3D-printing of alginate/gelatin scaffold loading tannic acid@ZIF-8 for wound healing: In vitro and in vivo studies.

In the present study, ZIF-8 metal-organic framework (MOF) modified with Tannic acid (TA@ZIF-8) was synthesized and impregnated in alginate-gelatin (Alg-Gel) hydrogel. The Alg-Gel scaffolds containing 0, 5, and 10 % of TA@ZIF-8 were fabricated through the 3D printing method specifically denoted as Alg-Gel 0 %, Alg-Gel 5 %, and Alg-Gel 10 %. XRD, FTIR, FESEM, and EDX physically and chemically characterized the synthesized ZIF-8 and TA@ZIF-8 MOFs. Besides, Alg-Gel containing TA@ZIF-8 prepared scaffolds and their biological activity were also evaluated. SEM images verified the nano-size formation of MOFs. Improved swelling and decreased degradation rates after adding TA@ZIF-8 were also reported. Increased compression strength from 0.628 to 1.63 MPa in Alg-Gel 0 % and Alg-Gel 10 %, respectively, and a 2.19 increase in elastic modulus in Alg-Gel 10 % scaffolds were exhibited. Biological activity of scaffolds, including Live-dead and Cell adhesion, antibacterial, in-vivo, and immunohistochemistry assays, demonstrated desirable fibroblast cell proliferation and adhesion, increased bacterial growth inhibition zone, accelerated wound closure and improved expression of anti-inflammatory cytokines in Alg-Gel 10 % scaffolds. The findings of this study confirm that Alg-Gel 10 % scaffolds promote full-thickness wound healing and could be considered a potential candidate for full-thickness wound treatment purposes.

Generation of Haploid Spermatids on Silk Fibroin-Alginate-Laminin-Based Porous 3D Scaffolds.

In vitro production of sperm is a desirable idea for fertility preservation in azoospermic men and prepubertal boys suffering from cancer. In this study, a biocompatible porous scaffold based on a triad mixture of silk fibroin (SF), alginate (Alg), and laminin (LM) is developed to facilitate the differentiation of mouse spermatogonia stem cells (SSCs). Following SF extraction, the content is analyzed by SDS-PAGE and stable porous 3D scaffolds are successfully prepared by merely Alg, SF, and a combination of Alg-SF, or Alg-SF-LM through freeze-drying. Then, the biomimetic scaffolds are characterized regarding the structural and biological properties, water absorption capacity, biocompatibility, biodegradability, and mechanical behavior. Neonatal mice testicular cells are seeded on three-dimensional scaffolds and their differentiation efficiency is evaluated using real-time PCR, flow cytometry, immunohistochemistry. Blend matrices showed uniform porous microstructures with interconnected networks, which maintained long-term stability and mechanical properties better than homogenous structures. Molecular analysis of the cells after 21 days of culture showed that the expression of differentiation-related proteins in cells that are developed in composite scaffolds is significantly higher than in other groups. The application of a composite system can lead to the differentiation of SSCs, paving the way for a novel infertility treatment landscape in the future.

Sulfated carboxymethyl cellulose and carboxymethyl κ-carrageenan immobilization on 3D-printed poly-ε-caprolactone scaffolds differentially promote pre-osteoblast proliferation and osteogenic activity.

The lack of bioactivity in three-dimensional (3D)-printing of poly-є-caprolactone (PCL) scaffolds limits cell-material interactions in bone tissue engineering. This constraint can be overcome by surface-functionalization using glycosaminoglycan-like anionic polysaccharides, e.g., carboxymethyl cellulose (CMC), a plant-based carboxymethylated, unsulfated polysaccharide, and κ-carrageenan, a seaweed-derived sulfated, non-carboxymethylated polysaccharide. The sulfation of CMC and carboxymethylation of κ-carrageenan critically improve their bioactivity. However, whether sulfated carboxymethyl cellulose (SCMC) and carboxymethyl κ-carrageenan (CM-κ-Car) affect the osteogenic differentiation potential of pre-osteoblasts on 3D-scaffolds is still unknown. Here, we aimed to assess the effects of surface-functionalization by SCMC or CM-κ-Car on the physicochemical and mechanical properties of 3D-printed PCL scaffolds, as well as the osteogenic response of pre-osteoblasts. MC3T3-E1 pre-osteoblasts were seeded on 3D-printed PCL scaffolds that were functionalized by CM-κ-Car (PCL/CM-κ-Car) or SCMC (PCL/SCMC), cultured up to 28 days. The scaffolds' physicochemical and mechanical properties and pre-osteoblast function were assessed experimentally and by finite element (FE) modeling. We found that the surface-functionalization by SCMC and CM-κ-Car did not change the scaffold geometry and structure but decreased the elastic modulus. Furthermore, the scaffold surface roughness and hardness increased and the scaffold became more hydrophilic. The FE modeling results implied resilience up to 2% compression strain, which was below the yield stress for all scaffolds. Surface-functionalization by SCMC decreased Runx2 and Dmp1 expression, while surface-functionalization by CM-κ-Car increased Cox2 expression at day 1. Surface-functionalization by SCMC most strongly enhanced pre-osteoblast proliferation and collagen production, while CM-κ-Car most significantly increased alkaline phosphatase activity and mineralization after 28 days. In conclusion, surface-functionalization by SCMC or CM-κ-Car of 3D-printed PCL-scaffolds enhanced pre-osteoblast proliferation and osteogenic activity, likely due to increased surface roughness and hydrophilicity. Surface-functionalization by SCMC most strongly enhanced cell proliferation, while CM-κ-Car most significantly promoted osteogenic activity, suggesting that surface-functionalization by CM-κ-Car may be more promising, especially in the short-term, for in vivo bone formation.

