Biomechanical aspects of bone microstructure in vertebrates : potential approach to paleontological investigations

The biomechanical or biophysical principles can be applied to study biological structures in their modern or fossil form. Bone is an important tissue in paleontological studies as it is a commonly preserved element in most fossil vertebrates, and can often allow its microstructures such as lacuna and canaliculi to be studied in detail. In this context, the principles of Fluid Mechanics and Scaling Laws have been previously applied to enhance the understanding of bone microarchitecture and their implications for the evolution of hydraulic structures to transport fluid. It has been shown that the microstructure of bone has evolved to maintain efficient transport between the nutrient supply and cells, the living components of the tissue. Application of the principle of minimal expenditure of energy to this analysis shows that the path distance comprising five or six lamellar regions represents an effective limit for fluid and solute transport between the nutrient supply and cells; beyond this threshold, hydraulic resistance in the network increases and additional energy expenditure is necessary for further transportation. This suggests an optimization of the size of bone’s building blocks (such as osteon or trabecular thickness) to meet the metabolic demand concomitant to minimal expenditure of energy. This biomechanical aspect of bone microstructure is corroborated from the ratio of osteon to Haversian canal diameters and scaling constants of several mammals considered in this study. This aspect of vertebrate bone microstructure and physiology may provide a basis of understanding of the form and function relationship in both extinct and extant taxa.

Characterisation of mesenchymal cells from osteophytes in osteoarthritis

Osteophytes form through the process of chondroid metamorphosis of fibrous tissue followed by endochondral ossification. Osteophytes have been found to consist of three different mesenchymal tissue regions including endochondral bone formation within cartilage residues, intra-membranous bone formation within fibrous tissue and bone formation within bone marrow spaces. All these features provide evidence of mesenchymal stem cells (MSC) involvement in osteophyte formation; nevertheless, it remains to be characterised. MSC from numerous mesenchymal tissues have been isolated but bone marrow remains the “ideal” due to the ease of ex vivo expansion and multilineage potential. However, the bone marrow stroma has a relatively low number of MSC, something that necessitates the need for long-term culture and extensive population doublings in order to obtain a sufficient number of cells for therapeutic applications. MSC in vitro have limited proliferative capacity and extensive passaging compromises differentiation potential. To overcome this barrier, tissue derived MSC are of strong interest for extensive study and characterisation, with a focus on their potential application in therapeutic tissue regeneration. To date, no MSC type cell has been isolated from osteophyte tissue, despite this tissue exhibiting all the hallmark features of a regenerative tissue. Therefore, this study aimed to isolate and characterise cells from osteophyte tissues in relation to their phenotype, differentiation potential, immuno-modulatory properties, proliferation, cellular ageing, longevity and chondrogenesis in in vitro defect model in comparison to patient matched bone marrow stromal cells (bMSC). Osteophyte derived cells were isolated from osteophyte tissue samples collected during knee replacement surgery. These cells were characterised by the expression of cell surface antigens, differentiation potential into mesenchymal lineages, growth kinetics and modulation of allo-immune responses. Multipotential stem cells were identified from all osteophyte samples namely osteophyte derived mesenchymal stem cells (oMSC). Extensively expanded cell cultures (passage 4 and 9 respectively) were used to confirm cytogenetic stability and study signs of cellular aging, telomere length and telomerase activity. Cultured cells at passage 4 were used to determine 84 pathway focused stem cell related gene expression profile. Micro mass pellets were cultured in chondrogenic differentiation media for 21 days for phenotypic and chondrogenic related gene expression. Secondly, cell pellets differentiated overnight were placed into articular cartilage defects and cultured for further 21 days in control medium and chondrogenic medium to study chondrogenesis and cell behaviour. The surface antigen expression of oMSC was consistent with that of mesenchymal stem cells, such as lacking the haematopoietic and common leukocyte markers (CD34, CD45) while expressing those related to adhesion (CD29, CD166, CD44) and stem cells (CD90, CD105, CD73). The proliferation capacity of oMSC in culture was superior to that of bMSC, and they readily differentiated into tissues of the mesenchymal lineages. oMSC also demonstrated the ability to suppress allogeneic T-cell proliferation, which was associated with the expression of tryptophan degrading enzyme indoleamine 2,3 dioxygenase (IDO). Cellular aging was more prominent in late passage bMSC than in oMSC. oMSC had longer telomere length in late passages compared with bMSC, although there was no significant difference in telomere lengths in the early passages in either cell type. Telomerase activity was detectable only in early passage oMSC and not in bMSC. In osteophyte tissues telomerase positive cells were found to be located peri vascularly and were Stro-1 positive. Eighty-four pathway-focused genes were investigated and only five genes (APC, CCND2, GJB2, NCAM and BMP2) were differentially expressed between bMSC and oMSC. Chondrogenically induced micro mass pellets of oMSC showed higher staining intensity for proteoglycans, aggrecan and collagen II. Differential expression of chondrogenic related genes showed up regulation of Aggrecan and Sox 9 in oMSC and collagen II in bMSC. The in vitro defect models of oMSC in control medium showed rounded and aggregated cells staining positively for proteoglycan and presence of some extracellular matrix. In contrast, defects with bMSC showed fragmentation and loss of cells, fibroblast-like cell morphology staining positively for proteoglycans. For defects maintained in chondrogenic medium, rounded, aggregated and proteoglycan positive cells were found in both oMSC and bMSC cultures. Extracellular matrix and cellular integration into newly formed matrix was evident only in oMSC defects. For analysis of chondrocyte hypertrophy, strong expression of type X collagen could be noticed in the pellet cultures and transplanted bMSC. In summary, this study demonstrated that osteophyte derived cells had similar properties to mesenchymal stem cells in the expression of antigen phenotype, differential potential and suppression of allo-immune response. Furthermore, when compared to bMSC, oMSC maintained a higher proliferative capacity due to a retained level of telomerase activity in vitro, which may account for the relatively longer telomeres delaying growth arrest by replicative senescence compared with bMSC. oMSC behaviour in defects supported chondrogenesis which implies that cells derived from regenerative tissue can be an alternative source of stem cells and have a potential clinical application for therapeutic stem cell based tissue regeneration.

