Corrosion and bone healing of Mg-Y-Zn-Zr-Ca alloy implants: Comparative in vivo study in a non-immobilized rat femoral fracture model.

Biodegradable magnesium (Mg) alloys exhibit improved mechanical properties compared to degradable polymers while degrading in vivo circumventing the complications of permanent metals, obviating the need for surgical removal. This study investigated the safety and efficacy of Mg-Y-Zn-Zr-Ca (WZ42) alloy compared to non-degradable Ti6Al4V over a 14-week follow-up implanted as pins to fix a full osteotomy in rat femurs and as wires wrapped around the outside of the femurs as a cerclage. We used a fully load bearing model allowing implants to intentionally experience realistic loads without immobilization. To assess systemic toxicity, blood cell count and serum biochemical tests were performed. Livers and kidneys were harvested to observe any accumulation of alloying elements. Hard and soft tissues adjacent to the fracture site were also histologically examined. Degradation behavior and bone morphology were determined using micro-computed tomography scans. Corrosion occurred gradually, with degradation seen after two weeks of implantation with points of high stress observed near the fracture site ultimately resulting in WZ42 alloy pin fracture. At 14 weeks however, normal bone healing was observed in femurs fixed with the WZ42 alloy confirmed by the presence of osteoid, osteoblast activity, and new bone formation. Blood testing exhibited no significant changes arising from the WZ42 alloy compared to the two control groups. No recognizable differences in the morphology and more importantly, no accumulation of Mg, Zn, and Ca in the kidney and liver of rats were observed. These load bearing model results collectively taken, thus demonstrate the feasibility for use of the Mg-Y-Zn-Zr-Ca alloy for long bone fracture fixation applications.

In vivo characterization of magnesium alloy biodegradation using electrochemical H2 monitoring, ICP-MS, and XPS.

The effect of widely different corrosion rates of Mg alloys on four parameters of interest for in vivo characterization was evaluated: (1) the effectiveness of transdermal H2 measurements with an electrochemical sensor for noninvasively monitoring biodegradation compared to the standard techniques of in vivo X-ray imaging and weight loss measurement of explanted samples, (2) the chemical compositions of the corrosion layers of the explanted samples by XPS, (3) the effect on animal organs by histology, and (4) the accumulation of corrosion by-products in multiple organs by ICP-MS. The in vivo biodegradation of three magnesium alloys chosen for their widely varying corrosion rates - ZJ41 (fast), WKX41 (intermediate) and AZ31 (slow) - were evaluated in a subcutaneous implant mouse model. Measuring H2 with an electrochemical H2 sensor is a simple and effective method to monitor the biodegradation process in vivo by sensing H2 transdermally above magnesium alloys implanted subcutaneously in mice. The correlation of H2 levels and biodegradation rate measured by weight loss shows that this non-invasive method is fast, reliable and accurate. Analysis of the insoluble biodegradation products on the explanted alloys by XPS showed all of them to consist primarily of Mg(OH)2, MgO, MgCO3 and Mg3(PO4)2 with ZJ41 also having ZnO. The accumulation of magnesium and zinc were measured in 9 different organs by ICP-MS. Histological and ICP-MS studies reveal that there is no significant accumulation of magnesium in these organs for all three alloys; however, zinc accumulation in intestine, kidney and lung for the faster biodegrading alloy ZJ41 was observed. Although zinc accumulates in these three organs, no toxicity response was observed in the histological study. ICP-MS also shows higher levels of magnesium and zinc in the skull than in the other organs. STATEMENT OF SIGNIFICANCE: Biodegradable devices based on magnesium and its alloys are promising because they gradually dissolve and thereby avoid the need for subsequent removal by surgery if complications arise. In vivo biodegradation rate is one of the crucial parameters for the development of these alloys. Promising alloys are first evaluated in vivo by being implanted subcutaneously in mice for 1month. Here, we evaluated several magnesium alloys with widely varying corrosion rates in vivo using multiple characterization techniques. Since the alloys biodegrade by reacting with water forming H2 gas, we used a recently demonstrated, simple, fast and noninvasive method to monitor the biodegradation process by just pressing the tip of a H2 sensor against the skin above the implant. The analysis of 9 organs (intestine, kidney, spleen, lung, heart, liver, skin, brain and skull) for accumulation of Mg and Zn revealed no significant accumulation of magnesium in these organs. Zinc accumulation in intestine, kidney and lung was observed for the faster corroding implant ZJ41. The surfaces of explanted alloys were analyzed to determine the composition of the insoluble biodegradation products. The results suggest that these tested alloys are potential candidates for biodegradable implant applications.

Biomimetic Rotated Lamellar Plywood Motifs by Additive Manufacturing of Metal Alloy Scaffolds for Bone Tissue Engineering.

