Collagen Mineralization Research Articles

Alteration of mineral crystallinity and collagen cross-linking of bones in osteopetrotic toothless (tl/tl) rats and their improvement after treatment with colony stimulating factor-1

A common feature of various types of mammalian osteopetroses is a marked increase in bone mass accompanied by spontaneous bone fractures. The toothless (tl/tl) rat osteopetrotic mutation is characterized by drastically reduced bone resorption due to a profound deficiency of osteoclasts and their precursors. An altered bone morphology has also been observed. The mutants cannot be cured by bone marrow transplantation, but skeletal defects are greatly reduced after treatment with colony stimulating factor 1 (CSF-1). The objectives of this study were to characterize mineral and collagen matrices in cancellous and compact bone isolated from long bones of 6-week-old normal littermates, tl/tl osteopetrotic mutants and mutants (tl/tl) treated with CSF-1. There were no differences in bone mineral content, but a significant decrease in the crystallinity of mineral evaluated by the method based on electron paramagnetic resonance spectrometry was observed in all bones of tl/tl mutants as compared to that of controls. Within the collagen matrix, slight decreases in the labile cross-links, but significant increases in the content of the stable cross-links, pyridinoline, and deoxypyridinoline, were observed in both cancellous and compact bone of osteopetrotic mutants. In tl/tl mutants treated with human recombinant CSF-1, the normalization of the crystallinity of bone mineral as well as collagen cross-links was found. Our results indicate that remodeling of bone matrix in tl/tl mutants is highly suppressed, but that after treatment with CSF-1, this activity recovers significantly. Taken together, these data provide further support for the hypothesis that CSF-1 is an essential factor for normal osteoclast differentiation and bone remodelling.

Tracing the pathway between mutation and phenotype in osteogenesis imperfecta: isolation of mineralization-specific genes.

The brittleness of bone in people with lethal (type II) osteogenesis imperfecta, a heritable disorder caused by mutations in the type I collagen genes, arises from the deposition of abnormal collagen in the bone matrix. The inability of the abnormal collagen to participate in mineralization may be caused by its failure to interact with other bone proteins. Here, we have designed a strategy to isolate the genes important for mineralization of collagen during bone formation. Cells isolated from 16-day embryonic chick calvaria and seeded post-confluence in culture deposited a mineralized matrix over a period of 2 weeks. Chick skin fibroblasts seeded and cultured under the same conditions did not mineralize. Using RT-PCR, we prepared short cDNAs (approximately 300 bp) corresponding to the 3' ends of mRNA from fibroblasts and separately from the mineralizing calvarial cells. Subtractive cDNA hybridization generated a pool of cDNAs that were specific to mineralizing calvarial cells but not to fibroblasts. Screening of 100,000 plaques of a chick bone ZAP Express cDNA library with this pool of mineralizing-specific cDNAs identified ten clones which comprised full-length cDNAs for the bone proteins osteopontin (eight of the ten positives), bone sialoprotein II (one of the ten positives), and cystatin (one of the ten positives). cDNAs for type I collagen, fibronectin, alkaline phosphatase, house-keeping genes, and other genes expressed in fibroblasts were not identified in this preliminary screen. The pool of short cDNAs is likely to comprise cDNAs for further bone-specific genes and will be used to screen the entire bone cDNA library of 4.2 million clones.

