1.

Introduction

The closure of incisions in tissues involves passing a needle through the tissues, close to the edges, pulling them together and relying on tissue and suture strength to hold them tight. Suturing is a delicate, demanding, and time-consuming procedure that requires significant technical skill.^1,2 Sutured sites have an increased risk for leaks, infections, and scarring.^3,4

Laser-tissue bonding has been introduced in an attempt to solve these problems. There are two methods of laser-bonding of incisions,^5–7 which differ by their biochemical mechanisms: photothermal bonding and photochemical bonding.

(A) Photothermal bonding. There are two basic photothermal procedures: laser welding and laser soldering. In laser-welding, a spot on the approximated edges of an incision is heated by laser light absorption in the tissue or in a coloring agent (which changes the penetration depth of the light).^8–10 The spot is heated to temperature $T$ for time $t$, causing adhesion. The full length of the incision is then bonded, spot by spot. In laser-soldering, a biological bonding agent, such as, albumin or chitosan,^11,12 is applied at the approximated edges and heated spot by spot. The heating denatures proteins and links them with collagen, thus generating a clot that seals the incision.¹³

Laser bonding, and specifically the introduction of a biological agent (e.g., albumin), generates a watertight seal and accelerates healing with very little scarring. In addition, it improves the immediate mechanical properties (e.g., tensile strength) of the target tissues after bonding. During the last few years, there has been significant progress in the development of laser bonding methods. Photothermal bonding has already been demonstrated in animal^14,15 and clinical⁹ works.

(B) Photochemical bonding. A chemical dye that is applied at the incision is sensitized by visible laser light. The irradiation drives a chemical reaction that initiates bonding through protein crosslinking.^16–18 With this approach, significant bond strengths have been achieved in many tissues. Photochemical bonding has also been demonstrated in animal experiments (including ones on the cornea) and clinically.^16,19

These two bonding methods have a potential of being widely used clinically. But suturing is obviously the standard method still used for closing incisions.

Our analysis of previous works showed that many researchers calculated the heat distribution generated by laser heating, under various experimental protocols, using different lasers, heating setups, and solder compounds.^20–22 However, very little work was done on monitoring and controlling the temperature of each heated spot on the incision.

The lack of temperature control caused underheating or overheating, and in both cases, the bonding strength was insufficient. Attempts have been made in the past to develop such a control, but more work is still needed. One pioneering work had a camera adapted for temperature-control of laser bonding and was successfully used clinically.^9,14 Yet, none of these approaches has been widely used.

We developed a fiber-optic system that used a middle-infrared (mid-IR) detector to measure the temperature ($T$) of the heated spot, during the irradiation time ($t$). A feedback loop was used to control the laser output so that $T$ was kept constant. We concentrated on laser soldering and used albumin as a solder. In addition to the experiments, we also carried out theoretical calculations of the temperature distribution during irradiation.^20,23–27 We tested our calculations at different values of $T$ and $t$ and found optimal conditions for $T\approx 60°\mathrm{C}$ and $t\approx 10\mathrm{s}$. This was also verified experimentally, and strong and watertight bonding, without noticeable thermal damage, was obtained.²⁸

Using this information, we succeeded in soldering incisions in vitro in skin,²⁹ bowel,³⁰ dura,³¹ and trachea.³² In experiments on skins of large farm pigs in vivo, the wound healing was faster than that obtained after standard suturing, and there were practically no scars.³³ Clinical laser-soldering protocols were also carried out on incisions in the abdomen skins of patients, after laparoscopic cholecystectomy. Again, strong bonding with very little scarring was obtained.^24,34

Recently, we used robotic laser soldering for bonding incisions in mouse skins in vitro and obtained a high bond strength.³⁵ Using this technique, we were able to deliver a measured amount of heat for a predefined amount of time, repeatably, thereby obtaining standardization and allowing for remote (e.g., daVinci System) operation or even an unaided one.

