Utility of Thermal Cross-Linking in Stabilizing Hydrogels with Beta-Tricalcium Phosphate and/or Epigallocatechin Gallate for Use in Bone Regeneration Therapy

β-tricalcium phosphate (β-TCP) granules are commonly used materials in dentistry or orthopedic surgery. However, further improvements are required to raise the operability and bone-forming ability of β-TCP granules in a clinical setting. Recently, we developed epigallocatechin gallate (EGCG)-modified gelatin sponges as a novel biomaterial for bone regeneration. However, there is no study on using the above material for preparing hydrogel incorporating β-TCP granules. Here, we demonstrate that vacuum heating treatment induced thermal cross-linking in gelatin sponges modified with EGCG and incorporating β-TCP granules (vhEc-GS-β) so that the hydrogels prepared from vhEc-GS-β showed high stability, β-TCP granule retention, operability, and cytocompatibility. Additionally, microcomputed tomography morphometry revealed that the hydrogels from vhEc-GS-β had significantly higher bone-forming ability than β-TCP alone. Tartrate-resistant acid phosphatase staining demonstrated that the number of osteoclasts increased at three weeks in defects treated with the hydrogels from vhEc-GS-β compared with that around β-TCP alone. The overall results indicate that thermal cross-linking treatment for the preparation of sponges (precursor of hydrogels) can be a promising process to enhance the bone-forming ability. This insight should provide a basis for the development of novel materials with good operativity and bone-forming ability for bone regenerative medicine.

Production and Characterization of a 316L Stainless Steel/β-TCP Biocomposite Using the Functionally Graded Materials (FGMs) Technique for Dental and Orthopedic Applications

Metallic biomaterials are widely used for implants and dental and orthopedic applications due to their good mechanical properties. Among all these materials, 316L stainless steel has gained special attention, because of its good characteristics as an implantable biomaterial. However, the Young’s modulus of this metal is much higher than that of human bone (~193 GPa compared to 5–30 GPa). Thus, a stress shielding effect can occur, leading the implant to fail. In addition, due to this difference, the bond between implant and surrounding tissue is weak. Already, calcium phosphate ceramics, such as beta-tricalcium phosphate, have shown excellent osteoconductive and osteoinductive properties. However, they present low mechanical strength. For this reason, this study aimed to combine 316L stainless steel with the beta-tricalcium phosphate ceramic (β-TCP), with the objective of improving the steel’s biological performance and the ceramic’s mechanical strength. The 316L stainless steel/β-TCP biocomposites were produced using powder metallurgy and functionally graded materials (FGMs) techniques. Initially, β-TCP was obtained by solid-state reaction using powders of calcium carbonate and calcium phosphate. The forerunner materials were analyzed microstructurally. Pure 316L stainless steel and β-TCP were individually submitted to temperature tests (1000 and 1100 °C) to determine the best condition. Blended compositions used to obtain the FGMs were defined as 20% to 20%. They were homogenized in a high-energy ball mill, uniaxially pressed, sintered and analyzed microstructurally and mechanically. The results indicated that 1100 °C/2 h was the best sintering condition, for both 316L stainless steel and β-TCP. For all individual compositions and the FGM composite, the parameters used for pressing and sintering were appropriate to produce samples with good microstructural and mechanical properties. Wettability and hemocompatibility were also achieved efficiently, with no presence of contaminants. All results indicated that the production of 316L stainless steel/β-TCP FGMs through PM is viable for dental and orthopedic purposes.

Enhancement of Bone-Forming Ability on Beta-Tricalcium Phosphate by Modulating Cellular Senescence Mechanisms Using Senolytics

Various stresses latently induce cellular senescence that occasionally deteriorates the functioning of surrounding tissues. Nevertheless, little is known about the appearance and function of senescent cells, caused by the implantation of beta-tricalcium phosphate (β-TCP)—used widely in dentistry and orthopedics for treating bone diseases. In this study, two varying sizes of β-TCP granules (<300 μm and 300–500 μm) were implanted, and using histological and immunofluorescent staining, appearances of senescent-like cells in critical-sized bone defects in the calvaria of Sprague Dawley rats were evaluated. Parallelly, bone formation in defects was investigated with or without the oral administration of senolytics (a cocktail of dasatinib and quercetin). A week after the implantation, the number of senescence-associated beta-galactosidase, p21-, p19-, and tartrate-resistant acid phosphatase-positive cells increased and then decreased upon administrating senolytics. This administration of senolytics also attenuated 4-hydroxy-2-nonenal staining, representing reactive oxygen species. Combining senolytic administration with β-TCP implantation significantly enhanced the bone formation in defects as revealed by micro-computed tomography analysis and hematoxylin-eosin staining. This study demonstrates that β-TCP granules latently induce senescent-like cells, and senolytic administration may improve the bone-forming ability of β-TCP by inhibiting senescence-associated mechanisms.

