Validation of a Cleanroom Compliant Sonication-Based Decellularization Technique: A New Concept in Nerve Allograft Production.

Defects of the peripheral nervous system are extremely frequent in trauma and surgeries and have high socioeconomic costs. If the direct suture of a lesion is not possible, i.e., nerve gap > 2 cm, it is necessary to use grafts. While the gold standard is the autograft, it has disadvantages related to its harvesting, with an inevitable functional deficit and further morbidity. An alternative to autografting is represented by the acellular nerve allograft (ANA), which avoids disadvantages of autograft harvesting and fresh allograft rejection. In this research, the authors intend to transfer to human nerves a novel technique, previously implemented in animal models, to decellularize nerves. The new method is based on soaking the nerve tissues in decellularizing solutions while associating ultrasounds and freeze-thaw cycles. It is performed without interrupting the sterility chain, so that the new graft may not require post-production γ-ray irradiation, which is suspected to affect the structural and functional quality of tissues. The new method is rapid, safe, and inexpensive if compared with available commercial ANAs. Histology and immunohistochemistry have been adopted to evaluate the new decellularized nerves. The study shows that the new method can be applied to human nerve samples, obtaining similar, and, sometimes better, results compared with the chosen control method, the Hudson technique.

Blocking Tumor-Educated MSC Paracrine Activity Halts Osteosarcoma Progression.

Purpose: Human osteosarcoma is a genetically heterogeneous bone malignancy with poor prognosis despite the employment of aggressive chemotherapy regimens. Because druggable driver mutations have not been established, dissecting the interactions between osteosarcoma cells and supporting stroma may provide insights into novel therapeutic targets.Experimental Design: By using a bioluminescent orthotopic xenograft mouse model of osteosarcoma, we evaluated the effect of tumor extracellular vesicle (EV)-educated mesenchymal stem cells (TEMSC) on osteosarcoma progression. Characterization and functional studies were designed to assess the mechanisms underlying MSC education. Independent series of tissue specimens were analyzed to corroborate the preclinical findings, and the composition of patient serum EVs was analyzed after isolation with size-exclusion chromatography.Results: We show that EVs secreted by highly malignant osteosarcoma cells selectively incorporate a membrane-associated form of TGFß, which induces proinflammatory IL6 production by MSCs. TEMSCs promote tumor growth, accompanied with intratumor STAT3 activation and lung metastasis formation, which was not observed with control MSCs. Importantly, intravenous administration of the anti-IL6 receptor antibody tocilizumab abrogated the tumor-promoting effects of TEMSCs. RNA-seq analysis of human osteosarcoma tissues revealed a distinct TGFß-induced prometastatic gene signature. Tissue microarray immunostaining indicated active STAT3 signaling in human osteosarcoma, consistent with the observations in TEMSC-treated mice. Finally, we isolated pure populations of EVs from serum and demonstrated that circulating levels of EV-associated TGFß are increased in osteosarcoma patients.Conclusions: Collectively, our findings suggest that TEMSCs promote osteosarcoma progression and provide the basis for testing IL6- and TGFß-blocking agents as new therapeutic options for osteosarcoma patients. Clin Cancer Res; 23(14); 3721-33. ©2017 AACR.

Does chronic raise of metal ion levels induce oxidative DNA damage and hypoxia-like response in patients with metal-on-metal hip resurfacing?

