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Review Article

Corneal Stroma Regeneration: New Approach for the Treatment of Cornea Disease

El Zarif, M OD, MSc∗,‡,§,¶; Alió del Barrio, JL MD, PhD, FEBOS-CR†,‡; Arnalich-Montiel, Francisco MD, PhD, FEBOS-CR†,||; De Miguel, María P. PhD∗∗; Makdissy, Nehman PhD§; Alió, Jorge L. MD, PhD, FEBOphth†,‡

Author Information
Asia-Pacific Journal of Ophthalmology: November-December 2020 - Volume 9 - Issue 6 - p 571-579
doi: 10.1097/APO.0000000000000337
  • Open


Human corneal transplantation with donor corneas has been the only option for treating corneal blindness for decades. Despite recent improvements in surgical techniques, donor corneal transplantation continues to be plagued by risks of suboptimal optical outcomes, visual loss, immune rejection, and the consequent graft failure. The demand for adequate donor corneas is increasing faster than the number of donors, leaving thousands of patients around the world waiting for possible treatment.1–3

Due to these facts, the development of alternative approaches such as corneal bioengineering to obtain corneal substitutes that could have similar characteristics to the human donor cornea becomes important today. Efforts have been made to replicate the corneal stroma in the laboratory to find an alternative to classical corneal transplantation, but due to the highly complex ultrastructure of the corneal stroma, the laboratory construction of the corneal stroma with enough transparency or strength for clinical use has not yet been achieved.4–6

Synthetic scaffold-based designs have raised some concerns about their potential to induce inflammatory responses caused by the involved biomaterials.7 As an alternative, in the last decade the acellular corneal stroma has been proposed as a scaffold and several corneal decellularization techniques have been described, which provide an acellular corneal extracellular matrix (ECM).8 These scaffolds have gained increasing interest as they provide an ideal natural environment for the growth and differentiation of cells (either transplanted donor cells or migrating host cells).7 Besides this, the removal of all immunogenic cellular component could open the field of xenotransplantation by using donor tissue from other animals such as the pig, that shares important similarities with the human cornea.9

In recent years, corneal stromal cell therapy has attracted much interest. Looking for extraocular sources of mesenchymal stem cells (MSCs), studies have shown that MSCs are capable of surviving and differentiating into adult human keratocytes in vitro and in vivo,10,11 even in xenogeneic scenarios in vivo, they do not induce any inflammatory reaction.7,12 MSCs have also shown immunomodulatory properties in syngeneic, allogenic, and even xenogeneic scenarios.13 Bone marrow MSC, umbilical cord MSC, and corneal stromal stem cells (CSSCs) form an alternative source to differentiate MSCs.11,14,15 Adipose-derived adult stem cells (ADASCs) form a wide source of MSCs (Fig. 1) and satisfy many requirements for their application to human corneal therapy.11,12 Embryonic stem cells have great potential to differentiate MSCs, but their use also involves important ethical issues. The use of induced pluripotent stem cell technology has opened a new and very promising field for future research; it has shown to exert immunomodulatory properties in the cornea similar to those observed with MSC,16 since theoretically they are cells with the ability to generate adult keratocytes in vitro.17

Microscopic appearance (phase-contrast photograph) of human adipose-derived adult stem cell in culture (×100 magnification).

Pioneering human clinical studies demonstrated published evidence that human ADASCs form an abundant extraocular source, are capable of surviving and differentiating in vivo into adult human keratocytes, and have demonstrated their ability to produce new collagen within the host stroma,18 as it was previously demonstrated in experimental animal models.12,19

This report aims to offer an integrated overview, based on evidence, about the current stage of human studies on the subject of regenerative advanced corneal stromal therapies in the treatment of corneal disease and, particularly, in keratoconus.


