LeRoy Klein

Contribution of collagen and mineral to the elastic-plastic properties of bone

Tension testing of wet bovine haversian cortical bone demonstrated marked plastic behavior. Progressive surface decalcification of this bone with dilute hydrochloric acid resulted in progressive decreases in the tension yield point and the ultimate stress with no change in the yield strain or ultimate strain unless decalcification was complete. The slope of the plastic region remained identical throughout decalcification. These findings are consistent with an elastic-perfectly plastic model for the mineral phase of bone tissue in which the mineral contributes the major portion of the tension yield strength. The slope or stiffness of the plastic region of the stress-strain curve is a function only of the properties of collagen, which itself plays a minor role in the tension yield strength of bone.

Critical biological determinants of incorporation of non-vascularized cortical bone grafts. Quantification of a complex process and structure

Our goal in this study was to evaluate the effects of and the interaction between the hypothesized principal determinants of the incorporation of grafts: antigenicity and treatment of the graft. We implanted fresh and frozen cortical bone grafts that were matched for both major and non-major histocompatibility complex antigens (syngeneic grafts), matched for major but not for non-major histocompatibility complex antigens (minor mismatch), and mismatched for both major and non-major histocompatibility complex antigens (major mismatch). We used a rat model with an eight-millimeter segmental defect in the femur. The construct was stabilized with a plastic plate, threaded Kirschner wires, and cerclage wires. We evaluated the grafts at one, two, and four months after implantation. We measured the immune response; assessed the incorporation of the graft with use of histological examination, biomechanical testing, and quantitative isotopic kinetics; and statistically analyzed the effects of and the interactions among three independent variables: time, the degree of matching for major histocompatibility complex antigens, and the treatment of the graft (whether it was fresh or frozen). These three independent variables had profound effects on the pattern, rate, and quality of the incorporation of the graft. Two-way and three-way interactions among these variables were also noted. Serial changes in every dependent variable were observed with time. Systemic antibody specific for donor antigens was measurable only in the serum of animals that had a major mismatch, but freezing markedly attenuated the systemic antibody response. Revascularization was profoundly affected by histocompatibility-antigen matching; the syngeneic grafts were revascularized more quickly and to a greater degree than the grafts with either a minor or a major mismatch. Freezing significantly (p < 0.001) reduced the revascularization of the syngeneic grafts but had no discernible effect on the grafts with a minor mismatch. CLINICAL RELEVANCE: The findings of this investigation are clinically important because they help to explain the unpredictability of incorporation of cortical bone grafts. The graft that is most commonly implanted clinically, the frozen (or processed) mismatched allograft, had the least predictable process of incorporation. However, our findings suggest that the process of incorporation may be manipulated; for example, by the addition or removal of cells and, indirectly, of cytokines.

Isotopic evidence for resorption of soft tissues and bone in immobilized dogs.

UNLABELLED Various experimental methods for producing bone and ligament atrophy have yielded contradictory results. These methods include denervation, immobilization (both internal and external), and disarticulation. We studied a model of internal skeletal fixation for twelve weeks in dogs that were chronically prelabeled with 3H-tetracycline, 45Ca, and 3H-proline. Bone resorption was analyzed by the loss of 3H-tetracycline, and bone and soft-tissue mass were analyzed by the radiochemical and chemical analysis of calcium and collagen. The strength of the anterior cruciate ligament was studied in tension to failure when a fast rate of deformation was applied. Failure of the femur-ligament-tibia complex occurred through the insertion of the ligament into the tibia for both the experimental and the control limbs. Loss of collagen was greater in the tibia and femur than in the lateral meniscus and anterior cruciate ligament, and correlated with a mechanical failure via bone. No evidence for collagen replacement in atrophied tissues was found, but one-half of the resorbed calcium was conserved. The marked loss of 3H-tetracycline indicated that bone atrophy was the result of increased resorption of bone rather than decreased bone formation. CLINICAL RELEVANCE We have demonstrated significant atrophy of the soft tissues (lateral meniscus and anterior cruciate ligament) as well as of bone in immobilized joints of dogs. It is likely that the decrease in strength of the bone-ligament-bone complex is related to this atrophy of soft tissues and bone around the joint.

