Effects of whole bone scaling on isolated osteoblast behaviour are unknown. With two orders of magnitude range in body mass, dog breeds are well-suited to determine such relationships.
Femoral heads from three canine hip replacement surgeries were collected. Bone fragments were washed in PBS+AB/AM, trypsin-digested and incubated in 0.2% collagenase. Cells from resultant supernatant were seeded in DMEM+10% FCS+AB/AM at 37 °C, 5% CO2, grown until confluence and then seeded (n=6) at a density of 1.3×104 cells/cm2. We determined cell proliferation after 4 days with crystal violet staining and basal alkaline phosphatase (TNAP) activity 1, 7 and 14 days post-confluence.
Median cell number reached 52.5×103 [30.1−72.5×103] cells/cm2 with doubling time of 3.5 [1.6−4.9] days. We found that cells from the 1 year-old Briard had greater cell count and shorter doubling time than the 8 year-old Labrador (P<0.001 for both cases). TNAP activity at day 1 post-confluence was increased in the Labrador sample when compared to the 1 year-old Briard (0.01 [0.01−0.02] vs 0.03 [0.02−0.04] U/min per mg protein; P<0.001). This difference wasnt found at subsequent time-points since TNAP activity in the Labrador sample decreased at day 7 post-confluence. TNAP is a key enzyme involved in mineralization and we are now optimising culture conditions to stimulate matrix mineralisation activity.
Age is an important factor in skeletal maturation/senescence: growth plates in dogs close at 1218 months old and seniority is reached at 513 years depending on dog size and breed. These data provide an important step towards cell culture optimization for canine osteoblasts highlighting the importance of obtaining skeletally mature samples in comparative biology studies.
|Mass (Kg)||Sex||Age (years)||Breed|
|Canine 1||39||Male||5.53||German shepherd|
14 May 2016 - 17 May 2016