Galectin-3 as a novel regulator of osteoblast-osteoclast interaction and bone homeostasis.

Bone tissue undergoes permanent and lifelong remodeling with a concerted action of bone-building osteoblasts and bone-resorbing osteoclasts. A precise cooperation between those two cell types is critical in the complex process of bone renewal. Galectin-3 is a member of the ß-galactoside-binding lectin family playing multiple roles in cell growth, differentiation and aggregation. As it has been described to be expressed in bone, galectin-3 might influence bone homeostasis by regulating the function and/or interplay of osteoblasts and osteoclasts. Here, we investigated the role of galectin-3 in osteoclastogenesis and osteoblast-osteoclast interactions. Bone histomorphometric analysis and µCT measurements revealed a decreased trabecular bone volume and an increased osteoclast number in 12weeks old male galectin-3 knockout mice compared to wildtype littermates. Galectin-3 deficient bone marrow cells displayed a higher osteoclastogenic capacity in ex vivo differentiation assays, associated with elevated TRAF6 mRNA levels, suggesting an intrinsic inhibition of osteoclastogenesis by galectin-3 interfering with RANKL-mediated signaling. Furthermore, the addition of extracellular galectin-3 to murine or human osteoclastogenesis assays inhibited osteoclast formation and osteoclast numbers were higher in co-culture assays with galectin-3 deficient osteoblasts. In conclusion, our data suggest the secretion of galectin-3 as a novel mechanism for osteoblasts to control osteoclastogenesis and to maintain trabecular bone homeostasis independently of the RANKL/OPG-axis.

Development of an ex vivo cavity model to study repair strategies in loaded intervertebral discs.

PURPOSE: The aim of this study was to compare two approaches for the delivery of biomaterials to partially nucleotomised intervertebral discs in whole organ culture under loading. Such models can help to bridge the gap between in vitro and in vivo studies by assessing (1) suitability of biomaterial delivery and defect closure methods, (2) effect of mechanical loading and (3) tissue response. METHODS: Mechanical performance of bovine discs filled with a hyaluronan-based thermoreversible hydrogel delivered through the annulus fibrosus (AF) or the bony endplate (EP) was evaluated under cyclic axial loading in a bioreactor. The loading protocol was optimised to achieve physiological disc height changes in nucleotomised discs. A loading regime of 0.06 ± 0.02 MPa, 0.1 Hz, 6 h daily was applied on the nucleotomised discs. Disc height and stiffness were tracked for 5 days, followed by histological analyses. RESULTS: Creation of a defect is less demanding for AF approach, while sealing is superior with the EP approach. Dynamic compressive stiffness is reduced following nucleotomy, with no significant difference between the two approaches. Disc height loss was higher, disc height recovery was lower and region around the defect with reduced cell viability was smaller for AF-approached than EP-approached discs. CONCLUSIONS: Two alternative methods for biomaterial testing in whole organ culture under loading were developed. Such models bring insights on the ability of the biomaterial to restore the mechanical behaviour of the discs. From a clinical perspective, the cavity models can simulate treatment of nucleotomy after disc herniation in young patients, whereby the remaining nucleus pulposus is still functional and therefore at high risk of re-herniation, though the defect may differ from the clinical situation.

Deciphering mechanical regulation of chondrogenesis in fibrin-polyurethane composite scaffolds enriched with human mesenchymal stem cells: a dual computational and experimental approach.

Fibrin-polyurethane composite scaffolds support chondrogenesis of human mesenchymal stem cells (hMSCs) derived from bone marrow and due to their robust mechanical properties allow mechanical loading in dynamic bioreactors, which has been shown to increase the chondrogenic differentiation of MSCs through the transforming growth factor beta pathway. The aim of this study was to use the finite element method, mechanical testing, and dynamic in vitro cell culture experiments on hMSC-enriched fibrin-polyurethane composite scaffolds to quantitatively decipher the mechanoregulation of chondrogenesis within these constructs. The study identified compressive principal strains as the key regulator of chondrogenesis in the constructs. Although dynamic uniaxial compression did not induce chondrogenesis, multiaxial loading by combined application of dynamic compression and interfacial shear induced significant chondrogenesis at locations where all the three principal strains were compressive and had a minimum magnitude of 10%. In contrast, no direct correlation was identified between the level of pore fluid velocity and chondrogenesis. Due to the high permeability of the constructs, the pore fluid pressures could not be increased sufficiently by mechanical loading, and instead, chondrogenesis was induced by triaxial compressive deformations of the matrix with a minimum magnitude of 10%. Thus, it can be concluded that dynamic triaxial compressive deformations of the matrix is sufficient to induce chondrogenesis in a threshold-dependent manner, even where the pore fluid pressure is negligible.

