Method of tissue repair

- 28036 Madrid

ES 2 201 167 T3

DESCRIPTION

Welding for tissue repair.

Technical field

The present invention relates to methods for joining living tissues, including veins, arteries, microvessels, tubes, nerves, organ tissues and biological surfaces, such as: peritoneum, omentum, fascia, shin, artificial tissues, and to pharmaceutical products useful in joining these tissues.

Fundamentals of the technique

The joining of tissues such as: veins, arteries, microvessels, tubes, nerves, tissues and biological surfaces such as the peritoneum and the skin, has been carried out mainly clinically, to date, by suturing and microsuturation.

Microsuturation requires considerable skill and is a time-consuming procedure. Often, the tissues that have been joined by microsuturation form considerable scar tissue. Some of the difficulties encountered with microsaturation can be better understood by considering the example of reattaching damaged peripheral nervous tissue.

Peripheral nerves

Electrical signals that control the body's organs and transmit information from one part to another to the central nervous system (CNS) travel along peripheral nerves. The structure of these peripheral nerves is analogous to telephone cables. On a telephone cable there is a strong, protective outer coating that protects all internal components. The copper wires are often bundled together in separate insulating tubes that lead to different systems. Each of the internal copper wires is a single line that can transmit electricity in any direction and has an insulating coating around it so that it does not interfere with the lines near it.

A peripheral nerve (Figure 1) has an outer membrane consisting of connective tissue such as collagen. This membrane (epineurium) protects and supports the independent nerve bundles together. The bundles of nerves that lie within this membrane are called fascicles. These fascicles also have a surrounding collagen-based membrane and their job is to bundle nerve axons together supplying a similar area of ​​the body. Within the fasciculus membrane the axons are surrounded by loose connective tissue. Axons are a long extension of a cell body that is contained within the CNS in the spinal column or in the brain. Sensory axons transmit to the CNS and motor axons transmit from the CNS. Nerve metabolism is supported by the vascular system from both outside the nerve and along the center of the nerve.

Peripheral nerves can have very small diameters. For example, the mature median nerve in the wrist is approximately 1 cm in diameter and contains an average of forty fascicles, each of which can contain up to 4,500 axons. When a peripheral nerve is cut, all axons distal to the wound change their properties as the flow of axons from the cell body is cut off. Even when the nerve is reconnected, these axons continue to degenerate distally. Schwann cells that normally wrap around axons as an isolation guide regenerating axons. Joining nerves as precisely as possible by aligning the corresponding bundles allows axons to regenerate more efficiently.

Nerve operations have been facilitated using magnification and special microsurgical equipment. It is necessary that precise repairs be carried out at the fascicular level, ensuring that regeneration is along the correct bundle, leading those supplied axons to the original area. The current peripheral nerve repair technique uses microsuturation (Figure 2). This technique requires a specialized surgeon, fully dedicated, since the microsuturation of just one of the many fascicles with three or more microsutures (using say a 70 micrometer diameter needle and 30 micrometer wire) can take very long operating times.

Microsuturation is currently used clinically where techniques are available. Unfortunately, there are relatively few surgeons who have the manipulative skills necessary to operate at high magnification. Even a reasonable microsuturation technique results in long operating times with added damage to internal axons as the sutures penetrate the thin perineurial isolation sheath. The use of sutures results in some scarring of the repair due to foreign body reaction. There is also evidence indicating that, in the long run, scar tissue formation and scar maturation can lead to attached nerve deficiency.

Work has been developed on the use of lasers only in the realization of nerve junctions. One of the problems with laser welding has been the fact that the intact gel-like nerve tissue of the axons is actually under pressure within the bundle. When the fascicle is cut, this material is extruded. This can lead to direct laser welding forming over the nerve tissue rather than surrounding the fasciculus membrane, causing nerve damage. To date, welding has typically been done using infrared lasers such as CO2 lasers that feature water absorption for energy transfer. Tissue preparation prior to welding relies on the overlap of nerve membranes. This is difficult due to extrusion of gel-like axons and can thus lead to denaturation of nerve axon material. The affected tissue tends to scar, and the fibrous tissue that proliferates as a result is a poorer conductor of electricity than nerve tissue. Joints formed to date, as described in the prior art, using laser welding, have typically lacked strength. These laser bonds alone tend to fail so microsaturation in addition to welding has been used to strengthen these bonds.

