Photobiomodulation studies on diabetic wound healing: An insight into the inflammatory pathway in diabetic wound healing

Abstract

Diabetes mellitus remains a global challenge to public health as it results in non‐healing chronic ulcers of the lower limb. These wounds are challenging to heal, and despite the different treatments available to improve healing, there is still a high rate of failure and relapse, often necessitating amputation. Chronic diabetic ulcers do not follow an orderly progression through the wound healing process and are associated with a persistent inflammatory state characterised by the accumulation of pro‐inflammatory macrophages, cytokines and proteases. Photobiomodulation has been successfully utilised in diabetic wound healing and involves illuminating wounds at specific wavelengths using predominantly light‐emitting diodes or lasers. Photobiomodulation induces wound healing through diminishing inflammation and oxidative stress, among others. Research into the application of photobiomodulation for wound healing is current and ongoing and has drawn the attention of many researchers in the healthcare sector. This review focuses on the inflammatory pathway in diabetic wound healing and the influence photobiomodulation has on this pathway using different wavelengths.

Abbreviations

ADP

adenosine diphosphate

ADSC

adipose‐derived stem cell

AGE

advanced glycation end product

AGER

receptor for advanced glycation end products

AKT

protein kinase B

AP‐1

activator protein‐1

ATP

adenosine triphosphate

bFGF

basic fibroblast growth factor

cAMP

cyclic adenosine monophosphate

CAT

catalase

CCO

cytochrome C oxidase

CFU

colony‐forming unit

Cox‐2

cyclooxygenase‐2

DAMPs

damage‐associated molecular patterns

DFU

diabetic foot ulcer

DM

diabetes mellitus

DM1

type 1 diabetes mellitus

DM2

type 2 diabetes mellitus

EC

endothelial cell

ECM

extracellular matrix

FoxO1

forkhead box protein O1

GaAIAs

gallium‐ aluminum‐arsenate laser

GRO‐a

growth‐regulated protein alpha

GRO‐B

growth‐regulated protein beta

ha‐ADS

human allogeneic adipose‐derived stem cells

HGF

human gingival fibroblast

HIF

hypoxia‐inducible factor

HIF‐1a

hypoxia‐inducible factor 1‐alpha

HMOX1

heme oxygenase 1

ICAM‐1

intercellular adhesion molecule 1

IDF

International Diabetes Federation

IkBa

NF‐kappa‐B inhibitor alpha

IL

interleukin

IL‐10

interleukin‐10

IL‐12

interleukin‐12

IL‐18

interleukin‐18

INF‐y

Interferon gamma

iNOS

inducible nitric oxide synthase

IRS‐1

insulin receptor substrate‐1

LED

light‐emitting diode

MAPK

mitogen‐activated protein kinase

MCP‐1

monocyte chemoattractant protein‐1

MMP

matrix metalloproteinase

NAD

nicotinamide adenine dinucleotide

NADPH

nicotinamide adenine dinucleotide phosphate

NF‐κB

nuclear factor kappa‐light‐chain‐enhancer of activated B cells

NIR

near‐infrared

NO

nitric oxide

NPWT

negative pressure wound therapy

OGTT

oral glucose tolerance test

PBM

photobiomodulation

PDGF

platelet‐derived growth factor

PG

prostaglandin

PGI2

prostacyclin

PI3K

phosphoinositide 3‐kinases

PKC

protein kinase C

RAGE

receptor for AGE

ROS

reactive oxygen species

SDF‐1a

stromal cell‐derived factor‐1 alpha

siRNA

short interfering RNA

SOD

superoxide dismutase

STAT

signal transducer and activator of transcription

TGF

transforming growth factor

TGFa

transforming growth factor alpha

TIMP

tissue inhibitors of metalloproteinase

TLR

toll‐like receptor

TNF

tumor necrosis factor

TNF‐α

tumour necrosis factor alpha

VEGF

vascular endothelial growth factor

VEGF‐A

vascular endothelial growth factor A

WHO

World Health Organisation

1. INTRODUCTION

Globally, diabetes mellitus (DM) is reported as one of the main contributors to mortality. There were approximately 537 million adults between the ages of 20 and 79 living with diabetes in 2021, with an estimated rise to 783 million by 2045. ¹ Africa is forecast to experience the most significant increase in diabetes prevalence among the International Diabetes Federation (IDF) regions, with projections showing a surge of 134% to 55 million individuals by 2045. ¹ The World Health Organisation (WHO) forecasts that by 2030 diabetes will rank among the top seven deadliest causes of mortality, underscoring its significant societal impact and the urgent need for improved public health measures and effective management strategies. ² Additionally, it is projected that diabetes will claim the lives of more than 6.7 million individuals aged 20–79 within that year. DM is a chronic metabolic disease characterised by hyperglycemia. ³ Diabetes develops due to the body's insufficient insulin production (type 1 DM [DM1]) or its improper utilisation of insulin (type 2 DM [DM2]). ⁴ , ⁵

Individuals with DM1 may experience atherosclerosis, leading to potential heart attacks and strokes. Insulin contributes to reducing glucose production in the liver. ⁶ In DM1, the inhibition that normally controls liver gluconeogenesis and glycogenolysis is removed, causing an increase in these activities and leading to hyperglycemia. ⁷ In the absence of glucose, cells begin to metabolise fatty acids. This process leads to ketogenesis, generating ketone bodies that acidify the blood, resulting in diabetic ketoacidosis. ⁸ While DM1 results from an autoimmune attack on beta‐cells, DM2 is associated with a broad spectrum of metabolic dysfunctions and highlights the dysfunction that can occur within cell signalling and metabolic pathways. ⁹ Characterised by an impairment in glucose regulation within the body, DM2 affects the circulatory, immune and nervous systems. ¹⁰ , ¹¹ Its primary cause is inadequate insulin production by the pancreas for cell glucose management, and a secondary cause is the continuous inflammation that hampers insulin creation in pancreatic islet β cells. ¹² , ¹³

People living with DM are at risk of developing several life‐threatening complications. Chronic ulcers are one of DMs more common and debilitating complications. As a consequence of DM, damage is imposed on blood vessels and nerves, causing patients to experience sensory loss, particularly in the lower limbs, and as a result, small wounds frequently go unnoticed. Due to the underlying pathologies, these wounds later develop into chronic, non‐healing diabetic foot ulcers (DFUs). ¹⁴ Approximately 60% of individuals with diabetes are likely to experience neuropathy, which may ultimately result in a foot ulcer. ¹⁵ The lifetime risk of developing a DFU ranges from 19% to 34%, with recurrence rates as high as 65% at 3–5 years. ¹⁶ Severe cases of chronic wounds may necessitate amputation, leading to disabilities, decreased quality of life and mortality, placing further strain on patients and healthcare systems. ¹⁷ Estimates suggest that DFUs account for about 20% of hospital admissions in diabetic patients. ¹⁸

During the healing process, DFUs fail to advance through the typical stages, commonly becoming stuck in the inflammatory phase of wound healing. ¹⁹ The healthcare sector has focused on wound healing research, particularly on factors that delay or prevent wound healing, with particular attention on diabetes and infections. ¹⁹ , ²⁰ Current treatments for chronic wounds remain somewhat ineffective, with a high rate of failure and relapse. ¹⁷ It has been estimated that a lower limb, or part thereof, is lost every 30 s worldwide due to DM, and that up to 50% of diabetic patients die within 2 years of amputation, with a 5‐year mortality rate of 50%–70%. ¹⁶ , ²¹ This demonstrates the need to research and develop new therapies directed at efficient and rapid wound healing. Studies have been conducted with regards to photobiomodulation (PBM), and many of these have demonstrated the benefits on wound healing ²² ; however, these benefits have not been properly optimised. This review focuses on the inflammatory pathway in diabetic wound healing and the influence PBM has on this pathway at different wavelengths.

