Chondrogenesis of mesenchymal stromal cells on the 3D printed polycaprolactone/fibrin/decellular cartilage matrix hybrid scaffolds in the presence of piascledine

Abstract Nowadays, cartilage tissue engineering (CTE) is considered important due to lack of repair of cartilaginous lesions and the absence of appropriate methods for treatment. In this study, polycaprolactone (PCL) scaffolds were fabricated by three-dimensional (3D) printing and were then coated with fibrin (F) and acellular solubilized extracellular matrix (ECM). After extracting adipose-derived stem cells (ADSCs), 3D-printed scaffolds were characterized and compared to hydrogel groups. After inducing the chondrogenic differentiation in the presence of Piascledine and comparing it with TGF-β3 for 28 days, the expression of genes involved in chondrogenesis (AGG, COLII) and the expression of the hypertrophic gene (COLX) were examined by real-time PCR. The expression of proteins COLII and COLX was also determined by immunohistochemistry. Glycosaminoglycan was measured by toluidine blue staining. 3D-printed scaffolds clearly improved cell proliferation, viability, water absorption and compressive strength compared to the hydrogel groups. Moreover, the use of compounds such as ECM and Piascledine in the process of ADSCs chondrogenesis induction increased cartilage-specific markers and decreased the hypertrophic marker compared to TGF-β3. In Piascledine groups, the expression of COLL II protein, COLL II and Aggrecan genes, and the amount of glycosaminoglycan showed a significant increase in the PCL/F/ECM compared to the PCL and PCL/F groups. Graphical abstract


Introduction
Nowadays, tissue engineering is widely regarded as a viable alternative modality for the treatment of various tissue defects, particularly in the context of injuries to cartilage and osteoarthritis.The primary cause of the inability of cartilage damage to undergo self-repair is the absence of blood vessels, lymph, and nerves, as well as the limited proliferative capacity of chondrocytes, which exist in small quantities [1,2].
The development of scaffolds in the field of cartilage tissue engineering (CTE) is of paramount importance, as they play a critical role in promoting cell growth and facilitating the regeneration of cartilage tissue.Optimal 3D scaffold should possess biocompatibility, biodegradability, suitable mechanical characteristics and high degree of porosity with interconnected pores that enhance cell attachment, cell growth and facilitate nutrient transport [3,4].The morphology of the scaffold, which is specifically tailored for the purpose of tissue regeneration, exerts a significant influence on the extent of cell adhesion, migration, proliferation, and differentiation.Furthermore, the scaffold must also possess the capability to support the growth of new tissue [5].3D printing is a technique of additive manufacturing (AM) that encompasses a wide range of biomaterials and polymers, facilitating an increase in chondrocyte viability [6].
Biomaterials utilized for the construction of scaffolds are categorized into two groups: natural and synthetic [7].By combining natural and synthetic components appropriately, one can fabricate a suitable scaffold [5].
Polycaprolactone (PCL) exhibits notable attributes such as high mechanical properties and flexibility, good biocompatibility, uncomplicated processability, low melting point (60 °C) and non-toxic degradation products [9,10].In order to improve the cellular behavior of PCL, its combination with natural polymers and protein can be used.
Fibrin (F) provides an optimal environment for the provision of nutrients and oxygen to cells, demonstrating commendable biocompatibility and biodegradability [11].Fibrin serves as a natural matrix for cells, facilitating effective cell adhesion [12].Fibrinogen and thrombin, which are easily obtainable from blood, serve as autologous sources for scaffold fabrication, thereby reducing the likelihood of scaffold rejection [13].However, it is essential to note that fibrin exhibits low mechanical and physical properties.As a result, synthetic polymers such as PCL should be employed in conjunction with fibrin in order to enhance its mechanical characteristics, taking into account the pivotal role played by mechanical and physical properties in scaffold construction [5].
Tissue engineering frequently employs diverse sources of stem cells, including bone marrow stem cells (BM-MSCs) and adipose-derived stem cells (ADSCs) [14].ADSCs have garnered significant attention due to their availability and substantial potential for chondrogenesis.Adipose tissue is replete with multipotent stem cells that makes it an appropriate candidate for tissue engineering.These stem cells possess the capacity to maintain their differentiation potential in various environments and scaffolding media [15].
On the other hand, growth factors play a crucial role in the proliferation, apoptosis and differentiation of stem cells [16].Among the various growth factors, the transforming growth factor β (TGF-β) family holds a position of utmost significance in promoting the development of cartilage (chondrogenesis).However, the effectiveness of TGF-β as an inducer is limited due to its high cost, short half-life (approximately 30 min), ability to initiate apoptosis and cell death, inflammation, formation of bony outgrowths within the joint (osteophyte formation), and the enlargement of specialized cells (hypertrophy) [16,17].For this reason, in many studies, researchers have gone to replace herbal medicines instead of growth factors.Piascledine is an herbal supplement derived from avocado and soybean seed oils (ASU).The key components found in Piascledine are campesterol, phytosterols, β-sitosterol and stigmasterol, which quickly penetrate cells [18].
Piascledine exhibits chondroprotective properties, anabolic and anti-catabolic properties and inhibits the destruction of cartilage cells in osteoarthritis.It stimulates collagen and aggregate synthesis by inhibiting inflammatory cytokines, including IL-1, IL-6, IL-8, PEG2, and TNF via NF-κβ [19].Indeed, Piascledine mitigates cartilage damage caused by inflammation by reducing the production of IL-1, PGE2, and MMP-3.Consequently, the utilization of Piascledine may assist in the differentiation of stem cells within the culture medium [20].
In addition to the use of herbal medicines, in recent years the use of protein compounds such as decellularized extracellular matrix (DECM) has played a significant role in cartilage tissue engineering studies.ECM is widely recognized as a crucial component for cell adhesion, proliferation, and differentiation.Generally, cells tend to exhibit improved functionality when situated within the ECM [5].A previous investigation conducted by Setayeshmehr et al. [21] successfully developed bioinks that are compatible with cells, utilizing solubilized decellularized cartilage matrix (SDCM) and poly (vinyl alcohol)-Norbornene (PVA-Nb) for the purpose of cartilage bioprinting applications.The study demonstrated that the combination of SDCM and PVA-Nb could enhance the printability of the bioinks and promote cell viability, while also exerting control over the swelling ratio of the 3D-printed constructs.Hybrid printing, which involves the incorporation of a hydrogel into a 3D-printed reinforcing polymeric material such as PCL, may provide a solution to the issue of poor mechanical properties typically associated with natural hydrogels such as collagen.The resulting hybrid constructs can exhibit moderately high mechanical strength due to the reinforcing nature of the synthetic polymer structure [22].Li et al. [23] the construction of a hybrid scaffold using 3D printing techniques, where PCL served as the backbone and was subsequently embedded with meniscus extracellular matrix containing kartogenin.The study presented that the fabricated scaffolds were capable of promoting meniscus renewal through the release of kartogenin.Another study conducted by Lee et al. [24] involved the development of hybrid constructs by coating 3D-printed PCL/ tricalcium phosphate (PCL/TCP) scaffolds with bone decellularized ECM (bdECM) in order to enhance osteoinductivity and osteoconductivity.The researchers showed that mandibular ossification was significantly improved when adipose-derived stem cells (ADSCs) were seeded onto bdECM-coated PCL/TCP scaffolds.Jang et al. [25] successfully designed a 3D hybrid construct using PCL and introduced a cell-laden hydrogel (a mixture of alginate/ADSCs/chondrocytes) into the scaffold.The study revealed that chondrogenic differentiation was enhanced in vitro and cartilage regeneration was significantly improved in vivo.In the field of cartilage tissue engineering, researchers are actively striving to identify factors that can induce chondrogenesis of ADSCs in order to produce cartilage of higher quality at a reduced cost.While Piascledine (ASU) has been used for the treatment of osteoarthritis, there are currently no reports on its in vitro application to induce ADSC chondrogenesis in a 3D cell culture environment.
This study aimed to develop an innovative hybrid construct using 3D printed PCL and injected a cell-laden hydrogel (a mixture of fibrin/ECM/ADSCs) into the scaffold, to prepare a hybrid 3D-printed PCL/F/ECM scaffold as an ideal construct for cartilage engineering with suitable mechanical and biological properties.Due to limitations of using TGF-β in cartilage differentiation and previous studies on the effects of Piascledine on chondrogenesis induction in ADSCs, the effect of this herbal factor on the scaffolds was compared with TGF-β3.This new approach further aims to optimize scaffold structure in order to imitate the proper cartilage characteristics.

