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Tendons and ligaments are jointed to bones via an enthesis that is essential to the proper function of the muscular and skeletal structures. The aim of the study is to investigate the effect of mechanical stress on the enthesis. We used ex vivo models in organ cultures of rat Achilles tendons with calcaneus including the enthesis. The organ was attached to a mechanical stretching apparatus that can conduct cyclic tensile strain. We made the models of 1-mm elongation (0.5 Hz, 3% elongation), 2-mm elongation (0.5 Hz, 5% elongation), and no stress. Histological evaluation by Safranin O staining and Toluidin Blue and Picro Sirius red staining was conducted. Expression of sex-determining region Y-box 9 (Sox9), scleraxis (Scx), Runt-related transcription factor 2 (Runx2), and matrix metalloproteinase 13 (Mmp13) were examined by real-time polymerase chain reaction and immunocytochemistry. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end-labeling and live/dead staining and was conducted for evaluation of the apoptosis and cell viability. The structure of the enthesis was most maintained in the model of 1-mm elongation. The electronic microscope showed that the enthesis of the no stress model had ill-defined borders between fibrocartilage and mineralized fibrocartilage, and that calcification of mineralized fibrocartilage occurred in the model of 2-mm elongation. Sox9 and Scx was upregulated by 1-mm elongation, whereas Runx2 and Mmp13 were upregulated by 2-mm elongation. Apoptosis was inhibited by low stress. The results of this study suggested that 1-mm elongation can maintain the structure of the enthesis, while 2-mm elongation promotes degenerative changes.

An asymmetric total synthesis of tupichilignan A was achieved using enantioenriched donor–acceptor (D–A) cyclopropanes. The key steps include an asymmetric cyclopropanation using the Hayashi–Jorgensen catalyst, an oxy-homo-Michael reaction of a bicyclic D–A cyclopropane, an α-benzylation of a γ-lactone, a decarboxylation furnishing a trans-α,β-dibenzyl-γ-lactone, a configurational inversion of a hydroxy chiral center via oxidation, and a final reduction, all of which occur with high stereoselectivity. The spectral data of the diastereomer previously identified in the literature as tupichilignan A were found to be inconsistent with the reported data for the natural product. On the basis of the spectral data of both of the synthesized diastereomers, the structure of tupichilignan A was revised, and the absolute configuration of the 7 position of tupichilignan A was changed from R to S.

A highly regioselective and stereoselective 1,5-addition of alkyl groups to enantioenriched donor–acceptor cyclopropanes 1 and bicyclic cyclopropanes such as 6-aryl-1-methoxycarbonyl-3-oxabicyclo[3.1.0]hexan-2-ones 4 using a Grignard reagent with a catalytic amount of Cu(OTf)2 (0.1 equivalent) afforded optically active diesters 2 or trans-α,β-disubstituted γ-butyrolactones 5 with an excellent level of stereoinduction. An excess of the Grignard reagent is necessary to perform the 1,5-alkylation with high yields. In the reaction of the enantioenriched bicyclic lactone 4, a highly stereoselective 1,5-addition and subsequent highly trans-selective protonation of the magnesium enolate via keto-enol isomerization furnished the trans-α,β-disubstituted-γ-lactones with three contiguous chiral centers with excellent enantioselectivity. Based on the results, we also proposed the mechanism through cluster ion pairs or a simple ion pair to rationalize the high stereoselectivity of the reaction.

The interactions of bone cells with their surrounding extracellular microenvironment may be mediated by integrins, a family of heterodimeric glycoproteins consisting of alpha and beta subunits that noncovalently interact to form cell-substratum adhesion receptors. We previously described the integrins on calvarial bone cells in rats with use of polyclonal antibodies against some integrin subunits. In the present study, we expanded this initial characterization by employing a more complete panel of monoclonal antibodies to identify integrins on human bone cells. Minced fragments of trabecular bone obtained during total knee arthroplasty were grown in culture until bone cells became confluent. The cells then were dissociated, plated again, grown to confluence, and assayed for alkaline phosphatase activity, response of cyclic adenosine monophosphate to stimulation with parathyroid hormone, and osteocalcin content. The percentage of the cells that adhered to various substrates was measured; 60-70% adhered to type-I collagen, fibronectin, vitronectin, and poly-D-lysine; 40-50% adhered to type-IV collagen, laminin, and gelatin; and only 10% adhered to fibrinogen. Flow cytometric analysis with anti-integrin monoclonal antibodies and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitates of the human bone cells revealed high levels of alpha 1 beta 1, alpha 3 beta 1, alpha 5, beta 1 and alpha v beta 5 integrins and much lower levels of alpha 2 beta 1, alpha 4 beta 1, alpha v beta 1, and alpha v beta 3 integrins. This description of the integrin repertoire of cultured human bone cells represents the first step toward an understanding of the role played by integrins in the growth, maintenance, and repair of bone.