Single-cell transcriptome analysis reveals the effectiveness of cytokine priming irrespective of heterogeneity in mesenchymal stromal cells.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) are recognized as a potential cell-based therapy for regenerative medicine. Short-term inflammatory cytokine pre-stimulation (cytokine priming) is a promising approach to enhance regenerative efficacy of MSCs. However, it is unclear whether their intrinsic heterogenic nature causes an unequal response to cytokine priming, which might blunt the accessibility of clinical applications. METHODS: In this study, by analyzing the single-cell transcriptomic landscape of human bone marrow MSCs from a naïve to cytokine-primed state, we elucidated the potential mechanism of superior therapeutic potential in cytokine-primed MSCs. RESULTS: We found that cytokine-primed MSCs had a distinct transcriptome landscape. Although substantial heterogeneity was identified within the population in both naïve and primed states, cytokine priming enhanced the several characteristics of MSCs associated with therapeutic efficacy irrespective of heterogeneity. After cytokine-priming, all sub-clusters of MSCs possessed high levels of immunoregulatory molecules, trophic factors, stemness-related genes, anti-apoptosis markers and low levels of multi-lineage and senescence signatures, which are critical for their therapeutic potency. CONCLUSIONS: In conclusion, our results provide new insights into MSC heterogeneity under cytokine stimulation and suggest that cytokine priming reprogrammed MSCs independent of heterogeneity.

Mixed Reality Improves 3D Visualization and Spatial Awareness of Bone Tumors for Surgical Planning in Orthopaedic Oncology: A Proof of Concept Study.

Introduction: In orthopedic oncology, computer navigation and 3D-printed guides facilitate precise osteotomies only after surgical exposure. Before surgeries start, it is challenging to mentally process and superimpose the virtual medical images onto patients' anatomy for preoperative surgical planning. Mixed Reality (MR) is an immersive technology merging real and virtual worlds, and users can interact with digital objects in real time. Through Head-Mounted Displays, surgeons directly visualize holographic models that overlaid on tumor patients. The technology may facilitate surgical planning before skin incisions. Methods: Nine bone tumor patients were included (July 2021 - Dec 2022). There were six primary bone sarcomas, two benign bone tumors, and one revision pelvic prosthesis. MR applications were created using patients' preoperative medical images. The surgeon examined each patient clinically using the conventional method of viewing 2D images and MR via HMD, Hololens 2. A Likert-Scale (LS) questionnaire and The National Aeronautics and Space Administration-Task Load Index (NASA-TLX) score were used to evaluate and compare the effectiveness of surgical planning and the surgeon's clinical cognitive workload for the two methods. Results: The qualitative survey of the LS questionnaire suggested that the MR group had superior spatial awareness of tumors and was considered more effective as a preoperative planning tool than the conventional group. For NASA-TLX scores, the overall cognitive workload was lower in MR 3D hologram group than in the 2D Group for preoperative clinical assessment. When using MR technology with HMDs, the surgeon reported no discomfort. Conclusion: MR technology may improve 3D visualization and spatial awareness of bone tumors in patients' anatomies and may facilitate surgical planning before skin incisions in orthopedic oncology surgery. With less cognitive load and better ergonomics, surgeons can focus on patients and surgical tasks with MR technology. Further studies must investigate whether MR technology improves clinical outcomes.

Intermediate-term results and risk factors analysis of tumor endoprosthesis in paediatric patients after the resection of lower extremity bone sarcoma.

