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Long bone nonunion in the context of congenital pseudarthrosis or carcinologic resection (with intercalary bone allograft implantation) is one of the most challenging pathologies in pediatric orthopedics. Autologous cancellous bone remains the gold standard in this context of long bone nonunion reconstruction, but with several clinical limitations. We then assessed the feasibility and safety of human autologous scaffold-free osteogenic 3-dimensional (3D) graft (derived from autologous adipose-derived stem cells [ASCs]) to cure a bone nonunion in extreme clinical and pathophysiological conditions. Human ASCs (obtained from subcutaneous adipose tissue of 6 patients and expanded up to passage 4) were incubated in osteogenic media and supplemented with demineralized bone matrix to obtain the scaffold-free 3D osteogenic structure as confirmed in vitro by histomorphometry for osteogenesis and mineralization. The 3D "bone-like" structure was finally transplanted for 3 patients with bone tumor and 3 patients with bone pseudarthrosis (2 congenital, 1 acquired) to assess the clinical feasibility, safety, and efficacy. Although minor clones with structural aberrations (aneuploidies, such as tri or tetraploidies or clonal trisomy 7 in 6%-20% of cells) were detected in the undifferentiated ASCs at passage 4, the osteogenic differentiation significantly reduced these clonal anomalies. The final osteogenic product was stable, did not rupture with forceps manipulation, did not induce donor site morbidity, and was easily implanted directly into the bone defect. No acute (<3 mo) side effects, such as impaired wound healing, pain, inflammatory reaction, and infection, or long-term side effects, such as tumor development, were associated with the graft up to 4 years after transplantation. We report for the first time that autologous ASC can be fully differentiated into a 3D osteogenic-like implant without any scaffold. We demonstrated that this engineered tissue can safely promote osteogenesis in extreme conditions of bone nonunions with minor donor site morbidity and no oncological side effects., Competing Interests: The authors have no conflicts of interest to disclose.

Adipose tissue was only recently considered as a potential source of mesenchymal stem cells (MSCs) for bone tissue engineering. To improve the osteogenicity of acellular bone allografts, adipose MSCs (AMSCs) and bone marrow MSCs (BM-MSCs) at nondifferentiated and osteogenic-differentiated stages were investigated in vitro and in vivo. In vitro experiments demonstrated a superiority of AMSCs for proliferation (6.1±2.3 days vs. 9.0±1.9 days between each passage for BM-MSCs, respectively, P<0.001). A significantly higher T-cell depletion (revealed by mixed lymphocyte reaction, [MLR]) was found for AMSCs (vs. BM-MSCs) at both non- and differentiated stages. Although nondifferentiated AMSCs secreted a higher amount of vascular endothelial growth factor [VEGF] in vitro (between 24 and 72 h of incubation at 0.1-21% O(2)) than BM-MSCs (P<0.001), the osteogenic differentiation induced a significantly higher VEGF release by BM-MSCs at each condition (P<0.001). After implantation in the paraspinal muscles of nude rats, a significantly higher angiogenesis (histomorphometry for vessel development (P<0.005) and VEGF expression (P<0.001)) and osteogenesis (as revealed by osteocalcin expression (P<0.001) and micro-CT imagery for newly formed bone tissue (P<0.05)) were found for osteogenic-differentiated AMSCs in comparison to BM-MSCs after 30 days of implantation. Osteogenic-differentiated AMSCs are the best candidate to improve the angio-/osteogenicity of decellularized bone allografts., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Introduction: We present here a surgical technique using a navigation system and an oscillating saw for the resection of a pelvic bone tumor combined with an allograft reconstruction., Step 1 Preoperative Planning: The surgeon and radiologist together delineate the tumor on each magnetic resonance imaging (MRI) slice; then the surgeon defines target planes for tumor resection and transfers them to the allograft., Step 2 Patient Positioning and Surgical Exposure: With the patient in the lateral decubitus position, combine ilioinguinal with iliocrural and obturator surgical approaches to expose the ilium., Step 3 Navigated Tumor Resection: Perform the osteotomies using the navigation system to guide the saw blade, following predefined target planes; perform a biopsy., Step 4 Navigated Allograft Cutting: Perform the osteotomies using the navigating saw, following the same target planes as used for the tumor resection., Step 5 Pelvic Reconstruction: Fix the graft and cement a femoral stem in place; then reinsert all detached tendons and elevated muscles., Results & Preop/postop Images: Editor's note: This technique is based on preliminary work that has not been presented in a peer-reviewed case series publication., What to Watch for: IndicationsContraindicationsPitfalls & Challenges., (Copyright © 2011 by The Journal of Bone and Joint Surgery, Incorporated.)

