Introduction

Collagen is one of the best biomaterials because of its good biocompatibility, negligible immunogenicity and biodegradability. It was opined that immune response of collagen depends upon the source from which it is collected (Wu et al. 2004). Most of the collagen scaffolds used in tissue engineering belongs to porcine or bovine origin. Bovines are prone to different zoonotic diseases like bovine spongiform encephalopathy (BSE or Mad Cow disease), transmissible spongiform encephalopathy (TSE) and foot and mouth disease (FMD). The use of collagen from this species may transmit the dreadful diseases to the recipient. Therefore, collagen of bovine origin has limited use (Pati et al. 2010). Keeping in view of certain disadvantages, source of bovine collagen may be replaced by caprine (goat) collagen because domestic goat is a dietary source of milk and meat in the Indian subcontinent. Though, caprine are susceptible of scrapie, but such prions do not cause any diseases in the human (Manuelidis et al. 2009). So caprine cadaver tissues may be considered as a safer source for preparation of collagen matrices.

Tissue decellularization is the process of removal of cells and debris to produce acellular extracellular matrix (ECM) framework. Various biological detergents have been used for decellularization of tissues. However, complete decellularization requires a combination of all three biological, chemical and physical methods (Gilbert et al. 2006). Various biological detergents and chemical like sodium deoxycholate (Gangwar et al. 2013), hypertonic solutions (Gangwar et al. 2015), sodium dodecyl sulphate, Triton X-100 and DNase (Gangwar et al. 2006) have been used successfully for decellularization of tissues. However, these detergents impair the collagenous and non-collagenous proteins, GAGs and growth factors. Further, certain chemical and enzymes are responsible for residual cytotoxicity in the decellularized extracellular matrix (Choi et al. 2012).

The fruit of Sapindus mukorossi, a member of the family Sapindaceae, contains 10.1% saponins in the fruit pericarp. Soap nut pericarp contains triterpenoidal saponins namely oleanane, dammarane and tircullane (Mostafa et al. 2013). These saponins are secondary metabolites and used as herbal detergent (Güçlü-Üstündağ and Mazza 2007). It also has anti-inflammatory, antimicrobial, antifungal, anti-parasitic and antiviral properties (George and Shanmugam 2014). LD50 of saponins from Sapindus mukorossi is more than 5000 mg/kg in wistar rats and the result of dermal irritation test in rabbits showed that the average score of dermal irritation per day of each rabbit is zero after 14 days of continuous dermal irritation by the saponins from Sapindus mukorossi (Du et al. 2014). According to the classification standard of toxicity in “Hygienic Standard for Cosmetics” (Ministry of Public Health of China, 2002), the sample is classified as “practical nontoxic” and “non dermal irritation”. Critical micelle concentration (CMC) of Sapindus saponin is between 0.04 and 0.05 wt%, much lesser than synthetic surfactants like, Triton X100 (0.13) and SDS (0.224) (Balakrishnan et al. 2006). Due to the low CMC value of Sapindus saponin, it could be further explored for potential applications as biodegradable surfactant in preparation of biological scaffolds. The saponins present in this herbal detergent have been used for decellularization of caprine aorta (Goyal et al. 2021).

Partial circumferential, full thickness esophageal abnormalities can develop due to esophageal perforation or tumor ablation, or at the time of surgical correction of esophageal stricture (Totonelli et al. 2013). Treatment of esophageal cancer often involves the removal of affected part. Tissue engineered esophageal scaffolds may be used for reconstruction of esophageal defects with promising results (Luc et al. 2014). Several methods of decellularization of esophagus using toxic biological detergents and other chemicals have been described. But there is a need to replace the use of these cytotoxic detergents and other agents used for decellularization of tissues.

