Subchondral bone response to injected adipose-derived stromal cells for treating osteoarthritis using an experimental rabbit model

Although articular cartilage is the target of osteoarthritis (OA), its deterioration is not always clearly associated with patient symptoms. Because a functional interaction between cartilage and bone is crucial, the pathophysiology of OA and its treatment strategy must focus also on subchondral bone. We investigated whether adipose-derived stromal cells (ASCs) injected into a joint at two different concentrations could prevent subchondral bone damage after the onset of mild OA in a rabbit model. We measured both volumetric and densitometric aspects of bone remodeling. Although OA can stimulate bone remodeling either catabolically or anabolically over time, the accelerated turnover does not allow complete mineralization of new bone and therefore gradually reduces its density. We measured changes in morphometric and densitometric bone parameters using micro-CT analysis and correlated them with the corresponding parameters in cartilage and meniscus. We found that ASCs promoted cartilage repair and helped counteract the accelerated bone turnover that occurs with OA.     

Subchondral bone and ligament changes precede cartilage degradation in guinea pig osteoarthritis

There is increasing recognition that osteoarthritis (OA) is a complex disease involving the whole synovial joint, rather than the articular cartilage alone, however its aetiology and pathogenesis is not understood. Our initial studies revealed elevated turnover of bone and ligament collagen in human and mouse OA, respectively. To investigate the relative appearance of pathology in cartilage, bone and ligament, we studied the progression of spontaneous OA in the Dunkin-Hartley (DH) guinea pig knee, and compared with age-matched control Bristol Strain 2 (BS2) knees. The classical radiographic OA score of the DH knees compared to BS2 knees was 2-fold higher at 24 weeks of age. The patella perimeter and subchondral bone density was significantly greater in the DHs at 24 and 36 weeks compared to BS2. The femoral intercondylar notch width was found to be significantly lower in the DHs at 24 and 36 weeks, compared to BS2, indicating bone remodelling at the cruciate ligament (CL) insertion site. We found significantly greater laxity of the DH anterior CL at 12, 16 and 20 weeks compared to BS2. This elevated laxity was associated with increased remodelling of the CLs, based on markers of collagen turnover, and occurred prior to bone and cartilage pathology. We propose that the laxity of the CL leads to remodelling of the subchondral bone, and intercondylar notch, due to a change in load through the joint. Remodelling of the CLs and bone occurs prior to and concomitant with histopathological changes in the articular cartilage respectively, demonstrating the fundamental role of the ligament and subchondral bone in the aetiology of knee OA.     

Subchondral bone in osteoarthritis: insight into risk factors and microstructural changes

Osteoarthritis (OA) is a major cause of disability in the adult population. As aprogressive degenerative joint disorder, OA is characterized by cartilage damage,changes in the subchondral bone, osteophyte formation, muscle weakness, andinflammation of the synovium tissue and tendon. Although OA has long been viewed as aprimary disorder of articular cartilage, subchondral bone is attracting increasingattention. It is commonly reported to play a vital role in the pathogenesis of OA.Subchondral bone sclerosis, together with progressive cartilage degradation, iswidely considered as a hallmark of OA. Despite the increase in bone volume fraction,subchondral bone is hypomineralized, due to abnormal bone remodeling. Somehistopathological changes in the subchondral bone have also been detected, includingmicrodamage, bone marrow edema-like lesions and bone cysts. This review summarizesbasic features of the osteochondral junction, which comprises subchondral bone andarticular cartilage. Importantly, we discuss risk factors influencing subchondralbone integrity. We also focus on the microarchitectural and histopathological changesof subchondral bone in OA, and provide an overview of their potential contribution tothe progression of OA. A hypothetical model for the pathogenesis of OA isproposed.     

Reduced expression of PMCA1 is associated with increased blood pressure with age which is preceded by remodelling of resistance arteries

Hypertension is a well‐established risk factor for adverse cardiovascular events, and older age is a risk factor for the development of hypertension. Genomewide association studies have linked ATP2B1, the gene for the plasma membrane calcium ATPase 1 (PMCA1), to blood pressure (BP) and hypertension. Here, we present the effects of reduction in the expression of PMCA1 on BP and small artery structure and function when combined with advancing age. Heterozygous PMCA1 null mice (PMCA1^Ht) were generated and conscious BP was measured at 6 to 18 months of age. Passive and active properties of isolated small mesenteric arteries were examined by pressure myography. PMCA1^Ht mice exhibited normal BP at 6 and 9 months of age but developed significantly elevated BP when compared to age‐matched wild‐type controls at ≥12 months of age. Decreased lumen diameter, increased wall thickness and increased wall:lumen ratio were observed in small mesenteric arteries from animals 9 months of age and older, indicative of eutrophic remodelling. Increases in mesenteric artery intrinsic tone and global intracellular calcium were evident in animals at both 6 and 18 months of age. Thus, decreased expression of PMCA1 is associated with increased BP when combined with advancing age. Changes in arterial structure precede the elevation of BP. Pathways involving PMCA1 may be a novel target for BP regulation in the elderly.     