2. WOUND HEALING

Wound healing is a sophisticated and varied process that aids the body in restoring the integrity of injured tissue. ²³ Under normal circumstances, wounds heal flawlessly via a progression of overlapping phases, but wounds related to health conditions such as diabetes and obesity are problematic. Wound healing is a complex process that involves the collaboration of many cell types and their products, which interact with each other and the extracellular matrix (ECM). ²⁴ These cells include platelets, macrophages, fibroblasts, epithelial cells and endothelial cells (ECs). ²⁴ , ²⁵ , ²⁶ Wound healing is also influenced by proteins and glycoproteins such as cytokines, chemokines, growth factors, inhibitors and cellular receptors. ²⁷

Under normal physiological conditions, there are four phases of wound healing (haemostasis, inflammation, proliferation and remodelling) that involve several cells and molecules responsible for regaining a healthy barrier (Figure 1). ²⁸ Haemostasis occurs immediately after injury and lasts for several hours. Blood vessels constrict to reduce blood flow. Platelets aggregate to form a clot, and fibrin is deposited to stabilise the clot. ²⁹ , ³⁰ , ³¹ , ³² Following this, the wound progresses to the inflammatory stage, which persists for 1–4 days. Immune cells such as neutrophils and macrophages migrate to the wound site to remove debris, pathogens and damaged tissue. This phase is characterised by redness, swelling, heat and pain. ³³ , ³⁴ , ³⁵ , ³⁶ , ³⁷ The proliferation phase, which lasts 4–21 days, forms new tissue as fibroblasts produce collagen and ECM. Angiogenesis occurs to supply the new tissue with nutrients and oxygen. Granulation tissue forms, and epithelial cells migrate over the wound bed to cover the new tissue. ³⁸ , ³⁹ , ⁴⁰ The fourth and final phase, known as the remodelling or maturation phase, is marked by ECM remodelling and the formation of scar tissue. ⁴¹ This phase can last for 21 days to a year. ⁴² Any error or delay in these prominent phases may result in impaired healing. Chronic wounds are characterised by their inability to advance through the usual healing stages, primarily due to a prolonged inflammatory phase and interruptions in the proliferative and remodelling stages. ²⁶ Chronic wounds retain pro‐inflammatory macrophages without changing into anti‐inflammatory forms due to improper or delayed progression through these stages, inhibiting wound healing. ⁴³ , ⁴⁴ , ⁴⁵ , ⁴⁶ , ⁴⁷

FIGURE 1.

Summary of the primary stages of wound healing following injury. ECM, extracellular matrix; FGF, fibroblast growth factor; IL‐1, interleukin‐1; IL‐6, interleukin‐6; MMPs, matrix metalloproteinase; NF‐κB, nuclear factor kappa‐light‐chain‐enhancer of activated B cells; PDGF, platelet‐derived growth factor; TGF‐β, transforming growth factor‐β; TIMPs, tissue inhibitors of metalloproteinase; TLRs, toll‐like receptor; TNF‐α, tumour necrosis factor alpha; VEGF, vascular endothelial growth factor.

3. INFLAMMATION AND DIABETIC WOUND HEALING

Diabetes significantly complicates wound healing, rendering such wounds chronic. Chronicity is caused by prolonged inflammation, proliferation and remodelling, which often leads to tissue fibrosis or unhealed wounds. ⁴⁸ Long‐term poorly controlled diabetes causes nerve fibre damage through the formation of advanced glycation end products (AGEs), activation of protein kinase C (PKC), increased reactive oxygen species (ROS), nitric oxide (NO) blocking, DNA damage and chronic inflammation. ⁴⁹ AGEs are natural chemicals that accumulate from embryonic onset and are part of normal ageing. ⁵⁰ The non‐enzymatic Maillard reaction creates AGEs by reducing sugar carbonyl groups like glucose and protein, lipids and nucleic acid amino groups. The early and middle steps of the Maillard reaction produce reversible intermediate products such as Schiff bases and Amadori products, followed by classic rearrangement that generates AGEs and cross‐links proteins. ⁵¹

Oxidative stress results from ROS production exceeding antioxidant defences. ⁵² Oxidative stress can affect the nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) pathway in diabetes through the PKC family, which includes serine/threonine kinases engaged in various cellular processes. ⁵³ Diabetes is characterised by an increase in ROS production and a weakened antioxidant defence system due to excessive blood glucose, insulin resistance and mitochondrial abnormalities. ⁵⁴ The overproduction of ROS can damage proteins, lipids and DNA, causing cell dysfunction and disrupting physiological functioning. ⁵⁴ , ⁵⁵ The frequent mitochondrial dysfunction observed in diabetes is linked to increased ROS emission. ⁵⁵ NO promotes vasodilation, which increases blood flow to the wound site, delivering oxygen and nutrients for tissue repair. ⁵⁶ Diabetes‐related hyperglycemia causes oxidative stress, which reduces endothelial NO production. ⁵⁴ Reduced NO availability impairs vasodilation, thereby reducing blood flow and oxygen to the wound site and slowing healing. NO blocking involves the inactivation of NO by ROS and down‐regulation of NO‐producing enzymes. ⁴⁸ , ⁵⁴ As a result, diabetic wounds show impaired angiogenesis, reduced collagen synthesis and a diminished immune response, all of which are critical for effective wound healing. ⁴⁸

In individuals with diabetes, elevated glucose levels and free fatty acids may trigger the activation of PKC, which subsequently leads to the activation of NF‐κB and increases the expression of pro‐inflammatory cytokines, chemokines and adhesion molecules. ⁵⁷ Oxidative stress not only prompts the phosphorylation and degradation of the NF‐κB inhibitor protein, inhibitors of κappa B alpha (IKBα), but also directly activates NF‐κB via the activation of inhibitor of nuclear factor‐κB kinase (IKK). ⁵⁸ This results in the release of active NF‐κB dimers, which translocate into the nucleus and activate target gene expression. It is not completely clear how diabetes affects local inflammatory pathways. A persistent increase in pro‐inflammatory cytokines in diabetes indicates a tendency towards inflammatory states and the development of inflammatory complications. ⁵⁹ Cells that act as the first line of defence, such as neutrophils, have diminished functional activity and altered cytokine release profiles. ⁶⁰ A delay in wound healing increases the chance of wound infection, which further slows the transition from the inflammatory to proliferative phase. ³⁶ Infections also accelerate the production of ROS and AGEs, further escalating inflammation and oxidative stress at the wound site. AGEs are associated with dysfunctionality of ECs, insulin resistance and vascular inflammation. ⁶¹ NF‐κB activation by ROS during hyperglycemia promotes pro‐inflammatory cytokines and attracts immune cells to the inflammation site. ⁶² Additionally, hyperglycemia‐induced AGEs can activate NF‐κB, exacerbating inflammation in diabetic states. AGEs activate NF‐κB signalling by interacting with their receptor, receptor for AGE (RAGE)/receptor for advanced glycation end products (AGER), resulting in the increased secretion of pro‐inflammatory cytokines and ROS. ⁶³ , ⁶⁴

