Passaged Articular Chondrocytes From the Superficial Zone and Deep Zone Can Regain Zone-Specific Properties After Redifferentiation

Abstract

Background: Bioengineered cartilage is a developing therapeutic to repair cartilage defects. The matrix must be rich in collagen type II and aggrecan and mechanically competent, withstanding compressive and shearing loads. Biomechanical properties in native articular cartilage depend on the zonal architecture consisting of 3 zones: superficial, middle, and deep. The superficial zone chondrocytes produce lubricating proteoglycan-4, whereas the deep zone chondrocytes produce collagen type X, which allows for integration into the subchondral bone. Zonal and chondrogenic expression is lost after cell number expansion. Current cell-based therapies have limited capacity to regenerate the zonal structure of native cartilage.

Hypothesis: Both passaged superficial and deep zone chondrocytes at high density can form bioengineered cartilage that is rich in collagen type II and aggrecan; however, only passaged superficial zone-derived chondrocytes will express superficial zone-specific proteoglycan-4, and only passaged deep zone-derived chondrocytes will express deep zone-specific collagen type X.

Study design: Controlled laboratory study.

Methods: Superficial and deep zone chondrocytes were isolated from bovine joints, and zonal subpopulations were separately expanded in 2-dimensional culture. At passage 2, superficial and deep zone chondrocytes were seeded, separately, in scaffold-free 3-dimensional culture within agarose wells and cultured in redifferentiation media.

Results: Monolayer expansion resulted in loss of expression for proteoglycan-4 and collagen type X in passaged superficial and deep zone chondrocytes, respectively. By passage 2, superficial and deep zone chondrocytes had similar expression for dedifferentiated molecules collagen type I and tenascin C. Redifferentiation of both superficial and deep zone chondrocytes led to the expression of collagen type II and aggrecan in both passaged chondrocyte populations. However, only redifferentiated deep zone chondrocytes expressed collagen type X, and only redifferentiated superficial zone chondrocytes expressed and secreted proteoglycan-4. Additionally, redifferentiated deep zone chondrocytes produced a thicker and more robust tissue compared with superficial zone chondrocytes.

Conclusion: The recapitulation of the primary phenotype from passaged zonal chondrocytes introduces a novel method of functional bioengineering of cartilage that resembles the zone-specific biological properties of native cartilage.

Clinical relevance: The recapitulation of the primary phenotype in zonal chondrocytes could be a possible method to tailor bioengineered cartilage to have zone-specific expression.

Keywords: bioengineered cartilage; phenotypic memory; tissue engineering; zonal chondrocytes.

Figure 1:. Effects of monolayer expansion on SZC and DZC.

(A) Calculated expansion yield fold at each passage and (B) % attachment of P0 and P2 SZC and DZC. (C) Confocal microscopy showing F-actin organization and cell shape (Gray = phalloidin for F-actin; blue = Hoechst for nuclei), (D) Area, and (E) Circularity of passaged SZC and DZC. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2:. The effects of passaging on chondrogenic, fibroblastic and zone-specific mRNA levels.

Real-time RT-PCR demonstrating a decrease in (A) chondrogenic mRNA levels and increased (B) Fibroblastic mRNA levels. Passaging decreases mRNA levels of (C) SZ-specific genes PRG4 and CLU, and (D) DZ-specific genes COLX and CHAD. mRNA levels were expressed as a percent of P0 SZ mRNA levels. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3:. The effect of redifferentiating SZC and DZC on chondrogenic and zone-specific gene expression.

Real-time RT-PCR demonstrating re-expression of (A) chondrogenic and a decrease in (B) fibroblastic mRNA levels, after redifferentiating P2 SZC and DZC. In addition, redifferentiation showed re-expression of mRNA levels of (C) SZ-specific genes in P2 SZC, and (D) DZ-specific in P2 DZC. mRNA levels are expressed as a percent of SZ P0 mRNA levels. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4:. Matrix accumulation and mechanical properties of redifferentiated P2 SZC and DZC.

(A) Toluidine Blue and H&E staining of passaged SZC and DZC at 11 and 21 days of redifferentiation. (B) GAG and (C) collagen accumulated per dry weight at 11 and 21 days. Mechanical indentation testing outputs (D) thickness and (E) Bulk Modulus of tissues at day 21. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5:. Matrix and zone-specific protein accumulation of redifferentiated P2 SZC and DZC.

Confocal microscopy images showing matrix accumulation of (A) ACAN and COL2 and decreased matrix accumulation of (B) COL1, a marker of fibroblastic differentiation. Additionally, the re-acquisition of DZ-specific (C) COLX and SZ-specific protein (D) PRG4. Specific proteins are shown in grayscale and tissues were counterstained with Hoechst for Nuclei (Blue). (E) PRG4 secretion of SZ and DZ tissue at days 11 and 21. *p < 0.05, **p < 0.01, ***p < 0.001.