Inhibition of collagen deposition in the extracellular matrix prevents the establishment of a stroma supportive of hematopoiesis in long-term murine bone marrow cultures.

Long-term production of murine hematopoietic cells in vitro is dependent on establishment of a complex microenvironment consisting of a variety of stromal cells and an extensive extracellular matrix which includes collagen, fibronectin, laminin, proteoglycans, and other undefined components adherent to the culture dishes. Cis-4-hydroxyproline (CHP), a relatively specific inhibitor of collagen secretion, was used to examine the role of extracellular collagen deposition in supporting hematopoiesis in long-term C57B1/6J mouse bone marrow cell cultures. Throughout the 10-wk culture period, all culture dishes contained either 0, 10, 25, or 50 micrograms/ml of CHP. All medium and nonadherent cells were removed at weekly intervals and replaced with fresh medium containing the previous concentrations of CHP. Nonadherent cells were assayed weekly for total cells and pluripotent, erythroid, megakaryocytic, and granulocytic-macrophage progenitor cells. Dishes were killed at selected intervals to assess protein and collagen synthesis in the adherent layer. Adherent cell numbers, as judged by microscopic examination and DNA assays, correlated inversely with CHP concentrations used and paralleled degree of collagen synthesis inhibition. The decreased hemopoietic progenitor cell production correlated closely with percent inhibition of collagen synthesis and stromal cellularity. The CHP concentrations tested were not directly toxic to hemopoietic progenitor cells. These studies demonstrate that collagen deposition in the extracellular matrix of murine bone marrow cell cultures is essential to the establishment of a functional stromal microenvironment that is supportive of long-term hematopoiesis.

Collagen heterogeneity in normal rabbit cornea. I. Isolation and biochemical characterization of the genetically-distinct collagens.

Limited proteolysis of preparations of mature rabbit cornea with pepsin allows the recovery of approximately 90% of the tissue collagen in soluble form. Using recently described selective precipitation techniques, the soluble cornea collagen can be resolved into two major fractions. The predominant fraction, which accounts for about 95% of the solubilized collagen, was identified by means of solubility properties, electrophoretic and chromatographic properties, as well as amino acid analyses of its constituent chains, as Type I collagen. The alternate fraction, which accounts for virtually all of the remainder of the solubilized cornea collagen, was identified by means of the same criteria as collagen comprised of the A and B chains. In addition, the stoichiometry of the latter chains in preparations of cornea collagen indicate that in the cornea these chains are likely to participate in the formation of only one type of collagen molecule with the chain composition, AB2. And finally, the demonstration that Type I and the AB2 collagens are the predominant forms of collagen recoverable from the mature cornea strongly suggests that molecules comprised of the A and B chains are not necessarily confined to basement membrane structures.

Evaluation of a subcutaneous glucose sensor out to 3 months in a dog model.

OBJECTIVE: To advance the feasibility of an implantable long-term glucose sensor with bioprotective sensor membranes and test protocols using a somatostatin analog (octreotide). RESEARCH DESIGN AND METHODS: Implantable sensors were constructed with one of eight bioprotective membranes and screened in vitro for stable response to glucose. Sensors were implanted subcutaneously into nondiabetic mongrel dogs and monitored at 4-min intervals via radiotelemetry. When implanted sensor responses showed evidence of tracking blood glucose after glucagon challenge (8-21 days postimplant), a glucose infusion protocol was used to assess performance. Sensor data were collected every 4 s after octreotide inhibition of endogenous insulin release. Reference plasma glucose samples were taken every 4-10 min. RESULTS: Preimplant in vitro testing of sensors verified linearity to 33.3 mM glucose and response times to 90% of equilibrium in 2-7 min. Ten implanted sensors tracked glucose for 20-114 days, during which 25 separate glucose infusion studies were conducted. The resulting regression data yielded a mean slope of 0.99 +/- 0.06, an intercept of 0.24 +/- 0.53 mM glucose, and a correlation coefficient 0.98 +/- 0.01. Long-term sensor stability was not judged adequate for clinical application, although two sensors tracked within +/- 15% for 33 and 42 days. In vivo oxygen delivery was shown to affect sensor performance. On explant, two of eight tested bioprotective membranes were found to be biostable and to fully protect the sensor's enzyme membrane. The foreign body capsule was adequately vascularized adjacent to the sensor up to 91 days postimplant. Sensor units eventually failed because of electronic problems (package leakage) or because of biodegradation or biofouling of test bioprotective membranes. CONCLUSION: Further development of this type of sensor may provide diabetic patients with a better means of monitoring blood glucose.