Through activation of the NF‐κB pathway, elevated AGE levels contribute to a prolonged inflammatory response and apoptosis associated with diabetic impaired wound healing. This increases mitochondrial oxidative stress, decreases adenosine triphosphate (ATP) production, alters mitochondrial morphology and changes mitochondrial dynamics. ⁶⁵ , ⁶⁶ Tumour necrosis factor alpha (TNF‐α), cyclooxygenase‐2 (Cox‐2) and interleukin‐6 (IL‐6) are mediated through NF‐κB. NF‐κB regulates pro‐inflammatory genes and is active in several pro‐inflammatory conditions (Figure 2). In Table 1, the key inflammatory cytokines and transcription factors implicated in diabetic wound healing are listed. TNF‐α and IL‐1β are primary cytokines that govern immune responses and trigger the release of secondary cytokines such as IL‐6 and IL‐8 from monocytes and macrophages. IL‐1β is a major player in a wide array of auto‐inflammatory diseases and acts as a key promoter of systemic and tissue inflammation in DM. ⁸⁰ Mirza et al. ⁸¹ showed that the expression of IL‐1β was increased in diabetic wounds and that inhibiting the IL‐1β pathway resulted in improved healing via stimulating a switch from the pro‐inflammatory to a reparative monocyte–macrophage phenotype. ⁸¹

FIGURE 2.

Hyperglycemia‐induced formation of non‐enzymatic advanced glycosylation products (AGEs) contributes to the inflammatory response in diabetic wound healing. The binding of AGE to their receptors (RAGE) activates the receptors for the cytokines interleukin‐1 (IL‐1), tumour necrosis factor alpha (TNF‐α) and growth factors. As a result, reactive oxygen species (ROS) is increased which causes damage to cellular structures and induces an imbalance in nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) regulation that contributes to the pathogenesis of diabetes. Pro‐inflammatory cytokines (IL‐1, cyclooxygenase‐2 [Cox‐2], interleukin‐6 [IL‐6] and TNF‐α) and NF‐κB activities are enhanced during the hyperglycemic state. As a result, there is increased ROS production, impaired vasculogenesis and degradation of the extracellular matrix (ECM), resulting in impaired wound healing.

TABLE 1.

Inflammatory cytokines, transcription factors and cells involved in diabetic wound healing and their roles.

Cytokine/transcription factor	Main source	Role
TNF‐α	Monocytes, macrophages, natural killer cells, adipocytes and CD4+ lymphocytes	Induce apoptosis and necrotic cell death. 67 Alters endothelial permeability. 68 Alters distribution of adhesion receptors involved in cell–cell adhesion. 69 Cell proliferation, apoptosis, anti‐infection, pro‐inflammation and cytokine production. 70
IL‐1	Macrophages and epithelial cells	Enhances ICAM‐1 synthesis. 71 Enhances vascular cellular adhesion molecule‐1 by glomerular endothelial cells. 72 Induces de novo synthesis and expression of ICAM‐1 by glomerular mesangial cells and renal tubular epithelia. 72
IL‐6	T cells and macrophages	Generate inflammation by controlling differentiation, migration, proliferation and cell apoptosis. 36 Regulates a wide range of immune activities. 36 Produces cell adhesion molecules, acute phase protein and facilitates the release of other cytokines in response to inflammatory stimuli. 73
IL‐8	Expressed in renal tubular epithelia	Induces functional chemokine receptor expression in human mesangial cells. 74 Produces other inflammatory cytokines (including IL‐1 and TNF‐α). 75 Up‐regulation of ICAM‐1, as well as apoptosis of endothelial cells. 75
TGF‐β	T‐cells and macrophages	Inhibit production of pro‐inflammatory cytokines. 76
HIF	Stimulated by angiotensin II and PDGFs)	Plays a role in the cellular response to hypoxia. 77 Plays a crucial role in the angiogenesis of tumours and mammalian development. 78
STAT	Activated by membrane receptor‐associated JAKs	Control fundamental cellular processes, such as cell survival, cellular proliferation and cell differentiation. 79
NF‐κB	Activated by inducible degradation of IκBα triggered through its site‐specific phosphorylation by a multi‐subunit IκB kinase (IKK) complex	Regulator of the acute phase of inflammation. 79

Abbreviations: HIF, hypoxia‐inducible factor: ICAM‐1, intercellular adhesion molecule 1; IL‐1, interleukin 1; IL‐6, interleukin 6, IL‐8, interleukin 8; NF‐κB, nuclear factor kappa‐light‐chain‐enhancer of activated B cells; PDGFs, platelet‐derived growth factors; STAT, signal transducer and activator of transcription; TGF‐β, transforming growth factor‐β; TNF‐α, tumour necrosis factor alpha.

Diabetes leads to higher TNF‐α levels, which prolong the inflammatory phase and delay wound healing. TNF‐α is released by inflammatory cells and adipocytes in response to chronic inflammation. TNF‐α can be an important diabetic marker and levels can be used to predict glycaemic control in obese diabetic patients. A study by Alzamil ⁸² showed that diabetes was strongly associated with elevated levels of TNF‐α, which was significantly elevated in patients with higher glycated HbA1c values. Data indicates that diabetes is strongly associated with elevated levels of TNF‐α, which is related to insulin resistance. This is because TNF‐α induces insulin receptor substrate‐1 (IRS‐1) serine phosphorylation, which inhibits peripheral insulin action and causes insulin resistance. ⁸³ The role of TNF‐α in diabetes is not fully understood and is still under investigation.

TNF‐α is known to be a central regulator of inflammatory and immune‐mediated events. ⁸⁴ It is mostly produced in peripheral and/or adipocyte tissue, and induces tissue‐specific inflammation through its involvement in ROS generation and the activation of various transcriptional pathways. ⁸⁴ It stimulates inflammation by attaching to its receptor and activating intracellular signalling pathways. TNF‐α activation stimulates NF‐κB and activator protein‐1 (AP‐1) transcription factors. These two transcription factors regulate signalling and inflammation. ⁸⁵ TNF‐α also plays a role in the generation of prostaglandins (PGs) and the induction of Cox‐2. During persistent inflammation, TNF‐α triggers caspases, leading to apoptosis.

