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1. Developing Bioactive-Nanograined Metals With Bacterial Biofilm Inhibition Properties For Orthopedic Devices

   Kelkar, Shaunak; Saleh, Bahram; Yusa, Fumie; Suzuki, Yohei; Komatsu, Takafumi; Webster, Thomas (January 2021, Orthopaedic Research Society)

   null (Ed.)

   INTRODUCTION: Orthopedic implants are important therapeutic devices for the management of a wide range of orthopedic conditions. However, bacterial infections of orthopedic implants remain a major problem, and not an uncommon one, leading to an increased rate of osteomyelitis, sepsis, implant failure and dysfunction, etc. Treating these infections is more challenging as the causative organism protects itself by the production of a biofilm over the implant’s surface (1). Infections start by the adhesion and colonization of pathogenic bacteria such as Staphylococcus aureus (SA), Staphylococcus epidermidis (SE), Escherichia coli (E. coli), Methicillin-Resistant Staphylococcus aureus (MRSA), and Multi-Drug Resistant Escherichia coli (MDR E. coli) on the implant’s surfaces. Specifically, Staphylococcus comprises up to two-thirds of all pathogens involved in orthopedic implant infections (2). However, bacterial surface adhesion is a complex process influenced by several factors such as chemical composition, hydrophobicity, magnetization, surface charge, and surface roughness of the implant (3). Considering the intimate association between bacteria and the implant surface, we measured the effect of stainless-steel surface properties on bacterial surface attachment and subsequent formation of biofilms controlling above mentioned factors. METHODS: The prominent bacteria responsible for orthopedic implant infections (SA, SE, E. coli, MRSA, and MDR E. coli) were used in this study. We were able to control the grain size of medical grade 304 and 316L stainless steel without altering their chemical composition (grain size range= 20μm-200nm) (4). Grain size control affected the nano-topography of the material surfaces which was measured by an Atomic Force Microscope (AFM). Grain sizes, such as 0.2, 0.5, 1, 2, 3, 9, and 10 μm, were used both polished and non-polished. All the stainless-steel samples were cleaned by treating with acetone and ethanol under sonication. Triplicates of all polished and non-polished samples with different grain sizes were subjected to magnetization of DM, 0.1T, 0.5T, and 1T, before seeding them with the bacteria. Controls were used in the form of untreated samples. Bacterial were grown in Tryptic Soy Broth (TSB). An actively growing bacterial suspension was seeded onto the stainless-steel discs into 24-well micro-titer plates and kept for incubation. After 24 hours of incubation, the stainless-steel discs were washed with Phosphate Buffer Saline (PBS) to remove the plankton bacteria and allow the sessile bacteria in the biofilm to remain. The degree of development of the bacterial biofilms on the stainless-steel discs were measured using spectrophotometric analysis. For this, the bacterial biofilm was removed from the stainless steel by sonication. The formation of biofilms was also determined by performing a biofilm staining method using Safranin. RESULTS SECTION: AFM results revealed a slight decrease in roughness by decreasing the grain size of the material. Moreover, the samples were segregated into two categories of polished and non-polished samples, in which polishing decreased roughness significantly. After careful analysis we found out that polished surfaces showed a higher degree for biofilm formation in comparison to the non-polished ones. We also observed that bacteria showed a higher rate for biofilm formation for the demagnetized samples, whereas 0.5T magnetization showed the least amount of biofilm formation. After 0.5T, there was no significant change in the rate of biofilm formation on the stainless-steel samples. Altogether, stainless steel samples containing 0.5 μm and less grainsize, and magnetized with 0.5 tesla and stronger magnets demonstrated the least degree of biofilm formation. DISCUSSION: In summary, the results demonstrate that controlling the grain size of medical grade stainless steel can control and mitigate bacterial responses on, and thus possibly infections of, orthopedic implants or other implantable devices. The research was funded by Komatsuseiki Kosakusho Co., Ltd (KSJ: Japan) SIGNIFICANCE/CLINICAL RELEVANCE: Orthopedic implants that more than 70% of them are made of metals (i.e., stainless steel, titanium, and cobalt-chromium alloys) are failing through loosening and breakage due to their limited mechanical properties. On the other hand, the risk of infection for these implants and its financial burden on our society is undeniable. We have seen that our uniformly nanograined stainless steel shows improved mechanical properties (i.e., higher stiffness, hardness, fatigue) as compared to conventional stainless steel along with the reduction of biofilm formation on its surface. These promising results made us to peruse the development of nanograined titanium and cobalt-chromium alloys for resolving the complications of orthopedic implants. 