To address at least some of the shortcomings of laser welding, various glues have been used in the formation of the welds. These fluid, low-protein tails tend to run between the joining nerve ends, which can result in nerve bundle axoplasm damage and also impede regeneration. They are also applied around the joint, which is then circumferentially welded.

ES 2 201 167 T3

These junctions later show thick scarring, which causes narrowing of the nerve. On the other hand, the unions tend to be weak.

The welding techniques available so far also tend to lack precision. Factors influencing the accuracy of this approximation adversely include differences in: the consistency of the glue used; the opening of the needle or other device used to apply the glue; and the pressure exerted in the application of the glue.

WO-A-91/04073 describes laser tissue welding with dye-enhanced welds and uses a viscous fibrinogen solution for tissue welding.

WO-A-92/14513 describes filler material for use in tissue welding, specifically collagen preparations in water containing 0.5-10% collagen by weight.

Neither of these two publications mentioned solves the problems identified.

The present invention provides liquid or solid welding compositions (claim 21), their use (claim 1) and their preparation methods (claim 15).

The welding of the invention is useful in a method of joining tissue comprising:

aligning and confining edges of tissue to be joined;

apply a weld, from one side to the other of the aligned and confined edges; and exposing the weld to a source of energy under conditions that provide a transfer of energy from the source to the weld to cause the weld to bond to the tissue surface adjacent to the edges, thereby providing a weld that supports the edges. together.

In addition to causing the weld to bind to the underlying tissue protein, energy transfer can affect the structure of the weld itself leading to the joint within the weld and an enhancement of the strength of the weld and therefore the Union.

Solder droplets are typically used where the solder is a fluid solder, and are "painted" across the edges.

The solder can also be provided as a preformed solid strip.

The power source is typically a laser.

A variety of tissue types can be joined using this method. The method is applicable to anastomosis of biological tubes including: veins, arteries, lymphatic vessels, nerves, efferent vessel, fallopian tubes, bile ducts, digestive tract tubes, ureter, urethra, tear ducts, bronchi and any other of such body tubes as well as for repairs of incisions or tears of biological organs such as kidneys, liver or spleen, or of biological surfaces such as the peritoneum and the skin. It will be understood, therefore, that the method can be used in a variety of attachment situations including the attachment of cylindrical anastomoses and the closure of linear defects such as incisions.

When tissue repair is with respect to nerve tissue or other tissue tubes where the contents of the tube need to be protected from damage, it is especially important that the weld does not concentrate on the edges that are being joined as this can damage extruded tissue. . Rather, the weld should be distributed along the planar or tubular surface on which the discontinuity lies.

Where the tissue to be repaired is essentially a hollow body tube such as a blood vessel, the repair may further comprise inserting a hollow, thin-walled weld cylinder into the tube under repair so that the cylinder extends over the cut parts of the tube. Typically, while supporting the cut tube and barrel assembly together, energy from the power source is directed through the tube wall to attach the barrel to the ends of the tube. The cylinder may incorporate a colorant, as will be described hereinafter, to attract energy to the cylinder for more efficient welding. The repair is completed by applying at least one bead of solder across the edges to the outer surface and treating the applied solder as described above.

When the repair is with respect to tissue surfaces such as the peritoneum, it will be understood that it is less important to avoid concentration of weld on the edges.

The method can also be modified to repair other discontinuities in tissue surfaces such as holes, which result from accidents or surgery. In this form of the invention the weld may be extended or pre-cut to conform to the shape of the repair site, and the edges of the repair site may not need to be aligned or confined for the repair to be made.

