Decellularized Caprine Conchal Cartilage towards Repair and

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Decellularized Caprine Conchal Cartilage towards Repair and Regeneration of Damaged Cartilage Piyali Das, Yogendra Pratap Pratap Singh, Siddhartha Narayan Joardar, Bikash Kanti Biswas, Rupnarayan Bhattacharya, Samit Kumar Nandi, and Biman B. Mandal ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00078 • Publication Date (Web): 24 Apr 2019 Downloaded from http://pubs.acs.org on April 24, 2019

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ACS Applied Bio Materials

Decellularized Caprine Conchal Cartilage towards Repair and Regeneration of Damaged Cartilage Piyali Das1†, Yogendra Pratap Singh2†, Siddhartha Narayan Joardar4, Bikash Kanti Biswas5, Rupnarayan Bhattacharya6, Samit Kumar Nandi1**, Biman B. Mandal2,3* Department of Veterinary Surgery and Radiology, West Bengal University of Animal and Fishery Sciences, Kolkata – 700037, West Bengal, India 2 Biomaterial and Tissue Engineering Laboratory, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati – 781039, Assam, India. 3 Centre for Nanotechnology, Indian Institute of Technology Guwahati, Guwahati – 781039, Assam, India. 4 Department of Microbiology, West Bengal University of Animal and Fishery Sciences, Kolkata – 700037, West Bengal, India 5 Directorate of Research, Extension and Farms, West Bengal University of Animal and Fishery Sciences, Kolkata – 700037, West Bengal, India 6 Department of Plastic Surgery, R. G. Kar Medical College & Hospital, Kolkata-700004, West Bengal, India 1

Corresponding author: *[email protected], [email protected] Co-corresponding author: **[email protected] †Authors contributed equally

Abstract Repair and regeneration of nasal and auricular cartilage thrust significant challenges in reconstructive surgery. The burgeoning clinical requirement, is yet to endorse a satisfactory cartilage replacement matrix. In this regard, we have bioengineered cross-linked decellularized caprine conchal cartilage (DC) as biocompatible, durable and non-toxic matrices. The DC matrices exhibited reduced DNA and sulphated glycosaminoglycan (sGAG) with minimal effect on the collagen content. Further, histology and scanning electron micrographs revealed significant loss of cellular bodies; besides presence of compact matrix consisting of intricate collagen fibers, when compared to unprocessed matrices. In vitro biological assessment of the matrices exhibited increased chondrocyte proliferation and viability with significantly higher DNA, sGAG and total collagen content. The matrices showed a 3-fold increase in the expression of cartilage specific genes, namely, aggrecan, collagen-II and sox-9 and exhibited minimal in vitro immunogenicity. Further, in vivo assessment was performed by xenografting these caprine matrices in a rabbit model. The retrieved matrices showed well organized structural and cellular orientation with

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extracellular matrix formation after 3 months of implantation. No significant infiltration of plasma cells, macrophage, lymphocytes and immature fibroblasts were recorded. Therefore, these affordable, resourceful, xenocompatible matrices offer a potential alternate in the repair and regeneration of nasal and auricular cartilages. Keywords: Reconstructive surgery; decellularization; extracellular matrix (ECM); xenogenic cartilage matrix, caprine

1. Introduction Cartilages are avascular, aneural and characterized by chondrocytes embedded in the predominantly collagen and proteoglycan matrix. In spite of being flexible and tough, the cartilage is highly prone to detrimental acute and/or chronic injuries. The injuries may originate from the alleviation of congenital abnormalities through auricular corrective surgery or augmentative nasal surgery.1 The intrinsic low regenerative property and lack of blood supply, retards the pace of cartilage growth and regeneration. Again, the small lacunae which enclose the chondrocytes curb their rapid migration to the damaged area, and impede satisfactory healing response after injury or disease.2 Therefore, increasing the efficiency of cartilage restoration and regeneration is claiming worldwide scientific attention, especially for applications in medical, orthopedics and otorhinolaryngology. The focus is now directed towards the development of tissue engineering and/or biomaterials based constructs for further large-scale practices. The gold standard technique in cartilage reconstruction is the application of autografts. However, there are severe disadvantages in the implementation of autologous cartilage. When derived from rib, nasal septum or auricle, the implantation sites are handicapped by donor–site morbidity, multistage surgical procedures, postoperative dysfunctions, or pain and meagre cosmetic benefits.3 Alternatively, allograft transplantation leads to graft rejection and disease transmission from the donor.4 The limitations in the availability of the human tissues/ cells have led researchers to invest in other cell source. Although, the applications of mesenchymal stem cells (MSCs) have gained much attention but the chances of a patient’s safety and functional recovery still remain questionable.5-6 Therefore, the implementation of tissue engineering, which combines the concepts of medicine, cell biology and engineering, is an emergent and comprehensive technology. It offers alternatives that enable structural, morphological, and mechanical functionality for regeneration of


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native tissues. In order to provide a suitable niche or base for 3D environment for the cells to proliferate and adhere, scaffold materials ranging from natural collagen and alginate, to the synthetic polyhydroxyacids and polyethylene glycol (PEG) hydrogels have been abundantly explored in cartilage tissue engineering.7-8 Although, various scaffolds are developed and broadly used to reconstruct cartilage, still the fabrication of a suitable scaffold with a highly organized microenvironment mimicking the native cartilage remains a major challenge. Acellular scaffolds derived from extracellular matrix (ECM) of native tissues are able to provide such a microenvironment and are gaining attention, especially as xenografts. Due to low regenerative capacity, transplantation (particularly xenotransplantation) is considered the best option for the repair of cartilage defects. Xenogenic donor cells or tissues could be initially transformed through genetic engineering techniques and then modified and grown under controlled conditions; followed by harvesting in quantities sufficient to satiate the growing medical demand.9 In the attempt to offer various clinicians a product with minimum recourse to immunosuppressant, specialized techniques must be adopted.1 Decellularization or chemical cross linking of the xenogenic cartilage tissue can ensure the complete removal of the antigenic cellular materials to avert any sort of immune reactions post-implantation. Concurrently, non-immunogenic ECM must remain unaltered to preserve the biomechanical features of the matrix eliciting efficient invasion of the tissue cells.10 Decellularized xenogenic organs are being successfully utilized in clinical applications; in areas as diverse as artificial acellular dermal matrix, knee meniscus constructs, temporomandibular joint disc, tendon Anterior Cruciate Ligament (ACL), heart valves, bladder etc.11-12 Extensive research on acellular xenogenic constructs from various animal sources is being carried out worldwide. For instance, cartilages from bovine, porcine and human sources have been rendered acellular and competed successful biomaterials for cartilage tissue engineering. However, there is a distinct dearth of reports on the processing and application of caprine cartilages. The caprine conchal cartilage can provide an easily quantifiable and relatively economical cell source that can support weight, provide solid anchorage and offer low risk of disease transmission. As of now, decellularization of tracheal, articular, nasal, meniscal cartilages and intervertebral discs have been successfully performed. On the contrary, very few reports have been found of research with ear/auricular cartilage which is an elastic cartilage type.13 Elastic cartilages are more challenging to decellularize due to their thick elastic fibers which are absent in hyaline and fibrous cartilage.