Collagen-Based Scaffolds

The arrangement of corneal collagen is the most difficult structure to reproduce either by cellular remodeling or by direct manufacturing in the laboratory. Many physical/engineering methods have been devised to attempt to control the organization of collagen, but none of them have been able to duplicate native corneal tissue structure. In general, they can neither match the mechanical properties nor recreate the local nanoscale organization. Although non–collagen-based materials (such as silk fibroin, gelatin hydrogels, chitosan, or synthetic polymers) seek to achieve higher strength than collagen-based scaffolds,11 at the same time they lack the theoretical higher biocompatibility that the collagen has, as these synthetic materials are not present in the normal cornea. For this reason, collagen-based scaffolds have gained more attention due to their excellent biocompatibility with the recipient corneal stroma, and because they provide an optimal scaffold to support cellular growth (either from donor implanted or host migrated cells).

Currently, the most promising approach that is reaching clinical practice is the use of decellularized corneal stroma. Multiple decellularized corneal sections can be obtained from a single donor cornea, and even xenogeneic donors (such as pigs) have been suggested for human transplantation through the application of these decellularization methods.7,9 Decellularized human corneal sections (Fig. 2A, B) that were recellularized with human ADASC have been assayed in experimental animals (Fig. 2C)7,20 and most importantly, in patients suffering from corneal stromal debilitating diseases.18,21–23 Survival of the transplanted cells with differentiation into corneal keratocytes and complete integration of the implant with naturally mimicking strength and total transparency with no rejection episodes was achieved in both preclinical and clinical studies (Fig. 2). Nevertheless, this approach still requires donor tissue and does not supply an unlimited source of cornea prostheses for their use in clinical patients.

Decellularized human corneal stroma lamina implantation into a rabbit's corneal stroma in vivo. Hematoxylin-eosin staining showing decellularized (A and B) and human adipose-derived adult stem cell recellularized (C) lamina perfectly integrated into the rabbit's corneal stroma 3 months after implantation.

In addition to the above-discussed decellularized corneal laminas (which represent naturally aligned collagen bundles), other methods have been proposed to mimic the corneal stroma structure, such as collagen vitrification.24 In this regard, a phase 1 clinical trial with vitrified collagen-based scaffolds grafted onto the anterior stroma was performed and demonstrated a suboptimal performance when compared with regular corneal donor tissue.2 Collagen 3D bioprinting25 has accomplished outcomes showing material with optical and chemical properties similar to human cornea but weaker tensile strength,26 among other limitations. Also, allogenic small incision lenticule extraction (SMILE) lenticule implantation has been assayed in both animals and humans, demonstrating to be a feasible approach, which can offer also possibilities for the correction of refractive errors and the improvement of corneal thickness, with potential applications in debilitating diseases such as corneal ectasias.27–29

Stem Cell Therapy Without Scaffold

Corneal MSC implantation has been assayed by different approaches: direct intrastromal transplantation, implantation at the ocular surface, intravenous, or at the anterior chamber, where cellular migration toward the inner stroma is expected.11 This cellular implantation without a carrier aims to remodel or generate new ECM within the corneal stroma.

Direct in vivo injection of stem cells inside the corneal stroma has been assayed in some studies, demonstrating the differentiation of the stem cells into adult keratocytes without evidence of immune rejection. The differentiation of human ADASC in functional human keratocytes (Fig. 3) was demonstrated in vivo using the rabbit as a model,12 and such cells, once implanted in the corneal stroma, express not only collagens type I and VI (the main components of corneal ECM), but also keratocyte-specific markers such as keratocan or aldehyde dehydrogenase, without inducing an immune or inflammatory response.30 Du et al31 reported restoration of corneal transparency and thickness in lumican-null mice 3 months after intrastromal transplant of human CSSC. They also confirmed that human keratan sulfate was deposited in the mouse stroma and the host collagen lamellae were reorganized, concluding that delivery of human CSSCs to the scarred human stroma may alleviate corneal scars without requiring invasive surgery.31 Very similar findings were reported by Liu et al who utilized human umbilical cord MSCs using the same animal model.32 Coulson-Thomas et al found that in a mouse model for mucopolysaccharidosis, transplanted human umbilical cord MSCs participate both in extracellular glycosaminoglycans turnover and enable host keratocytes to catabolize accumulated glycosaminoglycan products.33

Confocal microscopy image of keratocan production by human adipose-derived adult stem cell in culture. Cellular nuclei appear with blue fluorescence due to 4′,6-diamidino-2-phenylindole staining.

