Eye aesthetics and regenerative medicine

Contribution of autologous platelet gel (RegenWound®) in the treatment of severe corneal ulcers: first 10 cases.

Prof. Merieme HAROUCH (1) *, MD. Safaa JIHAD, Younes ALLOU (2).
(1) Casablanca Laser Vision Center, Casablanca Cornea-Keratoconus Center
(2) Medical Biotechnology Laboratory, Faculty of Medicine and Pharmacy, Rabat - Mohammed V. * Contact: m.harouch@lasik.ma


Severe corneal ulcers remain a challenge for ophthalmologists worldwide, due to the associated risk of corneal blindness or eye perforation.
In this series of preliminary cases, we have tried to study the use of the autologous platelet-rich plasma combined with autologous thrombin serumas an autologous platelet gel, to stimulate the healing process in a physiological way.
In this study, ten patients with corneal ulcers of different origins were treated with autologous platelet gel and we investigated the healing process and visual acuity.
These preliminary results suggest that the use of autologous platelet gel appears to be a promising therapeutic option for treating severe corneal ulcers.


Chronic corneal ulcers refractory to standard treatments remain a challenge for ophthalmologists worldwide, due to the associated risk of corneal blindness or eye perforation.
New medical and surgical approaches have been developed in recent years, including blood-derived products such as platelet-rich plasma (PRP).
PRP is now widely used in many areas of regenerative medicine to promote healing.
Its non-transfusion use was first reported in the 1980s in maxillofacial surgery, and has since been extended to fields such as aesthetic medicine, plastic surgery, ophthalmology, orthopedic surgery and cardiovascular surgery [1], as well as dental implant surgery, muscle and/or tendon repair, and chronic wound healing in plastic and reconstructive surgery [4-5]. [4-5]
The rationale behind using PRP for therapeutic applications is to mimic the biological healing process that normally occurs in the human body after injury [21].
Preparing PRP involves removing red and white blood cells, which delay healing, and concentrating platelets, thereby increasing the factors useful for healing [22].
As there are numerous PRP preparation protocols, differing in preparation devices, centrifugation conditions and operator dexterity, PRP is used to qualify biological products that vary widely in platelet concentration, quality and growth factor content, and level of contamination by pro-inflammatory red and white blood cells [23].
This wide variability in PRP preparations poses a problem when it comes to drawing accurate conclusions from the literature to guide PRP production and determine indications for use.
This has led to the development of PRP classification systems to facilitate clinical study reporting [24].
To meet the need for standardized PRP preparation, RegenLab has developed the polymer-gel separation system (Fig.4), used in this study, which efficiently recovers platelets and removes red and white blood cells in an automated closed-loop system.
In ophthalmology, PRP eye drops are effective in many indications, such as dry eye (DED) or postoperative dormant corneal ulcers [13].
However, for non-healing corneal ulcers, treatment options are limited and surgery has, until now, often been the only option, or techniques such as amniotic membrane grafting or conjunctival flap.
We describe here a new alternative, based on the use of PRP and autologous thrombin serum (ATS) to obtain an autologous platelet gel that will stimulate the healing process in a physiological way.
When liquid PRP is injected into tissues, it coagulates thanks to the supply of calcium ions by the interstitial fluid.
Nevertheless, for certain therapeutic applications, such as wound and ulcer treatment, healthcare providers need to obtain PRP gels or clots.
Coagulation of citrated PRP can be induced by activators such as thrombin, calcium solution or a combination of both.
There is often confusion between platelet activation and coagulation activation. There is a misconception that PRP must be activated to trigger the release of growth factors.
This belief stems from early in vitro experiments in which the addition of activators such as calcified thrombin was required to extract growth factors from platelets [25].
Platelets are not simple vesicles filled with growth factors, but functional entities that release growth factors in a controlled manner in response to local signals.
Thus, in vitro, in the absence of activation, platelets do not release their growth factors. High doses of calcified thrombin are therefore required to induce complete platelet degranulation and uncontrolled growth factor release.
Endogenous platelet activation occurs when PRP is injected into the patient's tissues [26].
Platelets are physiologically activated by contact with extracellular matrix proteins (e.g. collagen) at the injection site. At each stage of the healing process, platelets secrete different cocktails of growth factors, in response to local signals, to stimulate organized tissue repair.
Exogenous activation is only required to obtain a gel PRP that coagulates rapidly at the injection site or to obtain a gel PRP that coagulates rapidly at the injection site or to obtain a fibrin clot, platelet-enriched autologous fibrin sealant or suturable fibrin membranes [27,28].
These types of products are used, for example, to treat hard-to-heal wounds [29]. This prevents the PRP from spreading and ensures localized action.
In this study, we recommend the use of autologous serum containing autologous thrombin at physiological levels to activate PRP, either alone or in combination with a pharmaceutical-grade calcium solution (Fig.1).
Example of autologous platelet gel (Fig.1)
This serum is prepared from the patient's blood using a specific device that also uses gel separator technology, but in a tube without anticoagulant.
The use of autologous thrombin serum, combined with PRP, enables the physiological formation of a fibrin clot in which platelets secrete growth factors in a controlled and sequential manner throughout the process of replacing the clot with new tissue.
This article highlights the benefits of solid and membrane platelet-rich plasma in non-healing corneal defects.
in non-healing corneal defects, with a description of the first 10 consecutive cases to receive autologous platelet gel therapy with their indication, results and outcome.

