Advancements in 3D-printable polysaccharides, proteins, and synthetic polymers for wound dressing and skin scaffolding - A review.

This review investigates the most recent advances in personalized 3D-printed wound dressings and skin scaffolding. Skin is the largest and most vulnerable organ in the human body. The human body has natural mechanisms to restore damaged skin through several overlapping stages. However, the natural wound healing process can be rendered insufficient due to severe wounds or disturbances in the healing process. Wound dressings are crucial in providing a protective barrier against the external environment, accelerating healing. Although used for many years, conventional wound dressings are neither tailored to individual circumstances nor specific to wound conditions. To address the shortcomings of conventional dressings, skin scaffolding can be used for skin regeneration and wound healing. This review thoroughly investigates polysaccharides (e.g., chitosan, Hyaluronic acid (HA)), proteins (e.g., collagen, silk), synthetic polymers (e.g., Polycaprolactone (PCL), Poly lactide-co-glycolic acid (PLGA), Polylactic acid (PLA)), as well as nanocomposites (e.g., silver nano particles and clay materials) for wound healing applications and successfully 3D printed wound dressings. It discusses the importance of combining various biomaterials to enhance their beneficial characteristics and mitigate their drawbacks. Different 3D printing fabrication techniques used in developing personalized wound dressings are reviewed, highlighting the advantages and limitations of each method. This paper emphasizes the exceptional versatility of 3D printing techniques in advancing wound healing treatments. Finally, the review provides recommendations and future directions for further research in wound dressings.

Reinforcement of Hydrogels with a 3D-Printed Polycaprolactone (PCL) Structure Enhances Cell Numbers and Cartilage ECM Production under Compression.

Hydrogels show promise in cartilage tissue engineering (CTE) by supporting chondrocytes and maintaining their phenotype and extracellular matrix (ECM) production. Under prolonged mechanical forces, however, hydrogels can be structurally unstable, leading to cell and ECM loss. Furthermore, long periods of mechanical loading might alter the production of cartilage ECM molecules, including glycosaminoglycans (GAGs) and collagen type 2 (Col2), specifically with the negative effect of stimulating fibrocartilage, typified by collagen type 1 (Col1) secretion. Reinforcing hydrogels with 3D-printed Polycaprolactone (PCL) structures offer a solution to enhance the structural integrity and mechanical response of impregnated chondrocytes. This study aimed to assess the impact of compression duration and PCL reinforcement on the performance of chondrocytes impregnated with hydrogel. Results showed that shorter loading periods did not significantly affect cell numbers and ECM production in 3D-bioprinted hydrogels, but longer periods tended to reduce cell numbers and ECM compared to unloaded conditions. PCL reinforcement enhanced cell numbers under mechanical compression compared to unreinforced hydrogels. However, the reinforced constructs seemed to produce more fibrocartilage-like, Col1-positive ECM. These findings suggest that reinforced hydrogel constructs hold potential for in vivo cartilage regeneration and defect treatment by retaining higher cell numbers and ECM content. To further enhance hyaline cartilage ECM formation, future studies should focus on adjusting the mechanical properties of reinforced constructs and exploring mechanotransduction pathways.

Applied Compressive Strain Governs Hyaline-like Cartilage versus Fibrocartilage-like ECM Produced within Hydrogel Constructs.

The goal of cartilage tissue engineering (CTE) is to regenerate new hyaline cartilage in joints and treat osteoarthritis (OA) using cell-impregnated hydrogel constructs. However, the production of an extracellular matrix (ECM) made of fibrocartilage is a potential outcome within hydrogel constructs when in vivo. Unfortunately, this fibrocartilage ECM has inferior biological and mechanical properties when compared to native hyaline cartilage. It was hypothesized that compressive forces stimulate fibrocartilage development by increasing production of collagen type 1 (Col1), an ECM protein found in fibrocartilage. To test the hypothesis, 3-dimensional (3D)-bioprinted hydrogel constructs were fabricated from alginate hydrogel impregnated with ATDC5 cells (a chondrogenic cell line). A bioreactor was used to simulate different in vivo joint movements by varying the magnitude of compressive strains and compare them with a control group that was not loaded. Chondrogenic differentiation of the cells in loaded and unloaded conditions was confirmed by deposition of cartilage specific molecules including glycosaminoglycans (GAGs) and collagen type 2 (Col2). By performing biochemical assays, the production of GAGs and total collagen was also confirmed, and their contents were quantitated in unloaded and loaded conditions. Furthermore, Col1 vs. Col2 depositions were assessed at different compressive strains, and hyaline-like cartilage vs. fibrocartilage-like ECM production was analyzed to investigate how applied compressive strain affects the type of cartilage formed. These assessments showed that fibrocartilage-like ECM production tended to reduce with increasing compressive strain, though its production peaked at a higher compressive strain. According to these results, the magnitude of applied compressive strain governs the production of hyaline-like cartilage vs. fibrocartilage-like ECM and a high compressive strain stimulates fibrocartilage-like ECM formation rather than hyaline cartilage, which needs to be addressed by CTE approaches.