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2. NanoSUS (Ultra Fine Grained Stainless-Steel) for Orthopedic Implants

   Bahram Saleh, Ada Vernet-Crua (February 2021, Orthopaedic Research Society)

   Statement of Purpose: Orthopedic implants are important therapeutic devices for the management of a wide range of orthopedic conditions. However, bacterial infections of orthopedic implants remain a major problem, and not an uncommon one, leading to an increased rate of osteomyelitis, sepsis, implant failure and dysfunction, etc. Treating these infections is more challenging as the causative organism protects itself by the production of a biofilm over the implant’s surface (1). Infections start by the adhesion and colonization of pathogenic bacteria such as Staphylococcus aureus (SA), Staphylococcus epidermidis (SE), Escherichia coli (E. coli), Methicillin-Resistant Staphylococcus aureus (MRSA), and Multi-Drug Resistant Escherichia coli (MDR E. coli) on the implant’s surfaces. Specifically, Staphylococcus comprises up to two-thirds of all pathogens involved in orthopedic implant infections (2). However, bacterial surface adhesion is a complex process influenced by several factors such as chemical composition, hydrophobicity, magnetization, surface charge, and surface roughness of the implant (3). Considering the intimate association between bacteria and the implant surface, we measured the effect of stainless-steel surface properties on bacterial surface attachment and subsequent formation of biofilms controlling above mentioned factors. Method: The prominent bacteria responsible for orthopedic implant infections (SA, SE, E. coli, MRSA, and MDR E. coli) were used in this study. We were able to control the grain size of medical grade 304 and 316L stainless steel without altering their chemical composition (grain size range= 20μm-200nm) (4). Grain size control affected the nano-topography of the material surfaces which was measured by an Atomic Force Microscope (AFM). Grain sizes, such as 0.2, 0.5, 1, 2, 3, 9, and 10 μm, were used both polished and non-polished. All the stainless-steel samples were cleaned by treating with acetone and ethanol under sonication. Triplicates of all polished and non-polished samples with different grain sizes were subjected to magnetization of DM, 0.1T, 0.5T, and 1T, before seeding them with the bacteria. Controls were used in the form of untreated samples. Bacterial were grown in Tryptic Soy Broth (TSB). An actively growing bacterial suspension was seeded onto the stainless-steel discs into 24-well micro-titer plates and kept for incubation. After 24 hours of incubation, the stainless-steel discs were washed with Phosphate Buffer Saline (PBS) to remove the plankton bacteria and allow the sessile bacteria in the biofilm to remain. The degree of development of the bacterial biofilms on the stainless-steel discs were measured using spectrophotometric analysis. For this, the bacterial biofilm was removed from the stainless steel by sonication. The formation of biofilms was also determined by performing a biofilm staining method using Safranin. Results: AFM results revealed a slight decrease in roughness by decreasing the grain size of the material. Moreover, the samples were segregated into two categories of polished and non-polished samples, in which polishing decreased roughness significantly. After careful analysis we found out that polished surfaces showed a higher degree for biofilm formation in comparison to the non-polished ones. We also observed that bacteria showed a higher rate for biofilm formation for the demagnetized samples, whereas 0.5T magnetization showed the least amount of biofilm formation. After 0.5T, there was no significant change in the rate of biofilm formation on the stainless-steel samples. Altogether, stainless steel samples containing 0.5 μm and less grainsize, and magnetized with 0.5 tesla and stronger magnets demonstrated the least degree of biofilm formation. Conclusion: In summary, the results demonstrate that controlling the grain size of medical grade stainless steel can control and mitigate bacterial responses on, and thus possibly infections of, orthopedic implants or other implantable devices. The research was funded by Komatsuseiki Kosakusho Co., Ltd (KSJ: Japan) 

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3. Data: Sucrose-mediated formation and adhesion strength of Streptococcus mutans biofilms on titanium

   https://doi.org/10.18126/D1BG-NWTG  

   Butera, Tony; Waldman, Laura J.; Grady, Martha E. (January 2022, Materials Data Facility)

