SVF cell sheets: A new multicellular material-based strategy for promoting angiogenesis and regeneration in diced cartilage grafts

SVF cell sheets: A new multicellular material-based strategy for promoting angiogenesis and regeneration in diced cartilage grafts | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article SVF cell sheets: A new multicellular material-based strategy for promoting angiogenesis and regeneration in diced cartilage grafts Yangchen Wei, Yi Wei, Cong Xie, Zhengyang Li, Li Li, Yan Chen, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4479766/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Autologous diced cartilage, while biocompatible and easy to shape, is limited in clinical application due to its high resorption rate and challenges in establishing timely and effective neovascularization post-surgery. In this study, we produced SVF cell sheets from adipose-derived stromal vascular fraction (SVF) via enzymatic digestion, employing a temperature-sensitive culture system. Our in vivo and in vitro experiments validated that SVF cell sheets, when wrapped around granular cartilage, exhibited a notable promotion of cartilage regeneration and mitigated granular cartilage resorption in a rabbit diced cartilage graft model. Our findings demonstrate that SVF cell sheets facilitated effective neovascularization and timely cartilage block formation by secreting VEGF and Ang-1 while also suppressing the expression of pyroptotic proteins like NLRP3, Caspase1, and GSDMD. As a biofilm, derived from a multicellular source, SVF cell sheets hold promise in promoting neovascularization and cartilage regeneration in diced cartilage grafts while also preventing chondrocyte pyroptosis, presenting a potential novel approach for autologous diced cartilage transplantation. Biological sciences/Biotechnology/Regenerative medicine Biological sciences/Stem cells/Mesenchymal stem cells diced cartilage stromal vascular fractions cell sheet angiogenesis cartilage regeneration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Autologous cartilage transplantation is a common method for the clinical repair of craniofacial malformations and defects [ 1 ] . Autologous diced cartilage refers to autologous cartilage tissues from donor sites, such as the nasal septum, auricle, and costal cartilage, which are cut into fine particles of 0.5-1.0 mm 3 . Because autologous diced cartilage is easy to obtain, can be arbitrarily shaped, and has good histocompatibility [ 2 ] , it has been increasingly used as a filler and repair material in the fields of plastic surgery and repair in recent years. However, cartilage tissue has no blood vessels, lymphatic vessels, and nerves, and its self-repair ability is extremely limited. The lack of a true blood supply affects the survival and long-term effects of grafts. In the application of autologous diced cartilage for heterotopic transplantation, compared with large massive cartilage, diced cartilage has better chondrocyte viability, more obvious vascularization of pericartilage tissues, and less cartilage absorption [ 3 ] . Therefore, promoting the vascularization of autologous diced cartilage grafts and reducing graft absorption are keys to improving cartilage survival and regeneration. Clinically, wrapping materials such as autologous perichondrium or autofascia can be used to provide a relatively stable transplantation environment for diced cartilage grafts, which is conducive to promoting angiogenesis, inhibiting inflammation, and reducing absorption, so as to increase the survival of cartilage grafts and stabilize long-term surgical outcomes [ 4 , 5 ] ; the disadvantages are donor injury and limited fascial sources [ 6 ] . As an exogenous perichondrial analogue, cell sheets show great potential for application in tissue defect repair because they can protect intercellular junctions and the extracellular matrix [ 7 , 8 ] . Adipose-derived stromal vascular fractions (SVFs) are a group of heterogeneous cells that include adipose-derived stem cells, endothelial progenitor cells (EPCs), hematopoietic stem cells, pericytes, vascular smooth muscle cells, etc. [ 9 ] . Studies on cartilage regeneration have shown that SVFs can promote cartilage regeneration and repair [ 10 , 11 ] . In our previous study, rabbit adipose-derived SVFs cultured in vitro were mixed with autologous diced cartilage and then injected for transplantation in vivo, and the results showed that SVFs can promote angiogenesis in and the survival of diced cartilage grafts [ 12 ] . Takeuchi et al. reported that, compared to cell injection, cell sheet transplantation consistently increased the cell survival time, and there were fewer apoptotic cells in the cell sheet than in the cell suspension [ 13 ] . Costa et al. reported that SVF cell sheets have strong angiogenic potential and found that when implanted in a rat hindlimb ischemia model, SVF cell sheets significantly improved the restoration of blood flow in the hindlimbs [ 14 ] .However, no study has reported whether SVF cell sheets are superior to SVF cells in promoting angiogenesis in diced cartilage grafts. Recent studies have shown that chondrocyte pyroptosis-mediated cartilage inflammation and matrix degradation are involved in the development of articular cartilage damage [ 15 ] . Human adipose-derived stem cells can inhibit the chondrocyte pyroptosis signaling pathway, inhibit chondrocyte inflammatory cascades, and promote cartilage proliferation and regeneration [ 16 ] . However, whether chondrocyte pyroptosis occurs after the heterotopic transplantation of autologous diced cartilage and whether SVF cell sheets containing adipose-derived stem cells can inhibit chondrocyte pyroptosis and increase the survival of and regeneration in cartilage grafts have not been reported. The aim of this study was to investigate the beneficial effect of SVF cell sheets on angiogenesis in diced cartilage grafts and the potential effect of SVF cell sheets on inhibiting chondrocyte pyroptosis. 2. Results 2.1 SVF cell sheet characteristics and angiogenic effects in in vitro experiments After adherent growth, the rabbit adipose-derived SVF cells formed oblong fusiform or polygonal structures after approximately 10 days (Fig. 1a). HE staining showed that the SVF cells were arranged in a long fusiform and vortex shape and that the cell distribution was uniform (Fig. 1b). Flow cytometry revealed that the SVF included adipose stem cells (ADSCs) and EPCs[12]. At day 2 of culture in cell sheet-induction medium, observation under an inverted phase-contrast microscope showed that the cells in the temperature-sensitive culture dish had reached 100% confluence. When the induction culture was continued until day 14, a membranous tissue structure formed at the bottom of the culture dish. The culture dishes were incubated at 20-25 °C for approximately 40 min, and a white translucent membrane (SVF cell sheet) was obtained (Fig. 1c). SEM images of the SVF cell sheets showed that the SVF cells were connected and fused to each other and were tightly arranged, with a large amount of extracellular matrix deposited between the cells (Fig. 1d). The expression of the proangiogenic factors Ang-1 and VEGF in the three groups of cells was assessed by IF (green fluorescence (positive signal) indicated Ang-1 and VEGF expression, and blue indicated nuclei). The results showed that in the SVF cell-sheet group, the number of cells with positive signals for Ang-1 and VEGF (green fluorescence) was significantly greater than that in the blank group (P <0.01) and the SVF cell group (P <0.05); moreover, the number of cells in the SVF cell group was greater than that in the blank group (P <0.01) (Fig. 2a-d). The qRT-PCR and WB results showed that, in the SVF cell-sheet group, the messenger RNA (mRNA) and protein expression levels of Ang-1 and VEGF were significantly higher than those in the blank group (P <0.01) and SVF cell group (P<0.05); the expression levels in the SVF cell group were higher than those in the blank group (P<0.01) (Fig. 2e- i). These findings suggest that compared with SVF cells, SVF cell sheets could further upregulate the gene and protein expression of angiogenic factors. In vitro tube formation assays showed that cells in the blank group grew in cords rather than in tubes; in the SVF cell and SVF cell-sheet groups, after 6 h, the cells attached to the Matrigel and gradually extended their pseudopodia and made contact with the surrounding cells, forming a three-dimensional (3D) reticular lumen-like structure similar to blood vessels, with tube formation rates of 20.3% and 21.5%, respectively (Fig. 3). These results indicated that the SVF cell sheets had a greater proangiogenic effect than did the SVF cells. 2.2 An SVF cell sheet promotes cartilage regeneration in diced cartilage grafts New Zealand rabbits were euthanized at 1, 3, and 6 months after surgery by intravenous injection of 1% sodium pentobarbital 100 mg/kg, the bilateral diced cartilage grafts on the back were removed, and the appearance and morphological characteristics of the grafts were observed. At 1 month after surgery, the diced cartilage grafts in the blank group were slightly soft when touching, with some unfused diced cartilage scattered around the incision; the texture of the grafts in the perichondrium group was slightly tough, and a small amount of unfused diced cartilage could be observed around the central incision; and the diced cartilage grafts in the SVF cell-sheet group were basically fused and slightly tough when touching. At 3 months after surgery, the diced cartilage grafts in the blank group and the perichondrium group were basically fused, with an irregular appearance, and were relatively tough when touching; in the SVF cell-sheet group, the diced cartilage grafts were completely fused and were tough when touching. At 6 months after surgery, the diced cartilage grafts in the three groups were completely fused and were slightly hard when touching, and connective tissue fibrous membrane wrapping and neovascular growth were seen along the periphery (Fig. 4a). The wet weight of the grafts in each group was measured before transplantation and at 1, 3, and 6 months after transplantation. The results showed that in the blank group, the wet weights of cartilage grafts at 1 month, 3 months and 6 months after surgery were not significantly different from those before transplantation (P>0.05); at 3 and 6 months after surgery, the wet weights of cartilage grafts in the perichondrium group and SVF cell-sheet group were greater than those of cartilage grafts in the blank group (P <0.05), and the wet weight of grafts in the SVF cell-sheet group was slightly greater than that in the perichondrium group (p <0.05) (Table 1). To understand the regenerative activity of chondrocytes, specific staining of cartilage tissue blocks was performed. Safranin fast green staining results showed that at 3 months after surgery, cells at the edge of diced cartilage grafts in the SVF