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.

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.

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.

Cell sourcing for bone tissue engineering : amniotic fluid stem cells have a delayed, robust differentiation compared to mesenchymal stem cells

Cell based therapies for bone regeneration are an exciting emerging technology, but the availability of osteogenic cells is limited and an ideal cell source has not been identified. Amniotic fluid-derived stem (AFS) cells and bone-marrow derived mesenchymal stem cells (MSCs) were compared to determine their osteogenic differentiation capacity in both 2D and 3D environments. In 2D culture, the AFS cells produced more mineralized matrix but delayed peaks in osteogenic markers. Cells were also cultured on 3D scaffolds constructed of poly-e-caprolactone for 15 weeks. MSCs differentiated more quickly than AFS cells on 3D scaffolds, but mineralized matrix production slowed considerably after 5 weeks. In contrast, the rate of AFS cell mineralization continued to increase out to 15 weeks, at which time AFS constructs contained 5-fold more mineralized matrix than MSC constructs. Therefore, cell source should be taken into consideration when used for cell therapy, as the MSCs would be a good choice for immediate matrix production, but the AFS cells would continue robust mineralization for an extended period of time. This study demonstrates that stem cell source can dramatically influence the magnitude and rate of osteogenic differentiation in vitro.

Limitations and challenges in MSC delivery for bone regeneration

Objective: Regeneration of osseous defects by tissue-engineering or cell delivery approach provides a novel means of treatment utilizing cell biology, materials sciences, and molecular biology. The concept of in vitro explanted mesenchymal stem cells (MSCs) with an ability to induce new bone formation has been demonstrated in some small animal models. However, contradictory results have been reported regarding the regenerative capacity of MSCs after ex vivo expansion due to the lack of the understanding of microenvironment for MSC differentiation in vivo. ----- ----- Methods: In our laboratory tissue-derived and bone marrow-derived MSCs have been investigated in their osteogenesis. Cell morphology and proliferation were studied by microscopy, confocal microscopy, FACS and cell counting. Cell differentiation and matrix formation were analysed by matrix staining, quantitative PCR, and immunohistochemistry. A SCID skull defect model was used for cell transplantation studies.----- ----- Results: It was noted that tissue-derived and bone marrow-derived MSCs showed similar characteristics in cell surface marker expression, mesenchymal lineage differentiation potential, and cell population doubling. MSCs from both sources could initiate new bone formation in bone defects after delivery into a critical size defects. The bone forming cells were from both transplanted cells and endogenous cells from the host. Interestingly, the majority of in vitro osteogenic differentiated cells did not form new bone directly even though mineralized matrix was synthesized in vitro by MSCs. Furthermore, no new bone formation was detected when MSCs were transplanted subcutaneously.----- ----- Conclusion: This study unveiled the limitations of MSC delivery in bone regeneration and proposed that in vivo microenvironment needs to be optimized for MSC delivery in osteogenesis.

Spatial and temporal variations of mechanical properties and mineral content of the external callus during bone healing

After bone fracture, various cellular activities lead to the formation of different tissue types, which form the basis for the process of secondary bone healing. Although these tissues have been quantified by histology, their material properties are not well understood. Thus, the aim of this study is to correlate the spatial and temporal variations in the mineral content and the nanoindentation modulus of the callus formed via intramembranous ossification over the course of bone healing. Midshaft tibial samples from a sheep osteotomy model at time points of 2, 3, 6 and 9 weeks were employed. PMMA embedded blocks were used for quantitative back scattered electron imaging and nanoindentation of the newly formed periosteal callus near the cortex. The resulting indentation modulus maps show the heterogeneity in the modulus in the selected regions of the callus. The indentation modulus of the embedded callus is about 6 GPa at the early stage. At later stages of mineralization, the average indentation modulus reaches 14 GPa. There is a slight decrease in average indentation modulus in regions distant to the cortex, probably due to remodelling of the peripheral callus. The spatial and temporal distribution of mineral content in the callus tissue also illustrates the ongoing remodelling process observed from histological analysis. Most interestingly the average indentation modulus, even at 9 weeks, remains as low as 13 GPa, which is roughly 60% of that for cortical sheep bone. The decreased indentation modulus in the callus compared to cortex is due to the lower average mineral content and may be perhaps also due to the properties of the organic matrix which might be different from normal bone.

