Management of critical-sized bone defects in the treatment of fracture-related infection: a systematic review and pooled analysis.

PURPOSE: This systematic review determined the reported treatment strategies, their individual success rates, and other outcome parameters in the management of critical-sized bone defects in fracture-related infection (FRI) patients between 1990 and 2018. METHODS: A systematic literature search on treatment and outcome of critical-sized bone defects in FRI was performed. Treatment strategies identified were, autologous cancellous grafts, autologous cancellous grafts combined with local antibiotics, the induced membrane technique, vascularized grafts, Ilizarov bone transport, and bone transport combined with local antibiotics. Outcomes were bone healing and infection eradication after primary surgical protocol and recurrence of FRI and amputations at the end of study period. RESULTS: Fifty studies were included, describing 1530 patients, the tibia was affected in 82%. Mean age was 40 years (range 6-80), with predominantly male subjects (79%). Mean duration of infection was 17 months (range 1-624) and mean follow-up 51 months (range 6-126). After initial protocolized treatment, FRI was cured in 83% (95% CI 79-87) of all cases, increasing to 94% (95% CI 92-96) at the end of each individual study. Recurrence of infection was seen in 8% (95% CI 6-11) and amputation in 3% (95% CI 2-3). Final outcomes overlapped across treatment strategies. CONCLUSION: Results should be interpreted with caution due to the retrospective and observational design of most studies, the lack of clear classification systems, incomplete data reports, potential underreporting of adverse outcomes, and heterogeneity in patient series. A consensus on classification, treatment protocols, and outcome is needed to improve reliability of future studies.

Insights into treatment and outcome of fracture-related infection: a systematic literature review.

INTRODUCTION: Standardized guidelines for treatment of fracture-related infection (FRI) are lacking. Worldwide many treatment protocols are used with variable success rates. Awareness on the need of standardized, evidence-based guidelines has increased in recent years. This systematic literature review gives an overview of available diagnostic criteria, classifications, treatment protocols, and related outcome measurements for surgically treated FRI patients. METHODS: A comprehensive search was performed in all scientific literature since 1990. Studies in English that described surgical patient series for treatment of FRI were included. Data were collected on diagnostic criteria for FRI, classifications used, surgical treatments, follow-up protocols, and overall outcome. A systematic review was performed according to the PRISMA statement. Proportions and weighted means were calculated. RESULTS: The search yielded 2051 studies. Ninety-three studies were suitable for inclusion, describing 3701 patients (3711 fractures) with complex FRI. The population consisted predominantly of male patients (77%), with the tibia being the most commonly affected bone (64%), and a mean of three previous operations per patient. Forty-three (46%) studies described FRI at one specific location. Only one study (1%) used a standardized definition for infection. A total of nine different classifications were used to guide treatment protocols, of which Cierny and Mader was used most often (36%). Eighteen (19%) studies used a one-stage, 50 (54%) a two-stage, and seven (8%) a three-stage surgical treatment protocol. Ten studies (11%) used mixed protocols. Antibiotic protocols varied widely between studies. A multidisciplinary approach was mentioned in only 12 (13%) studies. CONCLUSIONS: This extensive literature review shows a lack of standardized guidelines with respect to diagnosis and treatment of FRI, which mimics the situation for prosthetic joint infection identified many years ago. Internationally accepted guidelines are urgently required to improve the quality of care for patients suffering from this significant complication.

Giant-cell-rich pseudotumour in Paget's disease.

Paget's disease (PD) of the bone is a disorder of bone remodelling that may be polyostotic or monostotic. Although development of a sarcoma in PD is well-recognised, it is less well recognised that pseudosarcomas in bone and soft tissue can also arise in this condition. In this report we document the case of a large giant-cell-rich pseudotumour that developed in the tibia and overlying soft tissues in a case of polyostotic PD. Bone and soft tissues were highly vascular and contained abundant haemorrhage with focal areas of new bone formation and a diffuse infiltrate of osteoclastic giant cells. The lesion has not recurred or produced metastases 3 years after removal. Clinicians should be aware that a benign giant-cell-rich pseudotumour can develop in PD and that it needs to be distinguished from other giant-cell-rich tumours.

Providing an Evidence Base for Tissue Sampling and Culture Interpretation in Suspected Fracture-Related Infection.

