Cells inside a 3D matrix (such as tissue extracellular matrix or biomaterials) sense their insoluble environment through specific binding interactions between their adhesion receptors and ligands present on the matrix surface. Despite the critical role of the insoluble matrix in cell regulation, there exist no widely-applicable methods for quantifying the chemical stimuli provided by a matrix to cells. Here, we describe a general-purpose technique for quantifying in situ the density of ligands for specific cell adhesion receptors of interest on the surface of a 3D matrix. This paper improves significantly the accuracy of the procedure introduced in a previous publication by detailed marker characterization, optimized staining, and improved data interpretation. The optimized methodology is utilized to quantify the ligands of integrins α 1 β 1, α 2 β 1 on two kinds of matched porous collagen scaffolds, which are shown to possess significantly different ligand density, and significantly different ability to induce peripheral nerve regeneration in vivo. Data support the hypothesis that cell adhesion regulates contractile cell phenotypes, recently shown to be inversely related to organ regeneration. The technique provides a standardized way to quantify the surface chemistry of 3D matrices, and a means for introducing matrix effects in quantitative biological models.

Multispectral multiphoton microscopy of ex vivo transected peripheral nerves treated with a series of collagen scaffolds of increasing cross-linking density is applied to quantify how biomaterial properties modulate the wound healing response and induce regeneration.

The adult mammal responds to severe injury of most organs spontaneously by wound contraction and scar formation, rather than by regeneration. In severe skin wounds, the ability of porous collagen scaffolds to induce regeneration was found to correlate strongly with a reduction in wound contraction rate. Here, we present quantitative evidence of a similar positive relationship between the extent of disruption of tissue contraction and quality of peripheral nerve regeneration in transected rat peripheral nerves. Our observations suggest that porous collagen scaffolds enhance regeneration both in injured adult skin and peripheral nerves by disrupting the formation of a contractile cell capsule at the edges of the wound. Preliminary observations made with other injured organs support the hypothesis that capsules or clusters of contractile cells impose a universal mechanical barrier during wound healing which, if disrupted appropriately, enhances the quality of induced regeneration in a wider range of organs.

Acute or chronic injury to an organ is followed by a spontaneous healing process. Injury to the mammalian fetus is reversible during early stages of gestation; the spontaneous wound response is capable of restoring the structure and function of the original organ (regeneration). In contrast, the unimpaired response of adults to severe injury is an irreversible process leading to closure of the injured site by contraction and formation of scar, a nonphysiological tissue (repair). The consequences of irreversible healing at the organ scale are far reaching: they often result in an essentially nonfunctional organ.

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PURPOSE:Bioadhesives have had limited use in ophthalmic surgery. Problems with these adhesives have included inadequate tensile strength and difficulty with their application to the tissue site. We evaluated a scaffold-enhanced cyanoacrylate bioadhesive composite as an alternative to sutures in ophthalmic surgery, including strabismus procedures.METHODS AND MATERIALS:The bioadhesive composite consisted of 2-octyl-cyanoacrylate combined with either a poly(L-lactic-co-glycolic acid) (PLGA) scaffold or a rehydrated porcine small intestine submucosa (SIS) scaffold. Extraocular rectus muscle and sclera were obtained from rabbits (n = 40) and were used, with these bioadhesive composites, to produce rectus muscle-to-sclera, sclera-to-sclera, and rectus muscle-to-rectus muscle adhesions. Control adhesions were created with cyanoacrylate only. The breaking load of the tissue repair was measured with a material strength-testing machine.RESULTS:In all cases, the scaffold-enhanced cyanoacrylate adhesions were significantly stronger (P < 0.001) than the cyanoacrylate alone. The rectus muscle-to-sclera adhesions were greater than the in vivo forces reported for the horizontal rectus muscles in humans in extreme gaze.CONCLUSION:This scaffold-enhanced bioadhesive composite produced initial muscle-sclera adhesions with strength satisfactory for strabismus surgery. It also may be applicable to other categories of ophthalmic surgery as a substitute for sutures.