cell sheet and perichondrium groups were oblate and existed alone, indicating the presence of new chondrocytes; the chondrocytes near the center of the cartilage were round and oval, and two or three isogenic cell population aggregates were observed. At 6 months after surgery, the number of new chondrocytes in the perichondrium group gradually increased; in the SVF cell-sheet group, large amounts of new cartilage and red cartilage matrix were observed, multiple chondrocytes were present in the lacuna, and new cartilage had gradually matured. In the blank group, there was no significant increase in the number of new chondrocytes or red cartilage matrix at 3 months after surgery compared with 1 month after surgery, and the amount of new chondrocytes at 6 months after surgery was slightly lower than that at 1 month after surgery (Fig. 4b). After Masson staining, the collagen fibers in the cartilage matrix were stained blue. At 1 month after surgery, the differences in the amount of blue stained cartilage matrix in the 3 groups were not significant. At 3 and 6 months after surgery, the blue staining area and staining intensity of the cartilage matrix in the perichondrium group and the SVF cell-sheet group were significantly greater than those in the blank group; the blue staining area and staining intensity of the cartilage matrix in the SVF cell-sheet group were higher than those in the perichondrium group, suggesting that the SVF cell sheets can increase the secretion of cartilage collagen (Fig. 4c). After toluidine blue staining, proteoglycan, the main component of the cartilage matrix, was stained blue-purple, and the staining intensity was related to the amount of proteoglycans. At 1 month after surgery, the differences in the amount of blue-purple stained cartilage tissues in the 3 groups were not significant. At 3 and 6 months after surgery, the blue-purple staining area and staining intensity of the cartilage tissues in the perichondrium group and the SVF cell-sheet group were significantly greater than those in the blank group; the blue-purple staining area and staining intensity of the cartilage tissues in the SVF cell-sheet group were greater than those in the perichondrium group, suggesting that SVF cell sheets can increase cartilage matrix secretion (Fig. 4d). These cartilage tissue-specific staining results suggest that compared with autologous perichondrium, SVF cell sheets better promote the survival of and regeneration in grafted cartilage. 2.3 SVF cell sheets promote angiogenesis in diced cartilage grafts To understand the effects of SVF cell sheets on vascularization around diced cartilage grafts, IHC was performed to assess the expression levels of Ang-1 and VEGF in cartilage tissues of the three groups, and the results showed that in the SVF cell-sheet group and perichondrium group, the chondrocytes surrounding the cartilage tissue and the tissue surrounding the cartilage block were intensely stained brownish yellow and dark brown. At 1, 3, and 6 months after surgery, the number of positive cells for both Ang-1 and VEGF was significantly higher in the perichondrium group than in the blank group, and the expression of Ang-1 and VEGF in the SVF cell-sheet group was further upregulated compared to that in the perichondrium group (Fig. 5a and 5b). The qRT-PCR and WB results showed that at 1, 3, and 6 months after surgery, the mRNA and protein expression levels of Ang-1 and VEGF in the SVF cell-sheet group and the perichondrium group were greater than those in the blank group (P <0.01) and that the levels in the SVF cell-sheet group were greater than those in the perichondrium group (P <0.05) (Fig. 5c-g). These results indicate that SVF cell sheets upregulate the gene and protein expression of angiogenesis-related factors, thereby facilitating angiogenesis in cartilage grafts. HE staining was used to observe the adipose tissue and vascularization around diced cartilage grafts. At 1 month after surgery, compared with those in the blank group, small amounts of connective and adipose tissues and blood vessel formation between diced cartilage grafts were observed in the perichondrium and SVF cell-sheet groups (P <0.05). At 3 and 6 months after surgery, in the blank group, small amounts of connective tissue and adipose tissue formed around the diced cartilage grafts, and a small amount of neovasculature could be observed. Compared with those in the blank group, in the perichondrium group, there was more connective and adipose tissue around the diced cartilage grafts, and more neovasculature could be observed around the cartilage (P<0.05). In the SVF cell-sheet group, a large amount of connective and adipose tissue and a large amount of neovasculature formed around the diced cartilage grafts (P <0.05) (Fig. 6a, b). The results indicate that the SVF cell sheets better promoted the formation of neovasculature in the cartilage grafts. 2.4 Effect of SVF cell sheets on pyroptosis in cartilage tissues To understand the viability and survival of chondrocytes in grafts 6 months after diced cartilage transplantation, TUNEL staining was performed, and the results showed that in the blank group, the TUNEL-positive cells (green stained) were located at the center and surrounding area of the diced cartilage grafts; in the SVF cell-sheet group and the perichondrium group, most TUNEL-positive cells were located in the center of diced cartilage grafts, and the percentage of TUNEL-positive cells was significantly lower than that in the blank group (both P<0.01); moreover, the percentage of TUNEL-positive cells in the SVF cell-sheet group was lower than that in the perichondrium group (P <0.01), suggesting that SVF cell sheets can reduce the cell death of cartilage tissues (Fig. 7a, c). To further elucidate the cause of cell death, the protein expression levels of NLRP3, Caspase1 and GSDMD, which are involved the canonical pyroptosis pathway, in the cartilage tissues of diced cartilage grafts were assessed by IHC, and the results confirmed that pyroptosis occurred in the chondrocytes. However, the protein expression of components of the pyroptosis signaling pathway was lower in the perichondrium group than in the blank group (P <0.05), and the expression in the SVF cell-sheet group was even lower (P <0.01). These findings suggest that SVF cell sheets reduced chondrocyte pyroptosis in the grafts, which is beneficial for cartilage regeneration (Fig. 7b, d). 3. Discussion The regeneration process of cartilage is based on intact perichondrium. The perichondrium is divided into two layers: the outer layer is mainly composed of connective tissue, which plays a supporting and protective role, and the inner layer, i.e., the germinal layer, which contains mesenchymal stem cells that can differentiate into chondrocytes [ 17 ] . The blood vessels attached to the perichondrium provide nutrients to cartilage and facilitate its growth, development and regeneration [ 18 ] . Some scholars have compared the grafting effects of Surgicel and autologous deep temporal fascia-wrapped diced cartilage grafts and found that the survival rate of cartilage blocks wrapped with Surgicel was low, while autologous deep temporal fascia wrapping can reduce inflammation in cartilage grafts and maintain the activity of chondrocytes, which are beneficial for cartilage graft survival [ 19 ] . However, due to the limited source of autologous perichondrium and the existence of donor site injury, the construction of perichondrium analogues to replace autologous perichondrium to promote the regenerative repair of cartilage grafts has been a research focus in the field of plastic surgery. The SVF comprises a group of cell populations obtained from adipose tissue digested with collagenase, and the cell composition can change with cell culture [ 20 ] . Currently, adipose-derived stem cells and EPCs are important components of the SVF [ 21 ] . Our previous studies also confirmed that rabbit adipose-derived SVF contains these two types of cells and that the injection of SVF cell suspensions promoted angiogenesis and the survival of diced cartilage grafts [ 12 ] . This study demonstrated for the first time that using SVF cell sheets to wrap diced cartilage grafts could more effectively promote angiogenesis and cartilage regeneration in cartilage grafts; notably, this method improved the survival rate of the grafts. Some studies have reported that SVF cells can secrete large amounts of angiogenic growth factors, such as VEGF, transforming growth factor-β (TGF-β), and basic fibroblast growth factor (bFGF), and that EPCs can differentiate into endothelial cells to participate in vascular budding and directly participate in vascular reconstruction [ 22 ] . At present, SVF cells are mostly used to promote the revascularization of adipose tissue after autologous fat transplantation [ 23 ] . In contrast to the traditional way of obtaining cells by digesting intercellular junctions with trypsin, cell sheets retain a large amount of extracellular matrix secreted by autologous cells, providing a microenvironment for the proliferation and differentiation of cells very similar to that in the body; thus, there is a greater potential for the application in the field of tissue defect repair. Temperature-sensitive culture systems with temperature-sensitive materials can regulate cell adhesion and detachment by altering the temperature, and because of their simple operation and easy control, these systems have become the main method for preparing tissue-engineered cell sheets [ 24 ] . To this end, in this study, a temperature-sensitive culture system was used to construct a perichondrial analogue, i.e., SVF cell sheets. The SVF cell sheets obtained by this method adhered and grew well at 37°C; when the temperature was lowered to room temperature and maintained for 40 min, almost all cells detached in the form of tightly connected cell sheets that remained active and continued to adhere and grow. SEM revealed that SVF cells were arranged in multiple layers in the sheet and that the intercellular junctions and extracellular matrix were tightly fused to each other. In vitro studies revealed that the expression levels of Ang-1 and VEGF were greater in SVF cell sheets than in SVF cells. Ang-1 and VEGF are currently recognized as the strongest proangiogenic factors. During angiogenesis, VEGF synergizes with Ang-1 to play an important role in promoting neovasculature formation, maturation and stability [ 25 – 27 ] . The in vitro tube formation assay revealed that the tube formation rate of SVF cell sheets was greater than that of SVF cells, suggesting that the neovascularization-promoting effect of SVF cell sheets was greater than that of the SVF cells, which is consistent with the results of Li et al. [ 21 ] . Recently, Li et al. found that compared with the direct injection of SVF cells, SVF cell sheet transplantation resulted in higher expression of the angiogenesis-related protein VEGF and stronger pro-neovascularization, which