Decellularized vascular matrix material -TEVG coated with PRP for anti-degradation and anti-inflammation

doi:10.21203/rs.3.rs-4352075/v1

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Research Article

Decellularized vascular matrix material -TEVG coated with PRP for anti-degradation and anti-inflammation

https://doi.org/10.21203/rs.3.rs-4352075/v1

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Background

Vascular regeneration is closely associated with inflammation response and the degradation rate of implants. Platelet-rich plasma (PRP) contains various cytokines and proteins, and autologous PRP can be used to treat implants to reduce inflammation response.

Objective

To reduce the immune rejection response and degradation rate of implants in vivo by adding different derivatives of PRP.

Methods

TEVG were separately mixed with PBS, FIB, PGF, and PGF-blend to prepare different ECM implants for cell co-culture and subcutaneous transplantation experiments in rats. Tissue morphology was observed through HE, MASSON staining, and scanning electron microscopy. The impact of TEVG on macrophages was observed through cell immunofluorescence and WB. Subcutaneous transplantation in rats was assessed through HE and MASSON staining, immunofluorescence staining for CD206, CD86 to observe cell quantity and M2/M1 ratio.

Results

PBS, FIB, PGF, and PGF-blend exhibited unique morphologies under scanning electron microscopy. Both in vitro and in vivo studies showed an increase in M2/M1 ratio with PGF and PGF- coated, increasing water absorption capacity, and slowing down the metabolism of ECM materials in the body. Additionally, PRP downregulates multiple inflammation-related genes, reducing inflammatory response .

Conclusion

PGF and PGF- can reduce the immune rejection response of TEVG subcutaneous transplantation and decrease its degradation rate by reducing collagen loss in the implants.

tissue-engineered vessels

macrophages

smooth muscle cells

PRP

Tissue-engineered blood vessels bring great hope to address the clinical shortage of small-caliber vessels. The team led by Laura utilized PGA materials and smooth muscle cells for co-culturing for 8 weeks to obtain vascular matrix materials, which have achieved success in animal experiments and entered clinical trials ^[1]. Compared to the clinically applied polymeric vascular graft material ePTFE, this cell-derived ECM material exhibits better regenerative plasticity and lower infection risk ^[2]. Through subcutaneous transplantation of this decellularized vascular matrix material-TEVG in rats, it was found that the material underwent significant morphological changes after 3 weeks of subcutaneous implantation, with substantial cell infiltration observed as early as the first week, predominantly consisting of inflammatory cells, closely related to tissue degradation metabolism ^[3]. Material degradation and inflammation response pose significant challenges to vascular tissue engineering. To reduce inflammation response during vascular material transplantation and enhance its in vivo regenerative function, studies have shown that cell-derived TEVG materials, by removing the cellular components within the material, can significantly reduce the body's immune rejection response ^[4]. Additionally, TEVG materials mimic the ECM structure of native blood vessels, exhibiting better regenerative plasticity. Although TEVG can remove a large amount of immunogenicity through decellularization, excessive removal of cellular remnants during TEVG formation can lead to protein loss, and issues with residual PGA in TEBV materials can also cause inflammatory reactions in the body ^[5]. Past efforts to remove immunogens from materials have resulted in TEVG materials becoming fragile. How to reduce the immune rejection response of vascular scaffold materials in the body and increase the degradation period of materials in vivo has become another major challenge in vascular transplantation. Studies have shown that early infiltrating cells into vascular scaffold materials are mainly inflammatory cells, closely associated with tissue degradation metabolism. In order to reduce inflammation response during vascular material transplantation and enhance its in vivo regenerative function, some scholars have used immunosuppressants such as cyclosporine and methylprednisolone to suppress the host's immune response to the material, thereby reducing inflammation response after vascular transplantation ^[6]. It is also possible to mitigate inflammation response after vascular transplantation by regulating the expression and release of inflammation-related factors such as tumor necrosis factor (TNF), interleukin (IL)-1, IL-6, etc. It is preferable to use natural ECM as vascular transplantation materials, as this material possesses natural biocompatibility and bioactivity, and can also reduce immune rejection and inflammation response ^[7]. However, these methods are complex and costly, posing significant limitations in future clinical applications. In the application process, whether there is an effective and efficient method to regulate the interaction between the graft and the host to reduce transplant immune response remains to be conclusively determined.

