Stephen Graves

Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow

Previous studies have provided evidence for the existence of adult human bone marrow stromal stem cells (BMSSCs) or mesenchymal stem cells. Using a combination of cell separation techniques, we have isolated an almost homogeneous population of BMSSCs from adult human bone marrow. Lacking phenotypic characteristics of leukocytes and mature stromal elements, BMSSCs are non-cycling and constitutively express telomerase activity in vivo. This mesenchymal stem cell population demonstrates extensive proliferation and retains the capacity for differentiation into bone, cartilage and adipose tissue in vitro. In addition, clonal analysis demonstrated that individual BMSSC colonies exhibit a differential capacity to form new bone in vivo. These data are consistent with the existence of a second population of bone marrow stem cells in addition to those for the hematopoietic system. Our novel selection protocol provides a means to generate purified populations of BMSSCs for use in a range of different tissue engineering and gene therapy strategies.

Synthetic bone graft substitutes

Replacement of extensive local bone loss is a significant clinical challenge. There are a variety of techniques available to the surgeon to manage this problem, each with their own advantages and disadvantages. It is well known that there is morbidity associated with harvesting of autogenous bone graft and limitations in the quantity of bone available. Alternatively allografts have been reported to have a significant incidence of postoperative infection and fracture as well as the potential risk of disease transmission. During the past 30 years a variety of synthetic bone graft substitutes has been developed with the aim to minimize these complications. The benefits of synthetic grafts include availability, sterility and reduced morbidity. The present article examines the relevance of synthetic bone graft substitutes, their mechanical properties and clinical application.

Differential Cell Surface Expression of the STRO-1 and Alkaline Phosphatase Antigens on Discrete Developmental Stages in Primary Cultures of Human Bone Cells

Human osteoblast‐like cells can be readily cultured from explants of trabecular bone, reproducibly expressing the characteristics of cells belonging to the osteoblastic lineage. Dual‐color fluorescence‐activated cell sorting was employed to develop a model of bone cell development in primary cultures of normal human bone cells (NHBCs) based on the cell surface expression of the stromal precursor cell marker STRO‐1 and the osteoblastic marker alkaline phosphatase (ALP). Cells expressing the STRO‐1 antigen exclusively (STRO‐1+/ALP−), were found to exhibit qualities preosteoblastic in nature both functionally by their reduced ability to form a mineralized bone matrix over time, as measured by calcium release assay, and in the lack of their expression of various bone‐related markers including bone sialoprotein, osteopontin, and parathyroid hormone receptor based on reverse trancriptase polymerase chain reaction (PCR) analysis. The majority of the NHBCs which expressed the STRO‐1−/ALP+ and STRO‐1−/ALP− phenotypes appeared to represent fully differentiated osteoblasts, while the STRO‐1+/ALP+ subset represented an intermediate preosteoblastic stage of development. All STRO‐1/ALP NHBC subsets were also found to express the DNA‐binding transcription factor CBFA‐1, confirming that these cultures represent committed osteogenic cells. In addition, our primer sets yielded four distinct alternative splice variants of the expected PCR product for CBFA‐1 in each of the STRO‐1/ALP subsets, with the exception of the proposed preosteoblastic STRO‐1+/ALP− subpopulation. Furthermore, upon re‐culture of the four different STRO‐1/ALP subsets only the STRO‐1+/ALP− subpopulation was able to give rise to all of the four subsets yielding the same proportions of STRO‐1/ALP expression as in the original primary cultures. The data presented in this study demonstrate a hierarchy of bone cell development in vitro and facilitate the study of bone cell differentiation and function.

