Fatty acid oxidation in bone tissue and bone cells in culture. Characterization and hormonal influences.

Fatty acid oxidation and its hormonal modulation were investigated in cultured rat calvaria and in cultivated cell populations. The latter were obtained from calvaria of newborn rats by sequential time-dependent digestion with collagenase, yielding eight cell populations: the early ones containing mainly fibroblasts, the middle ones being osteoblast-like, and late ones osteoblast-osteocyte-like. In calvaria, fatty acid oxidation was increased by adding 0.1 mM- and 1.0 mM-palmitate to the medium, containing 10% (v/v) fetal-calf serum. No effect was found after parathyrin addition in vitro or when injected in vivo. All cell populations obtained by sequential digestion were found to oxidize palmitate, whereby the osteoblast-like cells showed a lower oxidation rate than the other populations. Both parathyrin and calcitonin had no effect on fatty acid oxidation. 1,25-Dihydroxycholecalciferol at 1-100 nM and 24,25-dihydroxycholecalciferol at 100 nM increased oxidation primarily in the population enriched with osteoblast-like cells. Insulin at 1.6 microM diminished it in the cell populations enriched with osteoblast-like cells and in the late bone-cell fraction. However, glucagon had no effect. The energy provided by fatty acid oxidation in this system is approx. 40-80% of glucose metabolism, suggesting that this event may be of importance in the energy metabolism of bone.     

Phosphoproteins of chicken bone matrix. Proof of synthesis in bone tissue

Twelve-day-old embryonic chick mandibles were cultured in vitro for 6 days. Measurements of the weights of the explants, their mineral and protein components, and the EDTA-extractable proteins established that bone tissue synthesizes O-phosphoserine- and O-phosphothreonine-containing phosphoproteins which are similar to those present in embryonic and postnatal chicken bone matrix. The synthesis of the phosphoproteins was further confirmed by the demonstration that radioactively labeled O-phosphoserine and O-phosphothreonine were identified in bone and in the EDTA-extractable phosphoproteins after pulse-labeling chick mandibles in vitro with radioactively labeled serine and threonine, respectively.     

Studies on bone enzymes. The assay of acid hydrolases and other enzymes in bone tissue.

1. Nine acid hydrolases, cytochrome oxidase, alkaline phenylphosphatase and catalase were demonstrated in 0.25m-sucrose homogenates of newborn-rat calvaria. The acid hydrolases were: acid phenylphosphatase, acid beta-glycerophosphatase, beta-glucuronidase, beta-N-acetylglucosaminidase (beta-N-acetylaminodeoxyglucosidase), acid ribonuclease and acid deoxyribonuclease, showing optimum activity at about pH5; cathepsin, beta-galactosidase and hyaluronidase, with optimum activity at about pH3.6. 2. The main kinetic characters of these enzymes have been studied and methods for their quantitative assay have been worked out. The activities present in bone are given and compared with those found in liver. 3. Acid-phosphatase activity was assayed with phenyl phosphate and beta-glycerophosphate as substrates: activities with these two substrates appeared to be due to two different enzymes. Acid phenylphosphatase is particularly labile and is readily inactivated by various physical or chemical agents.     

Biomechanics in bone tissue engineering

Biomechanics may be considered as central in the development of bone tissue engineering. The initial mechanical aspects are essential to the outcome of a functional tissue engineering approach; so are aspects of interface micromotion, bone ingrowths inside the scaffold and finally, the mechanical integrity of the scaffold during its degradation. A proposed view is presented herein on how biomechanical aspects can be synthesised and where future developments are needed. In particular, a distinction is made between the mechanical and the mechanotransductional aspects of bone tissue engineering: the former could be related to osteoconduction, while the latter may be correlated to the osteoinductive properties of the scaffold. This distinction allows biomechanicians to follow a strategy in the development of a scaffold having not only mechanical targets but also incorporating some mechanotransduction principles.     

Direct extraction and assay of bone tissue collagenase and its relation to parathyroid-hormone-induced bone resorption.

A method has been developed for the quantitative extraction of collagenase from as little as one 19-day-fetal-mouse calvarium. About 20-40 munits of collagenase are extracted per mg of tissue, all in a latent form that, after proper activation, shows the typical properties of mammalian collagenase. Culturing the calvaria for 2 days with parathyroid hormone (PTH) increases their procollagenase content up to 3-fold and induces bone resorption. Both PTH effects are prevented by cycloheximide, but not by indomethacin. Calcitonin inhibits resorption without affecting the PTH-induced procollagenase synthesis. The role of this synthesis is discussed in relation to the mechanisms of bone resorption.     

