Critical-sized bone defects remain challenging in the clinical setting. Autologous bone grafting remains preferred by clinicians. However, the use of autologous tissue is associated with donor-site morbidity and limited accessibility to the graft tissue. Advances in the development of synthetic bone substitutes focus on improving their osteoinductive properties. Whereas osteoinductivity has been demonstrated with ceramics, it is still a challenge in case of polymeric composites. One of the approaches to improve the regenerative properties of biomaterials, without changing their synthetic character, is the addition of inorganic ions with known osteogenic and angiogenic properties. We have previously reported that the use of a bioactive composite with high ceramic content composed of poly(ethyleneoxide terephthalate)/poly(butylene terephthalate) (1000PEOT70PBT30, PolyActive, PA) and 50% beta-tricalcium phosphate (β-TCP) with the addition of zinc in a form of a coating of the TCP particles can enhance the osteogenic differentiation of human mesenchymal stromal cells (hMSCs) (3). To further support the regenerative properties of these scaffolds, inorganic ions with known angiogenic properties, copper or cobalt, were added to the coating solution. β-TCP particles were immersed in a zinc and copper or zinc and cobalt solution with a concentration of 15 or 45 mM. 3D porous scaffolds composed of 1000PEOT70PBT30 and pure or coated β-TCP were additively manufactured by 3D fibre deposition. The osteogenic and angiogenic properties of the fabricated scaffolds were tested in vitro through culture with hMSCs and human umbilical vein endothelial cells, respectively. The materials were further evaluated through ectopic implantation in an in vivo mini-pig model. The early expression of relevant osteogenic gene markers (collagen-1, osteocalcin) of hMSCs was upregulated in the presence of lower concentration of inorganic ions. Further analysis will focus on the evaluation of ectopic bone formation and vascularisation of these scaffolds after implantation in a mini-pig ectopic intramuscular model

Biphasic calcium phosphates (BCP) are the most frequently used materials because of their mineral analogy with bio-mineral part of bones. Their chemical synthesis can be modulated by doping, in order to respond to the biological needs. We present here the biological responses induced by copper ions in solution, to characterize its cytotoxicity and antibacterial activity. We also investigate the antibacterial property of Cu-doped BCP (Ca10 Cu0.1 (PO4)6 (OH)1.8 O0.2) on a strain of clinical interest: S. aureus, compared to undoped BCP. The sol-gel route has been used to prepare the BCP ceramics. Human BMC (Bone Marrow Cells) were obtained from metaphysal cancellous bone collected during hip arthroplasty and used for cytotoxicity evaluations. A strain of Staphylococcus aureus isolated from an osteoarticular infection after total knee arthroplasty was used to evaluate antibacterial activities. Results indicate that 3 ppm of copper ions leads to the death of all cultured bacteria in 24 hours and 25 ppm caused the death of all cells in 15 days. Regarding BCP, the undoped bioceramics increased the bacterial growth compared to a control without bioceramic. After 16 hours of contact, the copper ions released by the Cu-doped BCP induced a significant decrease of the bacterial concentration, indeed no viable bacteria were found. These materials seem to be a promising alternative for the preparation of multifunctional bone substitutes