Different tissues and organs have been embodied with different and exclusive factors such as tissue’s cellularity, lipid content, density and thickness, therefore it is difficult to concentrate on any single decellularizing agent whatsoever. However, it is quite understandable that every agent causing cellular disruption will also alter the ECM composition and cause ultrastructure destruction. Therefore, in order to obtain a complete acellular xenogenic cartilage matrix with minimum alterations, a combination treatment of alcohol, gamma irradiation and glutaraldehyde has been implicated in our study. Alcohols have been largely practiced as an active agent for decellularization because of its activity in removing lipid layers causing tissue dehydration.14-16 In fact, alcohols (e.g., methanol, ethanol and isopropanol) remove lipids and adipose tissues more effectively than lipases.17 On the other hand, exposure to the gamma radiation not only helps in sterilization but also affects the entire tissue causing cell damage.18-19 It acts evenly on entire tissue and/or organ regardless of its density and thickness which helps to acquire the desired result. Next, glutaraldehyde has been found to be a potential chemical agent which not only helped in crosslinking the collagenous tissue20 but also simultaneously increased the mechanical strength of the tissue as well as decreased enzymatic degradation. Masking of antigenicity is an essentially important factor to reduce the immunogenicity prior to xeno-transplantation. In a previous study, cross linking by glutaraldehyde has been reported to mask the antigenic properties of tissues successfully.21 It has been extensively used in many previous experiments for chemical cross linking which includes cross-linking of bioprosthetic heart valves21 and bovine cartilaginous matrix from nasal, articular site.20 Since, there are some risks over using excess dose of this agent, for instance, leaching out of this product to the surrounding area may cause toxic effects or tissue necrosis,21 hence, the standardization of the exact minimal dose is very crucial. The current study focuses on the processing and development of a suitable matrix from caprine conchal cartilage as potential xenogenic implant for nasal and auricular cartilage reconstructive surgery. We used a combination of alcohol, gamma irradiation and glutaraldehyde treatment for the decellularization and cross-linking of the conchal cartilage. The processed matrices were assessed for decellularization, characterized in vitro for biological studies using rabbit primary auricular chondrocytes and tested for in vitro immunogenicity using macrophages. Further, we assessed the in vivo xenocompatibility of the caprine derived matrices in a rabbit model. 2. Materials and Methods


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2.1 Isolation, decellularization and processing of conchal cartilage Fresh conchal cartilages were harvested from a newly slaughtered young goat from a local abattoir. The obtained cartilages were cleaned, dissected free of skin, cut into small pieces and osmotically pretreated with sterile deionized H2O for 24 hours at room temperature (RT) with 3 – 4 changes. Next, the cartilages were defatted and dehydrated by placing in absolute (95-100 %) ethanol for next 72 hours at 4 °C, with 2-3 changes. The alcohol treatment was followed by 25 kGy gamma irradiation with Co-60 isotope. Further, the matrices were washed and cross–linked with certain percentage of glutaraldehyde (0.25%, 0.5%, 1%, 1.5% to 2%) (GA, Sigma-Aldrich, USA) solution following earlier established protocol20 with some modifications in a sterile closed container for 48 hours at 4 °C. They were then washed entirely with PBS (pH 7.4) by constant shaking and agitation with 4 – 5 changes to get rid of any chemicals. One part of cartilage was kept unaltered to be used as a control group. Thus, samples were categorized as ‘treated’ and ‘untreated’ matrices depending on its treatment with or without glutaraldehyde (including alcohol treatment and exposure to gamma irradiation). The matrices were then stored with 1% antibiotics (penicillin and streptomycin) for further use. 2.2 Biochemical and histological assessment of the DC matrix The extraction of genomic DNA was performed using the traditional phenol- chloroform DNA extraction method.22 Briefly, DC matrices were minced, homogenized and submerged in sterile PBS. To each ~100 mg of sample, 920 µL of DNA extraction buffer, 50 µL of 10% SDS and 30 µL of proteinase K solution were added and incubated for a minimum of 6 hours at 37 °C. Next, equal volume of equilibrated phenol was mixed and centrifuged (1700 g for 10 minutes at RT). The upper clear layer was moved to another tube and mixed with equal volume of chloroform (Chloroform: isoamyl alcohol (24:1)). Next, 0.1 volume of 3 M sodium acetate was added followed by 2 volume of ice-cold 100% ethanol and the precipitated DNA was dissolved in TE buffer. DNA content was quantitated spectrophotometrically (Perkin Elmer, Lambda 25) by recording OD at 260/280 nm. Collagen content of the DC matrices was performed with hydroxyproline Assay Kit (SigmaAldrich, USA) according to the manufacturer’s instruction.23 Briefly, acid hydrolysis of the tissue homogenates was performed by incubating tissue homogenate with equal volume of concentrated hydrochloric acid (HCl) in a tightly capped tube and incubated for 3 hours at 120 °C. Supernatant



from the digestion was dried (60 °C oven) in a 96-well plate followed by addition of 100 µl of the Chloramine T/Oxidation Buffer Mixture (5 min incubation at RT). To this was added, equal volume of 4-(dimethylamino)benzaldehyde (DMAB) working reagent (90 minutes incubation at 60 °C) and OD at 560 nm was recorded. The sGAGs content was determined with Dimethylmethylene Blue (DMMB) assay as described previously.24 In brief, DMMB reagent solution (pH 3) was prepared according to standard protocol along with standard solution of chondroitin-4-sulfate (Sigma-Aldrich, USA). 20 µl of standard solution and digested DC matrices were added into the 96 well microplate where 200 µl of DMMB solution was pipetted to each sample and OD recorded at 525 nm. For histological examination, matrices were fixed in 10% neutral buffered formalin (NBF), paraffin embedded in wax and sectioned using microtome (Leica, RM2235, Germany) to a thickness of 5 µm. The staining of the obtained sections was done by haematoxylin and eosin (H&E, Sigma-Aldrich, USA) intended for monitoring cellular orientations and morphological analysis. 2.3 Field Emission Scanning Electron Microscopy (FESEM) The architecture and surface morphology of decellularized matrices were studied using FESEM (Zeiss, Germany). Fixation of the native and decellularized (1% GA treated) matrices was done using 2.5% (v/v) glutaraldehyde (24 hours at RT) followed by dehydration using series of ethanol and finally dried. The cross-sections of the dried matrices were sputter coated with gold and imaged at an operating voltage of 2kV. The obtained images were then processed by NIH ImageJ software. 2.4 In vitro recellularization and functional assessment of the DC matrix 2.4.1 Isolation and culture of rabbit auricular chondrocytes Auricular chondrocytes were isolated from ear of New Zealand rabbits according to a modified protocol by Singh et al.25 Briefly, the obtained ear was cleansed, freed of its skin, cut into fragments, washed with sterile PBS and treated with antibiotic solution (Invitrogen, USA). The fragments were digested by 0.2% protease XIV (Sigma-Aldrich USA), 0.05% collagenase (type IA, Sigma-Aldrich, USA) for overnight (37 °C and 5% CO2). The enzyme digest was centrifuged


