a method for preparation of etched collagen fibers that support neurite outgrowth

A Method for Preparation of Etched Collagen Fibers that Support Neurite Outgrowth

Eric Wong,' David Christiansen,* Aram Rizvi,' Herbert M. Geller,+ and Frederick H. Silver'

*Biomaterials Center, Department of Pathology and +Department of Pharmacology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey

Use of collagenous substrates for growth of attachment-dependent and attachmentindependent cells have been reported in the literature. Growth of fibroblasts and epithelial cells on collagen sponges and fibers has been previously studied in our laboratory. This article reports the result of studies of neurite outgrowth on etched collagen fibers in cell culture. Collagen fibers with a mean diameter of about 134 p m were etched on glass coverslips using a collagenase solution until individual fibrils of 1 p m in diameter were observed. The kinetics of etching were optimized at a calcium chloride concentration of 5 mM and a collagenase activity of 300 unit/mL. At room temperature an etching time of 10 h maximized the number of fibrils exposed per fiber. Cell culture studies on etched collagen fibers indicate that neurites from rat fetal cortical neurons elongate along the longitudinal axes of collagen fibrils. In some in- stances the cell body observed was not on top of the collagen fibrils but situated between two parallel fibrils. Results of these experiments indicate that growth of neurites along collagen fibrils in cell culture may be a useful model to evaluate neurite extension in the presence of neurotrophic factors as well as to study optimization of substrates for nerve regeneration.

INTRODUCTION

Collagen is an important component of neural connective tissue; however, its role in the peripheral nervous system and central nervous system (CNS) differ. In mammalian peripheral nerve, type I collagen is found in the epinerium, perineurium, and endoneurium; type 111 is more apparent in perineurium and endoneurium; and types IV and V collagens are concentrated in the endoneurium. Transection of rat sciatic nerve reveals collagen fibrils around the bands of Bungner. Endoneurial collagen has been postulated to protect peripheral nerve fibers against compressive forces3 while resistance to tensile stretching has been attributed to the epine~rium.~

In the mammalian CNS, types I and III collagen are in the pia mater and types IV and V collagens are located in the small blood vessels or capillaries as well as the pia materhpinal cord junction.' However, only after an insult such as glioma formation' or mechanical trauma to the cerebral cortex6 is collagen observed in the parenchyma.

Collagenous matrices have been shown to support the growth of a variety of cell types7-'' and under certain conditions they have been reported to modulate cell shape and cytoskeleton. Cellular alignment" not only reflects the chemistry of the substrate but also reflects the physical na- ture of the surface13 and may ultimately affect the locomo- tive behavior of different cell types. I4-I6

Requests for reprints should be sent to F. H. Silver, Biomaterials Center, UMDNJ- Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854.

Journal of Applied Biomaterials, Vol. 1, 225-232 (1990) 0 1990 John Wiley & Sons, Inc. CCC 1045-4861/90/030225-08$04.00

In cell culture, collagen has long been considered as a favorable substrate for neuritic outgrowth. Retinal neurons from chick embryo17 and sympathetic neurons from rat18 ex- tend neurites over a collagen substrate. Neurites from rat embryonic olfactory bulb and retina grow faster on air-dried collagen than on laminin. I9 Kleitman et a1." also observed that the density of neuritic outgrowth from embryonic retinal explants was higher on air-dried collagen than on Schwann cells, laminin, or extracted extracellular matrix.

Successful growth of neurites on air-dried collagen sug- gests that it may be possible to direct neurite growth in the presence of an oriented collagen substrate. This article de- scribes a technique for growing neurites on small diameter collagen fibers that are etched using a collagenase solution. The growth of neurites from cortical neurons of fetal rat on etched fibers was monitored using light microscopic techniques. Results of this study indicate that directed neurite outgrow can be achieved along the axes of oriented collagen fibers. Etched collagen fibers may act as a suitable scaffold for the regeneration of nervous tissue.

MATERIALS AND METHODS

Preparation of Collagen Fibers

A 1% (w/v) aqueous collagen dispersion was made by adding 1 g of type I collagen extract (Devro Inc., Somerville, NJ) to 100 mL of distilled water at pH 2.0. The pH of the water was adjusted by adding 1 N HCl. The dispersion was then blended at high speed for 3 min, followed by deaera- tion under vacuum at a pressure of 100 mTorr or less. The dispersion was transferred to a syringe connected to plastic tubing with an inner diameter of 0.28 mm. A constant and