BACKGROUND: The recent consensus definition for the diagnosis of fracture-related infection (FRI) includes the identification of indistinguishable microorganisms in at least 2 surgical deep-tissue specimens as a confirmatory criterion. However, this cut-off, and the total number of specimens from a patient with suspected FRI that should be sent for microbiological testing, have not been validated. We endeavored to estimate the accuracy of different numbers of specimens and diagnostic cut-offs for microbiological testing of deep-tissue specimens in patients undergoing surgical treatment for possible FRI. METHODS: A total of 513 surgical procedures in 385 patients with suspected FRI were included. A minimum of 2 surgical deep-tissue specimens were submitted for microbiological testing; 5 or more specimens were analyzed in 345 procedures (67%). FRI was defined by the presence of any confirmatory criteria other than microbiology. Resampling was utilized to model the sensitivity and specificity of diagnostic cut-offs for the number of surgical specimens yielding indistinguishable microorganisms and for the total number of specimens. The likelihood of detecting all clinically relevant microorganisms was also assessed. RESULTS: A diagnostic cut-off of at least 2 of 5 specimens with indistinguishable microorganisms identified by culture was 68% sensitive (95% confidence interval [CI], 62% to 74%) and 87% specific (95% CI, 81% to 94%) for the diagnosis of FRI. Two out of 3 specimens were 60% sensitive (95% CI, 55% to 66%) and 92% specific (95% CI, 88% to 96%). Submitting only 3 deep-tissue specimens risked missing clinically relevant microorganisms in at least 1 in 10 cases. CONCLUSIONS: The present study was the first to validate microbiological criteria for the diagnosis of FRI, supporting the current confirmatory diagnostic criteria for FRI. Analysis of at least 5 deep-tissue specimens in patients with possible FRI is recommended. LEVEL OF EVIDENCE: Diagnostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Performance of automated multiplex polymerase chain reaction (mPCR) using synovial fluid in the diagnosis of native joint septic arthritis in adults.

AIMS: This study aimed to assess the performance of an automated multiplex polymerase chain reaction (mPCR) technique for rapid diagnosis of native joint septic arthritis. PATIENTS AND METHODS: Consecutive patients with suspected septic arthritis undergoing aseptic diagnostic joint aspiration were included. The aspirate was used for analysis by mPCR and conventional microbiological analysis. A joint was classed as septic according to modified Newman criteria. Based on receiver operating characteristic (ROC) analysis, the area under the ROC curve (AUC) values of the mPCR and the synovial fluid culture were compared using the z-test. A total of 72 out of 76 consecutive patients (33 women, 39 men; mean age 64 years (22 to 92)) with suspected septic arthritis were included in this study. RESULTS: Of 72 patients, 42 (58%) were deemed to have septic joints. The sensitivity of mPCR and synovial fluid culture was 38% and 29%, respectively. No significant differences were found between the AUCs of both techniques (p = 0.138). A strong concordance of 89% (Cohen's kappa: 0.65) was shown. The mPCR failed to detect Staphylococcus aureus (n = 1) and Streptococcus pneumoniae (n = 1; no primer included in the mPCR), whereas the synovial fluid culture missed six microorganisms (positive mPCR: S. aureus (n = 2), Cutibacterium acnes (n = 3), coagulase-negative staphylococci (n = 2)). CONCLUSION: The automated mPCR showed at least a similar performance to the synovial fluid culture (the current benchmark) in diagnosing septic arthritis, having the great advantage of a shorter turnaround time (within five hours). Cite this article: Bone Joint J 2019;101-B:288-296.

Getting it right first time: The importance of a structured tissue sampling protocol for diagnosing fracture-related infections.