Subchondral bone osteoblasts can induce chondrocyte mineralization during osteoarthritis and this process is relevant to cartilage degradation

Pathological mineralization of articular cartilage is a characteristic feature of osteoarthritis (OA); however, the underlying mechanisms, and their relevance to cartilage degeneration, are not clear. The involvement of subchondral bone changes in OA have been reported previously with the characterization of abnormal subchondral bone mineral density (BMD), osteiod volume, altered bone mechanical parameters and an increase in bone turnover markers. A number of osteoarthritic animal models have demonstrated that subchondral bone changes often precede cartilage degeneration. In this study site specific localization of mineralization markers were detected in the OA cartilage. Chondrocytes and osteoblasts derived from OA cartilage and subchondral bone showed a significant increase in the mRNA expressions of mineralization markers. Interestingly, osteoblasts from OA subchondral bone could significantly decrease cartilage matrix expression; whereas, increase mineralization of chondrocytes (Figure 1). Osteogenic factors, such as CBFA1, ALP, and type X collagen (Col-X), were detected in chondrocytes under mineralization conditions (Figure 2). Furthermore, chondrocyte mineralization was followed by increased mRNA and protein levels of MMP-2, MMP-9 and MMP-13, all of which are detrimental to cartilage integrity in vivo. The data reported here suggests that the upregulation of subchondral bone-mineralization, typical of OA progression, causes cartilage mineralization, and that the mineralization of chondrocytes induce increased MMP levels with a subsequent degradation of the articular cartilage.

Application of Mesenchymal Stem Cells for repair and regeneration of cartilage and bone

Australian efforts to provide orthopaedic surgeons with living, load-bearing scaffolds suitable for current joint (knee and hip) replacement surgery, non-union fracture repair, and miniscal and growth plate cartilage regeneration are being lead by teams at the Institute for Medical and Veterinary Science and Women's and Children's Hospital in Adelaide; the Peter MacCallum and St Vincent's Medical Research Institutes in Melbourne; and the Mater Medical Research Institute and new Institute for Health and Biomedical Innovation at QUT, Brisbane. In each case multidisciplinary teams are attempting to develop autologous living tissue constructs, utilising mesenchymal stem cells (MSC), with the intention of effecting seamless repair and regeneration of skeletal trauma and defects. In this article we will briefly review current knowledge of the phenotypic properties of MSC and discuss the potential therapeutic applications of these cells as exemplified by their use in cartilage repair and tissue engineering based approaches to the treatment of skeletal defects.

Aggravation of ADAMTS and MMP production and ERK1/2 pathway in the interaction of osteoarthritic subchondral bone osteoblasts and articular cartilage chondrocytes : a possible pathogenic role in osteoarthritis

Introduction: Degradative enzymes, such as A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) and matrix metalloproteinases (MMPs), play key roles in osteoarthritis (OA) development. The aim of the present study was to investigate if cross-talk between subchondral bone osteoblasts (SBOs) and articular cartilage chondrocytes (ACCs) in OA alters the expression and regulation of ADAMTS5, ADAMTS4, MMP-1, MMP-2, MMP-3, MMP-8, MMP-9 and MMP-13, and also to test the possible involvement of mitogen activated protein kinase (MAPK) signaling pathway during this process. Methods: ACCs and SBOs were isolated from normal and OA patients. An in vitro co-culture model was developed to study the regulation of ADAMTS and MMPs under normal and OA joint cross-talk conditions. MAPK-ERK inhibitor, PD98059 was applied to delineate the involvement of specific pathway during this interaction process. Results: Indirect co-culture of OA SBOs with normal ACCs resulted in significantly increased expression of ADAMTS5, ADAMTS4, MMP-2, MMP-3 and MMP-9 in ACCs, whereas co-culture of OA ACCs led to increased MMP-1 and MMP-2 expression in normal SBOs. The upregulation of ADAMTS and MMPs under these conditions was correlated with activation of the MAPK-ERK1/2 signaling pathway and the addition of the MAPK-ERK inhibitor, PD98059, reversed the overexpression of ADAMTS and MMPs in co-cultures. Conclusion: In summary, we believe, these results add to the evidence that in human OA, altered bi-directional signals transmitted between SBOs and ACCs significantly impacts the critical features of both cartilage and bone by producing abnormal levels of ADAMTS and MMPs. Furthermore, we have demonstrated for the first time that this altered cross-talk was mediated by the phosphorylation of MAPK-ERK1/2 signaling pathway.

