Treatment of venous ulcers with growth factors : systematic review and meta-analysis

OBJECTIVE
To identify evidence about the effects of growth factor application on venous ulcer healing.


METHOD
Systematic review and meta-analysis, including Randomized Clinical Trials. Searches: Ovid MEDLINE, EMBASE, CINAHL, Cochrane CENTRAL, LILACS, Web of Science, Digital Library of Theses and Dissertations; Google Scholar and list of references.


RESULTS
802 participants were recruited from the 10 included studies: 472 in the intervention group (growth factors) and 330 as control. The relative risk for the complete healing outcome was 1.06 [95% CI 0.92-1.22], p = 0.41. Participants who received Platelet-Rich Plasma and Epidermal Growth Factor showed a slight tendency to achieve complete healing, but without statistical relevance (p <0.05). Most of the studies were classified as moderate risk of bias.


CONCLUSION
The effect of the application of growth factors for complete healing in venous ulcers is not clear, and clinical trials with methodological quality are required for more accurate recommendations.


INTRODUCTION
Tissue repair involves a series of complex, dynamic and sequential interactions involving cells of the epidermis, dermis, extracellular matrix, and plasma proteins (1) .The growth factors are responsible for inducing simultaneous effects on several cell types and provoking a series of biological functions in several tissues, positively interfering in the healing process, which makes its use an attractive therapeutic possibility in several segments of Medicine (2)(3) .
Growth factors act in a way to inhibit or stimulate the target cell's gene expression in the wound.By transmitting modulatory signals, growth factors regulate stimulatory and inhibitory growth processes, such as proliferation, differentiation, migration, and adhesion (4) .In addition, they also act to promote cell chemotaxis, being able to induce the migration of several cells, besides stimulating the angiogenesis and the synthesis of the extracellular matrix (2) .
Thus, the whole process involving tissue repair occurs continuously and dynamically, dictated by numerous growth factors and other cytokines, such as tumor necrosis factor (TNF-α) and interleukins (5) .When there is an imbalance between pro and antiinflammatory cytokines, the wound is chronified, which remains in a persistent and uncontrolled inflammatory phase (6) .At the same time, there is a low mitogenic activity, as well as the presence of fibroblasts with premature senescence and degradation of the cell matrix (6) .Insufficient bioavailability of growth factors, either by decreased synthesis and/or excessive degradation, is a hallmark of chronic wounds (4,7) .Therefore, the longer the duration of the ulcer, the worse its prognosis in relation to healing (8) .
The application of growth factors has been cited as a favoring factor for the tissue repair of chronic ulcers of different etiologies, thus enabling the individual to return to his or her habitual activities (7) .Considering the prevalence of venous ulcers, high recurrence rates and long treatment, it is mandatory to seek alternative therapies that aid tissue repair and promote the return of the affected individual to their normal daily activities in the shortest time possible (9) .
A previous search for evidence of the effectiveness of growth factors performed in the major electronic search databases for systematic reviews (Cochrane Library, Joanna Briggs Institute Library, Center for Review and Dissemination (CRD) of York University and PubMed) revealed little scientific production about growth factors for the treatment of venous ulcers.Two systematic reviews addressing the efficacy of growth factors (present in Platelet-Rich Plasma -PRP) on chronic ulcers (10)(11) ; and two others that addressed the efficacy of PRP in diabetic ulcers (12)(13) .However, none of the reviews evaluated growth factors alone.In addition, only one of them evaluated the application of PRP in venous ulcers (10) .However, this review does not contemplate the evaluation of other growth factors, in addition to having included studies only until January 2015.In addition, two reviews (12)(13) contemplate studies only with diabetic ulcers, whose etiology and treatment approach considerably differs from venous ulcers.
In view of the above, the treatment of venous ulcers with products or preparations containing growth factors should be investigated for its efficacy.

OBJECTIVE
To identify evidence in the scientific literature about the effects of the application of growth factors on venous ulcers healing, compared to other therapies.

