The effect of calcium phosphate biodegradable coatings of titanium implants on cell differentiation and apoptosis in rat bone tissue after experimental fracture

Abstract

BACKGROUND: The effectiveness of bone repair is determined by the balance of proliferative and destructive factors in the fracture union site. It can be enhanced by using various nanostructured materials possessing osteoinductive properties, in particular titanium implants with biodegradable calcium phosphate coatings. The effects of these coatings on the state of stem cells, their differentiation and distribution in the repair zone is unknown. OBJECTIVE: To study the dynamics of proliferation, differentiation, and apoptosis of stem cells after experimental fracture followed by implantation of titanium implants with calcium phosphate coatings. METHODS: The localization of proliferation (PCNA) and differentiation (CD44 and osteocalcin) factors and apoptotic molecules (MDM2, p53, caspase-3) was studied in a rat femoral fracture model with implant placement. Titanium implant screws with bioactive calcium phosphate and hydroxyapatite coatings formed by plasma electrolytic oxidation were used in the study. Experimental rats were arranged into three groups (15 animals per group): control group; rats implanted with uncoated implants; and rats implanted with coated implants. Control rats were subject to a similar fracture as experimental ones and were allowed to heal conservatively. Rats from all groups were sampled on days 7, 14, and 30 after injury. RESULTS: Low-differentiated PCNA-, osteocalcin-, and CD44-immunopositive cells were localized around the implant in the inner layer of the periosteum, layer of outer circumferential lamellae, and connective tissue lining of haversian canals. The spatial density of cells expressing the above proliferation and differentiation factors, as well as that of MDM2-immunoreactive cells, increased on day 7 and decreased by day 30 after injury. The spatial density of apoptotic cells reached the maximum on day 14 after injury. They were mainly found in the inner layer of the periosteum and outer circumferential lamellae. p53- and caspase-3-positive cells occurred on the surface of the concentric lamellae surrounding haversian canals and under the periosteum. Their spatial density decreased by day 30 after injury. CONCLUSIONS: Calcium phosphate coatings stimulate cell proliferation at early stages of fracture restoration and apoptotic cell death at later stages. Coating components may provide positional information guiding the differentiation of mesenchymal stromal cells. A change in the activity of apoptotic factors, osteocalcin, and CD44 is caused by gene induction in response to the diffusion of calcium phosphate compounds from coating to surrounding tissue.