Fabrication of chitosan/alginate/hydroxyapatite hybrid scaffolds using 3D printing and impregnating techniques for potential cartilage regeneration.

Three-dimensional (3D) printed hydrogel scaffolds enhanced with ceramics have shown potential applications for cartilage regeneration, but leaving biological and mechanical properties to be desired. This paper presents our study on the development of chitosan /alginate scaffolds with nano hydroxyapatite (nHA) by combining 3D printing and impregnating techniques, forming a hybrid, yet novel, structure of scaffolds for potential cartilage regeneration. First, we incorporated nHA into chitosan scaffold printing and studied the printability by examining the difference between the printed scaffolds and their designs. Then, we impregnated alginate with nHA into the printed chitosan scaffolds to forming a hybrid structure of scaffolds; and then characterized the scaffolds mechanically and biologically, with a focus on identifying the influence of nHA and alginate for potential cartilage regeneration. The results of compression tests on the scaffolds showed that the inclusion of nHA increased the elastic moduli of scaffolds; while the live/dead assay illustrated that nHA had a great effect on improving attachment and viability of ATCD5 cells on the scaffolds. Also, our results illustrated scaffolds with nHA impregnated in alginate hydrogel enhanced the cell viability and attachment. Furthermore, antibacterial activity of hybrid scaffolds was characterized with results indicating that the chitosan scaffolds had favourable antibacterial ability, which was further enhanced with the impregnated nHA. Taken together, our study has illustrated that chitosan/HA/alginate hybrid scaffolds are promising for cartilage regeneration and the methods developed to create hybrid scaffolds based on 3D printing and impregnating techniques, which can also be extended to fabricating scaffolds for other tissue engineering applications.

Cartilage Tissue Engineering Approaches Need to Assess Fibrocartilage When Hydrogel Constructs Are Mechanically Loaded.

Chondrocytes that are impregnated within hydrogel constructs sense applied mechanical force and can respond by expressing collagens, which are deposited into the extracellular matrix (ECM). The intention of most cartilage tissue engineering is to form hyaline cartilage, but if mechanical stimulation pushes the ratio of collagen type I (Col1) to collagen type II (Col2) in the ECM too high, then fibrocartilage can form instead. With a focus on Col1 and Col2 expression, the first part of this article reviews the latest studies on hyaline cartilage regeneration within hydrogel constructs that are subjected to compression forces (one of the major types of the forces within joints) in vitro. Since the mechanical loading conditions involving compression and other forces in joints are difficult to reproduce in vitro, implantation of hydrogel constructs in vivo is also reviewed, again with a focus on Col1 and Col2 production within the newly formed cartilage. Furthermore, mechanotransduction pathways that may be related to the expression of Col1 and Col2 within chondrocytes are reviewed and examined. Also, two recently-emerged, novel approaches of load-shielding and synchrotron radiation (SR)-based imaging techniques are discussed and highlighted for future applications to the regeneration of hyaline cartilage. Going forward, all cartilage tissue engineering experiments should assess thoroughly whether fibrocartilage or hyaline cartilage is formed.

Extrusion-based printing of chitosan scaffolds and their in vitro characterization for cartilage tissue engineering.

