SVF Cell Sheets as a New Multicellular Material-Based Strategy for Promoting Angiogenesis and Regeneration in Diced Cartilage Grafts

Abstract

Autologous diced cartilage, while biocompatible and easy to shape, is limited in clinical application due to its high adsorption rate and challenges in establishing timely and effective neovascularization postsurgery. In this study, the authors produced SVF cell sheets from adipose-derived stromal vascular fraction (SVF) through 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 adsorption 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 can replace perichondrium and promote the expression of proangiogenic growth factors Ang-1 and VEGF, thereby promoting local microvascular regeneration, reducing chondrocyte pyroptosis, and promoting the formation of cartilage blocks. This strategy provides a potential new method for autologous cartilage grafting, which will help solve the dilemma of limited sources of cartilage tissue in clinical practice and provide natural autologous cartilage filling materials for the treatment of craniofacial defects.

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 to 1.0 mm³. 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 increased morbidity at the donor site and limited fascial sources.⁶

As an exogenous perichondrial analog, cell sheets show great potential for application in tissue defect repair because they can conserve intercellular junctions and provide 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 and others.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 could promote angiogenesis and reduce cartilage absorption in the diced cartilage grafts.¹² Takeuchi et al¹³ reported that, compared with 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. Costa et al¹⁴ 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. However, no studies have reported whether SVF cell sheets are superior to perichondrium in promoting angiogenesis in diced cartilage grafts. In addition, previous research in this field aims to use autologous diced cartilage grafting to avoid problems such as deformation and absorption of whole cartilage. We plan to use SVF cell sheets to promote autologous diced cartilage regeneration, and compare them with perichondrium to prove their superiority in promoting cartilage tissue regeneration.

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. It also provides a potential new method for autologous slice cartilage grafting, which will help solve the dilemma of limited sources of cartilage tissue in clinical practice and provide natural autologous cartilage filling materials for the treatment of craniofacial defects.¹⁷

METHODS

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 Specifically, the adipose tissue of rabbit groin was collected and trypsinized with 1 mg/mL for 1 hour, the cell suspension was filtered using a sterile strainer and the supernatant was centrifuged, and the cell suspension was made with DMEM medium containing 10% fetal bovine serum, and inoculated in a Petri dish for culture. The experiments were approved by The Experimental Animal Department of Nanhua University [SYXK (Xiang 2020-0002)], and reported in accordance with ARRIVE guidelines. The temperature-sensitive culture dish is a technology for producing biofilms that can separate (80%–90%) of adherent cells from the surface of the culture dish in the form of membrane sheets under low-temperature conditions. An SVF cell suspension that had been passaged to the second generation was seeded uniformly (1×10⁶/cm²) 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, placed in a 37 °C, 5% CO₂ incubator for 14 days, and the cell-sheet induction medium was changed every day. Then, the dish was placed at 20 to 25 °C for ∼40 minutes, 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.

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 calculated density of SVF cell membrane constructed by the author under the microscope was 1×10⁶ cells/cm². Therefore, the number of cells used in the blank group and SVF cells group was equal to the SVF cell membrane density. The cells used for in vitro experiments are divided into 3 groups. In each group, rabbit ear chondrocytes (1×10⁶ cells/cm²) are first seeded in a Petri dish, and the same number of the respective cells are then added to the Transwell chamber for coculture. In the blank group, rabbit ear chondrocytes (1×10⁶ cells/cm²) are added to the Transwell chamber. In the SVF cell group, SVF cells (1×10⁶ cells/cm²) are added to the Transwell chamber. In the SVF cell-sheet group, the obtained SVF cell sheet is spread flat at the bottom of the Transwell chamber. The cells in the 3 groups were all cultured in low-glucose DMEM containing 10% FBS+1% antibiotics (penicillin and streptomycin) and in a 5% CO₂ incubator at 37 °C for 48 hours.

