Post implantation assessment of scaffolds.
This was done by histological assessment, mechanical testing, SEM and Immunohistochemistry
Histological assessment
The control and samples were fixed in 10% neutral buffered formalin and embedded in paraffin. Sections were stained with HE, DAPI, MT stain, AB pH 2.5, Toludine blue staining, to demonstrate cellular recruitment. Immunohistochemistry was performed for VAP and CD90+expression. Sections (4µ) were taken on the positively charged slides (Pathnsitu biotech, India). Before processing, slides were washed with D/W containing 0.05% tween 20 followed by antigen retrieval using sodium citrate pH 7.4. Serum blocking was done by goat serum and slides were incubated with VAP (Invitrogen, India) and CD90 (Invitrogen, India) overnight at 40C followed by wash with D/W containing 0.05% tween 20. Staining was done using anti mouse secondary antibody conjugate with Alexa 488 (Molecular Probes, USA) and counterstained using DAPI. Slides were mounted with DAKO mounting media. Stained sections were viewed under fluorescent (Nikon eclipse Ti Japan)
Mechanical testing
Mechanical testing was performed by tensometer, while measuring under tensile loader at room temperature The tensometer applied tensile force to strips of biocompatible ear pinna scaffolds treated with M2, M3 and M5 successively.
SEM analysis:
SEM was performed on cross sections of recellularized M2 (Magnification X 3500), M3 (Magnification X 3000) and M5 (Magnification X 1400) scaffold to investigate cellular architecture and its porosity.
Statistical AnalysisData are reported as the mean ± SD. Differences between three or more groups were assessed using one-way ANOVA. Significance level p = 0.05 was set for all the tests. In the figures, statistical significance is denoted as ∗ for p -value ≤ 0.05, ∗∗ for p -value ≤ 0.001, ∗∗∗ for p -value ≤ 0.0001.
  1. Results
  2. Macroscopic appearance of scaffold
The auricular-shaped scaffold produced by decellularizing goat ear pinna cartilage had a very similar anatomical shape to human auricle with an almost identical size. The engineered auricle was resilient and smooth and had maintained its structural integrity and texture (Figure 2B).
Histology
Histological examinations of the control and decellularized scaffolds were done at every 10th cycle with HE, AB pH-2.5, DAPI, and MT to ascertain whether the cellular components were totally removed and histoarchitecture was preserved.
HE staining of the scaffold showed that complete decellularization was achieved at 40th cycle for M5 and for M1-M4 80th cycle were needed for complete decellularization as shown in Figure 3A with progressive reduction in sequential cycles. Methods M2, M3 and M5 showed negligible nuclear material compared to M1 and M4. Control cartilage showed differentiated chondrocyte with lacunae and nucleus was better organized (Figure E-A)
DAPI staining used to crosscheck the decellularization process revealed complete removal of cellular material in all methods (Figure 3B). Control revealed abundant bluish stained nucleus (Figure 3E-B)
MT staining showed intact appearance of collagen and ECM in decellularized goat ear pinna cartilage scaffolds (Figure 3C) with no obvious disruptions to histoarchitecture. Scaffolds treated with methods 2 and 3 showed better preservation of collagen and ECM compared to other methods. Control showed normal bundle of collagen and ECM (Figure 3E-C).
AB pH-2.5 staining demonstrated that the major structural components via GAGs showed no disruption following treatment with preserved architecture. (Figure 3D). Control showed well organized ECM and GAGs (Figure 3E-D).
DNA quantification:
The DNA content of samples in experimental group showed reduction with each subsequent cycle compared to control (Figure 4A). Scaffolds processed with Method 2 showed the most reduction while Methods 1 and 4 yielded the least reductions in DNA content.
Mechanical testing:
Elasticity, stress rupture and ultimate stress limit of control and decellularized scaffold were confirmed by mechanical testing (Table 1). The mechanical testing analysis showed that the ultimate stress, strain and elasticity for control and decellularized ear scaffold were not significantly different. Ultimate stress and tensile strength in samples treated by M2 and M5 were similarly good. Significant elasticity was seen in samples treated by M2 and M5. Overall M2 and M3 showed better retention of mechanical properties compared to M1, M4 and M5.