Researchers have looked to cartilage tissue engineering to address the lack of cartilage regenerative capability related to cartilage disease/trauma. For this, a promising approach is extrusion-based three-dimensional (3D) printing technique to deliver cells, biomaterials, and growth factors within a scaffold to the injured site. This paper evaluates the printability of chitosan scaffolds for a cartilage tissue engineering, with a focus on identifying the influence of drying technique implemented before crosslinking on the improvement of chitosan printability. First, the printability of chitosan with concentrations of 8%, 10%, and 12% (w/v) was evaluated and 10% chitosan was selected for further studies. Then, different drying methods, including air drying, warm drying, and vacuum drying followed by crosslinking, were used to study their effect on the mechanical properties of the 10% chitosan scaffolds. Our compression testing results showed the highest elastic modulus for the scaffolds crosslinked with the air-drying technique; as a major part of experiemtn, pore sizes were studies and scaffolds with smaller pore sizes showed higher elastic modulus. Additionally, the geometrical features of scaffolds were examined using a scanning electron microscopy (SEM) technique. The morphology of scaffolds, dried with the aformentioned methods, was assess using SEM images to evaluate the dimensional stability of scaffolds. Chondrocyte cells cultured on the 3D-printed chitosan scaffolds dried using the air-drying technique showed high cell attachment while retaining round cellular morphology. Also, the results of the cytotoxicity test indicated that there was proper biocompatibility of the chitosan for the ATDC5 cells. Results showed that the drying method plays a decisive role in the mechanical and biological behavior of chitosan scaffolds. Considering biological and mechanical properties, the proposed 3D-printed chitosan scaffold can be of a potential structure for cartilage tissue engineering applications.

Electrospinning of Scaffolds from the Polycaprolactone/Polyurethane Composite with Graphene Oxide for Skin Tissue Engineering.

Creating scaffolds for skin tissue engineering remain challenging in terms of their mechanical and biological properties. In this paper, we present a study on the nanocomposite polyurethane (PU)/polycaprolactone (PCL) scaffolds with graphene oxide (GO), which were fabricated by using electrospinning method, for potential skin tissue engineering. For this, homogenous and soft PU nanofibers containing varying percent of polycaprolactone (12% and 15%) and nano GO (0.5-4%) were electrospun, respectively, and then characterized by different techniques/assays in vitro. For the scaffold characterization, scanning electron microscopy (SEM) and Fourier-transform infrared spectroscopy (FTIR) were used. The SEM results show the spun scaffolds have 3D porous structure (90%) with the fiber diameter increased with the GO concentration, while the FTIR results confirmed the presence of PU, PCL, and Go in the scaffolds. Also, the biocompatibility, via the cytotoxicity, of the scaffolds was examined by MTT assay with the human skin fibroblast cells, along with their wettability in terms of contact angle. Our results show that the scaffolds are biocompatible to the skin fibroblast cell, illustrating their potential use in skin tissue engineering. Also, our results illustrate that the addition of GO to the PU/PCL composite can increase the wettability (or hydrophilicity) and biocompatibility of scaffolds. Combined together, the nanocomposite PU/PCL scaffolds with GO are promising as biocompatible constructs for skin tissue engineering.

Encapsulation of rat cardiomyoblasts with alginate-gelatin microspheres preserves stemness feature in vitro.

INTRODUCTION: The emergence of numerous tissue engineering and regenerative medicine techniques cell encapsulation paves a way to heal and restore the function of various injured tissues mainly cardiovascular system. Here, we aimed to investigate the role of alginate-gelatin encapsulation on the dynamic of rat cardiomyoblasts in vitro. MATERIALS AND METHODS: Rat cardiomyoblasts cell line H9C2 were enclosed by using alginate-gelatin microspheres and incubated for 7 days. MTT method was used to examine cell viability. The level of genes associated with cardiomyoblasts maturation MYL7, NPPA, NKX2-5, and GATA4 real-time PCR. ELISA was used to measure the protein levels of Bcl-2 and Bax factor post-encapsulation. The level of SOD, GPx, and TAC was detected by biochemical analyses. Western blotting was performed to measure the content of AMP-activated protein kinase. RESULTS: We found that encapsulation was able to increase the viability of rat cardiomyocytes after 7 days. The decreased level of Bcl-2 (p < 0.001) coincided with non-significant differences in the level of Bax (p > 0.05). The transcription level of all genes MYL7, NPPA, NKX2-5, and GATA4 were found to down-regulate compared to the control non-treated cells (p < 0.05). No significant differences were found regarding the level of SOD, GPx, and TAC compared to the control (p>0.05). According to western blotting, revealed a reduced level of AMPK following 7-day incubation of rat cardiomyoblasts (p < 0.05). CONCLUSION: Data confirmed that the encapsulation of rat cardiomyoblasts with alginate-gelatin microspheres maintained the cells multipotentiality.

The effect of alginate-gelatin encapsulation on the maturation of human myelomonocytic cell line U937.

