Growth patterns and outcomes of growth hormone therapy in patients with acrodysostosis.

INTRODUCTION: Severe short stature is a feature of acrodysostosis, but data on growth are sparse. Treatment with recombinant human growth hormone (rhGH) is used in some centers to increase final height, but no studies have been published so far. Our objective was to conduct a multicenter, retrospective, cohort study to investigate growth in individuals with both types of acrodysostosis, treated with rhGH or not; we used the new nomenclature to describe acrodysostosis, as this disease belongs to the large group of inactivating PTH/PTHrP signaling disorders (iPPSD); acrodysostosis refers to iPPSD4 (acrodysostosis type 1 due to PRKAR1A mutations) and iPPSD5 (acrodysostosis type 2, due to PDE4D mutations). METHODS: We present auxological data from individuals with genetically characterized iPPSD4, and participants with clinical features of iPPSD5. RESULTS: We included 20 and 17 individuals with iPPSD4 and iPPSD5, respectively. The rhGH-treated iPPSD4 patients (n = 9) were smaller at birth than those who did not receive rhGH (median - 2.2 SDS vs. - 1.7 SDS); they showed a trend to catch-up growth during rhGH therapy (median 0.5 SDS in the first year). The rhGH-treated patients (n = 5) reached a better final height compared to those who did not receive rhGH (n = 4) (median - 2.8 SDS vs. - 3.9 SDS), suggesting that rhGH is efficient to increase height in those patients. The difference in target height to final height ranged between 1.6 and 3.0 SDS for iPPSD4 not treated with rhGH (n = 4), 2.1-2.8 SDS for rhGH-treated iPPSD4 (n = 5), 0.6-5.5 SDS for iPPSD5 not treated with rhGH (n = 5) and 2.5-3.1 for rhGH-treated iPPSD5 (n = 2). CONCLUSION: Final height may be positively influenced by rhGH in patients with acrodysostosis/iPPSD. Our rhGH-treated cohort started therapy relatively late, which might explain, at least in part, the limited effect of rhGH on height.

A modified rhTGF-beta1 and rhBMP-2 are effective in initiating a chondro-osseous differentiation pathway in bone marrow cells cultured in vitro.

Rat bone marrow cells were cultured in vitro in a collagen-gel medium at 0.5% fetal bovine serum concentration for 10 days in the presence of recombinant human transforming growth factor-beta-1, genetically engineered to contain a collagen binding domain (rhTGF-beta1-F2), or a commercial rhTGF-beta1. To compare the effects of TGF-betas with other growth factors in which the osteogenic capacity has been widely documented, a recombinant human bone morphogenetic protein (rhBMP-2) was evaluated. Once serum conditions compatible with growth were re-established, the selected cells were cultured for 6 more days in the presence of the growth factor. In the last 2 days, dexamethasone (dex) and beta-glycerophosphate (beta-GP) were added to promote osteogenesis. After this 16-day period, cells were placed into diffusion chambers or demineralized bone matrix (DBM) implants, and implanted subdermally on the backs of rats for 28 days. Biochemical, histological, and immunohistochemistry analysis provided evidence of cartilage (commercial rhTGF-beta1-treated cells), osteoid (rhTGF-beta1-F2-treated cells), and bone tissues (rhBMP-2 treated cells), inside the diffusion chambers, whereas bone, cartilage, and osteoid were observed inside the DBM implants under any of the three growth factors effect. Our study advances the technology capable of selecting a cell population from bone marrow that, in the presence of rhTGF-beta1 or rhBMP-2 in vitro, achieves chondro-osteogenic potential in vitro and in vivo.

Low-phytic acid corn improves nutrient utilization for growing pigs.