A subcutaneous glucose sensor with improved longevity, dynamic range, and stability of calibration.

OBJECTIVE: To evaluate the lifetime, response time, linearity, glucose range, and calibration stability of two different types of continuous glucose sensor implants in a dog model. RESEARCH DESIGN AND METHODS: Glucose sensors based on the enzyme electrode principle that are coupled to a radio transmitter were evaluated on the bench top, sterilized, and then implanted subcutaneously in nondiabetic mongrel dogs. A multichannel radio receiver and PC data processor were used to record the sensor glucose data. Initial early reliable sensor responsivity was recognized by a vigorous hyperglycemic excursion after an intramuscular injection of glucagon. Periodically the dogs were made temporarily diabetic by blocking pancreatic insulin secretion by subcutaneous injection of a synthetic somatostatin (octreotide). By using exogenous insulin injection followed by intravenous glucose infusion, glucose levels were manipulated through the entire clinical range of interest: 2.2-38.9 mmol/l (40-700 mg/dl). Every 5-10 min, reference blood glucose samples were obtained and run in our hospital clinical laboratory. The glucose sensor data was evaluated by linear least squares optimization and by the error grid method. RESULTS: Beginning as early as postimplant day 7, the in vivo performances of sensors were evaluated by using glucose infusion studies performed every 1-4 weeks. Bench-top and in vivo 90% response-time sensors were in the range of 4-7 min during sensor lifetime. Best-performing sensors from both types are summarized as follows. The earlier-stage technology was less linear with a dynamic range of no more than 22 mmol/l glucose, had a best-case recalibration interval of 18 days, and had a maximum lifetime of 94 days. The improved later-stage technology sensors, which were constructed with the addition of bioprotective and angiogenic membranes, were linear over the full extended range of clinical interest (2.2-38.9 mmol/l [40-700 mg/dl glucose]), had a best-case recalibration interval of 20 days, and had a maximum lifetime of >160 days. CONCLUSIONS: Stable clinically useful sensor performance was demonstrated as early as 7 days after implantation and for a sensor lifetime of 3-5 months. This type of subcutaneous glucose sensor appears to be promising as a continuous and painless long-term method for monitoring blood glucose. Specifically sensors with top-layer materials that stimulate angiogenesis at the sensor/tissue interface may have better dynamic measurement range, longer lifetimes, and better calibration stability than our previously reported sensors.

Laboratory evaluation of new reusable blood glucose sensor.

An enzyme-electrode sensor designed specifically for pocket-portable self-monitoring of blood glucose is described. The sensing device in this instrument is unique because it is reusable for at least 30 days, at which time it is easily replaced by placing a new enzyme-membrane cartridge over the electrode. As little as 7 microliters of undiluted whole blood, plasma, or serum is applied directly to the sensor, and glucose is automatically determined in 30 s. No manual timing or wiping step is required after sample application. On eight production instruments, plasma glucose concentration was determined (n = 20) at 57, 125, 246, and 347 mg/dl. The average coefficient of variation for the 80 determinations for each instrument ranged from 2 to 5%, averaging 3.7%. The instrument is inherently linear, independent of hematocrit, and without oxygen limitation when dissolved oxygen concentration is greater than 35 mmHg. No interferences were found from plasma constituents, heparin, or acetaminophen.

Resistance of the stromal cell in murine long-term bone marrow cultures to damage by ionizing radiation.

The ability of bone marrow stromal cells to survive and function after exposure to ionizing radiation remains controversial. Therefore, we used the murine long-term bone marrow culture system to analyze the effects of single doses of ionizing radiation (9-500 Gy) on the function of a preexisting, nearly confluent stroma that was supportive of hematopoiesis. Hematopoiesis ceased promptly in all the irradiated cultures and did not recover unless fresh marrow cells were inoculated. Radiation doses less than or equal to 100 Gy caused no obvious morphologic change in the cells. Total RNA, total protein, and collagen synthesis declined by 35%-60% within two days after even 9 Gy; but radiation doses up to 100 Gy caused minimal or no additional decline. Although RNA synthesis recovered nearly to normal within three weeks after radiation doses less than 100 Gy, total protein and collagen synthesis remained suppressed. Normal adherent layers irradiated with 9-50 Gy supported long-term hematopoiesis by fresh Sl/Sld marrow cells, although Sl/Sld marrow did not demonstrate sustained hematopoiesis when cultured in plain culture dishes or over normal stroma irradiated with 200 Gy. Thus, bone marrow stromal cells in long-term cultures did not show evidence of substantial cell death over at least the six-week period studied after irradiation with as much as 100 Gy, and they maintained hematopoietic supportive functions when irradiated with up to at least 50 Gy.