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(2000 rpm for 5 minutes) to isolate auricular chondrocytes and viability was chcked using trypan blue dye (Sigma-Aldrich, USA). Chondrocytes were cultured and maintained in DMEM and cells of passage three (P3) were used for all experiments. 2.4.2 Cell proliferation and viability Proliferation of chondrocytes was measured by means of alamar blue assay in accordance to previously published reports26-27 and manufacturer’s protocol. Briefly, samples were incubated in alamar blue dye (Invitrogen, USA) at 37 ºC. At predefined time points, absorbance was measured at 570 nm and 600 nm in a multiplate reader (Tecan Infinite Pro 200). Chondrocytes cultured on TCP were used as control. Further, to assess the viability of chondrocytes they were washed and stained with calcein AM (Sigma-Aldrich, USA) and visualized using fluorescence microscope. 2.4.3 Biochemical assessment of in vitro recellularized matrices For biochemical analysis, the wet weight (WW) for all samples was determined. The matrices for DNA and GAG estimation were digested using papain solution.28 The content of total DNA was estimated by means of PicoGreen DNA assay kit (Invitrogen, USA) in accordance to manufacturer’s protocol. Briefly, the digested matrices were diluted using 1X TE buffer and to this Quant-iT PicoGreen reagent (1:200 dilution) was added. Fluorescence was measured at excitation/emission: 480/528 nm. DMMB (1,9-dimethylmethylene blue, Sigma-Aldrich, USA) assay was used for estimation of sulfated GAG (sGAG).24 For the assay, samples were added to DMMB reagent (1:4 ratio) and OD was recorded at 525 nm. For estimation of collagen, the matrices were digested with pepsin cocktail (4 °C for 48 hours) according to a modified Hride Tullberg-Reinert method.29 In brief, the obtained solution of the digested matrices was dried in cell culture plate followed by addition of Sirius red dye (incubated at 37 °C for 1 hour). The wells were washed with 0.01 N HCL followed by resolving sample dye complex with 0.1 N NaOH and measuring absorbance at 550 nm. 2.4.4 Gene expression analysis RNA was isolated using TRIzol reagent (Sigma-Aldrich, USA) and cDNA was prepared using cDNA reverse transcription kit (Applied Biosystems, USA). Real time PCR was executed using SYBR Green dye (Invitrogen, USA) in a 7500 real time PCR system (Applied Biosystems, USA). A set reaction volume of composed of SYBR Green mix, forward primer, reverse primer and



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cDNA was prepared and run with the settings of holding stage (2 minutes at 50 °C, 10 minutes at 95 °C) and cycling stage (40 cycles of 15 s at 95 °C, and 45 s at 60 °C). Analysis was performed according to comparative Ct method (2-∆∆Ct) with normalization to glyceraldehyde-3-phosphatedehydrogenase (GAPDH). Table 1 lists the sequence of primers used. Table 1. Sequence of primers for real time PCR. Gene

Sequence

Accession No.

Aggrecan

F 5ʹ-CCCAACCAGCCTGACAACTT-3ʹ

NM_001164652.1

R 5ʹ-CCTTCTCGTGCCAGATCATCA-3ʹ Sox-9

F 5ʹ-TTCCGCGACGTGGACAT-3ʹ

NM_213843.1

R 5ʹ-GGCGGCAGGTACTGGTCAAACTC-3ʹ Collagen II

F 5ʹ-CAGGTGAAGGTGGGAAACCA-3ʹ

AF201724.1

R 5ʹ-ACCCACGAGGCCAGGA-3ʹ GAPDH

F 5ʹ-TCGGAGTGAACGGATTTGG-3ʹ

NM_001206359.1

R 5ʹ-CCAGAGTTAAAAGCAGCCCT-3ʹ

2.4.5 Macrophage stimulation and quantification of TNF-α release Macrophages were stimulated with the DC matrix and release of tumor necrosis factor alpha (TNFα) was recorded according to previously published reports.26,

30

RAW 264.7 cells (murine

macrophage cell line) were plated at 105 cells/cm2 in 12-well plate and incubated overnight. The matrices were cultured with these cells for 6 hours and the media was assayed for secreted TNFα. The amount of TNF-α release was measured by ELISA as per the manufacturer’s protocol (Invitrogen, USA). Cells cultured on TCP without matrices were used as negative control, while lipopolysaccharide (LPS from E. coli, Sigma-Aldrich, USA) (500 ng/ml) was taken as a positive control. 2.5 In vivo xenotransplantation of the DC matrix in rabbit model


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The in vivo animal experimentation were carried out in randomly selected 17 New Zealand white rabbits, weighing 1.5–2 kg of either gender, for duration of 3 months. The experiment was conducted in accordance with ‘‘Principles of laboratory animal care” from the Institutional Animal Ethical Committee (IAEC), West Bengal University of Animal and Fishery Sciences (WBUAFS), West Bengal, India (Permit No. 319/CPCSEA dated 14.02.2011). Prior to operation, all the animals were anaesthetized using an intramuscular injection of xylazine hydrochloride (5 mg/ kg) (Xylaxin®, Indian Immunologicals, India) and ketamine hydrochloride (25 mg/kg) (Ketalar®, Parke-Davis, India). For the implantation of the samples, 5 mm cuts were formed in the subcutaneous pockets of the dorsal surface in mid thoraco-lumbar region which was shaved and wiped with 70 % ethanol. The study included 2 groups: group 1- 1% GA ‘treated’ cartilages (size 2.5 cm X 1.5 cm) (10 rabbits) and group 2- native or ‘untreated’ cartilages of same size (7 rabbits). All the animals were maintained in separate cages under proper laboratory conditions. Before transplantation, it was ascertained that there was no microbial contamination in the materials to avoid unwanted infection post-surgery. The samples were retrieved after specified timepoints for histological examination and biochemical studies. 2.6 Histological and biochemical assessment of xenotransplanted matrix At previously decided time points (i.e., 45 and 90 days), implanted matrices along with surrounding tissues were collected. For histology, the tissue biopsies were fixed, dehydrated, embedded and sectioned. The sections were then stained with H&E for assessment of host tissue reactions with the xenografted cartilage tissue. Some specific staining methods were also used for the study of ECM. For example, Masson’s trichrome staining method was used for the staining of collagen ﬁbers.31 Similarly, reticular fibers and elastic fibers were separately stained with Gridley’s modified silver impregnation method and Weigert’s Resorcin Fuschin method respectively following manufacturer’s protocol.32-33 Bright field microscope (Leica DM 2000, Germany) was used to capture images. Quantitative analysis of ECM formation in the retrieved biopsies was studied by estimating the content of DNA, sGAG, collagen, elastin, fibronectin, transforming growth factor (TGF) β, β1 and β2 using specific kit protocols. The content of DNA, sGAG and collagen was performed according to protocols mentioned previously (Section 2.2). Elastin, fibronectin, TGF β, β1 and β2 were estimated by means of specific ELISA kits (MyBioSource and Bioassay Technologies