Thirty-five crossbred barrows averaging 14.5 kg initial BW were used in a 5-wk experiment to compare the P availability and nutritional value of a low-phytate hybrid corn (LPC, 0.26% total P, 0.08% phytic acid P) homozygous for the lpa 1-1 allele with a nearly isogenic normal hybrid corn (NC, 0.25% total P, 0.20% phytic acid P). The pigs were fed individually twice daily in metabolism pens. Three semipurified diets were created in which corn was the only source of phytate. Diet 1 contained 72% NC, 0.15% estimated available P (aP) and 0.55% Ca. Diet 2 contained 72% LPC, 0.24% aP, and 0.55% Ca. The only differences between Diets 1 and 2 were the source of corn and the levels of aP. No inorganic P (iP) was added to these diets in order to measure the animal response to the different levels of aP in the corn hybrids. Diet 3 was NC Diet 1 supplemented with iP to equal the level of aP in LPC Diet 2. Diets 4 and 5 were practical corn-soybean meal diets formulated with each corn to meet all minimum nutrient requirements and contained 0.30% aP and 0.65% Ca. For the semipurified diets, pigs fed LPC Diet 2 had higher (P < 0.01) growth performance, bone breaking strength, P absorption and retention, Ca absorption and retention, and N retention than pigs fed NC Diet 1. However, when the NC diet was supplemented with iP to equal the aP in the LPC diet, most criteria were similar (P > or = 0.2), indicating an equal nutritional value for both corn hybrids after adjusting for phytate level. The only treatment difference, other than P excretion, between the practical corn diets supplemented with soybean meal was a higher (P < 0.05) bone breaking strength for pigs fed LPC Diet 5 compared with NC Diet 4. The use of LPC in pig diets reduced P excretion in swine waste by 50 and 18.4% in the semipurified and practical diets, respectively, compared with NC. Using our in vitro procedure designed to simulate the digestive system of the pig, the availability of P for pigs was estimated at 56% for LPC and 11% for NC.

Effects of low phytic acid corn on phosphorus utilization, performance, and bone mineralization in broiler chicks.

In vivo and in vitro experiments were conducted to determine whether P in a low-phytate corn (LPC) containing the lpa 1-1 allele is more available than P in a near-isogenic wild-type corn hybrid (NC). The LPC was analyzed to contain 0.18% nonphytate P and 0.26% total P (TP), whereas NC contained 0.05% nonphytate P and 0.25% TP. For these studies, nonphytate P was considered to be available P (AP). In the in vivo study, 150 1-d-old male chicks were randomly assigned to five treatments (six pens of five chicks each) for 21 d. The dietary treatments included: A) a diet containing 60% NC, 0.2% AP, and 0.8% Ca; B) a diet containing 60% LPC, 0.28% AP, and 0.8% Ca; C) an NC diet similar to Diet A, but with KH2PO4 added to increase the AP to 0.28% to match the AP in Diet B; D) an LPC diet containing 0.45% AP and 1% Ca; and E) an NC diet supplemented with KH2PO4 to provide 0.45% AP and 1% Ca. Diets A, B, and C were semipurified diets, with corn being the sole source of phytate. The only differences between Diets A and B were the source of corn and the amount of AP present in the diets. The levels of AP in these diets were deficient in order to measure the animal response to the different levels of AP. Diets D and E were typical corn-soybean meal diets, and were formulated to contain an optimal level of AP. Performance and bone ash were similar (P > 0.05) in chicks fed Diets B and C and in chicks fed Diets D and E. Chicks fed LPC diets (B and D) retained more P (P < 0.05) than chicks fed NC diets (C and E). Chicks fed Diet B had significantly higher (P < 0.05) Ca retention compared with chicks fed Diet A. An in vitro digestion procedure that simulated the physiological conditions of the gastrointestinal tract of broilers was used to determine P release from LPC and NC. Results showed that 65% (1,420 mg/kg) of the TP in LPC was released, compared with 23% (543 mg/kg) from NC. Results of these experiments indicate that the P in LPC is more available than the P in NC, and reducing the phytate content did not compromise the nutritional value of LPC. The increased P retention in chicks fed LPC suggests that substituting LPC for NC leads to a reduction in manure P. Also, the in vitro procedure accurately predicted differences in in vivo P availability between the two corns.

Origin and seed phenotype of maize low phytic acid 1-1 and low phytic acid 2-1.