INTRODUCTION: Fracture-related infection (FRI) is an important complication following surgical fracture management. Key to successful treatment is an accurate diagnosis. To this end, microbiological identification remains the gold standard. Although a structured approach towards sampling specimens for microbiology seems logical, there is no consensus on a culture protocol for FRI. The aim of this study is to evaluate the effect of a structured microbiology sampling protocol for fracture-related infections compared to ad-hoc culture sampling. METHODS: We conducted a pre-/post-implementation cohort study that compared the effects of implementation of a structured FRI sampling protocol. The protocol included strict criteria for sampling and interpretation of tissue cultures for microbiology. All intraoperative samples from suspected or confirmed FRI were compared for culture results. Adherence to the protocol was described for the post-implementation cohort. RESULTS: In total 101 patients were included, 49 pre-implementation and 52 post-implementation. From these patients 175 intraoperative culture sets were obtained, 96 and 79 pre- and post-implementation respectively. Cultures from the pre-implementation cohort showed significantly more antibiotic use during culture sampling (P =  0.002). The post-implementation cohort showed a tendency more positive culture sets (69% vs. 63%), with a significant difference in open wounds (86% vs. 67%, P =  0.034). In all post-implementation culture sets causative pathogens were cultured more than once per set, in contrast to pre-implementation. Despite stricter tissue sampling and culture interpretation criteria, the number of polymicrobial infections was similar in both cohorts, approximately 29% of all culture sets and 44% of all positive culture sets. Significantly more polymicrobial cultures were found in early infections in the post-implementation cohort (P =  0.048). This indicates a better yield in the new protocol. CONCLUSION: A standardised protocol for intraoperative sampling for bacterial identification in FRI is superior than an ad-hoc approach. It has a positive effect on both surgeon and microbiologist by increasing awareness about the problem at hand. This resulted in more microbiologically confirmed infections and more certainty when identifying causative pathogens.

Biliary events and an increased risk of new onset irritable bowel syndrome: a population-based cohort study.

BACKGROUND: Prospective data are lacking to determine if irritable bowel syndrome (IBS) is a risk factor for cholecystectomy, or if biliary disease and cholecystectomy predisposes to the development of IBS. AIM: To test the hypothesis that IBS and biliary tract disease are associated. METHODS: Validated symptom surveys sent to cohorts of Olmsted County, MN, (1988-1994) with follow-up in 2003. Medical histories were reviewed to determine any 'biliary events' (defined by gallstones or cholecystectomy). Analyses examined were: (i) time to a biliary event post-initial survey and separately and (ii) risk of IBS (Rome II) in those with vs. without a prior biliary event. RESULTS: A total of 1908 eligible subjects were mailed a follow-up survey. For analysis (i) of the 726 without IBS at initial survey, 44 (6.1%) had biliary events during follow up, in contrast to 5 of 93 (5.4%) with IBS at initial survey (HR 0.8, 95% CI 0.3-2.1). For analysis (ii) of the 59 subjects with a biliary event at initial survey, 10 (17%) reported new IBS on the follow-up survey, while in 682 without a biliary event up to 1.5 years prior to the second survey, 58 (8.5%) reported IBS on follow-up (OR = 2.2, 95% CI 1.1-4.6, P = 0.03). CONCLUSION: There is an increased risk of new IBS in community subjects who have been diagnosed as having a biliary event.

Histopathology in Periprosthetic Joint Infection: When Will the Morphomolecular Diagnosis Be a Reality?

The presence of a polymorphonuclear neutrophil infiltrate in periprosthetic tissues has been shown to correlate closely with the diagnosis of septic implant failure. The histological criterion considered by the Musculoskeletal Infection Society to be diagnostic of periprosthetic joint infection is "greater than five neutrophils per high-power field in five high-power fields observed from histologic analysis of periprosthetic tissue at ×400 magnification." Surgeons and pathologists should be aware of the qualifications introduced by different authors during the last years in the histological techniques, samples for histological study, cutoffs used for the diagnosis of infection, and types of patients studied. Recently, immunohistochemistry and histochemistry studies have appeared which suggest that the cutoff point of five polymorphonuclear neutrophils in five high-power fields is too high for the diagnosis of many periprosthetic joint infections. Therefore, morphomolecular techniques could help in the future to achieve a more reliable histological diagnosis of periprosthetic joint infection.

The value of quantitative histology in the diagnosis of fracture-related infection.