Materials
PCL (Mw: 80,000 g/mol), Dulbecco's Modified Eagle Medium (DMEM), trypsin EDTA, sodium pyruvate, dexamethasone, Premix proline, ascorbate −2-phosphate, bovine serum albumin, linoleic acid, insulin transferrin selenium (ITS), type I collagenase, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich.Pure powder of avocado-soybean was obtained from Perarin Pars Co., TGF-β 3 (Sigma-Aldrich -T-9705), and fetal bovine serum (FBS) were purchased from Invitrogen.Penicillin and streptomycin were obtained from Gibco.Fibrinogen and thrombin were obtained from Cryoprecipitate, and fresh frozen plasma (FFP) was procured from the Blood Bank of Isfahan Province (Isfahan, Iran) following the approval of the Ethics Committee of the Faculty of Medicine, Isfahan University of Medical Science (397810).

Preparation of solubilized decellularized cartilage matrix (SDECM)
SDECM was obtained according to a modified method used in our previous study [1,2].Articular cartilage biopsies were obtained from the knee joints of three calves.
Cartilage was frozen and thawed at −20 °C to +25 °C three times, then frozen with liquid nitrogen and powdered using a freeze-mill (SPEX SamplePrep, Metuchen, NJ, USA) to obtain devitalized cartilage (DC).The DC was placed into dialysis tubes (3500 MWCO) and then bags were treated in a hypertonic salt solution (HSS) under slight stirring 8 hr at +25 °C.The bags were washed twice with Triton X-100 (0.01% v/v), then exposed with Benzonase (0.0625 KU mL −1 ) at 37 °C for 8 hr, further treated using sodium lauroyl sarcosine (SLS, 1% v/v) for 8 hr, and finally washed using 40% v/v ethanol followed by PBS before rinsing with distilled water for 24 h.Decellularized cartilage matrix (DECM) was solubilized using a HCl/pepsin solution.More specifically, DECM was mixed at a concentration of 10 mg DECM/mL HCl in 0.1 M HCl, which contained pepsin at a concentration of 1 mg/ml.The solution was stirred for 3 days at 25 °C, and the solution was then brought back to physiological pH using 1 M NaOH.The resulting solution was then centrifuged at 10,000 g for 3 min, and the supernatant was collected.The SDECM was frozen, lyophilized, cryo-milled (freeze-mill SPEX SamplePrep, Metuchen, NJ, USA) and stored at −80 °C.