laboratories) according to manufacturers’ protocol. Briefly, the retrieved matrices were cleansed, weighed, homogenized and centrifuged (1500 g for 15 minutes). Carefully, the supernatant was taken and further assayed according to the principle of sandwich ELISA. The biochemical estimations were performed by measuring the OD values in an ELISA reader (ECIL, India) using already supplied pre-coated ELISA plates. 2.7 Statistical analysis All statistical analysis was carried out with at least 3 replications of each sample categories unless otherwise specified. Data were analyzed using statistical software OriginPro 8 (Originlab Corporation, USA) and represented as mean ± standard deviation at both significant (*p ≤ 0.05) and highly significant (**p ≤ 0.01) level. The statistical significance of each experiment within the data set and among the groups was calculated using one-way analysis of variance (ANOVA) by Tukey’s test. 3. Results 3.1 Isolation, decellularization and processing of conchal cartilage Caprine conchal cartilage was isolated according to protocol mentioned in section 2.1. Decellularization of the cartilage was performed using ethanol, gamma irradiation and a range of glutaraldehyde concentration according to Holman and Beisang, 1984,20 which later on was standardized to 1% glutaraldehyde depending on the results of histology and biochemical estimations. Figure 1 shows the schematics of the study illustrating the harvesting, processing and decellularization of the matrices.


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Figure 1. Schematics of harvesting (A-B), processing (C) and decellularization (D) of conchal cartilage. 3.2 Biochemical and histological assessment of DC matrix Quantification of decellularized and native or unprocessed cartilage showed a striking difference in DNA content. Analysis of DNA content (Table 2, Figure 2A-C) within the DC matrix revealed the lower genomic DNA (6.03 X 10-3 ± 1.91 µg/mg WW and 21.67 X 10-3 ± 4.04 µg/mg WW in 1% and 0.25% GA treated cartilage samples respectively) in comparison to native or untreated cartilages (47.92 X 10-3 ± 2.77 µg/mg WW) (p ≤ 0.05). Similarly, GAG content of the native and decellularized cartilage was performed and it was found that GAG concentration was significantly reduced in 1% GA processed decellularized cartilage (6.56 ± 1.13 µg/mg WW) than 0.25% GA ‘treated’ (11.47 ± 1.08 µg/mg WW) and its native or untreated counterpart (17.74 ± 2.40 µg/mg WW) respectively (p ≤ 0.05). Hydroxyproline is one of the specific components of collagen fibers and its estimation indicated that the decellularization process had nominal effect on the collagen content of the cartilage. The collagen content was found to be 48.23 ± 2.56 µg/mg, 50.2 ± 1.73 µg/mg and 50.60 ± 1.4 µg/mg in 0.25%, 1% GA treated and untreated cartilage extracts respectively. Therefore, no significant difference was found in collagen content between the groups. Microscopic observation following H&E staining of decellularized cartilages revealed that the strategy for making them acellular by increasing concentration of glutaraldehyde was effective (Figure 2D-F). Upon gross inspection, it was clearly noticed that removal of cells and nuclear bodies were remarkably reduced in 1% and 0.25% GA treated cartilage. In case of 0.25% treated, degenerative stage of chondrocytes was observed while in 1% treatment, significant loss of cellular bodies was noticed. On the other hand, histological analysis of conchal cartilage sections before treatment with glutaraldehyde depicted a fibro-cartilaginous structure characterized by formation of well-organized chondroblasts and chondrocytes.



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Figure 2. Biochemical properties and H&E staining of glutaraldehyde treated (0.25 % and 1 %) and native cartilage. (A) DNA content, (B) sGAG content, (C) collagen content, (D) presence of cellular elements in native/untreated cartilage, (E) after 0.25% glutaraldehyde treatment and (F) after 1% glutaraldehyde treatment. Data are shown as mean ± SD, n=3 (*p ≤ 0.05). Scale bar represents 100 μm. Table 2. Biochemical assessment of the matrices quantifying ECM components. Sample

Component content /[μg * (mg wet weight-1)] DNA

sGAG

Collagen

0.25% GA treated cartilage

21.67 X 10-3 ± 4.04* 11.47 ± 1.08*

48.23 ± 2.56

1% GA treated cartilage

6.03 X 10-3 ± 1.91*

50.20 ± 1.73

Native/ untreated cartilage

47.92 X 10-3 ± 2.77* 17.74 ± 2.40*

6.56 ± 1.13*

50.60 ± 1.40

(Data are shown as mean ± SD, n=3, *p ≤ 0.05) 3.3 Field Emission Scanning Electron Microscopy (FESEM) The FESEM analysis revealed the microstructure and surface morphology of the decellularized cartilage. The decellularized cartilage displayed a loosely dense matrix comprising of fine, intact collagen fibers alike to that of native cartilage (Figure 3). The native cartilage exhibited thick


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intertwined elastic fibers that were embedded in a homogeneous grid of collagen. Further, empty lacunar structures in the decellularized cartilage were observed with maintained characteristic collagen matrix.

Figure 3. Field emission scanning electron microscopy (FESEM) of cartilage (A, C) before decellularization; (B, D) after decellularization. Scale bar = 2 µm. 3.4 In vitro recellularization and functional assessment of the DC matrix 3.4.1 Cell proliferation and viability For assessment of in vitro cellular proliferation and viability of the DC matrix, it was seeded with primary rabbit auricular chondrocytes. The matrix supported chondrocyte proliferation as indicated by alamar blue reduction assay after 14 days of culture. As compared to the control TCP, DC matrices showed significantly higher chondrocyte proliferation (p ≤ 0.01) on day 14 (Figure 4A). Further, calcein AM fluorescent imaging showed the viability of these chondrocytes on the DC matrix. The proliferating chondrocytes (stained green) formed a dense tissue over the DC matrix as compared to TCP (Figure 4B). On day 1, the spherical morphology of most of the cells indicate that they have recently adhered to the matrix but not completely spread. However, after14 days, we see typical chondrocyte morphology of the cells with prominent increase in cell number.



