Introduction

Inadequate availability of organs has recently become a serious problem in regenerative medicine and many researchers are trying to find a way to overcome it; as far as regards to the claim of the United Network for Organ Sharing (UNOS) in America, less than 33% of candidates receive a transplant. In addition to applying autotransplantation and xenotransplantation, tissue engineering has introduced a method which is using decellularized human or animal organs and tissues and then recellularize acellular organ and tissue to reestablish function (Wang et al. 2020).

Decellularized scaffolds

Decellularized tissues, like bovine pericardia, are natural scaffolds for tissue engineering which contain preserved extracellular matrix (ECM) (Mirsadraee et al. 2006; Rijal 2017; Heuschkel et al. 2019). These natural scaffolds imitate the ECM perfectly and widely used in many clinical applications for the reconstruction of damaged tissues. These scaffolds also have recently been used in 3D (3-dimensional) and whole organ engineering (Mirsadraee et al. 2006; Heuschkel et al. 2019). It widely used for reconstruction of valvular heart defects and post-infarction septal defects, mitral valve annulus failures, and obstruction of outflow.

Different types of scaffolds

Veritas Collagen Matrix®, Tutopatch®, Peri-Guard®, Collagen Matrix®, Integra® are the examples of commercialized pericardia scaffolds (Swetha et al. 2010; Badylak et al. 2011; Keane et al. 2015; Rijal 2017). In addition, non-cardiac usages of the pericardium in tracheoplasty surgeries and treatment of vaginal and abdominal wall defects have been reported. Still, there is a need to improve methods of decellularization to retain natural structure and function of decellularized ECM as well as modification of its natural properties to be more stable and biocompatible scaffold with anti-inflammatory and antibacterial effects as well as promoting the reconstruction abilities.

Modifiaction of scaffold surface to facilitate the cell adhesion

Modification of decellularized tissues with different agents reported as an appropriate method to improve the needed characteristics of these scaffolds. For example, the laser was used for surface modification of the decellularized extracellular cartilage matrix for cartilage tissue engineering successfully (Goldberg-Bockhorn et al. 2018). In another study decellularized cornea developed by organic acid treatment for improved corneal regeneration (Lin et al. 2019).

In regenerative medicine, most cells must be attached to a bed in order to proliferate and function. So, attachment is a vital factor which are taken into consideration by researchers (Saltzman et al. 1991; Lih et al. 2016). So, increasing cell attachment and biocompatibility of decellularized pericardia tissue can be considered as a particularly appealing goal in tissue regeneration.

Fibronectin and laminin are some molecules that have been used in cell attachment (Saltzman et al. 1991). Sometimes coating the bed for increasing its hydrophilicity is a solution to improve and have suitable cell attachment and growth (Lih et al. 2016). In some researches, cryogel is considered as an active analogue of the natural ECM which provides an appropriate condition for attachment and proliferation of cells (Akilbekova et al. 2018). Elebring et al. decellularized a porcine pancreas and recellularized acellular scaffolds with human fetal pancreatic stem cells. After 14 days, proliferation and attachment of cells were observed which shows their success in pancreas decellularization (Elebring et al. 2017). In other study, a rotating bioreactor was used for human umbilical vein endothelial cells seeding onto the decellularized artery to determine whether the decellularized Porcine carotid artery is conducive for cell attachment or not. Ultimately, they conclude that decellularized Porcine carotid artery improves cell adhesion which demonstrating its potential use as a small-diameter vascular graft (Ho 2018).

Scrophularia striata is a medicinal plant which belongs to the genus Scrophularia and widely use in Iranian folk medicine for infectious diseases and inflammatory (Kerdar et al. 2018). Nowadays, aqueous extraction of Scrophularia striata has shown biological activities including antimicrobial, antioxidant, anti-inflammatory, anticancer, antifungal, antibacterial, anti-asthmatic, anti-parasitic, neuroprotective anxiolytic, and anti-depressant in different studies (El-Naggar and Beal 1980; Boros and Stermitz 1990; Del Carmen Recio et al. 1994; Oh 2009; Haddadi et al. 2019). Naserzadeh et al. showed that by using this plant, the antibacterial activity of gold nanoparticles was increased due to the presence of polyphenol compound in aqueous extraction (Naserzadeh et al. 2019). Nepitrin, Quercetin, acteoside and cinnamic acid are the main compounds identified in Scrophularia striata extract. Moreover, n-hexane, caryophyllene oxide, spathulenol, α-cadinol and docosane were the main compounds identified in the essential oil of Scrophularia striata. Antimicrobial activity of Scrophularia striata was proved by Ethanolic, aqueous, methanolic and ethyl acetate extractions from this plant (Haddadi et al. 2019).