Effect of pentoxifylline in rat and swine models of hepatic fibrosis: role of fibroproliferation in its mechanism

Fibroproliferation was studied in two animal models of liver disease. Oral feeding of yellow phosphorus to pigs reproducibly results in fibrosis after 8 weeks of feeding, extensive fibrosis after 12 weeks and cirrhosis after 16 weeks of yellow phosphorus. Bile duct ligation was used to induce cirrhosis in the rat. Fibroproliferation was assessed as uptake of tritiated thymidine into fibroblasts which had been incubated with monocyte-conditioned medium obtained from monocytes of pigs treated with yellow phosphorus or bile duct-ligated rats and compared to the corresponding controls. Fibrosis was assessed by collagen content of liver sections obtained from the two animal models. The collagen content was determined by quantitation of Sirius red/Fast green-stained liver sections. In both animal models collagen content was significantly elevated at the conclusion of the treatment. Collagen content of liver sections of yellow phosphorus-treated animals were elevated (40 ± 2.7, n = 15) compared to mineral oil-treated controls (23 ± 1.2, n = 12) and collagen levels in the bile duct-ligated rat model liver sections were elevated (31.2 ± 1.6, n = 6) compared to sham-operated controls (21.6 ± 0.7, n = 6). The results of the fibroproliferation assay indicate that monocytes obtained from pigs treated with yellow phosphorus produce fibroproliferative factors during the development of fibrosis. This is in contrast to the bile duct-ligated rat model where no differences were observed in the production of fibroproliferative factors in the bile duct-ligated rats compared to sham operated controls suggesting that this may not be a key event in this model of fibrosis. Pentoxifylline treatment of the yellow phosphorus induced swine model of hepatic fibrosis has been associated with a marked improvement in fibrosis. In this study treatment of fibrotic pigs with pentoxifylline was associated with an improvement in liver function tests, a reduction of collagen content of liver sections, and reduction in fibroproliferation in pigs receiving yellow phosphorus treatment. Fibroproliferative factors were produced during the development of fibrosis in the swine model of fibrosis and their effect was blocked by pentoxifylline administered in vivo. This is in contrast to the bile duct-ligated rat model where pentoxifylline treatment was not associated with improvement in liver function tests or reduction of collagen content of liver sections and did not alter the fibroproliferative activity of monocyte-conditioned media. Taken together these results suggest that fibroproliferation and increased synthesis of collagen are key events in the yellow phosphorus-induced pig model of hepatic fibrosis and that the action of pentoxifylline in this animal model is likely to be related to its effects on fibroproliferation with a subsequent effect on collagen production. This is in contrast to the bile duct-ligated rat model where the results from this study suggest that fibroproliferation per se does not appear to be a key event and where pentoxifylline treatment does not alter fibroproliferative activity nor alter the course of fibrosis in this animal model.

Effects of strength and endurance training on bone mineral density and collagen metabolism in elderly women

Effects of strength and endurance training on bone mineral density and collagen metabolism in elderly women

Dexamethasone promotes von kossa-positive nodule formation and increased alkaline phosphatase activity in costochondral chondrocyte cultures

This study examined the effect of dexamethasone on von Kossa-positive nodule formation and alkaline phosphate specific activity of costochondral chondrocytes at two distinct stages of maturation. The nodules formed by the more mature growth zone chondrocyte cultures contained von Kossa-positive deposits in the extracellular matrix that had a punctate morphology. The nodules formed by the less mature resting zone cells also contained von Kossa-positive deposits, but differentiation was delayed by three-to-five days compared to the growth zone cell cultures. Dexamethasone stimulated the number of nodules formed and shortened the length of time required for von Kossa-positive nodule formation in both types of cultures. During the first 48 h of exposure to dexamethasone, alkaline phosphatase specific activity in the cell layer of both resting zone and growth zone cultures was increased in a dose-dependent manner. At 12 days post-confluence and thereafter, enzyme activity was inhibited in the dexamethasone-treated cultures. Changes in matrix vesicle alkaline phosphatase specific activity reflected those changes seen in the cell layer after dexamethasone treatment, but with higher magnitude, suggesting that one effect of dexamethasone might be to regulate matrix vesicle function. With the exception of one culture, the chondrocytes did not synthesize type X collagen under any of the experimental conditions used. Fourier transform infrared spectroscopy (FTIR) failed to detect the presence of calcium phosphates in any of the cultures exposed to dexamethasone except one. These results demonstrate that dexamethasone promotes early differentiation events, including nodule formation and increased alkaline phosphatase activity, in costochondral chondrocyte cultures. The failure to detect type X collagen synthesis and mineralization in both dexamamethasone-treated and control cultures suggests that these cultures lack the factors necessary for terminal differentiation and mineralization.