Improving the damage accumulation in a biomechanical bone remodelling model

We extend, reformulate and analyse a phenomenological model for bone remodelling. The original macrobiomechanical model (MBM), proposed by Hazelwood et al. [J Biomech 2001; 34:299–308], couples a population equation for the cellular activities of the basic multicellular units (BMUs) in the bone and a rate equation to account for microdamage and repair. We propose to account for bone failure under severe overstressing by incorporating a Paris-like power-law damage accumulation term. The extended model agrees with the Hazelwood et al. predictions when the bone is under-stressed, and allows for suitably loaded bones to fail, in agreement with other MBM and experimental data regarding damage by fatigue. We numerically solve the extended model using a convergent algorithm and show that for unchanging loads, the stationary solution captures fully the model behaviour. We compute and analyse the stationary solutions. Our analysis helps guide additional extensions to this and other BMU activity based models.     

Gene expression analyses of subchondral bone in early experimental osteoarthritis by microarray

Osteoarthritis (OA) is a degenerative joint disease that affects both cartilage and bone. A better understanding of the early molecular changes in subchondral bone may help elucidate the pathogenesis of OA. We used microarray technology to investigate the time course of molecular changes in the subchondral bone in the early stages of experimental osteoarthritis in a rat model. We identified 2,234 differentially expressed (DE) genes at 1 week, 1,944 at 2 weeks and 1,517 at 4 weeks post-surgery. Further analyses of the dysregulated genes indicated that the events underlying subchondral bone remodeling occurred sequentially and in a time-dependent manner at the gene expression level. Some of the identified dysregulated genes that were identified have suspected roles in bone development or remodeling; these genes include Alp, Igf1, Tgf β1, Postn, Mmp3, Tnfsf11, Acp5, Bmp5, Aspn and Ihh. The differences in the expression of these genes were confirmed by real-time PCR, and the results indicated that our microarray data accurately reflected gene expression patterns characteristic of early OA. To validate the results of our microarray analysis at the protein level, immunohistochemistry staining was used to investigate the expression of Mmp3 and Aspn protein in tissue sections. These analyses indicate that Mmp3 protein expression completely matched the results of both the microarray and real-time PCR analyses; however, Aspn protein expression was not observed to differ at any time. In summary, our study demonstrated a simple method of separation of subchondral bone sample from the knee joint of rat, which can effectively avoid bone RNA degradation. These findings also revealed the gene expression profiles of subchondral bone in the rat OA model at multiple time points post-surgery and identified important DE genes with known or suspected roles in bone development or remodeling. These genes may be novel diagnostic markers or therapeutic targets for OA.     

Visfatin aggravates transverse aortic constriction-induced cardiac remodelling by enhancing macrophage-mediated oxidative stress in mice

Previous studies have reported that visfatin can regulate macrophage polarisation, which has been demonstrated to participate in cardiac remodelling. The aims of this study were to investigate whether visfatin participates in transverse aortic constriction (TAC)-induced cardiac remodelling by regulating macrophage polarisation. First, TAC surgery and angiotensin II (Ang II) infusion were used to establish a mouse cardiac remodelling model, visfatin expression was measured, and the results showed that TAC surgery or Ang II infusion increased visfatin expression in the serum and heart in mice, and phenylephrine or hydrogen peroxide promoted the release of visfatin from macrophages in vitro. All these effects were dose-dependently reduced by superoxide dismutase. Second, visfatin was administered to TAC mice to observe the effects of visfatin on cardiac remodelling. We found that visfatin increased the cross-sectional area of cardiomyocytes, aggravated cardiac fibrosis, exacerbated cardiac dysfunction, further regulated macrophage polarisation and aggravated oxidative stress in TAC mice. Finally, macrophages were depleted in TAC mice to investigate whether macrophages mediate the regulatory effect of visfatin on cardiac remodelling, and the results showed that the aggravating effects of visfatin on oxidative stress and cardiac remodelling were abrogated. Our study suggests that visfatin enhances cardiac remodelling by promoting macrophage polarisation and enhancing oxidative stress. Visfatin may be a potential target for the prevention and treatment of clinical cardiac remodelling.     