Mitogen-activated protein kinase 2 regulates physiological and pathological bone turnover.

The objective of this study was to investigate the role of the serine-threonine kinase mitogen-activated protein kinase 2 (MK2) in bone homeostasis. Primary bone cell cultures from MK2(+/+) and MK2(-/-) mice were assessed for osteoclast and osteoblast differentiation, bone resorption, and gene expression. Bone architecture of MK2(+/+) and MK2(-/-) mice was investigated by micro-computed tomography and histomorphometry. Ovariectomy was performed in MK2(+/+) and MK2(-/-) mice to assess the role of MK2 in postmenopausal bone loss. Osteoclastogenesis, bone resorption, and osteoclast gene expression were significantly impaired in monocytes from MK2(-/-) compared to MK2(+/+) mice. Mechanistically, loss of MK2 causes impaired DNA binding of c-fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) to tartrate-resistant acid phosphatase (TRAP) and the calcitonin receptor gene promoter. In addition, MK2(-/-) mice showed an age-dependent increase in trabecular bone mass and cortical thickness, fewer osteoclasts, and lower markers of bone resorption than MK2(+/+) mice. Furthermore, MK2(-/-) mice were protected from ovariectomy-induced bone loss. Osteoblastogenesis and bone formation were unchanged in MK2(-/-) mice, whereas osteoblast expression of osteoprotegerin (OPG) and serum levels of OPG were higher in MK2(-/-) than in MK2(+/+) mice. Loss of MK2 effectively blocks bone resorption and prevents the development of postmenopausal bone loss. Small-molecule inhibitors of MK2 could thus emerge as highly effective tools to block bone resorption and to treat postmenopausal bone loss.

The combined effects of limited nutrition and high-frequency loading on intervertebral discs with endplates.

STUDY DESIGN: Whole ovine caudal intervertebral discs were cultured under simulated-physiologic or high-frequency loading and either sufficient or limited nutrition for 7 days. OBJECTIVE: To study the effect of high-frequency loading under sufficient or limited glucose conditions and to investigate the additive effects of load and nutrition on cell survival, gene expression, and cell activity after 7 days of culture. SUMMARY OF BACKGROUND DATA: Limited nutrition and certain mechanical stimuli are generally believed to be etiologic factors for disc degeneration. Although these effects and their interactions have been demonstrated in cell culture, no investigations have been reported in entire discs. METHODS: Discs were maintained in a whole organ culture bioreactor system under simulated-physiologic (0.2 Hz) or high-frequency (10 Hz) loading, in media with either limited (2 g/L) or sufficient (4.5 g/L) glucose concentration. After 7 days, cell viability, relative gene expression, newly synthesized chondroitin sulfate content, glycosaminoglycan synthesis rate, and disc morphology were assessed after culture and compared with fresh tissue. RESULTS: Culture under either limited glucose or high-frequency loading conditions led to a significant drop in cell viability. Combined treatment with limited glucose and high-frequency loading resulted in an additive increase in cell death in both the anulus fibrosus and nucleus pulposus and in an increase in MMP13 gene expression. CONCLUSION: Supporting in vivo studies and cell culture experiments, high-frequency loading simulating vibration conditions shows detrimental effects on intervertebral disc cells in whole organ culture. The effect on cell viability was exacerbated by limited nutrition culture. However, neither frequency nor limited glucose affected cell metabolism, measured by glycosaminoglycan synthesis rate. Longer culture periods may be required to detect changes at the extracellular matrix level.

Fixation compliance in a mouse osteotomy model induces two different processes of bone healing but does not lead to delayed union.

Delayed unions are a problematic complication of fracture healing whose pathophysiology is not well understood. Advanced molecular biology methods available with mice would be advantageous for investigation. In humans, decreased fixation rigidity and poor reduction are generally associated with delayed unions. In this study, these two factors were combined to observe their effect on bone healing in mice. Two plates with locking screws, one with 14 the bending stiffness of the other, were used to stabilize a 0.45mm gap osteotomy. muCT, radiographs, 4pt-bending tests and histological analysis demonstrated that the different plate types led to two different healing pathways. The less flexible bridging plate induced only intramembranous ossification whereas the more flexible bridging plate induced a mixture of endochondral and intramembranous ossification. However, the different plates led to a delay in healing of only 3-5 days in the period between 14 and 21 post-operative days. In mice, considerable fixation flexibility is necessary to induce secondary bone healing similar to that which occurs in humans, but this was not sufficient to induce a substantial delay in bone healing as would be expected in humans.

Effect of limited nutrition on in situ intervertebral disc cells under simulated-physiological loading.

