Fabrication and Application of Rose Bengal-chitosan Films in Laser Tissue Repair

Summary

Sutures are usually needed to repair tissue during surgical procedures. However, their application can be problematic as they are invasive and may damage tissue. The fabrication and application methods of a novel tissue adhesive are here reported. This adhesive film is laser-activated and does not require the use of sutures.

Abstract

Photochemical tissue bonding (PTB) is a sutureless technique for tissue repair, which is achieved by applying a solution of rose bengal (RB) between two tissue edges^1,2. These are then irradiated by a laser that is selectively absorbed by the RB. The resulting photochemical reactions supposedly crosslink the collagen fibers in the tissue with minimal heat production³. In this report, RB has been incorporated in thin chitosan films to fabricate a novel tissue adhesive that is laser-activated. Adhesive films, based on chitosan and containing ~0.1 wt% RB, are fabricated and bonded to calf intestine and rat tibial nerves by a solid state laser (λ=532 nm, Fluence~110 J/cm², spot size~0.5 cm). A single-column tensiometer, interfaced with a personal computer, is used to test the bonding strength. The RB-chitosan adhesive bonds firmly to the intestine with a strength of 15 ± 6 kPa, (n=30). The adhesion strength drops to 2 ± 2 kPa (n=30) when the laser is not applied to the adhesive. The anastomosis of tibial nerves can be also completed without the use of sutures. A novel chitosan adhesive has been fabricated that bonds photochemically to tissue and does not require sutures.

Protocol

1. Chitosan Adhesive Preparation

1. The chitosan powder is soluble in acetic acid solution; for the preparation of a stock solution of acetic acid (2% v/v), add 10 ml glacial acetic acid to 490 ml deionized water (DI-H₂O).
2. For the preparation of a stock solution of Rose Bengal (RB) (0.01% w/v) in acetic acid, weigh 5 mg of RB in a vial wrapped with aluminum foil to avoid photobleaching. Add 0.5 ml of DI-H₂O to dissolve the powder then add 49.5 ml of the acetic acid stock solution (described in section 1.1).
3. For the preparation of chitosan solution (1.7% w/v), weigh 0.85 g of chitosan powder (medium MW, 85% degree of acetylation) and add to 50 ml of RB acetic acid solution. The control was prepared by dissolving the chitosan in acetic acid solution without RB.
4. Add a magnetic stirrer to the chitosan mixture and stir at room temperature. To ensure complete dissolution of the components, stir the chitosan in acetic acid + RB for two weeks and the chitosan in acetic acid for 5 days (control). When chitosan is dissolved, the solution pH increases from ~2.6 to ~3.9. Note that RB is poorly soluble in acidic pH and it requires an increase in the pH of the solution and an extended stirring time to dissolve.
5. Centrifuge the solutions at 3270 x g for 1 hr and collect the supernatant using, for example, a sterile 10 ml syringe. Make sure not to disturb the impurity pellet. During this step, the solution is cleaned from macroscopic impurities and insoluble matter.
6. Inject the (centrifuged) chitosan solution on a flat perpex slab using a sterile syringe. The thickness of the dried film depends on the ratio between the dispensed amount of solution and the surface area over which the solution is spread. Ensure that the solution is free from air bubbles and evenly spread on the slab. To obtain thin films with ~13 μm thickness, inject ~1 ml solution over 12 cm² surface area (dimensions~3x4 cm, Figure 1A).
7. Cover the Perspex slab with a screen, made of aluminum foil for example, to avoid photobleaching of the solution. Ensure that enough air ventilation is guaranteed to dry the solution under the screen.
8. Leave the solution to dry at room temperature (25 °C) and pressure (1 atm) till the resulting film is insoluble in water and does not swell macroscopically. This is typically achieved in 2 weeks.
9. Detach the films carefully using a thin and flat spatula: these films can be easily peeled off the Perspex slab without damaging them.
10. Measure the thickness of the films using a micrometer.
11. Cut the adhesive in rectangular strips with a clean sharp scissor and place them between sterile glass slides to preserve their flat shape. Wrap the slides in aluminum foil for light shielding and store them at room temperature. Label the adhesives with or without RB as "rose adhesive" or "chitosan adhesive" respectively.
12. The rose adhesive films can be sterilized using ethanol (90%) or with gamma radiation (0.1 KGy/h for 24 hr)^4,5.

