The Effects of a Crosslinked, Modified Hyaluronic Acid (xCMHA-S) Gel on Equine Tendon Healing.

OBJECTIVE: To assess the effects of a crosslinked, modified hyaluronic acid (xCMHA-S) gel on equine tendon healing using an in vivo surgical model. STUDY DESIGN: In vivo experimental study. ANIMALS: Adult horses (n = 5). METHODS: Full thickness bilateral forelimb window tenectomies were surgically created in both forelimb superficial digital flexor tendons and xCMHA-S gel was implanted intraoperatively into the right forelimb lesion of each horse whereas the left forelimb served as the untreated control. Healing was monitored by serial ultrasound examinations every 14 days over the course of the 84 day study. In addition, gross pathology, scanning electron microscopy for fiber diameter, and histological scoring were completed on tendon samples harvested after euthanasia at 84 days. RESULTS: Ultrasound assessment demonstrated a significant decrease in mean lesion size of treated (0%) compared to control (30%) tendons at 84 days. Mean (±SD) cumulative histologic tendon scores for control tendons (17.7 ± 2.7) were significantly higher than treated tendons (13.6 ± 1.9), indicating less advanced healing in the control group. Tendon cell density was increased and neovascularization, intensity of inflammation, and uniformity of fiber diameter were increased in control compared to treated tendons. There were no differences in fibroblast shape, levels of intralesional hemorrhage, linearity of collagen fibers, or collagen fiber diameter or distribution between treated and control tendons. CONCLUSION: Tendons treated with xCMHA-S gel at the time of model induction had superior histologic healing scores and sonographically smaller lesions compared to controls, suggesting that xCMHA-S gel may aid the natural healing process.

Single-lumen and multi-lumen poly(ethylene glycol) nerve conduits fabricated by stereolithography for peripheral nerve regeneration in vivo.

BACKGROUND: The use of nerve conduits to facilitate nerve regrowth after peripheral nerve injury is limited to defects less than 3 cm. The purpose of this study is to determine the capability of novel single and multi-lumen poly(ethylene glycol) (PEG) conduits manufactured by stereolithography to promote peripheral nerve regeneration. MATERIALS AND METHODS: Eight Sprague Dawley rats with sharp transection injuries of the sciatic nerve were randomly assigned to receive single-lumen or multi-lumen PEG conduits to bridge a 10-mm gap. Sciatic nerve and conduit samples were harvested after 5 weeks, and axon number, myelin thickness, fiber diameter, and g-ratio were analyzed. The contralateral intact nerve was also harvested for comparison. RESULTS: Partial nerve regeneration was observed in three out of four single-lumen conduits and one out of four multi-lumen conduits. Axon number in the single-lumen regenerated nerve approached that of the contralateral intact nerve at 4,492 ± 2,810.0 and 6,080 ± 627.9 fibers/mm(2), respectively. The percentage of small fibers was greater in the single-lumen conduit compared with the intact nerve, whereas myelin thickness and g-ratio were consistently greater in the autologous nerve. Axon regrowth through the multi-lumen conduits was severely limited. CONCLUSION: Single-lumen stereolithography-manufactured PEG nerve conduits promote nerve regeneration, with regenerating axon numbers approaching that of normal nerve. Multi-lumen conduits demonstrated significantly less nerve regeneration, possibly due to physical properties of the conduit inhibiting growth. Further studies are necessary to compare the efficacy of the two conduits for functional recovery and to elucidate the reasons underlying their differences in nerve regeneration potential.

Mucoadhesive hyaluronic acid-based films for vaginal delivery of metronidazole.

Bacterial vaginosis is a prevalent women's health issue that affects millions of women worldwide every year; however, current treatments are often messy, inconvenient, and ineffective. Therefore, we developed a new hyaluronic acid-based film to deliver metronidazole that would be more effective, more convenient, and at a pH similar to that of the normal vaginal environment. Films were made by crosslinking modified hyaluronic acid to create a hydrogel, in which metronidazole or metronidazole benzoate and methylcellulose were incorporated, and the hydrogel was dried to a thin film. Through release testing, coupled with assessments of handleability, tensile strength, and mucoadhesion, it was determined that the films have the potential to remain in the vaginal environment for an extended time period and gradually release the drug for at least 6 days, which is a typical treatment length. As such, the films present a viable alternative to current treatment methods, allowing for both easy handling and a single treatment while eliminating the issues of pH and overall inconvenience.

Novel Cross-Linked Ocular Bandage Gel Improves Reepithelialization After Photorefractive Keratectomy: A Randomized, Masked Prospective Study.