Runx2 overexpression in bone marrow stromal cells accelerates bone formation in critically-sized femoral defects

The repair of large non-unions in long bones remains a significant clinical problem due to high failure rates and limited tissue availability for auto- and allografts. Many cell-based strategies for healing bone defects deliver bone marrow stromal cells to the defect site to take advantage of the inherent osteogenic capacity of this cell type. However, many factors, including donor age and ex vivo expansion of the cells, cause bone marrow stromal cells to lose their differentiation ability. To overcome these limitations, we have genetically engineered bone marrow stromal cells to constitutively overexpress the osteoblast specific transcription factor Runx2. In the present study, we examined Runx2-modified bone marrow stromal cells, delivered via poly(caprolactone) scaffolds loaded with type I collagen meshes, in critically-sized segmental defects in rats compared to unmodified cells, cell-free scaffolds and empty defects. Runx2 expression in bone marrow stromal cells accelerated healing of critically-sized defects compared to unmodified bone marrow stromal cells and defects receiving cell-free treatments. These findings provide an accelerated method for healing large bone defects which may reduce recovery time and the need for external fixation of critically-sized defects.

Enhancing in vivo vascularized bone formation by cobalt chloride-treated bone marrow stromal cells in a tissue engineered periosteum model

The periosteum plays an indispensable role in both bone formation and bone defect healing. In this study we constructed an artificial in vitro periosteum by incorporating osteogenic differentiated bone marrow stromal cells (BMSCs) and cobalt chloride (CoCl(2))-treated BMSCs. The engineered periostea were implanted both subcutaneously and into skull bone defects in SCID mice to investigate ectopic and orthotopic osteogenesis and vascularization. After two weeks in subcutaneous and four weeks in bone defect areas, the implanted constructs were assessed for ectopic and orthotopic osteogenesis and vascularization by micro-CT, histomorphometrical and immunohistochemical methods. The results showed that CoCl(2) pre-treated BMSCs induced higher degree of vascularization and enhanced osteogenesis within the implants in both ectopic and orthotopic areas. This study provided a novel approach using BMSCs sourced from the same patient for both osteogenic and pro-angiogenic purposes in constructing tissue engineered periosteum to enhance vascularized osteogenesis.