Phytic acid (myo-inositol-1, 2, 3, 4, 5, 6-hexakisphosphate or Ins P(6)) typically represents approximately 75% to 80% of maize (Zea mays) seed total P. Here we describe the origin, inheritance, and seed phenotype of two non-lethal maize low phytic acid mutants, lpa1-1 and lpa2-1. The loci map to two sites on chromosome 1S. Seed phytic acid P is reduced in these mutants by 50% to 66% but seed total P is unaltered. The decrease in phytic acid P in mature lpa1-1 seeds is accompanied by a corresponding increase in inorganic phosphate (P(i)). In mature lpa2-1 seed it is accompanied by increases in P(i) and at least three other myo-inositol (Ins) phosphates (and/or their respective enantiomers): D-Ins(1,2,4,5,6) P(5); D-Ins (1,4,5,6) P(4); and D-Ins(1,2,6) P(3). In both cases the sum of seed P(i) and Ins phosphates (including phytic acid) is constant and similar to that observed in normal seeds. In both mutants P chemistry appears to be perturbed throughout seed development. Homozygosity for either mutant results in a seed dry weight loss, ranging from 4% to 23%. These results indicate that phytic acid metabolism during seed development is not solely responsible for P homeostasis and indicate that the phytic acid concentration typical of a normal maize seed is not essential to seed function.

The number and generative capacity of human B lymphocyte progenitors, measured in vitro and in vivo, is higher in umbilical cord blood than in adult or pediatric bone marrow.

The lack of human B lymphocyte development in beige/nude/XID (bnx) mice is in sharp contrast to the robust development observed in another immune deficient strain, the NOD/SCID mouse. The ability to generate human B lymphocytes in the NOD/SCID, but not bnx mouse has been hypothesized to be caused by differences in the microenvironments or systemic cytokine concentrations. In the current studies we report that the differences in development can be primarily attributed to the source of the progenitors transplanted into the mice. The prior studies in bnx mice used cultured pediatric or adult bone marrow (BM) as the source of the CD34+ cells, whereas the NOD/SCID studies have predominantly used fresh or cultured umbilical cord blood (UCB). We have analyzed BM and UCB for the number of human CD34+/CD38- cells capable of in vitro B lymphocyte development, and have found a lower frequency of B lymphocyte generation in BM. The individual B lymphocyte clones that developed from bone marrow produced 100-fold fewer cells than the UCB-derived clones. In agreement with the in vitro studies, human B lymphocytes developed in bnx mice from both CD34+ and CD34+/CD38- cells isolated from human umbilical cord blood, but not from equivalent numbers of CD34+ and CD34+/CD38- progenitors from bone marrow. Therefore, the lower generative capacity, and frequency of B lymphocyte precursors in human marrow may be responsible for the previous results that showed a lack of B lymphocyte development in bnx mice.

Factors which affect the calcification of tissue-derived bioprostheses.

Mineralization of implanted bioprostheses poses a major clinical problem. Crosslinking of collagenous matrices, a process used to render tissues relatively inert and nonbiodegradable, seems to encourage calcification. Residual, noncovalently bound glutaraldehyde, as well as glutaraldehyde crosslinks which can be degraded with time, seem to play a role in this connection. Our findings demonstrate the need to carefully remove noncovalently or labile-associated glutaraldehyde by thorough rinsing or neutralization before implantation. Components of a valve prosthesis such as cusps and aortic wall, which are known to vary in their proportions of collagen, elastin, and noncollagenous proteins and to calcify to different extents, can both be prevented from calcifying if treated with a biphosphonate before implantation. Calcification can also be reduced by selective enzymatic removal of noncollagenous materials. In addition to the age of rats, animals usually used to evaluate calcification, the strain of animal can markedly affect the response. The Fischer-344 rat, a highly inbred animal, will not calcify exhaustively rinsed implants. Our findings suggest that multifactorial approaches may have to be combined to generate the most ideal bioprostheses. These should include careful removal of noncovalently bound glutaraldehyde, neutralization of the nonbifunctionally reacted residues, removal of lipids and noncollagenous proteins (and possibly the more antigenic nonhelical collagen telopeptides), as well as inclusion of agents such as biphosphohates, which by interfering with crystal growth prevent the accumulation of mineral in the interstices of the tissue.