Cox‐2, an inducible enzyme in macrophages, fibroblasts and immune cells, regulates inflammation through the production of PGs. Cox‐2 is up‐regulated during inflammation and is regulated by different cytokines, including IL‐1β, IL‐6, TNFα and various growth factors. ⁸⁶ In diabetes, the up‐regulation of Cox‐2 increases the synthesis of various prostanoids, which interfere with the regulation of vasomotor function and are implicated in the inhibition of apoptotic cell death. ⁸⁷ Cox‐2 is involved in the production of four principal bioactive PGs namely PGE2, prostacyclin (PGI2), PGD2 and PGF2α. These PGs regulate cell proliferation, cell differentiation, vascular tone and energy metabolism. ⁸⁸ PG levels and profiles fluctuate drastically during inflammation.

IL‐6 is a significant pro‐inflammatory cytokine that induces inflammation and insulin resistance in DM2 and protects against and amplifies inflammation. ⁸⁹ IL‐6 is a well‐known hallmark of DM because of its dysregulation. ⁸⁹ Patients with both DM1 and DM2 have elevated IL‐6 serum levels. ⁸⁸ , ⁹⁰ IL‐6 affects adipocytes, ECs, smooth muscle cells, fibroblasts, lymphocytes and macrophages throughout the wound healing inflammatory and tissue remodelling phases. ⁹¹ This makes IL‐6 an ideal marker since it aids wound healing and is elevated in diabetic ulcer fluid.

Higher serum levels of TNF‐α, monocyte chemoattractant protein‐1 (MCP‐1), matrix metalloproteinase (MMP)‐9 and fibroblast growth factor (FGF)‐2 have also been associated with non‐healing DFUs. ⁹² Diabetics' elevated pro‐inflammatory cytokines increase MMP production and decrease tissue inhibitors of metalloproteinase (TIMP), which degrades the ECM, reduces collagen synthesis and fibroblast proliferation and impairs cellular migration. Treatments that can reduce this hyper‐inflammatory state aid in their healing and progression through the stages of wound healing. ⁹³

4. CURRENT PHARMACOLOGICAL INTERVENTIONS EMPLOYED FOR THE TREATMENT OF DIABETIC FOOT ULCERS

Innovative treatment modalities are critical to improve healing outcomes and reduce the risk of complications, such as amputation. Several interventions employed to manage DFUs include systemic and oral pharmacotherapies, physical therapies and topical therapies (Figure 3). Current diabetic wound treatments include antibiotics to avoid infections, tissue excision and blood glucose management. ⁹⁴ Papain‐based formulations, clostridial collagenase, larval therapy and hydrogels are used in topical wound care to debride the necrotic tissue. ⁹⁵ Research has demonstrated that immunomodulatory agents such as honey can reduce the time needed for debridement and wound healing, while also eliminating bacteria from DFUs. ⁹⁶ Non‐surgical topical autolytic, enzymatic and biological therapy is also available. However, the efficacy of these methods is not well‐documented, largely due to the prolonged time needed for effective debridement. ⁹⁷ In severe ulcers, topical medications generally fail to penetrate deeper tissues. Frequent topical antibiotic use can lead to antibiotic‐resistant microorganisms and contact dermatitis due to allergic reactions to treatment components. ⁹⁸ , ⁹⁹

FIGURE 3.

Management strategies for diabetic wound care. These include the use of systemic and oral medications, physical therapies and topical treatments to promote healing. Advanced therapies, such as negative pressure wound therapy or bioengineered tissue, are also utilised to speed up the healing process.

Systemic and oral medications are easy to administer, which improves patient compliance, a feature crucial for those dealing with multiple health concerns. ¹⁰⁰ Early healing depends on their infection‐fighting ability. Their broad‐spectrum antibacterial effect helps in the early stages of treatment if identifying the responsible bacteria is difficult. ⁷⁶ Cilostazol, pirfenidone, low‐molecular‐weight heparin, hyperbaric oxygen therapy, prostacyclin analogues and ketanserin are among the treatments. ¹⁰¹ Despite its simplicity, systemic and oral medication have drawbacks. Systemic medications can influence tissues and systems not directly engaged in wound healing, causing adverse effects, while oral medications can cause gastrointestinal distress, which can be especially problematic for diabetes patients. ⁷⁶ , ¹⁰⁰ , ¹⁰¹ Patients with diabetes often take multiple medications, increasing the risk of adverse interactions and the complexity of diabetic wound management regimens can lead to poor adherence, reducing treatment efficacy. ⁷⁷

Various physical therapy techniques promote tissue regeneration and speed up diabetic wound healing. Options such as ultrasound therapy, electrical stimulation and laser therapy are among the available therapies. Every treatment option operates through a distinct mechanism, whether it is enhancing blood flow or minimising the risk of infection in the wound. ¹⁰² Negative pressure wound therapy (NPWT) involves administering controlled negative pressure at the wound site. A vacuum pump and hermetically sealed dressing help this procedure. The negative pressure drains fluid, reduces swelling and promotes granulation tissue growth. NPWT significantly accelerates diabetic wound healing by improving blood circulation, decreasing bacterial presence and stimulating tissue growth. ¹⁰³ It maintains a moist environment that speeds wound healing, outperforming dressings and topicals. ¹⁰⁴ Although NPWT proves to be an effective instrument in managing diabetic wounds, its applicability is not universal across all patient profiles or wound categories. Weighing factors include the specific wound site, existing infections and the patients' overall health condition. Patients may experience discomfort and skin irritation, and there is a need for specialised training to give the treatment.

Nanotechnology represents another therapeutic avenue, promising a transformative solution to the treatment of diabetic wounds. Benefits of this technology include faster healing, precise medication distribution and improved monitoring. ¹⁰⁵ However, we cannot overlook challenges like the risk of toxicity, financial considerations and regulatory obstacles. Using hydrogel frameworks and short interfering RNA (siRNA) technologies to locally modify gene expression to improve healing is another potential treatment. ¹⁰⁶ This technique uses hydrogels' biocompatibility and moisture‐retention and siRNA's gene‐controlling abilities to target key wound repair mechanisms. Hydrogels regulate siRNA release to inhibit genes over time. Customising hydrogels allows the therapy to target wound‐healing cells. ¹⁰⁶ Although biocompatible, some hydrogel compositions may cause immunological responses or toxicity, especially with long‐term use, and optimising siRNA release to ensure therapeutic efficacy without overdosing is challenging. ¹⁰⁶ , ¹⁰⁷ Financial investment and mass production of hydrogel‐siRNA systems for clinical use are also key obstacles.

For diabetic wound healing, gene therapy, neuropeptide delivery, stem cell therapy and PBM are studied. Gene therapy is promising for diabetic wound healing due to the intricacy of repair processes damaged in diabetes. By correcting fundamental genetic dysfunctions that hinder wound repair, it may lead to more lasting and infinite remedies. ¹⁰⁸ Gene therapy has many benefits, but also raises ethical questions about gene manipulation and genetic integrity, as well as safety concerns about unforeseen effects and long‐term repercussions. ¹⁰⁹ Therapies such as neuropeptides present an innovative and comprehensive strategy for enhancing the healing of diabetic wounds. They provide notable benefits such as stimulating the formation of new blood vessels, facilitating the movement of cells critical for repair, regulating inflammatory responses and managing pain. ¹¹⁰ However, several obstacles accompany the application of neuropeptides, including financial burden, difficulties in obtaining these therapies, the possibility of negative side effects, and the need for further in‐depth research over extended periods.