METHOD
This is a systematic review with meta-analysis, according to the recommendations proposed by the Cochrane Collaboration (14) .
The research question, elaborated according to the P.I.C.O.strategy (14) , was thus determined: What is the effect of the application of growth factors on venous ulcers healing compared to other therapies?
The clinical outcome evaluated was the total number of healed ulcers.Inclusion criteria were: Randomized Clinical Trials (RCTs) that address the use of growth factors associated or not with other therapies for the treatment of venous ulcers; studies covering the total number of healed ulcers.Exclusion criteria: ongoing studies and research protocols; articles that associate growth factors with the skin graft; studies that included ulcers of multiple etiologies without subgroup analysis.
The search in the electronic databases took place on October 6, 2017, by two independent reviewers.The search for studies originating from other sources occurred in September and October 2017.The repository of theses and dissertations was accessed nationally -Brazilian Digital Library of Theses and Dissertations, Google Scholar, list of references of included studies, as well as the list of articles related to each article included, through the PubMed platform.There was no restriction on language or year of publication.
Thesauri MeSH, DeCs, ENTREE and Cinahl Titles, as well as appropriate free terms, were rescued for the construction of specific search strategies for each database.The search strategy in the MEDLINE database via Ovid was elaborated from the Cochrane strategy of high sensitivity for identification of Randomized Clinical Trials (14) .The thesauri and free terms were adapted to the elaboration of the other strategies according to the specificities of each database.The strategies used are listed in Chart 1.
MeSH and DeCs thesauri were used to search other sources, as well as free terms combined with the Boolean operators AND and OR.
After exclusion of the duplicates, the studies were independently analyzed by two reviewers in relation to the title and abstract.The relevant studies have been fully recovered and the eligibility criteria.The degree of agreement between the two reviewers was established by the Kappa measure (Biostat ® 5.0), and the index reached was 0.83.Disagreements were discussed at a consensus meeting and disagreements were resolved with the collaboration of the reviewer and senior researcher.
The bias risk assessment was performed according to the recommendations of the Cochrane Collaboration (14) .The studies were evaluated in six domains (selection-sequence randomization bias, selection bias -allocation stealth, performance bias-masking of participants and investigator, detection bias -evaluator masking, friction bias -incomplete results at high loss rates, with losses of up to 20% being considered: low,> 20% ≤30%: moderate,> 30%: high risk; reporting bias -selective reporting, incomplete data publication, e.g., missing means and/or Standard Deviation for the rated outcome.Each study was classified as low, moderate or high risk of bias according to the domains evaluated.In order to evaluate the masking domain of the researchers and participants, it was considered low risk both the double-blind declared studies and the non-blinded studies in this question (14) .To evaluate the mastery domain of the evaluator was considered low risk only when the author declared the blinding in this item (16) .The GRADE system (14,17) (Grading of Recommendations Assessment, Development and Evaluation) was used to evaluate the quality of evidence.
The relative risk was calculated for the variables, considering a Confidence Interval of 95%.Additional analyzes were performed by subgroups by type of growth factors and their relative risk for the outcome of complete healing.The heterogeneity was evaluated statistically using the Chi-square test and considering fixed-effect analysis when heterogeneity less than 50%.To perform the meta-analysis, the statistical software Review Manager (RevMan) version 5.3 was used.
This review followed the recommendations proposed by PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyzes).The present review presents some updated results of a more comprehensive review, whose protocol is registered in the PROSPERO database (International Prospective Register of Ongoing Systematic Reviews) under the number CRD42016038390.

