The influence of fibrin based hydrogels on the chondrogenic differentiation of human bone marrow stromal cells

Mesenchymal Stem Cells (MSC) are frequently incorporated into osteochondral implants and cell seeding is often facilitated with hydrogels which exert a profound influence on the chondrogenic differentiation of MSC. An attempt was made to elucidate this effect by comparing the chondrogenic differentiation of Bone Marrow Stromal Cells (BMSC) in fibrin and fibrin alginate composites. A biphasic osteochondral model which simulated the native in vivo environment was employed in the study. In the first stage of the experiment, BMSC was encapsulated in fibrin, Fibrin Alginate 0.3% (FA0.3) and 0.6% (FA0.6). Chondrogenic differentiation within these cell-hydrogel pellets was compared against that of standard cell pellets under inductive conditions and the matrices which supported chondrogenesis were used in the cartilage phase of biphasic constructs. Neo-cartilage growth was monitored in these cocultures. It was observed that hydrogel encapsulation influenced mesenchymal condensation which preceded chondrogenic differentiation. Early cell agglomeration was observed in fibrin as compared to fibrin alginate composites. These fibrin encapsulated cells differentiated into chondrocytes which secreted aggrecan and collagen II. When the alginate content rose from 0.3 to 0.6%, chondrogenic differentiation declined with a reduction in the expression of collagen II and aggrecan. Fibrin and FA0.3 were tested in the cartilage phase of the biphasic osteochondral constructs and the former supported superior cartilage growth with higher cellularity, total Glycosaminoglycan (GAG) and collagen II levels. The FA0.3 cartilage phase was found to be fragmented and partially calcified. The use of fibrin for cartilage repair was advocated as it facilitated BMSC chondrogenesis and cartilaginous growth in an osteochondral environment.

Influences of age and mechanical stability on volume, microstructure, and mineralization of the fracture callus during bone healing : Is osteoclast activity the key to age-related impaired healing?

Earlier studies have shown that the influence of fixation stability on bone healing diminishes with advanced age. The goal of this study was to unravel the relationship between mechanical stimulus and age on callus competence at a tissue level. Using 3D in vitro micro-computed tomography derived metrics, 2D in vivo radiography, and histology, we investigated the influences of age and varying fixation stability on callus size, geometry, microstructure, composition, remodeling, and vascularity. Compared were four groups with a 1.5-mm osteotomy gap in the femora of Sprague–Dawley rats: Young rigid (YR), Young semirigid (YSR), Old rigid (OR), Old semirigid (OSR). Hypothesis was that calcified callus microstructure and composition is impaired due to the influence of advanced age, and these individuals would show a reduced response to fixation stabilities. Semirigid fixations resulted in a larger ΔCSA (Callus cross-sectional area) compared to rigid groups. In vitro μCT analysis at 6 weeks postmortem showed callus bridging scores in younger animals to be superior than their older counterparts (pb0.01). Younger animals showed (i) larger callus strut thickness (pb0.001), (ii) lower perforation in struts (pb0.01), and (iii) higher mineralization of callus struts (pb0.001). Callus mineralization was reduced in young animals with semirigid fracture fixation but remained unaffected in the aged group. While stability had an influence, age showed none on callus size and geometry of callus. With no differences observed in relative osteoid areas in the callus ROI, old as well as semirigid fixated animals showed a higher osteoclast count (pb0.05). Blood vessel density was reduced in animals with semirigid fixation (pb0.05). In conclusion, in vivo monitoring indicated delayed callus maturation in aged individuals. Callus bridging and callus competence (microstructure and mineralization) were impaired in individuals with an advanced age. This matched with increased bone resorption due to higher osteoclast numbers. Varying fixator configurations in older individuals did not alter the dominant effect of advanced age on callus tissue mineralization, unlike in their younger counterparts. Age-associated influences appeared independent from stability. This study illustrates the dominating role of osteoclastic activity in age-related impaired healing, while demonstrating the optimization of fixation parameters such as stiffness appeared to be less effective in influencing healing in aged individuals.

