Evaluation of antibiotic and cell-based therapy in preventing S. epidermidis-induced nonunion in rats.

Methicillin-resistant S. epidermidis (MRSE) is responsible for biofilm-related infections (Montanaro,2011; Romanò, 2013) and fracture nonunion, as recently demonstrated by our group (Lovati, 2016).The present study aims to investigate the efficacy of antibiotic or cell-based therapies in preventingbacterial infections and nonunion establishment.Under anesthesia, femoral fractures were performed in 30 rats, then the site of injury was injectedwith a clinical-derived MRSE strain and, finally, synthesized with stainless steel plates. Rats weredifferently treated as follows: MRSE-infected controls (IC); systemically-injected vancomycin (s-VANC);local vancomycin-enriched hydrogel (l-HYD); systemically-injected BMSCs (s-BMSCs); and locallyinjectedBMSCs (l-BMSCs).After 6 weeks, pro-inflammatory cytokines, quantitative micro-CT, histological and microbiologicalanalyses were carried out to investigate the host response to the different treatments.Half of the s-BMSCs rats died closely to the systemic cell injection, thus excluded for further analyses.Our results for the IC group were consistent with previously published data (Lovati, 2016), showingsigns of osteomyelitis and nonunion development. In s-VANC and l-HYD groups, micro-CT detected agood bony bridging and the microbiological counts were significantly lower with respect to the othergroups. Our study suggests that the association of s-VANC and l-HYD is an effective treatment toprevent biofilm-induced nonunions. Differently, we cannot positively support cell therapies for thispurpose due to the high risk related to the systemic cell injection, thus requiring further studies to beeventually proposed in clinics

Phenotypic and genomic identification of Staphylococcus epidermidis GOI1153754-03-14 isolated from an infected orthopedic prosthesis

Introduction: Staphylococcus epidermidis GOI1153754-03-14 is able to colonize orthopedic implants and to cause septic non-unions, as validated in a recent in vivo study (Lovati, 2016). To pore over the mechanisms leading to the biofilm formation on metallic implants, in the present study, we carried out the phenotypic and genotypic characterization of the clinical isolate S. epidermidis GOI1153754-03-14.Materials and Methods: The antimicrobial susceptibility and minimum inhibitory concentration (MIC) of the strain were evaluated through the Vitek2 System (Biomerieux), as well as its ability to form biofilm in vitro through a spectrophotometric assay (Stepanovich, 2000).The genomic DNA was extracted by Bacterial Genomic DNA Isolation Kit (Norgen Biotek Corp.). Libraries were prepared with the ThruPLEX DNA-seq (Rubicon Genomics) and then sequenced on the Illumina MiSeq platform through the MiSeq Reagent Kit v3 (600-cycles) to produce 300 bp paired-end reads (Illumina Inc.). Reads were quality-trimmed and gene annotated thanks to the RAST software (Aziz, 2008).Results: The antimicrobial susceptibility along with the MIC values are reported in Table 1. The outputs resulted in 51 contigs (Average = 50,720.6 Mb) with 396X fold average coverage. The total genome is 2,586,753 bp long with a GC content of 31.84% and an N50 value of 7 bp. The whole genome is composed by 2,467 protein-encoding genes and 64 RNAs (55 tRNAs and 9 rRNAs). The entire genome sequence has been deposited in the European Nucleotide Archive (ENA) under the accession no. FWCG01000000 (Bottagisio, 2017).Discussion: The genotypic and phenotypic characterization of the S. epidermidis GOI1153754-03-14 will enable a better comprehension of the mechanisms involved in the biofilm formation on orthopedic implants paving the way for innovative preventative and therapeutic strategies. Moreover, the sequence of this clinical strain is mandatory to develop dedicated proteomics analysis in order to highlight functional mechanism of biofilm formation

Nerve Repair Using Decellularized Nerve Grafts in Rat Models. A Review of the Literature

Peripheral nerve regeneration after severe traumatic nerve injury is a relevant clinical problem. Several different strategies have been investigated to solve the problem of bridging the nerve gap. Among these, the use of decellularized nerve grafts has been proposed as an alternative to auto/isografts, which represent the current gold standard in the treatment of severe nerve injury. This study reports the results of a systematic review of the literature published between January 2007 and October 2017. The aim was to quantitatively analyze the effectiveness of decellularized nerve grafts in rat experimental models. The review included 33 studies in which eight different decellularization protocols were described. The decellularized nerve grafts were reported to be immunologically safe and able to support both functional and morphological regeneration after nerve injury. Chemical protocols were found to be superior to physical protocols. However, further research is needed to optimize preparation protocols, including recellularization, improve their effectiveness, and substitute the current gold standard, especially in the repair of long nerve defects

Exploring multielement nanogranular coatings to forestall implant-related infections