A typical nerve repair using the method of the invention is one in which the edges are ends of a cut peripheral nerve bundle to be joined together or one end of a nerve bundle and the substitute nerve graft material bundle . The latter situation is particularly applicable where nerve repair is required but a section of the nerve under repair has been severely damaged or is unavailable, such that the available ends of the bundle are too far from each other to be directly attached. The actual nature of the damage sustained by the nerve and whether the repair is a primary or secondary repair are factors that affect recovery but in any case the edges of the nerve bundles to be joined are cleanly cut at right angles prior to to the union.

Application of the solder as a strip or strips, with space in between for natural coadaptation of the surfaces themselves allows the nerve under repair to be revascularized. Circumferential welding, by comparison, can present the body's natural healing process and thus reduce the necessary capillary blood access to the repair area. Laser welding and suturing techniques ultimately rely on the body to regenerate connective tissue to support the nerve together after failure of either the weld or suture connections and are replaced by the healing procedure. The present authors have shown in in vivo experiments that successful regeneration can be achieved by the methods of the present invention without restriction on the movement of surrounding tissue after the operation. In the case of nerve repair operation in human patients, it is routine to initially restrict the movements of the joints of the operated limbs to promote reduction

ES 2 201 167 T3 tension across the repair site.

Typical biodegradable biological welds useful in the method of the invention include protein welds.

It is envisioned that other biomolecules found in nature can be used as alternatives. Additional biological, biodegradable polypeptide analogs could be used. Biological, biodegradable polypeptide analogs useful in the invention include synthetic polypeptides and other molecules capable of forming a viscous "tail" that does not react adversely within tissue undergoing repair.

The protein solder can be a solid solder composition or a fluid solder.

Flowable protein solder compositions useful in strip soldering typically comprise between 100 and 120 mass% protein relative to water. Preferably, the fluid protein welds comprise between 100 and 110 mass% protein relative to water.

The fluid solder strip is typically 50 to 200 µm thick. Its length is selected to adapt the joint to be formed but is typically on the order of 2 to 3 mm in length. Typically it is painted from one side of the joint to the other.

Solid protein solder compositions useful in strip soldering typically comprise between 120 and 230 mass% protein relative to water. Preferably the strip comprises 170 to 230% by mass protein and more preferably about 210% by mass.

It will be understood that different proteins will have different degrees of solubility in water or appropriate solutions which, in turn, will affect the optimal concentration of protein in the composition for different protein welds. Appropriate ranges for particular proteins in both solid and fluid welds can be determined based on the known properties of the proteins.

Typically, the solid protein solder composition is provided as a preformed strip. Solid solder strips are easier to handle than fluid solders. Under the wet conditions inherent in surgery, fluid welds can run making it difficult for the laser to denature the weld before it has spread. Solid solder strips can have a similar or stiffer consistency. Typically they are placed across the junction with microforceps. In one form of the invention, the solder strips are envisioned to be substantially rectangular in shape. However, differently shaped strips may be required in different repair situations. It may also be desired to provide a plurality of strips bonded together for effective repair of a large, or substantial, number of repair sites.

The protein solder can comprise a single protein of which albumin is a typical example or alternatively the solder can comprise more than one protein.

Albumin exhibits desirable qualities for the formation of solid solder strips since it has a high proportion of β sheet structure, which gives the strips rigidity. Fibrin is another example of a protein with significant β-sheet structure. Incorporating α-helical protein into the weld can help to make the strips more malleable and therefore retain a smoother profile that is particularly well suited for joining rib ends. An example of a suitable proportion of α-helical protein is between 1 and 10% by weight of the protein used. About 5% is a preferred amount. Collagen, tropoelastin, and elastin are examples of suitable α-helical proteins.

The protein used in welding is selected to minimize the risk of adverse host reactions and should therefore preferably be an autologous protein for the host or a foreign protein of low antigenicity.

Proteins can be obtained from any suitable source. Recombinantly or synthetically produced proteins can be used as well as purified naturally occurring proteins.