Structural Relationship Between the Primary Crystal Formations and the Matrix Macromolecules in Different Hard Tissues. Discussion of a General Principle

For many years we have investigated the earliest crystal formations of different developing hard tissues (matrix vesicle, bone, dentine, enamel, etc.) by different electron microscopic measurements. It was observed that primarily Ca-phosphate (apatite) "chains," composed of nanometer sized particles (dots, islands), exist, which coalesce rapidly to needles. For the mineralization of collagen (e.g., bone, dentine) the center to center distances between the dots in the mineral chains represent the distances between nucleating sites, so-called "active sites" of collagen which bind primarily Ca for a subsequent nucleation. For the mineralization of noncollagen macromolecules (e.g., enamel) the same principle of mineral nucleation at such "active sites" exists being represented indirectly by corresponding center to center distances between the dots in the mineral chains.

Repair of experimental calvarial defects with Bio-Oss particles and collagen sponges in a rabbit model.

Various materials have been used for reconstruction of both acquired and congenital calvarial defects. Unfortunately, each has its limitations. Autologous bone grafts have irregular rates of resorption that may require secondary corrective surgery, and individual harvest sites have limited stores that can necessitate additional donor locations. Alloplastic materials have unlimited quantities and volume stability but they may not become incorporated and are associated with a higher incidence of infection. The optimal bone substitute should stimulate new bone formation and permanently supplant the temporary space filler, thereby reconstituting the surgical defect. We evaluated 2 newly available bone substitutes, resorbable natural bone mineral (Bio-Oss particles) and a combination of collagen and natural bone mineral collagen combination (Bio-Oss sponges), to repair calvarial defects in an adult, male, New Zealand white rabbit model. We found that the particulate Bio-Oss material resorbed and then underwent the normal physiological stages of bone remodeling. The collagen and Bio-Oss combination was replaced by new bone ingrowth. These materials may have potential for use in the reconstruction of skull defects.

Bound phosphoproteins enhance mineralization of alkaline phosphatase-collagen complexes in vivo

Phosphoproteins (PP) covalently bound to a collagenous matrix have been reported to promote its mineralization in vitro. It was the aim of the present study to determine whether PP also enhance the mineralization of collagen in vivo. To this end, collagen slices were prepared from demineralized bovine cortical bone. Following immobilization of rat dentin phosphoprotein (PP) to the slices, bovine intestinal alkaline phosphatase (ALP) was bound according to the SATA-MHS coupling method. Controls were without enzyme. The slices were implanted into skin pockets prepared over the skull of female Wistar rats (6-10 weeks old). After 3-31 days the implants were removed and analyzed for calcium and phosphate content. It was shown that slices of PP-treated bone collagen mineralized more rapidly and to a greater extent than bone collagen slices without PP. Controls remained free of mineral. It is concluded that mineralization of collagenous matrices, induced by alkaline phosphatase, is enhanced by bound phosphoproteins following implantation in subcutaneous connective tissue.

