High-strength biodegradable zinc alloy implants with antibacterial and osteogenic properties for the treatment of MRSA-induced rat osteomyelitis.

Implant-related infections caused by drug-resistant bacteria remain a major challenge faced by orthopedic surgeons. Furthermore, ideal prevention and treatment methods are lacking in clinical practice. Here, based on the antibacterial and osteogenic properties of Zn alloys, Ag and Li were selected as alloying elements to prepare biodegradable Zn-Li-Ag ternary alloys. Li and Ag addition improved the mechanical properties of Zn-Li-Ag alloys. The Zn-0.8Li-0.5Ag alloy exhibited the highest ultimate tensile strength (>530 MPa). Zn-Li-Ag alloys showed strong bactericidal effects on methicillin-resistant Staphylococcus aureus (MRSA) in vitro. RNA sequencing revealed two MRSA-killing mechanisms exhibited by the Zn-0.8Li-0.5Ag alloy: cellular metabolism disturbance and induction of reactive oxygen species production. To verify that the therapeutic potential of the Zn-0.8Li-0.5Ag alloy is greater than that of Ti intramedullary nails, X-ray, micro-computed tomography, microbiological, and histological analyses were conducted in a rat femoral model of MRSA-induced osteomyelitis. Treatment with Zn-0.8Li-0.5Ag alloy implants resulted in remarkable infection control and favorable bone retention. The in vivo safety of this ternary alloy was confirmed by evaluating vital organ functions and pathological morphologies. We suggest that, with its good antibacterial and osteogenic properties, Zn-0.8Li-0.5Ag alloy can serve as an orthopedic implant material to prevent and treat orthopedic implant-related infections.

Complementary and synergistic effects on osteogenic and angiogenic properties of copper-incorporated silicocarnotite bioceramic: In vitro and in vivo studies.

Promoting bone regeneration to treat bone defects is a challenging problem in orthopedics, and developing novel biomaterials with both osteogenic and angiogenic activities is sought as a feasible solution. Here, copper-silicocarnotite [Cu-Ca5(PO4)2SiO4, Cu-CPS] was designed and fabricated. In this study, the Cu-CPS ceramics demonstrated better mechanical, osteogenic, and angiogenic properties in vitro and in vivo than pure CPS one. Particularly, CPS with 1.0 wt% CuO (1.0Cu-CPS) exhibited the best performance. Additionally, hydroxyapatite with 1.0 wt% CuO (1.0Cu-HA) was used to explore the respective effects of copper and silicon (Si). According to the in vitro results, it indicated that Cu enhanced the osteogenic activity of CPS ceramics although Si played a dominate role in the osteogenic process. Moreover, Cu could promote an early stage of angiogenesis, and the complementary effect of Si and Cu was found in the late phase. Furthermore, the in vivo results illustrated that the synergistic effect of Cu and Si improved bone and vessel regeneration during the degradation of Cu-CPS scaffolds (P < 0.05). Therefore, Cu-CPS ceramics could improve osteogenesis and angiogenesis through the simultaneous effects of Cu and Si, thus, offering a promising treatment option in orthopedic application for bone tissue regeneration.

Antibacterial effect of a copper-containing titanium alloy against implant-associated infection induced by methicillin-resistant Staphylococcus aureus.

Implant-associated infection (IAI) induced by methicillin-resistant Staphylococcus aureus (MRSA) is a devastating complication in the orthopedic clinic. Traditional implant materials, such as Ti6Al4V, are vulnerable to microbial infection. In this study, we fabricated a copper (Cu)-containing titanium alloy (Ti6Al4V-Cu) for the prevention and treatment of MRSA-induced IAI. The material characteristics, antibacterial activity, and biocompatibility of Ti6Al4V-Cu were systematically investigated and compared with those of Ti6Al4V. Ti6Al4V-Cu provided stable and continuous Cu2+ release, at a rate of 0.106 mg/cm2/d. Its antibacterial performance against MRSA in vitro was confirmed by plate counting analysis, crystal violet staining, and scanning electron microscopic observations. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis demonstrated that Ti6Al4V-Cu suppressed biofilm formation, virulence, and antibiotic-resistance of MRSA. The in vivo anti-MRSA effect was investigated in a rat IAI model. Implants were contaminated with MRSA solution, implanted into the femur, and left for 6 weeks. Severe IAI developed in the Ti6Al4V group, with increased radiological score (9.6 ± 1.3) and high histological score (10.1 ± 1.9). However, no sign of infection was found in the Ti6Al4V-Cu group, as indicated by decreased radiological score (1.3 ± 0.4) and low histological score (2.3 ± 0.5). In addition, Ti6Al4V-Cu had favorable biocompatibility both in vitro and in vivo. In summary, Ti6Al4V-Cu is a promising implant material to protect against MRSA-induced IAI.

In vitro and in vivo studies of Zn-Mn biodegradable metals designed for orthopedic applications.