RESULTS
311 reports were retrieved after the exclusion of the duplicates and the application of the eligibility criteria.9 articles containing results from 10 studies were included in this review, as shown in the flowchart (Figure 1).
Chart 2 presents the characterization of the studies included in the analysis, as well as the bias risk assessment of each study.
The country that most produced research involving growth factors was the United States, responsible for more than half of the studies included in the analysis; all studies were published in the English language.) OR (MH "Systematic Random Sample") OR "randomized") OR ("placebo") OR ("randomly") OR (MH "Multicenter Studies") OR (MH "Intervention Trials") OR ("trial")) AND ((MH "Platelet-Derived Growth Factor") OR ("platelet-derived growth factor") OR (MH "Platelet-Rich Plasma") OR (MH "Epidermal Growth Factors") OR (MH "Growth Substances+") OR (MH "Vascular Endothelial Growth Factors+") OR ("recombinant platelet-derived growth factor") OR ("fibroblast growth factor") OR ("recombinant fibroblast growth factor") OR ("recombinant endothelial growth factor") OR ("transforming growth factor") OR ("recombinant transforming growth factor") OR ("recombinant epidermal growth factor") OR (MH "Blood Platelets")) AND ((MH "Venous Ulcer") OR (MH "Leg Ulcer+") OR ("venous leg ulcer") OR ("varicose ulcer"))) Cochrane Eight hundred and two participants with venous ulcers were recruited to participate in the 10 studies included in this review.The majority of the participants received compression therapy as adjunctive therapy, having only one study (18) that did not use compression bandaging.472 participants were treated with growth factors and 330 received standard treatment and/or placebo.236 (50%) healed completely in the "treated with growth factors" group and 151 (45.75%) in the "control" group.
With the exception of 1 study (18) , all of them considered chronic venous ulcers with more than 6 months of evolution at admission, with a mean ulcer duration of 14.32 months (SD ± 14.11) in the group that received growth factors and 16.04 months (SD ± 19.97) in the control group.In relation to the size, the group that received growth factors had ulcers with a mean of 8,722 cm 2 (SD ± 2.93) and the control group, a mean of 8,504 cm 2 (SD ± 4.50).
Only study I (25) was classified as low risk of bias in all domains.The J study (26) was classified as high risk for not reporting its results completely.Studies A, B, C, D, E, F, G and H were classified as being moderate risk of bias due to failures in the description of randomization method and/ or secrecy of allocation or by failures in the description of masking applied in the research (18)(19)(20)(21)(22)(23)(24) .
Chart 3 presents the description of the technologies evaluated, outcomes of interest and time of follow-up of each study.Ten studies were included in a meta-analysis for the overall complete healing outcome.We also performed analyzes by subgroups of growth factors to verify the effectiveness of each type of growth factor presented.
The forest plot shows the relative risk, 95% Confidence Intervals and Risk Classification of studies bias in a global manner, and also by the growth factor involved (Figure 2).The overall complete healing rate was similar between the groups that received growth factors and the control group (p=0.41).Participants treated with PRP and EGF presented a slight tendency to achieve complete healing, but without statistical relevance.Most of the studies presented moderate quality of evidence in the domains related to the randomization and allocation process.The Summary of Results presents the classification of evidence according to the GRADE System (Chart 4).