Emerging treatments like stem cell therapy may improve diabetic wound healing by improving tissue regeneration and reducing inflammation. ¹¹¹ However, regulatory, ethical and tumour development issues must be overcome. Despite these challenges, the prospect of stem cell therapy in the management of diabetic wounds is encouraging, as continuous research facilitates further innovations. ¹¹¹ , ¹¹²

5. IMPACT OF PBM ON DIABETIC WOUND HEALING AND INFLAMMATORY PROCESSES

PBM is of interest to many researchers as it enhances tissue repair, decreases pain and inflammation and prevents cell and tissue damage. ¹¹³ Other advantages of PBM include enhanced remodelling and repair of bone, restoration of normal neural function and modulation of the immune system. ¹¹³ , ¹¹⁴ PBM is a low‐risk and non‐invasive medical therapy for conditions such as diabetic chronic wounds and burns. PBM involves the use of low‐powered non‐ionising forms of light from lasers, light‐emitting diodes (LEDs), and broad‐band light with appropriate filters in the visible and near‐infrared (NIR) spectrum, and leads to physiological changes and therapeutic benefits to enhance the repair of tissue. ¹¹⁴ PBM is capable of promoting EC migration, proliferation and organisation for angiogenesis, infiltration of inflammatory cells to speed up healing and immune surveillance, as well as increase fibroblast matrix synthesis and wound contraction. ²⁷ , ¹¹⁴ PBM is widely used on different animal models, in vitro, and clinically, using different wavelengths and irradiances (power density) to induce wound healing; however, the challenge is that an optimal set of parameters has not been identified. ¹¹⁵

The effects of low‐dose laser treatments for wound healing were first described over 50 years ago by Endre Mester, who reported that the application of low‐dose laser treatments promoted wound healing and regrowth of hair in mice. ¹¹⁶ Niels Ryberg Finsen's turn‐of‐the‐20th‐century work on red and blue light on lupus vulgaris using concentrated light radiation led to low‐dose light therapy. ¹¹⁷ This study was recognised for the Nobel Prize in Medicine and Physiology in 1903. Light Amplification by Stimulated Emission of Radiation (LASER) was invented by Theodore Maiman on the basis of theoretical work conducted by Albert Einstein in 1917. ¹¹⁸ Light energy as a wound therapy tool was then rediscovered.

Exposure of cells or tissue to PBM results in the absorption of light (photon) energy that leads to an alteration in their physical and chemical properties. This is also known as photochemical and photobiological responses. ¹¹⁴ When applied with the appropriate treatment parameters, PBM serves as an effective strategy for wound healing. Wavelength affects how deeply light can penetrate through tissue. ¹¹⁵ The depth of penetration is higher for red light than it is for blue, violet, yellow and green light. NIR and infrared wavelengths penetrate deeper than red light. Wavelengths that are shorter than 450 nm can only penetrate to a depth of 200–1000 μm. ¹¹⁹ Wavelengths within the mid‐visible range, specifically from green to orange, have the ability to penetrate to a depth of 1–3 mm. ¹¹⁹ Wavelengths within the red to NIR range have the ability to penetrate to a depth of 1–3 cm. ¹¹⁹ This is a sufficient penetration depth to initiate healing of DFUs.

The extent to which light may permeate the skin and induce biological responses is contingent upon its wavelength. Dungel et al. ¹²⁰ examined the therapeutic benefits of pulsed LED light at different wavelengths on diabetic mouse excision wound healing. ¹²⁰ Pulsed LED was used with an irradiance of 40 mW/cm² with wavelengths of 470 nm (blue), 540 nm (green) or 635 nm (red). The study findings showed that red and green light accelerated wound healing, while blue light did not. Laser Doppler imaging showed that wavelength‐dependent light absorption enhanced wound perfusion. Low‐wavelength green to blue light increased wound surface temperature, but red light, which penetrates deeper tissue, boosted core body temperature. ¹²⁰ The deepest penetration and biological stimulation occur in NIR wavelengths. Zhang et al. 2016, ¹²¹ examined how much light penetrates biological tissues at 900–1650 nm. No matter the transmission or reflection geometry, deep tissue had the maximum spatial contrast in the 1300–1375 nm wavelength range. In strongly pigmented tissue, like the liver, 1550–1600 nm was also prominent. ¹²¹

Other important parameters to consider include power density or irradiance (mW/cm²), and energy density or fluence (J/cm²). Some research groups recommend using a fluence of 1–4 J/cm² and an irradiance of less than 100 mW/cm² for continuous exposure throughout similar time periods. ¹²² However, studies using higher fluences have shown beneficial effects. ¹²²

A well‐known molecular mechanism of PBM is based on the absorption of light in the red and NIR spectrum by a key enzyme of the respiratory chain within the mitochondria, cytochrome C oxidase (CCO). ¹²³ , ¹²⁴ , ¹²⁵ This enzyme mediates the transfer of electrons from cytochrome C to molecular oxygen. CCO absorbs incident photons and initiates a photochemical cascade. This results in increased generation of ATP and intracellular ROS within the electron transport chain. PBM alters mitochondrial CCO activities, activating IKK, a key regulator of NF‐κB activation. IKK is increased due to the generated ROS and causes NF‐κB to translocate into the nucleus and promote the transcription of target genes such as IL‐2, IL‐6, IL‐8, inducible nitric oxide synthase (iNOS), Cox‐2 and MMP‐9. ⁵⁸ , ¹²⁶

To demonstrate that PBM can improve mitochondrial bioenergetics and stimulate the rate of wound healing in diabetes (Table 2), Mehrvar et al. ¹²⁷ used PBM at a wavelength of 670 nm and energy density of 4.5 J/cm² for 5 days. Their findings reported that there was a 43% decrease in wound area on the ninth day in treated diabetic mice. Also, there was a 75% increase in redox ratio. Meireles et al., ¹²⁸ compared two wavelengths, 660 and 780 nm (total energy density of 20 J/cm²), on third‐degree burns created on the dorsum of streptozotocin‐ induced diabetic rats. When PBM was used daily, up to 21 days, a wavelength of 660 nm was more effective than 780 nm. However, 780 nm did affect the early stages and development of inflammation. PBM at 660 nm influenced the inflammatory response by increasing mononuclear cells, granulation tissue and neo‐angiogenesis. ¹²⁸

TABLE 2.

Various effects of photobiomodulation (PBM) on inflammation in diabetic wound healing.

