Table Of ContentPreparation of Collager&lycosaminoglycan
Copolymers for Tissue Regeneration
Lila J. Chamberlain and loannis V.Yannas
1. Introduction
Certain analogs of the extracellular matrix (ECM) have been shown to pos-
sess surprising morphogenetlc activity during healing of lessons m various ana-
tomlcal sites This chapter describes methods for synthesis of the two ECM
analogs that have been studied most extensively. The reader 1s referred to
descriptions of these methods m the original literature (Z-3) The blologlcal
actlvlty of ECM analogs has been revlewed elsewhere (4).
One of these analogs, referred to as the skm regeneration template (SRT),
has Induced regeneration of dermis in full-thickness skin wounds m the guinea
pig model (2,.5-7), the porcme model (8), and m humans (S,!Wl). Smce it IS
well known that the dermis of the adult mammal does not regenerate spontane-
ously (12,13), the SRT 1s required for dermal regeneration in all commonly
encountered skm wounds that are sufficiently deep to have compromised the
dermis. The SRT 1s currently used as a dermal regeneration treatment for
patients who have sustained deep burns or deep mechanical trauma, mcludmg
trauma from elective surgery, and who would otherwise have been treated with
autografts (10) In the clinical settmg or m animal models, the SRT 1s apphed
on wounds as a bllayer graft; the proximal layer 1s the highly porous ECM
analog and the distal layer 1s a slhcone film (Fig. 1). The latter has no blologl-
cal actlvlty, but serves as a temporary dressing that protects the proximal layer
from dehydration and bacterial invasion, and also converts the bdayer mto a
mechamcally competent sheet, capable of being handled conveniently and
sutured on the patlent’s tissues
Another ECM analog, referred to as the nerve regeneration template (NRT),
has induced regeneration of a functional peripheral nerve across a 15-mm gap
From Methods m Molecular Me&me, Vol 18 Tmsue Engmeenng Methods and Protocols
E&ted by J R Morgan and M L Yarmush 0 Humana Press Inc , Totowa, NJ
3
4 Chamberlain and Yannas
Silicf
18 Tissue
Matrix
Fig. 1. SRT as it would be implanted in the skin wound model
Nerve Stump Collagen-GAG Matrix
Fig. 2. NRT, ensheathed by an implantation tube, as it would be implanted in the
peripheral nerve wound model.
in the rat sciatic nerve model (14). In this model, the highly porous ECM ana-
log is used to till the lumen of a tube, made either of silicone (nondegradable)
or collagen (biodegradable); the nerve stumps are inserted into the tube and are
prevented from being displaced further by two sutures at each stump (Fig. 2).
If the silicone tube is used without an ECM analog, the maximum gap distance
that can be bridged by a functional peripheral nerve in this animal model is
only 10 mm (15). The structure of the NRT has been determined by selection
of the network structure that resulted in maximum regenerative activity in the
sciatic nerve model, using a gap of 10 mm (3,16). The selection of the collagen
tube instead of the silicone tube, based on superior regenerative activity, has
also been made in the same animal model (17).
Another ECM analog, similar in structure to the SRT and the NRT, has been
shown capable of regenerating the canine knee meniscus (18).
The structure of these biologically active ECM analogs has been character-
ized on the scale of the nanometer, as well as on the scale of the micrometer. In
the former scale, both ECM analogs referred to above are graft copolymers of
type I collagen and chondroitin 6-sulfate, which are crosslinked covalently,
and can therefore be described as insoluble macromolecular networks. On the
larger scale, the analogs are highly porous matrices that are characterized in
terms of the pore volume fraction, the average pore diameter and the average
orientation of pore channel axes. Being insoluble, the ECM analogs cannot be
isolated and characterized structurally using common biochemical techniques
CoNagen-Glycosaminoglycan Copolymers 5
Table I
Structural Properties of Two Regeneration Templates
Design parameter of Skm regeneration Nerve regeneration
ECM analog template (SRT) template (NRT)
Type I collagen/chondromn
6-sulfate, (w/w) 9812 98/2
Degradation half-life, wk 1.5 6-8
Average pore diameter, pm 2(1-l 25 5-l 0
Pore channel orientation Random Axral
for the structural analysts of protems. However, structural methodology that
has been used to characterize synthetic polymeric networks, including infrared
and Raman spectroscopy, rubber elasticity analysis of network structure and
various forms of microscopy, have been employed m the charactertzatton of
the ECM analogs . The structures of the SRT (2) and the NRT (3,16) (Table 1)
have been identified by selecting the analogs of maximum activity from a large
number of ECM analogs with related structure Inspection of Table 1 shows
that the NRT is significantly different from the SRT regarding network cross
lmk density, average pore diameter, and average ortentation of pore channel
axes (Fig. 3).
