Influence of testosterone and dihydrotestosterone on bone-matrix induced endochondral bone formation

Summary A bone matrix-induced endochondral bone development model has been used to study the effects of androgens on different stages of bone development in castrated young adult rats. Androgen treatment, especially with dihydrotestosterone (DHT) for 7 days, inhibited³⁵SO₄ incorporation by the developing cartilage in the induced plaques. Castrated control animals maintained for 11 days after implantation of bone matrix showed significantly lower calcium levels in the induced implant than was observed earlier in the unoperated controls. DHT treatment for 11 days caused dramatic increases in levels of calcium in the implants. Testosterone had little effect. When androgen treatment was continued for 21 days, while levels of alkaline phosphatase in the implants were unaffected, levels of calcium in the implants were significantly higher than on day 11 for both castrated control and androgen-treated animals. Peak alkaline phosphatase activity (day 10) is known to precede peak calcium mineralizing activity (day 12) in this model and it is also known that calcium levels remain high thereafter. Evaluation of calcium and alkaline phosphatase levels in the proximal tibial metaphyses of castrated control and androgen-treated groups of animals showed no changes after 11 days treatment. Prolonged treatment (21 days) elevated the levels of alkaline phosphatase whereas no change was observed in calcium levels in the tibial metaphyses. These findings demonstate that androgens stimulate mineralization and that DHT is more active when used for short periods of time and in early stages of bone development in matrix-induced implants.     

Changes in lipids during matrix: Induced endochondral bone formation

Summary The changes in lipids occurring during the process of endochondral ossification have been characterized by studying the discrete phases of matrix-induced endochondral bone formation in the rat. Calcium-acidic phospholipid-phosphate complexes were shown to increase in concentration during cartilage calcification (day 9) and to peak in content during early bone formation (day 11–13), the times during which the rate of mineral deposition, as indicated by the change in ash weight was greatest. These data support the hypothesis that the calcium-acidic phospholipid-phosphate complexes play a role in thein vivo initiation of hydroxyapatite deposition. The overall lipid composition of the induced matrix newly formed cartilage (days 7–9) was comparable to that of normal cartilage, with the phospholipid composition matching that of chondrocyte plasma membranes. Times of vascular invasion and formation of marrow cavities were marked by elevated total lipid and triglyceride contents.     

TRANSFORMATION OF FIBROBLASTS BY ALLOGENEIC AND XENOGENEIC TRANSPLANTS OF DEMINERALIZED TOOTH AND BONE

Xenogeneic transplants of powdered, dehydrated, demineralized matrix of bone and tooth were well tolerated in three species of rodents. Differences between the species were found in competence of fibroblasts to be transformed into cartilage and bone in vivo by these preparations. Rat fibroblasts were most susceptible to transformation of this sort; they were transformed by demineralized dentin of guinea pig, mouse, and rat, and to a limited extent, by a specimen of decalcified human bone.     

The potent bone-resorbing mediator of Actinobacillus actinomycetemcomitans is homologous to the molecular chaperone GroEL.

Actinobacillus actinomycetemcomitans is a Gram-negative bacterium implicated in the pathology of localized juvenile periodontitis, a condition involving rapid destruction of alveolar bone. We have established that gentle extraction of this bacterium in saline releases a proteinaceous fraction (which we have termed surface-associated material [SAM] which has potent osteolytic activity in the murine calvarial bone resorption assay. Fractionation of the SAM has now revealed that activity is associated with a 62-kD protein. This bone-resorbing activity can be blocked by a monoclonal antibody (raised to the whole bacterium) that is claimed to recognize a protein homologous to the Escherichia coli molecular chaperone GroEL. Purification of this bone-resorbing protein to homogeneity has been achieved by a combination of anion exchange, gel filtration, and ATP-affinity chromatography and the NH2-terminal sequence shows > 95% homology to E. coli GroEL. This GroEL homologue is found in the SAM of A. actinomycetemcomitans but is not found in the osteolytically active SAM from other Gram-negative or Gram-positive bacteria. The GroEL protein from E. coli, but not from Mycobacterium tuberculosis and Mycobacterium leprae, also showed activity in the bone resorption assay. We believe this to be the first observation that a molecular chaperone has the capacity to stimulate the breakdown of connective tissue.     

