The Combined Use of Platelet-Rich Plasma Clot Releasate and Allogeneic Human Umbilical Cord Mesenchymal Stem Cells Rescue Glucocorticoid-Induced Osteonecrosis of the Femoral Head

Yanxue Wang, Shuo Luan, Ze Yuan, Shaoling Wang, Shengnuo Fan, Chao Ma, Shaoling Wu, Yanxue Wang, Shuo Luan, Ze Yuan, Shaoling Wang, Shengnuo Fan, Chao Ma, Shaoling Wu

Abstract

Glucocorticoid-induced osteonecrosis of the femoral head (ONFH) is a refractory disease. The treatment options for ONFH, especially nonsurgical ones, merit further investigation. To evaluate the combinatorial therapeutic effects of platelet-rich plasma clot releasate (PRCR) and umbilical cord mesenchymal stem cells (UC-MSCs) on glucocorticoid-induced ONFH, a dexamethasone (DEX)-treated cell model and a high-dose methylprednisolone (MPS)-treated rat model were established. Cell counting kit-8 (CCK-8) assay was performed in vitro to determine the optimum dosage of PRCR for UC-MSC viability. The effects of PRCR, UC-MSCs, and PRCR + UC-MSCs on cell viability, apoptosis, migration, and differentiation capacities of DEX-treated bone marrow mesenchymal stem cells (BMSCs) and human umbilical vein endothelial cell (HUVECs) were explored via Transwell assays. Western blotting was conducted to evaluate the expression levels of RUNX2, VEGF, caspase-3, and Bcl-2 in the coculture systems. Ultrasound-guided intra-articular PRCR, UC-MSCs, and PRCR + UC-MSC injections were performed on the ONFH model rats. Microcomputed tomography, histological and immunohistochemical analyses, tartrate-resistant acid phosphatase (TRAP) staining, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were used to assess the therapeutic effects of PRCR and UC-MSCs on bone loss and necrosis induced by high-dose MPS. Results of this study revealed that the in vitro application of PRCR, UC-MSCs, and PRCR + UC-MSCs reversed the impaired proliferation and migration capacities and resisted apoptosis of BMSCs and HUVECs induced by DEX. Moreover, the PRCR and UC-MSC application significantly improved the alkaline phosphatase (ALP) and alizarin red (ALR) staining of BMSCs and tube formation capacity of HUVECs and promoted the protein expression of RUNX2 in BMSCs and VEGF in HUVECs. Similarly, in the ONFH rat model, the intra-articular injection of UC-MSCs and PRCR improved the subchondral bone mass parameters; promoted the expression of ALP, RUNX2, and VEGF; suppressed osteoclast overactivity; and resisted cell apoptosis. The combination of PRCR and UC-MSCs shows promising therapeutic effects in treating glucocorticoid-induced ONFH. The current study provides important information on intra-articular therapy, paving the way for the clinical management of ONFH in the future.

Conflict of interest statement

The authors declare that they have no competing interests.

Copyright © 2022 Yanxue Wang et al.

