Application of magnetic resonance microscopy to tissue engineering: a polylactide model.

Absorbable polymers are unique materials that find application as temporary scaffolds in tissue engineering. They are often extremely sensitive to histological processing and, for this reason, studying fragile, tissue-engineered constructs before implantation can be quite difficult. This research investigates the use of noninvasive imaging using magnetic resonance microscopy (MRM) as a tool to enhance the assessment of these cellular constructs. A series of cellular, polylactide constructs was developed and analyzed using a battery of tests, including MRM. Distribution of rat aortic smooth muscle cells within the scaffolds was compared as one example of a tissue engineering MRM application. Cells were loaded in varying amounts using static and dynamic methods. It was found that the cellular component was readily identified and the polymer microstructure readily assessed. Specifically, the MRM results showed a heterogeneous distribution of cells due to static loading and a homogenous distribution associated with dynamic loading, results that were not visible through biochemical tests, scanning electron microscopy, or histological evaluation independently. MRM also allowed differentiation between different levels of cellular loading. The current state of MRM is such that it is extremely useful in the refinement of polymer processing and cell seeding methods. This method has the potential, with technological advances, to be of future use in the characterization of cell-polymer interactions.

A hydrogel material for plastic and reconstructive applications injected into the subcutaneous space of a sheep.

Soft tissue reconstruction using tissue-engineered constructs requires the development of materials that are biocompatible and support cell adhesion and growth. The objective of this study was to evaluate the use of macroporous hydrogel fragments that were formed using either unmodified alginate or alginate covalently linked with the fibronectin cell adhesion peptide RGD (alginate-RGD). These materials were injected into the subcutaneous space of adult, domesticated female sheep and harvested for histological comparisons at 1 and 3 months. In addition, the alginate-RGD porous fragments were seeded with autologous sheep preadipocytes isolated from the omentum, and these cell-based constructs were also implanted. The results from this study indicate that both the alginate and alginate-RGD subcutaneous implants supported tissue and vascular ingrowth. Furthermore, at all time points of the experiment, a minimal inflammatory response and capsule formation surrounding the implant were observed. The implanted materials also maintained their sizes over the 3-month study period. In addition, the alginate-RGD fragments supported the adhesion and proliferation of sheep preadipocytes, and adipose tissue was present within the transplant site of these cellular constructs, which was not present within the biomaterial control sites.

In vivo characterization of a porous hydrogel material for use as a tissue bulking agent.

Tissue engineered biomaterial constructs are needed for plastic and reconstructive applications. To successfully form a space-filling tissue, the construct should induce a minimal inflammatory response, create minimal or no fibrotic capsule, and establish a vascular bed within the first few days after implantation to ensure survival of the implanted cells. In addition, the biomaterial should support cellular adhesion and induce tissue ingrowth. A macroporous hydrogel bead using sodium alginate covalently coupled with an arginine, glycine, and aspartic acid-containing peptide was created. A 6-month subcutaneous rat model study was performed to determine if the implanted material induced tissue ingrowth throughout the implantation area and maintained a three-dimensional vascular bed. The implanted materials produced a vascular bed, minimal inflammation and capsule formation, and good tissue ingrowth throughout the experiment. The material retained its bulking capacity by demonstration of no significant change of the cross-sectional area as measured from the center of the implants after the 2-week time point. In addition, the granulation tissue formed around the implant was loosely organized, and the surrounding tissue had integrated well with the implant. These results indicate that this material has the desired properties for the development of soft-tissue-engineering constructs.

Absorbable mesh aids in development of discrete, tissue-engineered constructs.

Absorbable mesh was investigated as a potential containment material in which to house discrete, small, tissue-engineered constructs. The mesh was fashioned into bags of varying shapes and consistent volumes. Cells were cultivated on porous, collagen beads, and the tissue constructs were placed into the bags. The mechanical integrity of the bags and feasibility of the design was tested in vitro. The bags successfully maintained their integrity as the cells developed on the collagen matrices. Furthermore, their porosity allowed access of nutrients and waste products to and from the developing tissue. Having demonstrated feasibility of processing, the next step is to optimize the cell culture specifications and materials design.

A structural basis for drug-induced long QT syndrome.