The biomechanics of the decellularized scaffolds should be taken in to consideration before preparation of any synthetic or biological scaffold. The mechanical strength of tissues is primarily due to presence of collagen and elastin in the extracellular matrix (Armentano et al. 1991). Regeneration of injured tissue depends on microarchitecture and porosity of the scaffold. Mechanical properties of the any scaffolds are inversely proportional to porosity. So the scaffold should be designed in such a way that it mimics the biomechanical characteristics of the adjacent native tissue (Liu et al. 2006). The tissues treated with deoxycholate showed superior mechanical properties, maintenance of the extracellular matrix and a lower DNA content than those treated with Triton X-100 (Ozeki et al. 2006). Combination of 0.5% sodium dodecyl sulphate and Triton X-100 is effective in decellularization, albeit with a loss of tensile strength (Bhrany et al. 2006).

Therefore, the present study was planned with the aim to analyze the efficacy of aqueous extract of the fruit pericarp of Sapindus mukorossi (an herbal detergent) for decellularization of esophageal tissues of caprine origin and to assess its in vitro cytocompatibility.

Materials and methods

Chemicals and reagents

Chemicals used in the present study were obtained from Merck (Dramstadt, Germany) unless otherwise indicated.

Preparation of soap nut pericarp extract

Sapindus mukorossi (Soap nut) fruit pericarp extract (10 wt %) was prepared according to the protocol developed by Köse and Bayraktar (2016). Briefly, Sapindus mukorossi fruits were purchased from market and validated at the College of Horticulture and Forestry of our University. After shed drying, fruit pericarp was segregated from seed (Fig. 1) and finely powdered. Ten gram of fruit pericarp powder was immersed in 100 ml phosphate buffer saline (PBS) for 24 h at 23 °C. This mixture was mechanically agitated on magnetic stirrer for 6 h at room temperature, filtered and centrifuged at 6000 rpm for 30 min. Supernatant was collected and used as stock solution to prepare 5% and 2.5% concentrations to be used for further studies.

Fig. 1
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Dry fruit pericarp separated from fruits of Sapindus mukorossi

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Fig. 2
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Native caprine esophagus (A) and decellularized tubular esophagus revealed milky white appearance due to progressive cell removal (B)

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Procurement and cleaning of caprine esophagus

Caprine esophagus is easily available and can be procured from local abattoir (Fig. 2A) 12Cm long cervical part of esophagi was collected aseptically from six different bucks in sterile phosphate buffered saline (PBS) admixed with (0.1% amikacin) and proteolytic inhibitor (0.25% EDTA). After removing adherent blood and debris, the samples were thoroughly washed using sterile PBS (pH 7.4). The period between procurement and the starting of protocol was about 4 h.

Decellularization of caprine esophagus

After initial washing, the native esophagus samples were transferred to 100 mL of 2.5, 5.0 and 10% extract of soap nut pericarp (SPE) and subsequently agitated on magnetic stirrer for 72 h at room temperature. Initially the tissue samples were retrieved randomly at 12, 24, 48 and 72 h time intervals to analyze the decellularization efficiency of different concentrations of soap nut pericarp extract by histological examination. The concentration of SPE at which the esophageal tissues were completely decellularized preserving ECM’s microarchitecture was selected for characterization via 4,6-diamidino-2-phenylindole.2Hcl (DAPI) staining, scanning electron microscopy (SEM) examination, deoxyribo nucleic acid (DNA) quantification, mechanical tensile testing, SDS-PAGE, fourier transform infrared (FTIR) spectroscopy and cytocompatibility using primary chicken embryo fibroblast cells proliferation assay.

Verification of cell removal

Histology

For histological observations, tissue samples (n = 6) were collected in neutral buffered 10% formalin saline. Thereafter, the tissues were dehydrated in graded ethanol, transferred in xyline, embedded in paraffin and 5 micron thick paraffin sections were cut. All tissue sections were stained with hematoxylin and eosin (H&E) and Masson’s trichrome for microscopic evaluation as per the scoring system developed by Goyal et al. (2021). Briefly, number of nuclei per square micrometer: More (+++), moderate (++), mild (+) and no nuclei material (−); cellular debris: more debris (+++), moderate debris (++), mild debris (+), no debris (−); arrangement of collagen fibers: more compact (+++), mildly loose (++), moderately loose (+), heavily loose (−); porosity: highly (+++), moderate (++) and mild porous (+).