In this study we evaluate the effect of the scrophularia striata extract on cell attachment and biocompatibility of decellularized bovine pericardia. At first, extraction of scrophularia striata and decellularization of bovine pericardia were prepared. Next, decellularized tissue was incubated in scrophularia striata solution. Isolated human adipose-derived mesenchymal stem cells were cultured on tissues and analysis were done.

Materials and methods

Plant isolation and extraction

Scrophularia Striata plant in spring in Ilam Province (Zagros) collected and dried in the shade. The plant approved by experts of Shahid Chamran University of Ahvaz. To extract the effective material from the plant, 200 g of powdered plant incubated in 1000 cc 70% alcohol for 3 days. Then, the extract was filtered and vacuum dried as far as possible. After that, solution with concentrations of 2%, 5%, and 10% weight—volume prepared.

The bovine pericardia

The Dashtyari breed's male bovine pericardia (two-year-old) was collected after the animals were sacrificed (Iran-Shahrekord slaughterhouse). The adipose tissue was removed and the samples placed in a bottle containing 1000 cc phosphate-buffered saline (PBS). Then, it was transferred to laboratory.

Decellularization method

All samples were placed in roller bottles (Hybridization Incubator GFL-7610) and incubated by SDS 1% (Biochem CAS: 151-21-3) for 48 h at 40 °C. The detergent was changed every 12 h. The decellularized tissues were washed at 4 °C for 12 h twice with distilled water. Then, the samples were washed three times for 8 h with PBS (Alizadeh et al. 2019).

Histological study

Hematoxylin and eosin staining (H&E) used for the morphological assessment of pericardium after decellularization. All the samples were fixed in paraformaldehyde 4% (Merck CAS 30525–89-4) and after that, the tissue processing was performed and samples were blocked in paraffin and have been cut in 5 µm thickness by microtome (Leitz 1512) and stained with hematoxylin and eosin (Rezakhani et al. 2020).

Remaining DNA assay (Hoechst staining)

The Hoechst 33258 staining (Sigma Aldrich. USA) was used for DNA evaluation before and after tissue decellularization. The stain was prepared for stock working dilution 1:5000 (1 mg/ml concentration). All samples were processed with a tissue processor and blocked with paraffin. Afterward, 5–7 μm slices were cut by microtome (Leitz 1512). Then, deparaffinization in 60 °C incubator PBS was used for washing and stained for 30 s by working diluted stain. Finally, DAKO fluorescent microscope was used to image recording (Li et al. 2018).

Treatment of the decellularized pericardium with Scrophularia Striata extract

The decellularized tissues after washing and sterilization prior to use were incubated an hour in solution with concentrations of 1%, 2.5%, and 5% weight – volume of Scrophularia Striata.

Isolation and culture of human adipose-derived mesenchymal stem cells (Ad-hMSCs)

The human adipose tissue of the inguinal section has been harvested from 15 hernias patients. The tissues were transferred to the laboratory,washed in PBS for three times,cut to the 3 × 3 mm and digested with collagenase I in 37 °C. Then, it was neutralized with DMEM (Bioidea) + 10%FBS (Gibco). The cell suspension was centrifuged at 1200 rpm for 5 min and the supernatant was discarded. Afterwards the cells were transferred to the culture flask and incubated with DMEM + 10%FBS + 1% Pen-strep medium in culture incubator (Memert). After 3 passages, cells were used for the MTT assay (Alizadeh et al. 2021).

MTT assay

The MTT test was based on tetrazolium salt which was broken down by living cells with mitochondrial succinate dehydrogenase. This enzyme breaks down the tetrazolium and converts it to insoluble purple crystals. During the test, 900 μl of the cell culture medium (DMEM (Sigma) with 10% FBS (Gibco) + 1% (penicillin–streptomycin) were added to a 24-well plate and 105 stem cells (Ad-MSCs) of human dispersed in it. Then, it was incubated for 24 h in 37 °C with 5% CO2 incubator (Memert). Next, the samples (decellularized bovine pericardia) were cut into 5 × 5 mm and placed inside the wells. The test was performed 48 h. 5 mg/ml MTT solution was prepared and ultimately, 100 μl MTT solution was added to each well and incubated for 4 h at 37° C (the final concentration of MTT in each well should be 0.5 mg/ml). Then, the content of the wells was removed and 200 μl DMSO was added to each well. After 30 min, the content transferred to a 96-well plate and 570 to 630 nm ELISA Reader beam was used to absorbance (Stat fax-2100, USA). The cell survival percentage was calculated by using its formula for each concentration (Koopman et al. 1994; Darzynkiewicz and Traganos 1998; Alizadeh et al. 2020).

