Modified Dendrimer Cross-Linked Collagen-Based Matrices Using PureCol.

Princz MA, Sheardown H.

Abstract

Dendrimer cross-linking has been achieved with pepsin digested over 80% type-I bovine collagen to create strong hydrogels with good cell compatibility. Herein we investigate the use of commercially available collagen-based products with the dendrimer cross-linking technology. Specifically PureCol(®) (PC), a 97% bovine type-I collagen, human collagen (HC) and human extracellular matrix (hECM) were concentrated, and then cross-linked with polypropyleneimine octaamine generation two dendrimers using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) chemistry. PC gels with 30 and 20 mg/ml bovine collagen were fabricated, and despite similar concentrations to >80% type-I bovine collagen dendrimer cross-linked gels (CG), PC gels demonstrated increased swelling and decreased stability, as determined with collagenase digestion. The highly purified bovine (PC) and human sourced-collagen (HC) gels were similar in performance, but not as stable as the CG gels, which may correlate to the manufacturer’s collagen purification and storage. Finally, the addition of hECM components to PC to create PC-hECM gels, resulted in a looser gel network, compared to heparinized dendrimer cross-linked bovine >80% type-I collagen gels (CHG). However, all collagen-based gels supported 3T3 fibroblast cell growth over 4 days, indicating these gels may be suitable for tissue-engineering applications.

However pepsinized type IV collagen depleted of the 7s domain may be prepared, as described by Timpl et al. (1983). Timpl, R., S. Johansson, V. van Delden, I. Oberbaumer, and M. Hook. 1983.Characterization of protease resistant fragments of laminin mediating attachment and spreading of rat hepatocytes. J. Biol. Chem. 258:8922-8927.

Answer: Using rotary shadowing technique under transmission electron microscopy, it was found that PureCol collagen consisted of approximately 80 % monomers, 13 % dimers, trimers, and oligomers with the remaining 7% collagen fragments.

Answer:
See reference below:   http://www.nature.com/labinvest/journal/v88/n2/full/3700703a.html#fig5

In summary:

Methods

Preparation of Collagen Gel:

Collagen type I gels were prepared by mixing appropriate volumes of collagen solution with 10X PBS) and 1 N NaOH in pre-cooled eppendorf tubes. The final concentrations of the collagen in the gel matrix were varied from 0.7 to 2.5 mg/ml to obtain an appropriate collagen sieve diameter. An average diameter of 5–7 μm is considered as optimal for the present application (average bacterial diameter, 2 μm and Ped-2E9 cell diameter, 10 μm). After addition of the reagents the gel solutions were kept at 37°C for 15–30 min to complete the gelation.

Determination of Gel Matrix Pore Size
by Cryo-Scanning Electron Microscopy:

To determine the pore size of the gel matrix sieves (with different collagen concentrations from 0.7 to 2 mg/ml), we performed a cryo-scanning electron microscopy (SEM) analysis with minimal
dehydration of the gel matrix. The gel preparations were mounted on a slit holder and plunged into liquid nitrogen slush. A vacuum was pulled and the samples were transferred to the Gatan Alto 2500 (Pleasanton, CA, USA) pre-chamber (cooled to approximately −60°C). After the samples were cut with a microtome with a cold knife, the samples were sublimated at −85°C for 30 min followed by sputter coating for 100 s with platinum. The samples were then transferred to the microscope cryostage (approximate temperature: −130°C) for imaging. The SEM images were taken with an FEI NOVA
nanoSEM (FEI, Hillsboro, OR, USA) using ET and TLD detectors operating at 5 kV accelerating voltage, with approximately 6 mm working distance and the
magnifications were varied between × 2500 and × 50 000.

Results

The collagen matrix microenvironment was fabricated such that the pore diameter of the gel matrix sieve is maintained between 5 and 7 μm. The cryo-SEM photographs confirmed that a collagen gel concentration of 1.5 mg/ml optimally achieves the desired pore size for the purposes of this study.

On average:

1.0 mg/ml,    give ~ 10 μm pore size

1.5 mg/ml,   give ~ 6 μm pore size

2.0 mg/ml,    give ~ 2 μm pore size

A: The simple answers to these two questions are “Yes”.  The collagen molecule in PureCol, Nutragen and VitroCol were prepared from native collagen matrix by pepsin treatment under controlled conditions to remove the non-helical portion, telo-peptides, only and the helical portion is intact. In this case, the enzymatic active sites for MMP (Matrix Metalloproteinase), such as for Mammalian Collagenase Matrix Metalloproteinase 8 (MMP-8), on the molecule was preserved. These pepsin treated collagen products should be behaved as a native intact collagen.

2. Since glycine and lysine amino acids can form cross links
between protein monomers, were these amino acids modified?  No.

3. Which protein expression system was used: bacteria, yeast
or mammalian cells?  Bacteria

Electrospinning of Collagen #1

Electrospinning of Collagen #2

Electrospinning of collagen #3

Answer: For most applications, a general molecular weight of the alpha chains in SDS-PAGE is roughly 100kDa.  The triple helix would then be 300kDa.

Answer:  A study was done to determine the density of PureCol, bovine collagen at a protein concentration of 3.0 mg/ml.  The results indicated that the density was 1.0.

From the viscosity of the collagen, one might think that it was higher, but the structure of collagen increases the viscosity, but not the density.