Platelet-rich plasma (PRP) primarily contains proteins such as platelets, fibrinogen, fibronectin, growth factors, cytokines, and other bioactive molecules. These components play important roles in stimulating cell proliferation, promoting angiogenesis, and reducing inflammation processes, without significantly increasing economic burden on patients. To date, PRP has been widely applied in tissue engineering fields such as cartilage tissue engineering ^[8], bone tissue engineering ^[9], skin regeneration ^[10], and neural tissue engineering ^[11]. In recent years, the application of PRP in vascular tissue engineering has also gained attention. For instance, our recent study found that PRP can promote the adhesion and migration ability of smooth muscle cells on PGA materials, thereby promoting the generation of collagen in the later stages ^[12]. PRP can be extracted from the patient's own blood or from compatible relatives, effectively addressing the issue of material biocompatibility during TEVG application. However, the impact of PRP on subcutaneous transplantation of ECM-treated TEVG sources has not been discovered yet. The long-term effects of vascular transplantation are significantly correlated with the inflammation response and degradation rate within the implanted body ^[13]. Strategies to reduce the degradation rate of vascular transplants and provide a sufficient time window for later vascular cell ingrowth include surface modification of materials. For example, Yilgor P. et al. designed a drug delivery system with controlled release functionality, such as the BMP-2/BMP-7 system, which releases anti-inflammatory, antibacterial, or pro-angiogenic drugs to achieve anti-inflammatory effects on transplants ^{[14, 15]}. However, such designs are complex and difficult to scale up for clinical application. Studies have shown that PRP can reduce the production of inflammatory cytokines IL-17A and IL-1β, thereby reducing the body's inflammation response, primarily used in skin repair and regeneration ^[16]. Whether PRP affects the inflammation response of TEVG materials in vivo and whether PRP improves the degradation metabolism process of vascular scaffold materials in vivo remain unknown.

After decellularizing TEBV, we obtained decellularized vascular extracellular matrix TEVG, which was freeze-dried at low temperatures. The TEVG was then treated with PBS, FIB, PGF, and PGF- respectively. After enzymatic digestion, the materials were observed for macrophage polarization in vitro. Additionally, the materials combined with PBS, FIB, PGF, and PGF- were separately transplanted subcutaneously into rats. Samples were retrieved at 1, 2, and 3 weeks, and macrophage CD206, CD86, and DAPI immunofluorescence staining were conducted to observe the M2/M1 situation. The number of inflammatory cells was observed through HE staining, and the collagen content at each time point was calculated through MASSON staining. The hydroxyproline assay was performed to verify the collagen situation using experimental samples from each week. Finally, transcriptome analysis was conducted to explore the possible mechanisms of PRP action during subcutaneous transplantation.

All experiments were conducted in strict compliance with the ethical guidelines of Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, under ethics approval numbers: GDREC2019285h (RI) and KY2023-192-01.

Nonwoven PGA material was procured from Biomedical Structures, located in Rhode Island, Warwick, USA. Smooth muscle cells (SMCs) were isolated from bovine’s aortic tissue, which were obtained from a local abattoir.

Acquisition, Decellularization, and Dissolution of TEBV to Form TEVG

The TEBV was obtained through dynamic cultivation of bovine SMCs and nonwoven PGA material using a peristaltic pump for 8 weeks. The decellularization method for TEBV referenced the work of the Alessandro F. Pellegata team ^[5]. The decellularized TEBV-TEVG was freeze-dried and stored at -80°C for later use. The DNA content was determined using a DNA extraction kit (Thermo Fisher Scientific, USA) according to the manufacturer's instructions.

The preparation of TEVG solution followed previous methods ^[17]. In summary, TEVG was digested into a solution by adding pepsin at a concentration of 10 mg/ml (w/v). The digestion process was carried out under shaking conditions at 37°C. Once the TEVG was completely dissolved into a solution, it was neutralized with 0.1N NaOH at a volume of 1/10 and 10×PBS at a volume of 1/9 to achieve a pH of 7.4. The solution was then stored at 4°C for further use.

Preparation of PRP

PRP was prepared using the traditional two-step centrifugation method. Initially, blood was collected from rats using anticoagulant tubes containing sodium citrate (KWS, China). Subsequently, two-step centrifugation was performed at 300 g for 15 minutes followed by 600 g for 10 minutes to obtain a larger volume of platelet-rich plasma (PRP) fraction. Platelet counts were conducted for both whole blood and PRP under an optical microscope, ensuring a platelet concentration in PRP approximately five times that of whole blood ^[18]. Platelets were stained and analyzed using the Richter staining method (Biosharp, China). Finally, PRP underwent repeated freeze-thaw cycles, followed by centrifugation to obtain cell-free PRP solution. A portion of the solution was used directly for subsequent experiments, while another portion was subjected to calcium ion activation to remove fibrin components, and the remaining PRP was stored at -80°C for further use.

Isolating and Identifying SMCs

For the isolation of SMCs, approximately 5 cm-long segments of bovine aorta were obtained from the Nan-Hai Abattoir in Foshan, Guangdong Province. Obtained media layer of the aorta, notable for its thicker width compared to human aortas, was preferred for subsequent experiments. The medial layer of bovine blood vessels, taken from the middle portion as much as possible, was cut into small pieces approximately 0.2cm * 0.2cm in size. Primary culture of smooth muscle cells was performed using a tissue adhesion method. The mesenteric tissues were evenly attached to T25 culture flasks (Corning, USA) and incubated in a 37°C incubator with 5% CO2 (Thermo, USA). DMEM/F12 medium (Corning, USA) containing 20% FBS (Gibco, USA) was added. After 4 hours, the flasks were left untouched for 1 week to prevent tissue floating and cell crawling.

