Aims

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

Adipose tissue is an attractive source of mesenchymal stem cells (MSCs) as it is largely dispensable and readily accessible through minimally invasive procedures such as lipoaspiration. Until recently MSCs could only be isolated in a process involving ex-vivo culture. Pericytes (CD45−, CD146+, and CD34−) and adventitial cells (CD45−, CD146−, CD34+) represent two populations of MSCs (collectively termed perivascular stem cells or PSCs) that can be prospectively purified using fluorescence activated cell sorting (FACS). We performed FACS on lipoaspirate samples from n=129 donors to determine the frequency and yield of PSCs and to establish patient and processing factors that influence yield. The mean number of stromal vascular fraction (SVF) cells from 100ml of lipoaspirate was 37.8×106. Within the SVF, mean cell viability was 82%, with 31.6% of cells being heamatopoietic (CD45+). Adventitial cells and pericytes represented 31.6% and 7.9% of SVF cells respectively. As such, 200ml of lipoaspirate would theoretically yield 24.5 million MSCs –a sufficient number to enable point-of-care delivery for use in several orthopaedic applications. The yield and prevalence of PSCs were minimally affected by donor age, sex and BMI. Storing lipoaspirate samples for up to 72 hours prior to processing had no significant deleterious effects on MSC yield or viability. Our study confirms that pure populations of MSC-precursors (PSCs) can be prospectively isolated from adipose tissue, in sufficient quantities to negate the necessity for culture expansion while widening possible applications to include trauma, where a time delay between extraction and implantation excludes their use

Introduction. Nonunions pose complications in fracture management that can be treated using electrical stimulation (ES). Bone marrow mesenchymal stem cells (BMMSCs) are essential in fracture healing, although the effects of different clinical ES waveforms available in clinical practice on BMMSCs cellular activities is unknown. Materials and Methods. We compared Direct Current (DC), Capacitive Coupling (CC), Pulsed Electromagnetic wave (PEMF) and Degenerate Wave (DW) by stimulating human-BMMSCs for 5 days for 3 hours a day. Cytotoxicity, cell proliferation, cell-kinetics and cell apoptosis were evaluated after ES. Migration and invasion were assessed using fluorescence microscopy and affected gene and protein expression were quantified. Results. DW had the greatest proliferative and least apoptotic and cytotoxic effects compared to other waveforms and unstimulated cells after 5 days of ES (p < 0.001). DC, DW and CC resulted in significantly more cells in S phase and G2/M phase (p < 0.01) compared to the unstimulated BMMSCs. CC and DW caused more cells to invade collagen and showed increased MMP-2 and MT1-MMP expression (p < 0.001) compared to the other waveforms and unstimulated BMMSCs. DC increased cellular migration in a scratch-wound assay and all ES waveforms increased migration gene expression with DC having the greatest effect (p < 0.01). Conclusion. The ES waveform is vital in influencing BMMSCs cellular activities. Migration and invasion were increased by ES which suggests that the recruitment of BMMSCs to the healing site during a fracture could be increased by ES

Prosthetic joint infection is one of the most challenging complications of joint alloplasty and the diagnosis remains difficult. The aim of the study was to investigate the bacterial flora in surgical samples from 22 prosthetic patients using a panel of culture-independent molecular methods including broad range 16S rRNA gene PCR, cloning, sequencing, phylogeny, quantitative PCR (qPCR), and fluorescence in situ hybridization (FISH). Concomitant samples were cultured by standard methods. Molecular methods detected presence of bacteria in samples from 12 of 22 patients. Using clone libraries a total of 40 different bacterial species were identified including known pathogens and species not previously described in association with prosthetic joint infections. The predominant species were Propionibacterium acnes and Staphylococcus epidermidis; polymicrobial infections were found in 9 patients. Culture-based methods showed bacterial growth in 8 cases with the predominant species being S. epidermidis. Neither anaerobic bacteria (including P. acnes) nor any of the species not previously described in implant infections were isolated. Additionally, 7 of the 8 culture positive cases were monomicrobial. Overall, the results of culture-based and molecular methods showed concordance in 11 cases (hereof 9 negative by both methods) and discrepancy in 6 cases. In the remaining 5 cases, culture-based methods identified only one species or a group of bacteria (e.g., coagulase negative staphylococci or coryneform rods), while culture-independent molecular methods were able to detect several distinct bacterial species including a species within the group identified by culture. A qPCR assay was developed to assess the abundance of Propionibacterium while S. aureus was quantified by a published S. aureus qPCR assay. These quantifications confirmed the findings from the clone library approach and showed the potential of qPCR for fast detection of bacteria in orthopedic samples. Additionally, both single cells and microcolonies were visualized using FISH and confocal scanning laser microscopy. In conclusion, the molecular methods detected a more diverse bacterial flora in prosthetic joint infections than revealed by standard culture-based methods, and polymicrobial infections were more frequently observed. The pathogenesis of these microorganisms and their role in implant-associated infections needs to be determined

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

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.