Greater heterogeneity of the bone mineralisation density distribution and low bone matrix mineralisation characterise tibial subchondral bone marrow lesions in knee osteoarthritis patients.

Tibial subchondral bone marrow lesions (BMLs) identified by MRI have been recognised as potential disease predictors in knee osteoarthritis (KOA), and may associate with abnormal bone matrix mineralisation and reduced bone quality. However, these tissue-level changes of BMLs have not been extensively investigated. Thus, the aim of this study was to quantify the degree of subchondral bone matrix mineralisation (both plate and trabeculae) in relation to histomorphometric parameters of bone remodelling and osteocyte lacunae (OL) characteristics in the tibial plateau (TP) of KOA patients with and without BMLs (OA-BML and OA No-BML, respectively) in comparison to nonOA cadaveric controls (CTL). Osteochondral (cartilage-bone) tissue was sampled from the BML signal region within the medial compartment for each OA-BML TP, and from a corresponding medial region for OA No-BML and CTL TPs. The tissue samples were embedded in resin, and sections stained with Von-Kossa Haematoxylin and Eosin (H&E) for quantitation of static indices of bone remodelling. Resin blocks were then further polished, and carbon-coated for quantitative backscattered electron imaging (qBEI) to determine the bone mineralisation density distribution (BMDD), as well as OL characteristics. It was found that OA-BML contained higher osteoid volume per tissue volume (OV/TV; %) and per bone volume (OV/BV; %) in both subchondral plate and trabecular bone compared to OA No-BML and CTL. The BMDD of OA-BML in both subchondral plate and trabecular bone was shifted toward a lower degree of mineralisation. Typically, an increase in both the heterogeneity of mineralisation density (Ca Width; wt%Ca) and the percentage of lower calcium (Ca Low; % B.Ar) in trabecular bone with OA-BML versus CTL was observed. Further, unmineralised OL density (#/mm2) in subchondral plate was distinctly higher in OA-BML samples compared to CTL. The KOA patients with and without BMLs had significantly decreased density of mineralised OL (#/mm2) in trabecular bone compared to CTL. Taken together, these findings indicate that tibial BMLs in advanced KOA patients are characterised by significantly hypo-mineralised subchondral bone compared with CTL. These differences associated with evidence of increased bone remodelling in OA-BML, and may influence the mechanical properties of the subchondral bone, with implications for the overlying cartilage.

Bone matrix microdamage and vascular changes characterize bone marrow lesions in the subchondral bone of knee osteoarthritis.

INTRODUCTION: Bone marrow lesions (BMLs) in the subchondral bone in osteoarthritis (OA) are suggested to be multifactorial, although the pathogenic mechanisms are unknown. Bone metabolism and cardiovascular risk factors associate with BML in epidemiologic studies. However, there are no studies at the tissue level investigating the relationship between these processes and BML. The aim of this study was to investigate the relationship between BMLs in the tibial plateau (TP) of knee OA and bone matrix microdamage, osteocyte density and vascular changes. METHODS: TP were obtained from 73 patients at total knee replacement surgery and BMLs were identified ex vivo in TP tissue using MRI. Comparator 'No BML' tissue was from matched anatomical sites to the BMLs. Quantitative assessment was made of subchondral bone microdamage, bone resorption indices, osteocyte cellularity, and vascular features. RESULTS: Several key parameters were different between BML and No BML tissue. These included increased microcrack burden (p = .01, p = .0001), which associated positively with bone resorption and negatively with cartilage volume, and greater osteocyte numerical density (p = .02, p = .01), in the subchondral bone plate and subchondral trabeculae, respectively. The marrow tissue within BML zones contained increased arteriolar density (p = .04, p = .0006), and altered vascular characteristics, in particular increased wall thickness (p = .007) and wall:lumen ratio (wall thickness over internal lumen area) (p = .001), compared with No BML bone. CONCLUSIONS: Increased bone matrix microdamage and altered vasculature in the subchondral bone of BMLs is consistent with overloading and vascular contributions to the formation of these lesions. Given the important role of BMLs in knee OA, these contributing factors offer potential targets for the treatment and prevention of knee OA.

MALDI mass spectrometry imaging of N-glycans on tibial cartilage and subchondral bone proteins in knee osteoarthritis.

