poly(ethylene glycol) hydrogels: a cell

I also thank all of them for the friendly and lively atmosphere that has enriched my time in the group. Human fibroblasts adhered, invaded, and apparently proliferated in artificial hydrogel matrices in a three-dimensional manner. Neurite outgrowth was dependent on the concentration of matrix-bound heparin, suggesting that heparin was necessary to immobilize neuroactive adhesion and/or growth factors in the hydrogel.

General Introduction

Proteoglycans represent another important class of adhesion molecules that should be particularly mentioned in the context of the project presented in this thesis. An important feature in the context of the material presented in this doctoral thesis is that proteoglycans' glycosaminoglycan side chains (such as highly negatively charged heparin) provide binding sites for adhesion molecules -such as laminin474/L and growth factors. In the context of this thesis project, aprotinin was used to determine a negative control (Chapter 5) for the plasmin degradability of the protein f A l-ent-PEG materials presented in Chapter 4.

Sprout axons must be able to reach the distal part of the projection for nerve regeneration and eventual reinnervation of the target organ to occur. The fact that the presence of the control peptide did not support cell adhesion strongly suggests that the observed adhesion of hFF to the grafted RGDSG surface was indeed integrin-mediated and therefore biologically specific.

Figure 2.1 A·D: Spontaneous Regeneration of Periphcral Nerves after Axotomy

DESIGN, SYNTHESIS, AND CHARACTERIZATION OF PROTEIN- graft-PEG HYDROGELS

PEGylated recombinant protein pentamers [A 1 or I B J (presumably protein I AJ-grafr-hexaPEGacrylate or protein I Bl-grafr-hexaPEGacrylate). was purified from excess PEG-diacrylate by Ni++ affinity chromatography: Briefly. after raising the pH to 7.9 by adding 1N NaOH. the solution of reaction products was applied to beds of His·Bind'" resin with sufficient binding capacity for the PEGacrylated target protein. The products of the subsequent multimerization of DsaI-digested segments 1 c I for recombinant protein pentamer [B] can be seen in lane 1 of the agarose gel shown in Figure 4.1 This was one of the reasons for focusing exclusively on protein pentamer species in subsequent experiments.

As a result, acid hydrolysis 01 is for the detection of acid munitions. Asparagine and glutamine n:sides were converted to aspartic acid and glutamic acid. respectively.. the number of those residues detected reflects the number of original asparagine and aspartic acid residues. or glutamine and glutamic acid residues: s. in the combined protein. ii) Cysteine ​​residues do not match the analysis method used. PEGycrylation of the recombinant protein pentamer was not completed unless TCEP was first used to break the disulfide bonds and release the protein thiols for reaction with the PEG diacrylate (Figure 4.3.a. The exact nature of the protein (various))[A] - The types of grqft-multiPEGacrylate product are not so important in terms of the biological characterization of the eventual protein[A]-grafr-PEG hydrogels, since the only two cross-linked matrix components were always proteinlA] and PEG.

The IO1 polyacrylamide SDS-PAGE gel shown illustrates the increase in molecular weight of recombinant protein pentamer [AJ due to PEGylation with a series of PEGs of increasing molecular weight. Elution at such high salt concentrations is evidence of the strong affinity of recombinant protein pentamer [Al for heparin, indicating that the heparin binding site was not steric. These results confirm the affinity of recombinant protein pentamer rAJ's heparin binding site for heparin.

Initial design goals were achieved by the "selective" grafting of PEG-diacrylate onto recombinant protein pentamer IAl to obtain protein[A]-gr«ft-PEG(3400)acrylate and -PEG(6000)acrylate.

Figure 4.2.a: Bactcrial I~xprcssion and Purification of Recomhinant Protein Pentamer 1 Al

CHAPTERS

14·15169 Cross-linking of protein graft-PEGacrylate around hFF-fibrin clusters was induced in situ by exposure to a transmitted light source through the cover of the culture well. During the inhibition studies, the respective inhibitor concentrations were maintained for four days by daily replacement of the cell culture medium with freshly prepared medium at the original inhibitor concentrations. During the inhibition studies, the relevant inhibitor concentrations were maintained by daily replacement of the old cell culture medium with freshly prepared medium at the original inhibitor concentrations.

To quantify the penetration depth of the outgrowth, the area of ​​the original hFF-fibrin cluster in the midplane was measured, as well as the area of ​​the hFF outgrowth, defined by the tips of the hFF branches in the midplane of focus. The highly branched nature of the hFF outgrowths suggests that the cells were involved in a proliferative mode of penetration. Comparison of the outgrowth of hFF-fibrin clusters in surrounding protein[Al-gr(itf-PEG(3400) and -PEG(6000) gels (Figure 5.11) underlines the importance of mechanical strength for sustained cell growth.

This demonstrates that the inhibition of growth and shrinkage of hFF clusters was not due to toxicity and cell death. These results indicate that cell-derived serine-protease activity (such as plasminogen activation and induction of fibrinolysis) is a relevant mode of penetration into the matrix of protein-f,'raft-PEG hydrogels. Cell adhesion sites and plasmin substrate sites are available within the protein scaffold of the protein-graft-PEGacrylate precursor, as well as sites for attachment of PEG acrylate to this scaffold.