Preferably, when welding with a laser that produces energy at a suitable wavelength is to be used, the composition includes a substance, such as a colorant, that absorbs energy at the wavelength produced by the laser with which it is to be used. use solder. It is preferable to choose the combination such that the dye or other substance absorbs the energy transmitted by the laser efficiently but the underlying tissue to be bonded absorbs the energy transmitted poorly. The colorant or other substance favors making the solder specific to the solder used which, in turn, helps minimize accidental tissue heating damage to the underlying tissue.

The binding procedure, where protein welds are used, features protein molecules that are available for cross-linking. This occurs when protein molecules are unfolded. In laser irradiation of, for example, a solder containing albumin and indocyanine green at a junction of nerve tissue, albumin molecules are heated through energy transfer from the indocyanine green molecules, allowing unfold and bond to each other and to the immediate tissue surface such as the fasciculus membrane.

Dyes that contrast with the tissues being repaired may also be helpful in making the weld easier to see. An example of a colorant with this property is Indocyanine Green.

When the laser used is a CO2 laser, a dye will not favor the transfer of energy, since the transfer of energy is by absorption of water.

The energy provided by the power source should be sufficient to bond the solder, to form the solder while minimizing damage to the underlying tissue. The temperature required to denature a protein weld is typically at least 50 ° C and can exceed 100 ° C. A preferred range is 50 ° to 90 ° C. A particularly preferred range is 80 ° to 90 ° C.

The treatment time for each bond to be made may vary depending on factors such as environmental conditions, altitude, and of course the nature of the tissue to be bonded. The duration of treatment is typically short. A 30 second pass per laser treatment of a 0.4 mg strip is an example of the time involved, although it will be understood that shorter or longer treatment times may be required. It will be understood that the weld

ES 2 201 167 T3 solid takes longer to denature than fluid solder.

In a second aspect the present invention provides a protein tissue welding composition comprising water soluble protein and a suitable water containing solvent for the protein.

In a third aspect the present invention provides a kit for use in tissue bonding comprising, in a preferably sterile package, a plurality of protein tissue welding strips and / or shapes of the second aspect of the invention. Preferably a plurality of strip lengths and / or shape sizes are included in the package.

The kit preferably includes means for sterile handling of the strips. The kit also preferably includes means for measuring the strips.

The kit may also comprise a power source such as a fiber coupled laser system.

Brief description of the drawings

Figure 1 shows the structure of a peripheral nerve in schematic form.

Figure 2 shows the attachment of a peripheral nerve by prior art microsuturing techniques.

Figures 3 a) and b) show in schematic form joining of a bundle of ribs with a) fluid welding and b) solid strips.

Figure 4 shows the repair site of a 0.3 mm diameter tibial nerve immediately after: a) diode laser strip welding, and b) microsuturing.

Figure 5a shows a rat tibial nerve weld by the laser welding methods of the present invention. The weld and membrane are denatured but no significant axon change has occurred (Giemsa x100).

Figure 5b shows a rat sciatic nerve attached by microsaturation using 10-0 nylon. Localized axonal and perineural damage occurs.

Figure 6 schematically shows the joining of a blood vessel using internal biodegradable solid solder cylinder and external solid solder strips.

Figure 7 schematically shows a cross section of a repaired nerve bundle.

Figure 8 shows the method used to measure the tensile strength of repaired ribs.

Figure 9 shows a solid welding strip positioned on a rat tibial nerve cut just prior to laser welding.

Figure 10a shows regeneration of myelinated axons in a laser nerve repair that has regenerated for 3 months.

Figure 10b shows fibrous tissue around a suture in a sutured nerve that has regenerated for 3 months.

Figure 11 shows muscle action potential results for repaired nerves.

Best method of carrying out the invention

Tissue repair is carried out using a laser to activate a protein weld applied across the edges of the tissue to be joined. This weld is denatured under laser irradiation and bonds with itself and the immediate membrane to form the bond. The procedure is shown schematically in Figures 3 and 7 for a nerve bundle repair. The solder is applied in longitudinal strips from one side of the joint to the other.

Nerve repair

Repair of severed nerve tissue is accomplished by placing weld across the cut edges and exposing the weld to laser as described above. To repair nerve tissue without damaging the nerve contents it is desirable to avoid concentrating the weld on the edges, as the extruded nerve contents can be damaged. Rather, the weld should be distributed across the planar or tubular surface on which the discontinuity lies.