The first clinical trial in clinical cases of keratoconus with implantation of autologous ADASCs decellularized and/or recellularized corneal stromal laminas, and confirmed preliminarily the safety and efficacy of the cellular therapy of the human corneal stroma.18,22,23,34 According to the clinical and preclinical available evidence, the direct intrastromal implantation of MSC within the cornea achieves the production of new ECM but is not expected to be quantitatively enough to be able to restore the thickness of a severely diseased human cornea, like in advanced keratoconus cases. However, the direct injection of stem cells may provide a promising treatment modality for the early treatment of corneal dystrophies, including keratoconus, in cases of corneal stroma progressive opacification in the context of systemic metabolic disorders, and for the modulation of corneal scars.

MSC Exosomes

It is important to highlight that the therapeutic effect of stem cells in a damaged tissue is not always directly related to the potential differentiation of the stem cells since multiple mechanisms might simultaneously contribute to this therapeutic action, for example, the secretion of paracrine growth factors capable of stimulating the host tissue (in which case the direct cellular differentiation of the stem cells might not be relevant and could even be nonexistent).30,35,36 It has been demonstrated that MSCs secrete paracrine factors such as vascular endothelial growth factor, platelet-derived growth factor, hepatocyte growth factor, among others13 that seem to promote cell migration and keratocyte survival by apoptosis inhibition, and upregulate the expression of ECM component genes in keratocytes, subsequently enhancing corneal re-epithelialization and stromal wound healing.37 Because of this, it has been suggested that the direct treatment of the corneal stroma with the MSC exosomes containing these growth factors could potentially achieve the same benefits of the cellular therapy, but without providing the cellular component itself.38 Shojaati et al showed that exosomes isolated from the culture media of human CSSCs offer similar immunosuppressive properties and significantly reduced stromal scarring in wounded corneas in vivo.39


Femtosecond Laser–Assisted Refractive Stromal Lenticule Addition

Donor stomal lenticules can be obtained from corneo-scleral eye bank buttons using a refractive lenticule extraction procedure with a 500-kHz VisuMax femtosecond laser (Carl Zeiss Meditec, Jena, Germany) and cryopreserved or stored in organ culture to be subsequently used for implantation. The implantation of these stromal lenticules has been proposed as a way to reverse the effect of previous laser refractive surgery,40,41 to treat different types of ametropia,42 and as a therapy for cornea ectatic disorders such as keratoconus, where progressive stromal thinning causes irregular astigmatism and refractive instability.

The myopic correction algorithm for SMILE produces positive meniscus lenticules, thicker in the center and gradually becoming thinner toward the periphery. The implantation of these lenticules induces an increase in anterior corneal curvature and has been used to correct hyperopia in humans. An inverted thickness profile that gradually becomes thicker from the center toward the periphery can also be produced using a hyperopic algorithm for SMILE. The implantation of these negative meniscus-shaped lenticules in an ex vivo study on human corneas causes a reproducible flattening of the central cornea and an increase of the stromal thickness, both desirable effects of any procedure for treating keratoconus eyes.43

Mastropasqua et al44 were able to replicate these results in 10 advanced keratoconus eyes. They coined this procedure “stromal lenticule addition keratoplasty” as a feasible and effective technique for stromal remodeling in advanced central keratoconus. The lenticules programmed for 8.00 diopter (D) of hyperopic correction with a 6-mm optical zone was introduced in a femtosecond intrastromal pocket at 120 μm from the corneal surface. No intraoperative or relevant postoperative complications were reported, and 9 of 10 showed improvement in corrected distance visual acuity 6 months after implantation, ranging from 1 to 3 lines. Mean central keratometry significantly decreased by a mean of 5 D, corneal thickness increased by 50 μm, and there was a significant improvement of corneal asphericity. The lenticule-host inferfaces could be identified using in vivo confocal microscopy, but keratocyte morphology appeared normal and extracellular tissue presented normal transparency.44

