3D-bioprinted GelMA/gelatin/amniotic membrane extract (AME) scaffold loaded with keratinocytes, fibroblasts, and endothelial cells for skin tissue engineering.

Gelatin-methacryloyl (GelMA) is a highly adaptable biomaterial extensively utilized in skin regeneration applications. However, it is frequently imperative to enhance its physical and biological qualities by including supplementary substances in its composition. The purpose of this study was to fabricate and characterize a bi-layered GelMA-gelatin scaffold using 3D bioprinting. The upper section of the scaffold was encompassed with keratinocytes to simulate the epidermis, while the lower section included fibroblasts and HUVEC cells to mimic the dermis. A further step involved the addition of amniotic membrane extract (AME) to the scaffold in order to promote angiogenesis. The incorporation of gelatin into GelMA was found to enhance its stability and mechanical qualities. While the Alamar blue test demonstrated that a high concentration of GelMA (20%) resulted in a decrease in cell viability, the live/dead cell staining revealed that incorporation of AME increased the quantity of viable HUVECs. Further, gelatin upregulated the expression of KRT10 in keratinocytes and VIM in fibroblasts. Additionally, the histological staining results demonstrated the formation of well-defined skin layers and the creation of extracellular matrix (ECM) in GelMA/gelatin hydrogels during a 14-day culture period. Our study showed that a 3D-bioprinted composite scaffold comprising GelMA, gelatin, and AME can be used to regenerate skin tissues.

Enhanced sciatic nerve regeneration with fibrin scaffold containing human endometrial stem cells and insulin encapsulated chitosan particles: An in vivo study.

BACKGROUND: Based on recent advances in tissue engineering and stem cell therapy in nervous system diseases treatments, this study aimed to investigate sciatic nerve regeneration using human endometrial stem cells (hEnSCs) encapsulated fibrin gel containing chitosan nanoparticle loaded by insulin (Ins-CPs). Stem cells and also Insulin (Ins), which is a strong signaling molecule in peripheral nerve regeneration, play an important role in neural tissue engineering. METHODS: The fibrin hydrogel scaffold containing insulin loaded chitosan particles was synthesized and characterized. Release profiles of insulin from hydrogel was determined through UV-visible spectroscopy. Also, human endometrial stem cells encapsulated in hydrogel and its cell biocompatibility were assigned. Furthermore, the sciatic nerve crush injury was carried out and prepared fibrin gel was injected at the crush injury site by an 18-gage needle. Eight and twelve weeks later, the recovery of motor and sensory function and histopathological evaluation were assessed. RESULTS: The in vitro experiments showed that the insulin can promote hEnSCs proliferation within a certain concentration range. Animals' treatment confirmed that developed fibrin gel containing Ins-CPs and hEnSCs significantly improves motor function and sensory recovery. Hematoxylin and Eosin (H&E) images provided from cross-sectional and, longitudinal-sections of the harvested regenerative nerve showed that regenerative nerve fibers had been formed and accompanied with new blood vessels in the fibrin/insulin/hEnSCs group. CONCLUSION: Our results demonstrated that the prepared hydrogel scaffolds containing insulin nanoparticles and hEnSCs could be considered as a potential biomaterial aimed at regeneration of sciatic nerves.

3D printing of complicated GelMA-coated Alginate/Tri-calcium silicate scaffold for accelerated bone regeneration.

Polymer-based composite scaffolds are an attractive class of biomaterials due to their suitable physical and mechanical performance as well as appropriate biological properties. When such composites contain osteoinductive ceramic nanopowders, it is possible, in principle, to stimulate the seeded cells to differentiate into osteoblasts. However, reproducibly fabricating and developing an appropriate niche for cells' activities in three-dimensional (3D) scaffolds remains a challenge using conventional fabrication techniques. Additive manufacturing provides a new strategy for the fabrication of complex 3D structures. Here, an extrusion-based 3D printing method was used to fabricate the Alginate (Alg)/Tri-calcium silicate (C3S) bone scaffolds. To improve physical and biological attributes, scaffolds were coated with gelatin methacryloyl (GelMA), a biocompatible viscose hydrogel. Conducting a combination of experimental techniques and molecular dynamics simulations, it is found that the composition ratio of Alg/C3S governs intermolecular interactions among the polymer and ceramic, affecting the product performance. Investigating the effects of various C3S amounts in the bioinks, the 90/10 composition ratio of Alg/C3S is known as the optimum content in developed bioinks. Accordingly, the printability of high-viscosity inks is boosted by improved hierarchical interactions among assemblies, which in turn leads to better nanoscale alignment in extruded macroscopic filaments. Conducting multiple tests on specimens, the GelMA-coated Alg/C3S scaffolds (with a composition ratio of 90/10) were shown to have improved mechanical qualities and cell adhesion, spreading, proliferation, and osteogenic differentiation, compared to the bare scaffolds, making them better candidates for further future research. Overall, the in-silico and in vitro studies of GelMA-coated 3D-printed Alg/C3S scaffolds open new aspects for biomaterials aimed at the regeneration of large- and complicated-bone defects through modifying the extrusion-based 3D-printed constructs.

