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Appendix

Preparation of Collagen-Glycosaminoglycan Copolymers for Tissue Regeneration Lila J. Chamberlain and Ioannis V. Yannas

Adapted from Chamberlain, L. J. and I. V. Yannas. 1998. Preparation of collagen-glycosaminoglycan copolymers for tissue regeneration. Methods of Molecular Medicine, edited by Morgan, J. R. and M. L. Yarmush. Tolowa, NJ: Humana Press.

Introduction

Certain analogs of the extracellular matrix (ECM) have been shown to possess regenerative activity during healing of lesions in various anatomical sites. This chapter describes methods for synthesis of the two ECM analogs that have been studied most extensively. The reader is referred to descriptions of these methods in the original literature (Yannas et al. 1980, 1989; Chang et al. 1990). The biologi-cal activity of ECM analogs has been reviewed elsewhere (Yannas 1995). Updated methodology for collagen scaffolds has been published (Soller et al. 2012).

One of these analogs, referred to as the dermis regeneration template (DRT; re-ferred to below also as SRT), has induced regeneration of dermis in full-thickness skin wounds in the guinea pig model (Yannas et al. 1981, 1982, 1989; Murphy et al. 1990), the porcine model (Orgill et al. 1996), and in humans (Yannas et al. 1981; Burke et al. 1981; Heimbach et al. 1988; Stem et al. 1990). Since it is well known that the dermis of the adult mammal does not regenerate spontaneously (Billingham and Medawar 1951, 1955), the DRT is required for dermal regeneration in all com-monly encountered skin wounds that are sufficiently deep to have compromised the dermis. The DRT is currently used as a dermal regeneration treatment for pa-tients who have sustained deep burns or deep mechanical trauma, including trauma from elective surgery, and who would otherwise have been treated with autografts (Heimbach et al. 1988). In the clinical setting or in animal models, the SRT is ap-plied on wounds as a bilayer graft; the proximal layer is the highly porous ECM analog and the distal layer is a silicone film. The latter has no biological activity, but serves as a temporary dressing that protects the proximal layer from dehydration and bacterial invasion, and also converts the bilayer into a mechanically competent sheet, capable of being handled conveniently and sutured on the patient’s tissues.

270 Appendix

Materials

Preparation of Collagen-GAG Suspension

1. Type I collagen, from bovine tendon, (Integra LifeSciences, Plainsboro, NJ), in the form of hydrated fibrillar granules, is divided into 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.

2. Cooled overhead blender (Granco overhead blender, Granco, Kansas City, MO), including a cooling system (Brinkman cooler model RC-2T, Brinkman, West-bury, NY). The blender is used to mix the collagen-glycosaminoglycan (GAG) suspension, which must be kept at 4 °C during the entire preparation.

3. 0.05 M acetic acid solution: Add 8.7 mL glacial acetic acid (Mallinckrodt Chemi-cal, Paris, KY) to 3 L dH2O. This solution has a shelf life of approx. 1 week.

4. Peristaltic pump (Manostat Cassette Pump, cat. No. 75-500-0.00, Manostat, New York).

5. 0.11 % w/v chondroitin 6-sulfate solution: Dissolve 275 mg chondroitin 6-sulfate (from shark cartilage, cat. No. C-4384, Sigma, St. Louis, MO) in 250 mL 0.05 M acetic acid solution. The chondroitin 6-sulfate is stored at 4 °C and has a shelf life of 1 day. The chondroitin 6-sulfate powder is stored in a desiccator at 4 °C.

Formation of Matrix Pore Structure

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.

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 Grade Silastic, Dow Corning, MI)4. Liquid nitrogen: 160-L canister

271Materials

5. Axial freezing bath: This custom-made device is required to freeze the suspen-sion for a nerve regeneration template (Loree 1988). To achieve the appropriate 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 tub-ing to cool the transfer fluid inside the bath (Silicone Oil, Slytherm 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 (CirTi Genesis). Required to sublimate the ice crystals, leaving behind a highly porous matrix structure.

