Efficacy of Plasma Rich in Growth Factors for Healing Chronic Skin Ulcers: A Systematic Review of Randomized Controlled Trials With Meta-Analysis

A cutaneous wound is a loss of substance that disturbs the skin’s normal anatomical structure and functional integrity.¹ Healthy tissue undergoes a normal wound healing process through consecutive physiological stages, including hemostasis, inflammation, proliferation, and remodeling.² Underlying conditions and diseases may hamper regeneration of the skin, however, leading to a chronic profile that prolongs patient recovery.³ Unfortunately, in some cases current guidelines and therapeutic options for wound care management do not achieve the desired efficacy, and other treatment modalities are required.⁴ 

Platelet-based therapies such as PRP have gained the attention of wound care specialists.^5,6 However, the efficacy and safety of clinical application of PRP remain controversial.⁷ According to a systematic review and meta-analysis published by Li et al⁸ in 2019, PRP is based on the withdrawal of a small blood volume that is centrifuged and fractioned to separate a platelet-rich fraction that is used as an autologous therapeutic agent to promote wound healing. However, different preparation protocols (eg, speed and number of spin cycles) and systems are available.⁹ These differences in preparation protocols result in significant differences in the characteristics of the PRP in terms of platelet concentration, leukocyte inclusion, presence and type of anticoagulant, and method of platelet activation.^10,11 Additionally, PRP is manually prepared and is patient specific. As highlighted by Anitua et al¹² in a letter to the editor published in 2011, it is not uncommon to find inconsistent and controversial results when evaluating the efficacy and safety of PRPs. 

There is considerable interest in assessing the characteristics of PRP to quantify variability between patients and determine the most adequate system. This information is then related to the clinical outcomes with the objective of assessing the clinical significance of PRP compositional variations. Different data collection systems are available to enable the clinicians to achieve this objective by collecting real-world data. Systematic reviews may be helpful in assessing the evidence of the clinical efficacy of a specific type of PRP through the combined analysis of multiple clinical trials that have been performed using this type of PRP in a specific clinical application. 

PRGF-Endoret (BTI Biotechnology Institute [hereinafter PRGF technology]) is a specific type of PRP that is leukocyte-free and moderately enriched in platelets. It is classified as pure PRP, specifically P2-x-Bβ category,¹⁰ and 24-00-1111 according to 2 extensively proposed classifications. PRGF, which shows a pro-angiogenic, pro-proliferative, chemotactic, immunomodulatory, and extracellular matrix synthesis–promoting action in vitro (Figure 1), has been widely applied in different medical fields such as traumatology, ophthalmology, maxillofacial surgery, and dermatology.^13-16 The preparation protocol of the PRGF has not changed substantially over the years.^17,18 This is of interest in the context of a systematic review that aims to assess the clinical trials that have been performed over time. Therefore, with the goal of synthetizing the scientific evidence reported in RCTs evaluating the effectiveness of PRGF in chronic ulcer healing, the authors of the current study conducted a systematic review with meta-analysis. By using explicit and reproducible methods, the present systematic review restructured and reanalyzed the available evidence on the effect of PRGF in chronic ulcer healing. This approach overcame the limitations associated with experimental design and the heterogeneity of PRP composition.

The increasing evidence for wound healing with the use of PRGF prompted the current systematic review to determine whether the additional use of PRGF can improve healing in chronic skin ulcers compared with conventional therapy. Efficiency (ie, probability of complete wound healing, infection risk, pain reduction) and cost savings were the parameters used to evaluate this hypothesis.

This systematic review with meta-analysis of RCTs was guided by the Cochrane Collaboration recommendations¹⁹ and performed using the PRISMA reporting guideline.²⁰ The review protocol was registered in PROSPERO (identifier: CRD42022359164).

Study question
The goal of this review was to answer the following question: Can the additional use of PRGF improve the healing of chronic skin ulcers compared with conventional therapy?

Study design
The study design was based on the PICO framework. The participants (P) included patients with chronic cutaneous ulcers. The intervention (I) was a PRP product that corresponded to PRGF technology or accomplished the preparation protocol of PRGF. The comparison (C) was conventional therapy for the treatment of chronic skin ulcers. The outcomes (O) were percentage wound area reduction at last follow-up, probability of complete healing of chronic ulcers at the end of the study, pain reduction, infection risk, and cost savings.