After 2 weeks, cells were passaged to the P3. Approximately 5 × 10^4 cells were collected on a cell scraper (Biosharp, China) and subjected to immunofluorescence staining according to the manufacturer's instructions. The cells were treated with antibodies (calponin, α-SMA, 1:500, Abcam, USA) and DAPI working solution (Solarbio, China) for visualization ^[19].

Scanning electron microscope (SEM)

Samples from different treatment groups PBS (TEVG treated with PBS), FIB (TEVG treated with 2 g/ml fibrinogen), PGF (TEVG treated with PRP containing fibrinogen precursor), and PGF- (TEVG treated with PRP without fibrinogen precursor) were fixed with 3 ml of 2.5% glutaraldehyde. The samples were then rinsed with 0.1 mol/L sodium dimethylaminomethylphosphonate buffer (pH 7.4) and kept in buffer solution at 4°C overnight. After 24 hours, and the TEVG of different groups were soaked in 1% citric acid for 1 hour and then repeatedly washed with buffer solution ^[20]. Finally, observation was conducted using a scanning electron microscope (S-3500N, Japan).

Growth Factor Release Experiment

To determine the time course of growth factor, release from PRP on TEVG materials, we used PDGF-BB and VEGF as assay markers. TEVG samples weighing 10µg dry weight were soaked in PGF and PGF-24h solutions and then placed in a 96-well plate (Corning, USA) for incubation, with 200µl of TEVG solution added to each well. Culture media were collected at various time points and stored at -80°C. According to the manufacturer's instructions (DuoSet®, USA), the media were stored at -80°C for 2, 4, 6, 8, 10, 12, and 14 days and subsequently quantified using ELISA to determine the concentrations of PDGF-BB and VEGF released from the test materials.

Material Water Absorption Test

The mass water absorption of the material refers to the percentage of water absorbed by the material when saturated, relative to the dry mass of the material. After treatment, samples from different experimental groups were freeze-dried, and the dry weight was measured as M. These samples were then placed in ddH2O, and the new weight was measured as m. The formula for calculating the material water absorption rate is: (m - M) / M.

CCK-8 Experiment

To begin, SMCs were seeded in a 96-well plate at a density of 4000 cells per well. Subsequently, cells were treated with different experimental groups of materials (TEVG solution at concentrations of 50 ng/ml, 100 ng/ml, 150 ng/ml, 200 ng/ml, and 250 ng/ml) for 24 hours. Following the instructions provided with the CCK-8 assay kit (Dojindo, Japan), 10% CCK-8 reagent was added and the plate was incubated at 37°C for 1.5 hours. The absorbance of the culture medium at 450nm was then measured using an enzyme-linked immunosorbent assay (ELISA) reader, and comparisons were made.

Extracorporeal Isolation and Cultivation Experiment of Macrophages

Macrophages were isolated from the femurs of healthy 6-week-old male SD rats ^[21]. Bone marrow was flushed out from the femurs using PBS. After settling, the bottom layer of fragmented bones was removed, and the remaining cell clumps were treated with a 0.3% sodium chloride solution to eliminate red blood cells. Following centrifugation, the cell pellets were resuspended in 1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) for macrophage suspension culture. After one week, cells were treated with 20 ng/ml of M-CSF. After 24 hours, macrophages and SMCs were treated with PBS (200 ng/ml TEVG solution), FIB (TEVG solution containing 2 g/ml fibrinogen), PGF (TEVG solution containing fibrinogen precursor), and PGF- (TEVG solution without fibrinogen precursor) for 24 hours before conducting cell immunofluorescence and WB experiments.

Cell Immunofluorescence Experiment:

Cells were treated with 4% paraformaldehyde for 10 minutes. After treated with 0.1% Triton X-100 in PBS, cells were incubated with 1% BSA dissolved in PBS for 30 minutes, followed by addition of primary antibodies against Calponin (CNN, 1:300, Abcam, USA), α-Smooth Muscle Actin (α-SMA, 1:300, Abcam, USA), CD206 (1:250, Abcam, USA), and INOS (1:250, Abcam, USA) at 4°C overnight. The next day, after washing three times with PBST and avoiding light exposure, corresponding secondary antibodies were incubated with the cells at a concentration ten times that of the primary antibodies. Finally, cells were treated with an anti-quenching mounting medium containing DAPI (Merck, China) for 1 minute and imaged using a confocal microscope (Thermo Fisher Scientific, USA).

