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The Bone & Joint Journal
Vol. 95-B, Issue 9 | Pages 1269 - 1274
1 Sep 2013
Uppal HS Peterson BE Misfeldt ML Della Rocca GJ Volgas DA Murtha YM Stannard JP Choma TJ Crist BD

We hypothesised that cells obtained via a Reamer–Irrigator–Aspirator (RIA) system retain substantial osteogenic potential and are at least equivalent to graft harvested from the iliac crest. Graft was harvested using the RIA in 25 patients (mean age 37.6 years (18 to 68)) and from the iliac crest in 21 patients (mean age 44.6 years (24 to 78)), after which ≥ 1 g of bony particulate graft material was processed from each. Initial cell viability was assessed using Trypan blue exclusion, and initial fluorescence-activated cell sorting (FACS) analysis for cell lineage was performed. After culturing the cells, repeat FACS analysis for cell lineage was performed and enzyme-linked immunosorbent assay (ELISA) for osteocalcin, and Alizarin red staining to determine osteogenic potential. Cells obtained via RIA or from the iliac crest were viable and matured into mesenchymal stem cells, as shown by staining for the specific mesenchymal antigens CD90 and CD105. For samples from both RIA and the iliac crest there was a statistically significant increase in bone production (both p < 0.001), as demonstrated by osteocalcin production after induction. . Medullary autograft cells harvested using RIA are viable and osteogenic. Cell viability and osteogenic potential were similar between bone grafts obtained from both the RIA system and the iliac crest. Cite this article: Bone Joint J 2013;95-B:1269–74


Bone & Joint Research
Vol. 5, Issue 4 | Pages 106 - 115
1 Apr 2016
Gruber HE Ode G Hoelscher G Ingram J Bethea S Bosse MJ

Objectives. The biomembrane (induced membrane) formed around polymethylmethacrylate (PMMA) spacers has value in clinical applications for bone defect reconstruction. Few studies have evaluated its cellular, molecular or stem cell features. Our objective was to characterise induced membrane morphology, molecular features and osteogenic stem cell characteristics. Methods. Following Institutional Review Board approval, biomembrane specimens were obtained from 12 patient surgeries for management of segmental bony defects (mean patient age 40.7 years, standard deviation 14.4). Biomembranes from nine tibias and three femurs were processed for morphologic, molecular or stem cell analyses. Gene expression was determined using the Affymetrix GeneChip Operating Software (GCOS). Molecular analyses compared biomembrane gene expression patterns with a mineralising osteoblast culture, and gene expression in specimens with longer spacer duration (> 12 weeks) with specimens with shorter durations. Statistical analyses used the unpaired student t-test (two tailed; p < 0.05 was considered significant). Results. Average PMMA spacer in vivo time was 11.9 weeks (six to 18). Trabecular bone was present in 33.3% of the biomembrane specimens; bone presence did not correlate with spacer duration. Biomembrane morphology showed high vascularity and collagen content and positive staining for the key bone forming regulators, bone morphogenetic protein 2 (BMP2) and runt-related transcription factor 2 (RUNX2). Positive differentiation of cultured biomembrane cells for osteogenesis was found in cells from patients with PMMA present for six to 17 weeks. Stem cell differentiation showed greater variability in pluripotency for osteogenic potential (70.0%) compared with chondrogenic or adipogenic potentials (100% and 90.0%, respectively). Significant upregulation of BMP2 and 6, numerous collagens, and bone gla protein was present in biomembrane compared with the cultured cell line. Biomembranes with longer resident PMMA spacer duration (vs those with shorter residence) showed significant upregulation of bone-related, stem cell, and vascular-related genes. Conclusion. The biomembrane technique is gaining favour in the management of complicated bone defects. Novel data on biological mechanisms provide improved understanding of the biomembrane’s osteogenic potential and molecular properties. Cite this article: Dr H. E. Gruber. Osteogenic, stem cell and molecular characterisation of the human induced membrane from extremity bone defects. Bone Joint Res 2016;5:106–115. DOI: 10.1302/2046-3758.54.2000483


Bone & Joint Research
Vol. 2, Issue 6 | Pages 112 - 115
1 Jun 2013
Ismail HD Phedy P Kholinne E Kusnadi Y Sandhow L Merlina M

