Chondrocyte transplantation using a collagen bilayer matrix for cartilage repair.

We have developed a novel, two-layered, collagen matrix seeded with chondrocytes for repair of articular cartilage. It consists of a dense collagen layer which is in contact with bone and a porous matrix to support the seeded chondrocytes. The matrices were implanted in rabbit femoral trochleas for up to 24 weeks. The control groups received either a matrix without cells or no implant. The best histological repair was seen with cell-seeded implants. The permeability and glycosaminoglycan content of both implant groups were nearly normal, but were significantly less in tissue from empty defects. The type-II collagen content of the seeded implants was normal. For unseeded implants it was 74.3% of the normal and for empty defects only 20%. The current treatments for articular injury often result in a fibrous repair which deteriorates with time. This bilayer implant allowed sustained hyaline-like repair of articular defects during the entire six-month period of observation.

Comparative in vitro analysis of topical hemostatic agents.

Hemostatic agents are broadly utilized across the surgical specialties. While specific characteristics desirable for individual applications may vary, these agents generally are expected to aid the patient's coagulation system in the rapid development of an occlusive clot. Six commonly utilized hemostatic agents were quantitatively compared in terms of their ability to mediate platelet aggregation, deposition and activation, and initiate gross clot formation in a series of in vitro tests. Three types of collagen sponges (Actifoam, Helistat, Instat), microfibrillar collagen (Avitene), a gelatin sponge (Gelfoam), and oxidized regenerated cellulose (Surgicel) were studied. Platelet aggregation: Citrated platelet rich plasma (PRP) was contacted with hemostatic agents both with and without thrombin in a stirred cuvette and the platelet count was measured over 5 min. Platelet deposition: To approximate arterial wounds, PRP was perfused in a controlled manner through hemostatic agents, and the effluent platelet count was measured over time. Platelet activation: ATP secretion from PRP contacted with hemostatic agents in a stirred cuvette was measured at 1 and 5 min. Clot formation: The clotting time of nonanticoagulated whole blood contacted with the hemostatic agents was measured. Materials which depleted platelets most rapidly and effectively in the perfusion system (Avitene, Helistat, and Actifoam) were also the most effective inducers of platelet aggregation and secretion. Clot formation (likely to be platelet dependent) was also more rapid in this group. An overall activity ranking combined the relative performance of each hemostatic agent on the various tests: Actifoam approximately Avitene > Helistat >> Gelfoam > Instat > Surgicel. The activity ranking generally reflects the materials used in these agents (collagen > gelatin > oxidized regenerated cellulose), as well as their processing (chemical crosslinking in collagen sponges may lower activity). Such in vitro assessment of the relative platelet and coagulation activities of hemostatic agents might serve as a useful screening tool before investigating properties which require more expensive animal or clinical testing.

Effects of growth-factor-enhanced culture on a chondrocyte-collagen implant for cartilage repair.

The effects of incubation and addition of growth factors to a chondrocyte-seeded collagen implant for cartilage repair were studied. Type I collagen matrices seeded with lapine articular chondrocytes and unseeded controls cultured in the presence and absence of fibroblast growth factor and insulin for 2, 6, and 9 weeks were subjected to biomechanical, biochemical, and histological analysis. Aggregate modulus of elasticity of seeded implants decreased by half at 6 weeks, then rose by a factor of 10 above initial values. Permeability of seeded implants and their controls decreased steadily. Glycosaminoglycan content peaked at 6 weeks, coinciding with the greatest number of chondrocytes and mitotic activity in seeded implants. Chondrocytes remained phenotypically stable and metabolically active; they incorporated glycosaminoglycan into the extracellular matrix, and formed an organized pericellular environment despite the predicted resorption of the collagen matrix. Adding fibroblast growth factor and insulin tripled the rate of cell turnover and doubled the glycosaminoglycan content of seeded implants, but had no effect on their material properties. In vitro incubation for 6 weeks in the presence of fibroblast growth factor and insulin creates a metabolically and mitotically active chondrocyte-collagen composite for implantation into articular cartilage defects.

Collagen-based devices for soft tissue repair.