Effects of rhBMP-2 Loaded Hydroxyapatite Granules/beta-Tricalcium Phosphate Hydrogel (HA/β-TCP hydrogel) Composite on a Rat Model of Coccygeal Intervertebral Fusion.

The effects and inflammation-related side effects of bone morphogenetic protein (BMP)-2 on posterior lumbar interbody fusion are controversial. One of the potential causes for the inconsistent results is the uncontrolled release of BMP-2 from the collagen carrier. Therefore, BMP delivery systems which support effective bone regeneration while attenuating side effects are strongly sought for. We developed NOVOSIS putty (NP), a novel composite material of hydroxyapatite (HA), beta-tricalcium phosphate (β-TCP)-hydrogel, and BMP-2, which can sustainably release BMP-2 over two weeks. This study investigated the effects and side effects of NP compared with those of collagen sponge (CS) containing BMP-2 using a rat coccygeal intervertebral fusion model. The fusion rates of NP with low and high doses of BMP-2 were significantly higher than those of an iliac bone (IB) graft, but those of CS with low and high doses of BMP-2 were not different from those of the IB graft. Furthermore, the incidences of ectopic bone formation and soft tissue swelling were significantly lower in the NP group than in the CS group. The HA/β-TCP hydrogel carrier enabled superior bone induction with low-dose BMP-2 and decreased the incidence of side effects caused by high-dose BMP-2 compared with that of the collagen carrier.

The Role of Bone Grafts in Preventing Medication-Related Osteonecrosis of the Jaw: Histomorphometric, Immunohistochemical, and Clinical Evaluation in Animal Model

Objective: To evaluate the effects of inorganic bovine bone graft (Lumina Bone, Criteria, Brazil) and beta-tricalcium phosphate (β-TCP) graft (ChronOS, Synthes, Brazil) in rats with the risk of developing post-extraction medication-related osteonecrosis of the jaw (MRONJ). Methods: Eighteen male Wistar rats weighing 350 to 450 g were induced to develop MRONJ using zoledronic acid for 5 weeks. In the sixth week, the right maxillary first molar was extracted. The animals in Group I (G1) did not receive bone grafts after tooth extraction, while Group II (G2) animals received inorganic bovine bone grafts, and Group III (G3) animals received beta-tricalcium phosphate (β-TCP) grafts. Clinical evaluation and histomorphometric and immunohistochemical analyses were performed. ANOVA and Tukey’s statistical tests were used and a level of significance was considered to be 5%. Results: In the clinical evaluation, animals from G2 and G3 did not present clinical manifestations of osteonecrosis, unlike the control group (G1) animals, which presented necrotic bone tissue exposure in all samples. In the histomorphometric evaluation, animals in G3 showed greater formation of bone tissue (66%) and less formation of bone lacuna (18%) than animals in G1 (58%/32%) and in G2 (59%/27%) ( P < 0.05). Moderate (++) immunostaining was observed in G2 and G3 for RANKL, TRAP, and OC, while G1 showed moderate (++) labeling for OC and mild (+) immunostaining for TRAP and RANKL. Conclusions: Greater formation of bone tissue and fewer bone lacunae were found in animals treated with β-TCP. In clinical evaluation, bone graft groups presented with the clinical manifestation of MRONJ and showed higher intensity of immunostaining for TRAP and RANKL. Despite the limitations of experimental animal studies, the results of this work may assist in the development of future clinical research for the prevention of MRONJ.

Activation of NLRP3 Inflammasome Complexes by Beta-Tricalcium Phosphate Particles and Stimulation of Immune Cell Migration in vivo

Beta-tricalcium phosphate (β-TCP) serves as a bone substitute in clinical practice because it is resorbable, biocompatible, osteointegrative, and osteoconductive. Particles of β-TCP are also inflammatory mediators although the mechanism of this function has not been fully elucidated. Regardless, the ability of β-TCP to stimulate the immune system might be useful for immunomodulation. The present study aimed to determine the effects of β-TCP particles on NLR family pyrin domain containing 3 (NLRP3) inflammasome complexes. We found that β-TCP activates NLRP3 inflammasomes, and increases interleukin (IL)-1β production in primary cultured mouse dendritic cells (DCs) and macrophages, and human THP-1 cells in caspase-1 dependent manner. In THP-1 cells, β-TCP increased also IL-18 production, and NLRP3 inflammasome activation by β-TCP depended on phagocytosis, potassium efflux, and reactive oxygen species (ROS) generation. We also investigated the effects of β-TCP in wild-type and NLRP3-deficient mice in vivo. Immune cell migration around subcutaneously injected β-TCP particles was reduced in NLRP3-deficient mice. These findings suggest that the effects of β-TCP particles in vivo are at least partly mediated by NLRP3 inflammasome complexes.