Assay of bone resorption in vivo with3H-tetracycline

Abstract3H-Tetracycline (3H-TC) was used to quantify resorption in whole bones of growing rats and dogs. After repeated isotopic labeling of actively growing embryos or neonates,3H-TC was observed to be distributed homogeneously and in equilibrium with45Ca. A rapid and large loss of3H-TC and a small loss of45Ca occurred during the early weeks of rapid bone growth, suggesting thatabsolute amounts of45Ca resorbed from bone, as reflected by losses of3H-TC, are five to ten times greater than thenet amounts of45Ca lost from bone. Minimal loss of3H-TC occurred due to nonspecific physicochemical exchange in vivo or in vitro (5%) except with nonradioactive tetracycline, and3H-TC was not greatly exchanged or reused (10%) in vivo. The data are considered in terms of local and systemic conservation of calcium.

Lowry assay of dilute protein solutions containing high concentrations of Triton X-100

Abstract Lowrys method (1) for protein determination is subject to interference from the nonionic detergent Triton X-100 (2,3) which is used in high concentrations (1–5%) to solubilize membrane proteins or enzymes (4–6) and structural acidic proteins (7). Hartree (3) could reduce the errors caused by 0.1% Triton X-100 by a modification of Lowrys method. However, when protein solutions containing 0.2% or more of the detergent are mixed with the Folin-Ciocalteu reagent (1) a precipitate forms that interferes with the assay. We could reduce this interference to an insignificant level either by centrifuging the precipitate and incorporating Triton X-100 in both the reagent blank and standards, or by removing the detergent prior to the assay. This report presents two simple procedures for the Lowry assay of dilute protein samples containing 1–5% Triton X-100.

Comparative response to parathyroid hormone in hyperthyroidism and hypothyroidism.

The effects of exogenous parathyroid hormone, administered for 3 days, were compared in six hyperthyroid and six hypothyroid subjects. Maximum increments were much greater in hyperthyroid than in hypothyroid subjects for serum calcium (3.5 mg/100 ml versus 1.6 mg/100 ml), urine calcium (476 mg versus 79 mg), urine hydroxyproline (56 mg versus 11 mg), and urine phosphorus (671 mg versus 192 mg). Maximum decrease in serum phosphorus (minus0.9 mg/100 ml versus minus 0.1 mg/100 ml) was also greater in hyperthyroid subjects. Serum parathyroid hormone immunoreactivity was significantly higher in hypothyroid subjects (0.48 ng/ml) that either normals (0.21 ng/ml) or hyperthyroid subjects (0.19 ng/ml). The data support the concept that excess thyroid hormone sensitizes and deficient thyroid hormone blunts the responsiveness of bone to parathyroid hormone. This may lead to a state of hypoparathyroidism in hyperthyroidism and hyperparathyroidism in hypothyroidism.

Biological and physical properties of autogenous vascularized fibular grafts in dogs.

The biological and biomechanical properties of normal fibulae, fibulae that had had a sham operation, and both vascularized and non-vascularized autogenous grafts were studied in dogs at three months after the operation. The study was designed to quantify and correlate changes in these properties in orthotopic, stably fixed, weight-bearing grafts and to provide a baseline for additional studies of allografts. The grafts were eight centimeters long and internally fixed. The mechanical properties of the grafts were studied by torsional testing. Metabolic turnover of the grafts was evaluated by preoperative labeling of the dogs with 3H-tetracycline for resorption of bone mineral and with 3H-proline for turnover of collagen. Cortical bone area and porosity were measured. Postoperative formation of bone was evaluated by sequential labeling with fluorochrome. The vascularized grafts resembled the fibulae that had had a sham operation and those that had not had an operation with regard to the total number of osteons and the remodeling process, as measured both morphometrically and metabolically. The vascularized grafts were stronger and stiffer than the non-vascularized grafts and were not different from the bones that had had a sham operation. In contrast, the non-vascularized grafts were smaller, weaker, less stiff, and more porotic, had fewer osteons, and demonstrated increased turnover and resorption compared with the vascularized grafts, the bones that had had a sham operation, and the bones that had not been operated on.

