The effect of skeletal maturity on the regenerative function of intrinsic ACL cells

Abstract

Anterior cruciate ligament (ACL) injuries are an important clinical problem, particularly for adolescent patients. The effect of skeletal maturity on the potential for ACL healing is as yet unknown. In this study, we hypothesized that fibroblastic cells from the ACLs of skeletally immature animals would proliferate and migrate more quickly than cells from adolescent and adult animals. ACL tissue from skeletally immature, adolescent, and adult pigs and sheep were obtained and cells obtained using explant culture. Cell proliferation within a collagen-platelet scaffold was measured at days 2, 7, and 14 of culture using AM MTT assay. Cellular migration was measured at 4 and 24 h using a modified Boyden chamber assay, and cell outgrowth from the explants also measured at 1 week. ACL cells from skeletally immature animals had higher proliferation between 7 and 14 days (p<0.01 for all comparisons) and higher migration potential at all time points in both species (p<0.01 for all comparisons). ACL cells from skeletally immature animals have greater cellular proliferation and migration potential than cells from adolescent or adult animals. These experiments suggest that skeletal maturity may influence the biologic repair capacity of intrinsic ACL cells.

Figure 1

(A) Cell number of IMMATURE, ADOLESCENT, and ADULT porcine composites at days 2, 7, and 14. Mean absorbance in the MTT assay correlated directly with cell number. Values are expressed as mean ± SD, with n = 8. * Indicates a significant difference between IMMATURE and ADOLESCENT cell (p = 0.010), # indicates a significant difference between IMMATURE and ADULT cells (p = 0.044). (B) Cell number of IMMATURE, ADOLESCENT, and ADULT ovine composites at days 2, 7, and 14. Mean absorbance in the MTT assay correlated directly with cell number. Values are expressed as mean ± SD, with n = 8. *Indicates a significant difference between IMMATURE and ADOLESCENT cells (p = 0.0091), # indicates a significant difference between IMMATURE and ADULT cells (p < 0.0001), ♠ indicates a significant difference between ADOLESCENT and ADULT cells (p = 0.007).

Figure 2

Ovine cell provisional scaffold contraction. Percent of day 0 area is represented at days 0, 2, 7, and 14. Note the increased rate of contraction in the ADOLESCENT and ADULT scaffolds when compared with the scaffolds seeded with IMMATURE ACL cells (p < 0.0001 for both comparisons at day 14). Error bars represent one standard deviation.

Figure 3

Area covered by cells growing from ACL explants cultured in complete medium at week 1. The values represent the square area of the culture dish covered by the outgrowing layer of cells from each individual ACL explant. Values are expressed as area of outgrowth ± SD, where * indicates IMMATURE and ADOLESCENT cell coverage was significantly different from ADULT outgrowth, p < 0.001 and p < 0.05.

Figure 4

(A) Cell migration of IMMATURE, ADOLESCENT, and ADULT porcine cells at 4 and 24 h. Results reported as optical density, which correlates directly with cell number using this assay. Values are expressed as mean ± SD, where n = 8. * Indicates IMMATURE cells migrated significantly more than ADOLESCENT and ADULT cells at 4 h, p < 0.001. ^# indicates IMMATURE cells migrated significantly more than ADOLESCENT (p < 0.01) and ADULT cells (p < 0.001) at 24 h. (B) Cell migration of IMMATURE, ADOLESCENT, and ADULT sheep cells at 4 h. Results reported as optical density, which correlates directly with cell number using this assay. Values are expressed as mean ± SD, where n = 8. * Indicates IMMATURE cells migrated significantly faster than cells from ADOLESCENT and ADULT ACLs at 4 and 24 h, p < 0.01.