Mark Siegrist

Cyclic tensile strain and cyclic hydrostatic pressure differentially regulate expression of hypertrophic markers in primary chondrocytes

Endochondral ossification is regulated by many factors, including mechanical stimuli, which can suppress or accelerate chondrocyte maturation. Mathematical models of endochondral ossification have suggested that tension (or shear stress) can accelerate the formation of endochondral bone, while hydrostatic stress preserves the cartilage phenotype. The goal of this study was to test this hypothesis by examining the expression of hypertrophic chondrocyte markers (transcription factor Cbfa1, MMP-13, type X collagen, VEGF, CTGF) and cartilage matrix proteins under cyclic tension and cyclic hydrostatic pressure. Chondrocyte-seeded alginate constructs were exposed to one of the two loading modes for a period of 3 h per day for 3 days. Gene expression was analyzed using real-time RT-PCR. Cyclic tension upregulated the expression of Cbfa1, MMP-13, CTGF, type X collagen and VEGF and downregulated the expression of TIMP-1. Cyclic tension also upregulated the expression of type 2 collagen, COMP and lubricin, but did not change the expression of SOX9 and aggrecan. Cyclic hydrostatic pressure downregulated the expression of MMP-13 and type I collagen and upregulated expression of TIMP-1 compared to the unloaded controls. Hydrostatic pressure may slow chondrocyte differentiation and have a chondroprotective, anti-angiogenic influence on cartilage tissue. Our results suggest that cyclic tension activates the Cbfa1/MMP-13 pathway and increases the expression of terminal differentiation hypertrophic markers. Mammalian chondrocytes appear to have evolved complex mechanoresponsive mechanisms, the effects of which can be observed in the histomorphologic establishment of the cartilaginous skeleton during development and maturation.

Cyclic compression of articular cartilage explants is associated with progressive consolidation and altered expression pattern of extracellular matrix proteins

In this study, we investigated the biosynthetic response of full thickness, adult bovine articular cartilage explants to 45 h of static and cyclic unconfined compression. The cyclic compression of articular cartilage resulted in a progressive consolidation of the cartilage matrix. The oscillatory loading increased protein synthesis ([35S]methionine incorporation) by as much as 50% above free swelling control values, but had an inhibitory influence on proteoglycan synthesis ([35SO4] incorporation). As expected, static compression was associated with a dose-dependent decrease in biosynthetic activity. ECM oligomeric proteins which were most affected by mechanical loading were fibronectin and cartilage oligomeric matrix protein (COMP). Static compression at all amplitudes caused a significant increase in fibronectin synthesis over free swelling control levels. Cyclic compression of articular cartilage at 0.1 Hz and higher was consistently associated with a dramatic increase in the synthesis of COMP as well as fibronectin. The biosynthetic activity of chondrocytes appears to be sensitive to both the frequency and amplitude of the applied load. The results of this study support the hypothesis that cartilage tissue can remodel its extracellular matrix in response to alterations in functional demand.

Development of mechanically stable alginate/chondrocyte constructs: effects of guluronic acid content and matrix synthesis

The purpose of this study was to investigate factors which enhanced the compressive properties of alginate/chondrocyte constructs. Firstly, we studied the effect of biochemical composition (high, mid and low guluronic acid content) and sterilization method on alginate properties. Secondly, we studied the biosynthetic characteristics of chondrocytes in three different alginate compositions and performed mechanical tests to determine whether the synthesis of cartilage matrix components could significantly enhance the compressive properties. 2% alginate solutions containing an initial cell density of 4 × 106 cells/ml were cast into cylinders and cultured for seven weeks. Compression tests, biochemistry, immunohistochemistry and electron microscopy were performed at fixed intervals during the seven‐week culture period. The dynamic modulus, peak strain, and peak stress were maximum for alginate with the highest guluronic acid content. The presence of cells and their respective matrix components enhanced the equilibrium modulus of the constructs for all types of alginate, though this effect was small. Alginate containing the middle amount of guluronic acid resulted in constructs which were both mechanically stable and which promoted synthesis of cartilage matrix proteins. In experiments and applications in which the mechanical integrity of the alginate is important, the composition and purity of the alginate and its method of sterilization should be selected with care.

