Introduction. Intraosseous transcutaneous amputation prostheses (ITAP) provide an alternative means of attaching artificial limbs for amputees. Conventional stump-socket devices are associated with soft tissue complications including; pressure sores and tissue necrosis. ITAP resolves these problems by attaching the exo-prosthesis transcutaneously to the skeleton. The aim of this study is to increase the attachment of dermal fibroblasts to titanium alloy in vitro. Fibronectin (Fn) and laminin 332 (Ln) enhance early cell growth and adhesion. We hypothesize that silanized dual coatings of fibronectin and laminin (SiFnLn) will be more durable when compared with adsorbed dual coating (AdFnLn), and will enhance early fibroblast growth and adhesion compared to single coatings. Methods. The kinetics of dual single and dual protein coating attachment onto titanium alloy was quantified on silanized 10mm diameter discs using radiolabelled Fn (125I-Fn) and Ln (125I-Ln). Sixty discs were polished, sterilized and silanized. Coating durability was assessed when soaked in fetal calf serum (FCS) for 0, 1, 24, 48 and 72hrs. Data was compared to un-silanized Ti discs with the same coatings. Five thousand human dermal fibroblasts were seeded on discs (n = 6) of Ti polished alone (Pol), Ti with adsorbed fibronectin (AdFn), Ti with adsorbed laminin (AdLn), Ti adsorbed dual coating (AdFnLn), Ti silanized (Si), Ti silanized with fibronectin (SiFn), Ti silanized with laminin (SiLn), Ti silanized with a dual coating (SiFnLn) for 24hrs. In order to measure cell adhesion fibroblasts were fixed, vinculin stained using mouse vinculin antibody and alexa fluor. Axiovision Image Analysis software was used to measure cell area, vinculin focal adhesion markers per cell and per unit cell area. Data was analysed in SPSS and significance was assumed at the 0.05 level. Results. Silanized dual coatings bonded to Ti alloy in significantly larger quantities compared with adsorbed coatings at all time points (all p values < 0.05). Fibroblasts cultured on dual coatings were significantly larger, produced more vinculin markers per cell, and per unit cell area compared with single coatings. Cells on SiFnLn were larger with more numerous vinculin markers per cell, and per unit cell area compared with AdFnLn (p<0.05). Conclusion. This study has demonstrated that covalently bonding both fibronectin and laminin to Ti alloy provides a durable, dual coating that enhances early fibroblast growth and attachment compared with either protein coating alone in vitro. Our study showed that there is non-competitive binding of laminin on Ti surfaces in the presence of fibronectin. Dual coatings may be applied to the skin-penetrating region of transcutaneous devices to improve the skin seal and this may have positive implications for the development of ITAP

Scar tissue formation secondary to acute muscle injury, surgical wounding and compartment syndrome can result in significant functional impairment and predispose to further injury. The source of fibroblasts, and the molecular mechanisms driving their activation and persistence in skeletal muscle fibrosis are not known. We hypothesized that cells expressing PDGFRβ become fibroblasts in response to injury and that targeting αv integrins in these cells reduces skeletal muscle fibrosis. We used double-fluorescent reporter mice to demonstrate that cells expressing PDGFRβ become activated myofibroblasts in response to cardiotoxin (CTX) induced skeletal muscle injury. Following injury, PDGFRβ+ cells moved from perivascular locations into the interstitium in a distribution characteristic of fibroblasts, and showed marked induction of fibroblastic genes including αSMA and collagen1 (all p<0.0001). To confirm that αv integrins present on PDGFRβ cells critically regulate skeletal muscle fibrosis we used Itgavflox/flox;PDGFRβ-Cre mice (transgenic mice in which αv integrins are ‘knocked-down’ in PDGFRβ+ cells). These mice were significantly protected from CTX induced fibrosis (p<0.01). To demonstrate potential clinical utility of targeting αv integrins, we used a small molecule inhibitor of αv integrins (CWHM12). Treatment with CWHM12 significantly reduced fibrosis when delivered from the time of injury (p<0.01) and when delivered after the fibrotic response had become established (p<0.01). We have identified a core pathway regulating fibrosis in skeletal muscle. Pharmacologic inhibition of αv integrins has potential clinical utility in the treatment and prevention of skeletal muscle fibrosis