may be attributed to the 3D tissue-like structure formed by SVF cell sheets, enhancing their paracrine effect; therefore, they are more conducive to promoting neovascularization [ 21 ] . The results of the animal experiments in this study showed that at 3 and 6 months after surgery, the wet weight of the grafts in the SVF cell-sheet group was slightly greater than that in the perichondrium group; cartilage tissue-specific staining revealed that in the SVF cell-sheet group, chondrocyte proliferation was active, and cellular matrix and collagen secretion were robust, confirming that compared with autologous cartilage perichondrial wrapping, SVF cell sheet wrapping is more favorable to cartilage regeneration in grafts. In this study, for SVF cell sheets, the expression of the pro-angiogenic factors Ang-1 and VEGF was stronger than that using autologous perichondrium, and more peripheral adipose tissue and neovasculature formation around the grafts were observed at 3 and 6 months after surgery, suggesting that the SVF cell sheets better promoted the vascularization of cartilage grafts. The reason may be that the SVF cell sheets contain a large number of EPCs, which are conducive to neovasculature formation [ 12 ] . In clinical practice, the heterotopic transplantation of autologous diced cartilage grafts also faces the problem because at the transplantation site, an immune-inflammatory response occurs, resulting in a decrease in viability and death of the transplanted chondrocytes [ 3 , 28 ] . In recent years, a new method of programmed cell death, GSDMD-mediated pyroptosis, has been discovered. GSDMD-mediated pyroptosis is closely related to inflammation and characterized by NLRP3 inflammasome activation [ 29 ] , which activates caspase-1 and catalyzes the cleavage of the effector protein GSDMD to produce the active fragment GSDMD-N, which is mosaiced in the cell membrane to form pores, allowing extracellular fluid to enter cells, which eventually leads to cell lysis and death [ 30 ] . Some studies have shown that chondrocyte pyroptosis may play a key role in the pathological process of osteoarthritis [ 31 ] . Adipose-derived mesenchymal stem cells can inhibit the chondrocyte pyroptosis signaling pathway to delay the progression of rat osteoarthritis, suggesting that adipose-derived stem cell inhibition of chondrocyte pyroptosis promotes the repair of cartilage tissue damage [ 16 ] . The canonical pyroptosis pathway is dependent on the activation of caspase-1, which can be mediated by danger-associated molecular patterns (DAMPs) [ 32 ] . Cartilage grafting occurs in a sterile environment, and when cartilage tissue is cut in vitro, DAMPs are produced, which in turn activate NLRP3. Therefore, this study investigated the expression of characteristic molecules of the canonical pyroptosis pathway. This study revealed for the first time that chondrocyte pyroptosis also occurred after autologous diced cartilage graft transplantation and that compared to autologous perichondrium, SVF cell sheets further inhibited chondrocyte pyroptosis and reduced chondrocyte death, which may be beneficial for graft regeneration and repair. Because the SVF contains a large number of adipose-derived mesenchymal stem cells, we speculate that the protective effect of SVF cell sheets might be mediated by adipose-derived stem cells. This study has limitations. For example, without the participation of a scaffold, the strength of the constructed cell sheets may be insufficient, thus limiting the subsequent repair and reconstruction of tissue or organs to some extent [ 33 ] . Future studies will optimize the culture conditions to obtain SVF cell sheets with better performance. Due to the heterogeneity of SVF cells, the molecular mechanisms underlying the promotion of angiogenesis, improvements in the blood supply of cartilage tissue and the inhibition of chondrocyte pyroptosis by SVF cell sheets need to be further investigated. 4. Methods 4.1 Preparation of SVF cell sheets and ultrastructural analysis Rabbit adipose-derived SVF cells were extracted and identified following the method reported in our previous study [ 12 ] . The experiments were approved by The Experimental Animal Department of Nanhua University (SYXK (Xiang 2020-0002)), and reported in accordance with ARRIVE guidelines. An SVF cell suspension that had been passaged to the 2nd generation was seeded uniformly (1x10 6 /cm 2 ) in a temperature-sensitive culture dish (35 mm, UpCell, Thermo Fisher Scientific), and after the cells had completely adhered to the dish, sheet-forming induction solution (Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 50 mg/L ascorbic acid) was added to the cells, followed by incubation for 14 days. Then, the dish was placed at 20–25°C for approximately 40 min, allowing SVF cell sheets to detach from the surface of the culture dish. The SVF cell sheets were fixed in 2.5% glutaraldehyde and washed with phosphate-buffered saline (PBS). The cells were then dehydrated in a graded ethanol series. The samples were gold-plated and observed using a scanning electron microscope. 4.2 Coculture and grouping of SVF cells and chondrocytes Rabbit ear chondrocytes were purchased from Wuhan Cloud Clone Technology Co., Ltd. (CS1261Rb01). After passage in culture, second-generation chondrocytes were used in subsequent experiments. The cells for in vitro experiments were divided into three groups. In the blank group, rabbit ear chondrocytes (2x10 6 cells/cm 2 ) were cultured alone. In the SVF cell group, SVFs (1x10 6 cells/cm 2 ) and chondrocytes were cocultured at a ratio of 1:1. In the SVF cell-sheet group, the obtained SVF cell sheets were cocultured with rabbit ear chondrocytes (1x10 6 cells/cm 2 ). The cells in the three groups were all cultured in low-glucose DMEM containing 10% FBS + 1% antibiotics (penicillin and streptomycin) and in a 5% CO 2 incubator at 37°C for 48 h. 4.3 Preparation and implantation of diced cartilage grafts Eighteen 6-month-old New Zealand white rabbits weighing 2.0-2.5 kg were obtained from the Experimental Animal Department of Nanhua University after the animal experiments were approved (SYXK (Xiang 2020-0002)). There was one rabbit per cage. After the rabbits were fed standard chow for 1 week, subcutaneous adipose tissue was extracted from the rabbits following the method reported in our previous studies; this tissue was used to prepare SVF cells. This study was approved by the Laboratory Animal Management and Ethics Committee of the First Affiliated Hospital of Nanhua University, and all procedures complied with the rules for the Management of Laboratory Animals of the People's Republic of China. Rabbits were anesthetized by the intraperitoneal injection of 5% urethane (5 mL/kg). After disinfection, the unilateral ear was removed from the root; the skin, fascia and perichondrium of the ear were removed; and the ear was soaked in saline containing gentamicin for 10 min. The ear cartilage was cut into approximately 1.0 mm 3 pieces and divided into three equal parts. For in vivo experiments, the animals were divided into three groups. In the blank group, rabbits were grafted with unwrapped diced cartilage; in the perichondrium group, rabbits were grafted with diced cartilage wrapped with autologous perichondria; and in the SVF cell-sheet group, rabbits were grafted with diced cartilage wrapped with SVF cell sheets. Before implantation, the wet weight of all diced cartilage was measured with an electronic scale. The skin on the back of each rabbit was prepared and routinely disinfected. Symmetrical areas to the right and left of the posterior midline were selected, three subcutaneous incisions were made, the diced cartilage grafts were transplanted into the subcutis, the wound was sutured, and erythromycin ointment was applied to the wound surface. The grafts were marked with a marker pen. Rabbits were randomly euthanized by ear intravenous injection of 3% pentobarbital sodium 100 mg/kg at 1, 3, and 6 months after surgery (6 at each time point). The diced cartilage grafts on the back were removed, and the gross condition was observed. The soft tissue from the surface of the diced cartilage grafts was removed, and the wet weight was measured. 4.4 Histological analysis and immunohistochemical analysis Cartilage tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Hematoxylin-eosin (HE) staining, Masson staining, safranin fast green staining, and toluidine blue staining were performed on 5-µm thick tissue sections according to the manufacturers’ instructions. All the samples were dehydrated, mounted with neutral gum, and observed under a microscope. IHC was performed according to standard protocols. In brief, antigen extraction was performed after tissue sections were dewaxed and dehydrated. Next, the tissue sections were treated with 0.3% hydrogen peroxide and then incubated with the following primary antibodies at 4°C overnight: anti-vascular endothelial growth factor (VEGF) (1:50, Abcam, Cat# ab52917), anti-angiopoietin-1 (Ang-1) (1:50, Proteintech, Cat# 23302-1-AP), anti-NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) (1:200, Proteintech, Cat# 27458-1-AP), anti-Caspase1 (1:200, Proteintech, Cat# 22915-1-AP), and anti-gasdermin D (GSDMD) (1:200, Proteintech, Cat# 20770-1-AP). A horseradish peroxidase-labeled secondary antibody was added, and the sections were incubated for 60 min. 3,3′-Diaminobenzidine (DAB) was used for color development, and the sections were counterstained with hematoxylin, cleared, mounted, and observed under a microscope. 4.5 Terminal deoxyribonucleotide transferase [TdT]-mediated dUTP nick end labeling (TUNEL) assay Paraffin sections of rabbit cartilage tissues were dewaxed and dehydrated.Chondrocyte death was assessed using the TUNEL assay. An in situ cell death detection kit (Shanghai Yisheng Biotechnology Co., Ltd. 40306ES50) was used according to the manufacturer’s instructions. Nuclei were stained with 4',6-diamino-2-phenylindole (DAPI, Wellbio, China). TUNEL-positive cells were detected and imaged using fluorescence microscopy. 4.6 Immunofluorescence (IF) detection After dewaxing and dehydrating the cell sections, they were blocked with 5% bovine serum albumin (BSA) for 60 min at room temperature. The sections were fully covered by anti-VEGF (1:50, Proteintech, Cat# 19003-1-AP) and anti-Ang-1 (1:50) antibodies, incubated in a refrigerator at 4°C overnight, washed with PBS, incubated with a secondary antibody in the dark for 90 min, incubated with DAPI at room temperature for 10 min, and mounted with glycerol. The cells were observed under a fluorescence microscope. 