Figures

Figure 1
Figure 1
Effect of different concentrations of PRCR on UC-MSC proliferation. CCK-8 assay indicated that the UC-MSCs treated with 2% PRCR achieved maximum proliferation. n = 3; ∗P < 0.05, compared to the other group. PRCR: platelet-rich plasma clot releasate; UC-MSCs: umbilical cord mesenchymal stem cells; CCK-8: cell counting kit-8 assay.
Figure 2
Figure 2
In vitro effects of PRCR, UC-MSCs, and PRCR + UC-MSCs on the proliferation, migration, and osteogenesis differentiation of DEX-treated BMSCs in the coculture systems. (a) Cocultured with PRCR, UC-MSCs, and PRCR + UC-MSCs, the cell viability of DEX-treated BMSCs was examined by CCK-8 assay. The treatment groups, including the PRCR, UC-MSC, and PRCR + UC-MSC groups, significantly promoted the BMSC proliferation inhibited by DEX (P < 0.05). (b) Migration activities of DEX-treated BMSCs were examined by Transwell assay in different coculture conditions, followed by quantitative analysis. The treatment groups significantly promoted the BMSC migration inhibited by DEX (P < 0.05). (c, d) Representative images of ALP staining (day 7) and ALR staining (day 14) under osteogenic medium in different coculture conditions, followed by quantitative analysis. The treatment groups significantly promoted the BMSC osteogenic differentiation inhibited by DEX (P < 0.05). (e) Western blotting was conducted to evaluate the RUNX2 expression. Higher levels of RUNX2 protein were detected in all the treatment groups compared to the DEX group (P < 0.05) (n = 5, #Comparisons between the DEX group and other groups, P < 0.05; ∗Comparisons between the two groups, P < 0.05). PRCR: platelet-rich plasma clot releasate; UC-MSCs: umbilical cord mesenchymal stem cells; BMSCs: bone marrow mesenchymal stem cells; DEX: dexamethasone; CCK-8: cell counting kit-8 assay; ALP: alkaline phosphatase; ALR: alizarin red.
Figure 3
Figure 3
In vitro effects of PRCR, UC-MSCs, and PRCR + UC-MSCs on the proliferation, migration, and capillary-like tube formation of DEX-treated HUVECs in the coculture systems (a) Cocultured with PRCR, UC-MSCs, and PRCR + UC-MSCs, the cell viability of DEX-treated HUVECs was examined by CCK-8 assay. These three treatment groups showed significant promotion of the HUVEC proliferation inhibited by DEX (P < 0.05). (b) Migration activities of DEX-treated HUVECs were examined by Transwell assay in different coculture conditions, followed by quantitative analysis. The treatment groups showed significant promotion of the HUVEC migration inhibited by DEX (P < 0.05). (c) Representative images of capillary-like tube formation in different coculture conditions, followed by quantitative analysis. The treatment groups showed significant promotion of the HUVEC angiogenesis differentiation inhibited by DEX (P < 0.05). (d) Western blotting was conducted to evaluate the VEGFA expression. Higher levels of VEGFA protein were detected in all treatment groups compared to the DEX group (P < 0.05) (n = 5, #Comparisons between the DEX group and other groups, P < 0.05; ∗Comparisons between the two groups, P < 0.05). CCK-8: cell counting kit-8 assay; VEGF: vascular endothelial growth factor; GADPH: glyceraldehyde-3-phosphate dehydrogenase; PRCR: platelet-rich plasma clot releasate; UC-MSCs: umbilical cord mesenchymal stem cells; DEX: dexamethasone; GADPH: glyceraldehyde-3-phosphate dehydrogenase.
Figure 4
Figure 4
Effects of PRCR, UC-MSCs, and PRCR + UC-MSCs on DEX-treated BMSCs and HUVEC apoptosis. (a, b) Representative images of TUNEL-positive cells (apoptotic cells) of DEX-treated BMSCs and HUVEC apoptosis in different coculture conditions. (c) Quantitative analysis of apoptosis rates of BMSCs and HUVECs, respectively. The PRCR and PRCR+UC-MSCs rescued the DEX-induced apoptotic effects (P < 0.05). (d) Western blotting was conducted to evaluate the expression of the apoptosis-related proteins. The PRCR, UC-MSC, and PRCR + UC-MSC groups significantly blocked caspase-3 activation and increased Bcl-2 expression compared to the DEX group (n = 5, #Comparisons between the DEX group and other groups, P < 0.05; ∗Comparisons between the two groups, P < 0.05). PRCR: platelet-rich plasma clot releasate; UC-MSCs: umbilical cord mesenchymal stem cells; DEX: dexamethasone; HBMSCs: human bone marrow mesenchymal stem cells; HUVECs: human umbilical vein endothelial cells; TUNEL: terminal transferase-mediated deoxyuridine triphosphate (dUTP) nick-end labeling; DAPI: 4′, 6-diamidino-2-phenylindole; GADPH: glyceraldehyde-3-phosphate dehydrogenase.
Figure 5
Figure 5
The time-course of animal experiments (ONFH model establishment and intra-articular injection schedule). ∗50 μL injected for each hip joint. PRCR: platelet-rich plasma clot releasate; UC-MSCs: umbilical cord mesenchymal stem cells; LPS: lipopolysaccharide (LPS); MPS: methylprednisolone.
Figure 6
Figure 6
Effects of PRCR, UC-MSCs, and PRCR + UC-MSCs on bone tissue protection and necrosis restoration in the ONFH rat model. (a) Visualized intra-articular injection using ultrasonic guidance, the white arrows indicated the needle pathway. (b, c) Representative micro-CT images of femoral heads (coronal, transverse, and sagittal images) from different treatment groups followed by quantitative analysis. The PRCR, UC-MSC, and PRCR + UC-MSC treatments improved the microstructural parameters changes and bone loss compared to the MPS group (P < 0.05). (d) Hematoxylin and Eosin (HE) staining of femoral heads from different groups. (e) Immunohistochemical staining of RUNX2, ALP, and VEGF of different groups followed by quantitative analysis. The treatment groups significantly promoted the expression of RUNX2, ALP, and VEGF compared to the MPS group (P < 0.05). (f) Representative tartrate-resistant acid phosphatase (TRAP) staining images showing the distributions of osteoclasts in the femoral heads from different groups, followed by quantitative analysis. The black arrows indicate the osteoclasts. The number of TRAP-positive cells in the treatment groups was significantly lower compared to the MPS group (P < 0.05). (g) Representative TUNEL staining images of femoral heads from different groups, followed by quantitative analysis. The apoptotic cell numbers in the treatment groups were significantly decreased compared to the MPS group (P < 0.05) (n = 5 for each group. #Comparisons between the DEX group and other groups, P < 0.05; ∗Comparisons between the two groups, P < 0.05). Tb.Th: trabecular thickness; Tb.Sp: trabecular separation; BV/TV: bone volume per tissue volume; Tb.N: trabecular number; PRCR: platelet-rich plasma clot releasate; UC-MSCs: umbilical cord mesenchymal stem cells; DEX: dexamethasone; TUNEL: terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick-end labeling; VEGF: vascular endothelial growth factor; ALP: alkaline phosphatase; MPS: methylprednisolone.

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