Additive manufacturing presents opportunities to treat bone defects using biomimetic tissue scaffolds. Past investigations have explored modulating scaffold mechanical properties through varying materials and geometric motifs. Herein, we applied the rotated plywood structure of bone tissue to a 3D printed scaffold with the goal of improving mechanical performance compared to an orthogonal mesh design commonly used in tissue scaffold applications. The scaffolds were subjected to uniaxial compression followed by scanning electron microscopy and microcomputer tomography. The uniaxial compression test was characterized through elastic modulus (mean 1.32 GPa biomimetic, 0.196 GPa orthogonal, p < 0.001), ultimate compressive strength (mean 16.546 MPa biomimetic, 6.309 MPa orthogonal design, p < 0.001), and ultimate compressive strain values (4.867% biomimetic, 9.000% orthogonal, p < 0.005). Correlation of microfracture imaging to bulk scaffold mode of failure suggest that utilizing the biomimetic plywood design not only improved mechanical performance, but also reduced asymmetrtic buckling, plastic deformation, and fracture propagation similar to bone tissue.

Nanostructured silicate substituted calcium phosphate (NanoSiCaPs) nanoparticles - Efficient calcium phosphate based non-viral gene delivery systems.

Nanostructured ceramic particles, particularly, nanoparticles of calcium phosphate (CaP) remain an attractive option among the various types of non-viral gene delivery vectors studied because of their safety, biocompatibility, biodegradability, and ease of handling as well as their adsorptive capacity for DNA. We have accordingly developed an enhanced version of nanostructured calcium phosphates (NanoCaPs), by substituting known amounts of silicate for phosphate in the hydroxyapatite (HA) lattice (NanoSiCaPs). Results indicate that in addition to the excellent transfection levels exhibited by un-substituted NanoCaPs alone in vitro, an additional 20-50% increase in transfection is observed for NanoCaPs containing 8.3-50mol% silicate aptly called NanoSiCaPs, owing to its rapid dissolution properties enabling nanoparticles escaping the lysosomal degradation. However, high silicate substitution (>50mol%) resulted in a drastic decline in transfection as the synthesized NanoCaPs deviated far from the characteristic hydroxyapatite phase formed as evidenced by the materials characterization results.

Visual H2 sensor for monitoring biodegradation of magnesium implants in vivo.

A visual sensor for H2 was used to transdermally monitor H2 that originated from biodegrading magnesium (Mg) alloys implanted subcutaneously in mice. The visual sensor consisted of a thin film of H2-sensitive material (MoO3 and Pd catalyst) coated on a flexible plastic sheet that was pressed against the mouse skin directly above the implant. Although the H2 levels permeating through the skin during the degradation process were very low, the sensor changed color to give a three dimensional (3D) visualization of H2 permeation. The correlation between the visual sensor response and measurements made with an electrochemical H2 microsensor on several magnesium alloys demonstrates that the visual sensor has the capability to monitor in real-time the dissolution rate of implants in vivo. This detection method is noninvasive, easy to implement, effective and potentially low cost compared to electrochemical detection. STATEMENT OF SIGNIFICANCE: Biodegradable Mg implants offer advantages over permanent implants such as stainless steel that are used for broken bone repair. Mg alloys gradually dissolve, avoiding the need for removal by a later surgery if complications arise. Here we report a visual H2 sensor that can be used in the research laboratory to monitor the corrosion process in vivo during animal testing of different Mg alloys. The sensor consists of a plastic sheet with a thin coating that changes color in the presence of H2 gas. The sensor is easily used by taping it on the skin over the Mg implant. The color change gives a map of the H2 level permeating from the degrading Mg through the skin above it. This low cost, simple method of monitoring the dissolution of biodegradable implants would greatly facilitate the development of the biodegradable materials, especially in animal studies where in vivo biodegradation is tested.

Binder-jetting 3D printing and alloy development of new biodegradable Fe-Mn-Ca/Mg alloys.

