Timothy M. Parker

Elastic protein-based polymers in soft tissue augmentation and generation

Five elastic protein-based polymers, designed as variations of polymer I, (GVGVP)251, elicited different responses when injected as subcutaneous implants in the guinea pig, a preclinical test used to evaluate materials for soft tissue augmentation and specifically for correction of urinary incontinence. All six polymers, prepared using recombinant DNA technology, expressed at good levels using transformed E. coli fermentation. These E. coli-produced polymers were purified for the first time to the exacting levels required for use as biomaterials where a large quantity could disperse into the tissues in a few days. Time periods of 2 and 4 weeks were used. Polymer I functioned as a bulking agent around which a fine fibrous capsule formed. Inclusion of (GVGVAP)8, a chemoattractant toward monocytes and elastin-synthesizing fibroblasts in the sequence of polymer I, resulted in an appropriate tissue response of invasion of macrophages. Inclusion of lysine residues, for lysyl oxidase cross-linking, suggested a possible remodeling of the implant toward fibers. Most promising however, when the cell attachment sequence, GRGDSP, was added to polymer I, the implant elicited tissue generation with a normal complement of collagen and elastic fibers, spindle-shaped histiocytes and angiogenesis. If this response is retained over time, the desired soft tissue augmentation and generation will have been achieved. Our working hypothesis is that on formation of elastin, with a half-life of the order of 70 years, a long lasting soft tissue augmentation would result rather than scar tissue as occurs with Contigen, the currently approved injectable implant for soft tissue augmentation.

Mechanics of elastin: molecular mechanism of biological elasticity and its relationship to contraction

Description of the mechanics of elastin requires the understanding of two interlinked but distinct physical processes; the development of entropic elastic force and the occurrence of hydrophobic association. Elementary statistical-mechanical analysis of AFM single-chain force-extension data of elastin model molecules identifies damping of internal chain dynamics on extension as a fundamental source of entropic elastic force and eliminates the requirement of random chain networks. For elastin and its models, this simple analysis is substantiated experimentally by the observation of mechanical resonances in the dielectric relaxation and acoustic absorption spectra, and theoretically by the dependence of entropy on frequency of torsion-angle oscillations, and by classical molecular-mechanics and dynamics calculations of relaxed and extended states of the beta-spiral description of the elastin repeat, (GVGVP)n. The role of hydrophobic hydration in the mechanics of elastin becomes apparent under conditions of isometric contraction. During force development at constant length, increase in entropic elastic force resulting from decrease in elastomer entropy occurs under conditions of increase in solvent entropy. This eliminates the solvent entropy change as the entropy change that gives rise to entropic elastic force and couples association of hydrophobic domains to the process. Therefore, association of hydrophobic domains within the elastomer at fixed length stretches interconnecting dynamic chain segments and causes an increase in the entropic elastic force due to the resulting damping of internal chain dynamics. Fundamental to the mechanics of elastin is the inverse temperature transition of hydrophobic association that occurs with development of mechanical resonances within fibrous elastin and polymers of repeat elastin sequences, which, with design of truly minimal changes in sequence, demonstrate energy conversions extant in biology and demonstrate the special capacity of bound phosphates to raise the free energy of hydrophobic association.

Prevention of postlaminectomy epidural fibrosis using bioelastic materials.