Eligibility criteria
RCTs were included according to the following selection criteria: individuals of any age and sex with chronic cutaneous ulcers managed with PRGF compared with such ulcers managed with conventional therapy. Exclusion criteria included out of scope studies, duplicate studies, non-RCTs, reviews, case reports, retrospective observational studies, preclinical studies (including animal or in vitro reports), and PRP treatments other than PRGF.

Outcomes
The primary outcome was percentage wound area reduction (comparing baseline and posttreatment status). Secondary outcomes were the probability of complete healing in chronic ulcers at the end of the study, pain reduction, infection risk, and cost savings.

Data sources and search strategy
A comprehensive systematic literature search was conducted. RCTs in which PRGF technology was evaluated for the management of cutaneous wounds were included. Systematic online searches were performed using the PubMed, Cochrane Library, and Scopus databases, with no language or time period restrictions, up to September 1, 2022. The search terms were as follows: (“platelet rich plasma” OR “plasma rich in growth factors” OR “platelet-rich” OR “PRGF”) AND (“ulcers” OR “chronic ulcer” OR “skin ulcer” OR “leg ulcer” OR “foot ulcer” OR “decubitus ulcer” OR “pressure ulcer” OR “ischemic ulcer” OR “venous ulcer” OR “varicose ulcer” OR “stasis ulcer” OR “diabetic ulcer” OR “chronic wound”) (MeSH Terms). Articles identified through reference list and bibliographic searches were also considered for data collection based on their title. Additional eligible RCTs were also identified through reference lists of articles and bibliographic searches.

Study selection
Two reviewers (A.P. and M.H.A.) performed the study selection. First, the title and abstract of the studies were screened to exclude those that did not comply with the research question of interest. Second, the full text of eligible articles or articles for which information in the title and abstract was insufficient to make a decision was obtained. Third, full-text publications were checked to identify studies that met all inclusion criteria. Publications that did not meet the inclusion criteria were excluded, and the reasons for the exclusion were recorded (Supplementary eTable; see article PDF). Disagreements were resolved based on discussion with the chief investigator (E.A.) and a vote by the 3 study authors.

The obtention of PRGF was based on a single step and anticoagulated blood centrifugation at low speed, PRP fractioning (excluding the buffy coat), and activation with calcium chloride. PRP was considered to be PRGF if it was prepared following the Anitua protocol or with medical devices and commercially available kits from BTI Biotechnology Institute, Vitoria, Spain.

Data extraction
Relevant data concerning the study, participants, treatment protocol, and main outcomes were extracted into an Excel (Microsoft) spreadsheet. The following data from each study were extracted by 2 reviewers (A.P. and M.H.A.): (a) primary author and year of publication, number of participants, and patient age; (b) ulcer etiology; (c) method of PRGF application; (d) dose; (e) follow-up; (f) main outcomes; and (g) conclusions. Data were entered into RevMan (version 5.4; The Cochrane Collaboration, 2020).

Risk of bias assessment
The methodologic quality of each trial was investigated using risk of bias assessment in accordance with the Cochrane Handbook for Systematic Reviews of Interventions.²¹ The assessment was performed by 2 reviewers (A.P. and M.H.A.). The RoB 2 was used to assess the included RCTs. The RoB 2 tool assesses bias in 5 domains: the randomization process, deviations from the intended interventions, the missing outcomes data, measurement of the outcome, and the selection of the reported result. Each study was classified as “low risk,” “some concerns,” or “high risk” of bias. 

An overall rating of low risk of bias indicated that all the domains were judged to be at low risk. An overall rating of some concerns indicated that at least 1 domain had some concerns, but none of the domains was at high risk. An overall rating of high risk of bias indicated that either at least 1 domain was at high risk, or the extent of domains with some concerns reduced the confidence in the outcome. Any disagreement was resolved through a discussion with the chief investigator (E.A.) and voting by the 3 researchers.