BACKGROUND: Mechanical failure of the endoprostheses is a concern in paediatric patients with primary bone sarcoma. Their long-term results are variable in the Asian population, thus we aim to investigate the outcome by assessing the mechanical failure, its risk factors and the functional results. METHODS: We retrospectively reviewed 38 paediatric patients (mean 13.29, range 6-18) with primary bone sarcoma of lower extremity undergone chemotherapy and limb salvage surgery with tumor endoprosthesis between 2003 and 2016. All hospital notes were reviewed for any type of failures. Risk factors for implant loosening like stem size, remaining bone length, stem length, extracortical bone bridge ingrowth (EBBI), the ratio of resected bone length to whole bone length, bone stem ratio and custom-made versus modular were analyzed. The limb function was recorded by Musculoskeletal Tumor Society (MSTS) score. Median follow-up time was 7.42 years (3.0-15.4 years) and minimum follow-up for surviving patients was 2 years. RESULTS: Endoprosthesis survivorship, according to Kaplan Meier was 94.7%, 85.4% and 66.2% at 2, 5 and 10 years respectively. Type II failure occurred in three patients (7.9%). Type III failure occurred in four patients (10.5%). Type IV failure occured in two patients (5.2%). Only EBBI independently predicted implant loosening (p = .007). Risk factors like stem size, remaining bone length, stem length, the ratio of resected bone length to whole bone length and custom-made versus modular were not associated with increase in implant loosening (p > .05). The mean stem size was 9.41 mm in asymptomatic group, comparable with 9.22 mm in the failure group (p = .79). The MSTS score was 29.62. CONCLUSIONS: Our data suggests that paediatric Chinese patients with small body built had good and excellent mid-term results in implant survival and limb function respectively. EBBI is important in preventing loosening in tumor endoprosthesis. In contrast to the reported higher failure risk with stem size <12 mm, we found no increased loosening rate with smaller stem size endoprosthesis. LEVEL OF EVIDENCE: Class III.

Review and Future/Potential Application of Mixed Reality Technology in Orthopaedic Oncology.

In orthopaedic oncology, surgical planning and intraoperative execution errors may result in positive tumor resection margins that increase the risk of local recurrence and adversely affect patients' survival. Computer navigation and 3D-printed resection guides have been reported to address surgical inaccuracy by replicating the surgical plans in complex cases. However, limitations include surgeons' attention shift from the operative field to view the navigation monitor and expensive navigation facilities in computer navigation surgery. Practical concerns are lacking real-time visual feedback of preoperative images and the lead-time in manufacturing 3D-printed objects. Mixed Reality (MR) is a technology of merging real and virtual worlds to produce new environments with enhanced visualizations, where physical and digital objects coexist and allow users to interact with both in real-time. The unique MR features of enhanced medical images visualization and interaction with holograms allow surgeons real-time and on-demand medical information and remote assistance in their immediate working environment. Early application of MR technology has been reported in surgical procedures. Its role is unclear in orthopaedic oncology. This review aims to provide orthopaedic tumor surgeons with up-to-date knowledge of the emerging MR technology. The paper presents its essential features and clinical workflow, reviews the current literature and potential clinical applications, and discusses the limitations and future development in orthopaedic oncology. The emerging MR technology adds a new dimension to digital assistive tools with a more accessible and less costly alternative in orthopaedic oncology. The MR head-mounted display and hand-free control may achieve clinical point-of-care inside or outside the operating room and improve service efficiency and patient safety. However, lacking an accurate hologram-to-patient matching, an MR platform dedicated to orthopaedic oncology, and clinical results may hinder its wide adoption. Industry-academic partnerships are essential to advance the technology with its clinical role determined through future clinical studies.

Complex joint-preserving bone tumor resection and reconstruction using computer navigation and 3D-printed patient-specific guides: A technical note of three cases.