Purpose: To compare, in a masked manner, a novel cross-linked hyaluronic acid ocular bandage gel (OBG) versus standard-of-care bandage contact lens (BCL) plus artificial tears with respect to safety and effectiveness in healing epithelial defects created for photorefractive keratectomy (PRK). Methods: This was a randomized, reading center-masked, exploratory study. Forty-five patients (myopic without significant anisometropia) scheduled for bilateral PRK (9-mm epithelial defect) were randomized post-PRK to treatment with OBG 8 times daily for 3 days, followed by 4 times daily for 11 days (Group 1); OBG 4 times daily for 14 days (Group 2); or BCL and artificial tears (Control). A masked reading center used image analysis of digital slit lamp photos of the fluorescein-stained cornea to evaluate defect size during the 14-day postoperative follow-up period. Effectiveness endpoints were (1) time to complete closure of the corneal defect and (2) proportion of patients with complete healing on day 3 postoperatively, whose defect remained closed. Safety assessments included findings for adverse events and vision, Standard Patient Evaluation of Eye Dryness (SPEED™) Questionnaire, slit lamp, intraocular pressure, and fundus examinations. Results: The proportion of patients with complete healing at 3 days was 73.3%, 86.7%, and 66.7% of patients in Groups 1, 2, and Control, respectively. On day 2, the mean wound size was 6%-26% smaller in Groups 1 and 2 compared with Control. No safety concern arose. SPEED scores were not significantly different across groups. Conclusion: OBG offers a well-tolerated and effective therapy for quickly reepithelializing the cornea following trauma, disease, or surgery.

Correction: Effect of Carboxymethylation on the Rheological Properties of Hyaluronan.

[This corrects the article DOI: 10.1371/journal.pone.0162849.].

Mammalian cell-seeded hydrogel microarrays printed via dip-pin technology.

Although significant advances have been made in the development of DNA and protein microarrays, less effort has been put toward developing mammalian cell microarrays. Such cellular microarrays may be useful in examining the effects of biological or chemical agents on cells, particularly in drug development and toxicological applications. Here, mammalian cell-seeded hydrogel microarrays were created using two different commercial microarrayers, with four different pin types. Human dermal fibroblasts were used here as a model cell type, seeded within polyethylene glycol-based hydrogels similar to those under investigation as tissue engineering scaffolds, which serve as synthetic extracellular matrices for the cells. Spot sizes of the hydrogels were found to vary with pin type. Multiple touches on a slide following a single dip in the reservoir print solution led to decreasing spot size with each touch; therefore, subsequent microarrays were printed with single touches after a dip. Individual pins of the same type and tip diameter had significantly different spot sizes, likely due to wear of the pins at the tip. However there was high run-to-run reproducibility between subsequent microarrays. Cell viability varied with pin type, and the number of cells per spot varied with cell density in the print solution, as expected.

Ocular translational science: A review of development steps and paths.

Developing successful drug delivery methods is challenging for any tissue, and the eye is no exception. Translating initial concepts into advanced technologies treating diseases in preclinical models and finally into functional and marketable products for humans can be particularly daunting. While referring to specific ophthalmic companies and products, this review considers key exchanges that lead to successful translation. By building on basic science discoveries in the academic setting, applied science can perform proof-of-concept work with simple, benchtop experiments. Eventually, simple models need to be translated to more robust ones where cells, tissues, and entire organisms are incorporated. Successful translation also includes performing due diligence of the intellectual property, understanding the market needs, undertaking clinical development, meeting regulatory requirements, and eventually scale up manufacturing. Different stages of the translation can occur in different environments, including moving from academia to industry, from one company to another, or between veterinary and human applications. The translation process may also rely on contract organizations to move through the complex landscape. While the path to a commercial, marketable product may not look the same each time, it is important to design a development plan with clear goals and milestones to keep on track.

Topical Cross-Linked HA-Based Hydrogel Accelerates Closure of Corneal Epithelial Defects and Repair of Stromal Ulceration in Companion Animals.