DAPI staining: (4, 6-diamidino-2-phenylindole 2Hcl)

Native and decellularized esophageal samples were collected in neutral buffered 10% formalin saline. The sectioned tissues were loaded on the amino propyl tri-ethoxy silane (APTES) coated slides, dewaxed in xylene, rehydrated in graded ethanol. The slides were rinsed with 0.2% Tris buffered saline Tween 20 (TBST) for 3 times and 200 µl DAPI staining solution was poured on each slides and incubated for 15 min in dark at 37 °C. The unbind DAPI solution slides was removed by rinsing the slides again with 0.2%TBST for 3 times, 3–5 min each and cover slips were mounted with gel mount. This stain binds strongly to adenine–thymine rich regions in DNA and emits blue fluorescence under fluorescent microscope.

Scanning electron microscopy (SEM)

The native and decellularized esophageal samples were examined under scanning electron microscope using Jeol JSM-840 model. Briefly, small pieces of samples were fixed into freshly prepared karnovasky fixative at 4 °C for 3–4 days. After proper fixation, tissue samples were washed with phosphate buffer (pH 7.2), dehydrated in ethanol and finally treated with Hexamethyldisilazane (HMDS). The dried samples were mounted on aluminum stubs with the help of adhesive carbon tape, coated with gold and, finally images were recorded and analyzed.

DNA extraction and quantification

Deoxyribo nucleic acid was extracted from native and decellularized esophagus as per the protocol explained by Green and Sambrook (2017). Briefly, tissue samples (25 mg each) were homogenized and incubated for 2 h at 37 °C in water bath. Thereafter, 50 μL of Proteinase K solution was added and left overnight at 56 °C. After centrifugation at 10,000 rpm for 10 min, supernatants were collected and equal volume of Tris-saturated phenol was added in each sample. Supernatants were collected after centrifugation and equal volume of chloroform: isoamyl alcohol (24:1) was added to these supernatants. Samples were again centrifuged as previously and aqueous phase was collected. The DNA was precipitated when 3 M sodium acetate and absolute ethanol (1:10) was added to aqueous phase. After centrifugation the precipitate is collected in the form of pellets, washed in 70% ethanol, air dried, dissolved in deionised water. The extracted DNA was used for quantification of DNA using Nanodrop 1000 spectrophotometer (Thermo Scientific, USA). Further, to identify the remnants of DNA in decellularized tissues, samples were separated by electrophoresis on agarose gel with ethidium bromide at 60 V for 1 h. After staining, DNA molecules were examined under ultraviolet trans-illumination (Fluor Shot PRO II SC850, Shanghai Bio-Tech Co., Ltd, China).

Molecular weight analysis

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed for molecular weight analysis of the native and decellularized esophageal samples (Laemmli 1970) (12 h, 24 h, 48 h and 72 h). In brief, 100 mg of each test sample was triturated with 10% SDS (1 mL), centrifuged at 10,000 rpm for 10 min and supernatant was collected. An equal volume of 1 × non-reducing sample buffer was added to the supernatant solution. Thereafter, SDS-PAGE of these samples was performed using 4% stacking gel and 10% resolving gels at 50 mA/gel. The gels were stained with 0.5% Coomassie brilliant blue R-250 dye dissolved in 30% methyl alcohol and 10% acetic acid for three hours. After staining, it was unstained with the solution containing 30% methanol and 10% acetic acid. The protein bands of different molecular weight in the gel were calibrated using known molecular weight marker and values were expressed in kDa.