Aims: This study aimed to investigate the role of quantitative histological analysis in the diagnosis of fracture-related infection (FRI). Patients and Methods: The clinical features, microbiology culture results, and histological analysis in 156 surgically treated nonunions were used to stratify the likelihood of associated infection. There were 64 confirmed infected nonunions (one or more confirmatory criteria: pus, sinus, and bacterial growth in two or more samples), 66 aseptic nonunions (no confirmatory criteria), and 26 possibly infected nonunions (pathogen identified from a single specimen and no confirmatory criteria). The histological inflammatory response was assessed by average neutrophil polymorph (NPs) counts per high-power field (HPF) and compared with the established diagnosis. Results: Assuming a cut-off of over five neutrophils per high-power field to diagnose septic nonunion, there was 80% sensitivity and 100% specificity (accuracy 90%). Using a cut-off of no neutrophils seen in any high-power field to diagnose aseptic nonunion, there was a sensitivity of 85% and a specificity of 98% (accuracy 92%). Conclusion: Histology can be used in a bimodal fashion as a diagnostic test for FRI. The presence of more than five NPs/HPF had a positive predictive value for infected nonunion of 100%, while the complete absence of any NPs is almost always indicative of an aseptic nonunion (positive predictive value of 98%). Cite this article: Bone Joint J 2018;100-B:966-72.

The effect of local antibiotic prophylaxis when treating open limb fractures: A systematic review and meta-analysis.

OBJECTIVES: As well as debridement and irrigation, soft-tissue coverage, and osseous stabilization, systemic antibiotic prophylaxis is considered the benchmark in the management of open fractures and considerably reduces the risk of subsequent fracture-related infections (FRI). The direct application of antibiotics in the surgical field (local antibiotics) has been used for decades as additional prophylaxis in open fractures, although definitive evidence confirming a beneficial effect is scarce. The purpose of the present study was to review the clinical evidence regarding the effect of prophylactic application of local antibiotics in open limb fractures. METHODS: A comprehensive literature search was performed in PubMed, Web of Science, and Embase. Cohort studies investigating the effect of additional local antibiotic prophylaxis compared with systemic prophylaxis alone in the management of open fractures were included and the data were pooled in a meta-analysis. RESULTS: In total, eight studies which included 2738 patients were eligible for quantitative synthesis. The effect of antibiotic-loaded poly(methyl methacrylate) beads was investigated by six of these studies, and two studies evaluated the effect of local antibiotics applied without a carrier. Meta-analysis showed a significantly lower infection rate when local antibiotics were applied (4.6%; 91/1986) than in the control group receiving standard systemic prophylaxis alone (16.5%; 124/752) (p < 0.001) (odds ratio 0.30; 95% confidence interval 0.22 to 0.40). CONCLUSION: This meta-analysis suggests a risk reduction in FRI of 11.9% if additional local antibiotics are given prophylactically for open limb fractures. However, due to limited quality, heterogeneity, and considerable risk of bias, the pooling of data from primary studies has to be interpreted with caution.Cite this article: M. Morgenstern, A. Vallejo, M. A. McNally, T. F. Moriarty, J. Y. Ferguson, S. Nijs, WJ. Metsemakers. Bone Joint Res 2018;7:447-456. The effect of local antibiotic prophylaxis when treating open limb fractures: A systematic review and meta-analysis. DOI: 10.1302/2046-3758.77.BJR-2018-0043.R1.

International survey among orthopaedic trauma surgeons: Lack of a definition of fracture-related infection.

INTRODUCTION: Fracture-related infection (FRI) is one of the most challenging musculoskeletal complications in orthopaedic-trauma surgery. Although the orthopaedic community has developed and adopted a consensus definition of prosthetic joint infections (PJI), it still remains unclear how the trauma surgery community defines FRI in daily clinical practice or in performing clinical research studies. The central aim of this study was to survey the opinions of a global network of trauma surgeons on the definitions and criteria they routinely use, and their opinion on the need for a unified definition of FRI. The secondary aims were to survey their opinion on the utility of currently used definitions that may be at least partially applicable for FRI, and finally their opinion on the important clinical parameters that should be considered as diagnostic criteria for FRI. METHODS: An 11-item questionnaire was developed to cover the above-mentioned aims. The questionnaire was administered by SurveyMonkey and was sent via blast email to all registered users of AO Trauma (Davos, Switzerland). RESULTS: Out of the 26'563 recipients who opened the email, 2'327 (8.8%) completed the questionnaire. Nearly 90% of respondents agreed that a consensus-derived definition for FRI is required and 66% of the surgeons also agreed that PJI and FRI are not equal with respect to diagnosis, treatment and outcome. Furthermore, "positive cultures from microbiology testing", "elevation of CRP", "purulent drainage" and "local clinical signs of infection" were voted the most important diagnostic parameters for FRI. CONCLUSION: This international survey infers the need for a consensus definition of FRI and provides insight into the clinical parameters seen by an international community of trauma surgeons as being critical for defining FRI.