2. Adhesive Optical Attenuation

1. Fix the rose adhesive inside a quartz cuvette.
2. Record its attenuated spectrum in the range of 400 - 800 nm using a dual-beam spectrophotometer⁴.
3. Assuming the validity of Beer's law, calculate the attenuation length of the adhesive as follows: I = I₀e^-Ax where I₀ is the incident beam intensity, 1/A is the attenuation length, and x is the film thickness.
4. Repeat steps 2.1 to 2.3 for the chitosan adhesive to serve as a control.

3. In Vitro Application: Laser Tissue Bonding on Intestine

1. Harvest calf intestine immediately after animal euthanasia and store at -80 °C.
2. Prior to use, defrost and hydrate the tissue by immersing it in DI-H₂O for 10 min.
3. Bisect the tissue into sections (2 x 1 cm) using a #10 blade under an operating microscope (x10). Keep the sections moist using DI-H₂O.
4. Bring the incision stumps close together (end to end). Wipe excess water off the surface using cotton tips.
5. Place a rose adhesive strip (10 x 6 mm) across the incision on the serosa layer with microforceps ensuring full contact with the intestine (Figure 2).
6. The tissue adhesion of the rose adhesive is activated by a diode-pumped solid state laser, which is coupled to a multimode optical fiber (core diameter 200 μm)⁴.
7. Set the power level of the laser to 180 mW and irradiate the adhesive for ~6 min with a beam spot size of ~5 mm, using the parameters listed in Table 1. Spot-irradiate the adhesive ensuring that each spot is irradiated for ~5 seconds before moving the beam to the adjacent spot. This procedure guarantees that the laser beam scans the whole surface area of the adhesive several times.
8. To assess the tissue bonding strength, clamp a sample to a calibrated single column tensiometer (Instron, MA, USA) using mechanical grips. Move the grips at 22 mm/min until the two tissue stumps separate⁴.
9. Place a rose adhesive on the tissue as described in step 3.5. Do not irradiate the sample and measure the tissue bonding strength as described in step 3.8. This sample serves as a control.

4. In Vivo Application: Tibial Nerve Anastomosis

1. Sedate the Wistar rat with isoflurane/O₂ mixture (4% during induction, 2% thereafter)⁵. Subcutaneously inject Buprenex (0.03 mg/kg) to provide intra-operative analgesic activity and alleviate possible pain or discomfort.
2. The following surgical procedure should be performed under an operating microscope (x10). Make an oblique skin incision about 3 cm long in the dorso-lateral part of the right thigh and expose the tibial nerve with a muscle-splitting approach through the gluteal muscles².
3. Partially dissect and trim the adventitia of the tibial nerve using straight micro-scissors and absorb excess water with sterile gauze or cotton tips.
4. Cut the tibial nerve with micro-scissors.
5. Position a sterile chitosan strip (dimensions 5 x 4 mm) underneath the tibial nerve using micro-forceps.
6. Approximate the nerve stumps end-to-end with micro-forceps over the rose adhesive strip.
7. The strip fully adheres to the nerve forming a collar, which can assist with the rotation of the nerve during the laser irradiation of the strip.
8. Activate the rose adhesive with the laser as described in steps 3.6) and 3.7).
9. Trim the redundant rose adhesive from the collar of the operated nerve.
10. Close the muscles and skin with five 3-0 sutures⁵.
11. Standard post-operative recovery procedures should be followed including daily inspection for dehiscence or pus formation and analgesic administration, if required.

5. Representative Results

The films that are obtained are bright rose in color, thin and have a smooth surface (Figure 1). They are also flexible and can be rolled into tubes of small diameter without causing tears or any other apparent damage (Figure 1B). The rose adhesive has two absorption peaks at 530 and 562 nm; the green laser is thus strongly absorbed by the adhesive and the corresponding attenuation length at 532 nm is ~12 μm (Figure 3). In contrast, chitosan films without RB attenuates weakly the laser (1/A ~162 μm), likely due to scattering. It appears from the spectra plot that no significant aggregation of RB occurs in the films.

The rose adhesive bonds firmly to the intestine upon laser irradiation (Figure 2) achieving a typical maximum load at failure of 0.9 ± 0.4 N (n=30). The adhesive strength was estimated as the maximum load divided by the adhesive surface area, namely, 15 ± 6 kPa (n=30). The non-irradiated rose adhesive bonded significantly less to tissue (2 ± 2 kPa, n=30, unpaired t-test p<10⁶). The in-vivo anastomosis of the tibial nerve was successfully achieved following the application and bonding of the rose adhesive in conjunction with the green laser. One week postoperatively, the operated nerve was in continuity and the repair strength was 17 ± 9 kPa (n=10).