2. Materials
2.1. Preparation of Collagen-GAG Suspension
Type I collagen, from bovine tendon, (Integra LifeSciences, Plamsboro, NJ), m
the form of hydrated fibrillar granules, is divided mto 14-g aliquots and stored at
0°C Freeze-thaw cycles during storage should be avoided. If dry collagen IS
used, it should be kept refrigerated at 4°C
Cooled overhead blender (Granco overhead blender, Granco, Kansas City, MO),
mcludmg a coolmg system (Brmkman cooler model RC-2T, Brmkman, West-
bury, NY) The blender is used to mix the collagen-glycosammoglycan (GAG)
suspension, which must be kept at 4°C during the entire preparation.
0 05 Macetic acid solution Add 8 7 mL glacial acetic acid (Mallmckrodt Chemt-
cal, Paris, KY) to 3 L dH,O This solution has a shelf life of approx 1 wk
Peristaltic pump (Manostat Cassette Pump, cat no 75-500-0.00, Manostat,
New York)
0 11% w/v chondroitm 6-sulfate solution Dissolve 275 mg chondromn 6-s&
fate (from shark cartilage, cat no C-4384, Sigma, St Louis, MO) m 250 mL
0.05 M acetic acid solution. The chondroitm 6-sulfate solution is stored at 4°C
and has a shelf life of 1 d The chondroitm 6-sulfate powder is stored m a desic-
cator at 4°C
Chamberlain and Yannas
Fig. 3. Environmental scanning electron micrographs of the (A) skin regeneration
template (scale bar = 100 pm) and the (B) nerve regeneration template (scale bar = 20 pm).
2.2. Formation of Matrix Pore Structure
2.2.1. Skin Regeneration Template
1. Freeze dryer (VirTis Genesis, VirTis, Gardiner, NY). Required to freeze the sus-
pension and to sublimate the ice crystals, leaving behind a highly porous matrix
structure. The freeze dryer is equipped with trays that are pressed against the
chamber shelves when placed in the freeze dryer. These trays ensure good con-
tact between the cooled shelf and the product, and are important for proper pore
formation in the skin regeneration template.
2.2.2. Nerve Regeneration Template
1. Polyvinylchloride (PVC) tubing (0.125 in. id, 0.25 in. od), cut into 12-cm lengths.
2. Silicone processing tubes (model 602-235 medical grade Silastic, 0.058 in. id,
0.077 in. od, Dow-Corning, Midland, MI) cut into 15-cm lengths.
3. Silicone adhesive (Medical GradeSilastic, Dow-Corning, MI).
4. Liquid nitrogen: 160-L canister.
5. Axial freezing bath: This custom-made device (Fig. 4) is required to freeze the
suspension for a nerve regeneration template (19). To achieve the appropriate
Collagen- Glycosaminoglycan Copolymers
Fig. 4. Schematic of the custom-made axial freezing bath (19). Not to scale. (A)
Electric timing motor. (B) Gear drive chain. (C) Drive gear. (D) Idler pulley. (E)
String. (F) Guide rod. (G) PNS Graft tube carrier. (H) PNS Graft tubes. (I) Counter-
weight. (J) Thermometer. (K) Thermocouple. (L) Temperature controller. (M) Insu-
late glass vessel. (N) Silicone heat transfer fluid (0) LN2 Solenoid valve. (P) N2
Throttle valve. (Q) One-quarter inch copper cooling (R) Circulator.
pore structure, the suspension is injected into tubes and lowered into a freezing
bath. The freezing apparatus consists of a liquid nitrogen-controlled cooling sys-
tem and a gear train arrangement, which allows for variable lowering velocities.
The cooling system uses liquid nitrogen, traveling through coiled copper tubing,
to cool the heat transfer fluid inside the bath (Silicone Oil, Syltherm XLT Heat
Transfer Liquid, Dow Corning, MI). A simple temperature controller is used to
regulate the flow of liquid nitrogen. The freezing bath is insulated with hard
Styrofoam and capped with an acrylic disk.
6. Freeze dryer (VirTis Genesis). Required to sublimate the ice crystals, leaving
behind a highly porous matrix structure.