Using a different approach, Pradhan et al45 proposed femtosecond laser–assisted small incision sutureless intrastromal lamellar keratoplasty as an alternative to corneal transplantation in keratoconus and presented the 1-year follow-up result in a single patient. As in the stromal lenticule addition keratoplasty procedure, sutureless intrastromal lamellar keratoplasty showed an improvement of the uncorrected and best-corrected visual acuity and a reduction of 7 D in the maximum keratometry with a considerable thicker lenticule (up to 332 μm in the central cornea).

Stem Cell Therapy of the Corneal Stroma for Advanced Keratoconus

Surgical Procedure

The results of the implantation of autologous ADASCs in the corneas of 5 patients from group 1 (G1) with advanced keratoconus have been reported.12,18,46,47 From simple liposuction performed with local anesthesia, 250 mL of human autologous adipose tissue was obtained, followed by the identification, isolation, characterization, and culture of adipose tissue mesenchymal-derived stem cells. A femtosecond laser was used in single-pass mode to dissect the corneas in the mid-stroma of 5 eyes with advanced keratoconus. Each of these eyes received an injection of 3 × 106 cells of autologous ADASCs in 1 mL of the phosphate-buffered solution into the stromal pocket, using a 25-G cannula. The surgical procedure and its outcomes were described in a previous publication.18

Five patients’ eyes of group 2 (G2) with advanced keratoconus received 120 μm decellularized human corneal stroma laminas only, and another 4 eyes from group 3 (G3) received the implantation of 120 μm human corneal stroma recellularized with autologous ADASCs (1 × 106cells/1 mL of phosphate-buffered solution, half million on each side).23,34 The corneal laminas were obtained from human corneas that were not suitable for corneal transplantation. The corneas of patients from G2 and G3 were dissected by femtosecond laser in the mid-stroma of the thinnest point measured by anterior segment optical coherence tomography. The protocol of decellularization and the surgical procedures were described in previous publications.7,23,34

The confocal microscope, the Rostock Cornea Module of the Heidelberg Retina Tomograph 3, was used to observe the evolution and the morphological change of implanted ADASCs and the decellularized/ADASC recellularized human corneal stromal laminas.48,49


No complications such as haze or infection were observed during the 3-year follow-up. Full corneal transparency was recovered within the first postoperative day in all patients of the G1 (Fig. 4A). Meanwhile, in G2 and G3, the implanted laminas showed mild early haziness during the first postoperative month. Corneal recovery and full transparency were observed within the third postoperative month in all patients (Fig. 4B, C). All patients were followed at 1 day, 1 week, and at 1, 3, 6, 12, and 36 months after operation.34

Biomicroscopic findings at 36 months after operation. A, Group 1, case 2 shows the transparency of the cornea. B, Group 2, case 9 notices the transparency of the implanted decellularized lamina. Red arrows present the periphery of the lamina. C, Group 2, case 7 shows some scattered, faint, patchy “islands” of haze (yellow arrows). These paracentral areas of haze did not have any impact on the visual outcomes. D, Group 3, case 10 indicates the periphery of the recellularized lamina (red arrows). Notice the transparency of the implanted tissue.

All cases improved at 36 months: 1 to 2 lines in LogMAR scale in their unaided distance visual acuity, corrected distance visual acuity, and rigid contact lens distance visual acuity in comparison with the preoperative values.34

A significant improvement in the refractive sphere was observed at 36 months. However, refractive cylinder presented minor changes in the 3 groups.

