Effect of strontium-containing on the properties of Mg-doped wollastonite bioceramic scaffolds.

BACKGROUND: Bone scaffold is one of the most effective methods to treat bone defect. The ideal scaffold of bone tissue should not only provide space for bone tissue growth, but also have sufficient mechanical strength to support the bone defect area. Moreover, the scaffold should provide a customized size or shape for the patient's bone defect. METHODS: In this study, strontium-containing Mg-doped wollastonite (Sr-CSM) bioceramic scaffolds with controllable pore size and pore structure were manufactured by direct ink writing 3D printing. Biological properties of Sr-CSM scaffolds were evaluated by apatite formation ability, in vitro proliferation ability of rabbit bone-marrow stem cells (rBMSCs), and alkaline phosphatase (ALP) activity using ß-TCP and Mg-doped wollastonite (CSM) scaffolds as control. The compression strength of three scaffold specimens was probed after completely drying them while been submerged in Tris-HCl solution for 0, 2,4 and 6 weeks. RESULTS: The mechanical test results showed that strontium-containing Mg-doped wollastonite (Sr-CSM) scaffolds had acceptable initial compression strength (56 MPa) and maintained good mechanical stability during degradation in vitro. Biological experiments showed that Sr-CSM scaffolds had a better apatite formation ability. Cell experiments showed that Sr-CSM scaffold had a higher cell proliferation ability compared with ß-TCP and CSM scaffold. The higher ALP activity of Sr-CSM scaffold indicates that it can better stimulate osteoblastic differentiation and bone mineralization. CONCLUSIONS: Therefore, Sr-CSM scaffolds not only have acceptable compression strength, but also have higher osteogenesis bioactivity, which can be used in bone tissue engineering scaffolds.

Partially Melted Ti6Al4V Particles Increase Bacterial Adhesion and Inhibit Osteogenic Activity on 3D-printed Implants: An In Vitro Study.

BACKGROUND: A porous Ti6Al4V implant that is manufactured using selective laser melting (SLM) has broad potential applications in the field of orthopaedic implants. The pore structure of the SLM porous Ti6Al4V implant allows for cell migration and osteogenic differentiation, which is favorable for bone ingrowth and osseointegration. However, it is unclear whether the pore structure and partially melted Ti6Al4V particles on a SLM porous Ti6Al4V implant will increase bacterial adhesion and, perhaps, the risk of implant-related infection. QUESTIONS/PURPOSES: (1) Is there more bacterial adhesion and colonization on SLM porous Ti6Al4V implants than on polished orthopaedic implants? (2) Do partially melted Ti6Al4V particles on SLM porous Ti6Al4V implants reduce human bone mesenchymal stem cells (hBMSCs) adhesion, viability, and activity? METHODS: To determine bacterial adhesion and biofilm formation, we incubated five different Ti6Al4V discs (polished, grit-blasted, plasma-sprayed, particle SLM porous, and nonparticle SLM porous discs) with methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli. Bacterial coverage on the surface of the five different Ti6Al4V discs were evaluated based on scanning electron microscopy (SEM) images quantitatively. In addition, a spread-plate method was used to quantitatively evaluate the bacterial adhesion on those implants. The biofilm formation was stained with crystal violet and semi-quantitatively determined with a microplate reader. The morphology and adhesion of hBMSCs on the five Ti6Al4V discs were observed with SEM. The cell viability was quantitatively evaluated with a Cell Counting Kit-8 assay. In addition, the osteogenic activity was determined in vitro with a quantitatively alkaline phosphatase activity assay and alizarin-red staining. For semiquantitative analysis, the alizarin-red stained mineralized nodules were dissolved and determined with a microplate reader. RESULTS: The polished discs had the lowest MRSA adhesion (8.3% ± 2.6%) compared with grit-blasted (19.1% ± 3.9%; p = 0.006), plasma-sprayed (38.5% ± 5.3%; p < 0.001), particle (23.1% ± 2.8%; p < 0.001), and nonparticle discs (15.7% ± 2.5%; p = 0.003). Additionally, when comparing the two SLM discs, we found that particle discs had higher bacterial coverage than nonparticle discs (23.1% ± 2.8% versus 15.7% ± 2.5%; p = 0.020). An E. coli analysis showed similar results, with the higher adhesion to particle SLM discs than to nonparticle discs (20.7% ± 4.2% versus 14.4% ± 3.6%; p = 0.011). In addition, on particle SLM porous discs, bacterial colonies were localized around the partially melted Ti6Al4V particles, based on SEM images. After a 7-day incubation period, the cell viability in the particle group (optical density value 0.72 ± 0.05) was lower than that in the nonparticle groups (optical density value: 0.87 ± 0.08; p = 0.003). Alkaline phosphatase activity, as a marker of osteogenic differentiation, was lower in the particle group than in the nonparticle group (1.32 ± 0.12 U/mL versus 1.58 ± 0.09 U/mL; p = 0.012). CONCLUSION: Higher bacterial adhesion was observed on SLM porous discs than on polished discs. The partially melted Ti6Al4V particles on SLM porous discs not only enhanced bacterial adhesion but also inhibited the osteogenic activity of hBMSCs. Postprocessing treatment is necessary to remove partially melted Ti6Al4V particles on an SLM implant before further use. Additional studies are needed to determine whether an SLM porous Ti6Al4V implant increases the risk of implant-related infection in vivo. CLINICAL RELEVANCE: As implants with porous Ti6Al4V made using SLM are being designed, our preliminary findings suggest that postprocessing treatment is needed to remove partially melted Ti6Al4V particles before further use. In addition, the depth of the porous structure of the SLM implant should not exceed the maximum depth of bone ingrowth because the host immune defense cannot prevent bacterial adhesion without integration.