Quality of evidence
The overall certainty of evidence was evaluated by 2 reviewers (A.P. and M.H.A.) using the GRADE approach.²¹ The GRADEpro Guideline Development Tool (McMaster University and Evidence Prime; available from gradepro.org) was used to assess the quality of the body of retrieved evidence.²² In case of disagreement, a discussion with the chief investigator (E.A.) and voting by the 3 researchers were used to resolve the disagreement.

Statistical analysis
The RevMan (version 5.4) software (The Nordic Cochrane Centre, Copenhagen, Denmark) was used to perform the meta-analysis. The following outcomes were included in the meta-analysis: percentage wound area reduction (primary outcome) and the probability of complete healing in chronic ulcers (secondary outcome). To identify percentage wound area reduction, mean differences were calculated from the data generated by each included RCT. The probability of complete healing in chronic ulcers was assessed as a dichotomous variable using the Mantel-Haenszel test and recorded as RR (95% CI). A pooled RR was interpreted as follows: if the risk ratio is greater than 1, then the event is more likely to occur if there was exposure. If the risk ratio is less than 1, then the event is less likely to occur if there was exposure. These formal results were interpreted together with visually assessed between-study variability by looking at the similarity of the individual point estimates and the degree of overlap in the 95% CIs. Heterogeneity among the selected studies was assessed using the I² statistic, with values greater than or equal to 75% indicating substantial heterogeneity. When no significant heterogeneity was found, the fixed-effects model was used; the random-effects model was used when significant heterogeneity was detected. The results were shown in a forest plot of interventions. Due to the low number of studies, sensitivity analysis and reporting bias assessment by funnel plot could not be performed.

Summary of the literature search
The flow diagram of the selected studies is shown in Figure 2. The initial electronic literature search yielded 986 publications. After duplicate removal, 649 studies were selected for screening. The titles and abstracts of these publications were screened, resulting in 113 studies potentially eligible for inclusion. After reading the full-text publications, 108 studies were excluded because they did not fulfill the inclusion criteria (Supplementary eTable). The remaining 5 studies were included in the qualitative synthesis and the quantitative meta-analysis.

Study characteristics
Only RCTs that evaluated the use of PRGF for the management of chronic cutaneous ulcers were included in this systematic review. Male and female participants with chronic wounds of different etiologies, including pressure ulcers and venous ulcers, were included.^23-27 Table 1 summarizes the main characteristics and outcomes of the selected studies. All selected trials were conducted in Spain between 2008 and 2018. The number of participants in each study ranged from 8 to 100. 

The selected studies included an experimental group (E) consisting of PRGF treatment for wound management, except for 1 study that included an additional experimental group (E3) that combined the use of PRGF and hyaluronic acid.²⁵ The selected studies included a control group (C) consisting of conventional wound treatment. The PRGF use evaluated in the selected studies consisted of weekly applications, except for 1 study that included an experimental group consisting of a single dose at baseline (E1) and additional experimental groups consisting of 2 total doses, 1 at day 0 and another at day 15 (E2 [standard wound care and PRGF], E3).²⁵ For the current study, the data selected for the meta-analysis were those of group E2, who received 2 doses at days 0 and 15. The conventional use of wound treatment consisted of weekly applications of ulcer debridement and wound care with physiological saline as the cleansing agent (along with the application of liquid hydrogels, sterile gauzes, or additional dressings), except for 1 study that applied the standard care every 3 days²⁵ and a different study in which standard care was applied 2 to 3 times a week.²³ The follow-up period of the selected studies ranged from 5 weeks to 24 weeks.^23-27

Risk of bias of included trials
The included studies were selected for qualitative analysis and subjected to a risk of bias assessment (Figure 3). The item “Randomization process bias” was judged low risk in 4 studies that provided a random sequence generation description and judged to have some concerns in 1 study. The item “Deviations from the intended interventions bias” was judged to have some concerns in all the studies. The item “Missing outcome data bias” was judged low risk in all the studies. The item “Measurement of the outcome” was judged low risk in all the studies. The item “Selection of the reported result” was judged low risk in all the studies. After the assessment, the overall risk of bias in all the studies was judged to be “some concerns.”