Magnetic resonance imaging (MRI) is a non-invasive technique routinely used to investigate pathological changes in knee osteoarthritis (OA) patients. MRI uniquely reveals zones of the most severe change in the subchondral bone (SCB) in OA, called bone marrow lesions (BMLs). BMLs have diagnostic and prognostic significance in OA, but MRI does not provide a molecular understanding of BMLs. Multiple N-glycan structures have been observed to play a pivotal role in the OA disease process. We applied matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) of N-glycans to formalin-fixed paraffin-embedded (FFPE) SCB tissue sections from patients with knee OA, and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) was conducted on consecutive sections to structurally characterize and correlate with the N-glycans seen by MALDI-MSI. The application of this novel MALDI-MSI protocol has enabled the first steps to spatially investigate the N-glycome in the SCB of knee OA patients.

Allogeneic Mesenchymal Precursor Cells Promote Healing in Postero-lateral Annular Lesions and Improve Indices of Lumbar Intervertebral Disc Degeneration in an Ovine Model.

STUDY DESIGN: In-vivo ovine model of intervertebral disc degeneration (IVD) to evaluate treatment with stem cells. OBJECTIVE: To determine if stem cells delivered to the nucleus pulposus (NP) or the annulus fibrosus (AF) of degenerated lumbar IVDs leads to improved indices of disc health. SUMMARY OF BACKGROUND DATA: Previous studies assessing the efficacy of stem cell injections into degenerated IVDs have reported positive findings. However, studies have been limited to small animals, targeting solely the NP, with short term follow-up. METHODS: Mesenchymal precursor cells (MSC) were obtained from the iliac crest of 8-week-old sheep. IVD degeneration was induced by postero-lateral annulotomy at three lumbar levels in eight 2-year-old sheep. Six months later, each degenerated IVD was randomized to one of three treatments: Injection of MSC into (i) previously incised AF (AFI), (ii) NP (NPI), or (iii) no injection (negative control, NC). The adjacent IVD received injection of phosphate buffered saline into NP (positive control, PC). Radiographs and magnetic resonance imaging scans were obtained at baseline, 6, 9, and 12 months. Discs were harvested at 12 months for biochemical and histological analyses. RESULTS: IVD degeneration was consistently observed postannulotomy, and characterized by reduced disc height index (DHI), disc height (DH), glycosaminoglycan (GAG) content, and increased grade of disc degeneration.Six months after stem cell injection, DHI and DH had recovered in AFI and NPI groups when compared with NC group (P < 0.01). Mean Pfirrmann grade improved from 3.25 to 2.67 (AFI group) and from 2.96 to 2.43 (NPI group). Mean histopathological grade improved for both AFI (P < 0.002) and NPI (P < 0.02) groups. Both AFI and NPI groups demonstrated spontaneous repair of the postero-lateral annular lesion. CONCLUSION: In this large animal model, injection of MSCs into the annulus fibrosus or the nucleus pulposus of degenerated IVD resulted in significant improvements in disc health. LEVEL OF EVIDENCE: N/A.

Temporal changes in bone composition, architecture, and strength following estrogen deficiency in osteoporosis.

Using an ovariectomized (OVX) ovine model, we provide an analysis of the timing of changes in bone following estrogen deficiency. The expression of genes known to regulate osteoclastogenesis, matrix production, and mineralization, as measured by real-time RT-PCR, was significantly increased by 12 months; and increased expression was maintained through to 31 months post-OVX compared to controls. FTIR spectroscopy confirmed that mineralized crystals were less mature than in controls 12 months post-OVX and were even less so by 31 months. The mineral-to-matrix ratio was significantly reduced by 31 months, while the ratio of mature to immature collagen cross-linking was initially increased at 12 months and subsequently reduced at 31 months post-OVX. In contrast, trabecular number, thickness, and separation were unchanged at 12 months. Significant reductions in trabecular number and thickness and a significant increase in trabecular separation were observed 31 months after OVX. Most notably perhaps these combined changes led to a significant reduction in the compressive strength of trabecular bone after 31 months. The results indicate that there is an initial increase in bone turnover, which is accompanied by a change in bone composition. This is followed by a continued increase in bone resorption and relative reduction in bone formation, leading to deterioration in bone microarchitecture. Ultimately, these cumulative changes led to a significant reduction in the compressive strength of bones following 31 months of estrogen deficiency. These findings provide important insight into the time sequence of changes during osteoporosis.

Increased proportion of hypermineralized osteocyte lacunae in osteoporotic and osteoarthritic human trabecular bone: implications for bone remodeling.

