Graft of 3D Bioprinted Autologous Minimally Manipulated Homologous Adipose Tissue for the Treatment of Diabetic Foot Ulcer

Due to its complications, consequences, and growing disease burden across the globe, diabetes is one of the diseases most targeted by health authorities.¹ The International Diabetes Federation has estimated that approximately 537 million adults are currently living with diabetes, and by 2045, the number of patients with diabetes will have increased to 783 million.² Diabetes affects approximately 61 million people in Europe annually, with Turkey having the highest age-adjusted prevalence (14.5%) in adults, followed by Spain (10.3%) and Albania (10.2%).² Although better management and early detection have reduced morbidity and mortality in patients with diabetes,³ the complications associated with the condition continue to pose serious problems. Around 15% of patients with diabetes are still at risk of developing a foot ulcer in their lifetime, and the emergence of DFU significantly increases the risk of further ulcerations, lower extremity amputation, and mortality.⁴ Compared to patients without diabetes, those with DFU have an approximately 15- to 20-fold increased risk of lower extremity amputations.⁴ Of all amputation cases involving patients with diabetes, 85% are preceded by foot ulceration with infection or gangrene.^5-7 Various therapies, including biological skin substitutes and physical treatment options, have been developed over the past 20 years. Therefore, to manage this serious health problem, an important first step is to take all necessary measures to prevent the disease. Second, complications should be appropriately treated to prevent detrimental outcomes.

The management of DFU is a challenging issue. In addition to widely accepted treatment modalities such as debridement, infection control, balancing moisture, and offloading, there are also many supplemental modalities—including negative pressure wound closure, hyperbaric oxygen treatment, and epidermal growth factor injections—that are recommended for the treatment of non-healing wounds or to accelerate the healing process. However, there is no single treatment modality that can heal all chronic wounds, and most products have their own limitations, such as multiple applications being required, pain during application, excessive cost, or limitations in terms of application area.²

Autologous fat grafting is increasingly studied across multiple fields. Mojallal et al demonstrated that autologous fat grafting stimulated the neosynthesis of collagen fibers and improved the vascularization and thickness of the dermis and subcutaneous tissue.⁸ Adipose tissue obtained from lipoaspiration is a rich, ubiquitous, and easily accessible source of MSCs.⁹ Adipose-derived stem cells are abundantly present in fat tissue, which also contains various growth factors (eg, insulin-like growth factor, hepatocyte growth factor, transforming growth factor–β 1, and vascular endothelial growth factor), consequently stimulating angiogenesis, epithelialization, and wound remodeling through paracrine secretion during wound repair.⁸ ADSCs are multipotent precursor cells that have the ability to differentiate into cell lineages associated with the regeneration of tissues, such as fibroblasts, keratinocytes, and endothelial cells.¹⁰ Prohealing growth factors, anti-inflammatory cytokines, proangiogenic factors, and healing-related peptides present in autologous fat may also have a positive effect on the wound-healing process.¹¹ Many clinical trials have revealed that the local injection of autologous micro-fragmented adipose tissue improves the healing rate in patients with DFU.¹² This present study aimed to investigate the efficacy of AMHAT with 3D bioprinting technology in patients with DFU.

Ethical permissions and design of the study

After receiving approval from the ethics committee of Ankara City Hospital for the research protocol (06/01/2021: E1-20-1339), a total of 20 eligible patients were enrolled in this clinical prospective interventional pilot study. The primary endpoint was a reduction in the size of DFU at weekly evaluations after treatment for 12 weeks, and the secondary endpoints were the epithelialization rate of DFU
and the amount of granulation of the wound bed at the same post-treatment evaluation times. The study was initiated only after obtaining informed consent from all patients.

Clinical case site inclusion and exclusion criteria

The inclusion criteria were: over 18 years of age, presence of type 1 or type 2 diabetes and ulcer wounds in the lower extremities due to peripheral neuropathy, pedal transcutaneous oxygen pressure over 40 mm Hg or impaired foot pulse detected by the Doppler test, and ability and willingness to provide consent and comply with the study procedures and follow-up evaluations. Patients who had Charcot foot ulceration; those showing clinical signs of inflammation or infection in the ulcer area; those diagnosed with malignant tumors; those who had participated in another clinical study within the last 4 weeks; those taking corticosteroids, immunosuppressors, or cytotoxic agents; and those that were deemed unsuitable to participate in a clinical trial by investigators were excluded from the study. Patients with osteomyelitis or wound site infection were included in the study after controlling for infection by amputation or debridement.

In this pilot study, all liposuction procedures were performed under general anesthesia, and patients at high risk for complications due to anesthesia were not included. Patients with coagulation disorders were also excluded.