Modelling Tissue Behaviour Based on Hyperelasticity Theory

The tissues are during their physiological function, e.g., in the course of growth, adolescence, and aging, subjected to a cyclic mechanical loading and to large displacements and rotations as well. A tissue free of all external tractions is in a state that minimizes its internal power. In the course of aging of the tissues, for instance in the wall of the aorta, the vein, and also in the myocardium or heart valves, the decrease of the water content and increase of the collagen content occurs; while in compact and trabecular bone the contents of both mineral substances and collagen, undergo reduction. In accordance with it, the strain energy function and the constitutive equations of living tissue based on the hyperelasticity theory using rotationless strain were studied. On the base of the proposed eigenvalue decomposition of the rotationless strain tensor and hyperelasticity the strain energy function was formulated as depending on biological time of tissue. The quantity of strain energy function per unit of the biological time, which essentially characterizes the velocity of change of mechanical response of tissue in the course of its aging, was also defined. The coefficient of tissue aging is the further diagnostic parameter, which is independent of the rotationless strain tensor and expresses the relative change of mechanical response of tissue during the biological time. The corresponding constitutive equation of tissue depending on the biological time is also determined. On the base of the regression analysis the theoretical stress-strain curves for myocardium and blood vessels were determined. The numerical results reveal that the coefficient of aging progressively increases in hardening tissues (coronary artery, vena cava inferior) whereas at the softening tissues it has a relatively slow increase at the dependence on tissue aging.

Theoretical model for the scattering of light by dentin and comparison with measurements

A theoretical model of the scattering of light by dentin is presented. The model that results is a superposition of several scattering contributions, i.e., scattering by mineral crystals, collagen fibrils, and dentinal tubules. These tubules are oriented so that they cause an asymmetrical scattering process. The angular intensity functions are calculated for planes that are parallel or perpendicular to the plane of the tubules. The shape of the measured intensity function in the plane perpendicular to the tubules and the values of the scattering coefficient can be explained by the model that we present.

The post-translational chemistry and molecular packing of mineralizing tendon collagens

Using cross-linking stereochemistry as indicators, the molecular environment of two collagens in the turkey leg Achilles tendon were compared. The tendon from one year old turkeys was dissected into nonmineralized, fully mineralized and transitionally mineralized portions. Amino acid composition and cyanogen bromide peptide mapping of these portions indicated that the collagens were essentially type I throughout. The fully mineralized compartment had a lysine hydroxylation level similar to turkey or mammalian bone collagen. The non- and transitionally mineralized collagens had a significantly higher lysine hydroxylation, typical of tendon or ligament. However, unlike mammalian tendon, the collagen cross-links were essentially derived from the carboxy-terminal ends of the molecules. The predominant cross-link in this portion was pyridinoline having a high content of 0.95 +/- 0.09 res/mole of collagen. The cross-links in the fully mineralized collagen were also essentially derived from carboxy-terminal aldehyde. However, here significant amounts of the lysyl analog of pyridinoline and lysine-involved bifunctional cross-links were present. The molecular loci of pyridinoline in nonmineralized collagen and the lysyl analog of pyridinoline in mineralized collagen were found to be identical. The total trifunctional cross-link level in the mineralized collagen, 0.55 +/- 0.05 res/mole of collagen, was virtually identical to that observed in old mammalian bone and dentin, and in long term in vitro incubation studies of predentin. We have tentatively concluded that the post-translational chemistry and molecular environments are different in these two turkey tendon fibrils. However, a relative paucity of amino-terminal based cross-links is a feature they have in common. The possible involvement of the amino-terminal telopeptides in collagen mineralization is discussed.

Bioactive Ceramic Prosthetic Coatings

The concept of bioactive ceramic coatings on macroscopically smooth prostheses tries to reconcile opposing principles: the ceramic with beneficial bone tissue growth effects is used as a coating since it does not have sufficient strength and toughness to be used by itself as a prosthesis material. But strength is still an issue since the coating is the primary means of transferring stresses from prosthesis to surrounding tissues. The interface between the metal core and ceramic surface is then critical, since it essentially depends on the strength characteristics of the ceramic. Conversely, when the ceramic coating is used as a means to enhance bone-tissue formation around and into the prosthesis surface, thereby helping to establish a mechanical form of retention, the adhesion of ceramic coating to metallic substrate is not critical. The optimum characteristics of the ceramic are then those that produce the highest effect on bone-tissue growth rates immediately after surgery. The rate of bioactivity is related to the chemical reactivity of the material causing interfacial dissolution, precipitation, and ion exchange reactions. Furthermore, it also appears to depend on a substratum function affecting mineral precipitation, collagen deposition, and cellular differentiation and proliferation.

