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Interleukin-12 (IL-12) and IL-23 are thought to have central roles in inflammation and are critical to pathologies associated with inflammation-induced bone disorders. The deletion of IL-12p40 (a common subunit of IL-12 and IL-23) can improve bone regeneration. However, the relative roles of IL-12 and IL-23 in bone disorders are largely unknown. Methods: Ectopic bone formation and skull defect models were established to evaluate the relative roles of IL-12 and IL-23 in inflammatory bone disorders. Differences in bone mass among WT, IL-12p35-/-, and IL-12p40-/- mice (young and elderly) were detected by micro-CT. Osteogenic and osteoclastic activities were explored using ELISA, qRT-PCR, and histological analysis. Moreover, the mechanisms by which IL-12 and IL-23 regulated the differentiation of BMMSCs and RAW264.7 cells were explored using Alizarin Red and tartrate-resistant acid phosphatase staining in vitro. Apilimod was used to inhibit IL-12 and IL-23 production in vivo. Results: Mice deficient in IL-12p40 promoted bone formation and protected against aging-related bone loss. By contrast, bone loss was aggravated in IL-12-/- mice, suggesting that IL-23 may play a dominant role in inflammation-related bone disorders. Mechanistically, IL-12 and IL-23 coupled osteogenesis and osteoclastic activities to regulate bone homeostasis and repair. IL-23 deficiency increased bone formation and inhibited bone resorption. Finally, apilimod treatment significantly improved bone regeneration and calvarial defect repair. Conclusion: These data collectively uncover a previously unrecognized role of IL-23 in skeletal tissue engineering. Thus, IL-23 can act as a biomarker to predict diseases and treatment efficacy, and apilimod can be used as an effective therapeutic drug to combat inflammatory bone disorders.

The integration of three-dimensional (3D) bioprinted scaffold’s structure and function for critical-size bone defect repair is of immense significance. Inspired by the basic component of innate cortical bone tissue—osteons, many studies focus on biomimetic strategy. However, the complexity of hierarchical microchannels in the osteon, the requirement of mechanical strength of bone, and the biological function of angiogenesis and osteogenesis remain challenges in the fabrication of osteon-mimetic scaffolds. Therefore, we successfully built mimetic scaffolds with vertically central medullary canals, peripheral Haversian canals, and transverse Volkmann canals structures simultaneously by 3D bioprinting technology using polycaprolactone and bioink loading with bone marrow mesenchymal stem cells and bone morphogenetic protein-4. Subsequently, endothelial progenitor cells were seeded into the canals to enhance angiogenesis. The porosity and compressive properties of bioprinted scaffolds could be well controlled by altering the structure and canal numbers of the scaffolds. The osteon-mimetic scaffolds showed satisfactory biocompatibility and promotion of angiogenesis and osteogenesis in vitro and prompted the new blood vessels and new bone formation in vivo. In summary, this study proposes a biomimetic strategy for fabricating structured and functionalized 3D bioprinted scaffolds for vascularized bone tissue regeneration.

Background/Aims: Bone nonunion remains a challenge for orthopaedists. The technological advancements that have been made in precisely silencing target genes have provided promising methods to address this challenge. Methods: We detected the expression levels of the bone morphogenetic protein (BMP) inhibitors Chordin, Gremlin and Noggin using realtime PCR in bone mesenchymal stem cells (BMSCs) isolated from patients with normal fracture healing and those with bone nonunion. Moreover, we detected the expression of Chordin, Gremlin and Noggin during the osteogenic differentiation of human BMSCs (hBMSCs) using real-time PCR and Western blot. We delivered Chordin siRNA to hBMSCs using a previously reported cationic polymer, polyspermine imidazole-4,5-imine (PSI), as a pH-responsive and non-cytotoxic transfection agent. The apoptosis and cellular uptake efficiency were analysed by flow cytometry. Results: We identified Chordin as the most appropriate potential therapeutic target gene for enhancing the osteogenic differentiation of hBMSCs. Chordin knockdown rescued the osteogenic capacity of hBMSCs isolated from patients with bone nonunion. Highly efficient knockdown of Chordin was achieved in hBMSCs using PSI. Chordin knockdown promoted hBMSC osteogenesis and bone regeneration in vitro and in vivo. Conclusions: Our results suggest that Chordin is a potential target for improving osteogenesis and bone nonunion therapy and that responsive and non-toxic cationic polyimines such as PSI are therapeutically feasible carriers for the packaging and delivery of Chordin siRNA to hBMSCs.