Results of anterior segment optical coherence tomography: central corneal thickness (Fig. 5), thinnest point measured by Pentacam Scheimpflug corneal topography (Oculus Inc., Wetzlar, Germany) (Fig. 6), and cornea volume showed a significant increase in all groups. The mean value results were better in G2 and G3 compared with G1.34 Also, significant improvement was obtained in mean values in third-order aberration root mean square and high-order aberration root mean square, where the results were better when comparing G2 and G3 with G1.34

Corneal AS-OCT sections and pachymetry maps (Visante) in group 1, group 2, and group 3. A, Group 1, case 1, OCT at 12 months. Notice the perfect transparency of the cornea (left). Observe the pachymetry map (right). B, Group 2, case 7, OCT at 36 months. Observe the integration of the implanted decellularized lamina in the host stoma and the improvement in corneal density (left). The pachymetry map (right) shows the enhancement in the thickness of the cornea. C, Group 3, case 12, OCT at 36 months. The enhancement in the integration of the implanted lamina in the host stroma (red arrows), and an improvement in corneal density can be noticed. Yellow arrow indicates the border of the lamina. Observe the enhancement in the pachymetry map (right). AS-OCT indicates anterior segment optical coherence tomography; OCT, optical coherence tomography.
Corneal topography changes (Pentacam) among preoperation, 12 months, and 36 months in groups 1, 2, and 3. A, Comparative pachymetric examinations (right) among preoperation (middle) and 12 months (left) in group 1, case 4. Observe the modest enhancement in the pachymetric parameters in the inferior part of the cornea. B, Comparative pachymetric examinations (right) among preoperation (middle) and 12 months (left) in group 3, case 12. Observe the improvement of the corneal thickness. C, Comparative pachymetric examinations (right) among preoperation (middle) and 36 months (left) in group 2, case 6. Observe the improvement of the corneal thickness. D, Corneal topography (Pentacam) comparison (right) between preoperation (middle) and nearly 3 years (left) in group 2, case 5. The enhancement of the keratometric parameters can be seen.

Moreover, an improvement of 2 D in the mean values with the results of the anterior mean keratometry was obtained (Fig. 6). In maximum keratometry, a flattening of 3 D was obtained at 36 months. More results were recorded in previous publications.15,23,34

Confocal microscopy showed a morphological change with G1, where ADASCs appeared round in shape, more voluminous, and refringent up to 6 months regarding host keratocytes (Fig. 7A). Then, implanted cells changed from round to a fusiform shape similar to normal keratocytes (Fig. 7B). The cellular density showed a gradual statistically significant increase at the anterior, mid, and posterior host stroma when compared with preoperative values. In G2, decellularized laminas appeared acellular during the first month (Fig. 7C). Unlike the recellularized ones in G3, cell structures similar to corneal keratocytes were observed within the laminas (Fig. 7D). The cell density increased during the 12 months’ follow-up until reaching a statistically significant increase in the anterior, mid, and posterior surfaces of the decellularized and recellularized laminas (Fig. 7E–G), and in the anterior and posterior host stroma with all the cases (Fig. 7H).

Confocal microscopy findings up to 12 months. A, Group 1, case 1, count of keratocytes and adipose-derived adult stem cells (ADASCs) (red arrow) in the surgical plane at 3 months after surgery. ADASCs appear rounded, larger, and more refringent than the normal keratocytes. B, Group 1, case 2, 1 year, all corneal stromal cells in the surgical plane assimilate a similar shape (yellow arrows). C, Group 2, case 7, anterior surface of a decellularized lamina appears without cells at 1 month. Observe the presence of reflective dots’ structures (blue arrows). D, Group 3, case 13, anterior surface of a recellularized lamina at 1 month. Few ADASCs can be seen (marked in blue). E, Group 2, case 9, posterior surface of the decellularized lamina at 1 year. The cells assimilate a morphology identical to that of normal corneal stromal keratocytes (yellow arrows). F, Group 3, case 10, posterior surface of a recellularized lamina at 3 months. Observe ADASCs round in shape more luminous and refringent (green arrows). G, Group 3, case 13, anterior surface of a recellularized lamina at 12 months. High number of stromal cells can be seen. H, Anterior corneal stroma (up) in group 2, case 9, and posterior corneal stroma (down) in group 3, case 13, with abundant corneal stromal cells at 12 months.