The bone infection osteomyelitis (typically Staphylococcus aureus) requires a multistep treatment process including: surgical debridement, long-term systemic high-dose antibiotics, and often bone grafting. With antibiotic resistance becoming increasingly concerning, alternative approaches are urgently needed. Herein, we develop a one-step treatment for osteomyelitis that combines local, controlled release of non-antibiotic antibacterials (copper) within a proven regenerative scaffold. To maximise efficacy we utilised bioactive glass – an established material with immense osteogenic capacity – as a copper ion delivery reservoir. Copper ions have also been shown to stimulate angiogenesis and induce MSC differentiation down an osteogenic lineage. To eliminate grafting requirements, the copper-doped BG was incorporated into our previously developed collagen scaffolds to produce multifunctional antibacterial, osteogenic, and angiogenic scaffolds. Scaffolds were fabricated by freeze-drying a co-suspension of collagen and bioactive glass particles (+/− copper doping, referred to as CuBG and BG, respectively) at a range of different concentrations (0–300% w/w bioactive glass/collagen). Scaffolds demonstrated a 2.7-fold increase in compressive modulus (300% CuBG vs. 0%; p≤0.01), whilst maintaining >98% porosity. The 300% CuBG scaffolds showed significant antibacterial activity against Staphylococcus aureus (p≤0.001). In terms of osteogenesis, both 100% and 300% CuBG scaffolds increased cell-mediated calcium deposition on the scaffolds at day 14 and 28 (p≤0.05 and p≤0.001), as confirmed by alizarin red staining. 100% CuBG scaffolds significantly enhanced angiogenesis by increased tubule formation (p≤0.01) and VEGF protein production (p≤0.001) (all ≥n=3). In summary, this single-stage, off-the-shelf treatment for osteomyelitis shows potential to minimise bone grafting and antibiotic dependence, while reducing hospital stays and costs

Prosthetic Joint Infection (PJI) is a devastating complication that can occur after total joint replacement surgery. With increasing antimicrobial resistance, there is a need for non-antibiotic approaches to treat and prevent PJI. Doping calcium phosphates with antimicrobial ions shows promise for these purposes. This systematic review aims to search and summarise the evidence-base for the potential of calcium phosphates doped with different antimicrobial ions. A systematic review was conducted on PubMed, Embase, Web-Of-Science, Cochrane Library and Emcare of in vitro and animal studies on the antimicrobial activity of (co)substituted calcium phosphates according to PRIMSA guidelines.. The research protocol, listing search terms and in/exclusion criteria, was registered a priori at . https://doi.org/10.7910/DVN/HEP18U. Data was extracted regarding ions, micro-organisms and antimicrobial activity. The search retrieved 1017 hits of which 148 papers were included. The substitution of 33 different ions was reported. Silver (n= 46), zinc (n=39), copper (n=18) and magnesium (n=14) were the most commonly doped ions. 36 different micro-organisms were studied of which E. coli (n=109), S. aureus (n=99), and C. albicans (n=22) were the most common. 6 different outcomes were reported, most commonly the K-ratio (n=53), the log CFU (n=41) and the bacterial inhibition zone (n=39). A validated outcome for the evaluation of biofilm prevention was lacking. There was considerable heterogeneity in studied ions, micro-organisms and reported outcomes. A lack of clearly defined reporting guidelines in the field of antimicrobial materials has led to the use of clinically irrelevant micro-organisms and a general lack of consistency of the methods used and the reported results. Currently, there is no universally accepted measure for the effectiveness required from biomaterials for treatment and prevention of PJI

The use of implant biomaterials for prosthetic reconstructive surgery and osteosynthesis is consolidated in the orthopaedic field, improving the quality of life of patients and allowing for healthy and better ageing. However, there is the lack of advanced innovative methods to investigate the potentialities of smart biomaterials, particularly for the study of local effects of implant and osteointegration. Despite the complex process of osseointegration is difficult to recreate in vitro, the growing challenges in developing alternative models require to set-up and validate new approaches. Aim of the present study is to evaluate an advanced in vitro tissue culture model of osteointegration of titanium implants in human trabecular bone. Cubic samples (1.5×1.5 cm) of trabecular bone were harvested as waste material from hip arthroplasty surgery (CE AVEC 829/2019/Sper/IOR); cylindrical defects (2 mm Ø, 6 mm length) were created, and tissue specimens assigned to the following groups: 1) empty defects- CTR-; 2) defects implanted with a cytotoxic copper pin (Merck cod. 326429)- CTR+; 3) defects implanted with standard titanium pins of 6 µm-rough (ZARE S.r.l) -Ti6. Tissue specimens were cultured in mini rotating bioreactors in standard conditions, weekly assessing viability. At the 8-week-timepoint, immunoenzymatic, microtomographic, histological and histomorphometric analyses were performed. The model was able to simulate the effects of implantation of the materials, showing a drop in viability in CTR+, differently from Ti6 which appears to have a trophic effect on the bone. MicroCT and histological analysis supported the results, with lower BV/TV and Tb.Th values observed in CTR- compared to CTR+ and Ti6 and signs of matrix and bone deposition at the implant site. The collected data suggest the reliability of the tested model which can recreate the osseointegration process in vitro and can therefore be used for preliminary evaluations to reduce and refine in vivo preclinical models. Acknowledgment: This work was supported by Emilia-Romagna Region for the project “Sviluppo di modelli biologici in vitro ed in silico per la valutazione e predizione dell'osteointegrazione di dispositivi medici da impianto nel tessuto osseo”