Material and method

This prospective study was conducted on 10 consecutive patients with severe corneal ulcers (5 men and 5 women; mean age 60 [40-81].

Underlying causes of corneal ulcers included trauma, herpes simplex infection, severe dry eye, neurotrophic keratitis or systemic disease (see breakdown of cases in figure 2).

Autologous platelet gel transplantation was performed in all patients between June 2021 and June 2022, and mean follow-up was months [2-14].

All patients presented with incomplete or non-healing corneal ulcers after initial treatment, which included etiological (anti-infective) therapy, artificial tears, bandage contact lenses, steroids or PRP eye drops, steroids or PRP eye drops.

The ulcers were located in several places:

  • 6 were central or paracentral,
  • 3 peripherals,
  • 1 total
  • 4 of these ulcers were perforated. 
  • The mean depth of the remaining 6 cases was 136 μm [50-323 μm].
  • The mean ulcer diameter was 4.73 mm [4 mm -11 mm].

Fig.2 The different clinical cases treated.

Example of case n° 3.
Perforated ulcer associated with rheumatoid arthritis.
  • A. Fluorescein picture: Seidel test.
  • B. Perforated ulcer seen with slit lamp.
Example of case n°2:
Deep peripheral ulcer associated with rheumatoid arthritis (256 μm).
  • (a) Fluorocein picture, visible ulcer and episcleritis.
  • (b) slit lamp with view of both peripheral and local ulcers with episcleritis near the ulcers.
  • (c) OCT picture with visualization of the ulcer.


All patients were treated with autologous platelet gel.
The procedure is based on the preparation of two biological products.
First, PRP is prepared using RegenKit BCT (RegenLab, Le Mont-Sur-Lausanne, Switzerland) and as follows:
Blood is extracted into the RegenKit BCT tube, 10 mL of which is collected using a blood sampling kit connected directly to the tube.
As there is a vacuum in the tube, blood sampling is automatic.
Invert the tube three times to mix the blood with the anticoagulant (sodium citrate).
Place the tube in the RegenLab centrifuge for 5 minutes (1500 G).
After centrifugation, we achieve density-specific physical separation of the blood components using the separation gel, leaving platelets and plasma on top of the separation gel.
The tube is inverted at least 10 times to suspend any platelets that have settled on the gel.
Extraction of 5 to 6 mL of PRP into a 10 mL syringe.

Principle of operation of Regen Lab technology for PRP preparation.