Objectives. Nonunion is one of the most troublesome complications to treat in orthopaedics. Former authors believed that atrophic nonunion occurred as a result of lack of mesenchymal stem cells (MSCs). We evaluated the number and viability of MSCs in site of atrophic nonunion compared with those in iliac crest. Methods. We enrolled five patients with neglected atrophic nonunions of long bones confirmed by clinical examinations and plain radiographs into this study. As much as 10 ml bone marrow aspirate was obtained from both the nonunion site and the iliac crest and cultured for three weeks. Cell numbers were counted using a haemocytometer and vitality of the cells was determined by trypan blue staining. The cells were confirmed as MSCs by evaluating their expression marker (CD 105, CD 73, HLA-DR, CD 34, CD 45, CD 14, and CD 19). Cells number and viability were compared between the nonunion and iliac creat sites. Results. After three weeks, numbers of 6.08×10. 6. cells (. sd. 2.07) and 4.98×10. 6. cells (. sd. 1.15) were obtained from the nonunion site and the iliac crest, respectively, with viability of 87.1% (81.7% to 90.8%) and 89.8% (84.7% to 94.5%), respectively. No differences was found between the two sources of MSCs regarding cells number (p = 0.347) and viability (p = 0.175). Conclusions. Our findings showed the existence of MSCs in the site of atrophic nonunion, at a similar number and viability to those isolated from the iliac crest


The Bone & Joint Journal
Vol. 100-B, Issue 7 | Pages 966 - 972
1 Jul 2018
Morgenstern M Athanasou NA Ferguson JY Metsemakers W Atkins BL McNally MA

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.


The Bone & Joint Journal
Vol. 97-B, Issue 9 | Pages 1279 - 1283
1 Sep 2015
Mahale YJ Aga N

In this retrospective observational cohort study, we describe 17 patients out of 1775 treated for various fractures who developed mycobacterium tuberculosis (MTB) infection after surgery. The cohort comprised 15 men and two women with a mean age of 40 years (24 to 70). A total of ten fractures were open and seven were closed. Of these, seven patients underwent intramedullary nailing of a fracture of the long bone, seven had fractures fixed with plates, two with Kirschner-wires and screws, and one had a hemiarthroplasty of the hip with an Austin Moore prosthesis. All patients were followed-up for two years. In all patients, the infection resolved, and in 14 the fractures united. Nonunion was seen in two patients one of whom underwent two-stage total hip arthroplasty (THA) and the other patient was treated using excision arthoplasty. Another patient was treated using two-stage THA. With only sporadic case reports in the literature, MTB infection is rarely clinically suspected, even in underdeveloped and developing countries, where pulmonary and other forms of TB are endemic. In developed countries there is also an increased incidence among immunocompromised patients. In this paper we discuss the pathogenesis and incidence of MTB infection after surgical management of fractures and suggest protocols for early diagnosis and management.

Cite this article: Bone Joint J 2015;97-B:1279–83.


The Bone & Joint Journal
Vol. 97-B, Issue 4 | Pages 539 - 543
1 Apr 2015
Lawendy A Bihari A Sanders DW McGarr G Badhwar A Cepinskas G

Compartment syndrome, a devastating consequence of limb trauma, is characterised by severe tissue injury and microvascular perfusion deficits. We hypothesised that leucopenia might provide significant protection against microvascular dysfunction and preserve tissue viability. Using our clinically relevant rat model of compartment syndrome, microvascular perfusion and tissue injury were directly visualised by intravital video microscopy in leucopenic animals. We found that while the tissue perfusion was similar in both groups (38.8% (standard error of the mean (sem) 7.1), 36.4% (sem 5.7), 32.0% (sem 1.7), and 30.5% (sem 5.35) continuously-perfused capillaries at 45, 90, 120 and 180 minutes compartment syndrome, respectively versus 39.2% (sem 8.6), 43.5% (sem 8.5), 36.6% (sem 1.4) and 50.8% (sem 4.8) at 45, 90, 120 and 180 minutes compartment syndrome, respectively in leucopenia), compartment syndrome-associated muscle injury was significantly decreased in leucopenic animals (7.0% (sem 2.0), 7.0%, (sem 1.0), 9.0% (sem 1.0) and 5.0% (sem 2.0) at 45, 90, 120 and 180 minutes of compartment syndrome, respectively in leucopenia group versus 18.0% (sem 4.0), 23.0% (sem 4.0), 32.0% (sem 7.0), and 20.0% (sem 5.0) at 45, 90, 120 and 180 minutes of compartment syndrome in control, p = 0.0005). This study demonstrates that the inflammatory process should be considered central to the understanding of the pathogenesis of cellular injury in compartment syndrome.