Aims. The Intraosseous Transcutaneous Amputation Prosthesis (ITAP) may improve quality of life for amputees by avoiding soft-tissue complications associated with socket prostheses and by improving sensory feedback and function. It relies on the formation of a seal between the soft tissues and the implant and currently has a flange with drilled holes to promote dermal attachment. Despite this, infection remains a significant risk. This study explored alternative strategies to enhance soft-tissue integration. Materials and Methods. The effect of ITAP pins with a fully porous titanium alloy flange with interconnected pores on soft-tissue integration was investigated. The flanges were coated with fibronectin-functionalised hydroxyapatite and silver coatings, which have been shown to have an antibacterial effect, while also promoting viable fibroblast growth in vitro. The ITAP pins were implanted along the length of ovine tibias, and histological assessment was undertaken four weeks post-operatively. Results. The porous titanium alloy flange reduced epithelial downgrowth and increased soft-tissue integration compared with the current drilled flange. The addition of coatings did not enhance these effects. Conclusion. These results indicate that a fully porous titanium alloy flange has the potential to increase the soft-tissue seal around ITAP and reduce susceptibility to infection compared with the current design. Cite this article: Bone Joint J 2017;99-B:393–400

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

Aims

To determine whether platelet-rich plasma (PRP) injection improves outcomes two years after acute Achilles tendon rupture.

Introduction. Traumatized musculoskeletal tissue often exhibits prolonged time to healing, mostly due to low blood flow and innervation. Intermittent Pneumatic Compression (IPC) increases blood flow and decreases thromboembolic event after orthopedic surgery,[1] however little is known about healing effects.[2] We hypothesized that IPC could stimulate tissue repair: 1.) blood flow 2.) nerve ingrowth 3.) tissue proliferation and during immobilisation enhance 4.) biomechanical tissue properties. Methods. Study 1: In 104 male Sprague Dawley (SD) rats the right Achilles tendon was ruptured and the animals freely mobilized. Half the group received daily IPC-treatment, using a pump and cuff over the hindpaw that inflates/deflates cyclicly, 0–55mmHg (Biopress SystemTM, Flexcell Int.), and the other half received sham-treatment. Healing was assessed at 1,3,6 weeks by perfusion-analysis with laser doppler scanner (Perimed, Sweden), histology and biomechanical testing. Study 2: 48 male SD-rats were ruptured as above. Three groups of each 16 rats were either mobilized, immobilized or immobilized with IPC treatment. Immobilization was performed by plaster cast. Healing was assessed at 2 weeks with histology and biomechanical testing. Results. Study 1: At 3 and 6 weeks reperfusion increased by 21% and 23% (p< 0.05) after IPC-treatment, strengthened by the observation of elevated numbers of blood vessels and nerves. Fibroblast density was at all time points significantly increased in the IPC group. At three and six weeks the IPC treated tendons displayed an increased tissue organization confirmed by higher collagen I/III ratio in the IPC group. No differences (p = 0.10) were found regarding biomechanical strength. Study 2: Compared to mobilization, immobilization caused a downregulation (p<0.05) of all biomechanical and histological parameters, eg. maximum force decreased 80% and collagen III occurrence by 83%. However when immobilization was combined with IPC biomechanical and histological healing increased significantly compared to pure immobilization, eg. maximum force increased 63% and collagen III occurrence by 150%. Conclusion. This study demonstrated that IPC treatment can counteract biomechanical and morphological deficits caused by immobilization by enhancing proliferative soft tissue repair. Thus, IPC promotes tissue repair by stimulating tissue perfusion and nerve ingrowth as well as accelerating both fibroblast proliferation and collagen organization

