KEY CONCEPTS
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Corneal regeneration is a new approach in corneal surgical technique based on advanced therapies using stem cell therapy and bioengineering. In the future, the combination of both will make it possible to renovate the corneal stroma without the need for corneal transplantation.
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Based on experimental animal models and laboratory studies, published work has supported a translational approach to these new therapies.
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Clinical results show that the regeneration of corneal stroma is achievable using stem cells and that the integration of acellular corneal tissue into the human corneal stroma of patients with keratoconus is feasible to improve corneal thickness, biological function, and visual outcomes.
Background: From the Lab to the Patient
The stroma constitutes more than 90% of the corneal thickness. Many features of the cornea, including its strength, morphology, and transparency, are attributable to the anatomy and properties of the corneal stroma. Many diseases such as corneal dystrophies, scars, or ectatic disorders induce a distortion of corneal anatomy or physiology leading to loss of transparency and subsequent loss of vision. In the last decade, enormous efforts have been made to replicate the corneal stroma in the laboratory to find an alternative to classical corneal transplantation. However, this has still not been accomplished because of the extreme difficulty in mimicking the highly complex ultrastructure of the corneal stroma, with substitutes obtained that do not achieve either enough transparency or strength. ,
In the last few years, cell therapy of the corneal stroma using mesenchymal stem cells (MSCs) from either ocular or extraocular sources has gained considerable interest; studies show that MSCs are capable of differentiating into adult keratocytes in vitro and in vivo. Several authors, including from our research group, have demonstrated that these stem cells can not only survive and differentiate into adult human keratocytes in xenogeneic scenarios without inducing any inflammatory reaction, but also (1) produce new collagen within the host stroma, , (2) modulate preexisting scars by corneal stroma remodeling, , and (3) improve corneal transparency in animal models for corneal dystrophies by collagen reorganization, as well as in animal models of metabolopathies by the catabolism of accumulated proteins. MSCs have also shown immunomodulatory properties in syngeneic, allogeneic, and even xenogeneic scenarios. , The first clinical data on the safety and preliminary efficacy of cellular therapy of the corneal stroma from phase 1 human clinical trials are now available, , and cellular therapy may end up providing a real alternative treatment option for corneal diseases in the near future.
When considering existing scientific evidence, it appears that all types of MSCs behave similarly in vivo ( Table 36.1 ) and are thus able to achieve keratocyte differentiation and modulate the corneal stroma. It has also been recently reported that MSCs secrete paracrine factors such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), and transforming growth factor beta 1 (TGFβ1). Although the precise actions of the different growth factors for cornea wound healing are not fully understood, overall they appear to promote cell migration, keratocyte survival by apoptosis inhibition, and upregulation of the expression of extracellular matrix (ECM) component genes in keratocytes, subsequently enhancing corneal reepithelialization and stromal wound healing. MSCs can be obtained from many human tissues, including adipose tissue, bone marrow, umbilical cord, dental pulp, gingiva, hair follicle, cornea, and placenta. ,
| CSSC | BM-MSC | ADASC | UMSC | ESC | iPSC | |
|---|---|---|---|---|---|---|
| Keratocyte differentiation in vitro demonstrated | Yes | Yes | Yes | Yes | Yes | Yes |
| Keratocyte differentiation in vivo demonstrated | Yes | Yes | Yes | Yes | No | No |
| Possible autologous use | Yes/No | Yes | Yes | Yes/No | No | Yes |
Corneal stromal stem cells (CSSCs) are a promising source for cellular therapy as the isolation technique and culture methods have been optimized and refined. Presumably, they should be efficient in differentiating into keratocytes, as they are already committed to the corneal lineage. However, isolating autologous CSSCs is more technically demanding, considering the small amount of tissue from which they are obtained. Furthermore, this technique still requires a contralateral healthy eye, which is not always available (such as in bilateral disease). Therefore these drawbacks may limit its use in clinical practice. Allogeneic CSSC use requires living or cadaveric donor corneal tissue.