Quality of evidence
Overall evidence was qualified using the GRADE criteria for the outcomes included in the quantitative analysis: percentage wound area reduction (primary outcome) and probability of complete healing in chronic ulcers (secondary outcome) (Table 2). The certainty of evidence was high for both variables. There was no reason to downgrade the certainty for the variable percentage wound area reduction. The certainty of evidence of the variable of complete wound healing was downgraded by 1 level because the optimal information size was not met. However, based on strength of association the certainty was upgraded by 1 level, resulting in a judgment of high certainty.

Percentage wound area reduction
All the selected studies evaluated percentage wound area reduction. Of the total 197 patients included in the 5 studies, 104 (53%) were in the experimental group (PRGF) and 93 (47%) were in the control group (conventional wound treatment). As shown in Figure 4, there was a statistically higher percentage wound area reduction among patients treated with PRGF compared with control (overall mean difference, 56.90 [95% CI, 52.28-61.51), I² = 0%; P = .56). The final test for overall effect demonstrated a z score of 24.2, yielding a 2-sided P of less than .00001. That is, the absolute difference between the mean percentage wound area reduction in chronic ulcers treated with PRGF vs. conventional therapy was 56.9%.

Probability of complete wound healing
Three studies evaluated the probability of complete healing in chronic ulcers.^23,25,27 Of the 78 patients included in these studies, 42 (54%) were in the experimental group (PRGF) and 36 (46%) were in the control group (conventional wound treatment). The present meta-analysis showed that patients treated with PRGF had a statistically higher proportion of completely healed wounds (RR, 7.07 [95% CI, 1.84-27.16], I² = 0%; P = .53). The final test for overall effect demonstrated a z score of 2.85, yielding a 2-sided P of less than .004. According to these results, the use of PRGF resulted in a 607% increase in the proportion of completely healed wounds compared with the use of standard care (Figure 5).

Pain reduction
Two studies evaluated pain reduction.^23,24 At the end of the trial conducted by Burgos-Alonso et al,²³ the average CIVIQ score (Chronic Venous Insufficiency quality of life Questionnaire, a scale used to assess quality of life in patients with chronic venous insufficiency) was 77 points for the PRGF group and 50.8 for the control group. Additionally, pain reduction was measured using the visual analog scale at the end of the study, with a mean score of 6.6 for patients treated with PRGF and 0.01 for patients in the control group. Escamilla Cardeñosa et al²⁴ reported statistically significant differences in pain between the PRGF group and the control group, with a greater reduction in the PRGF group. At baseline, the mean (SD) pain level among patients in the experimental group was 5.55 (2.32), and after PRGF intervention it was 3.16 (2.54). Patients in the control group had a mean pain level of 5.47 (2.60) at baseline and 4.91 (2.46) at the end of the study. 

Infection risk
All the selected studies presented relevant information about infection risk. In the study by Aguirre Anda et al,²⁷ 2 patients in the control group presented with wound infection after 3 and 6 weeks. Important skin eczema of likely allergic origin occurred after 3 weeks in a third patient in the control group. No infections were observed in the PRGF group. However, Anitua et al²⁶ indicated that infection of the ulcer bed developed in 1 patient in the PRGF group and in 2 patients in the control group. In all cases, however, ulcer bed infection resolved in approximately 8 days with oral antibiotic treatment. No differences were found in adverse events between the treatment groups. In Burgos-Alonso et al,²³ 1 patient in the PRGF group and 1 patient in the control group exhibited signs of infection. Another patient in the control group reported a perilesional itch sensation that resolved spontaneously. There were no intergroup differences with respect to adverse events. Moreover, Escamilla Cardeñosa et al²⁴ indicated that an important finding in their study was that none of the ulcers in the PRGF group exhibited signs of infection. No information regarding infection in the control group was provided. No adverse effects were observed in either of the 2 treatment groups. Similarly, Ramos-Torrecillas et al²⁵ indicated that no signs of infection were observed in the pressure ulcers during the follow-up period in any of the study groups. It is not possible, however, to extract definite conclusions from these studies regarding the potential effect of PRGF on chronic ulcer infection risk due to the limited data available, clinical diversity, and methodological heterogeneity.

Cost of PRGF vs. conventional therapy
One study evaluated the cost savings compared the cost of PRGF and standard care. Burgos-Alonso et al²³ indicated that venous leg ulcers in the PRGF group involved a single treatment per week and healed more rapidly compared with such ulcers in the control group. However, PRGF treatment involved a higher mean (SD) cost (€163 [65.9]) compared with conventional treatment (€147.3 [29.7]); this difference was not significant.