Initiation of mineralization in bioprosthetic heart valves: Studies of alkaline phosphatase activity and its inhibition by AlCl3 or FeCl3 preincubations

The principal cause of the clinical failure of bioprosthetic heart valves fabricated from glutaraldehyde-pretreated porcine aortic valves is calcification. Other prostheses composed of tissue-derived and polymeric biomaterials also are complicated by deposition of mineral. We have previously demonstrated that: (a) Failure due to calcification of clinical bioprosthetic valves can be simulated by either a large animal circulatory model or subdermal implants in rodents. (b) Calcification of bioprosthetic tissue has complex host, implant, and mechanical determinants. (c) The initial calcification event in the rat subdermal model is the mineral deposition in devitalized cells intrinsic to the bioprosthetic tissue within 48 to 72 h, followed later by collagen mineralization. (d) Initiation of bioprosthetic tissue mineralization, like that of physiological bone formation, has "matrix vesicles" as early nucleation sites. (e) Alkaline phosphatase (AP), an enzyme also associated with matrix vesicles involved in bone mineral nucleation, is present in both fresh and fixed bioprosthetic tissue at sites of initial mineralization. (f) Certain inhibitors of bioprosthetic tissue calcification (e.g., Al3+, Fe3+) are localized to the sites at which alkaline phosphatase is present. On the basis of these results, we hypothesize that alkaline phosphatase is a key element in the pathogenesis of mineralization of bioprosthetic tissue. In the present studies, we focused on the relationship of AP to early events in calcification, and the inhibition of both calcification and AP activity by FeCl3 and AlCl3 preincubations. Subdermal implants of glutaraldehyde pretreated bovine pericardium (GPBP) were done in 3-week-old rats. AP was characterized by enzymatic hydrolysis of paranitrophenyl phosphate (pnpp), and by histochemical studies. Calcification was evaluated chemically (by atomic adsorption spectroscopy) and morphologically (by light microscopy). The results of these studies are as follows: (a) Extractable AP activity is present in fresh but not glutaraldehyde-pretreated bovine pericardial tissue. However, histochemical studies reveal active AP within the intrinsic devitalized cells of GPBP, despite extended glutaraldehyde incubation. (b) Extrinsic AP is rapidly adsorbed following implantation, with peak activity at 72 h (424 +/- 67.2 nm pnpp/mg protein/min enzyme activity [units]), but markedly lesser amounts at 21 days (96.8 +/- 3.9 units). (c) Simultaneously to the AP activity maximum, bulk calcification is initiated, with GPBP calcium levels rising from 1.2 +/- 0.1 (unimplanted) to 2.4 +/- 0.2 micrograms/mg at 72 h, to 55.6 +/- 3.1 micrograms/mg at 21 days, despite a marked decline in AP activity at this later time.(ABSTRACT TRUNCATED AT 400 WORDS)

A contribution with review to the description of mineralization of bone and other calcified tissues in vivo

This manuscript considers certain aspects of mineral deposition in bone and other vertebrate calcifying tissues in order to examine physical, chemical, and biological factors important in the mineralization process. The paper in a discussion format principally presents a new data and the formulation of concepts based on such data as well as a summary of background material as necessary review. Mineralization is found to occur at spatially independent sites throughout the organic extracellular tissue matrices. Matrix vesicles and collagen fibrils each may serve as independent nucleation centers for mineral with vesicle mineralization being local and collagen mineralization dominating the tissues as a whole. Collagen fibril organization is suggested to be such that hole zones are aligned in three dimensions, creating extensive channels for mineral accommodation. Nucleation occurs initially in hole zones and crystal growth leads to the development of plate-like mineral particles whose orientation, disposition, and sizes within fibrils are detailed. Effects of diffusion, crystallinity, and critical nucleation and growth events are described with respect to their influence on mineral deposition in bulk and local regions of tissue matrices.