Cite this article: Bone Joint J 2015;97-B:539–43


The Bone & Joint Journal
Vol. 98-B, Issue 8 | Pages 1132 - 1137
1 Aug 2016
Lawendy A Bihari A Sanders DW Badhwar A Cepinskas G

Aims

Compartment syndrome results from increased intra-compartmental pressure (ICP) causing local tissue ischaemia and cell death, but the systemic effects are not well described. We hypothesised that compartment syndrome would have a profound effect not only on the affected limb, but also on remote organs.

Methods

Using a rat model of compartment syndrome, its systemic effects on the viability of hepatocytes and on inflammation and circulation were directly visualised using intravital video microscopy.


The Bone & Joint Journal
Vol. 98-B, Issue 8 | Pages 1126 - 1131
1 Aug 2016
Shiels SM Cobb RR Bedigrew KM Ritter G Kirk JF Kimbler A Finger Baker I Wenke JC

Aims

Demineralised bone matrix (DBM) is rarely used for the local delivery of prophylactic antibiotics. Our aim, in this study, was to show that a graft with a bioactive glass and DBM combination, which is currently available for clinical use, can be loaded with tobramycin and release levels of antibiotic greater than the minimum inhibitory concentration for Staphylococcus aureus without interfering with the bone healing properties of the graft, thus protecting the graft and surrounding tissues from infection.

Materials and Methods

Antibiotic was loaded into a graft and subsequently evaluated for drug elution kinetics and the inhibition of bacterial growth. A rat femoral condylar plug model was used to determine the effect of the graft, loaded with antibiotic, on bone healing.


The Bone & Joint Journal
Vol. 97-B, Issue 8 | Pages 1144 - 1151
1 Aug 2015
Waki T Lee SY Niikura T Iwakura T Dogaki Y Okumachi E Kuroda R Kurosaka M

MicroRNAs (miRNAs ) are small non-coding RNAs that regulate gene expression. We hypothesised that the functions of certain miRNAs and changes to their patterns of expression may be crucial in the pathogenesis of nonunion. Healing fractures and atrophic nonunions produced by periosteal cauterisation were created in the femora of 94 rats, with 1:1 group allocation. At post-fracture days three, seven, ten, 14, 21 and 28, miRNAs were extracted from the newly generated tissue at the fracture site. Microarray and real-time polymerase chain reaction (PCR) analyses of day 14 samples revealed that five miRNAs, miR-31a-3p, miR-31a-5p, miR-146a-5p, miR-146b-5p and miR-223-3p, were highly upregulated in nonunion. Real-time PCR analysis further revealed that, in nonunion, the expression levels of all five of these miRNAs peaked on day 14 and declined thereafter.

Our results suggest that miR-31a-3p, miR-31a-5p, miR-146a-5p, miR-146b-5p and miR-223-3p may play an important role in the development of nonunion. These findings add to the understanding of the molecular mechanism for nonunion formation and may lead to the development of novel therapeutic strategies for its treatment.

Cite this article: Bone Joint J 2015; 97-B:1144–51.


The Journal of Bone & Joint Surgery British Volume
Vol. 93-B, Issue 4 | Pages 517 - 524
1 Apr 2011
Cox G McGonagle D Boxall SA Buckley CT Jones E Giannoudis PV

The scarcity of mesenchymal stem cells (MSCs) in iliac crest bone marrow aspirate (ICBMA), and the expense and time in culturing cells, has led to the search for alternative harvest sites. The reamer-irrigation-aspirator (RIA) provides continuous irrigation and suction during reaming of long bones. The aspirated contents pass via a filter, trapping bony fragments, before moving into a ‘waste’ bag from which MSCs have been previously isolated. We examined the liquid and solid phases, performed a novel digestion of the solid phase, and made a comparative assessment in terms of number, phenotype and differentiation capacity with matched ICBMA.

The solid fraction from the filtrate was digested for 60 minutes at 37°C with collagenase. Enumeration was performed via the colony-forming unit fibroblast (CFU-F) assay. Passage (P2) cells were differentiated towards osteogenic, adipogenic and chondrogenic lineages, and their phenotypes assessed using flow cytometry (CD33, CD34, CD45, CD73, CD90, and CD105).

