Comparing Rates of Radiographic Baseplate Loosening Between Cement and Cementless INFINITY Total Ankle Prostheses.

INTRODUCTION: Total ankle replacement is indicated for end-stage ankle osteoarthritis. Periprosthetic radiolucency, from separation between the bone and implant, is suggested to be indicative of potential prosthesis failure risk. Anchoring implants with cement may mitigate this. This study aimed to compare rates of periprosthetic radiolucency between patients with cement-affixed and cementless implants. METHODS: A retrospective analysis was performed on 28 patients who underwent total ankle replacement with the INFINITY Total Ankle System (Wright Medical, Memphis, Tennessee) implant between January 2016 and May 2022. Anteroposterior, mortise, and lateral ankle X-rays were reviewed by 2 independent reviewers for tibiotalar angle, talar tilt, and periprosthetic radiolucency. The Wilcoxon rank-sum test and chi-square test were performed. RESULTS: The incidence of periprosthetic radiolucency at the most recent follow-up did not significantly differ between cement and cementless groups (57.1% in both groups, P = 1.0). Follow-up tibiotalar and talar tilt angles were not significantly different (P > .51 for all comparisons). CONCLUSIONS: Patients with cemented implants had a similar incidence of periprosthetic radiolucency compared with patients with cementless prostheses. Understanding of long-term clinical outcomes will help inform optimal operative approaches for ankle prostheses. LEVELS OF EVIDENCE: Therapeutic, Level III, Retrospective cohort study.

Arthroscopy and Micronized Allogenic Cartilage Matrix Grafting for a Missed Talus Nonunion: A Case Report.

CASE: A 33-year-old man who had experienced polytraumatic injury from a motorcycle collision 5 months previously presented to the clinic with right ankle pain. On physical examination, anterior ankle tenderness was present, and imaging revealed a previously unrecognized nondisplaced talar body fracture with a chondral defect. The patient underwent arthroscopy of the right ankle, which influenced the senior author to perform a medial malleolar osteotomy and subsequent open reduction internal fixation with micronized allogenic cartilage matrix. CONCLUSION: Arthroscopy provides valuable information for the surgical planning of talar nonunions, and allogenic cartilage matrix graft may provide benefit with associated chondral defects.

Treatment of Femoral Shaft Fractures in Patients with Klippel-Trénaunay Syndrome: A Report of 2 Cases.

CASES: Long-bone fractures in patients with Klippel-Trénaunay syndrome (KTS), a rare disorder of the venous, lymphatic, and capillary system, are difficult to treat with many complications. Two patients diagnosed with KTS presented with closed femoral shaft fractures after low-energy falls. Conservative treatment, open reduction internal fixation, and intramedullary nailing resulted in painful nonunions. Ultimately, both patients achieved pain relief and the ability to ambulate after en bloc resection and reconstruction. CONCLUSIONS: These cases demonstrate the challenges in achieving bony union when treating long-bone fractures in KTS. The feasibility of undergoing extensive resection and reconstruction to regain function is best approached with a multidisciplinary team.

Tumor Resection Guided by Intraoperative Indocyanine Green Dye Fluorescence Angiography Results in Negative Surgical Margins and Decreased Local Recurrence in an Orthotopic Mouse Model of Osteosarcoma.

BACKGROUND: Surgical resection with negative margins is the foundation of extremity sarcoma management. Failure to achieve negative surgical margins can result in local recurrence (LR), a potentially devastating complication. Indocyanine green (ICG) is a US FDA-approved fluorophore previously used to guide carcinoma resections. We investigated the potential of ICG as an intraoperative guide during experimental sarcoma resection. METHODS: Fifty 6-week-old immunocompetent Balb/c female mice received left proximal tibia paraphyseal injections of 5 × 105 K7M2 murine osteosarcoma cells. Animals were separated into two groups (n = 25 each): (1) ICG-assisted surgical resection; and (2) no ICG-assisted resection. Resections were performed 4 weeks after primary tumor engraftment. All animals received 7.5 ug ICG via retro-orbital injection 12 h prior to surgery. ICG fluorescence measurements and clinical evaluations were performed 4 weeks after resection to detect LR. RESULTS: Eleven of 25 animals from each group developed gross tumors. Four weeks after resection, group 1 had 0/11 tumor recurrences, while group 2 had recurrences in 9/11 (81.8%) experimental mice (p < 0.0002) (Fig. 2). There was a 100% NPV in group 1, and no tumor recurrence with fluorescence-free margins after the primary surgery. Group 2 had a 100% positive predictive value for the development of an LR if any fluorescent signal was present at the surgical margin after resection. CONCLUSION: Intraoperative ICG guidance led to reliably negative surgical margins and a diminished LR rate. Given the benign safety profile of ICG and its prior clinical success, these results could be immediately translatable to the clinical realm.

Disulfiram reduces metastatic osteosarcoma tumor burden in an immunocompetent Balb/c or-thotopic mouse model.

