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. 2021 Jan 13;13(2 Suppl):508S–520S. doi: 10.1177/1947603520988153

Infrapatellar Fat Pads–Derived Stem Cell Is a Favorable Cell Source for Articular Cartilage Tissue Engineering: An In Vitro and Ex Vivo Study Based on 3D Organized Self-Assembled Biomimetic Scaffold

Chen-Chie Wang 1,2, Ing-Ho Chen 1,2,3, Ya-Ting Yang 1, Yi-Ru Chen 1,4, Kai-Chiang Yang 1,4,✉
PMCID: PMC8804804  PMID: 33435725

Abstract

Objective

Adipose tissue–derived stem cells (ASCs) are a promising source of cells for articular cartilage regeneration. However, ASCs isolated from different adipose tissue depots have heterogeneous cell characterizations and differentiation potential when cultured in 3-dimensional (3D) niches.

Design

We compared the chondrogenicity of ASCs isolated from infrapatellar fat pads (IPFPs) and subcutaneous fat pads (SCFPs) in 3D gelatin-based biomimetic matrix.

Results

The IPFP-ASC-differentiated chondrocytes had higher ACAN, COL2A1, COL10, SOX6, SOX9, ChM-1, and MIA-3 mRNA levels and lower COL1A1 and VEGF levels than the SCFP-ASCs in 3D matrix. The difference in mRNA profile may have contributed to activation of the Akt, p38, RhoA, and JNK signaling pathways in the IPFP-ASCs. The chondrocytes differentiated from IPFP-ASCs had pronounced glycosaminoglycan and collagen type II production and a high chondroitin-6-sulfate/chondroitin-4-sulfate ratio with less polymerization of β-actin filaments. In an ex vivo mice model, magnetic resonance imaging revealed a shorter T2 relaxation time, indicating that more abundant extracellular matrix was secreted in the IPFP-ASC–matrix group. Histological examinations revealed that the IPFP-ASC matrix had higher chondrogenic efficacy of new cartilaginous tissue generation as evident in collagen type II and S-100 staining. Conclusion. ASCs isolated from IPFPs may be better candidates for cartilage regeneration, highlighting the translational potential of cartilage tissue engineering using the IPFP-ASC matrix technique.

Keywords: adipose tissue–derived stem cells, subcutaneous fat pad, infrapatellar fat pad, cartilage regeneration, chondrogenic differentiation

Introduction

Adipose tissue–derived stem cells (ASCs) are a promising cell source in regenerative medicine and can be isolated from various adipose tissue depots. 1 In addition to affect the ASC yield, 2 comparative assessments of ASCs from subcutaneous, intramuscular, and visceral fat has revealed differences in differentiation potential.3,4 Infrapatellar fat pads (IPFPs), which are located between the joint capsule and synovium, were demonstrated to have the same cell origin as hyaline cartilage in the knee joint during embryonic development. 5 According to embryologic developmental theory, the stem cells in IPFPs may have similar stemness, especially chondrogenic potential, as the stem cells harvested from the superficial zone of hyaline cartilage. Moreover, IPFP-ASCs were reported to have chondrogenic capacity even when harvested from patients with osteoarthritis (OA). 6

Most of the studies related to the chondrogenic induction of ASCs from different sources have been based on the 2-dimensional (2D) monolayer culture model. In addition to donor factors and the adipose tissue depot, dimensionality, a physical cue, was found to influence the differentiation potential of ASCs. 7 More recently, ASCs have been shown to have different chondrogenic profiles when cultured in 2D versus 3D microenvironments. 8 Even when ASCs are cultured in a 3D environment such as a tissue-engineered scaffold, the nonuniform culture niche may complicate their spatial effects on cellular differentiation. Accordingly, the present study employed the biomimetic scaffold with a honeycomb-like structure to analyze the engineering of cartilage tissue from adipose tissues in IPFPs (Hoffa’s fat pads) and subcutaneous fat pads (SCFPs) and evaluated the regenerative capability of the ASCs in the 3D niche.

Materials and Methods

Tissue Harvesting, Cell Cultivation, and Characterization

Adipose tissues were harvested from SCFPs (for the isolation of SCFP-ASCs) and IPFPs (for the isolation of IPFP-ASCs) of the stifle joint of the hind legs of 5 New Zealand white rabbits (10 weeks old) with the approval of IACUC (102-IACUC-022). The adipose tissues were minced and digested in 0.1% collagenase type I (C0130, Sigma-Aldrich) solution at 37°C for 30 minutes. At 37°C and in 5% CO2, the isolated cells were cultivated in proliferation medium (high-glucose Dulbecco’s modified Eagle’s medium, D5648, Sigma-Aldrich) containing 10% fetal bovine serum (SH30071.03, Hyclone GE Healthcare Life Sciences, UT, USA) and 1% antibiotic solution. The population doubling time, flow cytometry analysis, colony-forming efficiency, and differentiation potentials of SCFP-ASCs and IPFP-ASCs were demonstrated, and the results were provided in the Supplemental Material (Supplemental Fig. S1).

