METHODS FOR ADHERING TISSUE SURFACES AND MATERIALS AND BIOMEDICAL USES THEREOF

Abstract

The present invention relates to methods for adhering tissue surfaces and materials and biomedical uses thereof. In particular the present invention relates to a method for adhering a first tissue surface to a second tissue surface in a subject in need thereof, comprising the steps of adsorbing a layer of nanoparticles on at least one of the tissue surfaces, and approximating the surfaces for a time sufficient for allowing the surfaces to adhere to each other. The present invention also relates to a method for adhering a material to a biological tissue in a subject in need thereof, comprising the steps of adsorbing a layer of nanoparticles on the surface of the material and/or the biological tissue and approximating the material and the biological tissue for a time sufficient for allowing the material and the biological tissue to adhere to each other.

Claims

1. A method for adhering a biological tissue surface to a second surface in a subject in need thereof comprising the steps of adsorbing a layer of nanoparticles on at least one of the biological tissue surface and the second surface and bringing the biological tissue surface and the second surface into contact for a time sufficient for the biological tissue surface and the second surface to adhere to each other.

2. The method of claim 1, wherein the biological tissue is selected from the group consisting of skin tissue, hair tissue, nail tissue, corneal tissue, tongue tissue, oral cavity tissue, esophageal tissue, anal tissue, urethral tissue, vaginal tissue, urinary epithelial tissue, salivary gland tissue, mammary gland tissue, lacrimal gland tissue, sweat gland tissue, prostate gland tissue, bulbourethral gland tissue, Bartholin's gland tissue, uterine tissue, respiratory and gastrointestinal tract goblet cell tissue, gastric mucosal tissue, gastric gland tissue, pancreatic tissue, spleen tissue, pulmonary tissue, pituitary gland tissue, thyroid gland tissue, parathyroid gland tissue, testicular tissue, ovarian tissue, respiratory gland tissue, gastrointestinal gland tissue, adrenal gland tissue, renal tissue, liver tissue, adipose tissue, duct cell tissue, gall bladder tissue, epidydimal tissue, vas deferens tissue, blood vessel tissue, lymph gland tissue, lymphatic duct tissue, synovial tissue, serosal tissue, squamous tissue, cochlear tissue, choroid plexus tissue, ependymal tissue, dural tissue, pia-arachnoid tissue, sclera tissue, retinal tissue, iris tissue, ciliary tissue, dental tissue, otic tissue, ligament tissue, tendon tissue, elastic cartilage tissue, fibrocartilage tissue, hyaline cartilage tissue, bone marrow tissue, intervertebral disc tissue, compact bone tissue, cancellous bone tissue, skeletal muscle tissue, cardiac muscle tissue, smooth muscle tissue, cardiac valve tissue, pericardial tissue, pleural tissue, peritoneal tissue, blood cell tissue, neuronal tissue, glial tissue, sensory transducer cell tissue, pain sensitive tissue, autonomic neuron tissue, peripheral nervous system tissue, cranial nerve tissue, ocular lens tissue, germ cell tissue, thymus tissue, placental tissue, fetal membrane tissue, umbilical tissue, stem cell tissue, mesodermal tissue, ectodermal tissue, endodermal tissue, autologous tissue, and allograft tissue or a combination thereof.

3. The method of claim 1, wherein the second surface is a material to be adsorbed to the biological tissue surface.

4. The method of claim 3, wherein the material is selected form the group consisting of membranes, scaffold materials, films, sheets, tapes, patches, meshes and medical devices.

5. The method of claim 3, wherein the material is made of metal, glass or polymers.

6. The method of claim 3, wherein the material is a hydrogel.

7. The method of claim 3, wherein the material comprises a biologically active agent, a pharmaceutical agent or a radiosensitizer.

8. The method of claim 3, wherein the material is loaded with a plurality of cells.

9. The method according to claim 1, wherein the nanoparticles are metal oxides, alumina, silica, kaolin, hydroxyapatite, calcium carbonate, silicates such as micas quartz, zeolites or clays such as hectorite, laponite, montmorillonite, bentonite, or smectite.

10. The method according to claim 1, wherein the nanoparticles are metal particles.

11. The method according to claim 1, wherein the nanoparticles are made of organic polymers.

12. The method according to claim 1, wherein the nanoparticles are made of polysaccharides.

13. The method according to claim 1, wherein the nanoparticles are applied as an aqueous suspension of nanoparticles.

14. The method according to claim 1, wherein the nanoparticles are applied as a suspension containing a solvent.

15. The method according to claim 1, wherein the nanoparticles are applied as a powder.

16. The method according to claim 1, wherein the nanoparticles are applied with a technique selected from the group consisting of coating, dipping, spraying, spreading and solvent casting.

17. The method according to claim 1, wherein the nanoparticles are deposited on the tissue with a means selected from the group consisting of a patch, a dressing, an elastoplast and a band-aid having a plurality of capsules able to release the nanoparticles when contacted by tissue.

18. The method according to claim 3, wherein the nanoparticles are previously adsorbed on the surface of the material.

19. The method according to claim 1, wherein the second surface is a biological tissue and the biological tissue surface and the second surface are connected by sutures, staples, mechanical fixators, or mesh.

20. The method according to claim 1, wherein one or both of said nanoparticles and said second surface includes a drug to control bleeding, treat infection or malignancy, or promote tissue regeneration.