In recent years, Zn-based materials provide a new option as biodegradable metals for orthopedic applications. To improve the low strength and brittle nature of pure Zn, small amounts of alloying element Mn (0.1, 0.4 and 0.8 wt.%) were added into Zn to fabricate binary Zn-Mn alloys. An extremely high elongation (83.96 ± 2.36%) was achieved in the resulting Zn-0.8 wt.%Mn alloy. Moreover, Zn-Mn alloys displayed significantly improved cytocompatibility as compared to pure Zn, according to cell proliferation and morphology analyses. More importantly, a significantly improved osteogenic activity was verified after adding Mn regarding ALP activity and osteogenic expression. Furthermore, Zn-0.8 wt.%Mn alloy scaffolds were implanted into the rat femoral condyle for repairing bone defects with pure Ti as control. Enhanced osteogenic activities were confirmed for Zn-0.8Mn alloy in contrast to pure Ti based on Micro-CT and histological results, and favorable in vivo biosafety of Zn-0.8Mn alloy was verified by H&E staining and blood tests. The exceptional mechanical performance and favorable osteogenic capability render Zn-Mn alloy a promising candidate material in the treatment of bone defects or fracture repair. STATEMENT OF SIGNIFICANCE: The element Mn, on the one hand, as an essential trace element in the human body, promotes cell proliferation, adhesion, spreading, and regulates bone metabolism; on the other hand, it could significantly improve the ductility of Zn alloys. Here, we systematically reported the biocompatibility and biofunctionality of binary biodegradable Zn-Mn alloys in the bone environment. The Zn-Mn alloys promoted MC3T3-E1 cell proliferation, adhesion, spreading, and osteogenic differentiation in vitro. Furthermore, a rat femoral condyle defect model was established; porous Zn-Mn alloy scaffolds were manufactured to repair the bone defects. Significant bone regenerations, considerable bone ingrowth, and desirable biosafety were confirmed in vivo. Therefore, biodegradable Zn-Mn with promising osteogenic properties may become new options for orthopedic implant materials.

Antibacterial activity of copper-bearing 316L stainless steel for the prevention of implant-related infection.

Implant-related infection (IRI) is a devastating complication in orthopedic procedures. Traditional materials used in orthopedics are susceptible to bacterial infection. In this study, we developed a copper-bearing 316L stainless steel (316L-Cu SS) for the prevention of IRI. This 316L-Cu SS allowed stable and continuous release of copper ions with a rate of 5.079 ng/cm2 /day. Compared with 316L stainless steel (316L SS), 316L-Cu SS exhibited a broad-spectrum antibacterial effect against Staphylococcus aureus, Escherichia coli, and Staphylococcus epidermidis with the bacterial reduction percentages of 95.2, 94.8, and 94.1%, respectively. The antibiofilm activity was confirmed by crystal violet assay, scanning electron microscopy, and confocal laser scanning microscopy. The in vivo antibacterial performance was tested on a rat model. When nails were treated with a low concentration of bacteria, 316L SS group exhibit a bone infection with a radiographic score of 8.9 ± 1.1 and a histological score of 10.4 ± 1.0, which were higher than 316L-Cu SS group (1.2 ± 0.2 and 0.9 ± 0.2), indicating IRI was reduced by 316L-Cu SS. When nails were treated with a high concentration of bacteria, IRI was also alleviated by 316L-Cu SS. Together, these results demonstrated that 316L-Cu SS is a promising material for preventing IRI.

High-Dose TGF-ß1 Impairs Mesenchymal Stem Cell-Mediated Bone Regeneration via Bmp2 Inhibition.

Transforming growth factor-ß1 (TGF-ß1) is a key factor in bone reconstruction. However, its pathophysiological role in non-union and bone repair remains unclear. Here we demonstrated that TGF-ß1 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-ß1 inhibited BMMSC osteogenesis and attenuated bone regeneration in vivo. Furthermore, different TGF-ß1 levels exhibited opposite effects on osteogenic differentiation and bone healing. Mechanistically, low TGF-ß1 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-ß1 levels. In addition, high TGF-ß1 doses increased tomoregulin-1 (Tmeff1) levels, resulting in the repression of Bmp2 and bone formation in mice. Treatment with the TGF-ß1 inhibitor SB431542 significantly rescued BMMSC osteogenesis and accelerated bone regeneration. Our study suggests that high-dose TGF-ß1 dampens BMMSC-mediated bone regeneration by activating canonical TGF-ß/smad3 signaling and inhibiting Bmp2 via direct and indirect mechanisms. These data collectively show a previously unrecognized mechanism of TGF-ß1 in bone repair, and TGF-ß1 is an effective therapeutic target for treating bone regeneration disability. © 2019 American Society for Bone and Mineral Research.

Comparison and characterization of enriched mesenchymal stem cells obtained by the repeated filtration of autologous bone marrow through porous biomaterials.

