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, 10 (1), 209

Systemic Administration of Quality- And Quantity-Controlled PBMNCs Reduces Bisphosphonate-Related Osteonecrosis of Jaw-Like Lesions in Mice

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Systemic Administration of Quality- And Quantity-Controlled PBMNCs Reduces Bisphosphonate-Related Osteonecrosis of Jaw-Like Lesions in Mice

Shinichiro Kuroshima et al. Stem Cell Res Ther.

Abstract

Background: Definitive treatment strategies for bisphosphonate-related osteonecrosis of the jaw (BRONJ) have not been developed. Cell-based therapy is an attractive treatment method for intractable diseases in the medical and dental fields; however, approval has been challenging in dentistry. Recently, we developed quality- and quantity (QQ)-controlled peripheral blood mononuclear cells (PBMNCs) that have anti-inflammatory and pro-angiogenesis effects. The aim of this study was to investigate the effects of QQ-controlled PBMNC transplantation on BRONJ-like lesions in mice.

Methods: To create high-prevalence BRONJ-like lesions, cyclophosphamide (CY) and zoledronate (ZA) were used with tooth extraction. Drug treatment was performed for 5 weeks. QQ-controlled PBMNC transplantation was performed immediately following tooth extraction of both maxillary first molars at 3 weeks after drug administration. Mice were euthanized at 2 weeks post-extraction. Histomorphometric and immunohistochemical analyses, microcomputed tomography assessment, and quantitative polymerase chain reaction evaluation were conducted using maxillae and long bones.

Results: ZA effects on long bones were noted, regardless of CY. Severely inhibited osseous and soft tissue wound healing of tooth extraction sockets was induced by CY/ZA combination therapy, which was diagnosed as BRONJ-like lesions. QQ-controlled PBMNC transplantation reduced BRONJ-like lesions by improving soft tissue healing with increased M1 and M2 macrophages and enhanced neovascularization in the connective tissue of tooth extraction sockets. QQ-controlled PBMNC transplantation also reduced inflammation by decreasing polymorphonuclear cells and TNF-α expression in the tooth extraction sockets. Additionally, QQ-controlled PBMNC transplantation partially improved osseous healing of tooth extraction sockets. Interestingly, only 20,000 QQ-controlled PBMNCs per mouse induced these transplantation effects. QQ-controlled PBMNC transplantation did not affect the systemic microenvironment.

Conclusions: Our findings suggest that transplantation of a small amount of QQ-controlled PBMNCs may become novel therapeutic or prevention strategies for BRONJ without any adverse side effects.

