Inducing articular cartilage phenotype in costochondral cells - PubMed (original) (raw)

Inducing articular cartilage phenotype in costochondral cells

Meghan K Murphy et al. Arthritis Res Ther. 2013.

Abstract

Introduction: Costochondral cells may be isolated with minimal donor site morbidity and are unaffected by pathologies of the diarthrodial joints. Identification of optimal exogenous stimuli will allow abundant and robust hyaline articular cartilage to be formed from this cell source.

Methods: In a three factor, two level full factorial design, the effects of hydrostatic pressure (HP), transforming growth factor β1 (TGF-β1), and chondroitinase ABC (C-ABC), and all resulting combinations, were assessed in third passage expanded, redifferentiated costochondral cells. After 4 wks, the new cartilage was assessed for matrix content, superficial zone protein (SZP), and mechanical properties.

Results: Hyaline articular cartilage was generated, demonstrating the presence of type II collagen and SZP, and the absence of type I collagen. TGF-β1 upregulated collagen synthesis by 175% and glycosaminoglycan synthesis by 75%, resulting in a nearly 200% increase in tensile and compressive moduli. C-ABC significantly increased collagen content, and fibril density and diameter, leading to a 125% increase in tensile modulus. Hydrostatic pressure increased fibril diameter by 30% and tensile modulus by 45%. Combining TGF-β1 with C-ABC synergistically increased collagen content by 300% and tensile strength by 320%, over control. No significant differences were observed between C-ABC/TGF-β1 dual treatment and HP/C-ABC/TGF-β1.

Conclusions: Employing biochemical, biophysical, and mechanical stimuli generated robust hyaline articular cartilage with a tensile modulus of 2 MPa and a compressive instantaneous modulus of 650 kPa. Using expanded, redifferentiated costochondral cells in the self-assembling process allows for recapitulation of robust mechanical properties, and induced SZP expression, key characteristics of functional articular cartilage.

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Figures

Figure 1

Figure 1

Neocartilage morphology and histology. (A) A flat disc-shaped construct was observed in control, hydrostatic pressure (HP) and chondroitinase ABC (C-ABC) treatments. The addition of transforming growth factor beta-1 (TGF-β1) resulted in a bowl-shaped construct. (B) Histological staining demonstrated the presence of collagen (picrosirius red) and glycosaminoglycans (Safranin-O/fast-green) in all groups. Immunohistochemistry demonstrated the presence of type II collagen, and the absence of type I collagen, independent of the treatment regimen. Immunohistochemical staining for superficial zone protein (SZP) demonstrated its presence in the superficial zone of articular cartilage, and its absence in costal cartilage. Engineered neocartilage demonstrated the presence of SZP independent of treatment regimen. Morphology scale bar = 5 mm, histology scale bar = 100 μm.

Figure 2

Figure 2

Biochemical composition of neocartilage normalized to tissue wet weight and DNA. (A) Chondroitinase ABC (C-ABC) and transforming growth factor beta-1 (TGF-β1) individually enhanced collagen density (per wet weight (ww)), and C-ABC/TGF-β1 and hydrostatic pressure (HP)/C-ABC/TGF-β1 treatments synergistically enhanced collagen density (per ww). (B) Reflecting phenotypic changes, TGF-β1 led to the greatest increase in collagen production per cell. (C), (D) TGF-β1 additionally enhanced the glycosaminoglycan (GAG) content, normalized to ww and DNA, while C-ABC and combined treatments containing C-ABC showed the lowest GAG/DNA. Data reported as mean ± standard deviation. All groups not connected by a common lowercase letter are significantly different (P <0.05).

Figure 3

Figure 3

Scanning electron microscopy images and fibril analysis in neocartilage. (A) Samples were fixed and imaged after 4 weeks culture. Scale bar = 1 μm. (B) Chondroitinase ABC (C-ABC), hydrostatic pressure (HP), and all combinatorial treatments significantly increased the fibril diameter. Fibril density was significantly increased over control in all treatments with the exception of hydrostatic pressure. Data reported as mean ± standard deviation. All groups not connected by a common lowercase letter are significantly different (P <0.05). TGF-β1, transforming growth factor beta-1.

Figure 4

Figure 4

Tensile and compressive mechanical properties of neocartilage. (A), (B) Chondroitinase ABC (C-ABC)and transforming growth factor beta-1 (TGF-β1) significantly increased tensile stiffness and strength, while tensile properties trended higher with hydrostatic pressure (HP). The C-ABC/TGF-β1 and HP/C-ABC/TGF-β1 treatments synergistically enhanced tensile strength. (C), (D) TGF-β1 also significantly increased compressive instantaneous and relaxation moduli. Combined C-ABC/TGF-β1 treatment synergistically increased instantaneous modulus. GAG depletion with C-ABC treatment did not compromise compressive properties below control. Data reported as mean ± standard deviation. All groups not connected by a common lowercase letter are significantly different (P <0.05).

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