BACKGROUND: When bone marrow is repeatedly filtered through porous material, the mesenchymal stem cells (MSCs) in the bone marrow can adhere to the outer and inner walls of the carrier material to become enriched locally, and this is a promising method for MSC enrichment. In this process, the enrichment efficiency of MSCs involved in the regulation of the cell ecology of postfiltration composites containing other bone marrow components is affected by many factors. This study compared the enrichment efficiency and characterized the phenotypes of enriched MSCs obtained by the filtration of autologous bone marrow through different porous bone substitutes. METHODS: Human bone marrow was filtered through representative porous materials, and different factors affecting MSC enrichment efficiency were evaluated. The soluble proteins and MSC phenotypes in the bone marrow before and after filtration were also compared. RESULTS: The enrichment efficiency of the MSCs found in gelatin sponges was 96.1% ± 3.4%, which was higher than that of MSCs found in allogeneic bone (72.5% ± 7.6%) and porous ß-TCP particles (61.4% ± 5.4%). A filtration frequency of 5-6 and a bone marrow/material volume ratio of 2 achieved the best enrichment efficiency for MSCs. A high-throughput antibody microarray indicated that the soluble proteins were mostly filtered out and remained in the flow through fluid, whereas a small number of proteins were abundantly (> 50%) enriched in the biomaterial. In terms of the phenotypic characteristics of the MSCs, including the cell aspect ratio, osteogenetic fate, specific antigens, gene expression profile, cell cycle stage, and apoptosis rate, no significant changes were found before or after filtration. CONCLUSION: When autologous bone marrow is rapidly filtered through porous bone substitutes, the optimal enrichment efficiency of MSCs can be attained by the rational selection of the type of carrier material, the bone marrow/carrier material volume ratio, and the filtration frequency. The enrichment of bone marrow MSCs occurs during filtration, during which the soluble proteins in the bone marrow are also absorbed to a certain extent. This filtration enrichment technique does not affect the phenotype of the MSCs and thus may provide a safe alternative method for MSC enrichment.

lncRNA ADAMTS9-AS2 Controls Human Mesenchymal Stem Cell Chondrogenic Differentiation and Functions as a ceRNA.

Long noncoding RNAs (lncRNAs) have emerged as key regulators of cell differentiation and development. However, potential roles for lncRNAs in chondrogenic differentiation have remained poorly understood. Here we identify lncRNA ADAMTS9 antisense RNA 2, ADAMTS9-AS2, which controls the chondrogenic differentiation by acting as a competing endogenous RNA (ceRNA) in human mesenchymal stem cells (hMSCs). We screen out ADAMTS9-AS2 of undifferentiated and differentiated cells during chondrogenic differentiation by microarrays. Suppression or overexpression of lncRNA ADAMTS9-AS2 correlates with inhibition and promotion of hMSC chondrogenic differentiation, respectively. We find that ADAMTS9-AS2 can sponge miR-942-5p to regulate the expression of Scrg1, a transcription factor promoting chondrogenic gene expression. Finally, we confirm the function of ADAMTS9-AS2 to cartilage repair in the absence of transforming growth factor ß (TGF-ß) in vivo. In conclusion, ADAMTS9-AS2 plays an important role in chondrogenic differentiation as a ceRNA, so that it can be regarded as a therapy target for cartilage repair.

Screen-enrich-combine circulating system to prepare MSC/ß-TCP for bone repair in fractures with depressed tibial plateau.

Aim: To evaluate the clinical efficacy of mesenchymal stem cell/ß-tricalcium phosphate composites (MSC/ß-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/ß-TCP (n = 16) or with ß-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 ß-TCP. At 18 months postimplantation, new bone ratio was significantly higher in patients treated with MSC/ß-TCP than in those treated with ß-TCP only (p = 0.000). Patients with MSC/ß-TCP implants had better functional recovery (p = 0.028). Conclusion: MSC/ß-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.

Bone Mesenchymal Stem Cell-Enriched ß-Tricalcium Phosphate Scaffold Processed by the Screen-Enrich-Combine Circulating System Promotes Regeneration of Diaphyseal Bone Non-Union.

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 ß-tricalcium phosphate (ß-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 ß-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 ß-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.

Mesenchymal stem cells and porous ß-tricalcium phosphate composites prepared through stem cell screen-enrich-combine(-biomaterials) circulating system for the repair of critical size bone defects in goat tibia.

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 ß-tricalcium phosphate (ß-TCP) composites (MSCs/ß-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/ß-TCP prepared by SECCS and the other six goats (the control group) were treated with pure porous ß-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/ß-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 ß-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/ß-TCP was performed, with the MSCs spreading adequately, proliferating actively, and retaining the osteogenic potential. In vivo, the defect repair by MSCs/ß-TCP with good medullary cavity recanalization and cortical remodeling was significantly superior to that of pure porous ß-TCP. CONCLUSIONS: The MSCs/ß-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.

A novel cytotherapy device for rapid screening, enriching and combining mesenchymal stem cells into a biomaterial for promoting bone regeneration.

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 (ß-TCP) granules were processed in the SECCS for 10-15 minutes, to produce MSC/ß-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 ß-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/ß-TCP composites for bone regeneration during surgery. The SECCS represents a convenient, rapid, low-cost, and safe method for bone regeneration.