Scaffold-free Three-dimensional Graft From Autologous Adipose-derived Stem Cells for Large Bone Defect Reconstruction: Clinical Proof of Concept.

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.

Is conservative treatment still defensible in grade III acromioclavicular dislocation? Are there predictive factors of poor outcome?

The optimal treatment of grade III acromioclavicular (AC) dislocation is still controversial. Recent studies recommend surgery at that stage whereas meta-analysis favours conservative management. The objective of the present investigation was to analyse a clinical series of non-operated grade III AC dislocations and to determine their functional status. Thirty-five patients treated conservatively with a grade III acromioclavicular dislocation were retrospectively reviewed. Simple shoulder test, Oxford shoulder and bilateral Constant shoulder score were used for assessment. Various predictive criteria of poor outcome, particularly scapular dyskinesis were taken into account for analysis. Overall mean and median Constant Score of the injured side were 92.9 and 94, whilst the contralateral shoulder values were respectively 94.9 and 95 (mean and median scores). Ten patients had scapular dyskinesis. Laterality, shoulder activity and scapular dyskinesis were not statistically related to worse outcome. Twenty-eight (80%) patients resumed normal activity within six months. All but two patients were subjectively very satisfied or satisfied. Conservative treatment provided satisfactory results whatever the shoulder activity. No risk factors were predictive of a poorer outcome. Conservative management should remain the first option to manage these injuries.

Local complications of massive bone allografts: an appraisal of their prevalence in 128 patients.

Bone allografts were used in our department since twenty-five years to reconstruct segmental bone loss and our data were retrospectively reviewed to assess the complications related to the use of a bone allograft. A consecutive series of 128 patients who received a bone allograft was analyzed. The minimal follow-up was 18 months. Fracture, nonunion, infection and explantation were investigated using a multivariate analysis and logistical regression. Kaplan-Meier survival of the allograft was performed, using allograft removal as the end point. Tumour disease was excluded from this study. Patients were followed up for an average 103 months. Bone tumour occurred in 78% of the patients whereas revision arthroplasty was the cause of implantation in 15% of them. Nonunion was the most prevalent complication, occurring in 35% of the grafts. For nonunion occurrence, the type of reconstruction was found to be a significant variable, the intercalary allograft being the most exposed. Primary bone autografting at the anastomotic site was not significant to prevent nonunion. Fracture of the allograft was the second most frequent complication with a prevalence of 16.4%. The length of the allograft and an osteoarticular allograft were two significant variables in that occurrence. Infection of the allograft was present with a rate of 5.4% of patients. Explantation of failed allografts occurred in 30% of them. The duration of the frozen storage of the allograft and the donor age of the allograft were not significant on any local complication occurrence. Bone allografts are a reliable material but a high rate of local complications must be anticipated.

Beware of the commercialization of human cells and tissues: situation in the European Union.

With this analysis we would like to raise some issues that emerge as a result of recent evolutions in the burgeoning field of human cells, tissues, and cellular and tissue-based product (HCT/P) transplantation, and this in the light of the current EU regulatory framework. This paper is intended as an open letter addressed to the EU policy makers, who will be charged with the review and revision of the current legislation. We propose some urgent corrections or additions to cope with the rapid advances in biomedical science, an extensive commercialization of HCT/Ps, and the growing expectation of the general public regarding the ethical use of altruistically donated cells and tissues. Without a sound wake-up call, the diverging interests of this newly established 'healthcare' industry and the wellbeing of humanity will likely lead to totally unacceptable situations, like some of which we are reporting here.

Analysis of predisposing factors for contamination of bone and tendon allografts.

Bone and tissue allografts are widely used in transplantation. The increasing demand for safe allografts must be met, while minimizing disease transmission. We analysed the incidence and potential risk factors of allograft contamination and the effectiveness of disinfection, by reviewing 22 years of tissue bank activity and 474 donor procurements. We also compared different disinfection procedures used over the 22 years. The overall contamination rate was 10.1%. Risk factors were related to the donor or procurement method. Immediate culture at the tissue recovery site diminished the rate of false positives by reducing later sample manipulation. High-virulence allograft contamination was mainly related to donor factors, while low-virulence contamination was related to procurement methods. Analysis of donor-related risk factors showed no statistical differences for age, sex, or cause of death. An intensive care unit stay was associated with less contamination with high-virulence microbes. Procurement in a setting other than an operating theatre was associated with higher contamination rate. Team experience reduced contamination. Pelvic and tendon allografts were most frequently contaminated. Proper disinfection considerably reduced the contamination rate to 3.6%. We conclude that procurement must be performed under aseptic conditions, with short delays, and by trained personnel. Grafts should be disinfected and packed as soon as possible.

The enhanced performance of bone allografts using osteogenic-differentiated adipose-derived mesenchymal stem cells.

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.

