Restoration of native Coronal Plane Alignment of the Knee (CPAK) phenotype is a strategy suggested to achieve better satisfaction. The aim of this study was to investigate the influence of changes in CPAK classification on patient-reported outcome measures (PROMs) and survivorship in a large cohort of manual mechanically aligned (MA) cemented TKAs. A retrospective analysis of 1062 consecutive cemented TKAs using MA philosophy at a single institution. Pre- and post-operative hip-knee-ankle radiographs were classified using the CPAK classification. Oxford Knee Score (OKS) and patient satisfaction (4-point-Likert scale) were collected prospectively. Implant survival data was obtained from our national arthroplasty database. We compared the outcomes of patients who maintained or changed their CPAK classification following TKA. Satisfaction was analysed using chi-square test, and OKS was analysed using Mann-Whitney test. Pre-operatively, most patients were CPAK type-I (38.8%). 85.5% of patients changed their CPAK type post-operatively, with CPAK type-V observed in 41.2% of these. Significantly better satisfaction (p=0.033) and OKS (p=0.021) were observed at one-year follow-up in patients who changed CPAK type, although the difference was below OKS minimally important clinical difference. There was no difference in satisfaction (p=0.73) and OKS (p=0.26) at one year between CPAK-V and non-V classifications. Post-operative CPAK type had no correlation with satisfaction and OKS. 12 TKAs (1.1%) were revised within 10 years (3 septic). In this large cohort of MA-TKA, excellent survivorship was observed at 10 years, with no demonstrable difference in outcome related to the final CPAK phenotype or change in phenotype

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

Introduction. 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. Methods. 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. Results. MSCs were isolated from all fractions. Using the CFU-F assay median number of colonies: ICBMA=8 (2–21), LBFBM-liquid=14 (0–53), LBFBM-solid=116 (23–171) per 200μl of sample; MSC frequency, as percentage of total cells, using flow-cytometry, provided similar results. Mesenchymal stem cells isolated from the LBFBM phases appeared to not be inferior to ICBMA in terms of osteogenic, chondrogenic or adipogenic differentiation. Passaged cells from all fractions had a phenotype consistent with other reported sources. Discussion. Intra-medullary cavities of long-bones are frequently accessed by the orthopaedic/trauma surgeon. This represents a ‘low-tech’ method of harvesting large numbers of MSCs with a favourable differentiation profile for autologous/allogenous uses

Introduction. 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. Methods. 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. Results. We found MSCs were in all fractions/patients. Using the CFU-F assay median number of colonies: ICBMA=8 (2–21), RIA-liquid=12 (4–41), RIA-solid=115 (67–200) per 200μl of sample. Total yield of cells was calculated from volume of sample: ICBMA=670 (228–4275), RIA-liquid=39000 (16500–83700), RIA-solid=9400 (7210–28475). MSC frequency as a percentage of total cells using flow-cytometry on fresh sample found similar frequencies. MSCs isolated from the RIA phases differentiated into osteogenic, chondrogenic and adipogenic lineages at least as well as ICBMA. Passaged (P2) cells, from all fractions/patients, had a phenotype consistent with other reported sources. Discussion. The RIA filtrate bag is typically discarded at operation. These results show that this ‘waste’ represents a significant source of MSCs that could be isolated for autologous/allogenous use. Concentration of the liquid-phase/brief enzymatic digestion of the solid-phase offers the possibility of large numbers of MSCs being obtained without/with minimal culture expansion

Aims

Frailty greatly increases the risk of adverse outcome of trauma in older people. Frailty detection tools appear to be unsuitable for use in traumatically injured older patients. We therefore aimed to develop a method for detecting frailty in older people sustaining trauma using routinely collected clinical data.