Cell Proliferation, Glycosaminoglycan Production, and Survival of SCFP-ASC- and IFPD-ASC-Differentiated Chondrocytes in 3D Matrix

This study fabricated 3D matrixes (gelatin-based scaffold with an interconnecting honeycomb-like structure) by using flow-focusing microfluidic technology, as described in a previous study. 8 The porosity of the scaffold was 97%, and the pore size was controlled to 100 μm. SCFP-ASCs and IPFP-ASCs in passages 3 to 5 were collected individually and resuspended in culture medium; 2 × 105 cells in 100 μL of medium were seeded into the 3D matrixes by using a micropipette.

SCFP-ASCs and IPFP-ASCs were seeded into 3D matrix and cultured in proliferation medium for 2 days. The medium was then replaced with a chondrogenic medium (HyClone AdvanceSTEM Chondrogenic Differentiation Medium, Cat. SH30889.02) and cultured for an additional 3 weeks. Cell proliferation was determined through total DNA quantification (DNeasy Blood and Tissue kit, 69504, QIAGEN, Germany) after culturing in chondrogenic medium for 1, 2, and 3 weeks (n = 6 for each group at each time point). The glycosaminoglycan (GAG) production of SCFP-ASC- and IPFP-ASC-differentiated chondrocytes in 3D matrix was measured using a 1,9-dimethyl-methylene blue (DMMB) assay (341088, Sigma-Aldrich). The amount of GAG secreted was normalized to the number of cells in accordance with the total DNA content. Each group was composed of 6 replicates.

After 3 weeks of cell cultivation in 3D matrix, cell survival was estimated by applying 10 nM calcein acetoxymethyl stain (C3099, Thermo Fisher Scientific) for 60 min and observing the staining using a microscope (ECLIPSE TE2000-U, Nikon).

mRNA Expression of SCFP-ASC- and IPFP-ASC-Differentiated Chondrocytes in 3D Matrix

The total RNA of the samples was extracted (74106, RNeasy Mini Kit, Qiagen), and cDNA was synthesized from the RNA by using SuperScript III RT (18080051, Thermo Fisher Scientific). Collagen type I (COL1A1), collagen type II (COL2A1), collagen type X (COL10), aggrecan (ACAN), runt-related transcription factor 2 (RUNX2), SRY-related HMG box 6 (SOX6), SOX9, chondromodulin-1 (ChM-1), vascular endothelial growth factor (VEGF), and melanoma inhibitory activity protein (MIA-3) were selected as the target genes for gene expression analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was employed as an endogenous housekeeping gene.

Western Blot Analysis

Chondrogenic-differentiation-related kinases were determined using western blotting. The samples were lysed in protein extraction buffer (CelLytic M, C2978, Sigma-Aldrich) containing protease inhibitor cocktail (cOmplete ULTRA Tablets, 04693159001, Roche). In each lane, 10 μg of total cell extract protein was separated through sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene difluoride membrane (Amersham Hybond P 0.45 PVDF, GE Healthcare Life Science). The membrane was incubated with primary antibodies against phospho-Akt (9271, Cell Signaling), Akt (9272, Cell Signaling), phospho-p38 (4631, Cell Signaling), p38 (9212, Cell Signaling), phospho-AMPK (2531, Cell Signaling), AMPK (2532, Cell Signaling), ERK (9102, Cell Signaling), phospho-ERK (9101, Cell Signaling), RhoA (total RhoA protein, 2117, Cell Signaling), JNK (9252, SAPK/JNK, Cell Signaling), and β-actin (A5441, Sigma-Aldrich). The membrane was subsequently probed with a horseradish-peroxidase-conjugated secondary antibody (A0545, Sigma-Aldrich). Immune complexes were detected using a SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). The bands of each target protein were calculated and revealed through relative densitometry. Each group had three replicates.

Histological Study and Immunohistochemical Staining against the Extracellular Matrix

After culturing for 1, 2, and 3 weeks, the samples were retrieved (Suppl. Fig. S2) and prepared for histologic examinations. The paraffin samples were cut into 8-μm slides sequentially and stained with hematoxylin and eosin (H&E, 3008-1 and 3204-2, Muto, Japan). Alcian blue (Muto, Japan) was used to detect the GAG production in the samples, and nuclear fast red was employed for counterstaining.

For immunohistochemical (IHC) staining, the samples were pretreated as described in a previous study and incubated overnight with antibodies against collagen type I (1:500, bs-0578R, Bioss, Woburn, USA), collagen type II (1:200, bs-0709R, Bioss), or S-100 (1:500, bs-1248R, Bioss). 9 Otherwise, the sections were incubated with anti-collagen type X antibody (1:2000, ab49945, Abcam, Cambridge, UK) at 37°C for 1 hour. The presence of antigens was illustrated by a brown color with 3,3′-diaminobenzidine (Super Sensitive Polymer-HRP Detection System, BioGenex, CA, USA). The cell nucleus was subsequently counterstained with Mayer’s hematoxylin.

