CGF treatment of leg ulcers: A randomized controlled trial

1 Introduction

Concentrated Growth Factors (CGF) is a second-generation mixture of autologous growth factors compared to platelet rich plasma (PRP). It represents a multifactorial stimulation system as it uses all phases of the blood. It is proposed for the ancillary treatment of chronic skin ulcers.

The advantages of CGF found in literature are the immediate hemostatic action, biocompatibility, fast epithelial regeneration (by stimulating angiogenesis and promoting the synthesis of collagen protein, so it also promotes tissue healing) and a high antimicrobial effect due to the high concentration of leukocytes [1, 2, 3, 4].

Progress in the management of chronic skin wounds has led to high rates of recovery [5, 6, 7] but further works are required in order to improve the effectiveness of treatment protocols and the comfort and safety of patients also.

The CGF represents an evolution of the PRP. Its application is progressively spreading in the clinical field. CGF and PRP are based on the release of platelet growth factors, mainly contained in alpha granules, which exert a stimulating action on the reparative processes of skin lesions. Few studies have been published, hence the literature provides a low level of similarity in cases and protocols [8,9].

2 Materials and Methods

A multi-centre randomized controlled trial has been performed for patients with mixed ulcers: i.e. on a population with venous insufficiency (whose diagnosis was evidenced by a clinical and an echo-doppler examination of venous district of the lower limbs, as well as by the typical ulcer location - the medial region of the ankle) and chronic obstructive arterial disease (whose diagnosis was also evidenced by a clinical and instrumental examination showing the second stage of Rutherford classification peripheral arterial disease, with an ankle / arm pressure index between 0.5 and 0.9 at the affected limb). The protocol consisted of the application of CGF in addition to a standard wound dressing and elastic compression, performed once a week, for a 12 weeks period (or less in case of ulcer healing) and followed by a 6 months follow-up. None of the patients enrolled underwent surgery for venous or arterial disease, as they were considered not suitable or they refused the treatment: these kinds of mixed ulcers are usually characterized by a significantly slower healing time compared with ulcers that are just venous. The trial was preliminarily approved by the Institutional Review Board Independent Ethics Committee (IRB-IEC) as the Ethics Committee of the Inter-University Centre of Phlebo-lymphology (CIFL), with a positive result (protocol: ER: ALL.2016.03A). The study was conducted by recruiting patients from among ten vascular centres of Southern Italy - reported in “Supporting Info”- under the control of the authors as members of the “SIMCRI Group for the study of CGF in the treatment of Mixed Ulcers”, and coordinated by the CIFL researcher’s group.

It should be noted that both comparative study groups have previously applied procedures in clinical use: specifically, we refer to CGF as a recent method of preparing platelet gel that enriches the dressing of growth factors present in platelet granules [10, 11, 12, 13, 14, 15].

It is also correct to specify that no significant methodological changes have been made after the start of the trial.

Patient enrollment took place between December 2016 and December 2017. The clinical trial and related evaluation analysis were completed in July 2018. The primary end-point was identified in the reduction of the surface of the ulcerative wounds within 12 weeks of treatment. Secondary end-points were the presence and variation of ulceration pain and the reduction of bacterial load during treatment.

2.2 Preliminary exclusion criteria

The exclusion criteria applied in the recruitment of patients were the following: recent outcomes of myocardial infarction, dialysis, a serious infection of the lesion (with extension to the surrounding tissues), reduced life expectancy, cachexia. Drugs already in use for other pathologies of each patient were not considered exclusion criteria, except in cases of significant modification of the therapy, necessary during the study itself. The reasons for the exclusion from the study were reported.

Figure 1

Consort checlist

Table 1

Clinical features of Group A and Group B patients with mixed limb ulcers.