3D printing of various biomaterials including titanium and stainless steel has been studied for treating patients with cranio-maxillofacial bone defect. The potential long term complications with use of inert biometals have opened the opportunities for use of biodegradable metals in the clinical arena. The authors previously reported that binder-jet 3D printing technique enhanced the degradation rates of biodegradable Fe-Mn alloy by creating engineered micropores rendering the system attractive as biodegradable implantable devices. In the present study, the authors employed CALPHAD modeling to systematically study and modify the Fe-Mn alloy composition to achieve enhanced degradation rates. Accordingly, Ca and Mg addition to Fe-35wt% Mn solid solution predicted increase in degradation rates. In order to validate the CALPHAD results, Fe - (35-y)wt% Mn - ywt% X (X=Ca, Mg, and y=0, 1, 2) were synthesized by using high energy mechanical alloying (HEMA). Sintered pellets of Fe-Mn-Ca and Fe-Mn-Mg were then subjected to potentiodynamic polarization (PDP) and live/dead cell viability tests. Sintered pellets of Fe-Mn, Fe-Mn-Ca, and Fe-Mn-Mg also exhibited MC3T3 murine pre-osteoblast cells viability in the live/dead assay results. Fe-Mn and Fe-Mn-1Ca were thus accordingly selected for 3D printing and the results further confirmed enhanced degradation of Ca addition to 3D printed constructs validating the theoretical and alloy development studies. Live/dead and MTT cell viability results also confirmed good cytocompatibility of the 3D-printed Fe-Mn and Fe-Mn-1Ca constructs. STATEMENT OF SIGNIFICANCE: Bone grafting is widely used for the treatment of cranio-maxillofacial bone injuries. 3D printing of biodegradable Fe alloy is anticipated to be advantageous over current bone grafting techniques. 3D printing offers the fabrication of precise and tailored bone grafts to fit the patient specific bone defect needs. Biodegradable Fe alloy is a good candidate for 3D printing synthetic grafts to regenerate bone tissue without eliciting complications. CALPHAD theoretical models were used to develop new Fe-Mn-Ca/Mg alloys to enhance the degradation rates of traditional Fe-Mn alloys. In vitro experimental results also showed enhanced degradation rates and good cytocompatibility of sintered Fe-Mn-Ca/Mg compacts. 3D printing of Fe-Mn and Fe-Mn-1Ca alloys further demonstrated their feasibility as potentially viable bone grafts for the future.

In vivo monitoring the biodegradation of magnesium alloys with an electrochemical H2 sensor.

UNLABELLED: Monitoring the biodegradation process of magnesium and its alloys in vivo is challenging. Currently, this process is monitored by micro-CT and X-ray imaging in vivo, which require large and costly instrumentation. Here we report a simple and effective methodology to monitor the biodegradation process in vivo by sensing H2 transdermally above a magnesium sample implanted subcutaneously in a mouse. An electrochemical H2 microsensor was used to measure the biodegradation product H2 at the surface of the skin for two magnesium alloys (ZK40 and AZ31) and one high purity magnesium single crystal (Mg8H). The sensor was able to easily detect low levels of H2 (30-400µM) permeating through the skin with a response time of about 30s. H2 levels were correlated with the biodegradation rate as determined from weight loss measurements of the implants. This new method is noninvasive, fast and requires no major equipment. STATEMENT OF SIGNIFICANCE: Biomedical devices such as plates and screws used for broken bone repair are being developed out of biodegradable magnesium alloys that gradually dissolve when no longer needed. This avoids subsequent removal by surgery, which may be necessary if complications arise. A rapid, non-invasive means for monitoring the biodegradation process in vivo is needed for animal testing and point of care (POC) evaluation of patients. Here we report a novel, simple, fast, and noninvasive method to monitor the biodegradation of magnesium in vivo by measuring the biodegradation product H2 with an electrochemical H2 sensor. Since H2 rapidly permeates through biological tissue, measurements are made by simply pressing the sensor tip against the skin above the implant; the response is within 30s.

Synthesis, Osteoblast, and Osteoclast Viability of Amorphous and Crystalline Tri-Magnesium Phosphate.

Magnesium phosphate implants may be used for bone void filling applications, potentially replacing traditionally studied bioceramics, which suffer from limited resorption and inferior mechanical properties compared to natural bone. In this study, amorphous and crystalline trimagnesium phosphates were synthesized and characterized utilizing a variety of analytical methods. In vitro solubility and cytotoxicity of the corresponding amorphous and crystalline phosphates were also analyzed. Amorphous magnesium phosphate was shown to be more soluble than the crystalline counterpart in vitro while inducing mineralization of an amorphous phosphate phase mimicking hydroxyapatite-type characteristic morphology on the substrate surface. The rapid mineralization of the amorphous magnesium phosphate was found to promote the proliferation and differentiation of osteoblast-like cells in comparison to the crystalline phase. Both magnesium phosphates hindered the differentiation of monocytes into osteoclasts. The combined effects of the spontaneous serum-mediated apatite-like mineralization, increased osteoblast differentiation and suspended osteoclast formation indicate that the amorphous magnesium phosphates may be promising bioactive materials for bone void repair applications.

In vitro and in vivo corrosion, cytocompatibility and mechanical properties of biodegradable Mg-Y-Ca-Zr alloys as implant materials.

