Results using Trabecular Metal™ augments in combination with acetabular impaction bone grafting in deficient acetabula

We examined whether the use of trabecular metal wedges to fill segmental defects is an effective method of socket reconstruction when used in combination with impaction grafting and implantation of a cemented socket. Fifteen hips in 14 patients underwent impaction grafting in combination with a TM wedge with a minimum of 2 years follow-up. All patients had their defects assessed using the Paprosky classification. Patients were reviewed with x-rays and migration of the implant was measured. Outcome scores were also collected. Mean follow-up was 39 months (25-83). The mean age at surgery was 67.8 (49-85) years. Seven of the patients had previously undergone impaction grafting with the use of a stainless steel rim mesh to constrain the graft. None of the patients had failed either clinically or radiologically.

2010–2011 Queensland floods : using Haddon's Matrix to define and categorise public safety strategies

Objective The 2010–2011 Queensland floods resulted in the most deaths from a single flood event in Australia since 1916. This article analyses the information on these deaths for comparison with those from previous floods in modern Australia in an attempt to identify factors that have contributed to those deaths. Haddon's Matrix, originally designed for prevention of road trauma, offers a framework for understanding the interplay between contributing factors and helps facilitate a clearer understanding of the varied strategies required to ensure people's safety for particular flood types. Methods Public reports and flood relevant literature were searched using key words ‘flood’, ‘fatality’, ‘mortality’, ‘death’, ‘injury’ and ‘victim’ through Google Scholar, PubMed, ProQuest and EBSCO. Data relating to reported deaths during the 2010–2011 Queensland floods, and relevant data of previous Australian flood fatality (1997–2009) were collected from these available sources. These sources were also used to identify contributing factors. Results There were 33 deaths directly attributed to the event, of which 54.5% were swept away in a flash flood on 10 January 2011. A further 15.1% of fatalities were caused by inappropriate behaviours. This is different to floods in modern Australia where over 90% of deaths are related to the choices made by individuals. There is no single reason why people drown in floods, but rather a complex interplay of factors. Conclusions The present study and its integration of research findings and conceptual frameworks might assist governments and communities to develop policies and strategies to prevent flood injury and fatalities.

Mechanical stimulation of the pro-angiogenic capacity of human fracture haematoma: Involvement of VEGF mechano-regulation

Compromised angiogenesis appears to be a major limitation in various suboptimal bone healing situations. Appropriate mechanical stimuli support blood vessel formation in vivo and improve healing outcomes. However, the mechanisms responsible for this association are unclear. To address this question, the paracrine angiogenic potential of early human fracture haematoma and its responsiveness to mechanical loading, as well as angiogenic growth factors involved, were investigated in vitro. Human haematomas were collected from healthy patients undergoing surgery within 72. h after bone fracture. The haematomas were embedded in a fibrin matrix, and cultured in a bioreactor resembling the in vivo conditions of the early phase of bone healing (20 compression, 1. Hz) over 3. days. Conditioned medium (CM) from the bioreactor was then analyzed. The matrices were also incubated in fresh medium for a further 24. h to evaluate the persistence of the effects. Growth factor (GF) concentrations were measured in the CM by ELISAs. In vitro tube formation assays were conducted on Matrigel with the HMEC-1 cell line, with or without inhibition of vascular endothelial growth factor receptor 2 (VEGFR2). Cell numbers were quantified using an MTS test. In vitro endothelial tube formation was enhanced by CM from haematomas, compared to fibrin controls. The angiogenesis regulators, vascular endothelial growth factor (VEGF) and transforming growth factor β1 (TGF-β1), were released into the haematoma CM, but not angiopoietins 1 or 2 (Ang1, 2), basic fibroblast growth factor (bFGF) or platelet-derived growth factor (PDGF). Mechanical stimulation of haematomas, but not fibrin controls, further increased the induction of tube formation by their CM. The mechanically stimulated haematoma matrices retained their elevated pro-angiogenic capacity for 24. h. The pro-angiogenic effect was cancelled by inhibition of VEGFR2 signalling. VEGF concentrations in CM tended to be elevated by mechanical stimulation; this was significant in haematomas from younger, but not from older patients. Other GFs were not mechanically regulated. In conclusion, the paracrine pro-angiogenic capacity of early human haematomas is enhanced by mechanical stimulation. This effect lasts even after removing the mechanical stimulus and appears to be VEGFR2-dependent.