Mineralized human primary osteoblast matrices as a model system to analyse interactions of prostate cancer cells with the bone microenvironment

Prostate cancer metastasis is reliant on the reciprocal interactions between cancer cells and the bone niche/micro-environment. The production of suitable matrices to study metastasis, carcinogenesis and in particular prostate cancer/bone micro-environment interaction has been limited to specific protein matrices or matrix secreted by immortalised cell lines that may have undergone transformation processes altering signaling pathways and modifying gene or receptor expression. We hypothesize that matrices produced by primary human osteoblasts are a suitable means to develop an in vitro model system for bone metastasis research mimicking in vivo conditions. We have used a decellularized matrix secreted from primary human osteoblasts as a model for prostate cancer function in the bone micro-environment. We show that this collagen I rich matrix is of fibrillar appearance, highly mineralized, and contains proteins, such as osteocalcin, osteonectin and osteopontin, and growth factors characteristic of bone extracellular matrix (ECM). LNCaP and PC3 cells grown on this matrix, adhere strongly, proliferate, and express markers consistent with a loss of epithelial phenotype. Moreover, growth of these cells on the matrix is accompanied by the induction of genes associated with attachment, migration, increased invasive potential, Ca2+ signaling and osteolysis. In summary, we show that growth of prostate cancer cells on matrices produced by primary human osteoblasts mimics key features of prostate cancer bone metastases and thus is a suitable model system to study the tumor/bone micro-environment interaction in this disease.

Cell interactions of osteoarthritic subchondral bone osteoblasts and articular chondrocytes aggravate MMP-2 and MMP-9 production through the mediation of ERK1/2 and JNK phosphorylation

Matrix Metalloproteinases (MMP) play a key role in osteoarthritis (OA) development. The aim of the present study was to investigate whether, the cross-talk between subchondral bone osteoblasts (SBOs) and articular cartilage chondrocytes (ACCs) in OA alters the expression and regulation of MMPs, and also to test the potential involvement of mitogen activated protein kinase (MAPK) signalling pathway during this process.

Bone densitometry for the assessment of osteoporosis

The primary clinical role of the non-invasive physical measurement of a bone, generally referred to as ‘bone densitometry,’ is to identify those subjects at risk of an osteoporotic fracture and their subsequent response to pharmaceutical intervention. The true ‘gold standard’ measurement of the mechanical integrity of a bone, and hence its fracture load, is a destructive test, generally performed by compressing either a regular shaped sample or whole bone.

Cross-talk of subchondral bone osteoblasts and articular cartilage chondrocytes : a new insight in understanding osteoarthritis pathogenesis