Crosslinking, Sterilization, and Hydration


1. Vacuum oven (Fisher Isotemp Vacuum Oven, Fisher Scientific, Boston, MA; VacTorr 150 Vacuum Pump, GCA/Precision Scientific, Chicago, IL).

2. Silicone adhesive (Silastic, Dow-Corning, MI); sterilize by autoclaving. 3. Sterile implements: 5-L plastic tub (approx. W11 × L14 × D4 in.) with Teflon

cover (does not need to seal, only cover the tub), gauze, Teflon working sur-face, forceps, metal spatulas, rulers, scalpel blade holder, and scalpel blades. Sterilize by autoclaving.

4. Laminar flow bench (Relialab, Tenney Engineering, Union, NJ). All sterile pro-cedures are performed in the laminar flow bench.

5. 0.05 M acetic acid solution: Add 2.9 mL glacial acetic acid (Mallinckrodt) to 1000 mL dH20. Sterilize by filtration using a 0.2-µm filter (cat. No. 8310, Costar Scientific, Cambridge, MA). This solution has a shelf life of approx. 1 week.

6. 0.25 % glutaraldehyde in 0.05 M acetic acid: Combine 10 mL of 25 % glutar-aldehyde and 3 mL of 3 mL glacial acetic acid. Add distilled water to 100 mL. Add an additional 900 mL of dH20. This solution has a shelf life of about 1 week, and is stored in a dark container at room temperature. Sterilize by filtra-tion using a 0.2-µm filter.

7. 4000 mL dH20: Sterilize by filtration using a 0.2-µm filter. 8. Teflon cutting template: Make a matrix-cutting template by cutting a piece of

Teflon the size and shape of the desired matrix sheet. Using this type of tem-plate the matrix is cut without tearing, and is ensured the proper size matrix sheet. Sterilize template by autoclaving.

9. Phosphate buffered saline (PBS) (cat. No. P-3813, Sigma), 1000 mL: Sterilize by filtration using a 0.2-µm filter.

10. 70 % isopropanol in dH20, 1000 mL: Sterilize by filtration using a 0.2-µm filter

272 Appendix


1. Implantation tubes1: For implantation, the nerve regeneration template is ensheathed by an implantation tube. Tubes that can be used include porous colla-gen tubes (1.5 mm id, 3.0 mm od, Integra), nonporous collagen tubes (1.5 mm id, 1.8 mm od, Integra), and silicone tubes (model 602-235 medical grade Silastic, 0.058 in. id 0.077 in. od, Dow-Corning).

2. Vacuum oven (Fisher Isotemp Vacuum Oven, Fisher Scientific; VacTorr 150 Vacuum Pump, GCA/Precision Scientific).

3. Sterile implements: several pair of forceps, scalpel blade holder, scalpel blades, ruler, specimen jars, and a Teflon working surface. Sterilize by autoclaving.

4. PBS (cat. No. P-3813, Sigma) 1000 mL: Sterilize by filtration using a 0.2-µm filter.

5. 70 % isopropanol in dH20, 1000 mL: Sterilize by filtration using a 0.2-µm filter

Methods

Preparation of Collagen-GAG Suspension

The technique for preparing the collagen-GAG suspension is identical for the SRT and NRT (after 2000 NRT has been prepared without GAG and otherwise had simi-lar structure to SRT). It is important that the collagen and GAG components remain refrigerated; therefore, the entire suspension preparation must take place at 4 °C.

1. Defrost a 14-g aliquot of frozen hydrated tendon collagen for 30–60 min at room temperature.

2. Turn on cooling system for blender and cool to 4 °C (takes about 30 min).3. Add 13.69 g of defrosted hydrated tendon collagen (or 3.6 of dry collagen), all

at once, to 600 mL of 0.05 M acetic acid in one blender, and blend at high speed setting (approx. 20,000 RPM) for 90 min.2

4. Calibrate the peristaltic pump to 40 mL/5 min.5. Add 120 mL of 0.11 % w/v chondroitin 6-sulfate solution dropwise to the blend-

ing collagen dispersion over 15 min, using the peristaltic pump (maintain blender at 4 °C and high-speed setting).