A telemetry-instrumentation system for monitoring multiple subcutaneously implanted glucose sensors.

An implantable potentiostat-radiotelemetry system for in vivo sensing of glucose is described. An enzyme electrode sensor measures the oxidation current of hydrogen peroxide formed by the stoichiometric conversion of glucose substrate and oxygen cofactor in an immobilized glucose oxidase layer. The sensor current is converted to a frequency and transmitted at programmable intervals (4, 32, 256 s) to a remote receiver. Low power CMOS circuitry is employed and device operation for up to 1.5 years is predicted using two series connected 250 mAh lithium cells. Crystal controlled RF frequencies uniquely identify each sensor allowing over 10 sensors within the same 10 m radius. A custom interface card allows a PC to program the receiver and handle the transmitted sensor data using software written in Microsoft C and QuickBasic. Software control allows on-the-fly sensor addition or subtraction to the sensor group being monitored. Over 10 sensors can be tracked long-term using the longest transmit interval, or four sensors can be tracked during short-term infusion studies when the transmit interval is reduced to 4 s. The design, construction, operation, and performance of the system hardware and software are described and evaluated.

Enzymatic glucose sensors. Improved long-term performance in vitro and in vivo.

We studied the long-term in vitro and in vivo performance of enzyme electrode glucose sensors. Single commercially produced enzyme-active membranes remained functional for estimating glucose in vitro for 14-36 months. These membranes were implanted subcutaneously in rats for 1 year and, upon explanation, remained functional for measuring glucose in vitro. Sensors with these membranes plus an additional outer membrane with lower glucose permeability allowed glucose monitoring in the low oxygen tension of subcutaneous tissue. These sensors were surgically implanted in three nondiabetic dogs. Each sensor implant was coupled to a radio transmitter to allow continuous long-term glucose monitoring in these awake unrestrained dogs. In vivo sensor performance was evaluated by intravenous glucose infusion, with reference blood glucose determinations made in the clinical laboratory. These subcutaneously implanted sensors tracked changes in plasma glucose for up to 12 weeks. The in vivo initial response for three sensor implants was approximately 35 sec (n = 8). Sensor peak response to glucose after bolus infusion ranged from 3 to 14 min. Stability of sensor sensitivity within +/- 15% for more than 1 month was demonstrated in two of the dogs. Sensor lifetime was limited not by loss of enzyme activity, but by biodegradation of the outermost polyurethane membrane. The findings suggest that long-term continuous monitoring of blood glucose using a subcutaneously implanted enzyme electrode sensor may be possible.

Fracture strengths of ceramic brackets subjected to mesial-distal archwire tipping forces.

This study tested the strength of ceramic orthodontic brackets subjected to mesial-distal tipping forces on five types of preadjusted, maxillary right central incisor ceramic twin brackets for both 0.018" and 0.022" slot sizes. Description of each bracket was by manufacturer's abbreviation-crystallinity-slot bracket, eg., AL-P-18, meaning Allure-polycrystalline-0.018" slot bracket. Thirty brackets of each type were used for a total of 300 brackets, each bonded to a porcelain denture tooth. A special apparatus was designed to hold the denture tooth, the wire, and the bracket in a standard position while an Instron machine applied a tipping force to the full size rectangular archwire at a distance of 7.0 mm lateral to the center of the bracket. The tipping force was applied until the bracket fractured. The fracture force, fracture angle, and fracture location were recorded. High fracture force values tended to accompany large fracture angles while low fracture force values tended to be associated with small fracture angles. The clinical significance was that the stronger ceramic brackets can be expected to withstand larger amounts of archwire tipping adjustments prior to bracket fracture. With the literature indicating the optimum force for tipping of maxillary incisors to be from 50 to 125 g, all the brackets are sufficiently strong to consistently withstand the suggested magnitude of archwire tipping forces. However, if excessive tipping forces were required by the clinician, ceramic brackets would be prone to fracture.

Evidence for the existence of an alpha 1(V) alpha 2(V) alpha 3(V) collagen molecule in human placental tissue.