Study Design. The use of elastic protein-based polymers for the prevention of epidural fibrosis following lumbar spine laminectomy was investigated in a rabbit model. Objectives. To determine the safety and efficacy of two bioelastic polymers in matrix and gel forms as interpositional materials in preventing postlaminectomy epidural fibrosis. Summary of Background Data. Postlaminectomy epidural fibrosis complicates revision spine surgery and is implicated in cases of “failed back syndrome.” Materials employed as mechanical barriers to limit tethering of neural elements by the fibrosis tissue have met with little success. A recent family of protein-based polymers, previously reported to prevent postoperative scarring and adhesions, may hold promise in treating this condition. Methods. Sixteen female New Zealand White rabbits underwent laminectomy at L4 and L6. Two polymer compositions, each in membrane and gel forms, were implanted at a randomly assigned level in four rabbits each, with the remaining level serving as an internal control. The animals were killed at 8 weeks, and qualitative and quantitative histology and gross pathologic examination were performed for both the control and the experimental sites to assess the polymers’ efficacy in preventing dorsal epidural fibrosis. Results. The use of the polymers caused no adverse effects. Compared to the control sites, both polymers in either gel or membrane form significantly reduced the formation of epidural fibrosis and its area of contact with the dura postlaminectomy. However, no significant difference in efficacy was detected between either the polymers or their respective forms in preventing epidural fibrosis. Conclusions. The selected compositions of biosynthetic, bioelastic polymers were safe and effective in the limiting the direct contact and consequent tethering of the underlying neural elements by the postlaminectomy epidural fibrosis in rabbits.

Comparison of electrostatic-and hydrophobic-induced pKa shifts in polypentapeptides. The lysine residue

Abstract Acid—base titrations at 37°C determined the p K a values in water and saline of twelve synthesized polypentapeptides poly( f K (GVGIP), f v (GKGIP), where f v + f K = 1 with f K ranging from 1 to 0.06 and where K is the lysine (NH + 3 /NH 2 ) residue. In water the p K a was 9.60 near f K = 0.9 decreasing to 9.20 at f K = 1 and to 8.18 at f K = 0.06. At 37°C in the NH 2 state, the polymers form a viscoelastic phase of 50% water by volume where for f K = 0 previous dielectric permittivity data place the dielectric constant near 65. For f K K a could not be explained by the usual electrostatic self-energy argument of decreased dielectric constant as Lys (K) is replaced by Val (V). Instead, an apolar—polar repulsive free energy of hydration is discussed which provides a basis for hydrophobic-induced p K a shifts.

Cell Adhesive Properties of Bioelastic Materials Containing Cell Attachment Sequences

The biocompatibility, conformational and inverse temperature transition properties of poly(Vall-Pro2-Gly3-Va14- Gly5), i.e., poly(VPGVG), and its 7-irradiation crosslinked matrix and the poly(VPGVG)-derived hydrophobicity scale are noted. Also noted are the capacities of varying the bioactive role of bioelastic materials; that is, the bioelastic materials can be designed (1) to exhibit a range of elastic moduli, (2) to exhibit different rates of degradation, (3) for various modes of drug release, (4) to perform numerous free energy transductions, (5) to contain functional enzyme sites, and (6) to contain functional cell attachment sequences that promote growth to confluence.

Synthesis, Characterizations, and Medical Applications of Bioelastic Materials

Bioelastic materials are elastomeric polypeptides composed of repeating sequences. They are a relatively new class of polymers that may also be called elastic protein-based polymers, having their origins in repeating sequences found in the mammalian elastic protein, elastin. The most striking and longest sequence between cross-links in pig and cow is the polypentapeptide-(PPP), poly(VPGVG) or (Val1-Pro2-Gly3-Val4-Gly5)n, where n is 11 (Sandberg et al., 1985; Yeh et al, 1987). Another repeat first found in porcine elastin is a polytetrapeptide-(PTP), poly(VPGG) or (Val1-Pro2-Gly3-Gly4)n, but this repeat has not been found to occur with n greater than 2 without substitution (Sandberg et al., 1981). The next most common recurring sequence in mammalian elastin is a polyhexapeptide-(PHP), poly(APGVGV) or (Ala1-Pro2-Gly3-Val4-Gly5-Val6)n where, with but a couple of isomorphous hydrophobic residue replacements such as Val by He or Leu, n is 8 in man (Indik et al., 1987). The monomers, oligomers, and high polymers of these repeats have been synthesized and conformationally characterized (Urry and Long, 1976). The high polymers of these repeating sequences have been cross-linked into sheets, rods, and tubes, and the PPP and PTP have been found to be elastomeric with the former being capable of an elastic modulus similar to that of the natural elastic fiber (Urry et al., 1976, 1981, 1982).