Western Blot (WB) Experiment

Macrophages and the aforementioned SMCs from each experimental group were harvested. Protein extraction was carried out using a protein extraction kit containing protease inhibitors and RIPA buffer (Solarbio, China). Total protein concentration was determined using a BCA assay kit (Biosharp, China) according to the manufacturer's instructions. The primary antibodies: anti-Calponin (CNN, 1:500; Abcam, USA), anti-α-Smooth Muscle Actin (α-SMA, 1:2000; Abcam, USA), anti-CD206 (1:1000; Abcam, USA), anti-INOS (1:500; Abcam, USA), anti-Tubulin (1:15000; Abcam, USA), and anti-GAPDH (1:10,000; Abcam, USA). Finally, chemiluminescent detection was performed using an ECL substrate (Pierce) on the Millipore membrane. The membrane was then washed with an exposure machine (Tanon, Shanghai, China).

Subcutaneous Implantation in Rats

The decellularized TEVGs were divided into four experimental groups as per the experimental requirements, namely PBS, FIB, PGF, and PGF- groups. The TEVG materials from each of the four experimental groups were evenly cut into pieces of approximately 1cm x 1cm in size. Subsequently, these pieces were implanted subcutaneously into rats.

HE and MASSON staining assay

After completing the above experiments, the samples were gently washed three times with PBS, followed by fixation in 10% formaldehyde solution. Subsequently, the samples were washed again with PBS. Then, the samples were embedded in paraffin and sectioned. Finally, they underwent respective staining procedures for Hematoxylin and Eosin (HE) staining and Masson's Trichrome staining analysis.

Tissue Immunofluorescence Staining Experiment

After tissue sections were prepared for tissue immunofluorescence staining, they were fixed overnight in 4% paraformaldehyde at 4°C, followed by PBS washing. Subsequently, the sections were dehydrated with graded ethanol and treated with butanol for 1 hour. Paraffin embedding was carried out by incubating the samples in a 65°C dry oven for 3 hours, followed by sectioning. The subsequent steps followed those of the cell immunofluorescence staining experiment. The primary antibodies used, CD206 and INOS, were both diluted to 1:200 (Abcam, USA).

Hydroxyproline Detection Experiment

Using the Hydroxyproline Detection Kit (Solarbio, China), samples were weighed and labeled according to the instructions. The tissues were finely minced to facilitate subsequent digestion. Then, 2 mL of extraction solution containing HCl was added to the weighed samples, and they were processed at 110°C for 4–6 hours. After cooling, the pH was adjusted to 6.8–7.4 using NaOH solution. Subsequently, the samples were centrifuged at 16000 rpm at 25°C for 20 minutes. The supernatant was then collected for analysis. The spectrophotometer was preheated for 30 minutes, the wavelength was adjusted to 560 nm, and distilled water was used for zero calibration. A standard curve was drawn using standard solutions, and the hydroxyproline mass fraction was calculated based on the detection results.

Transcriptome Sequencing Analysis

After one week of subcutaneous transplantation of samples from both the experimental group without PRP and the experimental group with PRP (i.e., the aforementioned PGF experimental group), transcriptome sequencing was performed on the two experimental samples (n = 5). After collecting the samples, RNA was extracted. The extracted RNA was concentrated and purified using ethanol. The purified RNA was then converted into cDNA and labeled for high-throughput sequencing, generating a large amount of RNA sequence data. Subsequently, preprocessing was performed to remove low-quality sequences and adapter sequences, followed by analysis ^[22].

Statistical Analysis

All reported values were averaged (n = 5) and expressed as mean ± standard deviation (SD). Assuming equal variance, significant differences were determined using a two-sample t-test. Values with p < 0.05 were considered statistically significant.

Experimental Procedure

In this experiment, blood was collected from rat hearts, and platelet-rich plasma (PRP) was obtained using a two-step low-speed centrifugation method. The PRP underwent repeated freeze-thaw cycles, followed by centrifugation to remove the sedimented portion containing red blood cells, white blood cells, and platelets, resulting in a PRP solution referred to as PGF. PGF was then mixed with a 10% calcium gluconate solution to activate the coagulation reaction of PRP via calcium ion stimulation, converting soluble fibrinogen into insoluble fibrin. The PRP solution without fibrinogen, obtained through centrifugation, was termed PGF-. Subsequently, tissue-engineered vascular grafts (TEVGs) were mixed with different experimental groups for subcutaneous transplantation experiments in rats.

Quality Assessment of Rat PRP and Material Characterization of TEVG from Different Experimental Groups

Scanning electron microscopy revealed the overall smooth surface of the material from the experimental group treated with PBS alone, with occasional normal creases and depressions visible on the material surface (A). In image B, TEVGs treated with FIB showed the presence of filamentous fibrin fibers throughout the layer compared to the untreated group, which appeared relatively loose, possibly related to the soaking concentration. Image C depicts the scanning electron microscopy of TEVGs after treatment with PRP solution-PGF, showing the presence of filamentous protein throughout the longitudinal section of TEVGs. Image D shows the scanning electron microscopy of TEVGs after treatment with PGF- without fibrinogen removal, with fewer proteins observed compared to image C, but denser and less structured compared to image B.