Biochemical analysis
The samples (n = 3) were digested using Tris-EDTA buffered solution containing 1 mg/ ml proteinase K, 1 μg/ml iodoacetamide and 18.5 μg/ml pepstatin A (Sigma-Aldrich) at 65 °C for 24 h.The CyQuant DNA assay kit (Molecular Probes, Oregon, USA) was utilized to quantify the DNA content of the samples according to the manufacturer's instructions, using a spectrofluorometer at 480/520 nm excitation/emission wavelength (Perkin Elmer LS50B plate reader) [3,4].The sulfated glycosaminoglycans (sGAG) content was determined using the 9-dimethylmethylene blue chloride (DMMB) dye (Sigma Aldrich) in PBE buffer (3.72 g/l Na 2 EDTA and 14.2 g/l Na 2 HPO4, pH 6.5) via a multi-well plate reader (Bio-TEK Instruments) at 520 nm.After plotting the standard curve, the sGAG concentration of the samples was obtained via the interpolation of its absorbance from the standard curve [4].The hydroxyproline assay kit (MAK008, Sigma-Aldrich) was employed to measure the hydroxyproline content of the samples according to the manufacturer's instructions.Briefly, 100 µl of HCl 12 M (Sigma) was used to digest 10 mg of lyophilized samples (n = 3 for each group) at 120 °C for 3 h [5,6].The absorbance of the hydroxyproline standard solution and digested samples were measured using a multi-well plate reader (Bio-TEK Instruments) at 520 nm.The hydroxyproline concentration of the samples was obtained via the interpolation of its absorbance from the standard curve [7].

Thrombin, Fibrinogen, and fibrin Preparation
To prepare thrombin, FFP content was melted at 37 °C for 10 min in a water bath.Subsequently, 16 ml of FFP and 10 ml of calcium gluconate were transferred to a sterile tube and incubated for 60-90 min at 37° C. Following the incubation period, the tubes underwent centrifugation for 10 min at 2200 rpm.The resulting clear supernatant from each tube containing thrombin was aliquoted into 1 ml tube and preserved at −80 °C.Cryoprecipitate, a rich and valuable source of fibrinogen, was utilized to prepare the fibrinogen.A cryoprecipitate bag was placed in a water bath for 10 min at 37 °C.The outer surface of the bag was sterilized with 70% alcohol, and its contents were extracted under sterile conditions using a 10 ml syringe [1,8].

Preparation of 3D printed scaffolds
PCL granules were melted in a heating cylinder at 100-130 °C, and 3D-printed scaffolds were fabricated utilizing a Bioplotter 3D printer system (Envisiontec GmbH, Gladbeck, Germany) [9].PCL was extruded through a heated nozzle in the form of a strand, which was plotted on a plate in a layer-by-layer deposition manner [9][10][11].The samples possessed a nominal fiber diameter of 300 µm (ascertained through the utilization of a 27 G nozzle size), a layer thickness ranging from 280 to 300 µm, and a fiber spacing of 700 µm.The 3D scaffolds were cut from the printed block into a square prism shape with 5 × 5 × 4 mm 3 [9][10][11][12].

Fabrication of PCL/fibrin/ECM scaffolds
ECM was mixed with the fibrinogen solution [13].3D-printed scaffolds were divided into three groups: PCL, PCL/Fibrin, and PCL/Fibrin/ECM.ADSCs were added to the ECM/fibrinogen solution (50 µL) and seeded on the PCL scaffolds in different groups such that cells filled the scaffold porosity.Then, thrombin (50 µL) was added to this complex and allowed to gel at 37 °C for 30 min [14,15].After scaffold characterization, Piascledine and TGF-β3 were added to the PCL/Fibrin/ECM group and chondrogenic induction was evaluated in these scaffolds.

Scanning electron microscopy of scaffolds
The morphology and interconnectivity of the porous scaffolds were observed through the utilization of various imaging instruments such as scanning electron microscope (SEM; JEOL JSM-5200), stereomicroscope (Olympus SZH10), and visual slide microscope (Carl Zeiss Mirax desk).One scaffold was randomly chosen from each group, cut in the middle using a razor blade, and mounted onto a stub.Subsequently, these cross-sections were coated with a thin layer of gold using a sputtering device for 5 min before being observed under by SEM (n = 3) [16,17].
In the assay of cell morphology, after 4 days of cell culturing on the scaffolds, they were first washed twice with PBS and fixed with 4% glutaraldehyde (Merck, Germany) for 2 h.They were then gradually dehydrated with the incremental concentrations of ethanol (50, 70, 80, 90, and 100% ethanol for 30 min each).After drying, PCL and PCL/F scaffolds containing fixed cells were coated with gold and evaluated via SEM [18,19].

Porosity measurements
The porosity of the scaffolds was evaluated using the liquid displacement method [20].Briefly, a scaffold was transported to a scaled cylindrical container filled with a known hexane volume (V 1 ).The increase in hexane volume as a result of the inclusion of the scaffold in the container was calculated as (V 2 ).After the adsorption of hexane in 5 min, the scaffold was extracted, and the remaining volume in the container was calculated (V 3 ).The percentage of porosity was determined through the following formula (1): where is the volume of pores in the scaffold, and (V 2 −V 3 ) is the total volume of the scaffold (n = 3).

Water absorption capacity
The scaffold specimens were cut from moulds according to ASTM-D-5964 standard (4 mm in diameter and 4 mm in height) into Petri dishes in a circular shape.At room temperature, the structures were first dehydrated, weighted, and immersed separately in 10 mL of 10 mM phosphate-buffered saline (PBS; pH=7.4).At a specified point in time, the specimens (n = 3) were removed from the solution, carefully put on a glass for 5 s to eliminate any excess water, and weighed immediately [17].The quantity of water in each scaffold was evaluated according to Equation (2): where W d and W w are the weights of the specimen before and after submersion in the medium, respectively [10].

