Evaluating Micronized Adipose Tissue Niche and Artificial Dermis Grafts Following Nonmelanoma Skin Cancer Excision: A Pilot Study

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NMSC, including basal cell carcinoma, is the most common human malignancy. Various approaches have been used to address the reconstruction of skin and soft tissue defects resulting from NMSC resection.^1,2 These methods include primary closures, skin grafts, and local flaps. Primary closure is constrained by the size of the defect it can effectively address. In such cases, local flaps present an advantage by minimizing scarring and seamlessly blending with the surrounding skin, providing a more natural outcome. However, the size of the defect or limitations in the arc of rotation can pose obstacles to the use of local flaps. Distortion of the facial contour may also develop, especially around the nose or the orbit, where NMSC often occurs. Another issue to consider is that local flaps may sometimes have poor blood supply or result in imperfect closures, leading to potential problems. Skin grafting can be an alternative option, with fewer complications. However, it is important to understand that even skin grafting often does not provide the desired cosmetic and functional outcomes. The primary concerns associated with skin grafting encompass issues such as volume deficiency, inadequate color, contour, and texture matching at the recipient site, as well as potential morbidity at the donor site.^3-8 Moreover, the requirement for skin grafting or flap surgery can impose a substantial strain on patients, particularly among the older population, due to the surgical nature of the procedures. Prioritizing methods that ensure maximum safety and minimal invasiveness, all with the goal of attaining favorable functional and aesthetic outcomes, is essential when planning and performing reconstruction.

Numerous artificial dermal substitutes have been developed for use without the need for covering small wounds with skin grafts.^9-15 Applying artificial dermis to manage skin and soft tissue defects offers the advantage of reducing wound contraction. Nevertheless, it could potentially lead to a delay in wound healing, because wound contraction is predominantly active in open wounds.^16,17

To overcome the limitations associated with the use of artificial dermis, the authors of the curremt study explored the use of tissue-engineered dermis composed of autologous cultured dermal fibroblasts or adipose-derived SVF cells implanted onto artificial dermis. Clinical studies were conducted to evaluate the efficacy of integrating autologous cells into synthetic dermal substitutes for wound coverage, with positive and promising outcomes.^18,19 Nonetheless, it is crucial to acknowledge that the clinical application of cultured human fibroblasts or SVF cells requires Food and Drug Administration approval, along with time-intensive procedures for cell culture or cell isolation. Additionally, obtaining the necessary equipment, space, and personnel for cell procurement further complicates the consideration of using these cells for clinical purposes.

A recent development involves the formulation of MAT using mechanical dissolution rather than enzyme-based methods. MAT comprises 3 primary constituents. The constituents involve cellular elements such as ASCs, fibroblasts, immune cells, and endothelial progenitor cells; ECMs; and cytokines. In vitro²⁰ and in vivo²¹ studies of MAT grafts have yielded promising outcomes in terms of protein synthesis, angiogenesis stimulation, and antioxidant properties. Given the constituents and regenerative potential of MAT, the authors of the current study hypothesized that MAT grafts may have the potential to promote wound healing. In 2022, a clinical trial study indicated promising results of 3D-printed MAT grafting on wound healing in patients with diabetes, who commonly experience compromised wound healing processes.²² Prior to that study, the effects of MAT grafting on wound recovery were documented in research conducted in laboratory settings and animal models. The 2022 clinical study marked the first investigation of MAT grafting in a clinical setting. The term MAT niche graft was coined to underscore its distinctive 3D-printed characteristic, which prominently fills the wound imperfection.²²

Recently, the authors of the current study devised a MAT niche graft for use in the management of skin and soft tissue defects created by NMSC resection as an alternative to artificial dermis grafts. This exploratory pilot study was designed to assess the possibility of using a MAT niche graft to manage NMSC.

The MAT niche grafting method and this study protocol were approved by the Institutional Review Board of Korea University Guro Hospital, Seoul, Republic of Korea (No. 2020GR0590 and No. 2015GR0181). The study was carried out in adherence with the principles of the Declaration of Helsinki, and informed consent was obtained from each patient.