Mutations in the HERG K(+) channel gene cause inherited long QT syndrome (LQT), a disorder of cardiac repolarization that predisposes affected individuals to lethal arrhythmias [Curran, M. E. , Splawski, I., Timothy, K. W., Vincent, G. M., Green, E. D. & Keating, M. T. (1995) Cell 80, 795-804]. Acquired LQT is far more common and is most often caused by block of cardiac HERG K(+) channels by commonly used medications [Roden, D. M., Lazzara, R., Rosen, M., Schwartz, P. J., Towbin, J. & Vincent, G. M. (1996) Circulation 94, 1996-2012]. It is unclear why so many structurally diverse compounds block HERG channels, but this undesirable side effect now is recognized as a major hurdle in the development of new and safe drugs. Here we use alanine-scanning mutagenesis to determine the structural basis for high-affinity drug block of HERG channels by MK-499, a methanesulfonanilide antiarrhythmic drug. The binding site, corroborated with homology modeling, is comprised of amino acids located on the S6 transmembrane domain (G648, Y652, and F656) and pore helix (T623 and V625) of the HERG channel subunit that face the cavity of the channel. Other compounds that are structurally unrelated to MK-499, but cause LQT, also were studied. The antihistamine terfenadine and a gastrointestinal prokinetic drug, cisapride, interact with Y652 and F656, but not with V625. The aromatic residues of the S6 domain that interact with these drugs (Y652 and F656) are unique to eag/erg K(+) channels. Other voltage-gated K(+) (Kv) channels have Ile and Val (Ile) in the equivalent positions. These findings suggest a possible structural explanation for how so many commonly used medications block HERG but not other Kv channels and should facilitate the rational design of drugs devoid of HERG channel binding activity.

Comparative study of seeding methods for three-dimensional polymeric scaffolds.

Development of tissue-engineered devices may be enhanced by combining cells with porous absorbable polymeric scaffolds before implantation. The cells are seeded throughout the scaffolds and allowed to proliferate in vitro for a predetermined amount of time. The distribution of cells throughout the porous material is one critical component determining success or failure of the tissue-engineered device. This can influence both the successful integration of the device with the host tissue as well as the development of a vascularized network throughout the entire scaffold volume. This research sought to compare different seeding and proliferation methods to select an ideal method for a polyglycolide/aortic endothelial cell system. Two seeding environments, static and dynamic, and three proliferation environments, static, dynamic, and bioreactor, were analyzed, for a total of six possible methods. The six seeding and proliferation combinations were analyzed following a 1-week total culture time. It was determined that for this specific system, dynamic seeding followed by a dynamic proliferation phase is the least promising method and dynamic seeding followed by a bioreactor proliferation phase is the most promising.

Human papilloma virus in melanoma biopsy specimens and its relation to melanoma progression.

OBJECTIVES: To evaluate melanoma biopsy specimens for human papilloma virus (HPV) and determine the relation between the presence of HPV, in vitro growth, and clinical progression of melanoma in the patients from whom the biopsy specimens were derived. SUMMARY BACKGROUND DATA: Ultraviolet radiation from sun exposure appears to be the primary causal agent in the development of cutaneous melanoma. However, other agents, including HPV, as observed in different epithelial carcinomas, may also play a role in melanoma development and progression. METHODS: Twelve melanoma biopsy specimens obtained from 12 patients with AJCC stage III and IV melanoma were stained with antibodies against gp-100 (HMB-45) and S-100 protein to confirm melanoma diagnosis and with a polyclonal HPV antibody. After mechanical dissociation, the melanoma specimen cells' ability to grow in vitro was assessed. Patients were evaluated for melanoma progression with physical examination, complete blood count, and liver function tests every 3 months and a chest radiograph every 6 months. RESULTS: All biopsy specimens were positive for S-100, and nine (75%) were positive for gp-100. Seven of 12 (58%) were positive for HPV by immunohistochemistry. In vitro, none of the HPV-negative tumor cells grew from the tumor biopsies, whereas five of seven (71%) of the HPV-positive melanoma tumor cells grew very well. All patients with HPV-positive tumor cells had recurrences and died of melanoma progression, whereas four of five (80%) patients with HPV-negative tumor cells remained alive and without melanoma recurrence. CONCLUSIONS: The presence of HPV was found in 58% of the biopsy specimens obtained from patients with stage III and IV melanoma and correlated with rapid melanoma progression. HPV may serve as a cofactor in the development of melanoma and may modulate a more aggressive phenotype in HPV-containing melanoma cells.

The development of an embedding technique for polylactide sponges.

