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. 2023 Dec;46(6):100571.
doi: 10.1016/j.bj.2022.11.004. Epub 2022 Nov 26.

Effect of three clinical therapies on cytokines modulation in the hip articular cartilage and bone improvement in rat early osteonecrosis of the femoral head

Shan-Ling Hsu  1 , Shun-Wun Jhan  1 , Chieh-Cheng Hsu  1 , Yi-No Wu  2 , Kay L H Wu  3 , Chun-En Aurea Kuo  4 , Hung-Wen Chiu  5 , Jai-Hong Cheng  6
Affiliations

Effect of three clinical therapies on cytokines modulation in the hip articular cartilage and bone improvement in rat early osteonecrosis of the femoral head

Shan-Ling Hsu et al. Biomed J. 2023 Dec.

Abstract

Background: Extracorporeal shockwave therapy (ESWT) and adipose-derived mesenchymal stem cells (ADSCs) have been used clinically for the treatment of osteonecrosis of the femoral head (ONFH). The study elucidated that ESWT, ADSCs, and combination therapy modulated pro-inflammatory cytokines in the articular cartilage and subchondral bone of early rat ONFH.

Methods: ESWT and ADSCs were prepared and isolated for treatment. Micro-CT, pathological analysis, and immunohistochemistry were performed and analysed.

Results: After treatments, subchondral bone of ONFH was improved in trabecular bone volume (BV/TV) (p < 0.001), thickness (Tb.Th) (p < 0.01 and 0.001), and separation (Tb.Sp) (p < 0.001) and bone mineral density (BMD) (p < 0.001) using micro-CT analysis. The articular cartilage was protected and decreased apoptosis markers after all the treatments. The expression of IL33 (p < 0.001), IL5 (p < 0.001), IL6 (p < 0.001), and IL17A (p < 0.01) was significantly decreased in the ESWT, ADSCs, and Combination groups as compared with ONFH group. The IL33 receptor ST2 was significantly increased after treatment (p < 0.001) as compared with ONFH group. The Combination group (p < 0.01) decreased the expression of IL6 better than the ESWT and ADSCs groups.

Conclusion: ESWT, ADSCs and combination therapy significantly protected articular cartilage and subchondral bone of early rat ONFH by modulating the expression of pro-inflammatory cytokines including, IL33 and its receptor ST2, IL5, IL6, and IL17A.

Keywords: Adipose-derived mesenchymal stem cells; Extracorporeal shockwave therapy; Osteonecrosis of the femoral head; Pro-inflammatory cytokines.

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Conflict of interest statement

Conflicts of interest All authors have no conflicts of interest.

Figures

Fig. 1
Fig. 1
The experimental design and extracorporeal shockwave therapy (ESWT) and adipose-derived mesenchymal stem cells (ADSCs) treatments. (A) The flowchart shows the timeline of the study, including the ONFH rat model, ESWT (0.25 mJ/mm2 with 4000 impulses, 4 Hz), ADSCs (1 × 106 cells) injection and when animals were sacrificed. (B) The injection of ADSCs into the joint of the femoral head by using ultrasound guidance (arrow). N = 6 for each group.
Fig. 2
Fig. 2
Micro-CT analysis in each group. (A) The images show the left femoral head in sagittal, transverse and 3D structure views. The red line indicates the region of interest. (B) The micro-CT scanning data for the subchondral bone of the femoral head show the trabecular bone volume fraction (BV/TV), trabecular separation (Tb.Sp), trabecular thickness (Tb.Th), and bone mineral density (BMD). ∗∗p < 0.01 and ∗∗∗p < 0.001 were by comparing with ONFH group. Scale bar = 1 mm. N = 6 for each group.
Fig. 3
Fig. 3
The pathological analysis. The histological images were captured to show the articular cartilage of the left femoral head in Sham, ONFH, ESWT and Combination groups. The hypertrophy chondrocytes are indicated as the arrow and were reduced after treatments with (A) haematoxylin–eosin (H&E) and scale bar = 100 μm, (B) safranin-O stain and scale bar = 100 μm, (C) caspase 3 and scale bar = 50 μm as well as (D) TUNEL activity and scale bar = 50 μm. N = 6 for each group. ∗p < 0.05 and ∗∗∗p < 0.001 were by comparing with Sham group and ###p < 0.001 was by comparing with ESWT group among the treatment groups.
Fig. 4
Fig. 4
The expression of IL33 and its ST2 receptor. Immunohistochemical staining was performed for (A) IL33 and (B) ST2 in the articular cartilage of the left femoral head (left) and the data was calculated after treatments (right). Scale bar = 50 μm. ∗∗∗p < 0.001 was by comparing with ONFH group and ###p < 0.001 was by comparing with ESWT group among the treatment groups. N = 6 for all groups.
Fig. 5
Fig. 5
The pro-inflammatory cytokines. Immunohistochemical staining was performed for (A) IL5, (B) IL6 and (C) IL17A in the articular cartilage of the left femoral head (left) and the data was calculated after treatments (right). ∗∗p < 0.01 and ∗∗∗p < 0.001 were by comparing with ONFH group and ##p < 0.01 was by comparing with ESWT group among the treatment groups. Scale bar = 50 μm. N = 6 for each group.
Supplemental Fig. 1
Supplemental Fig. 1
The application of ESWT on early ONFH of rat. ESWT is applied to two focal points of femur head. Each of the two points is received 0.25 mJ/mm2 energy flux density with 2000 impulses of ESWT and total is 4000 impulses. N = 6 for each group.
Supplemental Fig. 2
Supplemental Fig. 2
The cell surface makers of rat ADSCs are analysed as well as positive markers are CD29 (99.79 %) and CD 90 (89.24 %). The negative markers are RT1a, RT1b and CD45. The percentage of marker is calculated in red line and the black line displays isotype controls.
Supplemental Fig. 3
Supplemental Fig. 3
The anti-inflammatory cytokines. Immunohistochemical staining was performed for (A) IL4, and (B) IL10 in the articular cartilage of the left femoral head (left) and the data was calculated after treatments (right). ∗∗∗P < 0.001 was by comparing with Sham group and #P < 0.05 and ###P < 0.001 were by comparing with ONFH group among the treatment groups. Scale bar = 50 μm. N = 6 for each group.
See this image and copyright information in PMC

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