Fourier transform infrared (FTIR) spectroscopy

Fourier Transform Infrared spectra were obtained using Perkin-elmer spectrum version 10.03.06. The spectra were collected over a wave number spectral range of 400–4000 cm−1 to analyze any changes in the amide groups. Briefly, 1 mg of each native and decellularized esophagus tissue samples were triturated in chloroform to obtain the extract and this extract was smeared over FTIR prism. The graph was obtained denoting percent transmission on y-axis and wavelength on x-axis.

Estimation of hydroxyproline contents

The hydroxyproline content of native and decellularized oesophagus was estimated as per the procedure of Reddy and Enwemeka (1996). Briefly, tissue samples (100 mg) were homogenised by teflon pestle tissue homogenizers (JSGW, India), centrifuged at 5000 rpm for 10 min and supernatant was collected. At the same time standard hydroxylproline concentrations (2.5 µg, 5 µg, 10 µg, 15 µg, 20 µg in 100 µl) were prepared. Thereafter, 100 µl each of 0.05 M copper sulphate and 2.5 N sodium hydroxide was added in standard and test samples, and transferred in a water bath at 40ºC for 5 min. Afterwards, 100 µl of 6% hydrogen peroxide was added and again placed in the water bath for 10 min. After cooling, 400 µl of 3 N H2SO4 was poured and incubated with 200 µl of 5% p-dimethylaminobenzaldehyde solution. Hydroxyprolene was measured by taking absorbance of the OD550 light with the use of spectrophotometer (Shimadzu).

Mechanical testing

Uniaxial tensile test of the native and decellularized tubular caprine esophagus (n = 6) was accomplished using Tensile Testing Machine (Model AMT-SC). Just before testing, the thickness and width of each sample was measured at proximal, middle and distal position using Bernier caliper to calculate an average initial cross sectional area for each specimen. The tubular esophagus (length 8 cm) was loaded into the fixed lower base and upper fixture of the machine by self-tightening clamps. Samples were strained until failure at a constant rate of 5 mm/min at room temperature and the tests ended when the tissue fractured. At this point, Change in length of the sample (δL) and Force (F) was noted.

The stress (σ) was computed as: σ = F/A0, where F represents the applied force, and A0 is the initial cross sectional area (m2) of the esophageal tissues. The strain (ε), was calculated as: ε = δL/L0, where δL is the uniaxial displacement in length, and L0 is the initial length of the tissues along a longitudinal axis. Young’s modulus of elasticity (E) is the ratio of the stress to strain, which can be calculated as E = σ/ε. Stiffness was calculated as k = F/δL, where F represents the applied force and δL is the displacement in length. Stretch ratio is the ratio between the final length and initial length of the esophageal samples.

Cytocompatibility assay

Acellular esophageal matrix was used for seeding of P-CEFs and, isolation and culture of the P-CEFs was done as per the protocol used by Gangwar et al. (2015). Briefly, chicken embryo was collected from 9 to 12 days embryonated eggs in antibiotic containing HBSS. After decapitation and evisceration, the embryo was minced, enzymatically disintegrated with 0.25% trypsin and filtered. The filtrate was centrifuged at 3000 rpm for 15 min and supernatant was discarded. The cell pellet was washed and, suspended in DMEM-LG containing 10% FBS (Sigma–Aldrich Company Ltd., Dorset, UK) and antibiotics. The P-CEFs were plated with seeding density of 2.2 × 105 cells/cm2 in T-25cm2 flasks and placed in CO2 incubator. When the cells attain 80–90% confluency, the cells were passaged and washed twice with HBSS. Meanwhile, the decellularized esophageal scaffolds were washed with DMEM. The scaffolds were transferred in the wells of culture plates and seeded at the rate of 2 × 104 P-CEF cells/cm2. The culture plates were incubated at 37 °C in a humidified atmosphere of 5% CO2 and culture media was changed after 48 h. After 96 h the seeded scaffold samples were collected in 10% formalin and in 2% glutaraldehyde for histopathological and SEM studies, respectively.

