Introduction
Open surgical wounds or surgical wounds healing by secondary intention are a common and complex problem. These wounds frequently take a long time to heal, require regular dressing changes and present a significant morbidity to the patient and a significant financial burden to healthcare systems. They may need many modalities of treatment, are susceptible to secondary infection, and may also require prolonged hospitalization and/or further operations (Ref. Reference Chetter1). The requirement to manage exudate and avoid repeated wound dressing changes has led to a significant increase in the use of newer modalities of wound management such as negative pressure wound therapy (NPWT) (Ref. Reference Chetter2).
NPWT is currently used widely in many aspects of wound care and has been strongly promoted for use on complex wounds (Refs Reference Armstrong and Andros3, 4). NPWT involves the application of an airtight wound dressing through which a negative pressure is applied, often with any wound and tissue fluid drawn away from the area being collected into a canister. The amount of pressure applied using the therapy can vary and there is no universally agreed protocol for its use (Ref. Reference Peinemann and Sauerland5). A number of surgical and non-surgical specialties prescribe NPWT leading to its widespread implementation in both primary/community care and in tertiary care (Refs Reference Liu6, Reference Gao7).
NPWT is postulated to facilitate wound healing via several primary and secondary mechanisms. The proposed primary mechanisms of action include macro-deformation or wound shrinkage, micro-deformation at the foam-wound interface, fluid removal whilst maintaining a moist environment and stabilization of the wound environment. The proposed secondary mechanisms include alteration of the mechanotransduction pathways and alteration of the wound healing microenvironment including cellular proliferation, differentiation, cell migration, angiogenesis and neurogenesis. Many theories have been proposed to support these primary and secondary mechanisms at a molecular level and the aim of this systematic review is to summarize the currently available evidence regarding the molecular mechanisms of action of NPWT (Refs Reference Borgquist, Ingemansson and Malmsjö8, Reference Kairinos, Solomons and Hudson9, Reference Saxena10, Reference Yang, Chang and Webb11, Reference Adámková12, Reference Kim13, Reference Gerry14, Reference Orgill15).
Methods
Search strategy
Medline, Embase and Elton Bryson Stephens Company (EBSCO) databases, and Clinical trial registries were searched from inception to January 2023 using pre-specified key words (Supplementary file 1). Article screening and extraction was performed by two authors (BR and NS) using the Rayyan online screening and data tool (Ref. Reference Ouzzani16). The reference lists of the retrieved articles and similar review articles in the field were also searched to identify additional papers. Studies that examined the mechanism of action of NPWT in patients or in animal models in preclinical studies or in-vitro studies were included. We included studies that evaluated the effect of any form of NPWT on open surgical wounds including diabetic foot ulcers, pressure ulcers, surgical site infections (SSI), traumatic wounds and post-operative wounds. Studies which focussed on the effects of NPWT on primarily closed wounds or stoma creation were excluded. Case reports, non-English papers, editorials/commentaries, reviews, letters and papers with limited data on methodology were excluded. The study was registered in the PROSPERO database (CRD42022303088) and was performed according to Preferred Reporting Items for Systematic Reviews and Meta Analyses (PRISMA) guidelines (Ref. Reference Moher17).
Data extraction
The key details regarding the method and results were recorded on a bespoke data extraction sheet. Data extraction was conducted by two independent reviewers (BR and NS). Discrepancies were resolved by discussion amongst the authors and a tie-breaking vote from the authors not involved in the screening process. Data elements extracted included study name and year of publication, country, immune cell/mediator(s) described in the study, model (clinical studies, animal wound models or in-vitro),type of wound, specific device with control intervention, duration and time points of analysis, quantitative/qualitative outcomes, duration of follow-up, publication status, funding and conflict of interest.
Assessment of risk of bias (RoB)
Risk of Bias assessment was performed using the RoBANS tool (Ref. 18) for non-randomized studies, the COCHRANE's Risk of Bias 2(ROB-2) tool (Ref. Reference Sterne19) for randomized clinical studies, Office of Health Assessment and Translation (OHAT) tool (Ref. 20) for in-vitro studies or the Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) tool (Ref. Reference Hooijmans21) for animal model studies. The risk of bias assessment and quality assessment figures were produced with the help of the interactive online web application, ‘robvis’ (Ref. Reference McGuinness and Higgins22).