Background and Purpose: Massive bone allografts are used when surgery causes large segmental defects. Shape-matching is the primary criterion for selection of an allograft. The current selection method, based on 2-dimensional template comparison, is inefficient for 3-dimensional complex bones. We have analyzed a 3-dimensional (3-D) registration method to match the anatomy of the allograft with that of the recipient., Methods: 3-D CT-based registration was performed to match the shapes of both bones. We used the registration to align the allograft volume onto the recipient's bone. Hemipelvic allograft selection was tested in 10 virtual recipients with a panel of 10 potential allografts, including one from the recipient himself (trap graft). 4 observers were asked to visually inspect the superposition of allograft over the recipient, to classify the allografts into 4 categories according to the matching of anatomic zones, and to select the 3 best matching allografts. The results obtained using the registration method were compared with those from a previous study on the template method., Results: Using the registration method, the observers systematically detected the trap graft. Selections of the 3 best matching allografts performed using registration and template methods were different. Selection of the 3 best matching allografts was improved by the registration method. Finally, reproducibility of the selection was improved when using the registration method., Interpretation: 3-D CT registration provides more useful information than the template method but the final decision lies with the surgeon, who should select the optimal allograft according to his or her own preferences and the needs of the recipient.

Pelvic sarcoma is associated with a relatively poor prognosis, due to the difficulty in obtaining an adequate surgical margin given the complex pelvic anatomy. Magnetic resonance imaging and computerized tomography allow valuable surgical resection planning, but intraoperative localization remains hazardous. Surgical navigation systems could be of great benefit in surgical oncology, especially in difficult tumor location; however, no commercial surgical oncology software is currently available. A customized navigation software was developed and used to perform a synovial sarcoma resection and allograft reconstruction. The software permitted preoperative planning with defined target planes and intraoperative navigation with a free-hand saw blade. The allograft was cut according to the same planes. Histological examination revealed tumor-free resection margins. Allograft fitting to the pelvis of the patient was excellent and allowed stable osteosynthesis. We believe this to be the first case of combined computer-assisted tumor resection and reconstruction with an allograft.

Background and Purpose: Osseous pelvic tumors can be resected and reconstructed using massive bone allografts. Geometric accuracy of the conventional surgical procedure has not yet been documented. The aim of this experimental study was mainly to assess accuracy of tumoral resection with a 10-mm surgical margin, and also to evaluate the geometry of the host-graft reconstruction., Methods: An experimental model on plastic pelvises was designed to simulate tumor resection and reconstruction. 4 experienced surgeons were asked to resect 3 different tumors and to reconstruct pelvises. 24 resections and host-graft junctions were available for evaluation. Resection margins were measured. Several methods were created to evaluate geometric properties of the host-graft junction., Results: The probability of a surgeon obtaining a 10-mm surgical margin with a 5-mm tolerance above or below, was 52% (95% CI: 37-67). Maximal gap, gap volume, and mean gap between host and graft was 3.3 (SD 1.9) mm, 2.7 (SD 2.1) cm3 and 3.2 (SD 2.1) mm, respectively. Correlation between these 3 reconstruction measures and the degree of contact at the host-graft junction was poor., Interpretation: 4 experienced surgeons did not manage to consistently respect a fixed surgical margin under ideal working conditions. The complex 3-dimensional architecture of the pelvis would mainly explain this inaccuracy. Solutions to this might be to increase the surgical margin or to use computer- and robotic-assisted technologies in pelvic tumor resection. Furthermore, our attempt to evaluate geometry of the pelvic reconstruction using simple parameters was not satisfactory. We believe that there is a need to define new standards of evaluation.