Device

The Dr. INVIVO 3D bioprinter (ROKIT Healthcare, Inc.) was used to produce AMHAT of the same shape as the wound in each patient. This bioprinter is registered with the United States Food and Drug Administration as a class 1 medical device (Regulation no: 880.6430). The device also complies with the international standard “Medical Device Quality Management System ISO 13485.”

Collection and analysis of data

A schematic view of the study design is shown in Figure 1. All eligible patients were asked to provide informed consent and did. In addition to the initial screening visit prior to AMHAT treatment, 12 visits were planned for follow-up evaluations.

On the day of AMHAT treatment, patients were hospitalized for liposuction, and amputation or debridement procedures were also performed. Each patient’s wound was photographed and scanned after preparation (including cleaning, surgical debridement, and amputation, if required). At least 20 ml of abdominal fat was harvested from each patient using the general liposuction method, and AMHAT was prepared by gently filtering the harvested fat, which produced a customized (same size and shape of wound) matrix with the bioprinter that was applied to the cleaned DFU (Figure 2). The wounds were then covered with the first dressing (Renasys-G Non-Adherent Gauze [Smith & Nephew], a non-adherent dressing) and then a secondary dressing (saline-soaked cotton gauze).

The following procedures were performed at all follow-up visits (first to 12th week): patients were assessed for adverse events for at least 3 days after AMHAT treatment; patients were assessed for complications following treatment; patients’ wounds were assessed, photographed, and/or scanned; and the dressings (both non-adherent and saline-soaked gauze) were changed.

The following procedures were performed at the 12th visit: patients were assessed for adverse events and complications following treatment, wound closure/epithelialization rate was evaluated, and patients’ wounds were photographed and/or scanned.

The parameters evaluated consisted of wound area reduction by week (average of each patient), percentage of wound area reduction/wound closure at 12 weeks, wound size (cm²) reduction at each week, and granulation ratio (percentage) at each week (digitally calculated after digital plotting by the senior author).

All patients continued to take their regular medications for the management of diabetes and comorbidities. All patients were instructed to offload the treated foot. The non-adherent dressing was kept in place for at least 4 weeks, if possible, and replaced at weekly visits if required. Saline-soaked cotton gauze was used as a secondary dressing throughout the study period.

The study group consisted of 7 (35%) female and 13 (65%) male patients. Seven (35%) patients had a history of surgical intervention, while for the remaining 13 (65%), only daily dressing changes had been applied before hospitalization. Surgical procedures performed during the course of this study included debridement in 4 patients, forefoot amputations in 2 patients, and toe amputation in 1 patient. Plantar wounds were noted in nearly half of the study patients (9/20). In 2 patients, applications of AMHAT were performed in the operating room immediately following toe amputation. Patient characteristics and surgical history are detailed in Table 1.

The mean age of the patients was 60.70 years (median 63.0 years). Time between wound occurrence and admission to the authors' department was a mean of 48.75 days (median 35.0 days), and the mean wound size at the time of hospitalization was 7.529 cm² (median 5.65 cm²) (Table 2). One patient with a wound surface area of 0.5 cm² had tunneling of 1.2 cm in depth.

All but one of the wounds were completely epithelialized at the ninth week of follow-up visits, and the mean weekly wound sizes of the patients are shown in Table 3. Mean wound areas decreased significantly in the first 7 weeks compared to the pre-application period (P=.000, P=.000, P=.001, P=.002, P=.014, P=.015, and P=.014 for the first to the seventh week of follow-up visits, respectively) (Table 4).

Table 5 presents the differences in mean wound sizes between each consecutive week. When the decrease in the mean wound size for each evaluation week was compared, there were significant decreases in the first 7 weeks (P=.000, P=.002, P=.004, P=.014, P=.016, P=.045, and P=.038, respectively) (Table 6).

The mean time to the complete wound closure was 32.20 ± 23.862 days (median 24.50 days, range 7–84 days) There were no surgical complications, liposuction site infections, DFU site rejections, or DFU site infections. Mild subcutaneous bleeding was observed in 3 patients and was successfully controlled by elastic bandaging. No patient deaths occurred.

Figures 3 and 4 represent one case of AMHAT application in this present work. A 49-year-old male presented to the authors’ clinic with a wound that had a duration of 25 days. After serial debridement and wound therapy with conventional dressing methods, AMHAT application took place 11 days after hospitalization. The patient had no comorbidities except type 2 diabetes, and had used both long- and short-acting insulin for glucose regulation. Figure 3 shows the wound before AMHAT application. The area pointed out with an arrow in Figure 3B is the area of a split-thickness skin graft, which was applied 4 weeks after AMHAT application. Full epithelization of both parts of the wound was achieved at the fifthweek after treatment. Figure 4A was taken 5 months after treatment, and Figure 4B shows the wound 10 months after treatment without any scarring from AMHAT.