Osteoarthritis (OA) is the most common musculoskeletal disorder and represents a major health burden to society. In the course of the pathological development of OA, articular cartilage chondrocytes (ACCs) undergo atypical phenotype changes characterized by the expression of hypertrophic differentiation markers. Also, the adjacent subchondral bone shows signs of abnormal mineral density and enhanced production of bone turnover markers, indicative of osteoblast dysfunction. Collectively these findings indicate that the pathological changes typical of OA, involve alterations of the phenotypic properties of cells in both the subchondral bone and articular cartilage. However, the mechanism(s) by which these changes occur during OA development are not completely understood. The purpose of this project was to address the question of how subchondral bone osteoblasts (SBOs) and ACCs interact with each other with respect to regulation of respective cells’ phenotypic properties and in particular the involvement of mitogen activated protein kinase (MAPK) signalling pathways under normal and OA joint condition. We also endeavoured to test the influence of cross-talk between SBOs and ACCs isolated from normal and OA joint on matrix metalloproteinase (MMP) expression. For this purpose tissues from the knees of OA patients and normal controls were collected to isolate SBOs and ACCs. The cellular cross-talk of SBOs and ACCs were studied by means of both direct and indirect co-culture systems, which made it possible to identify the role of both membrane bound and soluble factors. Histology, immunohistochemistry, qRT-PCR, zymography, ELISA and western blotting were some of the techniques applied to distinguish the changes in the co-cultured vs. non co-cultured cells. The MAPK signalling pathways were probed by using targeted MAPK inhibitors, and their activity monitored by western blot analysis using phospho MAPK specific antibodies. Our co-culture studies demonstrated that OA ACCs enhanced the SBOs differentiation compared to normal ACCs. We demonstrated that OA ACCs induced these phenotypic changes in the SBOs via activating an ERK1/2 signalling pathway. The findings from this study thus provided clear evidence that OA ACCs play an integral role in altering the SBO phenotype. In the second study, we tested the influence of normal SBOs and OA SBOs on ACCs phenotype changes. The results showed that OA SBOs increased the hypertrophic gene expression in co-cultured ACCs compared to normal SBOs, a phenotype which is considered as pathological to the health and integrity of articular cartilage. It was demonstrated that these phenotype changes occurred via de-activation of p38 and activation of ERK1/2 signaling pathways. These findings suggest that the pathological interaction of OA SBOs with ACCs is mediated by cross-talking between ERK1/2 and p38 pathways, resulting in ACCs undergoing hypertrophic differentiation. Subsequent experiments to determine the effect on MMP regulation, of SBOs and ACCs cross-talk, revealed that co-culturing OA SBOs with ACCs significantly enhanced the proteolytic activity and expression of MMP-2 and MMP-9. In turn, co-culture of OA ACCs with SBOs led to abundant MMP-2 expression in SBOs. Furthermore, we showed that the addition of ERK1/2 and JNK inhibitors reversed the elevated MMP-2 and MMP-9 production which otherwise resulted from the interactions of OA SBOs-ACCs. Thus, this study has demonstrated that the altered interactions between OA SBOs-ACCs are capable of triggering the pathological pathways leading to degenerative changes seen in the osteoarthritic joint. In conclusion, the body of work presented in this dissertation has given clear in vitro evidence that the altered bi-directional communication of SBOs and ACCs may play a role in OA development and that this process was mediated by MAPK signalling pathways. Targeting these altered interactions by the use of MAPK inhibitors may provide the scientific rationale for the development of novel therapeutic strategies in the treatment and management of OA.

Ovine bone and marrow derived progenitor cells : isolation, characterization, and osteogenic potential

Recently, research has focused on bone marrow derived multipotent mesenchymal precursor cells (MPC) for their potential clinical use in bone engineering. Prior to clinical application, MPC-based treatment concepts need to be evaluated in preclinical, immunocompetent, large animal models. Sheep in particular are considered a valid model for orthopaedic and trauma related research. However, ovine MPC and their osteogenic potential remain poorly characterized. In the present study, ex vivo expanded MPC isolated from ovine bone marrow proliferated at a higher rate than osteoblasts (OB) derived from tibial compact bone as assessed using standard 2D culture. MPC expressed the respective phenotypic profile typical for different mesenchymal cell populations (CD14-/CD31-/CD45- /CD29+/CD44+/CD166+) and showed a multilineage differentiation potential. When compared to OB, MPC had a higher mineralization potential under standard osteogenic culture conditions and expressed typical markers such as osteocalcin, osteonectin and type I collagen at the mRNA and protein level. After 4 weeks in 3D culture, MPC constructs demonstrated higher cell density and mineralization, whilst cell viability on the scaffolds was assessed >90%. Cells displayed a spindle-like morphology and formed an interconnected network. Implanted subcutaneously into NOD/SCID mice on type I collagen coated polycaprolactone-tricalciumphosphate (mPCL-TCP) scaffolds, MPC presented a higher developmental potential than osteoblasts. In summary, this study provides a detailed in vitro characterisation of ovine MPC from a bone engineering perspective and suggests that MPC provide promising means for future bone disease related treatment applications.