Keywords: Bisphosphonate-related osteonecrosis of the jaw; Macrophages; Neovascularization; QQ-controlled PBMNCs; Tooth extraction; Transplantation; Wound healing.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Experimental procedures. a Experimental schedule to create high-prevalence BRONJ-like lesions. b Experimental schedule for transplantation of QQ-controlled PBMNCs. c Area of interests (AOIs) in long bones. AOIs, total area (TA; the red area) and bone area (BA; the blue area). d AOIs in the hard tissue of tooth extraction sockets (area surrounded by black dotted line). e AOIs in the soft tissue of tooth extraction sockets (area surrounded by black dotted line)
Fig. 2
Fig. 2
Effects of 5 weeks of cyclophosphamide (CY) and zoledronate (ZA) combination therapy on long bones. a Representative H-E-stained images (bar, 400 μm). Bone area per tissue area (BA/TA) was significantly increased in CY/ZA vs. vehicle control (VC) (**p < 0.01). b Representative TRAP-stained images (black arrowheads: osteoclasts on bone surface, BM: bone marrow, bar: 100 μm). The number of osteoclasts was significantly decreased in CY/ZA vs. VC (**p < 0.01). c Representative microCT images. Bone volume fraction (BVF) was significantly increased in CY/ZA vs. VC (***p < 0.001). Trabecular number (Tb.N) was the same in CY/ZA vs. VC, while trabecular thickness (Tb.Th) was significantly increased in CY/ZA vs. VC (***p < 0.001). Trabecular separation (Tb.Sp) was significantly decreased in CY/ZA vs. VC (*p < 0.05). Bone mineral density (BMD) was significantly increased in CY/ZA vs. VC (***p < 0.001). The Mann-Whitney U test was conducted to assess the non-parametric BA/TA data. Student’s t test was performed for all other analyzed parameters. n = 7/group
Fig. 3
Fig. 3
Validation of BRONJ-like lesions induced by CY/ZA combination therapy with tooth extraction. a Representative occlusal view of intra-oral photos. Bone exposure without epithelium occurred in 92.8% of tooth extraction sockets (areas surrounded by red arrows). No bone exposure was observed in the VC group. Both open wound area and perimeter were significantly larger in CY/ZA vs. VC (***p < 0.001). b Representative H-E stained images of tooth extraction sockets of distal roots (black dotted line: tooth extraction sockets; areas surrounded by red dotted line: necrotic bone with empty lacunae; Ep: epithelium; Ct: connective tissue; M2: second molar; PMN infiltration: polymorphonuclear cell infiltration; bar, 200 μm). BA/TA and living bone area were significantly decreased in CY/ZA vs. VC (***p < 0.001). Necrotic bone and empty lacunae were significantly increased in CY/ZA vs. VC (**p < 0.01). c Representative TRAP-stained images (Ct: connective tissue; bar, 100 μm). The number of osteoclasts was significantly decreased in CY/ZA vs. VC (***p < 0.001). d Trichrome-stained images of extraction sockets of maxillary mesial roots (white dotted line: extraction sockets of mesial roots; bar, 100 μm). Severe infiltration of polymorphonuclear cells (PMNs) in the tooth extraction sockets was observed in CY/ZA relative to VC (***p < 0.001). Student’s t-test was performed for all analyzed parameters. n = 7/group
Fig. 4
Fig. 4
Effects of QQ-controlled PBMNC transplantation on impaired osseous healing at 2 weeks post-extraction. a Representative occlusal view of intra-oral photos (red dotted line: open wounds, M2: second molar, M3: third molar). Both wound open area and perimeter were significantly decreased in QQ-controlled PBMNC vs. VC (**p < 0.01). b Serum ALP levels were the same between QQ-controlled PBMNC and VC. c Representative H-E-stained images of tooth extraction sockets of distal roots (black dotted line: tooth extraction sockets; areas surrounded by red dotted line: necrotic bone with empty lacunae; Ep: epithelium; Ct: connective tissue; M2: second molar; bar, 200 μm). BA/TA was the same between QQ-controlled PBMNC and VC. Living bone area was significantly increased in QQ-controlled PBMNC vs. VC (*p < 0.05). Osteocyte density was significantly increased in QQ-controlled PBMNC vs. VC (*p < 0.05). Necrotic bone area was decreased in QQ-controlled PBMNC vs. VC, however, no statistically significant differences were observed (p > 0.05). Empty lacunae were similar between QQ-controlled PBMNC and VC. d Representative TRAP-stained images (Ct: connective tissue; black arrowheads: osteoclasts on bone surface; bar, 200 μm). Number of osteoclasts was significantly increased in QQ-controlled PBMNC vs. VC (*p < 0.05). e Representative microCT images (white dotted line: mesial and distal roots of tooth extraction sockets; M2: second molar). Bone fill in tooth extraction sockets was the same, regardless of cell transplantation. Trabecular number (Tb.N) was similar between groups. Trabecular thickness (Tb.Th) was the same between groups. Trabecular separation (Tb.Sp) was almost identical, irrespective of QQ-controlled MNC therapy. Bone mineral density (BMD) did not change between groups. Student’s t test was performed for all analyzed parameters. n = 7/group; QQ: QQ-controlled PBMNC therapy
Fig. 5
Fig. 5
Effects of QQ-controlled PBMNC transplantation on compromised soft tissue healing at 2 weeks post-extraction. a Representative trichrome-stained images of tooth extraction sockets of distal roots (black dotted line: tooth extraction sockets; bar, 200 μm). Collagen production in the connective tissue of tooth extraction was significantly increased in QQ-controlled PBMNC vs. VC (***p < 0.001). Notably, production of type I collagen was significantly increased in QQ-controlled PBMNC vs. VC (*p < 0.05), whereas the production of type III collagen was the same between QQ-controlled PBMNC and VC. Polymorphonuclear cells (PMNs) were significantly decreased in QQ-controlled PBMNC vs. VC (***p < 0.001). b Representative CD31-immunostained images (red fluorescence: blood vessels; Ep: epithelium; Ct: connective tissue; bar, 50 μm). The number of blood vessels was significantly increased in QQ-controlled PBMNC vs. VC (***p < 0.001). Vessel density was significantly increased in QQ-controlled PBMNC vs. VC (***p < 0.001). c Representative F4/80- and CD206-immunostained images (red fluorescence: F4/80+ macrophage; orange fluorescence: F4/80+CD206+ cell; Ct: connective tissue; bar, 100 μm). The numbers of F4/80+CD206- and F4/80+CD206+-cells were significantly increased in QQ-controlled PBMNC vs. VC (***p < 0.01 and *p < 0.05, respectively). d Relative gene expression levels of IL-10, TGF-β, and IL-1β were the same between groups at both 72 hours and 2 weeks after cell transplantation. The relative gene expression level of TNF-α was significantly decreased in QQ-controlled PBMNC vs. VC at 72 hours and 2 weeks after cell therapy (*p < 0.05). The Mann-Whitney U test was used for IL-1β analysis. Student’s t test was performed for all other analyzed parameters. n = 7/group; QQ: QQ-controlled PBMNC therapy
Fig. 6
Fig. 6
Effects of QQ-controlled PBMNC transplantation on CY/ZA affected-long bones. a Representative H-E-stained images (bar, 400 μm). BA/TA was the same, irrespective of QQ-controlled PBMNC transplantation. b Representative TRAP-stained images (BM: bone marrow; bar, 100 μm). QQ-controlled PBMNC transplantation did not significantly alter the number of osteoclasts. c The relative gene expression levels of CTSK, TRAP, F4/80, TNF-α, and IL-10 were the same between QQ-controlled PBMNC and VC. The Mann-Whitney U test was used for IL-10 analysis. Student’s t test was performed for all other analyzed parameters. n = 7/group; QQ: QQ-controlled PBMNC therapy

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