Sex-specific compromised bone healing in female rats might be associated with a decrease in mesenchymal stem cell quantity

Introduction The clinically known importance of patient sex as a major risk factor for compromised bone healing is poorly reflected in animal models. Consequently, the underlying cellular mechanisms remain elusive. Because mesenchymal stem cells (MSCs) are postulated to regulate tissue regeneration and give rise to essential differentiated cell types, they may contribute to sex-specific differences in bone healing outcomes. Methods We investigated sex-specific variations in bone healing and associated differences in MSC populations. A 1.5 mm osteotomy gap in the femora of 8 male and 8 female 12-month-old Sprague-Dawley rats was stabilized by an external fixator. Healing was analyzed in terms of biomechanical testing, bridging and callus size over time (radiography at 2, 4, and 6 weeks after surgery), and callus volume and geometry by μCT at final follow-up. MSCs were obtained from bone marrow samples of an age-matched group of 12 animals (6 per gender) and analyzed for numbers of colony-forming units (CFUs) and their capacity to differentiate and proliferate. The proportion of senescent cells was determined by β-galactosidase staining. Results Sex-specific differences were indicated by a compromised mechanical competence of the callus in females compared with males (maximum torque at failure, p = 0.028). Throughout the follow-up, the cross-sectional area of callus relative to bone was reduced in females (p ≤ 0.01), and the bridging of callus was delayed (p 2weeks = 0.041). μCT revealed a reduced callus size (p = 0.003), mineralization (p = 0.003) and polar moment of inertia (p = 0.003) in female animals. The female bone marrow contained significantly fewer MSCs, represented by low CFU numbers in both femora and tibiae (p femur = 0.017, p tibia = 0.010). Functional characteristics of male and female MSCs were similar. Conclusion Biomechanically compromised and radiographically delayed bone formation were distinctive in female rats. These differences were concomitant with a reduced number of MSCs, which may be causative for the suboptimal bone healing.

Interleukin-1β stimulates human renal fibroblast proliferation and matrix protein production by means of a transforming growth factor-β-dependent mechanism

One of the hallmarks of progressive renal disease is the development of tubulointerstitial fibrosis. This is frequently preceded by macrophage infiltration, raising the possibility that macrophages relay fibrogenic signals to resident tubulointerstitial cells. The aim of this study was to investigate the potentially fibrogenic role of interleukin-1beta (IL-1beta), a macrophage-derived inflammatory cytokine, on cortical fibroblasts (CFs). Primary cultures of human renal CFs were established and incubated for 24 hours in the presence or absence of IL-1beta. We found that IL-1beta significantly stimulated DNA synthesis (356.7% +/- 39% of control, P <.003), fibronectin secretion (261.8 +/- 11% of control, P <.005), collagen type 1 production, (release of procollagen type 1 C-terminal-peptide, 152.4% +/- 26% of control, P <.005), transforming growth factor-beta (TGF-beta) secretion (211% +/- 37% of control, P <.01), and nitric oxide (NO) production (342.8% +/- 69% of control, P <.002). TGF-beta (1 ng/mL) and the phorbol ester phorbol 12-myristate 13-acetate (PMA, 25 nmol/L) produced fibrogenic effects similar to those of IL-1beta. Neither a NO synthase inhibitor (N(G)-methyl-l-arginine, 1 mmol/L) nor a protein kinase C (PKC) inhibitor (bis-indolylmaleimide 1, 1 micromol/L) altered the enhanced level of fibronectin secretion or DNA synthesis seen in response to IL-1beta treatment. However, addition of a TGF-beta-neutralizing antibody significantly reduced IL-1beta-induced fibronectin secretion (IL-1beta + IgG, 262% +/- 72% vs IL-1beta + alphaTGF-beta 156% +/- 14%, P <.02), collagen type 1 production (IL-1beta + IgG, 176% +/- 28% vs IL-1beta + alphaTGF-beta, 120% +/- 14%, P <.005) and abrogated IL-1beta-induced DNA synthesis (245% +/- 49% vs 105% +/- 21%, P <.005). IL-1beta significantly stimulated CF DNA synthesis and production of fibronectin, collagen type 1, TGFbeta, and NO. The fibrogenic and proliferative action of IL-1beta on CF appears not to involve activation of PKC or production of NO but is at least partly TGFbeta-dependent.