Nitric Oxide (NO) assay

NO was measured by the Griess staining method. In the time span of 48 h, the supernatant of the mesenchymal stem cells which was exposed to treated decellularized bovine pericardia, was collected. 400 µl of supernatant were deproteinized by adding 6 mg of zinc sulfate. The vials were centrifuged at 4 °C and 12,000 rpm for 12 min. 100 µl of the supernatant of the de-proteinized samples were added to the wells of the 96-well plate. Then,100 µl of Vanadium chloride, 50 µl of sulfanilamide, and 50 µl of N-(1- Naphthyl) ethylene diamine dihydrochloride (NEDD) were added to each well; the plate was incubated for 30 min at 37 °C. Next, 100 µl of standard sodium nitrate solution, with concentrations of 0, 6.25, 12.5 25, 50, 100, and 200 mM, were prepared. Afterwards Vanadium chloride, sulfanilamide, and NEDD were added to the standard wells similar to the approach used for the samples. The standards and the samples were read at 540 and 630 nm wavelengths by an ELISA reader (Stat Fax 2100, USA) (Ayala et al. 2018; Rezakhani et al. 2017).

SEM examination

To assay cell attachment, 48 h after culturing of mesenchymal stem cells on treated decellularized pericardia, the SEM examination was performed. All of the samples were fixed in paraformaldehyde 4% and freeze-dried and next, examined on surface and cross-section.

Statistical analysis of data

With regard to the various doses, the one-way ANOVA test and the Toky post-hoc test was used, and the data were statistically analyzed by the GraphPad Prism software (Version 8). Mean differences with a P ≤ 0.05 were considered to be statistically significant. Each assay was performed 3 times and the mean of the resulted data was analyzed.

Results

Histology of decellularized bovine pericardia (H&E staining)

H&E staining showed that the natural bovine pericardia as a connective tissue is the reach of collagen fiber bundles. Connective tissue and fibroblast cells were intact (Fig. 1A). In the decellularized tissue, the cells completely removed and the collagen bundles remained intact (Fig. 1B).

Fig. 1
figure 1

The H&E staining of decellularized bovine pericardium. A: native tissue, B: decellularized tissue

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Remaining DNA assay (Hoechst staining)

For detecting of DNA debris in decellularized tissue, the Hoechst staining was performed. Both native and decellularized samples were stained and examined (Fig. 2A and B). The Native sample was glowing blue, which means that all cells and the cell nucleus were intact. In the decellularized tissue, the field of microscope was so dark and cell nucleus and DNA debris were removed.

Fig. 2
figure 2

The Hoechst staining of decellularized bovine pericardia. A: native tissue, B: decellularized tissue

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Effect of Scrophularia striata on survival of Ad-hMSCs (MTT assay)

A comparison of the mean survival of Ad-hMSCs by MTT assay after 48 h showed an increase in cell survival after treating the tissue with the extract. With increment in concentration, cell survival significantly was increased (p ≥ 0.001). The survival of cells in 0.5%, 2.5%, and 5% groups was about 5, 10 and 15 folds higher in comparison to control groups by the significance of p ≥ 0.01, P ≥ 0.001, and p ≥ 0.001 respectively. There was such a significant difference between the treated groups. So that the viability of 2.5% sample was closely two folds higher than 0.5 group (p ≥ 0.001). In 5% group, the viability was almost three times higher than the viability of 0.5% group (p ≥ 0.001). The p-value of 2.5% and 5% groups was 0.001 and the viability in the 5% group was 1.5 folds higher than 2.5% group (p ≥ 0.001) (Fig. 3).

Fig. 3
figure 3

MTT assay for Ad-hMSCs after culturing in decellularized bovine pericardia

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Effect of Scrophularia striata on NO secretion

The results showed that NO secretion increased in cells which were cultured in treated decellularized bovine pericardium with Scrophularia striata in a dose-dependent manner significantly (p≥ 0.001).

There was no significant change in NO secretion between the control and 0.5% treated samples, but in other two treated samples (2.5% and 5%) the NO secretion was increased significantly (p≥ 0.001). NO secretion in 2.5% and 5% samples was three and five folds higher than the NO secretion in control group, respectively. NO secretion in 2.5 and 5% treated samples was 1.5 and 2.5 times higher than 0.5% samples. NO secretion in 5% treated samples was 1.5 folds higher than 2.5% treated samples (p≥ 0.001) (Fig. 4).

Fig. 4
figure 4

The NO secretion from Ad-hMSCs cells after culturing in treated decellularized bovine pericardium

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SEM examination

The SEM examination was performed to evaluate the attachment of the cells to the decellularized pericardium. The results showed that in decellularized pericardium treated with scrophularia striata extract, the cell attachment was increased in a dose-dependent manner. Also, morphology and size of the cells were different. In treated samples, the cells were more elongated (Fig. 5A). In 0.5% samples, the cells were compacted and attached to each other and the cell pedicles were not seen clearly (Fig. 5B). In 2.5% samples, the attached cells were elongated and cell pedicels were visible. In 5% samples, SEM showed that the cells completely were attached to the scaffold, cell pedicels were clearly visible and each cell covered a greater part of the scaffold.