1 Using a degradable, collagen implantation tube yields a superior regenerated nerve than when using a nondegradable, silicone tube for regeneration across a 10-mm gap in the rat sciatic nerve. This finding is based on histological data at 30 week (Chamberlain 1996).2 The blending time is critical because this is the step that induces swelling of the collagen fibrils and conversion of about 90 % of banded collagen fibers to unbanded structures (Forbes 1980). The blending procedure does not, however, remove the triple helical structure of collagen (Yannas et al. 1980). Banded collagen induces platelet aggregation; by removing the banding, the collagen fibrils do not aggregate platelets (Sylvester et al. 1989). Blending for shorter times, or at slower speeds, does not eliminate the banding to the desired extent.

273Methods

6. Blend the mixture for an additional 90 min on high-speed setting (approx. 20,000 RPM).

7. Pour out the collagen-GAG suspension and store in a capped bottle at 4 °C. The suspension has a shelf life of about 4 month.3 If stored more than 4 week, reblend for 15 min at low speed (approx. 10,000 RPM), in cooled blender (4 °C), before using.

Formation of the Matrix Pore Structure


1. Remove the air from the collagen-GAG suspension by placing it into a 1500 mL Erlenmeyer flask under vacuum for 10 min with agitation, or until bubbles are no longer visible.

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 suspension into an aluminum VirTis freeze-dryer tray.

The depth of the suspension can be varied to change the thickness of the result-ing dry matrix.

6. Place the suspension-filled tray on the freeze-dryer shelf, and close the cham-ber door. Be sure that the tray and the shelf are in good contact.

7. Wait for approx. 1 h (or longer if necessary) until the collagen-GAG suspension is frozen.4

8. Check the condenser temperature. It must be at − 50 °C or below before pro-ceeding to the next step.

9. After the suspension is 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 overnight (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 in the chamber and remove the dry collagen-GAG matrix in the form of a white, highly porous sheet.

3 Collagen-GAG suspension that is stored for over 4 month may be contaminated with fungus. It is recommended to prepare the suspension shortly before matrices are to be manufactured to avoid possible contamination.4 Freezing the collagen-GAG suspension on a cool, flat surface creates randomly oriented pore channels with approximately circular cross sections. The average pore diameter can be manipulat-ed by varying the shelf temperature. For optimum dermal regeneration the pores must be between 20 and 125 µm (Yannas et al. 1989). Freezing at − 45 °C typically results in pores with an average channel diameter of 70 ± 30 µm (Fig. 3a).

274 Appendix


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.

2. Flush silicone processing tubes (15 cm in length) with dH20, 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 tem-perature 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 collagen-GAG 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 tempera-ture to − 80 °C.5 It takes 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 10-cc syringe (Becton Dickinson model

5604, Becton Dickinson, Rutherford, NJ) and expel all the air bubbles. Attach a 25-gage needle (Becton Dickinson model 25G5/8, 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.

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 suspension 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 suspen-sion into the silicone tube via the needle.

10. Inject additional suspension until the silicone processing tube becomes pres-surized and expands to fill the entire PVC jacket. 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

5 The temperature of the axial freezing bath controls the average pore diameter. In the case of NRTs, pores with an average diameter of 5–10 µm were determined to be optimal for the regenera-tion of axons (Chang et al. 1990; Chang and Yannas 1992; Loree et al. 1989). Freezing at − 80 °C results in pore channels that average 5–10 µm in diameter (Fig. 3b). Higher freezing temperatures result in larger pore diameters (Loree 1988).