Matrix metalloproteinases during wound healing - a double edged sword

The extracellular matrix (ECM) provides a framework for cells and gives skin its tensile strength and elasticity. Loss of its integrity necessitates the clearing of damaged components and the deposition of firstly a provisional matrix and later remodelling of the ECM to support a functionally intact tissue. Matrix metalloproteinases (MMPs) are an important family of enzymes that function in the breakdown of the ECM and modulate the function of many biologically active molecules housed in the ECM. Through their enzymatic actions MMPs play a role in fundamental processes such as immune cell infiltration and ECM remodelling during wound repair. Their tight control is necessary for timely wound healing and excessive MMP activity participates in the development and persistence of chronic wounds, while reduced activity contributes to fibrosis. A number of inhibitors have been designed to target this activity and improve wound healing with limited success. Novel strategies are currently being investigated to improve wound healing by targeting MMP modulating molecules.

Controlling whole blood activation and resultant clot properties by carboxyl and alkyl functional groups on material surfaces : a possible therapeutic approach for enhancing bone healing

Most research virtually ignores the important role of a blood clot in supporting bone healing. In this study, we investigated the effects of surface functional groups carboxyl and alkyl on whole blood coagulation, complement activation and blood clot formation. We synthesised and tested a series of materials with different ratios of carboxyl (–COOH) and alkyl (–CH3, –CH2CH3 and –(CH2)3CH3) groups. We found that surfaces with –COOH/–(CH2)3CH3 induced a faster coagulation activation than those with –COOH/– CH3 and –CH2CH3, regardless of the –COOH ratios. An increase in –COOH ratios on –COOH/–CH3 and –CH2CH3 surfaces decreased the rate of coagulation activation. The pattern of complement activation was entirely similar to that of surface-induced coagulation. All material coated surfaces resulted in clots with thicker fibrin in a denser network at the clot/material interface and a significantly slower initial fibrinolysis when compared to uncoated glass surfaces. The amounts of platelet-derived growth factor-AB (PDGF-AB) and transforming growth factor-b (TGF-b1) released from an intact clot were higher than a lysed clot. The release of PDGF-AB was found to be correlated with the fibrin density. This study demonstrated that surface chemistry can significantly influence the activation of blood coagulation and complement system, resultant clot structure, susceptibility to fibrinolysis as well as release of growth factors, which are important factors determining the bone healing process.

A deconvolution method for deriving the transit time spectrum for ultrasound propagation through cancellous bone replica models