Description

FIGURES

[0150] FIGS. 1A-1B. The preparation of nanoparticles and heart drug delivery system. A) A biodegradable porous polysaccharide 3D-matrix glued on rat heart. B) Macroscopic view after 3 days post-surgery. The 3D-matrix was still present glued on the heart and partially degraded as expected. Scale bar: 6 mm.

[0151] FIG. 2. Wound liver repair. A 1 cm transversal wound was made on the right lobe liver and then was bound with the preparation of nanoparticles. Macroscopic view after 3 days post-surgery of liver. The arrows shown a thin horizontal scar. No visible adhesions or inflammation were observed at the site of the injury.

[0152] FIGS. 3A-3C. Peritoneum and mesh or drug delivery system. A crosslinked PVA sheet (A; blue arrows) or 3D-matrix (B; green arrow) was glued with a drop of the preparation of nanoparticles on parietal-peritoneum. C) PVA-sheet could not be removed by forceps. Scale bar: 6 mm.

[0153] FIGS. 4A-4E. Effect of the preparation of nanoparticles on intestine. A 10 cm jejunum was harvested from rat (A), mounted on a plastic tube (B) and was cut into two parts (C). Then the preparation of nanoparticles was applied on the section (D), bounded together (D). After 1 minute, the system was removed from the container with a forceps. As shown in (E), the anastomosis was bounded by the preparation of nanoparticles.

[0154] FIG. 5. The preparation of nanoparticles in skin wound repair. A 1 cm length horizontal incision was made on both left side and right side of the dorsal midline with a scalpel. The edges of the wound were glued and sealed with the preparation of nanoparticles (left side) or sutured (Ethicon 4/0). At Day 3 post-surgery, no wound leakages, infection or inflammatory reactions were observed with the preparation of nanoparticles. The macroscopic skin scar are similar in both wound closure treatment.

[0155] FIG. 6. Effect of the preparation of nanoparticles on intestine small intestine. A piece of 10 cm jejunum was harvested from rat and cut in two parts. A first part was open and a drop of the preparation of nanoparticles was placed onto the last 2 cm of the intestine. Then 2 cm from the second intestine sample was applied to the preparation of nanoparticles. The two parts of tissue are stuck together and not separated in during the traction test.

[0156] FIG. 7. In order to evaluate the effect of the preparation of nanoparticles on spleen repair, the rat spleen removed and was cut vertically in two parts, then one section was coated with the preparation of nanoparticles and the two edges were brought together during 1 min. As shown in the figure, the two edges were glued together.

[0157] FIG. 8. shows the NP-glue or Fe2O3 NP-glue macroscopic final results.

[0158] FIGS. 9A-9D. Hepatic resection. The right hepatic lobe was exposed (FIG. 9A) and a ⅔ of the lobe was totally transversally cutted (FIGS. 9B and 9C). The section was then covered by the NP-glue-coated PVA membrane (FIG. 9D).

[0159] FIG. 10 shows a construction composed by 2 layers of SIS matrix, then a 3D-matrix, and a layer of SIS.

[0160] FIG. 11 shows that the traction of the construct of FIG. 10 with a forceps did not remove the structure.

[0161] FIG. 12 shows the fixation of a mesh and a SIS® membrane.

[0162] FIG. 13 shows the preparation of Fe2O3-nanoparticles in skin wound repair.

[0163] FIG. 14 shows that the Fe2O3 particles were observed at the site of the Fe2O3-treatment in comparison with the sutured-wound where no particles were shown

[0164] FIG. 15 shows the comparison of NP-glue and Dermabond® treatment in skin wound repair.

[0165] FIG. 16: Comparison of repair of full-thickness skin injury by SiO2NP nanobridging, by suturing, and by cyanoacrylate glue. Horizontal incisions were made at the dorsal face of rats with a scalpel. SiO2NP solution drop was put onto a wound edge with a brush and the two wound edges were gently pressed into contact for about a minute. The permanent closure was achieved within a minute. At day 3 post-surgery, no wound leakages, infection or inflammatory reactions were observed after nanobridging with SiO2NP. The other wounds were closed with a non-resorbable suture (Ethicon 4/0) and 2-octyl cyanoacrylate (Dermabond®). The rat skin closure quality achieved with nanoparticles and the suture were comparable. For the cyanoacrylate glue, the wound edges were not bonded correctly and an inflammatory skin reaction was noted at the time of the surgery for glue. Scale bars 0.5 cm.

[0166] FIG. 17. Skin wound closure with Fe2O3NP solution. A full-thickness skin lesion was made at the dorsal face of rats and 4 μL of Fe2O3NP solution was soaked onto one edge of the wound. Two wound edges were gently pressed into contact for about a minute. A thin and aesthetic scar was observed. At day 7 post-injury, the histological sections stained with Hematoxylin Phloxin and Saffron evidenced the site of the injury as only a very thin line (blue arrows). Magnification (inset) of this area revealed a normal repair process and some particle aggregates along the wound closure.

