ABSTRACT
BACKGROUND:Upregulation of hedgehog protein signaling can increase the expression of osteoarthritis markers,Runx2,a disintegrin and metalloproteinase with thrombospondin motifs,collagen type X alpha 1,and matrix metalloproteinase 13,while inhibition of hedgehog proteins attenuates the severity of osteoarthritis.It is speculated that osteoarthritic chondrocytes can influence bone formation by affecting osteoblasts through the Indian hedgehog protein(IHH)signaling pathway. OBJECTIVE:To investigate the effect of human osteoarthritic chondrocytes on subchondral osteoblasts. METHODS:Tibial plateau specimens from patients with osteoarthritis were collected.Chondrocytes were extracted using enzymatic digestion,and osteoblasts were extracted using enzymatic pre-digestion + bone block method.Chondrocytes were identified by toluidine blue staining and immunofluorescence and osteoblasts were identified by alkaline phosphatase staining and immunofluorescence.Chondrocytes were cultured in sodium alginate beads to maintain chondrocyte phenotype and co-cultured with osteoblasts.The co-culture system was added with IHH signaling pathway inhibitor(cyclopamine,10 nmol/L)and activator(purmorphamine,10 nmol/L)separately.After 48 hours of co-culture,osteoblasts from each group were collected,mRNA expressions of Gli1,osteoprotegerin,Runx2,parathyroid hormone-related peptide,alkaline phosphatase,receptor activator of nuclear factor-kB ligand(RANKL)and osteocalcin were detected by qRT-PCR,and protein expressions of GLi1,oseoprotegerin and RANKL in osteoblasts were detected by western blot. RESULTS AND CONCLUSION:The mRNA expression levels of GLi1,osteoprotegerin and RUNX2 in osteoblasts were significantly increased,while the mRNA expression levels of parathyroid hormone-related peptide were decreased(P＜0.05)when co-cultured with human osteoarthritic chondrocytes.The mRNA and protein levels of Gli1 were significantly decreased after the addition of IHH signaling pathway inhibitor(cyclopamine)(P＜0.05),and the mRNA and protein levels of Gli1 were significantly increased after the addition of IHH signaling pathway activator(purmorphamine)(P＜0.05).Osteoprotegerin showed the same trend as Gli1 in the experiment.The osteoprotegerin/RANKL ratio followed the same trend as osteoprotegerin.To conclude,human osteoarthritic chondrocytes can promote the expression of Gli1,osteoprotegerin,Runx2 and other proteins in osteoblasts.The upregulation of osteoprotegerin is related to the IHH signaling pathway.Osteoarthritic chondrocytes can up-regulate the expression of osteoprotegerin in osteoblasts through the IHH signaling pathway and thus up-regulate the osteoprotegerin/RANKL ratio,which will contribute to bone formation in subchondral bone.
ABSTRACT
Objective:To delineate the histomorphological disparities of subchondral bone between hemophilic arthritis (HA) and osteoarthritis (OA) and to explore the mechanisms underpinning aberrant bone remodeling in HA.Methods:Fifteen male HA patients, aged 32.60±7.58 years (range 22-45), who underwent total knee arthroplasty at the Second Affiliated Hospital of Anhui Medical University from January 2021 to June 2023, were included. All patients had hemophilia A and tested negative for coagulation factor VIII antibodies. Simultaneously, fifteen male OA patients, aged 75.67±5.09 years (range 71-87), also underwent arthroplasty. Tibial plateau bones were extracted for micro-CT, which assessed morphological parameters. Histological changes in the subchondral bone plate (SBP) and trabecular bone were evaluated with HE and Safranin O-Fast Green staining. TRAP staining determined osteoclast differentiation levels, and VEGF-A and Osterix immunohistochemistry gauged angiogenesis and osteoblast differentiation.Results:Micro-CT revealed that HA patients had a BV/TV of 25.14%±0.70% (medial) and 22.31%±0.53% (lateral), Conn.D. of 4.20±0.10 1/mm 3 (medial) and 3.27±0.08 1/mm 3 (lateral), BMD of 0.288±0.006 g/cm 3 (medial) and 0.285±0.004 g/cm 3 (lateral), Tb.Th of 0.257±0.008 mm (medial) and 0.206±0.008 mm (lateral), Tb.N of 0.984±0.043 1/mm (medial) and 0.908±0.026 1/mm (lateral), and Tb.Sp of 0.683±0.008 mm (medial) and 0.808±0.010 mm (lateral). These parameters were significantly lower than those in the OA group except for Tb.Sp, which was higher ( P<0.001). Histological staining indicated that the HA group's SBP thickness was 177.43±6.42 μm (medial) and 117.96±5.08 μm (lateral) with significant differences observed ( P<0.001). TRAP staining showed that TRAP + osteoclasts accounted for 33.4%±3.1% (medial) and 25.1%±2.3% (lateral) in HA subchondral bone, again significantly different ( P<0.001). Immunohistochemical staining revealed VEGFA + cells at 34.1%±5.9% (medial) and 25.9%±3.7% (lateral), and Osterix + cells at 14.6%±1.4% (medial) and 5.8%±1.1% (lateral) in HA patients, differing significantly from the OA group ( P<0.001). Conclusion:The HA group exhibited more extensive subchondral bone destruction, thinner trabeculae, a nearly absent tidemark, higher osteoclast differentiation, increased angiogenesis, and reduced osteoblast differentiation, indicating severe osteoporosis, despite thicker SBP. These findings suggest that targeting abnormal bone remodeling and angiogenesis in HA could retard its progression and provide therapeutic benefits.