4.7 Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) Total RNA from cells or tissues was prepared, and complementary DNA (cDNA) was synthesized by reverse transcription. The following synthetic primers were used: β-actin, 5'- TGGCCGAGACTTTGATTGT-3' upstream, 5'-TTACACAAATGCGATGCTGCC-3' downstream; VEGF, 5'- TGCGGATCAUACCACCAG-3' upstream, 5'-CCGGTTTCTTGCGCTTTC-3' downstream; and Ang-1, 5'- GGCTTGGTTGCTCGTCAAAC-3' upstream, and 5'-CAGGACGCTGTTGTTGGTTG-3' downstream; the sizes of the amplicons were 170 bp, 158 bp and 81 bp, respectively. The following 30-µL reaction system was used for PCR: 15 µL of SYBR Green mix, 2 µL of primer mix, 11 µL of nuclease-free water, and 2 µL of cDNA. The samples were subjected to 95°C for 10 min and 40 cycles of 95°C for 15 s and 60°C for 30 s. Gene expression was analyzed by the 2 −ΔΔCt method using β-actin as an internal control. 4.8 Western blot (WB) Total protein from cells or tissues were extracted with 200 µl of radioimmunoprecipitation (RIPA) lysis buffer. Protein quantification was performed by the bicinchoninic acid (BCA) assay. Proteins were loaded and electrophoresed on a gel and then transferred to NC membranes by electroblotting. The membranes were placed in 5% skim milk powder blocking solution for 90 min at room temperature and then incubated at 4°C overnight with the following primary antibodies: anti-VEGF (1:2000), anti-Ang-1 (1:1000), and anti-β-actin (1:5000). The next day, the membranes were washed with PBST and then incubated with a horseradish peroxidase-labeled secondary antibody (1:5000) at room temperature for 90 min. Finally, the membrane was washed, and the protein bands were developed using enhanced chemiluminescence (ECL) reagent. 4.9 Angiogenesis experiment Microvasculature regeneration was detected via a tube formation assay [ 34 ] . In brief, Matrigel was incubated in a 4°C freezer overnight. A 96-well plate was precooled; 50 µl of Matrigel was added to each well, and the plate was placed in a 37°C incubator for 30 min to allow the gel to coagulate. The cells were digested with trypsin and counted to prepare a cell suspension, and approximately 10,000 cells were added to each well. After incubation at 37°C in a 5% CO 2 tissue culture incubator for 6 h, angiogenesis was observed under a light microscope, and the number of lumens and branch points formed was counted. 4.10 Statistical analysis Statistical analysis was performed using SPSS 27.0 software. The data are expressed as the mean ± standard deviation ( ± SD), and the differences among groups were analyzed by using the t test or one-way analysis of variance (ANOVA). P < 0.05 indicates a statistically significant difference. 5. Conclusion In this study, SVF cells were used to construct a multicellular material, SVF cell sheets, in temperature-sensitive culture dishes, and the SVF cell sheets were wrapped around diced cartilage grafts prior to heterotopic transplantation. The results indicated that the SVF cell sheets promoted the survival of and regeneration in cartilage grafts and that those effects may be related to the promotion of angiogenesis, improvements in blood supply to diced cartilage grafts and the inhibition of chondrocyte pyroptosis by SVF cell sheets. These findings broaden the understanding of the application of SVF cell sheets in cartilage regeneration. The SVF cell sheet wrapping technique may become a new strategy for autologous diced cartilage transplantation. Declarations Data availability All data generated or analysed during this study are included in this article or supplementary information files. Authors' contributions Yangchen Wei, Yi Wei and Cong Xie conceptualized the study, wrote the original draft, conducted formal analysis, and wrote, reviewed and edited the manuscript. Zhengyang Li, Li Li and Yan Chen reviewed and edited the manuscript. Yiping Wang developed methodology. Chiyu Jia contributed to study conceptualization, writing the original draft, and writing, reviewing and editing the manuscript. Hongju Xie and Junlin Liao supervised the study. Competing interests The author(s) declare no competing interests. Funding This work was supported by the National Natural Science Foundation of China (Grant No. 82002059), the Natural Science Foundation of Hunnan Province, China (Grant No. 2023JJ30542), the Clinical Medical Technology Innovation Guidance Project of Hunan Province, China (Grant No. 2021SK51826), the Health Commission of Hunan Province, China (Grant No. D202304108322), and the Key Scientific Research Project of Higher Education in Hainan Province, China (Grant No. Hnky2021ZD-16). Consent to participate Not applicable. Consent for publication Not applicable. References Wilkes, G. H., Wong, J. & Guilfoyle, R. Microtia reconstruction. Plast Reconstr Surg 134, 464e-479e, doi: 10.1097/PRS.0000000000000526 (2014). Erol, O. O. Injection of Compressed Diced Cartilage in the Correction of Secondary and Primary Rhinoplasty: A New Technique with 12 Years' Experience. Plast Reconstr Surg 140, 673e-685e, doi: 10.1097/PRS.0000000000003815 (2017). Kim, S. H., Suh, J. H. & Jang, Y. J. Histomorphological Findings of Cartilage and Surrounding Tissues According to Thickness and Manipulations in Rabbits. Aesthet Surg J 42, NP489-NP500, doi: 10.1093/asj/sjac028 (2022). Kemaloglu, C. A. & Tekin, Y. A comparison of diced cartilage grafts wrapped in perichondrium versus fascia. Aesthetic Plast Surg 38, 1164–1168, doi: 10.1007/s00266-014-0403-6 (2014). Cerkes, N. & Basaran, K. Diced Cartilage Grafts Wrapped in Rectus Abdominis Fascia for Nasal Dorsum Augmentation. Plast Reconstr Surg 137, 43–51, doi: 10.1097/PRS.0000000000001876 (2016). Daniel, R. K. & Calvert, J. W. Diced cartilage grafts in rhinoplasty surgery. Plast Reconstr Surg 113, 2156–2171, doi: 10.1097/01.prs.0000122544.87086.b9 (2004). Wang, C. et al. Differentiation of Urine-Derived Human Induced Pluripotent Stem Cells to Alveolar Type II Epithelial Cells. Cell Reprogram 18, 30–36, doi: 10.1089/cell.2015.0015 (2016). Gao, H., Li, B., Zhao, L. & Jin, Y. Influence of nanotopography on periodontal ligament stem cell functions and cell sheet based periodontal regeneration. Int J Nanomedicine 10, 4009–4027, doi: 10.2147/IJN.S83357 (2015). Ramakrishnan, V. M. & Boyd, N. L. The Adipose Stromal Vascular Fraction as a Complex Cellular Source for Tissue Engineering Applications. Tissue Eng Part B Rev 24, 289–299, doi: 10.1089/ten.TEB.2017.0061 (2018). Pak, J. et al. Clinical Protocol of Producing Adipose Tissue-Derived Stromal Vascular Fraction for Potential Cartilage Regeneration. J Vis Exp, doi: 10.3791/58363 (2018). Salikhov, R. Z. et al. The Stromal Vascular Fraction From Fat Tissue in the Treatment of Osteochondral Knee Defect: Case Report. Front Med (Lausanne) 5, 154, doi: 10.3389/fmed.2018.00154 (2018). Yin, H. et al. 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VEGF-induced BBB permeability is associated with an MMP-9 activity increase in cerebral ischemia: both effects decreased by Ang-1. J Cereb Blood Flow Metab 25, 1491–1504, doi: 10.1038/sj.jcbfm.9600148 (2005). Traktuev, D. O. et al. Robust functional vascular network formation in vivo by cooperation of adipose progenitor and endothelial cells. Circ Res 104, 1410–1420, doi: 10.1161/CIRCRESAHA.108.190926 (2009). Pallua, N., Serin, M. & Wolter, T. P. Characterisation of angiogenetic growth factor production in adipose tissue-derived mesenchymal cells. J Plast Surg Hand Surg 48, 412–416, doi: 10.3109/2000656X.2014.903196 (2014). Bartaula-Brevik, S., Pedersen, T. O., Finne-Wistrand, A., Bolstad, A. I. & Mustafa, K. Angiogenic and Immunomodulatory Properties of Endothelial and Mesenchymal Stem Cells. Tissue Eng Part A 22, 244–252, doi: 10.1089/ten.TEA.2015.0316 (2016). Shen, H. H. et al. NLRP3: A promising therapeutic target for autoimmune diseases. Autoimmun Rev 17, 694–702, doi: 10.1016/j.autrev.2018.01.020 (2018). Zu, Y., Mu, Y., Li, Q., Zhang, S. T. & Yan, H. J. Icariin alleviates osteoarthritis by inhibiting NLRP3-mediated pyroptosis. J Orthop Surg Res 14, 307, doi: 10.1186/s13018-019-1307-6 (2019). An, S., Hu, H., Li, Y. & Hu, Y. Pyroptosis Plays a Role in Osteoarthritis. Aging Dis 11, 1146–1157, doi: 10.14336/AD.2019.1127 (2020). Ghonime, M. G., Shamaa, O. R., Eldomany, R. A., Gavrilin, M. A. & Wewers, M. D. Tyrosine phosphatase inhibition induces an ASC-dependent pyroptosis. Biochem Biophys Res Commun 425, 384–389, doi: 10.1016/j.bbrc.2012.07.102 (2012). Zheng, R. et al. Regeneration of Subcutaneous Cartilage in a Swine Model Using Autologous Auricular Chondrocytes and Electrospun Nanofiber Membranes Under Conditions of Varying Gelatin/PCL Ratios. Front Bioeng Biotechnol 9, 752677, doi: 10.3389/fbioe.2021.752677 (2021). Hu, Y. et al. Wnt10b-overexpressing umbilical cord mesenchymal stem cells promote fracture healing via accelerated cartilage callus to bone remodeling. Bioengineered 13, 10313–10323, doi: 10.1080/21655979.2022.2062954 (2022). Table Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files UneditedWBimage.pdf Table1.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4479766","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":313550103,"identity":"5383f22b-94b1-4644-bc50-fb6bcbbe4962","order_by":0,"name":"Yangchen Wei","email":"","orcid":"","institution":"Center of Burn\u0026Plastic and Wound Repair,The First Affiliated Hospital, Hengyang Medical School,University of South China","correspondingAuthor":false,"prefix":"","firstName":"Yangchen","middleName":"","lastName":"Wei","suffix":""},{"id":313550104,"identity":"c84bf4b8-e78c-4cda-8420-8c17910e397a","order_by":1,"name":"Yi 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11:03:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4479766/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4479766/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58386507,"identity":"f799e5c6-06db-459e-90bc-06c137c958ca","added_by":"auto","created_at":"2024-06-14 18:44:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1039595,"visible":true,"origin":"","legend":"\u003cp\u003eSVF cells and SVF cell sheets\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4479766/v1/152a02f98a928bc7619365e6.png"},{"id":58387489,"identity":"c70f6329-52f7-493d-bcbc-15652bb61e38","added_by":"auto","created_at":"2024-06-14 18:52:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2496133,"visible":true,"origin":"","legend":"\u003cp\u003eIn vitro experiments to detect the expression of Ang-1 and VEGF in each group\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4479766/v1/a1bcce97b423f8cd8d707938.png"},{"id":58386510,"identity":"36b970ed-927c-49c1-ae6a-c71ea12e158e","added_by":"auto","created_at":"2024-06-14 18:44:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":776808,"visible":true,"origin":"","legend":"\u003cp\u003eIn vitro angiogenesis experiment for each group. Scale bar: 100μm\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4479766/v1/ab06415d0e5f069b8a3cc813.png"},{"id":58386511,"identity":"e2182996-3efc-49fa-8cb8-adf249a3747b","added_by":"auto","created_at":"2024-06-14 18:44:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":9339363,"visible":true,"origin":"","legend":"\u003cp\u003eSVF cell sheets promote cartilage regeneration in diced cartilage grafts\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4479766/v1/a9b1a21f6fd5c862c1f085fe.png"},{"id":58386512,"identity":"48ed11bc-d8fe-44d7-81fb-4b9809894c9b","added_by":"auto","created_at":"2024-06-14 18:44:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":10489389,"visible":true,"origin":"","legend":"\u003cp\u003eSVF cell sheets promote the expression of Ang-1 and VEGF in diced cartilage grafts\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4479766/v1/62fa8715241fd31b3e1b0394.png"},{"id":58386515,"identity":"09a24559-cd20-49ae-b6f7-b35180d9bf85","added_by":"auto","created_at":"2024-06-14 18:44:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2495698,"visible":true,"origin":"","legend":"\u003cp\u003eSVFs cell sheets promote angiogenesis\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-4479766/v1/68fbadc8deb7f36b00635c29.png"},{"id":58386513,"identity":"feb1b2f3-9b18-41d2-ba5a-463d0564c32c","added_by":"auto","created_at":"2024-06-14 18:44:48","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":4000500,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of SVF cell sheets on chondrocyte pyroptosis\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-4479766/v1/4da2761bfa76091d913b95a4.png"},{"id":69270925,"identity":"a26f020d-5484-4650-a243-fc1196098d04","added_by":"auto","created_at":"2024-11-18 15:17:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":37799392,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4479766/v1/999a2944-1b6d-471f-8533-a2fcca2c2b41.pdf"},{"id":58386508,"identity":"4ad77d62-b6d4-4d72-bcbe-9aa81ceb290f","added_by":"auto","created_at":"2024-06-14 18:44:48","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":867230,"visible":true,"origin":"","legend":"","description":"","filename":"UneditedWBimage.