Figure 4. (A) Alamar blue proliferation assay and (B) calcein AM staining of primary rabbit chondrocytes cultured on tissue culture plate (TCP) on (I) day 1, (II) day 7, and (III) day 14; and 1% glutaraldehyde (GA) treated cartilage on (IV) day 1, (V) day 7, and (VI) day 14. Scale bar = 200 µm (**p ≤ 0.01). 3.4.2 Biochemical assessment To characterize the in vitro cellular compatibility of the DC matrix, the DNA, GAG and total collagen contents were measured biochemically. The quantification of DNA showed the proliferation of cells on the DC matrix. The results showed significantly higher DNA content (6.31±1.65 µg/mg WW, p ≤ 0.05) on the DC matrix as compared to TCP (3.13 ± 0.60 µg/mg WW) after 14 days of culture (Figure 5A). Similarly, significantly higher GAG content (27.91 ± 2.02 µg/mg WW, p ≤ 0.01) was observed in DC matrixes compared to the TCP (16.99 ± 1.07 µg/mg WW) on day 14 (Figure 5B). The estimated GAG content included both the GAG secreted on the


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matrix as well as in media. Further, the quantification of collagen content showed a similar trend with significantly higher (69.88 ± 9.78 µg/mg WW, p ≤ 0.01) values for DC matrix on day 14 as compared to the TCP (42.83 ± 7.81 µg/mg WW) (Figure 5C). 3.4.3 Gene expression analysis The chondrogenic potential of primary rabbit auricular chondrocytes cultured on the DC matrix was confirmed by the expression of the cartilage-specific genes namely aggrecan, collagen-II and sox-9. The results showed that the expression of these genes was higher on the DC matrix after 14 days of culture as compared to the control TCP (Figure 5D-F). The expression of aggrecan showed a 8 fold increase as compared to a 3 fold increase in case of TCP (p ≤ 0.01) upon normalization with day 1 values. Similarly, the expression of collagen-II and sox-9 showed 9 fold and 13 fold increase on DC matrix as compared to 4 fold and 5 fold increase on TCP (p ≤ 0.01), respectively. This shows that DC matrices supported the chondrogenic potential of chondrocytes in vitro.

Figure 5. Biochemical properties of decellularized cartilage (A-C). (A) DNA content, (B) GAG content and (C) collagen content in decellularized cartilage constructs after 14 days, normalized by wet weight (WW). Real time PCR showing expression of cartilage related genes (D) aggrecan, (E) collagen-II and (F) sox-9. Data are shown as mean ± SD, n=3. (*p ≤ 0.05, **p ≤ 0.01). 3.4.4 Macrophage Stimulation and TNF-α Release



Macrophages are highly responsive to TNFα and are also major producers of TNFα, which plays a critical role in inflammation and regulation of the immune response. The ability of the DC matrix to stimulate the secretion of TNF-α was studied with murine macrophage cells (RAW 264.7). Post 6 hours of culture, the DC matrix showed significantly lower (∼4-fold) level of TNF-α compared to the LPS (positive control) (p ≤ 0.01) (Figure 6). However, the level of TNF-α for DC matrix was significantly higher than the native cartilage (p ≤ 0.05) and negative control TCP (p ≤ 0.01).

Figure 6. Quantification of released TNF-α stimulated by DC matrices when cultured with RAW 264.7. Tissue culture plate (TCP) was used as negative control and lipopolysaccharide (LPS) as positive control. The data are represented as mean ± SD (n = 2). (*p ≤ 0.05 and **p ≤ 0.01). 3.5 In vivo xenotransplantation of the DC matrix in rabbit model The macroscopic evaluation of implanted cartilage samples post 3 months of surgery was achieved by gross observation at the site of surgery, biocompatibility of the materials as well as the histological and biochemical studies. The representative pictures of surgery during the time of in vivo xenoimplantation of cartilages have been given in Figure 7A, B. The gross observation at the site of implantation depicted an interesting view in terms of host- implant biocompatibility and graft rejection. Macroscopic view revealed that 1% GA treated cartilage (Figure 7C) was found to be well organized with no sign of inflammation and tissue necrosis surrounding the area. On the other hand, native or untreated cartilage was found to be quite deformed with surrounding inflammation and necrosis of host tissues (Figure 7D).


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Figure 7. Gross pictures (A, B) during surgery showing in vivo xenoimplantation of cartilages. Post 3 months, macroscopic view revealed 1% GA treated cartilage (C) to be well organized with no sign of inflammation and tissue necrosis, whereas, native or untreated cartilage (D) showed necrosis of host tissue. Scale bar is 1 cm. 3.6.1 Histology assessment of in vivo samples In order to understand the biocompatibility of the decellularized cartilage with the host and to monitor the integration and orientation of the graft, in vivo implantation of the decellularized (1% GA treated) cartilage was performed in the dorsal subcutaneous pocket of rabbits. The cartilage along with tissue were retrieved and further processed for histological analysis. According to the sections stained with H&E staining, from the 45 days retrieved samples (Figure 8A), few clusters of immune cells were observed at the interface of the cartilages and the host tissues which also described a distinctive trait of immune cells implying early immune response triggered by the host.34 The supporting matrix also showed cellular vacuolation along with trabecular formation. The material was well attached to the dermal and subdermal tissues and without any sign of inflammatory reactions or angioinvasion in the matrix. Macrophages were restricted to the cartilage tissue adjoining site. Sections from 90 days retrieved samples (Figure 8B) depicted more well organized structural and cellular orientation with no infiltration with plasma cells, macrophage, lymphocytes and immature fibroblast. Figure 8C depicts the H&E of the native tissue used as control. Some specific staining methods were also used for the study of extracellular matrix (ECM). For example, Masson’s trichrome staining method was applied for the staining of collagen ﬁbers,31 which depicted a normal cellular structure and organized collagen arrangement in in situ position after 45 days (Figure 8D). Similarly, 90 days retrieved sections (Figure 8E) showed a well-organized collagen fibers interwoven as the architectural matrix between cells and other supported structures. The section showed a well spreading collagen mass among the chondrocytes and supporting matrix. Angiogenesis was quite normal. The cellular orientation



retained their normal architecture. Figure 8F depicts collagen staining of native tissue. The arrangements of elastic fibers were qualitatively analyzed by specifically staining the tissue samples according to the Weigert’s Resorcin Fuschin method.33 The results of the primarily retrieved samples (45 days) (Figure 8G) depicted well arrangement of elastic fibers among the tissue cells with satisfactory cellular matrix. Proliferations of elastic fibers were well understandable. Similarly, 90 days retrieved samples (Figure 8H) showed a prominent elastin mass surrounding the area. The cellular orientation, fibrous tissue proliferation and other supporting parts retained their normal structure. Peripheral orientation and organization of elastic fibers were in normal state without disturbing the structure and architectural pattern of the tissue. Figure 8I depicts the elastin staining of native tissue. Simultaneously, reticular fibers were also separately stained with Gridley’s modified silver impregnation method32 which depicted a compact cartilaginous structure consisting of chondrocytes, collagen fiber and reticular formation. The periphery of the section has a continuous relationship with fibrous structure. There are no inflammatory cells related to immunological reaction indicating healthy cartilaginous stroma (Figure 8J). The peri-margin perichondrium was found to be invaded by large number of reticular fibers indicating healthy cartilaginous structure (Figure 8K). Figure 8L depicts the reticulin staining of native tissue.