Diagnostic accuracy of serum inflammatory markers in late fracture-related infection: a systematic review and meta-analysis.

AIMS: To assess the diagnostic value of C-reactive protein (CRP), leucocyte count (LC), and erythrocyte sedimentation rate (ESR) in late fracture-related infection (FRI). MATERIALS AND METHODS: PubMed, Embase, and Cochrane databases were searched focusing on the diagnostic value of CRP, LC, and ESR in late FRI. Sensitivity and specificity combinations were extracted for each marker. Average estimates were obtained using bivariate mixed effects models. RESULTS: A total of 8284 articles were identified but only six were suitable for inclusion. Sensitivity of CRP ranged from 60.0% to 100.0% and specificity from 34.3% to 85.7% in all publications considered. Five articles were pooled for meta-analysis, showing a sensitivity and specificity of 77.0% and 67.9%, respectively. For LC, this was 22.9% to 72.6%, and 73.5% to 85.7%, respectively, in five articles. Four articles were pooled for meta-analysis, resulting in a 51.7% sensitivity and 67.1% specificity. For ESR, sensitivity and specificity ranged from 37.1% to 100.0% and 59.0% to 85.0%, respectively, in five articles. Three articles were pooled in meta-analysis, showing a 45.1% sensitivity and 79.3% specificity. Four articles analyzed the value of combined inflammatory markers, reporting an increased diagnostic accuracy. These results could not be pooled due to heterogeneity. CONCLUSION: The serum inflammatory markers CRP, LC, and ESR are insufficiently accurate to diagnose late FRI, but they may be used as a suggestive sign in its diagnosis.

Fracture-related infection: A consensus on definition from an international expert group.

Fracture-related infection (FRI) is a common and serious complication in trauma surgery. Accurately estimating the impact of this complication has been hampered by the lack of a clear definition. The absence of a working definition of FRI renders existing studies difficult to evaluate or compare. In order to address this issue, an expert group comprised of a number of scientific and medical organizations has been convened, with the support of the AO Foundation, in order to develop a consensus definition. The process that led to this proposed definition started with a systematic literature review, which revealed that the majority of randomized controlled trials in fracture care do not use a standardized definition of FRI. In response to this conclusion, an international survey on the need for and key components of a definition of FRI was distributed amongst all registered AOTrauma users. Approximately 90% of the more than 2000 surgeons who responded suggested that a definition of FRI is required. As a final step, a consensus meeting was held with an expert panel. The outcome of this process led to a consensus definition of FRI. Two levels of certainty around diagnostic features were defined. Criteria could be confirmatory (infection definitely present) or suggestive. Four confirmatory criteria were defined: Fistula, sinus or wound breakdown; Purulent drainage from the wound or presence of pus during surgery; Phenotypically indistinguishable pathogens identified by culture from at least two separate deep tissue/implant specimens; Presence of microorganisms in deep tissue taken during an operative intervention, as confirmed by histopathological examination. Furthermore, a list of suggestive criteria was defined. These require further investigations in order to look for confirmatory criteria. In the current paper, an overview is provided of the proposed definition and a rationale for each component and decision. The intention of establishing this definition of FRI was to offer clinicians the opportunity to standardize clinical reports and improve the quality of published literature. It is important to note that the proposed definition was not designed to guide treatment of FRI and should be validated by prospective data collection in the future.

Efficient and sustained gene expression in primary T lymphocytes and primary and cultured tumor cells mediated by adeno-associated virus plasmid DNA complexed to cationic liposomes.