Preparation and Implantation of Diced Cartilage Grafts

Eighteen 6-month-old New Zealand white rabbits weighing 2.0 to 2.5 kg were obtained from the Experimental Animal Department of Nanhua University after the animal experiments were approved [SYXK (Xiang 2020-0002)]. They were divided into 1, 3, and 6 months groups after surgery according to the expected experimental time, with 6 animals in each group. 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 minutes. The ear cartilage was cut into ∼1.0 mm³ pieces and divided into 3 equal parts. For in vivo experiments, the animals were divided into 3 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, 3 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.

Histologic 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.

Immunohistochemistry 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: antivascular endothelial growth factor (VEGF) (1:50, Cat# ab52917; Abcam), antiangiopoietin-1 (Ang-1) (1:50, Cat# 23302-1-AP; Proteintech), anti-NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) (1:200, Cat# 27458-1-AP; Proteintech), anti-Caspase1 (1:200, Cat# 22915-1-AP; Proteintech), and anti-gasdermin D (GSDMD) (1:200, Cat# 20770-1-AP; Proteintech). A horseradish peroxidase-labeled secondary antibody was added, and the sections were incubated for 60 minutes. 3,3′-Diaminobenzidine (DAB) was used for color development, and the sections were counterstained with hematoxylin, cleared, mounted, and observed under a microscope.

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 (40306ES50; Shanghai Yisheng Biotechnology Co. Ltd) 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.

Immunofluorescence (IF) Detection

After dewaxing and dehydrating the cell sections, they were blocked with 5% bovine serum albumin (BSA) for 60 minutes at room temperature. The sections were fully covered by anti-VEGF (1:50, Cat# 19003-1-AP; Proteintech) 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 minutes, incubated with DAPI at room temperature for 10 minutes, and mounted with glycerol. The cells were observed under a fluorescence microscope.

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, 158, 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 minutes and 40 cycles of 95 °C for 15 seconds and 60 °C for 30 seconds. Gene expression was analyzed by the $2^{-\mathrm{\Delta \Delta }{C}_t}$ method using β-actin as an internal control.

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 minutes 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 minutes. Finally, the membrane was washed, and the protein bands were developed using enhanced chemiluminescence (ECL) reagent.

Angiogenesis Experiment

Microvasculature regeneration was detected through a tube formation assay.¹⁸ 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 minutes to allow the gel to coagulate. The cells were digested with trypsin and counted to prepare a cell suspension, and ∼10,000 cells were added to each well. After incubation at 37 °C in a 5% CO₂ tissue culture incubator for 6 hours, angiogenesis was observed under a light microscope, and the number of lumens and branch points formed was counted.

Statistical Analysis

Statistical analysis was performed using SPSS 27.0 software (IBM Corp., Armonk, NY). The data were analyzed to assess differences among groups, and results were reported with a significance level set at P<0.05. The data are expressed as the mean±SD. Employing parametric tests analyzed by using the t test or one-way analysis of variance (ANOVA).

RESULTS

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 ∼10 days (Fig. 1A). Quantitative analysis revealed an average cell length of 25±5 μm and width of 12±3 μm, with an aspect ratio averaging 2:1. 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 endothelial progenitor cells (EPCs) (our research group has successfully extracted SVFs cells in the early stage, and the results of cytologic morphologic identification have been published).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 to 25 °C for ∼40 minutes, 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).

FIGURE 1.

SVF cells and SVF cell sheets. (A) SVF cells are spindle-shaped or polygonal (scale bar: 200 μm). (B) HE staining of SVF cells, showing spindle-shaped and swirling arrangement (scale bar: 100 μm). (C) Overview of SVF cell sheets. (D) Scanning electron microscopy observation of SVF cell sheets, showing cells interconnected and fused, arranged closely with abundant extracellular matrix deposition between cells (scale bar: 50 μm).