Poor osteoinductive potential of subcutaneous bone cement-induced membranes for tissue engineered bone.

BACKGROUND: Large segmental bone defects remain a challenge for reconstructive surgeons. A two-stage repair strategy may offer a potential solution. Here, we sought to evaluate the osteoinductive potential of bone cement-induced membranes in an ectopic site. METHODS: First, bone cements were inserted into the subcutaneous tissues of 16 rabbits to induce membrane formation. After 2, 4, 6 and 8 weeks, the induced membranes were harvested to assess their vascularization and osteoinductive potential. Next, bone cements were subcutaneously inserted into 12 rabbits for 4 weeks. These bone cements were then harvested from the newly formed membranes and replaced with granular porous ß-TCP, with or without bone mesenchymal stem cells. New bone formation was then evaluated after 3, 6 and 9 weeks. RESULTS: The highest level of blood vessel formation and bone morphogenetic protein-2 expression in the membranes were found at 4 weeks (p < 0.05). In addition, vascular endothelial growth factor concentration was highest after 2 weeks (p < 0.001), persisting until 8 weeks. However, the results showed little ectopic bone formation at these time points. CONCLUSION: While bone cement-induced membranes appear to provide a suitable environment for bone formation, they fail to drive osteoinduction in non-osseous sites for the purposes of bone tissue engineering.

Evaluation of osteoinduction and proliferation on nano-Sr-HAP: a novel orthopedic biomaterial for bone tissue regeneration.

Hydroxyapatite (HAP), a CaP compound similar to the mineral phase present in bone, has excellent biocompatibility but little osseous inductivity. In this study, we evaluated the novel nano-Sr-HAP, in which the calcium of hydroxyapatite was substituted with strontium, which acts as a bone-forming agent. Its biocompatibility and osteoinduction were assayed using marrow mesenchymal stem cells (MSCs) and osteoblasts (OBs) in vitro. We were able to demonstrate that nano-Sr-HAP supported increased OB cell adhesion, proliferation and viability up to 4 days in culture when compared with nano-HAP. MSCs cultured with nano-Sr-HAP showed higher alkaline phosphatase (ALP) activity. More extracellular mineralized nodules were found with nano-Sr-HAP compared to nano-HAP, especially in images of ALP staining. We suggest that nano-Sr-HAP powders possess osteoconductive and osteoinductive properties and have the potential to be used in the repair of bone defects caused by osteoporotic fractures.