DFUs can cause limb loss if not recognized early and appropriately treated.⁴ The long duration of the wound treatment process and the inconvenience of daily or periodic dressing changes can adversely affect patients’ mental health and quality of life.⁵ Therefore, in addition to appropriate wound treatment, the recovery period is also important.

Various advanced treatment modalities are recommended for the treatment of DFUs that do not respond to standard wound care approaches, including hyperbaric oxygen treatments, negative pressure closures, and the use of various growth factors.^12,13,14 However, these modalities often require multiple treatments. In the present study, DFUs were treated with a single AMHAT application. This method allows for many growth factors—such as insulin-like growth factor, hepatocyte growth factor, transforming growth factor–β 1, and vascular endothelial growth factor—to reach the wound site through ADSCs and MSCs found in adipose tissue, thus triggering angiogenesis, epithelialization, and wound healing.¹¹

Many studies in the literature have examined the healing rate and duration of wounds andachievement of a 75% epithelization rate was considered a success.^15,16 While planning the present study, the authors aimed to determine the times taken to achieve wound healing rates of 25%, 50%, and 75%.

Fat grafting has become very popular, especially in reconstructive surgery. Its positive contribution to wound healing has been proven by animal and human studies.^17-22 However, studies on its use in DFUs are limited.^17,18,22 In their randomized controlled study, Smith et al stated that AFG accelerated wound healing compared to the conventional method, but the difference was not significant.¹⁷ In another study conducted in rats, Wang et al found that it significantly accelerated wound healing.¹⁸ Lonardi et al, in which AFG was applied to amputation sites in diabetic patients, observed that AFG significantly increased recovery.¹⁹

In none of the studies mentioned above were the methods applied similar to those of AMHAT, as the fat used was not micronized. Additionally, the computerized 3D configuration of the product serves as a template containing homogenous fat particles. However, the aforementioned studies only involved autologous adipose tissue transfer. AMHAT application in DFUs was used on 20 patients for the first time in the present study, which makes this contribution unique in the field of AFG application to diabetic patients.

In the existing literature, when DFUs were treated with conventional methods, the maximum healing level was reported to be 75% over a healing time of 5 to 20 weeks.^15,16 In a series with a high healing level and short healing time, the mean wound area was reported to be less than 3 cm².^2,15,16 In the present study using AMHAT, the reduction in the mean wound size was evaluated using photography and computerized digital surface calculations. The data showed a statistically significant decrease in wound size according to the weekly evaluations of DFUs. Both the decrease in wound size between the pre-application and evaluation weeks and the decreases between each consecutive week were significant (Tables 4 and 6). Therefore, AMHAT was shown to contribute positively to wound healing in the treatment of DFUs that did not respond to standard treatment approaches.

An important advantage of AMHAT application is that it allows for both the surgical procedure required by the wound (debridement and/or amputation, if necessary) and the preparation of the matrix with liposuction to be performed at the same stage. This eliminates the need for repeat surgery.

Like all chronic wounds, most DFUs take a very long time to heal. The negative psychological and social effects of long-term treatments on patients are significant.¹⁶ In the present study, the mean recovery time (ie, time to complete wound epithelization) was 32.20 days (median 24.50 days). The results of the mean healing time also suggested that AMHAT application could positively contribute to the healing of DFUs.

In studies where granulation formation is targeted, scarring may occur in the wound with spontaneous healing, or graft scarring may be observed after grafting.²³ In the present study, part of the large plantar wound of a patient was grafted, and the other part was treated with AMHAT. While the scar of the graft was noticeable, there was no visible scarring at the place where AMHAT was applied.  This indicates that the results achieved with AMHAT were similar to the pre-wound appearance without any scarring (Figures 3 and 4).

The main limitations of the present study are the small number of patients and the non-randomized design. Larger patient series and prospective randomized studies are needed to confirm the validity of these results. The cost-effectiveness of the procedure should also be analyzed.

This study’s data indicate that AMHAT with 3D bioprinting technology is beneficial in terms of ease of application, total wound healing time, significant decreases in the wound surface area, no or decreased scarring compared to grafting and conventional treatment modalities, and a mean of 32 days and median of 24 days required for full healing. Although cost analysis was not part of the current study, the fact that no patient required repeat surgery suggests that this approach will also decrease costs. Future randomized prospective studies examining AMHAT application through the lens of cost analysis could provide further support for this treatment modality.