Existing data were obtained from the medical records of 21 patients treated for skin and soft tissue defects resulting from NMSC resections using either artificial dermis or MAT grafts between May 2020 and July 2022. Before deciding on a reconstruction approach after NMSC removal, comprehensive information about different methods—skin grafts, local flaps, and grafting with artificial dermis or the MAT niche—was offered to and discussed with each patient. Patients who underwent artificial dermis or MAT niche grafting were included in the study. Twenty of the 21 patients were followed for more than 1 year. Scar assessments were conducted on these 20 patients with extended follow-up periods.

Ten patients (5 males and 5 females) of the total cohort of 21 patients were treated with MAT niche grafts. The mean (SD) age of these 10 patients was 75.0 (7.3) years. Seven graft sites were located in facial regions, and 3 were situated in non-facial areas. Of these 10 patients, 7 were diagnosed with basal cell carcinoma, and the remaining 3 had a diagnosis of squamous cell carcinoma, sebaceous carcinoma, or malignant spindle cell tumor. The other 11 patients (8 males and 3 females) underwent treatment involving artificial dermis grafts. The mean age of these 11 patients was 71.2 (10.3) years. In patients treated with artificial dermis grafts, 8 graft sites were on facial regions and 3 were on non-facial areas. Eight of these patients had basal cell carcinoma, and 3 had squamous cell carcinoma.

Following surgical excision of the NMSC, the mean (SD) size of the defect area was 2.4 (1.3) cm² (range, 1.0 cm²–4.3 cm²) in the MAT niche group and 2.1 (1.3) cm² (range, 1.0 cm²–4.5 cm²) in the artificial dermis group. No statistically significant differences were observed in terms of patient demographics (Table 1).

Surgical technique

Under local anesthesia administered in a solution containing 1% lidocaine and 1:1000 epinephrine, NMSC was excised from subcutaneous tissue at a 45° angle. Bowl-shaped ablation specimens were created, incorporating a 5-mm clinically verified tumor-free boundary. To verify negative margins, a frozen section analysis was conducted during surgery. If any remaining tumors were identified, the procedure was performed again until the tumor was completely eradicated. Wound photographs were taken.

MAT niche grafting

Wound photographs were converted to 3D image files and processed using NewCreatorK software V1.57.41 (ROKIT Healthcare) to create layers 1 mm to 5 mm thick. The G-code instructions from this process were then utilized with the 3D Bioprinter (Dr. INVIVO; ROKIT Healthcare), with transmission facilitated through the aforementioned software program. This allowed for the precise creation of the desired 3D structures.

Adipose tissue extracted from the lower abdomen through liposuction was mechanically micronized in descending order of particle size using a micronizer filter (Adinizer; BSLrest). The resulting MAT was mixed with saline after being collected into a syringe. The mixture was then gently agitated with normal saline to initiate a washing and purification process. The MAT solution was prepared and separated into distinct layers through the vertical arrangement of syringes, resulting in concentrated MAT. This solution was then transferred to new syringes as bio-ink for the 3D bioprinter. The bioprinter first created a scaffold with polycaprolactone based on wound dimensions, after which dispenser deposited MAT bio-ink into the scaffold. This process occurred in a temperature-controlled setting and took approximately 10 minutes.

The resulting niche contained a MAT bio-ink interior and a polycaprolactone exterior. Cooling the print bed and niche to −15 °C through the bioprinter's system solidified the niche; this step took 5 to 10 minutes. After approximately 10 minutes, the procedure was completed by carefully placing the frozen niche onto the wound using forceps.

A sterile silicone wound dressing (Mepitel; Mölnlycke Health Care) was used to protect the wound. Dressings were changed every week for observation (Figure 1).

Artificial dermis grafting

Artificial dermis grafting involved the application of an Insuregraf (Hyundai Bioland). All aspects of wound management were established to be identical to those used for the MAT niche group.