275Methods

and sealed. Pressure should be kept on the syringe plunger until the needle is completely out of the tube. Check to make sure the silicone processing tube is still filling the entire PVC jacket.

11. Attach the drive gear to the electric timing motor on the axial freezing appara-tus. Place prepared PVC jackets, up to four at a time, on the tube carrier. 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 10− 4 m/s.6 Monitor the process of lowering to ensure that the tubes do not stick to the copper tubing in the freez-ing 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 PVC jackets are fully immersed in the freezing bath, turn-off the

timing motor and remove the tubes from the bath. Quickly separate the tubes and remove the conical plugs. Cut off the plugged end of the silicone tube and cut each PVC jacket assembly approximately in half with a sharp razor blade. This process provides more exposed surface for sublimation of the ice crystals. Lay the PVC jacket assemblies on a freeze dryer tray and place the tray in the − 20 °C freeze dryer. This step must be done as quickly as possible (within a minute) 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, wait for the con-denser temperature to reach − 45 °C before proceeding to the next step).

16. Turn on the vacuum pump and wait 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 in 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 assemblies, which con-tain the dry, white, highly porous matric inside the silicone processing tubes.

Crosslinking, Sterilization, and Hydration


1. After removing the dry collagen-GAG matrix from the freeze-dryer, inspect the matrix for any irregularities; using a scalpel blade, remove any regions that appear to be distinctly different in appearance from that expected of a very highly

6 By slowly lowering the suspension-filled tubes into the freezing bath, the pores form as axially aligned channels. The degree of axial alignment can be modified by changing the lowering veloc-ity. For example, quenching the tubes in the freezing bath ( V = 1 m/s) creates radial pores, while lowering the device into the bath much more slowly, say at 10−4 m/s, results in highly aligned axial pores (Forbes 1980; Yannas and Tobolsky 1967), which are preferable for regeneration (Chang et al. 1990; Chang and Yannas 1992; Loree et al. 1989).

276 Appendix

porous solid of uniform thickness. Usually these regions will be located near the pan edges. Take note of the difference between the pan side (the side that was in contact with the horizontal pan surface) and the air side (the side that was in con-tact with the environment) of the dry matrix. The pan side has a much smoother surface. Future steps will require distinguishing between the pan and air sides of the dry matrix.

2. Make an aluminum foil pouch large enough to fit the sheet of dry matrix. Take a large piece of foil and fold it in half. The folded edge is 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 into the pouch (one sheet of matrix per pouch) and leave the top open.

3. Place the matrix-filled pouch (top open) in the vacuum oven for dehydrother-mal (DHT) treatment.7 The conditions of treatment in the vacuum oven are: 30 mTorr, 105 °C, 24 h.

4. After 24 h, remove the pouch and immediately seal the top by folding the top edges of the foil pouch at least twice. If the matrix is not being prepared for immediate use, it can be stored in the foil pouch in a desiccator (upto 1 year) until needed. The matrix is now sterile and must be handled using sterile procedure from this point.

5. After the DHT process, or after storage, sterile silicone adhesive is placed on the dry matrix. Prepare a sterile field in the laminar flow hood and place in it all sterile implements, including the sterile silicone adhesive and the dry matrix (dropped in the sterile area out of the foil pouch; the pouch can be discarded). Prepare operator for sterile work in gown, cap, coat, and sterile gloves. Pour 1000 mL of 0.05 M sterile acetic acid solution into the sterile 5-L plastic tub. Cover tub loosely with the sterile Teflon cover.

6. Place the matrix on the sterile Teflon working surface with the air side up (the silicone adhesive is placed on the air side of the dry matrix). Squeeze a long bead of the viscous silicone fluid along one edge of the dry matrix sheet. Holding the matrix with one finger on the edge, spread the silicone in a thick layer, using straight sweeps of the spatula toward the opposite edge of the sheet. Use only one sweep of the spatula for each portion of the dry matrix. Wipe the spatula clean with sterile gauze. Another bead of silicone adhesive may be necessary in the canter of the dry matrix to reach the opposite edge, if the matrix sheet is large. Spread along the pore channels, if possible, so the matrix does not tear.