Model	Laser parameters	Result
Wounded diabetic mice model (db/db−/− mice)	Daily PBM for 5 days at 670 ± 10 nm GaAlAs LED arrays (75 cm2), 60 mW/cm2, 90 s and 4.5 J/cm2	Increased wound closure, ATP and proliferation 127
Streptozotocin‐induced diabetic Wistar rats with third‐degree burns	Daily PBM for 21 days at 660 or 780 nm (35 mW), 24 h, 20 J/cm2 and 5 J/cm 2	PBM at 660 nm increased mononuclear cells, granulation tissue and neo‐angiogenesis. PBM at 780 nm affected early inflammation 128
Induced diabetic human fibroblast cells	Single exposure at 830 nm, 10.5 mW/cm2 and 5 J/cm2	Decreased apoptosis and increased cell proliferation and intracellular NO and ROS 129
Isolated human skin fibroblasts	Single exposure at 660 nm, 10.22 mW/cm2 and 5 J/cm2	Increased fibroblast migration, proliferation, viability and collagen production 130
Streptozotocin‐induced female diabetic Wistar rats	PBM administered four times on Days 1, 3, 8 and 10 at 660 ± 2 nm (30 mW) with 1 J/cm2, and a single exposure with 4 J/cm2 on Day 1	Accelerated wound closure, increased acute inflammation, leukocyte chemotaxis and myofibroblasts 131
Streptozotocin‐induced diabetic Wistar rats	PBM immediately post‐wounding and every second day for 4 sessions at 808 nm, 20 W, 4 W, 50 Hz 0.1 W/cm2 and 10 J/cm2	Faster reepithelialization, diffuse acute inflammation, fibroblast proliferation and granulation tissue formation 132
Streptozotocin‐induced male diabetic Wistar rats	PBM 6 days a week for 15 days at 890 nm, 80 Hz, 1.92 W/cm2 and 0.324 J/cm2	Better oral glucose tolerance test, decreased colony‐forming units, increased bending stiffness, maximum force and stress high load of wounds 133
Male diabetic Wistar rats	PBM 6 days a week for 15 days at 890 nm, 80 Hz, 1.92 W/cm2 and 0.324 J/cm2	Increased wound strength, decreased inflammatory cells, improved granulation tissue formation and angiogenesis 134
Volunteers (eight patients) aged between 30 and 75 years old with diabetic and pressure ulcer grades II, III or IV	Daily PBM for 12 days at 660 nm, 100 mW, 12 s and 2 J/cm2	Improved granulation tissue, reduced TNF‐α gene expression and increased VEFG and TGF‐β gene expression 135
Cultured human gingival fibroblasts	660 nm, 70 mW, 528 s and 8 J/cm2	Decreased expression of TNF‐α, IL‐1β, IL‐6 and IL‐8 136
Diabetic wounded fibroblast cell model	Single exposure at 660 nm and 5 J/cm2 (11 mW/cm2)	Increased Cox‐2, decreased IL‐6 137
Male diabetic Wistar rats. Wounds infected with MRSA	PBM immediately post‐surgery and 6 days a week for 16 days at 890 nm ± 10, 80 Hz, 0.001 W/cm2, 200 s and 0.2 J/cm2 (total 1.8 J/cm2/session)	Increased mRNA levels in HIF‐1α, bFGF, SDF‐1α and VEGF‐A 138
Diabetic rats (DM2)	890 nm, 80 Hz, 0.324 J/cm2 and 0.001 W/cm2	Reduced CFUs wound areas, wound size 139
Isolated murine embryonic fibroblasts	810 nm, 0.3, 3 and 30 J/cm2, 5, 15 and 30 min	Increase in NF‐κB activation, cellular ATP levels 140
Male diabetic Wistar rats	640 nm, 40 mW, 0.04 cm2, 4 s and 0.16 J and 4 J/cm2 for 10 days	Increase in NF‐κB activity 141
ADSC‐fibroblast co‐culture cell model	Single exposure at 660 (11.12 mW/cm2) or 830 nm (10.78 mW/cm2) and 5 J/cm2	Enhanced cell, increased PI3K, AKT, SOD, CAT and HMOX1, reduced FoxO1 and DNA damage 142
Human immortalised keratinocytes, human monocyte‐like cell line, human skin fibroblasts, human umbilical vein endothelial cells and streptozotocin‐induced diabetic rat model	400–900 nm, 10 mW/cm2 and 0.5–4 J/cm2	Peak effects on cell proliferation, bimodal effects, enhanced cell migration 67

Abbreviations: ADSC, adipose‐derived stem cell; ATP, adenosine triphosphate; bFGF, basic fibroblast growth factor; CAT, catalase; CFU, colony‐forming unit; Cox‐2, cyclooxygenase‐2; DM2, type 2 diabetes mellitus; GaAIAs, gallium‐ aluminum‐arsenate laser; HMOX1, heme oxygenase 1; LED, light‐emitting diode; NF‐κB, nuclear factor kappa‐light‐chain‐enhancer of activated B cells; NO, nitric oxide; PI3K, phosphoinositide 3‐kinases; ROS, reactive oxygen species; SOD, superoxide dismutase; TGF‐β, transforming growth factor‐β, TNF‐α, tumour necrosis factor alpha; VEGF, vascular endothelial growth factor.

NIR light has been shown to influence in vitro wound healing by reducing pro‐inflammatory cytokines and effecting intracellular ROS and NO. An in vitro study by Houreld et al., ¹²⁹ focused on the influence of PBM at 830 nm at a fluence of 5 J/cm² on NO, ROS and pro‐inflammatory cytokines (IL‐1β, IL‐6 and TNF‐α). Their findings showed a decrease in apoptosis, IL‐1β and TNF‐α, and an increase in cellular proliferation. Additionally, an increase in intracellular NO and ROS was also observed in less than an hour (15 min) post‐PBM. There was no effect on IL‐6. ¹²⁹ ROS is important in wound healing and is involved in cell signalling pathways. However, it has a biphasic effect, being both beneficial and detrimental depending on their concentration. NO is significantly lowered in diabetic wounds and has been linked to non‐healing diabetic wounds. ¹⁴³ , ¹⁴⁴ PBM using red light has also shown beneficial effects on wound healing in vitro. A study conducted at a wavelength of 660 nm (5 J/cm²) revealed increased fibroblast migration, proliferation, viability and collagen production in diabetic wounded cells in vitro. ¹³⁰ Another study by Ayuk et al., ¹⁴⁵ showed up‐regulation of genes coding for cell adhesion molecules and ECM proteins, including COL11A1 and COL14A1 post‐PBM.

de Loura Santana et al., ¹³¹ investigated the effect of single (PBM at 660 nm, 30 mW and at an energy density of 4 J/cm²) and multiple/fractioned (wounds submitted to four treatments on Days 1, 3, 8 and 10 at 660 nm, 30 mW, with 1 J/cm²) irradiations on wound closure rate, inflammatory infiltrate type, myofibroblasts, collagen deposition and optical retardation of collagen in wounded streptozotocin‐induced rats. Irrespective of single or multiple irradiations, there was accelerated wound closure by 40% in the first 3 days. There was also an increase in acute inflammatory infiltration as measured by neutrophil count. A chronic infiltrate (increased T lymphocyte count) replaced the acute inflammatory infiltrate on Day 8. PBM increased leukocyte chemotaxis, which peaked on Day 3 regardless of irradiation protocol. The single‐dose treatment group had the best inflammatory response from Day 3 to 10. PBM‐treated groups showed more myofibroblasts, necessary for wound contraction, and better collagen organisation than the control group. Myofibroblasts in PBM‐treated wounds remained elevated from Day 15, while they decreased in control wounds. Diabetes causes tissue glycation, which disrupts collagen organisation. Collagen fibres were better organised by a single PBM treatment than by a fractionated dose, showing slightly less organised tissue than uninjured skin. The control group exhibited the worst degree of collagen organisation. The authors concluded that PBM in the immediate post‐operative period can improve tissue repair in diabetic patients by modulating the inflammatory process, increasing myofibroblasts and enhancing collagen organisation, and that applying PBM in the inflammatory phase was the most important factor. ¹³¹

