numbers of previous surgeries, which could have influenced the development of bleb fibrosis. However, this approach allowed us to compare the development of bleb fibrosis under different conditions and might enable better generalizability to clinical practice. It is also important to keep in mind that all patients underwent surgery due to a clinically significant bleb fibrosis. Therefore, it is not surprising that the histological results do not differ significantly, despite the patients having different types of glaucoma.

In conclusion, our study provides important insights into the biocompatibility of the XEN and Preserflo implants in humans. The histological analyses revealed consistent histological patterns across a diverse patient population, suggesting that the findings may be generalizable across a wide range of patients. Most importantly, no patient showed signs of a pronounced immune or foreign-body reaction, underlining the value of the XEN and Preserflo implants in the management of treatment-refractory glaucoma.