[0167] FIG. 18. Liver injury repair with Ludox® TM50 silica nanoparticles. A 6 mm deep horizontal incision was performed with a scalpel on a right hepatic rat lobe and nanoparticles solution was deposited to the bleeding injury with a pipette then the edges of the wound were brought together. After about 1 min, hemostasis was complete. Three days post-injury, macroscopic examination of the liver surface showed a thin fibrotic line at the site of the injury (Inset, blue arrows). Histological cross section of the wound (Hematoxylin Phloxin and Saffron stain), showed the formation of tissue repair from the liver surface (blue arrow) and along the wound (white dotted line)

EXAMPLE 1. SILICA PARTICLES TM50 AQUEOUS DISPERSIONS

[0168] Silica Ludox® TM-50 water solutions with concentration of 52 wt % at pH 9 and SiO2/Na.sub.2O ratio of 200-250 and radius of about 15 nm, were purchased from Aldrich and used as received.

EXAMPLE 2. SILICA PARTICLES AL30 (SILICA USED AS A POWDER AND AS A 30% AQUEOUS SOLUTION)

[0169] Silica AL30 particles were synthesized by hydrolysis and condensation of tetraethylorthosilicate (TEOS, 99+%) following a procedure adapted from Stöber (Stöber, W., Fink, A. & Bohn, E. Controlled growth of monodisperse silica spheres in the micron size range. J. Colloid Interface Sci. 26, 62-69 (1968)). 600 mL of absolute ethanol and 36 mL of ammonium hydroxide solution (35 wt. % in water) were added to a round bottom flask and stirred for 5 min. 18 mL of TEOS were then quickly poured and the resulting solution was stirred overnight at room temperature. Silica particles were retrieved by centrifugation (7600 rpm, 45 min) and washed with absolute ethanol and followed by four cycles of centrifugation-dispersion. Silica particles were eventually air dried over 6 hrs at 80° C. Particles characterization was performed using dynamic light scattering (DLS) and transmission electron microscopy (TEM). The particles hydrodynamic radius (DLS) was 80 nm and the polydispersity index 15%. The radius determined from TEM images analysis was 50 nm.

[0170] DLS measurements were performed with an ALV/CGS-3 Compact Goniometer System equipped with a 22 mW HeNe Laser on diluted dispersion (2.7 mg/100 mL) in deionized water. The detection angle was varied from 30° to 150° with a 10° step. TEM images were obtained with a Zeiss CEM 902 microscope. The observation was made on a 1 wt. % dispersion in deionized water on a 400 mesh copper grid with a carbon film.

EXAMPLE 3. Fe.SUB.2./O.SUB.3 .NANOPARTICLES

[0171] Fe.sub.2O.sub.3 magnetic nanoparticles of 20-40 nm diameter and surface area equal to 30-60 m.sup.2/g were purchased from Alfa Aesar (Iron III oxide, magnetic NanoArc®) were processed with citric acid following a slightly modified procedure previously reported in the literature by Pinho et al. (ACS Nano, 2010, 4, 5339-5349).

[0172] In particular, 0.5067 g of Fe.sub.2O.sub.3 nanoparticles were dispersed in 8 mL of milli-Q water via ultrasonication for 5 minutes. The dispersion was subsequently transferred in a glass reactor equipped with a glass anchor-shaped stirrer that was preloaded with 100 mL of 0.02 M citric acid and was left under mechanical stirring overnight. Following the collection of the particles in a flas, they were decanted using a magnet and washed three times with milli-Q water. The citrated particles were re-dispersed in 12 mL milli-Q water via ultrasonication and were peptized via the addition of 40 μL of 35 w/w NH.sub.4OH aqueous solution. The pH of the supernatant solution measured using a pH meter paper was found to be between 7 and 7.5.

EXAMPLE 4: NANO-HYDROXYAPATITE NANOPARTICULES

[0173] Nano-hydroxyapatite nanoparticules suitable for tissue engineering, in situ tissue regeneration, as well as for drug delivery such as described in patent WO/2012/028620, PCT/EP2011/064924 for bone reconstruction and by J C Fricain et al. in Biomaterials 2013 Volume 34, Issue 12, April 2013, Pages 2947-2959 (A nano-hydroxyapatite—Pullulan/dextran polysaccharide composite macroporous material for bone tissue engineering). These nanoparticles can be used directly as components of NP-Glue alone, or on the surface of a medical device/biomaterial/tissue engineering product. They can be chemical modified or doped with several elements such as magnesium or strontium as reported by Brook I et al in J Biomater Appl. 2012 September; 27(3):291-8, or by Hao Y et al in J Nanosci Nanotechnol. 2012 January; 12(1):207-12.

[0174] Briefly, nHA is synthesized by wet chemical precipitation at room temperature. Fifty-nanometer nHA nanoparticles were obtained and characterized by using transmission electron microscopy, Fourier-transformed infrared spectroscopy and X-ray diffraction.

EXAMPLE 5: LIVER INJURY

[0175] Traumatic injury or surgery may trigger extensive bleeding. However, conventional hemostatic methods have limited efficacy and may cause surrounding tissue damage. For cyanoacrylate, the side effects include a systemic inflammatory reaction to foreign body, ie, pain and fever, local tissue necrosis and inflammatory reaction to foreign body, thromboembolic complications and septic complications. In addition, adherence of the needle to wall of varix and occlusion of the sclerotherapy catheter by residual adhesive has been reported. In contrast, fibrin sealants have the advantage of being biocompatible and biodegradable. The fibrin clot is resorbed as a part of the normal wound-healing process. As such, they are generally not associated with inflammation, foreign body reactions, tissue necrosis, or extensive fibrosis.

[0176] In this study, we use the preparation of nanoparticles of Example 1 to evaluate the ability of this device in hemostasis and liver or spleen tissue regeneration.