Purpose: The purpose of this study was to determine the safety of topical ocular administration of a cross-linked, modified hyaluronic acid (xCMHA-S) hydrogel, and its effectiveness in accelerating repair and closure of acute and nonhealing corneal ulcers in companion animals as a veterinary treatment and its utility as a model for therapy in human corneal ulceration. Methods: Two concentrations of xCMHA-S (0.33% and 0.75%) were topically administered to the eyes of rabbits six times daily for 28 days to assess safety. Then, 30 dogs and 30 cats with spontaneous acute corneal ulcers were treated with either xCMHA-S (0.75%) or a non-cross-linked hyaluronic acid (HA) solution (n = 15 per group for each species), three times daily until the ulcer had healed. Finally, 25 dogs with persistent nonhealing corneal ulcers were treated with xCMHA-S (0.75%) twice daily until the ulcer had healed. Results: Both concentrations of the xCMHA-S hydrogel were well tolerated, safe, and nontoxic in the 28-day exaggerated dosing study in healthy rabbits. Topically applied xCMHA-S significantly accelerated closure of acute corneal stromal ulcers in dogs and cats compared with a non-cross-linked HA solution. Further, topical administration of the xCMHA-S aided in closure of nonhealing corneal stromal ulcers in dogs. Conclusions: Hyaluronic acid has previously been shown to aid in corneal wound repair. This study demonstrates that a cross-linked, modified HA hydrogel provides further benefit by accelerating time to corneal wound closure compared to a non-cross-linked HA solution in companion animals, and therefore may be beneficial in fulfilling an unmet need in humans.

Effect of Carboxymethylation on the Rheological Properties of Hyaluronan.

Chemical modifications made to hyaluronan to enable covalent crosslinking to form a hydrogel or to attach other molecules may alter the physical properties as well, which have physiological importance. Here we created carboxymethyl hyaluronan (CMHA) with varied degree of modification and investigated the effect on the viscosity of CMHA solutions. Viscosity decreased initially as modification increased, with a minimum viscosity for about 30-40% modification. This was followed by an increase in viscosity around 45-50% modification. The pH of the solution had a variable effect on viscosity, depending on the degree of carboxymethyl modification and buffer. The presence of phosphates in the buffer led to decreased viscosity. We also compared large-scale production lots of CMHA to lab-scale and found that large-scale required extended reaction times to achieve the same degree of modification. Finally, thiolated CMHA was disulfide crosslinked to create hydrogels with increased viscosity and shear-thinning aspects compared to CMHA solutions.

Adhesive and mechanical properties of hydrogels influence neurite extension.

Photopolymerizable polyethylene glycol (PEG) hydrogels conjugated with bioactive ligands were examined for their use as scaffolds in peripheral nerve regeneration applications. The bioactivity and mechanical properties of PEG hydrogels can be tailored through the integration of bioactive factors (adhesion ligands, proteolytic sites, growth factors) and the alteration of PEG concentrations, respectively. For peripheral nerve regeneration, it will be important to determine the type and concentration of the bioactive molecules required to improve neurite extension. In this study, cell adhesion ligands (RGDS, IKVAV, and YIGSR) were covalently attached to PEG hydrogels. Both the type and concentration of cell adhesion ligand used affected neurite extension. Extension from PC12 cells was greater on hydrogels with RGDS incorporated than IKVAV, and the optimal concentration for each ligand was different. Cells adhered to but did not extend neurites on hydrogels with YIGSR. Cells did not adhere to hydrogels containing RGES. Furthermore, different combinations of these ligands affected neurite extension to different degrees. The mechanical properties of the hydrogels also significantly affected neurite extension. PC12 cells grown on more flexible hydrogels exhibited the greatest degree of neurite extension. PEG hydrogels have thus been developed with varying biochemical and mechanical properties that may enhance nerve regeneration.

Biologic gels in tissue engineering.

There are many different types of scaffold materials now available for tissue engineering applications. Hydrogels form one group of materials that have been used in a wide variety of applications. These hydrogels can be formed using natural materials, synthetic materials, or some combination of the two. There are advantages and disadvantages to using each type of material, and detailed investigations into the effects on various aspects of cell behavior of chemical and physical properties of the materials are needed to make an informed decision as to which material is best suited for a given application. By combining appropriate scaffold materials, such as hydrogels, with cells and proper signaling for those cells, more commercial tissue engineering products will become available for general use.

Efficacy of a crosslinked hyaluronic acid-based hydrogel as a tear film supplement: a masked controlled study.

Keratoconjunctivitis sicca (KCS), or dry eye, is a significant medical problem in both humans and dogs. Treating KCS often requires the daily application of more than one type of eye drop in order to both stimulate tear prodcution and provide a tear supplement to increase hydration and lubrication. A previous study demonstrated the potential for a crosslinked hyaluronic acid-based hydrogel (xCMHA-S) to reduce the clinical signs associated with KCS in dogs while using a reduced dosing regimen of only twice-daily administration. The present study extended those results by comparing the use of the xCMHA-S to a standard HA-containing tear supplement in a masked, randomized clinical study in dogs with a clinical diagnosis of KCS. The xCMHA-S was found to significantly improve ocular surface health (conjunctival hyperaemia, ocular irritation, and ocular discharge) to a greater degree than the alternative tear supplement (P = 0.0003). Further, owners reported the xCMHA-S treatment as being more highly effective than the alternative tear supplement (P = 0.0024). These results further demonstrate the efficacy of the xCMHA-S in reducing the clinical signs associated with KCS, thereby improving patient health and owner happiness.