Demineralized bone matrix mediates differentiation of bone marrow stromal cells in vitro: effect of age of cell donor.

Bone maintenance requires a continuous source of osteoblasts throughout life. Its remodeling and regeneration during fracture repair is ensured by osteoprogenitor stem cells which are part of the stroma of the bone marrow (BM). Many investigators have reported that in cultured BM stromal cells there is a cell population that will differentiate along an osteogenic lineage if stimulated by the addition of osteogenic inducers, such as dexamethasone (dex), beta-glycerophosphate (beta-GP), transforming growth factor beta-1 (TGF-beta 1) and bone morphogenetic protein-2 (BMP-2). Here we report the effects of demineralized bone matrix (DBM) on the osteogenic differentiation of BM stromal cells in vitro, using morphological criteria, alkaline phosphatase (AP) activity, and calcium accumulation. DBM and DBM-conditioned medium (DBMcm) enhanced bone formation in the presence of dex and beta-GP, whereas DBM particles caused changes in the cell phenotype. Temporal expression of total and skeletal AP by BM stromal cells from 4-week-old rats showed a biphasic pattern enhanced by DBM and suggesting the presence of two cell populations. In one population, AP synthesis reaches a maximum during the first week in culture, following which cells either die or loose their ability to synthesize AP. A second, less abundant population begins to proliferate and synthesize AP during the second and third weeks. The synthesis of AP, which often decreases by the third week, can be maintained at high levels only if DBM is added to the cultures. BM stromal cells isolated from 24- and 48-week-old rats showed a decrease or loss of this biphasic AP expression pattern compared with cells isolated from 4-week-old rats. The addition of DBM to cultures derived from 24- and 48-week-old rats stimulated mostly the second cell population to synthesize AP, suggesting that DBM contains a factor(s) that acts on a specific bone marrow cell population by increasing the proliferation of active cells or inducing the differentiation of dormant cells.

Human endothelial cells are defective in diabetic vascular disease.

Diabetic vascular disease is characterized pathologically by endothelial cell (EC) hyperplasia and basement membrane (BM) thickening. One key question regarding the pathogenesis of diabetic vascular disease is whether the EC or BM or both are primarily defective and responsible for these pathological changes. Previous studies, which took the approach of creating artificial diabetic conditions, have been inconclusive. It is known, however, that the extracellular matrix may be altered by glycosylation as a result of hyperglycemia, thereby altering EC function. To begin to address this question and more closely mimic the situation in vivo, we characterized human diabetic EC harvested from insulin-dependent diabetic mothers (IDDM) at the cellular and molecular levels. Human EC were isolated from both normal and IDDM umbilical cords and cellular functions evaluated using standard assays of attachment (% attached cells), proliferation (cpm/cell), resistance to detachment under shear stress (number of cells remaining attached), and glucose uptake (cpm/2 X 10(4) cells). Gene expression of major BM components (collagen type IV, laminin beta 1, and laminin beta 2) was quantified by Northern analysis. Diabetic EC demonstrated increased proliferation (two- to eightfold compared to normals), were 20-40% less resistant to shear stress and took up glucose 10-15% more slowly than normal EC. Furthermore, Northern analysis showed that the expression of major BM components was increased by an average of 10-18% in diabetic cells compared to normal cells. These results were consistent with in vivo observations and previously published data.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative trial in volunteers to investigate possible ethanol-ranitidine interaction.

OBJECTIVE: To assess the effect of ranitidine on ethanol absorption after ethanol 0.5 g/kg is given in three single doses of 0.167 g/kg to simulate normal social drinking. DESIGN: A double-blind, placebo-controlled, crossover trial was performed in 16 healthy men. Ethanol serum concentrations were measured on day 6 of each of the three treatment periods (placebo, ranitidine 150 mg bid, or ranitidine 300 mg bid). METHODS: Ethanol 0.167 g/kg was administered followed by a standard meal at 1700. The last tablet of the test medication was given 15 minutes later. Thirty and 60 minutes after the first intake, the same amount of ethanol was given again. Serum ethanol concentrations were measured multiple times during the four-hour period following oral ingestion of the first dose. RESULTS: Comparison of median serum ethanol concentrations, the areas under the curve, peak and time to peak serum ethanol concentrations showed no significant differences during medication with placebo, ranitidine 150 mg bid, or ranitidine 300 mg bid. Peak ethanol concentrations (median values) were 153, 140, and 155 mg/L, respectively. CONCLUSIONS: This study shows that treatment with ranitidine, in a dose up to 300 mg bid, has no significant effect on serum ethanol concentrations, even when ethanol was given in divided doses to simulate normal patterns of social drinking. This implies that concomitant dosing with ranitidine will not increase the adverse effects of moderate doses of ethanol on concentration and psychomotor function.