The distribution and orientation of the cytoskeletons in ASC-differentiated chondrocytes in 3D matrix were analyzed by labeling with fluorescent dyes. The samples were embedded in optimal cutting temperature compound (Tissue-Tek O.C.T. Compound, Sakura Finetek), prepared, cut into 150-μm slices, and mounted on slides. To quench the autofluorescence of the gelatin scaffold, the slides were immersed in 0.4% NaBH4 solution for 10 minutes. The samples were subsequently incubated with primary anti-β-actin antibodies (1:150, A5441, Sigma-Aldrich) at room temperature for 1 hour and then the primary anti-α-tubulin antibodies (1:200, MA190017, Thermo) at 4°C overnight. The presence of the first antibody was detected using the second antibodies tagged with red fluorescent dye (1:200, goat anti-mouse IgG labeled with CF 647, Biotium, Inc.) and green fluorescent dye (1:200, ab150157, goat anti-rat IgG H&L Alexa Fluor 488, Abcam) at room temperature for 1 hour. Finally, to visualize the cell nucleus, the samples were stained with 4′,6-diamidino-2-phenyl-indole (300 mM, Sigma-Aldrich) for 10 minutes and inspected using a confocal microscope (Leica TCS SP5 II, Leica).

Isolation and Characterization of Proteoglycan

To identify the components of proteoglycans, we further analyzed the respective quantity of chondroitin-6-sulfate (C6S, MW larger than 140 kDa) and chondroitin-4-sulfate (C4S, 45 kDa) in proteoglycans by using Western blotting. The same number of SCFP-ASC or IPFP-ASC was seeded into a 3D matrix and cultured in chondrogenic medium for 1 to 3 weeks. In the predetermined time point, the cell/matrix construct was weighted, and proteoglycan was isolated by extraction solution. The extraction solution containing 4 M guanidine-HCl, 0.05 M sodium acetate (pH 6), and protease inhibitor was added to the samples at and shaken at 4°C overnight. The supernatant was transferred to a new tube, and the residual fluid was subjected to the same extraction process a second time. The supernatant from the second extraction was homogeneously mixed with the first extracts, and 400 μL of ethanol was added to 50 μL of the mixed supernatant at −80°C for 1 hour, after which the mixture was centrifuged at 4°C for 10 minutes. The pellet was washed twice using 95% ethanol at 4°C, reconstituted with digestion buffer (0.1 M Tris buffer, pH 8) to the concentration of 0.5 mg/µL. Finally, 1 μL of chondroitinase ABC (25 U/mL, C3667, Sigma-Aldrich) was added to 25 µL samples, and the mixture was maintained at 37°C overnight. The samples (10 µL/line) were subsequently treated with Western blot sample buffer at 100°C for 5 minutes and processed in 4% to 15% gradient gel for the Western gradient procedure (anti-chondroitin-6-sulfate monoclonal antibody, MAB2035; anti-chondroitin-4-sulfate monoclonal antibody, MAB2030, Millipore).

Ex Vivo Study in Mice with Severe Combined Immunodeficiency

SCFP-ASCs and IPFP-ASCs (2 × 105) were seeded in 3D matrix and cultured in proliferation medium for 3 days, followed by culturing in chondrogenic medium for an additional 4 days. CB-17/lcr mice with severe combined immunodeficiency (SCID, male, 6 weeks old, Biolasco, Taiwan) were used as the recipients. The cell-matrix constructs were implanted under general anesthesia through intraperitoneal injections of anesthetic (0.06 mL/10 g body weight; a mixture of 1 mL of Zoletil [50 mg/mL, Virbace], 0.1 mL of xylazine [Sun Star Chem and Pharm Corp.], and 3.9 mL of normal saline). After adequate skin preparation and sterilization, the SCFP-ASC matrix and IFPD-ASC matrix were implanted subcutaneously in the left side of the back of one mouse (n = 5 for each time point) using a straight-line incision on the right side of the back, with a pocket to the contralateral side. Negative controls were the blank 3D matrixes (n = 2 for each time point) embedded at the same subcutaneous locations. The incision was then closed with a nonabsorbable suture. The mice received postoperative injections of cephalosporin (4 mg/kg body weight) to prevent infection.

Ex Vivo Magnetic Resonance Imaging Inspection

Magnetic resonance imaging (MRI) was performed using a 7T MRI scanner (Bruker Biospec 70/30 MRI). Rapid acquisition and relaxation enhancement T1 (RARE-T1) and fast low-angle shot T2 (FLASH-T2) measurements were made at 0, 4, 6, 12, and 24 weeks after implantation. The T1 measurements were conducted with a repetition time (TR) of 1300 ms and echo time (TE) of 9 ms. The field of view (FOV) was encoded at 3.5 × 3.5 mm2 and 1-mm slice thickness. The T2 weighted images were attained with a TR of 558 ms and TE of 9.2 ms. The FOV and slice thickness settings were the same as for the T1 measurements. These imaging parameters were further calculated for evaluating the properties of regenerated cartilage tissues in the backs of the mice. 10

Retrieval of Cartilage-Like Tissue for Histological Examination

The mice were sacrificed at 2, 4, 6, and 24 weeks after implantation, and the implants were removed together with the covering surrounding tissue from the subcutaneous site. The samples were prepared for histological examinations as reported in the previous section. The sections were stained with H&E and Alcian blue and subjected to IHC staining against collagen type I and type II, type X antibodies, and S-100.