Data synthesis and analysis
Due to the diversity of the variables and immune markers being evaluated and the heterogeneity of the studies being reviewed, it was not possible to pool data and present findings as a meta-analysis. Instead, a descriptive summary was performed with aggregated data presented as a narrative synthesis. The narrative synthesis includes elements such as the immune cell or biomarker of interest, its context and the impact of NPWT on it and the relationship between the immune cell/biomarker and wound healing and the concordance between studies with respect to these findings. Also, each study's methodological and summary characteristics are presented in a separate table to include the author(s), institution, year of publication, sample size, study model, biomarkers/cell markers under review and key findings reported by authors.
Results
Out of 6397 potential studies, 33 studies were included in the systematic review. This included 19 clinical studies, 11 animal studies and 3 in-vitro studies. The exclusion of all the other studies has been outlined in Figure 1 in accordance with the PRISMA reporting guidelines. Out of the 11 animal models, 1 study was conducted in a rabbit model, 5 studies were conducted in murine models and 5 studies in porcine models. Thirteen studies had a high risk of bias and 3 studies had some concerns of bias. Ten clinical studies and 10 animal studies analysed tissue samples from wounds while 5 clinical studies analysed the wound effluent. Five clinical studies and one animal study also used serum samples to correlate the effect of NPWT on wounds. Twenty-eight studies focussed on the effect of NPWT on molecular and cellular biomarkers, while 5 focussed on the effect of NPWT on differential gene expression in wound or serum samples. Substrate analysis was carried out by a combination of quantitative and semiquantitative methods including enzyme-linked immunosorbent assay(ELISA), immunohistochemical(IHC) staining or Western blot analysis. Analysis of gene expression was predominantly carried out by RNA sequencing and/or reverse transcription-quantitative polymerase chain reaction(RT-qPCR). These findings are elaborated in Table 1.
Figure 1. Literature search and study selection.
Table 1. Characteristics of the included studies
Vascular Endothelial Growth Factor (VEGF) was the most frequently studied growth factor in relation to NPWT with 7 papers identified (Refs Reference Labler27, Reference Labler28, Reference Karam34, Reference Mu38, Reference Yang41, Reference Zhou45, Reference Erba49, Reference Jacobs50). Results from clinical studies were reported in 4 studies (Refs Reference Labler27, Reference Labler28, Reference Karam34, Reference Mu38). A significant increase in the local VEGF concentration was seen in clinical wounds treated with NPWT, and reports from animal studies concurred with these findings (Refs Reference Yang41, Reference Zhou45, Reference Erba49, Reference Jacobs50). This increase in VEGF has been postulated to contribute to the increased neovascularization and granulation tissue formation in patients treated with NPWT. VEGF was elevated in all 7 studies which studied its effects. Tumour necrosis Factor alpha(TNF α) was downregulated in 5 out of 8 studies and was the next most common biomarker that was studied (Refs Reference Stechmiller25, Reference Eisenhardt26, Reference Karam34, Reference Wang37, Reference Brownhill44, Reference Dong55). TNF α is considered as a pro-inflammatory cytokine and a potent inducing agent for the upregulation of cytokines, reactive oxygen species and apoptosis. Elevated levels of TNF α in the wound bed have been associated with chronic non healing wounds with reduced granulation tissue production. Transforming Growth Factor Beta (TGF β) was upregulated in 5 out of 7 studies that studied its effects. The data from the in vitro models included in this paper (Refs Reference Lu53, Reference McNulty54, Reference Dong55) suggest that it leads to increased granulation tissue production. NPWT induces the production of TGF-β1, which is crucial for the initiation of the proliferation phase of wound healing. The effect of NPWT on wound healing is mediated through various signals, including TGF-β-Smad, which further underscores the importance of TGF-β in this context. Fibronectin was upregulated in both studies which evaluated its effects (Refs Reference Arslan23, Reference Yang39). Equivocal results were obtained across all studies with respect to Interleukins (IL) and Matrix Metalloproteinases (MMP) including IL1β, IL 6, IL8, IL8, MMP 2, 3 and 9. The effects of NPWT on 43 other molecular biomarkers and 13 different gene expressions were analysed across included studies (Table 1).
Clinical/human studies
Nineteen clinical studies were conducted to assess the MOA of NPWT from 2003 to 2022 with study numbers varying from three to172 patients. Out of these, 12 studies compared the mechanisms of action between NPWT and standard dressings and other studies studied the MOA of NPWT alone. Eleven studies used granulation tissue samples from wound beds, 5 studies studied samples from wound effluents and 5 studies analysed peripheral blood samples. Ten studies had a high risk of bias, one study had some concerns of bias and eight studies had a low risk of bias. (Fig. 2a, 2b) The main cytokines of interest in these studies were VEGF,TNF α, Interleukin(IL)-6, IL – 8, IL 1B, and the family of matrix metalloproteinases (MMP) MMP-1, MMP-2, MMP-9, MMP-13. VEGF was upregulated in all four studies which studies it's effects TNF α was downregulated in four out of four studies, Fibronectin and TGF B1 were upregulated in both studies which studied their effects. There was no concordance regarding the impact of NPWT on the other cytokines, biomarkers and/or genes.