Decellularized scaffold prepared by method 2, 3 and 5 showed the significantly biophysical stable, elastic and load-bearing object. Elasticity, stress rupture and ultimate stress limit of control and decellularized scaffold were confirmed by mechanical testing and summarized in Table 1. The mechanical testing analysis showed that the ultimate stress, strain and elasticity for control and decellularized ear scaffolds was not significantly different. Strain of decellularized scaffold was 0.32±0.015 mm, 0.55±0.018 mm, 0.27±0.011 mm, 0.35±0.014 mm, 0.25±0.010 mm and 0.34±0.011 mm in control, method 1, method 2, method 3, method 4 and method 5 respectively. There was no significant difference (p<0.0001) in control and control and M3, M4. Ultimate stress (N/mm2) of decellularized scaffold was 60.17±1.20, 20.33± 1.80, 98.00± 2.00, 18.21± 1.00, 13.01± 1.00 and 20± 2.50 N/mm2 in control, method 1, method 2, method 3, method 4 and method 5 respectively. There was significant difference (p<0.0001) between control and M2, M4, M5 Ultimate stress and tensile strength in samples treated by M2 and M5 were good and similar. Significant elasticity was seen in samples treated by M2 and M5. Overall M2 and M3 showed better retention of mechanical properties compared to M1, M4 and M5. Deformation (mm) study of scaffold showed 6.5± 0.5, 5.5± 1, 5.5± 0.5 , 7.0± 1.5, 5.5± 0.5, and 8.5± 0.8 in control, method 1, method 2, method 3, method 4 and method 5 respectively (Graph 3). Elasticity (N/mm2) of decellularized scaffold was 21.90±17.12, 71.21± 18.000, 45.94± 20, 55.57± 11, 95.56± 18, and 72.17± 25 in control and all methods 1, 2, 3, 4, 5 respectively (p<0.05). Method 2 and method 5 showed no significant difference in elasticity. Overall method 2 and 3 maintained mechanical properties as compared to method 1, 4, 5. Decellularized scaffold prepared by method 2, 3 and 5 showed the significantly biophysical stable, elastic and load-bearing object. Moreover, scaffolds prepared by these methods have more stability, elasticity and flexibility to attain human shaped ear pinna. Hence scaffolds prepared by 2, 3 and 5 were selected for in vivo experiment.
Scanning electron microscopy (SEM) analysis:
Scanning electron microscopy of decellularized ear pinna scaffold (Figure 4B) by M2 showed 3D network of ECM in overall morphology (Magnification, 1000X), M3 sample (Magnification, 1700X) and M5 sample (Magnification, 1000X). SEM results proved that M2 and M3 showed an excellent result to maintain 3D network of ECM and overall morphological porosity as compared to M5.
Biocompatibility evaluation
Selection: Scaffolds achieved by M2, M3 and M5 showed well preserved ECM (collagen and GAG), porous microstructure, superior mechanical stability and elasticity compared to the decellularized ear pinna generated via M1 and M4. These could harbor potential to attain shape desired for the human ear pinna. Hence ear pinna generated by these methods was used for biocompatibility studies.
Method: The scaffolds (n=3) developed from M1 and M4 were transplanted into the peritoneal cavity of mice (Figure 5A) for four week.
HE staining used to monitor the recellularization process showed that the ear pinna supported the chondrocyte growth as manifest by visible nuclei (Figure 7A). Complete recellularization was achieved at 4 weeks after grafting, when the scaffolds showed well organized 3D ECM and collagen in MT staining (Figure 7B), presence of GAGs on AB pH2.5 staining (Figure 7C) and recruitment of live chondrocyte cell (Figure 7D) on toludine blue staining. IHC staining revealed expression of VAP representing vasculogenesis (Figure 7B) and CD 90+(Figure 7C) representing stromal markers and recruitment of chondrocyte specific progenitor cells.
Mechanical testing after biocompatibility study:
Grafted scaffolds showed good mechanical strain, stress, deformation and elasticity as compared to control. The values of mechanical properties of recellularized pinna was summarized in (Table 3 and Figure 6). The Graph 1 showed strain (mm) of recellurized pinna as 0.20±0.10 mm, 0.32±0.02 mm and 0.17±0.005 for method 2, method 3 and method 5 respectively. Ultimate stress (N/mm2) of control and In vivo decellularized ear pinna were similar. M5 decellularization showed highest ultimate stress bearing capacity which was decreased significantly after recellularization (Graph 2). Deformation (mm) did not show much alteration during decellularization and recellularization process in M2 and M5 (Graph 3). In vivo decellularized ear pinna generated by M3 showed highest deformation. The Graph 4 showed elasticity (N/mm2) of biocompatible scaffold was 25.01±23.40 and 20.90±12.35 as compared to control (21.90±17.12 N/mm2). M5 decellularization showed significant increase in elasticity, which was decreased to (20.9\(\pm 5.35)\ \text{after\ in\ vivo\ recellularization}\).