Next, autologous thrombin serum is prepared using RegenKit ATS as follows:
- Extraction of blood into the RegenKit ATS tube, 10mL is extracted using a blood sampling kit directly connected to the tube.
directly connected to the tube. As there is a vacuum in the tube, blood sampling is automatic.
- The tube is placed directly into the centrifuge for 5 minutes (1500G-force).
- After centrifugation, we obtain a density-specific physical separation of the blood components using the separation gel.
of blood components with the separating gel, leaving a clot above the gel containing activated autologous thrombin
- A cannula is inserted into the tube, piercing the stopper, and upward movements are made to break the fibrin clot and release autologous thrombin serum.
Principle of operation of Regen Lab technology for Autologous Thrombin Serum preparation.
After these preparations, a mixture of the two biological products must be made with specific ratios.
To obtain a gel, we followed these different steps:
1- Transfer the 5-6mL of PRP obtained into a sterile dish.
2- Add 1mL of autologous thrombin serum to the same sterile dish.
3- Add 0.5mL of injectable calcium gluconate solution (10%) to the same sterile cup.
sterile cutting
4- Allow the coagulation process to take effect for a few minutes (<10 minutes).
5- Platelet gel can be cut and applied to the specific surface.
The resulting fibrin membrane was applied under an operating microscope as a plug to seal the defect in 4 cases, as a cover to cover the entire cornea, limbus and conjunctiva in 2 cases, and as a combination of both methods in 4 cases (see image 3,4,5).
In all cases, no sutures were used, and only a contact lens was inserted. Patients were discharged with a sterile eye dressing for 3 days and a prescription for the following treatment:
  • PRP eye drops 1 drop every 3 hours
  • Preservative-free lubricant UID
  • Preservative-free topical antibiotic (4 times/day)
  • Topical or oral steroid if necessary (to be adapted to immunological cases)
Image 3. Example of the case n°7. The autologous platelet gel overlay on an ulcer due to a chemical acid injury. 
(a) Postoperative slit lamp image at day 1.
(b) OCT picture at day1 with visualization of the autologous platelet gel and the therapeutic lens.


Healing was achieved after a mean period of 10 days (D7-D22) for all cases.
The lens was removed after resorption of the membrane. There were no further interventions, except for one patient due to partial re-epithelialization.
Complete healing was achieved after the second procedure.
Patients were followed up at regular intervals on day 1, day 3, day 7 and day 10 after surgery, with clinical examination (symptoms, visual acuity and corneal staining) and AS-OCT (anterior segment - OCT).
(anterior segment - OCT) at each visit.
The dressing contact lens was removed after the membrane had been absorbed, on average day 9 (day 3-day 17).
All patients also received routine and/or specific care for their underlying conditions.
Corneal transparency after transplantation was at least partially maintained. Patients with limbal stem cell deficiency
Patients with limbal stem cell deficiency due to chemical burns or Meibomian gland dysfunction received 3 series of PRP drops (10 days each).
PRP drops (10 days each). 
A new procedure was performed on a single patient with a deep corneal perforation associated with rheumatoid arthritis.
The first membrane was resorbed on day three, but due to incomplete healing, the procedure was repeated, and complete healing was achieved on day fourteen after the second procedure.
In our study, healing was complete in all patients, with no recurrence during the follow-up period, which ranged from 2 to 14 months.
Subjective improvement in anterior segment irritation and pain was reported by 90 % of patients as early as the first day after surgery.
In all patients with initial corneal neovascularization, stabilization or reduction of neovascular invasion was achieved within 3 weeks. Analysis of this parameter is beyond the scope of this article.
No ulcer recurrence was reported at the end of the follow-up period, and all patients included in this study are subject to regular follow-up.