Metal-on-metal hip resurfacing (MOM-HR) represents a viable alternative to traditional arthroplasty. Nevertheless, in MOM coupling both metal nanoparticles and ions are released, whose toxicity remains a matter of concern. We investigated whether 'endogenous' chronic exposure to cobalt and chromium induced a state of oxidative stress, DNA damage and a hypoxia-like response in patients with well-functioning MOM-HR. Twenty-two patients with unilateral MOM-HR were recruited at long-term. Twenty-one osteoarthritic subjects were enrolled for comparison. Serum ion levels were measured and correlated with 8-hydroxydeoxyguanosine and circulating-free-DNA, as markers of oxidative DNA damage. Moreover, the hypoxia-inducible factor-1α, marker of hypoxic state, was evaluated. Ion concentrations were found to be 5-to-15 times higher in MOM-HR patients than in presurgery subjects (p < 0.001); circulating-free-DNA, 8-hydroxydeoxyguanosine, and hypoxia-inducible factor-1α levels were not significantly different between groups and did not correlate with ion levels. Analyzing the results according to gender, MOM-HR males had higher 8-hydroxydeoxyguanosine levels (p = 0.01) compared with MOM-HR females. Similarly, circulating-free-DNA values were higher in males than females, even if this difference did not reach statistical significance. This research is the first that attempted to investigate the long-term effects of ion dissemination in subjects with well-fixed MOM implants. A significant correlation between biomarkers increase and ion levels was not demonstrated. Nevertheless, both circulating-free-DNA and 8-hydroxydeoxyguanosine showed a tendency to increase in MOM-HR males. Further studies with a larger sample size should be performed to detect the clinical relevance of biomarkers increase especially in younger subjects, where a chronic moderately elevated exposure has to be faced. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 460-466, 2017.

Long-term systemic metal distribution in patients with stainless steel spinal instrumentation: a case-control study.

STUDY DESIGN: Case-control study. OBJECTIVE: To verify whether metal ions in the serum of patients bearing spinal stainless steel instrumentation were elevated over the long-term period after implantation of stainless steel prostheses and to determine whether these levels could predict potential unfavorable outcomes. SUMMARY OF BACKGROUND DATA: Instrumented spinal arthrodesis, the standard procedure to correct scoliosis, routinely remains in situ for the lifetime of the patient. Elevated metal ion levels have been reported at short-term follow-up, but the long-term status, possibly related to systemic toxic effects, is unknown. METHODS: Twenty-two patients treated for scoliosis with posterior spinal arthrodesis using stainless steel instrumentation were included. Minimum follow-up was 10 years. Oswestry Disability Index and visual analog scale were recorded. Chromium (Cr) and nickel (Ni) levels were measured (ng/mL) and compared with levels in a control group including 30 healthy subjects. A receiver-operating characteristic curve was calculated on the basis of the clinical assessment (pain and disability) and the x-ray picture; the cutoff values for the parameters were settled, and the ion-testing potential was considered as a surrogate marker for failure. RESULTS: The level of Cr was significantly increased in patients, compared with controls (P=0.018). A remarkable Cr release without any clinical-radiologic sign was recorded in some female patients. A high specificity (93%), positive likelihood ratio (7.00), and overall accuracy (77%) were calculated for Cr; these indicate a high risk of failure when the levels exceeded the cutoff value, which was 0.6 ng/mL. No significant difference between the groups was found for Ni (P=0.7). CONCLUSIONS: Cr testing is suggested as a reliable marker for the malfunctioning assessment and as a support for standard procedures, especially with doubtful diagnosis. Furthermore, high levels of Cr ions were observed in female patients. This finding deserves attention especially when counseling young fertile women.

Effect of acetabular cup design on metal ion release in two designs of metal-on-metal hip resurfacing.

