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.

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.

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.