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Figure 8. Histology of 1% GA treated DC matrices post 45 and 90 days of implantation. H&E staining (A-C), collagen staining by Masson’s Trichrome (D-F), elastin staining by Weigert’s Resorcin Fuschin method (G-I) and Gridley’s modified silver impregnation method for reticular fiber staining (J-L). Scale bar = 200 µm for 45 days and 90 days; and 100 µm for native tissue. 3.6.2 Biochemical assessment of in vivo samples In order to validate the outcome of the qualitative study of in vivo tissue samples by histopathology that gave a vivid idea about the cellular structure, morphology and host-graft orientation; quantification of the ECM components was simultaneously performed by estimating the concentration of DNA, GAG, collagen, elastin, fibronectin, TGF β, β1 and β2 (Table 3) using specific kit protocols. Post 3 months of xenotransplantation, tissues samples were excised out from the 1% GA ‘treated’ groups. As control, tissues from healthy animals (i.e. without any implant) were used. DNA content estimation showed no significant change (Figure 9A). Similarly, sGAG, collagen, elastin, fibronectin content was also quantified for understanding of the host-graft orientation and morphology of ECM more precisely. The sulfated GAG concentration was found to be almost in equal amount in both the groups which was statistically insignificant (Figure 9B). On the other hand, collagen content estimated using hydroxyproline assay kit (Figure 9C) was found to be significantly increased (p ≤ 0.01) in decellularized cartilage xeno-grafted tissues (93.58 ± 6.81 µg/mg WW) post 3 months compared to tissue retrieved from healthy animals (54.45 ± 4.90 µg/mg WW). The data also supported the results of histological analysis, where well organized collagen fibers were noticed following Masson’s Trichome staining. Similarly, concentration of elastin was significantly increased (p ≤ 0.05) (9.18 ± 1.93 µg/mg WW) in xenoimplanted tissue samples compared to control group (5.27 ± 1.16 µg/mg WW) (Figure 9D). Earlier it was observed by histology that elastic fibers were well accommodated throughout the area and normal deposition of elastin as well as other ECM components like collagen and reticulin were distinctly present which also described the proper cell-material interaction and acceptance of the material or graft. Concentration of fibronectin (Figure 9E) and TGFβ, β1 and β2 (Figure 9F) were also measured using specific ELISA kits and found to be in normal range with no significant difference from the control group.



GAG content (µg/mg )

DNA content (µg/mg)

40

30

20

10

12

Fibronectin content (µg/mg)

10 8 6 4 2 0 Xenotransplanted group Control group

40 30 20 10

4.5

(F)

4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0

Xenotransplanted group Control group

= 1% GA treated cartilage implanted group

**

100

80

60

40

20

0 Xenotransplanted group Control group

Xenotransplanted group Control group

(E)

*

(C)

50

0

0 Xenotransplanted group Control group

(D)

60

Collagen content (µg/mg)

(B)

50

60

Concentration (ng/mg)

(A)

Elastin content (µg/mg)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 39

50 40 30 20 10 0

TGFBeta

TGFBeta 1

TGF Beta 2

= Control group

Figure 9. Biochemical assay of decellularized (1% GA treated) cartilage implanted tissue samples and control showing (A) DNA content, (B) sGAG content, (C) collagen content, (D) elastin content, (E) fibronectin content, and (F) TGF β, β1 and β2 content. Data are depicted as mean ± SD, n = 5 (for experimental group) and n= 3 (control). (*p ≤ 0.05, **p ≤ 0.01). Table 3. Summary of the biochemical analysis of the retrieved tissues post 3 months of xenotransplantation with decellularized cartilages (1% GA treated) and control (native tissue without any implant). Unit

Estimations

1% GA treated group

amount (µg/mg WW)

Control group (native tissue w/o any implant)

DNA

49.42 ± 2.72

50.46 ± 2.81

sGAG

50.79 ± 4.33

53.60 ± 2.88

Hydroxyproline

93.58 ± 6.81**

54.45 ± 4.90 **

Elastin

9.18 ± 1.93*

5.27 ± 1.16*


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(ng/mg WW)

Fibronectin

3.36 ± 1.05

3.24 ± 0.75

TGF β

55.76 ± 3.83

58.36 ± 2.41

TGF β1

45.69 ± 2.86

48.25 ± 2.54

TGF β2

49.21 ± 2.82

50.66 ± 1.55

4. Discussion Replacement and /or reconstruction of cartilage tissue defects is a major challenge in the field of orthopedics and otorhinolaryngology due to its poor regeneration capacity and low blood provision. These shortcomings are being partially addressed by the marked improvement in biomaterials based tissue engineering applications as well as cell replacement therapy.3-4, 35 Of late, xenotransplantation has garnered commendable attention in tissue engineering due to unlimited supply of cells/matrix36 to overcome shortage of organs, cells and tissues in transplantation therapy.37 Conversely, chances of acute or chronic rejections and immunogenic affects have restricted its broader clinical application in reality. To overcome the latter issue, xenogenic decellularized tissues or organs are being extensively used for reconstruction and replacement of host tissues or organs1, 10 respectively. In cartilage tissue engineering, acellular xenogenic and allogenic cartilaginous matrix are being significantly used. The fact that applications of the caprine cartilage have not yet been undertaken in a substantial manner (unlike the porcine and bovine), stimulated us to explore its viability in depth. This is supported by the fact that its size, similarity and availability may enhance feasibility and application in cartilage tissue engineering. Previous reports have shown the use of glutaraldehyde for chemically cross-linking cartilage matrices along with tanning of organs.20 Active expression of antigens is suppressed by the effects of glutaraldehyde21 which in turn provides advantages in xenotransplantation, reducing the likelihood of infections and tissue rejections. On the other hand, there is a widespread usage of gamma irradiation for numerous applications, for example sterilization19 and organ (cartilage) decellularization15 etc. The possible mechanisms behind tissue decellularization by gamma irradiation lie on two main parameters. Destruction of DNA as a result of direct cytotoxicity is one of the major causes of cellular degeneration along with indirect cytotoxicity generated by reactive