Favoring osseointegration and avoiding bacterial contamination are the key challenges in the design of implantable devices for orthopedic applications. To meet these goals, a promising route is to tune the biointerface of the devices, that can regulate interactions with the host cells and bacteria, by using nanostructured antibacterial and bioactive coatings. Indeed, the selection of adequate metal-based coatings permits to discourage infection while avoiding the development of bacterial resistance and nanostructuring permits to tune the release of the antimicrobial compounds, allowing high efficacy and decreasing possible cytotoxic effects. In addition, metal-doped calcium phosphates-based nanostructured coatings permit to tune both composition and morphology of the biointerfaces, allowing to regulate host cells and bacteria response. To tune the biointerfaces of implantable devices, nanostructured coatings can be used, but their use is challenging when the substrate is heat-sensitive and/or porous. Here, we propose the use of Ionized Jet Deposition (IJD) to deposit metallic and ion-doped calcium phosphates materials onto different polymeric substrates, without heating and damaging the substrate morphology. 3D printed scaffolds in polylactic acid (PLA) and polyurethane (PU), and electrospun matrices in polycaprolactone (PCL) and PLA were used as substrates. Biogenic apatite (HA), ion doped (zinc, copper and iron) tricalcium phosphate (TCP) and silver (Ag) coatings were obtained on porous and custom-made polymeric substrates. Chemical analyses confirmed that coatings composition matches that of the target materials, both in terms of main phase (HA or TCP) and ion doping (presence of Cu, Zn or Fe ion). Deposition parameters, and especially its duration time, influence the coating features (morphology and thickness) and substrate damage. Indeed, SEM/EDS observations show the presence of nanostructured agglomerates on substrates surface. The dimensions of the aggregates and the thickness of the coating films increase increasing the deposition time, without affecting the substrate morphology (no porosity alteration or fibers damaging). The possible substrate damage is influenced by target and substrate material, but it can be avoided modulating deposition time. Once the parameters are optimized, the models show suitable in vitro biological efficacy for applications in bone models, regenerative medicine and infection. Indeed, HA-based coatings favor cells adhesion on printed and electrospun fibers. For antibacterial applications, the ion doped TCP coatings can reduce the bacterial growth and adhesion (E.coli and S.aureus) on electrospun matrices. To conclude, it is possible achieve different properties applying nanostructured coatings with IJD technique on polymeric substrates, modulating deposition conditions to avoid substrate damage