The acceptance of broadband ultrasound attenuation for the assessment of osteoporosis suffers from a limited understanding of ultrasound wave propagation through cancellous bone. It has recently been proposed that the ultrasound wave propagation can be described by a concept of parallel sonic rays. This concept approximates the detected transmission signal to be the superposition of all sonic rays that travel directly from transmitting to receiving transducer. The transit time of each ray is defined by the proportion of bone and marrow propagated. An ultrasound transit time spectrum describes the proportion of sonic rays having a particular transit time, effectively describing lateral inhomogeneity of transit times over the surface of the receiving ultrasound transducer. The aim of this study was to provide a proof of concept that a transit time spectrum may be derived from digital deconvolution of input and output ultrasound signals. We have applied the active-set method deconvolution algorithm to determine the ultrasound transit time spectra in the three orthogonal directions of four cancellous bone replica samples and have compared experimental data with the prediction from the computer simulation. The agreement between experimental and predicted ultrasound transit time spectrum analyses derived from Bland–Altman analysis ranged from 92% to 99%, thereby supporting the concept of parallel sonic rays for ultrasound propagation in cancellous bone. In addition to further validation of the parallel sonic ray concept, this technique offers the opportunity to consider quantitative characterisation of the material and structural properties of cancellous bone, not previously available utilising ultrasound.

Mesenchymal stem cell therapies for bone and tendon conditions

Bone, tendon, and cartilage are highly specialized musculoskeletal connective tissues that are subject to injury and degeneration. These tissues have relatively poor healing capabilities, and coupled with their variable response to established medical treatments, produce significant morbidity. Mesenchymal stem cells (MSCs) are capable of regenerating skeletal tissues and therefore offer great promise in the treatment of connective tissue pathologies. Adult MSCs are multipotent cells that possess the properties of proliferation and differentiation into all connective tissues. Furthermore, they can be gene modified to secrete growth factors and utilized in connective tissue engineering. Potential MSC-based therapies for bone and tendon conditions are reviewed in this chapter.

The heparan sulfate proteoglycan (HSPG) glypican-3 mediates commitment of MC3T3-E1 cells toward osteogenesis

Heparan sulfate (HS) sugar chains attached to core proteoglycans (PGs) termed HSPGs mediate an extensive range of cell-extracellular matrix (ECM) and growth factor interactions based upon their sulfation patterns. When compared with non-osteogenic (maintenance media) culture conditions, under established osteogenic culture conditions, MC3T3-E1 cells characteristically increase their osteogenic gene expression profile and switch their dominant fibroblast growth factor receptor (FGFR) from FGFR1 (0.5-fold decrease) to FGFR3 (1.5-fold increase). The change in FGFR expression profile of the osteogenic-committed cultures was reflected by their inability to sustain an FGF-2 stimulus, but respond to BMP-2 at day 14 of culture. The osteogenic cultures decreased their chondroitin and dermatan sulfate PGs (biglycan, decorin, and versican), but increased levels of the HS core protein gene expression, in particular glypican-3. Commitment and progress through osteogenesis is accompanied by changes in FGFR expression, decreased GAG initiation but increased N- and O-sulfation and reduced remodeling of the ECM (decreased heparanase expression) resulting in the production of homogenous (21 kDa) HS chain. With the HSPG glypican-3 expression strongly upregulated in these processes, siRNA was used to knockdown this gene to examine the effect on osteogenic commitment. Reduced glypican-3 abrogated the expression of Runx2, and thus differentiation. The reintroduction of this HSPG into Runx2-null cells allowed osteogenesis to proceed. These results demonstrate the dependence of osteogenesis on specific HS chains, in particular those associated with glypican-3.

The osteogenic transcription factor Runx2 regulates components of the fibroblast growth factor/proteoglycan signaling axis in osteoblasts

