Adipose-derived stem cells in diabetic foot care: Bridging clinical trials and practical application

Abstract

Diabetic foot ulcers (DFUs) pose a critical medical challenge, significantly im-pairing the quality of life of patients. Adipose-derived stem cells (ADSCs) have been identified as a promising therapeutic approach for improving wound healing in DFUs. Despite extensive exploration of the mechanical aspects of ADSC therapy against DFU, its clinical applications remain elusive. In this review, we aimed to bridge this gap by evaluating the use and advancements of ADSCs in the clinical management of DFUs. The review begins with a discussion of the classification and clinical management of diabetic foot conditions. It then discusses the current landscape of clinical trials, focusing on their geographic distribution, reported efficacy, safety profiles, treatment timing, administration techniques, and dosing considerations. Finally, the review discusses the preclinical strategies to enhance ADSC efficacy. This review shows that many trials exhibit biases in study design, unclear inclusion criteria, and intervention protocols. In conclusion, this review underscores the potential of ADSCs in DFU treatment and emphasizes the critical need for further research and refinement of therapeutic approaches, with a focus on improving the quality of future clinical trials to enhance treatment outcomes and advance the field of diabetic wound care.

Key Words: Adipose-derived stem cells, Diabetic foot ulcers, Wound healing, Clinical trials, Stem cell therapy, Wound care

Core Tip: This review critically analyzes the current state of research on the efficacy of adipose-derived stem cells (ADSCs) against diabetic foot ulcers (DFUs). It offers a comprehensive overview of diabetic foot classification, clinical management, and the ADSC therapy intricacies. Despite its potential, the field faces challenges like biased trial designs, ambiguous inclusion criteria and intervention strategies. The review highlights these issues and discusses the preclinical efforts to enhance ADSC efficacy. It emphasizes the necessity of more rigorous research and refinement of therapeutic approaches to improve treatment outcomes in diabetic wound care and guide future research.

INTRODUCTION

Diabetic foot ulcers (DFUs) are a common and severe complication in patients with diabetes, leading to increased rates of hospitalization and, in extreme cases, lower limb amputations[1]. Approximately 25% of individuals with diabetes are at risk of developing DFUs, primarily due to a complex combination of factors like neuropathy, impaired wound healing, and vascular diseases[2,3]. The prevalence of DFUs is particularly higher in males and more common in individuals with Type 2 diabetes[4]. These ulcers resulting from peripheral arterial disease, inadequate foot care, neuropathy, and related foot deformities often lead to serious conditions such as gangrene or deep infections. Moreover, approximately 85% of amputations in diabetic patients follow the onset of foot ulcers, highlighting the critical need for effective interventions[5].

While numerous reviews have explored the mechanisms underlying the efficacy of adipose-derived stem cells (ADSCs) to treat DFUs, a clear articulation regarding the results and applications of clinical trials in this area is lacking[6]. ADSCs have shown promise in promoting angiogenesis, epithelial repair, immune modulation, and inflammation reduction, positioning them as a potentially transformative approach for managing DFUs[7,8]. Nevertheless, several critical aspects of ADSC therapy for DFU remain undefined, particularly the specific cells used, optimal timing and dosage, method of administration, and criteria for post-treatment evaluation.

This review critically assesses the current landscape of ADSC research, particularly focusing on clinical trial outcomes and practical applications in diabetic foot treatment. It aims to provide a comprehensive view of the challenges and potential roles of ADSCs in DFU management by integrating these findings with current clinical practices in managing diabetic foot. This review sheds light on future research directions and clinical applications.

CLASSIFICATION AND CLINICAL MANAGEMENT OF DIABETIC FOOT WOUNDS

Diabetic foot, characterized by infections, ulcers, and destructive lesions on the lower limbs of patients with diabetes, often coexists with neuropathy and peripheral artery disease (PAD)[9]. Traditionally, etiological classification has helped identify causative factors, including neuropathy, trauma with secondary infection, and arterial occlusive disease, thereby guiding appropriate treatment strategies[10]. The pathophysiology of DFUs involves a complex interplay of these factors, resulting in various wound types such as diabetes-related ulcerative disease (DUDT), neuropathic, ischemic, and neuro-ischemic ulcers[11]. Despite the diversity of DFU types, research beyond etiological classification remains limited.

Recent treatment advancements, particularly the emerging role of ADSCs in clinical settings, offer new avenues for managing these complex wounds. However, a notable gap exists in the literature regarding the integration of ADSC therapy into the standard classification and treatment protocols for diabetic foot. This gap highlights the need for comprehensive studies evaluating the efficacy of ADSCs in different DFU types.

Studies using etiological classification suggest that patients with peripheral neuropathy are more likely to develop DFUs at a younger age, while those with PAD have an increased risk correlated with PAD severity[12]. In clinical practice, neuropathic ulcers heal faster than neuro-ischemic and ischemic ulcers, with healing times influence more by etiological factors than wound size[13].