7. Next, use the spatula at a 90° angle to remove the excess silicone and create a thin layer of approximately constant thickness (approx. 1 mm) Use single strokes to cover each area. Exercise care, since the dry matrix tears easily. If tearing occurs, it will be necessary to discard the torn portion of the coated sheet. Wipe the spatula often with the gauze to remove excess silicone adhesive.

7 DHT treatment in a vacuum oven at 105 °C for 24 h serves as a method of crosslinking (Yannas 1972; Yannas et al. 1975)and sterilizing (Yannas et al. 1980) the prepared matrices. Treatment at 105 °C does not affect the triple helical structure of the collagen, provided the moisture content at the beginning of DHT treatment is below 10 wt%, which is achieved by freeze-drying the matrices (Yannas et al. 1980).

277Methods

8. Immerse the matrix, silicone side up, in the acetic acid solution for 15 s. Flip the matrix over, silicone side down, and leave it for 20 h in the covered tub filled with acetic acid.

9. After 20 h in the acetic acid solution, the air bubbles must be removed from the hydrated matrix. Prepare operator for sterile procedure (as in step 5). Remove the air in the hydrated matrix by holding one edge of the matrix (wearing sterile gloves) and gently rubbing the hydrated matrix with one finger until air bubbles come out. Be careful not to tear the hydrated matrix.

10. Turn the matrix over so that the silicone side faces up; allow it to rehydrate in the acetic acid for 4 h. Remove any trapped air bubbles. After the 4-h rehydra-tion, remove the acetic acid from the tub with suction. If the matrix is allowed to dehydrate, the pores close and its activity will be lost.

11. Within 1 min or less after removing the acetic acid, begin pouring 1000 mL of sterile 0.25 % glutaraldehyde in 0.05 M acetic acid into the tub. Pour carefully, to avoid air bubbles. Remove any air bubbles by gently rubbing the foam (see step 9). Soak the matrix in the glutaraldehyde solution for 24 h in the covered tub at ambient temperature (20–22 °C).8

12. Remove the excess glutaraldehyde solution with suction. Rinse the matrix 3x by adding three 1000-ml sterile water rinses to the tub, allowing the matrix to soak for 10 min in each rinse, then remove the rinse water with suction. Add a fourth rinse of sterile water (1000 mL) to the tub, cover, and soak the matrix for 24 h (see step 9).

13. After the 24-h period, the matrix must be cut and stored. Remove the hydrated, porous matrix from the water and place it, silicone side down, on the Teflon sheet. Be careful not to let the matrix fold. Cut away any jagged edges with a scalpel. Place the Teflon cutting template on the matrix and cut around it gently with a scalpel. Do not press down on the template while cutting. Hold only the edges of the matrix. This step must be completed within less than 1 min to pre-vent dehydration (which leads to pore closure and deactivation of the matrix).

14. For immediate use, place the cut matrix in a storage container filled with sterile PBS. The matrix can be stored in this medium for 1 day. For longer term stor-age, place the cut matrix into a storage container with 70 % sterile isopropanol. Store at 4 °C in this medium up to 30 day. Place the matrix in PBS for 12–24 h prior to use as an implant.

8 Glutaraldehyde is used to covalently crosslink the matrix in addition to crosslinking introduced by DHT treatment and may be omitted if a lower level of crosslink density (leading to a more rapid degradation rate in vivo) is desired (26; Yannas et al. 1980). For the SRT, a 24-h treatment in glutaraldehyde resulted in a degradation rate favorable for dermal regeneration. The maximum allowable degradation rate is 140 enzyme unites, as determined by using an in vitro collagenase digestion assay (Yannas et al. 1989). Glutaraldehyde treatment is not used for the NRT, since a rapidly degrading collagen-GAG matrix was found to be optimal for peripheral nerve regenera-tion (Chang 1988; Chang and Yannas 1992; Chang et al. 1990). Although the SRT has a higher crosslink density because of glutaraldehyde treatment, the degradation half-life of the SRT is much shorter than the NRT speculatively due to a significantly lower collagenolytic activity in a nerve lesion compared to a skin lesion (Table 1).