Background: The need for safe bone allografts is increasing and preservation of femoral heads from patients being operated on with hip arthroplasty should be encouraged. However, should we preserve femoral heads from patients operated on for neck fracture as tissue mechanical quality may not be satisfactory?, Material and Methods: We compared the evolution of stiffness and compactness of fresh-frozen morselized bone obtained from osteoarthrotic femoral heads and those from neck fractures. Both materials were also compared after freeze-drying and irradiation. We used 6 osteoarthrotic and 6 neck-fracture femoral heads to prepare 4 batches of morselized bone. 18 samples from each batch were impacted in a contained cylinder. Frozen bone grafts were tested after thawing at room temperature for 2 hours and freeze-dried grafts were tested after 30 minutes of rehydration., Results: The stiffness of fresh-frozen neck fracture bone was lower than that of fresh-frozen osteoarthrotic bone at 150 impactions. The stiffness of freeze-dried irradiated bone was higher than that of the fresh-frozen bone and did not differ between osteoarthrotic and neck-fracture bone., Interpretation: Solvent-treated freeze-dried bone from femoral heads procured during arthroplasty for sub-capital hip fractures represents a valuable source of material for allografts, addressing concerns regarding serological testing, medical history and bone quality.

Background: Defatted bone chips with or without freeze-drying and irradiation have mechanical advantages as compared to fresh-frozen controls in in vitro models of impaction. These improved results have been ascribed to replacement of viscous bone marrow by saline and embrittlement of the freeze-dried bone by irradiation., Material and Methods: To determine which of these hypotheses is correct, we compared the development of stiffness and compactness of morselized bone graft that had been: 1) fat-reduced with saline, and 2) fresh-frozen, solvent-detergent defatted, 3) freeze-dried irradiated and 4) not irradiated. We used 12 osteoarthrotic femoral heads to prepare these four batches of morselized bone, and impacted 18 samples from each batch in a cylinder. The frozen bone grafts were tested after thawing at room temperature for 2 hours and the freeze-dried grafts were tested after 30 minutes of rehydration. We monitored the development of compactness and stiffness of the material during impaction., Results: The stiffness of the freeze-dried irradiated bone was greater than that of the other three series after 10, 50 and 150 impactions. The freeze-dried bone chips that were not irradiated and the chips defatted with saline alone were less stiff than the fresh-frozen control after 150 impactions., Interpretation: The brittleness of freeze-dried irradiated bone, caused by loss of the capacity to absorb energy in a plastic way, increases the compactness and stiffness of the morselized grafts. Washing bone with saline alone or treating bone with solvent-detergent but no irradiation had no similar mechanical advantage and the bone did not impact better than fresh-frozen undefatted bone in our model.

Processed freeze-dried irradiated allografts seem to be used less than instead of fresh-frozen allografts for impaction bone grafting in revision hip arthroplasties. Although biologically acceptable, their use is discouraged because of their questionable mechanical properties following freeze-drying and irradiation procedures. To address this question, we impacted freeze-dried grafts in 6 cadaveric femurs and loaded with a cemented Charnley prosthesis. The routinely used fresh-frozen allografts were used as controls in the contralateral side. These constructs were compared simultaneously in a walking hip simulator for their stability during 900,000 loading cycles. The mechanical parameters were axial inducible displacement and subsidence of the implant. The former parameter was lower in the implant mounted on freeze-dried impacted grafts than that mounted on the fresh-frozen bone. The latter parameter was also lower in the freeze-dried group. At the end of the test, we found no implant loosening in either group and their 'pull out' resulted in cement-prosthesis debonding, which showed the mechanical integrity of the impacted grafts. Freeze-dried grafts provide more stable fixation of the stem than fresh-frozen morselized grafts, when tested in a hip simulator.