A biphasic scaffold design combined with cell sheet technology for simultaneous regeneration of alveolar bone/periodontal ligament complex

This study describes the design of a biphasic scaffold composed of a Fused Deposition Modeling scaffold (bone compartment) and an electrospun membrane (periodontal compartment) for periodontal regeneration. In order to achieve simultaneous alveolar bone and periodontal ligament regeneration a cell-based strategy was carried out by combining osteoblast culture in the bone compartment and placement of multiple periodontal ligament (PDL) cell sheets on the electrospun membrane. In vitro data showed that the osteoblasts formed mineralized matrix in the bone compartment after 21 days in culture and that the PDL cell sheet harvesting did not induce significant cell death. The cell-seeded biphasic scaffolds were placed onto a dentin block and implanted for 8 weeks in an athymic rat subcutaneous model. The scaffolds were analyzed by μCT, immunohistochemistry and histology. In the bone compartment, a more intense ALP staining was obtained following seeding with osteoblasts, confirming the μCT results which showed higher mineralization density for these scaffolds. A thin mineralized cementum-like tissue was deposited on the dentin surface for the scaffolds incorporating the multiple PDL cell sheets, as observed by H&E and Azan staining. These scaffolds also demonstrated better attachment onto the dentin surface compared to no attachment when no cell sheets were used. In addition, immunohistochemistry revealed the presence of CEMP1 protein at the interface with the dentine. These results demonstrated that the combination of multiple PDL cell sheets and a biphasic scaffold allows the simultaneous delivery of the cells necessary for in vivo regeneration of alveolar bone, periodontal ligament and cementum. © 2012 Elsevier Ltd.

Fast fourier analysis of structural organization in decellularized cartilage-on-bone laminates

Denaturation of tissues can provide a unique biological environment for regenerative medicine application only if minimal disruption of their microarchitecture is achieved during the decellularization process. The goal is to keep the structural integrity of such a construct as functional as the tissues from which they were derived. In this work, cartilage-on-bone laminates were decellularized through enzymatic, non-ionic and ionic protocols. This work investigated the effects of decellularization process on the microarchitecture of cartiligous extracellular matrix; determining the extent of how each process deteriorated the structural organization of the network. High resolution microscopy was used to capture cross-sectional images of samples prior to and after treatment. The variation of the microarchitecture was then analysed using a well defined fast Fourier image processing algorithm. Statistical analysis of the results revealed how significant the alternations among aforementioned protocols were (p < 0.05). Ranking the treatments by their effectiveness in disrupting the ECM integrity, they were ordered as: Trypsin> SDS> Triton X-100.

Biomimetic tubular nanofiber mesh and platelet rich plasma-mediated delivery of BMP-7 for large bone defect regeneration.

There is a growing need for successful bone tissue engineering strategies and advanced biomaterials that mimic the structure and function of native tissues carry great promise. Successful bone repair approaches may include an osteoconductive scaffold, osteoinductive growth factors, cells with an osteogenic potential and capacity for graft vascularisation. To increase osteoinductivity of biomaterials, the local combination and delivery of growth factors has been developed. In the present study we investigated the osteogenic effects of calcium phosphate (CaP)-coated nanofiber mesh tube-mediated delivery of BMP-7 from a PRP matrix for the regeneration of critical sized segmental bone defects in a small animal model. Bilateral full-thickness diaphyseal segmental defects were created in twelve male Lewis rats and nanofiber mesh tubes were placed around the defect. Defects received either treatment with a CaP-coated nanofiber mesh tube (n = 6), an un-coated nanofiber mesh tube (n=6) a CaP-coated nanofiber mesh tube with PRP (n=6) or a CaP-coated nanofiber mesh tube in combination with 5 μg BMP-7 and PRP (n = 6). After 12 weeks, bone volume and biomechanical properties were evaluated using radiography, microCT, biomechanical testing and histology. The results demonstrated significantly higher biomechanical properties and bone volume for the BMP group compared to the control groups. These results were supported by the histological evaluations, where BMP group showed the highest rate of bone regeneration within the defect. In conclusion, BMP-7 delivery via PRP enhanced functional bone defect regeneration, and together these data support the use of BMP-7 in the treatment of critical sized defects.