[0177] Both the procedure and the animal treatment complied with the Principles of Laboratory Animal Care formulated by the French National Society for Medical Research. Male Wistar rats (8 weeks) were anesthetized with sodium pentobarbital solution. A ventral midline laparotomy (5 cm) was performed. The right hepatic lobe was exposed and a 1 cm horizontal injury was performed with a scalpel and the preparation of nanoparticles was applied on the section. The two edges were brought together during 1 min and then the laparotomy was closed in two layers with a Vicryl 4/0. The animal was monitored during the acute post-surgery, in order to detect a bleeding syndrome. After 3 days post-surgery, animals were euthanized. The liver was exposed, photographed, and the injured area was removed with the surrounding tissue for histological studies.

[0178] Results: No inflammatory reactions or adhesions were observed after 3 days post-surgery. The emplacement of the injury was objective by a thin-scar line.

EXAMPLE 6: 3D SCAFFOLDS FIXATION ON HEART

[0179] The success of cell therapy depends of the ability to ensure the cell delivery within the target tissue. In cardiac or muscle ischemic injury, the research works aims to replace the cell lost. Genes, growth factors and cell-based therapies were developed. In cell-therapy, typical ways to deliver cells are intravenous, intracoronary or endocardial injection. In all case, a limited cell grafting is observed. To overcome this limitation, scaffold-delivery systems were developed. Scaffolds fixation to the infarct-area is a technical challenge. To avoid material slipping, the device was fixed to the heart with sutures or glue such as cyanoacrylate in function of the material composition.

[0180] In order to evaluate the ability of the preparation of nanoparticles to fix a scaffold to the heart and overcome the cytotoxicity of the cyanoacrylate glue, we implanted a 3D-biodegradable polysaccharide scaffold (Le Visage et al., Tissue Engineering 2012, 18(1-2):35-44) in beating heart position in rat with the preparation of nanoparticles of Example 1. In the previous study, the use of a scaffold promoted local cellular engraftment and survival but application was a main limitation in small and large animals (pig was tested).

[0181] Both the procedure and the animal treatment complied with the Principles of Laboratory Animal Care formulated by the French National Society for Medical Research. A 8 weeks Wistar rats were used for this evaluation. After anesthesia, tracheal intubation and mechanical ventilation, the thorax was open, the heart individualized. A 6 mm 3D-polysaccharide scaffold was glued with the preparation of nanoparticles onto the heart. The thorax was then closed (Ethicon 4/0). 3D-scaffold fixation was evaluated after 3 days.

[0182] After 3 days, the macroscopic evaluation does not evidenced inflammation. The 3D-scaffold was still visible onto the heart and the degradation of the polysaccharide scaffold occurred.

EXAMPLE 7: THE PREPARATION OF NANOPARTICLES TISSUE FIXATION

[0183] Intestine

[0184] Anastomotic leakage still remains a major complication in general surgery and can result in significant morbidity, mortality. Several patient related risk factors for leakage of intestinal anastomoses (corticosteroids, perioperative blood transfusion) or technical surgical factors (tight knotting of sutures, staplers, and intraluminal devices) have been identified to influence the outcome of gastrointestinal anastomoses. These considerations lead to the reinforcement of the anastomosis by glue. Tissue adhesive offers a reduction of suture material and, therefore, could ameliorate healing of intestinal anastomoses. Several studies already showed less inflammation and damage as well as a better blood supply in intestinal wound healing without sutures or staples. Different tissue adhesives were used but the outcome was poor due to their toxicity.

[0185] In order to evaluate the effect of the preparation of nanoparticles of Example 1 on intestine small intestine, a piece of 10 cm jejunum was harvested from rat and cut in two equal parts. A first part (5 cm length) was open and a drop of the preparation of nanoparticles was placed onto the last 2 cm of the intestine. Then 2 cm from the second intestine sample was applied to the preparation of nanoparticles. The two parts of tissue are stuck together and not separated in during the traction test.

[0186] Vessel

[0187] The same procedure was performed with vessel. The tissue was open and a drop of the preparation of nanoparticles of Example 1 was placed onto a piece of vessel. Then another part of vessel coated the preparation of nanoparticles. The two parts of tissue are stuck together and not separated in during the traction test.

[0188] Spleen Repair

[0189] In order to evaluate the effect of the preparation of nanoparticles of Example 1 on spleen repair, the rat spleen removed and was cut vertically in two parts, then one section was coated with the preparation of nanoparticles and the two edges were brought together during 1 min. As shown in the figure, the two edges were glued together.

EXAMPLE 8: SCAFFOLDS FIXATION ONTO PERITONEUM

[0190] Abdominal wall repair by an open or laparoscopic approach is one of the most common operations performed in general surgery. Fixation of mesh is typically performed to minimize risk of recurrence either in open or laparoscopic hernia repair. Mesh fixation with staples has been implicated as a cause of chronic inguinal pain (0.7% to 62.9%). This related to reaction of suture fixation, displacement of the mesh. To overcome this side-effect, the use of adhesives for mesh fixation is increasing.

[0191] In order to evaluate the effect of the preparation of nanoparticles of Example 1 onto peritoneum, PVA scaffolds or 3D-polysaccharide matrix were fixed onto ex vivo parietal peritoneum abdominal wall with the preparation of nanoparticles. After 1 min, the scaffold and the 3D matrix could not be removed by the forceps.