Human adult adipose tissue is a good source of autologous extraocular stem cells and fulfils many requirements: easy accessibility to the tissue, high cell retrieval efficiency, and the ability of its adipose-derived adult mesenchymal stem cells (h-ADASCs) to differentiate into multiple cell types (keratocytes, osteoblasts, chondroblasts, myoblasts, hepatocytes, neurons, etc.). This cellular differentiation occurs because of the effect of very specific stimulating factors or environments for each cell type, avoiding the mixure of multiple cell lines within the same niche.
Bone marrow MSCs (BM-MSCs) are the most widely studied MSCs, presenting a similar profile to ADASCs, but their extraction requires a bone marrow puncture, which is a complicated and painful procedure sometimes requiring general anesthesia.
Umbilical MSCs (UMSCs) present an attractive alternative, but their autologous use is currently limited as the umbilical cord is not generally stored after birth.
Embryonic stem cells (ESCs) have great potential, but also present significant ethical issues. However, the use of induced pluripotent stem cell (iPSC) technology could solve such problems, and their capability for generating adult keratocytes has already been proven in vitro.
Finally, it is important to note that the therapeutic effect of MSCs in damaged tissue is not always related to the potential differentiation of the MSCs in the host tissue. Multiple mechanisms might contribute simultaneously to this therapeutic effect: secretion of paracrine trophic and growth factors capable of stimulating resident stem cells, reduction of tissue injury, and activation of immunomodulatory effects. As such, the direct cellular differentiation of the MSCs might not be relevant and could even be nonexistent. , ,
We will review the different types of stem cells (mesenchymal and others) that have been proposed for the regeneration of the corneal stroma, as well as the current in vitro or in vivo evidence. Finally, we will review the different surgical approaches that have been suggested (in vivo) for the application of stem cell therapy to regenerate the corneal stroma.
Translation to a New Type of Advanced Corneal Therapy
STEM CELL SOURCES USED FOR CORNEAL STROMA REGENERATION
Bone Marrow Mesenchymal Stem Cells
Park et al. reported that human BM-MSCs differentiate in vitro into keratocyte-like cells when they are grown in specific keratocyte differentiation conditions. They demonstrated the strong expression of keratocyte markers such as lumican and aldehyde dehydrogenase (ALDH) along with the loss of expression of MSC markers such as α-smooth muscle actin. However, they could not demonstrate an evident expression of keratocan in these differentiated cells. Trosan et al. showed that mice BM-MSCs cultured in corneal extracts and insulin-like growth factor-I (IGF-I) efficiently differentiate into corneal-like cells with expression of corneal-specific markers such as cytokeratin 12, keratocan, and lumican. The survival and differentiation of human BM-MSCs into keratocytes has also been demonstrated in vivo when these cells are transplanted within the corneal stroma. Keratocan expression was observed without any sign of immune or inflammatory response.
Adipose-Derived Adult Mesenchymal Stem Cells
Human ADASCs (h-ADASCs) cultured in vitro ( Fig. 36.1A,B ) under keratocyte differentiation conditions express collagens and other corneal-specific matrix components. This expression is quantitatively similar to that achieved by differentiated human CSSCs (h-CSSCs).
The differentiation of h-ADASCs into functional human keratocytes has also been demonstrated in vivo for the first time, in a previous study by our group using the rabbit as a model. These cells, once implanted intrastromally, express not only collagens type I and VI (the main components of corneal ECM), but also keratocyte-specific markers such as keratocan or ALDH, without inducing an immune or inflammatory response. These findings were later reproduced and confirmed by other authors in several research papers.
Umbilical Cord Mesenchymal Stem Cells
Human MSCs isolated from neonatal umbilical cords have exhibited similar differentiation behavior to other types of MSCs when transplanted inside the corneal stroma in vivo, expressing keratocyte-specific markers such as keratocan without inducing immune or rejection responses. Liu et al. reported that the injection of these cells within the corneal stroma of lumican null mice improved corneal transparency and increased stromal thickness with reorganized collagen lamellae and also improved host keratocyte function through enhanced expression of keratocan and ALDH in these mice. These data are encouraging, although to date, the autologous use of umbilical cord mesenchymal stem cells (UCMSCs) is not possible as the umbilical cord from new births is not generally stored.