Ophthalmic Uses of a Thiol-Modified Hyaluronan-Based Hydrogel.

Significance: Hyaluronic acid (HA, or hyaluronan) is a ubiquitous naturally occurring polysaccharide that plays a role in virtually all tissues in vertebrate organisms. HA-based hydrogels have wound-healing properties, support cell delivery, and can deliver drugs locally. Recent Advances: A few HA hydrogels can be customized for composition, physical form, and biomechanical properties. No clinically approved HA hydrogel allows for in vivo crosslinking on administration, has a tunable gelation time to meet wound-healing needs, or enables drug delivery. Recently, a thiolated carboxymethyl HA (CMHA-S) was developed to produce crosslinked hydrogels, sponges, and thin films. CMHA-S can be crosslinked with a thiol-reactive crosslinker or by oxidative disulfide bond formation to form hydrogels. By controlled crosslinking, the shape and form of this material can be manipulated. These hydrogels can be subsequently lyophilized to form sponges or air-dried to form thin films. CMHA-S films, liquids, and gels have been shown to be effective in vivo for treating various injuries and wounds in the eye in veterinary use, and are in clinical development for human use. Critical Issues: Better clinical therapies are needed to treat ophthalmic injuries. Corneal wounds can be treated using this HA-based crosslinked hydrogel. CMHA-S biomaterials can help heal ocular surface defects, can be formed into a film to deliver drugs for local ocular drug delivery, and could deliver autologous limbal stem cells to treat extreme ocular surface damage associated with limbal stem cell deficiencies. Future Directions: This CMHA-S hydrogel increases the options that could be available for improved ocular wound care, healing, and regenerative medicine.

A Crosslinked HA-Based Hydrogel Ameliorates Dry Eye Symptoms in Dogs.

Keratoconjunctivitis sicca, commonly referred to as dry eye or KCS, can affect both humans and dogs. The standard of care in treating KCS typically includes daily administration of eye drops to either stimulate tear production or to hydrate and lubricate the corneal surface. Lubricating eye drops are often applied four to six times daily for the life of the patient. In order to reduce this dosing regimen yet still provides sufficient hydration and lubrication, we have developed a crosslinked hydrogel based on a modified, thiolated hyaluronic acid (HA), xCMHA-S. This xCMHA-S gel was found to have different viscosity and rheologic behavior than solutions of noncrosslinked HA. The gel was also able to increase tear breakup time in rabbits, indicating a stabilization of the tear film. Further, in a preliminary clinical study of dogs with KCS, the gel significantly reduced the symptoms associated with KCS within two weeks while only being applied twice daily. The reduction of symptoms combined with the low dosing regimen indicates that this gel may lead to both improved patient health and owner compliance in applying the treatment.

Formulation Changes Affect Material Properties and Cell Behavior in HA-Based Hydrogels.

To develop and optimize new scaffold materials for tissue engineering applications, it is important to understand how changes to the scaffold affect the cells that will interact with that scaffold. In this study, we used a hyaluronic acid- (HA-) based hydrogel as a synthetic extracellular matrix, containing modified HA (CMHA-S), modified gelatin (Gtn-S), and a crosslinker (PEGda). By varying the concentrations of these components, we were able to change the gelation time, enzymatic degradation, and compressive modulus of the hydrogel. These changes also affected fibroblast spreading within the hydrogels and differentially affected the proliferation and metabolic activity of fibroblasts and mesenchymal stem cells (MSCs). In particular, PEGda concentration had the greatest influence on gelation time, compressive modulus, and cell spreading. MSCs appeared to require a longer period of adjustment to the new microenvironment of the hydrogels than fibroblasts. Fibroblasts were able to proliferate in all formulations over the course of two weeks, but MSCs did not. Metabolic activity changed for each cell type during the two weeks depending on the formulation. These results highlight the importance of determining the effect of matrix composition changes on a particular cell type of interest in order to optimize the formulation for a given application.

Comparing predictive drug nephrotoxicity biomarkers in kidney 3-D primary organoid culture and immortalized cell lines.