In selected extremity bone sarcomas, joint-preserving surgery retains the natural joints and nearby ligaments with a better function than in traditional joint-sacrificing surgery. Geometric multiplanar osteotomies around bone sarcomas were reported with the advantage of preserving more host bone. However, the complex surgical planning translation to the operating room is challenging. Using both Computer Navigation and Patient-Specific Guide may combine each technique's key advantage in assisting complex bone tumor resections. Computer Navigation provides the visual image feedback of the pathological information and validates the correct placement of Patient-Specific Guide that enables accurate, guided bone resections. We first described the digital workflow and the use of both computer navigation and patient-specific guides (NAVIG) to assist the multiplanar osteotomies in three extremity bone sarcoma patients who underwent joint-preserving bone tumor resections and reconstruction with patient-specific implants. The NAVIG technique verified the correct placement of patient-specific guides that enabled precise osteotomies and well-fitted patient-specific implants. The mean maximum deviation errors of the nine achieved bone resections were 1.64 â€‹± â€‹0.35 â€‹mm (95% CI 1.29 to 1.99). The histological examination of the tumor specimens showed negative resection margin. At the mean follow-up of 55 months (40-67), no local recurrence was noted. There was no implant loosening that needed revision. The mean MSTS score was 29 (28-30) out of 30 with the mean knee flexion of 140° (130°-150°). The excellent surgical accuracy and limb function suggested that the NAVIG technique might replicate the surgical planning of complex bone sarcoma resections by combining the strength of both Computer Navigation and Patient-Specific Guide. The patient-specific approach may translate into clinical benefits. The translational potential of this article: The newly described technique enhances surgeons' capability in performing complex joint-preserving surgery in bone sarcoma that is difficult to be achieved by the traditional method. The high precision and accuracy may translate into superior clinical outcomes.

Long-term outcome of vascularized iliac bone grafting for osteonecrosis of femoral head: A retrospective study with 17-year follow-up.

AIMS: This study aims to investigate the long-term results of vascularized iliac bone grafting (VIBG) for osteonecrosis of the femoral head (ONFH). The primary outcome is the long-term survivorship of VIBG, using conversion to total hip arthroplasty as an end-point. Secondly, this study will also analyse the patient or disease factors influencing the long-term survivorship of VIBG. METHODS: Forty-two patients (50 hips) underwent VIBG for ONFH in our institute between September 1995 and November 2013. Only patients with a follow-up of at least 5 years were included. The risk factors, surgical complications and VIBG survivorship were recorded. The stage of ONFH was classified according to the Ficat staging of the pre-operative radiographs. VIBG was only performed to patients with ONFH of Ficat stage II and stage III. Patients with hip arthritis (Ficat stage IV) did not receive VIBG and thus excluded from the study. Long-term survivorship of VIBG is measured by conversion to total hip arthroplasty. RESULTS: Twenty-eight hips (56%) had surviving VIBG for the duration of follow-up. The overall mean graft survival was 12.2 ± 7.8 years (0.4-24.0). Steroid and alcohol-induced osteonecrosis were more predominant in the graft-failure group, which had a hazard ratio of 2.33 and 2.07 respectively for graft failure (p = 0.047). In terms of complication, there was one case of groin wound infection which required surgical debridement. CONCLUSION: At a long-term follow-up of 17 years, our results showed that VIBG is effective in treating patients with pre-collapse (Ficat Stage II) and early post-collapse (Ficat stage III) in ONFH. Alcoholics and patients with steroid are at a higher risk of graft failure, so VIBG should be performed cautiously in these patients. VIBG is an intermediate operation until osteoarthritis sets in, either by the progression of ONFH or natural degenerative change.

Local Recurrence After Minimally Invasive Curettage For Primary Giant Cell Tumor of Bone With Perioperative Bisphosphonate Is Comparable to Open Curettage: Retrospective Comparison With 9-Year Follow-Up.

PURPOSE: To compare the long-term oncological outcome of minimally invasive curettage (MIC) with conventional open curettage (OC). METHODS: We studied patients with primary giant cell tumor of bone (GCTB) of extremities who underwent intralesional tumor curettage and cementation and perioperative bisphosphonates from February 2003 to June 2016. All cases were histology-confirmed diagnoses of GCTB. Recurrent GCTB, malignant GCTB, cases in the axial skeleton (pelvis and spine), or cases with bone grafting of the curetted cavity were excluded. The local recurrence-free (LR-free) estimates of the OC and MIC groups were compared. The hazard ratio of a local recurrence was calculated for the various factors of the patients, disease, and treatment. RESULTS: At a mean follow-up of 8.8 years, the overall LR rate was 24.2% (8 out of 33 patients). There was no statistical difference in LR in MIC and OC groups (27.8 % vs 20%; P = .6). The mean time to LR was 33.1 months (8 to 75). The operative time was comparable in both MIC and OC groups. None of the risk factors studied led to a significantly higher hazard of LR. CONCLUSIONS: At a long-term follow-up of 9 years, MIC showed similar LR-free survival to OC. Combining bisphosphonates and MIC with a less invasive approach showed reasonable LR-free survival in long-term follow-up. LEVEL OF EVIDENCE: Level III, retrospective cohort study.

