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Blood Transfus. 2010 April; 8(2): 107–112.
PMCID: PMC2851214

Blood components for topical use in tissue regeneration: evaluation of corneal lesions treated with platelet lysate and considerations on repair mechanisms

Abstract

Background

The fields of application of topically administered platelet derivatives are numerous and increasing. The use of this blood component is based on the fact that it contains growth factors and proteins of the clotting system. Studies carried out so far have been aimed at identifying these substances, assaying their content in the various types of platelet concentrate used, determining the in vivo and in vitro mechanisms of action, and trying to standardise the production methods. However, much still remains to be discovered, not only about the growth factors, but also about all those cytokines and biochemical mediators that are involved in the processes of tissue regeneration.

Methods

We studied the use of platelet lysate, obtained from platelet-rich plasma which had been frozen, for the treatment of corneal ulcers caused by neurotrophic keratitis and of epithelial and stromal loss following physical or chemical trauma. The platelet lysate was administered in the form of eye drops to patients who had not responded to conventional therapy and who were at risk of corneal scarring.

Results

The results were satisfactory in terms of both tissue regeneration and healing time. The clinical follow-up showed a clear reduction in the time of regeneration of the damaged epithelium and stabilisation of the repair process. The epithelial defects disappeared completely in all the treated eyes within 6 to 32 days, with the time depending on the type of lesion and the severity of the damage.

Conclusions

The cornea reacts to damage by releasing numerous substances, including cytokines, growth factors, proteases and neuropeptides in order to restore its anatomical integrity. A change in the balance between inhibitory and stimulating substances can lead to the development of complications. Fast, correct re-epithelialisation is fundamental for the formation of new, transparent tissue. The use of non-gelified platelet-rich plasma was found to be effective in all cases with loss of epithelium, such as post-herpetic corneal ulcers or ulcers occurring following trauma or exposure to caustic substances.

Keywords: platelet-rich plasma, corneal ulcers, growth factors, platelet lysate

Attempts to influence the processes of tissue repair and wound healing are as old as the history of mankind and, therefore, medicine. It is known that both the Egyptians and the Ancient Greeks had the idea of using a glue to heal wounds, applying vegetable resins to the wounds. The first scientific studies focused on the use of human fibrin, given the marked adhesive properties of this compound. More recently, wars and battles were the stimulus to the search for new solutions. However, the study of the biological preparation called fibrin glue and its clinical use began to spread in the 1970s: since then, thousands of articles on its properties have been published throughout the world18. The use of platelet-derived blood components and their like in clinical practice began in the last decade of the last century in the field of dentistry9,10 and spread rapidly throughout the world, being incorporated into various areas of modern medicine. There are numerous fields of application: from dental implants to maxillofacial surgery1118, from the treatment of soft tissue lesions1925 to consolidation and filling of bone lesions26,27, from heart surgery to abdominal surgery, to the treatment of difficult-to-heal wounds28. It is now well accepted that highly concentrated platelets in the form of a platelet gel are the source of release of growth factors and numerous other important biological mediators29,30. Cryoprecipitate is rich in fibrinogen, fibronectin, factor VIII, and other clotting factors, which are fundamental for the formation of the fibrin scaffold and the process of haemostasis31.

The underlying biological mechanisms and molecular processes involved are only partly understood. Nevertheless, it is not uncommon that empirical procedures are adopted in medical practice with success before science is able to provide answers to the many questions concerning them or that medications traditionally used among indigenous populations are found to contain potent pharmacological agents. A thorough review of the extensive literature on platelet derivatives reveals various inconsistencies that are not easy to explain. Furthermore, alongside euphoric and triumphal claims, much more moderate, if not clearly critical, opinions have been expressed. This is because, although the beneficial effects of platelet derivatives on tissue regeneration have now been recognised by the entire scientific community, the data presented are very heterogeneous32. Various methods have been described for producing platelet derivatives: in test-tubes, in traditional bags, by apheresis with cell separators, with the help of various different instruments3337. Thus the final platelet concentration varies, the manipulation methods differ, the methods of application differ and the types of activators differ3840. There is clearly a need for validated, shared standards.