In the technique of impaction bone grafting, implant stability depends on the mechanical properties of the impacted morselized grafts. Although the procedure is usually performed with fresh-frozen femoral heads, there is still some concern about their supply and safety. Bone processing is a potential solution, but the mechanical properties of this material during and after impaction need to be determined. We used 6 osteoarthrotic femoral heads to prepare two paired batches of morselized bone. One batch was morselized and frozen. The other batch was chemically treated, morselized, freeze-dried and then gamma-irradiated. We impacted 18 samples from each batch in a contained cylinder. Freeze-dried bone grafts were tested after 30 minutes of rehydration. The changes in the compactness and stiffness of the material were monitored during the impaction. The compaction of the freeze-dried bone was faster than that of their fresh-frozen control. The maximal stiffness reached by both materials was the same (55 MPa), but the freeze-dried grafts required three to four times fewer impactions to achieve that stiffness. After 3, 10 and 50 impactions the freeze-dried bone was stiffer than the fresh-frozen bone. As it is easier to impact, the freeze-dried bone may be mechanically more efficient than the fresh-frozen bone in surgical conditions. Moreover, the processed bone meets the highest safety standards, as regards the risk of disease transmission.

The mechanical properties of the impacted material is important when using impaction bone grafting in revision arthroplasty. We did an in vitro study to monitor the stiffness and compactness of morsellized bone grafts during impaction. Using human osteoarthrotic femoral heads as the bone source we prepared 3 types of morselized grafts with the same bone mill: (1) purely cancellous grafts, (2) cortico-cancellous grafts and (3) cortico-cancellous bone with cartilage remnants. 5 g of bone samples were progressively impacted up to 150 times in a contained cavity. All types of grafts became stiffer and stiffer during the first 30 impactions. After 30 impactions, the grafts' compactness continued to increase slowly without concomitant changes in their stiffness. Over-impaction was not useful, but did not jeopardize the implant stability. The presence of cartilage remnants considerably compromised the increase in stiffness. In contrast, the addition of cortical bone from the femoral neck did not affect the impaction and resultant stiffness.

In order to minimize the risk of contamination associated with tissue transplantation, tissue banks commonly chemically treat the tissues whenever possible. As viral inactivation uses agents lethal to microorganisms, it is imperative to assure that chemically inactivated tissue remains biocompatible. In vitro assays can be an effective means to assess the acute cytotoxicity of chemically treated human allografts. We have used different types of cells cultured in the presence of treated tissue extract. A standard cell line, a human fibroblast (WI38), which was the same for all the samples, was chosen. In addition, as the banked tissues (bone and fascia lata) were prepared to be used in bone or as a dura mater substitute, two other cell types were also used: an osteoblastic cell line (SaOS-2) and a neuronal cell line (Neuro 2A). Cytotoxic assessment was performed by qualitative evaluation of cell morphology based on confluence, granulation, vacuolization and swelling analysis. In addition, quantitative methods based on the use of neutral red (NR) and 3- (4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) were assayed. Qualitative and quantitative evaluation of fascia lata and bone extracts did not show deleterious effects on cell cultures. These results show that in vitro methods can be appropriate to select a non-toxic procedure before it is used in the human body and that several strong chemical treatments can result in a tissue suitable for human.

We compared the reliability of computer-assisted radiographic analysis (CARA) and visual evaluation of radiographs to assess host-graft junctions. 68 host bone/allograft junctions were obtained from an ongoing study on bone allografting in sheep. At 6 months, the grafted tibias were explanted and healing of the host-graft junctions were macroscopically determined. 49 junctions were macroscopically healed, whereas 19 had not united. 51 (0.8) of the junctions were correctly classified by radiographs, while 63 (0.9) of the junctions were correctly classified by CARA (p = 0.03). These findings warrant further evaluation in a clinical setting.

