High seeding density of human chondrocytes in agarose produces tissue-engineered cartilage approaching native mechanical and biochemical properties

Abstract

Animal cells have served as highly controllable model systems for furthering cartilage tissue engineering practices in pursuit of treating osteoarthritis. Although successful strategies for animal cells must ultimately be adapted to human cells to be clinically relevant, human chondrocytes are rarely employed in such studies. In this study, we evaluated the applicability of culture techniques established for juvenile bovine and adult canine chondrocytes to human chondrocytes obtained from fresh or expired osteochondral allografts. Human chondrocytes were expanded and encapsulated in 2% agarose scaffolds measuring Ø3–4 mm × 2.3 mm, with cell seeding densities ranging from 15 to 90 × 106 cells/mL. Subsets of constructs were subjected to transient or sustained TGF-β treatment, or provided channels to enhance nutrient transport. Human cartilaginous constructs physically resembled native human cartilage, and reached compressive Young’s moduli of up to ~250 kPa (corresponding to the low end of ranges reported for native knee cartilage), dynamic moduli of ~950 kPa (0.01 Hz), and contained 5.7 percent wet weight (%/ww) of glycosaminoglycans (≥ native levels) and 1.5 %/ww collagen. We found that the initial seeding density had pronounced effects on tissue outcomes, with high cell seeding densities significantly increasing nearly all measured properties. Transient TGF-β treatment was ineffective for adult human cells, and tissue construct properties plateaued or declined beyond 28 days of culture. Finally, nutrient channels improved construct mechanical properties, presumably due to enhanced rates of mass transport. These results demonstrate that our previously established culture system can be successfully translated to human chondrocytes.

Mechanical properties and structure‐function relationships of human chondrocyte‐seeded cartilage constructs after in vitro culture

“…While a few studies have characterized the frictional and shear properties of tissue engineered articular cartilage constructs cultured with bovine or porcine cells, this study is the first to perform such analysis on human chondrocyte‐seeded implants. Additionally, only a few previous studies have characterized the compressive properties of human chondrocyte seeded implants . Overall, the results of this study provide a deeper understanding of the spectrum of mechanical behavior exhibited by tissue engineered human articular cartilage.…”

Nutrient Channels Aid the Growth of Articular Surface-Sized Engineered Cartilage Constructs

“…69 The current study, in tandem with our recent and promising results from constructs seeded with young and adult human chondrocytes (ages 17 to 35), suggests that engineering articular surfacesized constructs with human cells is a feasible strategy for treating advanced OA. 49 Study 1 showed that premature channel filling was detrimental to constructs (Fig. 4C, D and 5C), whereas Study 2 constructs with repunched channels exhibited the highest functional properties (Fig.…”

“…12,[74][75][76][77] With regard to (b), we have recently observed that young adult human chondrocytes, isolated from expired osteochondral allografts from a tissue bank, exhibit chondrogenic potential nearly comparable to that of immature bovine chondrocytes. 49 Therefore, we plan to further investigate this source of human cells in our effort to translate our tissue engineering approach to the clinic.…”

“…Thus, other studies have used passaged bovine, canine, or human chondrocytes. 34,[41][42][43][44][45][46][47][48][49] A direct comparison of passaged versus primary bovine chondrocytes in agarose could elucidate whether results derived with primary bovine cells are applicable for passaged cells.…”

Optimizing nutrient channel spacing and revisiting TGF-beta in large engineered cartilage constructs

“…Further, juvenile chondrocytes have shown promise for clinical applications in allogeneic transplantation (Adkisson et al, 2010). Ongoing and future studies by our group seek to translate results in bovine model systems to human chondrocytes (Cigan et al, 2016; O'Connell et al, 2015). …”