TREM-1 Deficiency Can Attenuate Disease Severity without Affecting Pathogen Clearance

Triggering receptor expressed on myeloid cells-1 (TREM-1) is a potent amplifier of pro-inflammatory innate immune reactions. While TREM-1-amplified responses likely aid an improved detection and elimination of pathogens, excessive production of cytokines and oxygen radicals can also severely harm the host. Studies addressing the pathogenic role of TREM-1 during endotoxin-induced shock or microbial sepsis have so far mostly relied on the administration of TREM-1 fusion proteins or peptides representing part of the extracellular domain of TREM-1. However, binding of these agents to the yet unidentified TREM-1 ligand could also impact signaling through alternative receptors. More importantly, controversial results have been obtained regarding the requirement of TREM-1 for microbial control. To unambiguously investigate the role of TREM-1 in homeostasis and disease, we have generated mice deficient in Trem1. Trem1−/− mice are viable, fertile and show no altered hematopoietic compartment. In CD4+ T cell- and dextran sodium sulfate-induced models of colitis, Trem1−/− mice displayed significantly attenuated disease that was associated with reduced inflammatory infiltrates and diminished expression of pro-inflammatory cytokines. Trem1−/− mice also exhibited reduced neutrophilic infiltration and decreased lesion size upon infection with Leishmania major. Furthermore, reduced morbidity was observed for influenza virus-infected Trem1−/− mice. Importantly, while immune-associated pathologies were significantly reduced, Trem1−/− mice were equally capable of controlling infections with L. major, influenza virus, but also Legionella pneumophila as Trem1+/+ controls. Our results not only demonstrate an unanticipated pathogenic impact of TREM-1 during a viral and parasitic infection, but also indicate that therapeutic blocking of TREM-1 in distinct inflammatory disorders holds considerable promise by blunting excessive inflammation while preserving the capacity for microbial control.

Collagen Fibrillogenesis by Chondrocytes in Alginate

Collagen is the primary structural component in connective tissue. The poor mechanical properties of most cell-seeded cartilage grafts used for cartilage repair can be attributed to the low level of collagen synthesized compared with native cartilage. In this study, the synthesis and assembly of collagen by chondrocytes in hydrogels were investigated, with particular attention paid to the role of cross-link formation in this process. Primary bovine chondrocytes were seeded in alginate and collagen synthesis was assessed in the presence and absence of beta-aminopropronitrile (BAPN), a potent inhibitor of the enzyme lysyl oxidase and collagen cross-link formation. Cultures on days 21, 35, and 49 were evaluated by stereology, biochemistry, and real-time reverse transcriptase-polymerase chain reaction. All measures of collagen synthesis (except hydroxyproline) significantly increased in the presence of 0.25 mM BAPN. By 35 days of culture, the average collagen fibril diameter was 62 +/- 10 nm in control cultures and 109 +/- 20 nm with BAPN supplementation. The collagen volume density increased from 5 +/- 3% in control cultures to 17 +/- 1% in the presence of BAPN. Likewise, the expression of cartilage-specific collagens (type II and XI) and aggrecan increased significantly as a result of BAPN culture. These findings demonstrate the prominent role of collagen cross-linking in collagen fibrillogenesis and suggest approaches by which collagen synthesis and assembly could be controlled in tissue-engineered constructs.

Description of an ancient social bee trapped in amber using diagnostic radioentomology

The application of non-invasive imaging technologies using X-radiation (diagnostic radioentomology, ‘DR’) is demonstrated for the study of amber-entombed social bees. Here, we examine the external and internal morphology of an Early Miocene (Burdigalian) stingless bee (Apinae: Meliponini) from the Dominican Republic using non-destructive X-ray microtomography analysis. The study permits the accurate reconstruction of features otherwise obscured or impossible to visualize without destroying the sample and allows diagnosis of the specimen as a new species, Proplebeia adbita Greco and Engel.