The use of absorbable polymeric biomaterials is increasing in the field of tissue engineering. These polymeric scaffolds provide mechanical strength and shape as the engineered tissue forms. Histological analysis is an important part of the development of an appropriate polymeric construct, because it allows the analysis of the cell/material interaction. Unfortunately, routine paraffin processing often degrades these absorbable polymers, and routine staining can dissolve the remnants. This research sought to develop a histological procedure that would retain the polymer structure. Two processing procedures, paraffin and glycol methacrylate, were tested on three in vitro groups of poly-L-lactide sponges, high cell density seeding, low cell density seeding, and a control. The paraffin processing caused shrinkage and degradation of the polymer, and staining dissolved the remnants. The glycol methacrylate processing minimized damage to the polymer even after staining.

Parameters affecting cellular adhesion to polylactide films.

Absorbable biomaterials have been recently incorporated into the field of tissue engineering. Little work has been performed, even with the clinically acceptable absorbables, concerning their tissue promoting capability or lack, thereof. Furthermore, the relative attractions of cells to these implants may be largely disguised by the presence of serum. This research involved the development of an adhesion assay to compare the adhesion behavior of two cell types to two different polylactides in a serum free environment. The results showed that the attachment behavior depends not only on the cell or the polymer but a combination of the two.

Cellular ingrowth and thickness changes in poly-L-lactide and polyglycolide matrices implanted subcutaneously in the rat.

Highly porous matrices of poly-L-lactide (PL) and polyglycolide (PG), 24, 50, or 95 mg/cc in the form of 10 x 10 x 3 mm wafers, were implanted subcutaneously (two per rat) in the flanks of 8-12-week-old female Lewis rats (n = 120). Matrices were harvested, two rats per week, for 15 weeks and examined histologically. At weeks 1 and 2, a thin fibrous capsule was present and matrices showed capillary beds and host-cell infiltration along the implant margins. By week 4, the PL specimens had some arterioles while the PG specimens still had only capillary beds. At week 7, PL had well developed arterioles, venules, and capillaries while PG began to show modest vascular beds of capillaries only. In terms of cellular ingrowth, PL remained unchanged from 7 to 15 weeks. Giant cell formation was observed wherever polymer was present. There was a loss of thickness and cell mass for both matrices over time (PG > PL) despite initial host-cell ingrowth. As both polymers degraded and were absorbed, the ingrown cells mass regressed. There was little remaining PG at 15 weeks, leaving no trace of cells that previously had ingrown and no evidence of scar tissue.

Development of technologies aiding large-tissue engineering.

There are many clinical situations in which a large tissue mass is required to replace tissue lost to surgical resection (e.g., mastectomy). It is possible that autologous cell transplantation on biodegradable polymer matrices may provide a new therapy to engineer large tissue which can be used to treat these patients. A number of challenges must be met to engineer a large soft tissue mass. These include the design of (1) a structural framework to maintain a space for tissue development, (2) a space-filling matrix which provides for localization of transplanted cells, and (3) a strategy to enhance vascularization of the forming tissue. In this paper we provide an overview of several technologies which are under development to address these issues. Specifically, support matrices to maintain a space for tissue development have been fabricated from polymers of lactide and glycolide. The ability of these structures to resist compressive forces was regulated by the ratio of lactide to glycolide in the polymer. Smooth muscle cell seeding onto polyglycolide fiber-based matrices has been optimized to allow formation of new tissues in vitro and in vivo. Finally, polymer microsphere drug delivery technology is being developed to release vascular endothelial growth factor (VEGF), a potent angiogenic molecule, at the site of tissue formation. This strategy, which combines several different technologies, may ultimately allow for the engineering of large soft tissues.

Nonpeptidal P2 ligands for HIV protease inhibitors: structure-based design, synthesis, and biological evaluation.

Design and synthesis of nonpeptidal bis-tetrahydrofuran ligands based upon the X-ray crystal structure of the HIV-1 protease-inhibitor complex 1 led to replacement of two amide bonds and a 10 pi-aromatic system of Ro 31-8959 class of HIV protease inhibitors. Detailed structure-activity studies have now established that the position of ring oxygens, ring size, and stereochemistry are all crucial to potency. Of particular interest, compound 49 with (3S,3aS,6aS)-bis-Thf is the most potent inhibitor (IC50 value 1.8 +/- 0.2 nM; CIC95 value 46 +/- 4 nM) in this series. The X-ray structure of protein-inhibitor complex 49 has provided insight into the ligand-binding site interactions. As it turned out, both oxygens in the bis-Thf ligands are involved in hydrogen-bonding interactions with Asp 29 and Asp 30 NH present in the S2 subsite of HIV-1 protease. Stereoselective routes have been developed to obtain these novel ligands in optically pure form.