Role of skeletal macrophages in fracture repair: A systematic review.

In the field of bone research, the importance of the function of skeletal macrophages (sMΦ) and their crucial role in immune homeostasis and bone regeneration has been extensively studied. The aim of the present systematic review was to summarize the role of sMΦ in bone fracture healing and to evaluate their potential for immunoregulatory therapy in bone regeneration. A systematic literature search of PubMed and Embase® was performed to retrieve studies on the role of sMΦ in bone injury repair. The Systematic Review Centre for Laboratory animal Experimentation tool was used to assess the risk of bias of the studies included. A total of four articles were included in the present review. A relatively high risk of bias was identified in the included articles as none of the assessors in these studies were blinded. sMΦ were defined by the surface markers F4/80+, Mac-2- / low, TRAP-, CD169+, Ly6G- and CD115low. All of the studies provided support for the essential role of sMΦ in intramembranous ossification or endochondral ossification during fracture healing. F4/80+Mac-2-CD169+ sMΦ are a promising therapeutic target for immunoregulatory therapy of bone repair due to their essential role in bone formation and homeostasis. Future studies aimed at profiling and modulating sMΦ to promote bone regeneration are required.

Segmental composite porous scaffolds with either osteogenesis or anti-bone resorption properties tested in a rabbit ulna defect model.

A functional biomaterial with a therapeutic effect is desirable as an adjuvant therapy to enhance bone formation and prevent local recurrence of bone tumours, especially when the resection margins are not identifiable. In this study, novel composite materials were developed with dual properties of osteopromotion and bone resorption to mimic the tumour inhibition effect, including water-soluble phosphorylated chitosan (P-chitosan) for increasing osteoblasts activity and disodium (1 → 4)-2-deoxy-2-sulphoamino-ß-d-glucopyranuronan (S-chitosan) for inhibiting bone resorption activity. First, P-chitosan and S-chitosan were respectively incorporated into two kinds of PLGA/TCP-based scaffold, i.e. PLGA-TCP-P-chitosan (P/T/P-chitosan) and PLGA-TCP-S-chitosan (P/T/S-chitosan) scaffolds. We subsequently tested combined scaffolds of PLGA-TCP-P-S-P-chitosan (P/T/PSP-chitosan) made of P/T/P-chitosan and P/T/S-chitosan to assess their integral effect, on enhancement of bone formation with P/T/P-chitosan and inhibition of tissue regeneration with P/T/S-chitosan, in an established rabbit ulnar bone defect model to imitate bone resection post-bone tumour. To compare bone healing in the defects, the P/T/P-chitosan group was regarded as a bone formation enhancement group, while the P/T group served as a control. Bone mineral density (BMD) in the P/T/P-chitosan and P/T/PSP-chitosan groups were found to be significantly higher than those in the P/T group, while that in the P/T/P-chitosan group was greater than that in the P/T/PSP-chitosan group (p < 0.05). These findings demonstrated that P/T/PSP-chitosan scaffolds possessed more osteogenic potential than the P/T scaffold but less osteogenic effect than the P/T/P-chitosan scaffold, as the S-chitosan component inhibited the activities of osteoblasts for bone formation. These findings implied a dual function of the designed P/T/PSP-chitosan for further preclinical validation and potential applications in the prevention of local recurrence and for enhancing bone repair after bone tumour resection. Copyright © 2013 John Wiley & Sons, Ltd.

Genome-Wide Transcriptome Profiling of the Neoplastic Giant Cell Tumor of Bone Stromal Cells by RNA Sequencing.