Suturing is a standard procedure in cataract treatment, corneal surgery, or corneal transplantation (penetrating keratoplasty). It is inexpensive, reliable, and readily available.³ However, the tissue is very delicate and presents a considerable challenge. One complication in corneal transplantation is astigmatism, due to nonuniform tensions generated by the sutures around the cut, resulting in corneal deformation.⁴ Chemical adhesives have also been used in corneal transplantation, but they can have inadequate tensile strength, they are toxic, and suffer from other problems.^36,37

In our previous experiments on laser soldering of corneal incisions in piglets, we proved that our noncontact and temperature-controlled soldering method, based on a ${\mathrm{CO}}_2$-laser, generated an immediate, reproducible, and watertight bond.^21,29,38

One goal of the current research was to maximize the burst pressure $P_{\mathrm{B}}$ of soldered corneal incisions. The second goal was to check if there is any change in the corneal shape of the piglets due to the soldering process. To fulfill the first goal, we found the optimal conditions needed to generate immediate very high $P_{\mathrm{B}}$ ex vivo. We report here these optimal conditions. To achieve the second goal, we used these optimal conditions, assuming that the same $P_{\mathrm{B}}$ will be obtained in live piglets. We allowed the eyes to heal for 6 weeks and observed the corneal shape, using optical coherence tomography (OCT) and histopathology. We did not observe changes in the shape of the cornea as a result of the soldering. Obtaining immediate strong bonding on corneal incisions, without deforming the retina, will be an important step toward bringing laser soldering of corneal incisions from the laboratory to clinical use.

2.

Materials and Methods

2.4.

Surgical Protocols

By observing the process through a microscope (Fig. 1), the surgeon followed these protocols:

• 1. A linear incision of length $\approx 5\mathrm{mm}$ was made on the cornea. Each incision was made in one of two modes: (I) perpendicular to the corneal surface [Fig. 2(A1); this cut was used only in the ex vivo experiments], or (II) slanted with respect to the corneal surface [Fig. 2(A2); this cut was used both in the in vivo and the ex vivo experiments].

• 2. Only for the laser-soldering cases: a layer of solder of thickness $\approx 500\mu \mathrm{m}$ was spread over the approximated edges of the incision and some solder was spread between the edges.

• 3. The distal tip of the handpiece was brought to a height 3 to 5 mm above the incision, and the laser beam heated a spot on the incision to the optimal values of $T$ and $t$. The tip was then moved to a neighboring spot along the incision, according to one of two patterns: (I) linear heating [Fig. 2(B1)] or (II) zigzag heating [Fig. 2(B2)]. During our experiments, we used for example a vertical incision-zigzag heating protocol or a slanted incision-linear heating protocol. In most cases, we used a slanted incision-zigzag heating protocol.

Fig. 1

The laser soldering protocol, using the laser soldering system.

Fig. 2

Soldering protocols: (A1) Cross section of a perpendicular (vertical) incision. (A2) Cross section of a slanted incision. (B1) Top view of a linear heating along an incision. (B2) Top view of a zigzag-pattern heating along an incision. The dotted area marks the places where albumin was applied. The circles mark the heated-spot diameter. The broken arrows mark the direction of the heating spot by spot. This figure is not in scale, the cut edges have been moved far apart, for clarity.

3.

Results

3.1.

Ex-Vivo Experiments

Several soldering protocols were tested (Fig. 2) for soldering of incisions on the cornea. The results (Table 1) showed that for the slanted incision-zigzag heating protocol the average $P_{\mathrm{B}}$ for 14 eyes was $(875\pm 274)\mathrm{mmHg}$. It is an order of magnitude higher than the average value of $(92\pm 8.4)\mathrm{mmHg}$, obtained for 18 eyes, where the vertical incision-linear heating protocol (Table 1) was used. The latter is also higher than the clinically accepted value of $P_{\mathrm{B}}=80\mathrm{mmHg}$ for sutured corneal incisions.^21,37

Table 1

Burst-pressure (PB) measured after ex vivo soldering of incisions of length ≈(5±1)  mm in porcine corneas, using different soldering protocols.