Heparan sulfate proteoglycans cooperate with basic fibroblast growth factor (bFGF/FGF2) signaling to control osteoblast growth and differentiation, as well as metabolic functions of osteoblasts. FGF2 signaling modulates the expression and activity of Runt-related transcription factor 2 (Runx2/Cbfa1), a key regulator of osteoblast proliferation and maturation. Here, we have characterized novel Runx2 target genes in osteoprogenitors under conditions that promote growth arrest while not yet permitting sustained phenotypic maturation. Runx2 enhances expression of genes related to proteoglycan-mediated signaling, including FGF receptors (e.g., FGFR2 and FGFR3) and proteoglycans (e.g., syndecans [Sdc1, Sdc2, Sdc3], glypicans [Gpc1], versican [Vcan]). Runx2 increases expression of the glycosyltransferase Exostosin-1 (Ext1) and heparanase, as well as alters the relative expression of N-linked sulfotransferases (Ndst1 = Ndst2 > Ndst3) and enzymes mediating O-linked sulfation of heparan sulfate (Hs2st > Hs6st) or chondroitin sulfate (Cs4st > Cs6st). Runx2 cooperates with FGF2 to induce expression of Sdc4 and the sulfatase Galns, but Runx2 and FGF2 suppress Gpc6, thus suggesting intricate Runx2 and FGF2 dependent changes in proteoglycan utilization. One functional consequence of Runx2 mediated modulations in proteoglycan-related gene expression is a change in the responsiveness of bone markers to FGF2 stimulation. Runx2 and FGF2 synergistically enhance osteopontin expression (>100 fold), while FGF2 blocks Runx2 induction of alkaline phosphatase. Our data suggest that Runx2 and the FGF/proteoglycan axis may form an extracellular matrix (ECM)-related regulatory feed-back loop that controls osteoblast proliferation and execution of the osteogenic program.

Osteogenic differentiation of murine embryonic stem cells is mediated by fibroblast growth factor receptors

The mechanisms involved in the control of embryonic stem (ES) cell differentiation are yet to be fully elucidated. However, it has become clear that the family of fibroblast growth factors (FGFs) are centrally involved. In this study we examined the role of the FGF receptors (FGFRs 1-4) during osteogenesis in murine ES cells. Single cells were obtained after the formation of embryoid bodies, cultured on gelatin-coated plates, and coaxed to differentiate along the osteogenic lineage. Upregulation of genes was analyzed at both the transcript and protein levels using gene array, relative-quantitative PCR (RQ-PCR), and Western blotting. Deposition of a mineralized matrix was evaluated with Alizarin Red staining. An FGFR1-specific antibody was generated and used to block FGFR1 activity in mES cells during osteogenic differentiation. Upon induction of osteogenic differentiation in mES cells, all four FGFRs were clearly upregulated at both the transcript and protein levels with a number of genes known to be involved in osteogenic differentiation including bone morphogenetic proteins (BMPs), collagen I, and Runx2. Cells were also capable of depositing a mineralized matrix, confirming the commitment of these cells to the osteogenic lineage. When FGFR1 activity was blocked, a reduction in cell proliferation and a coincident upregulation of Runx2 with enhanced mineralization of cultures was observed. These results indicate that FGFRs play critical roles in cell recruitment and differentiation during the process of osteogenesis in mES cells. In particular, the data indicate that FGFR1 plays a pivotal role in osteoblast lineage determination.

Stimulation of osteogenic and angiogenic ability of cells on polymers by pulsed laser deposition of uniform akermanite-glass nanolayer

Polymer biomaterials have been widely used for bone replacement/regeneration because of their unique mechanical properties and workability. Their inherent low bioactivity makes them lack osseointegration with host bone tissue. For this reason, bioactive inorganic particles have been always incorporated into the matrix of polymers to improve their bioactivity. However, mixing inorganic particles with polymers always results in inhomogeneity of particle distribution in polymer matrix with limited bioactivity. This study sets out to apply the pulsed laser deposition (PLD) technique to prepare uniform akermanite (Ca2MgSi2O7, AKT) glass nanocoatings on the surface of two polymers (non-degradable polysulfone (PSU) and degradable polylactic acid (PDLLA)) in order to improve their surface osteogenic and angiogenic activity. The results show that a uniform nanolayer composed of amorphous AKT particles (∼30nm) of thickness 130nm forms on the surface of both PSU and PDLLA films with the PLD technique. The prepared AKT-PSU and AKT-PDLLA films significantly improved the surface roughness, hydrophilicity, hardness and apatite mineralization, compared with pure PSU and PDLLA, respectively. The prepared AKT nanocoatings distinctively enhance the alkaline phosphate (ALP) activity and bone-related gene expression (ALP, OCN, OPN and Col I) of bone-forming cells on both PSU and PDLLA films. Furthermore, AKT nanocoatings on two polymers improve the attachment, proliferation, VEGF secretion and expression of proangiogenic factors and their receptors of human umbilical vein endothelial cells (HUVEC). The results suggest that PLD-prepared bioceramic nanocoatings are very useful for enhancing the physicochemical, osteogenic and angiogenic properties of both degradable and non-degradable polymers for application in bone replacement/regeneration.