Statistical Analysis

Data are expressed as mean ± standard deviation. Statistical analyses were conducted using analysis of variance with post hoc Dunnett’s or Scheffe’s multiple comparison tests. A P value of less than 0.05 was considered statistically significant.

Results

Cell Proliferation, GAG Production, and Survival of SCFP-ASCS and IPFP-ASCS in 3D Matrix

The amount of total DNA was not changed significantly at the various time points for either SCFP-ASCs or IPFP-ASCs ( Fig. 1A ), which may have revealed that the 3D matrix was filled with cells. In addition, no significant difference was discovered in the total DNA between the SCFP-ASCs and IPFP-ASCs. The DMMB assay revealed that the SCFP-ASCs had a stable GAG/DNA ratio ( Fig. 1B ). In contrast, the GAG/DNA ratio of the IPFP-ASCs increased over time and was significantly higher than that of the SCFP-ASCs (P < 0.05 at weeks 1 and 2; P < 0.01 at week 3).

Figure 1.

Figure 1.

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The SCFP-ASCs and IPFP-ASCs were seeded in gelatin-based 3D matrix and evaluated. (A) Total DNA content did not increase over time, which revealed that the 3D matrix was full of cells. (B) DMMB assay revealed that the IPFP-ASCs had a significantly higher GAG/DNA ratio than the SCFP-ASCs. (C) Surviving cells (green fluorescence) distributed uniformly among the internal porous spaces of the 3D matrix (red fluorescence); abundant cells were found within the internal spaces of the 3D matrix (scale = 100 µm). ASCs, adipose tissue–derived stem cells; IPFP, infrapatellar fat pads; SCFP, subcutaneous fat pads; DMMB, 1,9-dimethyl-methylene blue; GAG, glycosaminoglycan.

The distribution and survival of cells in 3D matrix were evaluated using calcein acetoxymethyl staining. Gelatin has autofluorescence, and thus, the color red represented gelatin matrixes whereas green indicated live cells under fluorescent microscope inspection. Live cells were distributed uniformly in the internal porous spaces of the 3D matrix, with no empty spaces observed ( Fig. 1C ).

mRNA Expression of SCFP-ASC- and IPFP-ASC-Derived Chondrocytes

Relative to undifferentiated cells, the IPFP-ASC-derived chondrocytes had higher ACAN mRNA expression than the SCFP-ASC-derived chondrocytes in 3D matrix at week 1 (P < 0.05) ( Fig. 2 ). Although COL2A1 was upregulated in the SCFP-ASC-derived chondrocytes, the IPFP-ASC-derived chondrocytes had extremely high COL2A1 expression (P < 0.001 for all time points). The SCFP-ASC-derived chondrocytes exhibited high COL1A1 expression that was significantly different from that of the IPFP-ASC-derived chondrocytes (P < 0.05 at weeks 2 and 3). However, a high COL10 level was discovered in the IPFP-ASC-derived chondrocytes at weeks 1 and 2 (both Ps < 0.05). The IPFP-ASC-derived chondrocytes also had higher expression of SOX6 (weeks 1 and 2; both Ps < 0.01), SOX9 (week 1; P < 0.01), ChM-1 (weeks 1, 2, and 3; P < 0.01), and MIA-3 (P < 0.05 at weeks 1 and 2, P < 0.01 at week 3) and lower expression of VEGF (weeks 2 and 3; P < 0.05), RUNX2 (P < 0.05 at week 2, P < 0.01 at week 3) than the SCFP-ASC-derived chondrocytes in 3D matrix.

Figure 2.

Figure 2.

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mRNA expression profile of the ASC-derived chondrocytes in 3D matrix. The IPFP-ASC-derived chondrocytes had higher ACAN mRNA expression than did the SCFP-ASC-derived chondrocytes at week 1 and extremely high COL2A1 expression. By contrast, the SCFP-ASC-differentiated chondrocytes exhibited significantly higher COL1A1 expression at weeks 2 and 3. Although the IPFP-ASC-derived chondrocytes had high COL10 level at weeks 1 and 2, they also had significantly higher SOX6 (weeks 1 and 2), SOX9 (week 1), ChM-1 (weeks 1, 2, and 3), and MIA-3 (weeks 1, 2, and 3) levels and lower VEGF (weeks 2 and 3) and RUNX2 (weeks 2 and 3) levels than the SCFP-ASC-derived chondrocytes in 3D matrix. ASCs, adipose tissue–derived stem cells; IPFP, infrapatellar fat pads; SCFP, subcutaneous fat pads.