Pts serie n.100	Group A n. 53	Group B n. 47
Male/Female	M = 18 /F =35	M = 16 / F =31
Mean Age	M = 62+/-9	M = 68+/-5
	F = 71+/-12	F = 69+/-10
Wound Size (cm2)	24+/-16	22+/-9

The exclusion criteria expected for the study was the following: extension or onset of severe ulcer infection and amputation or revascularization surgery.

3 Randomization

Patients were randomized by selecting enrolled patients for Group A (CGF supplementary treatment) and Group B (standard treatment as control group), by a software-generated procedure provided by a computerized randomization program with audit trial. Randomization was implemented by the single coordinator for randomization and was controlled by the principal investigator at University of Naples “Federico II”. Randomization was also indicated in the patient record.

3.3 Pain assessment

Pain assessment was performed at enrolment and at each dressing change by evaluating pain intensity according to the Wong-Baker scale, as in Fig.2, using its numerical values, and recording on the personal data sheet of each patient its reported intensity, as well as the characteristics of location, duration, concomitance and associated symptoms.

Figure 2

Wong-Baker scale for pain measurement.

Patients, however, were allowed to treat pain with home therapies using non-steroidal anti-inflammatory drugs, when necessary.

4 Results

The treatment of wound dressings, according to the procedures for group A and group B of patients, associated with elastic compression, was correctly performed for 12 weeks in all recruited patients. In group A, complete wound healing was found in all patients (53/53) during the first 12 weeks of observation: in 11 patients. (20.7%) after 5 weeks, in 14 patients (26.4%) in 6 weeks, in 10 patients (18.8%) in 7 weeks, in 8 patients (15.0%) in 8 weeks, in 7 patients (13.2%) in 9 weeks, in 2 patients (3.7%) in 10 weeks, and in 1 patient (1.8%) in 11 weeks. Ulcers healing in group B occurred in the first phase of the study (12 week interval from first observation) only in 32 out of this 47 patients (68% of cases), with the following timing: in 2 patients (4.2%) in the first 5 weeks; in 1 patient (2.1%) in 6 weeks, in 3 patients (6.3%) in 8 weeks, in 5 patients (10.5%) in 9 weeks, in 7 patients (14.8%) in 10 weeks, in 6 patients (13.9%) in 11 weeks, in 8 patients (18.6%) in 12 weeks, while in 15 patients (31.9%) wound healing was achieved over 12 weeks.

Evolution of the healing times in the two groups of treated patients is described in Fig. 2.Comparing the two groups in consecutive weeks, the surface of the ulcers did not show a significant reduction in comparative control after the first week (p = 0.014), while it was possible to evaluate significant reductions in all consecutive comparative controls occurred between the first and second week (p = 0.004), and then between subsequent weeks (p <0.001).

The overall evaluation of pain intensity by means of the Visual Analogue Scale (VAS), using the Wong-Baker scale, initially homogeneous in the two study groups (5.38 +/- 1.35 in Group A and 5.16 +/- 1.94 in group B) showed a significant improvement (p <0.001) of pain symptoms in Group A patients compared to those of Group B, starting from the second week of treatment (Group A = 2.88 +/- 0.83, Group B = 5.26+ /-1.14), progressively improved in the following weeks (Tab. III), with the complete disappearance of pain that accompanied the healing of the ulcer at an average 3.4 weeks.

The evaluation of wound infection degree in the two groups of treated patients is described in Tab. IV. (Tab. IV)

No complications or undesirable effects of GCF (such as skin irritation, pain or allergic reactions) have been observed in any of the Group A patients.