WONG ET AL. 226

continuous pressure was applied to the syringe with a syringe pump resulting in the extrusion of collagen fibers into a buffer solution containing 135 mM NaCl, 30 mM TES (N-tris[hydroxylmethyl]methyl-2-aminoethane sul- phonic acid) (Sigma Chemical Co., St. Louis, MO), and 30 mM NaHPO, at pH 7.5. Air-dried collagen fibers were crosslinked by severe dehydration as described previously.21 The composition of the collagen was characterized by so- dium dodecyl sulfate polyacrylamide gel electrophoresis as alpha 1(1), alpha l(ll), beta, gamma and higher molecular weight components of type I collagen after reduction, and heat denaturation. The results of amino acid analysis were consistent with the composition of bovine type I collagen as described previously.2’

Fibers formed in this manner were then placed onto 18 mm diameter circular glass coverslips (Fisher Scientific, Pittsburgh, PA) and were allowed to air dry for at least 12 h. Both ends of the fibers were glued onto the coverslips using Silicone medical grade adhesive (Dow Coming, Inc., Mid- land, MI). The fibers were crosslinked by severe dehydration for 72 h at 110°C and then sterilized by exposure to ultraviolet irradiation under a laminar air flow bench. The duration of irradiation did not exceed 48 h to avoid denaturation of collagen.

Preparation of Etching Solutions

Crosslinked collagen fibers attached to coverslips were placed in a solution containing 50 mM Tris-HC1 (Sigma Chemical Co.) with different amounts of collagenase (Sigma Chemical Co.), and CaCl, (Fisher Scientific) at pH 7.6 and 37°C to expose individual collagen fibril bundles. Con- centration of collagenase solutions tested ranged from 100 units/mL to 900 units/mL and CaCl, concentrations studied ranged from 0 mM to 100 mM. The fibers were examined at intervals of one hour under an Olympus CK2 phase contrast microscope.

Etching of Collagen Fibers

Glass coverslips with crosslinked collagen fibers were placed in a 12 well tissue culture plate (Costar Inc., Cambridge, MA). The fibers were etched in an etching solution containing 0-900 units/mL collagenase, 0-100 mM CaCl,, and 50 mM Tris-HC1 at pH 7.6. When fine collagen fibers (fibril bundles) were visualized under a phase-contrast microscope, an equal volume of 10 mM EDTA (Sigma Chemical Co., St. Louis, MO) was added to the etchant in order to stop the action of collagenase. Etched collagen fibers were irradiated for up to 48 h using ultraviolet light prior to cell culture studies.

The glass coverslips were rinsed twice with Dulbecco’s Modified Eagle’s Medium (Gibco Laboratories, Grand Island, NY) containing 10% fetal calf serum, 20 units/mL penicillin, 20 pg/mL streptomycin, and 0.05 pg/mL amphotericin-B (Gibco Laboratories, Grand Island, NY) ab- breviated below as DMEM/FCS/A. Prior to addition of neu-

rons, the glass coverslips were incubated at 37°C with 5% CO, for at least 3 h.

To confirm the presence of etched collagen fibers, sev- eral glass coverslips were stained with Picrosirius red and examined for birefringence under a Leitz Laborlux 12 Pol microscope equipped with a polarizer and analyzer.

Preparation of Neurons

Fetal brains were dissected aseptically from etherized preg- nant Sprague-Dawley rats at day 15 of gestation, following the procedure described by Ventimiglia and Geller.22 Fetal corti- ces were isolated in DMEM/FCS/A under a dissecting microscope. The cortical tissues were then cut into small pieces. After the meninges were removed, the tissues were enzy- matically dissociated into individual cells in DMEM/FCS/A with 100 units/mL collagenase and 0.05% trypsin (Gibco Laboratories, Grand Island, NY). The suspension was incubated at 37°C water bath for 30 min, followed by cen- trifugation at 1000 rpm for 5 min. The supernatant was discarded and the cells were treated with DMEM/FCS/A with 80 Kunitz units/mL DNAse (Sigma Chemical Co., St. Louis, MO) and 200 BAEE units/mL soybean trypsin in- hibitor (Sigma Chemical Co., St. Louis, MO). Cells were resuspensed in DMEM/FCS/A and triturated 10 times with a Pasteur pipette to form a cell suspension. After centrifuging at 1000 rpm for 5 min, the supernatant was discarded and the cells were washed with DMEM/FCS/A twice. The concentration of cells in the suspension was estimated using a hemocytometer. Approximately 100 000 cells were placed into each well of the tissue culture plate containing a coverslip with etched collagen fibers.

lmmunocytochemistry

Cell cultures were examined under a phase contrast microscope for neurite extension. Cells on coverslips were then fixed for 5 min at -20°C in an acid-alcohol solution containing 95% ethanol and 5% acetic acid. The coverslips were rinsed three times in DMEM and once in DMEM with 10% horse serum. Neurofilament specific monoclonal antibody RT9723 was added to the cells on coverslips, and incubated for 25 min at 4°C. The coverslips were rinsed four times in DMEM. Fluorescein-conjugated goat anti-mouse IgG (Cappel Research Products, West Chester, PA) was added to the cells on coverslips, followed by incubation for 25 min at 4°C. The coverslips were rinsed four times in distilled water, and mounted onto glass slides in glycerol containing 0.1 % p-phenylenediamine to prevent fading of immunofluorescence.24 The edge of the coverslip was sealed using clear nail enamel.

