Fabrication of an injectable acellular adipose matrix for soft tissue regeneration

po Laboratory of Tissue Engineering and Biomedical Materials, University of Science, Ho Chi Minh City, Viet Nam a r t i c l e i n f o Article history: Received 27 June 2020 Received in revised form 7 October 2020 Accepted 15 October 2020 Available online 17 October 2020 composition, including various bioactive molecules, such as collagen, elastin, laminin, fibronectin and glycosaminoglycan (GAG) iomaterial design and regeneration. xenogeneic tissue r, small intestine, The elimination of e cellular compo- which physical, chemical, and/or biological means are applied to remove cells from their ECM, thereby minimizing inflammatory response or immune-mediated implant rejection [5]. Recently, ECM isolated from adipose tissue has been examined for biomaterial fabrication and its potential as a stem cell- supporting scaffold in regenerative therapies [6e9]. Adipose tis- sue is a connective tissue made of stromal cells, neurovascular * Corresponding author. 227 Nguyen Van Cu Street, Ward 4, District 6, HoChi Minh City, 700000, Viet Nam. E-mail address: tlbha@hcmus.edu.vn (H.L.B. Tran). Contents lists availab Journal of Science: Advanc .e l Journal of Science: Advanced Materials and Devices 6 (2021) 1e10Peer review under responsibility of Vietnam National University, Hanoi.fold for cell attachment and development and for maintaining and supporting tissue growth and regeneration. ECM has a complex nents, is the critical checkpoint to be overcome in ECM utilization. Decellularization is the primary strategy for ECM preparation, in1. Introduction Biomaterials composed of an extracellular matrix (ECM) have been widely used in reconstructive surgical applications, especially in regenerative medicine for tissue and organ replacement. ECM is the vital factor in tissue construction, serving as a structural scaf- [1e3]. Therefore, ECM is highly interesting for b and fabrication, especially at aiming for implant ECM is harvested from a variety of allogeneic or sources, including the dermis, urinary bladde mesothelium, pericardium and heart valves [4]. immunogenic factors, mainly caused by nativnc-nd/4.0/).Keywords: Extracellular matrix Decellularization Injectable Biomaterial Tissue engineeringhttps://doi.org/10.1016/j.jsamd.2020.10.002 2468-2179/© 2020 The Authors. Publishing services b license ( b s t r a c t The acellular adipose matrix (AAM) represents a promising source of biomaterial for applications in soft tissue regeneration. In this study, we aimed to prepare an injectable AAM to serve as a ready-to-use allograft. A fabrication procedure including harvesting, delipidating, and decellularizing was estab- lished. Accordingly, we proposed a mechanical disruption during delipidation and a final homogeniza- tion to produce the AAM powder in an injectable form. Our results demonstrated that mechanical disruption would enhance the efficiency of fat removal from the tissue and would significantly shorten the delipidation duration. A decellularization strategy composed of TriseHCl and Sodium Dodecyl Sulfate was developed for adipose tissue. The evidence for a complete cellular remnant removal was shown through Hematoxylin and Eosin staining and DNA measurements. The AAM powder was prepared by mechanical disruption under reduced temperatures, which resulted in an AAM particle size in the range of 100 mm. In vitro cytotoxicity testing via extraction demonstrated that AAM had no toxicity on human adipose derived stem cells (hADSCs). Further experiments showed a positive support of the AAM extraction on the migration and proliferation of hADSCs, which indicated its role as a modulator for cell recruitment and proliferation during the tissue remodeling process. Subcutaneous injection of AAM presented a gradual degradation and integration with the surrounding tissues. In the following 8 week post-injection period, AAM supported the in vivo adipogenesis observed in the peripheral side along the implant. Overall, the results of this study demonstrated that the fabrication protocol for the acellular adipose matrix can be applied to injectable materials, which provide proper biocompatibility and po- tential applicability as off-the-shelf material for soft tissue regeneration. © 2020 The Authors. Publishing services by Elsevier B.V. on behalf of Vietnam National University, Hanoi. This is an open access article under the CC BY-NC-ND license ( National University, Ho Chi Minh Citb Department of Human Physiology and Animal Biotechnology, Faculty of Biology e Biotechnology, University of Science, Ho Chi Minh City, Viet Nam c y, Viet NamOriginal Article Fabrication of an injectable acellular adi regeneration My Thi Ngoc Nguyen a, b, c, Ha Le Bao Tran a, b, c, * a journal homepage: wwwy Elsevier B.V. on behalf of Vietnam d/4.0/).se matrix for soft tissue le at ScienceDirect ed Materials and