Thèse de : Prof. Dr. J.M. MBUYI MUAMBA

Studies on the organic matrix of bone; 1984

Promoteur: Prof. Dr. W. MERLEVEDE

Summary:

In the first part of this monograph, an overview of the general methodology of bone matrix studies was described. Special consideration has been given to the size of the bone particles and to the cut-off of the dialysis tube during EDTA extraction and demineralization. Indication of bone particle size and dialysis tube cut-off may permit a better understanding of analytical results. Two methods of determination of bone matrix components were improved. (1) polyethylen glycol (PAG 6000) was incorporated to immunodiffusion agar gels and was found to enhance the visualization of the immune precipitates. This permitted to detect 3 plasma proteins (IgE, IgD and α1 acid glycoprotein) which were not previously described in bone. IgE, α1 acid glycoprotein and α2 HS-glycoprotein were found to be concentrated in the bone more than in serum by factors between 11 and 525. (2) using Alcian Blue 8GX, glycosaminoglycans (GAG’s) and proteoglycans (PG’s) were determined in the bone extracts. The results obtained showed that GAG’s and PG’s make up about 7.35% of the total noncollagenous fraction, value which is in agreement with other results of GAG’s reported in literature (Leaver, 1979).

As stated in the introductory note, the aims of the present thesis were to investigate some physiological and pathological variations of bone matrix. Essential results are summarized in the synoptic tables 1, 2 and 3. Table 1 gives the results of matrix; table 2 lists the results of EDTA extractability and collagenase susceptibility, and table 3 compiles the findings of the gradient density fractionation of bone.

1) Matrix compositions of bone and skin from rats were compared in terms of size, and collagen and non collagenous components. Bone matrix was smaller than skin matrix (p<0,001) and contained higher amounts of sialic and uromic acids (p<0,001). The EDTA extractability of bone was lower while its collagenase susceptibility was higher than those of skin (p<0,001).

2) The age and sex-related changes of bone matrix were assessed in wistar rats. Bone matrix size and chemical composition were affected more by aging than by sex. The changes in bone matrix composition were more marked in the noncollagenous fraction than in collagen. The bone matrix size becomes smaller with increase of age. The sialic acid content consistentently increased while uronic acid decreased with aging. In general, collagen/noncollagenous component ratios showed a downward trend with aging. Decrease of the EDTA extractability and collagenase susceptibility were also observed with advancing age. The radiogrammetric and photonabsorptiometric measurements of bone mass in rats disclosed a continuous growth of bone mass with aging.

3) The effects of pregnancy on bone matrix proteins were investigated in wistar rats. Pregnancy brings about a significant decrease of the matrix size (p<0,05) and increase of EDTA extractability of matrix components (p<0,05), this latter being marked especially for collagen (p<0,007).

4) Bone matrix variations were assessed in humain bone according to the anatomical localization and types of bone (spony or compact bone). Rib, iliac crest and femur were used in this respect. Significant variations were observed in the matrix size and bone matrix composition. Matrix size of femur than in rib or in iliac crest (p<0,05-p<0,01). Spongy bone had slightly higher extractability value, and lower collagen/noncollagenous component ratios than compact bone.

5) Bone tissue maturation was studied in human bone using the density gradient fractionation technique. Significant differences were found between spongy and compact bone, spongy bone containing a higher proportion of little mineralized osteons than compact bone while this latter contained higher proportion of fully mineralized osteons than spongy bone (p<0,01). A decrease of the matrix size and EDTA extractability from bone fractions of increasing mineral density was noted. Among the matrix components of these bone fractions sialic acid consistensly increased while uronic acid decreased, relative to thez increase of mineral density.

6) Significant interspecies differences were found in the matrix size and composition of rat, bovine and human bones. The extractability of rat, bone was higher than that of bovine (p<0,01) and human bones (p<0,001). Inversely, the matrix size of rat bone was smaller than that of bovine bone and more markedly for human bone (p<0,001). Bone matrix and EDTA extracts from rats contained higher amounts of non collagenous proteins (p<0,001) and loxer amounts of collagen (p<0,05 – p<0,001). The collagen/noncollagenous component ratios were lower in rats than in bovine or human bones (p<0,001).

