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Orthopaedic Proceedings
Vol. 95-B, Issue SUPP_10 | Pages 17 - 17
1 Feb 2013
Smith I Milto K Doherty C Amyes S Simpson A Hall A
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Staphylococcus aureus is a highly virulent pathogen and is implicated in approximately 50% of cases of septic arthritis. Studies investigating other S. aureus-related infections have suggested that alpha (Hla), beta (Hlb) and gamma (Hlg) toxins are key virulence factors. In particular, the ‘pore-forming’ alpha toxin is believed to be most potent. In this study, we have assessed the influence of alpha toxin on in situ chondrocyte viability. Osteochondral explants were harvested from the metacarpophalangeal joints of 3-year-old cows and placed into flasks containing Dulbecco's Modified Eagle's Medium. The flasks were then inoculated with the following isogenic ‘knockout’ strains of S. aureus: DU5946 (Hla+Hlb-Hlg-) or DU1090 (Hla-Hlb+Hlg+). The explants were incubated (37°C) and stained after 18, 24 and 40hrs with chloromethylfluorescein di-acetate and propidium iodide, labelling living chondrocytes green and dead cells red, respectively. Axial sections were imaged by confocal microscopy and the percentage cell death obtained using Volocity 4 software. The alpha toxin-producing S. aureus caused rapid cell death, with 24.8+/−3.7% at 18hrs and 44.6+/−7.2% at 24hrs. At 40hrs, there was significantly more chondrocyte death (87.4+/−3.6%; p<0.001) compared to the alpha toxin knockout strain (4.1+/−1.7%; means +/− SEM; n=4). In situ chondrocyte viability was significantly compromised by alpha toxin, with beta and gamma toxins having minimal effect. Further work will clarify the exact mechanism through which this important toxin induces chondrocyte death. Thereafter, it is hoped that targeted treatments can be developed to reduce the extent of cartilage destruction during, and after, an episode of septic arthritis


Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_XXXVII | Pages 571 - 571
1 Sep 2012
Nawaz S Bentley G Briggs T Carrington R Skinner J Gallager K Dhinsa B
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Chondral injuries of the knee are extremely common and present a unique therapeutic challenge due to the poor intrinsic healing of articular cartilage. These injuries can lead to significant functional impairment. There are several treatment modalities for articular osteochondral defects, one of which is autologous chondrocyte implantation. Our study evaluates the mid to long term functional outcomes in a cohort of 828 patients who have undergone an autologous chondrocyte implantation procedure (either ACI or MACI), identifying retrospectively factors that may influence their outcome.

The influence of factors including age, sex, presence of osteoarthritis and size and site of lesion have been assessed individually and with multivariate analysis. All patients were assessed using the Bentley Functional Score, Visual Analogue Score and the Cincinnati Functional Score. Assessment were performed pre-operatively and of their status in 2010. The majority of patients had several interim scores performed at varying intervals.

The longest follow-up was 12 years (range 24 to 153 months) with a mean age of 34 years at time of procedure. The mean defect size was 486 mm2 (range 64 to 2075 mm2). The distribution of lesions was 51% Medial Femoral Condyle, 12.5% Lateral Femoral Condyle, 18% Patella (single facet), 5% Patella (Multifacet) and 6% Trochlea. 4% had cartilage transplant to multiple sites. 30% failed following this procedure at a mean time of 72 months. 52% patients stated a marked improvement in their functional outcomes within the first two years. 49% stated an excellent result following their procedure.

High failure rate was noted in those with previous cartilage regenerative procedures, transplants occurring on the patella, particularly if involving multifacets. Multiple site cartilage transplantation was also associated with a high failure rate.

Autologous chondrocyte implantation is an effective method of decreasing pain and increasing function, however patient selection plays clear role in the success of such procedure.


