MSCs have long promised benefits of synthesising bone/cartilage, treating non-unions and potentially accelerating fracture repair. This potential has been tempered by MSC scarcity in the ‘gold-standard’ iliac crest bone marrow aspirate (ICBMA) and the resulting need to expand numbers via cell-culture. Culture of MSCs is time-consuming, expensive and results in cells with a reduced differentiation capacity. The reamer-irrigator-aspirator (RIA) is an innovation designed to reduce intra-medullary (IM) pressures during reaming of long-bones via continuous irrigation and suction. Aspirated contents are passed via a coarse filter, which traps bony-fragments before moving into a ‘waste’ bag - from which MSCs have been previously isolated. We examined liquid and solid phases found in this ‘waste’, 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 filtrate ‘waste’ bag from RIA reaming (6 patients) was filtered (70μm) and the solid fraction digested for 60min (37°C) with collagenase. MSCs were isolated from liquid & solid fractions and from 10ml matched ICBMA. Enumeration of MSCs was achieved via colony-forming-unit-fibroblast (CFUF) assay and flow-cytometry on fresh sample using CD45low, CD271+. MSCs were cultured by virtue of their plastic adherence and passaged in standard, non-haematopoietic media. Passage (P2) cells were differentiated towards osteogenic, adipogenic and chondrogenic lineages with their phenotype assessed with flow cytometry CD33 CD34 CD45 CD73 CD90 CD105.Introduction
Methods
Although chondrocytes have been used for autologous implantation in defects of articular cartilage, limited availability and donor-site morbidity have led to the search for alternative cell sources. Mesenchymal stem cells from various sources represent one option. The infrapatellar fat-pad is a promising source. Advantages include low morbidity, ease of harvest and ex-vivo evidence of chondrogenesis. Expansion of MSCs from human fat-pad in FGF-2 has been shown to enhance chondrogenesis. To further elucidate this process, we assessed the role of TGF-?3, FGF-2 and oxygen tension on growth kinetics of these cells during expansion. Infrapatellar fatpads were obtained from 4 donors with osteoarthritis. Cells were expanded in various media formulations (STD, FGF, TGF and FGF/TGF) at both 20% and 5% oxygen tensions. Colony forming unit fibroblast assays were performed for each expansion group and assessed with crystal violet staining. Cell aggregates from each group underwent chondrogenic differentiation in 5% and atmospheric oxygen tension. Pellets were analyzed on day 21. 5% Oxygen tension during expansion increased the colony size for both FGF and FGF/TGF groups. Cells expanded in FGF/TGF proliferated more rapidly. Biochemical analysis revealed that cells expanded in FGF-2 had higher glycosaminoglycan synthesis rates, a marker for chondrogenesis. Differentiation at 5% pO2 led to higher levels of sGAG but its effect was generally less potent compared to expansion in FGF-2.Methods
Results
Iliac crest bone marrow aspirate (ICBMA) is frequently cited as the ‘gold-standard’ source of MSCs. Mesenchymal stem cells have been shown to reside within the intramedullary (IM) cavities of long-bones and a comparative assessment with ICBMA has not yet been performed. Aspiration of the IM cavities of 6 patients' femurs with matched ICBMA was performed. The long-bone-fatty-bone-marrow (LBFBM) aspirated was filtered (70μm) and the solid fraction digested for 60min (37°C) with collagenase. Enumeration was performed via the colony-forming-unit-fibroblast (CFU-F) assay and using the CD45low CD271+ phenotype via flow-cytometry. Passaged (P2) cells were differentiated towards osteogenic, adipogenic and chondrogenic lineages with their phenotype assessed using flow-cytometry CD33 CD34 CD45 CD73 CD90 CD105.Introduction
Methods