Western Blotting

When chondrogenic medium was provided to the cells, the IPFP-ASCs had higher phospho-Akt/Akt and phospho-p38/p38 ratios than those of the SCFP-ASCs in 3D matrix ( Fig. 3 ). In both groups, the phospho-p38/p38 ratio was decreased at week 3; however, the IPFP-ASCs still had a higher phospho-p38/p38 ratio than the SCFP-ASCs at week 3. No differences were discovered in the phospho-AMPK/AMPK or phospho-ERK/ERK ratio between these 2 types of ASCs under chondrogenic induction in 3D matrix. Regarding RhoA signaling, The IPFP-ASCs had significant higher RhoA activity than the SCFP-ASCs at week 1, whereas no differences were found at week 2 or 3. Similarly, the IPFP-ASCs had significantly higher JNK activity than the SCFP-ASCs at week 1.

Figure 3.

Figure 3.

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The IPFP-ASCs had higher phospho-Akt/Akt and phospho-p38/p38 ratios than did the SCFP-ASCs throughout the 3-week chondrogenic induction period. No differences were discovered in the phospho-AMPK/AMPK or phospho-ERK/ERK ratio between these two types of ASC. The IPFP-ASCs had significantly higher RhoA and JNK activities than did the SCFP-ASCs at week 1 under chondrogenic induction. ASCs, adipose tissue–derived stem cells; IPFP, infrapatellar fat pads; SCFP, subcutaneous fat pads.

Hematoxylin and Eosin Staining and Immunostaining of Extracellular Matrix and Immunofluorescent Staining of Cytoskeletal Elements

Histological examinations showed that both the SCFP-ASCs- and IPFP-ASC-differentiated chondrocytes were distributed uniformly in the 3D matrix, and H&E and Alcian blue staining showed abundant extracellular matrix (ECM) deposition ( Fig. 4A ). Cell aggregations were discovered and found to be positive to collagen type II and S-100 but negative to collagen type I immunostaining. The IPFP-ASC-derived chondrocytes had particularly strong signals in collagen type II staining performed at week 3. The SCFP-ASC-derived chondrocytes had weak signals in collagen type X staining.

Figure 4.

Figure 4.

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Histological and IHC inspections were conducted on the SCFP-ASCs and IPFP-ASC matrices under chondrogenic induction. (A) H&E staining revealed that both the SCFP-ASC- and IPFP-ASC-differentiated chondrocytes were distributed uniformly in the 3D matrix. Alcian blue staining showed abundant ECM deposition. Clusters were positive to collagen type II and S-100 but negative to collagen type I staining. The IPFP-ASC-derived chondrocytes had strong signals in collagen type II staining at week 3, whereas the SCFP-ASC-derived chondrocytes showed weak signals in collagen type X staining (scale = 100 µm). (B) The SCFP-ASC-derived chondrocytes had cortical β-actin and uniformly distributed α-tubulin in the cytoplasm, and these cells had more polymerized β-actin filaments with a directional orientation in α-tubulins when compared with those of the IPFP-ASC-derived chondrocytes (scale = 50 µm). ASCs, adipose tissue–derived stem cells; IPFP, infrapatellar fat pads; SCFP, subcutaneous fat pads; IHC, immunohistochemistry; H&E, hematoxylin and eosin; ECM extracellular matrix.

Regarding the cytoskeletons, β-actin filaments (red appearance) were mainly cortical actins, whereas α-tubulin (green appearance) was distributed uniformly and occupied most of the cytoplasm for the SCFP-ASC-derived chondrocytes ( Fig. 4B ). The β-actin filaments were more polymerized with a directional orientation in α-tubulins in the SCFP-ASC-differentiated chondrocytes than in the IPFP-ASC-derived chondrocytes.

Characterization of Proteoglycans and C6S/C4S Ratio

Proteoglycans were isolated, reacted with antibodies, and analyzed using Western blotting ( Fig. 5A ). The C6S/C4S ratio in the SCFP-ASC-derived chondrocytes did not change over time, remaining at approximately 1. In contrast, the C6S/C4S ratio was higher than 1 and increased gradually from week 1 to weeks 2 and 3 for the IPFP-ASC-differentiated chondrocytes ( Fig. 5B ).

Figure 5.

Figure 5.

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Analysis of C6S/C4S ratio in SCFP-ASC- and IPFP-ASC-derived chondrocytes in 3D matrix. (A) Total proteoglycans were reacted with C6S or C4S antibodies and analyzed using Western blotting. C6S (MW larger than 140 kDa) and C4S (45 kDa) were detected. (B) The SCFP-ASC-derived chondrocytes had a similar C6S/C4S ratio (at approximately 1) at all time points. By contrast, the IPFP-ASC-differentiated chondrocytes had a higher C6S/C4S ratio at week 1, and the ratio increased between week 1 and weeks 2 and 3. ASCs, adipose tissue–derived stem cells; IPFP, infrapatellar fat pads; SCFP, subcutaneous fat pads; MW, molecular weight.

Ex Vivo MRI Inspection

To investigate the MRI signal change from newly generated cartilage-like tissue, the difference in chondrogenicity for ASCs obtained from different sources in 3D matrix in a SCID mice model was evaluated. T2 fast low-angle shot (FLASH) ( Fig. 6A ) and T1 rapid acquisition with relaxation enhancement (RARE) ( Fig. 6B ) MRI were performed at weeks 0, 4, 6, 12, and 24. The IPFP-ASC–matrix group had slightly lower T2 signal intensity than the SCFP-ASC–matrix group did ( Fig. 6C ). Regarding the T1 RARE images, the signal intensity of the IPFP-ASC–matrix group increased gradually, whereas the signal intensity of the SCFP-ASC–matrix group decreased significantly over time ( Fig. 6D ). The IPFP-ASC–matrix group had significantly higher signal intensity than the SCFP-ASC–matrix group after week 4.

