Safety assessment of a three‐dimensional‐printed autologous omentum patch: An application on the kidneys as a new treatment approach

Abstract Recently, the field of regenerative medicine has made great strides in the development of new treatments for various organ dysfunctions. One of the most promising new approaches is the use of three‐dimensional (3D) printing and autologous tissues. In this study, we evaluated the safety of a 3D‐printed autologous omentum patch to kidneys using large animals. A total of seven micropigs underwent transplantation of the 3D‐printed autologous omentum patch. Twelve weeks after transplantation, the safety was evaluated by measuring body weight, blood, and the renal resistive index. In addition, biopsy samples were histologically analyzed. The results showed no surgical complications, renal functional hematological changes, or inflammatory responses. Therefore, this study provides important insights into direct therapy to kidneys with a 3D‐printed patch made of autologous tissue. Furthermore, it has the potential for the development of new therapies for various organ dysfunction.


INTRODUCTION
Because the kidneys are complex organs with different types of cells with unique structures, it is very difficult to recover damaged kidneys with conventional medicine. 1 To solve this problem, new approaches using biosources have been studied, and treatments using stromal vascular fraction, stem cells, and progenitor cells have been shown to have therapeutic effects. 2 In addition, there are many studies on methodological aspects for kidney treatment. Imafuku et al. confirmed the effect of restoring kidney function by applying an allogenic mesenchymal stem cell (MSC) sheet to the injured kidney. 3 In addition, hydrogel and autologous kidney progenitor cell mixture were injected into the kidney's parenchymal region and applied to clinical trials. 4 Recently, the delivery method to the subcapsular layer has been reported for the longterm presence of therapeutic sources. It has the advantage of reducing secondary damage caused by injection in the kidneys. 5 Tissue-engineered patch transplantation, which applies a therapeutic source to the renal parenchymal region (near the cortex) and subcapsular layer, is a delivery method that produces a direct effect on the glomerulus which plays an important role in renal function. 6,7 The omentum is a subcutaneous and visceral adipose tissue that has been used medically for a long time because it contains various biosources for the repair and regeneration of internal organs. 8,9 Furthermore, micronized adipose tissue is emerging as a new paradigm in regenerative medicine because adjusting the size of adipose tissue facilitates the use of biological components in adipose tissue, including growth factors and cells. 10 These tissue engineering treatment sources have therapeutic efficacy by adjusting to repair the microstructure of the surrounding environment and producing paracrine effects of growth factors and cytokines. For these reasons micronized fat is used in dermatology, plastic surgery, and orthopedics as a source of regeneration. 11 In the previous rodent-model study, the patch made of micronized omentum reduced tubular injury by reducing the gene expression of the renal injury marker, such as Havcr1, Lcn2, and Ccl2. 12 In addition, the patch showed an effect on reducing renal interstitial fibrosis. After ureteral obstruction, fibrosisrelated genes were observed to be downregulated by the omentum patch, and lower fibrosis was observed in the omentum patch implant group compared to the fibrin patch group. Many studies have been reported that MSCs have antifibrotic effects. 13 In this vein, using omentum as a source of MSCs can be a potential treatment for renal fibrosis.
In this study, we applied the omentum patch to large animals to evaluate the safety. The autologous omentum, which was harvested in the operating room, micronized and it was immediately produced to a patch without additional culture process. The 3D bioprinter was used to fabricate the patch; hence, the precise patch could be produced at high speed. There was no immune response because autologous tissue was used, and patch transplantation did not induce any renal damage or disease. It was a preliminary clinical trial to establish a safe and fast surgical procedure. Therefore, this study provides evidence that the omentum patch and transplantation process are safe.