EXAMPLE 9: SKIN WOUND CLOSURE

[0192] Wound closure using suture materials is an integral part of the surgical process. Sutures are natural or synthetic textile biomaterials widely used in wound closure, to ligate blood vessels and to draw tissues together. Sutures consist of a fiber or fibrous structure with a metallic needle attached at one of the fiber ends and they can be classified into two broad categories: absorbable and non-absorbable. The most crucial requirements of sutures materials are physical and mechanical properties, handling properties, biocompatibility and antimicrobial nature and all these properties are interrelated. The choice of suture material also impacts on the wound healing process. One additional feature which is esthetically needed in the sutures is the scar prevention. Prevention of scarring is the major challenge to the wound healing process.

[0193] Bioadhesives (natural or synthetic materials) can be used for soft tissue repair to create a seal preventing leakage of biological fluids or to reinforce anatomic integrity. These products are widely used by many surgeons and some dermatological or plastics surgeons as an attractive alternative to sutures and staples, and a convenient and practical wound sealant.

[0194] Both the procedure and the animal treatment complied with the Principles of Laboratory Animal Care formulated by the French National Society for Medical Research. Adult Wistar male rats weighing 250 g (Wi/Wi, Charles-Rivers, France) were anesthetized by intraperitoneal injection of sodium pentobarbital solution (30 mg/kg, Centravet, France). The back was shaved, disinfected, and then draped in a sterile fashion. A 1 cm length horizontal incision was made on both left side and right side of the dorsal midline with a scalpel. The edges of the wound were glued and sealed with the preparation of nanoparticles of Example 1 (left side) or sutured (Ethicon 4/0).

[0195] The wound was observed after 1, 3 and 4 days after surgery. After 4 days, the rats were euthanized by an intraperitoneal injection of sodium pentobarbital (60 mg/kg) and the scar and the 0.5 cm of surrounding tissue was excised, gently rinsed in saline, fixed in a 4% paraformaldehyde solution, dehydrated, and embedded in paraffin. Seven-micron-thick sections were obtained (Leitz Wetzlar microtome, France), stained with Hemalun-eosin, and photographed using Q Capture Pro Software (Qimaging, Canada).

[0196] During the surgical procedure, the preparation of nanoparticles was easily applied on the wound. During the follow-up, no wound leakages, infection or inflammatory reaction were observed.

EXAMPLE 10: PREPARATION OF MEMBRANES

[0197] The NP-Glue powder (EXAMPLE 2) and the Fe2O3 NP-glue (EXAMPLE 3) were poured onto the wet PVA membrane. The powder in excess was removed by a gentle PVA/powder membrane shaking. FIG. 8 shows the NP-glue or Fe2O3 NP-glue macroscopic final results.

EXAMPLE 11: HEPATIC RESECTION

[0198] Hepatic resection has been increasing in frequency in the management of metastatic or primary neoplasms of the liver. Although mortality for this procedure has steadily decreased, the associated morbidity remains high. Morbidity is mainly associated with operative time and blood loss, especially in jaundiced and cirrhotic patients. During hepatic resection, control of bleeding from various sources is the most important problem faced by surgeons.

[0199] In this experiment, we evaluated the ability of a prepared NP-glue-coated PVA membrane (Example 9) to control the bleeding after the hepatectomy. Both the procedure and the animal treatment complied with the Principles of Laboratory Animal Care formulated by the French National Society for Medical Research. Male Wistar rats (8 weeks) were anesthetized with sodium pentobarbital solution. A ventral midline laparotomy (5 cm) was performed. The right hepatic lobe was exposed (FIG. 9A) and a ⅔ of the lobe was totally transversally cutted (FIGS. 9B and 9C). The section was then covered by the NP-glue-coated PVA membrane (FIG. 9D). The bleeding was evaluated and then, the laparotomy was closed in two layers with a Vicryl 4/0. The animal was monitored during the acute post-surgery, in order to detect a bleeding syndrome.

EXAMPLE 12: PREPARATION OF A MULTILAYER

[0200] Tissue constructs are indicated for use in general surgical procedures for the reinforcement and repair of soft tissue where weakness exists, including, but not limited to, defects of the thoracic wall, suture line reinforcement, muscle flap reinforcement, hernia repair, soft tissue reconstructive procedures including plastic and reconstructive surgical applications, and for reinforcement of soft tissues which are repaired by suture or suture anchors.

[0201] They are also used in research 3D cell culture, in order to mimic the natural cell microenvironment, in biomaterial fields or in tissue engineering and regenerative medicine. Generally, a combination of cells/drugs and materials are needed to improve/replace the biological/physiological functions. One technical challenge is the fixation of these construct onto the target organ.

[0202] To evaluate the ability of the NP-glue (Example 2) to fix a 3D system, we elaborate a construction composed by 2 layers of SIS matrix, then a 3D-matrix, and a layer of SIS. The four layers are then bonded together with the NP-glue (Example 2), and implanted subcutaneously using NP-glue as a fixation system.

[0203] The construct was then used in an ex vivo experiment. The device was glued onto the peritoneum with the NP-glue (Example 2). As shown in FIG. 11, traction with a forceps did not remove the structure.

[0204] Same results were observed with mesh. For this experiment the mesh was glued with a NP-glue (Example 2) and then fixed onto peritoneum (FIG. 12). The same procedure was used for the SIS® membrane (FIG. 12).