Our Scaffold-Enhanced Biological Adhesive (SEBA) system was investigated as an alternative to sutures or adhesives alone for repair of wounds. Two scaffold materials were investigated: (i) a synthetic biodegradable material fabricated from poly(L-lactic-co-glycolic acid); and (ii) a biologic material, small intestinal submucosa, manufactured by Cook BioTech. Two adhesive materials were also investigated: (i) a biologic adhesive composed of 50%(w/v) bovine serum albumin solder and 0.5mg/ml indocyanine green dye mixed in deionized water, and activated with an 808-nm diode laser; and (ii) Ethicon’s Dermabond, a 2-octyl-cyanoacrylate. The tensile strength and time-to-failure of skin incisions repaired in vivo in a rat model were measured at seven days postoperative. Incisions closed by protein solder alone, by Dermabond alone, or by suture, were also tested for comparison. The tensile strength of repairs formed using the SEBA system were 50% to 65% stronger than repairs formed by suture or either adhesive alone, with significantly less variations within each experimental group (average standard deviations of 15% for SEBA versus 38% for suture and 28% for adhesive alone). In addition, the time-to-failure curves showed a longevity not previously seen with the suture or adhesive alone techniques. The SEBA system acts to keep the dermis in tight apposition during the critical early phase of wound healing when tissue gaps are bridged by scar and granulation tissue. It has the property of being more flexible than either of the adhesives alone and may allow the apposed edges to move in conjunction with each other as a unit for a longer period of time and over a greater range of stresses than adhesives alone. This permits more rapid healing and establishment of integrity since the microgaps between the dermis edges are significantly reduced. By the time the scaffolds are sloughed from the wound site, there is greater strength and healing than that produced by adhesive alone or by wounds following suture removal. This hypothesis is supported by the data of this study, as well as, the acute tensile strength data of Part I of this study.

An ex vivo study was conducted to determine the effect of the irregularity of the scaffold surface on the tensile strength of repairs formed using our Scaffold-Enhanced Biological Adhesive (SEBA). Two different scaffold materials were investigated: (i) a synthetic biodegradable material fabricated from poly(L-lactic-co-glycolic acid); and (ii) a biological material, small intestinal submucosa, manufactured by Cook BioTech. The scaffolds were doped with protein solder composed of 50%(w/v) bovine serum albumin solder and 0.5mg/ml indocyanine green dye mixed in deionized water, and activated with an 808-nm diode laser. The tensile strength of repairs performed on bovine thoracic aorta, liver, spleen, small intestine and lung, using the smooth and irregular surfaces of the above scaffold-enhanced materials were measured and the time-to-failure was recorded.The tensile strength of repairs formed using the irregular surfaces of the scaffolds were consistently higher than those formed using the smooth surfaces of the scaffolds. The largest difference was observed on repairs formed on the aorta and small intestine, where the repairs were, on average, 50% stronger using the irregular versus the smooth scaffold surfaces. In addition, the time-to-failure of repairs formed using the irregular surfaces of the scaffolds were between 50% and 100% longer than that achieved using the smooth surfaces of the scaffolds. It has previously been shown that distributing or dispersing the adhesive forces over the increased surface area of the scaffold, either smooth or irregular, produces stronger repairs than albumin solder alone. The increase in the absolute strength and longevity of repairs seen in this new study when the irregular surfaces of the scaffolds are used is thought to be due to the distribution of forces between the many independent micro-adhesions provided by the irregular surfaces.