Hypermineralized osteocyte lacunae (micropetrosis) have received little research attention. While they are a known aspect of the aging human skeleton, no data are available for pathological bone. In this study, intertrochanteric trabecular bone cores were obtained from patients at surgery for osteoporotic (OP) femoral neck fracture (10F, 4M, 65-94 years), for hip osteoarthritis (OA; 7F, 8M, 62-87 years), and femora at autopsy (CTL; 5F, 11M, 60-84 years). Vertebral trabecular bone cores were also obtained from the vertebra of autopsy cases (CVB; 3F, 6M, 53-83 years). Specimens were resin-embedded, polished, and carbon coated for quantitative backscattered electron imaging (qBEI), energy dispersive X-ray (EDX) spectrometry, and imaging analysis. Bone mineralization (Wt %Ca) was not different between OP, OA, and CTL; but was greater in femoral CTL than in CVB. The percent of hypermineralized osteocyte lacunae relative to the total number (HL/TL) was greater in OP and OA than in CTL. However, relative to bone mineral area, OP was characterised by increased hypermineralized osteocyte lacunar number density (Hd.Lc.Dn), whereas OA was characterised by decreased osteocyte lacunar number density (Lc.Dn) and total osteocyte lacunar number density (Tt.Lc.Dn). Lc.Dn was higher in CVB than in femoral CTL. The calcium-phosphorus ratio (R(Ca/P)) was not different between hypermineralized osteocyte lacunae and bone matrix in each group. In addition, this study focused on the phenomenon of osteocyte lacunae hypermineralization using qBEI. Seven morphological types of osteocyte lacunae hypermineralization were described according to the presence of one or several hypermineralized spherites, associated or not with a hypermineralized lacunar ring. This study has described, for the first time, the morphology of hypermineralized osteocyte lacunae in OP and OA human bone. Further studies are suggested to investigate the functional influence of hypermineralized osteocyte lacunae on bone remodeling and bone biomechanical properties.

Application of in vivo micro-computed tomography in the temporal characterisation of subchondral bone architecture in a rat model of low-dose monosodium iodoacetate-induced osteoarthritis.

INTRODUCTION: Osteoarthritis (OA) is a complex, multifactorial joint disease affecting both the cartilage and the subchondral bone. Animal models of OA aid in the understanding of the pathogenesis of OA and testing suitable drugs for OA treatment. In this study we characterized the temporal changes in the tibial subchondral bone architecture in a rat model of low-dose monosodium iodoacetate (MIA)-induced OA using in vivo micro-computed tomography (CT). METHODS: Male Wistar rats received a single intra-articular injection of low-dose MIA (0.2 mg) in the right knee joint and sterile saline in the left knee joint. The animals were scanned in vivo by micro-CT at two, six, and ten weeks post-injection, analogous to early, intermediate, and advanced stages of OA, to assess architectural changes in the tibial subchondral bone. The articular cartilage changes in the tibiae were assessed macroscopically and histologically at ten weeks post-injection. RESULTS: Interestingly, tibiae of the MIA-injected knees showed significant bone loss at two weeks, followed by increased trabecular thickness and separation at six and ten weeks. The trabecular number was decreased at all time points compared to control tibiae. The tibial subchondral plate thickness of the MIA-injected knee was increased at two and six weeks and the plate porosity was increased at all time points compared to control. At ten weeks, histology revealed loss of proteoglycans, chondrocyte necrosis, chondrocyte clusters, cartilage fibrillation, and delamination in the MIA-injected tibiae, whereas the control tibiae showed no changes. Micro-CT images and histology showed the presence of subchondral bone sclerosis, cysts, and osteophytes. CONCLUSIONS: These findings demonstrate that the low-dose MIA rat model closely mimics the pathological features of progressive human OA. The low-dose MIA rat model is therefore suitable to study the effect of therapeutic drugs on cartilage and bone in a non-trauma model of OA. In vivo micro-CT is a non-destructive imaging technique that can track structural changes in the tibial subchondral bone in this animal model, and could also be used to track changes in bone in preclinical drug intervention studies for OA treatments.

An update on primary hip osteoarthritis including altered Wnt and TGF-ß associated gene expression from the bony component of the disease.

The study of primary hip OA is continuing to redefine what was once considered a stagnant pathology as one of dynamic change, occurring over a long period of time involving the many composite tissue types of the joint including the bone. Examination of the inverse relationships evident between OA and fracture cohorts, including individuals with osteoporosis (OP), indicates an imbalance in formation and resorption in the bony component of both pathologies. This review contains an overview of primary OA followed by an assessment of differential gene expression and altered cellular characteristics identified in the bony compartments of primary hip OA, with a focus on the wingless mouse mammary tumor virus integration (Wnt) and TGF-ß signalling pathways. The studies reviewed here suggest that OA is a systemic disease involving the bone and validate the assessment of molecular changes to further investigate this complex disease.

Osteal tissue macrophages are intercalated throughout human and mouse bone lining tissues and regulate osteoblast function in vitro and in vivo.