Figure 6.

Figure 6.

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ASCs were seeded in 3D matrix and cultured for 3 days in proliferation medium and an additional 4 days in chondrogenic medium before implantation in SCID mice. (A) T2 FLASH and (B) T1 RARE MRI was performed at weeks 0, 4, 6, 12, and 24. (C) The IPFP-ASC–matrix group had slightly lower T2 signal intensity than did the SCFP-ASC–matrix group (D). Regarding the T1 RARE image, the 2 groups achieved the same level of signal intensity at week 4. The signal intensity of the IPFP-ASC–matrix group increased gradually, whereas the signal intensity of the SCFP-ASC–matrix group decreased significantly over time. ASCs, adipose tissue–derived stem cells; IPFP, infrapatellar fat pads; SCFP, subcutaneous fat pads; SCID, severe combined immunodeficiency; FLASH, fast low-angle shot; RARE, rapid acquisition with relaxation enhancement.

Histological Inspection of the Retrieved Samples

Under histological examination, it was evident in the sections of cartilage-like samples that the internal spaces in the 3D matrix were filled with cells. Alcian blue staining revealed that considerable ECM was secreted by the resident cells ( Fig. 7 ). The SCFP-ASC- and IPFP-ASC-differentiated chondrocytes had positive collagen type II and S-100 but negative collagen type I and collagen X staining. The IPFP-ASC-differentiated chondrocytes had stronger positive collagen type II and S-100 staining at week 2, and their staining resembled that of the SCFP-ASC-differentiated chondrocytes at week 4. However, SCFP-ASC-differentiated chondrocytes showed a condensed ECM with more pronounced staining for collagen type II at week 6. The 3D matrix degraded gradually, and residual materials were visualized after 6 months. The newly formed cells and ECM grew further and fused after the degradation of the 3D matrix.

Figure 7.

Figure 7.

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The ASC–matrix groups were retrieved from SCID mice for histological examination. H&E and Alcian blue staining revealed that the internal space of the 3D matrix was full of cells and a large amount of ECM. Both the SCFP-ASC- and IPFP-ASC-differentiated chondrocytes secreted collagen type II and S-100 stain; collagen type I and collagen X staining was not detected. The IPFP-ASC-differentiated chondrocytes had stronger positive collagen type II and S-100 staining at week 2 and similar staining to the SCFP-ASC-differentiated chondrocytes at week 4. The 3D matrix degraded gradually, and the newly formed cells and ECM further grew and fused with the residual matrix after 6 months (scale = 100 µm). ASCs, adipose tissue–derived stem cells; IPFP, infrapatellar fat pads; SCFP, subcutaneous fat pads; H&E, hematoxylin and eosin; ECM, extracellular matrix; SCID, severe combined immunodeficiency.

Discussion

ASCs are a promising source of cells for the treatment of OA and other cartilage lesions. 11 However, the heterogeneity of cell proliferation, stemness, and differentiation capacity are major concerns related to ASCs. Otherwise, ASCs have been demonstrated to have different chondrogenic profiles when cultured in 2D and 3D niches.7,8 Therefore, we compared the chondrogenicity of ASCs isolated from IPFPs and SCFPs in 3D biomimetic matrixes.

Compared with the SCFP-ASC, the IPFP-ASC-derived chondrocytes had a higher GAG/DNA ratio ( Fig. 1B ). The ASCs from both SCFPs and IPFPs grew and became cell aggregates in the 3D matrix ( Fig. 1C ). In one study, the formation of cell spheroids enhanced the chondrogenic potential of ASCs. 12 In the IPFP-ASC group, the spheroid structure was composed of more cells and more ECM than that in the SCFP-ASC group, which indicated that the ASCs isolated from IPFPs had a superior spheroid structure, possibly benefiting chondrogenesis.

The inherited potential of angiogenesis is another concern when using ASCs as a cell source for cartilage tissue engineering. 13 Hyaline cartilage is avascular tissue in which nutrition is mainly provided by joint fluid diffusion. If the engineered cartilage has angiogenesis potential, neovascularization may invade the newly formed cartilage and lead the hyaline cartilage to differentiate into hypertrophied cartilage, impairing the tissue’s ability to sustain mechanical loading. Pires et al. 14 reported that SCFP-ASCs harvested from a patient with OA expressed more endothelial cell marker CD31 than did IPFP-ASCs. Another study has mentioned that ASCs are unsuitable for cartilage tissue engineering because they secrete angiogenic factors. 13 Nevertheless, all other studies have employed a 2D culture system rather than simulated cellular subsistence in a 3D biological environment. Our study first revealed that mRNA expression of VEGF was not upregulated when SCFP-ASCs were cultured in 3D matrix with chondrogenic induction, and the IPFP-ASCs had lower VEGF expression than did the SCFP-ASCs ( Fig. 2 ). IPFP-ASC-differentiated chondrocytes also had higher ACAN (at week 1), COL2A1, SOX6 (at weeks 1 and 2), SOX9 (at week 1), ChM-1, and MIA-3 (at weeks 1 and 2) expression and lower COL1A1, COL10, and RUNX2 expression. SOX6 is known to drive ACAN expression by securing the binding of SOX9 to a down-upstream target such as COL2A1. 15 Furthermore, MIA-3 can stabilize cartilage differentiation by regulating signaling processes during differentiation. 16 Likewise, ChM-1 was shown to maintain a chondrocyte’s phenotype and prevent hypertrophy. 17 The pattern of mRNA expression revealed that the IPFP-ASCs were a superior cell source for articular cartilage repair.