Material water absorption rate testing of the four experimental groups (PBS, FIB, PGF, PGF-) revealed an increase in water absorption rate compared to PBS treatment, with statistically significant differences observed among the three groups. The PGF group exhibited the highest water absorption rate, followed by the PBS group, with PGF- and FIB groups in between. This trend correlates with the protein content observed under electron microscopy, where higher protein content corresponds to higher water absorption rates (E).

Using a growth factor detection kit for rats, the release of growth factors over time was examined in experimental groups with added PRP-derived solutions. Results showed sustained release of VEGF and PDGF-BB in both PGF and PGF- groups over two weeks, with no significant decrease in VEGF levels. However, the release curve of PDGF-BB showed a downward trend in the PGF- group on the tenth day, possibly related to the removal of fibrinogen. Natural plasma proteins have a sustained-release effect on growth factors, and the removal of individual proteins can affect the rate of growth factor release (F and G).

The figure labeled as I depicts an overview of TEBV, while figure J displays the HE and Masson staining results of TEBV and TEVG. The staining did not reveal complete cellular structures, and the framework structure of TEVG remained largely unchanged. The results shown in figure H indicate that the DNA content after decellularization is significantly less than 50 ng/mg.

Blood was collected from rat hearts using blood collection tubes containing anticoagulants, resulting in bright red blood (K). After Wright's staining, platelets were stained light purple, with fewer platelet-stained positive areas in whole blood (L). Image L-right shows Wright's staining of PRP, revealing numerous light purple stained areas. Statistical analysis showed that the platelet count in PRP was 4.10 ± 0.13 times that of whole blood, consistent with PRP application standards (M).

Phenotypic Identification of SMCs

The results of the cell immunofluorescence experiment revealed that the SMCs used here expressed smooth muscle surface markers CNN and α-SMA (A). Positive expression of CNN and α-SMA proteins was also observed in the WB experiment. Semi-quantitative analysis of the WB experiment was performed in Figures C and D, respectively, revealing no significant differences between the experimental groups. Figure E depicts the treatment of smooth muscle cells with solutions of TEVG dissolved in varying concentrations. CCK8 results on the third day showed that solutions of different concentrations of TEVG had no toxicity to SMCs. Cell proliferation experiment revealed that from a concentration of 50ng/ml onwards, SMCs exhibited better proliferative capacity. However, this proliferation trend was statistically significant only at a concentration of 200ng/ml, which is why this concentration was used consistently in our other experiments."

Effect of TEVG of different groups on Macrophage Phenotype, Light Microscopy and WB Experiment

After 24 hours of co-culture of TEVG solution from different experimental groups with macrophages in vitro, it was observed that A depicts primary cells freshly isolated from bone marrow, showing various morphological features such as spindle-shaped, flattened, and round cells. After 24 hours of M-CSF treatment, cells exhibited larger cell volume, transparent cytoplasm, round or oval-shaped nuclei, and some cytoplasmic protrusions (B). Macrophages treated with PBS control showed mostly round cells (C), with fewer elongated cells. The morphology of macrophages in the FIB group appeared similar to PBS. Comparing the macroscopic images of macrophages treated with PGF and PGF-, it was observed that there were more elongated cells compared to the PBS and FIB experimental groups.

WB detected the effect of extracellular materials on macrophage polarization in vitro. The results revealed that the PBS and FIB groups exhibited higher expression of M1 marker INOS compared to the PGF and PGF- groups (D-G), with statistically significant differences observed. The expression of M2 marker CD206 was highest in the PGF- and PGF groups. The CD206/INOS ratio was as follows: PGF > PGF- > PBS/FIB groups. Thus, it can be inferred that the presence of fibrous proteins alone has minimal effect on the M2/M1 ratio of macrophages.

Effects of TEVG of different experimental groups on macrophage phenotypes, Cell immunofluorescence staining

After co-culturing the materials with macrophages for 24 hours, immunofluorescence staining was performed for M2-CD206 and M1-INOS. The results showed that the control groups, PBS and FIB, exhibited relatively weaker M2 expression and stronger M1 expression, while PGF and PGF- showed stronger M2 expression and weaker M1 expression compared to both control groups (A). Analysis of fluorescence intensity from the images revealed that the expression intensity of INOS was stronger in PBS and FIB compared to PGF and PGF- (B), While CD206 expression was higher in PGF and PGF- (C). Calculation of M2/M1 ratio showed higher values in the experimental groups PGF and PGF-, with statistical differences observed, while no statistical differences were found between PBS and FIB, PGF and PGF- (D).

HE Staining

HE reveals the cell infiltration status of the transplanted material. Results indicate that, in the first week, there is relatively high cell infiltration observed in both the PBS and FIB control groups, while the center of PGF and PGF- groups shows significant areas devoid of cell infiltration (A). Cell counting statistics reveal that the PGF group has the least number of cells, with statistically significant differences compared to the other three groups. There is no statistically significant difference in cell numbers between the PBS and FIB groups (B). In the second and third weeks, cell infiltration is observed in the material center, but there is no significant difference in the quantity of cell infiltration observed.

