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.

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

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

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

Bone is the second most commonly transplanted tissue worldwide, with over four million operations using bone grafts or bone substitute materials annually to treat bone defects. However, significant limitations affect current treatment options and clinical demand for bone grafts continues to rise due to conditions such as trauma, cancer, infection and arthritis. The need for a novel, cost effective treatment option for osteochondral defects has therefore never been greater. As an emerging technology, three-dimensional (3D) bioprinting has the capacity to deposit cells, extracellular matrices and other biological materials in user-defined patterns to build complex tissue constructs from the “bottom up”. Through use of extrusion bioprinting and fused deposition modelling (FDM) 3D printing, porous 3D scaffolds were successfully created in this study from hydrogels and synthetic polymers. Mesenchymal stem cells (MSCs) seeded onto polycaprolactone scaffolds with defined pore sizes and porosity maintained viability over a 7-day period, with addition of alginate hydrogel and scaffold surface treatment with NaOH increasing cell adhesion and viability. MSC-laden alginate constructs produced via extrusion bioprinting also maintained structural integrity and cell viability over 7 days in vitro culture. Growth within osteogenic media resulted in successful osteogenic differentiation of MSCs within scaffolds compared to controls (p<0.001). MSC spheroids were also successfully created and bioprinted within a novel, supramolecular hydrogel with tunable stiffness. In conclusion, 3D constructs capable of supporting osteogenic differentiation of MSCs were biofabricated via FDM and extrusion bioprinting. Future work will look to increase osteochondral construct size and complexity, whilst maintaining cell viability

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

Background. Impaction bone grafting with milled human allograft is the gold standard for replacing lost bone stock during revision hip surgery. Problems surrounding the use of allograft include cost, availability, disease transmission and stem subsidence (usually due to shear failure of the surrounding allograft). Aims. To investigate various polymers for use as substitute allograft. The ideal graft would be a composite with similar mechanical characteristics as allograft, and with the ability to form de novo bone. Methods. High and low molecular weight (MW) forms of three different polymers (polylactic acid (PLA), poly (lactic-co-glycolic) acid (PLGA) and polycaprolactone (PCL)) were milled, impacted into discs, and then tested in a custom built shear testing rig, and compared to allograft. A second stage of the experiment involved the addition of skeletal stem cells (SSC) to each of the milled polymers, impaction, 8 days incubation, and then tests for cell viability and number, via fluorostaining and biochemical (WST-1, DNA) assays. Results. The shear strengths of both high/ low MW PLA, and high/low MW PLGA were significantly higher than those of milled allograft but high and low MW PCL was poor to impact, and had significantly lower shear strengths. Fluorostaining showed good cell survival on high MW PLA, high MW PCL and both high and low MW PLGA. These findings were confirmed on both DNA and WST-1 assays. Conclusions. High MW PLA as well as high and low MW PLGA performed well both in mechanical testing and cell compatibility studies. These three polymers are good contenders to produce a living composite for use as substitute human allograft in impaction bone grafting

Introduction. Infection of endoprostheses is a serious complication in orthopedic surgery. As silver is known for its antibactierial effects, silver-coated endoprostheses have gained increased attention to decrease infection rates. However, cytotoxic effects of silver on bone cells have not been investigated in detail. We aimed to investigate whether silver nano-/microparticles and ionic silver exert cytotoxic effects on osteoblasts and osteoclasts in vitro and to correlate potential effects with the antibacterial effect on Staph. epidermidis. Methods. Murine osteoclasts (OC) and murine osteoblasts (OB) were treated with silver particles (avg. sizes: 50nm, 3μm, 30μm, 8μg/ml–500μg/ml) and Ag+NO3- (0.5μg/ml–500μg/ml). Silver treatment started on day 3 to prevent interference with cell adhesion. XTT assays were performed to assess cell viability. Tartrate resistant acidic phosphatase (TRAP) activity and alkaline phosphatase (ALP) activity served as measures for OC and OB differentiation, respectively. The release of silver ions from silver particles was quantified with atomic emission spectometry (AES). Titanium particles (avg. sizes: 50nm and 30μm) were used as controls to investigate whether potential silver effects were particle- or ion-mediated. The antimicrobial activity of silver ions and particles was tested with Staph. epidermidis agar inhibition assays. Results. Ionic silver had the strongest impact on cell differentiation and viability of OC and OB (OC differentiation: mean IC50 = 5 μg/ml, OC viability: mean IC50 = 14 μg/ml, OB differentiation: mean IC50 = 1 μg/ml, OB viability: mean IC50 = 1 μg/ml). Silver nanoparticles decreased cell differentiation and viability in a dose dependent manner (OC differentiation: mean IC50 = 5μg/ml, OC viability: mean IC50 = 14μg/ml, OB differentiation: mean IC50 = 1μg/ml, OB viability: mean IC50 = 1μg/ml). Silver microparticles as well as titanium nano- and microparticles had no effect on cell differentiation and viability. AES showed a size and dose dependent release of silver ions from silver nano- and microparticles. Agar inhibition assays showed a dose correlation of the antibacterial effect of silver with the cytotoxic effects on OB and OC. Conclusion. Silver nanoparticles and silver ions exert dose-dependent cytotoxic effects on OB and OC in vitro resulting in a severe alteration of cell differentiation and viability. The effect of silver on OB and OC seems to be mediated primarily by silver ions and correlates with the substance's antibacterial effects. The cytotoxicity of silver nanoparticles is mediated primarily by the size-dependent liberation of silver ions. Disturbance of OB and OC survival may have deleterious effects on the osseointegration of orthopedic implants. Further in vivo studies are needed to investigate the osseointegration of silver coated implants prior to their widespread clinical application

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.