Pressure effect on inverse temperature transitions: biological implications

Abstract Elastic protein-based polymers of the form poly [ f x (VPGXG), f v (VPGVG)], where f x and f v are mole fractions with f x + f v =1, exhibit inverse temperature transitions in the form of a phase separation in which folding and aggregation of polymer chains into more-ordered states of the condensed (coacervate) phase occurs on raising the temperature. When X = Trp, Phe or Tyr, an increase in pressure causes a substantial increase in the temperature of the transition. The data are interpreted to indicate that water molecules surrounding the aromatic side chains of Trp(W), Phe(F) or Tyr(Y) occupy less volume than water molecules in bulk water. The calculated volume change for poly[0.8(GVGVP), 0.2(GFGVP)], for example, on going from coacervate phase, where hydrophobic associations have largely eliminated waters of hydrophobic hydration, to be dispersed in water where the hydrophobic moieties are surrounded by water is 80 cm 3 /mol of mean pentamers or some 400 cm 3 /mol of (GFGVP). The results provide an understanding of the source of pressure effects in biological systems and for the capacity to design materials capable of exhibiting baromechanical transduction.

Protein-Based Materials with a Profound Range of Properties and Applications: The Elastin ΔTt Hydrophobic Paradigm

The term protein-based polymer has its origin in a symposium containing that name organized by D.L. Kaplan, M.T. Marron, and D.A. Tirrell (1990). As the term is used here, it refers to polymers comprised of repeating peptide sequences in which the repeats may be as few as two residues or as many as hundreds of residues. In the latter case, properties of protein-based polymers and globular proteins naturally merge. For the former case, there are now known many examples of repeating peptide sequences in proteins. These occur most commonly in proteins that fill structural roles as opposed to the frequently discussed catalytic, transport, or transductional roles of globular proteins.

Non-linear hydrophobic-induced pKa shifts: Implications for efficiency of conversion to chemical energy

Abstract By using one Asp or one Glu per thirty residues in a polytricosapeptide capable of exhibiting a hydrophobic folding and assembly transition and stepwise converting a set of the five Val residues (most proximal to the Asp or Glu residue) to more-hydrophobic Phe residues, a non-linear hydrophobic-induced p K a shift was observed with a Δ p K a of 0.4 (Asp) and 0.3 (Glu) on addition to 2 Phe residues per 30mer but with a Δ p K a of 4.7 (Asp) and 2.7 (Glu) on going from 4 Phe/30mer to 5 Phe/30mer. As a shift in p K a can be equivalent to the conversion to chemical energy from whatever energy input — mechanical, chemical, electrochemical, pressure or light — which effects a change in hydrophobicity, the non-linear hydrophobic-induced p K a shift means increased efficiency of energy conversion with increased hydrophobicity of the protein-based polymer.

Poly (VAL1-PRO2-ALA3-VAL4-GLY5): A Reversible, Inverse Thermoplastic

Initial characterization of the sequential polypentapeptide, poly (VPAVG) an analog of the polypentapeptide of elastin poly (VPGVG), is reported. It undergoes a concentration dependent inverse temperature transition, in which it is soluble in water below 25°C and aggregates on raising the temperature. When crosslinked by 20 Mrads of γ-irradiation to give X20-poly (VPAVG), an elastomeric matrix is formed. At 25°C, this material exhibits an elastic modulus of 1.5 x 105 dynes/cm2, similar to that found at that temperature for X20-poly (VPGVG). On raising the temperature to 37°C, however, the elastic modulus increases three orders of magnitude to 3 × 108 dynes/cm2 which is two orders of magnitude greater than obtained for X20-poly (VPGVG). This reversible hardening on raising the temperature defines X20-poly (VPAVG) as a reversible, inverse thermoplastic. This property makes it an interesting new material to add to the already described set of bioelastic materials. The potential applications and extensions of applications made possible by this additional bioelastic material are briefly considered.