EXAMPLE 13: PREPARATION OF Fe.SUB.2.0.SUB.3.-NANOPARTICLES IN SKIN WOUND REPAIR

[0205] A 1 cm full-thickness length horizontal incision was made on both left side and right side of the dorsal midline with a scalpel. The edges of the wound were glued with a brush and sealed with the preparation of Fe.sub.20.sub.3-nanoparticles of Example 3 (right side) or sutured (Ethicon 4/0). 1 min after particle deposition, the wound was sealed. At Day 3 post-surgery, no wound leakages, infection or inflammatory reactions were observed with the preparation of nanoparticles. The macroscopic skin scars were similar in both wound closure treatment and the Fe2O3 particles were observed with a 7 Tesla whole body MRI (blue arrow) (FIG. 14). The Fe2O3 particles were observed at the site of the Fe2O3-treatment in comparison with the sutured-wound where no particles were shown (FIG. 15).

EXAMPLE 14: COMPARISON OF NP-GLUE AND DERMABOND® TREATMENT IN SKIN WOUND REPAIR

[0206] After a 1 cm full-thickness length horizontal incision, the wound were glued and sealed with nanoparticles (right side) or plastic surgery clinical (Dermabond®). At Day 3 post-surgery, an inflammatory reaction was observed with the clinical glue. In comparison, the treated wound preparation of nanoparticles was almost repaired.

EXAMPLE 15: ORGAN REPAIR, HEMOSTASIS, AND IN VIVO BONDING OF MEDICAL DEVICES BY AQUEOUS SOLUTIONS OF NANOPARTICLES

Experimental Section

[0207] Silica SiO2NP nanoparticles were prepared using Stöber et al. method.sup.[17] In particular 600 mL of absolute ethanol and 36 mL of ammonium hydroxide solution (35 wt. % in water) were added to a round bottom flask and stirred for 5 min. 18 mL of TEOS were then quickly poured and the resulting solution was stirred overnight at room temperature. Silica particles were retrieved by centrifugation (7600 rpm, 45 min) and washed with absolute ethanol and followed by four cycles of centrifugation-dispersion. Silica particles were eventually air dried over 6 hours at 80° C. Particles characterization was performed using dynamic light scattering (DLS) and transmission electron microscopy (TEM). The particles hydrodynamic radius (DLS) was 80 nm and the polydispersity index 15%. The radius determined from TEM images analysis was about 50 nm (Supporting Information, Fig. S4). The particles were dispersed in milli-Q waters at 30 wt %. Silica Ludox® TM-50 water solutions with concentration of 52 wt % of silica particles at pH 9 with particle radius of about 15 nm was purchased from Aldrich and used as received.

[0208] Iron oxide Fe2O3NP nanoparticle solutions were prepared using commercially available (NanoArc® purchased from Alfa Aesar) magnetic Fe.sub.2O.sub.3 nanoparticles of 20-40 nm diameter and surface area equal to 30-60 m.sup.2/g. In particular, 0.5067 g of Fe.sub.2O.sub.3 nanoparticles were dispersed in 8 mL of milli-Q water via ultrasonication for 5 minutes. The dispersion was subsequently transferred in a glass reactor equipped with a glass anchor-shaped stirrer that was preloaded with 100 mL of 0.02 M citric acid and was left under mechanical stirring overnight. Following the collection of the particles in a flask, they were decanted using a magnet and washed three times with milli-Q water. The citrated particles were re-dispersed in 12 mL milli-Q water via ultrasonication and were peptized with 40 μL of 35% w/w NH.sub.4OH aqueous solution to obtain a 42.2 g/L concentration of the initial Fe2O3 particles. The pH of the solution measured using a pH meter paper was found to be between 7 and 7.5. Supporting Information Fig. S5 shows TEM image of Fe2O3NP nanoparticles.

[0209] Results and Discussion:

[0210] Stopping bleeding (hemostasis), preventing body fluid leakages, wound closing, and organ repair are everyday challenges in medical and surgical practice..sup.[1] Sutures and staples are standard and efficient tools. Still, suturing can be demanding in inaccessible body regions or within minimally invasive surgery. Unfortunately, sutures are traumatic to tissues especially soft tissues such as liver.sup.[2], spleen.sup.‘3’, kidney.sup.[4] or lune.sup.[5]. During last decades synthetic or biological tissue adhesives that rely on in situ polymerization or cross-linking reactions emerge as a complementary technique..sup.[1c, 6] However, tissue adhesives currently available in clinical practice present significant inherent limitations such as toxicity, insufficient strength and/or excessive swelling..sup.[1c, 6c, 7] Biomimetic approaches and new chemistries that yield polymer materials with adaptable adhesion strength are under development..sup.[6b, 6e, 8] In practice, gluing or sealing with polymers remains a complex process: it requires both stringent storage and preparation conditions before in vivo glue application or in vivo initiation and control of chemical polymerization or cross-linking reactions.

[0211] Recently, a novel approach to adhesion of hydrogels has been proposed..sup.[9] It relies on the use of aqueous nanoparticle solutions in place of polymer adhesives. The method does not require a chemical reaction: a droplet of nanoparticle solution is spread on a gel surface and gel pieces are brought into contact. Nanoparticles, which are adsorbed to gel surfaces act as connectors between the pieces and assure adhesion. The adhesion strength is brought by macromolecules of the gel that are adsorbed onto the nanoparticles. Under constraint, adsorbed layers are able to reorganize, dissipate energy and prevent interfacial fracture propagation. The approach is not limited to synthetic hydrogels, and the adhesion was shown ex vivo for two slices of calf liver using a silica nanoparticle solution.