Resorbable composite scaffolds for craniofacial bone tissue engineering

Aim: Bone loss associated with trauma, osteo-degenerative diseases and tumors has tremendous socioeconomic impact related to personal and occupation disability and health care costs. In the present climate of increasing life expectancy with an ensuing increase in bone-related injuries, orthopaedic surgery is undergoing a paradigm shift from bone-grafting to bone engineering, where a scaffold is implanted to provide adequate load bearing and enhance tissue regeneration. We aim to develop composite scaffolds for bone tissue engineering applications to replace the current gold standard of autografting. ---------- Methods: Medical grade polycaprolactone-tricalcium phosphate (mPCL/TCP) scaffolds (80/20 wt%) were custom made using fused deposition modelling to produce 1x1.5x2 cm sized implants for critical-sized pig cranial implantations, empty defects were used as a control. Autologous bone marrow stromal cells (BMSCs) were extracted and precultured for 2 weeks, dispersed within fibrin glue and injected during scaffold implantation. After 2 years, microcomputed tomography and histology were used to assess bone regenerative capabilities of cell versus cell-free scaffolds. ---------- Results: Extensive bone regeneration was evident throughout the entire scaffold. Clear osteocytes embedded within mineralised matrix and active osteoblasts present around scaffold struts were observed. Cell groups performed better than cell-free scaffolds. ---------- Conclusions: Bone regeneration within defects which cannot heal unassisted can be achieved using mPCL/TCP scaffolds. This is improved by the inclusion of autogenous BMSCs. Further work will include the inclusion of growth factors including BMP-2, VEGF and PDGF to provide multifunctional scaffolds, where the three-dimensional (3D) template itself acts as a biomimetic, programmable and multi-drug delivery device.

Resorbable composite scaffolds for bone tissue engineering

Bone loss associated with trauma osteo-degenerative diseases and tumors has tremendous socioeconomic impact related to personal and occupation disability and health care costs. Bone grafting is often critical to surgical therapies. Autogenous bone is presently the preferred grafting material; however, this holds several disadvantages such as donor site morbidity. In the present climate of increasing life expectancy with an ensuing increase in bone-related injuries, orthopaedic surgery is undergoing a paradigm shift from bone-grafting to bone engineering, where a scaffold is implanted to provide adequate load bearing and enhance tissue regeneration. Our group at Queensland University of Technology (QUT) have developed, characterised and tested polycaprolactone/ tricalcium phosphate (PCL/TCP) composite scaffolds for low load-bearing bone defects. These scaffolds are being further developed for application in higher load bearing sites. Our approach emphasizes the importance of the biomaterials’ structural design, the scaffold architecture and structural and nutritional requirements for cell culture. These first-generation scaffolds made from medical grade PCL (mPCL) have been studied for more than 5 years within a clinical setting 1. This paper describes the application of second-generation scaffolds in small and large animal bone defect models and the ensuing bone regeneration as shown by histology and µCT.

Adapting to the work-life interface : the influence of individual differences, work and family on well-being, mental health and work engagement