Neurite extension on etched collagen fibers was examined using a Zeiss Axioplan fluorescence microscope equipped with Normarski differential interference contrast optics, an epifluorescence illuminator with fluorescein and rhodamine filters, and a mounted camera with automatic focusing. Pho- tographs were taken using Kodak T-MAX films.

PREPARATION OF ETCHED COLLAGEN FIBERS 22i

RESULTS When a wet collagen fiber was placed onto a coverslip: the fiber flattened out and the fibrils settled to the bottom because of gravity, attaching to the coverslip (Fig. 2). Non- fibrous material remained at the surface and became irregu- larly shaped when air-dried. The fibril bundles attached ta the coverslip became exposed (Fig. 2) after etching by collagenase. These fibrils were observed after staining with Pi- crosirius red.25 Birefringent collagen fibers were observed under polarized light (Fig. 3).

The purpose of these studies was to investigate the ability of a collagen fiber substrate to support directed growth of neurites. Collagen fibers formed by extrusion into fiber formation buffer consist of axially oriented collagen fibrils in a nonoriented matrix (Fig. la). These fibers had a mean diameter of 134 pm (Fig. Ib).

, matrix

10 -

1

. . . . . . .

64 73 81 89 98 106 l t 4 123 131 140 148 156 165 173 181 190

Initial Fiber Diameter

(b)

Figure 1. (a) Diagram illustrating substructure of extruded collagen fiber prior to etching showing axially oriented collagen fibrils within a nonoriented matrix. (b) Graph showing distribution of diameters of collagen fibers. Mean fiber diameter was observed to be 134 pm with a standard deviation of ? 24 pm. Arrows are drawn showing 2 one standard deviation (small vertical line) from the mean (large vertical line).

228 WONG ET AL

o etching

Partially etched

Etched A n n n n

Over etched

Figure 2. Diagramatic representation of the etching process. Initially, collagen fibrils are observed to settle to the bottom of a flattened fiber on glass coverslips because of gravity. When the fiber is partially etched, fibrils become more visible at the surface of the fiber. As the etching process continues, more collagen fibrils are eaten away by collagenase until only one layer of fibrils remain on the coverslip. Neurons are plated onto the coverslips at this step because the monolayer of fibrils optimizes our visualization of neurites along the fibrils. When overetched, the coverslip has few or no visible fibrils.

The activity of collagenase is controlled by calcium ions. When calcium ions are absent, the rate of etching of collagen fibers at all collagenase concentrations studied was slow (Figs. 4-6), with a maximum of 40 fibrils/fiber after 10 h of etching and 100 fibrils/fiber after 20 h of etching with 900 unit/mL of collagenase. The extent of etching appeared optimum at a calcium chloride concentration of 5 mM (Fig. 5). Calcium concentrations below 5 mM resulted in slow etching rates while above 5 mM rapid digestion of fibrils was observed.

At low concentrations of calcium ions (5 mM), the number of collagen fibrils were examined at 1 h intervals by phase contrast microscopy during the etching process (Fig. 6). At a collagenase concentration of 300 units/mL, fibril bundles began to appear 3 h after the start of etching. The number of fibrils reached a peak between the 9th and 11th hours, when the upper portion of the fiber was removed by collagenase. After the 12th hour, the number of fibril bundles gradu- ally decreased because of excessive etching.

The optimum etching conditions based on Figures 5 and 6 were observed to be at a calcium concentration of 5 mM, collagenase concentration of 300 units/mL and an etching time of 10 h at 22°C. The conditions were used in the cell culture experiments described below. After etching, individual collagen fibrils had diameters of about 1 pm.

When cortical neurons were added to the fibrils in culture, neurite outgrowth from these neurons was observed as early as 12 h after plating. These neurites elongated along the axes of the fibrils. Observations using Nomarski differential interference contrast microscopy (Fig. 7a) showed that the cell body of a neuron is sometimes situated between two parallel fibrils. Two neurites were seen extending from

Figure 3. Polarized light micrograph showing morphology of etched fiber. Fiber etched with collagenase (300 units/mL) for 10 h at room temperature is highly birefringent after staining with Picrosirius red. Oriented fibrils with diameters of about 1 pm are observed. Bar equals 10 pm.