Hollow Body Tube Repair

When repairing hollow body tubes it is preferable to insert an internal welding cylinder into the tube so that the discontinuity lies in the middle. The cut ends of the tube are placed on opposite ends of the welding cylinder. The arrangement is shown in Figure 6. Laser treatment can then be carried out to cause the cylinder to bond to the tube being attached while supporting the arrangement in place. If there is a good fit between the tube and the cylinder this laser stage may not be required. The joint is completed by the addition of external welding as for rib repairs.

Tissue surface repairs

Surfaces, such as the peritoneum, are tissue planes where sutureless joints can be achieved by applying solder across the discontinuities to be joined and soldered as described above. In this case it is less important to avoid concentration of solder on the edges.

Laser and welding system suitable for fasicular nerve repair

To denature the protein weld, a GaAs / GaA1As laser diode with a nominal power of 250 mW (Spectra Diode Labs, San Jose, California) is used. The laser light is coupled onto a 100 µ / m diameter core optical fiber that is manually supported on a fiber support. The diode is operated continuously at 75 mW during laser welding. Because this laser is Class 3b, and is not safe for the eyes, protective goggles must be worn at all times when using this laser.

A suitable protein solder is a mixture of water, albumin, and indocyanine green dye (ICG) (Becton Dickinson, Missouri). Indocyanine green has a maximum absorption coefficient at a wavelength of 805 nm of 2 x 10⁵M^-1 cm^-1 . The percentages of albumin and dye compared to water were 110% and 0.6% respectively for fluid welding. 210% albumen was used in the preparation of solder strips. It is noteworthy that the ICG dye appears to preferentially bind with albumin ensuring that heat is efficiently transferred to denature the protein solder.

Laser welding technique

When performing surgery, a surgical microscope or some form of magnification is preferable. For a laser weld repair of a tubular joint, a portion of thin gauze material is placed under the joint to aid in a rotational technique. The fabric edges are prepared from

ES 2 201 167 T3 according to standard techniques for the type of tissue and geometry of the repair.

Using microforceps, the edges are aligned and stitched together. A 2mm long strip of fluid solder is "painted" lengthwise across the edge joint using a 30 gauge needle freshly coated on the solder. Alternatively a strip weld is placed across the joint using microforceps. The solid strip repair method is simpler. A solid strip is supported on special microforceps and placed across the joint parallel to the length of the structures to be joined. The laser output is then directed to the solid strip and the solid solder changes color indicating denaturation, which causes it to adhere to the underlying tissue membrane. The procedure is repeated with additional strips to ensure a strong surface bond.

The diode laser output from 100 µm optical fiber is then used in a continuous 30 second pass to denature solid solder into a strip solder. At a diode output power of 75 mW, the solid solder strip turns brown on the surface and opaque underneath from the single pass, indicating denaturation. When fluid soldering is used denaturation occurs more quickly. A two second laser pass may be sufficient to denature the fluid weld. Typically a second layer of fluid solder is applied to the strip to increase the strength of the solder and the two second laser pass is repeated. The gauze is then used under the seam with the microforceps to rotate the seam so that other strips can be applied.

Preparation of fluid protein solder

Composition

Albumin (fraction V powder from Sigma, St. Louis, Missouri) at least 100% to 110% by weight compared to water.

Indocyanine green (Becton Dickinson, Missouri) about 0.6% by weight compared to water.

- Water (injection grade)

Procedure: A solution of ICG in water was prepared in a minitube. Albumin was added to the tube. Albumin and solution were mixed using a vortex mixer. This causes the structure of the protein to change leading to the binding of protein molecules to one another rather than to water molecules.

Solid protein solder preparation

Composition

Albumin (fraction V powder from Sigma, St. Louis, Missouri) 210% by weight compared to water.

Indocyanine green (Becton Dickinson, Missouri) about 0.6% by weight compared to water.