Cellular responses to silicone and polyurethane prosthetic surfaces.

Prosthetic devices composed of silicone or polyurethane are commonly used in surgery. These devices elicit a soft tissue reaction which may frequently be complicated by capsule formation. Histologically the capsule comprises both cellular (fibroblasts and endothelial cells (EC)) and matrix components (predominantly collagen type I). We hypothesized that the function of the cellular elements is altered by exposure to prosthetic materials and that this alteration contributes to capsule formation. To test this hypothesis, we utilized specific in vitro assays of cell function (attachment, proliferation, matrix gel contraction), which closely mimic in vivo cellular events, in order to define the responses of EC and fibroblasts to prosthetic surfaces (foam polyurethane, flat silicone, and textured silicone). Morphologic changes were evaluated by scanning electron microscopy (SEM). Attachment of both cell types to all prosthetic surfaces was significantly decreased compared to control (HUVEC: control, 55 +/- 1; foam polyurethane, 19 +/- 4*; flat silicone, 25 +/- 3*; textured silicone, 36 +/- 1*; fibroblast: control, 93 +/- 6; foam polyurethane, 21 +/- 4*; flat silicone, 57 +/- 5*; textured silicone, 44 +/- 5* (*P < 0.05 = significant; units, percentage spread)). Fibroblast proliferation was significantly decreased on foam polyurethane (0.1 +/- 0.03*) and textured silicone (0.18 +/- 0.05*), but not on flat silicone (0.79 +/- 0.2; control = 0.96 +/- .2). In contrast, HUVEC proliferation was significantly decreased on both silicone surfaces but not on polyurethane (units, cpm/cell; control, 0.26 +/- 0.05; foam polyurethane, 0.15 +/- 0.05; flat silicone, 0.08 +/- 0.03*; textured silicone, 0.02 +/- 0.01*).(ABSTRACT TRUNCATED AT 250 WORDS)

New evidence and new hope concerning endothelial seeding of vascular grafts.

Endothelial cell (EC) seeding of prosthetic bypass grafts has been promoted as a method of improving graft patency. However, an efficient and reliable method of seeding vascular prostheses with ECs is lacking due to inefficient harvesting of ECs and poor attachment and proliferation of cells on the prosthetic surfaces. To investigate the effect of a commonly used prosthetic surface on EC attachment and proliferation, we measured the attachment and proliferation of ECs on polytetrafluoroethylene (PTFE) grafts uncoated or coated with gelatin, laminin, fibronectin, collagen type I and/or III, or RGD (arginine-glycine-aspartate)-containing peptide. EC attachment and proliferation were both significantly decreased on the untreated PTFE graft surface. Conversely, coating of PTFE with fibronectin, RGD, laminin, or gelatin significantly (p less than 0.05) improved the attachment of ECs, with the most striking increases occurring with laminin and gelatin. Similarly, all matrix components in this study improved EC proliferation compared with untreated PTFE, with RGD and gelatin producing the most significant improvement. PTFE adversely effects EC attachment and proliferation. These properties can be improved by treating PTFE graft surfaces with extracellular matrix components in relatively low concentrations. Future investigations are needed to determine whether there are combinations and concentrations of matrix components that will optimize these cellular functions on vascular prostheses.

Distribution of griseofulvin in the rat: comparison of the oral and topical route of administration.