Stem cell–related gene expression in clonal populations of mesenchymal stromal cells from bone marrow

Decline in the frequency of potent mesenchymal stem cells (MSCs) has been implicated in ageing and degenerative diseases. Increasing the circulating stem cell population can lead to renewed recruitment of these potent cells at sites of damage. Therefore, identifying the ideal cells for ex vivo expansion will form a major pursuit of clinical applications. This study is a follow-up of previous work that demonstrated the occurrence of fast-growing multipotential cells from the bone marrow samples. To investigate the molecular processes involved in the existence of such varying populations, gene expression studies were performed between fast- and slow-growing clonal populations to identify potential genetic markers associated with stemness using the quantitative real-time polymerase chain reaction comprising a series of 84 genes related to stem cell pathways. A group of 10 genes were commonly overrepresented in the fast-growing stem cell clones. These included genes that encode proteins involved in the maintenance of embryonic and neural stem cell renewal (sex-determining region Y-box 2, notch homolog 1, and delta-like 3), proteins associated with chondrogenesis (aggrecan and collagen 2 A1), growth factors (bone morphogenetic protein 2 and insulin-like growth factor 1), an endodermal organogenesis protein (forkhead box a2), and proteins associated with cell-fate specification (fibroblast growth factor 2 and cell division cycle 2). Expression of diverse differentiation genes in MSC clones suggests that these commonly expressed genes may confer the maintenance of multipotentiality and self-renewal of MSCs.

Osteoarthritic cartilage chondrocytes alter subchondral bone osteoblast differentiation via MAPK signalling pathway involving ERK1/2

Osteoarthritic subchondral bone is characterized by abnormal bone density and enhanced production of bone turnover markers, an indication of osteoblast dysfunction. Several studies have proposed that pathological changes in articular cartilage influence the subchondral bone changes, which are typical of the progression of osteoarthritis; however, direct evidence of this has yet to be reported. The aim of the present study was to investigate what effects articular cartilage cells, isolated from normal and osteoarthritic joints, may have on the subchondral bone osteoblast phenotype, and also the potential involvement of the mitogen activated protein kinase (MAPK) signalling pathway during this process. Our results suggest that chondrocytes isolated from a normal joint inhibited osteoblast differentiation, whereas chondrocytes isolated from an osteoarthritic joint enhanced osteoblast differentiation, both via a direct and indirect cell interaction mechanisms. Furthermore, the interaction of subchondral bone osteoblasts with osteoarthritic chondrocyte conditioned media appeared to significantly activate ERK1/2 phosphorylation. On the other hand, conditioned media from normal articular chondrocytes did not affect ERK1/2 phosphorylation. Inhibition of the MAPK–ERK1/2 pathways reversed the phenotype changes of subchondral bone osteoblast, which would otherwise be induced by the conditioned media from osteoarthritic chondrocytes. In conclusion, our findings provide evidence that osteoarthritic chondrocytes affect subchondral bone osteoblast metabolism via an ERK1/2 dependent pathway.

Addressing issues in sparseness, ecological bias and formulation of the adjacency matrix in Bayesian spatio-temporal analysis of disease counts