Giant cell tumor of bone (GCTB) is the most common non-malignant primary bone tumor reported in Hong Kong. Failure of treatment in advanced GCTB with aggressive local recurrence remains a clinical challenge. In order to reveal the molecular mechanism underlying the pathogenesis of this tumor, we aimed to examine the transcriptome profiling of the neoplastic stromal cells of GCTB in this study. RNA-sequencing was performed on three GCTB stromal cell samples and one bone marrow-derived MSC sample and 174 differentially expressed genes (DEGs) were identified between these two cell types. The top five up-regulated genes are SPP1, F3, TSPAN12, MMP13, and LGALS3BP and further validated by qPCR and Western Blotting. Knockdown of SPP1 was found to induce RUNX2 and OPG expression in GCTB stromal cells but not the MSCs. Ingenuity pathway analysis (IPA) of the 174 DEGs revealed significant alternations in 23 pathways; variant calling analysis revealed 1915 somatic variants of 384 genes with high or moderate impacts. Interestingly, four canonical pathways were found overlapping in both analyses; from which VEGFA, CSF1, PLAUR, and F3 genes with somatic mutation were found up-regulated in GCTB stromal cells. The STRING diagram showed two main clusters of the DEGs; one cluster of histone genes that are down-regulated in GCTB samples and another related to osteoblast differentiation, angiogenesis, cell cycle progression, and tumor growth. The DEGs and somatic mutations found in our study warrant further investigation and validation, nevertheless, our study add new insights in the search for new therapeutic targets in treating GCTB. J. Cell. Biochem. 118: 1349-1360, 2017. © 2016 Wiley Periodicals, Inc.

Differential expression of filamin B splice variants in giant cell tumor cells.

Giant cell tumor of bone (GCT) is the most commonly reported non-malignant bone tumor in Hong Kong. This kind of tumor usually affects people aged 20-40 years. Also, it is well known for recurrence locally, especially when the tumor cannot be removed completely. Filamins are actin-binding proteins which contain three family members, filamin A, B and C. They are the products of three different genes, FLNA, FLNB and FLNC, which can generate various transcript variants in different cell types. In this study, we focused on the effects of FLNBv2 and FLNBv4 toward GCT cells. The only difference between FLNBv2 and FLNBv4 is that FLNBv4 does not contain hinge 1 region. We found that the relative abundance of FLNBv4 varies among different GCT cell lines while the expression level of FLNBv4 in normal osteoblasts was only marginally detectable. In the functional aspect, overexpression of FLNBv4 led to upregulation of RANKL, OCN, OPG and RUNX2, which are closely related to GCT cell survival and differentiation. Moreover, FLNBv4 can have a negative effect on cell viability of GCT cells when compare with FLNBv2. In conclusion, splicing variants of FLNB are differentially expressed in GCT cells and may play a role in the proliferation and differentiation of tumor cells.

Patient-specific instrument can achieve same accuracy with less resection time than navigation assistance in periacetabular pelvic tumor surgery: a cadaveric study.

PURPOSE: Inaccurate resection in pelvic tumors can result in compromised margins with increase local recurrence. Navigation-assisted and patient-specific instrument (PSI) techniques have recently been reported in assisting pelvic tumor surgery with the tendency of improving surgical accuracy. We examined and compared the accuracy of transferring a virtual pelvic resection plan to actual surgery using navigation-assisted or PSI technique in a cadaver study. METHODS: We performed CT scan in twelve cadaveric bodies including whole pelvic bones. Either supraacetabular or partial acetabular resection was virtually planned in a hemipelvis using engineering software. The virtual resection plan was transferred to a CT-based navigation system or was used for design and fabrication of PSI. Pelvic resections were performed using navigation assistance in six cadavers and PSI in another six. Post-resection images were co-registered with preoperative planning for comparative analysis of resection accuracy in the two techniques. RESULTS: The mean average deviation error from the planned resection was no different ([Formula: see text]) for the navigation and the PSI groups: 1.9 versus 1.4 mm, respectively. The mean time required for the bone resection was greater ([Formula: see text]) for the navigation group than for the PSI group: 16.2 versus 1.1 min, respectively. CONCLUSIONS: In simulated periacetabular pelvic tumor resections, PSI technique enabled surgeons to reproduce the virtual surgical plan with similar accuracy but with less bone resection time when compared with navigation assistance. Further studies are required to investigate the clinical benefits of PSI technique in pelvic tumor surgery.