Masson staining

Masson staining reveals changes in collagen content of the materials subcutaneously transplanted after being processed by different experimental groups, further characterizing the degradation rate of ECM in vivo. From the visual images of staining, it is evident that the blue staining area of PGF and PGF- at 1–3 weeks is significantly larger than that of the PBS and FIB groups (A). Statistical analysis of the positive staining area of collagen from the first to the third week reveals that, compared to the FIB group, the positive staining area of Masson staining for PGF- is larger and statistically significant. There is also a statistical difference between PGF and PBS; however, no significant difference is observed between PBS and FIB or between PGF and PGF- (B).

Figure C illustrates that the hydroxyproline experiment data from the subcutaneous transplantation experiment in rats for three weeks reveal that the collagen content of PGF and PGF- experimental groups is higher than that of the PBS group from the first to the third week. The collagen content of the FIB group is slightly higher than that of the PBS group but without statistical difference. Compared to the non-transplanted TEVG, the decrease in collagen content for PGF and PGF- experimental groups is smaller in the first week. In the second week, compared to the ECM group, the collagen content decreases by approximately 35% for the PBS and FIB groups, and by approximately 26% for the PGF and PGF- groups. The decrease in the third week is relatively smaller compared to the second week, indicating a significant decline in the extracellular matrix in the second week.

M1 and M2 Cell Immunofluorescence Staining

After analyzing the results of tissue immunofluorescence staining following subcutaneous implantation of the materials, moderate CD206 cell infiltration was observed in all four experimental groups in weeks 2–3, with fewer CD86-positive cells (A). Among these four groups, PBS and FIB showed a higher number of CD86 cells. Analysis of the CD206/CD86 ratio for each group across the three time points revealed no statistical difference in the first week (B). However, in the second and third weeks, both the PGF and PGF- experimental groups exhibited higher CD206/CD86 ratios, with statistically significant differences (C, D), while no statistical differences were observed between PBS and FIB, PGF and PGF- groups.

Transcriptome Sequencing Analysis

Transcriptome sequencing analysis was performed on the subcutaneously implanted SECM in rats after one week. The results revealed 189 upregulated genes and 178 downregulated genes (A, B). Analysis of the top 100 differentially expressed genes showed that PRP downregulated various inflammation-related genes such as CFB, FGB, and IL17a, which are primarily involved in regulating inflammatory responses and macrophage phagocytic function. Among the upregulated genes, several genes associated with coagulation function were prominently upregulated, including F2 and F9.

According to the literature, M2 macrophages exhibit an elongated morphology and play a crucial role in promoting tissue repair and anti-inflammatory effects during later stages of cellular activity. They play a significant role in inflammation by secreting anti-inflammatory factors such as IL-10, TGF-β, which inhibit inflammatory responses, thereby reducing tissue damage and the progression of inflammatory diseases. M2 macrophages are involved in tissue repair and regeneration processes, facilitating the repair and reconstruction of damaged tissues. They promote angiogenesis, collagen synthesis, and cell proliferation, thereby facilitating tissue healing. Moreover, M2 macrophages modulate immune responses, influencing the activation and differentiation of T cells, thereby promoting immune balance maintenance. Additionally, M2 macrophages secrete various cytokines and growth factors, such as VEGF and PDGF, which play crucial roles in angiogenesis and tissue regeneration ^[23]. Platelet-rich plasma (PRP) has been widely used in clinical practice, but its application in tissue-engineered blood vessels is limited. PRP can be autologously obtained and utilized, but whether it can modify the transplant material to reduce in vivo inflammatory reactions and increase the degradation metabolism time of the material remains unknown. To address this issue, tissue-engineered blood vessels (TEBV) obtained through tissue engineering culture were subjected to decellularization to remove cellular components. Subsequently, this material was freeze-dried and subjected to different experimental group treatments. Through the detection of growth factor content in the culture medium, we found that this composite material could continuously release growth factors PDGF-BB and VEGF for up to 15 days. PRP is rich in various growth factors ^[24]. PDGF-BB and VEGF are known to promote cellular proliferation activity ^{[25, 26]}.

We investigated the toxicity and proliferation activity of decellularized vascular graft materials on smooth muscle cells by designing different experimental groups. By comparing the OD450 data on the third day, we found that ECM concentrations ranging from 50ng/ml to 250ng/ml could promote the proliferation of smooth muscle cells. At a concentration of 200ng/ml, the TEVG exhibited a significant promotion effect on smooth muscle cells. Therefore, we determined this concentration for subsequent co-culture experiments with macrophages. Macrophages are among the earliest immune cells recruited to the graft site during the immune response and graft degradation and regeneration processes, playing a crucial role ^[27]. The TEVG materials prepared using the aforementioned decellularization method primarily consist of ECM proteins, predominantly collagen. These proteins play a crucial role in facilitating host self-regeneration and reconstruction. This regeneration process is closely associated with the phenotype of macrophages. Research has shown that decellularized ECM exhibits favorable M2 macrophage polarization characteristics in vivo ^[28]. The effect of PRP combined with ECM on the M2/M1 phenotype of macrophages, both in vivo and in vitro, remains unknown. During our processing, we observed an increase in the water absorbency of ECM materials treated with FIB, PGF, and PGF-. This increase is speculated to be the result of the interaction of several proteins ^[29]. In vitro studies indicate that the PBS group alone exhibits significant M1 polarization, presumably related to the presence of undegraded PGA material in TEVG. Both the PGF and PGF- experimental groups promote M2 polarization. The conclusion aligns with the study findings on the effect of PRP alone on macrophages ^[30].