Changes in lipids during matrix: induced endochondral bone formation

The changes in lipids occurring during the process of endochondral ossification have been characterized by studying the discrete phases of matrix-induced endochondral bone formation in the rat. Calcium-acidic phospholipid-phosphate complexes were shown to increase in concentration during cartilage calcification (day 9) and to peak in content during early bone formation (days 11-13), the times during which the rate of mineral deposition, as indicated by the change in ash weight was greatest. These data support the hypothesis that the calcium-acidic phospholipid-phosphate complexes play a role in the in vivo initiation of hydroxyapatite deposition. The overall lipid composition of the induced matrix newly formed cartilage (days 7-9) was comparable to that of normal cartilage, with the phospholipid composition matching that of chondrocyte plasma membranes. Times of vascular invasion and formation of marrow cavities were marked by elevated total lipid and triglyceride contents.     

Dissociative extraction and reconstitution of extracellular matrix components involved in local bone differentiation.

Subcutaneous implantation of demineralized diaphyseal bone matrix in allogeneic rats results in the local induction of endochondral bone differentiation. We have explored the potential of three dissociative extractants, 4 M guanidine hydrochloride (Gdn . HCl), 8 M urea/1 M NaCl, and 1% NaDodSO4 at pH 7.4, containing protease inhibitors to solubilize putative inductive molecules in the bone matrix. Extraction of bone matrix with any one of these extracts resulted in the loss of the bone inductive property. The solubilized extracts were then reconstituted with the residue by dialysis against water. The various reconstituted matrices were bioassayed for bone inductive potential by quantitation of alkaline phosphatase activity and 45Ca incorporation on day 12 after implantation. There was complete recovery of biological activity after reconstitution of the residues with each of the three extracts. Polyacrylamide gel electrophoresis of the extracts revealed similar protein profiles. Gel filtration of the 4 M Gdn. HCl extract on Sepharose CL-4B showed a heterogeneous broad peak. When fractions of that peak containing proteins less than 50,000 daltons were reconstituted with inactive 4 M Gdn . HCl-treated bone matrix and then implanted, new bone was induced. These observations demonstrate the dissociative extraction and successful biological reconstitution of bone inductive macromolecules in demineralized bone matrix.     

Riboflavin nutritional status and flavoprotein enzymes in normal and genetically diabetic KK mice

Riboflavin nutritional status in normal control Swiss albino (SA) and genetically diabetic (KK) mice aged 8–9 mo was assessed by determining the glutathione reductase activity in erythrocyte hemolysates, with and without addition of flavin adenine dinucleotide (FAD); the ratio (activity with added FAD)/(activity without FAD) was expressed as the activity coefficient (AC). AC values from 0.9 to 1.3 were considered normal and those greater than 1.3 were regarded as evidence of riboflavin deficiency. Among SA mice, 35% were found to be riboflavin deficient. Among KK mice, 83% were riboflavin deficient. The difference in prevalance was significant (p < 0.01). Supplementation of riboflavin to deficient KK mice returned their AC values to normal. Based upon AC values, both SA and KK mice were classified into normal (nondeficient) and riboflavin-deficient groups. Glutathione reductase (GR) activity in erythrocyte hemolysates and liver supernatants was significantly lower in both deficient SA and KK than in normal SA and KK mice. Treatment of deficient KK with riboflavin restored the enzyme activity in both preparations to normal. In contrast to the finding in erythrocytes, the hepatic GR activity was not increased by FAD in vitro either in normal or deficient mice. Hepatic mitochondrial succinate dehydrogenase (SDH) activity was significantly decreased in riboflavin-deficient KK mice. The enzyme activity was increased several-fold above normal in deficient KK mice supplemented with riboflavin. In conclusion, the data suggest that genetic diabetes increases the prevalence of riboflavin deficiency, which in turn causes a decrease in the erythrocyte and hepatic GR and hepatic SDH activities. This deficiency can be corrected by riboflavin supplementation, with subsequent augmentation of GR and SDH activities, indicating that these enzymes are dependent on the riboflavin nutritional status of the animal.