The main objective of this PhD was to further develop Bayesian spatio-temporal models (specifically the Conditional Autoregressive (CAR) class of models), for the analysis of sparse disease outcomes such as birth defects. The motivation for the thesis arose from problems encountered when analyzing a large birth defect registry in New South Wales. The specific components and related research objectives of the thesis were developed from gaps in the literature on current formulations of the CAR model, and health service planning requirements. Data from a large probabilistically-linked database from 1990 to 2004, consisting of fields from two separate registries: the Birth Defect Registry (BDR) and Midwives Data Collection (MDC) were used in the analyses in this thesis. The main objective was split into smaller goals. The first goal was to determine how the specification of the neighbourhood weight matrix will affect the smoothing properties of the CAR model, and this is the focus of chapter 6. Secondly, I hoped to evaluate the usefulness of incorporating a zero-inflated Poisson (ZIP) component as well as a shared-component model in terms of modeling a sparse outcome, and this is carried out in chapter 7. The third goal was to identify optimal sampling and sample size schemes designed to select individual level data for a hybrid ecological spatial model, and this is done in chapter 8. Finally, I wanted to put together the earlier improvements to the CAR model, and along with demographic projections, provide forecasts for birth defects at the SLA level. Chapter 9 describes how this is done. For the first objective, I examined a series of neighbourhood weight matrices, and showed how smoothing the relative risk estimates according to similarity by an important covariate (i.e. maternal age) helped improve the model’s ability to recover the underlying risk, as compared to the traditional adjacency (specifically the Queen) method of applying weights. Next, to address the sparseness and excess zeros commonly encountered in the analysis of rare outcomes such as birth defects, I compared a few models, including an extension of the usual Poisson model to encompass excess zeros in the data. This was achieved via a mixture model, which also encompassed the shared component model to improve on the estimation of sparse counts through borrowing strength across a shared component (e.g. latent risk factor/s) with the referent outcome (caesarean section was used in this example). Using the Deviance Information Criteria (DIC), I showed how the proposed model performed better than the usual models, but only when both outcomes shared a strong spatial correlation. The next objective involved identifying the optimal sampling and sample size strategy for incorporating individual-level data with areal covariates in a hybrid study design. I performed extensive simulation studies, evaluating thirteen different sampling schemes along with variations in sample size. This was done in the context of an ecological regression model that incorporated spatial correlation in the outcomes, as well as accommodating both individual and areal measures of covariates. Using the Average Mean Squared Error (AMSE), I showed how a simple random sample of 20% of the SLAs, followed by selecting all cases in the SLAs chosen, along with an equal number of controls, provided the lowest AMSE. The final objective involved combining the improved spatio-temporal CAR model with population (i.e. women) forecasts, to provide 30-year annual estimates of birth defects at the Statistical Local Area (SLA) level in New South Wales, Australia. The projections were illustrated using sixteen different SLAs, representing the various areal measures of socio-economic status and remoteness. A sensitivity analysis of the assumptions used in the projection was also undertaken. By the end of the thesis, I will show how challenges in the spatial analysis of rare diseases such as birth defects can be addressed, by specifically formulating the neighbourhood weight matrix to smooth according to a key covariate (i.e. maternal age), incorporating a ZIP component to model excess zeros in outcomes and borrowing strength from a referent outcome (i.e. caesarean counts). An efficient strategy to sample individual-level data and sample size considerations for rare disease will also be presented. Finally, projections in birth defect categories at the SLA level will be made.

Botanical matrix

Botanical matrix is a graphic map produced via a process involving an initial site installation (350 m contour transect), a botanical survey and photographic documentation of species. The site is a housing subdivision at Point Henry, on the SE coast of Western Australia which is a landscape which is host the most botanically diverse vegetation found worldwide - known locally as 'kwongan'. Notoriously difficult vegetation to measure and map, kwongan is a visual 'engima', for paradoxically it appears to the lay person as visually bland and highly homogenous. There is thus is a critical need for the development of new forms of representation which overcome the barriers between the perception and reality of this botanical condition. Botanical Matrix is one result of the author's research which seeks to address this important problem.

The mechanical response of the ovine lumbar anulus fibrosus to uniaxial, biaxial and shear loads

Analytical and computational models of the intervertebral disc (IVD) are commonly employed to enhance understanding of the biomechanics of the human spine and spinal motion segments. The accuracy of these models in predicting physiological behaviour of the spine is intrinsically reliant on the accuracy of the material constitutive representations employed to represent the spinal tissues. There is a paucity of detailed mechanical data describing the material response of the reinforced­ground matrix in the anulus fibrosus of the IVD. In the present study, the ‘reinforced­ground matrix’ was defined as the matrix with the collagen fibres embedded but not actively bearing axial load, thus incorporating the contribution of the fibre-fibre and fibre-matrix interactions. To determine mechanical parameters for the anulus ground matrix, mechanical tests were carried out on specimens of ovine anulus, under unconfined uniaxial compression, simple shear and biaxial compression. Test specimens of ovine anulus fibrosus were obtained with an adjacent layer of vertebral bone/cartilage on the superior and inferior specimen surface. Specimen geometry was such that there were no continuous collagen fibres coupling the two endplates. Samples were subdivided according to disc region - anterior, lateral and posterior - to determine the regional inhomogeneity in the anulus mechanical response. Specimens were loaded at a strain rate sufficient to avoid fluid outflow from the tissue and typical stress-strain responses under the initial load application and under repeated loading were determined for each of the three loading types. The response of the anulus tissue to the initial and repeated load cycles was significantly different for all load types, except biaxial compression in the anterior anulus. Since the maximum applied strain exceeded the damage strain for the tissue, experimental results for repeated loading reflected the mechanical ability of the tissue to carry load, subsequent to the initiation of damage. To our knowledge, this is the first study to provide experimental data describing the response of the ‘reinforced­ground matrix’ to biaxial compression. Additionally, it is novel in defining a study objective to determine the regionally inhomogeneous response of the ‘reinforced­ground matrix’ under an extensive range of loading conditions suitable for mechanical characterisation of the tissue. The results presented facilitate the development of more detailed and comprehensive constitutive descriptions for the large strain nonlinear elastic or hyperelastic response of the anulus ground matrix.