A mouse model of luciferase-transfected stromal cells of giant cell tumor of bone.

A major barrier towards the study of the effects of drugs on Giant Cell Tumor of Bone (GCT) has been the lack of an animal model. In this study, we created an animal model in which GCT stromal cells survived and functioned as proliferating neoplastic cells. A proliferative cell line of GCT stromal cells was used to create a stable and luciferase-transduced cell line, Luc-G33. The cell line was characterized and was found that there were no significant differences on cell proliferation rate and recruitment of monocytes when compared with the wild type GCT stromal cells. We delivered the Luc-G33 cells either subcutaneously on the back or to the tibiae of the nude mice. The presence of viable Luc-G33 cells was assessed using real-time live imaging by the IVIS 200 bioluminescent imaging (BLI) system. The tumor cells initially propagated and remained viable on site for 7 weeks in the subcutaneous tumor model. We also tested in vivo antitumor effects of Zoledronate (ZOL) and Geranylgeranyl transferase-I inhibitor (GGTI-298) alone or their combinations in Luc-G33-transplanted nude mice. ZOL alone at 400 µg/kg and the co-treatment of ZOL at 400 µg/kg and GGTI-298 at 1.16 mg/kg reduced tumor cell viability in the model. Furthermore, the anti-tumor effects by ZOL, GGTI-298 and the co-treatment in subcutaneous tumor model were also confirmed by immunohistochemical (IHC) staining. In conclusion, we established a nude mice model of GCT stromal cells which allows non-invasive, real-time assessments of tumor development and testing the in vivo effects of different adjuvants for treating GCT.

Use of Computer Navigation in Orthopedic Oncology.

The use of computer navigation was first described in the surgical resection of pelvic tumors in 2004. It was developed to improve surgical accuracy with the goal of achieving clear resection margins and better oncologic results. During the past few years, there has been tremendous advancement of computer-assisted tumor surgery (CATS) in the field of orthopedic oncology. Currently, CATS with image fusion offers preoperative three-dimensional surgical planning and allows surgeons to reproduce the intended bone resections in musculoskeletal tumors. The technique is reported to be useful in technically demanding resections, such as in pelvic and sacral tumors; joint-preserving intercalated and multiplanar tumor resection; and complex reconstruction with custom computer-aided design prostheses or allografts. This article provides an up-to-date review of the recent developments and key features in CATS, its current status in clinical practice, and future directions in its development.

Comparison of the anti-tumor effects of denosumab and zoledronic acid on the neoplastic stromal cells of giant cell tumor of bone.

Denosumab and Zoledronic acid (ZOL) are two antiresorptive drugs currently in use for treating osteoporosis. They have different mechanisms of action but both have been shown to delay the onset of skeletal-related events in patients with giant cell tumor of bone (GCT). However, the anti-tumor mechanisms of denosumab on the neoplastic GCT stromal cells remain unknown. In this study, we focused on the direct effects of denosumab on the neoplastic GCT stromal cells and compared with ZOL. The microscopic view demonstrated a reduced cell growth in ZOL-treated but not in denosumab-treated GCT stromal cells. ZOL was found to exhibit a dose-dependent inhibition in cell growth in all GCT stromal cell lines tested and cause apoptosis in two out of three cell lines. In contrast, denosumab only exerted a minimal inhibitory effect in one cell line and did not induce any apoptosis. ZOL significantly inhibited the mRNA expression of receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) in two GCT stromal cell lines whereas their protein levels remained unchanged. On the contrary, denosumab did not regulate RANKL and OPG expression at both mRNA and protein levels. Moreover, the protein expression of Macrophage Colony-Stimulating Factor (M-CSF), Alkaline Phosphatase (ALP), and Collagen α1 Type I were not regulated by denosumab and ZOL either. Our findings provide new insights in the anti-tumor effect of denosumab on GCT stromal cells and raise a concern that tumor recurrence may occur after the withdrawal of the drug.

Joint-preserving tumor resection and reconstruction using image-guided computer navigation.