We have used cationic liposomes to facilitate adeno-associated virus (AAV) plasmid transfections of primary and cultured cell types. AAV plasmid DNA complexed with liposomes showed levels of expression several fold higher than those of complexes with standard plasmids. In addition, long-term expression (> 30 days) of the gene, unlike the transient expression demonstrated by typical liposome-mediated transfection with standard plasmids, was observed. Southern analysis of chromosomal DNA further substantiated the hypothesis that the long-term expression was due to the presence of the transgene in the AAV plasmid-transfected group and not in the standard plasmid-transfected group. AAV plasmid-liposome complexes induced levels of transgene expression comparable to those obtained by recombinant AAV transduction. Primary breast, ovarian, and lung tumor cells were transfectable with the AAV plasmid DNA-liposome complexes. Transfected primary and cultured tumor cells were able to express transgene product even after lethal irradiation. High-level gene expression was also observed in freshly isolated CD3+, CD4+, and CD8+ T cells from normal human peripheral blood. Transfection efficiency ranged from 10 to 50% as assessed by intracellular interleukin-2 levels in interleukin-2-transfected cells. The ability to express transgenes in primary tumor and lymphoid cells may be applied toward tumor vaccine studies and protocols which may eventually permit highly specific modulation of the cellular immune response in cancer and AIDS.

Chronic sternal osteomyelitis complicating primary manubriosternal septic arthritis.

We present a 40-year-old woman complaining of worsening chest pain. She was previously treated with a prolonged course of antibiotics for suspected sternal osteomyelitis. Radiological investigations were suggestive of ongoing inflammation within the manubriosternal joint. Formal surgical debridement yielded evidence corroborating the diagnosis although microbiological samples were negative. Prolonged empirical treatment with intravenous antibiotics leads to a complete resolution of symptoms. Primary culture-negative manubriosternal septic arthritis is rare, and suboptimal treatment, particularly if confused with other conditions such as synovitis, acne, pustulosis, hyperostosis and osteitis (SAPHO) syndrome, compounds its considerable morbidity.

Single-stage treatment of chronic osteomyelitis with a new absorbable, gentamicin-loaded, calcium sulphate/hydroxyapatite biocomposite: a prospective series of 100 cases.

AIMS: Chronic osteomyelitis may recur if dead space management, after excision of infected bone, is inadequate. This study describes the results of a strategy for the management of deep bone infection and evaluates a new antibiotic-loaded biocomposite in the eradication of infection from bone defects. PATIENTS AND METHODS: We report a prospective study of 100 patients with chronic osteomyelitis, in 105 bones. Osteomyelitis followed injury or surgery in 81 patients. Nine had concomitant septic arthritis. 80 patients had comorbidities (Cierny-Mader (C-M) Class B hosts). Ten had infected nonunions. All patients were treated by a multidisciplinary team with a single-stage protocol including debridement, multiple sampling, culture-specific systemic antibiotics, stabilisation, dead space filling with the biocomposite and primary skin closure. RESULTS: Patients were followed up for a mean of 19.5 months (12 to 34). Infection was eradicated in 96 patients with a single procedure and all four recurrences were successfully managed with repeat surgery. Adverse events were uncommon, with three fractures, six wound leaks and three unrelated deaths. Outcome was not dependant on C-M host class, microbial culture, wound leakage or presence of nonunion. CONCLUSION: This single-stage protocol, facilitated by the absorbable local antibiotic, is effective in the treatment of chronic osteomyelitis. It offers a more patient-friendly treatment compared with other published treatment options. Cite this article: Bone Joint J 2016;98-B:1289-96.

Pharmacokinetics of gentamicin eluted from a regenerating bone graft substitute: In vitro and clinical release studies.

OBJECTIVES: Deep bone and joint infections (DBJI) are directly intertwined with health, demographic change towards an elderly population, and wellbeing.The elderly human population is more prone to acquire infections, and the consequences such as pain, reduced quality of life, morbidity, absence from work and premature retirement due to disability place significant burdens on already strained healthcare systems and societal budgets.DBJIs are less responsive to systemic antibiotics because of poor vascular perfusion in necrotic bone, large bone defects and persistent biofilm-based infection. Emerging bacterial resistance poses a major threat and new innovative treatment modalities are urgently needed to curb its current trajectory. MATERIALS AND METHODS: We present a new biphasic ceramic bone substitute consisting of hydroxyapatite and calcium sulphate for local antibiotic delivery in combination with bone regeneration. Gentamicin release was measured in four setups: 1) in vitro elution in Ringer's solution; 2) local elution in patients treated for trochanteric hip fractures or uncemented hip revisions; 3) local elution in patients treated with a bone tumour resection; and 4) local elution in patients treated surgically for chronic corticomedullary osteomyelitis. RESULTS: The release pattern in vitro was comparable with the obtained release in the patient studies. No recurrence was detected in the osteomyelitis group at latest follow-up (minimum 1.5 years). CONCLUSIONS: This new biphasic bone substitute containing antibiotics provides safe prevention of bone infections in a range of clinical situations. The in vitro test method predicts the in vivo performance and makes it a reliable tool in the development of future antibiotic-eluting bone-regenerating materials.Cite this article: M. Stravinskas, P. Horstmann, J. Ferguson, W. Hettwer, M. Nilsson, S. Tarasevicius, M. M. Petersen, M. A. McNally, L. Lidgren. Pharmacokinetics of gentamicin eluted from a regenerating bone graft substitute: In vitro and clinical release studies. Bone Joint Res 2016;5:427-435. DOI: 10.1302/2046-3758.59.BJR-2016-0108.R1.