The ocular surface comprises the cornea, conjunctiva and overlying tear film.
The homeostasis of the ocular surface depends on the anatomical and physiological integrity of all its components.
Corneal healing is a complex, multi-faceted process involving inflammation, cell death, proliferation, migration and scarring.
Corneal healing is a complex, multifaceted process involving inflammation, cell death, proliferation, migration and differentiation, as well as remodeling of the extracellular matrix [6].
The resulting release of growth factors (e.g. KGF, PDGF and HGF) and cytokines triggers the transformation of epithelial and stromal cells into myofibroblasts and determines the outcome of an epithelial lesion. [7]
Plasma contains many components necessary for cell survival, such as nutrients, vitamins, hormones, electrolytes, growth factors (IGF and HGF) and proteins. 
These proteins, essential to the coagulation process and the formation of fibrin polymers, serve as scaffolding for cell adhesion, migration, proliferation and differentiation into new tissues. [12]
Platelet-rich plasma (PRP) is the fraction obtained by centrifuging whole blood. [3]. 
Platelets play a central role in hemostasis, as they aggregate to form a pro-coagulant surface and generate thrombin and fibrin. They contain growth factors such as VEGF, PDGF and TGF-β, as well as cytokines.
PRP promotes tissue regeneration and repair, and influences cell reactivity in the areas of angiogenesis and inflammation.
angiogenesis and inflammation.
This property is very useful when rapid healing and tissue regeneration are required, for example in the case of corneal ulcers.
Whether in solid or membrane form, PRP is in solid or membrane form, PRP is used to fill defects or provide adhesive support.
These powerful healing properties are due to the fresh platelet content of PRP (whether solid or activated), which is almost two to three times higher than in whole blood, or four times higher in PRP eye drops. eye drops.
Growth factors are released progressively from these fresh platelets over a period of 7 to 10 days. [8,9,10]
This treatment resulted in complete healing, eliminating the need for costly and time-consuming surgery.
costly and time-consuming.
Its main advantage is to preserve partial or total corneal transparency, by stabilizing or reducing corneal thickness. This objective can also be achieved by with a conjunctival flap, as demonstrated by our patient with a corneal perforation.
Evidence for the efficacy of autologous platelet gel was also reported in a recent article published by a Mexican team on 3 cases of non-healing corneal ulcers. [11]
Pro-inflammatory cytokines, such as interleukin 1-2-8, interferon and tumor necrosis factor, are released by ocular surface epithelial cells in the event of corneal damage [13], are released by ocular surface epithelial cells in the event of corneal damage [13].
Inflammation is an aggravating factor, and controlling it is essential to avoid further damage to the corneal stroma.
Autologous fibrin gel and platelet-rich plasma (PRP) both contain hepatocyte growth factor (HGF) and other factors. widely used in tissue repair and regenerative medicine.
They protect tissues from inflammatory damage by decreasing gene expression of COX-1, COX-2, and almost completely block cellular production of PGE2 and expression of COX proteins [14, 15, 16,17].
Numerous studies have shown that emergency penetrating keratoplasty to treat perforated ulcers is associated with a higher rate of graft failure is associated with a higher rate of graft failure than keratoplasty performed several weeks or months after injury.
Given the impact of inflammation on corneal healing, management of corneal perforations caused by inflammation or infection should not only focus on tectonics, but also resolve the ongoing inflammation-induced destruction of corneal stroma, leading to inflammation-induced corneal stroma scarring leading to perforation. [18, 19, 20]
With scaffolding, growth factors and anti-inflammatory properties, the use of autologous platelet gel is an ideal substrate for corneal grafting, to repair deep ulcers and perforations, with or without inflammation-mediated tissue destruction.


Autologous platelet gel is a simple, straightforward surgical technique that should become part of the therapeutic arsenal for ocular surface diseases.
However, the indications for this technique need to be clarified, as it cannot correct all the secondary conditions associated with limbal destruction.
The autologous combination of platelet-rich plasma and thrombin serum in the form of a platelet gel has proven its healing properties in the repair of corneal and ocular surface ulcers, but a larger randomized controlled clinical trial is needed to validate these preliminary results.
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