Document Gjm0XKw3gzZ5jZykknVKmv5n

B.F.GOODRICH Ci-IMMiO'.H CGHl'AKY In f_<? r-0tr i ? ;< L 1 on Ce;.'iV:,cr'0:'.He pee j o : John MeI son Loca tion: Cleveland Da to: May 21, 1959 1' roni: L . B . Cr ido. r Subject: Review of Hand Problem Prior to our meeting on May 29thto^K?VW^t!e^^ffl!''bf this problem, I thought it would be well to set dt-m on paper some, of our current thinking about the results obtained since ur last review. 1 would als like to make some comments about the direction in which ur future research relating to this problem should be headed. With respect t ur findings since aur last review, I have enclosed a summary of Dr. Eirichs objectives and cnclusins including the results from both his 1968 and 1969 reports. Hopefully, this will relieve you f some of the agony of sorting out these facts from the rather lengthy, narrative description of his work. There are several significant facts that have been barn ut of this work. Firstly, is his discovery that slight imbalances in calcium and phosphate ions have a substantial effect on the'soluability f hydroxyapatite (HAP). This finding is particularly significant when one considered the possible anesthetic action of YC1 monomer on the osteoblast and osteoclast cells. Another significant result from Dr. giriebs ' work (although a major portion of this phase was done at the Development Center) is that it has been established that some small amount (508-10QOppm) of VC1 is bound to collagen. Eirich's postulation that this occurs at chain ends is a reasenable rationalisation but remains to be established. The method that he has developed for measuring decalcification and recalcification of bone tissue can be used by Ir. Motzkiia in her studies involving living bone tissue. Dr. Eirich's theory regarding the effects of VCl monomer on the function of the lipid portions f living cells can perhaps be explored by this method. There has also been a considerable advancement in our over all knowledge of the metabolism of bone tissue from the various articles and reviews published in the literature during the past year. Most significant of these is the work showing that the. initial growth of KAP occurs in "holes" in the collagen fibrils. A number of agents are effective in blocking recalcification of bone tissue by reacting with the Z-NH2 2ro,1Ps in collagen. A theory has been proposed that the hydrophobic character of the blocking agents is effective in excluding water from the "holes" in the fibrils thus preventing the initial growth of HAP. Perhaps even more important are the new analytical methods that are suggested for measuring decalcification, in vivo, by monitoring the level of hydroxyproline in urine. It has now been established that major disruptions in bone metabolism can be related to gross changes (increase) in the hydroxy proline level in the urine. This applies to test? in a wide varietv of test animals as well as humans. I have also 20680001 MAY 2 3 1969 Pa ge 2 In our nice tins on May 29, I think it would be well if we could also arrive at soma decisions regarding the type of proof required for a conclusive solution to this problem. One of the things that I have been concerned about is that we can go on ed infinitum in isolating components that can have an effect on the lysing of bone tissue but unless we can see this effect in the x-rays of test animals we will never be convinced that we have found a cause for the problem. The MCA results clearly show that only a small percentage of the total population exposed are vulnerable to this problem. If we can expect this same frequency in test animals the expectations of producing a positive result detectable by x-ray are quite remote. It is my feeling that a more sensitive indicator is needed. If we could, for example, show that exposure to various agents (VC1 monomer, VC1 peroxide, poly scraping, etc.) causes increases in hydroxyproline in the urine or increased calcium or phosphate levels in solid excrement we would then have a positive indicator that we have isolated the material that effects bona matabolism. The results of this type of testing would also be available in a much shorter period of time. As a minimum it should be incorporated in our present test program at Kettering. Data relating to the use of these test methods in problems involving the resorption of bone tissue are now available. We also have in hand the analytical .test methods for this type of study. listribiition W.E. McCormick D.L. Dowe11 P.H. Lawrence C.H. Lufter F.L. Ramp. R.D. Scott Dr. R.H. Wilson R.J. Wolf File dmr 9U L.B. Crider *0 o Cd CD O oo Summary of Dr. Eirich's work contained in 1968 report: Ob jectives 1. Determine the absorption of gelatin on hydroxy apatite (HAP) 2. Measure binding of calcium and .phosphate ions on gelatin. 