A hydrogen tethered inhibitor of matrix metalloproteinases

During wound repair, the balance between matrix metalloproteinases (MMPs) and their natural inhibitors (the TIMPs) is crucial for the normal extra cellular matrix turnover. However, the over expression of several MMPs including MMP-1, 2, 3, 8, 9 and MMP-10, combined with abnormally high levels of activation or low expression of TIMPs, may contribute to excessive degradation of connective tissue and formation of chronic ulcers. There are many groups exploring strategies for promoting wound healing involving delivery of growth factors, cells, ECM components and small molecules. Our approach for improving the balance of MMPs is not to add anything more to the wound, but instead to neutralise the over-expressed MMPs using inhibitors tethered to a bandage-like hydrogel. Our in vitro experiments using designed synthetic pseudo peptide inhibitors have been demonstrated to inhibit MMP activity in standard solutions. These inhibitors have also been tethered to polyethylene glycol hydrogels using a facile reaction between the linker unit on the inhibitor and the gel. After tethering the inhibition of MMPs diminishes to some extent and we postulate that this arises due to poor diffusion of the MMPs into the gels. When the tethered inhibitors were tested against chronic wound fluid obtained against patients we observed over 40% inhibition in proteolytic activity suggesting our approach may prove useful in rebalancing MMPs within chronic wounds.

Autocrine fibroblast growth factor 2 increases the multipotentiality of human adipose-derived mesenchymal stem cells

Multipotent mesenchymal stem cells (MSCs), first identified in the bone marrow, have subsequently been found in many other tissues, including fat, cartilage, muscle, and bone. Adipose tissue has been identified as an alternative to bone marrow as a source for the isolation of MSCs, as it is neither limited in volume nor as invasive in the harvesting. This study compares the multipotentiality of bone marrow-derived mesenchymal stem cells (BMSCs) with that of adipose-derived mesenchymal stem cells (AMSCs) from 12 age- and sex-matched donors. Phenotypically, the cells are very similar, with only three surface markers, CD106, CD146, and HLA-ABC, differentially expressed in the BMSCs. Although colony-forming units-fibroblastic numbers in BMSCs were higher than in AMSCs, the expression of multiple stem cell-related genes, like that of fibroblast growth factor 2 (FGF2), the Wnt pathway effectors FRAT1 and frizzled 1, and other self-renewal markers, was greater in AMSCs. Furthermore, AMSCs displayed enhanced osteogenic and adipogenic potential, whereas BMSCs formed chondrocytes more readily than AMSCs. However, by removing the effects of proliferation from the experiment, AMSCs no longer out-performed BMSCs in their ability to undergo osteogenic and adipogenic differentiation. Inhibition of the FGF2/fibroblast growth factor receptor 1 signaling pathway demonstrated that FGF2 is required for the proliferation of both AMSCs and BMSCs, yet blocking FGF2 signaling had no direct effect on osteogenic differentiation. Disclosure of potential conflicts of interest is found at the end of this article.

A peptidomimetic inhibitor of matrix metalloproteinases containing a tetherable linker group

Successful wound repair and normal turnover of the extracellular matrix relies on a balance between matrix metalloproteinases (MMPs) and their natural inhibitors (the TIMPs). When over-expression of MMPs and abnormally high levels of activation or low expression of TIMPs are encountered, excessive degradation of connective tissue and the formation of chronic ulcers can occur. One strategy to rebalance MMPs and TIMPs is to use inhibitors. We have designed a synthetic pseudopeptide inhibitor with an amine linker group based on a known high-affinity peptidomimetic MMP inhibitor have demonstrated inhibition of MMP-1, -2, -3 and -9 activity in standard solutions. The inhibitor was also tethered to a polyethylene glycol hydrogel using a facile reaction between the linker unit on the inhibitor and the hydrogel precursors. After tethering, we observed inhibition of the MMPs although there was an increase in the IC50s which was attributed to poor diffusion of the MMPs into the hydrogels, reduced activity of the tethered inhibitor or incomplete incorporation of the inhibitor into the hydrogels. When the tethered inhibitors were tested against chronic wound fluid we observed significant inhibition in proteolytic activity suggesting our approach may prove useful in rebalancing MMPs within chronic wounds.

