Harrie Weinans

Trabecular bone's mechanical properties are affected by its non-uniform mineral distribution.

The bone remodeling process takes place at the surface of trabeculae and results in a non-uniform mineral distribution. This will affect the mechanical properties of cancellous bone, because the properties of bone tissue depend on its mineral content. We investigated how large this effect is by simulating several non-uniform mineral distributions in 3D finite element models of human trabecular bone and calculating the apparent stiffness of these models. In the linear model we assumed a linear relation between mineral content and Youngs modulus of the tissue. In the exponential model we included an empirical exponential relation in the model. When the linear model was used the mineral distribution slightly changed the apparent stiffness, the difference varied between an 8% decrease and a 4% increase compared to the uniform model with the same BMD. The exponential model resulted in up to 20% increased apparent stiffness in the main load-bearing direction. A thin less mineralized surface layer (28 microm) and highly mineralized interstitial bone (mimicking mineralization resulting from anti-resorptive treatment) resulted in the highest stiffness. This could explain large reductions in fracture risk resulting from small increases in BMD. The non-uniform mineral distribution could also explain why bone tissue stiffness determined using nano-indentation is usually higher than finite element (FE)-determined stiffness. We conclude that the non-uniform mineral distribution in trabeculae does affect the mechanical properties of cancellous bone and that the tissue stiffness determined using FE-modeling could be improved by including detailed information about mineral distribution in trabeculae in the models.

Mechanical control of human osteoblast apoptosis and proliferation in relation to differentiation

Bone cells respond to mechanical stimulation. This is thought to be the mechanism by which bone adapts to mechanical loading. Reported responses of bone cells to mechanical stimuli vary widely and therefore there is no consensus on what mechanisms of mechanotransduction are physiologically relevant. We hypothesize that the differentiation stage of osteoblastic cells used to study responses to strain in vitro determines the outcome of applied loading. A human fetal osteoblast cell line was triggered to differentiate in culture to the advanced state of mineralization by addition of the osteogenic factors dexamethasone and b-glycerophosphate. Osteoblast cultures were subjected to increasing levels of cyclic, equibiaxial stretch at different stages of differentiation. We show that differentiation of human osteoblasts affects their responses to stretch in vitro. In 7-day osteoblast cultures, stretch results in decreased cell numbers as cells are triggered into apoptosis, independent of the stretch level (between 0.4–2.5%). In more mature cultures, apoptosis is not affected by the same treatment. Stretching differentiating cultures at day 14 actually increases proliferation. This is the first study reporting on differentiation-dependent mechanical control of osteoblast proliferation and apoptosis and is fundamental in understanding mechanotransduction processes in bone. The tight regulation of these responses by differentiation implies the significance of the differentiation stage of osteoblasts for the translation of mechanical signals and corroborates with the putative role of the osteoblastic lineage as mechanotransducer in bone.n

Stimulation of osteogenic differentiation in human osteoprogenitor cells by pulsed electromagnetic fields: an in vitro study.

BackgroundAlthough pulsed electromagnetic field (PEMF) stimulation may be clinically beneficial during fracture healing and for a wide range of bone disorders, there is still debate on its working mechanism. Mesenchymal stem cells are likely mediators facilitating the observed clinical effects of PEMF. Here, we performed in vitro experiments to investigate the effect of PEMF stimulation on human bone marrow-derived stromal cell (BMSC) metabolism and, specifically, whether PEMF can stimulate their osteogenic differentiation.MethodsBMSCs derived from four different donors were cultured in osteogenic medium, with the PEMF treated group being continuously exposed to a 15 Hz, 1 Gauss EM field, consisting of 5-millisecond bursts with 5-microsecond pulses. On culture day 1, 5, 9, and 14, cells were collected for biochemical analysis (DNA amount, alkaline phosphatase activity, calcium deposition), expression of various osteoblast-relevant genes and activation of extracellular signal-regulated kinase (ERK) signaling. Differences between treated and control groups were analyzed using the Wilcoxon signed rank test, and considered significant when p < 0.05.ResultsBiochemical analysis revealed significant, differentiation stage-dependent, PEMF-induced differences: PEMF increased mineralization at day 9 and 14, without altering alkaline phosphatase activity. Cell proliferation, as measured by DNA amounts, was not affected by PEMF until day 14. Here, DNA content stagnated in PEMF treated group, resulting in less DNA compared to control.Quantitative RT-PCR revealed that during early culture, up to day 9, PEMF treatment increased mRNA levels of bone morphogenetic protein 2, transforming growth factor-beta 1, osteoprotegerin, matrix metalloproteinase-1 and -3, osteocalcin, and bone sialoprotein. In contrast, receptor activator of NF-κB ligand expression was primarily stimulated on day 14. ERK1/2 phosphorylation was not affected by PEMF stimulation.ConclusionsPEMF exposure of differentiating human BMSCs enhanced mineralization and seemed to induce differentiation at the expense of proliferation. The osteogenic stimulus of PEMF was confirmed by the up-regulation of several osteogenic marker genes in the PEMF treated group, which preceded the deposition of mineral itself. These findings indicate that PEMF can directly stimulate osteoprogenitor cells towards osteogenic differentiation. This supports the theory that PEMF treatment may recruit these cells to facilitate an osteogenic response in vivo.