2.3. Cross/inking, Sterilization, and Hydration
2.3.1. Skin Regeneration Template
1. Vacuum oven (Fisher Isotemp Vacuum Oven, Fisher Scientific, Boston, MA;
VacTorr 150 Vacuum Pump, GCAlPrecision Scientific, Chicago, IL).
8 Chamberlain and Yannas
2 Slhcone adhesive (SIlastic, Dow-Cornmg, MI), sterilize by autoclavmg
3 Stenle implements 5-L plastic tub (approx W 11 x L14 x D4 m ) with Teflon cover
(does not need to seal, only cover the tub), gauze, Teflon working surface, forceps,
metal spatulas, rulers, scalpel blade holder, and scalpel blades Sterilize by autoclaving
4 Lammar flow bench (Rehalab, Tenney Engineering, Umon, NJ) All sterile pro-
cedures are performed m the lammar flow bench
5 0 05 M acetlc acid solution Add 2 9 mL glacial acetic acid (Mallmckrodt) to
1000 mL dH,O Sterilize by filtration using a 0 2-p filter (cat. no 83 10, Costar
Scientific, Cambridge, MA) This solution has a shelf life of approx 1 wk
6 0 25% glutaraldehyde m 0 05 M acetic acid* Combme 10 mL of 25% glutaralde-
hyde and 3 mL glacial acetic acid Add distilled water to 100 mL Add an addl-
tlonal900 mL of dH,O This solution has a shelf hfe of about 1 wk, and 1s stored
m a dark contamer at room temperature Sterilize by filtration using a 0 2-pm
filter
7 4000 mL dH,O: Sterlhze by filtration usmg a 0 2-pm filter
8 Teflon cutting template. Make a matrix-cutting template by cuttmg a piece of
Teflon the size and shape of the desired matrix sheet. Using thrs type of template
the matrix 1s cut without tearing, and is ensured the proper size matrix sheet
Sterilize template by autoclavmg
9 Phosphate buffered salme (PBS) (cat no. P-3813, Sigma), 1000 mL Sterlhze by
filtration using a 0.2~pm filter
10 70% lsopropanol m dH,O, 1000 mL Sterdize by filtration using a 0 2-w filter
2.3.2. Nerve Regeneration Template
1 Implantation tubes (see Note 1). For implantation, the nerve regeneration tem-
plate 1s ensheathed by an lmplantatlon tube (Fig. 2) Tubes that can be used
include porous collagen tubes (1.5 mm Id, 3 0 mm od, Integra), nonporous col-
lagen tubes (1.5 mm id, 1 8 mm od, Integra), and silicone tubes (model 602-235
medical grade SIlastic, 0 058 m id, 0 077 m od, Dow-Corning)
2 Vacuum oven (Fisher Isotemp Vacuum Oven, Fisher Sclentlfic, VacTorr 150
Vacuum Pump, GCA/Preclslon Scientific)
3 Sterile implements several pair of forceps, scalpel blade holder, scalpel blades,
ruler, specimen Jars, and a Teflon working surface Sterilize by autoclavmg
4 PBS (cat no P-38 13, Sigma), 1000 mL Sterilize by filtration using a 0 2-pm filter
5 70% lsopropanol m dHzO, 1000 mL Sterdize by filtration using a 0 2-p filter
3. Methods
3.1. Preparation of Collagen4A G Suspension
The techmque for preparing the collagen-GAG suspension 1si dentical for the
SRT and NRT It 1s important that the collagen and GAG components remam
refrigerated; therefore, the entire suspension preparation must take place at 4OC
1 Defrost a 14-g ahquot of frozen hydrated tendon collagen for 3&60 mm at room
temperature
Collagen-Glycosam~noglycan Copolymers 9
2 Turn on coolmg system for blender and cool to 4°C (takes about 30 mm)
3 Add 13 69 g of defrosted hydrated tendon collagen (or 3 6 g of dry collagen), all
at once, to 600 mL of 0.05 Macettc acid in one blender, and blend at high speed
setting (approx 20,000 RPM) for 90 mm (see Note 2)
4 Calrbrate the peristaltic pump to 40 mL/5 mm
5 Add 120 mL of 0 11% w/v chondrottin 6-sulfate solution dropwtse to the blend-
mg collagen dtsperston over 15 mm, using the pertstaltrc pump (mamtam blender
at 4°C and htgh-speed setting)
6 Blend the mixture for an addmonal90 mm on high-speed settmg (approx 20,000 RPM)
7 Pour out the collagenGAG suspenston and store m a capped bottle at 4°C The suspen-
sion has a shelf hfe of about 4 mo (see Note 3) If stored more than 4 wk, reblend for