Fabrication and Biological Activity of 3D-Printed Polycaprolactone/Magnesium Porous Scaffolds for Critical Size Bone Defect Repair.

Polycaprolactone (PCL) is widely used in bone tissue engineering due to its biocompatibility and mechanical strength. However, PCL is not biologically active and shows poor hydrophilicity, making it difficult for new bones to bind tightly to its surface. Magnesium (Mg), an important component of natural bone, exhibits good osteo-inductivity and biological activity. Therefore, porous PCL/Mg scaffolds, including pure PCL, PCL/5%Mg, PCL/10%Mg, and PCL/15%Mg, were prepared to elucidate whether the porous structure of scaffolds and the bioactivity of PCL may be enhanced via 3D printing and incorporation of Mg powder. Compared with the control group (pure PCL only), the hydrophilicity of composite PCL/Mg scaffolds was greatly increased, resulting in the scaffolds having decreased water contact angles. Tests for adhesion and proliferation of rat bone marrow mesenchymal stem cells (rBMSCs) indicated that the PCL/10%Mg scaffold showed superior compatibility. Furthermore, as indicated by alkaline phosphatase (ALP) activity and semiquantitative analysis of alizarin red staining, PCL/10%Mg scaffolds exhibited significantly stronger osteogenic activity than the other scaffolds. Animal experiments demonstrated that PCL/10%Mg scaffolds displayed pro-osteogenic effects at an early stage (4 weeks) and produced more new bone mass 8-12 weeks following implantation, compared with the control group. Visceral and blood parameter analyses indicated that PCL/10%Mg scaffolds did not exert any noticeable toxic effects. PCL/10%Mg composite scaffolds were found to promote bone defect repair at an early stage with good cytocompatibility. This finding revealed a new concept in designing bone tissue materials, which showed potential as a clinical treatment for bone defects.

Anodic fabrication and bioactivity of Nb-doped TiO2 nanotubes.

We report anodic formation of Ti-Nb-O nanotubes on top of a Ti35Nb alloy, and in vitro bioactivity and stem cell response of the anodic nanotubes. It was found that the amorphous Ti-Nb-O nanotubes presented a significantly enhanced in vitro bioactivity (in simulated body fluids) compared to those of undoped TiO2 nanotubes and porous Ti-Nb-O without nanotubular structure. Similar to undoped TiO2 nanotubes, the Ti-Nb-O nanotubes also promote mesenchymal stem cell adhesion and fast formation of extracellular matrix (ECM) materials. The above findings make it possible to further explore the biological properties, such as cell proliferation and drug delivery, of a variety of Ti-alloy-based oxide nanotubes.

Osteoblast/bone-tissue responses to porous surface of polyetheretherketone-nanoporous lithium-doped magnesium silicate blends' integration with polyetheretherketone.

The porous surface of a polyetheretherketone (PK)-nanoporous lithium-doped magnesium silicate (NLS) blend (PKNLS) was fabricated on a PK surface by layer-by-layer pressuring, sintering, and salt-leaching. As controls, porous surfaces of a PK/lithium-doped magnesium silicate blend (PKLS) and PK were fabricated using the same method. The results revealed that porosity, water absorption, and protein absorption of the porous surface of PKNLS containing macropores and nanopores were obviously enhanced compared to PKLS and PK containing macropores without nanopores. In addition, PKNLS, with both macroporostiy and nanoporosity, displayed the highest ability of apatite mineralization in simulated body liquid, indicating excellent bioactivity. In vitro responses (including adhesion, proliferation, and differentiation) of MC3T3E1 cells to PKNLS were significantly enhanced compared to PKLS and PK. In vivo implantation results showed that new bone grew into the macroporous surface of PKNLS, and the amount of new bone for PKNLS was the highest. In short, PKNLS integration with PK significantly promoted cells/bone-tissue responses and exhibited excellent osteogenesis in vivo, which might have great potential for bone repair.

Long-Term Prevention of Bacterial Infection and Enhanced Osteoinductivity of a Hybrid Coating with Selective Silver Toxicity.

