The reepithelialization and revascularization processes of denuded sclera after pterygium excision are not well-known. Some studies have implicated the centripetal migration of undifferentiated conjunctival cells and possible conjunctival epithelial stem cell involvement, but the exact roles of these cells and the mechanisms modulating the migration and differentiation of cells in the repair process are controversial.18
In addition, the aggressive recurrence of pterygium after surgical excision suggests the involvement of an unknown cellular mechanism in the pathogenesis and wound healing processes. In a previous study, we conclusively demonstrated the involvement of bone marrow-derived cells in the pathogenesis of pterygium.6
This study shows that CD34, c-kit, AC133, and STRO-1 are strongly expressed in amniotic membrane applied to bare sclera after pterygium removal. The leukocyte antigen, CD34, is known to be expressed on the surfaces of hematopoietic progenitor cells, vascular endothelial cells, and some fibroblasts.12
During the repair process after pterygium excision, round or spindle-shaped, CD34-positive mononuclear cells were observed on the stromal side of the amniotic membrane. Cells that stained positively for CD34 clustered in small, vessel-like shapes in some regions of amniotic membrane. Whether this vessel formation was due to the vasculogenic transformation of progenitor cells or to the angiogenesis of existing vessels is unclear. A recent study on staining patterns in vessels indicated that CD34 is expressed on endothelial cells in tumors and fetal tissue, but not during the angiogenic microvascular process in wounded tissue.19
This result suggests that CD34-positive endothelial cells involved in wound healing originate from bone marrow. Therefore, our findings provide evidence of bone marrow-derived progenitor cell involvement in the healing process.
AC133 is a marker for endothelial progenitor and neuronal stem cells and is rapidly downregulated during cell differentiation.20
Abundant AC133-positive cells were observed on removed amniotic membranes by morphological analysis, and were round to spindle-shaped, similar to CD34-positive cells. The specific roles of these cells are uncertain; however it is possible that these cells may differentiate into endothelial cells, since a previous study showed that a subset of CD34-positive cells express AC133 as a true functional marker of endothelial progenitor cells.21
In cross sections, we found that amniotic membranes were covered by one or two layers of small cuboidal epithelial cells that were morphologically similar to conjunctival basal epithelial cells. Additionally, aberrant c-kit expression was localized within the epithelium that had grown over the amniotic membrane. These findings are consistent with a report that corneal basal epithelial cells immunostain for SCF.5
These regenerated epithelial cells are an important source of a variety of cytokines, and chemotactic and growth factors, including SCF.22
Thus, our results provide more evidence that regenerated epithelial cells, indeed, contribute to the healing process. On a flat mount, c-kit-positive mononuclear cells appeared round or spindle-shaped and resembled mast cells or tissue macrophages. These findings are consistent with reports that mast cells are expressed in pterygial tissue and that their numbers significantly increase during the wound healing process.23,24
These cells may participate in the scarring caused by fibroblast proliferation or in angiogenesis by providing vascular endothelial growth factor.25
The results of our study suggest that bone marrow-derived progenitor cells are involved in healing and recurrence after pterygium excision. The basic mechanism of how bone marrow-derived progenitor cells migrate from the bone marrow into injured sites at a restricted vessel is unclear. A recent study indicated that tissue hypoxia might fundamentally govern progenitor cell recruitment and retention.26
Vascular disruption due to surgical excision is likely to cause a hypoxic zone on the ocular surface, in which the activation of trafficking-related factors, such as hypoxia inducible factor-1α (HIF-1α), facilitate progenitor cell recruitment in ischemic tissue requiring repair.
In addition to hypoxia, several other mechanisms may lead to progenitor cell release in response to tissue damage. Surgical injury might induce a rapid influx of not only inflammatory cells, but also of abundant growth factors and chemokines, such as IL-6 and matrix metalloprotease-9.27
These may stimulate bone marrow and enhance the migration of stem cells into the damaged ocular surface to repair damaged lesions via a rich vascular arcade of limbus. In addition, substance p, induced by surgical pain, may play a role in progenitor cell recruitment.28
Bone marrow-derived cells may be important for the renewal of resident stem cells, required for the maintenance of an organ or for the repair of tissue damage. Moreover, migrated bone marrow-derived cells would be activated by chemotactic factors released by certain inflammatory cells and keratocytes and gain the ability to differentiate into vascular endothelial cells, fibroblasts, and epithelial cells to recover the damaged surface.
Our findings also explain the features of recurrence after pterygium removal. After surgical excision, if abnormal microenvironments affected the wound healing process, then infiltrating progenitor cells would differentiate and proliferate more, thus contributing to alterations of fibroblasts and limbal stem cells. In addition, we observed many STRO-1-positive cells (a fibroblast characteristic) on the amniotic membrane. The inappropriate proliferation of bone marrow-derived circulating fibroblasts contributes to the genesis of subepithelial fibrosis and, thus, accelerates recurrence. This hypothesis is compatible with a finding of our previous study, which is that much stronger immunoreactivity to the progenitor cell markers is present in recurrent pterygium.6
Thus, stem cells may play a role as indicators of pterygium recurrence, and it is reasonable to believe that the more stem cells present, the higher the pterygium recurrence rate will be after removal.
The application of amniotic membrane for ocular surface reconstruction was found to be a useful therapeutic method with various advantages.29,30
It provides a healthy basement membrane for epithelial proliferation and contains various proteins that inhibit proteinases, which are destructive to tissue after the initial injury. The reported clinical outcomes after amniotic membrane graft for pterygia have demonstrated good results, although some studies reported high recurrence rate.30-33
The reasons for this variability are unclear, but many kinds of growth factors in amniotic membrane might induce excessive fibrovascular proliferation and pterygium recurrence. In a previous study, alkali-burned corneas, treated with amniotic membrane coverage for three days, showed enhanced wound healing, compared with those treated with coverage for seven days.34
Therefore, we reasoned that covering the bare sclera with the temporary amniotic membrane patch may alleviate possible adverse effects caused by permanent amniotic membrane grafting. Additionally, when amniotic membrane covers nerves exposed in the cornea, the application of amniotic membrane relieves pain. Based on these findings, we applied the amniotic membrane stromal-surface-down to the exposed sclera and observed many infiltrating cells, including bone marrow-derived progenitor cells and inflammatory cells, over the amniotic membrane. Thus, a temporary amniotic membrane patch could effectively prevent recurrence by absorbing excessively infiltrating stem cells and relieving pain by means of a biological wound-dressing effect.
To the best of our knowledge, our findings demonstrate, for the first time, the possibility that bone-marrow-derived cells are involved in the process of wound healing and in the recurrence of pterygium after surgical excision. Additional experiments are required to identify the factors that mediate recruitment, proliferation, and differentiation of progenitor cells to reveal the pathogenesis of pterygium and the wound healing process after pterygium removal. We hope that the clinical applications of this new concept will accelerate the healing process in a controlled fashion in the treatment of this disease.