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4479766/v1/cc2669a15d52c855f3042cd3.pdf"},{"id":58386509,"identity":"60739609-775d-4010-9bec-a586868e1e37","added_by":"auto","created_at":"2024-06-14 18:44:48","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11229,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4479766/v1/43a5d665ec723f18f093747f.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"SVF cell sheets: A new multicellular material-based strategy for promoting angiogenesis and regeneration in diced cartilage grafts","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAutologous cartilage transplantation is a common method for the clinical repair of craniofacial malformations and defects\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Autologous diced cartilage refers to autologous cartilage tissues from donor sites, such as the nasal septum, auricle, and costal cartilage, which are cut into fine particles of 0.5-1.0 mm\u003csup\u003e3\u003c/sup\u003e. Because autologous diced cartilage is easy to obtain, can be arbitrarily shaped, and has good histocompatibility\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e, it has been increasingly used as a filler and repair material in the fields of plastic surgery and repair in recent years. However, cartilage tissue has no blood vessels, lymphatic vessels, and nerves, and its self-repair ability is extremely limited. The lack of a true blood supply affects the survival and long-term effects of grafts. In the application of autologous diced cartilage for heterotopic transplantation, compared with large massive cartilage, diced cartilage has better chondrocyte viability, more obvious vascularization of pericartilage tissues, and less cartilage absorption\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Therefore, promoting the vascularization of autologous diced cartilage grafts and reducing graft absorption are keys to improving cartilage survival and regeneration. Clinically, wrapping materials such as autologous perichondrium or autofascia can be used to provide a relatively stable transplantation environment for diced cartilage grafts, which is conducive to promoting angiogenesis, inhibiting inflammation, and reducing absorption, so as to increase the survival of cartilage grafts and stabilize long-term surgical outcomes\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e; the disadvantages are donor injury and limited fascial sources\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAs an exogenous perichondrial analogue, cell sheets show great potential for application in tissue defect repair because they can protect intercellular junctions and the extracellular matrix\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Adipose-derived stromal vascular fractions (SVFs) are a group of heterogeneous cells that include adipose-derived stem cells, endothelial progenitor cells (EPCs), hematopoietic stem cells, pericytes, vascular smooth muscle cells, etc.\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Studies on cartilage regeneration have shown that SVFs can promote cartilage regeneration and repair\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. In our previous study, rabbit adipose-derived SVFs cultured in vitro were mixed with autologous diced cartilage and then injected for transplantation in vivo, and the results showed that SVFs can promote angiogenesis in and the survival of diced cartilage grafts\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Takeuchi et al. reported that, compared to cell injection, cell sheet transplantation consistently increased the cell survival time, and there were fewer apoptotic cells in the cell sheet than in the cell suspension\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Costa et al. reported that SVF cell sheets have strong angiogenic potential and found that when implanted in a rat hindlimb ischemia model, SVF cell sheets significantly improved the restoration of blood flow in the hindlimbs\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e.However, no study has reported whether SVF cell sheets are superior to SVF cells in promoting angiogenesis in diced cartilage grafts.\u003c/p\u003e \u003cp\u003eRecent studies have shown that chondrocyte pyroptosis-mediated cartilage inflammation and matrix degradation are involved in the development of articular cartilage damage\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Human adipose-derived stem cells can inhibit the chondrocyte pyroptosis signaling pathway, inhibit chondrocyte inflammatory cascades, and promote cartilage proliferation and regeneration\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. However, whether chondrocyte pyroptosis occurs after the heterotopic transplantation of autologous diced cartilage and whether SVF cell sheets containing adipose-derived stem cells can inhibit chondrocyte pyroptosis and increase the survival of and regeneration in cartilage grafts have not been reported. The aim of this study was to investigate the beneficial effect of SVF cell sheets on angiogenesis in diced cartilage grafts and the potential effect of SVF cell sheets on inhibiting chondrocyte pyroptosis.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cp\u003e2.1 SVF cell sheet characteristics and angiogenic effects in in vitro experiments\u003c/p\u003e\n\u003cp\u003eAfter adherent growth, the rabbit adipose-derived SVF cells formed oblong fusiform or polygonal structures after approximately 10 days (Fig. 1a). HE staining showed that the SVF cells were arranged in a long fusiform and vortex shape and that the cell distribution was uniform (Fig. 1b). Flow cytometry revealed that the SVF included adipose stem cells (ADSCs) and EPCs[12]. At day 2 of culture in cell sheet-induction medium, observation under an inverted phase-contrast microscope showed that the cells in the temperature-sensitive culture dish had reached 100% confluence. When the induction culture was continued until day 14, a membranous tissue structure formed at the bottom of the culture dish. The culture dishes were incubated at 20-25 \u0026deg;C for approximately 40 min, and a white translucent membrane (SVF cell sheet) was obtained (Fig. 1c). SEM images of the SVF cell sheets showed that the SVF cells were connected and fused to each other and were tightly arranged, with a large amount of extracellular matrix deposited between the cells (Fig. 1d).\u003c/p\u003e\n\u003cp\u003eThe expression of the proangiogenic factors Ang-1 and VEGF in the three groups of cells was assessed by IF (green fluorescence (positive signal) indicated Ang-1 and VEGF expression, and blue indicated nuclei). The results showed that in the SVF cell-sheet group, the number of cells with positive signals for Ang-1 and VEGF (green fluorescence) was significantly greater than that in the blank group (P \u0026lt;0.01) and the SVF cell group (P \u0026lt;0.05); moreover, the number of cells in the SVF cell group was greater than that in the blank group (P \u0026lt;0.01) (Fig. 2a-d). The qRT-PCR and WB results showed that, in the SVF cell-sheet group, the messenger RNA (mRNA) and protein expression levels of Ang-1 and VEGF were significantly higher than those in the blank group (P \u0026lt;0.01) and SVF cell group (P\u0026lt;0.05); the expression levels in the SVF cell group were higher than those in the blank group (P\u0026lt;0.01) (Fig. 2e- i). These findings suggest that compared with SVF cells, SVF cell sheets could further upregulate the gene and protein expression of angiogenic factors.\u003c/p\u003e\n\u003cp\u003eIn vitro tube formation assays showed that cells in the blank group grew in cords rather than in tubes; in the SVF cell and SVF cell-sheet groups, after 6 h, the cells attached to the Matrigel and gradually extended their pseudopodia and made contact with the surrounding cells, forming a three-dimensional (3D) reticular lumen-like structure similar to blood vessels, with tube formation rates of 20.3% and 21.5%, respectively (Fig. 3). These results indicated that the SVF cell sheets had a greater proangiogenic effect than did the SVF cells.\u003c/p\u003e\n\u003cp\u003e2.2 An SVF cell sheet promotes cartilage regeneration in diced cartilage grafts\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;New Zealand rabbits were euthanized at 1, 3, and 6 months after surgery by intravenous injection of 1% sodium pentobarbital 100 mg/kg, the bilateral diced cartilage grafts on the back were removed, and the appearance and morphological characteristics of the grafts were observed. At 1 month after surgery, the diced cartilage grafts in the blank group were slightly soft when touching, with some unfused diced cartilage scattered around the incision; the texture of the grafts in the perichondrium group was slightly tough, and a small amount of unfused diced cartilage could be observed around the central incision; and the diced cartilage grafts in the SVF cell-sheet group were basically fused and slightly tough when touching. At 3 months after surgery, the diced cartilage grafts in the blank group and the perichondrium group were basically fused, with an irregular appearance, and were relatively tough when touching; in the SVF cell-sheet group, the diced cartilage grafts were completely fused and were tough when touching. At 6 months after surgery, the diced cartilage grafts in the three groups were completely fused and were slightly hard when touching, and connective tissue fibrous membrane wrapping and neovascular growth were seen along the periphery (Fig. 4a). The wet weight of the grafts in each group was measured before transplantation and at 1, 3, and 6 months after transplantation. The results showed that in the blank group, the wet weights of cartilage grafts at 1 month, 3 months and 6 months after surgery were not significantly different from those before transplantation (P\u0026gt;0.05); at 3 and 6 months after surgery, the wet weights of cartilage grafts in the perichondrium group and SVF cell-sheet group were greater than those of cartilage grafts in the blank group (P \u0026lt;0.05), and the wet weight of grafts in the SVF cell-sheet group was slightly greater than that in the perichondrium group (p \u0026lt;0.05) (Table 1).