With the increasing incidence of diabetic foot cases, a shift toward severity-based classification has emerged to better capture clinical manifestations[14]. The International Working Group on the Diabetic Foot (IWGDF) 2023 guidelines recommend the Site, Ischemia, Neuropathy, Bacterial Infection, and Depth system for healthcare communication and outcome auditing, the IDSA/IWGDF classification for infected ulcers, and the Wound, Ischemia, and foot Infection system for PAD while acknowledging the absence of a single system for predicting individual ulcer outcomes[15,16].

Despite these advancements in classification, treatment protocols have largely remained consistent, as demonstrated by various guidelines over the years[17-19] (Figure 1). These protocols typically begin with diagnosing different types of diabetic foot conditions and employ standard wound care as the primary treatment, complemented by adjunctive treatments when necessary[20].

Figure 1 Standard clinical protocols for diabetic foot. This figure illustrates a detailed algorithm for managing diabetic foot complications, with a focus on infection, ischemia resulting from peripheral arterial disease, and Charcot neuroarthropathy. A: Initial evaluation: Incorporates diagnostic testing and imaging to guide therapeutic decisions; B: Infection management: Encompasses a spectrum from antibiotic therapy and localized wound care to hospitalization for advanced cases. Surgical intervention is considered for non-responsive infections; C: Ischemia treatment: Strategies include revascularization and angioplasty, with amputation reserved for critical cases; D: Charcot neuroarthropathy management: Ranges from conservative orthopedic approaches to surgical correction, based on disease progression and treatment response; E: Ulceration and deformity management: Emphasizes comprehensive wound care, off-loading techniques, and surgical options when necessary. The decision-making process within the algorithm is dynamic, adjusting based on the ulcer’s response to treatment and incorporating adjunctive therapies such as hyperbaric oxygen or cellular and tissue-based products where appropriate.

In summary, while traditional classifications and treatment protocols provide a foundational approach to diabetic foot management, integrating innovative therapies like ADSCs, still in the phase of clinical exploration and application, could significantly advance care strategies for these complex wounds. These findings suggest the critical need for more focused research on effectively integrating ADSC therapy into existing treatment paradigms and its potential impact on different DFU types.

MECHANISMS OF ADSC TREATMENT FOR DIABETIC FOOT

ADSCs, a specific type of mesenchymal stem cells (MSCs), have garnered significant attention in regenerative medicine due to their multipotency, ease of harvest, and substantial proliferative capacity[21]. Unlike other stem cells, such as those derived from bone marrow or embryonic tissues, ADSCs are more abundant and pose fewer ethical concerns[22]. Originating from adipose tissue, ADSCs are capable of differentiating into various cell types, including adipocytes, osteoblasts, and chondrocytes[23], as shown in Figure 2.

Figure 2 Therapeutic mechanisms of adipose-derived stem cells in diabetic foot Ulcer Healing. This figure delineates the multifaceted role of adipose-derived stem cells in the healing process of diabetic foot ulcers, highlighting their interaction with various cellular and molecular pathways. This includes anti-inflammatory effects, promotion of angiogenesis, and facilitation of epidermal regeneration. VEGF: Vascular endothelial growth factor.

A crucial aspect of ADSCs in treating DFUs is their immunomodulatory function and the release of growth factors like vascular endothelial growth factor (VEGF) and basic fibroblast growth factor, which are essential for tissue repair. This is in contrast to traditional treatments for DFUs that mainly focus on symptom management rather than underlying cellular repair. ADSCs stand out by addressing the root causes of DFU complications, such as poor blood flow and damaged skin tissue.

In managing vascular damage, a key challenge in DFU treatment, ADSCs release factors promoting blood vessel growth. For instance, their paracrine effects releasing VEGF contrast with traditional methods, which primarily involve physical wound care and do not directly stimulate angiogenesis[24]. ADSCs have been shown to enhance wound healing in diabetic animal models by activating vascular endothelial cells and stimulating angiogenesis through VEGF and other vasoactive factors[25].

Furthermore, ADSCs facilitate the involvement of keratinocytes in forming the wound epidermis, a vital process in wound healing[26]. Molecular mechanisms, including cell signaling pathways like serine/threonine-specific protein kinase (AKT) and Notch, play crucial roles in the pathophysiology of DFUs. Furthermore, media conditioned with ADSCs accelerate the migration and proliferation of endothelial cells through the activation of the AKT pathway[27]. ADSCs also regulate healing in DFUs through signaling pathways related to chemokine receptors and the modulation of the Notch pathway, highlighting the key roles of these pathways in cellular migration[28].

ADSCs also reduce inflammation and tissue damage, which are typically not fulfilled by conventional DFU therapies. ADSCs modulate immune responses by balancing pro-inflammatory and regulatory T cells and reducing oxidative stress, a capacity not seen in traditional DFU treatments[29]. ADSCs also influence macrophage polarization, mediated by Toll-like receptors expressed on their surface, shifting the macrophage phenotype from a pro-inflammatory M1 type to an anti-inflammatory M2 type, which is crucial in promoting wound healing[30].

In summary, ADSC-based therapy presents a comprehensive approach to tackle the complexities of DFUs, encom-passing angiogenesis, skin formation, cell migration, and immune regulation. This multifaceted mechanism sets ADSCs apart from other stem cell types and traditional treatments, offering a potentially transformative method for managing DFUs.