INTRODUCTION. In order to address high failure rates following rotator cuff repairs, a greater understanding is required of the underlying structural changes so that treatments can be appropriately targeted and biomarkers of failure can be identified. As collagen is the primary constituent of tendon and determines force transmission, collagen structural changes may affect responses to loading. For example changes in collagen 1 and 5 are associated with the hyperelastic Ehlers-Danlos syndrome, which is diagnosed by looking for pathopneumonic altered collagen fibres or ‘collagen flowers’ in skin using transmission electron microscopy (TEM). To date no study has been performed on the microstructure of torn human rotator cuff tendons using TEM. It was hypothesized that normal, small and massive human rotator cuff tendons tears will have altered microscopic structures. The unique study aimed to use TEM to compare the ultrastructure of small and massive rotator cuff tears, to normal rotator cuff tendons. METHODS. Samples from 7 human rotator cuff tendons repairs were obtained, including 4 massive (>5 cm) and 3 small (< 1 cm) tears, and 3 matched normal controls with no history of connective tissue disorders. Specimens were fixed in 4% glutaraldehyde in 0.1M phosphate buffer, processed and examined blind using routine TEM examination. To assess whether changes in the relative expression of collagen 1 and 5 (COL1A1, COL5A1 and COL5A2) occurred in all tears, qPCR was performed on another 6 phenotypically matched patients. RESULTS. The basic structure of the normal tendon consisted of tightly packed clumps of dense packed parallel running collagen fibers with few fibroblasts and small amounts of fine filamentous material between clumps. In contrast, torn samples were more variable with areas of less dense packing of collagen fibers and larger areas of filamentous material plus variable numbers of lipid droplets both within the fibroblast and between the collagen bundles. There was also evidence of twisting and random orientation of individual collagen fibers. All torn tendons showed evidence of a proportion of the fibers within the collagen bundles being enlarged with a serrated outline, similar in appearance to ‘collagen flowers’. Clear differences between the small and massive tears were not identified. qRT-PCR of torn rotator cuff tendon specimens demonstrated no altered collagen expression compared to normal tendons. DISCUSSION. This novel study has identified the previously unreported presence of atypical collagen fibers with focal swelling resulting in the appearance of ‘collagen flowers’ in torn rotator cuff tendons only. This appearance is considered pathognomonic of Ehlers-Danlos syndrome, classical type 1 and 2. Torn tendons also showed an increase in filamentous material, and infiltration with fat droplets. These novel findings may offer insight into the mechanisms of structural damage that contribute to rotator cuff failure. Further examination is required, to evaluate the significance of these observations

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

We hypothesise that the Masquelet induced membrane used for the reconstruction of large bone defects were likely to involve mesenchymal stem cells (MSCs), given the excellent resultant skeletal repair. This study represents the first characterisation in humans of the induced membrane formed as a result of the Masquelet technique. Methods. Induced membranes and matching periosteum were harvested from 7 patients. Cytokines (BMP2, VEGF, SDF1) and cell lineage markers (CD31, CD271, CD146) were studied by immunohistochemisty. Flow cytometry was used to measure the cellularity and cellular composition. MSCs were enumerated using a colony forming unit fibroblast assay. In expanded cultures, a 96-gene array card was used to assess their transcriptional profile. Alkaline phophatase, alizarin red and calcium assays were employed to measure their in vitro osteogenic potential. Results. Membrane was more cellular(p=0.028), had more MSC phenotype(p=0.043) compared to matched periosteum. The molecular profiles were similar, except for 2-fold abundance of SDF-1 in membrane (p=0.043)compared to periosteum. Membrane and periosteum had a similar proportion of endothelial cells and CFU-F colonies; expanded MSCs from both sources were highly osteogenic. Discussion. These results indicate that the induced membrane possesses a rich source of MSC and therefore our findings support the view that the induced membrane plays an active role in bone regeneration

We aimed to investigate the effect of Seprafilm®, a synthetic biomembran, on the intra-articular adhesion formation in an experimental arthrofibrosis model. Twenty male white rabbits were randomly allocated into two groups of 10 animals in each. A standard surgical procedure aiming at the development of arthrofibrosis and including medial parapatellar arthrotomy, lateral eversion of the patella, partial synovectomy and debridement of anterior of supracondylar area and patella joint surface by scalpel was performed on all rabbits' right knees. Group 1 rabbits served as controls, and in Group 2 rabbits a Seprafilm®, barrier placed into the described area. In both groups, after surgery, knee joint was immobilized by a no.5 wire suture passing from the ankle and groin and keeping the joint in 140° of flexion. At 6th week, all animals were sacrificed and adhesion formation was evaluated both macroscopically and histo-pathologically. All data were semi-quantified and analyzed statistically by Fisher's exact test. While all rabbits in control group displayed different rates of adhesion macroscopically (62.5% severe, 25% moderate, 12.5% mild), none in the study group had it. The average macroscopic adhesion score was 2.5 ± 0.75 in control group, and 0 in Seprafilm® group. Histopathologic evaluation also revealed microscopic adhesion in all rabbits in control group, but none in Seprafilm® group. Fibroblast proliferation in Seprafilm® group (100% mild) was significantly lower than in control group (62.5% severe, 37.5% moderate) (p<0.05). In conclusion, use of Seprafilm® as a mechanical barrier may be of value against the formation of arthrofibrosis in risky knees such as septic and traumatic ones