- Water (injection grade)

Procedure: The ICG was dissolved in the water and the albumin was added to this solution in a minitube. This combination was mixed using a vortex mixer and a needle. The combination was mixed (for about 3 minutes) until a homogeneous, malleable, green paste was made. The phase of the mixture changed under this mixing technique to provide a nearly solid composition, with protein primarily for protein binding rather than protein for water binding. The system was no longer a solution at this stage. The protein paste was pliable and can be shredded for up to about 30 minutes after mixing.

After this time the paste hardened due to dehydration and became too hard to cut.

The resulting strips were between 50 and 100 µm thick, approximately 0.6 mm wide, and 1.5 to 3.5 mm long. It will be understood that where the strips are used in composing rib fascicles, the desired width and length are dictated by the dimensions of the fascicles. The width, thickness and length mentioned herein are suitable for use with a rat tibial nerve having a diameter of 0.2 to 0.8 mm. The ratio of strip width to rib circumference is typically:

Width ~ 1/5 circumference

Example 1

A 75 mW diode laser coupled with 100 μm core optical fiber operating at a wavelength of 800 nm, along with a protein solder has been used to make solder cut rat tibial nerve strips, reducing the long time required for microsurgical nerve repair. Welding is produced by selective laser denaturation of protein-based weld containing the indocyanine green dye. The operating time for laser welding was 10 +/- 5 min. (n = 24) compared with 23 +/- 9 min (n = 13) for microsuturation. The laser welding technique resulted in patent welds with a tensile strength of 15 +/- 5 g, while the microsutured ribs had a tensile strength of 40 +/- 10 g. Pathological histology of the laser welded nerves, conducted immediately after surgery, indicated adhesion of the weld to the outer membrane, with minimal damage to the inner axons of the nerves. An in vivo study, with a total of fifty-seven adult male wistar rats, compared tibial nerves repaired by laser welding with conventional microsuture repair. Twenty-four laser welded nerves and thirteen sutured nerves were characterized at three months and showed successful regeneration with mean Composite Muscle Action Potential (CMAP) of 2.4 +/- 0.7 mV and 2.7 +/- 0.8 mV , respectively. The pathological histology of the in vivo study confirmed the comparable regeneration of axons in nerves operated with laser and suture. A faster, less damaging, and longer lasting laser-based anastomotic technique is presented.

Materials and methods

1. Animals

A total of fifty-seven young adult male Wistar rats, weighing between 400 and 550 g initially, were used in this study. Thirty-four rats received laser weld repair and the remaining twenty-three received standard microsuture repair, as detailed below. Five rats from each repair method were used for measurements of tensile strength and light microscopy immediately after surgery and subjected to 6

ES 2 201 167 T3 took the remaining thirty-seven rats to a functional recovery study using electrophysiology and pathological histology.

two. Laser welding system

To denature the protein weld, a GaAs / GaA1As laser diode with a nominal power of 250 mW (Spectra Diode Labs, San Jose, California) was used. The laser light was coupled onto a 100 µm diameter core optical fiber that was manually supported on a fiber support. The diode laser was mounted on a heat sink and the diode current and temperature were controlled by an SDL-800 conductive diode. The diode was operated continuously at 75 mW during laser welding, corresponding to a maximum power density of 955 W / cm in the tissue. The laser output power was measured with a Scientech current meter (Boulder, Colorado). Because this laser is Class 3b, and not safe for the eyes, protective goggles were worn at all times when using this laser.

The solder used in this study was an albumin-based protein mixture, which also contained indocyanine green dye (ICG) (Becton Dickinson, Missouri). Indocyanine green has a maximum absorption coefficient at a wavelength of 805 nm of 2 x 10⁵ M^-1 cm^-1. It is noteworthy that this dye appears to preferentially bind with proteins ensuring that heat is efficiently transferred to denature the protein solder.