Embryonic Stem Cells
Current experience with these human pluripotent stem cells for corneal stromal regeneration is much more limited. Chan et al. reported that differentiation of these cells into a keratocyte lineage can be induced in vitro, demonstrating upregulation of keratocyte markers including keratocan.
To the best of our knowledge, no in vivo studies with these cells have been performed in the field of regenerative medicine for the corneal stroma. The use of these cells also raises many ethical issues, and together with the lack of in vivo data, discourages their current use in a clinical setting.
Induced Pluripotent Stem Cells
As already discussed, the use of ESCs has been partially abandoned because of ethical concerns, especially since the discovery of iPSCs, which are derived from adult cells. In 2012 Shinya Yamanaka from Japan and John B. Gurdon from the UK received the Nobel Prize in Physiology or Medicine for discovering that mature, specialized cells can be reprogrammed to an immature or stem cell state and then redirected to the required cell lineage using specific factors and environmental stimuli. iPSCs promise to be the future of tissue and cellular engineering.
Regarding their application in the regeneration of the corneal stroma, human iPSCs have demonstrated the capability for differentiating into neural crest cells (the embryonic precursor to keratocytes). By culturing them on cadaveric corneal tissue, their keratocyte differentiation is promoted by the acquisition of a keratocyte-like morphology to express markers similar to corneal keratocytes. iPSC-derived MSCs have also been shown to exert immunomodulatory properties in the cornea similar to those observed with BM-MSCs. To the best of our knowledge, no studies have been published reporting the capability of iPSCs for differentiating into adult keratocytes in vivo in the animal model.
Corneal Stromal Stem Cells
The limbal palisades of Vogt form a niche that contains both limbal epithelial stem cells (LESCs) and CSSCs. CSSCs express genes typical of descendants of the neural ectoderm such as PAX6, adult stem cell markers such as ABCG2, and MSC markers such as CD73 and CD90. , They exhibit clonal growth, self-renewal properties, and a potential for differentiation into multiple distinct cell types. Unlike keratocytes, h-CSSCs undergo extensive expansion in vitro without losing their ability to adopt a keratocyte phenotype. , These corneal MSCs have a demonstrated potential for differentiation into corneal epithelium and adult keratocytes in vitro. , When cultured on a substratum of parallel aligned polymeric nanofibers, h-CSSCs produce layers of highly parallel collagen fibers with packing and fibril diameter indistinguishable from that of the human stromal lamellae. The ability of h-CSSCs to adopt a keratocyte function has been even more striking in vivo. When injected into the mouse corneal stroma, h-CSSCs express keratocyte mRNA and protein, replacing the mouse ECM with human matrix components. These injected cells remain viable for many months, apparently becoming quiescent keratocytes.
These experimental data have raised interest in this novel cell-based therapy for corneal stromal diseases; however, before its application in clinical practice, its efficacy and safety need to be well proven in human clinical trials, and other limitations such as the high laboratory costs and potential therapeutic efficacy differences among different donors have to be given serious consideration.
CORNEAL STROMA REGENERATION TECHNIQUES: EARLY APPLICATION IN CLINICAL PRACTICE
All these types of stem cells have been used in various ways in a variety of research projects to find the optimal procedure for regenerating the human corneal stroma. Corneal MSC implantation has been assayed and studied by direct intrastromal transplantation or after implantation on the ocular surface, intravenously, and into the anterior chamber where cellular migration within the stroma is to be expected. Different cellular carriers have been analyzed to enhance the potential benefits of this therapy.
Ocular Surface Implantation of Stem Cells
Surface implantation of MSCs would be the optimal approach for ocular surface reconstruction and corneal epithelium/limbal stem cell niche regeneration. However, surface implantation of MSCs would play a role in the prevention or modulation of anterior stromal scars after an ocular surface injury (such as a chemical burn). As discussed previously, MSCs secrete paracrine factors that enhance corneal reepithelialization and stromal wound healing. Thus the benefit of MSCs on the ocular surface may be more justified by these paracrine effects rather than by direct differentiation of the MSCs into epithelial cells. In this respect, Di et al. assayed subconjunctival injections of BM-MSCs in diabetic mice and reported an increased corneal epithelial cell proliferation as well as an attenuated inflammatory response mediated by tumor necrosis factor-α–stimulated gene 6 (TSG6).