DISCUSSION
It is known that the classification of evidence quality, according to the GRADE System, evaluates five domains, the first domain being the risk of bias of the studies involved.In this review, most studies were classified as moderate risk of bias, with the most frequent failure being to describe the method of randomization and/or secrecy of allocation used.In an attempt to improve the quality of the research reports, the CONSORT (Consolidated Standards of Reporting Trials) (27)(28) .Every article resulting from a clinical trial must follow the recommendations described in the statement, which include details about the masking process, method of randomization and secrecy of allocation, among others (27)(28) .Failure to comply with the recommendations inevitably leads to a reduction in the quality of the study's evidence and a real difficulty in interpreting the evidence found.Considering the data presented by the 10 studies analyzed, it was verified that the application of growth factors in venous ulcers presented favorable results in most of the studies, however, with little statistical relevance, small sample size and studies classified as moderate risk of bias.Therefore, the evidence generated must be interpreted with caution.
According to the year of publication of the studies, it is noticed that initially there was a greater emphasis in studies containing recombinant growth factors.From 2000, the studies that used Platelet-Rich Plasma as a primary source for the application of growth factors in venous ulcers.However, in the last decade, only 2 Randomized Clinical Trials (18)(19) were conducted.A hypothesis for the low production of RCTs could involve the little encouraging results obtained so far.Another issue could be related to the association of factors and growth to compression therapy, present in most studies.Compression therapy is already consecrated as a gold standard for the treatment of venous ulcers due to its effectiveness (29) .Therefore, it was expected that the ulcers presented good healing rates in both groups, which could explain the simple positive effect of growth factors on venous ulcers when compared to the promising results obtained in wounds of other etiologies.
Thus, when analyzing the forest plot, it was observed that the incidence of global complete healing was similar between the groups that received growth factors and the control group.The confidence intervals of all the studies analyzed and the diamond chart crossed the statistical null line indicating statistically insignificance of this result.However, it is important to emphasize that the groups that received treatment with any growth factor did not present inferior results to the control groups.
Four studies evaluated the use of PRP for the treatment of venous ulcers (18)(19)21,24) .Thus, when analyzing the summary estimate regarding the treatment of venous ulcers using Platelet-Rich Plasma, there was a slight tendency to achieve complete healing in the group treated with PRP compared to the control group, however, without statistical relevance (p=0.08). It is oteworthy that the studies presented small samples and were classified as moderate risk of bias.However, there is increasing interest in the tissue regenerative potential of PRP, several studies worldwide have reported positive results using PRP in chronic ulcers (30- 31,33-34) .A non-randomized comparative study published in 2017 evaluated 40 participants with venous ulcers, evaluating the PRP associated with compression therapy for 6 weeks.The PRP group had significant wound area reduction rates (p<0.0001)(34) .However, few Randomized Clinical Trials were reported using PRP for treatment of venous ulcers (31) .
Other reviews have evaluated the potential of PRP for the healing of chronic ulcers.A review published in 2011 included 4 Randomized Clinical Trials where Platelet-Rich Plasma was evaluated in chronic ulcers (mostly diabetic and pressure ulcer), the outcome for the complete healing outcome was significant in favor of PRP (p=0.01) (11).In contrast, a Cochrane review published in 2016 analyzed 8 Randomized Clinical Trials that also used PRP for leg ulcers (diabetic, venous and mixed etiologies), where it was found that there is insufficient evidence to safeguard the beneficial factor of PRP (10) .
When analyzing the KGF applied in venous ulcers, it was observed that there was no beneficial effect for the complete healing outcome.The relative risk of venous ulcer healing completely with the use of KGF was 0.93, with no statistical significance.It is noted that the two studies analyzed were published by the same author who evaluated different doses of the drug in an attempt to establish an effective dosage.A review published in 2012 discussed on the potential of KGF for tissue regeneration (35) .The authors concluded that although KGF showed positive results in in vitro or animal models, the result was not replicated in clinical studies, causing a scientific disappointment and consequent discontinuation of some studies (35) .In recent years, studies involving KGF have focused on the treatment of burns due to its anti-scar potential (36) .