Orthopaedic Proceedings
Vol. 104-B, Issue SUPP_6 | Pages 6 - 6
1 Jun 2022
Turnbull G Shu W Picard F Clarke J
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As arthroplasty demand grows worldwide, the need for a novel cost-effective treatment option for articular cartilage (AC) defects tailored to individual patients has never been greater. 3D bioprinting can deposit patient cells and other biomaterials in user-defined patterns to build tissue constructs from the “bottom-up,” potentially offering a new treatment for AC defects. Novel composite bioinks were created by mixing different ratios of methacrylated alginate (AlgMA) with methacrylated gelatin (GelMA) and collagen. Chondrocytes and mesenchymal stem cells (MSCs) were then encapsulated in the bioinks and 3D bioprinted using a custom-built extrusion bioprinter. UV and double-ionic (BaCl2 and CaCl2) crosslinking was deployed following bioprinting to strengthen bioink stability in culture. Chondrocyte and MSC spheroids were also bioprinted to accelerate cell growth and development of ECM in bioprinted constructs. Excellent viability of chondrocytes and MSCs was seen following bioprinting (>95%) and maintained in culture, with accelerated cell growth seen with inclusion of cell spheroids in bioinks (p<0.05). Bioprinted 10mm diameter constructs maintained shape in culture over 28 days, whilst construct degradation rates and mechanical properties were improved with addition of AlgMA (p<0.05). Composite bioinks were also injected into in vitro osteochondral defects and crosslinked in situ, with maintained cell viability and repair of osteochondral defects seen over a 14-day period. In conclusion, we developed novel composite bioinks that can be triple-crosslinked, facilitating successful chondrocyte and MSC growth in 3D bioprinted scaffolds and in vitro repair of an osteochondral defect model. This offers hope for a new approach to treating AC defects


Orthopaedic Proceedings
Vol. 99-B, Issue SUPP_18 | Pages 10 - 10
1 Nov 2017
Roberts S Borjesson A Sophocleous A Salter D Ralston S
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The arcOGEN study identified the 9q33.1 locus as associated with hip osteoarthritis (OA) in females. TRIM32 lies within this locus and may have biological relevance to OA; it encodes a protein with E3 ubiquitin ligase activity. Sanger sequencing of TRIM32 in the youngest 500 female patients with hip OA from the arcOGEN study identified genetic polymorphisms in the proximal promoter, and 3'untranslated region of TRIM32 that are disproportionately represented in female patients with hip OA compared to the control population. Reduced expression of TRIM32 was identified in femoral head articular chondrocytes from patients with hip OA compared to control patients. Trim32 knockout resulted in increased aggrecanolysis in murine femoral head explants. Murine chondrocytes deficient in Trim32 exhibited increased expression of mature chondrocyte markers following anabolic cytokine stimulation, and increased expression of hypertrophic chondrocyte markers following catabolic cytokine stimulation. Trim32 knockout mice demonstrated increased cartilage degradation and tibial subchondral bone changes after surgically-induced knee joint instability. Increased cartilage degradation and medial knee subchondral bone changes were also identified in aged Trim32 knockout mice. These results further implicate TRIM32 in the genetic predisposition to OA, and indicate a role for TRIM32 in the joint degeneration evident in OA. These results support the further study of TRIM32 in the pathophysiology of OA and development of novel therapeutic strategies to manage OA


Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_XXXIII | Pages 11 - 11
1 Jul 2012
Smith I Cyrulik K Amyes S Simpson A Hall A
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In some centres, serial bedside aspirations, in association with intravenous antibiotics, are still an accepted treatment for septic arthritis (Mathews, Postgraduate Medical Journal, 2008). However, there is a risk that bacterial products remain in the joint, even when the bacteria have been destroyed. We have conducted a study to ascertain whether bacterial products alone have an effect on in situ chondrocyte viability. A hip aspirate (25μl), containing Staphylococcus aureus, from a patient with septic arthritis was added to 5ml culture medium and incubated (37°C) for 48hrs. The solution was then centrifuged (3400g for 10mins) and the supernatant removed. Cartilage explants were harvested from a bovine metacarpophalangeal joint, placed into the bacterial supernatant and incubated at 37°C. Explants were removed at hourly intervals over a 6-hour period and stained with the fluorescent probes chloromethylfluorescein di-acetate (10μM) and propidium iodide (10μM) to label living chondrocytes green and dead cells red respectively. Following imaging of cartilage by confocal microscopy, the percentage cell death at each time point was obtained using Volocity 4 software. Chondrocyte death increased markedly with time: 0.04% at 2hrs, 28% at 4hrs and 39% at 6hrs. This study shows that bacterial products rapidly penetrate the cartilage matrix and have a damaging effect on in situ chondrocyte viability. Further work will clarify the contributions made by the various toxic components in the culture supernatant, but these data support the need to remove the bacteria and their products aggressively as part of the treatment of septic arthritis