We examined the influence of ethylene oxide (EO) and gamma irradiation on the osteoinductive capacity of demineralized bone. Demineralized bone powder prepared from Wistar rats was exposed to EO (55 degrees C or 40 degrees C) or gamma irradiation (25 KGy) or was preserved in ethanol. Sterilely-prepared bones served as controls. The powder was packed in a gelatin capsule and implanted for 6 weeks in muscles of 6-week-old female rats. Exposure of demineralized bone particles to EO 55 degrees C resulted in an almost complete loss of osteoinductivity. Irradiated bones lost about 40% of their osteoinductive capacity, while sterilization with EO at 40 degrees C resulted in only a slight alteration of the osteoinductivity, as assessed by the recovered weight ratio, calcium content, alkaline phosphatase activity measurements and histomorphometry. Ethanol treatment had no influence on the new bone yield when compared to controls. As EO exposure at 40 degrees C is a true sterilization procedure, it can be recommended in a clinical setting for its small effect on osteoinductive capacity as assessed experimentally in rats.

Plaster of Paris implants containing vancomycin (60 mg/g of carrier) were prepared in order to be used as local delivery system for the treatment of bone infections. The regulation of the release rate was performed by coating the carrier with a polylactide-co-glycolide polymer composed by 10% (w/w) polyglycolic acid and 90% (w/w) racemic poly (D,L-lactic acid). The release of the antibiotic from the biodegradable matrix was evaluated in vitro. From this investigation, it is clear that the drug elution depends on the coating depth. After a burst effect occurring on the first day of the experiment, therapeutic concentrations were measured during one week when uncoated implants were used. The coating allowed decrease of the burst effect and extended efficient release to more than five weeks when the implants were embedded with six layers (162 microns) of PLA45GA10. This delivery system was implanted into the femoral condyle of rabbits. It was shown that the in vivo release was also closely regulated by the coating depth. In all bone tissues (bone marrow and cortical bone) surrounding the pellets, the drug concentration exceeded the Minimum Inhibitory Concentration for the common causative organisms of bone infections (Staphylococcus aureus) for at least four weeks without inducing serum toxic levels. Due to its cheapness, facility of use and sterilization, biocompatibility and biodegradability, plaster of Paris coated with PLA45GA10 polymer giving a controlled release of vancomycin appears to be a promising sustained release delivery system of antibiotics for the treatment of bone and joint infections.

The aim of this study was to find a safe, effective sterilization method that does not destroy the bone-inductive capacity of demineralized bone implants. Five sterilizing agents were tested in rats. Implants procured and processed under sterile conditions served as controls. New bone formation was evaluated by determining dry weight, calcium content, and Sr-85 incorporation of the induced ossicles. Glutaraldehyde solution, formaldehyde gas, and ethylene oxide destroyed almost all the bone-inductive capacity. Irradiation by 2.5 Mrads Co-60 resulted in a loss of about half of the inductive capacity. Merthiolate (0.18 per cent) was the only sterilizing agent that did not reduce the bone-inductive capacity of the demineralized implants. Because merthiolate is not sporicidal, gamma irradiation appears to be the most appropriate sterilizing agent for demineralized bone in clinical use.

Three procedures to obtain bone inductive implants were tested heterotopically in 3-month-old allogeneic rats: 1) antigen-extracted HCl-decalcified at 4 degrees C, autolysed implant (AAA bone); 2) HCl-decalcified implant at 4 degrees C; 3) HCl-decalcified implant at room temperature. Each type of implant was either deep-frozen at -35 degrees C for at least 2 months or immediately freeze-dried. The bone inductive capacity of the differently HCl-decalcified cortical bone implant was evaluated at 2 months by isotopic strontium incorporation and by ash-weight measurements. Bone HCl-decalcification alone, either at 4 degrees C or at room temperature, gave a higher new bone yield than the freeze-dried AAA bone. The type or short-term preservation technique had no effect on the osteoinductive capacity of either of the differently treated implants, AAA bone expected.