[0212] We propose that the principle of adhesion by particle nanobridging could be applied to wound closure. Nevertheless, decades of research on polymer tissue adhesives show how challenging it is to achieve an adequate adhesion in the presence of blood and this within a short time compatible with clinical practice. Moreover, adhesive joints have to withstand after closure constraints of in vivo conditions such as tissue motions or body fluids flow. Here, we demonstrate in vivo the applicability of silica nanoparticle aqueous solutions to repair in rats with two very different types of tissues such as skin and liver. We also show that iron oxide nanoparticles can be used to achieve strong and rapid wound closure and repair. Iron oxide nanoparticles are metabolized and, as an additional boon, they could provide a contrast in magnetic resonance imaging enabling clinical in situ observations..sup.[10]

[0213] Wound closure is not the only area of applications that could benefit from adhesion brought by nanoparticles. For example, hepatic resection has been increasing in frequency in the management of metastatic or primary neoplasms of the liver. Although mortality for this procedure has steadily decreased, the morbidity mainly associated with operative time and blood loss remains high especially in cirrhotic patients. During hepatic resection, control of bleeding is a crucial problem faced by surgeons..sup.[2, 5a, 7c, 11] We show here that particle nanobridging can provide means for rapid and permanent hemostasis after rat liver resection. To this end a polymer synthetic film was coated by nanoparticles by adsorbing nanoparticles onto its surface and spread to cover the intensely bleeding liver section. Strong adhesion and permanent hemostasis were achieved within a minute.

[0214] Similarly, to illustrate possibilities of nanobridging to attach prosthetic, medical and tissue engineering devices to organs in the wet and moving environment, we permanently fastened a 3D tissue-engineering scaffold to the beating rat heart using an aqueous solution of silica nanoparticle.

[0215] In order to optimize adsorption onto tissue surface it is advantageous to avoid using nanoparticles that are stabilized by polymer layers. Indeed, grafted or adsorbed polymers can be effectively repelled by intercellular (macro) molecules and thus prevent adsorption of particles onto tissue surface. Thus nanoparticles that have been optimized to circulate in the body are to be avoided. Two types of nanoparticles were thus used in this study. Silica nanoparticles (SiO2NP) with radius of about 50 nm were synthesized by Stöber method and applied as a solution in deionized water at concentration of 30 wt % (pH=8.5) or, when indicated, as a powder. Iron oxide Fe.sub.2O.sub.3 nanoparticles (Fe2O3NP) were purchased from Alfa Aeser, stabilized by citric acid, peptized and used in aqueous solution in milli-Q water at 42 g/L.

[0216] All procedures and animal treatment were in accordance with the Principles of Laboratory Animal Care issued by the National Society for Medical Research (authorization no. 006235 from French ministry of agriculture). For cutaneous wounds, the selection of the closure device depends essentially on the depth of the wound. For superficial lacerations, use of suture, adhesive tapes and cyanoacrylate adhesives such as 2-octyl-cyanoacrylate, N-butul-2-cyanoacrylate-methacryloxysulfolane, N-butyl-2-cyanoacrylate) are the current methods of choice in humans..sup.[1c] For deep wounds, closure suturing is the clinical gold standard (FIG. 16)..sup.[1a, 1b, 12] Indeed, cyanoacrylate adhesives cannot be properly used in this situation since they form a rigid joint and in contact with living cells they provoke local tissue reaction (toxicity and/or inflammation).

[0217] We anticipated that thanks to their size nanoparticles should not affect substantially the natural wound healing process and should not lead to formation of a rigid macroscopic barrier. We therefore aimed for repair by nanobridging of full thickness cutaneous incisions and compared resulting healing with that of sutured incisions in Wistar rats. Because the healing depends of the thickness of the skin and of the local skin state of tension,.sup.[1a, 13] we investigated an incisional wound model in two different sites: the thin abdominal skin and the thick dorsal skin and results were comparable.

[0218] Dorsal wounds of 1.5 cm in length and 3 mm in depth were nanobridged by Stöber silica (SiO2NP), commercial silica suspension Ludox TM50, or iron oxide (Fe2O3NP) nanoparticles solutions and compared to a standard suture by non-resorbable clinical thread (4/0, Ethicon) and commercial cyanoacrylate glues (Dermabond®, Histacryl®). Nanoparticle solutions were spread with a brush (n=6) or a micropipette (n=5) on one edge of the wound and two edges were brought together manually and pressed into contact. By using a micropipette, we could vary the volume of nanoparticle solution spread (from 2 μl to 15 μl). Excess solution, which rose to the wound surface, was removed with a compress. The wound edges were maintained in contact manually for less than one minute after which time the wound has closed. For all animals, wounds did not reopen during the follow up. The macroscopic results evidenced no pathological inflammation or necrosis (FIGS. 16 and 17). For all tested nanoparticles, scars were aesthetic, a feature which bodes well for many areas of skin surgery. Furthermore, nanobridging allows easily repositioning and adjusting wound edges to obtain an optimal alignment. Repositioning is in principle possible for suturing, but it requires removal of suture by trained personnel and increases operation time, adds local trauma that delays healing.