Statistical analysis

DNA content, collagen content and tensile strength of native and decellularized esophageal samples was compared by independent sample t-test.

Results

Decellularization of esophageal tissues was evaluated by macroscopic examination. The processed tissues became milky white at 72 h interval (Fig. 2B).

Histology

Histological images of native (Fig. 3A) and 2.5, 5.0 and 10% SPE processed caprine esophagus at different time intervals are presented in Fig. 3B (H&E; 4X; scale bar 200 μm) and 3C (Masson’s trichrome; 4X; scale bar 200 μm). Microscopic evaluation of samples at different time intervals is presented in Table 1. Staining with haematoxylin and eosin revealed the nuclei and cellular debris in 2.5% processed samples collected at different time intervals. Collagen fibres were compactly arranged. The samples processed in 5% solution at 72 h interval revealed negligible nuclei with preservation of ECM microarchitecture and debris was absent. The collagen fibres were mildly thick and loose with moderate porosity. The samples ran in 10% SPE showed complete decellularization but ECM’s microarchitecture was completely disturbed. Collagen fibres were loosely arranged. On the basis of histological findings, 5% SPE was selected for further studies.

Fig. 3
figure 3

A Microscopic image of native esophagus after H&E staining showing mucosa, submucosa and muscular layers, Magnification ×40 , scale bar represents 200 µm; B Comparison of H&E stained microscopic images of caprine esophagus processed using 2.5% a–d, 5.0% e–h and 10% i–l extract of soap nut pericarp (SPE) at 12, 24, 48 and 72 h, Magnification ×4 , scale bar represents 200 µm; C Masson’s trichrome stained microscopic images of caprine esophagus processed in 5.0% extract of soap nut pericarp (SPE) at 12, 24, 48 and 72 h, Magnification ×4 , scale bar represents 200 µm; D Representative images depicting DAPI stained native caprine esophagus tissues showing cold blue fluorescence (a) and processed samples using 5% SPE showing evidence of gradual decreased in number of nuclei at 24 h(b) 48 h (c) and absence of nuclei at 72 h interval (d), Magnification ×4 , scale bar represents 200 µm; E SEM images of native caprine esophagus (a, b) and decellularized esophagus (c, d); F DNA content (ng/mg of tissue) in native and decellularized caprine esophagus, P < 0.01, n = 6 (a) and Ethidium Bromide stained agarose gel image showing DNA band W2 = Native, W3 = Decellularized esophagus (b)

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Table 1 Histologic observations of native and SPE processed caprine esophagus in different concentrations of SPE at different time intervals
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DAPI staining

DAPI stained native esophageal samples showed cold blue fluorescence which depicts the nuclear DNA. The soap nut pericarp extract (5%) processed esophageal tissues showed gradual decrease in cold blue fluorescence at 24 h and 48 h interval. However, the samples collected at 72 h showed complete removal of nuclear components (Fig. 3D).

Scanning electron microscopy

Scanning electron microscopic images of native esophageal samples showed undulating luminal surface and external surface resembled a foam configuration over the collagen fibers. The luminal surface of decellularized esophageal matrix showed randomly oriented collagen fibres with large interconnected pores and cells were absent. However, the external surface was more textured with fibrous structures and collagen fibres were well preserved (Fig. 3E).

DNA extraction and quantification

Tissue specimens were taken from native and decellularized esophagus for DNA quantification. Quantitative evaluation of DNA demonstrated a significant (P < 0.05) reduction of DNA content in the decellularized esophagus as compared to native tissues. Quantity of DNA in native and decellularized esophagus was 55.40 ± 2.3 ng/mg of tissue and 02.70 ± 0.6 ng/mg of tissue, respectively (Fig. 3F). Agarose gel electrophoresis of native and decellularized esophagus was performed to study the DNA fragmentation after processing in 5% SPE. DNA extract from native esophagus tissue showed distinct band which indicates a large DNA fragment (Fig. 3f Lane W2). Decellularized esophageal scaffold extract did not show any DNA band indicative of removal of all DNA fragments from the tissues (Fig. 3F Lane W3).