278 Appendix


1. Make an aluminum foil pouch for each PVC jacket assembly (containing the silicone processing tube and the matrix). Take a large piece of foil and fold it in half. The folded edge is 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 PVC jacket assembly into the pouch (one tube per pouch) and leave the top open. The PVC jacket is left in place to protect the matrix from damage during handling and storage.

2. Prepare foil pouches (see step 1) for the implantation tubes. Place each implan-tation tube in a pouch, leaving the top open.

3. Place the matrix-filled pouches (top open) and the implantation tube pouches (top open) in the vacuum oven for the DHT treatment.9 The conditions of treat-ment in the vacuum oven are: 30 mTorr, 105 °C, 24 h.

4. After 24 h, remove each pouch and immediately seal the top by folding the top edges of the foil pouch, at least twice. If the matrix is not being prepared for immediate use, it can be stored in the foil pouch in a desiccator (up to 1 year) until needed. The matrix is now sterile and must be handled using sterile proce-dure from this point.

5. After the DHT process is complete, the matrix can be cut and hydrated for use. Prepare a sterile field and place in it all-sterile implements, including the PVC jacket assemblies and implantation tubes. Prepare the operator for sterile work in gown, cap, coat, and sterile gloves.

6. Under sterile conditions, trim each implantation tube to a length of 20 mm, using a scalpel.

7. Remove the matrix from the silicone processing tube by making a careful slit with the scalpel down the length of the silicone tube and gently pulling out the matrix with forceps. Discard the silicone processing tube.

8. Trim off any crushed or otherwise damaged pieces of the dry matrix. Cut the remaining portion of the dry matrix into 10-mm segments. The exact length depends on the experimental design for use for use of the implant.

9. Insert each 10-mm segment of matrix into the center of a trimmed implantation tube.

10. Place each implant into a specimen jar filled with sterile PBS for hydration and short term (less than 2 day) storage. If implants will not be used immediately, store at 4 °C in 70 % isopropanol for up to 30 day. Transfer the implants to ster-ile PBS solution 1 day prior to implantation.

9 After approx. 2000 the structure of the NRT was prepared in the Yannas laboratory at MIT nearly identically to that of SRT/DRT with respect to pore size and crosslinking treatment. Also, the GAG component was omitted (See Yannas I.V. 2015. Tissue and Organ Regeneration in Adult. Second Edition. New York: Springer).

279References for the Appendix

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Index

© Springer Science+Business Media, LLC 2015I. V. Yannas, Tissue and Organ Regeneration in Adults,DOI 10.1007/978-1-4939-1865-2

AAnatomical boundaries, 57–59, 61Anatomically well-defined wound, 7, 47, 61Animal models, 5, 6, 15, 51, 59, 94, 107, 227,

235, 245, 253, 259experimental, 70, 255

BBasement membrane, 2, 14, 28, 40, 43, 45, 46,

48, 89, 92, 95, 121, 132, 136, 153, 158, 184, 197, 203, 207

approaches to, 43composition of, 40penetration of, 40physiological, 184regeneration of, 31, 40, 43scaffolding of, 14structure of, 96, 97synthesis of, 42synthesis of skin, 97–101, 103, 108tubular, 30, 31, 42, 79, 148, 150

Biodurabletubes, 166

CCollagen-binding integrins (CBI), 24, 245, 246Collagen surface, 244, 256, 264, 265Contractile cell assembly, 218, 219, 221, 222,