Antibacterial and osteogenic design is required for ideal orthopedic implants. The excellent antimicrobial performance of silver nanoparticles (AgNPs) has attracted interest for the treatment of implant-related infections. However, the dose-dependent cytotoxicity of silver and its negative impact on bone implants restrict the further use of AgNPs coatings. Therefore, a hybrid coating containing polydopamine (PDA), hydroxyapatite (HA), AgNPs, and chitosan (CS) is prepared. Organic chelators CS and PDA that have promising biocompatibility are used to prevent the rapid release of silver ions from the AgNPs coating. The double chelating effect of PDA and CS significantly reduces silver ion release from the hybrid coating. The coating exhibits excellent anti-biofilm efficiency of 91.7%, 89.5%, and 92.0% for Staphylococcus aureus, Staphylococcus epidermidis, and Escherichia coli, respectively. In addition, the coating can significantly stimulate osteogenic differentiation of MC3T3-E1 cells and promote bone-implant osseointegration in vivo as compared to that in the control group. The longitudinal biosafety of the coating is confirmed in vivo by histological evaluation and blood tests. The results of this study indicate that the hybrid coating exhibits antibacterial properties as well as allow bone-implant osseointegration, thereby providing insight into the design of multifunctional implants for long-term orthopedic applications.

[Clinical study of lumbar fusion by hybrid construct of stem cells technique and biodegradable material].

OBJECTIVE: To explorer the effectiveness of enriched bone marrow stem cells technique for lumbar fusion. METHODS: With the randomization and control principles, 2 graft materials [Enrichment bone marrow mesenchymal stem cells hybridized with beta-tri calcium phosphate (composite graft group), autologous iliac crest bone graft (autograft group)] were compared in posterior lumbar fusion procedures. 56 patients with degenerative disc disease, lumbar instability or spinal stenosis, were included. The volume of cells suspension in pre- and post-enrichment and the number of nucleated cells (NCs) were identified. The number of osteoprogenitor cells was estimated by counting the colony-forming units which express alkaline phosphatase (CFUs/ALP+). Then the efficiency of the enrichment was evaluated. Clinical follow-up with roentgenogram and Oswestry scale scores was performed for outcome evaluation. RESULTS: (249 +/- 31) ml bone marrow per patient from bilateral iliac crests was aspirated peri-operatively. About (43 +/- 11) ml enriched bone marrow was collected. The number of NCs was concentrated from (15.9 +/- 3.3) x 10(6)/ml to (44.1 +/- 10.8) x 10(6)/ml, CFUs/ALP+ was significantly increased from (118 +/- 86)/ml to(486 +/- 305)/ml. The follow-up was about (26.3 +/- 7.5) months. There was no significant differences in age, gender, disease and fusion segments between the two groups. The fusion rate was 93.3% and 96.2% for composite graft group and autograft group, respectively (chi2 = 0.2146, P = 0.6432). There was no difference in operation time between the two group (t = 0.5243, P = 0.6022), but blood loss in composite graft group was more than that in autograft group (t = 6.4664, P < 0.01). Cell salvage for auto-transfusion could transfuse back half of the blood loss during operation. No hematoma or chronic soreness in the bone marrow donor sites of composite graft group occurred, but a little exudation or moderate swelling in the wound happened in 4 cases which disappeared under medical treatment. Meanwhile, 15.4% patients had hematoma in the iliac bone donor site and 26.9% patients had chronic soreness, but no case had wound problem in autograft group. As for Oswestry scale scores, there was no significant difference between the two groups. CONCLUSIONS: The enrichment technique of autologous bone marrow stem cells can greatly increase the concentration of MSCs. It is a rapid and safe method used peri-operatively. The composite material of enriched MSCs and porous beta-TCP is a good bone substitute in posterior spinal fusion.

Dual modulation of bone formation and resorption with zoledronic acid-loaded biodegradable magnesium alloy implants improves osteoporotic fracture healing: An in vitro and in vivo study.