Regarding PDGF, the two studies included in this analysis were conducted by the same research group, where the authors evaluated in one study the efficacy of recombinant PDGF at a dosage of 100μm/g applied twice a week and in another study, your daily application (22) .Analyzing the results of the meta-analysis, it was observed that there were no differences between the groups that received PDGF and control group for the complete healing outcome (p=0.44).Other studies have already been carried out using PDGF for the treatment of chronic ulcers, but the results were not very encouraging (37)(38) .
Only one study evaluated the efficacy of TGF for venous ulcers, the study evaluated 12 ulcers in the use of TGF-impregnated collagen based dressing; 12 in use of pure collagen matrix and 12 only with petrolatum gauzedressing (25) .The complete healing rate of TGF-treated ulcers was not higher than that of ulcers treated with collagen matrix or petrolatum gauze dressing (p=0.80).Although TGF is a fundamental factor for wound healing, when there is an imbalance of this growth factor and its isomers an exacerbated scar formation can occur, increasing the chance of formation of hypertrophic scar and keloid (39)(40) .Therefore, it is necessary to establish safely the ideal dosage that is capable of promoting a better scarring without exceeding in the formation of the scar.
Analyzing the EGF results in the forest plot, it is possible to observe that although it presents a relative risk of 2.87 in favor of the use of EGF this result is not significant (p=0.17),besides being classified as low quality of evidence.It is worth remembering that this study was the only one that received the classification of high risk of bias due to incomplete data reporting.However, interest in this growth factor grows in the scientific community.A review of growth factors for treatment of chronic ulcers has reported that topically applied EGF, such as cream or even injected, has been studied by several research groups especially in Asia and Cuba (6) .Other studies using EGF in chronic ulcers reported positive results.A prospective study published in 2014 evaluated 33 venous ulcers treated with EGF embedded in a collagen-based dressing and found a significant beneficial EGF result (41) .Two clinical trials evaluated the successful regenerative potential of EGF in diabetic ulcers (42)(43) .Therefore, the use of EGF seems to be an interesting and promising alternative for the treatment of chronic ulcers, such as venous etiology.
In the US, the only one approved by the Food and Drug Administration (FDA) for use in chronic ulcers to date was Becaplermin/ Regranex® (PDGF).EGF (Heberprot-P®) has been successfully used in Asia, Central and South America, expanding its worldwide acceptance to Europe, mainly for use in diabetic ulcers (6) .In Brazil, no growth factor has approval for use in wounds, as medicine, by the Brazilian Agency of Sanitary Surveillance (ANVISA -Agência Nacional de Vigilância Sanitária) until the present moment.This fact hinders the development of research to evaluate the efficacy of these recombinant growth factors in the Brazilian setting.
However, obtaining and preparing Platelet-Rich Plasma autologous has a relatively low degree of difficulty and has been shown to be safe in previous studies (30)(31) .The preparation of the PRP does not require sophisticated equipment, with only a small centrifuge and the preparation material.Several techniques for obtaining PRP have been described in the literature (44)(45) .However, there is a need for follow-up of a multiprofessional team, with the involvement of at least the medical professional and nurse to apply the technology safely.
Most of the studies included in the present review evaluated patients with chronic ulcers with evolution for less than 2 years.Demographic studies conducted in Brazil have shown that most of the Brazilian venous ulcers exceed 10 years of evolution (46)(47)(48) .In addition, the size of Brazilian venous ulcers easily surpasses the average found in the studies analyzed in the present review.A study, published in 2012, showed that 39.2% of patients had ulcers over 24 cm 2 (46) .
Another aggravating factor in the Brazilian setting is the limited material resource found in the public health system.Despite the recommendation for using compression therapy as first-line treatment for venous ulcers, in practice, few patients have access to this technology.According to a study carried out in a public clinic in the Rio de Janeiro State, only 1.5% of patients use compression therapy (47) .A comparative study including 40 participants applied the autologous PRP in the test group and only the compression therapy in the control group, obtaining significant results (p <0.05) favorable to PRP (34) .The application of autologous PRP in the Brazilian context where compression therapy does not present much scope, either due to a lack of material, financial resources or even intolerance by patients, may be a possibility of treatment.
However, further studies with methodological quality are needed to validate or not the application of growth factors for the treatment of venous ulcers in the Brazilian setting.