BACKGROUND: Joint-preserving surgery is performed in select patients with bone sarcomas of extremities and allows patients to retain the native joint with better joint function. However, recurrences may relate to achieving adequate margins and there is frequently little room for error in tumors close to the joint surface. Further, the tumor margin on preoperative CT and/or MR images is difficult to transpose to the actual extent of tumor in the bone in the operating room. QUESTIONS/PURPOSES: We therefore determined whether joint-preserving tumor surgery could be performed accurately under image-guided computer navigation and determined local recurrences, function, and complications. METHODS: We retrospectively studied eight patients with bone sarcoma of extremities treated surgically by navigation with fused CT-MR images. We assessed the accuracy of resection in six patients by comparing the cross sections at the resection plane with complementary prosthesis templates. Mean age was 17 years (range, 6-46 years). Minimum followup was 25 months (mean, 41 months; range, 25-60 months). RESULTS: The achieved resection was accurate, with a difference of 2 mm or less in any dimension compared to that planned in patients with custom prostheses. We noted no local recurrence at latest followup. The mean Musculoskeletal Tumor Society score was 29 (range, 28-30). There were no complications related to navigation planning and procedures. There was no failure of fixation at the remaining epiphysis. CONCLUSIONS: In selected patients, the computer-assisted approach facilitates precise planning and execution of joint-preserving tumor resection and reconstruction. Further followup assessment in a larger study population is required in these patients. LEVEL OF EVIDENCE: Level IV, therapeutic study. See Instructions for Authors for a complete description of levels of evidence.

Computer-assisted tumor surgery in malignant bone tumors.

BACKGROUND: Small recent case series using CT-based navigation suggest such approaches may aid in surgical planning and improve accuracy of intended resections, but the accuracy and clinical use have not been confirmed. QUESTIONS/PURPOSES: We therefore evaluated (1) the accuracy; (2) recurrences; and (3) function in patients treated by computer-assisted tumor surgery (CATS). METHODS: From 2006 to 2009, we performed CATS in 20 patients with 21 malignant tumors. The mean age was 31 years (range, 6-80 years). CT and MR images for 18 cases were fused using the navigation software. Reconstructed two-dimensional/three-dimensional images were used to plan the bone resection. The achieved bone resection was compared with the planned one by assessing margins, dimensions at the level of bone resection, or fitting of CAD custom prostheses. Function was assessed with the Musculoskeletal Tumor Society (MSTS) score. The minimum followup was 31 months (mean, 39 months; range, 5-69 months). RESULTS: Histological examination of all resected specimens showed a clear tumor margin. The achieved bone resection matched the planned with a difference of ≤ 2 mm. The achieved positions of custom prostheses were comparable to the planned positions when merging postoperative with preoperative CT images in five cases. Three of the four patients with local recurrence had tumors at the sacral region. The mean MSTS score was 28 (range, 23-30). CONCLUSION: CATS with image fusion allows accurate execution of the intended bone resection. It may be beneficial to resection and reconstruction in pelvic, sacral tumors and more difficult joint-preserving intercalated tumor surgery. Comparative clinical studies with long-term followup are necessary to confirm its efficacy. LEVEL OF EVIDENCE: Level IV, therapeutic study. See Guidelines for Authors for a complete description of levels of evidence.

p63 regulates cell proliferation and cell cycle progression­associated genes in stromal cells of giant cell tumor of the bone.