Holan et al. suggested MSC application to the ocular surface using nanofiber scaffolds . They reported that BM-MSCs grown on these scaffolds can enhance reepithelialization and suppress neovascularization and local inflammatory reaction when applied to an alkali-injured eye in a rabbit model, and these results were comparable to those obtained with LESCs; both were better than the results obtained with ADASCs. The same group suggested that these results might be improved when these nanofiber scaffolds seeded with rabbit BM-MSCs are covered with cyclosporine-A (CSA)–loaded nanofiber scaffolds, observing an even greater scar suppression and healing results with the combination of both nanofibers (MSC and CSA).
Topical application of a suspension of autologous ADASCs has been reported in an isolated clinical case report in which authors describe the healing of a neurotrophic ulcer unresponsive to conventional treatment. The lack of further scientific evidence for this delivery method since 2012 raises questions about its real efficacy.
Finally, Basu et al. suggested the delivery of MSCs using fibrin glue . They resuspended CSSCs in a solution of human fibrinogen, and this was added onto a wounded ocular surface with thrombin on the wound bed. Using this method, they demonstrated the prevention of corneal scarring in the mouse model together with the generation of new collagen organization indistinguishable from that of native tissue. This group is enrolled in a clinical trial to validate these findings, using autologous and heterologous CSSCs from limbal biopsies for cases of chemical burns, neurotrophic ulcers, and established scars. Preliminary reports showed an improvement in visual parameters, corneal epithelialization, corneal neovascularization, and corneal clarity.
Intrastromal Implantation of Stem Cells Alone
Direct in vivo injection of stem cells into the corneal stroma has been assayed in several studies, demonstrating the differentiation of stem cells into adult keratocytes without signs of immune rejection. In our study, we also demonstrated the production of human ECM by immunohistochemistry when h-ADASCs were transplanted inside the rabbit cornea ( Figs. 36.1A,B and 36.2A,B ). As expected, collagen types I and VI were found expressed in the rabbit corneal stroma, as well as in the transplanted h-ADASCs. Collagen types III and IV, not normally expressed in the corneal stroma, were not detected either in the host corneal stroma or in the transplanted h-ADASCs (see Fig. 36.2C ). Du et al. reported restoration of corneal transparency and thickness in lumican null mice (thin corneas, haze, and disruption of normal stromal organization) 3 months after intrastromal transplant of h-CSSCs. They also confirmed that human keratan sulfate was deposited in the mouse stroma, and the host collagen lamellae were reorganized, concluding that delivery of h-CSSCs to the scarred human stroma may alleviate corneal scars without requiring surgery. Very similar findings were reported by Liu et al. who utilized h-UMSCs using the same animal model. Coulson-Thomas et al. found that, in a mouse model for mucopolysaccharidosis, transplanted h-UMSCs participate in extracellular glycosaminoglycans (GAG) turnover and enable host keratocytes to catabolize accumulated GAG products.
Recently, our group has published the first clinical trial in which the preliminary safety and efficacy of the cellular therapy of the human corneal stroma is reported. , In this pilot clinical trial, we implanted autologous ADASCs (obtained by elective liposuction) in a midstromal femtosecond laser-assisted lamellar pocket in patients with advanced keratoconus (see Fig. 36.1A,B ). No signs of inflammation or rejection were observed, confirming all previous evidence reported in the animal model. ,
Intrastromal Implantation of Stem Cells Together With a Biodegradable Scaffold
To enhance the growth and development of the stem cells injected into the corneal stroma, transplantation together with biodegradable synthetic ECM has been performed. Espandar et al. injected h-ADASCs with a semisolid hyaluronic acid hydrogel into the rabbit corneal stroma and reported better survival and keratocyte differentiation of the h-ADASCs when compared with their injection alone ( Fig. 36.3A,B ). Ma et al. used rabbit ADSCs with a polylactic-co-glycolic (PLGA) biodegradable scaffold in a rabbit model of stromal injury in which they observed newly formed tissue with successful collagen remodeling and less stromal scarring (see Fig. 36.3A–C ). At 3 months post implantation, a high extrusion rate of the implant was observed (see Fig. 36.3D,E ). Initial data show that these scaffolds may enhance stem cell effects on corneal stroma, although further research is required and warranted.