Materials and methods

The lesions treated in this study were corneal ulcers of varied pathogenesis. Platelet concentrates have been used in ophthalmology in macular hole surgery for some time4144. There are numerous ocular and systemic pathologies, such as viral keratides, chemical-induced lesions or burns to the surface of the eye, drug-induced toxicity, corneal surgery, multiple sclerosis and diabetes, which can cause neurotrophic keratopathy45. Although many medical and surgical techniques have been proposed for the treatment of neurotrophic keratopathy, the management of this condition remains problematic.

Given the known beneficial effects of platelet concentrates on tissue regeneration, we treated patients with corneal lesions with the topical application of non-gelified, autologous platelet-rich plasma (PRP) administered in the form of eye-drops.

We evaluated two clinical situations which could be susceptible to the use of PRP in the form of eye drops: neurotrophic keratopathy, which has a broad range of clinical manifestations, such as persistent epithelial defects, superficial punctate keratitis, dystrophic corneal ulcers and keratopathy with loss of epithelial-stromal tissue due to chemical or physical trauma. We were referred all patients with corneal lesions that did not re-epithelialise after 1 week of conventional treatment. The aim of the treatment was to avoid formation of scar tissue and consequent permanent corneal opacity. During a period of 7 years, we treated 120 eyes in 103 patients aged between 21 and 78 years old (mean, 52 years), who did not respond to conventional treatment (therapeutic contact lenses, topical artificial tears, eye packs and antibiotic eye-drops) (Table I).

Table I
Pathologies treated

The working protocol was that patients were recruited by ophthalmologists on the basis of the above-described characteristics and, after assessment of the blood count and written, informed consent, a venous blood sample was taken. The treatment lasted from 7 to 14 days, depending on the clinical indications. On the basis of the intended duration of the treatment, about 20–30 mL of venous blood were collected into Vacutainer test-tubes containing anticoagulant (EDTA). The test-tubes were kept plugged and centrifuged at a low rate (for 5 minutes at a velocity of 1,200 to 1,600 rpm depending on the number of platelets and the haematocrit found in the initial blood count). The test-tubes were opened individually under a laminar flow hood and the supernatant PRP aspirated using 1 mL insulin syringes with a removable needle. From 0.6 – 0.7 mL of supernatant were collected into each syringe, avoiding contamination by red blood cells. The optimal concentration of platelets was considered to be about 500x103 platelets/μL. The syringes prepared in this way were delivered to the ophthalmology ward or directly to the patient, transported in refrigerated containers and stored in a freezer for the entire duration of the treatment. The contents of a single syringe, kept in a refrigerator at +4°C, were used each day, being applied to the ocular lesion as normal eye-drops.

A platelet count and tests of sterility were conducted on each preparation (Table II).

Table II
Working protocol

All the patients were evaluated before the treatment, after 1 week, 2 weeks and 4 weeks. The evaluation consisted of an ophthalmological examination, including corrected visual acuity, biomicroscopy, photography of the anterior segment, staining of the anterior segment with fluorescein, confocal corneal microscopy. The patients were instructed to keep the syringe in a refrigerator and, after having removed the needle, apply the eye-drops several times a day, (on average, from 5 to 10 times) together with netilmicin eye-drops four times a day. An antibiotic is prescribed routinely by ophthalmologists for these types of lesions, independently of whether or not PRP eye-drops are used.