[0219] The presence of nanoparticles applied by brush or micropipette does not modify the first stages of healing process namely vascular clot formation and inflammation that prevent bleeding and remove cells and dying tissue..sup.[14] As for sutures, the granulation tissue formed a new connective matrix serving as a migration structure for the cells. For Fe2O3 particles, Hematoxyline-Phloxine-Ponceau staining reveals the presence of small amount of aggregates (FIG. 17). Controlling particle aggregation is important. Indeed, when powders of silica nanoparticle rather than solutions were spread, the particle agglomerates limit the adequate wound closure and healing.

[0220] Cauterization, sutures or hemostatic sealants can treat surface lacerations of soft and wet tissues deeply penetrated by blood such as liver, spleen or kidney..sup.[2, 11a-d, 11g] However, use of these techniques for deep wounds closure is very challenging. A 1.5 cm long and 6 mm deep horizontal incision on a right hepatic rat lobe was performed with a scalpel. To repair, SiO.sub.2 or Fe.sub.2O.sub.3 nanoparticle solutions were deposited to the bleeding injury area with a pipette. The two edges of the wound were brought manually together and kept in contact. After about 1 min, hemostasis was complete and the injury stayed closed. In control experiments, the mechanical pressure did not lead to any permanent hemostasis in the absence of nanoparticle-solution. The rats were monitored during the acute post-surgery and no bleeding syndrome was detected (n=3). At day 3 post-surgery, stereo-macroscopic observation of the liver showed a thin scar tissue (FIG. 18). Histological studies revealed the presence of thin granulation tissue between the two edges of the injury. Nanobridging not only assured hemostasis, biliostasis and wound closure, but also liver function that was not affected by the application of nanoparticle solutions. Alat and Asat enzymes were measured to be, respectively, 26 U/L and 81 U/L before surgery and 24 U/L and 74 U/L 3 days after repair by SiO2NP. The total bilirubin was in the normal range (1.4 μmol/L and 1.5 μmol/L, respectively, before and 3 days post-surgery).

[0221] For hemostasis after hepatectomy we propose employing nanoparticles in order to firmly attach membranes onto bleeding liver section. To illustrate the potential of such an approach we used a poly(vinylalcohol) (PVA) membrane with a surface coated with silica nanoparticles..sup.[15] PVA film was swollen in phosphate buffer saline solution. The coating was realized by spreading SiO2NP powder on a surface of the swollen film. The unattached silica particles were removed by gently shaking the film. A ventral midline laparotomy (5 cm) was performed on a Wistar rat. The right hepatic lobe was exposed and resection of ⅔ of the lobe was totally transversally cut and the coated membrane was lightly pressed for few seconds against the bleeding section (FIG. 18). Hemostasis was immediately obtained. After 15 min of monitoring, the abdominal wall was closed with a Vicryl 4/0 and the rat was monitored during the acute post-surgery without any evidence of a bleeding syndrome. As shown in FIG. 18, neither pathologic inflammation nor bleeding was observed at the site of injury three days after surgery. In control experiments no hemostatic seal could be achieved with PVA membrane in the absence of SiO2 coating.

[0222] For a membrane fixed onto a liver tissue to stop bleeding, the organ motions were limited. In many clinical situations, it is important to secure membranes, medical devices or tissue engineering constructs to organs that undergo important contractions such as the beating heart..sup.[8a, 16] The application of adhesives is thus much more demanding and, when possible; suturing or cyanoacrylate glues are employed despite their toxicity and difficulty to apply at wet conditions. To check whether the adhesion brought by nanoparticles can withstand stringent in vivo conditions and prevent device slipping, we evaluated the ability of nanobridging to fix a scaffold onto the beating heart of rats. Rats were thus anesthetized and a tracheal intubation and mechanical ventilation were performed. The thorax was opened, and a drop of the silica Ludox® TM50 was spread on the surface of the heart with a brush. A 3D-scaffold of 6 mm in diameter made of a porous polysaccharide biodegradable hydrogel optimized for cell therapy.sup.[16] was brought into contact with the surface coated by nanoparticles and stayed firmly fixed resisting heart contractions and wet environment. After 3 days, the thorax was re-opened and the 3D-scaffold was still visible onto the heart (FIG. 19). The macroscopic evaluation does not evidence any sign of inflammation and as expected the degradation of the polysaccharide scaffold started to occur.

[0223] In summary, we demonstrated that rapid and strong adhesion by aqueous solutions of nanoparticles can be advantageously used in very different clinical situations. For skin wounds a remarkable aesthetic healing was obtained and repair procedure does not require any specific preparation or training. Bleeding control and tissue repair by nanobridging shown here in the case of liver could be used on spleen, kidney, heart, and lungs surgeries. When tight sealing is needed nanobridging could complement anastomosis and classical suturing protocols. The possibility of securing medical devices could open new applications in repair and regenerative medicine. From chemistry standpoint, the principle illustrated here is not limited to silica and iron oxide nanoparticles and they are many possible choices of sizes, forms and surface chemistries. In particular, nanoparticles with intrinsic biological effects such as silver nanoparticles for skin infection or drug delivery systems could provide useful options. Translation to clinical practice will require careful safety and toxicity investigations. A better understanding of biological mechanisms of the adhesion by nanobridging will guide the design of future-generation tissue adhesives.

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