Devices sevier .com/locate/ jsamdNational University, Hanoi. This is an open access article under the CC BY-NC-ND structures, and specific ECM components. Adipose tissue can be obtained in large quantities using liposuction techniques. Accord- ing to a report by the American Society of Plastic Surgeons in December 2016, liposuction was the second most popular cosmetic surgery performed in the United States, with more than 222,000 procedures performed annually. Significant quantities of human adipose tissues are routinely discarded as medical waste during these routine operations [10]. Especially, adipose tissue collected from liposuction is usually done in healthy patients, thereby reducing the risk of infections. Adipose tissues collected from this liposuction procedure become a potential and easily available source for biomaterial fabrication. Publications demonstrated that adipose tissue derived ECM had the most significant potential to promote de novo adipogenesis and, hence, to promote long-term retention. They also identified several qualities that would enhance the adipoinductive properties of grafted material, including minimal in vivo immunogenicity, the ability to induce angiogenesis in vivo [8]. ECM-based biomaterials have been uti- lized not only in adipose tissue engineering but also in soft tissue engineering and even in bone regenerative medicine [11e13]. This study aims to establish an effective method to prepare human acellular adipose matrix (AAM) in an injectable form for allograft production. Accordingly, human adipose tissue harvested from liposuction was treated through a series of successive deli- pidation and decellularization steps using Isopropanol, Tris- Hydrochloride and Sodium Dodecyl Sulphate (SDS). A homoge- nous AAM powder in an injectable form was generated and investigated for its effects on human adipose derived stem cells in terms of cytotoxicity, migration, and proliferation. AAM was also injected into mice to explore their feasibility and potential as a safe and ready-to-use allograft. 2. Experimental 2.1. Acellular adipose matrix (AAM) fabrication 2.1.1. Harvesting adipose tissue Fresh human adipose tissue was obtained from healthy female patients who underwent abdominal liposuction surgery at Xuan Truong Paradise Plastic Aesthetic (Ho Chi Minh City, Vietnam). Liposuction procedures performed on patients were in accordance with the ethical standards of XuanTruong Paradise Plastic Aesthetic (No. 2604/QÐ-TMXT) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. A written consent was obtained from all patients. The samples were preserved in an 1X Phosphate Buffer Saline (PBS, Gibco, USA) supplemented with 400 UI/ml Penicillin and 400 mg/ml Streptomycin (SigmaeAldrich, USA). Frozen gel packs are used to keep the samples cold during delivery. The freshly isolated adipose tissue samples are separated from the preservation buffer and washed three times with PBS to remove blood compo- nents. Adipose tissue samples were frozen at
86 C for further processing. 2.1.2. Delipidation of adipose tissue Frozen adipose tissue samples were thawed at 37 C for the AAM fabrication, which included two successive delipidation and decellularization procedures. Initially, 10 mL of the liposuction sample underwent a mechanical disruption using a bead ruptor homogenizer (Omni e Bead Ruptor 24, USA) to facilitate fat removal. A ruption protocol was set up optimizing the ruption speed (from 0.8 m/s to 3.9 m/s) and the number of cycles (1 cycle ¼ 30 s, delay time ¼ 10 s). After disruption, the sample temperature and the outcoming oil volume were measured by a M.T.N. Nguyen and H.L.B. Tranthermometer (JENWAY, UK) and a 10-mL cylinder, respectively. 2After mechanical processing, samples were incubated in 99.9% isopropanol (Merck, Germany) (fluid ratio between sample and Isopropanol was 1 : 4). The incubation period was investigated to achieve a complete removal of the lipid content. 2.1.3. Decellularization of adipose tissue Before decellularization, the samples were washed three times with PBS to eliminate residual Isopropanol. The samples were treated in 10 mM TriseHCl (pH 8) and 0.1% SDS according to our previous publication [3]. In this study, samples were exposed to 10mM TriseHCl (pH 8) for either 8 h or 24 h, then incubated in 0.1% SDS for 12 h. Finally, the treated samples were washed with PBS for 24 h to remove residual reagents. After decellularization, AAM was frozen at