ID	Bonding method	Eye ID	PB (mmHg)	Eye ID	PB (mmHg)	Eye ID	PB (mmHg)	PB (mmHg) average
1	Make a vertical incision, spread albumin, solder the cut in a linear motion.	1	69	4	100	7	83	$89.5\pm 10.4$
		2	84	5	98	8	96
		3	91	6	95
2	Make a vertical incision, spread albumin, and solder the cut in a zigzag motion.	9	83	13	98	17	97	$92\pm 8.4$
		10	81	14	81	18	85
		11	103	15	96
		12	99	16	95
3	Make a slanted incision, spread albumin, and solder the cut in a zigzag motion.	19	1025	24	1350	29	626	$875\pm 274$
		20	1024	25	650	30	800
		21	1020	26	420	31	660
		22	1130	27	644	32	846
		23	1300	28	754

We also did laser welding of incisions ex vivo: we used either one of our heating protocols, without any solder, as preliminary experiments in preparation for the in vivo experiments. The average $P_B$ measurements in these cases (not shown) exhibited bond strengths of $\sim 30\pm 10\mathrm{mmHg}$ in 15 porcine eyes, which is far below the acceptable value of 80 mmHg.

We compared our results by calculating the one-sample $T$-test of each experiment with the accepted value of 80 mmHg for suturing. We obtained $P$-values that are smaller than 2%, 0.1%, and 0.01%, for the soldering methods 1, 2, and 3, as shown in Table 1, respectively. Calculating the unpaired two-sample $T$-tests for either permutation of the average $P_{\mathrm{B}}$ of any two of these bonding methods, yielded $P\text{-}\text{values}<0.01$. These results are statistically significant.

OCT and histopathology of the soldered incisions in these eyes showed well-approximated tissues, and no noticeable corneal shape distortions at the area of the cut (not shown).

Furthermore, qualitative comparisons of the radii-of-curvature of corneas soldered by our protocols, to those that had been welded, were carried out using OCT and histopathology imaging. They exhibited no noticeable changes in the radii of curvature, suggesting no or minimal generation of optical aberrations to the cornea by the laser soldering protocol. These are not shown for the ex vivo results but are shown representatively for the in vivo results, in Fig. 3.

Fig. 3

Each row shows three images of a cross section of a bonded incision: (a) histopathology, (b) OCT at high magnification, and (c) OCT at low magnification. The top row represents a soldered incision in the cornea of piglet-D (Table 2). It was soldered using the slanted incision-zigzag heating protocol. The center row represents welded incision in the cornea of piglet-G (Table 2). It was welded using the slanted incision-zigzag heating protocol. The bottom line represents the cornea of piglet-K (Table 2). This piglet belonged to the control group where a slanted incision was not laser heated.

Table 2

Experimental results for laser-soldering of piglet corneal-incisions, in vivo.