15 mm at low speed (approx 10,000 RPM), in cooled blender (4”C), before usmg
3.2. Formation of the Matrix Pore Structure
3 2 1 Skrn Regeneration Template
1 Remove the air from the collagen-GAG suspenston by placmg it mto a 1500-mL
Erlenmeyer flask under vacuum for 10 mm wtth agttatton, or unttl bubbles are no
longer vistble
2 Set the shelf temperature of the freeze-dryer to -45°C
3 Turn on the condenser of the freeze-dryer.
4 Allow at least 1 h for the shelf temperature to reach -45°C
5. Pour the collagen-GAG suspenston mto an alummum VirTts freeze-dryer tray
The depth of the suspension can be varied to change the thickness of the resultmg
dry matrix
6 Place the suspenston-filled tray on the freeze-dryer shelf, and close the chamber
door Be sure that the tray and the shelf are m good contact
7 Wait for approx 1 h (or longer as necessary) until the collagenGAG suspenston
1s frozen (see Note 4)
8 Check the condenser temperature It must be at -50°C or below before proceed-
mg to the next step
9 After the suspension 1s frozen, turn on the freeze-dryer vacuum pump Make sure
the chamber door makes a good seal
10 Once the vacuum IS below 200 mtorr, increase the shelf temperature to 0°C
11 Leave overmght (at least 15 h).
12 Increase the shelf temperature to 20°C
13. When the chamber reaches 20°C turn off the vacuum pump and condenser
Release the vacuum m the chamber and remove the dry collagen-GAG mati~x in
the form of a white, highly porous sheet
3.2 2. Nerve Regenerabon Template
1 Prepare vented PVC Jackets by heating 12-cm sections of flexible PVC tubing at
105°C for 2 h, to straighten Puncture each tube with a 25 gage needle at 90-degree
Intervals around the tube, spaced 1 cm apart for the length of the tube
10 Chamberlain and Yannas
................... . ...
.........
Collagen-GAG
Suspens10n \
9
Conical Plug Conical Plug
Pressurized, Silicone /
Processing Tube
Fig. 5. Preparation of the PVC jacket assemblies for freezing the NRT. (A) Dia-
gram of PVC assembly during injection of the collagenGAG suspension. (B) Final
PVC jacket assembly, ready for freezing. Not to scale.
2. Flush silicone processing tubes (15 cm in length) with dHzO, and let dry.
3. Seal one end of each silicone processing tube with silicone adhesive. Inject a
cylindrical plug of adhesive, approx 5 mm in length, into the end of silicone tube
and allow the excess to stay on the outside of the tube. The excess is important
for adhesion and can be cut off later. Let cure for 24 h at room temperature to a
tack-free, elastomeric state.
4. Prepare for use a 160-L liquid nitrogen tank for the bath cooling system.
5. Remove the air from the collagenGAG suspension by placing into a 1500-mL
Erlenmeyer flask under vacuum for 10 min with agitation, or until bubbles are no
longer visible.
6. Turn on the cooling system of the axial freezing bath and set the bath temperature
to -80°C (see Note 5). It will take approx 45 min of liquid nitrogen cooling for
the bath to reach this temperature.
7. Insert each plugged silicone processing tube into a prepared PVC jacket.
8. Draw collagen-GAG suspension into a IO-cc syringe (Becton Dickinson model
5604, Becton Dickinson, Rutherford, NJ) and expel all the air bubbles. Attach a
25-gage needle (Becton Dickinson model 25G518, Becton Dickinson) to the
syringe and insert the needle carefully into the plugged end of the silicone tube.
The needle should be inserted far enough so that a needle length of about 3-5 mm
extends beyond the Silastic plug into the tube (Fig. 5A).
9. Inject collagen-GAG suspension until the tube is full and no air remains in the
tube. Pinch the free end of the silicone processing tube against the wall of the
PVC jacket using a conical, plastic plug (the end of a pipet tip works well). Insert
the plug far enough so that the silicone processing tube is sealed, and no suspen-
Collagen-Glycosaminoglycan Copolymers 11
Conical Plug
$
?