CLINICAL TRIALS OF ADSC THERAPY FOR DIABETIC FOOT

Clinical trial landscape

As of December 2023, an extensive search through the International Clinical Trials Registry Platform and Clinical Trials identified 38 clinical trials focused on ADSCs for treating diabetic foot, highlighting significant trends as detailed in Supplementary material. The majority of these trials are Phase 2, with 10 trials focusing on initial efficacy assessment. Moreover, in six trials, a trend of the combination of Phase 1 and 2 was observed, suggesting a growing interest in assessing both safety and early efficacy simultaneously. However, only six Phase 3 trials have been conducted, indicating a nascent stage in advanced efficacy evaluations.

Geographically, the maximum number of research is conducted in South Korea (9 trials) and the United States (8 trials), reflecting their robust medical infrastructures and funding capabilities. The treatment methods in the trails varied, predominantly non-invasive or minimally invasive, with injection methods being the most common in 20 trials. The use of gel sheets and innovative techniques like fibrin glue and biometrics in 17 trials demonstrates a diverse range of treatment modalities being explored.

The diversity in trial designs is notable, ranging from randomized parallel group designs, predominantly open-label, to single-group and observational studies. This variety raises questions about the comparability and generalizability of the results. The diverse methodologies, including the use of various cell types (autologous in 21 early-stage trials for safety and allogeneic in 16 Later-stage trials for efficacy and scalability), diverse dosing strategies, and a wide range of participant numbers (up to 290 in some trials), suggest that the findings may be context- or patient-specific. This variation in trial design and methodology implies that conclusions from one study may not apply to all DFU cases, highlighting the need for standardization in future research.

The primary endpoints of these trials often focus on wound healing, size, healing duration, and safety, with less attention given to crucial aspects of diabetic foot pathology like angiogenesis and perfusion. Secondary endpoints, including quality of life and pain assessment, underline the importance of these patient-centric outcomes. However, the varying focus of these goals across trials can result in a fragmented understanding of the full potential of ADSC therapy in treating DFUs.

In summary, while the increase in ADSC-related trials indicates a growing interest in this treatment modality, the heterogeneity in trial designs and methodologies underscores the need for more standardized and comprehensive approaches to better understand the efficacy and generalizability of ADSC therapy in treating DFU.

Clinical trials outcome

As of December 2023, 20 clinical trial outcomes, some of which are included among the 38 trials mentioned in Table 1 and others not part of these 38 trials, have been reported for ADSC therapy in the treatment of diabetic foot. Collectively, these studies indicated no safety concerns or adverse reactions, suggesting a favorable safety profile for ADSCs. Among these, the GENNAI27 trial (NCT03276312) involving 114 patients conducted over six months investigated autologous micro-fragmented adipose tissue in minor diabetic foot amputations and reported a significant reduction in hospitalization duration (16.2 d less, P = 0.025). This finding highlights the efficacy of ADSCs not only in healing DFUs but also in managing complications like minor amputations and ischemia.

Table 1 Outcome of clinical trials on adipose-derived stem cells treatment for diabetic foot.