Action	Piglet	OCT	Histology
Group I solder: slanted incision, zigzag heating	A	1. Slanted prolonged penetrating cut.	1. Well-bonded corneal cut.
		2. Hyperreflective anterior corneal side, encompassing roughly the outer 40% to 70% of the wound length from the entry at the front corneal surface to the exit at the back corneal surface.	2. Mildly thinned epithelium at the bonded cut.
		3. Small area of the posterior stromal hyperreflectivity near the endothelial side of the wound.	3. The basal cell layer shows mild folding.
			4. Thin epithelium-to-endothelium stromal scar.
			5. The scar shows mild hypercellularity and neovascularization.
			6. The Descemet’s membrane is disrupted and the gap between its edges is occupied by a triangular scarring with the base toward the inner surface of the cornea.
	B	1. Same as the OCT results for piglet A.	Same as the histology results for piglet A.
		2. Same as piglet A but only 50% of the wound is encompassed.
		3. Prolonged posterior stromal hyperreflective area near the wound endothelium.
	C	1, 3. Same as the OCT results for piglet A.	Same as the histology results for piglet A.
		2. 40%.
	D	1. Same as the OCT results for piglet A.	1, 2, 3, 4. Same as the histology for piglet A.
		2. 70%.	5. The scar shows mild hypercellularity and extensive neovascularization.
		3. Area of the posterior stromal hyperreflectivity near the endothelial side of the wound, bulging into the anterior chamber.	6. The Descemet’s membrane is disrupted and is covered by endothelium.
Group-II weld: no albumin, slanted incision, zigzag heating	E	1, 2. Same as the OCT results for piglet D.	Same as the histology results for piglet D.
		3. Minimal posterior stromal hyperreflectivity near the wound endothelium within some localized stromal thinning.
	F	1, 2, 3. Same as the OCT results for piglet D.	Same as histology results for piglet D.
	G	1, 3. Same as OCT results for piglet D.	1, 2, 4, 5, 6. Similar to the histology for piglet A.
		2. 50% to 70%.	3. Not seen.
			Extensive inflammation is present at the cornea peripheral to the bonded perforated cut and limbus at this side.
	H	Experimental failure	Experimental failure
Group-III control: slanted cut, no albumin, no laser heating.	I	1. Same as the OCT results for piglet D.	1, 2, 4, 5, 6. Similar to the histology for piglet A.
		2. 20% to 30%.	3. Not seen.
		3. Same as the OCT results for piglet D, with slight bulging into the anterior chamber.	There is adhesion between the inner surface of the scar and the anterior part of the iris.
	J	1, 2, 3. Same as the OCT results for piglet I,	Same as the histology results for piglet I.
	K	1, 2, 3. Same as the OCT results for piglet I.	Same as the histology results for piglet I.
		Massive excess hyperreflective tissue bulging into the anterior chamber, anterior to a separate defined anterior chamber structure, separated from the cornea by a thin membranous limit.
	L	1, 2. Same as the OCT results for piglet I.	1, 2, 3, 4, 5. Similar to the histology for piglet A.
		3. Same as the OCT results for piglet D.	6. Same as the histology results for piglet D.

4.

Discussion

Eyes that underwent corneal surgery must be able to withstand burst pressures that can be developed in various physiological responses, such as sneezing or coughing. As mentioned, the acceptable $P_{\mathrm{B}}$ for bonded corneal incisions is at least 80 mmHg.^36,37,41 The first aim of this work is to achieve this, or higher $P_{\mathrm{B}}$, immediately after the surgery. Toward this goal, we tested different irradiation and incision protocols. These included the vertical incision-linear heating, the vertical incision-zigzag heating, and the slanted incision-zigzag heating protocols, as presented in Table 1.

It can be observed in Table 1 that in the ex vivo experiments the slanted incision-zigzag heating protocol generated nearly a 10-fold higher $P_{\mathrm{B}}$ than the generally acceptable one. This is probably because the spreading of the albumin on a larger area and because the laser heating was done on this larger area (on the surface and in the bulk). The vertical incision–linear heating and the vertical heating-zig zag protocols generated $P_{\mathrm{B}}$ that was still higher than the quoted minimally acceptable value of $P_{\mathrm{B}}$.^36,37,41

We assumed that the same $P_{\mathrm{B}}$ values will apply for the in vivo cases. It is important to notice that the incisions in the control group eventually closed since these incisions are self-sealing. However, as mentioned, with no laser bonding the $P_{\mathrm{B}}$ was only 30 mmHg, which is far too low. Therefore, only the soldered incisions achieved a water-tight seal of very high strength, immediately after surgery, whereas the welded or nonheated incisions did not.

Though a more thorough comparison may be needed, the bonding quality obtained with the $1.9\text{-}\mu \mathrm{m}$ laser is better than the one obtained with a ${\mathrm{CO}}_2$ laser.²⁹ This is most probably due to the difference in penetration depths and a more uniform heating along the depth of the incision.