Osteoporotic fracture (OPF) remains a major clinical challenge for skeletal regeneration. Impaired osteogenesis and excessive remodeling result in prolonged and poor quality of fracture healing. To augment bone formation and inhibit excessive resorption simultaneously, we constructed a biodegradable magnesium-based implant integrated with the anti-catabolic drug zoledronic acid (ZA); this implant exhibits controllable, sustained release of magnesium degradation products and ZA in vitro. The extracts greatly stimulate the osteogenic differentiation of rat-bone marrow-derived mesenchymal stem cells (rBMSCs), while osteoclastogenesis is inhibited by ZA. Implantation of intramedullary nails to fix femur fracture in ovariectomy-induced osteoporotic rats for up to 12 weeks demonstrates magnesium implants alone can enhance OPF repair through promoting callus formation compared to conventional stainless steel, while the combinatory treatment with local ZA release from implant coating further increases bone regeneration rate and callus size, remarkably improves bone quality and mechanical strength and suppresses osteoclasts and bone remodeling, due to the synergistic effect of both agents. The slow and uniform degradation of the implant ensures a steady decrease in bending force, which meets clinical requirements. In summary, biodegradable magnesium-based implants can locally co-deliver magnesium degradation products and zoledronic acid in a controlled manner, and can be superior alternatives for the reconstruction of osteoporosis-related fracture. STATEMENT OF SIGNIFICANCE: Management of osteoporotic fracture has posed a major challenge in orthopedics, as the imbalance between diminished osteogenesis and excessive bone remodeling often leads to delayed and compromised fracture repair. Among various efforts expended on augmenting osteoporotic fracture healing, herein we reported a new strategy by engineering and utilizing a biodegradable magnesium-based implant integrated with local drug delivery, specifically, zoledronic acid (ZA)-loaded polylactic acid/brushite bilayer coating on a biodegradable Mg-Nd-Zn-Zr alloy (denoted as Mg/ZA/CaP), aiming to combine the favorable properties of Mg and zoledronic acid for simultaneous modulation of bone formation and bone resorption. In vitro and in vivo studies demonstrated its superior treatment efficacy along with adequate degradation. It stimulated new bone formation while suppressing remodeling, ascribed to the local release of magnesium degradation products and zoledronic acid. To our knowledge, the enhanced fracture repair capability of Mg-based implants was for the first time demonstrated in an osteoporotic fracture animal model. This innovative biodegradable Mg-based orthopedic implant presents great potential as a superior alternative to current internal fixation devices for treating osteoporotic fracture.

Mg-based bone implants show promising osteoinductivity and controllable degradation: A long-term study in a goat femoral condyle fracture model.

In this work, the in vivo biocompatibility and biodegradation behavior of Mg-Nd-Zn-Zr alloy (denoted as JDBM) screws were studied using a goat femoral condyle fracture model. Blood analysis indicates that the liver and kidney functions of goats were not affected by JDBM, JDBM coated with brushite (denoted as JDBM-DCPD) and PLA implants. Radiographic analysis shows that JDBM-DCPD screw has lower degradation rate than JDBM. Histological images show that compared with PLA, JDBM and JDBM-DCPD show superior effect to promote more new bone formation. JDBM-DCPD group has more new bone formation than JDBM group, indicating good osteoinductivity of DCPD coating. JDBM group show higher osteogenic factors level (BMP2, ALP and OC) in peri-implant callus tissue than PLA group. Long-term (18months) in vivo implants Micro-CT result shows that the degradation of JDBM-DCPD screw may be slower than desirable, and the thickness of DCPD coating could be further adjusted to match the degradation to the bone recovery.

Nano-copper-bearing stainless steel promotes fracture healing by accelerating the callus evolution process.