One of the most reproducible effects of PBM is an overall reduction in inflammation. Akyol and Güngörmüş, ¹³² studied the effect of PBM on the healing of skin incisions in streptozotocin‐induced Wistar rats. On the left side an incision (15 mm) was made using a diode laser at high‐power (808 nm, 4 W, 50 Hz), and on the right side two incisions (15 mm) were performed using a steel scalpel and a high‐power diode laser. Wounds on the left side of each rat received PBM (808 nm, 0.1 W/cm², 20 s, 2 J/cm² per session immediately post‐surgery and on Days 2, 4, 6 and 8; total dose 10 J/cm²), and scalpel incisions were used as controls. The rats were organised into three experimental groups, Group 1: scalpel (control), Group 2: diode and Group 3: diode combined with PBM. Results from their study showed reepithelialization was faster in Group 2 (diode) and Group 3 (diode and PBM) as compared to Group 1 (control) on the 10th day. Reepithelialization was at the edge of the wound in the control, but was covering less than half of the wound in Groups 2 and 3. There were also differences in inflammation. Groups 1 (control) and 2 (diode) showed acute inflammation, while Group 3 (diode and PBM) showed diffuse acute inflammation, fibroblast proliferation and granulation tissue development. Day 20 showed no differences between groups, and persistent inflammation was noted. Since three wounds were created in the same animal, systemic effects could have occurred and cannot be ruled out. The authors concluded that diode laser and PBM post‐surgery incisions caused the least tissue damage and resolved inflammation the fastest. ¹³²

PBM has been shown to decrease bacterial load, improve healing, and advance wounds through the inflammatory phase in diabetic rats. Asghari et al., ¹³³ evaluated the combined influences of PBM (pulsed wave laser 890 nm, 80 Hz, 1.92 W/cm², 0.324 J/cm²; 6 days a week for 15 days) and metformin (50 mg/kg; daily for 15 days) on microbial flora and biomechanical wound parameters in diabetic streptozotocin‐induced Wistar rats. Rats that received combined PBM and metformin, or PBM alone, exhibited statistically improved oral glucose tolerance test (OGTT) outcomes than placebo and metformin alone. PBM alone significantly reduced colony‐forming units (CFUs) at Day 7, while metformin and PBM had little effect. Metformin increased bending stiffness and stress high load, while PBM increased bending stiffness, maximal force and wound stress high load. Interestingly, combined PBM and metformin did not affect biochemical wound parameters measured. PBM was noted to have accelerated the process of wound healing significantly and to have reduced CFUs. ¹³³

A similar study was conducted by Bagheri et al. ¹³⁴ with the aim of determining if PBM and metformin administered alone or in combination affected inflammation and proliferation in streptozotocin‐induced wounded diabetic Wistar rats. Rats were treated the same as in the previous study; however, treatment was stopped on Day 7. PBM increased wound strength during the inflammatory phase (Day 4) and the proliferation phase (Day 7) of wound healing, as did metformin. PBM, metformin and combination wound treatments reduced inflammatory cells and enhanced granulation tissue formation and angiogenesis during the inflammatory and proliferative phases. Combination treatment with PBM and metformin showed better results than either treatment alone. On Days 4 and 7, metformin alone increased M2 macrophages, while PBM alone decreased them relative to controls. The study found that PBM alone and in conjunction with metformin accelerates diabetic wound repair during the inflammatory and proliferative stages. ¹³⁴

PBM at 660 nm was found to down‐regulate the translation of the inflammatory factor TNF‐α in diabetic patients with grades III and IV pressure ulcers. Ruh et al. ¹³⁵ performed PBM daily for 12 days using visible red light (660 nm, 100 mW with 2 J/cm² per session) and studied gene expression of inflammatory factors IL‐6 and TNF‐α, and growth factors VEGF and TGF‐β before and after PBM treatment. Their analysis of the lesion tissue revealed an improvement of up to 50% in the granulation tissue. Gene expression of IL‐6 was not significantly different before and after treatments; however, TNF‐α gene expression was reduced, and VEFG and TGF‐β gene expression increased. ¹³⁵

The effect of PBM (660 nm, 70 mW and 8 J/cm²) on hyperglycemia‐induced inflammation in human gingival fibroblasts (HGFs) was investigated. ¹³⁶ Fibroblast cells cultured in high‐glucose (35 mM) medium expressed more pro‐inflammatory cytokines (TNF‐α, IL‐1β, IL‐6 and IL‐8), but when exposed to PBM, expression levels decreased to the same level as normoglycemic control cells. Cyclic adenosine monophosphate (cAMP) signalling was assumed to affect pro‐inflammatory cytokines. ¹³⁶ In a similar study by Shaikh‐Kader et al., ¹³⁷ conducted in a diabetic wounded fibroblast cell model, PBM (660 nm, 11 mW/cm², 5 J/cm²) increased Cox‐2 48 h post‐PBM and decreased IL‐6 at 24 and 48 h. The in vitro studies by Lee et al., ¹³⁶ and Shaikh‐Kader et al., ¹³⁷ found a decrease in IL‐6 using a fluence of 8 J/cm² and 5 J/cm², respectively, while the study by Ruh et al., ¹³⁵ using a total fluence of 24 J/cm² did not find an effect in vivo, with all three studies using the same wavelength of 660 nm. The difference in effects may be as a result of the different fluencies used.

Studies have shown that PBM reduces pro‐inflammatory cytokine levels, which in turn reduces inflammation in the wound area. Ebrahimpour‐Malekshah et al., ¹³⁸ evaluated the effects of PBM (890 nm, 80 Hz, 3.46 J/cm²) and human allogeneic adipose‐derived stem cells (ha‐ADS) individually and in combination (PBM + ha‐ADS) for 8 days in a diabetic wounded rat model infected with methicillin‐resistant Staphylococcus aureus (MRSA). The findings showed that rats that received combination therapy (PBM + ha‐ADS), as well as PBM alone, had increased levels of mRNA for hypoxia‐inducible factor‐1α (HIF‐1α), bFGF, stromal cell‐derived factor‐1α (SDF‐1α) and vascular endothelial growth factor A (VEGF‐A).