Figure 2. Risk of Bias Assessment of the included studies: (a) RoBANS for non-randomized studies, (b) SYRCLE tool for animal studies, (c) RoB-2 tool for randomized studies.
Animal studies
Eleven animal studies were included in this review out of which five studies used murine models, five studies used porcine models and one study used rabbit models. The sample size ranged from six to 56 animals. Three studies had a high risk of bias, two studies had some concerns and six studies had a low risk of bias. (Fig. 2c) All studies used tissue samples and two studies also used serum samples in addition for analysis. The main cytokines of interest in these studies were TNF α, FGF-2, TGFB1, PDGF and VEGF. Three out of three studies reported the upregulation of VEGF following NPWT. Two studies reported the upregulation of TNF α while one study reported its upregulation following NPWT. The results of most of the included animal studies suggest that many of the cytokines/chemokines and genes are upregulated following the upregulation of NPWT.
In vitro studies
Three studies studied the mechanisms of action of NPWT using in vitro models using murine fibroblasts (Ref. Reference Lu53), human fibroblasts in a 3D fibrin matrix (Ref. Reference McNulty56) and a combination of PMNs, HL 60 cell lines and Macrophages (Ref. Reference Dong55) respectively. Each study examined a completely different set of biomarkers (Table 1). Two studies conducted their experiments in a cell culture under negative pressure. Two studies also reported the upregulation of TGF-B under the effect of NPWT (Refs Reference Lu53, Reference McNulty56). The risk of bias assessment using the OHAT tool revealed a low risk of bias for one study, some concerns of bias and high risk of bias for the other two studies.
Gene expression changes
The effect of NPWT on 17 different gene expressions was assessed in this systematic review (Table 1). Since no two studies evaluated the effects of similar gene expressions, it was not possible to collate these findings. The results of the included studies have suggested that the genes induced by NPWT were associated with cell proliferation and inflammation, and the most down-regulated genes were linked to epidermal differentiation. NPWT has also been postulated to aid differential gene expression to influence re-epithelialization and angiogenesis (Ref. Reference Frear30). NPWT was also observed to alter multiple proteins in the granulation tissue to aid antioxidant pathways and detoxification (Ref. Reference Liu57) The gene ontology enrichment analysis performed in one of the studies was consistent with a number of previous studies showing that the wound healing process was associated with altered extracellular matrix deposition (Ref. Reference Maione58), cytoskeletal deregulation (Ref. Reference Jozic59), dyslipidemia (Ref. Reference Yazdanpanah60) and prolonged inflammation response (Ref. Reference Gouin and Kiecolt-Glaser61). They also unexpectedly found some signalling pathways that seemed weakly relevant to the curative effect of wounds in the enrichment analysis of Kyoto Encyclopaedia of Genes and Genomes (KEGG) signalling pathways, such as thyroid hormone synthesis, thyroid hormone signalling pathway, human T-cell leukaemia virus 1 infection and African trypanosomiasis (Refs Reference Capewell62, Reference McGill63, Reference Shimauchi and Piguet64).
Discussion
This systematic review summarizes the current understanding of the mechanism of action of NPWT based on studies published over the last 20 years. The effect of NPWT was assessed in 33 studies which included 19 clinical studies, 11 animal models and 3 in-vitro studies. Given that more than 43 different molecular biomarkers and 17 different gene expressions were analysed across all studies, there was some clear concordance in actions on several markers studied and variation between studies with respect to the effects on other biomarkers/genes following NPWT (Table 2).
Table 2. Variation in outcomes following NPWT on common biomarkers of interest
It has been postulated that NPWT produces hypoxia driven immunomodulation, local and/or systemic attenuation of the acute inflammatory response, angiogenesis and cell recruitment which combine to produce the clinical effects of NPWT (Refs Reference Orgill15, Reference Scherer51). However, the specific mechanisms of action by which these are achieved continue to be controversial. This is mainly because of the limited concordance among these studies to enable conclusions with regard to the specific mechanisms involved. The previous systematic review in this topic (Ref. Reference Glass65) suggested that human studies supported angiogenesis via VEGF, cell recruitment predominantly via IL-8 and reduced MMP expression, animal models suggested an anti-inflammatory response via IL-10, VEGF, FGF-2, CGRP and substance P and in vitro models suggested increased granulation tissue formation. They also reported that human studies predominantly studied cytokine and MMP data while growth factor data were predominantly derived from animal studies and in vitro models. However, the effect of NPWT on the differential gene expressions was not explored in this review. First insights into the molecular mechanisms behind NPWT suggested that NPWT also induces gene expression changes at the wound bed. These changes were postulated to range from 10-fold induction to 27-fold suppression (Refs Reference Yang41, Reference Nuutila66, Reference Liu67).