Over the last decades, biodegradable metals emerged as promising materials for various biomedical implant applications, aiming to reduce the use of permanent metallic implants and, therefore, to avoid additional surgeries for implant removal. However, among the important issue to be solved is their fast corrosion - too high to match the healing rate of the bone tissue. The most effective way to improve this characteristic is to coat biodegradable metals with substituted calcium phosphates. Tricalcium phosphate (β-TCP) is a resorbable bioceramic widely used as synthetic bone graft. In order to modulate and enhance its biological performance, the substitution of Ca2+ by various metal ions, such as strontium (Sr2+), magnesium (Mg2+), iron (Fe2+) etc., can be carried out. Among them, copper (Cu2+), manganese (Mn2+), zinc (Zn2+) etc. could add antimicrobial properties against implant-related infections. Double substitutions of TCP containing couples of Cu2+/Sr2+ or Mn2+/Sr2+ ions are considered to be the most perspective based on the results of our study. We established that single phase Ca3−2x(MˊMˊˊ)x(PO4)2 solid solutions are formed only at x ≤ 0.286, where Mˊ and Mˊˊ—divalent metal ions, such as Zn2+, Mg2+, Cu2+, Mn2+, and that in case of double substitutions, the incorporation of Sr2+ ions allows one to extend the limit of solid solution due to the enlargement of the unit cell structure. We also reported that antimicrobial properties depend on the substitution ion occupation of Ca2+ crystal sites in the β-TCP structure. The combination of two different ions in the Ca5 position, on one side, and in the Ca1, Ca2, Ca3, and Ca4 positions, on another side, significantly boosts antimicrobial properties. In the present work, zinc-lithium (Zn-Li) biodegradable alloys were coated with double substituted Mn2+/Sr2+ β-TCP and double substituted Cu2+/ Sr2+ β-TCP, with the scope to promote osteoinductive effect (due to the Sr2+ presence) and to impart antimicrobial properties (thanks to Cu2+ or Mn2+ ions). The Pulsed Laser Deposition (PLD) method was applied as the coating's preparation technique. It was shown that films deposited using PLD present good adhesion strength and hardness and are characterized by a nanostructured background with random microparticles on the surface. For coatings characterization, Fourier Transform Infrared Spectroscopy, X-ray Diffraction, and Scanning Electron Microscopy coupled with Energy Dispersive X-ray and X-ray Photoelectron Spectroscopy were applied. The microbiology tests on the prepared coated Zn-Li alloys were performed with the Gram-positive (Staphylococcus aureus, Enterococcus faecalis) and Gram-negative (Salmonella typhimurium, Escherichia coli) bacteria strains and Candida albicans fungus. The antimicrobial activity tests showed that Mn2+/Sr2+ β-TCP -coated and Cu2+/Sr2+ β-TCP coated Zn-Li alloys were able to inhibit the growth of all five microorganisms. The prepared coatings are promising in improving the degradation behavior and biological properties of Zn-Li alloys, and further studies are necessary before a possible clinical translation

The discussion will focus on new approaches to reduce bacterial adhesion on the surface of polymethylmethacrylate (PMMA) in contact with bone, comparing the clinical and engineering point of view. One possibility is to encourage and speed up direct interaction with the bone, for example by adding a bioactive phase in the cement (hydroxyapatite, glass and bioactive glass ceramic). A widespread strategy is also the addition of different types of antibiotics (gentamicin, tobramycin vancomycin, etc.), although they are known to have some drawbacks: not complete release, resistant strain development. Another strategy could be represented by the PMMA-based composite cements loaded with a completely inorganic filler consisting of a bioactive glass doped with ions whose bioactivity mechanism is well-known and encompasses a chemical and biological interaction with tissues promoting osteoinduction. Bioactive glasses can be doped with antibacterial ions (silver, copper, etc.) preserving their biocompatibility and bioactivity and, at the same time, acquiring antibacterial properties. Thus, it is possible to produce composite cements that combine the properties of the polymer matrix with those of the inorganic filler, overcoming the main problems associated with the use of antibiotics. An additional possibility is the addition of essential oils, vegetable oils with remarkable antibacterial properties

As near-infrared (NIR) photothermal agents, copper sulfide nanoparticles (CuSNP) offer several advantages over plasmonic gold nanoparticles (GNP), the most widely used photothermal nanotransducers in biomedical applications. CuSNP exhibit strong optical absorption at NIR wavelengths (650–1100 nm) and convert it into heat due excitation of electronic transitions or plasmonic photoexcitation. In contrast with GNP, CuSNP are degradable, readily prepared, inexpensive to produce, efficiently cleared from the body and their photothermal efficiency is less sensitive to the dielectric constant of the surrounding medium. We explored the feasibility of CuSNP to function as degradable NIR nanotransducers within fibrin-based cellular scaffolds, paying great attention to the stability and photothermal efficiency of the composite. We tested in vitro and in vivo whether NIR-responsive fibrin hydrogels comprising CuSNP (CuSNP hydrogels) are reliable platforms for triggering transgene expression in cells harboring a gene circuit activatable by heat and dependent of rapamycin. NIR laser irradiation of the CuSNP hydrogels increased local temperature and, in the presence of rapamycin, triggers the gene switch based on the promoter of the highly heat-inducible HSP70B gene (HSPA7). After implantation of such a cell-containing CuSNP hydrogel, transgenic expression can be remotely triggered by NIR-irradiation. Interestingly, we found that CuSNP hydrogels induce remodeling activity in stem cells and stimulate an angiogenic response. In short, CuSNP hydrogels offer compelling features for tissue engineering applications, as fully degradable implants with enhanced integration capacity in host tissues that can provide for remote control in the deployment of therapeutic gene products