Ref.	Country	Cell type	Condition	Intervention	Cell count	Follow-up	Enrollment	Outcome
Mrozikiewicz-Rakowska et al[55], 2023	Poland	Allogeneic	Ulcer	Hydrogel	2.5 × 106 cells	49 d	47	The time required for a 50% reduction in wound size was 17.6 ± 1.5 d (P = 0.029)
Lee et al[41], 2012	Korea	Allogeneic	Ischemia	Intramuscular injection	5 × 106 cells	6 months	15	66.7% of patients showed clinical improvement, with enhanced pain relief, increased claudication walking distance, and the development of vascular collateral networks
Moon et al[33], 2019	Korea	Allogeneic	Ulcer	Hydrogel	1 × 106 cells per film	12 wk	59	Wound closure reached 73% by week 8 and increased to 82% by week 12, with a median closure time of 28.5 d
Yastı et al[80], 2023	Türkiye	Autologous	Ulcer	3D-AMHAT	15-25 mL of fat	12 wk	20	All wounds, except one, were fully epithelialized by the ninth week, with a mean time to complete closure of 32.20 d
Bajuri et al[81], 2023	Malaysia	Autologous	Ulcer	3D-AMHAT	20 mL of fat	12 wk	10	Within 12 wk, complete healing was observed in 70% of the patients
Bura et al[31], 2014	France	Autologous	Ischemia	Intramuscular injection	1 × 108 cells	6 months	7	All ulcers showed 100% improvement after 6 months, with three completely healed
Uzun et al[82], 2021	Türkiye	Allogeneic	Ulcer	Injection	6 × 106 cells	4 years	20	90% healing, with no adverse reactions
Nolan et al[83], 2022	United Kingdom	Autologous	Ulcer	Intramuscular injection	/	4 wk	18	Increased mean microvessel density by + 32% to + 45% at 1 wk (P = 0.035)
Dreifuss et al[84], 2017	United States	Autologous	Pedal atrophy	Injection	/	12 months	23	Dermal thickness increased significantly post-injection, persisting through 24 months (P < 0.05)
Kress et al[85], 2023	United States	Autologous	Ulcer	Injection	13.9 mL of fat	9.3 months	10	Achieved full clinical recovery with no reulceration
Carstens et al[53], 2021	United States	Autologous	Ulcer	Subcutaneous injection	3 × 107 cells	12 months	63	At 6 months, 51 subjects achieved complete closure, and 8 had closure of ≥ 75%
Nilforoushzadeh et al[86], 2020	Iran	Autologous	Ulcer	Engineered skin graft	/	21 wk	5	There was a significant increase (P ≤ 0.05) in both skin thickness and vascular bed density
Lonardi et al[87], 2019	Italy	Autologous	Minor amputations	Injection	10-30 mL of fat	6 months	114	At 6 months, 80% of the feet showed healing (P = 0.0064)
Carstens et al[42], 2017	United States	Autologous	Ischemia	Intramuscular injection	/	12 months	10	All patients showed clinical improvement (rest pain, claudication, ABI)
Zollino et al[88], 2019	Italy	Autologous	Ulcer	Injection	15 mL of fat	24 wk	16	The average wound healing time was 17.5 ± 7.0 wk (P < 0.036)
Raposio et al[43], 2018	Italy	Autologous	Ischemia	Subcutaneous injection	5 × 105 cells	6 months	7	After 6 months, all patients demonstrated complete wound healing
Namgoong et al[89], 2010	Korea	Autologous	Ulcer	Hydrogel	30-50 mL of fat	16 wk	20	Eight out of 10 patients (80%) achieved complete wound healing after 16 wk, with a reduction in wound area by 4.3 ± 1.0 cm² (P = 0.043)
Han et al[90], 2010	Korea	Autologous	Ulcer	Subcutaneous injection	4.0-8.0 × 106 cells	8 wk	54	100% healing was achieved
Smith et al[91], 2020	United Kingdom	Autologous	Ulcer	Injection	2 mL of fat/cm²	21 months	18	There was no difference between any of the groups in terms of clinical outcomes
Gennai et al[92], 2021	Italy	Autologous	Minor amputations	Injection	10-30 mL of fat	6 months	114	The average hospital length of stay was 16.2 d (P = 0.025)

ABI: Ankle/brachial index.

However, limitations such as sample sizes and varied follow-up durations pose challenges to the generalizability and long-term effects. For example, the small cohort reported by Bura et al[31] (NCT01211028) might not adequately represent the broader patient population. Additionally, the varied follow-up durations, ranging from as short as 49 d to as long as four years, raise questions about the long-term effects and sustainability of ADSC therapy.

The diverse efficacy results, ranging from no difference to 100% healing, highlight the need for standardized measures and study designs for better comparability.

Innovative approaches, like the dual-dose strategy (low-cell group, 1 × 10⁶ cells/kg and high-cell group, 2 × 10⁶ cells/kg) in the study of Soria-Juan et al[32] (NCT04466007) highlights the importance of exploring different dosing regimens to determine optimal treatment protocols. Furthermore, studies such as that of Moon et al[33] (NCT02619877) show promise in utilizing allogeneic ASC-hydrogel complexes. They report closure rates of 73% in the treatment group vs 47% in controls at 8 wk, which increase to 82% and 53%, respectively, by 12 wk, thereby indicating the potential for innovative treatment modalities.

In summary, these clinical trials illuminate the therapeutic potential of ADSCs in treating DFUs and associated complications. However, the diverse outcomes and identified limitations emphasize the need for more comprehensive future studies. Research focusing on determining optimal dosages, refining administration methods, and establishing precise patient selection criteria is crucial. Addressing these areas could significantly advance the clinical management of DFUs, enhancing patient outcomes and potentially expanding the treatment spectrum to more effectively tackle the broader complications associated with diabetic foot conditions.

CONSIDERATIONS ABOUT ADSCS IN THE TREATMENT OF DIABETIC FOOT

Safety and efficacy of ADSCs

The application of ADSCs in treating DFUs shows potential but requires careful assessment of both safety and effectiveness. To date, clinical trials employing ADSCs for DFUs have not reported any adverse reactions, suggesting a positive safety profile. However, the long-term safety implications, particularly for allogeneic ADSCs, require more in-depth investigation.

Allogeneic ADSCs exhibit phenotypic stability over in vitro passages, retaining their multilineage differentiation and proliferation capabilities[34]. These cells do not express type II human leukocyte antigen, reducing the risk of immunological rejection following allogenic transplantation[35,36]. This aspect also sidesteps the ethical dilemmas typically associated with embryonic stem cells. In terms of clinical efficacy, a recent study has shown that ADSCs promote wound healing similar to allogeneic non-diabetic cells, indicating a preference for allogeneic ADSCs in clinical practice[37].

A major concern in the clinical use of any MSC is the risk of tumorigenicity, particularly for cells expanded in vitro[38]. This risk is intricately linked to genomic instability, necessitating stringent checks for transformation potential prior to clinical application. Long-term patient follow-up is essential, given the extended nature of cancer progression, to thoroughly ascertain safety. Moreover, a study has also demonstrated that ADSC transplantation does not promote tumor-related angiogenesis in distant tumors nor exacerbate tumor growth or metastasis[39].