Bone defects are a common challenge in clinic, usually warranting bone grafts. However, current strategies to obtain effective graft materials have many drawbacks. Mesenchymal stem cell (MSC)-based therapy is a promising alternative. We designed an innovative appliance named the stem cell screen–enrich–combine(-biomaterials) circulating system (SECCS). In this study, 42 patients who required bone graft underwent SECCS-based treatment. Their bone marrow samples and beta-tricalcium phosphate (β-TCP) granules were processed in the SECCS for 10–15 minutes, to produce MSC/β-TCP composites. These composites were grafted back into bone defect sites. The results showed 85.53% ± 7.95% autologous MSCs were successfully screened, enriched, and seeded on the β-TCP scaffolds synchronously. The cell viability remained unchanged after SECCS processing. Clinically, all patients obtained satisfactory bone healing. Thus, without in vitro culture, the SECCS can produce bioactive MSC/β-TCP composites for bone regeneration during surgery. The SECCS represents a convenient, rapid, low-cost, and safe method for bone regeneration.

Aim: To evaluate the clinical efficacy of mesenchymal stem cell/β-tricalcium phosphate composites (MSC/β-TCP) prepared with a screen-enrich-combine circulating system (SECCS) in patients with depressed tibial plateau fractures. Materials & methods: Bone defects in depressed tibial plateaus were filled with MSC/β-TCP (n = 16) or with β-TCP only (n = 23). Enrichment efficiency and effect of enrichment on cell viability were evaluated. Clinical results were assessed by imaging examination and Lysholm score. Results: SECCS effectively integrated MSCs with β-TCP. At 18 months postimplantation, new bone ratio was significantly higher in patients treated with MSC/β-TCP than in those treated with β-TCP only (p = 0.000). Patients with MSC/β-TCP implants had better functional recovery (p = 0.028). Conclusion: MSC/β-TCP prepared by SECCS were effective in the treatment of bone defects in patients with depressed tibial plateau fractures, promoted bone regeneration and improved joint function recovery.

Transforming growth factor-β1 (TGF-β1) is a key factor in bone reconstruction. However, its pathophysiological role in non-union and bone repair remains unclear. Here we demonstrated that TGF-β1 was highly expressed in both C57BL/6 mice where new bone formation was impaired after autologous bone marrow mesenchymal stem cell (BMMSC) implantation in non-union patients. High doses of TGF-β1 inhibited BMMSC osteogenesis and attenuated bone regeneration in vivo. Furthermore, different TGF-β1 levels exhibited opposite effects on osteogenic differentiation and bone healing. Mechanistically, low TGF-β1 doses activated smad3, promoted their binding to bone morphogenetic protein 2 (Bmp2) promoter, and upregulated Bmp2 expression in BMMSCs. By contrast, Bmp2 transcription was inhibited by changing smad3 binding sites on its promoter at high TGF-β1 levels. In addition, high TGF-β1 doses increased tomoregulin-1 (Tmeff1) levels, resulting in the repression of Bmp2 and bone formation in mice. Treatment with the TGF-β1 inhibitor SB431542 significantly rescued BMMSC osteogenesis and accelerated bone regeneration. Our study suggests that high-dose TGF-β1 dampens BMMSC-mediated bone regeneration by activating canonical TGF-β/smad3 signaling and inhibiting Bmp2 via direct and indirect mechanisms. These data collectively show a previously unrecognized mechanism of TGF-β1 in bone repair, and TGF-β1 is an effective therapeutic target for treating bone regeneration disability. © 2019 American Society for Bone and Mineral Research.

Bone non-union after fracture, considered a therapeutic challenge for orthopedics, always needs a reversion surgery, including autograft transplantation (AGT). However, adverse events related to autograft harvest cannot be ignored. Our group designed a novel system called the bone marrow stem cell Screen-Enrich-Combine Circulating System (SECCS) by seeding mesenchymal stem cells (MSCs) into β-tricalcium phosphate (β-TCP) during surgery to thereafter rapidly process bioactive bone implantation. In this retrospective case-control study, 30 non-union patients who accepted SECCS therapy and 20 non-union patients who accepted AGT were enrolled. By SECCS therapy, the MSC-enriched β-TCP particles were implanted into the non-union gap. During the enrichment procedure, a significant proportion of MSCs were screened and enriched from bone marrow into porous β-TCP particles, and the cells possessed the capacity for three-line differentiation and were CD90+/CD105+/CD34-/CD45-. Approximately 82.0±10.7% of MSCs were enriched from 60 mL bone marrow without damaging cell viability, and approximately 11,444.0±6,018 MSCs were transplanted per patient. No implant-related infections occurred in any case. After 9 months of follow-up, 27 patients (90%) in the SECCS group acquired clinical union, compared with 18 patients (90%) in the AGT group (clinical union time, P = 0.064), and postoperative radiographic union score at 9 months post-operation was similar between the two groups. In conclusion, the SECCS could concentrate a large proportion of MSCs from bone marrow to acquire enough effective cells for therapy without in vitro cell culture. Bone substitutes processed by SECCS demonstrated encouraging promotion of bone regeneration and showed a satisfactory clinical curative effect for diaphyseal bone non-union, which was non-inferior to AGT.