Bronfenbrenner.s Bioecological Model, expressed as the developmental equation, D f PPCT, is the theoretical framework for two studies that bring together diverse strands of psychology to study the work-life interface of working adults. Occupational and organizational psychology is focused on the demands and resources of work and family, without emphasising the individual in detail. Health and personality psychology examine the individual but without emphasis on the individual.s work and family roles. The current research used Bronfenbrenner.s theoretical framework to combine individual differences, work and family to understand how these factors influence the working adult.s psychological functioning. Competent development has been defined as high well-being (measured as life satisfaction and psychological well-being) and high work engagement (as work vigour, work dedication and absorption in work) and as the absence of mental illness (as depression, anxiety and stress) and the absence of burnout (as emotional exhaustion, cynicism and professional efficacy). Study 1 and 2 were linked, with Study 1 as a cross-sectional survey and Study 2, a prospective panel study that followed on from the data used in Study1. Participants were recruited from a university and from a large public hospital to take part in a 3-wave, online study where they completed identical surveys at 3-4 month intervals (N = 470 at Time 1 and N = 198 at Time 3). In Study 1, hierarchical multiple regressions were used to assess the effects of individual differences (Block 1, e.g. dispositional optimism, coping self-efficacy, perceived control of time, humour), work and family variables (Block 2, e.g. affective commitment, skill discretion, work hours, children, marital status, family demands) and the work-life interface (Block 3, e.g. direction and quality of spillover between roles, work-life balance) on the outcomes. There were a mosaic of predictors of the outcomes with a group of seven that were the most frequent significant predictors and which represented the individual (dispositional optimism and coping self-efficacy), the workplace (skill discretion, affective commitment and job autonomy) and the work-life interface (negative work-to-family spillover and negative family-to-work spillover). Interestingly, gender and working hours were not important predictors. The effects of job social support, generally and for work-life issues, perceived control of time and egalitarian gender roles on the outcomes were mediated by negative work-to-family spillover, particularly for emotional exhaustion. Further, the effect of negative spillover on depression, anxiety and work engagement was moderated by the individual.s personal and workplace resources. Study 2 modelled the longitudinal relationships between the group of the seven most frequent predictors and the outcomes. Using a set of non-nested models, the relative influences of concurrent functioning, stability and change over time were assessed. The modelling began with models at Time 1, which formed the basis for confirmatory factor analysis (CFA) to establish the underlying relationships between the variables and calculate the composite variables for the longitudinal models. The CFAs were well fitting with few modifications to ensure good fit. However, using burnout and work engagement together required additional analyses to resolve poor fit, with one factor (representing a continuum from burnout to work engagement) being the only acceptable solution. Five different longitudinal models were investigated as the Well-Being, Mental Distress, Well-Being-Mental Health, Work Engagement and Integrated models using differing combinations of the outcomes. The best fitting model for each was a reciprocal model that was trimmed of trivial paths. The strongest paths were the synchronous correlations and the paths within variables over time. The reciprocal paths were more variable with weak to mild effects. There was evidence of gain and loss spirals between the variables over time, with a slight net gain in resources that may provide the mechanism for the accumulation of psychological advantage over a lifetime. The longitudinal models also showed that there are leverage points at which personal, psychological and managerial interventions can be targeted to bolster the individual and provide supportive workplace conditions that also minimise negative spillover. Bronfenbrenner.s developmental equation has been a useful framework for the current research, showing the importance of the person as central to the individual.s experience of the work-life interface. By taking control of their own life, the individual can craft a life path that is most suited to their own needs. Competent developmental outcomes were most likely where the person was optimistic and had high self-efficacy, worked in a job that they were attached to and which allowed them to use their talents and without too much negative spillover between their work and family domains. In this way, individuals had greater well-being, better mental health and greater work engagement at any one time and across time.

Adult human articular chondrocytes in a microcarrier-based culture system : expansion and redifferentiation

xpanding human chondrocytes in vitro while maintaining their ability to form cartilage remains a key challenge in cartilage tissue engineering. One promising approach to address this is to use microcarriers as substrates for chondrocyte expansion. While microcarriers have shown beneficial effects for expansion of animal and ectopic human chondrocytes, their utility has not been determined for freshly isolated adult human articular chondrocytes. Thus, we investigated the proliferation and subsequent chondrogenic differentiation of these clinically relevant cells on porous gelatin microcarriers and compared them to those expanded using traditional monolayers. Chondrocytes attached to microcarriers within 2 days and remained viable over 4 weeks of culture in spinner flasks. Cells on microcarriers exhibited a spread morphology and initially proliferated faster than cells in monolayer culture, however, with prolonged expansion they were less proliferative. Cells expanded for 1 month and enzymatically released from microcarriers formed cartilaginous tissue in micromass pellet cultures, which was similar to tissue formed by monolayer-expanded cells. Cells left attached to microcarriers did not exhibit chondrogenic capacity. Culture conditions, such as microcarrier material, oxygen tension, and mechanical stimulation require further investigation to facilitate the efficient expansion of clinically relevant human articular chondrocytes that maintain chondrogenic potential for cartilage regeneration applications.