While current evidence supports the safety of allogeneic ADSC therapy, particularly in the absence of serious adverse reactions in short-term studies, ongoing vigilance is recommended to reinforce this safety profile.

Timing of ADSC use in diabetic foot treatment

The application of ADSCs in managing diabetic foot conditions requires careful consideration of clinical factors. ADSC therapy is not recommended for Charcot foot and active diabetic foot infection, aligning with treatment guidelines for diabetic foot. However, its potential is evident in other aspects of diabetic foot care. For instance, in patients with ischemic-type DFUs, where standard clinical protocols prioritize vascular reconstruction surgery to restore blood perfusion[40], ADSC therapy can be a viable alternative, especially for those with PAD who are ineligible for revascularization. Several clinical trials have shown that local muscular injections of ADSCs can effectively alleviate PAD symptoms, enhance collateral circulation, and improve tissue perfusion[31,41-43]. Moreover, ADSC therapy could be highly beneficial in individuals with non-healing ulcers, particularly those who have not shown signs of improvement after 4-6 wk of standard care and where angiography confirms the infeasibility of revascularization[44]. Furthermore, for neuropathic, ischemic, and neuro-ischemic foot ulcers, where standard wound care fails to yield significant improvement—typically characterized as less than a 50% reduction in wound area after 4 wk—ADSC therapy can prioritized as an adjunct treatment[19].

Patients most likely to benefit from ADSC therapy include those with long-standing, non-responsive ulcers despite conventional treatments, those with ulcers complicated by underlying vascular insufficiencies, and patients where surgical options are limited due to other comorbidities. In such scenarios, ADSCs offer an innovative approach to stimulate healing and improve overall wound outcomes.

In summary, while the integration of ADSC therapy in diabetic foot care shows promise, its application should be tailored to specific patient needs and conditions. Future research and clinical practice should focus on identifying and refining the criteria for patient selection to maximize the benefits of this advanced therapeutic option.

Methods of administration in ADSC therapy for diabetic foot

The administration of ADSCs in diabetic foot treatment utilizes various delivery methods, each with distinct advantages and drawbacks. These methods include intravenous infusion, arterial, muscular, or local injections (Table 2).

Table 2 Comparison of adipose-derived stem cells administration methods for diabetic foot treatment.

Method	Advantages	Disadvantages
Intravenous infusion	Targets multiple areas	Cells may get trapped in the lungs
Arterial injection	Direct delivery to the foot	Technically challenging and risk of blood vessel issues
Muscular injection	Directly reaches wound, good for poor blood flow, improves cell survival and nerve regeneration	Less effective for wounds away from muscle
Local injection	High cell concentration at the wound	Risk of uneven cell spread and clumping

Intravenous infusion enables the systemic distribution of ADSCs, potentially targeting multiple affected areas. However, a major drawback of this method is the risk of stem cells getting trapped in the lungs, limiting the number of cells reaching the target site in the foot[45,46]. Arterial injection delivers ADSCs directly into the blood flow toward the foot, potentially enhancing the targeting of the affected area. However, the technical difficulty of the procedure and the risk of vascular complications are the major concerns of arterial injections. Muscular injection, the most common method, is preferred due to its direct delivery of cells near the wound site. This method enhances cell survival and functionality by providing better access to nutrition and oxygen and is particularly beneficial for patients with diabetes with compromised peripheral blood flow[47]. However, it may be less effective for wounds not in close proximity to muscle tissue. Local injection involves directly injecting ADSCs into or around the ulcer to ensure a high concentration of cells at the wound site. However, this method may result in uneven cell distribution and localized cell accumulation, potentially compromising treatment efficacy[48].

The choice of syringe and needle size is also crucial in the injection technique. Jesuraj et al[49] recommend a 27-gauge needle to optimize stem cell viability while ensuring effective delivery and minimizing tissue trauma. However, Onishi et al[50] demonstrated that ADSCs maintain viability and function even when passed through a 30-gauge needle at a controlled flow rate[50]. Thus, using a 27–30 gauge needle with an injection speed maintained at or below 4 mL/min is advised to maximize cell viability and treatment efficacy.

For ischemic-type DFUs, muscular injection is currently preferred[32], whereas, for neuropathic, ischemic, and neuro-ischemic foot ulcers, both muscular injection and adjunctive therapy are considered viable[51]. Moreover, muscular injection has been reported to facilitate peripheral nerve regeneration and mitigate denervated muscle atrophy[52].

These studies suggest that clinicians should carefully consider the type of ulcer, the patient’s overall condition, and the specific advantages and limitations of each method when deciding on the most appropriate ADSC administration technique.

Dosage in ADSC therapy for diabetic foot

Determining the appropriate dosage of ADSCs is critical in treating DFUs, with body weight and wound area being key factors. Current practices in determining ADSC dosage for DFUs vary widely in clinical trials, with dosages ranging from 5 × 10⁵ to 1 × 10⁸ cells, highlighting the need for personalized therapy[43,53,54].