Background Efficacious bone substitute is essential for the treatment of a critical size bone defect. Currently, the bone substitutes commonly used in clinical practice lack osteogenic capacity and the therapeutic efficacy is not ideal. Herein, a novel stem cell screen-enrich-combine(−biomaterials) circulating system (SECCS) was introduced to provide the substitutes with osteogenic ability. The stem cell screening, enrichment, and recombination with substitutes could be integrated during the surgical operation. Using SECCS, the bioactive mesenchymal stem cells (MSCs) and porous β-tricalcium phosphate (β-TCP) composites (MSCs/β-TCP) were rapidly prepared for critical size bone defect repair and validated in goat models of critical size tibia defects. Methods Twelve goats with right hind limb tibia defects of 30 mm were randomly divided into two groups: six (the experimental group) were treated with MSCs/β-TCP prepared by SECCS and the other six goats (the control group) were treated with pure porous β-TCP. The repair effect was assessed by x-ray, computed tomography (CT), micro-CT, histology and histomorphology 6 months after the operation. In addition, the enrichment efficacy of MSCs and the characteristics of the MSCs/β-TCP prepared by SECCS were evaluated. Results The SECCS could compound about 81.3 ± 3.0% of the MSCs in bone marrow to the porous β-TCP without affecting the cell viability. The average number of MSCs for retransplantation was 27,655.0 ± 5011.6 for each goat from the experimental group. In vitro, satisfactory biocompatibility of the MSCs/β-TCP was performed, with the MSCs spreading adequately, proliferating actively, and retaining the osteogenic potential. In vivo, the defect repair by MSCs/β-TCP with good medullary cavity recanalization and cortical remodeling was significantly superior to that of pure porous β-TCP. Conclusions The MSCs/β-TCP prepared through SECCS demonstrated significant therapeutic efficacy in goat models of critical size bone defect. This provides a novel therapeutic strategy for critical size bone defects caused by severe injury, infection, and bone tumor resection with a high profile of safety, effectiveness, simplicity, and ease.

Small-interfering RNA (siRNA) provides a rapid solution for drug design and provides new methods to develop customizable medicines. Polyethyleneimine 25 kDa (PEI25kDa) is an effective transfection agent used in siRNA delivery. However, the lack of degradable linkage causes undesirable toxicity, hindering its clinical application. We designed a low-molecular-weight cross-linked polyethylenimine named PEI-Et (Mn:1220, Mw:2895) by using degradable ethylene biscarbamate linkage with lower cytotoxicity and higher knockdown efficiency than PEI25kDa in delivery Chordin siRNA to human bone mesenchymal stem cells (hBMSCs). Suppression of Chordin by using anti-Chordin siRNA delivered by PEI-Et improved bone regeneration in vitro and in vivo associated with the bone morphogenetic protein-2 (BMP-2) mediated smad1/5/8 signaling pathway. Results of this study suggest that Chordin siRNA can be potentially used to improve osteogenesis associated with the BMP-2-mediated Smad1/5/8 signaling pathway and biodegradable biscarbamate cross-linked low-molecular-weight polyethylenimine (PEI-Et) is a therapeutically feasible carrier material to deliver anti-Chordin siRNA to hBMSCs.

Cytotherapy for bone regeneration has not been widely used clinically. A new method based on enriched bone-marrow-derived mesenchymal stem cells (MSCs) combined with porous beta-tricalcium phosphate (beta-TCP) was used for posterior spinal fusion in 41 patients. The aim of the present study was to assess the clinical feasibility of peri-operative bone marrow stem cell enrichment and their combination with tricalcium phosphate. About 252 ml marrow per patient was harvested from bilateral iliac crest, the enriched MSCs were produced by a cell processor peri-operatively, then combined with porous beta-TCP granules by a negative pressure and a short-time incubation in the meantime of conventional operation, which were finally implanted back into the patient. About 45 ml enriched MSC suspension was collected, and 78+/-16% of MSCs were recovered. By enrichment technique, the number of colony-forming units which expressed alkaline phosphatase (CFUs-ALP+, to estimate the prevalence of MSCs) was increased 4.3 times; the increasing folds of bone marrow nucleated cells (NCs) and MSCs had a positive correlation. The natural log (ln) of MSC number declined with age, and also, the MSC number of younger subjects (or =40 years) was more than that of older ones (40 years), but none for NCs. The number of NCs and MSCs was not different significantly between men and women. However, the patients with thoracolumbar fracture (TLF) had significantly more MSCs than those with degenerative disc disease (DDD), but not for NCs. On the other hand, enriched MSCs could adhere to the wall of porous beta-TCP within 2h combination, and proliferate well during culture in vitro. After 34.5 months, 95.1% cases had good spinal fusion results. None of the samples before grafting was positive in bacterial culture. Only four patients had a little exudation or moderate swelling in their wounds, and recovered with conservative treatment.