Differences between in vitro viability and differentiation and in vivo bone-forming efficacy of human mesenchymal stem cells cultured on PCL-TCP scaffolds

Human mesenchymal stem cells (hMSCs) possess great therapeutic potential for the treatment of bone disease and fracture non-union. Too often however, in vitro evidence alone of the interaction between hMSCs and the biomaterial of choice is used as justification for continued development of the material into the clinic. Clearly for hMSC-based regenerative medicine to be successful for the treatment of orthopaedic trauma, it is crucial to transplant hMSCs with a suitable carrier that facilitates their survival, optimal proliferation and osteogenic differentiation in vitro and in vivo. This motivated us to evaluate the use of polycaprolactone-20% tricalcium phosphate (PCL-TCP) scaffolds produced by fused deposition modeling for the delivery of hMSCs. When hMSCs were cultured on the PCL-TCP scaffolds and imaged by a combination of phase contrast, scanning electron and confocal laser microscopy, we observed five distinct stages of colonization over a 21-day period that were characterized by cell attachment, spreading, cellular bridging, the formation of a dense cellular mass and the accumulation of a mineralized extracellular matrix when induced with osteogenic stimulants. Having established that PCL-TCP scaffolds are able to support hMSC proliferation and osteogenic differentiation, we next tested the in vivo efficacy of hMSC-loaded PCL-TCP scaffolds in nude rat critical-sized femoral defects. We found that fluorescently labeled hMSCs survived in the defect site for up to 3 weeks post-transplantation. However, only 50% of the femoral defects treated with hMSCs responded favorably as determined by new bone volume. As such, we show that verification of hMSC viability and differentiation in vitro is not sufficient to predict the efficacy of transplanted stem cells to consistently promote bone formation in orthotopic defects in vivo.

Physical function and health-related quality of life of older adults undergoing hospital rehabilitation : how strong is the association?

The purpose of this research is to report preliminary empirical evidence regarding the association between common physical performance measures and health-related quality of life (HRQoL) of hospitalized older adults recovering from illness and injury. Frequently, these patients do not return to premorbid levels of independence and physical ability. Rehabilitation for this population often focuses on improving physical functioning and mobility with the intention of maximizing their HRQoL for discharge and thereafter. For this reason, longitudinal use of physical performance measures as an indicator of improvement in physical functioning (and thus HRQoL) is common. Although this is a logical approach, there have been mixed results from previous investigations into the association between common measures of physical function and HRQoL amongst other adult patient populations.1,2 There has been no previous investigation reporting the association between HRQoL and a variety of common physical performance measures in hospitalized older adults.

Ovine cortical osteoblasts outperform bone marrow cells in an ectopic bone assay

Reviewing the available literature, one could conclude that marrow-derived mesenchymal stem cells (BMSCs) are the ‘gold standard’ source for bone tissue engineering applications, due to their multilineage differentiation potential and easy accessibility. However, comprehensive studies comparing their osteogenic potential with bone-derived osteoblasts (OBs) to justify the preferred application of BMSCs based on performance are few. To address these shortfalls, in the present study, ovine BMSCs and OBs seeded onto scaffolds were characterized in vitro and transplanted subcutaneously into NOD/SCID mice in combination with and without recombinant human bone morphogenetic protein 7 (rhBMP-7). It was hypothesized that cell origin, ossification type and degree of vascularization and ossification depends on the nature and commitment of transplanted cells and stimulating growth factors, such as rhBMP-7. After retrieval, specimens were analysed by biomechanical testing, µCT analysis, scanning electron microscopy/energy-dispersive X-ray spectroscopy and histo- and immunohistochemistry for osteocalcin, type II collagen and BrdU. The results showed a high degree of cell survival and proliferation ectopically, resulting in active contribution to endochondral osteogenesis. When compared to BMSCs, OBs showed a higher degree of bone deposition while OB-derived bone was of higher maturation. Stimulation with rhBMP-7 increased the rate of bone synthesis for both BMSCs and OBs, additionally promoting neovascularization and osteoclast activity. These results suggest that the origin and commitment of transplanted cells highly influence the type and degree of ossification, that rhBMP-7 represents a powerful adjuvant for bone tissue-engineering applications, and that mature bone is an adequate alternative cell source for bone tissue-engineering applications.

Bone tissue engineering

Bone is a complex, living, constantly changing tissue. Bone consists of cancellous and cortical bone. This architecture allows the skeleton to perform its essential mechanical functions.