Integrin-mediated interactions between human bone marrow stromal precursor cells and the extracellular matrix

To date, the precise interactions between bone marrow stromal cells and the extracellular matrix that govern stromal cell development remain unclear. The integrin super-family of cell-surface adhesion molecules represents a major pathway used by virtually all cell types to interact with different extracellular matrix components. In this study, purified populations of stromal precursor cells were isolated from the STRO-1-positive fraction of normal human marrow, by fluoresence-activated cell sorting, and then assayed for their ability to initiate clonogenic growth in the presence of various integrin ligands. Bone marrow-derived stromal progenitors displayed differential growth to fibronectin, vitronectin, and laminin, over collagen types I and III, but showed a similar affinity for collagen type IV. The integrin heterodimers alpha1beta1, alpha2beta1, alpha5beta1, alpha6beta1, alpha(v)beta3, and alpha(v)beta5 were found to coexpress with the STRO-1 antigen on the cell surface of CFU-F, using dual-color analysis. Furthermore, only a proportion of stromal precursors expressed the integrin alpha4beta1, while no measurable levels of the integrin alpha3beta1 could be detected. Subsequent adhesion studies using functional blocking antibodies to different integrin alpha/beta heterodimers showed that stromal cell growth on collagen, laminin, and fibronectin was mediated by multiple beta1 integrins. In contrast, cloning efficiency in the presence of vitronectin was mediated in part by alpha(v)beta3. When human marrow stromal cells were cultured under osteoinductive conditions, their ability to form a mineralized matrix in vitro was significantly diminished in the presence of a functional blocking monoclonal antibody to the beta1 integrin subunit. The results of this study indicate that beta1 integrins appear to be the predominant adhesion receptor subfamily utilized by stromal precursor cells to adhere and proliferate utilizing matrix glycoproteins commonly found in the bone marrow microenvironment and bone surfaces. Furthermore, these data suggest a possible role for the beta1 integrin subfamily during the development of stromal precursor cells into functional osteoblast-like cells.

Integrin Expression and Function on Human Osteoblast-like Cells

The integrin family of cell adhesion molecules are a series of cell surface glycoproteins that recognize a range of cell surface and extracellular matrix (ECM)‐associated ligands. To date, the precise role of individual integrin molecules in bone cell–ECM interactions remains unclear. Cell binding assays were performed to examine the ability of normal human bone cells (NHBCs) to adhere to different ECM proteins in vitro. NHBCs displayed preferential adhesion to fibronectin over collagen types I, IV, and vitronectin and showed low affinity binding to laminin and collagen type V. No binding was observed to collagen type III. The integrin heterodimers α1β1, α2β1, α3β1, α5β1, αvβ3, and αvβ5 were found to be constitutively expressed on the cell surface of NHBCs by flow cytometric analysis. The integrins α4β1 and α6β1 were not expressed by NHBCs. Subsequent binding studies showed that NHBC adhesion to collagen and laminin was mediated by multiple integrins where cell attachment was almost completely inhibited in the presence of a combination of function‐blocking monoclonal antibodies (Mabs) to α1β1, α2β1, α3β1, and β1. In contrast, the adhesion of NHBCs to fibronectin was only partially inhibited (50%) in the presence of blocking Mabs to α3β1, α5β1, and β1. The attachment of NHBCs to collagen, laminin, fibronectin, and vitronectin was also found to be unaffected in the presence of a function‐blocking Mab to αvβ3. The results of this study indicate that β1 integrins appear to be the predominant adhesion receptor subfamily utilized by human osteoblast‐like cells to adhere to collagen and laminin and in part to fibronectin.

Expression of Osteoclast Differentiation Signals by Stromal Elements of Giant Cell Tumors

The mechanisms by which primary tumors of the bone cause bone destruction have not been elucidated. Unlike most other lytic bone tumors, osteoclastomas, otherwise known as giant cell tumors (GCT), contain osteoclast‐like cells within the tumor stroma. A new member of the TNF‐ligand superfamily member, osteoclast differentiation factor (ODF/OPGL/RANKL/TRANCE), was recently identified. ODF was shown to directly stimulate osteoclastogenesis, in the presence of M‐CSF. In this study, the expression of ODF was examined in a number of tumor samples associated with bone lysis in vivo. In addition, we investigated expression of the ODF receptor on osteoclast precursors, RANK, as well as the ODF inhibitor osteoprotegerin (OPG), and another TNF‐ligand superfamily member, TRAIL, previously shown to abrogate the inhibitory effects of OPG. We report here the novel finding that GCT stromal cells contain abundant ODF mRNA, whereas the giant cell population exclusively expresses RANK mRNA. These results are consistent with the osteoclast‐mediated bone destruction by these tumors. We also report the expression of OPG and TRAIL mRNA in GCT samples. A comparison with other lytic and nonlytic tumors of bone showed that GCT express more ODF and TRAIL mRNA relative to OPG mRNA. In addition, GCT were found to express a number of cytokines previously reported to play central roles in osteoclastogenesis, namely, IL‐1, −6, −11, −17, as well as TNF‐α. Importantly, GCT were also found to express high levels of M‐CSF mRNA, a cytokine shown to be an essential cofactor of ODF, and a survival factor for mature and developing osteoclasts. Furthermore, expression of these molecules by stromal cells isolated from GCT continued in vitro. Thus GCT constitutively express all of the signals that are currently understood to be necessary for the differentiation of osteoclasts from precursor cells.