Figure 1. A) The RB-chitosan solution is casted on a flat Perspex slab (slab thickness~5 mm). Graph paper is placed underneath the slab to facilitate the dry-cast step. The water-insoluble film is obtained two weeks after casting. B) The rose adhesive is flexible and can be rolled into a small cylinder.

Figure 2. The rose adhesive is bonded to calf intestine after laser irradiation. Uniform irradiation is applied on the RB adhesive during tissue-bonding; however selected spots are shown in this picture to illustrate the photo-bleaching effect of the laser on the adhesive.

Figure 3. The absorption spectrum (in arbitrary units) of the rose adhesive shows two peaks at 530 and 562 nm. The green laser (λ= 532 nm) is thus strongly absorbed by the adhesive during the laser exposure. The chitosan adhesive without RB absorbs poorly the laser beam.

	n	Area (mm2)	Power (W)	Time (s)	Fluence (J/cm2)	I (W/cm2)	Max Load/Area (kPa)
Adhesive+Laser	30	60 ± 10	0.18 ± 0.03	365 ± 5	~110	~0.9	15 ± 1
Adhesive	30	60 ± 10	NA	NA	NA	NA	2 ± 2

Table 1. Legend. n, sample number; Area, surface area of the rose adhesive (mean ± maximum error); Power, laser power (mean ± maximum error); Time, irradiation time (mean ± maximum error); Fluence, average laser fluence; I, estimated irradiance; Max Load/Area, maximum load at failure of the repaired tissue divided by the adhesive surface area, (mean ± SE).

Discussion

The rose adhesive fabrication is based on a simple dry-cast process, though the dissolution of RB in acidic pH requires prolonged stirring of the chitosan solution. It is important to let the solution dry up until it becomes a water-insoluble film. This happens when the weight water content is ~10% in the dried film⁶. Insoluble films are usually obtained 2 weeks after dry-casting at standard conditions of temperature and pressure (~25 °C and ~1 atm). The mechanism of tissue bonding is not clear yet, although it was observed that RB diffuses from the adhesive into the adjacent tissue, allowing the laser to photo-activate it efficiently at the tissue interface. It may be speculated that the RB ability of producing singlet oxygen, upon light irradiation, may play a role in crosslinking tissue collagen and chitosan via their amino groups^7,8. A recent study on the rose adhesive showed also that the maximum temperature achieved during the tissue-adhesive bonding was ~32 °C, supporting the hypothesis of a photochemical process⁴. Other sutureless techniques for tissue repair are currently investigated by several research groups. Laser tissue welding (LTW) for example has been successfully applied in ophthalmic surgery by Pini et al ⁹. In general though, LTW can cause collateral thermal damage as tissue temperature can reach 65-75 °C during laser irradiation. Cyanoacrylate glues are also applied clinically to close skin wounds instead of sutures; nevertheless they are generally not used for internal organ repair due to their toxicity ¹⁰. Although the rose adhesive (~15 KPa) has higher bonding strength than fibrin glues (~8 kPa) ¹¹, cyanoacrylate glues still provide the strongest adhesion (~150 kPa) ¹². The thickness of the rose adhesive requires particular attention during fabrication: a thick adhesive (≥20 μm) would, for example, prevent the laser from reaching the tissue interface and would weaken the bonding strength due to excessive absorption of the laser by the rose bengal. A thin adhesive (<10 μm), on the other hand, would increase the laser irradiance and fluence at the adhesive-tissue interface. However, care should be taken in reducing the film thickness in order to prevent excessive heating of tissue during laser irradiation. The rose adhesive induces no significant cytotoxic effects on human fibroblasts⁴ and has thus a promising use in repairing soft tissue inside the body, such as intestine and peripheral nerves. This adhesive can also have applications in tissue engineering: It can be integrated, for example, in a bandage with extracellular matrices to repair tissue and enhance wound healing without the aid of sutures¹³. It should be noted that we have not observed any significant adverse effects, including localized inflammation, to be associated with this procedure.

Materials

Name	Company	Catalog Number	Comments
Rose Bengal	Sigma-Aldrich	632-69-9
Chitosan (medium MW)	Sigma-Aldrich	10318AJ
Glacial acetic acid	Sigma-Aldrich	08050051	2% v/v in DI water
Magnetic stirrer	Heidolph	MR Hei-Mix S
Centrifuge	Beckman Coulter Inc.	Allegra X-12R
Spectrophotometer	Varian Inc., Agilent	Cary 4000 UV-Visible
Laser	CNI Laser	MGL-532
Micrometer	Mitutoyo	Series 227
Surgical microscope	Carl Zeiss, Inc.	OPMI