INTRODUCTION: The overall survival rate of patients with osteosarcoma (OS) and pulmonary metastases has remained stagnant at 15-30% for several decades. Disulfiram (DSF) is an FDA-approved aldehyde dehydrogenase inhibitor that reduces the metastatic phenotype of OS cells in vitro. Here we evaluate its in vivo efficacy, as compared to doxorubicin chemotherapy, in a previously-validated orthotopic model of metastatic OS. RESULTS: All treatment groups displayed a significantly reduced quantitative OS metastatic burden compared with controls. The metastatic burden of Lo DSF-treated animals was equivalent to the DXR group. Ninety-five percent of control animals displayed evidence of metastatic disease, which was significantly greater than all treatment groups. DISCUSSION: Disulfiram treatment resulted in a reduced burden of OS metastatic disease compared with controls. This was statistically-equivalent to doxorubicin. No additive effect was observed between these two therapies. MATERIALS AND METHODS: One-hundred twenty immunocompetent Balb/c mice received proximal tibia paraphyseal injections of 5 × 105 K7M2 murine OS cells. Therapy began three weeks after injection: saline (control), low-dose disulfiram (Lo DSF), high-dose disulfiram (Hi DSF), doxorubicin (DXR), Lo DSF + DXR, and Hi DSF + DXR. Transfemoral amputations were performed at 4 weeks. Quantitative metastatic tumor burden was measured using near-infrared indocyanine green (ICG) angiography.

Quantitative Primary Tumor Indocyanine Green Measurements Predict Osteosarcoma Metastatic Lung Burden in a Mouse Model.

BACKGROUND: Current preclinical osteosarcoma (OS) models largely focus on quantifying primary tumor burden. However, most fatalities from OS are caused by metastatic disease. The quantification of metastatic OS currently relies on CT, which is limited by motion artifact, requires intravenous contrast, and can be technically demanding in the preclinical setting. We describe the ability for indocyanine green (ICG) fluorescence angiography to quantify primary and metastatic OS in a previously validated orthotopic, immunocompetent mouse model. QUESTIONS/PURPOSES: (1) Can near-infrared ICG fluorescence be used to attach a comparable, quantitative value to the primary OS tumor in our experimental mouse model? (2) Will primary tumor fluorescence differ in mice that go on to develop metastatic lung disease? (3) Does primary tumor fluorescence correlate with tumor volume measured with CT? METHODS: Six groups of 4- to 6-week-old immunocompetent Balb/c mice (n = 6 per group) received paraphyseal injections into their left hindlimb proximal tibia consisting of variable numbers of K7M2 mouse OS cells. A hindlimb transfemoral amputation was performed 4 weeks after injection with euthanasia and lung extraction performed 10 weeks after injection. Histologic examination of lung and primary tumor specimens confirmed ICG localization only within the tumor bed. RESULTS: Mice with visible or palpable tumor growth had greater hindlimb fluorescence (3.5 ± 2.3 arbitrary perfusion units [APU], defined as the fluorescence pixel return normalized by the detector) compared with those with a negative examination (0.71 ± 0.38 APU, -2.7 ± 0.5 mean difference, 95% confidence interval -3.7 to -1.8, p < 0.001). A strong linear trend (r = 0.81, p < 0.01) was observed between primary tumor and lung fluorescence, suggesting that quantitative ICG tumor fluorescence is directly related to eventual metastatic burden. We did not find a correlation (r = 0.04, p = 0.45) between normalized primary tumor fluorescence and CT volumetric measurements. CONCLUSIONS: We demonstrate a novel methodology for quantifying primary and metastatic OS in a previously validated, immunocompetent, orthotopic mouse model. Quantitative fluorescence of the primary tumor with ICG angiography is linearly related to metastatic burden, a relationship that does not exist with respect to clinical tumor size. This highlights the potential utility of ICG near-infrared fluorescence imaging as a valuable preclinical proof-of-concept modality. Future experimental work will use this model to evaluate the efficacy of novel OS small molecule inhibitors. CLINICAL RELEVANCE: Given the histologic localization of ICG to only the tumor bed, we envision the clinical use of ICG angiography as an intraoperative margin and tumor detector. Such a tool may be used as an alternative to intraoperative histology to confirm negative primary tumor margins or as a valuable tool for debulking surgeries in vulnerable anatomic locations. Because we have demonstrated the successful preservation of ICG in frozen tumor samples, future work will focus on blinded validation of this modality in observational human trials, comparing the ICG fluorescence of harvested tissue samples with fresh frozen pathology.

Lung cells support osteosarcoma cell migration and survival.