Resident macrophages are an integral component of many tissues and are important in homeostasis and repair. This study examines the contribution of resident tissue macrophages to bone physiology. Using immunohistochemistry, we showed that a discrete population of resident macrophages, OsteoMacs, was intercalated throughout murine and human osteal tissues. OsteoMacs were distributed among other bone lining cells within both endosteum and periosteum. Furthermore, OsteoMacs were coisolated with osteoblasts in murine bone explant and calvarial preparations. OsteoMacs made up 15.9% of calvarial preparations and persisted throughout standard osteoblast differentiation cultures. Contrary to previous studies, we showed that it was OsteoMacs and not osteoblasts within these preparations that responded to pathophysiological concentrations of LPS by secreting TNF. Removal of OsteoMacs from calvarial cultures significantly decreased osteocalcin mRNA induction and osteoblast mineralization in vitro. In a Transwell coculture system of enriched osteoblasts and macrophages, we demonstrated that macrophages were required for efficient osteoblast mineralization in response to the physiological remodeling stimulus, elevated extracellular calcium. Notably, OsteoMacs were closely associated with areas of bone modeling in situ, forming a distinctive canopy structure covering >75% of mature osteoblasts on diaphyseal endosteal surfaces in young growing mice. Depletion of OsteoMacs in vivo using the macrophage-Fas-induced apoptosis (MAFIA) mouse caused complete loss of osteoblast bone-forming surface at this modeling site. Overall, we have demonstrated that OsteoMacs are an integral component of bone tissues and play a novel role in bone homeostasis through regulating osteoblast function. These observations implicate OsteoMacs, in addition to osteoclasts and osteoblasts, as principal participants in bone dynamics.

Differential gene expression of bone anabolic factors and trabecular bone architectural changes in the proximal femoral shaft of primary hip osteoarthritis patients.

Previous studies have shown a generalised increase in bone mass in patients with osteoarthritis (OA). Using molecular histomorphometry, this study examined the in vivo expression of mRNA encoding bone anabolic factors and collagen type I genes (COL1A1, COL1A2) in human OA and non-OA bone. Bone samples were obtained from the intertrochanteric (IT) region of the proximal femur, a skeletal site distal to the active site of disease, from individuals with hip OA at joint replacement surgery and from autopsy controls. Semi-quantitative reverse transcription-polymerase chain reaction analysis revealed elevated mRNA expression levels of alkaline phosphatase (p < 0.002), osteocalcin (OCN) (p < 0.0001), osteopontin (p < 0.05), COL1A1 (p < 0.0001), and COL1A2 (p < 0.002) in OA bone compared to control, suggesting possible increases in osteoblastic biosynthetic activity and/or bone turnover at the IT region in OA. Interestingly, the ratio of COL1A1/COL1A2 mRNA was almost twofold greater in OA bone compared to control (p < 0.001), suggesting the potential presence of collagen type I homotrimer at the distal site. Insulin-like growth factor (IGF)-I, IGF-II, and transforming growth factor-beta1 mRNA levels were similar between OA and control bone. Bone histomorphometric analysis indicated that OA IT bone had increased surface density of bone (p < 0.0003), increased trabecular number (Tb.N) (p < 0.0003), and decreased trabecular separation (Tb.Sp) (p < 0.0001) compared to control bone. When the molecular and histomorphometric data were plotted, positive associations were observed in the controls for OCN/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) versus bone tissue volume (r = 0.82, p < 0.0007) and OCN/GAPDH versus Tb.N (r = 0.56, p < 0.05) and a negative association was observed for OCN/GAPDH versus Tb.Sp (r = -0.64, p < 0.02). These relationships were not evident in trabecular bone from patients with OA, suggesting that bone regulatory processes leading to particular trabecular structures may be altered in this disease. The finding of differential gene expression, as well as architectural changes and differences in molecular histomorphometric associations between OA and controls, at a skeletal site distal to the active site of joint degeneration supports the concept of generalised involvement of bone in the pathogenesis of OA.

Evidence for reduced bone formation surface relative to bone resorption surface in female femoral fragility fracture patients.