Intrastromal Implantation of Stem Cells With a Decellularized Corneal Stroma Scaffold
The complex structure of the corneal stroma has still not been replicated and there are well-known drawbacks to the use of synthetic scaffold-based designs: (1) strong inflammatory responses induced with biodegradation and (2) nonspecific inflammatory response induced by all polymer materials.
Recently, several corneal decellularization techniques have been described, which provide an acellular corneal ECM (see Fig. 36.1C ). These scaffolds have gained attention in the last few years, as they provide a more natural environment for the growth and differentiation of cells when compared with synthetic scaffolds. In addition, components of the ECM are generally conserved among species and are well tolerated by xenogeneic recipients. Moreover, keratocytes are essential for remodeling the corneal stroma and for normal epithelial physiology. This highlights the importance of transplanting a cellular substitute together with the structural support (acellular ECM) to undertake these critical functions in corneal homeostasis. To the best of our knowledge, all attempts to repopulate decellularized corneal scaffolds have used corneal cells. However, as already discussed, these cells have significant drawbacks that limit their autologous use in clinical practice (damage to the donor tissue, lack of cells, and more difficulty generating cell subcultures), thus redirecting efforts to find an extraocular source of autologous cells. In a previous study by our group, we showed the perfect biointegration of human decellularized corneal stromal sheets (100-μm thickness) with and without h-ADASC colonization inside the rabbit cornea (see Fig. 36.2D–F ) and observed no rejection response despite the graft being xenogeneic. We also demonstrated the differentiation of h-ADASCs into functional keratocytes inside these implants in vivo, which then achieved their proper biofunctionalization (see Fig. 36.2D,E ). In our experience, decellularization of the whole (∼500 µm) corneal stroma (using sodium dodecyl sulfate anionic detergent) lacks efficacy, as it not possible to completely remove the whole cellular component. However, we demonstrated that this method completely removes the cellular component and preserves the tissue integrity of the corneal stroma when thinner lenticules are treated—a method that has been later confirmed by other authors with the use of electron microscopy. , , Others have also assayed the integration of decellularized pig articular cartilage ECM colonized with mice BM-MSCs in the rabbit corneal stroma and reported similar findings, although the transparency of these decellularized scaffolds was not clearly reported.
In our opinion, the implantation of MSCs together with decellularized corneal ECM would be the best technique to restore effectively the thickness of a diseased and severely weakened human cornea, because the implantation of MSCs alone only achieves limited new ECM formation and thickness restoration. , Moreover, with this technique, and by using autologous MSCs from a given patient, it is theoretically possible to transform allogenic grafts into functional autologous grafts, thus avoiding any risk of rejection. Following this research line, we have recently published the first clinical trial using these decellularized human corneal stroma scaffolds (120-µm thickness and 9.0-mm diameter laminas), with or without autologous ADASC recellularization, in patients with advanced keratoconus (see Fig. 36.1C,D ). ,
Decellularized tissues have the drawback of requiring specific laboratory equipment, although eye banks could potentially do this and deliver such grafts to different clinical centers. Keratophakia (intrastromal insertion of an allogeneic lenticule) was originally described by Barraquer in 1964 but was abandoned because of the unpredictability of the refractive outcome and the relatively high frequency of interface haze development. The lack of haze observed in our pilot clinical trial could be due to the absence of donor keratocytes that could potentially activate postoperatively and generate scar tissue. Moreover, rejection episodes have already been described after the implantation of allogeneic lenticules, a risk that is theoretically avoided by the use of decellularized grafts. It is reasonable to consider that as long as human decellularized tissue is used, there will be no risk for zoonotic diseases.