Improved cartilage integration and interfacial strength after enzymatic treatment in a cartilage transplantation model.

The objective of the present study was to investigate whether treatment of articular cartilage with hyaluronidase and collagenase enhances histological and mechanical integration of a cartilage graft into a defect. Discs of 3 mm diameter were taken from 8-mm diameter bovine cartilage explants. Both discs and annulus were either treated for 24 hours with 0.1% hyaluronidase followed by 24 hours with 10 U/ml collagenase or left untreated (controls). Discs and annulus were reassembled and implanted subcutaneously in nude mice for 5 weeks. Integration of disc with surrounding cartilage was assessed histologically and tested biomechanically by performing a push-out test. After 5 weeks a significant increase in viable cell counts was seen in wound edges of the enzyme-treated group as compared with controls. Furthermore, matrix integration (expressed as a percentage of the total interface length that was connected; mean ± standard error) was 83 ± 15% in the treated samples versus 44 ± 40% in the untreated controls. In the enzyme-treated group only, picro-Sirius Red staining revealed collagen crossing the interface perpendicular to the wound surface. Immunohistochemical analyses demonstrated that the interface tissue contained cartilage-specific collagen type II. Collagen type I was found only in a small region of fibrous tissue at the level of the superficial layer, and collagen type III was completely absent in both groups. A significant difference in interfacial strength was found using the push-out test: 1.32 ± 0.15 MPa in the enzyme-treated group versus 0.84 ± 0.14 MPa in the untreated controls. The study shows that enzyme treatment of cartilage wounds increases histological integration and improves biomechanical bonding strength. Enzymatic treatment may represent a promising addition to current techniques for articular cartilage repair.

ERK activation and αvβ3 integrin signaling through Shc recruitment in response to mechanical stimulation in human osteoblasts

Osteoblast growth and differentiation are critical processes for bone development and maintenance, and are regulated by both humoral and mechanical factors. Humoral (hormonal) factors can affect gene transcription via MAPkinases, e.g., extracellular signal‐regulated kinase (ERK). We studied whether the ERK pathway is also involved in processing mechanical inputs in human bone cells. Exposing MG63 cells to physiologically relevant levels of fluid flow resulted in ERK phosphorylation. Genistein blocked this response, indicating that it is dependent on tyrosine phosphorylation. Furthermore, αvβ3 integrins were activated in response to fluid flow, as shown by recruitment of adaptor molecule Shc and clustering of αvβ3 in focal adhesion‐like structures. Antibodies blocking formation of β1 or β3 integrin‐matrix interactions or RGD peptides could not inhibit fluid flow‐induced ERK phosphorylation, suggesting that formation of new integrin‐matrix interactions is not essential for this response and that other upstream mechanosensors regulate induction of ERK phosphorylation in response to fluid flow in human bone cells. J. Cell. Biochem. 87: 85–92, 2002.

Inhibition of Gsk3β in cartilage induces osteoarthritic features through activation of the canonical Wnt signaling pathway

OBJECTIVEnIn the past years, the canonical Wnt/β-catenin signaling pathway has emerged as a critical regulator of cartilage development and homeostasis. In this pathway, glycogen synthase kinase-3β (GSK3β) down-regulates transduction of the canonical Wnt signal by promoting degradation of β-catenin. In this study we wanted to further investigate the role of Gsk3β in cartilage maintenance.nnnDESIGNnTherefore, we have treated chondrocytes ex vivo and in vivo with GIN, a selective GSK3β inhibitor.nnnRESULTSnIn E17.5 fetal mouse metatarsals, GIN treatment resulted in loss of expression of cartilage markers and decreased chondrocyte proliferation from day 1 onward. Late (3 days) effects of GIN included cartilage matrix degradation and increased apoptosis. Prolonged (7 days) GIN treatment resulted in resorption of the metatarsal. These changes were confirmed by microarray analysis showing a decrease in expression of typical chondrocyte markers and induction of expression of proteinases involved in cartilage matrix degradation. An intra-articular injection of GIN in rat knee joints induced nuclear accumulation of β-catenin in chondrocytes 72 h later. Three intra-articular GIN injections with a 2 days interval were associated with surface fibrillation, a decrease in glycosaminoglycan expression and chondrocyte hypocellularity 6 weeks later.nnnCONCLUSIONSnThese results suggest that, by down-regulating β-catenin, Gsk3β preserves the chondrocytic phenotype, and is involved in maintenance of the cartilage extracellular matrix. Short term β-catenin up-regulation in cartilage secondary to Gsk3β inhibition may be sufficient to induce osteoarthritis-like features in vivo.