In a clinical trial, the dosage is calculated based on patient weight[32], which may not fully consider other critical factors such as wound size, depth, and the patient’s overall health condition. Innovative approaches, like hydrogel-based adjuncts, provide a framework for dosage customization. For instance, standard dosing for wounds up to 12 cm² involves 2.5 × 10⁶ cells in 1 mL of fibrin gel, with proportional increases for larger wounds[55]. Products like the Korean ALLO-ASC-DFU hydrogel, containing allogeneic ADSCs, demonstrate the practical application of this approach, incorporating 1 × 10⁶ ADSCs per 5 cm × 5 cm gel sheet[33].

Post-treatment assessment is crucial for evaluating the efficacy of ADSC therapy. Parameters for evaluating the treatment success encompass the assessment of the wound’s healing progress, the patient’s sensory function, and vascular health. The percentage change in DFU wound area after 4 wk of treatment serves as a reliable efficacy and healing predictor[40]. Sensory assessments for pressure or vibration perceptions are the other methods. Evaluation of pressure perceptions is performed using Semmes–Weinstein 10 g monofilaments, while 128 Hz tuning forks or fingertip touch assessments on the patient’s toe tips are used for vibration perceptions. Vascular status can be evaluated through changes in intermittent claudication history and pedal pulse palpation[44]. Further evaluations can incorporate additional instruments and examinations to ensure a comprehensive assessment.

Despite the advancements in dosage determination, there is a significant need for more standardized and evidence-based approaches. The current knowledge gap on optimal dosage for different DFU types and patient profiles hinders the ability to uniformly apply ADSC therapy effectively. Standardizing dosage determination would not only enhance treatment efficacy but also ensure patient safety. Future research must focus on establishing evidence-based guidelines for ADSC dosages, considering the multifaceted nature of DFUs and patient-specific factors. Such advancements in dosage determination are crucial for fully realizing the potential of ADSC therapy in diabetic foot care, offering a promise of more effective and personalized treatment strategies.

PROGRESS ON ENHANCING ADSC EFFICACY IN DIABETIC FOOT TREATMENT

Current research supports various approaches to enhance the therapeutic efficacy of ADSCs in DFU treatment, including methods to improve ADSC properties, combination therapies, bioengineering methods, and the use of ADSC-derived exosomes (Figure 3).

Figure 3 Multidimensional enhancement of adipose-derived stem cells for diabetic foot ulcer therapy. This figure presents a comprehensive overview of various strategies employed to enhance the therapeutic properties of adipose-derived stem cells (ADSCs) for the treatment of diabetic foot ulcers. The strategies are categorized based on their primary mechanisms of action and delivery methods. A: Preconditioning strategies: Platelet lysate increases keratinocyte migration. Deferoxamine upregulates neuroprotective and angiogenic factors. Salidroside counters hyperglycemia-induced suppression. Photobiomodulation boosts ATP, ROS, and protective gene expressions. Hypoxic Preconditioning stimulates angiogenesis. Overexpressing HPGDS enhances immunomodulation and growth factor production; B: Combination therapies: Exenatide-4 and ADSCs promote endothelial migration and proliferation. Platelet-rich plasma combined with ADSCs enhances angiogenesis. VAP-PLGA microspheres with ADSCs activate the PI3K/Akt/HIF-1a pathway, aiding in cellular survival. MSC/T/H/S product reduces inflammation and promotes tissue repair; C: Biomaterials and bioactive molecule application: ECM sheets and hydrogels provide a scaffold for ADSCs, augmenting angiogenesis. Pluronic F-127 hydrogels deliver ADSCs, enhancing the modulation of nerve regeneration. Dual-crosslinked hydrogel systems ensure cell viability and sustained release of therapeutic factors; D: Exosome and gene therapy approaches: Exosomes with miR-133a-3p accelerate Schwann cell proliferation and inhibit apoptosis. Exosomes within an ECM hydrogel target the ACE2 receptor, aiding in ischemic tissue repair. Exosomal miR-126-5p improves vascular cell migration. Exosomal miR-204-3p/HPK2 axis enhances angiogenesis. These diverse strategies underscore the potential of ADSCs in improving the therapeutic outcomes for diabetic foot ulcers through a variety of mechanisms, including enhancing cell viability, modulating immune responses, and promoting angiogenesis and tissue regeneration. ADSCs: Adipose-derived stem cells; VAP-PLGA microspheres: Velvet antler polypeptide PLGA microspheres; MSCs: Mesenchymal stem cells.

Enhancing ADSC properties

Studies have explored various methods to augment the healing capabilities of ADSCs. For instance, Hermann et al[56] found that incorporating platelet lysate into ADSC culture improved keratinocyte migration and viability. Carolina Oses et al[57] demonstrated that preconditioning ADSCs with Deferoxamine, a hypoxic mimetic, increased the secretion of neuroprotective and angiogenic factors. Similarly, Ariyanti et al[58] reported that salidroside pretreatment counters hyperglycemia-induced suppression of key wound healing factors in MSCs, such as Heme Oxygenase-1, Fibroblast Growth Factor 2, Hepatocyte Growth Factor, reducing intracellular reactive oxygen species (ROS) levels and apoptosis, enhancing their survival and migration under hyperglycemic conditions.

