Demineralized bone

One of the most sensitive bioassays for osteoblast and osteoclast activities in vivo is the use of ectopic models of bone formation and bone matrix resorption (38,39). Devitalized, demineralized bone powders (DBP) are subcutaneously implanted in young rats. There is a phenotypic conversion of connective cell tissue mesenchyme into cartilage. Subsequently this cartilage becomes calcified, vascularized and bone is deposited in two weeks. If mineral-containing bone particles (BP) are implanted, a different phenomenon is observed. Large multinucleated osteoclast-like cells are recruited to the site of implantation. There is a complete resorption of the BP four weeks after implantation. In collaboration with Dr. Julie Glowacki of the Harvard University School of Medicine, we took advantage of these procedures and used implants of normal DBP and BP into rats that had been maintained on the three experimental diets C, L, and D (40). 

Alkali treatment reduces shrinkage temperature (Fysh, 1958), and Courts (1960) suggests a minimum figure of approximately 44°C for limed ossein (demineralized bone). At this stage few intermolecular cross-links can be present because the material can be dissolved in water at 60°C. Gustavson (1962) has shown that treatment with periodate also lowers the shrinkage temperature (Ts) by as much as 20°C. This could suggest that alkali and periodate attack the same cross-link, but other side reactions under the conditions used will also have some effect. 

In the alkali process, demineralized bones (ossein) or cattle skins are usually used. The animal tissue is held in a calcium hydroxide (lime) slurry for a period of 1-3 months at 15-20°C. At the end of the liming, the stock is washed with cold water to remove as much of the lime as possible. The stock solution is then neutralized with acid (HGl, H2SO4, H3PO4) and the gelatin is extracted with water in an identical manner to that in the acid process. 

Bones are constantly dissolved by osteoclasts and remineralized by osteoblasts in response to mechanical forces. Osteoclasts possess an acidic compartment and pass demineralized bone products to the periosteum (Sect. 1). They develop in stress-induced bony microcracks and are activated by differentiation factors secreted by osteoblasts, especially after menopause. Menopausal osteoporosis is controlled by drugs that are a stable form of pyrophosphate (bisphosphonate) or cathepsin K inhibitors (Sect. 2). The calcium ion concentration of blood is raised by parathyroid hormone and a vitamin D derivative called calcitriol. Parathyroid hormone causes kidneys to excrete phosphate, retain calcium, and activate calcitriol production (Sect. 3). Calcitriol induces calcium transporter proteins in osteoclasts and intestinal epithelium, where they move calcium from bone or diet into blood (Sect. 4). The chapter concludes with a discussion of calcitonin which lowers blood calcium concentrations by reversing parathyroid hormone effects on the kidney and inhibiting osteoclast activity (Sect. 5). 

Despite the substantial amounts of Na+ ions released from demineralized bone and entering the cytosol in exchange for protons, excessive amounts of dihydrogen phosphate (HjPO also enter the cytosol. To compensate, there is a separate inward diffusion of disodium monohydrogen phosphate (NajHPO from the periosteum/bone marrow through Pit-2 transporters (Sect. 9.3.5) in the osteoclast basolateral membrane (Fig. 10.2). Nevertheless, the pH eventually falls below levels compatible with mitochondrial function, perhaps explaining the osteoclast s short half-life. 

Just as inflammation enhances bone resorption by increasing ODF production locally, the cessation of estrogen production at menopause causes osteoporosis systemically (ODF production increases). Menopausal women therefore suffer a net loss of bone because ODAR (RANK) is activated by ODF (RANKL) in the absence of a compensating increase in OCIF (OPG) production. The osteoclasts demineralize bone faster than the osteoblasts deposit it. 

Calcium ions are mostly present in bones or chelated to biological molecules. In blood plasma, only 1% of the calcium ions present are unbound 78% is bound to albumin, 8% to citrate, and 13% to other plasma proteins. The free calcium ions are prevented from precipitating by plasma pyrophosphate. Calcium ions are also stored in the endoplasmic reticulum (ER), mostly chelated to ER-resident proteins and phosphatidylser-ine. Free calcium ions may be released through transient receptor potential channels to the cytosol where it activates numerous physiological processes. If the free calcium ion concentration of blood plasma falls, parathyroid hormone (PTH) is secreted by the parathyroid gland cells. PTH speeds up the transport of demineralized bone products by osteoclasts. In the kidney, it increases the excretion of phosphate and decreases the excretion of calcium. PTH also acts on kidney cells to make calcitriol from vitamin D, which induces calcium transporters in the intestine and osteoclasts. PTH mediates these effects by activating G-protein-coupled receptors in the kidney and osteoclasts. 

In the acid process, the bones and skins are treated in a vessel containing a dilute solution of acid for a predetermined period of time. Then, the acid is washed out with cold water. In the alkali process, the demineralized bones (demineralization is mostly done with acid solutions to remove calcium and other salts from the bone to prepare the collagen-rich bone material known as ossein) are placed in liming pits and soaked in a lime suspension for longer than 60 days. For the hides or skins, a caustic soda solution is used for a shorter period of time. After this treatment, the raw material is washed thoroughly to remove any residual lime. The acid pretreatment is mostly used for skin, while the alkali pretreatment is mostly used for bones (Petersen and Yates, 1977). 

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