Compressive modulus
The compressive strength of each scaffold (5 × 5 × 4 mm 3 ) was determined using a universal testing machine (Lloyd LRX, UK) according to the ASTM D695 standard and a load cell of 500 N in a dry state at room temperature.The load was compressed vertically at a cross-head speed of 3 mm/min until the original thickness of the scaffold was reduced to about 70%.The compressive modulus was then calculated at a compressive strain of 20% from the slope of the linear portion of the stress-strain curve (n = 3) [21][22][23][24].

Experimental design
The study setup included three groups: a control group (PCL/F/E) consisting of ADSCs seeded on PCL/F/ECM scaffolds in the chondrogenic medium without ASU; and experimental groups (ASU), including a chondrogenic medium with 10 µg/ml ASU and the TGF group that received TGF-β (Table 1).The cells were kept in the chondrogenic medium for 28 days.

Isolation and culture of human ADSCs
After obtaining written consent in the operating room and following the approval of the Ethics Committee of the Faculty of Medicine, Isfahan University of Medical Science (397810), about 30 g of subcutaneous adipose tissue was obtained from three patients aged 25-40 years old under sterile conditions and transferred to the lab.The adipose tissue was digested with a solution of 0.075% type I collagenase at 37 °C for 30 min.Subsequently, complete cell culture medium [DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin (Gibco) was added to the cell suspension to neutralize the activity of the enzyme.The cell suspension was then centrifuged for 7 min at 1400 rpm, and the supernatant was removed along with adipocytes.Finally, the resulting cellular pellet was cultured in complete cell culture medium at 37 °C, in 5% CO 2 condition, in a humidified incubator.After 24 h, additional cells were eliminated by changing the medium [25,26].

Cell preparation
ADSCs were routinely grown in DMEM supplemented with 10% FBS, and 1% penicillin/streptomycin.The cells were cultured in a 75 cm 2 culture flask and maintained in a tissue culture incubator at 37 °C and 5% CO 2 .The culture medium was replaced every 2 days [27,28].After reaching about 90% confluence, the cells were detached by 0.025% trypsin and 0.01% EDTA in PBS solution and transferred to a centrifuge tube containing the culture medium.After centrifugation, the cells were re-suspended in fresh culture medium and counted before seeding onto scaffolds [28].

MTT assay
The scaffolds were prepared to a size of 5 × 5 × 4 mm 3 and were sterilized with 70% EtOH and UV light.The scaffolds were placed overnight in the culture medium.ADSCs were collected using a 2% collagenase digestion process from liposuction.ADSCs were cultured up to passage 2 and were collected by trypsin-EDTA treatment.The corresponding cell density (2 × 10 5 cells) was seeded in the scaffolds.The cell-seeded scaffolds were incubated at 37 °C in 5% CO 2 and cultured for MTT assay on the 1st, 4th, and 7th days (n = 3).The medium was replaced daily [25,26,29].The sample containing PCL scaffolds with the cells plus the culturing medium without any treatment was regarded as the control sample.The culture medium was first aspirated and replaced with 400 µL/well MTT solution at 0.5 mg/mL in a 24-well culture plate.Next, each plate was incubated at 37 °C for 30 min.The MTT solution was then aspirated, and 1 mL/well DMSO containing 125 mL/well of glycine buffer (pH=10) was added to dissolve the formazan crystals.Finally, after 5 min of rotary agitation, the absorbance of the DMSO solution at 540 nm was calculated using a Thermo Spectronic Genesis10 UV/visible spectrophotometer [17,25,30].Cell viability was calculated according to Equation (3): Cell viability % optical density OD of treated cells OD of the no ( )= ( ) The sample containing PCL scaffolds with the cells plus the culturing medium without any treatment was regarded as the control sample.

In vitro chondrogenic differentiation
Harvested hADSCs from passage 3 were re-suspended in the chondrogenic medium (high-glucose DMEM, supplemented with 100 µg sodium pyruvate, 10 µg/ml ASU, 100 nM dexamethasone, 1% ITS + Premix, 40 µg/ml proline, 50 µg/ml ascorbate-2-phosphate, and 1% penicillin-streptomycin, bovine serum albumin 0.5 mg/ml, linoleic acid 5 µg/ml).To load the cells on the PCL/F/ECM composite scaffold in the 24-well plate scaffold, 2 × 10 6 in 200 ml of the medium was loaded on each scaffold; then, the plate was incubated at 37 °C and 5% CO 2 for 2 hrs.Then, 500 µl of the chondrogenic medium was added to each well, with half of the medium replaced every two days.In addition, the cell-laden hydrogels and hybrid constructs were cultured for 28 days in culture medium containing 10 ng/ml TGF-β3 (named as TGF medium) or 10 μg/ ml ASU (ASU medium) to investigate chondrogenesis of the ADSCs treated by ASU compared to the TGF-β3.

RNA extraction and real-time polymerase chain reaction (PCR)
At first, scaffolds in different groups were washed with PBS and digested via trizol reagent (Invitrogen); then, total RNA was isolated by an RNase minikit (Qiagen).The RNA concentration was determined using a biophotometer (Eppendorf).Next, 100 ng of the extracted RNA was reverse-transcribed to cDNA using a cDNA synthesis kit (Fermentas) according to the manufacturer's instruction.The relative quantification of expression type II, X collagen, and aggrecan were measured using a Maxima SYBER ® RoxqPCR master mix kit (Fermentas) (Table 2).The genes were normalized against the reference gene (GAPDH).The expression level of each target gene was calculated via 2 -ΔΔct , as previously described [7].