Application of high voltage, high frequency pulsed electromagnetic field on cortical bone tissue

Over the last few decades, electric and electromagnetic fields have achieved important role as stimulator and therapeutic facility in biology and medicine. In particular, low magnitude, low frequency, pulsed electromagnetic field has shown significant positive effect on bone fracture healing and some bone diseases treatment. Nevertheless, to date, little attention has been paid to investigate the possible effect of high frequency, high magnitude pulsed electromagnetic field (pulse power) on functional behaviour and biomechanical properties of bone tissue. Bone is a dynamic, complex organ, which is made of bone materials (consisting of organic components, inorganic mineral and water) known as extracellular matrix, and bone cells (live part). The cells give the bone the capability of self-repairing by adapting itself to its mechanical environment. The specific bone material composite comprising of collagen matrix reinforced with mineral apatite provides the bone with particular biomechanical properties in an anisotropic, inhomogeneous structure. This project hypothesized to investigate the possible effect of pulse power signals on cortical bone characteristics through evaluating the fundamental mechanical properties of bone material. A positive buck-boost converter was applied to generate adjustable high voltage, high frequency pulses up to 500 V and 10 kHz. Bone shows distinctive characteristics in different loading mode. Thus, functional behaviour of bone in response to pulse power excitation were elucidated by using three different conventional mechanical tests applying three-point bending load in elastic region, tensile and compressive loading until failure. Flexural stiffness, tensile and compressive strength, hysteresis and total fracture energy were determined as measure of main bone characteristics. To assess bone structure variation due to pulse power excitation in deeper aspect, a supplementary fractographic study was also conducted using scanning electron micrograph from tensile fracture surfaces. Furthermore, a non-destructive ultrasonic technique was applied for determination and comparison of bone elasticity before and after pulse power stimulation. This method provided the ability to evaluate the stiffness of millimetre-sized bone samples in three orthogonal directions. According to the results of non-destructive bending test, the flexural elasticity of cortical bone samples appeared to remain unchanged due to pulse power excitation. Similar results were observed in the bone stiffness for all three orthogonal directions obtained from ultrasonic technique and in the bone stiffness from the compression test. From tensile tests, no significant changes were found in tensile strength and total strain energy absorption of the bone samples exposed to pulse power compared with those of the control samples. Also, the apparent microstructure of the fracture surfaces of PP-exposed samples (including porosity and microcracks diffusion) showed no significant variation due to pulse power stimulation. Nevertheless, the compressive strength and toughness of millimetre-sized samples appeared to increase when the samples were exposed to 66 hours high power pulsed electromagnetic field through screws with small contact cross-section (increasing the pulsed electric field intensity) compare to the control samples. This can show the different load-bearing characteristics of cortical bone tissue in response to pulse power excitation and effectiveness of this type of stimulation on smaller-sized samples. These overall results may address that although, the pulse power stimulation can influence the arrangement or the quality of the collagen network causing the bone strength and toughness augmentation, it apparently did not affect the mineral phase of the cortical bone material. The results also confirmed that the indirect application of high power pulsed electromagnetic field at 500 V and 10 kHz through capacitive coupling method, was athermal and did not damage the bone tissue construction.

Studies of bone cell adhesion and proliferation on ion implanted titanium discs

Argon ions were implanted on titanium discs to study its effect on bone cell adhesion and proli feration. Polished titanium discs were prepared and implanted with argon ions with different doses. Afterwards the samples were sterilized using UV light, inocu lated with human bone cells and incubated. Once fixed and rinsed, image analysis has been used to quantify the number of cells attached to the titanium discs. Cell proliferation tests were also conducted after a period of 120 hours. Cell adhesion was seen to be higher with ion im planted surface. SEM analysis has shown that the cells attached spread more on ion implanted surface. The numbers of cells attached were seen to be higher on implanted surfaces; they tend to occupy wider areas with healthier cells.