Although cutaneous tissue injury is inevitable over a lifetime, poor wound healing is increased in the presence of various risk factors, including rheumatism, diabetes, peripheral vascular disease, and previous scarring. In fact, it is believed that growth factor deficiencies within the damaged tissue may be partly responsible for the impaired healing process.¹ Likewise, metalloproteinase dysregulation and prolonged inflammation may also be involved in excessive degradation of the extracellular matrix, leading to delayed healing. 

Platelets play a fundamental role in the initiation and progression of skin wound healing. Platelets adhere, aggregate, and release numerous growth factors, morphogens, and cytokines that regulate the migration, proliferation, differentiation, and function of keratinocytes, fibroblasts, stem cells, and endothelial cells within cutaneous tissue.²⁸ Some of the growth factors released by platelets that are involved in wound healing include platelet-derived growth factor, transforming growth factor β1, vascular endothelial growth factor, fibroblast growth factor, epidermal growth factor, insulin-like growth factor 1, and hepatocyte growth factor.²⁹ The therapeutic potential of these bioactive proteins has been widely described in the scientific literature.³⁰ Thus, autologous platelet-based therapies are being widely used as alternatives or adjuvant modalities for chronic wound management.³¹

Several systematic reviews have analyzed the therapeutic efficacy and safety of PRPs in the management of chronic wounds.^32,33 The systematic review is a type of secondary study conducted from a defined research question to identify, evaluate, select, and synthesize pieces of evidence from primary studies that meet the predefined eligibility criteria.¹⁹ Most of these systematic reviews of PRP concluded that such therapy promotes wound healing and may be useful in the management of chronic ulcers.³⁴ However, many studies do not distinguish or analyze the specific characteristics of varying PRP protocols and tend to include different types of platelet-rich preparations within a unique meta-analysis.³⁵ In fact, some RCTs selected for the present systematic review have been previously included in other meta-analyses that evaluated different types of PRPs in the management of wound healing. These systematic reviews include PRGF and other PRPs combined in the same meta-analyses when evaluating percentage wound area reduction and the probability of complete healing in chronic ulcers.^4,32-42 Although the results reported by these studies are encouraging, sometimes the simultaneous analysis of different PRPs, with varying composition and preparation techniques, led to heterogeneous or controversial results, because the therapeutic potential of PRP depends significantly on the biological content of each preparation.^36-45 

The current systematic review included RCTs that evaluated the effect of PRGF on chronic skin ulcer healing. The PRGF obtention protocol includes the following characteristics: blood is collected into 9-mL tubes with 3.8% (wt/vol) trisodium citrate as anticoagulant.⁴⁶ Afterward, blood is subjected to a single centrifugation at 580 g for 8 minutes at room temperature (System V centrifuge; BTI Biotechnology Institute). The erythrocytes fall to the bottom of the tube due to a density gradient, while a buffy coat rich in leukocytes remains in the middle of the tube, and a plasma fraction is observable at the top of the tube. The 2 mL (F2) just above the buffy coat and the remaining plasma fraction at the top of the tube (F1) are then separately collected. The buffy coat is avoided to prevent pro-inflammatory events related to the presence of leukocytes.⁴⁷ F1 presents a platelet yield similar to that of peripheral blood, while F2 presents a 2-fold enriched concentration of platelets.^46,47 These 2 fractions are subsequently activated with calcium chloride and further processed to obtain different formulations, such as an injectable liquid, an autologous clot, and a fibrin membrane for wound care.⁴⁸

The results presented herein suggest that PRGF can improve chronic wound healing when compared with conventional wound treatment. The included RCTs demonstrate that both percentage wound area reduction and probability of complete healing in chronic ulcers increased significantly in response to PRGF. In addition, although the evidence remains inconclusive, the qualitative analysis showed that PRGF did not increase infection risk and did reduce wound pain. The main conclusions of this systematic review regarding the pros and cons of the application of PRGF for chronic ulcer healing are summarized in Table 3. These results are consistent with previous findings reported in numerous pilot studies and case series involving PRGF that were not included in the present systematic review.^49-73 These studies corroborate that the delivery of autologous growth factors, fibrin, and bioactive proteins through PRGF promotes rapid granulation tissue development and accelerates the epithelialization rate. 