The intrinsic biological and physiochemical characteristics of collagen have been exploited to prepare a variety of commercially available medical products spanning many medical specialties. The properties of purified collagen can be modified to obtain forms which comply to specific applications. In particular, collagen materials in the form of gels, matrices, and films have been applied, either alone or in combination with other agents, to address soft tissue repair. This review describes how the versatile properties of collagen have made it one of the most useful biomaterials for wound care applications.

Structural investigation of the covalent and electrostatic binding of yeast cytochrome c to the surface of various ultrathin lipid multilayers using x-ray diffraction.

X-Ray diffraction was used to characterize the profile structures of ultrathin lipid multilayers having a bound surface layer of cytochrome c. The lipid multilayers were formed on an alkylated glass surface, using the Langmuir-Blodgett method. The ultrathin lipid multilayers of this study were: five monolayers of arachidic acid, four monolayers of arachidic acid with a surface monolayer of dimyristoyl phosphatidylserine, and four monolayers of arachidic acid acid with a surface monolayer of thioethyl stearate. Both the phosphatidylserine and the thioethyl stearate surfaces were found previously to covalently bind yeast cytochrome c, while the arachidic acid surface electrostatically binds yeast cytochrome c. Meridional x-ray diffraction data were collected from these lipid multilayer films with and without a bound yeast cytochrome c surface layer. A box refinement technique, previously shown to be effective in deriving the profile structures of ultrathin multilayer lipid films with and without electrostatically bound cytochrome c, was used to determine the multilayer electron density profiles. The surface monolayer of bound cytochrome c was readily apparent upon comparison of the multilayer electron density profiles for the various pairs of ultrathin multilayer films plus/minus cytochrome c for all cases. In addition, cytochrome c binding to the multilayer surface significantly perturbs the underlying lipid monolayers.

Orientation and lateral mobility of cytochrome c on the surface of ultrathin lipid multilayer films.

We have previously shown that cytochrome c can be electrostatically bound to an ultrathin multilayer film having a negatively charged hydrophilic surface; furthermore, x-ray diffraction and absorption spectroscopy techniques indicated that the cytochrome c was bound to the surface of these ultrathin multilayer films as a molecular monolayer. The ultrathin fatty acid multilayers were formed on alkylated glass, using the Langmuir-Blodgett method. In this study, optical linear dichroism was used to determine the average orientation of the heme group within cytochrome c relative to the multilayer surface plane. The cytochrome c was either electrostatically or covalently bound to the surface of an ultrathin multilayer film. Horse heart cytochrome c was electrostatically bound to the hydrophilic surface of fatty acid multilayer films having an odd number of monolayers. Ultrathin multilayer films having an even number of monolayers would not bind cytochrome c, as expected for such hydrophobic surfaces. Yeast cytochrome c was covalently bound to the surface of a multilayer film having an even number of fatty acid monolayers plus a surface monolayer of thioethyl stearate. After washing extensively with buffer, the multilayer films with either electrostatically or covalently bound cytochrome c were analyzed for bound protein by optical absorption spectroscopy; the orientation of the cytochrome c heme was then investigated via optical linear dichroism. Polarized optical absorption spectra were measured from 450 to 600 nm at angles of 0 degrees, 30 degrees, and 45 degrees between the incident light beam and the normal to the surface plane of the multilayer. The dichroic ratio for the heme alpha-band at 550 nm as a function of incidence angle indicated that the heme of the electrostatically-bound monolayer of cytochrome c lies, on average, nearly parallel to the surface plane of the ultrathin multilayer. Similar results were obtained for the covalently-bound yeast cytochrome c. Furthermore, fluorescence recovery after photobleaching (FRAP) was used to characterize the lateral mobility of the electrostatically bound cytochrome c over the monolayer plane. The optical linear dichroism and these initial FRAP studies have indicated that cytochrome c electrostatically bound to a lipid surface maintains a well-defined orientation relative to the membrane surface while exhibiting measurable, but highly restricted, lateral motion in the plane of the surface.

Location of the heme-Fe atoms within the profile structure of a monolayer of cytochrome c bound to the surface of an ultrathin lipid multilayer film.