Immunocytochemistry
Scaffolds underwent immunohistochemical staining for types II and X collagen.Staining was also performed on 5 µm sections fixed using 4% paraformaldehyde for 20 min and then washed with PBS. 5 µm thick sections were placed on adhesive slides, deparaffinized at 60 C for one hour, and passed through xylol three times for 10 min.The sections were then rehydrated by serial application of decreasing concentrations of alcohol before being placed in distilled water and used for staining with monoclonal antibodies to type II (ab34712; Abcam) and X collagen (Ab14977182; Abcam).For antigen retrieval, the slides were incubated in hyaluronidase 8 mg/ml (Sigma) for 3 h at 37 °C.The nonspecific binding sites were blocked using 3% BSA blocking buffer, and the slides were incubated with diluted primary antibodies at 4 °C overnight.Following washing, the slides were labeled with secondary antibody (anti-mouse IgG, ab2891; Abcam) and co-stained with the pan-nuclear marker DAPI (1:1000 dilution).Representative images of each section were taken using an Olympus fluorescent microscope (Olympus BX51) equipped with a digital camera [31].

Histological analysis
After 28 days of culture, toluidine blue staining was performed to demonstrate the intensity of glycosaminoglycan synthesis.Constructs were fixed for 24 h at 4 °C in 10% buffered formalin in the PBS solution.Constructs were processed by increasing ethanol solutions before being cleared by xylene.Then, samples were embedded in paraffin and cut into 5 μm slices.Xylene-cleared sections were stained using Hematoxylin and Eosin (H&E).To stain the GAGs, toluidine blue staining (0.125%, 20 sec) was performed before samples were rinsed with distilled water, dehydrated, cleared, and mounted on microscope slides [7].

Statistical analysis
All the values are expressed as mean and standard deviation.Statistical analyses of different groups were performed by one-way analysis of variance (ANOVA), along with Tukey's post-hoc test, in SPSS 11.5.p value with p < 0.05 were regarded as statistically significant.

DNA, sGAG, and hydroxyproline content
ECM-derived biomaterials can be achieved from both native tissue or ECM deposited through in vitro cell culture.There is a possibility to decellularize ECM to completely remove all nucleic acids and cellular components or devitalize to inactive all residence cells within the ECM without fully removing them.Effective decellularization process consequences in an decellularized ECM that has similar biochemical composition as native tissue with the low immunogenicity [32].The DNA, sGAG, and Table 2. the genes and primer sequences used for real-time Pcr.
hydroxyproline contents of natural cartilage were determined to be 3.20 ± 0.20 μg/mg, 5.8 ± 0.20 μg/mg, and 8.50 ± 0.10 μg/mg sample, respectively (Figure 1).According to the results after decellularization, solubilization, and dialysis, the DNA content was further reduced to 0.2% of that of the original lyophilized cartilage (p < 0.05), and the GAG content further decreased to 12% of that of the natural cartilage.Furthermore, the hydroxyproline content was reduced by 35% compared to the natural cartilage (p < 0.05).In a study performed by Sutherland et al. [32] the porcine knee and hip articular cartilage were decellularized via osmotic shock, treatment with detergent, and enzymatic washes.Following the decellularization process an 86% and 55% reduction in DNA and GAG content, respectively, was measured.Nevertheless, there was no significant difference in hydroxyproline content compared to natural cartilage through decellularization.In the present study, the decellularization procedure was followed by solubilization and further purification process using dialysis in order to use the DCM as a hydrogel, which led to more reductions in DNA content (to 0.2%), GAG (to 12%) and hydroxyproline content (to 35%).Open pores with uniform shape and size were found in the 3D-printed PCL scaffolds with regular interconnected pores.In the other groups, porosity was decreased by the addition of fibrin and ECM to the PCL.Pore size is a key factor affecting the improvement of cell viability in the 3D porous network of the scaffolds.The size must be small enough to allow for cell attachment and large enough for the inflow of cells [33].Pore interconnectivity is considered a promising factor since it could contribute to cell migration into internal pores, improve the flow of nutrients and metabolic waste, and enhance the communication of cells in various pores during cell culture, while also providing sufficient space for cell growth [34,35].SEM micrographs for cell seeded PCL (Figure 2(C-iv)) and PCL/F/ECM (Figure 2(C-v)) exhibited more attached cells within the PCL/F/ ECM scaffolds.Therefore, the results indicated that PCL/F/ECM scaffolds were more cytocompatible than PCL scaffolds and highly practical for tissue regeneration with a suitable pore size for cell attachment.The results were also in agreement with previous studies indicating that PCL-ECM constructs could are suitable for tissue engineering applications [36,37].

Porosity measurements
In all the samples, porosity was higher than 72%, and a non-significant difference was detected for porosity in different groups.SEM results revealed that PCL scaffolds had a porosity of 80.2 ± 3.1% and a pore size of < 400 µm.The PCL/F and PCL/F/ ECM scaffolds were in the range of 250-400 µm pore size, with a porosity of 72.6 ± 2.8% and 75.5 ± 4.2%, respectively (n = 3) (Table 3).Similarly, in PCL/MECM scaffolds fabricated by Li et al. [37] the entire PCL structure was occupied with a porous MECM sponge.They reported a microporous structure in the pores between PCL fibers.Their results showed that PCL/MECM scaffolds exhibited a hierarchical pore microstructure and the ECM containing constructs presented less porous than PCL scaffolds.