Biomimetic 3D-printed PCL scaffold containing a high concentration carbonated-nanohydroxyapatite with immobilized-collagen for bone tissue engineering: enhanced bioactivity and physicomechanical characteristics.

A challenging approach of three-dimensional (3D)-biomimetic scaffold design for bone tissue engineering is to improve scaffold bioactivity and mechanical properties. We aimed to design and fabricate 3D-polycaprolactone (PCL)-based nanocomposite scaffold containing a high concentration homogeneously distributed carbonated-nanohydroxyapatite (C-nHA)-particles in combination with immobilized-collagen to mimic real bone properties. PCL-scaffolds without/with C-nHA at 30%, 45%, and 60% (wt/wt) were 3D-printed. PCL/C-nHA60%-scaffolds were surface-modified by NaOH-treatment and collagen-immobilization. Physicomechanical and biological properties were investigated experimentally and by finite-element (FE) modeling. Scaffold surface-roughness enhanced by increasing C-nHA (1.7 - 6.1-fold), but decreased by surface-modification (0.6-fold). The contact angle decreased by increasing C-nHA (0.9 - 0.7-fold), and by surface-modification (0.5-fold). The zeta potential decreased by increasing C-nHA (3.2-9.9-fold). Average elastic modulus, compressive strength, and reaction force enhanced by increasing C-nHA and by surface-modification. FE modeling revealed that von Mises stress distribution became less homogeneous by increasing C-nHA, and by surface-modification. Maximal von Mises stress for 2% compression strain in all scaffolds did not exceed yield stress for bulk-material. 3D-printed PCL/C-nHA60% with surface-modification enhanced pre-osteoblast spreading, proliferation, collagen deposition, alkaline phosphatase activity, and mineralization. In conclusion, a novel biomimetic 3D-printed PCL-scaffold containing a high concentration C-nHA with surface-modification was successfully fabricated. It exhibited superior physicomechanical and biological properties, making it a promising biomaterial for bone tissue engineering.

Artificial testis: a testicular tissue extracellular matrix as a potential bio-ink for 3D printing.

Testicular scaffolds may be an option for fertility preservation. The aim was to develop various procedures for the decellularization of testicular tissue and to design a bio-ink to construct a bioartificial testis. Ram testicular tissue fragments were decellularized using NaCl buffer, NaCl buffer-Triton, SDS and SDS-Triton. The removal of the cells from the tissues was confirmed by DAPI and H & E staining, as well as the evaluation of the DNA content. Alcian blue, Orcein and Masson's trichrome staining methods were also used to confirm that T-ECM was preserved intact. Then, the optimal decellularization protocol was selected to determine the parameters of the bio-ink and printing of the scaffold. The extracted T-ECM was used to print the hydrogel scaffold in combination with alginate-gelatin. The printability, morphological, mechanical and biological properties of the printed hydrogels were characterized. Decellularization of testicular tissue fragments using the NaCl buffer-Triton protocol was significantly more efficient than other decellularization methods in removing the cellular debris and preserving the T-ECM compounds. The 3D printed scaffold with 5% T-ECM showed a uniform surface morphology with high cell attachment and cyto-biocompatibility properties for spermatogonia stem cells in vitro and in vivo compared to other groups. It is concluded that T-ECM can be used as a biomimetic material to make an artificial testis with possible in vitro sperm production.