J. Sun (Taiyuan, CN)
The Second Hospital of Shanxi Medical University Department of OrthopaedicsPresenter Of 2 Presentations
23.3.6 - Deleted Ihh in Mesenchyme Promotes Chondrocyte Hypertrophy and Pathological Matrix Calcification
Abstract
Purpose
Indian hedgehog (Ihh) is known to control hypertrophic differentiation process of chondrocytes, its role from the mesenchyme cells to the early stages of chondrogenesis is unclear. To test the role of Ihh in the mesenchyme cells to the earlier stages of chondrogenesis, we have generated Prx1-Cre;Ihhfl/fl;Rosa26-ZsGreen1 mice to precisely delete Ihh in the mesenchyme cells. we can also track and isolate the deleted Ihh mesenchyme with EGFP. Thus, we can determine the role of Ihh from the mesenchyme to the earlier stage of chondrogenesis and bone formation in vivo.
Methods and Materials
The Prx1-Cre;Ihhfl/+;Rosa26-ZsGreen1 male mice were interbred with female Ihhfl/fl mice to obtain Prx1Cre;Ihhfl/fl;Rosa26-ZsGreen1 animals.Safranin-O staining was performed to assess glycosaminoglycan production and von Kossa staining was performed to evaluate the mineralization of bone. RNA in situ hybridization for Collagen X mRNA was performed using the RNAscope® 2.0 paraffin embedded (FFPE) Reagent Kit. The complete bone marrow cavity of the tibia was evaluated by micro-CT.
Results
In situ hybridization on paraffin sections prepared from tibia at P0, a strong Collagen X mRNA was found in the whole medullary cavities in the mutant tibia while collagen X mRNA was only presented in pre-hypertrophic zone in control. Hypertrophic chondrocytes are located at hypertrophic zone without ossification in control mice, while whole cartilage template is von Kossa positive staining in mutant mice (Fig 1). The GFP positive cells that were derived from deleted Ihh mesenchyme cells overlap with von Kassa and Osteocalcin positive staining area. Bone histomorphometric analyses indicated that deleted Ihh results in more and disorganized trabecular formation (Fig 2).
Conclusion
Our findings demonstrate that deleting Ihh in mesenchyme cells during early limb development promote chondrocyte hypertrophy and pathological matrix calcification
23.4.4 - Upregulation of IL-1β, -6 and TNF-α is responsible for the form of post-traumatic osteoarthritis in a porcine model
Abstract
Purpose
The mechanisms of PTOA following anterior cruciate ligament (ACL) injury are likely due to the biological and mechanical insults at the time of injury. These patients still remain at high risk for PTOA after treated with ACL reconstruction (ACL-R). We do not know if the cartilage degeneration after ACL-R is primarily due to abnormal mechanics, inflammation or both. “Idealized” ACLR model (restore normal joint kinematic) was used to test if inflammatory factors are responsible for PTOA.
Methods and Materials
Eighteen-month-old female mini-pigs were divided into “idealized” anterior cruciate ligament reconstruction (IACLR) group and control group (n=16 limbs per group). Real-time PCR, safranine O staining and indian ink staining were performed to verify whether animal models were successfully established or not. Multiple linear regression analysis was used to evaluate the correlation between levels of the inflammatory factors by the Luminex (including interferon [IFN]-γ, interleukin [IL]-1β, IL-4, IL-6, IL-8, IL-10, IL-12 and tumor necrosis factor [TNF]-α) and changes in cartilage histology (quantified by morphological scoring) after surgery.
Results
A significant OA cartilage damage with increased MMP-1, MMP-13 mRNA levels and reduced the level of aggrecan mRNA/protein was observed in IACLR groups. Gross morphology score from IACLR animals was dramatically increased compared with control. Moreover, IACLR animals significantly increased the levels of IL-1β, IL-4, IL-6 and TNF-α in the synovial fluid of the knee compared with control. The levels of IL-1β, IL-6 and TNF-α are correlated with morphological score of PTOA.
Conclusion
IACLR is well-known restoring normal joint kinematic model. These results demonstrated that inflammatory factors are independently responsible for the onset of PTOA.