Since this previous systematic review, more than 20 newer biomarkers, cytokines and genes have been studied across 19 more recent studies, the summary of which has been collated in this paper. The data summarized in this review confirms that NPWT-induced strain promotes a pro-angiogenic and pro-mitogenic phenotype in subjacent cell proliferation. NPWT induced cell deformation leads to proliferation as a consequence of cytoskeletal tension. Integrins, adhesive contacts within the cell matrix, act as strain gauges, triggering mechanoreceptor signalling pathways (Refs Reference Shyy and Chien68, Reference Wilkes69). Application of NPWT results in positive pressure at the wound bed and hence reduced blood flow in the tissue immediately adjacent to the filler material (Ref. Reference Wilkes70). NPWT enhances specific inflammatory gene expression at the acute phase associated with epithelial migration and wound healing. However, its continued use may inhibit epithelial differentiation (Ref. Reference Nuutila66). NPWT is also associated with an up-regulation of basic fibroblast growth factor (bFGF) and extracellular signal-regulated kinase (ERK) 1/2 signalling, which may be involved in promoting the NPWT-mediated wound healing response (Ref. Reference Yang41).
This systematic review has a few limitations. The inherent heterogeneity of the included studies makes the data unsuitable for meta-analysis. The clinical studies were mostly underpowered and were opportunistic as reported in the previous review. There was a significant variation in terms of the methodology, mainly concerning sample collection/storage, time interval from collection to analysis and techniques utilized to extract and study the biomarkers of interest. The data from a majority of human studies do not take into account extrinsic factors such as collection and storage of samples which do not account for degradation of biomarkers. Moreover, important clinical information including the use of antibiotics, immunosuppressants including corticosteroids or anti-biologicals was not included. Given the extensive number of biomarkers and genes analysed in the included studies, there was limited concordance to suggest a strong correlation between NPWT and regulation of many biomarkers. The time-points at which these biomarkers were studied also varied significantly among studies. It has also been suggested that the magnitude of negative pressure employed is likely to influence blood flow, which in turn influences the degree of hypoxia and reperfusion. This has been shown to alter the expression of mechanosensitive genes (Refs Reference Saxena10, Reference Glass and Nanchahal71).
There were some discrepancies between animal and human studies especially with respect to the regulation of MMP and IL-6. Although the animal studies address most of these issues, the extrapolation of this data to predict clinical biological response is not appropriate. Although in-vitro studies using human cell lines have the potential to circumvent these concerns, only three studies have been conducted over the last 10 years. Only two out of three studies studied the effects of NPWT on human derived cell lines and analysed a completely different set of biomarkers via different methodologies. Although we have a better understanding of the primary and secondary mechanisms of action of NPWT, namely: macrodeformation, cellular proliferation, differentiation, cell migration, angiogenesis and neurogenesis, a comprehensive temporal expression profile of most biomarker changes with NPWT remains elusive. However, VEGF was elevated in all 7 reports which had studied its effects. Tumour necrosis Factor alpha (TNF α) was downregulated in 5 out of 8 studies, Transforming Growth Factor Beta (TGF β) was upregulated in 4 out of 7 studies, and Fibronectin was upregulated in both studies which evaluated its effects.
In conclusion, NPWT stimulates modulation of numerous local and circulating cytokines and growth factor expressions to promote an anti-inflammatory profile. This is most likely achieved by downregulation of TNFα, upregulation of VEGF, TGF-β and fibronectin. This review has also identified many other biomarkers and gene expressions of interest with regard NPWT actions which may help to direct future research in this field.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/erm.2023.24.
Acknowledgements
The authors would like to thank the Academic vascular Surgical Unit at Hull Royal Infirmary for their support with this article.
Funding statement
None.
Competing interest
None.
Data availability statement
The data that support the findings of this study are available from the corresponding author,[BR], upon reasonable request. This paper was shortlisted for the ESVS Prize poster session at the Annual Meeting of the European Society of Vascular Surgery at Rome, Italy in September 2022.
Ethical standards
None.