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oxygen species.38 Meanwhile, alcohol not only dehydrated the samples but at the same time enhanced the degree of cellular loss by rapidly dissociating the lipids and phospholipids resulting in cell lysis by shrinkage.14-16 Initially, we started the procedure using 0.25% glutaraldehyde treatment as mentioned earlier20 but later on by various biochemical and histological experiments, it was observed that treatment with 1% glutaraldehyde in addition to gamma irradiation yielded the required result. Glutaraldehyde and gamma irradiation complemented each other in order to get a cross-linked, acellular cartilage matrix. Upon gross observation, a remarkable absence of visible cells were noticed in 1% GA treated cartilage tissues examined by H&E staining whereas, 0.25% GA treated sections showed degenerative stage of chondrocytes. Control sections showed a well maintained fibro-cartilaginous structure. Additionally, glutaraldehyde treatment augmented the toughness of the material rendering it suitable for surgical implantation.39 The effectiveness of the decellualrizing agent was also ascertained by quantitative estimation of DNA and it was found that there was a decrease of 87.42% in DNA content of 1% GA treated cartilage tissue compared to intact or untreated cartilage. Treatment with 0.25% GA also showed 54.78% reduction of cellular DNA. Therefore, significant removal of cellular content upon decellularization and cross linking by gamma irradiation and 1% glutaraldehyde over 0.25% of the same was verified. There have been numerous decellularization protocols reported for cartilaginous tissues. However, as the principle lies on the elimination of the cellular material it is essential to maintain the structural and functional components of ECM, and therefore the selection of suitable decellualrizing agent with minimal effective dose is critical.13 To chemically cross-link the cartilage matrices, glutaraldehyde has been commonly used as it helps in degeneration of cellular components that along with gamma irradiation contributes in reducing the likelihood of immune reactions in the application of these matrices. Collagen is an important organic component of ECM. Hydroxyproline, which is an essential amino acid in collagen, can be used to calculate the content of collagen. In the present study, the quantitative

estimation

of

hydroxyproline

indicated

no

significant

difference

after

decellularization. This strongly supports the hypothesis that the impact of glutaraldehyde was not only effective in cellular degeneration but also in maintaining the structural integrity of the engineered constructs. On contrary, proteoglycans, the other important organic ingredient of ECM was significantly reduced upon decellularization. 1% GA treated cartilage showed almost 63% decrease in sGAG content quantitatively whilst 0.25% GA treatment resulted in 35% reduction


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compared to the native cartilage. Consequently, the loss of sGAG not only reduces the chances of immunogenicity but it also plays important role in improving the growth of chondrogenic cells in the scaffold and hence supported matrix remodeling and restoration of the graft.35, 40 It has also been reported that water soluble cellular constituents are partly eliminated due to the decrease of sGAG which is responsible for expanding the water flow and dispersal of the decellularizing agent into the matrix.41 The results of microscopy confirmed that the histoarchitecture was mostly retained following decellularization. Moreover, the quantification of DNA, collagen and sGAG confirmed a reduction in the number of cells along with abatement of immune response of the matrix. FESEM analysis also confirmed that the matrix structure of the cartilage tissue was well retained in the decellularized matrix. Further, the DC matrixes were evaluated in vitro for their suitability and assessment to form a functional neo-cartilage tissue using rabbit auricular primary chondrocytes. To secure a fully functional cartilage, the revitalization of the matrix is a primary requirement. Thus, it is important that the decellularized matrices are non-cytotoxic, so cells can attach and survive. The in vitro evaluation of the DC matrix in our study revealed it to be non-cytotoxic, where the seeded chondrocytes were still viable after 14 days of culture. The DC matrix supported the attachment and proliferation of the chondrocytes while maintaining their typical round morphology. As for the regeneration of cartilage, the production of ECM components, sGAG and collagen is important.42 Results indicated a significantly increased ECM formation over time for DC matrix cultured with primary chondrocytes. This increase in GAG and collagen content of the revitalized matrix was 4.0 fold and 1.5 fold as compared to the non-seeded decellularized matrix. Previous reports have shown that the reduced GAGs in the decellularized matrices is advantageous,35 as it has been reported that higher amount of GAGs prevent chondrocyte adhesion.40 The process of cell adhesion is crucial for cell migration, therefore partial or even complete depletion of GAGs could be advantageous for cartilage revitalization. The results of our study indicates the enhanced depletion of GAG levels in decellularized cartilage as compared to the native cartilage, thus assisting in chondrocyte attachment and functional regeneration of the construct. Further, enhanced matrix production, as seen via biochemical analyses was consistent with the higher expression of cartilage-specific genes (aggrecan, collagen-II and sox-9). These markers are synthesized by chondrocytes in order to maintain the cartilage matrix.43 Thus, these genes directly reflect the ECM composition and their higher expression on the DC matrices showed their



capability to support and stimulate chondrocyte growth and matrix synthesis. Macrophages or the antigen presenting cells are the foremost cells to recognize invading pathogens in the body. Their encounter with pathogens stimulate them to produce cytokines namely IL-1 and TNF-α.44 Hence a macrophage activation study was conducted and the findings indicated significantly lower TNF-α production elicited by DC matrices compared to LPS (positive control). Thus, this minimal inflammatory activity in vitro of the DC matrices renders it safe for further use. Hence, further in vivo studies were performed to assess the xenocompatibility of the matrices. Xenogenic materials have been used in cartilage tissue engineering.1, 35, 45 Decellularization of cartilages with various chemical agents has also been implicated successfully15, 45-46 with most of the focus on in vitro analysis. The in vitro results alone are not the alternative of in vivo application because of distinguishable differences between in vivo host environments to that of an in vitro microenvironment. Therefore, in our study, we have correlated our in vitro analysis with the results of in vivo xenografting including qualitative and quantitative studies. Histological observations provide useful information about the interactive behavior of decellularized cartilage with surrounding host tissue. Decellularized matrix was well aligned and attached to the host tissue without any drastic infiltration of immunogenic cells leading to necrosis and graft rejection. Since, collagen is one of the vital compounds of ECM along with proteoglycans, elastin and reticulin; therefore the presence of sufficient amount of collagen in the implanted site demonstrates a wellorganized format of the material. In this experiment, collagen staining revealed an organized architecture of the material along with host tissue not displaying any abnormality which was also supported by the quantitative estimation of collagen content which was increased (~71%) prior to xenografting compared to normal animal. Besides, normal deposition of reticulin, elastin was also observed histologically which indirectly supported the fact of graft acceptance. Elastic fibers were richly disbursed in conchal cartilage compared to other cartilaginous matrix13 In vivo implantation of decellularized cartilage led to the accumulated incorporation of elastic fibers at the surgical site; which in turn led to successful remodeling of host tissue and revitalization of the xenografted cartilage matrix. On the other hand, the level of proteoglycan, DNA, fibronectin and TGF β, β1, β2 of both the control as well as experimental animals were found unaltered possibly due to successful acceptance of the graft and absence of any unusual inflammation or tissue damage. Fibronectin and TGF β are the components of ECM which play vital roles in cartilage formation and maintenance. Fibronectin, which is associated with integrin-based focal adhesion, also plays