Introduction: As we approach the post-antibiotic era, the development of innovative antimicrobial strategies that carry out their activities through non-specific mechanisms could limit the onset and spread of drug resistance. In this context, the use of nanogranular coatings of multielement nanoparticles (NPs) conjugated to the surface of implantable biomaterialsmight represent a strategy to reduce the systemicdrawbacks by locally confining the NPs effects against either prokaryotic or eukaryotic cells. Methods: In the present study, two new multielement nanogranular coatings combining Ag and Cu with either Ti or Mg were synthesized by a gas phase physical method and tested against pathogens isolated from periprosthetic joint infections to address their potential antimicrobial value and toxicity in an in vitro experimental setting. Results: Overall, Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli displayed a significantly decreased adhesion when cultured on Ti-Ag-Cu and Mg-Ag-Cu coatings compared to uncoated controls, regardless of their antibiotic resistance traits. A dissimilar behavior was observed when Pseudomonas aeruginosa was cultured for 30 and 120 minutes upon the surface of Ti-Ag-Cu and Mg-Ag-Cu-coated discs. Biofilm formation was mainly reduced by the active effect of Mg-Ag-Cu compared to Ti-Ag-Cu and, again, coatings had a milder effect on P. aeruginosa, probably due to its exceptional capability of attachment and matrix production. These data were further confirmed by the evaluation of bacterial colonization on nanoparticle-coated discs through confocal microscopy. Finally, to exclude any cytotoxic effects on eukaryotic cells, the biocompatibility of NPs-coated discs was studied. Results demonstrated a viability of 95.8% and 89.4% of cells cultured in the presence of Ti-Ag-Cu and Mg-Ag-Cu discs, respectively, when compared to negative controls. Conclusion: In conclusion, the present study demonstrated the promising antiadhesive features of both Ti-Ag-Cu and Mg-Ag-Cu coatings, as well as their action in hampering the biofilm formation, highlighting the safe use of the tested multielement families of nanoparticles as new strategies against bacterial attachment to the surface of biomedical implants

A Precautionary Approach to Guide the Use of Transition Metal-Based Nanotechnology to Prevent Orthopedic Infections

The increase of multidrug-resistant bacteria remains a global concern. Among the proposed strategies, the use of nanoparticles (NPs) alone or associated with orthopedic implants represents a promising solution. NPs are well-known for their antimicrobial effects, induced by their size, shape, charge, concentration and reactive oxygen species (ROS) generation. However, this non-specific cytotoxic potential is a powerful weapon effective against almost all microorganisms, but also against eukaryotic cells, raising concerns related to their safe use. Among the analyzed transition metals, silver is the most investigated element due to its antimicrobial properties per se or as NPs; however, its toxicity raises questions about its biosafety. Even though it has milder antimicrobial and cytotoxic activity, TiO2 needs to be exposed to UV light to be activated, thus limiting its use conjugated to orthopedic devices. By contrast, gold has a good balance between antimicrobial activity as an NP and cytocompatibility because of its inability to generate ROS. Nevertheless, although the toxicity and persistence of NPs within filter organs are not well verified, nowadays, several basic research on NP development and potential uses as antimicrobial weapons is reported, overemphasizing NPs potentialities, but without any existing potential of translation in clinics. This analysis cautions readers with respect to regulation in advancing the development and use of NPs. Hopefully, future works in vivo and clinical trials will support and regulate the use of nano-coatings to guarantee safer use of this promising approach against antibiotic-resistant microorganisms

Tissue engineering approaches to develop decellularized tendon matrices functionalized with progenitor cells cultured under undifferentiated and tenogenic conditions

Tendon ruptures and retractions with an extensive tissue loss represent a major clinical problem and a great challenge in surgical reconstruction. Traditional approaches consist in autologous or allogeneic grafts, which still have some drawbacks. Hence, tissue engineering strategies aimed at developing functionalized tendon grafts. In this context, the use of xenogeneic tissues represents a promising perspective to obtain decellularized tendon grafts. This study is focused on the identification of suitable culture conditions for the generation of reseeded and functional decellularized constructs to be used as tendon grafts. Equine superficial digital flexor tendons were decellularized, reseeded with mesenchymal stem cells (MSCs) from bone marrow and statically cultured in two different culture media to maintain undifferentiated cells (U-MSCs) or to induce a terminal tenogenic differentiation (T-MSCs) for 24 hours, 7 and 14 days. Cell viability, proliferation, morphology as well as matrix deposition and type I and III collagen production were assessed by means of histological, immunohistochemical and semi-quantitative analyses. Results showed that cell viability was not affected by any culture conditions and active proliferation was maintained 14 days after reseeding. However, seeded MSCs were not able to penetrate within the dense matrix of the decellularized tendons. Nevertheless, U-MSCs synthesized a greater amount of extracellular matrix rich in type I collagen compared to T-MSCs. In spite of the inability to deeply colonize the decellularized matrix in vitro, reseeding tendon matrices with U-MSCs could represent a suitable method for the functionalization of biological constructs, considering also any potential chemoattractant capability of the newly deposed extracellular matrix to recruit resident cells. This bioengineering approach can be exploited to produce functionalized tendon constructs for the substitution of large tendon defects

A new strategy for the decellularisation of large equine tendons as biocompatible tendon substitutes

Tendon ruptures and/or large losses remain to be a great clinical challenge and often require full replacement of the damaged tissue. The use of auto- and allografts or engineered scaffolds is an established approach to restore severe tendon injuries. However, these grafts are commonly related to scarce biocompatibility, site morbidity, chronic inflammation and poor biomechanical properties. Recently, the decellularisation techniques of allo- or xenografts using specific detergents have been studied and have been found to generate biocompatible substitutes that resemble the native tissue. This study aims to identify a novel decellularisation protocol for large equine tendons that would produce an extracellular matrix scaffold suitable for the regeneration of injured tendons in humans. Specifically, equine tendons were treated either with tri (n-butyl) phosphate alone, or associated to multiple concentrations of peracetic acid (1, 3 and 5 %), which has never before been tested in vitro. Samples were then analysed by histology and with biochemical, biomechanical, and cytotoxicity tests. The best decellularisation protocol, resulting from these examinations, was selected and the chosen scaffold was re-seeded with murine fibroblasts. Resulting grafts were tested for cell viability, histologic analysis, DNA and collagen content. The results identified 1 % tri (n-butyl) phosphate combined with 3 % peracetic acid as the most suitable decellularised matrix in terms of biochemical and biomechanical properties. Moreover, the non-cytotoxic nature of the decellularised matrix allowed for good fibroblast reseeding, thus demonstrating a biocompatible matrix that will be suitable for tendon tissue engineering and hopefully as substitutes in severe tendon damages