5 Discussion

The presence of a chronic non-healing ulcer is a condition of discomfort for each patient because of the painful symptomatology and the impairments that limit their daily habits in terms of social life or work. The healing of leg ulcers, that are mainly caused by venous insufficiency, makes favourable use of physical methods aimed at the reduction of venous ankle hypertension, whereby the patient extended position, the use of elastic stockings or bandages, combined with the cleaning of the ulcer, leads to healing of the ulcer in variable times [6,17]. The concomitance of obstructive arterial disease of the same limb (also of moderate degree, without critical ischemia) determines a further reduction of the oxygenation of peripheral tissues and determines a greater difficulty and prolongation of healing times, also due to the selective contraindications for elastic compression of the limb [16]. Healing of mixed ulcers of the lower limbs may take several weeks, and several trials indicate the average healing time over 24 weeks, with the frequent risk of early recurrences [20, 21, 22, 23].

The social cost of treatment must also involve the costs of home self- dressing, medical examinations and possible hospitalizations for specific procedures.Furthermore, most patients belong to the category of geriatric patients and may have numerous co-morbidities that influence the course of the wound and interfere with the healing pathway [24].

In fact, the limits of the study can be found in the multi-factoriality of pathologies that support mixed ulcers: they occur especially in the elderly population, both from the point of view of chronic venous insufficiency and for chronic peripheral artery disease, but also for the effect of pharmacological therapies (that each patient practiced at the same time during the study period for these pathologies) or for concomitant chronic pathologies such as diabetes, hypertension and coronary heart disease,: their time of onset, their severity of concomitant diseases, the quantity and quality of associated drug therapies could represent subgroups of study that have not been evaluated in this trial, but have not been considered preventively determinants for the sought therapeutic effect.

A recent controlled critical review, produced with the Cochrane Collaboration method [25], indicates moreover that there is limited evidence to support the fact that dressing modality and their frequency in the management of venous or mixed leg ulcers could be clinically significant on time of healing and on its stability: probably the limited number of trials, the lack of meta-analysis in this field and the poor quality in method does not allow to formulate recommendations with adequate levels of evidence, nor to define the comparative validity of the various types of treatment proposed.

Therapeutic effects of the application of autologous growth factors of blood derivation have found a wide space of application in several sectors of medicine and surgery (from odontostomatology to orthopedics, or plastic surgery and also vascular surgery), both in vitro and in vivo, with various mechanisms: induction of cell proliferation, stimulation of inflammatory cells, increase of collagen deposition and angiogenesis, through the enhancement of enzymatic fibrinolysis and stimulation of protein synthesis [26, 27, 28].

Further analytical and interpretative assessments of the role of autologous growth factors are beyond the scope of this article, but further information can be derived from future studies in this field.The regenerative properties of blood are related to the presence of platelets, leukocytes, mononuclear cells and growth factors. The platelets contain in fact numerous active substances that play an important role in tissue regeneration through the recruitment and activation of mesenchymal cells, such as osteoblasts, fibroblasts and endothelial cells. The active substances are located in different sub-cellular structures: alpha-granules, dense granules, lysosomes and micro-peroxisomes. Alpha-granules contain chemiotactic and mitogenic growth factors important in tissue regeneration, including PDGF, TGF-beta 1 and -beta 2, VEGF, EGF, IGF and FGF. Undoubtedly, personalized compression therapy (especially with low extensibility bandages, possibly through the execution of multi-layer type), in the context of mixed ulcers, and also for those exclusively of venous origin, represents a fundamental step in treatment, and it is reported as a high-grade recommendation in all the guidelines produced in the vascular field.

6 Conclusions

The results of this multicentric study on the effects of CGF in the treatment of mixed ulcers of the lower limbs showed a significant advantage in the use of CGF in association with cleansing and selective compression in the healing and stabilization of this kind of ulcers. Pain was also significantly reduced comparatively in the group of patients who were treated with CGF, and furthermore the cost / benefit analysis of the use of this product indicates significant advantages in favour of this procedure. In the treatment of difficult mixed ulcers, CGF has been therefore proved useful and effective in obtaining a faster, less painful, longer lasting and overall cheaper healing of the ulcer. For these reasons, the enhancement of treatment with CGF to all the chronic ulcerative manifestations with a tendency to chronicization is considered valid and suggested by the authors.