Incidence and risk factors for deep surgical site infection after primary total hip arthroplasty : a systematic review

Although deep surgical site infection (SSI) is a major complication of primary total hip arthroplasty (THA), there are conflicting data regarding the incidence of deep SSI, and no comprehensive evaluation of the associated risk factors has been undertaken. We performed a systematic review of the literature; undertaking computer-aided searches of electronic databases, assessment of methodological quality, and a best-evidence synthesis. The incidence of SSI ranged from 0.2% before discharge to 1.1% for the period up to and including 5 years post surgery. Greater severity of a pre-existing illness and a longer duration of surgery were found to be independent risk factors for deep SSI. There is a need for high-quality, prospective studies to further identify modifiable risk factors for deep SSI after THA.

Proinflammatory cytokines inhibit osteogenic differentiation from stem cells: implications for bone repair during inflammation

OBJECTIVE The effects of inflammation on bone development from mesenchymal stem cells (MSC) are unclear due to the difficulty in isolating MSC. The aim of this study was to develop a MSC isolation method and to determine the in vitro effects of interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) on their osteogenic differentiation. METHODS Murine MSC were isolated from the limbs of C57/Bl6 mice through collagenase digestion of bone and enriched as the Stem cell antigen (Sca-1)(+) CD31(-) CD45(-) population, using lineage immunodepletion, followed by fluorescence-activated cell sorting (FACS). They were differentiated along the osteoblast linage in the presence or absence of IL-1beta and TNFalpha. Mineralization was measured as was the expression of a number of osteogenic genes by quantitative polymerase chain reaction (PCR). RESULTS We show that osteogenic differentiation from the MSC population is suppressed by IL-1beta and TNFalpha. In addition to suppression of bone mineralization, both cytokines inhibited the differentiation-associated increases in alkaline phosphatase (ALP) activity and the gene expression for ALP, alpha1(I) procollagen, runt-related transcription factor 2 (Runx2) and osterix. However, only TNFalpha inhibited osteonectin and osteopontin mRNA expression and only IL-1beta reduced cell proliferation. CONCLUSIONS The convenient isolation technique enables the easy generation of sufficient MSC to permit the molecular analysis of their differentiation. We were thus able to show that the proinflammatory cytokines, IL-1beta and TNFalpha, can compromise bone development from this primary MSC population, although with some significant differences. The potential involvement of specific inflammatory mediators needs to be taken into account if optimal bone repair and presumably that of other tissues are to be achieved with MSC.

Five-year results of the ASR XL Acetabular System and the ASR Hip Resurfacing System: an analysis from the Australian Orthopaedic Association National Joint Replacement Registry.