Molecular weight analysis

SDS-PAGE of the supernatants obtained after centrifugation of homogenized tissues was performed to determine the residual protein material left over, after each successive interval. The protein bands of native and processed esophagus at different time intervals are presented in Fig. 4. The typical collagen pattern was represented in the native esophagus. Collagen proteins of the native tissues remained in the stacking gel. After processing in 5% SPE, the soluble proteins decreased progressively as shown in SDS-PAGE of native and decellularized matrix. Decellularized scaffolds lack the high molecular weight protein band.

Fig. 4
figure 4

SDS-PAGE of Caprine esophagus. Lane W1- ladder, Lane W2- Decellularised esophageal matrix-72 h, Lane W3- Esophageal matrix -48 h, Lane W4- Esophageal matrix -24 h, Lane W5-Esophageal matrix -12 h, Lane W6- Native esophagus

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Fourier transform infrared (FTIR) spectroscopy analysis

The FTIR spectra of native and decellularized esophagus indicate the presence of organic molecules of similar type in both the tissues. The absorption peak of amide A was strong and broad. In native tissues, peptide bond vibration develops amide A (3417 cm−1), B (2953 cm−1), I (1642 cm−1), II (1462 cm−1) and III (1218 cm−1) bands of collagen (Fig. 5A). In decellularized esophageal scaffolds, absorption peaks of amide A (3419 cm−1), B (2928 cm−1), I (1642 cm−1), II (1490 cm−1) and III (1216 cm−1) were observed (Fig. 5B).

Fig. 5
figure 5

Fourier transform infrared (FTIR) spectroscopy analysis of native caprine oesophagus (A) and decellularized oesophagus after processing in 5% aqueous extract of fruit pericarp of Sapindus mukorossi (B)

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Collagen quantification

The Collagen contents of native and acellular esophagus are presented in Fig. 6.The collagen content in native and acellular esophagus was 42.8 ± 2.18 µg/mg wet tissue and 41.2 ± 2.0 µg/mg wet tissue, respectively. Collagen quantification assay indicate no significant (P > 0.05) difference in its content between native and decellularized caprine esophagus.

Fig. 6
figure 6

Mean ± SE of collagen contents (µg/mg wet tissue) in native and acellular esophagus. Collagen content maintained in the acellular matrix compared to the native tissue, with a slight but not significant decrease

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Biomechanical strength

The values of different mechanical test parameters of native and decellularized esophageal tissues are showed in Table 2. Compared to native esophagus, decellularized esophagus demonstrated a significant (P > 0.05) reduction in stress and strain. However, elastic modulus of the decellularized esophagus scaffolds increased significantly as compared to native tissues. A non-significant (P < 0.05) increase in stiffness and decrease (P < 0.05) in stretch ratio was recorded in the decellularized esophagus scaffolds as compared to native counterparts.

Table 2 Mean ± SE of biomechanical properties of native and decellularized caprine esophagus
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Cytocompatibility assay

The cytocompatibility assay, performed using P-CEFs, showed that the scaffolds prepared by 5% SPE were cytocompatible. Microscopic examination of 96 h post-seeding esophageal scaffolds showed expression of ECM secreted by P-CEFs infiltrated within the scaffold at places (Fig. 7A). Scanning electron microscopic examination of P-CEFs cultured decellularized esophageal matrix revealed densely spread ECM secreted by P-CEFs on decellularized matrices indicative of effective binding of PCEFs with the scaffold prepared using 5% SPE (Fig. 7B).