Effective penetration of griseofulvin across the dermal barrier has been achieved using an anhydrous solvent system of benzyl alcohol (10%), acetone (40%), and isopropanol (50%). There were quantitative differences in the relative accumulation of griseofulvin in skin compared with internal organs, when the topical and oral routes of administration were compared. The topical route enhanced localized concentrations of griseofulvin at the site of application, and these persisted for several days. After daily topical application a steady state was reached at day 3, when the diffusion across the skin barrier and epidermal loss seemed to equal the total amount applied to the skin surface. The application of griseofulvin topically, required a much smaller amount of drug to achieve similar integumentary levels compared with the amount required orally.

Mechanism of crosslinking of proteins by glutaraldehyde. IV: In vitro and in vivo stability of a crosslinked collagen matrix.

The use of native or reconstituted collagen as a bioprothesis for tissue augmentation requires the introduction of exogenous synthetic crosslinks. The degree of crosslinking determines the rate of resorption or replacement of the implanted materials by the host. Since biophysical and chemical methods to quantify these crosslinks have in general been difficult to evaluate, we have developed in vitro enzymatic approaches which enable us to correlate the degree of crosslinking with the rates of enzymatic degradation. When the number of stable crosslinks formed is large it is essential to partially unfold the collagen fibrils by heating or by exposure to denaturing agents to enhance their susceptibility to hydrolysis. In the present study we demonstrate that increasing the number of reactive amino groups on collagen by coupling 1,6-diaminohexane to carboxyl groups using a water soluble carbodiimide can significantly enhance the number of crosslinks introduced by glutaraldehyde. We also show that the enzymatic method developed correlates well with the biodegradation of radiolabeled crosslinked collagenous tissues implanted subcutaneously in rats.

Inhibition of ectopic calcification of glutaraldehyde crosslinked collagen and collagenous tissues by a covalently bound diphosphonate (APD).

Calcification of collagen-derived prosthesis, such as glutaraldehyde crosslinked porcine heart valves or heart valves assembled out of bovine pericardium, presents a major clinical problem. Their subcutaneous implantation into young rats provides us with a reproducible method of assessing this form of ectopic calcification. Long-term implantation is essential, since some materials which do not calcify within the first month frequently exhibit a delayed calcific response. Crosslinked pericardium is much more likely to calcify than crosslinked tendon or reconstituted crosslinked pepsin extracted bovine type I collagen. The covalent binding of a diphosphonate to collagen and collagen-rich tissues can prevent calcification. The binding of this diphosphonate and its ability to inhibit calcification can be enhanced by increasing the number of amino groups on the collagen molecule. The degree of calcification is inversely related to the number of diphosphonate molecules covalently bound to collagen. Under standard conditions, chemical modifications appear to occur primarily on the surface of the collagen fibrils, as evidenced by the relationship between the number of molecules of APD bound and fibril diameter. The bound diphosphonate seems to interfere with crystal growth and prevent the formation of highly insoluble hydroxyapatite on the surface and interstices of the collagen fibrils.

Cellular events associated with the induction of bone by demineralized bone.

Implantation of demineralized bone (DB) in the form of powder or intact segments in extra skeletal sites stimulates new bone formation. Urist and co-workers presented substantial evidence that there is a noncollagenous protein that has the ability to induce bone formation. One aim of this study was to trace the process of bone formation when DB, in the form of perforated rectangular plates, is implanted subcutaneously in 2-month-old rats. A second objective was to determine whether cartilage cells play a role in the formation of bone in this model. Various DB plates with 0.25 mm diameter holes were implanted subcutaneously for 1-4 weeks in rats. One week after implantation, DB plates were covered by vascularized connective tissue that invaded the perforations. Aggregates of chondrocytes were observed within the holes and on periosteal surfaces in only a few specimens. Further cartilage proliferation was not observed, and by the 2nd week there was no evidence of endochondral bone formation. Where these cartilage-like cells were present, a thin layer of mineral was deposited around them; resorption and fibrous tissue infiltration followed. This aborted form of endochondral calcification was not followed spatially by bone formation. Patent vascularized channels were invaded by alkaline phosphatase-positive mononuclear cells and fibroblasts, and became enlarged by the enzymatic action of macrophages. The next step involved the calcification of DB plates adjacent to the wide spaces. Osteoclasts now appeared leading to the resorption of this recalcified matrix. The eroded and now enlarged lacunar surfaces were lined by newly formed bone and osteoblasts. This process continued so that, at the end of 4 weeks following implantation, the original DB plates were replaced by trabecular bone. Biochemical data on calcium and alkaline phosphatase levels in the implants paralleled the morphological observations.