However, translating these enhancements from a preclinical to a clinical setting presents several challenges. First, the scalability of these methods needs to be considered. Techniques that are feasible in a laboratory setting may not be practical or cost-effective when scaled up for clinical applications. Moreover, the safety profile of these enhanced ADSCs must be rigorously evaluated in humans, as alterations in cell properties could potentially lead to unforeseen side effects or immune reactions.

Zhao et al[59] confirmed the benefits of hypoxic preconditioning of ADSCs, which enhanced protection against oxygen-glucose deprivation and boosted angiogenesis through the VEGF/VEGFR2 and SDF-1a/CXCR4 axes. Additionally, Ouyang et al[60] discovered that the downregulation of hematopoietic prostaglandin D synthase in type 2 diabetic mouse wounds delays healing. In contrast, its overexpression in ADSCs accelerated wound healing in diabetic mice by reducing neutrophil and CD8 T-cell recruitment, promoting M2 macrophage polarization, and increasing growth factor production. Finally, Fallahi et al[61] highlighted the role of photobiomodulation in preserving mitochondrial function and therapeutic potential of diabetic stem cells by enhancing ATP levels and Matrix Metalloproteinases activity while reducing ROS and expression of PINK1 and PARKIN.

Combination therapies

The combination of ADSCs with other therapeutic agents has shown the potential to amplify their healing effects in diabetic foot treatment. However, understanding the synergistic benefits and possible risks of these combinations is crucial.

Seo et al[62]demonstrated that combining Exenatide-4 with ADSCs improved human endothelial cell migration, invasion, and proliferation, suggesting that Exenatide-4 synergistically enhanced the healing properties of ADSCs. Similarly, Ebrahim et al[63] found that a mixture of platelet-rich plasma and ADSCs accelerated diabetic wound healing by modulating the Notch pathway and promoting angiogenesis. This combination leverages the growth factors in platelet-rich plasma and the regenerative capacity of ADSCs, potentially offering a powerful approach to wound treatment.

Jiang et al[64] confirmed that Velvet antler polypeptide PLGA microspheres (VAP-PLGA microspheres) enhanced ADSC function in wound healing through the PI3K/Akt/HIF-1α pathway, suggesting that such biomaterials can act as scaffolds or carriers, improving the delivery and efficacy of ADSCs in the wound site. Yang et al[65] reported that an early inflammation combination product named MSC/T/H/S also showed promising results in reducing inflammation and promoting tissue repair.

However, combining ADSCs with other agents carries risks. Interactions between ADSCs and other compounds could potentially lead to unforeseen biological responses, potentially increasing the risk of abnormal tissue growth or immune reactions. The safety profile of these combinations needs thorough evaluation, especially concerning long-term effects and potential immune responses.

Additionally, the complexity of administering combination therapies poses challenges. Ensuring the optimal ratio of ADSCs to other agents, determining the best method of delivery, and monitoring for adverse effects require careful consideration and advanced clinical trial designs.

In summary, combining ADSCs with other agents presents an exciting frontier in DFU treatment; however, balancing the synergistic effects with potential risks is crucial. Future research should focus on optimizing these combinations for enhanced healing and rigorously evaluating their safety profiles to ensure the best outcomes for patients with DFUs.

Bioengineering approaches

Innovative bioengineering strategies are significantly improving the efficacy of ADSCs in treating DFUs. Several studies have demonstrated their development and effectiveness. For instance, Kato et al[66]developed ADSC sheets that secreted angiogenic growth factors and integrated them into new vessel structures. Lee et al[67] created a novel extracellular matrix (ECM) sheet dressing scaffold from human adipose tissue featuring angiogenic and bioactive factors. Kaisang et al[68] utilized Pluronic F-127 hydrogel to encapsulate ADSCs, accelerating wound closure by enhancing angiogenesis. Da Silva et al[69] demonstrated the effectiveness of gellan gum-hyaluronic acid hydrogels in delivering ADSCs to modulate inflammation and promote nerve regeneration. Han et al[70] developed a dual-crosslinked hydrogel system that maintained high cell viability post-extrusion, which is crucial for ADSC injection therapy. However, despite the promise of these hydrogel-based methods, their practical application depends on ease of use in clinical settings, stability during storage and transportation, and compatibility with existing wound care protocols.

Another critical consideration is the regulatory pathway for these bioengineered products. Obtaining approval from health authorities requires extensive testing to demonstrate safety and efficacy, which can be a lengthy and resource-intensive process. Additionally, training healthcare professionals in the proper application of these novel products will be essential for their successful integration into clinical practice.

In summary, bioengineering approaches hold great potential in enhancing ADSC therapy for DFUs. Nevertheless, their transition from the laboratory to the clinic involves overcoming significant hurdles related to scalability, practicality, regulatory approvals, and integration into existing healthcare systems. Future research and development should focus not only on refining these bioengineering techniques but also on addressing these practical considerations to facilitate their widespread adoption in clinical practice.