Evaluation

The recovery duration of the groups was compared, and both functional and cosmetic results were evaluated. Using the POSAS,²³ a combined subjective and objective assessment was carried out 1 year post reconstruction. Three scales were used to assess scar attributes: the PSAS, the OSAS (by the operator [SK Han]), and the OSAS (by 2 plastic surgeons). The PSAS was used to evaluate color, pliability, thickness, relief, itching, and pain. The OSAS evaluated by the operator assessed vascularization, pigmentation, pliability, thickness, relief, and scar contracture. Two plastic surgeons reviewed OSAS scores based on 12-month post-treatment photographs, assessing attributes easily judged from images. Each parameter was scored on a 10-point scale (1 = normal skin, 10 = the worst imaginable scar), with cumulative POSAS scores and overall scores indicating the skin condition.

Statistical analyses

Data are presented as mean (SD). Mann-Whitney U tests were used to compare patient age, wound size, complete healing time, PSAS scores, and OSAS scores between the 2 groups. The Pearson chi-square test was used for sex, cancer type, cancer location, and complications. A significance level of P < .05 was deemed statistically important. All statistical computations were executed using SPSS for Windows version 12.0 (IBM Corporation).

Both the MAT niche and the artificial dermis grafts exhibited successful integration with the wound beds, demonstrating the absence of failure (Figures 2, 3). The mean (SD) duration required for complete wound healing was 39.2 (11.4) days (range, 27–65 days) in the MAT niche group and 63.7 (34.8) days (range, 30–132 days) (P = .04) in the artificial dermis group.

In the MAT niche and artificial dermis groups, the sum of the PSAS scores was a mean of 11.4 (3.4) and 12.6 (4.1), respectively, with no significant difference between them (P = .62). Similarly, the mean holistic OSAS scores assigned by the operator were 8.9 (3.0) for the MAT niche group and 11.6 (3.2) for the artificial dermis group (P = .05). The corresponding mean OSAS scores allocated by the investigators were 5.8 (2.8) and 6.1 (2.3) for the MAT niche and artificial dermis groups, respectively (P = .59). Of the 21 parameters used for scar evaluation, the MAT niche group exhibited superior outcomes in 14. Nevertheless, the study revealed statistically significant effects in only 2 parameters based on the operator's assessments: scar relief (mean 1.6 [0.7] vs 2.2 [0.6]; P = .047) and scar contracture (mean 1.3 [0.5] vs 2.5 [1.0]; P = .011) (Table 2).

Irritant dermatitis was observed in 2 patients in each of the 2 groups. However, it responded well to conservative treatment and was effectively resolved. No notable adverse events or cancer recurrences besides these instances were recorded in either group during the follow-up period.

When considering reconstruction, it is advisable to opt for the least invasive and safest techniques available, with the aim of attaining favorable outcomes in both function and aesthetics. Moreover, the procedure should be uncomplicated in its execution and involve a concise operation duration. Elaborate surgeries can pose a challenge, particularly among older patients. Remarkably, the advancement of state-of-the-art technology has prompted the adoption of cellular and/or biological dermal approaches to enhance wound recovery.^24-27 This approach is uncomplicated, is time-efficient, and alleviates the surgical load on patients. The use of artificial dermis and/or cultured fibroblasts stands out as a prominent treatment option.

A variety of artificial dermal substitutes have been developed and used in clinical settings to mitigate wound contraction. Artificial dermis grafts for skin and soft tissue reconstruction offer several advantages over conventional treatment methods, including flap surgery. This technique is characterized by its simplicity and ease of execution, boasting a high success rate and short surgical time.^9-15 It obviates the need for major operations, thus alleviating the surgical burden on patients. Moreover, it preserves facial contours, minimizes discomfort during postoperative management, eliminates the need for painful dressing changes, and remains feasible even in cases of multiple NMSCs. Importantly, the approach circumvents donor site morbidity. Another notable advantage of using artificial dermis is that the graft closely resembles the surrounding skin, resulting in minimal color mismatch. The restoration of the epidermal portion through epithelialization, induced by the migration and proliferation of adjacent epidermal cells, including melanocytes, contributes to achieving a comparable density and activity of melanocytes and precursor melanocytes within the graft's epidermis, akin to those observed in the neighboring skin.²⁸ Nonetheless, numerous researchers have demonstrated that artificial dermis can potentially impede wound healing, because wound contraction predominantly occurs in open wounds.^16,17 The findings from a prior study similarly indicate that using artificial dermis to cover wounds decreased wound contraction but delayed the overall wound healing process.²⁸ In the same study, wound treatment through the topical application of fibroblasts demonstrated a notable acceleration in wound healing compared with artificial dermis treatment.