METHODS
This study included seven male 22-35-month-old micropigs (Sus scrofa) weighing 38.2-63.8 kg to confirm the safety of transplanting the omentum patch. After applying the patch in each kidney for 12 weeks, safety data were obtained. The animal study was approved by the Institutional Animal Care and Use Committee (IACUC) of Apures (#210205-001). First, clippers were used to shave the central and flank areas of the pigs' abdomens to prevent infection. The surgical sites were aseptically prepared with 2% povidone and 70% alcohol (repeated 3 times) and wrapped with a sterile surgical cloth. Then, an incision was made in the skin and muscle ~10 cm from the abdominal midline, and the abdominal cavity was accessed.
Omentum was harvested and refined for use as bio-ink in a 3D bioprinter. The micronization process was conducted using the Dr. INVIVO AI Regen KIT (#ARK-001, ROKIT Healthcare, Inc.). A 3D bioprinter, Dr. INVIVO (ROKIT Healthcare, Inc.), was used to produce patches for accurate and fast manufacturing in a sterile environment. Fibrin glue (Beriplast P; CSL Behring GmbH) was used to fabricate the patch. All devices used in this study were approved as medical grade, and all surgical procedures were performed in a sterile state in the operating

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Direct treatment of kidneys using the autologous omentum was safe, suggesting that patch transplantation to the kidneys could be a potential treatment for kidney injury.
room. The micronized autologous omentum, dispensed by the Dr. INVIVO device, was printed in a 30 × 40 × 0.8-mm (width × length × thickness) patch and transplanted into the kidney.
For postoperative assessment, the complete blood count (CBC) was measured. Blood samples were collected using ethylenediamine tetraacetic acid and serum-separating tubes and analyzed the CBC (Sysmex XN-1000) at a specialized animal diagnostic testing center (NEODIN BioVet Laboratory, Seoul, Korea). All blood samples were taken pre-and 12 weeks postoperatively for the CBC test (n = 7). For 5 weeks postoperative, the CBC was measured every week to monitor the immune response to transplantation (n = 3). After 12 weeks of transplantation, ultrasound (Sonoscape S8; SonoScape Medical Corp.) analysis including two-dimensional kidney size analysis was performed. The renal resistive index (RI) was calculated to evaluate the arterial blood velocity near the renal hilum or at the level of the corticomedullary junction using ultrasound and was obtained using the following formula: The animals were euthanized 12 weeks after patch transplantation, and their kidneys were analyzed. Small pieces of kidney tissue were removed for histopathological analysis before and after patch transplantation. First, the biopsy specimens were dehydrated using a series of ethanol solutions and embedded in paraffin. Next, the blocks were sectioned into 5-μm slides, and samples were stained with hematoxylin and eosin (H&E). Briefly, the nuclei were stained with hematoxylin at room temperature for 10 min, and eosin was used for differentiation. Then, the images were observed under a light microscope (BX51; Olympus). For immunohistochemistry analysis, we stained injury marker, CD68 (MCA2317GA; Bio-Rad). The clone BA4D5 was verified to be specific for porcine CD68. 14 This study showed statistical analyses as means, and the overall standard error of the mean was used.