Long-term reconstitution of mice after ex vivo expansion of bone marrow cells: differential activity of cultured bone marrow and enriched stem cell populations.

In this report, we evaluated the short-term expansion of murine bone marrow mononuclear cells (BMMNC) and enriched stem cell populations to determine the capacity of these cells for long-term rescue and engraftment to lethally irradiated recipients. In our study, nonadherent bone marrow mononuclear cell (NBM-MNC) and Thy1+Lin- stem cells populations were cultured with interleukin-3 (IL-3) or IL-3 plus stem cell factor (SCF) for periods up to 6 days. By day 6 of culture, the mononuclear cells (MNC) decreased to 6% of input cell number, whereas Thy1+Lin- cells increased by 2310%. Doses of 95,000; 100,000; 50,000; and 250,000 NBM-MNCs at 0, 1, 2, and 6 days of culture, respectively, rescued 50% of lethally irradiated mice. When 250,000 MNCs were cultured for 0, 1, 2, and 6 days, 71, 61, 100, and 50% of the animals survived lethal irradiation for greater than 24 weeks. In contrast, doses of 8,000 and 21,000 Thy1+Lin- cells cultured 0 and 1 day, respectively, yielded 50% survival rates. These same cells cultured for 6 days failed to rescue recipients even at high doses. Twenty thousand Thy1+Lin- cells cultured for 0, 1, 2, and 6 days, even in the presence of SCF, produced decreasing survival rates of 86, 43, 26, and 0%, respectively. The proliferative responses of these different populations in combination with their long-term rescue abilities indicated that the absolute number of long-term rescue units (LD50, 24 weeks) in the cultured Thy1+Lin- population decreased faster than in similarly cultured NBM-MNCs. Studies evaluating donor cell engraftment demonstrated that animals rescued with cultured Thy1+Lin- and NBM-MNCs maintained high levels of donor reconstitution [7]. The percent donor T cell engraftment did not significantly change between 2 and 17 months post-bone marrow transplantation (post-BMT). Therefore, those animals who received sufficient cells to survive lethal irradiation generally established and maintained high levels of donor engraftment. The data suggest a role for accessory cells and/or factors in the preservation of stem cell activity.

Enrichment of murine hematopoietic stem cells. Reconstitution of syngeneic and haplotype-mismatched mice.

A method is described to purify murine hematopoietic stem cells. The procedure involves fluorescence-activated cell sorting of nonadherent nucleated bone marrow cells for the presence of the antigen, Thy1.2, and the absence of the lineage-specific antigens, Lyt2, L3T4, Mac1, B220, and J11D.2. These Thy1.2+T-B-M-J- cells are 200-800-fold enriched in in vitro colony-forming units. Moreover, this narrow subset shows enhanced ability to form spleen colonies and engraft lethally irradiated mice. Data reported herein demonstrate that these purified pluripotent stem cell populations also have enhanced potential for the rescue of lethally irradiated haplotype-mismatched mice.

Optimizing electroporation parameters for a variety of human hematopoietic cell lines.

The parameters affecting electroporation of four human hematopoietic cell lines were investigated. The optimal conditions for electroporation are described for both transient and stable expression of foreign genes. A correlation exists between the levels of transient gene expression and stable transfection frequency. In addition, linear DNA yields higher stable transfection frequencies than supercoiled DNA. The cumulative results indicate that electroporation is a simple and useful method for obtaining transient and stable expression of foreign genes in human hematopoietic cells.