Anterior Chamber Injection of Stem Cells
Demirayak et al. reported that BM-MSCs and ADASCs, suspended in phosphate-buffered solution (PBS) and injected into the anterior chamber after a penetrating corneal injury in a mouse model, are able to colonize the corneal stroma and increase the expression of keratocyte-specific markers such as keratocan, with a demonstrated increase in keratocyte density by confocal microscopy. Conversely, the possible side effects of this MSC injection into the anterior chamber for the lens epithelium and trabecular meshwork are highly questionable, as it may induce scarring and a subsequent glaucoma. Considering this, the potential clinical use of this approach, in our opinion, is limited.
Intravenous Injection of Stem Cells
Systemic use of MSCs by intravenous injection has also been tested. Intravenous injection of BM-MSCs in mice after an allograft corneal transplant led to colonization of the transplanted cornea and conjunctiva but not the contralateral ungrafted cornea, simultaneously decreasing immunity and significantly improving allograft survival rate. Yun et al. recently reported similar findings with the intravenous injection of iPSC-derived MSCs and BM-MSCs after a surface chemical injury, where they observed that the corneal opacity, inflammatory infiltration, and inflammatory markers in the cornea were markedly decreased in the treated mice, without significant differences between both MSC types. In contrast, our group did not observe any benefit in corneal allograft survival and rejection rates after systemic injection of rabbit ADASCs prior to surgery, during surgery, and at various times after surgery in rabbits with vascularized corneas (model more similar to human corneal transplants than those reported in mice). A shorter graft survival compared with the nontreated corneal grafts was noted.
AUTOLOGOUS VERSUS ALLOGENIC MSC
A critical question for future clinical trials to further assess the feasibility of cellular therapy of the corneal stroma is whether the use of autologous MSCs is necessary and whether allogenic MSCs could achieve the same benefit without any risk of inflammation or rejection. If we consider all published evidence in the animal model in which human MSCs were implanted in the corneal stroma, despite being a xenogeneic transplant, no signs of rejection or inflammation have been reported. This coincides with the strong evidence on the immunomodulatory and immunosuppressive properties of MSCs, which help them to evade host immune rejection and to survive by inhibiting adhesion and invasion, and which induce cell death of inflammatory cells, partially because of a rich extracellular glycocalyx that contains TSG6. , TSG6 plays a critical role in the immunosuppressive properties exhibited by MSCs. , , Taken together, the use of allogenic MSCs would greatly simplify the clinical application of MSCs, as clinical application centers would not need any specific equipment and potential MSC banks could store and supply stem cells for use in patients. Low-cost systems are already available that are capable of enhancing the preservation of MSCs at hypothermic temperatures while maintaining their normal function, thereby widening the timeframe for distribution between the manufacturing site and the clinic and reducing the waste associated with the limited shelf life of cells stored in their liquid state. Funderburgh et al. recently reported that MSCs from different donors may have different immunosuppressive properties and, consequently, different abilities to regenerate and relieve stromal scars. Considering this important finding, the best donors could be selected by MSC banks to expand and supply only those MSCs with the highest immunosuppressive and regenerative capacity; if so, autologous cells would not be necessary. We should also consider that adult keratocytes obtained from autologous MSCs may carry the same genetic defect that led to the corneal disease, such as in the case of corneal dystrophy. In this scenario, the use of allogenic instead of autologous MSCs would be interesting. A recent study observed gene expression differences between the iPSC-derived keratocytes generated from fibroblasts of both keratoconic and normal human corneal stroma, influencing cellular growth and proliferation, confirming that, at least in keratoconus cases, adult cells obtained from MSCs may still not be functionally normal.