A multiscale mechanobiological model of bone remodelling predicts site-specific bone loss in the femur during osteoporosis and mechanical disuse

We propose a multiscale mechanobiological model of bone remodelling to investigate the site-specific evolution of bone volume fraction across the midshaft of a femur. The model includes hormonal regulation and biochemical coupling of bone cell populations, the influence of the microstructure on bone turnover rate, and mechanical adaptation of the tissue. Both microscopic and tissue-scale stress/strain states of the tissue are calculated from macroscopic loads by a combination of beam theory and micromechanical homogenisation. This model is applied to simulate the spatio-temporal evolution of a human midshaft femur scan subjected to two deregulating circumstances: (i) osteoporosis and (ii) mechanical disuse. Both simulated deregulations led to endocortical bone loss, cortical wall thinning and expansion of the medullary cavity, in accordance with experimental findings. Our model suggests that these observations are attributable to a large extent to the influence of the microstructure on bone turnover rate. Mechanical adaptation is found to help preserve intracortical bone matrix near the periosteum. Moreover, it leads to non-uniform cortical wall thickness due to the asymmetry of macroscopic loads introduced by the bending moment. The effect of mechanical adaptation near the endosteum can be greatly affected by whether the mechanical stimulus includes stress concentration effects or not.     

Tissue remodelling and increased DNA damage in patients with incompetent valves in chronic venous insufficiency

Chronic venous insufficiency (CVI), in which blood return to the heart is impaired, is a prevalent condition worldwide. Valve incompetence is a complication of CVI that results in blood reflux, thereby aggravating venous hypertension. While CVI has a complex course and is known to produce alterations in the vein wall, the underlying pathological mechanisms remain unclear. This study examined the presence of DNA damage, pro-inflammatory cytokines and extracellular matrix remodelling in CVI-related valve incompetence. One hundred and ten patients with CVI were reviewed and divided into four groups according to age (<50 and ≥50 years) and a clinical diagnosis of venous reflux indicating venous system valve incompetence (R) (n = 81) or no reflux (NR) (n = 29). In vein specimens (greater saphenous vein) from each group, PARP, IL-17, COL-I, COL-III, MMP-2 and TIMP-2 expression levels were determined by RT-qPCR and immunohistochemistry. The younger patients with valve incompetence showed significantly higher PARP, IL-17, COL-I, COL-III, MMP-2 and reduced TIMP-2 expression levels and a higher COL-I/III ratio. Young CVI patients with venous reflux suffer chronic DNA damage, with consequences at both the local tissue and systemic levels, possibly associated with ageing.     

Histomorphometric analysis of subchondral bone of the femoral head in osteoarthritis and osteoporosis

There have been reports both supporting and refuting an inverse relationship between hip fracture and hip osteoarthritis (OA). We have investigated this relationship using histomorphometric study of femoral head subchondral bone. We studied 74 subjects with hip fracture (74% females) and 24 subjects with osteoarthritis (45% females). By histomorphometric analysis of parafined sections, we analysed followed subhondral trabecular bone parameters bone volume (BV), bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th.), trabecular number (Tb.N.) and trabecular separation (Tb.S.). The subjects with osteoarthritis and subjects with hip fracture had BV/TV 31.3% and 19.6% respectively. BV/TV of osteoarthritis group was rather uniform whereas BV/TV of hip fracture group was greatly ranged and we divided it into three subgroups, 13.2%, 19.8% and 25.9% respectively. The OA group and hip fracture groups had Tb.Th. as followed 0.205 mm, 0.148 mm, 0.170 mm and 0.183 mm respectively. The OA group and hip fracture three subgroups had Tb.N. as followed 1.454/mm, 0.897/mm, 1.170/mm and 1.425/mm respectively. The OA group and hip fracture three subgroups had Tb.S. as followed 0.518 mm, 0.681 mm, 0.620 mm and 0.550 mm respectively. The results of our study support an inverse relationship between hip fracture and hip osteoarthritis.     

An analytical and numerical study of the stability of bone remodelling theories: dependence on microstructural stimulus.