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a crucial role in describing the micro-environmental niche for cellular functions both in normal and emergency conditions,47 while TGF β super family have been found to be involved in differentiation of chondrocytes from early to terminal stages, starting from proliferation, condensation, terminal differentiation to their maintenance.48 The retention of these bioactive molecules will be especially advantageous in regenerating cartilage as the avascular nature of cartilage limits its supply of appropriate growth factors and nutrients. The results of our study showed the revitalization and remodeling of the cartilage matrix; which in turn were helped synergistically by the surrounding host cells to migrate and proliferate abundantly. It also validated the immune-compatibility and applicability of the decellularized material for further broadened approach. Enhanced cellular proliferation and migration will be beneficial to matrix formation and biomechanical properties of decellularized cartilage matrix.49 In the reconstructive surgery, autografts and allografts are frequently being practiced. In order to get more efficient outcome, implementation of xenogenic materials with complete removal of host tissues may serve as a successful alternative in the transplantation biology. With this aim, the study was conducted to make caprine conchal cartilages acellular by chemical cross linking. The decellularized and cross linked materials not only loses the cellular DNA and proteoglycans but are also masked immunologically; this consequently helped in delayed or non-immunogenic response in the host body causing efficient tissue regeneration and graft acceptance. 5. Conclusion A variety of cartilage graft substitutes are available for application in cartilage tissue engineering, but none gives satisfactory outcome in clinical setting. The present study of decellularization and cross linking of caprine conchal cartilage with 1% glutaraldehyde is a successful means of removing cellular component while maintaining its collagenous matrix thus reducing risk of inflammatory reactions and disease transmission. Possible cytotoxicity and cellular compatibility of the matrix tested both in vitro and in vivo showed no inflammatory responses. Due to decellularization, although the amount of ECM is decreased, but the remaining collagen matrix still provides in vivo physiological niche in regards to gross mechanical strength. The recognized drawbacks of inadequate availability, donor-side morbidity, graft rejection, disease transmission and extrusion can be overcome by this newly developed acellular animal conchal cartilage. This



kind of intervention may open floodgates of novel alternative improvisation in the field of cartilage graft and plastic reconstruction surgery.

Acknowledgments SKN greatly acknowledges Department of Biotechnology, DBT (BT/PR7292/AAQ/534/2012 dated 05.08.2013), Government of India for generous funding. BBM wishes to acknowledge the generous funding support from Department of Biotechnology (DBT) and the Department of Science and Technology (DST), Government of India. Central Instruments Facility, IITG is acknowledged for high end instruments support. Furthermore, the authors wish to express their sincere thanks to the Honorable Vice Chancellor, West Bengal University of Animal and Fishery Sciences, Kolkata, India, for their generous and kind support to this work.


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Figure 1. Schematics of harvesting (A-B), processing (C) and decellularization (D) of conchal cartilage. 150x48mm (300 x 300 DPI)


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Figure 2. Biochemical properties and H&E staining of glutaraldehyde treated (0.25 % and 1 %) and native cartilage. (A) DNA content, (B) sGAG content, (C) collagen content, (D) presence of cellular elements in native/untreated cartilage, (E) after 0.25% glutaraldehyde treatment and (F) after 1% glutaraldehyde treatment. Data are shown as mean ± SD, n=3 (*p ≤ 0.05). Scale bar represents 100 μm. 149x92mm (250 x 250 DPI)



Figure 3. Field emission scanning electron microscopy (FESEM) of cartilage (A, C) before decellularization; (B, D) after decellularization. Scale bar = 2 µm. 149x103mm (200 x 200 DPI)


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Figure 4. (A) Alamar blue proliferation assay and (B) calcein AM staining of primary rabbit chondrocytes cultures on tissue culture plate (TCP) and 1% glutaraldehyde treated cartilage. Scale bar = 400 µm (**p ≤ 0.01). 149x125mm (200 x 200 DPI)



Figure 5. Biochemical properties of decellularized cartilage (A-C). (A) DNA content, (B) GAG content and (C) collagen content in decellularized cartilage constructs after 14 days, normalized by wet weight (WW). Real time PCR showing expression of cartilage related genes (D) aggrecan, (E) collagen-II and (F) sox-9. Data are shown as mean ± SD, n=3. (*p ≤ 0.05, **p ≤ 0.01). 149x77mm (250 x 250 DPI)


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Figure 6. Quantification of released TNF-α stimulated by DC matrices when cultured with RAW 264.7. Tissue culture plate (TCP) was used as negative control and lipopolysaccharide (LPS) as positive control. The data are represented as mean ± SD (n = 2). (*p ≤ 0.05 and **p ≤ 0.01). 149x105mm (200 x 200 DPI)



Figure 7. Gross pictures (A, B) during surgery showing in vivo xenoimplantation of cartilages. Post 3 months, macroscopic view revealed 1% GA treated cartilage (C) to be well organized with no sign of inflammation and tissue necrosis, whereas, native or untreated cartilage (D) showed necrosis of host tissue. Scale bar is 1 cm. 149x36mm (200 x 200 DPI)


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Figure 8. Histology of 1% GA treated DC matrices post 45 and 90 days of implantation. H&E staining (A-C), collagen staining by Masson’s Trichrome (D-F), elastin staining by Weigert’s Resorcin Fuschin method (G-I) and Gridley’s modified silver impregnation method for reticular fiber staining (J-L). Scale bar = 200 µm for 45 days and 90 days; and 100 µm for native tissue. 149x86mm (200 x 200 DPI)



Figure 9. Biochemical assay of decellularized (1% GA treated) cartilage implanted tissue samples and control showing (A) DNA content, (B) sGAG content, (C) collagen content, (D) elastin content, (E) fibronectin content, and (F) TGF β, β1 and β2 content. Data are depicted as mean ± SD, n = 5 (for experimental group) and n= 3 (control). (*p ≤ 0.05, **p ≤ 0.01). 149x101mm (200 x 200 DPI)


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Graphical abstract 149x88mm (200 x 200 DPI)


Decellularized Caprine Conchal Cartilage towards Repair and

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