The ultimate goal of the protein-groft-PEG hydrogel material, described in Chapters 4 and 5, was to be used as a matrix for nerve regeneration.

Figure 5.1: hFF Adhesion to Protein Pentamers

BACKGROUND

2.~n mg/ml heparin corresponded to an amount corresponding to 1 molecule of heparin per 62 heparin binding sites in the non-swollen protein-graft-PEG(3400) hydrogels. Here, 2.X3 mg/ml heparin corresponded to an amount corresponding to I-molecule heparin per 50 heparin binding sites in the non-swollen protein-graft-PEG(6000) hydrogels. Fibroblast or glial cell outgrowth, but lack of neurite outgrowth, from whole DRGs inside protein-graft-PEG hydrogels.

Lack of neurite outgrowth from dissociated DRG cell-fibrin clusters inside protein-graft-PEG hydrogels. Lack of neurite extension of PC12 cells embedded in fibrin and placed inside protein-graft-PEG hydrogels. Neurite-like extensions grew from surface-embedded chick embryonic DRGs on protein-graft-PEG hydrogels under different culture conditions (Figure 6.15.a,b).

Dependence of neurite outgrowth on protein-graft-PEG hydrogels Based on Figure 6.17.a, surface neurite outgrowth appeared to require both heparin and hep.bind.- YIGSR bound to 10% (m+l') protein-graft- PEG( 3400) hydrogel matrices. Alternatively, the inhibitory effect on neurite outgrowth may be due to an excessive concentration of hep.bind.- YIGSR peptide in the matrix. The resolution of this issue depends on the results of expected release studies of hep.bind.- YIGSR peptide from protein-graft-PEG hydrogels.

A likely explanation for the effect of PEG chain length on neurite outgrowth is that protein-graft-PEG(3400) hydrogels are expected to be more cross-linked.

Table 6.1: Culture Conditions for DRGs Half-Embedded In Protein-grait-PEG(3400) Hydrogels

SUMMARY, CONCLUSIONS, AND OUTLOOK

Before the PEGylation of protein pentamers, a crucial step towards obtaining protein-graft-PEG hydrogels was the reduction of protein thiols using TCEP, avoiding the use of thiol-containing reducing agents such as ~-mercaptoethanol or DTT, which involve just thiol exchange and requires repeated dialysis for -often incomplete- removal. Protein-gr~ft-PEG hydrogels represent a homogeneous, highly cross-linked matrix whose mechanical strength was sufficient to support cell attachment and migration. Protein-graft-PEG hydrogels were transparent and therefore allowed microscopic analysis of three-dimensional cell cultures.

The main component of protein;?raft-PEG hydrogels, protein;?raft-hexaPEGacrylate, as well as the degradation products of the hydrogels appeared to be nontoxic in contact with model tissue, hFF-fibrin clusters, as well as with embryonic chick DRGs. Another potential material shortcoming of the protein-graft PEG hydrogels could be hydrolytic degradation in bulk (among the acrylate esters), although hydrolysis compared to enzymatic degradation is likely insignificant under physiological conditions. If hFFs were to infiltrate the material, the right combination of adhesive and neuroinductive signals could enable neurite ingrowth into future variants of protein [A f-PEG hydrogels.

One way to modify current protein[A]-/?ratf-PEG hydrogels is to use the heparin bridging scheme to present additional adhesion and/or growth factors. Protein-grayt-PEG hydrogels could prove to be useful matrices for the delivery of stem cells that differentiate in response to local stimuli at the implant site. However, in terms of material design, as long as uncertainty remains regarding the utility (and choice) of minimally neuroactive oligopeptides (Figure 6.17) that may or may not actually be immobilized into the hydrogels via heparin, additional neuroactive domains could be incorporated. in the protein backbone of protein-gr({jt-PEG hydrogels) in the hope of promoting neurite outgrowth in three dimensions.

In fact, if a proven immobilization scheme can be established, adhesion sites in the backbone become indispensable, as any signal of choice can be delivered by linking it to a heparin analogue followed by direct binding to exclusively heparin-binding protein graft-PEG hydrogels.

A synthetic peptide containing the IKV A V sequence in the A chain of laminin mediates cell attachment, migration, and neurite outgrowth. - microscopic observations of the effects of localized crushes on the connective tissues of the peripheral nerve. Peripheral nerve regeneration through blind-terminated semipermeable guide channels: effect of molecular weight limitation.

Exogenous matrix precursors promote functional nerve regeneration through a 15-mm gap within a silicone chamber in rats. Peripheral nerve regeneration by entubulation repair: comparison of biodegradable nerve conduits versus polyethylene tubes and effects of laminin-containing gel. Dependence of nerve regeneration through rat muscle grafts on basement membrane availability and orientation.

Role of Schwann cells in peripheral nerve axon regeneration through muscle basal lamina grafts. Peripheral nerve regeneration via an 80-mm gap bridged with polyglycolic acid (PGA)-collagen tube filled with laminin-coated collagen fibers: histological and electrophysiological evaluation of regenerated nerves. Adult nerve-derived syngeneic Schwann cells seeded into semipermeable guide channels improve peripheral nerve regeneration.

Enhanced synthesis of brain-derived neurotrophic factor in the damaged peripheral nerve:. different mechanisms are responsible for the regulation of BDNF and NGF mRNA.