The purpose of this observational prospective cohort study was to evaluate the serum concentrations of cobalt (Co), chromium (Cr), and nickel (Ni) at a 2-year follow-up in patients operated on with a novel design of hip resurfacing: Romax resurfacing system (RRS). RRS is characterized by the presence of an acetabular notch which theoretically provides a wider range of motion and a reduced incidence of groin pain. The presence of radiolucencies and functional outcome, assessed using the Harris hip score (HHS) and the University of California Activity scale (UCLA), were secondary endpoints. Moreover, these results were compared with those obtained in our previous study from a similar cohort of patients implanted using the Birmingham Hip Resurfacing (BHR) system. At a 2-year follow-up, the serum levels of Co in patients operated on using the RRS were five times higher (p = 0.0002) than those found before surgery (Co, means: 1.04 and 0.20 ng/mL, respectively); similarly, Cr levels were 13 times higher (p < 0.0001) at a 2-year follow-up than before surgery (Cr, means: 1.69 and 0.13 ng/mL, respectively). Ni concentrations (0.42 and 0.78 ng/mL) were not significantly different (p = 0.16), even if they increased 86% after surgery. In the RRS patients, an inverse correlation was found between Co and Cr concentrations and length of follow-up (Co: r = -0.64, p = 0.0096; Cr: r= -0.45, p = 0.08). The serum levels of Co and Cr were not significantly different between RRS (Co: 1.04 ng/mL and Cr: 1.69 ng/mL) and BHR (Co: 1.39 ng/mL and Cr: 2.30 ng/mL) patients at 2 years (p = 0.95 and 0.26 for Co and Cr, respectively). Our results showed that RRS patients achieved an excellent clinical outcome with limited metal ion release.

How do metal ion levels change over time in hip resurfacing patients? A cohort study.

Metal-on-metal hip resurfacing (MOM-HR) is offered as an alternative to traditional hip arthroplasty for young, active adults with advanced osteoarthritis. Nevertheless, concerns remain regarding wear and corrosion of the bearing surfaces and the resulting increase in metal ion levels. We evaluated three cohorts of patients with Birmingham hip resurfacing (BHR) at an average follow-up of 2, 5, and 9 years. We asked whether there would be differences in ion levels between the cohorts and inside the gender. Nineteen patients were prospectively analyzed. The correlation with clinical-radiographic data was also performed. Chromium, cobalt, nickel, and molybdenum concentrations were measured by atomic absorption spectrophotometry. Chromium and cobalt levels demonstrated a tendency to decrease over time. Such tendency was present only in females. An inverse correlation between chromium, implant size, and Harris hip score was present at short term; it disappeared over time together with the decreased ion levels. The prospective analysis showed that, although metal ion levels remained fairly constant within each patient, there was a relatively large variation between subjects, so mean data in this scenario must be interpreted with caution. The chronic high exposure should be carefully considered during implant selection, particularly in young subjects, and a stricter monitoring is mandatory.

Relevance of deep decortication and vascularization in a case of post-traumatic femoral non-union treated with grafts, platelet gel and bone marrow stromal cells.

PURPOSE: A male patient suffering from non-union of the femoral diaphysis after a traumatic fracture was treated with deep decortication and grafted with lyophilized bone, platelet gel (PG) and autologous bone marrow stromal cells (BMSCs). After 40 days from surgery, he was re-operated, due to fracture secondary displacement, caused by inappropriate load during sports activity. In addition to radiographs, two bone biopsies were retrieved: this allowed for a histological evaluation of the early response of host bone to the graft. To our knowledge, there is no report describing such early tissue response. METHODS: A clinical-radiographic evaluation of the patient and a histomorphometric analysis of the bone biopsies were performed. RESULTS: An early reparative bone formation was observed adjacent to the osteointegrated graft. Non-resorbed bone chips and large islands of non-vital bone particles, surrounded by fibrous tissue, were observed in a zone of sclerotic diaphyseal bone, that is the process was delayed despite decortication. CONCLUSIONS: These findings support the concept, until now evidenced only by imaging, that bone chips added with PG and BMSCs are effective in shortening the healing time in fracture non-union. The clinical relevance of deep decortication and vascularization is emphasized. LEVEL OF EVIDENCE: Therapeutic studies-investigating the results of treatment, Level V.

Potential role of tartrate-resistant acid phosphatase 5b (TRACP 5b) as a surrogate marker of late loosening in patients with total hip arthroplasty: a cohort study.