7) The effect of arthritis was assessed both in experimental conditions (adjuvant arthritis) and rheumatoid arthritis (R.A). in adjuvant arthritis, marked increase of matrix exrtractability and size was found. Treatement with either Indomethacin or Clozic did not affect significantly the EDTA extractability and matrix size. The matrix composition was modified by the arthritis process. Marked decrease of the collagen and hexose contents was observed (p<0,01) while the sialic acid was increased in arthritic bone (p<0,05). The effects of antirheumatic treatment on individual components of matrix are variable, depending on the drug used and the bone site examined. In general Indomethacin further decreased the amounts of hexose, uronic and sialic acids in the metaphysic and increased these components in the diaphysis. Clozic increased the hexose and discreased the hydroxyproline in the diaphysis while a decrease of uronic acid was observed in the metaphysis. Noteworthy is the marked decrease of collagenase susceptibility brought about by arthritis. In human arthritis, an increase in the collagen content of bone matrix and a decrease of collagen extractability (p<0,05) were the only prominent features.

8) Bone matrix changes were studied in human osteoerthrosis. The EDTA extractability of osteoarthrotic bone was significantly increased as well as the amounts of noncollagenous component ratios were lower in osteorthrosis than in controls (p<0,001). Studies of the fractionation patterns of bone particles according to the mineralization degree of asteons showed a higher proportion of little mineralized osteons in osteoarthrosis, reltive to controls. A decreased collagenase susceptibility was also noted in osteoarthrotic bone.

9) Studies of bone matrix changes in human osteoporosis disclosed a discrease in the EDTA extractability and collagenase susceptibility and an increase in bone matrix size. The total amount of sialic acid also descreased (p<0,05). The collagen/noncollagenous component ratios were higher in osteoporotic bone particles according to the mineralization degree of osteons disclosed 2 subtypes: (1) a subtype with predominance of little mineralized osteons. In experimental osteoporosis (osteoporosis induced by high dosis of ketoconazole), a decrease of the sialic acid content as well as an increase of matrix size and collagen/noncollagenous component ratios were also observed. From thses studies of bone organic matrix in normal and pathological conditions the following conclusions can be made. Bone differs from other connective tissues by its matrix size. This gradually and markedly decreases with the increase of mineralization and aging reflecting a relationship between the bone matrix size and the mineralization degree. Changes in bone not only involve its matrix size but also its composition. Although collagen is the most abundant component of the matrix, the noncollagenous fraction seems to be more affected by bone matrix variations. Indeed, bone and skin contain the same amounts of collagen. However, their noncollagenous component contents markedly differ.

Sialoprotein and proteoglycan have been found to be in higher amount in bone than in skin. Similarly, the bone collagen content does not vary with aging while the noncollagenous proteins, especially sialoprotein considerably increases while proteoglycan decreases. Marked differences were also observed in the non collagenous fraction and not in the collagen content fraction of rib, iliac crest and femur. The changes in the bone collagen contet were observed in arthritic conditions where a decrease was noted in adjuvant arthritis and an increase in human arthritis. Apart from these changes in arthritis, other pathological conditions of osteoporosis and osteoathrosis displayed marked changes mainly in the noncollagenous fraction. Therefore, the use of collagen/noncollagenous component ratios sees to be an interesting index of alteration of bone matrix composition. As there exists an intimate relationship between the fort, function nd the internal organization of bone, the alteration of these ratios may be related to the alteration of the bone biomechanical function. In general, these ratios were found to be low in conditions where the biomechanical function was important such as in the femur relative to iliac crest and rib or preserved such as in osteoarthrosis relative to osteoporosis. The reverse ws true in conditions where the biomechanicam function was altered, such as in osteoporosis and rheumatoid arthritis where the ratios are higher relative to controls.

Our study has also demonstrated that the alteration of extractability of bone tissue may be related to the alteration of its turnover since increase in EDTA extractability occurred where bone turnover may de high, for example in young bone, in arthritic inflammation, and osteoarthrosis while it was decreased in osteoporosis and in old bone where a decrease in turnover may de expected. Studies of collagenase digestion showed that with aging, arthritis, osteoporosis, the collaagenase susceptibility was markedly reduced according to a mechanism which may be related to the nature and number of crosslinking in normal conditions and to the possible alteration of collagen structure in pathological conditions. The technique of bone fractionation according to the mineral density of osteons seems to be a suitable

method for assessing bone changes during the mineralization process. The quantitative and qualitative changes observed in bone matrix in the present study, both in physiopathological and pathological conditions may be considered as a step to further in this field. Through these studies we have learned that bone research is as herd the tissue itself.