MESENCHYMAL STEM CELL EXOSOMES
Exosomes are nanosized extracellular vesicles that originate from the fusion of intracellular multivesicular bodies with cell membranes and are released into extracellular spaces. They have been implicated in the ability of MSCs to repair damaged tissue. Shojaati et al. recently showed that exosomes isolated from the culture media of h-CSSCs had similar immunosuppressive properties and also significantly reduced stromal scarring in wounded corneas in vivo. This finding suggests that for some diseases, such as prevention or reduction of corneal scars, MSC exosomes may provide a non–cell-based therapy. Zhang et al. suggested that exosomes released by transplanted UCMSCs within the diseased cornea are able to enter into the diseased host corneal keratocytes and enhance their biological functions.. The authors experimented in vitro using mucopolysaccharidosis VII mice and discovered that UCMSC-secreted exosomes assisted in the recycling process of accumulated GAGs in the lysosomes of diseased cells. These findings open an exciting new field for research as the use of exosomes may overcome some of the limitations and risks associated with intrastromal cellular injection, given that exosomes can potentially be applied topically.
First Clinical Human Experience in Advanced Keratoconus Cases
Recently, our group performed the implantation of ADASCs and decellularized/recellularized laminas in 14 patients with advanced keratoconus. This clinical experience opened a new and exciting line of therapy for research. As mentioned, the production of new ECM by the implanted MSCs occurs but is not quantitatively enough to be able to restore the thickness of a severely diseased human cornea (as in extreme keratoconic corneas). Meanwhile the implantation of decellularized/recellularized laminae could restore the corneal thickness and the keratometric parameters. However, the direct injection of stem cells may provide a promising treatment modality for corneal dystrophies and corneal stroma progressive opacification in the context of systemic metabolic disorders, and for the modulation of corneal scarring.
STUDY APPROVAL, DESIGN, AND SUBJECTS
This investigation was a prospective series of consecutive cases. The study was conducted in strict adherence to the tenets of the Declaration of Helsinki and was registered in ClinicalTrials.gov (Code: NCT02932852).
Fourteen patients were enrolled in the study, were operated within an interval of 3 months, and were randomly distributed into three study groups: group 1 (G-1) patients were treated with autologous ADASC implantation ( n = 5); group 2 (G-2) received decellularized human corneal stroma transplantation ( n = 5), and group 3 (G-3) received autologous ADASC recellularized human corneal stroma transplantation ( n = 4).
Thirteen patients were included in the clinical follow-up. One patient from G-1 was lost after the first postoperative month because of inability to attend further follow-up for reasons unrelated to the study.
Inclusion and exclusion criteria were defined in previous articles. , , , , Clinical monitoring of the study of the patients was established for safety purposes at 1 week, and 1, 3, 6, 12, and 36 months for the purpose of the clinical outcomes of the investigation and to observe implant safety for a long time.
METHODOLOGY
Autologous ADASC Isolation, Characterization, and Culture
Patients underwent standard liposuction. Approximately 250 mL of mixed fat was obtained from each patient using local anesthesia. The adipose tissue was processed according to the methods described in the previous articles (see Fig. 36.1A,B ). ,
Laminas
Human corneal stroma of donor corneas with nonviable endothelium but with negative viral serology was used. The corneas were provided by the eye bank. The quality and safety standards for donation, procurement, testing, processing, conservation, storage, and testing of human cells and tissues were followed. Donor corneas were dissected with IntraLase iFS femtosecond laser (AMO, Santa Ana, CA), two to three consecutive laminas 120-µm thick and 9.0 mm in diameter were obtained. The decellularization protocol was based on previous publications (see Fig. 36.1 C). , , Twenty-four hours before implantation, the laminas for patients who received recellularized tissue were placed in tissue culture wells for recellularization with autologous ADASC (0.5 × 10 6 cells per 1 mL of PBS were cultured on each side of the laminas). Then the laminas were submerged in PBS at room temperature and transferred to implantation (see Fig. 36.1D ). , ,
Surgical Procedure: Autologous ADASC Implantation
The method for the implantation of the MSCs has been described previously. Topical anesthesia was used. A 60-kHz IntraLase iFS femtosecond laser (AMO Inc, Irvine, CA) was used in single-pass mode for the recipient corneal lamellar dissection. An intrastromal laminar cut of 9.5-mm diameter was created at medium depth of the thinnest preoperative pachymetry point measured by the Visante OCT (Carl Zeiss, Jena, Germany). Three million autologous ADASCs contained in 1 mL PBS were injected into the pocket ( Fig. 36.4A,B ).