ADSC-derived exosomes

The therapeutic potential of ADSC-derived exosomes has gained significant attention. Huang et al[71]showed that ADSC sheets loaded with exosomal Nuclear Factor I C silencing modulate the miR-204-3p/HIPK2 axis, reducing high-glucose-induced angiogenesis. Guo et al[72]revealed that miR-125b-5p derived from ADSC-Exos might play a pivotal role in ischemic muscle repair by targeting ACER2. Chen et al[73]demonstrated that ADSC extracellular vesicles carrying miR-130a-3p alleviate diabetic peripheral neuropathy by activating the NRF2/HIF1α/ACTA11 axis, thereby accelerating Schwann cell proliferation and inhibiting apoptosis. Additionally, Song et al[74] developed a novel ECM@exo, combining ADSC-derived exosomes with an ECM hydrogel. This system ensures sustained release and concentration at the wound site and has been shown to reduce inflammation and boost angiogenesis, collagen deposition, cell proliferation, and migration, thereby expediting the healing process in DFUs.

While preclinical research has highlighted the potential of ADSCs in DFU treatment through various methods, the comparative effectiveness of these approaches is yet to be fully explored. Future studies must prioritize evaluating factors that enhance ADSC properties to determine the most efficacious combination therapies and identify the superior bioengineering methods. Addressing these questions is essential for optimizing ADSC-based treatment strategies, thereby advancing the clinical management of DFUs.

Optimizing ADSC application in clinical settings for diabetic foot treatment

Improving disease models in preclinical studies is crucial for advancing ADSC therapy in diabetic foot treatment. Current research primarily relies on mouse or rat models to simulate diabetic conditions, but these models have significant limitations in replicating the complexities of human DFUs[75]. Therefore, human-like models, which closely resemble human skin structure and wound healing processes, such as pig skin models, are endorsed by the FDA for chronic wound studies[76]. These models could provide more accurate insights into ADSC efficacy and mechanisms.

In the realm of clinical trials, improving the quality of studies involving ADSCs is an urgent need. In its 2023 update, IWGDF has raised concerns regarding the current use of ADSCs as adjunctive therapy for DFUs[77]. Moreover, data on the cost-effectiveness and feasibility of ADSC treatments are not sufficient. Therefore, high-quality clinical trials should be conducted to address these challenges. Such trials should feature stringent blinding, randomized controls to reduce bias and extended follow-up periods for assessing long-term effects. They should also aim to determine optimal ADSC dosages for varying diabetic foot conditions, explore diverse treatment methodologies, establish maximum safe dosages, and assess the feasibility of repeated treatments. These efforts are pivotal for ensuring the clinical efficacy and safety of ADSC therapy in diabetic foot care.

ADSCs present a viable treatment option for patients with diabetic foot, particularly those ineligible for conventional surgical interventions. ADSC therapy, especially via muscular injections, should be considered for patients who are only candidates for palliative surgery[77]. In such cases, ADSCs can improve blood perfusion, especially in patients unable to undergo vascular reconstruction. In individuals with diabetes who show persistent non-rigid hammertoe ulcers or abundant calli that have not responded to non-surgical treatments, digital flexor tendon tenotomy may be considered. Similarly, for non-healing plantar forefoot ulcers, procedures such as Achilles tendon lengthening, metatarsal head resection, joint arthroplasty, or osteotomy can be explored. ADSC treatment can be attempted concurrently to com-plement these interventions, potentially aiding in wound healing without adversely affecting the disease progression. However, nerve decompression procedures are generally not recommended in these contexts[78]. ADSCs offer therapeutic potential for patients suffering from chronic neuropathic pain associated with DFUs. Studies suggest that MSCs can facilitate the recovery of motor and sensory nerve conduction velocities, restore the normal ultrastructure in peripheral nerves, increase blood flow in damaged nerves, and reduce thermal and mechanical allodynia[79].

Despite the promising potential of ADSC therapy in diabetic foot care, significant improvements are needed in both preclinical and clinical studies. Bridging the gap between laboratory research and clinical application necessitates further advancements and refinements in ADSC treatment methods. This evolution in treatment approach will be instrumental in enhancing patient outcomes and expanding the therapeutic repertoire for DFU management.

CONCLUSION

In summary, ADSC therapy shows promising potential in diabetic foot wound healing but remains in a developmental phase for routine clinical use. Preclinical research highlights the efficacy of ADSCs in diabetic foot care, but future studies are needed to explore the comparative effectiveness of various treatment approaches. Identifying optimal enhancement factors, combination therapies, and bioengineering methods is crucial for advancing ADSC-based treatments. Clinical trials have established a favorable safety profile for ADSCs, yet there is a need to refine dosage, administration, and patient selection for these treatments to be more effective. As traditional treatment protocols continue to serve as the foundation for diabetic foot management, the integration of ADSC therapy could offer significant advancements in treating these complex wounds, especially for patients with limited treatment options. Looking ahead, the future of ADSC therapy in diabetic foot care is promising, yet it necessitates further research and refinement. Bridging the gap between laboratory research and clinical practice will be essential for leveraging the full therapeutic potential of ADSCs and revolutionizing diabetic foot care.