Dystrophic calcification and mineralization during bone induction: biochemical differences.

The calcification of implants of glutaraldehyde-cross linked collagenous tissues and collagen was studied in young and old rats and compared to bone induction by non-crosslinked osteogenically active demineralized bone matrix (DBM). Glutaraldehyde-crosslinked implants of DBM, tendon, and cartilage calcified in young but not in old animals and accumulated only trace amounts of BGP (Bone Gla protein, osteocalcin). Alkaline phosphatase activity and BGP was high in implants of DBM and undetectable in crosslinked implants. To try and understand why bone formation is so significantly reduced in older Fischer 344 rats, we developed a system which consists of cylinders of DBM sealed at the ends with a Millipore filter. Cells originating from 20 day old embryo donors were introduced into the chambers prior to subcutaneousmplantation. After 4 weeks of implantation in 26 month old rats, the cylinders containing embryonic calvaria or muscle cells were found to be full of bone and/or cartilage.

Ectopic bone formation is enhanced in senescent animals implanted with embryonic cells.

Ectopic bone formation induced by the subcutaneous implantation of demineralized bone matrix (DBM) is very significantly reduced in older Fischer 344 rats. Cells originating from calvaria of 20-day-old embryo donors were introduced into cylinders of DBM sealed at the ends with a Millipore filter or collagen sponges prior to subcutaneous implantation. Cells within the chambers had access to vascular channels that could penetrate through the interstices of the DBM. After four weeks of implantation in 26-month-old rats, the cylinders were full of bone. This bone was assessed by histologic techniques, by calcium and bone gamma-carboxyglutamic acid (gla) protein (BGP) concentrations, and by alkaline phosphatase activity. Cylinders to which no cells were added produced no bone. Bone marrow cells enclosed in similar cylinders or injected weekly at the implantation site also enhanced new bone formation but to a much lesser extent. Embryonic muscle cells formed large amounts of cartilage and less bone. Fibroblasts were inactive in this system. Prior treatment of the DBM with trypsin inhibited the myoblast response but not that of calvaria cells.

Biochemical differences between dystrophic calcification of cross-linked collagen implants and mineralization during bone induction.

Ectopic calcification of diseased tissues or around prosthetic implants can lead to serious disability. Therefore, calcification of implants of glutaraldehyde-cross-linked collagenous tissues and reconstituted collagen was compared with mineralization induced by demineralized bone matrix (DBM). Whereas implants of DBM accumulated large amounts of calcium and a bone-specific gamma-carboxyglutamic acid protein (BGP or osteocalcin) following implantation in both young and older rats, implants of cross-linked pericardium calcified with only traces of BGP. Glutaraldehyde-cross-linked DBM failed to calcify after implantation in 8-month-old rats for 2-16 weeks. Implants of cross-linked type I collagen exhibited small calcific deposits 2 weeks postimplantation but calcium content eventually dropped to levels equal to those of soft tissues as the implants were resorbed. The calcium content of DBM implanted in 1- and 8-month-old rats reached comparable levels after 4 weeks, but the BGP content was approximately twice as high in the younger animals than in the older ones. Glutaraldehyde-cross-linked implants of DBM, tendon, and cartilage calcified significantly in young but not in old animals. This form of dystrophic calcification was associated with only trace amounts of BGP. Alkaline phosphatase activity was high in implants of DBM and undetectable in implants of cross-linked collagenous tissues. These results show that implants of glutaraldehyde-cross-linked collagenous tissues and reconstituted collagen calcify to different extents depending upon their origin and the age of the host, and that the mechanism of dystrophic calcification differs significantly from the process of mineralization associated with bone induction as reflected by alkaline phosphatase activity and BGP accumulation.