\u003c/p\u003e\n\u003cp\u003eTo understand the regenerative activity of chondrocytes, specific staining of cartilage tissue blocks was performed. Safranin fast green staining results showed that at 3 months after surgery, cells at the edge of diced cartilage grafts in the SVF cell sheet and perichondrium groups were oblate and existed alone, indicating the presence of new chondrocytes; the chondrocytes near the center of the cartilage were round and oval, and two or three isogenic cell population aggregates were observed. At 6 months after surgery, the number of new chondrocytes in the perichondrium group gradually increased; in the SVF cell-sheet group, large amounts of new cartilage and red cartilage matrix were observed, multiple chondrocytes were present in the lacuna, and new cartilage had gradually matured. In the blank group, there was no significant increase in the number of new chondrocytes or red cartilage matrix at 3 months after surgery compared with 1 month after surgery, and the amount of new chondrocytes at 6 months after surgery was slightly lower than that at 1 month after surgery (Fig. 4b). After Masson staining, the collagen fibers in the cartilage matrix were stained blue. At 1 month after surgery, the differences in the amount of blue stained cartilage matrix in the 3 groups were not significant. At 3 and 6 months after surgery, the blue staining area and staining intensity of the cartilage matrix in the perichondrium group and the SVF cell-sheet group were significantly greater than those in the blank group; the blue staining area and staining intensity of the cartilage matrix in the SVF cell-sheet group were higher than those in the perichondrium group, suggesting that the SVF cell sheets can increase the secretion of cartilage collagen (Fig. 4c). After toluidine blue staining, proteoglycan, the main component of the cartilage matrix, was stained blue-purple, and the staining intensity was related to the amount of proteoglycans. At 1 month after surgery, the differences in the amount of blue-purple stained cartilage tissues in the 3 groups were not significant. At 3 and 6 months after surgery, the blue-purple staining area and staining intensity of the cartilage tissues in the perichondrium group and the SVF cell-sheet group were significantly greater than those in the blank group; the blue-purple staining area and staining intensity of the cartilage tissues in the SVF cell-sheet group were greater than those in the perichondrium group, suggesting that SVF cell sheets can increase cartilage matrix secretion (Fig. 4d). These cartilage tissue-specific staining results suggest that compared with autologous perichondrium, SVF cell sheets better promote the survival of and regeneration in grafted cartilage.\u003c/p\u003e\n\u003cp\u003e2.3 SVF cell sheets promote angiogenesis in diced cartilage grafts\u003c/p\u003e\n\u003cp\u003eTo understand the effects of SVF cell sheets on vascularization around diced cartilage grafts, IHC was performed to assess the expression levels of Ang-1 and VEGF in cartilage tissues of the three groups, and the results showed that in the SVF cell-sheet group and perichondrium group, the chondrocytes surrounding the cartilage tissue and the tissue surrounding the cartilage block were intensely stained brownish yellow and dark brown. At 1, 3, and 6 months after surgery, the number of positive cells for both Ang-1 and VEGF was significantly higher in the perichondrium group than in the blank group, and the expression of Ang-1 and VEGF in the SVF cell-sheet group was further upregulated compared to that in the perichondrium group (Fig. 5a and 5b).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The qRT-PCR and WB results showed that at 1, 3, and 6 months after surgery, the mRNA and protein expression levels of Ang-1 and VEGF in the SVF cell-sheet group and the perichondrium group were greater than those in the blank group (P \u0026lt;0.01) and that the levels in the SVF cell-sheet group were greater than those in the perichondrium group (P \u0026lt;0.05) (Fig. 5c-g). These results indicate that SVF cell sheets upregulate the gene and protein expression of angiogenesis-related factors, thereby facilitating angiogenesis in cartilage grafts.\u003c/p\u003e\n\u003cp\u003eHE staining was used to observe the adipose tissue and vascularization around diced cartilage grafts. At 1 month after surgery, compared with those in the blank group, small amounts of connective and adipose tissues and blood vessel formation between diced cartilage grafts were observed in the perichondrium and SVF cell-sheet groups (P \u0026lt;0.05). At 3 and 6 months after surgery, in the blank group, small amounts of connective tissue and adipose tissue formed around the diced cartilage grafts, and a small amount of neovasculature could be observed. Compared with those in the blank group, in the perichondrium group, there was more connective and adipose tissue around the diced cartilage grafts, and more neovasculature could be observed around the cartilage (P\u0026lt;0.05). In the SVF cell-sheet group, a large amount of connective and adipose tissue and a large amount of neovasculature formed around the diced cartilage grafts (P \u0026lt;0.05) (Fig. 6a, b). The results indicate that the SVF cell sheets better promoted the formation of neovasculature in the cartilage grafts.\u003c/p\u003e\n\u003cp\u003e2.4 Effect of SVF cell sheets on pyroptosis in cartilage tissues\u003c/p\u003e\n\u003cp\u003eTo understand the viability and survival of chondrocytes in grafts 6 months after diced cartilage transplantation, TUNEL staining was performed, and the results showed that in the blank group, the TUNEL-positive cells (green stained) were located at the center and surrounding area of the diced cartilage grafts; in the SVF cell-sheet group and the perichondrium group, most TUNEL-positive cells were located in the center of diced cartilage grafts, and the percentage of TUNEL-positive cells was significantly lower than that in the blank group (both P\u0026lt;0.01); moreover, the percentage of TUNEL-positive cells in the SVF cell-sheet group was lower than that in the perichondrium group (P \u0026lt;0.01), suggesting that SVF cell sheets can reduce the cell death of cartilage tissues (Fig. 7a, c).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;To further elucidate the cause of cell death, the protein expression levels of NLRP3, Caspase1 and GSDMD, which are involved the canonical pyroptosis pathway, in the cartilage tissues of diced cartilage grafts were assessed by IHC, and the results confirmed that pyroptosis occurred in the chondrocytes. However, the protein expression of components of the pyroptosis signaling pathway was lower in the perichondrium group than in the blank group (P \u0026lt;0.05), and the expression in the SVF cell-sheet group was even lower (P \u0026lt;0.01). These findings suggest that SVF cell sheets reduced chondrocyte pyroptosis in the grafts, which is beneficial for cartilage regeneration (Fig. 7b, d).\u0026nbsp;\u003c/p\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eThe regeneration process of cartilage is based on intact perichondrium. The perichondrium is divided into two layers: the outer layer is mainly composed of connective tissue, which plays a supporting and protective role, and the inner layer, i.e., the germinal layer, which contains mesenchymal stem cells that can differentiate into chondrocytes\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. The blood vessels attached to the perichondrium provide nutrients to cartilage and facilitate its growth, development and regeneration\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Some scholars have compared the grafting effects of Surgicel and autologous deep temporal fascia-wrapped diced cartilage grafts and found that the survival rate of cartilage blocks wrapped with Surgicel was low, while autologous deep temporal fascia wrapping can reduce inflammation in cartilage grafts and maintain the activity of chondrocytes, which are beneficial for cartilage graft survival\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. However, due to the limited source of autologous perichondrium and the existence of donor site injury, the construction of perichondrium analogues to replace autologous perichondrium to promote the regenerative repair of cartilage grafts has been a research focus in the field of plastic surgery.\u003c/p\u003e \u003cp\u003eThe SVF comprises a group of cell populations obtained from adipose tissue digested with collagenase, and the cell composition can change with cell culture\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Currently, adipose-derived stem cells and EPCs are important components of the SVF\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Our previous studies also confirmed that rabbit adipose-derived SVF contains these two types of cells and that the injection of SVF cell suspensions promoted angiogenesis and the survival of diced cartilage grafts\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. This study demonstrated for the first time that using SVF cell sheets to wrap diced cartilage grafts could more effectively promote angiogenesis and cartilage regeneration in cartilage grafts; notably, this method improved the survival rate of the grafts.