80 C and dried by lyophilization (BenchTop, Scientific, USA). Lyophilized AAM was minced with scissors and milled by a bead ruptor homogenizer (Omni e Bead Ruptor 24, USA) under reduced temperature (-5 C e 0 C). The AAM particles were passed through a 100-mm strainer to remove the large particles. 2.2. Evaluation of acellular adipose tissue 2.2.1. Oil red O staining Native adipose tissue (nAT) samples and treated samples were cut into small pieces and fixed in 10% formalin for 1 h. The samples were incubated in 60% Isopropanol for 30 min, followed by Oil Red O staining for 1 h. The samples were washed twice with distilled water and observed under the microscope for the detection of oil droplets. Images were taken using an Olympus CKX-RCD micro- scope (Tokyo, Japan) equipped with a DP2-BSWmicroscope digital camera. 2.2.2. Hematoxylin & Eosin (H&E) staining nATand treated samples were fixedwith 10% formalin at 4 C for 24 h. They were dehydrated step-wise using ethanol, immersed in xylene, and embedded in paraffin. The paraffin sections were cut at 4 mm, deparaffinized, and stained with H&E. Images were taken using an Olympus CKX-RCD microscope (Tokyo, Japan) equipped with a DP2-BSW microscope digital camera. 2.2.3. DNA quantification AAM was digested by 100 ng/ml Proteinase K at 55 C for 24 h. The digested samples were then centrifuged at 13,000 rpm for 30 min. Afterward, the supernatants were purified with phenole- chloroformeisoamyl alcohol (25:24:1) followed by centrifugation at 13,000 rpm for 30 min. Finally, the DNA pellets were washed with 70% ethanol and dried by air. The DNA contents were quan- tified by measuring the absorbance at 260/280 nm in a Beckman Coulter DTX 880 Multimode Detector spectrophotometer (Full- erton, CA, USA). 2.3. In vitro effects of AAM on human adipose derived stem cells (hADSCs) 2.3.1. MTT assay for cytotoxicity testing on extract Human adipose derived stem cells (hADSCs) were used for cytotoxicity testing. hADSCs are available in our laboratory, which were isolated, cultured, and expanded according to our previous publication [14]. An in vitro MTT assay on the extract of test samples in the culture medium was performed to measure the effect of the extract on the relative viability of hADSCs. The extracts of the test sample and the positive control were prepared by 24-h incubation in a complete medium (DMEM-F12 (SigmaeAldrich, USA) sup- Journal of Science: Advanced Materials and Devices 6 (2021) 1e10plemented with 10% Fetal Bovine Serum (FBS, SigmaeAldrich, hemocytometer. ped with a DP2-BSW microscope digital camera and analyzed by weeks old, 25e30 g). Mice injected with saline only were used as the control group. After 2, 4, and 8 weeks, the mice were sacri- ficed for sample collection. The injected areas in the abdomen were the target for harvesting. The skin and the abdomen muscle under the skin were dissected, fixed in 10% formalin and stained with H&E. 2.5. Statistical analysis All data were presented as mean ± SD. The statistical signifi- cance was determined at each test group by ANOVA One way, which was analyzed using Prism 6 (GraphPad Software, Inc., San Diego, CA). A confidence level of 95% (p < 0.05) was considered asImageJ. 2.4. Animal injection The experiments were conducted following the National In- stitutes of Health guidelines for the care and use of laboratory an- imals (NIH Publication No. 85e23 Rev.1985). The animal experiment protocol was approved by the Animal Care and Use Committee of the University of Science in Ho Chi Minh City, Viet- nam (Registration No. 03/15-010-00). The AAM powder was mixed with physiological saline at a concentration of 0.05 g/ml for subcutaneous injection into abdominal areas of mice (Mus musculus var. albino, male, 6e82.3.3. Cell migration hADSCs were seeded into 6-well dishes (5  104 cells per well) and cultured overnight at 37 C, 5% CO2. A wound scratch was produced in the monolayer on each plate using a pipette tip (100e1000 mL tip). The adherent monolayer was then washed two times in PBS 1X to remove non-adherent cells. The AAM extract was prepared as given above and added to the wells. After 0 and 24 h, images of cell migration into the scratch area were captured with an Olympus CKX-RCD microscope (Olympus, Japan) equip-carding the MTT solution, 100 ml of ethanol/DMSO (1:1) (SigmaeAldrich, USA) was added and mixed well. The color that developed was quantified by measuring the absorbance at 575 nm using a microplate reader (Biochrom, USA). The data obtained were expressed as a Relative growth rate (RGR - %) according to the ISO 10993-5 instruction. 2.3.2. Cell proliferation hADSCs were cultured under AAM extract conditions for 1e11 days to examine the effect of the AAM extract on the cell prolif- eration. A complete medium and a serum free medium were used as the positive and negative control, respectively. hADSCs were trypsinized and reseeded into a 96-well plate (103 cells per well). The cells were cultured overnight by a complete medium for adherence and spreading on the culture surface. On the next day, the complete medium was replaced by either AAM extract, com- plete medium or serum free medium. Cell proliferation was evaluated by a cell number counting chamber using aUSA), 1X antibiotics (SigmaeAldrich, USA)) at 37 C (0.05 g/ml, ISO 10993-12). Liquid extracts were collected and placed on the sub- confluent monolayer of the cells. After incubation of cells with the extract at 37 C for 24 h, liquid extracts were replaced with a 100 ml MTT solution (0.5 mg/ml in culture medium) (Sigma- eAldrich, USA) and were incubated at 37 C for 4 h. After dis- M.T.N. Nguyen and H.L.B. Transtatistically significant. 