Typical accuracy and quality control of a process for creating CT-Based virtual bone models

A pragmatic method for assessing the accuracy and precision of a given processing pipeline required for converting computed tomography (CT) image data of bones into representative three dimensional (3D) models of bone shapes is proposed. The method is based on coprocessing a control object with known geometry which enables the assessment of the quality of resulting 3D models. At three stages of the conversion process, distance measurements were obtained and statistically evaluated. For this study, 31 CT datasets were processed. The final 3D model of the control object contained an average deviation from reference values of −1.07±0.52 mm standard deviation (SD) for edge distances and −0.647±0.43 mm SD for parallel side distances of the control object. Coprocessing a reference object enables the assessment of the accuracy and precision of a given processing pipeline for creating CTbased 3D bone models and is suitable for detecting most systematic or human errors when processing a CT-scan. Typical errors have about the same size as the scan resolution.

The effect of friction on indenter force and pile-up in numerical simulations of bone nanoindentation

Nanoindentation is a useful technique for probing the mechanical properties of bone, and finite element (FE) modeling of the indentation allows inverse determination of elasto-plastic constitutive properties. However, FE simulations to date have assumed frictionless contact between indenter and bone. The aim of this study was to explore the effect of friction in simulations of bone nanoindentation. Two dimensional axisymmetric FE simulations were performed using a spheroconical indenter of tip radius 0.6m and angle 90°. The coefficient of friction between indenter and bone was varied between 0.0 (frictionless) and 0.3. Isotropic linear elasticity was used in all simulations, with bone elastic modulus E=13.56GPa and Poisson’s ratio =0.3. Plasticity was incorporated using both Drucker-Prager and von Mises yield surfaces. Friction had a modest effect on the predicted force-indentation curve for both von Mises and Drucker-Prager plasticity, reducing maximum indenter displacement by 10% and 20% respectively as friction coefficient was increased from zero to 0.3 (at a maximum indenter force of 5mN). However, friction has a much greater effect on predicted pile-up after indentation, reducing predicted pile-up from 0.27m to 0.11m with a von Mises model, and from 0.09m to 0.02m with Drucker-Prager plasticity. We conclude that it is important to include friction in nanoindentation simulations of bone.

ERK-1/2 and p38 in the Regulation of Hypertrophic Changes of Normal Articular Cartilage Chondrocytes Induced by Osteoarthritic Subchondral Osteoblasts

Objective. Previous studies have shown the influence of subchondral bone osteoblasts (SBOs) on phenotypical changes of articular cartilage chondrocytes (ACCs) during the development of osteoarthritis (OA). The molecular mechanisms involved during this process remain elusive, in particular, the signal transduction pathways. The aim of this study was to investigate the in vitro effects of OA SBOs on the phenotypical changes in normal ACCs and to unveil the potential involvement of MAPK signaling pathways during this process. Methods. Normal and arthritic cartilage and bone samples were collected for isolation of ACCs and SBOs. Direct and indirect coculture models were applied to study chondrocyte hypertrophy under the influence of OA SBOs. MAPKs in the regulation of the cell–cell interactions were monitored by phosphorylated antibodies and relevant inhibitors. Results. OA SBOs led to increased hypertrophic gene expression and matrix calcification in ACCs by means of both direct and indirect cell–cell interactions. In this study, we demonstrated for the first time that OA SBOs suppressed p38 phosphorylation and induced ERK-1/2 signal phosphorylation in cocultured ACCs. The ERK-1/2 pathway inhibitor PD98059 significantly attenuated the hypertrophic changes induced by conditioned medium from OA SBOs, and the p38 inhibitor SB203580 resulted in the up-regulation of hypertrophic genes in ACCs. Conclusion. The findings of this study suggest that the pathologic interaction of OA SBOs and ACCs is mediated via the activation of ERK-1/2 phosphorylation and deactivation of p38 phosphorylation, resulting in hypertrophic differentiation of ACCs.