Topographic imaging of mineral and collagen in the calcifying turkey tendon

Topographic imaging, a method of providing a direct view of ultrastructure in three dimensions, has been newly applied to a study of mineral crystals and collagen from calcifying turkey leg tendon. Individual crystals obtained from intact tendon were observed as thin platelets of irregular shape having a relatively smooth surface. Mineralized collagen fibrils isolated singly or examined in thin tissue sections were found to exhibit the characteristic 64-70 nm period and were associated with platelets and needle-like mineral. The crystals were disposed in numerous fashions, notably as small groups of platelets within individual collagen hole zones, as a number of needle-like densities arranged parallel to one another, or as a combination of platelets and needles over entire stretches of single collagen fibrils. The topographic observations of crystals and crystal-collagen interaction clearly demonstrate the plate-like habit of the mineral in calcifying turkey tendon and suggest that these crystals are located both within and on the surface of collagen fibrils. In certain sites such as the collagen hole zones, the crystals appear organized in a specific manner, possibly with a preferred c-axial orientation. Crystals of hydroxyapatite prepared in vitro and examined topographically are similar in habit and texture to the crystals from tendon. When interpretation of this method is corroborated by other independent microscopic techniques, topographic imaging has widespread potential application in many fields of study in which structural surface features of biological tissues or non-biological materials are of interest at the electron microscope level.

Mineralization of Collagen and Elastic Fibers in Superficial Dystrophic Cutaneous Calcification: An Ultrastructural Study

The ultrastructural morphology of localized skin calcifications without associated diseases and with normal serum calcium and phosphate ion values is still unknown. In a case of superficial dystrophic calcinosis cutis (DCC), the role of collagen, elastin and ground substance in the process of calcification and the organization of the apatite crystals could be studied by light and electron microscopy despite technical difficulties in sectioning the hard tissue. Ultrastructural investigation revealed the nucleation of calcification being related to collagen and elastic fibers. No intracellular calcification was found. A flower-like arrangement of pleomorphic crystals was found around single collagen fibrils resembling the calcification of collagen seen in bone tissue. The elastic fibers showed a different pattern of calcification compared with other diseases (e.g. pseudoxanthoma elasticum) with known calcification of the elastic fibers. The process of mineralization was initially linked to the microfibrils of the elastic fiber.

The mineralization of collagen in vitro

The kinetics of crystallization of calcium phosphates on bovine tendon collagen (type I) has been studied using the constant composition method. During the experiments, the supersaturation was maintained constant by the potentiostatically controlled addition of solutions containing calcium and phosphate ions at the molar ratio of the phase being precipitated on the collagen substrate. In solutions supersaturated with respect to hydroxyapatite, this phase grows directly on the collagen surfaces with a typical plate-like apatitic morphology. The kinetics of the reaction reflects a polynucleation mechanism with a rate of growth dependent upo the fourth power of the relative supersaturation with respect to hydroxyapatite. At higher supersaturations, the constant composition results indicate the formation of octacalcium phosphate at the collagen surface but only following an initial induction period. This phase subsequently hydrolyzes to hydroxyapatite. Calculated surface energies of the nucleating phases are close to those anticipated for inorganic calcium salts. Crystal nuclei sizes, estimated using classical nucleation theory, verified the possibility of nucleation of both octacalcium phosphate and hydroxyapatite in the 64 nm collagen spacings.

Effects of acetylsalicylic acid on heterotopic bone resorption and formation in rats.