The expression of the proangiogenic factors Ang-1 and VEGF in the 3 groups of cells was assessed by immunofluorescence (IF) staining. 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) (Figs. 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) (Figs. 2E–I) (the full-length blot is shown in the S1). These findings suggest that compared with blank group, SVF cells and SVF cell sheets have the ability to further upregulate the gene and protein expression of angiogenesis factors. The fold change of protein synthesis between SVF cell sheets and SVF cells is smaller than 0.5. There is an effect at the gene level, but not at the protein level.

FIGURE 2.

In vitro experiments to detect the expression of Ang-1 and VEGF in each group. (A and B) Immunofluorescence staining images of Ang-1 and VEGF expression in each group of cells. (C and D) Semiquantitative analysis of the percentage of Ang-1 and VEGF fluorescent staining positive cells in each group. (E and F) mRNA expression levels of Ang-1 and VEGF in each group of cells. (G) Electrophoresis images of Ang-1 and VEGF protein expression in each group of cells. (H and I) Semiquantitative analysis of Ang-1 and VEGF protein expression in each group of cells (have been cropped, original gels are presented in Supplementary Fig. 1, Supplemental Digital Content 2, http://links.lww.com/SCS/H691). Compared with the blank group: **P<0.01; compared with the SVFs cell-sheet group: ^# P<0.05.

In vitro tube formation assays showed that cells in the blank group grew in cords rather than in tubes, the rate of the blank tube formation was 0; in the SVF cell and SVF cell-sheet groups, after 6 hours, the cells attached to the Matrigel and gradually extended their pseudopodia and made contact with the surrounding cells, forming a 3-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. The marginal difference of only 1.2% between SVF cell sheets and SVF cells suggests that there is no significant superiority of SVF cell sheets in forming vessels over SVF cells. This might be due to factors such as the in vitro environment and nutrition.

FIGURE 3.

In vitro angiogenesis experiment for each group. It was seen that the blank group had less angiogenesis, and the SVF cell group and SVF cell-sheet group had more angiogenesis, which proved that SVFs cell sheet could promote angiogenesis (scale bar: 100 μm).

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 morphologic 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 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 3 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, 3, 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) (Supplemental Table 1, Supplemental Digital Content 1, http://links.lww.com/SCS/H690).

FIGURE 4.

SVF cell sheets promote cartilage regeneration in diced cartilage grafts (n=6). (A) Macroscopic view of diced cartilage grafts retrieved at 1, 3, and 6 months postoperatively. (B) Safranin O-fast green staining of cartilage tissue (scale bar: 100 μm). (C) Masson staining of cartilage tissue (scale bar: 100 μm). (D) Toluidine blue staining of cartilage tissue (scale bar: 100 μm).

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 2 or 3 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.

Stromal Vascular Fraction 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 3 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 with that in the perichondrium group (Figs. 5A, B).

FIGURE 5.

SVF cell sheets promote the expression of Ang-1 and VEGF in diced cartilage grafts (n=6). (A and B) Immunohistochemical analysis of Ang-1 and VEGF in diced cartilage grafts in each group. (C and D) Real-time quantitative PCR detection of Ang-1 and VEGF mRNA expression levels in diced cartilage grafts in each group. (E and F) Semiquantitative analysis of Ang-1 and VEGF protein expression in diced cartilage grafts in each group. (G) Electrophoresis images of Ang-1 and VEGF protein expression in diced cartilage grafts in each group (have been cropped, original gels are presented in Supplementary Fig. 2, Supplemental Digital Content 2, http://links.lww.com/SCS/H691). Compared with the Blank group: *P<0.05, **P<0.01; compared with the perichondrium group: ^# P<0.05.

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) (Figs. 5C–G) (the full-length blot is shown in the S2). These results indicate that SVF cell sheets upregulate the gene and protein expression of angiogenesis-related factors, thereby facilitating angiogenesis in cartilage grafts.

Hematoxylin-eosin 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) (Figs. 6A, B). The results indicate that the SVF cell sheets better promoted the formation of neovasculature in the cartilage grafts.

FIGURE 6.