Discussion

In this study our aim was to use Scrophularia striata extract to improve biocompatibility of decellularized bovine pericardium. The biocompatibility is a critical characteristic of engineered biomaterials for designing an optimized novel biomeshes for clinical applications. In principle, the biological properties of implantable scaffold should be matched to the target tissues and able to promote cell proliferation and attachment (Badylak and Gilbert 2008; Williams 2008). Reconstruction and regeneration of damaged tissues can be considered as a key purpose of using biologic scaffolds in tissue engineering (O’Brien 2011). In fact, the grafted biomaterials must accelerate new blood vessels formation through the promotion of new ECM components production (Liang et al. 2004).

Bovine pericardia tissue has been broadly applied as a biomedical tool to restore cardiac valves and congenial disease treatment because of many desirable features including fibroserous structure which is made- up of collagen bundles and elastic fibers (Bielli et al. 2018). So, the decellularized bovine pericardium seems to be favorable in regenerative medicine approaches. The successful decellularization method can remove nuclear substances and cell debris to avoid immune rejection as well as the lowest level of ECM nature and structural damage that provides cell growth improvement (Wolf et al. 2014). Although, the efficiency of decellurization methods depends on tissues’ origin and materials and procedures which were used. The chemical decellularization protocol is using some detergents such as SDS which is widely used. In addition to collagenous and non-collagenous proteins damage that is caused by SDS, the remaining detergents decrease biocompatibility of the decellularized tissues. For this reason, some modifications in decellularization methods as well as the treatment with some materials such as medicinal plants extract for the treatment of decellularized scaffold after chemical decellularization could be efficient in increasing biocompatibility (Badylak et al. 2011).

Scrophularia striata is an Iranian herbal medicinal plant known as “Tashaneh Dari” from the Scrophulariaceae family. Many investigations have been reported the beneficial pharmacologic effects of striata including anti-inflammatory properties, cell proliferation promotion, and wound healing progress (Azadmehr et al. 2009; Monsef-Esfahani et al. 2010; Ghashghaii et al. 2017). Our results of this study suggested that the improvement of biocompatibility of decellularized bovine pericardia due to different concentrations of Scrophularia striata extracts. Here, decellularization process was achieved successfully by SDS 1% solution. As shown in the final histological evaluation (Figs. 1 and 2), the intact collagen bundles without fibroblast cells present in H&E and Hoechst images that confirmed the efficacy of the decellularization method. Our results were as similar as Heuschkel MA et al. study who reported complete decellularization using SDS1% (Heuschkel et al. 2019). In addition, Ad-hMSCs viability and NO secretion increased after treatment of decellularized tissue with different concentrations of herbal extracts (Figs. 4, 5). The MTT and NO secretion data supports the concepts of treated tissues biocompatibility elevation. The assay results showed that 5% weight—volume of Scrophularia Striata increased Ad-MSCs cell proliferation and NO production was much more than 1% and 2.5% weight—volume herbal extract (Gardin et al. 2015; Li et al. 2018; Heuschkel et al. 2019). The effect of presence this plant on cell viability and proliferation was investigated by Haddadi et al. They showed that because of using Scrophularia striata, by passing the time, the number of live cells and their growth will increase. Additionally, the higher the concentration of the plant (up to a limited value), the higher the cell viability and life span (Haddadi et al. 2019).

Fig. 5
figure 5

SEM examination, control (a), 0.5% (b), 2.5% (c) and 5% (d). The attached cells marked using red lines

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Moreover, the ability of cell for attachment increased after treatment with 1%, 2.5%, and 5% weight—volume of Scrophularia Striata resulted in the approperiate morphology and attachment of cells on the treated decellularized bovine pericardia, as shown in SEM data (Fig. 5). This finding has been proven by Haddadi et al. in 2019. SEM data showed that higher concentration of Scrophularia Striata, were associated with a higher cell attachment and migration (Haddadi et al. 2019).

Here, we introduced a novel decellularization procedure to enhance the biocompatibility of decellularized bovine pericardia by Scrophularia striata extraction. In the regard, this biocompatible natural scaffold will be able to increase regeneration related to the elevation of cell engraftment, proliferation, and attachment after applying in skin or heart disorders.

Conclusion

The results of this study indicated that Scrophularia striata in a dose-dependent manner increased significantly cell viability and cell attachment in decellularized bovine pericardia using SDS. In conclusion, treating or washing SDS decellularized bovine pericardia with Scrophularia striata extract can increases the biocompatibility of this scaffold. These findings could pave the way for the use of natural extracts of medicinal plants to reduce unwanted effects and make desired changes in decellularized tissues.