Synergetic effect of freeze-drying and gamma irradiation on the mechanical properties of human cancellous bone.

Freeze-drying and irradiation are common process used by tissue banks to preserve and sterilize bone allografts. Freeze dried irradiated bone is known to be more brittle. Whether bone brittleness is due to irradiation alone, temperature during irradiation or to a synergetic effect of the freeze-drying-irradiation process was not yet assessed. Using a left-right femoral head symmetry model, 822 compression tests were performed to assess the influence of sequences of a 25 kGy irradiation with and without freeze-drying compared to the unprocessed counterpart. Irradiation of frozen bone did not cause any significant reduction in ultimate strength, stiffness and work to failure. The addition of the freeze-drying process before or after irradiation resulted in a mean drop of 35 and 31% in ultimate strength, 14 and 37% in stiffness and 46 and 37% in work to failure. Unlike irradiation at room temperature, irradiation under dry ice of solvent-detergent treated bone seemed to have no detrimental effect on mechanical properties of cancellous bone. Freeze-drying bone without irradiation had no influence on mechanical parameters, but the addition of irradiation to the freeze-drying step or the reverse sequence showed a detrimental effect and supports the idea of a negative synergetic effect of both procedures. These findings may have important implications for bone banking.

Computer-Navigated Bone Cutting in the Resection of a Pelvic Bone Tumor and Reconstruction with a Massive Bone Allograft.

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.

Computer-assisted resection and reconstruction of pelvic tumor sarcoma.

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.

Formalin fixation could interfere with the clinical assessment of the tumor-free margin in tumor surgery: magnetic resonance imaging-based study.

OBJECTIVES: The tumor-free margin in bone and soft-tissue cancer is a key factor for subsequent treatment. While flattening and shrinkage of specimens after formalin fixation have been described in breast cancer, there are no data for bone and soft tissue sarcoma. Fixation could interfere with the accuracy of the assessment of the tumor-free margin. METHODS: The influence of formalin fixation was assessed on forelimb specimens in a preclinical porcine model. The specimens were subjected to magnetic resonance imaging before and after formalin fixation. Weight, width and height of the specimen were measured and different consecutive volumes (total, muscles, bones and fatty tissue) were obtained by segmentation. RESULTS: After formalin fixation, the weight increased, total volume and muscle volume slightly increased while bone did not change and fatty tissue decreased. The width of the specimens increased while their height decreased. CONCLUSIONS: Formalin fixation caused slight muscle expansion, fatty tissue shrinkage and flattening of the specimen. These changes could interfere with the assessment of the tumor-free margin in clinical practice.

Selection of massive bone allografts using shape-matching 3-dimensional registration.

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.

Histology can be predictive of the clinical course of a primary aneurysmal bone cyst.

INTRODUCTION: Aneurysmal bone cyst is a benign lesion occurring in young patients which frequently recurs after treatment. Biopsy is mandatory for the diagnosis of a putative aneurysmal bone cyst as this lesion can be secondary to another underlying process including a malignant bone tumour. The histopathological features of aneurysmal bone cysts have been examined with the goal of finding relevant criteria for predicting favourable evolution or recurrence of the disease. PATIENTS AND METHODS: Twenty-one biopsies of surgically treated aneurysmal bone cysts, from 21 patients, were analysed. Histomorphometry by two different methods (3,000- and 200-point-counting) and by two observers was performed to quantify the percentage of each tissue type in the cyst (cellular, fibrillar, osteoid). A healing index was developed by calculating a ratio of osteoid and fibrillar material divided by cellular tissue. Biopsies were also examined using two immunostains, cluster of differentiation 68 (CD68) and proliferating cell nuclear antigen (PCNA). RESULTS: The final outcome was healing for 16 aneurysmal bone cysts (healing group) and recurrence for the five others (recurrence group), after a mean follow-up of 4.43 years. The two groups differed significantly in the proportion of their cellular content and their healing index. The ratio of CD68 negative to CD68 positive cells was also significantly different between the two groups. CONCLUSION: Biopsy should be considered as a helpful prognostic factor for aneurysmal bone cyst.

Particle size influence in an impaction bone grafting model. Comparison of fresh-frozen and freeze-dried allografts.