Notably, fibroblasts stand out as pivotal mesenchymal cells crucially involved in the wound healing process.^29-32 Several studies have reported that fibroblasts expedited wound healing through accelerated migration to the wound area and the early synthesis of new skin.^33,34 Additionally, fibroblasts applied in a wound secrete various cytokines and ECMs, including collagen and glycosaminoglycan, thus facilitating wound healing.^33,35-39 However, treatment with fibroblast grafts showed much more severe wound contraction than treatment with artificial dermis grafts.⁴⁰ Grinnell⁴¹ and Spector⁴² demonstrated that fibroblasts possess the capacity to differentiate into myofibroblasts, which subsequently contributed to the development of wound contraction.

In the current study, the wound healing rate was markedly faster in patients who underwent MAT niche grafting than in those treated with the artificial dermis (P = .04). The principal objective of this study was not a formal statistical validation of MAT niche graft effects. The study assumed an exploratory stance, evaluating the potential of MAT niche grafts within the constraints of a limited sample size. Despite these limitations, the outcomes are encouraging, showing promising effects of MAT niche grafts. Likewise, in the domain of scar assessment, recipients of the MAT niche treatment had superior outcomes compared with those who underwent artificial dermis grafting. Despite the identification of statistical significance in only 2 parameters, these findings underscore the potential efficacy of MAT niche treatment. The inherent statistical weakness is attributed to the study's small sample size. A clinical study with a larger number of participants is needed to test the reliability of the statistical results. These findings suggest that MAT niche grafts have the potential to serve as an effective treatment alternative to artificial dermis grafting. Furthermore, in the current study the assessment of safety and tolerability yielded favorable results, because there were no adverse events. Hence, these results imply that MAT niche treatment could provide tangible advantages for patients.

The encouraging outcomes of the MAT niche graft can be attributed to the abundance of essential wound healing factors within it. First, the cellular constituents within the MAT niche, encompassing fibroblasts, endothelial cells, and ASCs, play a pivotal role in promoting tissue regeneration and remodeling. Previous research established that the rejuvenating effects primarily stemmed from ASCs within the SVF of adipose tissue. Various mechanical techniques, such as chopping and centrifugation, have been developed to extract ASCs from adipose tissue without enzymatic digestion.^43-45 It is hypothesized that these techniques may compact tissue and ASCs through the mechanical disruption of mature adipocytes and lipid-containing vesicles. Furthermore, these formulations, characterized by a dense population of ASCs, demonstrate substantial promise in the area of tissue regeneration.⁴⁶ In 2016, Ceserani et al²⁰ observed that MAT acquired through mechanical methods maintained angiogenic and anti-inflammatory properties in vitro, outperforming cultured ASCs. MAT is regarded as a promising and secure alternative to circumvent the intricate regulatory challenges linked to enzymatic treatment and cell culture. Second, ECM components and cytokines within the MAT niche construct a scaffold conducive to robust tissue regeneration. Furthermore, the 3D architecture of the MAT niche can expedite a volume replacement effect, hastening the healing process by stimulating cell homing or paracrine signaling effects. The current progress in 3D bioprinting technology has enabled our team to develop a dedicated MAT niche to address skin and soft tissue defects. This autograft MAT niche has the potential to avoid the challenges of SVF transplantation, bypassing the need for collagenase digestion and associated regulatory issues. By using minimally manipulated autologous adipose tissue, ethical considerations are met while avoiding concerns about immunological rejection from heterologous and/or allogeneic material.