Introduction. This study reports the results of percutaneous autologous bone marrow grafting in 62 patients with corticosteroids treatment who had one hip osteonecrosis treated with bone marrow (BM) injection and the other contralateral hip osteonecrosis with core decompression (CD) alone. Only patients with bilateral symptomatic osteonecrosis and with those hips at stage I or II (as defined by Steinberg) were included in this study. Material and Methods. Between 1988 and 1995, 62 consecutive patients (28 males and 34 females) were included in this study. These patients had a mean age of 31 years (range 18 to 34 years) at the time of the onset of symptoms. The average follow-up was 17 years (range, 15 to 20 years). An average of 152 + 16 milliliters of marrow was aspirated from the iliac crest. The number of stroma progenitor that was transplanted was estimated by counting the Fibroblast Colony Forming Units which express type I and type III collagen. The bone marrow graft obtained after concentration contained average 4889 + 716 progenitors per cubic centimeter (range 3515 to 6293 per cubic centimeter). Each hip received a mean number of thirty cubic centimeters of bone marrow graft (range 27 to 35 cubic centimeters). The average total number of CFU-F injected in each hip was therefore 147 × 103 cells (range 119 × 103 to 195 × 103 cells). Results. Clinical results were determined by the change in Harris hip scores from preoperative evaluation to the last follow-up visit, by the change in the radiographic progression and by the need of subsequent total hip arthroplasty. Bone marrow grafting afforded better reduction in pain, effected a reduction with time in the number of hips that progressed to collapse, and delayed the need for total hip replacement. Ten hips had collapsed and needed arthroplasty at the most recent follow-up after bone marrow grafting, compared to 45 after core decompression. For hips with collapse, the mean survival time before collapse was 71.2 months (43.35- 60.96; 95% CI) for the bone marrow graft group and 38.5 months for the control group (13.2–39.74; 95% CI). With the number available, there was a positive correlation (Spearman's test) between the duration of clinical survival before collapse and the number or concentration of CFU-F and CFU-GM in the graft in the CD group. These results are explained by the fact that bone marrow injection improved the repair process on MRI and on histology. Overall, 10 hips with bone marrow injection showed a total regression of the signal, 59 hips showed a partial reduction (42 with BM and 17 with CD) and 55 hips did not show a significant reduction (10 with BM and 45 with CD). Conclusion. Bone marrow grafting afforded better reduction in pain, reduction in the number of collapses, delayed the need for total hip replacement, and improved the repair process on MRI and on histology

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.

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 aim of this study was to report the incidence of arthrofibrosis of the knee and identify risk factors for its development following a fracture of the tibial plateau. We carried out a retrospective review of 186 patients (114 male, 72 female) with a fracture of the tibial plateau who underwent open reduction and internal fixation. Their mean age was 46.4 years (19 to 83) and the mean follow-up was16.0 months (6 to 80).

A total of 27 patients (14.5%) developed arthrofibrosis requiring a further intervention. Using multivariate regression analysis, the use of a provisional external fixator (odds ratio (OR) 4.63, 95% confidence interval (CI) 1.26 to 17.7, p = 0.021) was significantly associated with the development of arthrofibrosis. Similarly, the use of a continuous passive movement (CPM) machine was associated with significantly less development of arthrofibrosis (OR = 0.32, 95% CI 0.11 to 0.83, p = 0.024). The effect of time in an external fixator was found to be significant, with each extra day of external fixation increasing the odds of requiring manipulation under anaesthesia (MUA) or quadricepsplasty by 10% (OR = 1.10, p = 0.030). High-energy fracture, surgical approach, infection and use of tobacco were not associated with the development of arthrofibrosis. Patients with a successful MUA had significantly less time to MUA (mean 2.9 months; sd 1.25) than those with an unsuccessful MUA (mean 4.86 months; sd 2.61, p = 0.014). For those with limited movement, therefore, performing an MUA within three months of the injury may result in a better range of movement.

Based our results, CPM following operative fixation for a fracture of the tibial plateau may reduce the risk of the development of arthrofibrosis, particularly in patients who also undergo prolonged provisional external fixation.

Cite this article: Bone Joint J 2015;97-B:109–14.

Objectives

One commonly used rat fracture model for bone and mineral research is a closed mid-shaft femur fracture as described by Bonnarens in 1984. Initially, this model was believed to create very reproducible fractures. However, there have been frequent reports of comminution and varying rates of complication. Given the importance of precise anticipation of those characteristics in laboratory research, we aimed to precisely estimate the rate of comminution, its importance and its effect on the amount of soft callus created. Furthermore, we aimed to precisely report the rate of complications such as death and infection.

Heterotopic ossification occurring after the use of commercially available bone morphogenetic proteins has not been widely reported. We describe four cases of heterotopic ossification in patients treated with either recombinant bone morphogenetic protein 2 or recombinant bone morphogenetic protein 7. We found that while some patients were asymptomatic, heterotopic ossification which had occurred around a joint often required operative excision with good results.