233, 239, 241Contraction

antagonizes regeneration, 21blockade, 173, 237, 251, 252, 255–257,

263in injured axolotl, 212, 217in injured frog, 212, 213in rabbit ear, 18, 21, 69, 214, 235oral mucosa, 256

Criticalaxon elongation, 142, 144, 145, 162, 169,

177size, 19, 25

DDecellularized matrices, 21, 22, 104, 254

regenerative activity of, 260–266Defect closure rule, 79–81, 84, 86, 87, 91,

135, 213Degradable tubes, 167–169

based on natural polymers, 169, 170Dermis

papillary, 40physiological, 34, 183, 257regeneration, 24, 55, 127synthesis, 209

Dermis regeneration template (DRT), 2, 24, 130, 132, 182, 194, 228, 248, 254, 256, 261

Dermo-epidermal junction, 29, 40, 208Dermoepidermal junction, 103, 112, 118,

122–127

EEndoneurium, 30, 38, 148, 150, 151, 155, 176,

178, 203, 208regeneration, 33, 141, 202

Epidermis, 2, 3regeneration, 29, 30, 42

Epineuriumregeneration, 38, 159

Experimental volume, 27, 47, 48, 50, 52, 53, 57, 90

size of, 56, 57

332

FFibrotic liver, 55, 254, 266, 267Frog regeneration model, 85

HHair follicle regeneration, 18, 29, 35, 75

IInduced, 1

regeneration, 2, 3, 5, 25, 27, 34, 50, 236Injury, 257

acute, 1chronic, 3, 8, 15, 267

In vitro synthesis, 22, 23, 25, 48of an epidermis, 92, 94, 95, 130

In vivo synthesis, 98, 136, 153, 195Irreducible process, 181Irreversible

damage, 39injury, 6, 11, 13, 17, 21, 46

KKeratinocyte-seeded DRT, 126, 130, 131, 136

LLigand density, 49, 245

adhesion, 246, 248

MMinimum reactants, 179Myelin sheath, 31, 42, 178

regeneration, 43, 45

NNonregenerative, 3, 42

ears, 18endoneurial stroma is, 35–37tissues, 2, 27, 34, 39, 40, 45, 50, 54, 58,

91, 180, 205, 208

OOrgan synthesis, 207

PPerineurium

regeneration, 31, 33, 142, 156, 157Peripheral nerve regeneration, 6, 31, 49, 52,

55, 139, 154, 202, 222, 249experimental and clinical studies of, 21model of, 51theories of, 20vivo studies of, 141

RReaction diagrams, 179–181, 196

irreducible, 198tabulation of, 185, 194

Regenerationparadigm, 21, 24, 25, 253, 254, 261, 265theories, 20, 137

Regenerative, 3tissues, 27, 38, 42, 57

Reversible healing, 6, 268

SScaffold regeneration paradigm, 21, 24, 254,

261, 266Scar formation, 8, 10, 24, 80, 81, 83, 85, 87,

108, 225, 227, 231, 235, 236, 268due to deep vein injury, 13hypothetically inhibited, 18

Scarlessfetal healing, 258healing axolotl, 217, 258healing fetus, 19, 21healing oral mucosa, 217healing rabbit ear, 259

Scar structure, 20, 39Schwann cell seeding, 33, 37, 42, 162Sebaceous gland regeneration, 18, 35, 75,

123, 129Skin adenexa regeneration, 24, 89Skin adnexa regeneration, 132Skin regeneration, 13, 21, 28, 55, 58, 59, 90,

126, 253Spontaneous, 1

regeneration, 4, 5, 8, 10, 11, 21, 32, 37, 46, 56, 139

regenerative tissue, 34Stroma

regeneration, 2, 57, 211Surface biology, 237, 252

TTubulation, 20, 32, 37, 61, 139, 161, 168, 177

WWound, 7, 17, 47

chronic, 55contraction, 17, 19, 21, 24, 49, 70, 223,

226, 227, 236, 237, 248, 250, 255

Index

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