Protein-based tissue engineering in bone and cartilage repair

Bioactive proteins signal host or transplanted cells to form the desired tissue type. Matrix systems are utilized to locally deliver the proteins and to maintain effective protein concentrations. For some indications, a matrix is required to define the physical form of the regenerated tissue. Substantial progress has been made in bone tissue engineering in recent years, based on the results of controlled clinical studies using bone morphogenetic proteins. Ongoing research in this area centers on the design of additional delivery matrices to expand the clinical indications, using synthetic delivery systems that mimic biological qualities of the natural materials currently in use. Although a similar rationale exists for the regeneration of articular cartilage with bioactive factors, advancement in this area has not been as substantial.     

Protein- and gene-based tissue engineering in bone repair

A tissue engineering approach to bone regeneration includes the use of a scaffold, cells and bioactive factors alone or in various combinations. Several investigators have demonstrated enhanced bone formation when the tissue-engineered construct possesses traits inherent to autogenic bone grafts, namely osteoconductivity, osteoinductivity and osteogenicity. Use of the biodegradable polymer poly(lactide-co-glycolide) in combination with bone morphogenetic protein or primary cells genetically modified to release osteogenic protein have demonstrated the ability to induce osteogenic differentiation of, and subsequent mineralization by, muscle-derived cells and mesenchymal stem cells in both in vitro and in vivo applications.     

Cytochrome oxidase activity in bone tissue

Cytochrome oxidase activity was studied in articular and epiphyseal cartilage, in bone tissue and in callus. Changes in the activity of the enzyme were observed after ischaemic condition. Activity was the highest in the epiphyseal cartilage while in the other structures it decreased in the order of articular cartilage, chondroid tissue, newly formed woven bone, connective tissue cells, immature, preexisting lamellar bone tissue. It is assumed that differences in enzyme activity are due to differences in metabolic rate. The higher the activity of a structure in the intact state, the more marked was its decrease upon ischaemia. This implies that cells of higher metabolic rate are more sensitive to ischaemia.     

The relationship between bone marrow adipose tissue and bone metabolism in postmenopausal osteoporosis

Postmenopausal osteoporosis (PMOP) is a prevalent skeletal disorder associated with menopause-related estrogen withdrawal. PMOP is characterized by low bone mass, deterioration of the skeletal microarchitecture, and subsequent increased susceptibility to fragility fractures, thus contributing to disability and mortality. Accumulating evidence indicates that abnormal expansion of marrow adipose tissue (MAT) plays a crucial role in the onset and progression of PMOP, in part because both bone marrow adipocytes and osteoblasts share a common ancestor lineage. The cohabitation of MAT adipocytes, mesenchymal stromal cells, hematopoietic cells, osteoblasts and osteoclasts in the bone marrow creates a microenvironment that permits adipocytes to act directly on other cell types in the marrow. Furthermore, MAT, which is recognized as an endocrine organ, regulates bone remodeling through the secretion of adipokines and cytokines. Although an enhanced MAT volume is linked to low bone mass and fractures in PMOP, the detailed interactions between MAT and bone metabolism remain largely unknown. In this review, we examine the possible mechanisms of MAT expansion under estrogen withdrawal and further summarize emerging findings regarding the pathological roles of MAT in bone remodeling. We also discuss the current therapies targeting MAT in osteoporosis. A comprehensive understanding of the relationship between MAT expansion and bone metabolism in estrogen deficiency conditions will provide new insights into potential therapeutic targets for PMOP.     

Ultrastructural and morphometric alterations in bone marrow stromal tissue after 7 Gy irradiation.

To evaluate the response of marrow stroma to 7 Gy irradiation, femoral bone marrow was fixed by vascular perfusion (so as to avoid the artificial destruction of sinus endothelia), and was examined using light and electron microscopy with morphometric methods. The radiation caused a marked decrease in hematopoietic cell number (NHC) within 3 days post-irradiation, followed by total recovery of hematopoiesis, which occurred gradually over 28 days. An increased number of fat cells was seen by 7 days. During the whole course of hypoplasia and recovery, the continuity of sinus wall, three-dimensional reticular mesh work in hematopoietic parenchyma, gap junctions (GJ) between stromal cells, the adventitial cell cover of sinus wall (ACC), and the stromal cell numbers of reticular cells (RC), sinus endothelia (SE), and macrophages (MP) were maintained. The cellularity of stromal components of RC, SE, and MP seemed passively increased in contrast to a reduction in numbers of NHC. A similar tendency was observed (1) between NHC and ACC and (2) between GJ and the cellularity of fat cells, which had a statistical significant correlation (p less than 0.05; t-test). The mechanism of radio resistance in bone marrow stroma and the possible functional adaptation and cellular coordination after irradiation are discussed.