We have recently developed x-ray diffraction methods to derive the profile structure of ultrathin lipid multilayer films having one to five bilayers (e.g., Skita, V., W. Richardson, M. Filipkowski, A.F. Garito, and J.K. Blasie. 1987. J. Physique. 47:1849-1855). Furthermore, we have employed these techniques to determine the location of a monolayer of cytochrome c bound to the carboxyl group surface of various ultrathin lipid multilayer substrates via nonresonance x-ray diffraction (Pachence, J.M., and J.K. Blasie. 1987. Biophys. J. 52:735-747). Here an intense tunable source of x-rays (beam line X9-A at the National Synchrotron Light Source at the Brookhaven National Laboratory) was utilized to measure the resonance x-ray diffraction effect from the heme-Fe atoms within the cytochrome c molecular monolayer located on the carboxyl surface of a five monolayer arachidic acid film. Lamellar x-ray diffraction was recorded for energies above, below, and at the Fe K-absorption edge (E = 7,112 eV). An analysis of the resonance x-ray diffraction effect is presented, whereby the location of the heme-Fe atoms within the electron density profile of the cytochrome c/arachidic acid ultrathin multilayer film is indicated to +/- 3 A accuracy.

The location of cytochrome c on the surface of ultrathin lipid multilayer films using x-ray diffraction.

X-ray diffraction and spectroscopic techniques were used to characterize ultrathin fatty acid multilayers having a bound surface layer of cytochrome c. Three to six monolayers of arachidic acid were deposited onto an alkylated glass surface, using the Langmuir-Blodgett method. These fatty acid multilayer films were stored either in a 1 mM NaHCO3 pH 7.5 solution or a buffered 10 microM cytochrome c solution, pH 7.5. After washing extensively with buffer, these multilayer films were assayed for bound cytochrome c by optical spectroscopy. It was found that the cytochrome c bound only to the odd-numbered monolayer films (which have hydrophilic surfaces). The theoretical number of cytochrome c molecules bound to the ultrathin multilayer films having three or five monolayers was calculated as N = 1.2 x 10(13)/cm2 (assuming a hexagonally close-packed monolayer of protein), which would produce an optical density of 0.002 at a wavelength of 550 nm; for a three or five monolayer ultrathin film that was incubated with cytochrome c, OD550 approximately equal to 0.002. The protein was released from the film when treated with greater than 100 mM KCl solution, as would be expected for an electrostatic interaction. Meridional x-ray diffraction data were collected from the arachidic acid films with and without a bound cytochrome c layer. A box refinement technique, previously shown to be effective in deriving the profile structures of nonperiodic ultrathin films, was used to determine the multilayer electron density profiles. The electron density profiles and their autocorrelation functions showed that bound cytochrome c resulted in an additional electron dense feature on the multilayer surface, consistent with a bound cytochrome c monolayer. The position of the bound protein relative to the multilayer surface was independent of the number of fatty acid monolayers in the multilayer. Future studies will use these methods to investigate the structures of membrane protein complexes bound directly to the surface of multilayer films.

Low-angle neutron scattering analysis of Na/K-ATPase in detergent solution.

Purified Na/K-ATPase from guinea pig renal outer medulla has been delipidated and solubilized in Brij 58 (polyoxyethylene ether; C-16, E-20). At a concentration of 2 mg of Brij 58/mg of protein, about one-half the enzyme complement was solubilized and almost 50% of Na/K-ATPase activity was retained by the enzyme-micelle complex. Guinier plots of the neutron scattering profiles yielded no evidence of heterogeneity with respect to subunit composition or the state of aggregation in the solubilized oligomers. Contrast matching with D2O used to obtain estimates of the molecular weight of the micellar form of Na/K-ATPase gave a mean value of 310,000 +/- 42,700, which corresponds to an alpha 2 beta 2 tetramer. A Stuhrmann plot of the neutron scattering data yielded an estimated radius of gyration of 67 A. The Stuhrmann plot also indicated an asymmetrical distribution of neutron scattering density. On the basis of the Stuhrmann plot parameters, the estimated molecular weight, and the radius of gyration, a low-resolution model was formulated of the oligomeric unit of Na/K-ATPase.

A structural investigation of cytochrome c binding to photosynthetic reaction centers in reconstituted membranes.