\u003c/p\u003e \u003cp\u003eSome studies have reported that SVF cells can secrete large amounts of angiogenic growth factors, such as VEGF, transforming growth factor-β (TGF-β), and basic fibroblast growth factor (bFGF), and that EPCs can differentiate into endothelial cells to participate in vascular budding and directly participate in vascular reconstruction\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. At present, SVF cells are mostly used to promote the revascularization of adipose tissue after autologous fat transplantation\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. In contrast to the traditional way of obtaining cells by digesting intercellular junctions with trypsin, cell sheets retain a large amount of extracellular matrix secreted by autologous cells, providing a microenvironment for the proliferation and differentiation of cells very similar to that in the body; thus, there is a greater potential for the application in the field of tissue defect repair. Temperature-sensitive culture systems with temperature-sensitive materials can regulate cell adhesion and detachment by altering the temperature, and because of their simple operation and easy control, these systems have become the main method for preparing tissue-engineered cell sheets\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. To this end, in this study, a temperature-sensitive culture system was used to construct a perichondrial analogue, i.e., SVF cell sheets. The SVF cell sheets obtained by this method adhered and grew well at 37\u0026deg;C; when the temperature was lowered to room temperature and maintained for 40 min, almost all cells detached in the form of tightly connected cell sheets that remained active and continued to adhere and grow. SEM revealed that SVF cells were arranged in multiple layers in the sheet and that the intercellular junctions and extracellular matrix were tightly fused to each other. In vitro studies revealed that the expression levels of Ang-1 and VEGF were greater in SVF cell sheets than in SVF cells. Ang-1 and VEGF are currently recognized as the strongest proangiogenic factors. During angiogenesis, VEGF synergizes with Ang-1 to play an important role in promoting neovasculature formation, maturation and stability\u003csup\u003e[\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. The in vitro tube formation assay revealed that the tube formation rate of SVF cell sheets was greater than that of SVF cells, suggesting that the neovascularization-promoting effect of SVF cell sheets was greater than that of the SVF cells, which is consistent with the results of Li et al.\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Recently, Li et al. found that compared with the direct injection of SVF cells, SVF cell sheet transplantation resulted in higher expression of the angiogenesis-related protein VEGF and stronger pro-neovascularization, which may be attributed to the 3D tissue-like structure formed by SVF cell sheets, enhancing their paracrine effect; therefore, they are more conducive to promoting neovascularization\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe results of the animal experiments in this study showed that at 3 and 6 months after surgery, the wet weight of the grafts in the SVF cell-sheet group was slightly greater than that in the perichondrium group; cartilage tissue-specific staining revealed that in the SVF cell-sheet group, chondrocyte proliferation was active, and cellular matrix and collagen secretion were robust, confirming that compared with autologous cartilage perichondrial wrapping, SVF cell sheet wrapping is more favorable to cartilage regeneration in grafts. In this study, for SVF cell sheets, the expression of the pro-angiogenic factors Ang-1 and VEGF was stronger than that using autologous perichondrium, and more peripheral adipose tissue and neovasculature formation around the grafts were observed at 3 and 6 months after surgery, suggesting that the SVF cell sheets better promoted the vascularization of cartilage grafts. The reason may be that the SVF cell sheets contain a large number of EPCs, which are conducive to neovasculature formation\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn clinical practice, the heterotopic transplantation of autologous diced cartilage grafts also faces the problem because at the transplantation site, an immune-inflammatory response occurs, resulting in a decrease in viability and death of the transplanted chondrocytes\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. In recent years, a new method of programmed cell death, GSDMD-mediated pyroptosis, has been discovered. GSDMD-mediated pyroptosis is closely related to inflammation and characterized by NLRP3 inflammasome activation\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e, which activates caspase-1 and catalyzes the cleavage of the effector protein GSDMD to produce the active fragment GSDMD-N, which is mosaiced in the cell membrane to form pores, allowing extracellular fluid to enter cells, which eventually leads to cell lysis and death\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Some studies have shown that chondrocyte pyroptosis may play a key role in the pathological process of osteoarthritis\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. Adipose-derived mesenchymal stem cells can inhibit the chondrocyte pyroptosis signaling pathway to delay the progression of rat osteoarthritis, suggesting that adipose-derived stem cell inhibition of chondrocyte pyroptosis promotes the repair of cartilage tissue damage \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. The canonical pyroptosis pathway is dependent on the activation of caspase-1, which can be mediated by danger-associated molecular patterns (DAMPs)\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Cartilage grafting occurs in a sterile environment, and when cartilage tissue is cut in vitro, DAMPs are produced, which in turn activate NLRP3. Therefore, this study investigated the expression of characteristic molecules of the canonical pyroptosis pathway. This study revealed for the first time that chondrocyte pyroptosis also occurred after autologous diced cartilage graft transplantation and that compared to autologous perichondrium, SVF cell sheets further inhibited chondrocyte pyroptosis and reduced chondrocyte death, which may be beneficial for graft regeneration and repair. Because the SVF contains a large number of adipose-derived mesenchymal stem cells, we speculate that the protective effect of SVF cell sheets might be mediated by adipose-derived stem cells.\u003c/p\u003e \u003cp\u003eThis study has limitations. For example, without the participation of a scaffold, the strength of the constructed cell sheets may be insufficient, thus limiting the subsequent repair and reconstruction of tissue or organs to some extent\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Future studies will optimize the culture conditions to obtain SVF cell sheets with better performance. Due to the heterogeneity of SVF cells, the molecular mechanisms underlying the promotion of angiogenesis, improvements in the blood supply of cartilage tissue and the inhibition of chondrocyte pyroptosis by SVF cell sheets need to be further investigated.\u003c/p\u003e"},{"header":"4. Methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e4.1 Preparation of SVF cell sheets and ultrastructural analysis\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eRabbit adipose-derived SVF cells were extracted and identified following the method reported in our previous study\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. The experiments were approved by The Experimental Animal Department of Nanhua University (SYXK (Xiang 2020-0002)), and reported in accordance with ARRIVE guidelines. An SVF cell suspension that had been passaged to the 2nd generation was seeded uniformly (1x10\u003csup\u003e6\u003c/sup\u003e/cm\u003csup\u003e2\u003c/sup\u003e) in a temperature-sensitive culture dish (35 mm, UpCell, Thermo Fisher Scientific), and after the cells had completely adhered to the dish, sheet-forming induction solution (Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) containing 10% fetal bovine serum (FBS), 50 mg/L ascorbic acid) was added to the cells, followed by incubation for 14 days. Then, the dish was placed at 20\u0026ndash;25\u0026deg;C for approximately 40 min, allowing SVF cell sheets to detach from the surface of the culture dish. The SVF cell sheets were fixed in 2.5% glutaraldehyde and washed with phosphate-buffered saline (PBS). The cells were then dehydrated in a graded ethanol series. The samples were gold-plated and observed using a scanning electron microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e4.2 Coculture and grouping of SVF cells and chondrocytes\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eRabbit ear chondrocytes were purchased from Wuhan Cloud Clone Technology Co., Ltd. (CS1261Rb01). After passage in culture, second-generation chondrocytes were used in subsequent experiments.\u003c/p\u003e \u003cp\u003eThe cells for in vitro experiments were divided into three groups. In the blank group, rabbit ear chondrocytes (2x10\u003csup\u003e6\u003c/sup\u003e cells/cm\u003csup\u003e2\u003c/sup\u003e) were cultured alone. In the SVF cell group, SVFs (1x10\u003csup\u003e6\u003c/sup\u003e cells/cm\u003csup\u003e2\u003c/sup\u003e) and chondrocytes were cocultured at a ratio of 1:1. In the SVF cell-sheet group, the obtained SVF cell sheets were cocultured with rabbit ear chondrocytes (1x10\u003csup\u003e6\u003c/sup\u003e cells/cm\u003csup\u003e2\u003c/sup\u003e). The cells in the three groups were all cultured in low-glucose DMEM containing 10% FBS\u0026thinsp;+\u0026thinsp;1% antibiotics (penicillin and streptomycin) and in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator at 37\u0026deg;C for 48 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Preparation and implantation of diced cartilage grafts\u003c/h2\u003e \u003cp\u003e Eighteen 6-month-old New Zealand white rabbits weighing 2.0-2.5 kg were obtained from the Experimental Animal Department of Nanhua University after the animal experiments were approved (SYXK (Xiang 2020-0002)). There was one rabbit per cage. After the rabbits were fed standard chow for 1 week, subcutaneous adipose tissue was extracted from the rabbits following the method reported in our previous studies; this tissue was used to prepare SVF cells. This study was approved by the Laboratory Animal Management and Ethics Committee of the First Affiliated Hospital of Nanhua University, and all procedures complied with the rules for the Management of Laboratory Animals of the People's Republic of China.\u003c/p\u003e \u003cp\u003eRabbits were anesthetized by the intraperitoneal injection of 5% urethane (5 mL/kg). After disinfection, the unilateral ear was removed from the root; the skin, fascia and perichondrium of the ear were removed; and the ear was soaked in saline containing gentamicin for 10 min. The ear cartilage was cut into approximately 1.0 mm\u003csup\u003e3\u003c/sup\u003e pieces and divided into three equal parts. For in vivo experiments, the animals were divided into three groups. In the blank group, rabbits were grafted with unwrapped diced cartilage; in the perichondrium group, rabbits were grafted with diced cartilage wrapped with autologous perichondria; and in the SVF cell-sheet group, rabbits were grafted with diced cartilage wrapped with SVF cell sheets. Before implantation, the wet weight of all diced cartilage was measured with an electronic scale. The skin on the back of each rabbit was prepared and routinely disinfected. Symmetrical areas to the right and left of the posterior midline were selected, three subcutaneous incisions were made, the diced cartilage grafts were transplanted into the subcutis, the wound was sutured, and erythromycin ointment was applied to the wound surface. The grafts were marked with a marker pen. Rabbits were randomly euthanized by ear intravenous injection of 3% pentobarbital sodium 100 mg/kg at 1, 3, and 6 months after surgery (6 at each time point). The diced cartilage grafts on the back were removed, and the gross condition was observed. The soft tissue from the surface of the diced cartilage grafts was removed, and the wet weight was measured.