3. Surgery

Anesthesia was maintained during surgery using a mixture containing fluothane (4% during induction, 2% after) in O2 (1l / min). Using an OPMI 7 surgical microscope (Zeiss, West Germany) the sciatic nerve of the left leg was exposed at the sciatic notch so that the branches of the nerve could be distinguished. The tibial branch, just below the sciatic notch, was exposed from the surrounding subcutaneous tissue for a length of 1 cm. For a laser weld repair, a portion of thin gauze material was placed under the tibial nerve to promote rotation of the nerve, and for suture repair, a portion of plastic was placed under the nerve to allow easier suturing. The tibial nerve was then cut with serrated microscissors and left for 3 minutes for normal axoplasm extrusion to take place. This was then cut with the serrated microscissors as required, after which the nerve was repaired with either four laser welding strips or four 10-0 perineurial sutures.

The laser welding method involved aligning both stumps of the cut nerve with microforceps, then a 2 mm long strip of weld was "painted" lengthwise across the junction of the cut ends using a gauge needle. 30 recently coated on the weld (Figure 3a). The diode laser output from the 100 µ / m optical fiber was then used, in a continuous two second pass, to denature the weld into a strip weld. At a diode output power of 75 mW, the solder was observed to turn brown on the surface and opaque underneath from the single pass, indicating denaturation. A second layer of solder was applied to the strip and the two second laser pass was repeated. The gauze was then used under the nerve with the microforceps to rotate the nerve so that three other two-layer strips could be applied, each approximately 90 ° apart.

Seven rats were operated on with a more advanced version of the organic solder, which was still an albumin-based protein mixture but had the advantage of being dehydrated and cut into solid rectangular strips (Figure 9). The average surface area of ​​the solder strips was 1.5 +/- 0.5 mm² and the thickness was 0.15 +/- 0.01 mm. Four strips were placed along the tibial anastomized nerve and then irradiated with the same procedure adopted for fluid welding. The solid strip was fused with the perineurium of the tibial nerve by laser radiation, joining the extremities of the severed nerve.

For the entire operation, the anastomosis time was recorded and a photographic record was taken for later reference. The animals were placed in their cages without restriction of movement for 3 months.

Four. Immediate measurement of tensile strength and pathological histology

In ten of the operated rats, the 1 cm long portion of the laser repaired nerves was harvested and sutured immediately for tensile strength measurements. Fine silk was tied to each end of the tibial nerve. One end was then attached to a calibrated force transducer (FT30C, Grass Instruments, Quincy, Mass) and the other to a screw-driven translator (Figure 8). As the screw was turned the translator would stretch the nerve slowly and firmly. The applied voltage was observed on an oscilloscope connected to the output of the force transducer. Tension was applied until the nerve was cut, and the breaking force was recorded. The ribs were kept moist, since in drying, the tensile strength can be increased.

For optical microscopy, the tibial nerve anastomosis site was fixed in 5% formalin, dehydrated alcohol, embedded in paraffin, longitudinally sectioned and marked with either Masson or Giemsa trichrome.

5. Functional assessment: pathological histology and electrophysiology

Three months after the operation, the rats were re-anesthetized using the method described in section

3. The site was exposed and the tibial nerve anastomosis was observed. The other two branches of the sciatic nerve, the peroneal and sural nerves, were then cut so that only the tibial nerve branch of the sciatic nerve could conduct electrical stimulation of the sciatic nerve to the rear foot muscles. Two days later the rats were placed on their side and isolated from the table by a folded surgical bandage. An infrared lamp was used to keep her rectal temperature above 36 ° C.

A clinical electromyograph (Cadwell Sierra EMG / EP) was used for stimulation and recording. Two 25 to 10 mm gauge stimulation electrodes were placed separately on each side of the sciatic nerve above the sciatic notch, near the hip. The nerve was activated using rectangular pulses (0.1 to 0.3 ms, 0 to 30 mA, 1 Hz). Compound muscle action potentials (CMAPs) were recorded from the plantar muscles of the foot in response to supramaximal stimulation of the sciatic nerve. A series of three recording electrodes was used. A 25-gauge ground electrode was inserted subcutaneously between the electrodes.^1,2 stimulation and recording. I know

ES 2 201 167 T3 inserted a 30 gauge reference electrode into the end area of ​​the heel and a 30 gauge recording electrode was inserted into the plantar muscles of the foot. CMAPs were recorded and processed to determine their negative wave peak value.