In 2022, the potential of MAT niche grafting was reported for the management of diabetic foot ulcers.²² In that study, fibrin glue was used to consolidate and stabilize the MAT niche. The inclusion of fibrin glue facilitated application, sustained adherence, and secured the fixation of MAT onto the wound in an optimized arrangement tailored to the wound's specific shape, depth, and wound bed intricacies.^47,48 Nonetheless, the preparation of the MAT niche took more than 50 minutes, with the most time-consuming step being the solidification of the MAT niche. While alternative solidifying materials might accelerate the solidification of the MAT niche, the effect of using such alternatives on wounds could be uncertain, potentially impeding the wound healing process, especially in patients with compromised immune systems. Moreover, the inclusion of fibrin glue in MAT has the potential to diminish the efficacy of MAT by diluting its components, particularly in the context of small wounds. Consequently, in this study the authors endeavored to solidify MAT at a reduced temperature as an alternate approach. Lower temperatures could potentially influence multiple factors within MAT by altering its molecular structure.²² However, in 2014, Choudhery et al⁴⁹ found that adipose tissue could be cryopreserved effectively for later clinical use and that thawed ASCs, a vital part of MAT, retained functionality similar to that of freshly processed ASCs. Various other studies have also reported the successful cryopreservation of ASCs from freshly collected adipose tissue, with cryopreservation maintaining their capacity for proliferation and differentiation.^50-52 Moreover, an earlier clinical investigation evaluating the feasibility of using a low-temperature MAT niche in the management of diabetic foot ulcer exhibited a favorable outcome with low-temperature MAT niche grafting.^22,53

The authors of the current study highlight the following technical tip when applying MAT niche to wounds. Before bioprinting, MAT shows a property similar to that of a liquid state. Unlike fibrin glue added to the MAT niche, the low-temperature MAT niche quickly returns to its liquid-like state at room temperature. The MAT niche melts approximately 1 minute after being applied to a wound and may flow down without maintaining a 3D structure. It is important to fix MAT with a silicone dressing as soon as possible after application to the wound.

The authors of the current manuscript frequently encounter patients with cutaneous and connective tissue defects necessitating reconstruction in clinical practice. Choosing the right reconstruction method is pivotal for achieving favorable outcomes. While the current study was retrospective and nonrandomized in nature, its outcomes are promising. Notwithstanding the acknowledged constraints, the findings of the study indicate that MAT niche grafting could emerge as an appealing alternative to conventional reconstruction methods following NMSC resection. To the knowledge of the authors of the current study, this is the first clinical endeavor comparing MAT grafts with artificial dermis grafts.

This study had limitations. It was a retrospective pilot study with a limited patient cohort and carries the inherent constraints of retrospective designs. Furthermore, the choice of reconstruction method was patient-driven, which precluded randomization. The sample size for outcomes analysis was inadequate for deriving substantial conclusions. Consequently, more robustly structured prospective studies involving larger participant pools are necessary to definitively ascertain the effectiveness of MAT niche grafts.

The current study establishes the feasibility of exploring the effects of MAT niche grafting, which resulted in faster healing and enhanced cosmetic outcomes over a 12-month period following the excision of NMSC compared with artificial dermal grafts. However, of the 21 parameters evaluated, statistically significant effects were observed in only 2 parameters evaluated based on the operator's assessments, namely, scar relief and scar contracture. Additional research is required to comprehensively ascertain the effectiveness of the MAT niche graft technique.

Authors: Yu-Kyeong Yun, MD¹; Seung-Kyu Han, MD, PhD¹; In-Jae Yoon, MD¹; Sik Namgoong, MD, PhD¹; Seong-Ho Jeong, MD, PhD¹; Eun-Sang Dhong, MD, PhD¹; Jee-Hee Kim, PhD²; and Min-Chae Lee, MSc²

Affiliations: ¹Department of Plastic Surgery, Korea University College of Medicine, Seoul, Republic of Korea; ²R&D Center, ROKIT Healthcare, Seoul, Republic of Korea

Ethical Approval: This study was approved by the institutional review board of Korea University Guro Hospital (No. 2020GR0590 and No. 2015GR0181).

Disclosure: This study was supported by grants from ROKIT Healthcare: I2000041, Seoul, Republic of Korea. The authors disclose no financial or other conflicts of interest.

Correspondence: Seung-Kyu Han, MD, PhD; Department of Plastic Surgery, Korea University Guro Hospital, 148 Gurodong-ro, Guro-gu, Seoul 08308, Republic of Korea; pshan@kumc.or.kr

Manuscript Accepted: March 7, 2024