Results

The treatment of the corneal ulcers with autologous PRP was found to be effective: all those lesions in which the conventional protocol had failed were healed. Epithelial defects, whose clinical evolution was studied using confocal microscopy, disappeared completely in all the eyes in a period from 6 to 30 days. Re-epithelialisation occurred within 1 week and, in the following period, up to 1 month, complete functional maturation of the epithelium took place. In three cases (dystrophic ulcer) the epithelial defect recurred 1 week after interrupting the treatment with the autologous lysate. In one case, a 72-year old patients with an 8-month history of post-herpetic uveitis and keratopathy, we had to suspend treatment because of excessive vascularisation, which promptly resolved following local instillation of a glucocorticoid. In the patients with Sjögren’s syndrome and severe chronic ocular graft-versus-host disease, the daily instillation of autologous plasma, even platelet-poor plasma, immediately relieves ocular dryness and prevents the formation of small corneal lesions, certainly being more effective in this than artificial tears. Given the aseptic preparation of the product and careful maintenance of low temperature during its storage and administration, there were no cases of bacterial contamination, which is a potential risk of "home made" products.

Discussion and Conclusions

The cornea reacts to damage by releasing numerous substances, including cytokines, growth factors, proteases and neuropeptides, in order to restore anatomical integrity (re-epithelialisation, stromal repair, and re-innervation). In chronological order, the first process to occur is re-epithelialisation, through the stimulation of the proliferation, migration and differentiation of the adjacent epithelium. This process is initiated and controlled by the release of soluble factors from the epithelium itself, from keratocytes, and the lachrymal glands. The factors involved include those with stimulatory effects such as fibronectin, tumour necrosis factor-a, colecystokinin gene-related peptide, platelet-derived growth factor, interleukin 6, and those with inhibitory effects, such as hepatocyte growth factor and tumour growth factor.

A change in the balance between inhibitory and stimulating factors leads to the development of complications, while the aim is to obtain fast, correct re-epithelialisation. The factors released by the damaged epithelium also affect the stroma, causing an increase in the apoptosis of keratocytes, their activation, migration and deposition of collagen fibrils. Epithelial regrowth starts from the peripheral margins and is mediated by a complex interaction between cell proliferation, adhesion molecules and stimulation by growth factors. In the absence of complications, re-epithelialisation occurs in a few days, while complete functional and morphological maturation of the epithelium takes longer. Every change to the process of re-epithelialisation, whether this is a delay or a defect in epithelialisation, has a negative effect on the repair of the underlying stroma, thus significantly increasing the risk of corneal scarring and, therefore, of corneal opacity. Confocal microscopy, a technique that enables completely non-invasive in vivo study of the individual cells of the various layers of the corneal, used during the follow-up of the patients, showed how the keratocytes undergo intense proliferation and subsequent activation and migration in the first days up to 3 weeks after the application of the autologous PRP, while scar tissue is intensely infiltrated by lymphomonocytes and Langherans’ cells. The keratocytes activated in this way undergo myofibroblastic transformation and produce collagen fibres and substances essential for the process of restoring full integrity to the stroma.

It is, therefore, clear that a whole series of mechanisms are involved in the process of healing a wound, be it of the cornea or any other tissue, and that the action of growth factors (specifically, platelet-derived growth factor), although undoubtedly fundamental is not exclusive. What we know about the correlation between growth factors and platelets is that the former are stored in the alpha granules of the latter, together with other biochemical mediators4650. All these substances are normally present in the body; however, in particular environmental conditions and at high concentrations, they are able to manifest all their regenerative potential, triggering a beneficial chain reaction that starts and amplifies the process of resolution, stimulating effects such as angiogenesis, chemotaxis of macrophages, proliferation and migration of fibroblasts and the synthesis of collagen5156. The results we obtained in this study, using PRP with a relatively low platelet count, were excellent: the method was simple, safe, effective and economic. Although a gelified product enabling slow release of growth factors cannot be used in the eye, more than 90% of the treated patients had complete resolution of the corneal lesions. The concerns that could be raised are related to the fact that the growth factors can affect the balance between local inhibitory and stimulating factors, with a possible evolution towards fibrosis. In our substantial experience, gained in patients undergoing rigorous controls, we found only one case of hypervascularisation. Monitoring with confocal microscopy enabled any anomalous reactions to be detected early and appropriately managed. This working protocol, which was very simple and functional, would be easy to standardise in order to obtain a universally recognised and applied therapeutic product.

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