3. Study the effects of gelatin on the precipitation of HAP. 4. Measure the binding of VC1 monomer on gelatin. Conclusions 1. Gelatin is strongly absorbed on HAP. An absorption isotherm of gelatin on HAP was obtained. In the presence of VC1 monomer the initial absorption of gelatin on HAP is suppressed. . 2. The binding of calcium and phosphate ions to gelatin is small and not much dependent on pH. Ion binding is not effected by VC1 monomer. 3. The precipitation of HAP is only slightly affected by VC1 monomer. 4. The degree of binding of VC1 monomer on gelatin is so slight that it cannot be measured Summary of Dr. Eirich's work contained in 1969 report: Objectives 1. Study the effects of VC1 monomer on the precipitation of hydroxyapatite 2. Continue'work on measuring the direct binding of VC1 on collagen or gelatin. 3. Study changes in collagen structure induced by VC1 monomer. 4. Develope a method for measuring the rate of calcification and decalcification of bone tissue. 5. Complete basic study of decalcification of bone slices. 6. Study the affects of VC1 monomer on decalcification rate. m 2 3 *969 20G60003 Conclusions Page 2 1. Radioactive tracer methods were used to study the precipitation of HAP in vitro. The results obtained are at considerable variance with the data in the literature. It was noted that potassium ions in particular have a sustantial effect on the solubility of HAP. This finding is of utmost importance since it points to the possibility that minor biochemical imbalances of sodium and potassium could be caused by the action of VC1 monomer on the lipid portion of cells. 2. The amount of VC1 bound to collagen is of the order of 500-1000 ppm. This is approximately one VC1 per collagen chain end group. The binding of VC1 is stronger at neutral than at acid pH which also fits the picture of end-group binding. 3. VC1 monomer does not cause any disordering of collagen structure that can be observed by electron microsoope. 4. A method was devel oped for measuring the rate of calcification and decalcification of bone tissue using sedimentation methods. 5. Studies in vitro show two distinct regimes of decalcification, a slower rate followed by one that is faster. The initial slow rate is thought to be diffusion controlled. 6. Although it is too early to arrive at conclusive observations, the presence of VC1 monomer definitely seems to change the decalcification process. Summary of data from the literature Reconstituted collagen fibrils as well as collagen fibrils from a wide variety of tissues and denrineral ized. collagen from bone are effective nucleation calalysts, and are readily able to induce the formation of apatite crystals from solutions that do not form crystals in the absence of collagen. The manner in which collagen macromolecules are packed in native fibrils....steric relationships between amino acid groups....charge distribution.. ..space geometry...are factors that facilitate the induction of apatite crystals. The small apatite crystallites, initially deposited in collagen, in vitro, are located in specific regions of coLlagen fibrils which we now know to correspond to "holes" in the fibrils. There is no established relationship between the 700A repeat spacing in collagen fibrils and calcification. OOSOOO1 Page 3 The blocking of Z-NH2 groups with fluoro-2, 4-dinitrobenzene in vitro completely blocks calcification of collagen. Blocking is also accomplished with carbobenzoxy chloride. This reaction is reversible. The above does not establish that I-NH2 groups are themselves involved in nucleation. The bulky dinitrophenyl or carbobenzoxy groups could distort or sterically shield the other adjacent groups that are vital to recalcification. The recalcification of collagen having modified I-NH2 groups is effected by the exclusion of water by the modifying agents. The modification of the Z-NH2 groups by hydrophobic groups located in the "holes" can effect recalcification by excluding water from the holes. Methylation of the carboxyl groups in collagen inhibits in vitro recalcification of bone tissue. There are numerous references in the literature that osteomyelitis and collagen resorption in humans, rats and other test animals is reflected by increased urinary hydroxyproline. Some interesting work has been reported on the effects of estrogens on collagen matabolism. It is known that collagen in the uterus increases about 8 fold during pregnancy and resorption is complete about 3 weeks after post partum. ^-Estradiol has a marked effect on retarding the degradation of collagen during the first 4 days post partum. SOOOSSOZ