Sodium/hydrogen exchanger NHA2 in osteoclasts: subcellular localization and role in vitro and in vivo

NHA2 was recently identified as a novel sodium/hydrogen exchanger which is strongly upregulated during RANKL-induced osteoclast differentiation. Previous in vitro studies suggested that NHA2 is a mitochondrial transporter required for osteoclast differentiation and bone resorption. Due to the lack of suitable antibodies, NHA2 was studied only on RNA level thus far. To define the proteins role in osteoclasts in vitro and in vivo, we generated NHA2-deficient mice and raised several specific NHA2 antibodies. By confocal microscopy and subcellular fractionation studies, NHA2 was found to co-localize with the late endosomal and lysosomal marker LAMP1 and the V-ATPase a3 subunit, but not with mitochondrial markers. Immunofluorescence studies and surface biotinylation experiments further revealed that NHA2 was highly enriched in the plasma membrane of osteoclasts, localizing to the basolateral membrane of polarized osteoclasts. Despite strong upregulation of NHA2 during RANKL-induced osteoclast differentiation, however, structural parameters of bone, quantified by high-resolution microcomputed tomography, were not different in NHA2-deficient mice compared to wild-type littermates. In addition, in vitro RANKL stimulation of bone marrow cells isolated from wild-type and NHA2-deficient mice yielded no differences in osteoclast development and activity. Taken together, we show that NHA2 is a RANKL-induced plasmalemmal sodium/hydrogen exchanger in osteoclasts. However, our data from NHA2-deficient mice suggest that NHA2 is dispensable for osteoclast differentiation and bone resorption both in vitro and in vivo.

Inhibition of endogenous antagonists with an engineered BMP-2 variant increases BMP-2 efficacy in rat femoral defect healing

Bone morphogenetic proteins (BMP) have been used successfully by orthopedic clinicians to augment bone healing. However, these osteoinductive proteins must be applied at high concentrations to induce bone formation. The limited therapeutic efficacy may be due to the local expression of BMP antagonists such as Noggin that neutralize exogenous and endogenous BMPs. If so, inhibiting BMP antagonists may provide an attractive option to augment BMP induced bone formation. The engineered BMP-2 variant L51P is deficient in BMP receptor type I binding, but maintains its affinity for BMP receptor type II and BMP antagonists including Noggin, Chordin and Gremlin. This modification makes L51P a BMP receptor-inactive inhibitor of BMP antagonists. We implanted β-tricalcium phosphate ceramics loaded with BMP-2 and/or L51P into a critical size defect model in the rat femur to investigate whether the inhibition of BMP antagonist with L51P enhances the therapeutic efficacy of exogenous BMP-2. Our study reveals that L51P reduces the demand of exogenous BMP-2 to induce bone healing markedly, without promoting bone formation directly when applied alone.

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.

Sodium-Dependent Phosphate Transporters in Osteoclast Differentiation and Function

Osteoclasts are multinucleated bone degrading cells. Phosphate is an important constituent of mineralized bone and released in significant quantities during bone resorption. Molecular contributors to phosphate transport during the resorptive activity of osteoclasts have been controversially discussed. This study aimed at deciphering the role of sodium-dependent phosphate transporters during osteoclast differentiation and bone resorption. Our studies reveal RANKL-induced differential expression of sodium-dependent phosphate transport protein IIa (NaPi-IIa) transcript and protein during osteoclast development, but no expression of the closely related NaPi-IIb and NaPi-IIc SLC34 family isoforms. In vitro studies employing NaPi-IIa-deficient osteoclast precursors and mature osteoclasts reveal that NaPi-IIa is dispensable for bone resorption and osteoclast differentiation. These results are supported by the analysis of structural bone parameters by high-resolution microcomputed tomography that yielded no differences between adult NaPi-IIa WT and KO mice. By contrast, both type III sodium-dependent phosphate transporters Pit-1 and Pit-2 were abundantly expressed throughout osteoclast differentiation, indicating that they are the relevant sodium-dependent phosphate transporters in osteoclasts and osteoclast precursors. We conclude that phosphate transporters of the SLC34 family have no role in osteoclast differentiation and function and propose that Pit-dependent phosphate transport could be pivotal for bone resorption and should be addressed in further studies.