   Bacterial biofilms associated with implants remain a significant source of infections in dental, implant, and other healthcare industries due to challenges in biofilm removal. Biofilms consist of bacterial cells surrounded by a matrix of extracellular polymeric substance (EPS), which protects the colony from many countermeasures, including antibiotic treatments. Biofilm EPS composition is also affected by environmental factors. In the oral cavity, the presence of sucrose affects the growth of Streptococcus mutans that produce acids, eroding enamel and forming dental caries. Biofilm formation on dental implants commonly leads to severe infections and failure of the implant. This work determines the effect of sucrose concentration on biofilm EPS formation and adhesion of Streptococcus mutans, a common oral colonizer. Bacterial biofilms are grown with varying concentrations of sucrose on titanium substrates simulating dental implant material. Strategies for measuring adhesion for films such as peel tests are inadequate for biofilms, which have low cohesive strength and will fall apart when tensile loading is applied directly. The laser spallation technique is used to apply a stress wave loading to the biofilm, causing the biofilm to delaminate at a critical tensile stress threshold. Biofilm formation and EPS structures are visualized at high magnification with scanning electron microscopy (SEM). Sucrose enhanced the EPS production of S. mutans biofilms and increased the adhesion strength to titanium, the most prevalent dental implant material. However, there exists a wide range of sucrose concentrations that are conducive for robust formation and adhesion of S. mutans biofilms on implant surfaces. 

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4. 3D printing of drug-eluting bioactive multifunctional coatings for orthopedic applications

   https://doi.org/10.18063/ijb.v9i2.661  

   Adarkwa, E.; Roy, A.; Ohodnicki, J.; Lee, B.; Kumta, P. N.; Desai, S. (January 2023, International journal of bioprinting)

   Three-dimensional (3D) printing is implemented for surface modification of titanium alloy substrates with multilayered biofunctional polymeric coatings. Poly(lactic-co- glycolic) acid (PLGA) and polycaprolactone (PCL) polymers were embedded with amorphous calcium phosphate (ACP) and vancomycin (VA) therapeutic agents to promote osseointegration and antibacterial activity, respectively. PCL coatings revealed a uniform deposition pattern of the ACP-laden formulation and enhanced cell adhesion on the titanium alloy substrates as compared to the PLGA coatings. Scanning electron microscopy and Fourier-transform infrared spectroscopy confirmed a nanocomposite structure of ACP particles showing strong binding with the polymers. Cell viability data showed comparable MC3T3 osteoblast proliferation on polymeric coatings as equivalent to positive controls. In vitro live/dead assessment indicated higher cell attachments for 10 layers (burst release of ACP) as compared to 20 layers (steady release) for PCL coatings. The PCL coatings loaded with the antibacterial drug VA displayed a tunable release kinetics profile based on the multilayered design and drug content of the coatings. Moreover, the concentration of active VA released from the coatings was above the minimum inhibitory concentration and minimum bactericidal concentration, demonstrating its effectiveness against Staphylococcus aureus bacterial strain. This research provides a basis for developing antibacterial biocompatible coatings to promote osseointegration of orthopedic implants. 

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5. Preconditioning Layers Affect Osteoblastic Cell Adhesion to Orthopedic Implant Surfaces

   https://doi.org/10.1007/978-3-031-17445-2_9  

   Boyd, James D; Grady, Martha E (November 2022, Springer International Publishing)

   Bacteria can proliferate orthopedic implants, resulting in infection rates as high as 5%. A consistent problem across implantology is the development of surfaces which successfully promote the adhesion and propagation of healthy fibroblast and osteoblast cells while deterring formation of bacterial biofilms. Selecting surface configurations which favor cell adhesion will lead to decreased infection rates. Progress in identifying appropriate surface configurations is hindered by the lack of quantitative adhesion techniques capable of comparing adhesion of cells and biofilms directly. Recent advancements in adhesion techniques have allowed for quantitatively measured adhesion strengths of both bacterial biofilms and cell monolayers using the laser spallation technique. The quantified stress-based adhesion values allow surface and environmental factors that modulate both bacterial and cell adhesion to implant surfaces to be evaluated. During implantation, blood propagates wound sites completely coating implant surfaces. Quantitatively determining the impact of preconditioning layers that accumulate on the implant surface on cell adhesion is vital to predict implant behavior. Previous work has demonstrated that these preconditioning layers either negatively or neutrally impact bacterial adhesion to titanium implant surfaces. This study focuses on the impact that blood plasma and fibronectin coatings have on the adhesion of osteoblastic (MG 63) cells and fibroblasts to the same titanium surfaces. Adhesion results indicate that preconditioning layers and increased surface roughness positively impact cell adhesion. Incorporating the increased adhesion values for cell adhesion into the Adhesion Index demonstrates that increased surface roughness, coupled with natural wound healing preconditioning of surfaces, yields positive biocompatibility. 

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