Chromatography in native type V collagen from human placenta on phosphocellulose using nondenaturing conditions results in the partial resolution of two cellulose using nondenaturing conditions results in the partial resolution of two fractions. The first fraction contains each of the three type V alpha chains in approximately equal proportions and upon thermal denaturation exhibits a melting temperature of 33 degrees C. Fraction two contains the alpha 1 (V) and alpha 2 (V) chains in approximately a 2:1 ratio, respectively, and has a melting temperature of 35 degree C. These data indicate the presence of two molecular species of type V collagen in placenta, namely an alpha 1 (V) alpha 2 (V) alpha 3 (V) molecule and the previously described [alpha 1 (V)]2-alpha 2 (V) molecule.

The isolation and characterization of the cyanogen bromide peptides from the B chain of human collagen.

Cleavage of the collagen B chain with cyanogen bromide yields nine peptides which have been isolated and characterized with regard to molecular weight and amino acid composition. The peptides are recovered in equimolar quantities and account for the full amino acid complement of the chain as isolated following limited pepsin digestion of human placental tissue. These data thus confirm the unique composition of the chain and further indicate that the chain has been isolated in essentially pure form. The total number of amino acid residues (1018) observed in the cyanogen bromide peptides of the B chain indicate that it is comparable in length to the previously characterized collagen alpha chains. Thus, the apparent larger size of the B chain noted in previous studies may possibly be attributed to the relatively large quantities of hydroxylysine-linked carbohydrate, but more likely to the increased numbers of large hydrophobic amino acids in the B chain. Although the cyanogen bromide peptide pattern obtained in studies on the B chain serves to differentiate this chain from other known chains, some possible homologies between the B chain peptides and peptides derived from the alpha chains of type I, II, and III collagens are noted.

Identification of collagen chains as a function of cyanogen bromide peptide patterns using gel permeation high-performance liquid chromatography.

A gel permeation high-performance liquid chromatography system utilizing commercially-available silica-based gels has been developed for evaluation of the cyanogen bromide cleavage products derived from collagen a chains. The high efficiency and precision of the system permits unequivocal identification of various chains by inspection of the peptide elution pattern following a single run requiring 40 minutes. The system is sufficiently sensitive to permit analyses to be performed with as little 1.0 microgram of sample, although the columns utilized may accommodate samples as large as 1.0 mg making the system useful for preparative purposes as well.

Prediction of pocket-portable and implantable glucose enzyme electrode performance from combined species permeability and digital stimulation analysis.

Development of enzyme electrode sensors can be facilitated by the use of computer simulation modeling techniques. In this work, the model accounts for contacting solution effects, changes in species diffusion and partition coefficients across multiple membrane layer interfaces, cofactor limitation effects, enzyme loading, enzyme kinetics and decay, and other variations in electrode layer geometry. Experimentally determined single species membrane permeabilities are included in the digital simulations to predict the performance characteristics of a multilayered glucose oxidase based enzyme electrode. Information obtained includes range of sensor linearity under different substrate and cofactor concentration combinations, sensor time response and output as permeability parameters and enzyme loading are varied, sensor washout and interference effects, and prediction of decay of sensor enzyme concentration and resultant estimated sensor lifetime as a function of contacting solution conditions and sensor permeability.

Isolation and characterization of the cyanogen bromide peptides derived from the human alpha 2(V) collagen chain.

The human alpha 2(V) collagen chain when cleaved with cyanogen bromide yields ten peptides which can be recovered in approximately equimolar quantities. Characterization of the purified peptides with regard to molecular weight and amino acid composition establishes the uniqueness of the peptides and reveals that the alpha 2(V) chain recovered following limited pepsin digestion contain 956 amino acid residues. Possible homologies between the alpha 2(V) peptides and peptides derived from other collagen chains were noted. In addition, a high-performance liquid chromatography system is described for the separation of three of the alpha 2(V) chain peptides which were not resolved by using conventional separation techniques.

Evidence that the collagen in the culture medium of Chinese hamster lung cells contains components related at the primary structural level to the alpha1(V) collagen chain.