The origin of unstable bone remodelling simulations using strain-energy-based remodelling rules was studied mathematically in order to assess whether the unstable behavior was due to the mathematical rules proposed to characterize the processes, or to the numerical approximations used to exercise the mathematical predictions. A condition which is necessary for the stability of a strain-energy-based remodelling theory was derived analytically using the calculus of variation. The analytical result was derived using a simple elastic model which consists of a long beam loaded by an axial force and a bending moment. This loading situation mimics the coupling between local density and global density distributions seen in vivo. A condition necessary for a stable remodelling scheme is arrived at, but the conditions necessary to guarantee a stable remodelling scheme are not. In this remodelling scheme, the elastic modulus is proportional to volumetric density raised to an exponent n, and the microstructural stimulus is taken as the strain energy density divided by volumetric density raised to an exponent m. In order for a remodelling scheme to be stable in this loading situation, m must be greater than n. Finite-difference time-stepping is used to verify the predictions of the analytical study. These numerical studies appear to confirm the analytical studies. Physiologic interpretation of the behavior found with n greater than m indicates that this type of unstable behavior is unlikely to be observed in vivo. Since numerical approximations are not made in deriving this stability condition, we conclude that the mathematical rules proposed to characterize bone remodelling based on strain energy density should meet this condition to be relevant to physiologic bone remodelling.     

Prediction of mandibular bone remodelling induced by fixed partial dentures

Fixed partial dentures (FPD) or dental bridges have been extensively utilised in prosthodontic restoration. Despite considerable clinical success to date, there has been limited fundamental understanding of the biomechanical consequences induced by FPD treatment. It is noted that FPD construction significantly alters the biological and mechanical environment in the supporting bone region. Thus, the surrounding bones will be engaged to adapt to such a biomechanical change. This paper aims to address this critical issue by developing a new remodelling procedure induced by FPD restoration. Specifically, it relates the mechanical stimulus to the change in Hounsfield Unit (HU) value in terms of surface area density (SAD) of bony morphology, which allows direct correlation to clinical computerised tomography (CT) data. The procedure will provide prosthodontist with a new approach for assessing FPD treatment, thereby optimising FPD design for improving longevity and reliability of future FPD restoration.     

Age-related changes in human trabecular bone: Relationship between microstructural stress and strain and damage morphology

Accumulation of microdamage in aging and disease can cause skeletal fragility and is one of several factors contributing to osteoporotic fractures. To better understand the role of microdamage in fragility fracture, the mechanisms of bone failure must be elucidated on a tissue-level scale where interactions between bone matrix properties, the local biomechanical environment, and bone architecture are concurrently examined for their contributions to microdamage formation. A technique combining histological damage assessment of individual trabeculae with linear finite element solutions of trabecular von Mises and principal stress and strain was used to compare the damage initiation threshold between pre-menopausal (32-37 years, n=3 donors) and post-menopausal (71-80 years, n=3 donors) femoral cadaveric bone. Strong associations between damage morphology and stress and strain parameters were observed in both groups, and an age-related decrease in undamaged trabecular von Mises stress was detected. In trabeculae from younger donors, the 95% CI for von Mises stress on undamaged regions ranged from 50.7-67.9MPa, whereas in trabeculae from older donors, stresses were significantly lower (38.7-50.2, p<0.01). Local microarchitectural analysis indicated that thinner, rod-like trabeculae oriented along the loading axis are more susceptible to severe microdamage formation in older individuals, while only rod-like architecture was associated with severe damage in younger individuals. This study therefore provides insight into how damage initiation and morphology relate to local trabecular microstructure and the associated stresses and strains under loading. Furthermore, by comparison of samples from pre- and post-menopausal women, the results suggest that trabeculae from younger individuals can sustain higher stresses prior to microdamage initiation.     

Integrity under stress: Host membrane remodelling and damage by fungal pathogens

Membrane bilayers of eukaryotic cells are an amalgam of lipids and proteins that distinguish organelles and compartmentalise cellular functions. The mammalian cell has evolved mechanisms to sense membrane tension or damage and respond as needed. In the case of the plasma membrane and phagosomal membrane, these bilayers act as a barrier to microorganisms and are a conduit by which the host interacts with pathogens, including fungi such as Candida, Cryptococcus, Aspergillus, or Histoplasma species. Due to their size, morphological flexibility, ability to produce long filaments, secrete pathogenicity factors, and their potential to replicate within the phagosome, fungi can assault host membranes in a variety of physical and biochemical ways. In addition, the recent discovery of a fungal pore‐forming peptide toxin further highlights the importance of membrane biology in the outcomes between host and fungal cells. In this review, we discuss the apparent “stretching” of membranes as a sophisticated biological response and the role of vesicular transport in combating membrane stress and damage. We also review the known pathogenicity factors and physical properties of fungal pathogens in the context of host membranes and discuss how this may contribute to pathogenic interactions between fungal and host cells.     