STUDY DESIGN: Whole ovine caudal intervertebral discs (IVD) were cultured in sufficient and limited nutrition under simulated-physiologic loading for 7 and 21 days. OBJECTIVE: To study the effect of limited nutrition on disc cells embedded in their native tissue in short- and midterm whole organ disc culture. SUMMARY OF BACKGROUND DATA: Nutrient-limited induction of disc cell death in vitro has been demonstrated and is believed to be a factor in disc degeneration. Nutrient-limited cell death and its consequences, as it relates to degeneration, have not been investigated in the intact IVD. METHODS: Ovine IVDs with endplates were cultured for 7 and 21 days under simulated-physiologic loading, either in media with limited (2 g/L) or sufficient (4.5 g/L) glucose concentration. Cell viability, relative gene expression, newly synthesized chondroitin sulfate content, and matrix metalloproteinase (MMP) activity were measured after culture and compared to fresh tissue. RESULTS: In sufficient glucose media, cell viability was maintained through 7 days to 21 days of culture. In limited glucose, it dropped significantly to 62% in the anulus fibrosus and to 56% in the nucleus pulposus after 7 days and remained so until 21 days (63% in the anulus fibrosus and 52% in the nucleus pulposus). No significant differences were found between culture conditions for relative gene expression, newly synthesized chondroitin sulfate and inactive and active forms of MMP13 and MMP7. CONCLUSION: With this culture system, whole IVD explants could be maintained up to 21 days. Cell viability decreased to 50% to 60% under limited nutrition within days and remained so up to 3 weeks. The surviving cells did not compensate matrix production in this time frame.

Effect of TGF beta1, BMP-2 and hydraulic pressure on chondrogenic differentiation of bovine bone marrow mesenchymal stromal cells.

Bioactive factors, such as TGF beta and BMP-2, as well as mechanical factors i.e. compressive loading and hydraulic pressure, have been shown to induce and/or modulate chondrogenesis of bone marrow derived mesenchymal stromal cells (BMSCs). Since these factors are intracellularly transduced through different mechanisms, it is hypothesized that TGF beta, BMP-2 and hydraulic pressure may act synergistically on chondrogenic differentiation of BMSCs. Aggregates of bovine BMSC were cultured in the presence of 10 ng/ml TGF beta1, 50 ng/ml BMP-2 or both. Half of the samples were loaded for 4 hours per day with 0.5-3 MPa cyclic hydraulic pressure at 1 Hz. After 14 days of culture/loading, gene expression of chondrogenic genes was assessed. DNA as well as glycosaminoglycan (GAG) content of the pellets were analysed. Neither pressure nor BMP-2 had an influence on GAG/DNA content. However, cells responded to the presence of TGF beta1 with an up-regulation of chondrogenic genes and GAG/DNA of the aggregates increased compared to controls demonstrating the cells ability to respond to external stimuli. The used concentrations of BMP-2 and parameters for pressure were neither able to induce nor modulate chondrogenesis of bovine BMSCs and thus no synergistic effects were observed.

Inhibition of vertebral endplate perfusion results in decreased intervertebral disc intranuclear diffusive transport.

Impaired nutrition of the intervertebral disc has been hypothesized to be one of the causes of disc degeneration. However, no causal relationship between decreased endplate perfusion and limited nutrient transport has been demonstrated to support this pathogenic mechanism. To determine the importance of endplate perfusion on solute diffusion into the nucleus pulposus and to show causality of endplate perfusion on intranuclear diffusion in large animal lumbar intervertebral discs, diffusive transport into ovine lumbar intervertebral discs was evaluated after inhibiting adjacent vertebral endplate perfusion. Partial perfusion blocks were created in vertebrae close and parallel to both endplates of lumbar discs of anaesthetized sheep. To assess diffusivity of small molecules through the endplate, N2O was introduced into the inhalation gas mixture and concentrations of intranuclear N2O were measured for 35 min thereafter. Post mortem, procion red was infused through the spinal vasculature and perfusion through the endplate was assessed by quantifying the density of dye-perfused endplate vascular buds in histology sections. Perfusion of the endplates overlying the nucleus pulposus was inhibited by almost 50% in the partially blocked discs relative to the control discs. There was also a nine-fold decreased transport rate of intranuclear N2O in partially blocked discs compared with control discs. The density of perfused endplate vascular buds correlated significantly to the amount of transported intranuclear N2O (r2 = 0.52, P = 0.008). The vertebral endplate was demonstrated to be the main route of intravascular solute transport into the nucleus pulposus of intervertebral discs, and inhibition of endplate perfusion can cause inhibited solute transport into the disc intranuclear tissue.