We also note the relatively large standard deviation of the $P_{\mathrm{B}}$ results (Table 1). Our initial investigations traced this to how accurately the sample was cut. Introduction of “knifes” better than the standard scalpels may be needed. Femtosecond lasers were recently successfully adapted for ophthalmic laser surgery.⁴² This advancement has the potential to increase the reproducibility in the $P_{\mathrm{B}}$ results.

The in vivo experiments were performed under the assumption that the same immediate $P_{\mathrm{B}}$ would be maintained if the irradiation protocol of the optimized ex vivo experiments is repeated. The aim in the in vivo experiments was not to observe $P_{\mathrm{B}}$ but to observe the corneal shape, after the weeks-long recovery period.

We believe that, as in any change in stromal integrity, there should be some change in the curvature of the cornea. The quantification of the degree of corneal distortion was not performed, using corneal topographic scans, as it was not available to us. We therefore attempted to qualitatively assess the change using both OCT and histopathology measurements (e.g., Fig. 3). We found that the histology and the OCT findings in corneas reported in Table 2 are quite similar for the three study groups. These showed complete closure and negligible change in corneal shape. From this, we conclude that the optical aberrations generated by this procedure should be negligible.

The OCT and histopathology measurements also revealed that the differences in the outcome were mostly in the area of the posterior stromal hyperreflectivity, near the endothelial side of either wound. In the soldered group it was small, whereas it slightly increased in the welded group and further slightly increased in the control group. Yet, all these changes were small. We note the overall similarity in all the images we obtained, regardless of the group to which they belonged. This similarity between the groups suggests that if there was thermal damage in the welding and soldering samples, it was insignificant.

We observed inflammation only in one in vivo eye out of the 12 quoted. This inflammatory reaction may be attributed to an abnormal recovery and not to the surgical protocol itself.

The outcome of this work is a demonstration that laser-soldering of corneas is feasible and that it generates an immediate and high burst-pressure bond. This should be of benefit to future corneal surgical protocols. Perpendicular incisions should be used for corneal transplantation, and we showed that the acceptable $P_{\mathrm{B}}$ has been achieved in this case. As for cataract surgery, it can benefit from the slanted incision-zigzag heating protocol, due to the exceptionally high bonding strength obtained with this improved method of corneal incision-closure.

Some of the advantages of laser soldering of corneal incisions, compared to suturing, include the following. (1) Immediate closure of nonpenetrating corneal wounds, such as in deep anterior lamellar keratoplasty. (2) Negligible optical aberrations and very little scarring were observed, thus demonstrating an optically transparent tissue. This will be beneficial for reducing postsurgical complications such as astigmatism. (3) In the case of corneal transplantation, a much easier and faster closure of a full 360-deg corneal graft has the potential to be obtained. (4) Reduction of the postoperative foreign body (sutures and stitches) sensation and discomfort.⁴³ (5) Sealing of miniature cuts that may appear in the cornea as a result of a trauma. Such cuts are extremely hard to close, so the surgeons are relying on the corneal self-healing while accepting the deformations that are generated in the curvature of the cornea as a result.

All these important advantages could make laser soldering a very promising technique for various types of corneal surgery.

5.

Conclusions

Laser bonding of incisions was successfully demonstrated by several groups worldwide. However, it has not yet made a clinical impact.

We developed a fiber-optic temperature-controlled laser-soldering system for bonding corneal incisions, using albumin as a solder. The use of visible and mid-IR fibers made it possible to accurately monitor and control the temperature of each bonded spot on an incision and to do it rapidly. We used this system for bonding either vertical incisions or slanted incisions in corneas of piglets. A strong and watertight bonding was obtained immediately in both procedures. We did not observe a change in the corneal shape if the eye was soldered, welded, or left to self-heal.

The skill required to operate the laser soldering system is significantly lower than the one needed for suturing by an experienced surgeon. This is a noncontact method and a surgeon only needs to maintain the handpiece at a set distance above the cut and slowly move the distal end along the incision.

Future experiments will focus on corneal transplantation in large pigs, and the success in those experiments will enable us to start human clinical trials. We hope that in the future, laser soldering could substitute suturing in corneal surgery.