Pathological histology of the sutured and laser-welded nerves was carried out, after electrophysiological testing with the same procedure that was adopted in section 4.

Results

At the completion of the surgery, all the anastomoses were successful. The operating procedure was found to be easier by laser welding than by microsuturing. This resulted in shorter operating times for laser weld repairs {10 ± 5 min (n = 24)} than {23 ± 9 min (n = 13)} for microsuture repairs. The tensile strength of five ribs repaired by laser welding, immediately after the operation, was 15 ± 5 g and the tensile strength of the microsutured ribs, 40 ± 10 g.

Histopathological examination of the anastomotic sites immediately after surgery demonstrated that the albumin-based laser welding and ICG dye bonded well with the outer membrane of the nerve, the perineurium, while the internal axons remained unheated. In Figure 10a, a tibial nerve bundle weld produced by ICG albumin / dye diode laser welding is shown in section. Both the protein solder and the perineurium had been denatured forming the bond. On the underside of the link, the axoplasm had its normal wavy structure. Note that since the heating was concentrated in the dye, only denaturation of the weld and adjacent perineurium occurred.

One of the promising aspects of laser anastomosis is the potential for reduced damage to the axoplasm by eliminating the need for sutures. Figure 5b shows a section showing the effect of microsuturing nerve bundles using 10-0 nylon. This Giemsa-marked cut indicates axon extrusion at the junction, as well as localized axonal and perineurial damage due to the suture.

Pathological histology at 3 months showed regeneration of myelinated axons in laser nerve repairs (Figure 10a), without discontinuity of either the fibers and their envelopes or the fibrous perineurium. No evidence of inflammation or myelin phagocytosis was seen. Complete restoration, as assessed by light microscopy, of the histological integrity of the tibial nerve had been achieved by laser welding.

The sutured nerves also showed successful anastomosis with regeneration of myelinated axons, however, it was still evident that the nylon thread was surrounded by fibrous tissue, which created an obstacle to the directionality of the regenerated axons (Figure 10b).

The electrophysiological measurements of the in vivo study were performed on twenty-four rats repaired by laser welding and thirteen rats repaired by microsuture with three months of recovery. Of this group, all twenty-four laser weld anastomoses were patent as were the thirteen microsuture anastomoses. The mean amplitude of muscle action potentials resulting from supramaximal nerve stimulation above the repair site was 2.4 +/- 0.7 mV for the twenty-four laser-welded tibial nerves and 2.7 +/- 0.8 mV for the thirteen microsutured nerves. The normal muscle action potential produced by supramaximally tibial nerve stimulation was recorded at 8.7 ± 3 mV from ten rats (Figure 11). Discussion

Clinically, when a major peripheral nerve is cut, forty or more bundles may need to be reattached individually. With three or four microsutures per fascicle, suturing tends to be lengthy, as it must be meticulous. In a nerve graft, where two anastomoses are needed, the suturing time is doubled. We have searched for a suitable method of nerve anastomosis that can at least duplicate the end result but was significantly faster than the present hand-stitched microsuture technique. A plus of the described laser welding method was the demonstrated lack of change in axonal components below the denatured perineurial layer seen immediately after surgery. Three months later comparable regeneration was demonstrated by electrophysiological nerve conduction studies. Industrial applicability

The present invention has application in the field of surgery where it is applicable in joining tissue edges together, in end-to-end, side-to-end and side-to-side applications.

References

1) R. Malik, S. Ho and DB Church: A new method for recording and analyzing evoked motor potential from dogs. Journal of Small Animal Practice (1989) 30, 13-19.

2) R. Malik, S. Ho: Motor nerve conduction parameters in the cat. Journal of Small Animal Practice (1989) 30, 396-400.

3) Laser activated protein bands for peripheral nerve repair. A Lauto, R Trickett, R Malik, J Dawes, E Owen. European Biomedical Optical Week BIOS Europe 195. September 12-16, 1995 (Press Release).