PBM at 890 nm has been shown to significantly accelerate healing in ischemic MRSA‐infected delayed healing wounds in rats with DM2. Moradi et al., ¹³⁹ compared the effects of PBM (890 nm, 80 Hz, 0.324 J/cm² and 0.001 W/cm²) and allograft ADS treatment alone and in combination (PBM + ADS) on the maturation phase of delayed wound healing. The PBM + ADS and PBM alone groups had significantly less CFUs on Day 16. On Day 4, the PBM + ADS and PBM alone groups had considerably smaller wound areas than the control group and ADS alone. Furthermore, the wound area was decreased by all three treatments by Days 8 and 12. Compared to ADS treatment alone, PBM + ADS significantly reduced wound size. On Day 16, PBM + ADS reduced wound area more than PBM alone and had the highest closure rate, whereas the control group had the lowest. ¹³⁹ A similar study by Bagheri Tadi et al., ¹⁴⁶ showed that PBM alone (890 nm, 80 Hz and 0.2 J/cm²), ADS alone and PBM + ADS treatments substantially accelerate the inflammatory and proliferative phases of wound healing in a MRSA‐infected rat model. These interventions enhanced the number of blood vessels and fibroblasts, and new dermis and epidermis volume. On Days 4 and 8, treated groups had more granulation tissue and decreased inflammatory cells (including macrophages and neutrophils) than the controls. Oxidative stress indicators nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX1 and NOX4) were significantly down‐regulated in PBM + ADS‐treated rats on Day 8, while antioxidants catalase (CAT) and superoxide dismutase (SOD) were up‐regulated in all groups on Days 4 and 8. PBM at 890 nm and ADS alone, and in combination, modulated the inflammatory response; however, a combination treatment of PBM + ADS was found to be more effective than either PBM or ADS alone. ¹⁴⁶

PBM has the ability to increase mitochondrial respiration by increasing cellular ATP levels. It is important to note that PBM induces positive effects in numerous signalling pathways, including NF‐κB. These pathways regulate histamine release, PG production and Cox expression, all of which are involved in inflammation. In laser‐induced NF‐κB signalling pathways, ROS is crucial. ¹³⁹ According to Chen et al., ¹⁴⁰ NF‐κB activation in murine embryonic fibroblasts increased significantly in response to ROS generation at 810 nm and fluences over 0.003 J/cm². In addition, their research revealed an increase in cellular ATP levels, indicating that PBM also stimulates mitochondrial respiration. ¹⁴⁰ An increase in NF‐κB was also observed post‐PBM using 640 nm at 4 J/cm² during the initial phases of epithelial wound healing in traumatic ulcers created in Wistar rats. ¹⁴¹ Increased NF‐κB activity is essential for epithelialization and early wound healing. ¹⁴¹

PI3K/AKT is modulated by multiple stimuli and interacts with other signalling pathways. PI3K/AKT and forkhead Box Protein O1 (FoxO1) pathways play a major role in redox balance. Numerous diseases, including diabetes, are associated with PI3K/AKT dysregulation. An in vitro study using a wounded diabetic adipose‐derived stem cell (ADSC)‐fibroblast co‐culture cell model showed faster wound closure post‐PBM at 660 and 830 nm (5 J/cm²). Cell migration was insufficient and wound healing was delayed in control cell groups. ¹⁴² Both 660 and 830 nm irradiated groups had homogeneous cell movement and stained nuclei with spherical forms, indicating less DNA damage than control groups with irregular shapes. This study showed that PBM at 660 and 830 nm with 5 J/cm² increases PI3K and protein kinase B (AKT) and decreases FoxO1. Antioxidants (SOD, CAT and HMOX1) increased, indirectly confirming that excess free radicals were neutralised and oxidative stress was reduced, allowing for faster healing. ¹⁴²

Optimization of parameters remains a topic of interest in wound healing therapies. PBM using combination therapy appears to have superior anti‐inflammatory modulatory effects than solo therapies. Chen et al., ¹⁴⁷ used human immortalised keratinocytes (HaCaT), human monocyte‐like cell line (U937), human skin fibroblasts (WS1) and human umbilical vein endothelial cells (HUVECs) grown under hyperglycemic conditions (10 g/L glucose for 3 days), and a streptozotocin‐induced diabetic rat model to investigate 12 wavelengths (400–900 nm, 10 mW/cm² and 0.5–4 J/cm²) on DFUs in vitro and in vivo. Dose effect studies of PBM at various wavelengths (405, 425, 455, 495, 510, 530, 560, 660, 730, 805 and 850 nm), fluencies (0.5, 1, 2, 4, 6, 8 and 10 J/cm²) and power densities (10 and 40 mW/cm²) revealed that a power density of 10 mW/cm² presented better effects, and a fluence range of 0.5–2 J/cm² exhibited peak effects on cell proliferation. Diabetic HaCaT cells showed bimodal effects at 425 and 660 nm. The red‐infrared band had fewer peak effects than the green‐blue band in the other three diabetes cell types (425 nm in MUVECs, 495 nm in WS1 cells and 510 nm in U937 cells). The migration of diabetic HaCaT cells was significantly enhanced by PBM at 405, 510, 530 and 560 nm, while migration of WS1 cells was enhanced at 405 and 530 nm. The wounds were larger on Days 5 and 10 in the DM2 rat model as compared to wounds in healthy rats. PBM accelerated the recovery process across all wavelengths, but blood glucose levels remained unchanged. ¹³⁹ Inflammatory cell infiltration results showed PBM (425, 630 and 730 nm, 10 mW/cm², 4 J/cm²) bidirectional regulation of inflammation, which promoted the inflammatory response in the early stages (Day 3), and inhibited the inflammatory response in the late stages (PBM at all wavelengths on Day 15). ¹⁴⁷

6. CONCLUSION

The management of wound healing remains a delicate and complex process that is easily disrupted by dysregulation of essential biochemical pathways, resulting in disorderly forms of healing, particularly in pathological conditions such as diabetes. The transition from the inflammatory phase to the proliferative phase is a crucial stage in wound healing, and chronic diabetic ulcers are associated with a deficient transition. Studies in diabetic wound healing using PBM have been undertaken and will continue to be conducted, but optimised parameters remain unclear, making it difficult to employ PBM therapeutically in diabetic wound healing. Most PBM studies are performed in vitro and in animal models, and not enough human clinical trials have been conducted; however, evidence still exists on the beneficial effects of PBM and the inflammatory phase of wound healing. Properly optimised therapies that target and facilitate the resolution of inflammation and boost cellular proliferation and remodelling may provide a foundation for future therapeutic development to treat chronic wounds more effectively. PBM studies demonstrate the benefits of using the appropriate wavelength and fluence of light to reduce inflammation, accelerate essential cell proliferation and ECM deposition, and enhance tissue repair. Despite this, PBM is not used in mainstream medicine nor to its maximum potential, and this is largely due to a lack of comprehension of the therapy and the molecular mechanisms underlying it. More research on the anti‐inflammatory effect of PBM under diabetic conditions, and in human studies is required to advance the field of PBM and make it a more acceptable treatment for DFUs.

AUTHOR CONTRIBUTIONS

Tintswalo N. Mgwenya prepared the first draft of the manuscript. Nicolette N. Houreld contributed to writing the manuscript and designed the study. Heidi Abrahamse contributed to editing the manuscript.

CONFLICT OF INTEREST STATEMENT

All authors report no conflicts of interest.

Mgwenya TN, Abrahamse H, Houreld NN. Photobiomodulation studies on diabetic wound healing: An insight into the inflammatory pathway in diabetic wound healing. Wound Rep Reg. 2025;33(1):e13239. doi: 10.1111/wrr.13239