PVC Jacket
%
i
Liquid Collagen-
i- GAG Suspension
ilicone Processing Tube
iilicone
‘IProceswe
Freezing
Front
Frozen Collage;v \
GAG Suspension Tray-Tube
Interface
Frozen Collagen-
GAG Suspension
Freezing Bath
Heat Transfer Fhud
Fig. 6. Manufacturing assembly diagram for the peripheral nerve regeneration tem-
plate. (A) Freezing orientation. (B) Freeze-drying (sublimation) orientation. Not to scale.
sion can leak out. Insert another conical, plastic plug into the needle end of the
tube. The plug at the needle end should not block the flow of the suspension into
the silicone tube via the needle (Fig. 5A).
10. Inject additional suspension until the silicone processing tube becomes pressur-
ized and expands to fill the entire PVC jacket (Fig. 5B). The silicone tube will
inflate because of pressure from the injection of additional suspension. The end
of the needle should be inside the PVC jacket to help prevent pressure build up at
the needle tip. When the silicone tube has completely filled the PVC jacket, care-
fully remove the needle; simultaneously, press the conical plug into the end of
the tube until the silicone processing tube is pinched against the PVC jacket and
sealed. Pressure should be kept on the syringe plunger until the needle is com-
pletely out of the tube. Check to make sure the silicone processing tube is still
filling the entire PVC jacket (Fig. 5B).
11. Attach the drive gear to the electric timing motor on the axial freezing apparatus
(Fig. 4). Place prepared PVC jackets, up to four at a time, on the tube carrier (Fig. 4).
Place the tube carrier on the gear train and manually lower until the bottom of the
PVC jacket assembly is just touching the freezing bath. Start the motor and let
the tubes lower into the bath at a velocity of lo4 m/s (see Note 6; Fig. 6A).
12 Chamber/a/n and Yannas
Monitor the process of lowermg to ensure that the tubes do not stick to the copper
tubmg m the freezing bath
12 Turn on the freeze dryer and set the shelf temperature to -20°C
13 Turn on the condenser of the freeze dryer.
14 When the PVCJackets are fully immersed m the freezmg bath, turn off the ttmmg
motor and remove the tubes from the bath. Qmckly separate the tubes and remove
the comcal plugs Cut off the plugged end of the srhcone tube and cut each PVC
Jacket assembly approxtmately m half with a sharp razor blade Thts process
provides more exposed surface for subhmatton of the me crystals Lay the PVC
Jacket assemblies on a freeze dryer tray and place the tray m the -20°C freeze
dryer (Fig. 6B) This step must be done as quickly as possible (wtthm a mmute)
to ensure that the tubes stay completely frozen
15 Seal the chambers on the freeze dryer and close the vacuum outlet tube Check to
be sure the condenser temperature IS below -45°C (if not, watt for the condenser
temperature to reach 45°C before proceeding to the next step)
16 Turn on the vacuum pump and wart for the vacuum to reach 200 mtorr Make
sure that the chamber door IS sealed
17 Once the vacuum reaches 200 mtorr, increase the shelf temperature to 0°C Leave the
PVC Jacket assemblies m the freeze dryer for 17 h at this temperature and pressure
18 Increase the temperature to 25°C then turn off the vacuum pump and the con-
denser Release the vacuum and remove the PVC Jacket assembhes, whtch con-
tam the dry, whrte, hrghly porous matrix msrde the &cone processmg tubes
3.3. Cross/inking, Sterilization, and Hydration
3 3 1 Sk/n Regeneration Template
1 After removmg the dry collagenGAG matrix from the freeze-dryer, inspect the
matrix for any uregularmes, using a scalpel blade, remove any regtons that appear
to be dtstmctly different m appearance from that expected of a very htghly porous
solid of uniform thtckness Usually these regions ~111 be located near the pan
edges Take note of the dtfference between the pan side (the side that was m
contact with the horrzontal pan surface) and the an stde (the stde that was m
contact wtth the environment) of the dry matrix The pan stde has a much
smoother surface Future steps ~111 requrre dtstmguishmg between the pan and
an stdes of the dry matrtx.
2 Make an alummum foil pouch large enough to tit the sheet of dry matrix Take a
large piece of foil and fold it m half The folded edge 1s now the bottom of the
pouch. Take the left edges and fold, at least twice, to form a sealed side. Repeat
on the right side of the pouch Insert the dry matrix mto the pouch (one sheet of
matrix per pouch) and leave the top open
3. Place the matrix-filled pouch (top open) m the vacuum oven for dehydrothermal
(DHT) treatment (see Note 7). The condtttons of treatment m the vacuum oven
are’ 30 mtorr, lOS’C, 24 h
4 After 24 h, remove the pouch and tmmedtately seal the top by folding the top
edges of the for1 pouch at least twice If the matrtx IS not bemg prepared for