Ophthalmic surgery currently utilizes suture materials to repair wounds created during eye operations. Although effective, suture-based techniques can result in complications that further impair the patient's vision, such as retinal detachment and scleral perforation associated with strabismus (eye muscle) surgery. Two techniques currently under development avoid sutures altogether, yielding similar strength results, reduced operating time, and simpler methods of repair. The first of these techniques employs a light-activated scaffold-enhanced protein solder to re-adhere the tissue. The second technique utilizes commercially available bioadhesives that have been scaffold-enhanced to improve their handling characteristics. A comparison of these two techniques is given. Initial tensile strength results show a higher strength of repair when a scaffold is utilized, with significantly less variations within each experimental group. Repairs formed using the scaffold-enhanced cyanoacrylate adhesives were the strongest. The tensile strength of extraocular muscle-to-sclera adhesions was 72% stronger than cyanoacrylate alone (4.2 +/- 0.2 N vs. 2.4 +/- 0.4 N) and 78% stronger than native tissue (2.3 +/- 0.4 N). Sclera-to-sclera adhesions were 60% stronger than adhesions formed with cyanoacrylate alone (3.9 +/- 0.2 N vs. 2.5 +/- 0.4 N), while the tensile strength of extraocular muscle-to-extraocular muscle adhesions were 81% of native extraocular muscle tensile strength (5.6 +/- 0.2 N vs. 6.2 +/- 0.3 N), and 50% stronger than adhesions formed using cyanoacrylate alone (3.6 +/- 0.4 N). The data analysis and resulting conclusions favor the less invasive adhesive technique as an alternative for tissue reattachment during ophthalmic procedures. Future experiments will examine the optimization of application parameters and detail tensile strength time course studies.

The clinical acceptance of laser-tissue repair techniques is dependent on the reproducibility of viable repairs. Reproducibility is dependent on two factors: (i) the choice of materials to be used as the adhesive; and (ii) obtaining temperatures high enough to cause protein denaturation at the vital tissue interface without causing excessive thermal damage to the surrounding tissue. The use of a polymer scaffold as a carrier for the protein solder provides for uniform application of the solder to the tissue, thus allowing for pre-selection of optimal laser parameters. The scaffold also facilitates precise tissue alignment and ease of clinical application. In addition, the scaffold can be doped with various pharmaceuticals such as hemostatic and thrombogenic agents to aid wound healing.An ex vivo study was performed to correlate solder and tissue temperature with the tensile strength of arterial repairs formed using scaffold-enhanced light-activated surgical adhesives. Previous studies by our group using solid protein solder without the scaffold indicate that a solder/tissue, interface temperature of 65 degreesC is optimal. Using this parameter as a benchmark, laser irradiance was varied and temperatures were recorded at the surface and at the tissue interface of scaffold-enhanced protein solder using an infrared temperature monitoring system, designed by the researchers, and a type-K thermocouple, respectively.

Clinical adoption of laser tissue welding (LTW) techniques has been beleaguered by problems associated with thermal damage of tissue and insufficient strength of the resulting tissue bond. The magnitude of these problems has been significantly reduced with the incorporation of indocyanine green (ICG)-doped protein solders into the LTW procedure to form a new technique known as laser tissue soldering (LTS). With the addition of ICG, a secondary concern has arisen relating to the potential harmful effects of the degradation products of the chromophore upon thermal denaturation of the protein solder with a laser. In this study, two different food colorings were investigated, including blue #1 and green consisting of yellow #5 and blue #1, as alternative chromophores for use in LTS techniques. Food coloring has been found to have a suitable stability and safety profile for enteral use when heated to temperatures above 200 degrees C; thus, it is a promising candidate chromophore for LTS which typically requires temperatures between 50 degrees C and 100 degrees C. Experimental investigations were conducted to test the tensile strength of ex vivo repairs formed using solders doped with these alternative chromophores in a bovine model. Two commonly used chromophores, ICG and methylene blue (MB), were investigated as a reference. In addition, the temperature rise, depth of thermal coagulation in the protein solder, and the extent of thermal damage in the surrounding tissue were measured. Temperature rise at the solder/tissue interface, and consequently the degree of solder coagulation and collateral tissue thermal damage, was directly related to the penetration depth of laser light in the protein solder. Variation of the chromophore concentration such that the laser light penetrated to a depth approximately equal to half the thickness of the solder resulted in uniform results between each group of chromophores investigated. Optimal tensile strength of repairs was achieved by optimizing laser and solder parameters to obtain a temperature of approximately 65 degrees C at the solder/tissue interface. The two alternative chromophores tested in this study show considerable promise for application in LTS techniques, with equivalent tensile strength to solders doped with ICG or MB, and the potential advantage of eliminating the risks associated with harmful byproducts.