Orthopaedic Proceedings
Vol. 95-B, Issue SUPP_16 | Pages 84 - 84
1 Apr 2013
Koga T Niikura T Lee SY Dogaki Y Okumachi E Waki T Kurosaka M
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Introduction. The hematoma occurring at a fracture site is known to play an important role in fracture healing. Previously, we demonstrated that fracture hematoma contained multilineage mesenchymal progenitor cells. On the other hand, the process of fracture healing is associated by two different mechanisms, intramembranous and endochondral. However, there are no reports proving the details about cellular analysis in the process of endochondoral ossification. Hypothesis. We hypothesized that one of the cell origins for endochondral ossification after fracture was hematoma. Materials & Methods. Fracture hematoma was obtained during osteosynthesis. Hematoma-derived cells were isolated and cultured for 5-weeks of chondrogenic induction followed by 2-weeks hypertrophic induction using pellet culture system. The pellets were analyzed histologically and immunohistochemically. The gene expression levels of chondrogenic, hypertrophic, osteogenic and angiogenic markers were measured by real-time PCR. Results. The histological and immunohistochemical analysis revealed that the Hematoma-derived cells differentiated into hypertrophic chondrocytes through chondrocytes, and finally differentiate into calcifying chondrocytes. The same trend was seen in the gene expression using real-time PCR analysis. Discussion & Conclusions. Our results suggest that fracture hematoma may be an origin of cells which play key roles in the process of endochondoral ossification during fracture healing


Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_XXXVII | Pages 123 - 123
1 Sep 2012
Dhollander A Verdonk P Verdonk R Verbruggen G Almqvist K
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Aim. The present study was designed to evaluate the implantation of alginate beads containing human mature allogenic chondrocytes for the treatment of symptomatic cartilage defects in the knee. Methods. A biodegradable, alginate-based biocompatible scaffold containing human mature allogenic chondrocytes was used for the treatment of chondral and osteochondral lesions in the knee. Twenty-one patients were clinically prospectively evaluated with use of the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and a Visual Analogue Scale (VAS) for pain preoperatively and at 3, 6, 9, 12, 24 and 36 months of follow-up. Results. A statistically significant clinical improvement became apparent after 6 months and patients continued to improve during the 36 months of follow-up. Adverse reactions to the alginate/fibrin matrix seeded with the allogenic cartilage cells were not observed. Two of the procedures failed. One of the patients had loosening of the periosteal flap, which was attributed to a failure of the surgical procedure. The other failure case was the result of the poor quality and quantity of the repair tissue itself. Discussion. The results of this pilot study show that the alginate-based scaffold containing human mature allogenic chondrocytes is feasible for the treatment of symptomatic cartilage defects in the knee. The described technique provides clinical outcomes equal to those of other cartilage repair techniques


Orthopaedic Proceedings
Vol. 97-B, Issue SUPP_4 | Pages 1 - 1
1 May 2015
Davidson E White T Hall A
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Articular cartilage has very poor repair potential, however it has an extraordinary capacity to withstand physiological mechanical loads in an intact joint. The nature and extent of chondrocyte death in articular cartilage following many forms of injury (trephine, scalpel, osteotome, sutures and drilling) has been characterised, but the ability to bear mechanical injury from iatrogenic surgical interventions is still unknown. A standard arthroscopic probe was moved at varying physiological pressures along the articular cartilage of joint before staining with fluorescent dyes to allow live/dead cell imaging using laser confocal scanning microscopy and imaging software, Image J. Bovine metatarsal phalangeal joints and fresh human cadaveric femoral condyles were used. The probe caused statistically significant chondrocyte death in bovine cartilage (p=0.02). Mild pressure 5% cell death, moderate (standard arthroscopic technique pressure) 22% and severe pressure 38%. A similar result was seen in human tissue with 24% cell death at moderate pressure compared to a control (p=0.0699). The widely assumed benign arthroscopic probe produces significant cell death in articular cartilage when used at standard operating pressures


