Changes in Bone Structure Following Implantation of Biphasic and Triphasic Strontium Enriched Biomaterials in Animals with Experimental Osteoporosis. Summary of the Doctoral Thesis

Abstract

Bone regeneration is provided by complicated interaction between osteocytes, osteoblasts and osteoclasts, which is regulated by different morphogens, remodeling and transcription factors, inflammatory and anti-inflammatory cytokines. When bone physiological metabolisms is disrupted by a musculoskeletal disease such as osteoporosis or sever skeletal trauma, surgical treatment of the bone pathologies can lead to increased rate of the bone fracture, nonunion and metal implant dislocation. To overcome those complications, implants are enriched with different metal ions by creating physiologically active biomaterials to improve bone regeneration. One of them is strontium (Sr), which chemical properties are similar to calcium ions found in bone tissue. Incorporation of Sr during bone remodeling, has led to its local application in variety of biomaterials used in tissue engineering. Sr simultaneously can increase new bone formation and decrease osteoclast activity during bone resorption. However, to better understand Sr induced changes during bone remodeling, it is important to analyze interaction of biologically active factors within different bone condition. The aim of the study was to determine and describe bone remodeling, mineralization, growth, immunological and osteoclastogenesis related factors in healthy and osteoporotic rabbits bone after implantation of biphasic and triphasic Sr enriched biomaterials. Forty-six female rabbits were included in the study, where 10 of them composed a control group, but osteoporosis was induced in 36 by ovarectomy and glucocorticosteroids. Osteoporosis affected rabbits were further divided into groups, and bone defect in femur bone of right leg were filled with hydroxyapatite (HA) 30% and tricalcium phosphate (TCP) 70%, 5% Sr enriched HA_30/TCP_70, HA_70/TCP_30, Sr-HA_70/TCP_30 granules or left empty in sham group. Tissue samples were obtained 12 weeks after implantation, where bone samples were taken either form left non-operated leg. Histomorphometry was used to analyze peculiarities of bone area between healthy and osteoporotic rabbits. Immunohistochemistry was used to evaluate osteoprotegerin (OPG), nuclear factor kappa beta-105 (NFkB-105), osteocalcin (OC), bone morphogenetic protein-2/4 (BMP-2/4), collagen-1-alpha (Col-1α), matrix metalloproteinase-2 (MMP-2), tissue inhibitor of matrix metalloproteinase-2 (TIMP-2), interleukin-1 (Il-1) and interleukin-10 (Il-10) containing structures between healthy and osteoporotic rabbits. Bone structure of control group was formed by compact bone and osteon channels filled with blood vessels and cancellous bone with distinct bone trabeculae, osteocytes and red bone marrow filled with adipose tissue. Unlike, osteoporotic bone structure showed notably thinner compact bone, less osteon channels, where high amount of connective tissue was found. Also bone trabeculae were notably thinner and less frequent. And red bone marrow consisted more adipose tissue. Such bone structural changes were found between all osteoporotic animals. Trabecular bone area in healthy rabbits was 0.393 mm^2, while in osteoporotic rabbits it varied from 0.206 mm^2 to 0.242 mm^2, which was significantly less. Osteoporotic operated rabbit bone samples, regardless of used biomaterials, showed increased expression of Col-1α, BMP-2/4, TIMP-2 and Il-1 compared to healthy animals. Only Sr-HA_70/TCP_30 bone samples were able to increase expression of OC and NFkB-105, and HA_70/TCP_30 bone samples higher expression of Il-10, while number of OPG and MMP-2 positive bone cells were almost equal. Similarly, sham bone samples showed higher expression of Col-1α, TIMP-2 and Il-1 compared to healthy rabbits. Nevertheless, operated sham bone samples increased only expression of Col-1α compared to non-operated leg. Whereas Sr-HA_70/TCP_30 and HA_70/TCP_30 biomaterials induced higher expression of Col-1α, NFkB-105, OC, OPG, BMP-2/4, MMP-2, TIMP-2, Il-1, Il-10 compared to non-operated leg. Moreover, Sr enriched biomaterials compared to other study groups showed higher expression of osteogenesis, cellular activity, mineralization and remodeling related factors, while increasing osteoclastogenesis inhibitor factor OPG in osteoporotic bone condition. Distribution of Il-1 and Il-10 among osteoporotic bone samples was equal. Osteoporotic bone structure is characterized by miscellaneous expression of analyzed factors. Despite decreased bone remodeling capacity under osteoporotic conditions, our results reveals that tissue trauma and implanted biomaterials improve bone regenerative properties compared to healthy bone. Biomaterials induce stimulation of bone matrix remodeling, immunomodulatory response, osteoblastogenesis, mineralization and bone cell activity, while suppress osteoclastogenesis. Sr enriched biomaterials do not increase inflammatory adverse reaction and maintain similar level to trauma induced pro- and anti-inflammatory response. Sr-HA_70/TCP_30 biomaterials induce most noticeable osteoporotic bone tissue response.