Nevertheless, it should be considered that the inclusion of heterogeneous experimental units with specific characteristics could influence meta-analyses results. In the present systematic review, only 1 RCT evaluated the effect of PRGF on chronic pressure ulcers (n = 100),²⁵ whereas 3 RCTs assessed the effectiveness of PRGF on chronic venous ulcers (n = 102)^23,24,27 and the remaining RCT included multiple types of chronic ulcers (n = 14).²⁶ The population of these studies consisted mainly of people 65 years of age or older, but patient age varied notably between RCTs, ranging from a mean (SD) of 53 (20) years²⁶ to 82 (5) years.²⁵ Some other variables, including wound size or time of evolution of lesions, are rarely provided, but their effect on intervention outcomes could be significant. Only 2 studies reported on initial ulcer area, with Anitua et al²⁶ noting a mean (SD) area of 5.5 (4.8) cm2 for the intervention PRGF group and 8.9 (8.6) cm² for the control group, and Burgos-Alonso et al²³ reporting 7.1 (9.1) cm² for the intervention PRGF group and 8.9 (6.8) cm² for the control group. Ulcer duration was noted in 2 studies, with Anitua et al²⁶ reporting a mean ulcer duration of 68 (61) days in the PRGF group and 110 (164) days in the control group, and Aguirre Anda et al²⁷ reporting a mean duration of 123.9 (63.7) days in the PRGF group and 51.1 (30.8) days in the control group.

From a regulatory point of view, PRGF technology is classified as a nonindustrial, human-use biomedical product by the Spanish Agency of Medicines and Medical Devices (AEMPS).⁷⁴ Furthermore, within the European regulatory framework, it is covered by Directive 2002/98/EC of the European Parliament, which established the classification of non-replacement therapeutic use of autologous plasma and associated fractions, components, or derivatives as a medicinal product for human use.⁷⁵ In fact, based on data from both Spanish and European health budgets for wound care management, studies published in the past 10 years have reported the positive cost-effectiveness of PRGF when compared with the standard treatment.^39,58,76,77

The current study has several limitations. Although the included clinical trials allowed the analysis of the outcomes of percentage wound area reduction and the probability of complete healing in chronic ulcers, more RCTs are needed to assess the outcomes of pain, infection risk, and cost savings. All the included studies were performed in Spain; more clinical trials in other countries are needed. Homogeneous data collection for other outcomes, such as pain and cost savings, is an important consideration in designing future RCTs. Data on these outcomes could be recorded using validated scales or standardized measurements available in medical guidelines. The identification of homogeneous control therapies could contribute to reducing statistical heterogeneity. Additionally, in the present study no distinction was made between chronic ulcer etiology either between or within RCTs; thus, it was not possible to perform analyses stratified by ulcer type or stage. As a result, the current clinical efficacy of PRGF for a specific type of ulcer, such as venous ulcers, pressure ulcers, or diabetic ulcers, could not be determined. 

The adjuvant use of PRGF has been shown to significantly enhance both percentage wound area reduction and the probability of complete healing in chronic ulcers. More studies are needed to assess the effect of PRGF on pain and infection, and the cost-effectiveness of such treatment. 

Authors: Eduardo Anitua, MD, DDS, PhD^1,2; Ander Pino, PhD^1,2; and Mohammad H. Alkhraisat, MD, DDS, PhD^1,2

Affiliations: ¹University Institute for Regenerative Medicine and Oral Implantology (UIRMI), Vitoria, Spain; ²BTI Biotechnology Institute, Vitoria, Spain

Disclosure: E.A. is the scientific director, and A.P. and M.H.A. are researchers at BTI Biotechnology Institute, the company that developed the PRGF-Endoret technology. This research received no external funding.

Correspondence: Eduardo Anitua, PhD; BTI Biotechnology Institute, R&D, Jacinto Quincoces 39, Vitoria, Alava 01007 Spain; eduardo@fundacioneduardoanitua.org

Manuscript Accepted: April 25, 2024