The collagenous protein synthesized by cultured Chinese hamster lung (CHL) cells and present in the culture medium has been isolated after limited pepsin digestion and differential salt precipitation. Molecular size analysis of this material indicates that the CHL cell medium collagen contains chains which exhibit an apparent molecular mass of approximately 85,000 Da. When chromatographed on CM-cellulose under denaturing conditions, the reduced and alkylated CHL cell medium collagen chains elute slightly after the human alpha1(I) chain but well before the pepsin-derived alpha1(V) chain, which is the constituent chain present in the CHL cell cellular matrix collagen. Analysis of the peptides derived by CNBr cleavage of the CHL medium collagen chains by chromatography on CM-cellulose reveals, however, that these chains contain peptides which correspond both in size and in chemical properties to those derived from the alpha1(V) collagen chain, but clearly lack two peptides (alpha1(V)-CB4 and alpha1(V)-CB5) which are normally present in pepsin-derived alpha1(V) chains. Furthermore, analysis of the CHL cell culture medium collagenous material obtained without pepsin digestion indicates the presence of collagenous chains that exhibit after reduction a molecular mass of approximately 160,000 Da, which is smaller than the proposed size of the pro alpha1(V) collagen chain. These results demonstrate that the collagenous protein present in the culture medium of CHL cells is directly related at the primary structural level to the alpha1(V) collagen chain, and it is postulated that this material represents the large fragment derived from a collagenase cleavage of the [pro alpha1(V)]3 molecules present in the cell layer. Furthermore, these results and previous reports indicate that the only identifiable genetic type of procollagen chain synthesized by this cloned cell line in culture corresponds to the pro alpha1(V) chain.

Collagen molecules comprised of alpha 1(V)-chains (B-chains): an apparent localization in the exocytoskeleton.

Antibodies specific for alpha 1(V) chains and native collagen molecules containing the alpha 1(V) chain have been used to study the localization of alpha 1(V)-containing molecules in differentiating hyaline cartilage. Immunofluorescence data show that the undifferentiated mesenchyme contains significant quantities of these molecules throughout the cell-rich tissue matrix. Examination of fully differentiated hyaline cartilage reveals a unique staining pattern wherein the immunofluorescent material is restricted to the pericellular matrix within the chondrocyte lacunae. We conclude from these data in conjunction with other evidence that the Type V collagens function as components of an exocytoskeleton for connective tissue cells.

Synthesis of [pro alpha 1(IV)]3 collagen molecules by cultured embryo-derived parietal yolk sac cells.

Electron immunohistochemical studies demonstrate that cultured embryo-derived parietal yolk sac (ED-PYS) carcinoma cells synthesize type IV collagen. This material has been isolated and characterized. The collagen obtained after limited pepsin digestion from the medium in which the cells are grown is composed of homogeneous components with a molecular mass of approximately 95 000 daltons. When chromatographed on (carboxymethyl)cellulose under denaturing conditions, the chains elute as acidic components slightly before the human alpha 1(I) chain and coincident with the position of elution of the pepsin-derived human alpha 1(IV) chain. This analysis indicates the presence of a single type of collagen chain in the pepsin-derived ED-PYS synthesized material. In addition, the profile of cyanogen bromide (CNBr) cleavage products obtained from the pepsin-derived ED-PYS cell collagen chains is essentially identical with that derived from the human alpha 1(IV) chain. Isolation of the medium collagen in the absence of pepsin digestion reveals the presence of two high molecular weight components equivalent in size to procollagen alpha chains. However, both high molecular weight products yield CNBr cleavage products that correspond to those obtained from the pepsin-derived alpha 1(IV) chain. The ED-PYS cell-associated collagens obtained with or without the use of pepsin contain components that are essentially identical with those isolated from the culture-medium collagen. These data provide definitive evidence for the existence of type IV collagen molecules composed solely of alpha 1(IV) procollagen chains and further document the usefulness of ED-PYS cells for investigating the biosynthesis of basement membrane components.

Chinese hamster lung cells synthesize and confine to the cellular domain a collagen composed solely of B chains.

The acid-soluble collagen extracted from cultured Chinese hamster lung (CHL) cell layers has been isolated after limited pepsin digestion and differential salt fractionation. Polyacrylamide gel electrophoresis of this material under denaturing conditions showed the presence of collagen chains with an apparent molecular mass of 120,000 daltons both before and after reduction, indicating the absence of interchain disulfide bonds in the native molecule. When chromatographed on CM-cellulose under denaturing conditions, the majority (> 90%) of the CHL cell layer collagen chains eluted as relatively basic components slightly before the human alpha 2(I) chain and coincident with the human B chain. In addition, the CM-cellulose elution profiles of the cyanogen bromide peptides derived from the human B chain and from the CHL cell layer chain were essentially identical. Examination of CHL cells in culture by using affinity-purified antibody to human B chain revealed this collagen to be localized in an extracellular matrix surrounding the cells. Furthermore, analysis of the culture medium indicated the absence of any comparable collagen chain. These data provide additional evidence for the existence of a molecular form of collagen composed solely of B chains and suggest that this molecular form of collagen has an unusual affinity for the cell layer in this system.