Water absorption capacity
Water uptake is considered an essential factor in scaffold degradation.Water absorption assays are used to indirectly evaluate the interconnectivity between pores of manufactured scaffolds [10].The data related to the water absorption capacity of scaffolds is presented in Table 3.Consistent with previous studies [38], the water absorption of the PCL/F/ECM scaffolds was higher than that of PCL and F/PCL scaffolds.The results showed that the water absorption capacity was 58.3 ± 2.04% for the PCL scaffolds, 125.3 ± 3.5% for the PCL/F scaffolds, and 133.7 ± 4.1% for the PCL/F/ECM scaffolds (n = 3) (Table 3).As SDECM was added, water uptake was significantly enhanced (p < 0.05).Therefore, the structure could absorb the culture medium, facilitate the proper infiltration of nutrients, and increase cell viability.The scaffold's final water uptake ability is associated with the pore-interconnectivity, porosity, and quality of scaffold materials [39].
Furthermore, the high rate of scaffolds' water uptake with increasing the ECM can be interrelated with the high porosity and hydrophilicity of F/ECM, which allows the medium to reach the inner pores, leading to the easy diffusion of nutrients to promote cell growth, specifically in PCL/F/ECM scaffolds [34].In this study, the results of the water absorption test showed a significant increase in scaffolds containing fibrin and cartilage extracellular matrix, which was consistent with other studies [40].The presence of the cartilage matrix, which contained glycosaminoglycan, increased water absorption and scaffold hydrophilicity.In general, in tissue engineering, scaffolds that have a high water absorption capacity are important due to their effect on initial adhesion and cell migration and increasing cell viability [40,41].

Compressive modulus
In agreement with previous studies, ECM can offer a proper microenvironment for stem cell infiltration, differentiation, and regeneration [1,37].Furthermore, the embedded ECM can enhance the biomechanical properties of the PCL/F/ECM hybrid scaffolds.The ideal compressive module and mechanical properties can result in successful tissue regeneration [42].The compressive modulus of PCL/F scaffolds (4.39 ± 0.57 MPa) was slightly higher than that of the PCL scaffolds (3.09 ± 0.28 MPa), while the compressive modulus of the PCL/F/ECM scaffolds (5.11 ± 0.82 MPa) was significantly higher than those of the PCL and PCL/F scaffolds (p < 0.05) (Table 3).The PCL/F/ ECM scaffolds exhibited improved biomechanical characteristics compared to the PCL and PCL/F scaffolds (Figure 3), which was consistent with previous studies [43].It is proved that the mechanical properties of the scaffolds depend on their structure [44].The higher mechanical properties, namely compressive strength and elastic modulus, are justified by the fact that the micro-cracks on the surface of the struts are filled and the weak and fragile struts are coated, turning the polymers into strong and tough struts [43].
Although fibrin is a natural polymer with a high degradation rate, adding fibrin to scaffolds does not reduce the overall mechanical properties, yet it could increase the elastic modulus.Although the elastic modulus of the native articular cartilage is in the range between 130-573 kPa according to the previous studies [7], while the compression modulus of the PCL/F/ECM scaffold was superior to the native cartilage, the hybrid scaffold is tough enough to resist biomechanical loads and preserve the structural integrity in the defect.Thus, these scaffolds can be considered as an ideal scaffold for cartilage engineering.The results were in agreement with those of Choi et al. [45] study, indicating that ECM enhances hydrogel's mechanical and cellular properties.Thus, ECM can be used as a 3D interconnected structure to improve cartilage regeneration.It is well-recognized that the cartilage must tolerate the load, so it should have proper compression strength.

MTT assay
The cytocompatibility of fabricated constructs was evaluated by MTT assay.MTT is a valuable technique to assess the biocompatibility of the utilized materials for cell viability.The results of the MTT assay presented an increase in cell viability of the ADSCs at days 4 and 7 compared to day 1, which suggested that all the scaffolds were cell compatible consistent with previous studies [2,37].The integration of PCL scaffolds injected by cell laden fibrin and fibrin/ECM hydrogels significantly enhanced the proliferation of ADSCs after 7 days of cell culture compared to PCL scaffolds (Figure 4).The increased hydrophilicity and adhesive behavior of fibrin and ECM injected into the PCL scaffolds might be a possible reason for the improvement in cell proliferation.The porous structure of the scaffolds greatly contributes to the adhesion and proliferation of the cells [43].SDECM based scaffolds displayed outstanding cell growth and proliferation.Meanwhile, PCL scaffolds alone exhibited the lowest cell viability rate among the samples, because of the lack of ECM in their structure.
The physiochemical attributes of the scaffolds are affected by the presence of ECM in hydrogels [46].Accordingly, increased cell viability for the scaffolds containing ECM was significant compared to the other groups.The existing fibrin in the ECM structure is accountable for enhancing cellular adhesion.In addition, fibrin contributed to cellular growth [47].PCL is a hydrophobic polymer and does not strengthen cell attachment [48].Cartilage ECM is mainly composed of collagens (75%), GAG (17%), glycoproteins (<1%), and elastin (<1%) [35,49].However, compared with other groups, the cells in the PCL/F/ECM scaffolds exhibited the best proliferation rate.MTT results showed that the PCL/F/ECM scaffolds presented proper cytocompatibility.Li et al. [37] showed that the SMSCs could grow uniformly on the PCL/MECM scaffolds.They demonstrated that MECM injected between PCL fibers can offer a proper microenvironment for cell attachment and growth.The ADSC/chondrocyte growth in the alginate loaded on PCL constructions was much higher than in the ADSC/chondrocyte 2D co-culture in alginate.Jang et al. [50] concluded that an increase in cell viability could be attributed to the microenvironment differences created by the cell-laden hydrogel assemblies and 3D cell-laden PCL constructs.