Sildenafil attenuates hypoxic pulmonary remodelling by inhibiting bone marrow progenitor cells

The recruitment of bone marrow (BM)‐derived progenitor cells to the lung is related to pulmonary remodelling and the pathogenesis of pulmonary hypertension (PH). Although sildenafil is a known target in PH treatment, the underlying molecular mechanism is still elusive. To test the hypothesis that the therapeutic effect of sildenafil is linked to the reduced recruitment of BM‐derived progenitor cells, we induced pulmonary remodelling in rats by two‐week exposure to chronic hypoxia (CH, 10% oxygen), a trigger of BM‐derived progenitor cells. Rats were treated with either placebo (saline) or sildenafil (1.4 mg/kg/day ip) during CH. Control rats were kept in room air (21% oxygen) with no treatment. As expected, sildenafil attenuated the CH‐induced increase in right ventricular systolic pressure and right ventricular hypertrophy. However, sildenafil suppressed the CH‐induced increase in c‐kit⁺ cells in the adventitia of pulmonary arteries. Moreover, sildenafil reduced the number of c‐kit⁺ cells that colocalize with tyrosine kinase receptor 2 (VEGF‐R2) and CD68 (a marker for macrophages), indicating a positive effect on moderating hypoxia‐induced smooth muscle cell proliferation and inflammation without affecting the pulmonary levels of hypoxia‐inducible factor (HIF)‐1α. Furthermore, sildenafil depressed the number of CXCR4⁺ cells. Collectively, these findings indicate that the improvement in pulmonary haemodynamic by sildenafil is linked to decreased recruitment of BM‐derived c‐kit⁺ cells in the pulmonary tissue. The attenuation of the recruitment of BM‐derived c‐kit⁺ cells by sildenafil may provide novel therapeutic insights into the control of pulmonary remodelling.     

Alleviation of osteoarthritis by Trichostatin A, a histone deacetylase inhibitor, in experimental osteoarthritis

This study investigated the effects of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) on cartilage degradation in an experimental model of osteoarthritis (OA). Thirty-two male New Zealand rabbits underwent unilateral anterior cruciate ligament transection (ACLT) on left knee joints to induce OA and were randomly divided into two groups (n = 16), the TSA group was injected intra-articularly with 0.3 ml TSA [250 ng/ml in the dimethylsulphoxide (DMSO)], the OA group received DSMO since 4 weeks after operation once a week for 5 weeks. Rabbits were killed seven days after the last injection. Left knee cartilage was harvested for morphological, histological and genetic analysis. Another ten rabbits were used for normal control and received no injection. The TSA group showed less cartilage degradation as compared to the OA group assessed by morphological and histological evaluation. Gene expression of matrix metalloproteinase-1 (MMP-1), MMP-3, MMP-13, and interleukin-1 (IL-1) was increased significantly in the OA group compared to the normal group. The elevated expression was reduced by TSA. Our results suggest that TSA could be considered as a potential agent for treatment for OA.     

Comet assay for quantification of the increased DNA damage burden in primary human chondrocytes with aging and osteoarthritis

It is known that chondrocytes from joints with osteoarthritis (OA) exhibit high levels of DNA damage, but the degree to which chondrocytes accumulate DNA damage during “normal aging” has not been established. The goal of this study was to quantify the DNA damage present in chondrocytes obtained from cadaveric donors of a wide age range, and to compare the extent of this damage to OA chondrocytes. The alkaline comet assay was used to measure the DNA damage in normal cartilage from the ankle (talus) and the knee (femur) of cadaveric donors, as well as in OA chondrocytes obtained at the time of total knee replacement. Chondrocytes from younger donors (<45 years) had less DNA damage than older donors (>70 years) as assessed by the percentage of DNA in the comet “tail”. In donors between 50 and 60 years old, there was increased DNA damage in chondrocytes from OA cartilage as compared to cadaveric. Talar chondrocytes from 23 donors between the ages of 34 and 78 revealed a linear increase in DNA damage with age (R ² = 0.865, p < 0.0001). A “two‐tailed” comet assay was used to demonstrate that most of the accumulated damage is in the form of strand breaks as opposed to alkali‐labile base damage. Chondrocytes from young donors required 10 Gy irradiation to recapitulate the DNA damage present in chondrocytes from older donors. Given the potential for DNA damage to contribute to chondrocyte dysfunction and senescence, this study supports the investigation of mechanisms by which hypo‐replicative cell types accumulate high levels of damage.