Today, many attempts have been collected in the field of tissue engineering for reconstitution of injured bone marrow capacity by transplantation of functional cell source. By having a three-dimensional condition, microcapsules are appropriate candidates for cells transplantation to target sites. Here, we examined the effect of alginate-gelatin microcapsules on functional maturation of human myelomonocytic cell line U937 after 7 days in vitro. U937 cells were encapsulated by the mixture of alginate-gelatin and cultured for 7 days. Trypan blue staining was used to show cell survival rate. Morphological changes were determined by haematoxylin and eosin staining. The expression of monocyte (CD14) and leukocyte (CD33) factors were measured by flow cytometry. The functional maturation of encapsulated cells was shown by immunocytochemistry targeting myeloperoxidase (MPO) activity and level of CD68. Transcription level of adhesion molecules CD68L, CD18, CD11b, and CD49d/VLA was detected by real-time polymerase chain reaction. In vivo constitutive capacity of encapsulated U937 was investigated in rabbits via administration to bone marrow. We showed enhanced U937 viability and monocyte and band cell-like appearance 7 days after encapsulation. These changes coincided with increasing CD33 and CD14 levels and a decrease of CD15, confirming cell maturation (p < 0.05). High level of MPO and CD68-positive cells showed the functional maturation of U937 cells into neutrophils and macrophages. Compared with that of nonencapsulated cells, the level of adhesion factor was up-regulated. We found labelled cells in the peripheral blood after cell transplantation to bone marrow. These data suggest that alginate-gelatin encapsulation of U937 cells promotes functional leukopoiesis and monocytopoiesis.

Alginate-gelatin encapsulation of human endothelial cells promoted angiogenesis in in vivo and in vitro milieu.

Up to present, many advantages have been achieved in the field of cell-based therapies by applying sophisticated methodologies and delivery approaches. Microcapsules are capable to provide safe microenvironment for cells during transplantation in a simulated physiological 3D milieu. Here, we aimed to investigate the effect of alginate-gelatin encapsulation on angiogenic behavior of human endothelial cells over a period of 5 days. Human umbilical vein endothelial cells were encapsulated by alginate-gelatin substrate and incubated for 5 days. MTT and autophagy PCR array analysis were used to monitor cell survival rate. For in vitro angiogenesis analysis, cell distribution of Tie-1, Tie-2, VEGFR-1, and VEGFR-2 were detected by ELISA. In addition to in vitro tubulogenesis assay, we monitored the expression of VE-cadherin by Western blotting. The migration capacity of encapsulated HUVECs was studied by measuring MMP-2 and MMP-9 via gelatin zymography. The in vivo angiogenic potential of encapsulated HUVECs was analyzed in immune-compromised mouse implant model during 7 days post-transplantation. We demonstrated that encapsulation promoted HUVECs cell survival and proliferation. Compared to control, no significant differences were observed in autophagic status of encapsulated cells (p > 0.05). The level of Tie-1, Tie-2, VEGFR-1, and VEGFR-2 were increased, but did not reach to significant levels. Encapsulation decreased MMP-2, -9 activity and increased the VE-cadherin level in enclosed cells (p < 0.05). Moreover, an enhanced in vivo angiogenic response of encapsulated HUVECs was evident as compared to non-capsulated cells (p < 0.05). These observations suggest that alginate-gelatin encapsulation can induce angiogenic response in in vivo and in vitro conditions.

Barium-cross-linked alginate-gelatine microcapsule as a potential platform for stem cell production and modular tissue formation.

Influence of gelatine concentration and cross-linker ions of Ca2+ and Ba2+ was evaluated on characteristics of alginate hydrogels and proliferation behaviours of model adherent and suspendable stem cells of fibroblast and U937 embedded in alginate microcapsules. Increasing gelatine concentration to 2.5% increased extent of swelling to 15% and 25% for barium- and calcium-cross-linked hydrogels, respectively. Mechanical properties also decreased with increasing swelling of hydrogels. Both by increasing gelatine concentration and using barium ions increased considerably the proliferation of encapsulated model stem cells. Barium-cross-linked alginate-gelatine microcapsule tested for bone building block showed a 13.5 ± 1.5-fold expansion for osteoblast cells after 21 days with deposition of bone matrix. The haematopoietic stem cells cultured in the microcapsule after 7 days also showed up to 2-fold increase without adding any growth factor. The study demonstrates that barium-cross-linked alginate-gelatine microcapsule has potential for use as a simple and efficient 3D platform for stem cell production and modular tissue formation.