Collagen and elastin are the primary components of the extracellular matrix ^[31]. To validate the effect of PRP addition on the in vivo extracellular matrix (ECM) degradation, different experimental groups were implanted subcutaneously in rats. Through Masson's trichrome staining and hydroxyproline assay, collagen content was calculated for each time point. Results showed that collagen content in the experimental groups was higher than that in the control group during weeks 1–3. HE staining revealed that in the first week, the cell count in the transplantation area was higher in the PBS and FIB experimental groups compared to the PGF and PGF- experimental groups containing PRP. Immunofluorescence analysis of macrophages showed that the M2/M1 ratio was higher in the experimental groups than in the control group. Therefore, it can be concluded that PRP reduces the inflammatory response to vascular scaffold materials and promotes M2 polarization, thereby reducing the metabolic rate of ECM in rats. Despite the significant role of fibrinogen in ECM degradation, studies indicate that fibrinogen influences ECM degradation through various pathways, including regulating ECM stability, modulating the activity of degrading enzymes, participating in signal transduction regulation, and contributing to inflammation and repair processes ^[32]. However, in the results of this study, no significant impact of adding FIB on the degradation and inflammatory response of TEVG was observed. It is speculated that this may be related to the quantity of fibrinogen bound to TEVG. Scanning electron microscopy results showed that although fibrinogen was present throughout the TEVG layers, the overall quantity was sparse. Compared to the PGF experimental group, the protein network in the PGF group was denser.

This experiment found that the FIB group, simulating the physiological concentration of fibrinogen in rats, did not significantly affect the degradation of ECM or the polarization of macrophages. This could be attributed to the relatively low adsorption of fibrinogen onto the ECM induced by this in vitro simulation. Electron microscopy revealed that although fibrinogen structures different from pure ECM were visible throughout the layers, their overall abundance was limited. It appears that fibrinogen in plasma is not the primary factor in slowing ECM degradation during the PRP application process. However, the specific components involved in this series of actions remain unknown. From the results of the experiments on the four TEVG experimental groups, it can be observed that fibrinogen did not play a significant role in changes in inflammatory cell counts, macrophage phenotype conversion, or ECM degradation. In vitro macrophage polarization experiments showed that the FIB group exhibited similar results to the PBS group. Whether increasing fibrinogen can provide raw materials for phagocytes to reduce ECM degradation time remains uncertain and requires further experimentation.

To investigate further mechanisms, transcriptome sequencing revealed that compared to the PBS group, the PRP group showed upregulation of 189 genes and downregulation of 178 genes. Functional analysis of the top one hundred genes with significant differences revealed that the downregulated genes in the PRP group were mainly related to inflammation. For instance, the gene Cfb (complement factor B) encodes complement factor B, a component of the complement system. The complement system is part of the immune system, involved in immune responses, inflammatory reactions, and pathogen clearance. The primary role of complement factor B is to interact with other complement proteins during complement activation, forming a complement enzyme complex, thereby promoting pathogen lysis and clearance ^[33]. The down regulated gene CXCL6 encodes the cytokine CXCL motif chemokine 6 (CXCL6), which is a member of the chemokine family. CXCL6 plays a crucial role in inflammation, immune responses, and tissue repair. It acts as a chemoattractant for specific types of leukocytes, such as neutrophils and monocytes, promoting their migration to inflammatory sites and participating in the inflammatory process ^{[34, 35]}. In addition, among the downregulated genes, Il12rb2 (Interleukin 12 receptor subunit beta 2), Il17a (Interleukin 17a), MASP1 (Mannan-binding lectin serine peptidase 1), MAPK10 (Mitogen-activated protein kinase 10), and other genes are closely related to tissue inflammation and activation of phagocytic cell functions ^{[35, 36]}. In addition, among the downregulated genes, Il12rb2, Il17a, MASP1, MAPK10, and other genes are closely related to tissue inflammation and activation of phagocytic cell functions ^{[37, 38]}. Other upregulated genes include Cebpb (CCAAT/enhancer binding protein beta), F2 (Coagulation factor II), F9 (Coagulation factor IX), and other genes, primarily associated with cell proliferation and differentiation ^[39]. In the upregulated genes, the predominant downregulation appears to be in inflammation-related genes, which are primarily associated with the activity and chemotaxis of immune cells. Conversely, the upregulated genes associated with PRP are primarily related to coagulation processes. This implies that in future applications of vascular transplantation, attention should be paid to the potential risks associated with PRP.