The ability to reproduce strong repairs is essential to establishing the reliability of laser-tissue soldering techniques and advancing their use to the clinical setting. While some thermal damage is necessary to achieve a viable solder-tissue bond, excessive thermal damage leads to decreased flexibility and strength of the repair. In addition, if the temperature at the solder/tissue interface is too low, inadequate solder-tissue bonding will occur to provide a strong repair. This suggests the presence of an optimal temperature for laser-tissue repair. The choice of solder material presents another challenge to the reproducibility of strong repairs. The emerging use of chromophore-enhanced solder-doped polymer scaffolds offers numerous advantages over more traditional liquid and solid solders composed of serum albumin and an absorbing chromophore mixed in deionized water. Polymer scaffolds, fabricated from poly(L-lactic-co-glycolic acid) using a solvent casting and particulate leaching technique, are porous enough to absorb serum albumin and can also be doped with various hemostatic and thrombogenic agents to aid in tissue healing. Use of the polymer scaffolds allows one to combine the strength of solid solders and the flexibility of liquid solders without the common “runaway” problems. An in vitro study was performed to correlate tissue temperature with the tensile strength of arterial repairs formed using the chromophore-enhanced solder-doped polymer scaffolds. Laser irradiance was varied and the solder surface and solder/tissue interface temperatures were monitored by an IR temperature monitoring system and a type-K thermocouple, respectively. The solder/tissue interface temperature required for optimized tensile strength was determined to be 67 ± 5°C. This value was in agreement with previous studies using serum albumin solders alone, where the optimal solder/tissue interface temperature was found to be 65°C.

An investigation was conducted to assess the feasibility of using various synthetic polymers as scaffolds to traditional albumin protein solders with the aim of enhancing the mechanical strength of repairs formed during laser tissue soldering. Biodegradable polymer membranes of controlled porosity were fabricated with either polylactic acid (PLA), polyglycolic acid (PGA), or two different poly(L-lactic-co-glycolic acid) (PLGA) blend ratios, using a solvent-casting and particulate-leaching technique. In addition, membranes were prepared by combining each of the above mentioned polymers with poly(ethylene glycol) (PEG). The porous membranes provided a scaffold into which the traditional protein solder composition of serum albumin and indocyanine green dye mixed in deionized water was readily absorbed. Studies were conducted to assess the influence of various processing parameters on the strength of tissue repairs formed using the new adhesives. These parameters included the polymer type, the polymer/PEG blend ratio, the salt particle size, and the initial albumin weight fraction. No significant difference was observed between the four polymers investigated, however, variation of the polymer type and blend ratio is known to influence the degradation rate of the membranes. The addition of PEG to the films during the casting stage was observed to increase the flexibility of the membranes. Finally, repair strength increased significantly with an increase in the size of pores present in the membranes and with increased protein concentration.