BACKGROUND: Impaction bone grafting with large particles is considered as mechanically superior to smaller morsels. Interest of freeze-dried irradiated bone for impaction bone grafting has been observed with small particles. Influence of bone process on other particle sizes still needed to be assessed. MATERIAL AND METHODS: Twenty-four osteoarthrotic femoral heads were used to prepare fresh-frozen and freeze-dried irradiated cancellous bone. Each group was divided into four batches of different particle sizes, each batch containing 18 samples. The different particle sizes were obtained with a Retsch Cross Beater Mill SK 100, Noviomagus rotating bone mills with two sizes of rasps and a Luer bone rongeur. Bone grafts were impacted in a contained cylinder. Stiffness was monitored during impaction. RESULTS: Freeze-dried irradiated grafts showed higher stiffness than fresh-frozen bone whatever the size of the particles. Large particles obtained with the rongeur and the large rasp from the Noviomagus bone mill were mechanically superior than small particles up to 30 impactions. INTERPRETATION: Large particles offer better mechanical performance as a greater magnitude of force would be required to deform and break the particles. Freeze-dried irradiated bone brittleness reduces this advantage after 30 impactions. Large particles embrittlement leads to similar mechanical results as small particles at higher impaction rate. This may account for partial collapse of the graft layer in clinical situation when impaction rate is lower. This model supports the use of small particles obtained with thin rasp bone mill when freeze-dried irradiated bone for impaction bone grafting and large particles obtained with the Rongeur when fresh-frozen bone is available.

Surgical inaccuracy of tumor resection and reconstruction within the pelvis: an experimental study.

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.

An analysis of critical care staff's attitudes to donation in a country with presumed-consent legislation.

CONTEXT: Lack of knowledge and confidence among critical care staff in identifying potential donors and communicating with donor families may explain missed organ and tissue donations. OBJECTIVES: To elucidate attitudes of critical care staff toward donation and their knowledge, involvement, and self-reported skills and confidence levels with donation-related tasks. METHODS: Between January 2004 and May 2006, Donor Action Foundation's Hospital Attitude Survey was used to collect data from staff members in critical care units in our university hospital (study group) and 2 other Belgian university hospitals (control group). In total, 92 physicians, 433 nurses, and 26 other staff members participated. RESULTS: The 2 groups did not differ significantly with regard to donation-related attitudes. The study group had a significantly lower perception of the public's approval of organ donation than did the control group (P < .001). Nurses reported significantly less involvement than did physicians (P < .001) in caring for potential donors, communicating severe brain damage, explaining brain death, requesting donation, and contacting procurement staff. Probably because of previous training, physicians in the study group reported less need for additional training on donation-related issues than did control physicians. However, compared with physicians, nurses in the study group requested significantly more training on a number of donation tasks. Nurses with more than 5 years of critical care experience were consistently more confident with donation-related tasks. CONCLUSION: Support rates for donation are high overall, and previous training is associated with improved attitudes and decreased educational needs. Educational efforts tailor-made for nurses should increase nurses' confidence levels when confronted with potential donors and their next of kin.

Current limitations to the histopathological diagnosis of some frequently encountered bone tumours.

The final diagnosis of a bone tumour comes in many cases like the last piece of a puzzle which requires integration of clinical, imaging and pathological data. However there are situations in which a discrepancy exists between histology and imaging studies and where histology alone cannot be decisive. This paper reviews such situations.

Inaccuracy in selection of massive bone allograft using template comparison method.

The use of massive bone allografts is increasing year by year and selection method remains unchanged. Superposition of patient's radiograph over allograft image and comparison of distances is the gold standard. Experiment was led to test selection procedure of a major european tissue bank. Four observers were asked to select an allograft for 10 fictive recipients. Nine allografts were provided. To simulate a perfect allograft, recipient himself was inserted in the pool of allografts (trap graft). The 10 potential bone transplants were classified in four categories (from adequate to unacceptable). In addition, observers were asked to choose the three best grafts for a given recipient. Quadratic kappa measuring agreement on classification between two observers ranged between 0.74 (substantial) and 0.47 (moderate). Trap graft was quoted by observers as adequate four times (10%) and was cited eight times (20%) among the three best matching allografts. None of the observers discovered that recipient was among allograft panel. This study demonstrates that current selection method is inaccurate for hemipelvic allograft selection. New methods should be developed and tested to assist tissue banks in bone allograft selection.

The use of allografts in paediatric orthopaedic surgery.

Autograft harvesting in a growing child sometimes leads to disastrous consequences. Allograft can advantageously replace autograft in the majority of the cases. This overview presents the most frequently used allografts in paediatric orthopaedic surgery and discusses their benefits. Illustrative cases are presented to highlight specific indications.

Post-traumatic bone loss of the femur treated with segmental bone allograft and bone morphogenetic protein: a case report.

Reconstruction of a major bone loss remains a challenge for the orthopaedic surgeon. Most of the bone defects result from a bone tumour resection whereas a post-traumatic bone loss is more rare due to the numerous options available for bone fixation. However in high-energy trauma, the injury to bone may be so extensive as to justify removal of fragmented bone. A 57-year-old man presented with a severe injury at the thigh after a hunting accident, including a comminuted fracture of the femoral shaft. After thorough debridement, he was left with a large diaphyseal bone defect which was subsequently treated with a structural bone allograft, autogenous graft and rhBMP-7. Bone healing was achieved after several months.