Heparin Sulfation and Carbohydrate Length Affect Osteogenesis and Osteoclastogenesis

Objectives: Objectives: Heparin is a heterogeneous mixture of sulfated glycosaminoglycans of varied length (mers) and sulfation that has been shown to affect bone homeostasis in vitro and in vivo. Low-molecular-weight-heparins (LMWHs) can be fabricated with precise chain-length and sulfate position. Objective was to evaluate the effects of heparin, heparosan, and six LMWHs (8 or 18 mer) and different sulfations on osteogenesis and osteoclastogenesis.
Methods: Methods: MC3T3-E1 Subclone-4 cells were cultured in media containing 1 or 10μg/mL heparin or LMWHs and tested for cytotoxicity (4-hours), calcium accretion (alizarin-red, days 21 and 28), and collagen organization by picrosirius red (PSR, day 21). RAW 264.7 cells were cultured in media containing 1 or 10μg/mL heparin, heparosan or LMWHs and 30ng/mL receptor activator of nuclear factor kappa-B ligand (RANKl). Cells were stained for tartrate-resistant-acid-phosphatase (TRAP) for 4 days and osteoclasts counted. TUNEL assay was evaluated at day 4.
Results: Results: No materials were toxic at 4-hours. Alizarin-red staining indicated heparin and LMWH of 18 carbohydrates with specific sulfation increased calcium accretion at 10μg/mL compared to controls. Other combinations of mer length and sulfation were similar to controls. PSR data indicated heparin and specific sulfation-18mer resulted in collagen stained primarily red compared to other LMWHs and controls, which stained more yellow indicating a difference in collagen organization and maturation. Heparin and specific sulfation-18mer also resulted in significantly fewer osteoclasts and were smaller than controls. TUNEL demonstrated this was not due to LMWHs promoting apoptosis.
Conclusions: Conclusions: We have demonstrated that LMWH carbohydrate length and degree of sulfation affect osteogenesis and osteoclastogenesis in vitro. A mer length of 18 and specific sulfation led to increased osteogenesis and decreased osteoclastogenesis. These results are promising for possible development of treatments for bone restoration while inhibiting bone resorption.