Since osteoimmunology is gaining increasingly interest and evidence for involvement of complement in bone biology was found, the role of complement in bone biology and fracture healing was evaluated. After characterizing the bone phenotype, a fracture healing experiment with C3- and C5- deficient mice was performed. After osteotomy of the right femur and external fixation, healing was analyzed after 1, 3, 7 and 21 days. Bone characterization revealed a reduced number of osteoclasts in C5-deficient animals with a significantly reduced resorption activity. While bone mineral density was significantly higher in complement-deficient strains, stiffness was significantly reduced. After 21 days of fracture healing, C5-deficient animals showed reduced stiffness and a smaller callus volume compared to controls. Interestingly, C3- more than C5-deficient animals showed reduced bone formation. Altogether, bone phenotype of complement-deficient animals resembles a mild form of osteopetrosis. This might be due to the resorption defect seen in C5-deficient mice. A reason for the minor involvement of C3-deficient mice compared to the C5-deficient animals could be the cross-talk between the coagulation cascade with side activation of complement factor C5 by thrombin. These results indicate for the first time an essential role of complement in bone biology and fracture healing. Future studies should focus on the molecular basis of complement involvement and the osteoclastic resorption defect

Femoroacetabular impingement (FAI) is a significant cause of osteoarthritis in young active individuals but the pathophysiology remains unclear. Increasing mechanistic studies point toward an inflammatory component in OA. This study aimed to characterise inflammatory cell subtypes in FAI by exploring the phenotype and quantification of inflammatory cells in FAI versus OA samples. Ten samples of labrum were obtained from patients with FAI (confirmed pathology) during open osteochondroplasty or hip arthroscopy. Control samples of labrum were collected from five patients with osteoarthritis undergoing total hip arthroplasty. Labral biopsies were evaluated immunohistochemically by quantifying the presence of macrophages (CD68 and CD202), T cells (CD3), mast cells (mast cell tryptase) and vascular endothelium (CD34). Labral biopsies obtained from patients with FAI exhibited significantly greater macrophage, mast cell and vascular endothelium expression compared to control samples. The most significant difference was noted in macrophage expression (p<0.01). Further sub typing of macrophages in FAI using CD202 tissue marker revealed and M2 phenotype suggesting that these cells are involved in a regenerate versus a degenerate process. There was a modest but significant correlation between mast cells and CD34 expression (r=0.4, p<0.05) in FAI samples. We provide evidence for an inflammatory cell infiltrate in femoroacetabular impingement. In particular, we demonstrate significant infiltration of mast cells and macrophages suggesting a role for innate immune pathways in the events that mediate hip impingement. Further mechanistic studies to evaluate the net contribution and hence therapeutic utility of these cellular lineages and their downstream processes may reveal novel therapeutic approaches to the management of early hip impingement

Mesenchymal stem cells (MSCs) reside around blood vessels in all organs. This reservoir of progenitors can be ‘recruited’ in response to injury. The ability to manipulate stem cells therapeutically within injured tissue provides an attractive alternative to transplantation. Stem cells are regulated by neighbouring cells. We hypothesized that endothelial cells (ECs) influence MSC differentiation into bone and fat. MSCs were sorted from fat using fluorescent activated sorting. Their capacity to differentiate into bone, fat and cartilage was used to confirm MSC phenotype. MSCs and ECs were cultured in two-dimensions (standard culture dishes) and three-dimensions (vascular networks suspended in gel). Cocultures were exposed to osteogenic and adipogenic media. The role of EC-released factors on MSC differentiation was determined using a system in which cells share media but do not contact. Wnt pathway modulators were used to investigate the role of Wnt signalling. MSCs differentiated into bone, fat and cartilage. MSCs and ECs integrated in two- and three-dimensions. MSCs and ECs formed vessel-like structures in three-dimensions. When cultured with ECs, MSC differentiation to bone was accelerated while differentiation to fat was inhibited. This effect on osteogenesis was maintained when cells shared media but did not contact. Coculture with Wnt modulators confirmed that this effect is in part, mediated through Wnt signalling. Our data suggest that ECs influence MSC differentiation. Therapeutic targeting of EC-MSCs signalling may enable manipulation of MSCs in vivo avoiding the need for cell transplantation. This could enable trauma and orthopaedic patients who have healthy resident stem cells to self-repair