BACKGROUND Articular Surface Replacement (ASR) hip prostheses, which have metal-on-metal bearing surfaces, were manufactured by DePuy Orthopaedics (Warsaw, Indiana) for use in both conventional total hip arthroplasty and hip resurfacing. Both the ASR XL Acetabular System and the ASR Hip Resurfacing System were recently recalled worldwide by the manufacturer. This report summarizes an analysis by the Australian Orthopaedic Association National Joint Replacement Registry (AOANJRR) of the outcome of arthroplasties involving the ASR prostheses. METHODS The first recorded use of the ASR XL Acetabular System in Australia occurred in 2004, and the Registry recorded 4406 procedures involving this system through December 31, 2009. The first recorded use of the ASR Hip Resurfacing System in Australia occurred in 2003, and the Registry recorded 1167 procedures through December 31, 2009. The Kaplan-Meier method and proportional-hazard modeling were used to compare the revision rate of primary total hip arthroplasties involving the ASR XL Acetabular System with that of arthroplasties involving all other conventional prostheses as well as with that of arthroplasties involving all other conventional prostheses with a metal-on-metal-articulation. In addition, the revision rate of primary arthroplasties involving the ASR Hip Resurfacing System was compared with that of arthroplasties involving all other hip resurfacing prostheses. Patient demographics, prosthesis characteristics, and information regarding the type of revision and the reason for revision were also compared. RESULTS Arthroplasties involving both ASR designs had a significantly greater revision rate compared with those involving all other prostheses. The cumulative revision rate of arthroplasties involving the ASR XL Acetabular System at five years postoperatively was 9.3% (95% confidence interval [CI], 7.3% to 11.9%) compared with 3.4% (95% CI, 3.3% to 3.5%) for total hip arthroplasties involving all other conventional prostheses. The cumulative revision rate of arthroplasties involving the ASR Hip Resurfacing System at five years postoperatively was 10.9% (95% CI, 8.7% to 13.6%) compared with 4.0% (95% CI, 3.7% to 4.5%) for arthroplasties involving all other resurfacing prostheses. Arthroplasties involving the ASR XL Acetabular System had a greater rate of revision due to implant loosening and/or osteolysis and due to metal sensitivity compared with total hip arthroplasties involving all other conventional prostheses. Arthroplasties involving the ASR XL Acetabular System also had a significantly greater revision rate compared with total hip arthroplasties involving all other conventional metal-on-metal prostheses. Arthroplasties involving the ASR Hip Resurfacing System had a greater rate of revision due to metal sensitivity compared with total hip arthroplasties involving all other resurfacing prostheses. CONCLUSIONS ASR prostheses used in conventional hip arthroplasty and in hip resurfacing exhibited a greater revision rate compared with other prostheses in the AOANJRR. These results are consistent with those derived from other registries and from published studies of individual cohorts.

Relationship between body adiposity measures and risk of primary knee and hip replacement for osteoarthritis: a prospective cohort study

IntroductionTotal joint replacement is considered a surrogate measure for symptomatic end-stage osteoarthritis. It is unknown whether the adipose mass and the distribution of adipose mass are associated with the risk of primary knee and hip replacement for osteoarthritis. The aim of the present investigation was to examine this in a cohort study.MethodsA total of 39,023 healthy volunteers from Melbourne, Australia were recruited for a prospective cohort study during 1990 to 1994. Their body mass index, waist circumference, and waist-to-hip ratio were obtained from direct anthropometric measurements. The fat mass and percentage fat were estimated from bioelectrical impedance analysis. Primary knee and hip replacements for osteoarthritis between 1 January 2001 and 31 December 2005 were determined by data linkage to the Australian Orthopaedic Association National Joint Replacement Registry. Cox proportional hazards regression models were used to estimate the hazard ratios (HRs) for primary joint replacement associated with each adiposity measure.ResultsComparing the fourth quartile with the first, there was a threefold to fourfold increased risk of primary joint replacement associated with body weight (HR = 3.44, 95% confidence interval (CI) = 2.83 to 4.18), body mass index (HR = 3.44, 95% CI = 2.80 to 4.22), fat mass (HR = 3.51, 95% CI = 2.87 to 4.30), and percentage fat (HR = 2.99, 95% CI = 2.46 to 3.63). The waist circumference (HR = 2.77, 95% CI = 2.26 to 3.39) and waist-to-hip ratio (HR = 1.46, 95% CI = 1.21 to 1.76) were less strongly associated with the risk. Except for the waist-to-hip ratio, which was not significantly associated with hip replacement risk, all adiposity measures were associated with the risk of both knee and hip joint replacement, and were significantly stronger risk factors for knee.ConclusionsRisk of primary knee and hip joint replacement for osteoarthritis relates to both adipose mass and central adiposity. This relationship suggests both biomechanical and metabolic mechanisms associated with adiposity contribute to the risk of joint replacement, with stronger evidence at the knee rather than the hip.