Real-time PCR
ADSCs differentiation can be modified with various microenvironmental manipulations, such as topographical cues and chemical compositions [50].According to the viability assay and mechanical evaluation of the hybrid constructs, the (PCL/F/ECM) constructs were considered an optimum scaffold for further in vitro evaluations and the F/ECM scaffold was ignored for further evaluations.RT-PCR was performed to assess the expression levels of aggrecan (Acan), type II collagen (COLII), and type X collagen (COLX) in ADSCs cultured in Fibrin/ECM hydrogels (H) or in fibrin/ECM loaded on PCL 3D printed scaffolds (PCL/F/ECM) after 28 days of culture in the presence of Piascledine (ASU) or TGF-β3.Results showed that the expression of collagen II and aggrecan genes in the PCL/F/ECM 3D printed hybrid constructs was significantly increased compared to ADSCs culture in fibrin/ECM hydrogels in both the TGF and ASU media (Figure 5).In particular, PCL/F/ECM hybrid scaffolds presented a significantly higher chondrogenic gene expression than the Fibrin/ECM hydrogels.These results proposed that the culture of ADSCs in 3D printed hybrid scaffolds could display much improved chondrogenesis than ADSCs culture in the scaffold-free hydrogels.
It has been reported that the presence of Piascledine (ASU) in the culture medium for 21 days could induce the chondrogenesis of ADSCs in scaffolds [51].In this study, the chondrogenic induction potential of ASU and TGF-β3 was simultaneously compared after 28 days of culture.ASU possesses chondroprotective, anabolic, and anticatabolic properties.It inhibits the breakdown of cartilage and promotes cartilage repair by inhibiting several molecules and pathways.ASU stimulates the synthesis of collagen and aggrecan by inhibiting inflammatory cytokines such as IL-1, IL-6, IL-8, TNF, and PGE2 [52].Some of these effects have been described to be associated with the inhibition of NF-κβ nuclear translocation and an increase in the production of TGF-β in chondrocytes [53].The therapeutic role of ASU in treating osteoarthritis has been reported in several studies.Previous studies showed that ASU has a chondroprotective effect on chondrocytes [52].This study revealed an up-regulation in the gene expression profile of collagen type II and aggrecan compared to control groups, which are specific chondrogenic associated genes.
Henrotin YE et al. [53] stated that ASU can stimulate the expression of type II collagen in chondrocytes when cultured in a monolayer environment.This result suggests that ASU may be capable of inducing the endogenous production of TGF-βs in ADSCs, and as a result, chondrogenesis induction can occur.Furthermore, the expression of type X collagen, a hypertrophic factor, was considered a limitation during the differentiation of hADSCs to chondrocytes [54].Here, the expression of the hypertrophy-specific gene (collagen type X) on day 28 showed a significant decrease in ASU supplemented scaffolds compared to TGF-β3, consistent with previous studies' results [25,53,55].It should be noted that in order to induce chondrogenesis in stem cells and achieve hyaline cartilage, collagen X gene expression should be reduced to prevent the hypertrophy of the engineered cartilage.Our results indicate that Piascledine could significantly support the survival of differentiating ADSCs in scaffolds fabricated by 3D printing more effectively than scaffold free hydrogels.Furthermore, the use of Piascledine decreases the expression of collagen type X in 3D-printed scaffolds.This suggests that treatment using ASU in combination with cell-laden fibrin/ECM/3D-printed PCL scaffolds may be considered as an appropriate tool in CTE when compared to scaffold free hydrogels.

Immunohistochemistry (IHC) assay
IHC analyses showed the distribution of type II and type X collagen in the F/ECM hydrogels and cell laden Fibrin/ECM/3D-printed PCL scaffolds cultured in the presence of TGF-β3 and ASU.The results revealed that type II and X collagens expression in the PCL/F/ECM hybrid scaffolds was significantly higher than in the fibrin/ ECM hydrogels (p < 0.05) (Figure 6(A,B)).These data were quantified using Image J software (Image J 1.51, Java 1.6.0_24(64-bit).The average percentage of COLLII positive area was found to be significantly higher in TGF-β3 and ASU groups compared to the control groups (p < 0.05) (Figure 6(A-a)).
IHC showed that the expression of type X collagen was significantly decreased in the presence of ASU compared to the TGF-β3 (Figure 6(B-b)).Our results align with previous findings reported by other research groups, which have shown that medium containing TGF-β3 increased COLX protein expression significantly [56].Expression of COLX protein indicates hypertrophy in differentiated cells treated with TGF-β3.It has been previously shown that TGF-β3 increases cell hypertrophy and promotes cartilage to become bony [52].Deng et al. concluded that pre-loaded TGF-β3 in hydrogel promoted uniform GAG distribution in vivo, while collagen type II production did not occur without exogenous TGF-β3 supplementation [57].In the present study, Coll X protein was increased in differentiated cells in the medium containing TGF-β3 compared to ASU groups, which can suggest ASU as a proper alternative for TGF-β3 growth factor in cartilage regeneration.Past in-vitro and in-vivo studies have shown that ASU positively affects osteoarthritis [7], inhibits cartilage fracture, stimulates synthesis by inhibiting a number of molecules and pathways involved in osteoarthritis, and leads to collagen and Acan formation [8].It can also prevent the progression of osteoarthritis by inhibiting inflammatory cytokines such as IL-1, IL-6, IL-8, TNF, and PGE2 by modulating NF-кβ [9].In a previous study conducted on ASU, it was observed to induce more chondrogenic differentiation from hADSc in fibrin scaffolds than the 2D culture as control group [58].There was no evidence of the molecular mechanism of ASU on chondrogenic induction in stem cells.Yet, many studies have shown that ASU increased expression of COL II, Acan and SOX9 from chondrocytes in joint cartilage via inhibition of inflammatory molecules such as interleukins and TNF-α [59].