BACKGROUND: Osteosarcoma (OS) is the most common primary bone tumor, with a propensity to metastasize to the lungs. Five-year survival for metastatic OS is below 30%, and has not improved for several decades despite the introduction of multi-agent chemotherapy. Understanding OS cell migration to the lungs requires an evaluation of the lung microenvironment. Here we utilized an in vitro lung cell and OS cell co-culture model to explore the interactions between OS and lung cells, hypothesizing that lung cells would promote OS cell migration and survival. The impact of a novel anti-OS chemotherapy on OS migration and survival in the lung microenvironment was also examined. METHODS: Three human OS cell lines (SJSA-1, Saos-2, U-2) and two human lung cell lines (HULEC-5a, MRC-5) were cultured according to American Type Culture Collection recommendations. Human lung cell lines were cultured in growth medium for 72 h to create conditioned media. OS proliferation was evaluated in lung co-culture and conditioned media microenvironment, with a murine fibroblast cell line (NIH-3 T3) in fresh growth medium as controls. Migration and invasion were measured using a real-time cell analysis system. Real-time PCR was utilized to probe for Aldehyde Dehydrogenase (ALDH1) expression. Osteosarcoma cells were also transduced with a lentivirus encoding for GFP to permit morphologic analysis with fluorescence microscopy. The anti-OS efficacy of Disulfiram, an ALDH-inhibitor previously shown to inhibit OS cell proliferation and metastasis in vitro, was evaluated in each microenvironment. RESULTS: Lung-cell conditioned medium promoted osteosarcoma cell migration, with a significantly higher attractive effect on all three osteosarcoma cell lines compared to basic growth medium, 10% serum containing medium, and NIH-3 T3 conditioned medium (p <0.05). Lung cell conditioned medium induced cell morphologic changes, as demonstrated with GFP-labeled cells. OS cells cultured in lung cell conditioned medium had increased alkaline phosphatase staining. CONCLUSIONS: Lung endothelial HULEC-5a cells are attractants for OS cell migration, proliferation, and survival. The SJSA-1 osteosarcoma cell line demonstrated greater metastatic potential than Saos-2 and U-2 cells. ALDH appears to be involved in the interaction between lung and OS cells, and ALP may be a valuable biomarker for monitoring functional OS changes during metastasis.

Retinal Targets ALDH Positive Cancer Stem Cell and Alters the Phenotype of Highly Metastatic Osteosarcoma Cells.

Aldehyde dehydrogenase (ALDH) is a cancer stem cell marker. Retinoic acid has antitumor properties, including the induction of apoptosis and inhibition of proliferation. Retinal, the precursor of retinoic acid, can be oxidized to retinoic acid by dehydrogenases, including ALDH. We hypothesized that retinal could potentially be transformed to retinoic acid with higher efficiency by cancer stem cells, due to the higher ALDH activity. We previously observed that ALDH activity is greater in highly metastatic K7M2 osteosarcoma (OS) cells than in nonmetastatic K12 OS cells. We also demonstrated that ALDH activity correlates with clinical metastases in bone sarcoma patients, suggesting that ALDH may be a therapeutic target specific to cells with high metastatic potential. Our current results demonstrated that retinal preferentially affected the phenotypes of ALDH-high K7M2 cells in contrast to ALDH-low K12 cells, which could be mediated by the more efficient transformation of retinal to retinoic acid by ALDH in K7M2 cells. Retinal treatment of highly metastatic K7M2 cells decreased their proliferation, invasion capacity, and resistance to oxidative stress. Retinal altered the expression of metastasis-related genes. These observations indicate that retinal may be used to specifically target metastatic cancer stem cells in OS.

Chick embryo extract demethylates tumor suppressor genes in osteosarcoma cells.

BACKGROUND: Epigenetics is the study of changes in gene expression or cellular phenotype caused by mechanisms other than changes in the underlying DNA sequence. It is widely accepted that cancer has genetic and epigenetic origins. The idea of epigenetic reprogramming of cancer cells by an embryonic microenvironment possesses potential interest from the prospect of both basic science and potential therapeutic strategies. Chick embryo extract (CEE) has been used for the successful expansion of many specific stem cells and has demonstrated the ability to facilitate DNA demethylation. QUESTIONS/PURPOSES: The current study was conducted to compare the status of DNA methylation in highly metastatic and less metastatic osteosarcoma cells and to investigate whether CEE may affect the epigenetic regulation of tumor suppressor genes and thus change the metastatic phenotypes of highly metastatic osteosarcoma cells. METHODS: K7M2 murine OS cells were treated with CEE to determine its potential effect on DNA methylation, cell apoptosis, and invasion capacity. RESULTS: Our current results suggest that the methylation status of tumor suppressor genes (p16, p53, and E-cadherin) is significantly greater in highly metastatic mouse ostoesarcoma K7M2 cells in comparison with less metastatic mouse osteosarcoma K12 cells. CEE treatment of K7M2 cells caused demethylation of p16, p53, and E-cadherin genes, upregulated their expression, and resulted in the reversion of metastatic phenotypes in highly metastatic osteosarcoma cells. CONCLUSIONS: CEE may promote the reversion of metastatic phenotypes of osteosarcoma cells and can be a helpful tool to study osteosarcoma tumor reversion by epigenetic reprogramming. CLINICAL RELEVANCE: Demethylation of tumor suppressor genes in osteosarcoma may represent a novel strategy to diminish the metastatic potential of this neoplasm. Further studies, both in vitro and in vivo, are warranted to evaluate the clinical feasibility of this approach as an adjuvant to current therapy.