Of course, this study also has its limitations. For instance, unlike conventional ECM derived from cell sources, the TEVG used in this study contains some undegraded PGA material fragments, as observed in HE and MASSON staining. Whether this will lead to undesirable immune reactions requires further experimentation and validation.

The combination of PRP with decellularized TEVG materials provides a protective environment for TEVG, enhancing its water absorption capacity, increasing the proportion of reparative macrophages, reducing early inflammatory cell infiltration, and slowing down the metabolism of ECM materials in the body. Results from rat subcutaneous transplantation and sequencing show that PRP promotes a decrease in the number of M1 cells and an increase in the number of M2 cells, thereby increasing the M2/M1 ratio. This shifts the metabolism of the material in the body towards a reparative phenotype. Additionally, PRP downregulates multiple inflammation-related genes, reducing inflammatory response to the material and increasing the biocompatibility of the transplant material. This provides a favorable direction for subsequent in situ transplantation. Although PRP also increases the expression of genes associated with thrombosis, this may be related to the presence of fibrinogen and some growth factors derived from PRP sources. In summary, PRP effectively improves the biocompatibility of the material, providing a reference for the in vivo application of tissue-engineered blood vessels.

PRP Plate-rich-plasma

TEBV Acellular vascular scaffolds

TEVG Fetal Bovine Serum

CHAPS (3-cholamidopropyl)-dimethylammonio

SDS Sodium Dodecyl Sulfate

ECM Extracellular Matrix

SMC Smooth Muscle Cell

PGA Polyglycolic Acid

PDGF-BB Platelet Derived Growth Factor-BB

VEGF Vascular Endothelial Growth Factor

M1 Classical activated macrophages

M2 Alternatively activated macrophages

F2 Coagulation factor II

F9 Coagulation factor IX

Il12rb2 Interleukin 12 receptor subunit beta 2

Il17a Interleukin 17a

MASP1 Mannan-binding lectin serine peptidase 1

MAPK10 Mitogen-activated protein kinase 10

Cfb Complement factor b

Acknowledgements

Not applicable.

Author contributions

YDW and JYX contributed to manuscript writing and the production of all images; HJJ, CX, and QL contributed to the

Figs.1, 5; HHZ and XHS contributed to software analysis; all authors have

read and agreed to the published version of the manuscript.

Authors' information

Yin-Di Wu, Hong-Jing, Jiang, Doctor of Philosophy (PhD) student of School of Medicine, South China University of Technology.

Jian-Yi, Xu, Xu-Heng, Sun, Qing Liu, Cong Xiao, Master degree (M.D) of school of Medicine, South China University of Technology.

Hao-Hao, Zhou: M.D of Ji Hua Institute of Biomedical Engineering Technology, Ji Hua Laboratory.

Yue-Heng, Wu, Zhan-Yi, Lin, Professor and PhD of Guangdong Provincial People’s Hospital (Guangdong Academy of Medical Sciences, Guangdong Academy of Medical Sciences), South Medical University.

Funding

This work was supported by the Summit Program of The NSFC Incubation Program of GDPH (KY012021150).

Availability of data and materials

Not applicable.

Ethics approval and consent to participate

All animal experimental procedures were performed according to protocols approved by Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences.

Consent for publication

All authors give consent for the publication of manuscript in Journal of Nanobiotechnology.

Competing interests

The authors declare that they have no competing interests.

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Decellularized vascular matrix material -TEVG coated with PRP for anti-degradation and anti-inflammation

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Abstract

Background

Objective

Methods

Results

Conclusion

Figures

Introduction

Materials and Methods

Acquisition, Decellularization, and Dissolution of TEBV to Form TEVG

Preparation of PRP

Isolating and Identifying SMCs

Scanning electron microscope (SEM)

Growth Factor Release Experiment

Material Water Absorption Test

CCK-8 Experiment

Extracorporeal Isolation and Cultivation Experiment of Macrophages

Cell Immunofluorescence Experiment:

Western Blot (WB) Experiment

Subcutaneous Implantation in Rats

HE and MASSON staining assay

Tissue Immunofluorescence Staining Experiment

Hydroxyproline Detection Experiment

Transcriptome Sequencing Analysis

Statistical Analysis

Results

Experimental Procedure

Quality Assessment of Rat PRP and Material Characterization of TEVG from Different Experimental Groups

Phenotypic Identification of SMCs

Effect of TEVG of different groups on Macrophage Phenotype, Light Microscopy and WB Experiment

Effects of TEVG of different experimental groups on macrophage phenotypes, Cell immunofluorescence staining

HE Staining

Masson staining

M1 and M2 Cell Immunofluorescence Staining

Transcriptome Sequencing Analysis

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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