Fig. 7
figure 7

Light microscopic examination of H&E stained samples show evidence of different extent of P-CEF cells infiltration into the scaffold with expression of some extra-cellular matrix, Magnification ×40 , scale bar represents 200 µm (A) Scanning electron microscopic examination revealed growth of P-CEF cells on scaffold (B)

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Discussion

Congenital and acquired affections of esophagus like esophageal atresia, esophageal cancer, ingestion of caustic etc. require tissue graft for reconstruction of esophageal defect. Surgical interventions such as gastric and colonic interposition are standard, often complicated by stenosis with donor site morbidity (Totonelli et al. 2013). Soap nut pericarp extract is an herbal solution and it is nontoxic to host cells (Du et al. 2014). If traces of chemicals remain within the scaffold even after proper washings, they will be toxic to recipient’s host cells (Gilbert et al. 2006). With an effort to minimize the use of toxic chemicals/reagents/enzymes, in the present study caprine esophageal tissue was processed using 2.5. 5 and 10% extract of soap nut pericarp under continuous agitation on magnetic stirrer for 72 h at room temperature.

Macroscopic examination of decellularized esophageal tissues revealed milky white appearance which might be attributed to complete removal of cells. Decellularization efficiency was further evaluated by histology (H&E staining), DAPI (4, 6-diamidino-2-phenylindole 2Hcl) fluorescent staining, scanning electron microscopy and DNA quantification of the decellularized scaffold. Hematoxylin–eosin staining of decellularized samples showed complete decellularization of the native esophagus at 72 h interval. Sapindus mukorossi fruit pericarp extract (5%) was effective in cell extraction along with removal of residual cellular components without significant disturbances in extracellular matrix (ECM) morphology. There is about 10% saponin in fruit pulp of Sapindus mukorossi. Saponins present in the soap nut are natural non-ionic surfactant with a great cleaning capacity (Du et al. 2014). The samples processed in 5% solution at 72 h interval revealed negligible nuclei with preservation of ECM microarchitecture and debris was absent which may be comparable to SDS. Decellularization process should not disturb the natural architecture and composition of the extracellular matrix (Petersen et al. 2010). Sodium deoxycholate (SDC) and sodium dodecyl sulphate (SDS) effectively removes the cellular debris from the decellularized tissues, but SDC disturbs the native tissue architecture more as compared to SDS (Gilbert et al. 2006). In the present study, decellularized samples revealed almost complete removal of nuclear components in esophageal matrices as evidenced by absence of nuclei in H&E and DAPI stained sections, and significant (P < 0.05) reduction in DNA content as proposed by Crapo et al. (2011).

Scanning electron microscopy of native esophagus resembled a foam configuration over the collagen fibers. However, decellularized esophageal scaffold showed natural architecture of extracellular matrix with large interconnected pores and intact cells were absent. Analysis of decellularized scaffolds demonstrates that the decellularization process removes the cellular material and debris of the cells with preservation of confluent layer of ECM and mucosal structure (Totonelli et al. 2013).

The use of 5% SPE leads to extraction of approximately 94% of total DNA from the decellularized esophageal scaffolds. DNA quantification of these scaffolds supported the results of H&E staining, DAPI fluorescent staining and scanning electron microscopy (Simões et al. 2017). Decellularization process minimizes the DNA from the native tissues and acellular scaffolds can be implanted successfully in the recipients without any immune response (Syed et al. 2014).

Fourier transform infrared (FTIR) spectroscopy was performed to analyse the secondary structure of proteins present in the acellular matrices (Gupta et al. 2013). In FTIR spectrum of native and decellularized esophagus, change in the structure of collagen proteins was analysed using the amide A, B, I, II, III peaks. The amide A band (3294 cm−1) corresponds to H-bonded N–H stretching (Doyle et al. 1975). It was observed at 3417 cm−1 and 3419 cm−1 in native and decellularized tissues, respectively. The amide B band corresponds to CH2 asymmetric stretching (Abe and Krim 1972) and was found at 2953 and 2928 cm−1 in both native and decellularized tissues, respectively. The amide I band (1641–1658 cm−1) corresponds to C = O and C-N stretching (Zhou et al. 2018) as recorded at 1642 cm−1 for native and decellularized tissue. Absorption peak (1409 cm−1) is associated with C–H surface stretching and –C–O stretching (Wang et al. 2019) and was observed at 1462 cm−1 for both the tissues. The amide III band was observed at 1216 cm−1 for native and decellularized tissue confirming presence of hydrogen bonds (Muyonga et al. 2004).