The ideal corneal stroma constructed by bioengineering should be capable of mimicking the natural human cornea and maintaining the corneal stroma homeostasis. For these purposes, it has to contain both a cell source and a collagen scaffold.4–6 For the cellular component, several cell types from both ocular and extraocular sources have been investigated, being the human MSC the most widely tested so far. However, further development of cheaper and standardized induced pluripotent stem cell techniques may surpass MSC use.16 The use of decellularized cornea sections could be the most clinically relevant.7,8,20 However, it is still handicapped by the need for donor corneal tissue. The use of MSC exosomes (without their cellular component) reveals an exciting field of research as their use could overcome some of the limitations and risks associated with the direct delivery of stem cells to humans in vivo if exosomes could be applied topically.38

The popularization of SMILE as a refractive surgery option is providing a large amount of donor corneal tissue that could potentially be used for human transplantation. Liu et al and Zhao et al27,28 demonstrated after 2 years’ follow-up that small incision allogenic intrastromal lenticule implantation is feasible and safe for reshaping the cornea. The corneal healing remained stable and their results were confirmed by slit-lamp biomicroscopy and by confocal microscopy in vivo and corneal densitometry measurements. Additionally, the authors observed corneal reinnervation signs within the lenticule. The stromal addition of SMILE created lenticules that were proven ex vivo and in vivo (its feasibility, safety, and efficacy in the treatment of thinning disorders such as keratoconus).43–45 This procedure increases corneal thickness providing additional strength to the weakened cornea and anterior corneal flattening when using a negative meniscus-shaped lenticule.44 The transplantation of negative meniscus lenticules induced more flattening than the one observed with planar lenticules. However, the latter can be implanted in eccentric cones, whereas the former only in central cones (a much less common situation in keratoconus patients).34,44 The feasibility and safety of corneal stromal cell therapy for advanced keratoconic corneas were demonstrated for the first time. The appearance of new collagen formation within the surgical plane was also observed when MSCs were implanted. Although it was not enough to restore the thickness of the diseased corneas, this neo-collagen may play a role in the stromal remodeling of corneal dystrophies and corneal scars to enhance corneal transparency (Figs. 5A, 6A).18 The implantation of decellularized or recellularized human corneal stroma laminas effectively restores corneal thickness, and it potentially avoids any risk of graft rejection (while this risk is still present by the use of allogenic stromal implants). The increase in corneal thickness is lower when negative meniscus laminas are used compared with planar lenticules as they are thinner in the center.44 However, they achieve a larger flattening effect in central cones. It has also to be considered that negative meniscus-shaped lenticules may generate unacceptable aberrations within the visual axis in eccentric cones. Therefore, they might not be indicated in many cases of keratoconus.34,44

Synthetic collagen scaffolds are, by definition, a promising source of donor stromal tissue as they do not need human donor tissue and can be potentially available all over the world. However, their difficulties that are still present today, which are to mimic the transparency and strength of the human cornea, together with the high expenses for their production in the laboratory, preclude their presence in the real clinical practice for the next few years.


Cellular therapy with implantation of autologous ADASCs decellularized human corneal stroma, and allogenic SMILE lenticule corneal inlays have been shown to be a potentially effective therapy for keratoconus. Such promising findings open a new perspective in therapy for the corneal stroma based on corneal stromal regeneration and enhancement of corneal thickness, and topographic and visual parameters. Future studies are needed to expand on the potential application of these new therapies for the treatment of corneal stromal diseases.


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adipose-derived adult stem cells; cornea surgery; corneal bioengineering; corneal stem cell therapy; corneal transplant; keratoconus; stem cells

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