MSCs from the RIA phases were able to differentiate at least as well as those from ICBMA, and all fractions had phenotypes consistent with other established sources. The median number of colonies for the three groups was: ICBMA = 8.5 (2 to 86), RIA-liquid = 19.5 (4 to 90), RIA-solid = 109 (67 to 200) per 200 μl. The mean total yield of cells for the three groups was: ICBMA = 920 (0 to 4275), RIA-liquid = 114 983 (16 500 to 477 750), RIA-solid = 12 785 (7210 to 28 475).

The RIA filtrate contains large numbers of MSCs that could potentially be extracted without enzymatic digestion and used for bone repair without prior cell expansion.


Bone & Joint Research
Vol. 1, Issue 11 | Pages 289 - 296
1 Nov 2012
Savaridas T Wallace RJ Muir AY Salter DM Simpson AHRW

Objectives

Small animal models of fracture repair primarily investigate indirect fracture healing via external callus formation. We present the first described rat model of direct fracture healing.

Methods

A rat tibial osteotomy was created and fixed with compression plating similar to that used in patients. The procedure was evaluated in 15 cadaver rats and then in vivo in ten Sprague-Dawley rats. Controls had osteotomies stabilised with a uniaxial external fixator that used the same surgical approach and relied on the same number and diameter of screw holes in bone.


The Bone & Joint Journal
Vol. 95-B, Issue 6 | Pages 820 - 824
1 Jun 2013
Zsoldos CM Basamania CJ Bal GK

Gunshot injuries to the shoulder are rare and difficult to manage. We present a case series of seven patients who sustained a severe shoulder injury to the non-dominant side as a result of a self-inflicted gunshot wound. We describe the injury as ‘suicide shoulder’ caused by upward and outward movement of the gun barrel as the trigger is pulled. All patients were male, with a mean age of 32 years (21 to 48). All were treated at the time of injury with initial repeated debridement, and within four weeks either by hemiarthroplasty (four patients) or arthrodesis (three patients). The hemiarthroplasty failed in one patient after 20 years due to infection and an arthrodesis was attempted, which also failed due to infection. Overall follow-up was for a mean of 26 months (12 to 44). All four hemiarthroplasty implants were removed with no feasible reconstruction ultimately possible, resulting in a poor functional outcome and no return to work. In contrast, all three primary arthrodeses eventually united, with two patients requiring revision plating and grafting. These patients returned to work with a good functional outcome. We recommend arthrodesis rather than replacement as the treatment of choice for this challenging injury.

Cite this article: Bone Joint J 2013;95-B:820–4.


The Journal of Bone & Joint Surgery British Volume
Vol. 93-B, Issue 4 | Pages 510 - 516
1 Apr 2011
Sugata Y Sotome S Yuasa M Hirano M Shinomiya K Okawa A

Several bisphosphonates are now available for the treatment of osteoporosis. Porous hydroxyapatite/collagen (HA/Col) composite is an osteoconductive bone substitute which is resorbed by osteoclasts. The effects of the bisphosphonate alendronate on the formation of bone in porous HA/Col and its resorption by osteoclasts were evaluated using a rabbit model. Porous HA/Col cylinders measuring 6 mm in diameter and 8 mm in length, with a pore size of 100 μm to 500 μm and 95% porosity, were inserted into a defect produced in the lateral femoral condyles of 72 rabbits. The rabbits were divided into four groups based on the protocol of alendronate administration: the control group did not receive any alendronate, the pre group had alendronate treatment for three weeks prior to the implantation of the HA/Col, the post group had alendronate treatment following implantation until euthanasia, and the pre+post group had continuous alendronate treatment from three weeks prior to surgery until euthanasia. All rabbits were injected intravenously with either saline or alendronate (7.5 μg/kg) once a week. Each group had 18 rabbits, six in each group being killed at three, six and 12 weeks post-operatively. Alendronate administration suppressed the resorption of the implants. Additionally, the mineral densities of newly formed bone in the alendronate-treated groups were lower than those in the control group at 12 weeks post-operatively. Interestingly, the number of osteoclasts attached to the implant correlated with the extent of bone formation at three weeks.

In conclusion, the systemic administration of alendronate in our rabbit model at a dose-for-weight equivalent to the clinical dose used in the treatment of osteoporosis in Japan affected the mineral density and remodelling of bone tissue in implanted porous HA/Col composites.


The Bone & Joint Journal
Vol. 95-B, Issue 9 | Pages 1263 - 1268
1 Sep 2013
Savaridas T Wallace RJ Salter DM Simpson AHRW

Fracture repair occurs by two broad mechanisms: direct healing, and indirect healing with callus formation. The effects of bisphosphonates on fracture repair have been assessed only in models of indirect fracture healing.