PREPARATION OF ETCHED COLLAGEN FIBERS

3oounit/d cOll.genase T at 10 houn

229

400 -

300 -

200 -

100 -

0-

120 7 I 1001 80

0 10 20 30 Time (Hours)

Figure 4. Effect of collagenase activity on the extent and rate of etching. A plot of number of fibrils per fiber as a function of time in the presence of increasing amounts of collagenase. Below a collagenase concentration of 300 units/mL the extent of fibril etching is incomplete. Above a concentration of 600 unit/mL collagenase digestion results in total digestion of many fibrils.

the cell body, and running along the nearest fibril bundles. When the same neuron was examined for neurofilament immunoreactivity, only the cell body and the neurites were fluorescent (Fig. 7b). We observed that neurites and fibrils were hardly distinguishable under Normarski optics because they have similar dimensions. However, neurofilament immunoreactivity was detected only in neurites, which were seen running along the longitudinal axes of the fibrils. In some cases a group of neurites originating from a cluster of neurons was seen under Normarski optics, but only neurites and neurons were fluorescent for neurofilament.

1 2 cl LL . Y 0

T -1 OmM 5 mM 10 mi% 50 mM

Calcium Concentration

Figure 5. Effect of CaCI, concentration on the extent of etching. The number of fibrils/fiber after 10 h of etching at 300 units/mL of collagenase activity initially increases between 0 and 5 mM CaCI, and then decreases at levels of CaCI, above 5 mM.

DISCUSSION

Collagen is the primary structural protein found in mam- mals. Unlike other structural proteins, collagen has desir- able mechanical properties that enable it to act as a tissue scaffold in both soft and hard tissues.26 In soft tissues such as skin, collagen is elastic and it can absorb energy. In other tissues such as tendon, collagen is stiff and it can transmit forces. Depending on the fiber diameter, orientation, and degree of crosslinking, networks of collagen fibers can prevent mechanical failure of tissues, and act as a cell support and partition between chemical and functional units.”

Use of collagen as a support in cell culture is well docu- mented. It has become a widely used substrate for attachment- dependent and attachment-independent cells. Recently, collagen-directed migration of fibroblasts was observed in our laboratory. In cell culture experiments using a substrate consisting of collagen fibers, lamellopodia from fibroblasts were found oriented to the longitudinal axes of collagen fibers.*’ These fibroblasts with directionally oriented lamellopodia were seen mainly at the leading edge of fibroblast proliferation. Similarly, collagen-directed neurite extension was observed in our experiments presented in this article. Our findings suggest that very small diameter collagen fibrils can be produced from collagen fibers by etching using collagenase solutions. These fibrils appear to directionally orient the extension of neurites in cell culture. As a result, growth of neurites occurs along the fibril axes.

Successful clinical application of collagen has been docu- mented. Human skin epidermis has been grown in culture on collagen films and gels. These artificial skin grafts were later applied to bum victims. The grafts were found to protect bum patients from infection and to provide a physiologic barrier to prevent heat and moisture loss. 29-34 Analogously,

230 WONG ET AL.

T

0 10 20 30

Time (Hours) Figure 6. Effect of collagenase activity on the extent and rate of etching in the presence of CaCI,. At a calcium concentration of 5 mM, the number of fibrils exposed per collagen fiber is highest after 10 h of etching and in the presence of 300 units/mL of collagenase.

neurons may be grown on a substrate consisting of collagen fibrils, allowing the extension of neurites along the longitudinal axes of the fibrils. This artificial “neural bridge” may be used clinically in patients with central and peripheral nerve damage.

Recently, we have reported the use of small diameter type I collagen fibers in regeneration of rat sciatic nerve. A four-fold increase in the number of regenerating axons was

observed.35 Similar facilitated axonal regeneration in rat sciatic nerve was observed by other investigator^.^^ Our observations presented in this article may help us to explain the role of collagen fibers in the regeneration of rat sciatic nerve. Growth of neurites on collagen fibrils may also be used to evaluate neurite extension in the presence of neurotrophic factors such as nerve growth factor and neural-cell adhesion molecules.

Figure 7. Morphology of neurites grown in culture on etched collagen fibers. (a) Rat fetal cortical cells were isolated and grown in tissue culture for 12 h. At that time neurons with neurites extending from the cell body were visualized under Normarski phase contrast optics. (b) After addition of neurofilament specific monoclonal antibody RT97 and fluorescein-conjugated goat antimouse IgG, the same neuron and neurites are fluorescent.

PREPARATION OF ETCHED COLLAGEN FIBERS 231

Figure 7.

The authors are grateful to Mr. David Dickerman and Ms. Rita Hahn for their technical assistance. Support was provided by New Jersey Center for Advanced Biotechnology and Medicine and NIH Grant NS 25168 to H. G.

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Received October 19, 1989 Accepted May 1 , 1990

a method for preparation of etched collagen fibers that support neurite outgrowth

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