\u003c/p\u003e \u003cp\u003e \u003cb\u003e4.4 Histological analysis and immunohistochemical analysis\u003c/b\u003e Cartilage tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Hematoxylin-eosin (HE) staining, Masson staining, safranin fast green staining, and toluidine blue staining were performed on 5-\u0026micro;m thick tissue sections according to the manufacturers\u0026rsquo; instructions. All the samples were dehydrated, mounted with neutral gum, and observed under a microscope.\u003c/p\u003e \u003cp\u003eIHC was performed according to standard protocols. In brief, antigen extraction was performed after tissue sections were dewaxed and dehydrated. Next, the tissue sections were treated with 0.3% hydrogen peroxide and then incubated with the following primary antibodies at 4\u0026deg;C overnight: anti-vascular endothelial growth factor (VEGF) (1:50, Abcam, Cat# ab52917), anti-angiopoietin-1 (Ang-1) (1:50, Proteintech, Cat# 23302-1-AP), anti-NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) (1:200, Proteintech, Cat# 27458-1-AP), anti-Caspase1 (1:200, Proteintech, Cat# 22915-1-AP), and anti-gasdermin D (GSDMD) (1:200, Proteintech, Cat# 20770-1-AP). A horseradish peroxidase-labeled secondary antibody was added, and the sections were incubated for 60 min. 3,3\u0026prime;-Diaminobenzidine (DAB) was used for color development, and the sections were counterstained with hematoxylin, cleared, mounted, and observed under a microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.5 Terminal deoxyribonucleotide transferase [TdT]-mediated dUTP nick end labeling (TUNEL) assay\u003c/h2\u003e \u003cp\u003eParaffin sections of rabbit cartilage tissues were dewaxed and dehydrated.Chondrocyte death was assessed using the TUNEL assay. An in situ cell death detection kit (Shanghai Yisheng Biotechnology Co., Ltd. 40306ES50) was used according to the manufacturer\u0026rsquo;s instructions. Nuclei were stained with 4',6-diamino-2-phenylindole (DAPI, Wellbio, China). TUNEL-positive cells were detected and imaged using fluorescence microscopy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.6 Immunofluorescence (IF) detection\u003c/h2\u003e \u003cp\u003eAfter dewaxing and dehydrating the cell sections, they were blocked with 5% bovine serum albumin (BSA) for 60 min at room temperature. The sections were fully covered by anti-VEGF (1:50, Proteintech, Cat# 19003-1-AP) and anti-Ang-1 (1:50) antibodies, incubated in a refrigerator at 4\u0026deg;C overnight, washed with PBS, incubated with a secondary antibody in the dark for 90 min, incubated with DAPI at room temperature for 10 min, and mounted with glycerol. The cells were observed under a fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.7 Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR)\u003c/h2\u003e \u003cp\u003eTotal RNA from cells or tissues was prepared, and complementary DNA (cDNA) was synthesized by reverse transcription. The following synthetic primers were used: β-actin, 5'- TGGCCGAGACTTTGATTGT-3' upstream, 5'-TTACACAAATGCGATGCTGCC-3' downstream; VEGF, 5'- TGCGGATCAUACCACCAG-3' upstream, 5'-CCGGTTTCTTGCGCTTTC-3' downstream; and Ang-1, 5'- GGCTTGGTTGCTCGTCAAAC-3' upstream, and 5'-CAGGACGCTGTTGTTGGTTG-3' downstream; the sizes of the amplicons were 170 bp, 158 bp and 81 bp, respectively. The following 30-\u0026micro;L reaction system was used for PCR: 15 \u0026micro;L of SYBR Green mix, 2 \u0026micro;L of primer mix, 11 \u0026micro;L of nuclease-free water, and 2 \u0026micro;L of cDNA. The samples were subjected to 95\u0026deg;C for 10 min and 40 cycles of 95\u0026deg;C for 15 s and 60\u0026deg;C for 30 s. Gene expression was analyzed by the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method using β-actin as an internal control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.8 Western blot (WB)\u003c/h2\u003e \u003cp\u003eTotal protein from cells or tissues were extracted with 200 \u0026micro;l of radioimmunoprecipitation (RIPA) lysis buffer. Protein quantification was performed by the bicinchoninic acid (BCA) assay. Proteins were loaded and electrophoresed on a gel and then transferred to NC membranes by electroblotting. The membranes were placed in 5% skim milk powder blocking solution for 90 min at room temperature and then incubated at 4\u0026deg;C overnight with the following primary antibodies: anti-VEGF (1:2000), anti-Ang-1 (1:1000), and anti-β-actin (1:5000). The next day, the membranes were washed with PBST and then incubated with a horseradish peroxidase-labeled secondary antibody (1:5000) at room temperature for 90 min. Finally, the membrane was washed, and the protein bands were developed using enhanced chemiluminescence (ECL) reagent.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.9 Angiogenesis experiment\u003c/h2\u003e \u003cp\u003eMicrovasculature regeneration was detected via a tube formation assay\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. In brief, Matrigel was incubated in a 4\u0026deg;C freezer overnight. A 96-well plate was precooled; 50 \u0026micro;l of Matrigel was added to each well, and the plate was placed in a 37\u0026deg;C incubator for 30 min to allow the gel to coagulate. The cells were digested with trypsin and counted to prepare a cell suspension, and approximately 10,000 cells were added to each well. After incubation at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e tissue culture incubator for 6 h, angiogenesis was observed under a light microscope, and the number of lumens and branch points formed was counted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e4.10 Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using SPSS 27.0 software. The data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (\u003cspan class=\"InlineEquation\"\u003e\u003c/span\u003e\u0026plusmn;\u0026thinsp;SD), and the differences among groups were analyzed by using the t test or one-way analysis of variance (ANOVA). P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicates a statistically significant difference.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn this study, SVF cells were used to construct a multicellular material, SVF cell sheets, in temperature-sensitive culture dishes, and the SVF cell sheets were wrapped around diced cartilage grafts prior to heterotopic transplantation. The results indicated that the SVF cell sheets promoted the survival of and regeneration in cartilage grafts and that those effects may be related to the promotion of angiogenesis, improvements in blood supply to diced cartilage grafts and the inhibition of chondrocyte pyroptosis by SVF cell sheets. These findings broaden the understanding of the application of SVF cell sheets in cartilage regeneration. The SVF cell sheet wrapping technique may become a new strategy for autologous diced cartilage transplantation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this article or supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYangchen Wei, Yi Wei and Cong Xie conceptualized the study, wrote the original draft, conducted formal analysis, and wrote, reviewed and edited the manuscript. Zhengyang Li, Li Li and Yan Chen reviewed and edited the manuscript. Yiping Wang developed methodology. Chiyu Jia contributed to study conceptualization, writing the original draft, and writing, reviewing and editing the manuscript. Hongju Xie and Junlin Liao supervised the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (Grant No. 82002059), the Natural Science Foundation of Hunnan Province, China (Grant No. 2023JJ30542), the Clinical Medical Technology Innovation Guidance Project of Hunan Province, China (Grant No. 2021SK51826), the Health Commission of Hunan Province, China (Grant No. D202304108322), and the Key Scientific Research Project of Higher Education in Hainan Province, China (Grant No. Hnky2021ZD-16).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWilkes, G. 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Bioengineered 13, 10313\u0026ndash;10323, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/21655979.2022.2062954\u003c/span\u003e\u003cspan address=\"10.1080/21655979.2022.2062954\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"diced cartilage, stromal vascular fractions, cell sheet, angiogenesis, cartilage regeneration","lastPublishedDoi":"10.21203/rs.3.rs-4479766/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4479766/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAutologous diced cartilage, while biocompatible and easy to shape, is limited in clinical application due to its high resorption rate and challenges in establishing timely and effective neovascularization post-surgery. In this study, we produced SVF cell sheets from adipose-derived stromal vascular fraction (SVF) via enzymatic digestion, employing a temperature-sensitive culture system. Our in vivo and in vitro experiments validated that SVF cell sheets, when wrapped around granular cartilage, exhibited a notable promotion of cartilage regeneration and mitigated granular cartilage resorption in a rabbit diced cartilage graft model. Our findings demonstrate that SVF cell sheets facilitated effective neovascularization and timely cartilage block formation by secreting VEGF and Ang-1 while also suppressing the expression of pyroptotic proteins like NLRP3, Caspase1, and GSDMD. As a biofilm, derived from a multicellular source, SVF cell sheets hold promise in promoting neovascularization and cartilage regeneration in diced cartilage grafts while also preventing chondrocyte pyroptosis, presenting a potential novel approach for autologous diced cartilage transplantation.\u003c/p\u003e","manuscriptTitle":"SVF cell sheets: A new multicellular material-based strategy for promoting angiogenesis and regeneration in diced cartilage grafts","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-14 18:44:43","doi":"10.21203/rs.3.rs-4479766/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"08caac33-d57a-429d-82ec-923b08485935","owner":[],"postedDate":"June 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":33152205,"name":"Biological sciences/Biotechnology/Regenerative medicine"},{"id":33152206,"name":"Biological sciences/Stem cells/Mesenchymal stem cells"}],"tags":[],"updatedAt":"2024-11-18T15:08:49+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-14 18:44:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4479766","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4479766","identity":"rs-4479766","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}