Engineered bone tissue to recreate the continuity of damaged skeletal segments is one of the field of interest of tissue engineering. Trabecular titanium has very good mechanical properties and high in vitro and in vivo biocompatibility: it can be used in biomedical applications to promote osteointegration demonstrating that it can be successfully used for regenerative medicine in orthopaedic surgery (1). Purpose of this investigation was to evaluate the behavior of adipose tissue derived stem cells (hASCs) cultured on scaffolds of Trabecular TitaniumTM (Lima-Lto) (TT). hASCs are considered to be multipotent mesenchymal stem cells that are easily induced to differentiate into functional osteoblasts both in vitro and in vivo (2). The hASCs were obtained from the subcutaneous adipose tissue of healthy donors during total hip replacement procedures after digestion with collagenase. They were seeded on monolayer and on the TT scaffolds, and incubated at 37 degrees C in 5% CO2 with osteogenic medium or control medium. The expression of bone-related genes using RT-PCR, time course of alkaline phosphatase activity and morphological investigation with Scanning Electron Microscopy (SEM) were performed to evaluate the osteogenic differentiation of hASCs. Alkaline phosphatase activity, marker of the differentiation toward the osteogenic pattern, was significantly higher in hASCs grown with osteogenic medium than in cells grown with control medium, both in monolayer and TT scaffolds; moreover, also alkaline phosphatase of hASCs grown on TT scaffolds in the presence of control medium increased with time, differently from that of cells grown on monolayer. The osteogenic differentiated hASCs expressed the bone-related genes type I collagen, osteocalcin, Runx-2 and alkaline phosphatase. SEM observations showed that hASCs differentiated toward osteoblast-like cells: they produced a big amount of extracellular matrix that covered the surface of the porous scaffolds with bridges between the pore walls. These data suggest that hASCs are able to adhere to TT scaffolds, to acquire an osteoblastic phenotype and to produce abundant extracellular matrix, with but also without osteogenic medium. We can therefore conclude that this material carries osteinductive properties being responsible of ostegenic differentiation; consequently, this scaffold/cells construct is effective to regenerate damaged tissue and to restore the function of bone tissue

Background. Previous data from our institution show that more than half of all prosthetic joint infections are due to S. aureus. A significant proportion of these bacteria may have an endogenous source. Detecting and treating asymptomatic S. aureus nasal carriers preoperatively has been shown to reduce the risk of infection. Material and Methods. This is an ongoing prospective study that started in March/2009 and involves primary total knee or hip arthroplasties candidates. So far preoperative nasal swab cultures were performed in 211(61%) out of 347 patients operated until April/2010. Carriers are identified and randomly chosen for preoperative treatment consisting of nasal mupirocin twice a day and daily cloro-hexidine baths in the 5 days that precede surgery. Antibiotic prophylaxis is cefazolin 24hours adding a single vancomycin dose in MRSA carriers. Results. Around 30% (64/211) of patients presented Saureus nasal carriage and 6 of them (9.4%) were methicilin-resistant. Infection rate was lower in the non-carriers: 1.36% (2/147). The group of 33 untreated carriers so far has the higher prevalence of infection (6.06%; Odds ratio=4.677; p=0.098). In the 31 treated carriers there was only one infection to date (3.23%; Odds ratio=2.371; p=0.463). The 136 patients control group in which no preoperative nasal culture was made registered 4 infections (2.94%; Odds ratio=2.197; p=0.356). Five out of the nine infections involved S. aureus. We further isolated 3 S. epidermidis, 1 enterococcus and 2 gram-negatives. Discussion. It seems that two S. aureus infections were due to endogenous contamination as the infecting bacteria possesses the exact same phenotype as the colonizing preoperative one (one untreated and one treated carrier). In other two S. aureus cases the seeding appears exogenous (a preoperative negative culture and a infecting bacteria different than the preoperative screening). One infection was registered in a non-studied patient. Accordingly, exogenous contamination seems responsible for at least 6 out of 9 infections. Conclusion. These preliminary results suggest that S. aureus colonization may be an endogenous risk factor for prosthetic joint infection that can be influenced. However exogenous contamination is still a major risk factor in our institution

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.

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.

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.

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.