In Situ Quantification of Surface Chemistry in Porous Collagen Biomaterials.

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 ß 1, α 2 ß 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.

Common features of optimal collagen scaffolds that disrupt wound contraction and enhance regeneration both in peripheral nerves and in skin.

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.

Sutureless ophthalmic surgery: a scaffold-enhanced bioadhesive technique.

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.

A new technique of tissue repair for ophthalmic surgery.

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.

Effect of varying chromophores used in light-activated protein solders on tensile strength and thermal damage profile of repairs.

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.

Optimal parameters for arterial repair using light-activated surgical adhesives.

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 degrees C 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.

Use of an infrared temperature monitoring system to determine optimal temperature for laser-solder tissue repair.

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.

Biodegradable synthetic polymer scaffolds for reinforcement of albumin protein solders used for laser-assisted tissue repair.

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.