In addition to chondrogenic gene expression, signaling pathways such as Akt and p38 MAPK are known to be active during chondrogenesis.18,19 Compared with the SCFP-ASCs, the IPFP-ASCs exhibited higher Akt and p38 MAPK activities under chondrogenic induction, especially at weeks 1 and 2 ( Fig. 3 ). The phosphorylation of AMPK is known to be decreased during chondrogenic differentiation, and the activation of phospho-AMPK inhibited the gene expression of COL2A1, ACAN, and SOX9 in one study. 20 By contrast, inhibiting ERK phosphorylation attenuated SOX9 expression and chondrogenic gene expression in human ASCs. 21 Despite the SCFP-ASC- and IPFP-ASC-differentiated chondrocytes having differences in mRNA expression of the aforementioned genes, we did not discover a clear difference in AMPK activity. Additionally, no difference was found in the ERK phosphorylation between the SCFP-ASC and IPFP-ASC groups. RhoA/ROCK signaling regulates the chondrogenesis of ASCs through the control of L-SOX5, SOX6, and SOX9 expression in a context-dependent manner. 22 Lu et al. 23 reported that the raw materials of a 3D niche had an impact on the RhoA/Rock signaling pathway to ASCs, and regulation of ROCK1 and ROCK2 expression was not consistent under chondrogenic induction. Additionally, JNK activity was enhanced in bone marrow–derived mesenchymal stem cells (MSCs) during chondrogenesis. 24 We also discovered in this study that the IPFP-ASCs exhibited different RhoA and JNK activities to the SCFP-ASCs in week 1. Taken together, these findings revealed that under chondrogenic induction, the IPFP-ASCs had different Akt, p38, RhoA, and JNK activities during the early and middle stages (weeks 1 and 2) relative to the SCFP-ASCs.

SCFP-ASC- and IPFP-ASC-differentiated chondrocytes showed differences in the ECM-related mRNA expression ( Fig. 2 ) and chondrogenesis-related signaling pathway ( Fig. 3 ), and thus may also affect ECM production in the 3D niche ( Fig. 4A ). The IPFP-ASC-differentiated chondrocytes had abundant ECM depositions in the 3D matrix. In addition, high ACAN and COL2A1 expression in the IPFP-ASC-differentiated chondrocytes resulted in pronounced Alcian blue (GAG) and collagen type II and S-100 staining, especially at week 3. This finding revealed that the IPFP-ASC-differentiated chondrocytes had a similar composition of ECM to hyaline cartilage. 25 Akt and RhoA/ROCK signaling are known to modulate actin organization.18,26 Although disruption of actin filaments and inhibition of actin polymerization were found to induce a chondrogenic phenotype in human MSCs, 27 the role of tubulin during MSC differentiation is not fully understood. However, reorganization of the microtubule array was essential to the chondrogenesis of bovine MSCs. 28 We noticed that the chondrocytes derived from SCFP-ASCs had directional tubulin orientation, whereas the arrangement of the tubulin network was different in the IPFP-ASC-differentiated chondrocytes, revealing that the modulation of tubulin in chondrogenic induction may be influenced by the location from which ASCs are harvested ( Fig. 4B ).

Chondroitin sulfate (CS), the major component of ECM in articular cartilage, plays a critical role in maintaining structural integrity of ECM. The major proteoglycan in mature articular cartilage is C6S, and only a small amount of C4S has been found in well-developed diarthrodial joints. 29 Aging and inflammation decrease the C6S/C4S ratio, an index that may indicate chondrocyte vitality and phenotype maintenance. 30 In our study, the secretion of C6S was significantly higher than that of C4S at weeks 1, 2, and 3 in the IPFP-ASC group; the reverse was noted in the SCFP-ASC group, with the C4S concentration higher than the C6S concentration at all assayed times ( Fig. 5 ). The proportional C6S and C4S content was similar to that in the superficial zone, with a higher percentage of C6S in the IPFP-ASC group. Thus, the differentiated cells maintained the phenotype as chondrocytes in the superficial layer.