This study introduces a class of biodegradable Mg-Y-Ca-Zr alloys novel to biological applications and presents evaluations for orthopedic and craniofacial implant applications. Mg-Y-Ca-Zr alloys were processed using conventional melting and casting techniques. The effects of increasing Y content from 1 to 4 wt.% as well as the effects of T4 solution treatment were assessed. Basic material phase characterization was conducted using X-ray diffraction, optical microscopy and scanning electron microscopy. Compressive and tensile tests allowed for the comparison of mechanical properties of the as-cast and T4-treated Mg-Y-Ca-Zr alloys to pure Mg and as-drawn AZ31. Potentiodynamic polarization tests and mass loss immersion tests were used to evaluate the corrosion behavior of the alloys. In vitro cytocompatibility tests on MC3T3-E1 pre-osteoblast cells were also conducted. Finally, alloy pellets were implanted into murine subcutaneous tissue to observe in vivo corrosion as well as local host response through H&E staining. SEM/EDS analysis showed that secondary phase intermetallics rich in yttrium were observed along the grain boundaries, with the T4 solution treatment diffusing the secondary phases into the matrix while increasing the grain size. The alloys demonstrated marked improvement in mechanical properties over pure Mg. Increasing the Y content contributed to improved corrosion resistance, while solution-treated alloys resulted in lower strength and compressive strain compared to as-cast alloys. The Mg-Y-Ca-Zr alloys demonstrated excellent in vitro cytocompatibility and normal in vivo host response. The mechanical, corrosion and biological evaluations performed in this study demonstrated that Mg-Y-Ca-Zr alloys, especially with the 4 wt.% Y content, would perform well as orthopedic and craniofacial implant biomaterials.

In vitro degradation and cytotoxicity response of Mg-4% Zn-0.5% Zr (ZK40) alloy as a potential biodegradable material.

Mg-4 wt.% Zn-0.5 wt.% Zr (ZK40) alloy was studied as a candidate material for biodegradable metallic implants in terms of its biocorrosion resistance, mechanical properties and cytocompatibility. The corrosion characteristics of ZK40 alloy were assessed by potentiodynamic polarization and immersion testing in DMEM+10% FBS solution. Analysis of the degradation characteristics by potentiodynamic polarization measurements shows the corrosion rates of ZK40 alloy in as-cast and solution treatment (T4) condition were slightly higher than those of pure Mg or as-drawn AZ31. Determination of the corrosion rate by the weight loss technique reveals that the as-cast ZK40 resulted in slower degradation than other alloy specimens after 7 days of immersion but exhibited accelerated degradation after 14 and 21 days, respectively. T4-treated ZK40 exhibited stable degradation rates compared to as-cast ZK40 and close to those of pure Mg and AZ31 during immersion testing for 14 and 21 days. In order to examine the in vitro cytocompatibility of ZK40 alloy, live/dead cell viability assay and indirect MTT assay were performed using a murine osteoblast-like cell line (MC3T3). After 3 days of direct culture of MC3T3 on ZK40 alloys the live/dead assay indicated favorable cell viability and attachment. The degradation product of ZK40 also showed minimal cytotoxicity when assessed in indirect MTT assay. The mechanical properties of the as-cast and T4-treated ZK40 alloy were superior to those of pure Mg and comparable to as-drawn AZ31. Solution treatment did not significantly enhance the cytocompatibility and mechanical properties of ZK40 alloy. Overall, the ZK40 alloy exhibited favorable cytocompatibility, biocorrosion, and mechanical properties rendering it a potential candidate for degradable implant applications.

Novel processing of iron-manganese alloy-based biomaterials by inkjet 3-D printing.

The present work provides an assessment of 3-D printed iron-manganese biodegradable scaffolds as a bone scaffold material. Iron-based alloys have been investigated due to their high strength and ability to slowly corrode. Current fabrications of Fe-based materials generate raw material which must be machined into their desired form. By using inkjet 3-D printing, a technique which generates complex, customizable parts from powders mechanically milled Fe-30Mn (wt.%) powder was directly processed into scaffolds. The 3-D printed parts maintained an open porosity of 36.3% and formed a mixed phase alloy of martensitic ε and austenitic γ phases. Electrochemical corrosion tests showed the 3-D printed Fe-Mn to desirably corrode significantly more rapidly than pure iron. The scaffolds exhibited similar tensile mechanical properties to natural bone, which may reduce the risk of stress shielding. Cell viability testing of MC3T3-E1 pre-osteoblast cells seeded directly onto the Fe-Mn scaffolds using the live/dead assay and with cells cultured in the presence of the scaffolds' degradation products demonstrated good in vitro cytocompatibility compared to tissue culture plastic. Cell infiltration into the open pores of the 3-D printed scaffolds was also observed. Based on this preliminary study, we believe that 3-D printed Fe-Mn alloy is a promising material for craniofacial biomaterial applications, and represents an opportunity for other biodegradable metals to be fabricated using this unique method.