The cellular microenvironment is recognized to play a key role in stabilizing cell differentiation states and phenotypes in culture. This study addresses the hypothesis that preservation of in vivo-like tissue architecture in vitro produces a cell culture more capable of responding to environmental stimuli with clinically relevant toxicity biomarkers. This was achieved using kidney proximal tubules in three-dimensional organoid hydrogel culture, with comparisons to conventional monolayer kidney cell cultures on plastic. Kidney proximal tubule cultures and two immortalized kidney cell line monolayer cultures exposed to known nephrotoxic drugs were evaluated for inflammatory cytokines, nephrotoxicity-associated genes, Kim-1 protein, cytochrome enzymes, and characteristic cellular enzyme shedding. Significant similarities are shown for these traditional biomarkers of kidney toxicity between in vivo and 3-D organoid endpoints of drug toxicity, and significantly, a consistent lack of clinically relevant endpoints produced by traditional 2-D kidney cell cultures. These findings impact both in vitro bioreactor-based kidney functional and regenerative medicine models, as well as high-throughput cell-based drug screening validations.

A 3-D organoid kidney culture model engineered for high-throughput nephrotoxicity assays.

Cell-cell and cell-matrix interactions control cell phenotypes and functions in vivo. Maintaining these interactions in vitro is essential to both produce and retain cultured cell fidelity to normal phenotype and function in the context of drug efficacy and toxicity screening. Two-dimensional (2-D) cultures on culture plastics rarely recapitulate any of these desired conditions. Three dimensional (3-D) culture systems provide a critical junction between traditional, yet often irrelevant, in vitro cell cultures and more accurate, yet costly, in vivo models. This study describes development of an organoid-derived 3-D culture of kidney proximal tubules (PTs) that maintains native cellular interactions in tissue context, regulating phenotypic stability of primary cells in vitro for up to 6 weeks. Furthermore, unlike immortalized cells on plastic, these 3-D organoid kidney cultures provide a more physiologically-relevant response to nephrotoxic agent exposure, with production of toxicity biomarkers found in vivo. This biomimetic primary kidney model has broad applicability to high-throughput drug and biomarker nephrotoxicity screening, as well as more mechanistic drug toxicology, pharmacology, and metabolism studies.

Thiolated carboxymethyl-hyaluronic-Acid-based biomaterials enhance wound healing in rats, dogs, and horses.

The progression of wound healing is a complicated but well-known process involving many factors, yet there are few products on the market that enhance and accelerate wound healing. This is particularly problematic in veterinary medicine where multiple species must be treated and large animals heal slower, oftentimes with complicating factors such as the development of exuberant granulation tissue. In this study a crosslinked-hyaluronic-acid (HA-) based biomaterial was used to treat wounds on multiple species: rats, dogs, and horses. The base molecule, thiolated carboxymethyl HA, was first found to increase keratinocyte proliferation in vitro. Crosslinked gels and films were then both found to enhance the rate of wound healing in rats and resulted in thicker epidermis than untreated controls. Crosslinked films were used to treat wounds on forelimbs of dogs and horses. Although wounds healed slower compared to rats, the films again enhanced wound healing compared to untreated controls, both in terms of wound closure and quality of tissue. This study indicates that these crosslinked HA-based biomaterials enhance wound healing across multiple species and therefore may prove particularly useful in veterinary medicine. Reduced wound closure times and better quality of healed tissue would decrease risk of infection and pain associated with open wounds.

Fabrication of Off-the-Shelf Multilumen Poly(Ethylene Glycol) Nerve Guidance Conduits Using Stereolithography.

A manufacturing process for fabricating off-the-shelf multilumen poly(ethylene glycol) (PEG)-based nerve guidance conduits (NGCs) was developed that included the use of stereolithography (SL). A rapid fabrication strategy for complex 3D scaffolds incorporated postprocessing with lyophilization and sterilization to preserve the scaffold, creating an implantable product with improved suturability. SL is easily adaptable to changes in scaffold design, is compatible with various materials and cells, and can be expanded for mass manufacture. The fabricated conduits were characterized using optical and scanning electron microscopy, and measurements of swelling ratio, dimensional swelling factor, resistance to compression, and coefficient of friction were performed. Water absorption curves showed that the conduits after lyophilization and sterilization return easily and rapidly to a swollen state when placed in an aqueous solution, successfully maintaining their original overall structure as required for implantation. Postprocessed conduits at the swollen state were less slippery and therefore easier to handle than those without postprocessing. Suture pullout experiments showed that NGCs fabricated with a higher concentration of PEG were better able to resist suture pullout. NGCs having a multilumen design demonstrated a better resistance to compression than a single-lumen design with an equivalent surface area, as well as a greater force required to collapse the design. Conduits fabricated with a higher PEG concentration were shown to have compressive resistances comparable to those of commercially available NGCs. The use of SL with PEG and the manufacturing process developed here shows promise for improving the current state of the art in peripheral nerve repair strategies.