RESULTS
A patch made from autologous omentum was transplanted in seven animals under the renal capsules ( Figure 1). Autologous omental tissue was collected from the animals and processed as bio-ink. During this process, omentum and fibrin glue were mixed at an optimized ratio (60% of omentum) to fabricate the patch based on a previous optimization study. 12 A 30 × 40 × 0.8-mm (width × length × thickness) patch was produced using 3D bioprinting technology. The 3D bioprinter allowed the patch Resistive index (RI) = (peak systolic velocity − end diastolic velocity) (peak systolic velocity) F I G U R E 1 Surgical procedure of the omentum patch transplantation. The harvested omentum was fabricated to the patch using a 3D bioprinter. Then, the patch was transplanted into the renal capsule.
to be fabricated with a constant thickness, ensuring that the components were evenly distributed. A thin patch printed of 0.8 mm thickness was transplanted under the kidney capsule to minimize the effect of renal interstitial hydrostatic pressure. Part of the kidney capsule layer was cut for patch insertion, but no bleeding occurred during the transplantation process. The capsule layer was sealed with fibrin glue after the transplantation to prevent the patch from escaping. The safety of patch transplantation was evaluated 12 weeks after surgery. A CBC was performed using whole blood of pre-or postoperatively (n = 7; Table 1). Because graft rejection caused by immune response mainly occurs at the beginning of transplantation, we checked the CBC for the first 5 weeks (n = 3) (Table S1). From the data, we observed that all components of blood in the omentum patch groups showed no significant difference from normal ranges. 15,16 Biochemical profile analysis, including BUN and creatinine, was not sensitive because the contralateral kidney was preserved (data not provided).
Renal vascular resistance can be assessed by calculating the RI using ultrasound Doppler examination. Hence, this study noninvasively assessed the renal RI values associated with renal perfusion using Doppler ultrasonography. The mean RI values were 0.58 ± 0.03 (left) and 0.57 ± 0.02 (right ; Table S2). There was no significant difference in Doppler examinations compared to the normal reference value. 17,18 These results indicate that patch transplantation into the kidney did not induce resistance.
A small piece of the kidney was removed before and after patch transplantation. The biopsy samples were stained with H&E to check for any signs of disease. H&E staining is a useful tool for identifying patterns, shapes, and structures of different types of cells and tissues and is widely used in disease diagnosis. 19 The images confirmed that after patch transplantation, the kidney retained the normal histological structure of renal tubules and glomeruli ( Figure S1). In addition, the samples were stained with CD68, an injury marker, which recognizes the monocyte and macrophage lineage. 14 From the data, there were no abnormalities, such as immune inflammation and necrosis, after patch transplantation compared to before patch transplantation ( Figure S2). Therefore, all data suggest that patch transplantation is safe for animals without causing any damage or complications.

DISCUSSION
This study applied the omentum patch to the kidneys of porcine models. Large animal models are widely used to develop safe preclinical protocols because of their humanlike anatomic and physiological properties. 20,21 By transplanting an omentum patch in the kidneys of porcine models, we could optimize the conditions of the patch for safe transplantation, such as the size, thickness, and stiffness of the patch. In addition, a surgical protocol was developed to safely insert the patch under the kidney capsule  layer without bleeding. In previous study, we confirmed that the omentum patch had antifibrosis and antitubular injury effects on kidneys of chronic kidney disease rodent models. 12 In order to apply the patch to human clinical trials, we evaluated the safety of patch transplantation and found there were no immune responses or surgical complications after patch transplantation. Omentum was made in the form of a patch using 3D bioprinter after the micronization process. Because adipocytes and milky spots are randomly distributed in native omentum, the active ingredients can be applied unevenly when applied as a patch. In the previous study, the native omentum was micronized to form a uniform patch, and the patch was transplanted in the kidneys to provide evenly active biomaterials. 12 In addition, the patch was made by mixing with fibrin glue that is degradable in vivo. 22 Twelve weeks after transplantation, all patches had disappeared from the kidneys. As fibrin glue degraded after transplantation, the active biomaterials of omentum can be continuously supplied to the kidney through controlled release. Kesavan et al. 23 reported that a wound patch made of micronized fat fabricated using a 3D bioprinter showed high efficiency in wound healing. Because the wound patch containing many growth factors and cytokines could make the 3D regeneration environment, it was suitable to repair skin. The 3D bio-printed patch was applied on cartilage regeneration. 24 The cartilage powder and autologous fat were used to fabricate the patch, then the patch was applied to the cartilage defect of the osteoarthritis animal models. From the results, the hyaline cartilage was observed after 32 weeks of patch treatment. The patch can control the release by regulating the degradation time; hence, it can be useful for delivering substances into the body. For example, applying patch technology to islet cell transplants will be more effective as it can continuously release insulin. 25 However, there is a lack of quality and quantity studies of cells, such as MSCs, in micronized tissue. A further study for investigating micronized omentum in terms of cell quality is required.
This study demonstrates the feasibility of using an omentum patch in a porcine model. Furthermore, 3Dbioprinting technology, as a novel approach, was simple but could make a precise patch. These findings suggest that the autologous omentum patch and the surgical procedures of patch transplantation are safe to apply in clinical trials.