Study limitations
Although there was no restriction on the language of publication, it is understood that the search results may have failed to retrieve any potential study, mainly because it did not include bases outside the United States-Europe-Latin America axis.Another limiting factor of this review is the fact that the reviewers did not attempt to contact the authors of the included studies to clarify some items evaluated by the bias risk assessment instrument.The risk assessment of bias considered only the data available in the published material and in the respective protocols, when found.In addition, the present review evaluated only complete healing as an outcome, as this was the outcome most evaluated by the studies and easier homogenization in a meta-analysis.However, another review addressing other outcomes is already under way.

Contributions for the sectors of Nursing, Health or Public Policy
The present review provides recommendations for clinical practice on the use of growth factors in venous ulcers, supporting the decision making of both nurses and other professionals who wish to use this therapy in their patients.
There are recommendations for clinical practice, considering the application of growth factors as adjuvant therapy to compression therapy:

CONCLUSION
Evidence on the application of growth factors to the treatment of venous ulcers is still limited.The relative effect of the application of growth factors to complete healing on venous ulcers is unclear.There was a slight tendency to achieve complete healing when applied to Platelet-Rich Plasma and Epidermal Growth Factor, however, these findings were not relevant (p<0.05).However, most of the studies included in this analysis were classified as moderate risk of bias.
Thus, more robust studies, with greater power, higher methodological quality and greater casuistry are necessary to generate more precise recommendations on the use of growth factors for the treatment of venous ulcers.
Note: Risk of bias: Green color: Low risk; Yellow color: Moderate risk; Red color: High risk of bias.

Figure 2 -
Figure 2 -Global and subgroup meta-analysis charts for comparative analysis of the occurrence of complete healing outcome in venous ulcers with growth factor versus standard treatment/placebo Chart 4 -Summary of Results presenting the relative risk, number of participants and quality of evidence for each decrease factor evaluated according to the GRADE System COMPARISON BETWEEN TREATMENT WITH GROWTH FACTORS OR STANDARD TREATMENT/PLACEBO FOR VENOUS ULCERS FOR COMPLETE HEALING OUTCOME Population: Patients with venous ulcers.Intervention: Growth Factors.Setting: Hospital or outpatient.Comparator: Standard treatment/placebo.

Chart 1 -
Search strategies employed in their respective databases (((randomized controlled trial.ptor controlled clinical trial.ptor randomized.ab.OR placebo.ab.OR clinical trial as topic.sh.ORrandomly.ab.ORtrial.ti.) not (exp animals/ not humans.sh.)) and (platelet derived endothelial cell growth factor/ OR platelet derived growth factor/ OR platelet derived growth factor a/ OR platelet derived growth factor ab/ OR platelet derived growth factor ab/ OR platelet derived growth factor b/ OR recombinant platelet derived growth factor/ OR recombinant cytokine/ OR recombinant growth factor/ ORbecaplermin/ OR recombinant fibroblast growth factor/ OR recombinant growth factor/ OR recombinant keratinocyte growth factor/ ORsprifermin/ ORvelafermin/ OR recombinant fibroblast growth factor 19/ OR transforming growth factor/ OR transforming growth factor alpha/ OR transforming growth factor beta/ OR recombinant transforming growth factor.mp. ORexp epidermal growth factor/ ORexp recombinant epidermal growth factor/ ORexp fibroblast growth factor/ ORexp plateletrich plasma/ OR platelet-rich plasma.ab.OR autologous platelet-rich gel.mp.OR autologous platelet-rich plasma.mp.) and (exp leg ulcer/ OR venous leg ulcer*.ab.OR venous ulcer*.ab.OR varicose ulcer.mp.))CINAHL (((MH "Randomized Controlled Trials") OR (MH "Clinical Trials+") OR ("controlled clinical trial") OR (MH "Clinical Trial Registry") OR (MH "Random Assignment") OR (MH "Stratified Random Sample"

Study's ID Authors and year of publication Growth Factor tested Number of Participants Frequency of Application Masking Risk of Bias Test Control
Chart 2 -Distribution of studies according to the authors, growth factor tested, number of participants, frequency of application, masking and risk of bias Note: *9 reports included, however, 1 report reports results from 2 different studies, therefore, 10 studies were analyzed.Figure 1 -Flowchart of search and selection of studies To be continued Treatment

of venous ulcers with growth factors: systematic review and meta-analysis
Carvalho MR, Silveira IA, Oliveira BGRB.