Mammalian cytochrome c can effectively replace bacterial cytochrome c2 as the electron donor to the bacterial photosynthetic reaction center in either the natural chromatophore or a reconstituted reaction center/phospholipid membrane. In this paper, the reconstituted membrane was used to describe the nature of cytochrome c binding to the reaction center, the location of bound cytochrome c in the membrane profile and the perturbation of the reaction center and phospholipid profile structures indicated by cytochrome c binding. These structural studies utilized the combined techniques of X-ray and neutron diffraction.

The location of redox centers in biological membranes determined by resonance x-ray diffraction. I. Observation of the resonance effect.

We have developed resonance X-ray diffraction methods to locate for the first time intrinsic metal atoms associated with redox centers within biological membrane systems. The study of membranes containing dilute concentrations of resonant scatterers has been made possible by the development of synchrotron radiation sources of X-rays. The technique permits altering the scattering power of a particular atom relative to others by varying the incident X-ray energy. Thus, this method may be used to locate a metal atom within a complex integral protein without chemical modification of the membrane. We present resonance diffraction data taken with synchroton radiation for two different membrane systems: cytochrome oxidase incorporated into lipid vesicles and a photosynthetic reaction center-cytochrome c complex also reincorporated into lipid vesicles.

The location of redox centers in biological membranes determined by resonance x-ray diffraction. II. Analysis of the resonance diffraction data.

In the preceding paper (Stamatoff, J., Eisenberger, P., Blasie, J.K., Pachence, J.M., Tavormina, A., Erecinska, M., Dutton P.L. and Brown, G. (1982) Biochim. Biophys. Acta 679, 177-187), we described the observation of resonance X-ray scattering effects from intrinsic metal atoms associated with redox centers in membrane proteins on the lamellar X-ray diffraction from oriented multilayers of reconstituted membranes. In this paper, we discuss the possible methods of analysis of such data and present the results of our model refinement analysis concerning (a) the location of the cytochrome c heme iron atom in the profile structure of a reconstituted membrane containing a photosynthetic reaction center-cytochrome c complex and (b) the location of the heme a and a3 iron atoms in the profile structure of a reconstituted membrane containing cytochrome oxidase. The former results are of special importance because they provide a test of the validity of the resonance diffraction data and the methods of analysis, since the location of cytochrome c in the reaction center-cytochrome c membrane profile is known independently of the resonance diffraction experiments.

The reaction center profile structure derived from neutron diffraction.

Both reaction center protein from the photosynthetic bacteria Rhodopseudomonas sphaeroides and egg phosphatidylcholine can be deuterium labelled; the reaction center protein can be incorporated into the phosphatidylcholine bilayers forming a homogeneous population of unilamellar vesicles. The lipid profile and the reaction center profile within these reconstituted membrane profiles were directly determined to 32 A resolution using lamellar neutron diffraction from oriented membrane multilayers containing either deuterated or protonated reaction centers, and either deuterated or protonated phosphatidylcholine. The 32 A resolution reaction center profile shows that the protein spans the membranes, and has an asymmetric mass distribution along the perpendicular to the membrane plane. These results were combined with previously described X-ray diffraction results in order to extend the resolution of the derived reaction center profile to 9 A.

Structural studies on reconstituted reaction center-phosphatidylcholine membranes.

Reaction center protein, isolated from the photosynthetic bacterium Rhodopseudomonas sphaeroides R26 mutant, was incorporated into phosphatidylcholine bilayers forming a homogeneous population of unilamellar vesicles. Cytochrome c, added to preformed reaction center-phosphatidylcholine vesicles, rapidly reduced up to 90% of the laser-generated (BChl)2+ of the reaction center (with kinetics of electron transfer similar to those in the chromatophore membrane) which suggests that the portion of the reaction center which accommodates functional cytochrome c binding sites is exposed predominantly on the exterior of the vesicles. Unit cell electron density profiles were derived from lamellar X-ray diffraction from oriented reaction center-phosphatidylcholine membrane multilayers at varying lipid/protein ratios. The analysis of these profiles showed that the reaction center protein incorporates into the phosphatidylcholine membrane with unique sidedness and that the profile of the reaction center protein itself is asymmetric and spans the membrane.