Treatment for fractures requires internal fixation devices, which are mainly produced from stainless steel or titanium alloy without biological functions. Therefore, we developed a novel nano-copper-bearing stainless steel with nano-sized copper-precipitation (317L-Cu SS). Based on previous studies, this work explores the effect of 317L-Cu SS on fracture healing; that is, proliferation, osteogenic differentiation, osteogenesis-related gene expression, and lysyl oxidase activity of human bone mesenchymal stem cells were detected in vitro. Sprague-Dawley rats were used to build an animal fracture model, and fracture healing and callus evolution were investigated by radiology (X-ray and micro-CT), histology (H&E, Masson, and safranin O/fast green staining), and histomorphometry. Further, the Cu2+ content and Runx2 level in the callus were determined, and local mechanical test of the fracture was performed to assess the healing quality. Our results revealed that 317L-Cu SS did not affect the proliferation of human bone mesenchymal stem cells, but promoted osteogenic differentiation and the expression of osteogenesis-related genes. In addition, 317L-Cu SS upregulated the lysyl oxidase activity. The X-ray and micro-CT results showed that the callus evolution efficiency and fracture healing speed were superior for 317L-Cu SS. Histological staining displayed large amounts of fibrous tissues at 3 weeks, and cartilage and new bone at 6 weeks. Further, histomorphometric analysis indicated that the callus possessed higher osteogenic efficiency at 6 weeks, and a high Cu2+ content and increased Runx2 expression were observed in the callus for 317L-Cu SS. Besides, the mechanical strength of the fracture site was much better than that of the control group. Overall, we conclude that 317L-Cu SS possesses the ability to increase Cu2+ content and promote osteogenesis in the callus, which could accelerate the callus evolution process and bone formation to provide faster and better fracture healing.

Biodegradable Mg-Cu alloy implants with antibacterial activity for the treatment of osteomyelitis: In vitro and in vivo evaluations.

Treatment of chronic osteomyelitis (bone infection) remains a clinical challenge; in particular, it requires an implantable material with improved antibacterial activity. Here, we prepared biodegradable magnesium (Mg)-copper (Cu) alloys with different Cu contents (0.05, 0.1, and 0.25 wt%) and assessed their potential for treating methicillin-resistant Staphylococcus aureus-induced osteomyelitis. We evaluated the microstructures, mechanical properties, corrosion behavior, and ion release of the alloys in vitro, and their biocompatibility and antibacterial activity in vitro and in vivo. The antibacterial activity of the Mg-Cu alloys in vitro was demonstrated by microbiological counting assays, bacterial viability assays, biofilm formation observations, and the expression of biofilm, virulence, and antibiotic-resistance associated genes. The antibacterial activity of Mg-Cu alloys in vivo was confirmed by imaging examination, microbiological cultures, and histopathology. The biocompatibility of Mg-Cu alloys was confirmed by cell proliferation, vitality, and morphology assays in vitro and Cu(2+) or Mg(2+) ion assays, blood biochemical tests, and histological evaluation in vivo. The alloy containing 0.25 wt% Cu exhibited the highest antibacterial activity among the tested alloys, with favorable biocompatibility. Collectively, our results indicate the potential utility of Mg-Cu alloy implants with 0.25 wt% Cu in treating orthopedic infections.

A novel nano-copper-bearing stainless steel with reduced Cu(2+) release only inducing transient foreign body reaction via affecting the activity of NF-κB and Caspase 3.

Foreign body reaction induced by biomaterials is a serious problem in clinical applications. Although 317L-Cu stainless steel (317L-Cu SS) is a new type of implant material with antibacterial ability and osteogenic property, the foreign body reaction level still needs to be assessed due to its Cu(2+) releasing property. For this purpose, two macrophage cell lines were selected to detect cellular proliferation, apoptosis, mobility, and the secretions of inflammatory cytokines with the influence of 317L-Cu SS. Our results indicated that 317L-Cu SS had no obvious effect on the proliferation and apoptosis of macrophages; however, it significantly increased cellular migration and TNF-α secretion. Then, C57 mice were used to assess foreign body reaction induced by 317L-Cu SS. We observed significantly enhanced recruitment of inflammatory cells (primarily macrophages) with increased TNF-α secretion and apoptosis level in tissues around the materials in the early stage of implantation. With tissue healing, both inflammation and apoptosis significantly decreased. Further, we discovered that NF-κB pathway and Caspase 3 played important roles in 317L-Cu SS induced inflammation and apoptosis. We concluded that 317L-Cu SS could briefly promote the inflammation and apoptosis of surrounding tissues by regulating the activity of NF-κB pathway and Caspase 3. All these discoveries demonstrated that 317L-Cu SS has a great potential for clinical application.