Computer-aided molecular modeling of the binding site architecture for eight monoclonal antibodies that bind a high potency guanidinium sweetener.

Computer-aided molecular modeling of the antibody binding site of eight different monoclonal antibodies (mAb) that bind the intense sweetener ligand (N-(p-cyanophenyl)-N'-diphenylmethyl) guanidine acetic acid was completed using canonical loop structures and framework regions from known immunoglobulins as "parent structures" for the molecular scaffoldings. The models of the fragment variable (Fv) region of the mAb were analyzed for the presence and location of residues predicted to be involved in ligand binding. Several binding site tryptophan residues in these models were located in positions that support previous flurospectroscopic observations of the mAb-ligand complexation. Computer-aided renderings of the electrostatic potential at the van der Waals surface of the Fv region were compared and found to be consistent with the ligand binding specificity profiles for the different mAb. The Fv model of mAb NC6.8 was consistent with the binding site features determined in the Fab structure recently solved by X-ray diffraction techniques. These Fv models should provide an adequate basis for site-directed mutagenesis experiments in order to characterize interactive motifs in the mAb binding site.

Crystal structure at 1.9-A resolution of human immunodeficiency virus (HIV) II protease complexed with L-735,524, an orally bioavailable inhibitor of the HIV proteases.

L-735,524 is a potent, orally bioavailable inhibitor of human immunodeficiency virus (HIV) protease currently in a Phase II clinical trial. We report here the three-dimensional structure of L-735,524 complexed to HIV-2 protease at 1.9-A resolution, as well as the structure of the native HIV-2 protease at 2.5-A resolution. The structure of HIV-2 protease is found to be essentially identical to that of HIV-1 protease. In the crystal lattice of the HIV-2 protease complexed with L-735,524, the inhibitor is chelated to the active site of the homodimeric enzyme in one orientation. This feature allows an unambiguous assignment of protein-ligand interactions from the electron density map. Both Fourier and difference Fourier maps reveal clearly the closure of the flap domains of the protease upon L-735,524 binding. Specific interactions between the enzyme and the inhibitor include the hydroxy group of the hydroxyaminopentane amide moiety of L-735,524 ligating to the carboxyl groups of the essential Asp-25 and Asp-25' enzymic residues and the amide oxygens of the inhibitor hydrogen bonding to the backbone amide nitrogen of Ile-50 and Ile-50' via an intervening water molecule. A second bridging water molecule is found between the amide nitrogen N2 of L-735,524 and the carboxyl oxygen of Asp-29'. Although other hydrogen bonds also add to binding, an equally significant contribution to affinity arises from hydrophobic interactions between the protease and the inhibitor throughout the pseudo-symmetric S1/S1', S2/S2', and S3/S3' regions of the enzyme. Except for its pyridine ring, all lipophilic moieties (t-butyl, indanyl, benzyl, and piperidyl) of L-735,524 are rigidly defined in the active site.

The development of cyclic sulfolanes as novel and high-affinity P2 ligands for HIV-1 protease inhibitors.

Design and synthesis of a novel series of protease inhibitors incorporating conformationally constrained cyclic ligands for the S2-substrate binding site of HIV-1 protease is described. We recently reported urethanes of 3-tetrahydrofuranyl as P2 ligands for HIV-1 protease inhibitors. Subsequently, we have found that the urethane of 3(S)-hydroxysulfolane further increased the in vitro potency of these inhibitors. Furthermore, introduction of a small 2-alkyl group cis to the 3-hydroxyl group of either heterocyclic system further enhanced enzyme affinity. The cis-2-isopropyl group thus far offered optimum enhancement of the inhibitory properties. This led to the discovery of inhibitor 43 (IC50 3.5 nM, CIC95 50 +/- 14 nM) of comparable in vitro antiviral potency to the current clinical candidate 1 (Ro 31-8959) but of reduced molecular weight due to the exclusion of the P3 quinoline ligand. Also, it has been demonstrated that the octahydropyrindene derivative 34 is an effective replacement of the P1' decahydroisoquinoline derivative.

Parallel evoluton in lichen-forming fungi.

The asexual lichen-forming fungus Parmelia hypotropa has two common chemical races that differ sharply in biogeography and niche characteristics and appear to have been derived by morphologic parallelism from chemically identical races of the closely related Parmelia perforata, which is sexual. Among the lichen fungi many chemically variable asexual morphs, which conventional taxonomies treat as species, are probably polyphyletic like Parmelia hypotropa.