In a cohort study, the role of the active tartrate-resistant acid phosphatase (TRACP 5b), a marker of bone-resorbing osteoclasts, for the assessment of loosening after total hip arthroplasty (THA), was analyzed, as well as its correlation with osteolysis and multinucleated cell appearance in the retrievals. Eighty THA patients, who went consecutively to the orthopedic department, were asked to participate, and 54 accepted and were enrolled in the study. Finally, 46 subjects were analyzed, clinical-radiographic evaluation was considered the gold standard, serum TRACP 5b was blindly measured, and a cut-off was obtained, by performing a ROC Curve. Based on the gold standard, patients were split by 19 stable and 27 loosened subjects, and results were matched. TRACP 5b was significantly higher in loosened patients than in stable ones (p < 0.001). A good specificity (89.5%), positive predictive value (90.0%), and likelihood ratio (6.33) were calculated, that provided strong evidence of loosening with TRACP 5b levels higher than the cut-off. Moreover, TRACP 5b and osteolysis (Fisher's exact test, p = 0.03) were found significantly correlated. TRACP 5b has been proven a reliable marker, specifically related to resorbing-multinucleated cells, to ascertain late loosening in THA, and could support standard procedures, if confirmed by performing prospective studies.

Evaluation of restorative materials using a new perfusion system.

PURPOSE: 1) To test a new perfusion device able to alternate demineralizing/nondemineralizing solutions, as an acid attack system, and 2) to standardize the dentin demineralization procedure, in order to define the in vitro secondary caries inhibiting potential of different restorative materials. MATERIALS AND METHODS: A fluoride-containing adhesive/composite resin (group A), an experimental adhesive/composite resin (group B), and a glass-ionomer cement (group C) were used to restore 24 Class II cavities in extracted molars. Optimal conditions to obtain dentin demineralization inside the perfusion device were identified and applied to restored teeth. Dentin demineralization after perfusion was analyzed by microradiography. The output parameters were lesion morphology, dentin mineral volume percentage, and integrated mineral loss (Delta Z, % volume x microm) of the exposed (outer lesions) and marginal (inner lesions or caries inhibition zones, CIZs) dentin. RESULTS: Demineralization increased as follows: group A < group B < group C. Group A behaved better than group B, probably due to fluoride content, as indicated by Delta-Z values, higher number of CIZs, and few inner lesions. Group C showed a marginal protective effect, demonstrated by the frequent CIZs and Delta-Z positive values. This effect, however, was unable to reduce the high demineralization, probably due to the critical handling characteristics, inducingthe worst marginal adaptation. CONCLUSION: A new dynamic perfusion device was tested and a reproducible procedure was standardized in order to achieve in vitro conditions that could better simulate the pH changes of oral environment. A limited fluoride protective effect was demonstrated by using the perfusion system, whereas a perfect marginal adaptation was shown as a paramount factor to prevent restoration failure.

Serum ion levels after ceramic-on-ceramic and metal-on-metal total hip arthroplasty: 8-year minimum follow-up.

Alternative bearing surfaces for total hip arthroplasty, such as metal-on-metal and ceramic-on-ceramic, offer the potential to reduce mechanical wear and osteolysis. In the short and medium term, the second generation of metal-on-metal bearings demonstrated high systemic metal ion levels, whereas ceramic-on-ceramic bearings showed the lowest ones. We aimed to verify whether the long-term ion release in metal-on-metal subjects was still relevant at a median 10-year follow-up, and whether a fretting process at the modular junctions occurred in ceramic-on-ceramic patients and induced an ion dissemination. Serum levels were measured in 32 patients with alumina-on-alumina implants (group A), in 16 subjects with metal-on-metal implants (group B), and in 47 healthy subjects (group C). Group B results were compared with medium-term findings. Cobalt and chromium levels were significantly higher in metal-on-metal implants than in ceramic-on-ceramic ones and controls. Nevertheless, ion levels showed a tendency to decrease in comparison with medium-term content. In ceramic-on-ceramic implants, ion values were not significantly different from controls. Both in groups A and B, aluminum and titanium release were not significantly different from controls. In conclusion, negligible serum metal ion content was revealed in ceramic-on-ceramic patients. On the contrary, due to the higher ion release, metal-on-metal coupling must be prudently considered, especially in young patients, in order to obtain definitive conclusions.