Cyclosporin A does not affect the absolute rate of cortical bone resorption at the organ level in the growing rat.

The weanling rat, an animal model of rapid bone turnover, was used to evaluate the effects of various doses of cyclosporin A (CsA) on various bones during different time periods. Sprague-Dawley male rats were extensively prelabeled with 3H-tetracycline during 1–3 weeks of age. At 4 weeks of age, four groups of rats were given daily subcutaneous injections: vehicle or CsA—low dose (10 mg/kg), intermediary dose (20 mg/kg), or high dose (30 mg/kg) for 7, 14, or 28 days. Three different whole bones—the femur (low turnover), scapula (moderate turnover), and lumbar-6 vertebra (high turnover) were harvested intact at 4, 5, 6, and 8 weeks of age. The whole bones were assayed weekly for total dry defatted weight, calcium mass (formation), and loss of 3H-tetracycline (bone resorption) following treatment with CsA. Serum CsA levels, calcium creatinine, and alkaline phosphatase were measured weekly. Significant decreases in serum calcium and alkaline phosphatase were observed at 1 and 2 weeks, and were normalized by 4 weeks of treatment. No significant changes in serum creatinine were noted. For all three doses of CsA, no effect was observed on the absolute rate of cortical bone resorption of three different, whole bones over three time periods. Body weight and bone formation in treated animals was significantly smaller in a dose- and time-related fashion compared with control animals at sacrifice. However, compared with the initial control animals, body weights and bone masses of the final treated animals were much larger, suggesting that the smaller bone masses were due to insufficient growth and slow gain in bone mass. Our isotopic data demonstrate that CsA has no effect on the basal rate of bone resorption and decreases rate of bone formation, as observed globally at the whole bone level. Bone measurements at the organ level may lead to different interpretations from those observed at the tissue level.

Simultaneous Quantification of 3H-Collagen Loss and 1H-Collagen Replacement During Healing of Rat Tendon Grafts

Tail or Achilles tendons were taken from rats that were repeatedly prelabeled with 3H-proline so as to be in an isotopic steady state. Fresh and frozen isografts and allografts, plus frozen xenografts were implanted as functional Achilles grafts into rats and guinea pigs. At one month of implantation isografts showed a one to one replacement of resorbed old collagen with non-radioactive new collagen without a change in total collagen mass. Allografts had almost complete replacement with a small loss of collagen mass. After three months of implantation, isografts showed continued maintenance of collagen mass while allografts showed a continued decrease in replacement and total mass. The major effect of the immune response on collagen turnover in tendon allografts was to partially inhibit the production of new collagen with a resultant atrophy in collagen mass. Scar formation in tendon isografts or allografts was minimum although 50 to 64 per cent of the grafted tendon collagen turned over in three months time. The xenograft showed an almost complete loss of pre-existing collagen of tendon within a period of one month. The loss of morphological integrity of tendon occurred only in the xenograft. In tendon xenografts there was a marked increase in collagen destruction with almost complete inhibition of collagen synthesis. The grafting of a known amount of radioactive tendon to non-radioactive animals and its precise removal has permitted the simultaneous quantification of mature collagen destruction, new collagen replacement and change in collagen mass. Any loss in collagen mass as a result of grafting was due chiefly to insufficient or no replacement of the destroyed collagen with new collagen.

Determination of inorganic phosphorus in the presence of organic phosphorus and high concentrations of proteins

Abstract The phosphorus assay method of Lowry and Lopez has been modified to permit a rapid and accurate determination of inorganic phosphorus in the presence of organic phosphorus without prior deproteinization of the samples. The reduction of ammonium phosphomolybdate to molybdenum blue is carried out with ascorbic acid in the presence of acetate buffer, pH 4.0, containing 10% sodium dodecyl sulfate to prevent protein precipitation. The method is applicable to samples containing up to 30 mg protein per milliter of final reaction volume. Acid-labile phosphorus compounds present in serum are not hydrolyzed, and less than 5% phosphocreatine is split during the time necessary for the assay. The addition of SDS to the buffer does not affect the sensitivity and reliability of Lowry and Lopezs method.