Histological examination
The basophilic matrix following Toluidin Blue (TB) staining indicated chondrogenic induction in hADSCs in target groups (Figure 7(A)).Furthermore, this data was quantified utilizing image J software (Image J 1.51, Java 1.6.0_24(64-bit) (Figure 7(B)).TB dye has a notable affinity for acidic polysaccharides (such as glycosaminoglycans), and is therefore useful for the identification of chondrogenesis.Semi-quantitative findings of TB staining indicated the accumulation of acidic glycoproteins in the cell laden 3D printed scaffolds to be significantly higher than the hydrogel groups (p < 0.05) cultured in the presence of the TGF-β3 and Piascledine (Figure 7(A)).TB staining also showed that in the presence of TGF-β3 and Piascledine, the accumulation of GAGs in the intercellular matrix significantly increased compared to control groups (p < 0.05) (Figure 7(A)).Generally, the blue color of the 3D scaffolds became more intense compared to hydrogel groups.These results demonstrate that the 3D printed scaffold has a strong potential clinical application for CTE.Other studies have shown that Piascledine could enhance the differentiation of ADSCs in fibrin scaffolds [25].Piascledine was shown to stimulate collagen and proteoglycan synthesis in cultured chondrocytes [60].A few in vitro studies have shown that Piascledine could stimulate aggrecan production and restore it after IL-1β treatment, significantly increased the expression of type II collagen in comparison to TGF-β1 during the induction of chondrogenesis in ADSCs [61], and could increase the glycosaminoglycan content in the scaffolds containing rabbit chondrocytes significantly after implantation under the skin of mice [14].Overall, it has been shown that Piascledine components exert anti-inflammatory and pro-anabolic effects in chondrocytes and enhance chondroprotective properties by stimulating the production of type II collagen and GAG.

Conclusion
PCL scaffolds were fabricated by 3D printing.SDECM was obtained after decellularization and solubilization of articular cartilage biopsies from the knee joints of the calf.Fibrinogen and thrombin were obtained from the blood plasma.Fibrin and Fibrin/ECM hydrogels were injected into the PCL scaffolds to fabricate cell laden hybrid scaffolds.According to the viability assay and physicomechanical evaluation of the scaffolds, the cell laden fibrin/ECM 3D printed PCL scaffold (PCL/F/ECM) was considered as an optimum scaffold for further in vitro evaluations, and the cell laden Fibrin 3D printed PCL scaffold was ignored for further evaluations.The cell laden hydrogels and hybrid scaffold were cultured for 28 days in the culture medium containing TGF-β3 or ASU to investigate chondrogenesis of the ADSCs treated by ASU compared to the TGF-β3 on the fabricated scaffolds and also to evaluate the effect of 3D printed framework on chondrogenesis.The findings of gene expression, IHC, and histological study indicated the overexpression of the type II collagen gene as well as accumulation of type II collagen protein and acidic glycoproteins in the cell laden 3D printed scaffolds compared to the hydrogel groups cultured in the presence of the TGF-β3 and ASU.In addition, the results showed that the expression of type X collagen was significantly decreased in the presence of ASU compared to the TGF-β3.The supplementation of ASU during chondrogenesis of ADSCs in the PCL/F/ECM scaffolds was found to be an effective inducer to improve cell proliferation, survival and differentiation.ASU also increased the main and specific markers of cartilage and reduced hypertrophic factors, such as collagen X, compared to TGF-β3.

Figure 2 (
Figure2(A) demonstrates the macroscopic pictures of 3D-PCL, 3D-PCL/fibrin, and 3D-PCL/fibrin/ECM scaffolds.The SEM images of the 3D-printed PCL scaffolds indicated a macroporous arrangement with interconnected and well-organized pores produced within the scaffold (Figure2(B)).Open pores with uniform shape and size were found in the 3D-printed PCL scaffolds with regular interconnected pores.In the other groups, porosity was decreased by the addition of fibrin and ECM to the PCL.Pore size is a key factor affecting the improvement of cell viability in the 3D porous network of the scaffolds.The size must be small enough to allow for cell attachment and large enough for the inflow of cells[33].Pore interconnectivity is considered a promising factor since it could contribute to cell migration into internal pores, improve the flow of nutrients and metabolic waste, and enhance the communication of cells in various pores during cell culture, while also providing sufficient space for cell growth[34,35].SEM micrographs for cell seeded PCL (Figure2(C-iv))

Figure 1 .
Figure 1.analysis of key biomolecules during the decellularization and solubilization process of the processed ecm.a) dna content; B) GaG content; and c) collagen content of lyophilized cartilage and sdcm (data presented as mean ± sd; *p < 0.05; n = 4).

Figure 3 .
Figure 3. representative stress-strain curves of the scaffolds.

Figure 7 .
Figure 7. (a) representative toluidine blue staining of the glycosaminoglycan (scale bar 100 μm), and the results of glycosaminoglycan intensity in different groups 28 days after the culture of adscs.H (hydrogel), cont (control), Pias(piascledine), 3d (3d printed scaffold), tGf (tGf-β).data are presented as mean ± sd.error bars represent the standard deviation of the mean.asterisks indicate significant differences between groups.*p < 0.05.(B) Quantified data using image J software.

Table 1 .
abbreviations of groups and subgroups.