Fragility fractures, including neck of femur fractures, result from reductions in the amount, quality and architecture of bone. The aim of this study was to compare the cancellous bone structure, and static indices of bone turnover, in female patients, who had sustained fragility fracture at the femoral neck, with age-matched females without fragility fracture. Bone samples were taken from the intertrochanteric region of the proximal femur of female patients undergoing hip arthroplasty surgery for a subcapital fragility fracture of the femoral neck (#NOF) or from age-matched female control individuals at routine autopsy. Contiguous bone samples were analyzed for undecalcified histomorphometry and for mRNA expression. The histomorphometric data, which were normally distributed, indicated no difference between the mean values for any of the structural parameters in control and fracture samples. In particular, the bone volume (BV/TV) values were not different and did not change significantly with age in these cohorts of individuals aged >65 years. The static indices of bone turnover, eroded surface (ES/BS) and osteoid surface (OS/BS), were positively correlated with age in the >65-year-old control group (p < 0.055 and p < 0.03, respectively). The median values for these indices were not different between the fracture and control groups. However, both the median and the range of OS/BS values were increased for >65-year-old controls compared with a group of younger females aged <65 years, suggesting an increase in bone formation surface in older females in the proximal femur after 65 years of age. When the data were further interrogated, a reduction in the percentage osteoid surface to eroded surface quotient (OS/ES) was found for the fracture group compared with the age-matched control group suggesting a reduced adaptive modeling drift capability in the fracture group. In contiguous bone samples, increased median values for receptor activator of nuclear factor kappa beta (RANK) and interleukin-6 (IL-6) mRNA expression were observed in the fracture group. Study of cultured human osteoblasts showed that recombinant human IL-6 (rhIL-6) inhibited osteoblast differentiation, as measured by an increase in the immature osteoblast marker, STRO-1 and concomitantly decreased expression of the osteoblast maturation marker, alkaline phosphatase. Importantly, cells cultured in the presence of IL-6 showed significantly less mineral deposition in vitro compared with control cultures. These data suggest that perturbations in bone formation surface, relative to resorption surface, are potentially important in producing bone in the proximal femur with increased propensity to fracture.

Stability of RNA isolated from human trabecular bone at post-mortem and surgery.

To determine the reliability of gene expression studies in human post-mortem bone, it is important to evaluate the stability of RNA isolated from such tissues as a function of the post-mortem interval. The stability of total RNA and bone-specific mRNA species was examined in bone samples obtained from routine autopsies and at surgery. The optimal temperature for any storage and transport of the bone before RNA isolation was shown to be 4 degrees C, and RT-PCR analysis is the preferred technique for the analysis of gene expression in post-mortem bone as it tolerates partial RNA degradation. For gene expression studies in bone, post-mortem cases, with a post-mortem interval of less than 48 h, should be selected, and the time that bone is stored after retrieval at autopsy or surgery should be kept to a minimum. Overall, our findings indicate that with appropriate storage and handling, RNA can be reliably isolated from human bone obtained at post-mortem and surgery to study ex vivo the pattern of gene expression in healthy individuals and in patients with musculoskeletal diseases such as osteoporosis and osteoarthritis.

Increased expression of IL-6 and RANK mRNA in human trabecular bone from fragility fracture of the femoral neck.

Previous studies have implicated pro-inflammatory cytokines in the bone loss of estrogen deficiency. The aim of this study was to investigate the expression of key regulatory molecules of bone remodeling in the trabecular bone microenvironment in osteoporosis. Bone samples were taken from the intertrochanteric region of the proximal femur of patients undergoing total hip arthroplasty for a subcapital fragility fracture of the femoral neck (#NOF). For comparison, samples were taken from age-matched control individuals at routine autopsy. Expression of RANKL, RANK, osteoprotegerin (OPG), IL-6, IL-11, osteocalcin (OCN), and calcitonin receptor (CTR) messenger RNA (mRNA) species were analyzed and the data were nonparametrically distributed. The median expression of the proresorptive genes, RANK and IL-6, were significantly elevated in the fracture group compared to an age-matched control group (2.2 [1.9-2.9; 25th-75th percentiles] > 1.0 [0.4-2.1], P < 0.03; 3.9 [1.8-6.2] > 0.8 [0.7-1.5], P < 0.002, respectively). In contrast, there were no significant differences in expression of RANKL, OPG, CTR, or OCN mRNA between the #NOF and control groups. The median RANKL/OPG mRNA ratio was significantly greater in hip fracture bone than in bone from controls (4.8 [3.8-7.6] > 3.2 [2.1-4.0], P < 0.05). IL-6 mRNA levels associated strongly with RANKL mRNA levels in the #NOF group (r = 0.77, P < 0.001), but not in the control group. A strong positive association was found between IL-11 mRNA levels and RANKL mRNA levels in the #NOF group (r = 0.81, P < 0.001), consistent with the apparent coordinated regulation of IL-6 and IL-11 in bone samples from the #NOF group (r = 0.93, P < 0.0001). These data suggest a relative increase in the expression of the molecular promoters of osteoclast formation and activity in #NOF bone, which may lead to the imbalance between bone formation and resorption associated with fragility fracture.