Iliac crest bone marrow aspirate (ICBMA) is frequently cited as the ‘gold-standard’ source of MSCs. MSCs have been shown to reside within the intramedullary (IM) cavities of long-bones [Nelea, 2005] however a comparative assessment with ICBMA has not yet been performed and the phenotype of the latter compartment MSCs remains undefined in their native environment. Aspiration of the IM cavities of 6 patients' femurs with matched ICBMA was performed. The long-bone-fatty-bone-marrow (LBFBM) was filtered (70μm) to separate liquid and solid fractions and the solid fraction was briefly (60min, 37oC) digested with collagenase. MSC enumeration was performed using the colony-forming-unit-fibroblast (CFU-F) assay and quantification of cells with the CD45low CD271+ phenotype by flow-cytometry. [Jones 2002, Buhring 2007] MSCs were cultured and standard expansion media and passage 2 cells were differentiated towards osteogenic, adipogenic and chondrogenic lineages.Introduction
Methods
Therapeutic exploitation of MSCs in orthopaedics has been tempered by their scarcity within ‘gold-standard’ iliac crest bone marrow aspirate (ICBMA) and the resulting need to expand cells in vitro. This is time-consuming, expensive and results in cells with a reduced differentiation capacity. [Banfi 2000] The RIA is a device that provides continuous irrigation and suction during reaming of long bones. Aspirated contents pass via a filter, trapping bony-fragments, before moving into a ‘waste’ bag, from which MSCs have been previously isolated. [Porter 2009] We hypothesised that ‘waste’ RIA bag contains more MSCs than a standard aspirated volume of ICBMA (30 ml). We further hypothesised than a fatty solid phase within this ‘waste bag’ contains many MSCs trapped within the adipocyte-rich stromal network and hence requiring an enzymatic digestion for their efficient release [Jones 2006]. The discarded filtrate ‘waste’ bag that contained saline from marrow cavity irrigation procedure from RIA reaming (7 patients) was filtered (70μm) and the solid fraction digested for 60min (37oC) with collagenase. MSC enumeration was performed using the colony-forming-unit-fibroblast (CFU-F). Following culture in standard expansion media, passage 2 cells were differentiated towards osteogenic, adipogenic and chondrogenic lineages and their phenotype was assessed using flow cytometry. ICBMA from the same patients was used as controls.Introduction
Methods
More than 100,000 anterior cruciate ligament reconstructions are performed annually in the USA. The hamstrings and the patellar tendons are the most frequently used graft tissues. Up to ten percent of these grafts are deemed to have failed, generating considerable discussion in the literature regarding the ideal graft choice. A three-dimensional computational model, taking into account both material and geometrical non-linearities, would be useful in predicting the performance of different graft tissues and fixations. Unfortunately, the mechanical characteristics and parameters needed for such a model are complex and largely unknown. The aim of this study is to develop a method for measuring the geometrical properties needed as input for a three-dimensional tendon model. A laser-based, non-contact technique is used to generate a series of cross-sectional profiles along the length of the tendon. Unlike previously proposed methods, it is able to detect concavities and can be constructed using equipment commonly found in an engineering laboratory. A laser line generator (Stocker-Yale Lasiris SNF, Quebec, Canada) projects a horizontal line onto the sample. Images of the line are acquired with a digital video camera (Basler A631fc, Germany) as the tendon is rotated. These images are reassembled into 2-D slices using MatLab software. Multiple cross-sections can be combined to create three dimensional geometries. The new method was validated on objects of known shape (circular and hexagonal cylinders). The cross-sectional area measurement was found to be accurate to within 2.5%. The method was repeatable to within 1.7%. Six bovine flexor tendons have been analysed; concavities were evident in four of these. This method could be adapted to determine the surface geometries of other long and slender objects.
In contrast, the coronal femoral osteotomies had a worse healing response. The alignment of the fracture line perpendicular to the plane of motion of the joint exposes the repair tissue within it to increased shear and tensile stresses. This may play a negative role in the repair of these coronal defects when compared to sagittal osteotomies, which are relatively protected from the high transarticular pressures and showed a greater tendency to remodel their articular surface.
