Comparative study of collagen and gelatin in chitosan-based hydrogels for effective wound dressing: Physical properties and fibroblastic cell behavior.

The influence of collagen as an effective substitute for gelatin was investigated on properties of chitosan/gelatin hydrogels for fibroblasts growth and attachment for wound dressing applications. We synthesized hydrogels based on chitosan associated with collagen and gelatin biopolymers (in the ratio of 1:5 and 1:1, respectively). The hydrogels properties such as morphology, swelling ratio, mechanical characteristics, water vapor loss, water vapor transmission rate (WVTR), and biodegradation were analyzed. 1 × 105 human fibroblasts were seeded per ml of hydrogels and maintained for 7 days. Cell viability was assessed by using MTT. The presence of collagen caused reduced swelling ratio, and biodegradation rate compared to chitosan/gelatin hydrogels (p < 0.05). The introduction of collagen into chitosan hydrogels improved the mechanical strength compared to gelatin. Hydrogels with collagen possessed an optimum WVTR compared to the chitosan group and hydrogels with gelatin (p < 0.05). Analyzing the morphology of hydrogels revealed that the addition of collagen leads to a homogenous and interconnected structure. Collagen impregnation promoted cell survival and attachment compared with chitosan hydrogels after 7 days (p < 0.05). Collectively, these results demonstrated the potential of the chitosan/collagen hydrogels for wound dressing applications.

Gene regulatory and gene editing tools and their applications for retinal diseases and neuroprotection: From proof-of-concept to clinical trial.

Gene editing and gene regulatory fields are continuously developing new and safer tools that move beyond the initial CRISPR/Cas9 technology. As more advanced applications are emerging, it becomes crucial to understand and establish more complex gene regulatory and editing tools for efficient gene therapy applications. Ophthalmology is one of the leading fields in gene therapy applications with more than 90 clinical trials and numerous proof-of-concept studies. The majority of clinical trials are gene replacement therapies that are ideal for monogenic diseases. Despite Luxturna's clinical success, there are still several limitations to gene replacement therapies including the size of the target gene, the choice of the promoter as well as the pathogenic alleles. Therefore, further attempts to employ novel gene regulatory and gene editing applications are crucial to targeting retinal diseases that have not been possible with the existing approaches. CRISPR-Cas9 technology opened up the door for corrective gene therapies with its gene editing properties. Advancements in CRISPR-Cas9-associated tools including base modifiers and prime editing already improved the efficiency and safety profile of base editing approaches. While base editing is a highly promising effort, gene regulatory approaches that do not interfere with genomic changes are also becoming available as safer alternatives. Antisense oligonucleotides are one of the most commonly used approaches for correcting splicing defects or eliminating mutant mRNA. More complex gene regulatory methodologies like artificial transcription factors are also another developing field that allows targeting haploinsufficiency conditions, functionally equivalent genes, and multiplex gene regulation. In this review, we summarized the novel gene editing and gene regulatory technologies and highlighted recent translational progress, potential applications, and limitations with a focus on retinal diseases.

Digital Droplet PCR Method for the Quantification of AAV Transduction Efficiency in Murine Retina.

Many retinal cell biology laboratories now routinely use Adeno-associated viruses (AAVs) for gene editing and regulatory applications. The efficiency of AAV transduction is usually critical, which affects the overall experimental outcomes. One of the main determinants for transduction efficiency is the serotype or variant of the AAV vector. Currently, various artificial AAV serotypes and variants are available with different affinities to host cell surface receptors. For retinal gene therapy, this results in varying degrees of transduction efficiencies for different retinal cell types. In addition, the injection route and the quality of AAV production may also affect the retinal AAV transduction efficiencies. Therefore, it is essential to compare the efficiency of different variants, batches, and methodologies. The digital droplet PCR (dd-PCR) method quantifies the nucleic acids with high precision and allows performing absolute quantification of a given target without any standard or a reference. Using dd-PCR, it is also feasible to assess the transduction efficiencies of AAVs by absolute quantification of AAV genome copy numbers within an injected retina. Here, we provide a straightforward method to quantify the transduction rate of AAVs in retinal cells using dd-PCR. With minor modifications, this methodology can also be the basis for the copy number quantification of mitochondrial DNA as well as assessing the efficiency of base editing, critical for several retinal diseases and gene therapy applications.

Influence of gelatin and collagen incorporation on peroxidase-mediated injectable pectin-based hydrogel and bioactivity of fibroblasts.

Pectin has recently attracted increasing attention for biomedical and pharmaceutical applications. Due to the lack of adhesion molecules in polysaccharides, phenolic hydroxyl conjugated gelatin was added to enzymatically-gellable peroxidase-modified pectin derivative and compared with phenolic hydroxyl -pectin/collagen. Both pectin and gelatin were modified by tyramine hydrochloride in the presence of EDC/NHS. The phenolic hydroxyl -pectin/phenolic hydroxyl -gelatin, phenolic hydroxyl-pectin/collagen, and phenolic hydroxyl -pectin hydrogels were prepared using horseradish peroxidase and hydrogen peroxide. The hydrogels were characterized by gelation time analysis. Morphology, enzymatic biodegradation, mechanical and swelling properties as well as water vapor transmission rate were also evaluated. Fibroblasts were cultured for 7 days, and the survival rate was evaluated using conventional MTT assay. Hydrogels composed of Ph-pectin/Ph-gelatin showed decreased biodegradation rate, and WVTR and further improved mechanical performance in comparison with other groups. Both phenolic hydroxyl -pectin/collagen and phenolic hydroxyl -pectin/phenolic hydroxyl -gelatin hydrogels exhibited porous structures. The hydrogels composed of collagen promoted cell survival rate 1.4 and 3.5 times compared to phenolic hydroxyl -gelatin and phenolic hydroxyl -pectin based hydrogels at the end of 7 days, respectively (p < 0.001). The study demonstrated the potential of enzymatically-gellable pectin-based hydrogels as cost-effective frameworks for use in tissue engineering applications.

Gelatin improves peroxidase-mediated alginate hydrogel characteristics as a potential injectable hydrogel for soft tissue engineering applications.

To develop an efficient injectable alginate-based hydrogel for soft tissue engineering applications, phenol moiety (Ph) was introduced into alginate (Alg-Ph), and the influence of gelatin as cell adhesive molecule was evaluated on the peroxidase-mediated alginate hydrogel properties and cultured chondrocytic cell behavior. Addition of gelatin (1.5% w/v) to Alg-Ph (1.5% w/v) hydrogels (Alg-Ph/gelatin) regulated characteristics of the enzymatically gellable alginate hydrogel with increasing gelation time to 5.1 min (76%). Swelling ratio and degradation rates of the Alg-Ph/gelatin hydrogel also increased 60 and 100%, respectively, while the mechanical strength value was 35% less than the Alg-Ph hydrogel. Scanning electron microscopy images showed that the addition of gelatin could also increase uniformity of pore sizes inside the Alg-Ph/gelatin hydrogels. The chondrocyte cells maintained their original phenotype and revealed statistically more metabolic activities in the Alg-Ph/gelatin hydrogel. Hydrogels subscutaneously implanted in rats could also be identified readily without complete absorption and signs of toxicity or any untoward reactions after 1 month. Viable chondrocyte cells inside globular aggregates were seen as red colored areas in the cell-laden hydrogels. The study demonstrates that enzymatically gellable alginate/gelatin hydrogel has fair potential as a natural-based injectable hydrogel for soft tissue engineering applications.