A rodent model of rigid compression plate fixation of a standardised tibial osteotomy was used. Ten skeletally mature Sprague–Dawley rats received daily subcutaneous injections of 1 µg/kg ibandronate (IBAN) and ten control rats received saline (control). Three weeks later a tibial osteotomy was rigidly fixed with compression plating. Six weeks later the animals were killed. Fracture repair was assessed with mechanical testing, radiographs and histology.

The mean stress at failure in a four-point bending test was significantly lower in the IBAN group compared with controls (8.69 Nmm-2 (sd 7.63) vs 24.65 Nmm-2 (sd 6.15); p = 0.017). On contact radiographs of the extricated tibiae the mean bone density assessment at the osteotomy site was lower in the IBAN group than in controls (3.7 mmAl (sd 0.75) vs 4.6 mmAl (sd 0.57); p = 0.01). In addition, histological analysis revealed progression to fracture union in the controls but impaired fracture healing in the IBAN group, with predominantly cartilage-like and undifferentiated mesenchymal tissue (p = 0.007).

Bisphosphonate treatment in a therapeutic dose, as used for risk reduction in fragility fractures, had an inhibitory effect on direct fracture healing. We propose that bisphosphonate therapy not be commenced until after the fracture has united if the fracture has been rigidly fixed and is undergoing direct osteonal healing.

Cite this article: Bone Joint J 2013;95-B:1263–8.


Bone & Joint Research
Vol. 2, Issue 2 | Pages 26 - 32
1 Feb 2013
Neumann H Schulz AP Gille J Klinger M Jürgens C Reimers N Kienast B

Objectives

Osteochondral injuries, if not treated adequately, often lead to severe osteoarthritis. Possible treatment options include refixation of the fragment or replacement therapies such as Pridie drilling, microfracture or osteochondral grafts, all of which have certain disadvantages. Only refixation of the fragment can produce a smooth and resilient joint surface. The aim of this study was the evaluation of an ultrasound-activated bioresorbable pin for the refixation of osteochondral fragments under physiological conditions.

Methods

In 16 Merino sheep, specific osteochondral fragments of the medial femoral condyle were produced and refixed with one of conventional bioresorbable pins, titanium screws or ultrasound-activated pins. Macro- and microscopic scoring was undertaken after three months.


The Journal of Bone & Joint Surgery British Volume
Vol. 88-B, Issue 6 | Pages 783 - 789
1 Jun 2006
Sen C Eralp L Gunes T Erdem M Ozden VE Kocaoglu M

In this retrospective study we evaluated the method of acute shortening and distraction osteogenesis for the treatment of tibial nonunion with bone loss in 17 patients with a mean age of 36 years (10 to 58). The mean bone loss was 5.6 cm (3 to 10). In infected cases, we performed the treatment in two stages. The mean follow-up time was 43.5 months (24 to 96). The mean time in external fixation was 8.0 months (4 to 13) and the mean external fixator index was 1.4 months/cm (1.1 to 1.8). There was no recurrence of infection. The bone evaluation results were excellent in 16 patients and good in one, while functional results were excellent in 15 and good in two. The complication rate was 1.2 per patient.

We conclude that acute shortening and distraction osteogenesis is a safe, reliable and successful method for the treatment of tibial nonunion with bone loss, with a shorter period of treatment and lower rate of complication.


The Journal of Bone & Joint Surgery British Volume
Vol. 88-B, Issue 2 | Pages 232 - 237
1 Feb 2006
Saridis A Panagiotopoulos E Tyllianakis M Matzaroglou C Vandoros N Lambiris E

We reviewed 13 patients with infected nonunion of the distal femur and bone loss, who had been treated by radical surgical debridement and the application of an Ilizarov external fixator. All had severely restricted movement of the knee and a mean of 3.1 previous operations. The mean length of the bony defect was 8.3 cm and no patient was able to bear weight.

The mean external fixation time was 309.8 days. According to Paley’s grading system, eight patients had an excellent clinical and radiological result and seven excellent and good functional results. Bony union, the ability to bear weight fully, and resolution of the infection were achieved in all the patients. The external fixation time was increased when the definitive treatment started six months or more after the initial trauma, the patient had been subjected to more than four previous operations and the initial operation had been open reduction and internal fixation.