Orthopaedic Proceedings
Vol. 101-B, Issue SUPP_1 | Pages 5 - 5
1 Jan 2019
McLean M Akbar M McCall K Kitson S Crowe1 L Blyth M Smith I Rooney B Spencer S Leach W Campton L Gilchrist D McInnes I Millar N
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Tranexamic acid (TXA) is an anti-fibrinolytic medication commonly used to reduce peri-operative bleeding. Increasingly, topical administration as an intra-articular injection or peri-operative wash is being administered at concentrations between 10–100mg/ml. This study investigated effects of TXA on human periarticular tissues and primary cell cultures using clinically relevant concentrations. Tendon, synovium and cartilage obtained from routine orthopaedic surgeries were used ex vivo or cultured for in vitro studies using various concentrations of TXA. They were stained with 5-chloromethylfluorescein diacetate and propidium iodide and imaged using confocal microscopy to identify the proportion of live and dead cells. The in vitro effect of TXA on primary cultured tenocytes, synovial like fibroblast (FLS) cells and chondrocytes was investigated using cell viability assays (MTT), fluorescent microscopy and multi-protein apoptotic arrays for cell death. There was significant (p<0.01) increase in cell death in all tissue treated with 100mg/ml TXA, ex vivo. MTT assays revealed significant (p<0.05) decrease in cell viability following treatment with 50 or 100mg/ml of TXA within 4 hours of all cell types cultured in vitro. Additionally, there was significant (p<0.05) increase in cell apoptosis detected by fluorescent microscopy within 1 hour of exposure to TXA. Furthermore, multi-protein apoptotic arrays detected increased apoptotic proteins within 1 hour of TXA treatment in tenocytes and FLS cells. Our study provides evidence of TXA cytotoxicity to human peri-articular tissues ex vivo and in vitro at concentrations and durations of treatment routinely used in clinical environments. Clinicians should therefore show caution when considering use of topical TXA administration


Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_XXXVII | Pages 80 - 80
1 Sep 2012
Utzschneider S Lorber V Dedic M Paulus A Sievers B Jansson V
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Periprosthetic osteolysis depends on the biological activity of wear particles, but there is little known about the distribution of polyethylene wear particles (PE) in the surrounding joint tissue. The purpose of this study was to examine the localisation of wear particles of six different PEs, including four crosslinked polyethylenes (XPE), as well as their biological activity in the murine knee. Material and Methods. Wear particles of 4 XPE- and 2 UHMWPE-inserts were isolated (knee joint simulator). For all groups the particles were similar in size and shape (mean diameter 0.3–05μm; 20nm-nucleopore-filter; ISO; n = 100.000).56 female Balb/c mice were randomly assigned to six treatment groups and one control group: control (PBS), XPE1 (3×30 kGy Gamma, annealed/sequential irradiated), XPE2 (95 kGy E-beam, remelted), XPE3 (65 kGy E-beam, remelted), XPE 4 (50 kGy Gamma, remelted), UHMWPE 1, UHMWPE 2. 50 μl of each particle suspension [(0.1% vol/vol (particle volume/PBS volume) after removal of endotoxin] were injected into the left knee joint. After 1 week the mice were killed and a histological and immunhistochemical analysis of the knee joints was done (IL-1, TNF-, ICAM-1). For the immunhistochemistry the articular cartilage, the bone marrow and the synovial membrane were evaluated semiquantitatively (Kruskal-Wallis test; all pairwise multiple comparison procedure; Bonferoni correction; significance level: p<0.05). Results. All groups showed a thickened synovial layer with an increased cellular infiltration. The particles of XPE 1 and 2 were localised in the bone marrow as well as in the joint space. In contrast, the particles of XPE 3 and 4 were distributed in the synovial layer and in the bone marrow as well, but not in the joint space. The UHMWPE1 particles were mainly located in the bone marrow and joint space while the UHMWPE2 particles were mainly found in the bone marrow and the synovial layer. For all PE groups there was a higher cytokine expression compaired to control (p<0.0024) without any differences between the groups (bone marrow/synovial layer). The chondrocytes in the groups with XPE 1- and XPE 2-particles expressed more TNF- than in the control group and the other treatment groups (p = 0.000). Conclusion. XPEs lead to a similar inflammatory reaction in vivo compared to conventional polyethylenes. The high TNF- expression in the articular cartilage (groups XPE 1 and 2) might be explained by the localisation of the wear particles in the joint space in direct contact with the chondrocytes. Long-term studies are necessary to analyse the effects of different wear particle distributions in the joint surrounding tissue after knee and hip replacement. Furthermore it has to be investigated, whether their distribution in the joint space or their transport into the bone marrow is responsible for the level of the chronic inflammatory reaction


Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_XXXVII | Pages 61 - 61
1 Sep 2012
Gulihar A Taylor G Hadi M
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Background. There are several case reports of chondrolysis following joint arthroscopy. Continuous post-operative infusion of local anaesthetic solutions, especially 0.5% Bupivacaine, has been implicated as the causative factor in many of these cases. Recent in vitro studies have shown that even a single exposure of articular cartilage to different local anaesthetic solutions can cause apoptosis and mitochondrial dysfunction in chondrocytes leading to cell death. There is currently no study looking at methods to prevent this toxicity of local anaesthetic solutions to articular cartilage. Glucosamine has a protective and reparative effect on articular cartilage and a Cochrane review in 2007 found that it provides mild benefit in pain and function in patients with arthritis. Aims. Oncologic: To compare the effect of a single exposure, in vitro, of different local anaesthetic solutions on human articular cartilage. To investigate the protective and reparative effects of Glucosamine on articular cartilage exposed to 0.5% Bupivacaine. Methods. Chondral explants (n = 354) were obtained from femoral heads of 14 fracture neck of patients undergoing hemiarthroplasty. To compare the effect of local anaesthetics, each specimen was exposed to one of 8 test solutions for one hour. After this exposure, the specimens were washed and incubated in culture medium containing radio-labelled 35-sulphur for 16 hours. The unbound radioactivity was then washed off and the chondral specimens were digested with proteinase for 24 hours. The uptake of 35-S by each specimen was measured and this gave an estimate of proteoglycan metabolism. Test solutions: 1. 1% Lidocaine; 2. 2% Lidocaine; 3. 0.25% Bupivacaine; 4. 0.5% Bupivacaine,. 5. 0.5% Levo-Bupivacaine; 6. Control solution of M199 culture medium. 7. To investigate its protective effect, 100 micrograms of Glucosamine was added along with 0.5% Bupivacaine; 8. To investigate the reparative effect of Glucosamine, the specimen was exposed to 0.5% Bupivacaine for one hour. After washing, 100 mcg of Glucosamine was added to the culture medium in which the chondral specimen was incubated. Results. Compared to the control culture medium, the inhibition of proteoglycan metabolism was 54% with 1% Lidocaine (p<0.001), 75% with 2% Lidocaine (p<0.01), 50% with 0.25% Bupivacaine (p = 0.04), 78% with 0.5% Bupivacaine (p<0.001) and 73% with 0.5% Levo-Bupivacaine (p<0.001). Adding Glucosamine for protection reduced the toxicity of 0.5% Bupivacaine to 43%, compared to 78% without. However, Glucosamine was not able to repair the damage caused by 0.5% Bupivacaine, with inhibition of proteoglycan metabolism at 70% even after 16 hours of incubation. Conclusion. All local anaesthetic solutions tested were toxic to articular cartilage, 0.5% Bupivacaine being the worst offender. Higher concentrations were more harmful. The addition of Glucosamine to 0.5% Bupivacaine protected against its toxicity to articular cartilage but was not able to repair the damage caused


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).


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 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.


Bone & Joint Research
Vol. 1, Issue 8 | Pages 174 - 179
1 Aug 2012
Alfieri KA Forsberg JA Potter BK

Heterotopic ossification (HO) is perhaps the single most significant obstacle to independence, functional mobility, and return to duty for combat-injured veterans of Operation Enduring Freedom and Operation Iraqi Freedom. Recent research into the cause(s) of HO has been driven by a markedly higher prevalence seen in these wounded warriors than encountered in previous wars or following civilian trauma. To that end, research in both civilian and military laboratories continues to shed light onto the complex mechanisms behind HO formation, including systemic and wound specific factors, cell lineage, and neurogenic inflammation. Of particular interest, non-invasive in vivo testing using Raman spectroscopy may become a feasible modality for early detection, and a wound-specific model designed to detect the early gene transcript signatures associated with HO is being tested. Through a combined effort, the goals of early detection, risk stratification, and development of novel systemic and local prophylaxis may soon be attainable.


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.