Sequential Zonal Chondrogenic Differentiation of Mesenchymal Stem Cells in Cartilage Matrices

Abstract

Engineering approaches that mimic the process of fetal development have the potential to regenerate the zonal organization of articular cartilage. The objective of this work was to investigate the effect of sequential addition of zone-specific growth factors BMP-7, IGF-1, and IHH to TGF-β1 supplemented chondrogenic medium on zonal differentiation of human mesenchymal stem cells (hMSCs) encapsulated in an articular cartilage-derived matrix. First, fetal or adult bovine articular cartilage was decellularized, digested, and methacrylate-functionalized to produce an injectable macromer (CarMa, f-CarMa for fetal, a-CarMa for adult) for encapsulation of hMSCs. Next, the optimum matrix source and initial cell density for chondrogenic differentiation of hMSCs to the superficial and calcified zone phenotypes were determined by encapsulation of the cells in CarMa hydrogel and incubation in chondrogenic medium/TGF-β1 supplemented with BMP-7 and IHH, respectively. Then, the encapsulated hMSCs were pre-exposed to BMP-7 supplemented chondrogenic medium/TGF-β1 and the effect of sequential addition of IGF-1 and IHH to the medium on the expression of zone-specific markers was investigated. According to the results, f-CarMa and high cell densities enhanced differentiation of the encapsulated hMSCs to the superficial zone phenotype whereas a-CarMa and low cell densities enhanced differentiation to the calcified zone. The addition of IGF-1 to the chondrogenic medium/TGF-β1 stimulated differentiation of the encapsulated hMSCs, pre-exposed, to BMP-7, to the middle zone phenotype. The addition of IHH to the chondrogenic medium/TGF-β1 stimulated maturation of the encapsulated hMSCs, pre-exposed to BMP-7 and IGF-1, to the calcified zone phenotype. The results are potentially useful for engineering injectable, cellular hydrogels for regeneration of full-thickness articular cartilage.

Advances and prospects in biomimetic multilayered scaffolds for articular cartilage regeneration

“…Moeinzadeh et al encapsulated human MSCs (hMSCs) in injectable decellularized cartilage macromer hydrogel and cultured them in chondrogenic medium/TGF-β1/BMP-7 to form a superficial zone-like cellular construct. Furthermore, the hMSCs in the hydrogel were stimulated to undergo differentiation into cells with middle- and calcified-zone phenotypes in chondrogenic medium/TGF-β1/IGF-1 and chondrogenic medium/TGF-β1/IHH [ 62 ].…”

Challenges for Natural Hydrogels in Tissue Engineering

“…A major challenge in articular cartilage regeneration is creating hydrogels that mimic the physical and biochemical properties of different cartilage zones. In that regard, digested and decellularized articular cartilage is used as an injectable matrix in cartilage regeneration [13,35]. The articular cartilage is cut into small pieces, frozen in liquid nitrogen, milled, and sieved to obtain <1 mm fragments.…”

“…Human adult mesenchymal stem cells (hMSCs) harvested from the patient’s bone marrow are used extensively in cellular therapies [10,13,44]. Therefore, hMSCs are used to study cell adhesion to the surface of feather β-keratin hydrogels.…”

“…Oxygen, nutrients, proteins and other biomolecules readily permeate through the water-swollen network of hydrogels to nourish the encapsulated cells [5,6]. Synthetic, as well as natural hydrogels, are used extensively as preformed or injectable matrices in cellular tissue constructs [7,8,9,10,11,12,13]. Although synthetic hydrogels provide matrices with tunable properties, they lack the bioactive amino acid sequences required for adhesion, growth, and maturation of the encapsulated cells [14,15].…”

Decellularized Articular Cartilage Microparticles for Expansion of Mesenchymal Stem Cells and Zonal Regeneration of Articular Cartilage

“…We previously demonstrated for the first time that high cellularity, low matrix stiffness and combination of transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-7 (BMP-7) led to chondrogenic differentiation of MSCs to the superficial zone phenotype of articular cartilage; medium cellularity and stiffness, and combination of TGF-β1 and IGF-1 led to the middle zone phenotype; and low cellularity, high matrix stiffness and combination of TGF-β1 and hydroxyapatite (HA) led to the calcified zone phenotype [16,17]. Further, we recently demonstrated that MSCs encapsulated in digested, decellularized articular cartilage could sequentially be differentiated to the superficial zone phenotype, followed by the middle and calcified zones by sequential supplementation of the chondrogenic medium with BMP-7, insulin growth factor-1 (IGF-1) and Indian hedgehog (IHH) [18]. In addition, we showed that digested and decellularized fetal articular cartilage induced differentiation of MSCs to the superficial zone phenotype of chondrocytes whereas the adult cartilage induced differentiation to the calcified phenotype.…”