Imaging of activated macrophages in experimental osteoarthritis using folate-targeted animal single-photon-emission computed tomography/computed tomography

OBJECTIVEnEvaluation of macrophage activation may provide essential information about the etiology and progression rate of osteoarthritis (OA). Activated macrophages abundantly express folate receptor β (FRβ), which can be targeted using radioactive-labeled folic acid. This study was undertaken to investigate whether macrophage activation can be monitored in small animal models of OA using a folate radiotracer and to determine whether macrophage activation differs in different models of OA with different OA progression.nnnMETHODSnTwo rat models of OA were used: the mono-iodoacetate (MIA) model, which is a fast-progressing biochemically induced model, and the anterior cruciate ligament transection (ACLT) model, which induces OA at a slower pace. Images were obtained using high-resolution small animal single-photon-emission computed tomography/computed tomography. The specificity of the technique was tested by eradicating macrophages using clodronate-laden liposomes and blockade of FRβ by cold folic acid.nnnRESULTSnThe MIA model had high initial macrophage activation, with a peak after 2 weeks which disappeared after 8 weeks. The ACLT model showed less activation but was still active 12 weeks after induction. The technique allowed monitoring of the disease process over time, in which late stages of the disease showed less macrophage activation than early stages, especially in the fast-progressing MIA model of OA.nnnCONCLUSIONnOur findings indicate that macrophage activation in experimental OA can clearly be demonstrated and monitored by the folate radiotracer. The high resolution, high sensitivity, and high specificity of the technique allow clear localization of macrophage activity in a disease model that is not known for abundant macrophage involvement.

Physiological effects of oral glucosamine on joint health: Current status and consensus on future research priorities

The aim of this paper was to provide an overview of the current knowledge and understanding of the potential beneficial physiological effects of glucosamine (GlcN) on joint health. The objective was to reach a consensus on four critical questions and to provide recommendations for future research priorities. To this end, nine scientists from Europe and the United States were selected according to their expertise in this particular field and were invited to participate in the Hohenheim conference held in August 2011. Each expert was asked to address a question that had previously been posed by the chairman of the conference. Based on a systematic review of the literature and the collection of recent data, the experts documented the effects of GlcN on cartilage ageing, metabolic/kinetic and maintenance of joint health as well as reduction of risk of OA development. After extensive debate and discussion the expert panel addressed each question and a general consensus statement was developed, agreeing on the current state-of-the-art and future areas for basic and clinical studies. This paper summarizes the available evidence for beneficial effects of GlcN on joint health and proposes new insight into the design of future clinical trials aimed at identifying beneficial physiological effect of GlcN on joint tissues.

Platelet-rich plasma for chronic achilles tendinopathy: a double-blind randomised controlled trial with one year follow-up

Introduction Chronic Achilles tendinopathy occurs frequently and is very hard to treat. The disease involves local degeneration of tendon tissue, of which regeneration may be improved by injecting platelet-rich plasma (PRP), an increasingly used therapy for releasing growth factors into degenerative tendon. However, high-quality randomised clinical trials on this topic are lacking. The aim of this study was to evaluate the effect of a PRP injection in patients with chronic Achilles tendinopathy. Methods In this stratified, block randomised, double-blind, placebo-controlled trial at single center 54 patients aged 18–70 years were randomised in two treatment groups. Next to an eccentric training program the patients received a blinded injection containing either PRP group or saline (placebo group). Primary outcome, the objective and validated Victorian Institute of Sports Assessment-Achilles (VISA-A) score, was assessed and ultrasound examination was performed at baseline and all follow-up appointments. Results After randomisation into the PRP group (n=27) and the placebo group (n=27) there was a complete follow-up. After one year, the mean VISA-A score improved in both the PRP-group and the placebo group. There was no significant difference in increase between both groups (adjusted between-group difference, 5.5; 95% CI, −4.9 to 15.8, p=0.292). Ultrasonographic tendon structure improved significantly in both groups, but not significant different between both groups (adjusted between-group difference, 1.2 %, 95% CI, −4.1 to 6.6, p=0.647) Conclusion One-year follow-up analysis of the world’s first randomised controlled trial showed no evidence for the use of platelet-rich plasma injection in chronic Achilles tendinopathy. These findings are in line with our 6 months results (De Vos et al JAMA 2010).

Computer Simulations of Cancellous Bone Remodeling

The bone remodeling process is essential for the maintenance of our skeleton. It enables adaptation of the bone mass and architecture to changes in external loads (1,2), and it prevents accumulation of damage (3,4). Damage accumulation is prevented by a frequent turnover of the bone tissue by the bone remodeling process: old tissue is replaced by new tissue. Bone remodeling is performed by two types of cells: osteoclasts, which are multinucleated bone resorbing cells, and osteoblasts, which are bone-forming cells. Osteoclasts resorb packets of bone tissue, and osteoblasts replace the resorbed tissue with new mineralized bone tissue (see Fig. 1).