33. Results 3.1. Preparation of injectable acellular adipose matrix (iAAM) 3.1.1. Delipidation process The delipidation of adipose tissue was accomplished by a me- chanical disruption followed by a chemical extraction in Iso- propanol. In the mechanical disruption, it was found that the outcome oil volume significantly depends on the ruption speed. Mainly at 3.55 m/s and 3.9 m/s, an oil removal would be is achieved greater than 60% of the initial sample volume (Fig. 1A). A repeating ruption cycle also assisted in fat elimination, which was observed after 6 ruption cycles. However, a 6-cycle ruption at 3.9 m/s sharply increased the sample temperature, which exceeded 37 C (as reference temperature value e Fig. 1A Red dash line). It also indi- cated that the ruption protocol at 3.55 m/s for 6 cycles would seem to yield the highest removal of fat volume and to avoid provoking of the reference temperature. After being processed by the bead ruptor at 3.55 m/s for 6 cycles, the liposuction sample (Fig. 1B) became a homogeneous mixture (Fig. 1C). A large amount of oil could easily be separated and observed as the upper yellowish layer after centrifugation (Fig. 1D). The upper oil layer was discarded, and the interested fraction of adipose tissue ECM was harvested for further incubation in Iso- propanol. The treated samples were stained with Oil Red O to detected residual lipids. In the nAT sample, Oil Red O staining revealed the lipid droplets (stained red color) due to the solubility of the dye in the lipid. The extracellular matrix of nAT was not stained with oil red, and was clearly seen after delipidation. The results showed that, without mechanical processing, there was an abundant quantity of lipid droplets remaining in the samples after 6 and 24 h extraction in Isopropanol. The lack of lipid droplets in these samples was only determined after 48 h extraction. On the other hand, a remarkable decrease of lipid droplets was observed in treated samples after 1e3 h extraction in Isopropanol by utilizing disruption in the delipidation procedure. There were no lipid droplets detected in samples that underwent disruption and 6 h delipidation (Fig. 1E). 3.1.2. Decellularization process H&E images showed the morphology of nAT, which was composed of many unilocular cells with a very thin rim of cyto- plasm containing a single large lipid droplet and the nucleus along the edge. Extracellular fibers of adipose tissue were stained pink, whereas nuclei were stained purple (Fig. 2A). Cellular remnants were found in the adipose tissue treated with 10 mM TriseHCl for 8 h and 0.1% SDS for 12 h (T8S12) (Fig. 2B). The prolonged treatment in TriseHCl for 24 h before SDS incubation (T24S12) resulted in the absence of cellular nuclei, and exposed a fibrous matrix structure with a high interconnectivity left behind (Fig. 2C). The amount of residual DNA fragments was measured to determine the cell removal and the decellularization efficacy. As shown in Fig. 2D, the amounts of residual DNA varied between the decellularization protocols. An average of 445.3 ng/mg was found in nAT, while 120 ng/mg of DNA content was yielded after a T8S12 treatment. In correspondence with the histology result, the residual DNA of the T24S12 treated samples was determined as about 38.3 ng/mg, which result was lower than 50 ng/mg (p-value < 0.05, n ¼ 6) as an evidence for the DNA removal. 3.1.3. Preparation of AAM powder After decellularization, the acellular adipose matrix (AAM) was lyophilized and underwent a 3 cycle milling at 6 m/s 30 s per cycle, (delay time between cycles was 40 s). A cryo cooling unit was used Journal of Science: Advanced Materials and Devices 6 (2021) 1e10to prevent the increase of sample temperature during the milling M.T.N. Nguyen and H.L.B. Tranprocess. This process generated a fine powder (Fig. 3A) composed of irregularly shaped particles having a diameter of less than 100 mm (Fig. 3B,C). 3.2. In vitro cytotoxicity of AAM on hADSCs The AAM powder was incubated in a complete medium (0.01 gr/ml) at 37 C for 24 h to prepare the AAM extraction. hADSCs were cultured to reach the 80% confluence and exposed to AAM extraction for 24 h. MTT assay was conducted to reveal the effect of AAM extraction on the hADSC relative growth rate (RGR - %). Fig. 1. Delipidation process. A e Bead ruption effect on oil removal of adipose tissue (N ¼ 3). Adipose tissue before ruption. C e Tissue suspension after ruption at 3.55 m/s for 6 cycles. D Isopropanol treated samples. Scale bar ¼ 50 mm. Fig. 2. Evaluation of decellularized samples. A e H&E staining of native adipose tissue (nAT)