Objectives

Recently, high failure rates of metal-on-metal (MOM) hip implants have raised concerns of cobalt toxicity. Adverse reactions occur to cobalt nanoparticles (CoNPs) and cobalt ions (Co²⁺) during wear of MOM hip implants, but the toxic mechanism is not clear.

Objectives

Infection of implants is a major problem in elective and trauma surgery. Heating is an effective way to reduce the bacterial load in food preparation, and studies on hyperthermia treatment for cancer have shown that it is possible to heat metal objects with pulsed electromagnetic fields selectively (PEMF), also known as induction heating. We therefore set out to answer the following research question: is non-contact induction heating of metallic implants effective in reducing bacterial load in vitro?

Objectives

The cytotoxicity induced by cobalt ions (Co²⁺) and cobalt nanoparticles (Co-NPs) which released following the insertion of a total hip prosthesis, has been reported. However, little is known about the underlying mechanisms. In this study, we investigate the toxic effect of Co²⁺ and Co-NPs on liver cells, and explain further the potential mechanisms.

We investigated the effect of progesterone on the nerve during lengthening of the limb in rats. The sciatic nerves of rats were elongated by leg lengthening for ten days at 3 mm per day. On alternate days between the day after the operation and nerve dissection, the progesterone-treated group received subcutaneous injections of 1 mg progesterone in sesame oil and the control group received oil only. On the fifth, tenth and 17th day, the sciatic nerves were excised at the midpoint of the femur and the mRNA expression level of myelin protein P0 was analysed by quantitative real time polymerase chain reaction. On day 52 nodal length was examined by electron microscopy, followed by an examination of the compound muscle action potential (C-MAP) amplitude and the motor conduction velocity (MCV) of the tibial nerve on days 17 and 52. The P0 (a major myelin glycoprotein) mRNA expression level in the progesterone-treated group increased by 46.6% and 38.7% on days five and ten, respectively. On day 52, the nodal length in the progesterone-treated group was smaller than that in the control group, and the MCV of the progesterone-treated group had been restored to normal.

Progesterone might accelerate the restoration of demyelination caused by nerve elongation by activating myelin synthesis.

An increasing number of patients are treated by autologous chondrocyte implantation (ACI). This study tests the hypothesis that culture within a defined chondrogenic medium containing TGF-β enhances the reexpression of a chondrocytic phenotype and the subsequent production of cartilaginous extracellular matrix by human chondrocytes used in ACI. Chondrocytes surplus to clinical requirements for ACI from 24 patients were pelleted and cultured in either DMEM (Dulbecco’s modified eagles medium)/ITS+Premix/TGF-β1 or DMEM/10%FCS (fetal calf serum) and were subsequently analysed biochemically and morphologically.

Pellets cultured in DMEM/ITS+/TGF-β1 stained positively for type-II collagen, while those maintained in DMEM/10%FCS expressed type-I collagen. The pellets cultured in DMEM/ITS+/TGF-β1 were larger and contained significantly greater amounts of DNA and glycosaminoglycans. This study suggests that the use of a defined medium containing TGF-β is necessary to induce the re-expression of a differentiated chondrocytic phenotype and the subsequent stimulation of glycosaminoglycan and type-II collagen production by human monolayer expanded chondrocytes.