The effects of acetylsalicylic acid (ASA) on bone metabolism have been studied in a bone transplantation model using radioisotopic and biochemical parameters. Isografts (femora) from infant inbred rats, extensively prelabeled with collagen and mineral-tracing radioisotopes, were transplanted to muscle pouches in young male rats. Bones from the opposite side of the donor rats served as nonimplanted reference bones. The recipients were given 150 mg/kg/12 h of ASA by gavage for 18 days. The serum concentrations obtained were comparable with the recommended anti-inflammatory levels in humans. Twenty-four hours before being killed the animals were labeled again with other collagen and mineral radioisotopes. After 18 days of medication the resorption of the transplanted bone was inhibited by about 15% in the ASA treated rats compared with controls, as measured by the losses of collagen (14C-hydroxyproline) and mineral (strontium-85). Also, the net gains of mineral and collagen in the ASA-treated transplants were reduced by about 15% and 11% respectively compared with controls during the medication period. During the last 24 h of the study the rates of mineral incorporation (calcium-47 uptake) and collagen synthesis (3H-hydroxyproline) were reduced to an even greater degree in the ASA-treated transplants. These results indicate an inhibitory effect of ASA on bone metabolism.

Intrafibrillar water in cartilaginous tissues

A model mineralizing system was subjected to magnetic resonance microscopy to investigate how water proton transverse (T2) relaxation times and magnetization transfer ratios can be applied to monitor collagen mineralization. In our model system, a collagen sponge was mineralized with polymer-stabilized amorphous calcium carbonate. The lower hydration and water proton T2 values of collagen sponges during the initial mineralization phase were attributed to the replacement of the water within the collagen fibrils by amorphous calcium carbonate. The significant reduction in T2 values by day 6 (p < 0.001) was attributed to the appearance of mineral crystallites, which were also detected by x-ray diffraction and scanning electron microscopy. In the second phase, between days 6 and 13, magnetic resonance microscopy properties appear to plateau as amorphous calcium carbonate droplets began to coalesce within the intrafibrillar space of collagen. In the third phase, after day 15, the amorphous mineral phase crystallized, resulting in a reduction in the absolute intensity of the collagen diffraction pattern. We speculate that magnetization transfer ratio values for collagen sponges, with similar collagen contents, increased from 0.25 ± 0.02 for control strips to a maximum value of 0.31 ± 0.04 at day 15 (p = 0.03) because mineral crystals greatly reduce the mobility of the collagen fibrils.

Mineral balance during nutritional supplementation in adolescents with Crohn disease and growth failure

The adaptive response of whole body mineral metabolism and collagen turnover to nutritional supplementation was determined in adolescent males with Crohn disease and growth failure. Body calcium, magnesium, phosphorus, and nitrogen status was characterized in six patients before and after 3 weeks of nutritional supplementation and in five healthy age- and sex-matched controls by the metabolic balance technique; collagen turnover was assessed by urinary hydroxyproline excretion. Fecal calcium (P less than 0.05), magnesium (P less than 0.01), and nitrogen (P less than 0.01) losses were significantly greater in the Crohn disease patients compared with the controls; after dietary supplementation, only fecal magnesium excretion increased further (P less than 0.001). Approximately twofold increases in phosphorus (P less than 0.001), nitrogen (P less than 0.02), and calcium (P less than 0.05) retention occurred with nutritional supplementation, whereas the marked renal conservation of phosphorus (P less than 0.001) suggested that this mineral was a limiting nutrient. Urinary hydroxyproline excretion was reduced (P less than 0.05) compared with the control values; however, with nutritional supplementation, urinary hydroxyproline excretion increased significantly (P less than 0.02), suggesting that the soluble fraction of tissue collagen was rapidly turning over during nutritional rehabilitation. These findings suggest that the mineral deficiencies present in adolescents with Crohn disease and growth failure can be reversed with nutritional supplementation. Furthermore, the improvement in nutritional status is reflected in the restoration of collagen-containing tissues of the body. However, nutrient imbalances may be present during nutritional therapy, thereby preventing optimal recovery from malnutrition.