The consequences of 5% SPE solution on collagen content was quantified in the decellularized esophagus scaffolds. Collagen quantification assay indicate no significant change in the collagen content after decellularization of esophagus (Totonelli et al. 2013). Conversely, Dahl et al. (2003) reported slightly increased collagen content after decellularization because cells and possibly some proteoglycans no longer contribute to dry weight.

Biomechanical properties of decellularized scaffold are an important factor to find out the success rate of the scaffolds used for reconstruction of tissues (Ahim et al. 2019). The axial strain is a physiological property of oesophagus. Collagen and elastin present in the wall of the esophagus are responsible for mechanical function of the esophagus (Stavropoulou et al. 2009). Compared to native esophagus, decellularized esophagus demonstrated a significant reduction in stress and strain. Strain in longitudinal direction of native caprine esophagus and the esophagus decellularized by SPE was 64 ± 8% and 43 ± 6%, respectively. Decellularized scaffolds supported lesser stresses and tensile forces than native esophagus. Processing of tissues in 0.1% SDS may decrease in mechanical compliance under inflation loading (Azhim et al. 2014). Esophagus is very extensible up to an axial Green strain of 0.6. (Lu and Gregersen 2001). Elastic modulus of the decellularized esophagus scaffolds increased significantly (P > 0.05) as compared to native tissues. It showed that treatment of esophagus in 5% SPE did not affect the major architectural network of the collagen and elastin in the esophageal ECM. Luc et al. (2014) also noted an increase in elasticity modulus in longitudinal traction of porcine decellularized tubular esophageal scaffolds. Sodium dodecyl sulphate and Triton X-100 are also effective in decellularization of tissues while maintaining the major architectural network of the elastin and collagen in the ECM (Zou and Zhang 2012). A non-significant (P < 0.05) increase in stiffness and decrease (P < 0.05) in stretch ratio was recorded in the decellularized esophagus scaffolds as compared to native counterparts. Yang et al. (2006) evaluated the mechanical properties of rectangular specimens of porcine esophageal mucosa and the muscularis separately. Stresses of the mucosal and muscular specimens in longitudinal traction were 9.4 MPa and 1.5 MPa respectively.

The seeded scaffolds showed evidence of P-CEFs proliferation and viability on the matrices as reported by Rhee and Grinnell (2007). Further, histological section of the seeded scaffolds revealed extracellular matrix secreted by P-CEFs on the surface and between the collagen fibres of acellular esophageal matrices which imply that 5% SPE solution is nontoxic to the cells. Scanning electron microscopic examination also revealed adhesion of fibroblasts with the acellular matrices as reported by Divya and Nandakumar (2006). Aqueous extract obtained from Sapindus mukorossi fruit pericarp is nontoxic to recipient’s cells (Du et al. 2015). Traces of biological detergents and enzymes present in the scaffolds even after multiple washing may be toxic to host cells (Gilbert et al. 2006).

Conclusions

It was concluded that the 5% Sapindus mukorossi fruit pericarp extract is best for decellularization of caprine esophagus as evidenced by histological examination (H&E and Masson’s trichrome staining), scanning electron microscopy, fluorescent staining, DNA quantification and of the decellularized scaffold. The scaffolds maintained adequate mechanical strength and results of in vitro cytocompatibility were encouraging. These scaffolds could serve as a xenogenic biomaterial for reconstruction of esophageal defects.