SVFs cell sheets promote angiogenesis. (A) Observation of vascular formation around cartilage blocks in each group using HE staining (scale bar: 100 μm). (B) Semiquantitative analysis of angiogenesis around cartilage blocks in each group. Compared with the blank group: *P<0.05; compared with the perichondrium group: ^# P<0.05 (red arrows indicate neovascularization, blue arrows indicate connective tissue, and black arrows indicate adipose tissue).

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 (Figs. 7A, C).

FIGURE 7.

The effect of SVF cell sheets on chondrocyte pyroptosis (n=6). (A) TUNEL staining images of chondrocytes in each group of cartilage tissue (scale bar: 25 μm). (B) Immunohistochemical images of NLRP3, Caspase1, and GSDMD in each group of cartilage tissue (scale bar: 25 μm). (C) Quantitative analysis of TUNEL-positive rate in each group of cartilage tissue. (D) Semiquantitative analysis of NLRP3, Caspase1, and GSDMD-positive cells in each group of cartilage tissue. The data are expressed as mean±SD. *P<0.05 and **P<0.01, compared with the blank group. ^# P<0.05 and ^## P<0.01, compared with the perichondrium group.

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 (Figs. 7B, D).

DISCUSSION

The regeneration process of cartilage is based on intact perichondrium. The perichondrium is divided into 2 layers: the outer layer is mainly composed of connective tissue, which plays a supporting and protective role, and the inner layer, that is, the germinal layer, which contains mesenchymal stem cells that can differentiate into chondrocytes.¹⁹ The blood vessels attached to the perichondrium provide nutrients to cartilage and facilitate its growth, development, and regeneration.²⁰ Some scholars have compared the grafting effects of Surgicel and autologous deep temporal fascia-wrapped diced cartilage grafts and found that the survival rate (the portion of cartilage that has not been absorbed by the body after transplantation) 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.²¹ However, due to the limited source of autologous perichondrium and the existence of donor site injury, the construction of perichondrium analogs 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.²² Currently, adipose-derived stem cells and EPCs are important components of the SVF.²³ Our previous studies also confirmed that rabbit adipose-derived SVF contains these 2 types of cells and that the injection of SVF cell suspensions promoted angiogenesis and the survival of diced cartilage grafts.¹² Our findings demonstrate the feasibility of preparing SVF cells into SVF cell sheets and the feasibility of replacing perichondrium, as well as the advantages of reducing cell pyroptosis and increasing the expression of proangiogenic factors. Since SVF is a multicellular material, it is hypothesized that angiogenesis occurs through MSC-mediated paracrine effects involving proangiogenic factors such as Ang-1 and VEGF, while also being facilitated by ECs. However, this study did not include experiments designed to distinguish the relative significance of these effects, which will be addressed in future research.

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.²⁴ SVF cells also release anti-inflammatory cytokines such as interleukin-10 (IL-10), which can inhibit inflammasome activation and reduce pyroptosis. Under hypoxic conditions, SVF cells may also upregulate HIF-1α, leading to increased expression of angiogenic factors such as VEGF, thereby enhancing angiogenesis. At present, SVF cells are mostly used to promote the revascularization of adipose tissue after autologous fat transplantation.²⁵ 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.²⁶ To this end, in this study, a temperature-sensitive culture system was used to construct a perichondrial analog, that is, 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 minutes, 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 a long spindle shape 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.^27–29 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 al²³ Recently, Li et al²³ 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 proneovascularization, 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.

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 proangiogenic 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.^3,30 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,³¹ 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.³² Some studies have shown that chondrocyte pyroptosis may play a key role in the pathologic process of osteoarthritis.³³ 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).³⁴ 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 with 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.³⁵ Future research will design biomaterials to be compounded with SVF cell sheets to obtain elasticity and tension superior to perichondrium, and then complete the optimization of biomimetic membranes. 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.

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 before 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.

Supplementary Material

scs-36-1889-s001.docx ^{(16.3KB, docx)}

scs-36-1889-s002.pdf ^{(846.9KB, pdf)}