Infrapatellar Fat Pads-Derived Stem Cell Is a Favorable Cell Source for Articular Cartilage TE

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

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: They compared the chondrogenicity of ASCs isolated from infrapatellar fat pads (IPFPs) and subcutaneous fat pads (SCFPs) in a 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 the 3D matrix. The difference in mRNA profile may have contributed to the 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 a 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.

Adipose tissue–derived stem cells (ASCs) are a promising cell source in regenerative medicine and can be isolated from various adipose tissue depots. In addition to affecting the ASC yield, comparative assessments of ASCs from subcutaneous, intramuscular, and visceral fat has revealed differences in differentiation potential. 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. 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 the chondrogenic capacity even when harvested from patients with osteoarthritis (OA). 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. 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 niche1.

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.

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.

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 weeks 1 and weeks 2 and 3. ASCs, adipose tissue–derived stem cells; IPFP, infrapatellar fat pads; SCFP, subcutaneous fat pads; MW, molecular weight.

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.

1. Wang, C.-C., Chen, I.-H., Yang, Y.-T., Chen, Y.-R., & Yang, K.-C. (2021). 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. CARTILAGE. https://doi.org/10.1177/1947603520988153