Mechanical tension as a driver of connective tissue growth in vitro

We propose the progressive mechanical expansion of cell-derived tissue analogues as a novel, growth-based approach to in vitro tissue engineering. The prevailing approach to producing tissue in vitro is to culture cells in an exogenous “scaffold” that provides a basic structure and mechanical support. This necessarily pre-defines the final size of the implantable material, and specific signals must be provided to stimulate appropriate cell growth, differentiation and matrix formation. In contrast, surgical skin expansion, driven by increments of stretch, produces increasing quantities of tissue without trauma or inflammation. This suggests that connective tissue cells have the innate ability to produce growth in response to elevated tension. We posit that this capacity is maintained in vitro, and that order-of-magnitude growth may be similarly attained in self-assembling cultures of cells and their own extracellular matrix. The hypothesis that growth of connective tissue analogues can be induced by mechanical expansion in vitro may be divided into three components: (1) tension stimulates cell proliferation and extracellular matrix synthesis; (2) the corresponding volume increase will relax the tension imparted by a fixed displacement; (3) the repeated application of static stretch will produce sustained growth and a tissue structure adapted to the tensile loading. Connective tissues exist in a state of residual tension, which is actively maintained by resident cells such as fibroblasts. Studies in vitro and in vivo have demonstrated that cellular survival, reproduction, and matrix synthesis and degradation are regulated by the mechanical environment. Order-of-magnitude increases in both bone and skin volume have been achieved clinically through staged expansion protocols, demonstrating that tension-driven growth can be sustained over prolonged periods. Furthermore, cell-derived tissue analogues have demonstrated mechanically advantageous structural adaptation in response to applied loading. Together, these data suggest that a program of incremental stretch constitutes an appealing way to replicate tissue growth in cell culture, by harnessing the constituent cells’ innate mechanical responsiveness. In addition to offering a platform to study the growth and structural adaptation of connective tissues, tension-driven growth presents a novel approach to in vitro tissue engineering. Because the supporting structure is secreted and organised by the cells themselves, growth is not restricted by a “scaffold” of fixed size. This also minimises potential adverse reactions to exogenous materials upon implantation. Most importantly, we posit that the growth induced by progressive stretch will allow substantial volumes of connective tissue to be produced from relatively small initial cell numbers.

Mechanical influences on endothelial cell network formation in vitro

While both the restoration of the blood supply and an appropriate local mechanical environment are critical for uneventful bone healing, their influence on each other remains unclear. Human bone fracture haematomas (<72h post-trauma) were cultivated for 3 days in fibrin matrices, with or without cyclic compression. Conditioned medium from these cultures enhanced the formation of vessel-like networks by HMEC-1 cells, and mechanical loading further elevated it, without affecting the cells’ metabolic activity. While haematomas released the angiogenesis-regulators, VEGF and TGF-β1, their concentrations were not affected by mechanical loading. However, direct cyclic stretching of the HMEC-1 cells decreased network formation. The appearance of the networks and a trend towards elevated VEGF under strain suggested physical disruption rather than biochemical modulation as the responsible mechanism. Thus, early fracture haematomas and their mechanical loading increase the paracrine stimulation of endothelial organisation in vitro, but direct periodic strains may disrupt or impair vessel assembly in otherwise favourable conditions.