The SCFP-ASCs and IPFP-ASCs exhibited differing chondrogenic potential in the 3D matrix in vitro, resulting in different properties of the newly formed cartilaginous tissues ex vivo. The T2 FLASH MRI images of the mouse model revealed that the IPFP-ASC matrix construct had shorter T2 relaxation time than the SCFP-ASC–matrix group after postimplantation week 4 ( Fig. 6 ). Shorter T2 relaxation time was inferred to result in more abundant ECM secreted and a favorable chondrogenic phenotype being retained. 16 The T1 RARE images showed that the signal intensity of the SCFP-ASC–matrix group decreased quickly. The results revealed faster scaffold degradation in the SCFP-ASC–matrix group in the ex vivo model. Although both the SCFP-ASC- and IPFP-ASC-derived chondrocytes deposited ECM functionally ( Fig. 7 ), the quality of the ECM was difficult to evaluate using histological staining, and MRI could be employed to assess the focal adhesion of the ECM and scaffold in between. Correspondingly, the chondrocytes derived from IPFP-ASCs have a great potential for cartilage repairing even ex vivo.

A limitation of this study is the ectopic implantation of ACS-matrix in SCID mice. In articular cartilage, oxygen tension ranges from 53 mm Hg in the superficial layer to 7.6 mm Hg in the deep zone. 31 On the contrary, the oxygen tension is around 40 to 50 mm Hg in knee joint capsule and 40 to 60 mm Hg in subcutaneous space, respectively.32,33 Regarding the influences of oxygen tension on differentiation, MSCs can be programmed to chondrogenic cell fate by low oxygen tension. 34 Likewise, low oxygen tension is also known to enhance chondrogenesis of IPFP in agarose hydrogels in the presence of TGF-β3. 35 However, the well-vascularized environment in subcutaneous pocket may affect the phenotyping of tissue engineered cartilage. Therefore, orthotopic transplantation of the IPFP-ASC–matrix in a big animal model may provide solid evidence to support the findings of this study.

In conclusion, the IPFP-ASC–matrix had higher chondrogenic potential and efficacy of new cartilaginous tissue generation than the SCFP-ASC–matrix. The ASCs isolated from IPFPs are thus better than those isolated from SCFPs as a source of cells for cartilage regeneration.

Supplemental Material

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Footnotes

Supplementary material for this article is available on the Cartilage website at https://journals.sagepub.com/home/car.

Authors’ Note: This study was conducted in Taipei Tzu Chi Hospital and Taipei Medical University simultaneously.

Acknowledgments and Funding: This work was supported by the Ministry of Science and Technology, Taiwan (Grant number MOST106-2314-B-303-002) and Buddhist Tzu Chi Medical Foundation (TCMMP-106-05-02).

Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Informed Consent: No human subjects were enrolled in this study, and institutional review board approval was not required.

ORCID iD: Kai-Chiang Yang Inline graphic https://orcid.org/0000-0002-0979-4463

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Supplementary Materials

sj-pdf-3-car-10.1177_1947603520988153 – Supplemental material for Infrapatellar Fat Pads–Derived Stem Cell Is a Favorable Cell Source for Articular Cartilage Tissue Engineering: An In Vitro and Ex Vivo Study Based on 3D Organized Self-Assembled Biomimetic Scaffold
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Supplemental material, sj-pdf-3-car-10.1177_1947603520988153 for Infrapatellar Fat Pads–Derived Stem Cell Is a Favorable Cell Source for Articular Cartilage Tissue Engineering: An In Vitro and Ex Vivo Study Based on 3D Organized Self-Assembled Biomimetic Scaffold by Chen-Chie Wang, Ing-Ho Chen, Ya-Ting Yang, Yi-Ru Chen and Kai-Chiang Yang in CARTILAGE

sj-tif-1-car-10.1177_1947603520988153 – Supplemental material for Infrapatellar Fat Pads–Derived Stem Cell Is a Favorable Cell Source for Articular Cartilage Tissue Engineering: An In Vitro and Ex Vivo Study Based on 3D Organized Self-Assembled Biomimetic Scaffold
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Supplemental material, sj-tif-1-car-10.1177_1947603520988153 for Infrapatellar Fat Pads–Derived Stem Cell Is a Favorable Cell Source for Articular Cartilage Tissue Engineering: An In Vitro and Ex Vivo Study Based on 3D Organized Self-Assembled Biomimetic Scaffold by Chen-Chie Wang, Ing-Ho Chen, Ya-Ting Yang, Yi-Ru Chen and Kai-Chiang Yang in CARTILAGE

sj-tif-2-car-10.1177_1947603520988153 – Supplemental material for Infrapatellar Fat Pads–Derived Stem Cell Is a Favorable Cell Source for Articular Cartilage Tissue Engineering: An In Vitro and Ex Vivo Study Based on 3D Organized Self-Assembled Biomimetic Scaffold
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Supplemental material, sj-tif-2-car-10.1177_1947603520988153 for Infrapatellar Fat Pads–Derived Stem Cell Is a Favorable Cell Source for Articular Cartilage Tissue Engineering: An In Vitro and Ex Vivo Study Based on 3D Organized Self-Assembled Biomimetic Scaffold by Chen-Chie Wang, Ing-Ho Chen, Ya-Ting Yang, Yi-Ru Chen and Kai-Chiang Yang in CARTILAGE

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