Scanning electron microscopy (SEM) analysis:
Scanning electron microscopy of scaffold showed the 3D network of ECM cell structure in transplanted M2 (Magnification X 3500), M3 (Magnification X 3000) and M5 (Magnification X 1400) scaffold (Figure 5B). SEM analysis revealed that transplanted scaffolds maintained ECM collagen, elastic fibers and significantly showed bunch of chondrocyte cell recruitment. It showed very excellent results of regeneration of and recellularization with chondrocytic cells.
Discussion:
This study assessed the tissue engineered scaffold of ear pinna cartilage by decellularization using different chemical treatments methods. The study also extended to biocompatibility and recellularization of the scaffold on implantation into an animal model. Chemical and enzymatic detergents solubilize cell membranes and remove DNA thus proving to be effective agents to remove immunogenic tissue material.
These different decellularization methods proved efficacy to generate ideal ear pinna scaffold. Current decellularization protocols are designed to overcome the previous limitations (Utomo, Pleumeekers, & Nimeskern, 2015)(Cervantes et al., 2013). Rahman S et al presented a protocol to decellularize cartilage using trypsin followed by freeze and thaw cycles (Rahman et al., 2018). The present study found that antioxidant treatment followed by freeze and thaw cycles can efficiently remove the DNA content and also preserve the ECM and mechanical properties. This protocol does require more cycles (80 cycles) for decellularization compared to the enzymatic method. However, it has been found to generate stable human ear pinna shaped cartilage with better mechanical properties. It also provides high specificity for removal of antigenic material. On the other hand, enzymatic decellularization protocol was quick and was able to achieve complete decellularization at the 40th cycle. However, this rapid decellularization was at the cost of not maintaining the mechanical properties. The resultant scaffold was more elastic, thin and more time was needed to generate the desired human ear pinna.
The present study shows that scaffold prepared by Methods 2, 3 and 5 were ideal scaffolds with excellent porosity and mechanical properties that retain excellent anatomical ear pinna shape molded in different size and shapes- 55, 65, and 75mm.
The ECM was characterized by histochemistry in this study. HE staining revealed a well-maintained structure of the scaffold with preserved ECM. The residual DNA content after decellularization was greatly reduced compared to that found in native tissue. M5 showed faster decellularization compared to other methods. This is significant as DNA is a sensitive indicator of cell debris due to its high stability and as a marker strongly correlates with adverse host immunogenicity. MT and AB staining demonstrated no obvious disruption to the overall histo-architecture following treatment and were found to maintain collagen and GAGs structure receptively. The results of decellularization methods were promising and appreciable as the amount of collagen remain unchanged. This outcome is desirable as collagen molecules play a vital role in maintaining ECM and also determining tissue functions. SEM analysis reported a preserved ECM of ear pinna cartilage without evidence of any damaged area. Biocompatible testing conducted on animal models (albino mice) found satisfactory vasculogenesis and angiogenesis in graft (Figure. 5A). Biocompatibility and recellularization detected through MT and AB staining pH 2.5, revealed maintenance of ECM architecture in vivo. Scaffolds prepared by Methods 2, 3 and 5 subjected to surface topography confirmed good elasticity. Biocompatibility Biocompatible testing of grafted human ear pinna found fully vascularized. IHC staining with VAP revealed that the transplanted ear pinna express vasculogenesis and angiogenesis marker VAP, while CD90 + marker expression revealed that chondrogenic differentiation as shown in Figure.7 B and C. Expression of CD90+ is important with regard to enhanced collagen type II and chondrogenic differentiation. IHC staining revealed chondrogenic differentiation (Figure. 7B and C) with the graft showing cells in most places amidst abundant collagen matrix and proteoglycan content after four weeks. This was confirmed by Toluidine blue staining in (Figure 7A). Thus, this study provided a novel approach to generate ear pinna xenograft for clinical applications of auricle cartilage to overcome the present limitations. The decellularized scaffold is biodegradable, biocompatible, preserves ECM and it can reduce post-transplant management problem.