Giant cell tumor of bone (GCT) is a destructive neoplasm of uncertain etiology that affects the epiphyseal ends of long bones in young adults. GCT stromal cells (GCTSCs) are the primary neoplastic cells of this tumor and are the only proliferating cell component in long-term culture, which recruits osteoclast-like giant cells that eventually mediate bone destruction. The oncogenesis of GCT and factors driving the neoplastic stromal cells to proliferate extensively and pause at an early differentiation stage of pre-osteoblast lineage remain unknown. Overexpression of p63 was observed in GCTSCs and there is growing evidence that p63 is involved in oncogenesis through different mechanisms. This study aimed to understand the specific role of p63 in cell proliferation and oncogenesis of GCTSCs. We confirmed p63 expression in the mononuclear cells in GCT by immunohistochemical staining. By real-time PCR analysis, we showed a higher level (>15­fold) of TAp63 expression in GCTSCs compared to that in mesenchymal stem cells. Furthermore, we observed that knockdown of the p63 gene by siRNA transfection greatly reduced cell proliferation and induced cell cycle arrest at S phase in GCTSCs. We found that the mRNA expression of CDC2 and CDC25C was substantially suppressed by p63 knockdown at 24-72 h. Moreover, p63 was found to be recruited on the regulatory regions of CDC2 and CDC25C, which contain p53-responsive elements. In summary, our data suggest that p63 regulates GCTSC proliferation by binding to the CDC2 and CDC25C p53-REs, which may inhibit the p53 tumor suppressor activity and contribute to GCT tumorigenesis.

CCAAT/enhancer binding protein beta is up-regulated in giant cell tumor of bone and regulates RANKL expression.

Giant cell tumor (GCT) of bone is an aggressive non-cancerous tumor, which consists of multi-nucleated osteoclast-like giant cells, stromal cells, and monocytes. It is believed that stromal cells are the neoplastic component of this tumor. Expression of the receptor activator of nuclear factor kappa B ligand (RANKL) in the stromal cells stimulates the monocytes to form giant multi-nucleated osteoclast-like cells, causing bone over-resorption at the tumor site. Previously, our group has reported the up-regulation of RANKL in GCT of bone stromal cells, but the mechanism is unknown. Using stromal cell culture of GCT obtained from patients, we demonstrated the up-regulation of the transcriptional activator CCAAT/enhancer binding protein beta (C/EBPbeta). RANKL promoter studies revealed that C/EBPbeta over-expression induced RANKL promoter activity in a dose-dependent manner and a CCAAT-box within the region nt -357/-1 contributed to the basal transcription activity, with a possible C/EBPbeta binding element in the region nt -460/-358 leading to further induction. Furthermore, we also showed that C/EBPbeta bound to the RANKL promoter in GCT stromal cells in vivo by chromatin immunoprecipitation. To conclude, our study has shown that C/EBPbeta is a RANKL promoter activator in stromal cells of GCT of bone and we have proposed a model in which C/EBPbeta plays an important role in the osteolytic characteristics and pathological causes of GCT of bone.

Bisphosphonates reduce local recurrence in extremity giant cell tumor of bone: a case-control study.

BACKGROUND: Giant cell tumor (GCT) of bone is a benign but locally aggressive tumor that is characterized by the presence of mononuclear stromal cells and multinucleated giant cells. Although topical adjuvants have been used in the past, local recurrence following intralesional excision of GCT of bone continues to remain a problem. The use of bisphosphonates as an anti-osteoclastic agent in the management of osteolytic bone metastases is well accepted. Furthermore in vitro studies have shown that bisphosphonates also induce apoptosis in GCT stromal cells. Therefore our clinical study aims to investigate whether the administration of bisphosphonate as an adjuvant can further decrease local recurrence following the surgical treatment of GCT of bone. METHOD: A retrospective case-control study was performed between 1988 and 2004. Forty-four patients with histological diagnosed appendicular GCT were included. Intralesional curettage or wide excision of the lesions was followed with cementation or biological reconstruction. Additional intravenous and oral bisphosphonates were given peri-operatively to 24 patients who were treated between 1998 and 2004. The average follow-up of the control group was 115 months while that of the treatment group was 48 months. RESULTS: In the bisphosphonate treated group, 1 of 24 patients (4.2%) developed local recurrence. In the control group, 6 of 20 patients (30%) developed local recurrence. The difference in the recurrence rate was statistically significant between the bisphosphonate treatment group and the control group (Log Rank test p=0.056). The effect of reduction of local recurrence was significant in patients with stage III diseases. Patients treated with bisphosphonate did not report any untoward effects. CONCLUSION: Clinical use of bisphosphonates as an adjuvant therapy for giant cell tumor of bone demonstrated a lower local recurrence rate. The clinical response seems to be more promising in stage III diseases.