Laser tissue soldering has been investigated for several years by researchers in our laboratory as an alternative to conventional tissue fasteners, including sutures, staples and clips. Laser tissue soldering is a bonding technique in which protein solder is applied to the tissue surfaces to be joined, and laser energy is used to bond the solder to the tissue surfaces. Over the past four years we have been investigating the use of synthetic polymer membranes as a means for reinforcing the strength of tissue repairs formed using traditional laser tissue soldering techniques. The purpose of this study was to assess the influence of various processing parameters on the strength of tissue repairs formed using the reinforced solder. Biodegradable polymer membranes of specific porosity were fabricated by means of a solvent-casting and particulate-leaching technique, using three different poly(alpha ester)s: polyglycolic acid (PGA), polylactic acid (PLA) and poly(L-lactic-co-glycolic acid) (PLGA). In addition, several membranes were also prepared with poly(ethylene glycol) (PEG). The membranes were then doped with the traditional protein solder mixture of serum albumin and indocyanine green dye. Varied processing parameters included the polymer type, the PLGA copolymer blend ratio, the polymer/PEG blend ratio, the porosity of the polymer membrane and the initial albumin weight fraction. Variation of the polymer type had negligible effect on the strength of the repairs. Although it is known that alteration of the copolymer blend ratio of PLGA influences the degradation rate of the polymer, this variation also had no significant effect on the strength of the repairs formed. Increased membrane flexibility was observed when PEG was added during the casting stage. An increase in the porosity of the polymer membranes led to a subsequent increase in the final concentration of protein contained within the membranes, hence aiding in strengthening the resultant repairs. Likewise, an increase in the initial albumin weight fraction increased the strength of the resultant repairs.

An in vivo study was conducted to investigate the feasibility, mechanical function, and chronic biocompatibility of a new range of light-activated surgical adhesives for vascular anastomosis. Porcine carotid arteries (n=12) and femoral arteries (n=12) were exposed, and a 0.3 -0.6cm longitudinal incision was made in the arterial walls. The vessels were divided equally into two groups. Vessels belonging to the first group were repaired using a surgical adhesive, composed of a poly(L-lactic-co-glycolic acid) scaffold doped with the traditional protein solder mix of serum albumin and indocyanine green dye mixed in deionized water. The adhesive was applied across the incision and denatured using an 805-nm diode laser. Vessels belonging to the second group formed part of a control study, and were repaired using conventional suturing techniques. Blood flow was restored to the vessels immediately after the procedure and the incision sites were checked for patency. The strength and hemostatic abilities of the new surgical adhesives were evaluated in the context of arterial pressure, persistence of hemostasis and presence of any inflammatory reaction after 3 days. The adhesive technique compared favorably with the suture technique. Repairs formed with the adhesive technique were achieved more rapidly than suturing, and acute leakage was observed less frequently. Repairs closed by suture did not burst, but leaked at pressures significantly below those of vessels closed with the adhesive material. Finally, the adhesive technique produced better histology than the suture technique, suggesting that it holds great promise as an alternative to suturing.

Thermal damage of tissue is a major concern for all laser tissue repair techniques where the resulting strength of a repair is sensitive to slight alterations in tissue temperature as well as changes in the duration of exposure of the tissue to the laser beam. Too low of a temperature will prevent proper bonding between the surfaces while prolonged exposure of the tissue to the laser beam results in collateral thermal damage and decreased flexibility and strength of the repair. Temperature feedback systems that monitor the surface temperature of the repair site and adjust the laser irradiance accordingly increase the success rate of the technique. Knowledge of an optimal temperature for tissue soldering will also increase the reliability of the technique. The choice of solder material has been another challenge to the reproducibility of strong repairs. The emerging use of solder-doped polymer membranes as surgical adhesives offers numerous advantages over more traditional liquid and solid solders. Poly (L-lactic-co-glycolic acid) (PLGA), when used as a polymer scaffold, is porous enough to absorb serum albumin and can also be doped with various hemostatic and thrombogenic agents to aid tissue healing. An in vitro study was performed to correlate tissue temperature with the tensile strength of repairs formed using the solder-doped polymer membranes. Previous studies by our group indicate that a solder/tissue interface temperature of 65 degrees C is optimal. Using this parameter as a bench mark, laser irradiance was varied and the solder surface and solder/tissue interface temperatures were monitored by an IR temperature monitoring system, designed by the researchers, and a type K thermocouple, respectively.