Applying extrusion-based 3D printing technique accelerates fabricating complex biphasic calcium phosphate-based scaffolds for bone tissue regeneration.

BACKGROUND: Tissue engineering (TE) is the main approach for stimulating the body's mechanisms to regenerate damaged or diseased organs. Bone and cartilage tissues due to high susceptibility to trauma, tumors, and age-related disease exposures are often need for reconstruction. Investigation on the development and applications of the novel biomaterials and methods in bone tissue engineering (BTE) is of great importance to meet emerging today's life requirements. AIM OF REVIEW: Biphasic calcium phosphates (BCPs) offer a chemically similar biomaterial to the natural bone, which can significantly promote cell proliferation and differentiation and accelerate bone formation and reconstruction. Recent advancements in the bone scaffold fabrication have led to employing additive manufacturing (AM) methods. Extrusion-based 3D printing, known also as robocasting method, is one of the extensively used AM techniques in BTE applications. This review discusses materials and methods utilized for BCP robocasting. KEY SCIENTIFIC CONCEPTS OF REVIEW: Recent advancements and existing challenges in the use of additives for bioink preparation are critically discussed. Commercialization and marketing approach, post-processing steps, clinical applications, in-vitro and in-vivo evaluations beside the biological responses are also reviewed. Finally, possible strategies and opportunities for the use of BCP toward injured bone regeneration are discussed.

Commercialization and regulation of regenerative medicine products: Promises, advances and challenges.

The ultimate goal of regenerative medicine is to repair, regenerate, or reconstruct functional loss in failed tissues and/or organs. Although regenerative medicine is a relatively new field, multiple diverse research groups are helping regenerative medicine reach its objectives. All endeavors in this field go through in silico, in vitro, in vivo, and clinical trials which are prerequisites to translating such approaches from the bench to the bedside. However, despite such promise, there are only a few regenerative medicine approaches that have actually entered commercialization due to extensive demands for the inclusion of multiple rules, principles, and finances, to reach the market. This review covers the commercialization of regenerative medicine, including its progress (or lack thereof), processes, regulatory concerns, and immunological considerations to name just a few key areas. Also, commercially available engineered tissues, including allografts, synthetic substitutes, and 3D bioprinting inks, along with commercially available cell and gene therapeutic products, are reviewed. Clinical applications and future perspectives are stated with a clear road map for improving the regenerative medicine field.

Self-Oxygenation of Tissues Orchestrates Full-Thickness Vascularization of Living Implants.

Bioengineering of tissues and organs has the potential to generate functional replacement organs. However, achieving the full-thickness vascularization that is required for long-term survival of living implants has remained a grand challenge, especially for clinically sized implants. During the pre-vascular phase, implanted engineered tissues are forced to metabolically rely on the diffusion of nutrients from adjacent host-tissue, which for larger living implants results in anoxia, cell death, and ultimately implant failure. Here it is reported that this challenge can be addressed by engineering self-oxygenating tissues, which is achieved via the incorporation of hydrophobic oxygen-generating micromaterials into engineered tissues. Self-oxygenation of tissues transforms anoxic stresses into hypoxic stimulation in a homogenous and tissue size-independent manner. The in situ elevation of oxygen tension enables the sustained production of high quantities of angiogenic factors by implanted cells, which are offered a metabolically protected pro-angiogenic microenvironment. Numerical simulations predict that self-oxygenation of living tissues will effectively orchestrate rapid full-thickness vascularization of implanted tissues, which is empirically confirmed via in vivo experimentation. Self-oxygenation of tissues thus represents a novel, effective, and widely applicable strategy to enable the vascularization living implants, which is expected to advance organ transplantation and regenerative medicine applications.

3D-Printed Hydrogel-Filled Microneedle Arrays.

Microneedle arrays (MNAs) have been used for decades to deliver drugs transdermally and avoid the obstacles of other delivery routes. Hydrogels are another popular method for delivering therapeutics because they provide tunable, controlled release of their encapsulated payload. However, hydrogels are not strong or stiff, and cannot be formed into constructs that penetrate the skin. Accordingly, it has so far been impossible to combine the transdermal delivery route provided by MNAs with the therapeutic encapsulation potential of hydrogels. To address this challenge, a low cost and simple, but robust, strategy employing MNAs is developed. These MNAs are formed from a rigid outer layer, 3D printed onto a conformal backing, and filled with drug-eluting hydrogels. Microneedles of different lengths are fabricated on a single patch, facilitating the delivery of various agents to different tissue depths. In addition to spatial distribution, temporal release kinetics can be controlled by changing the hydrogel composition or the needles' geometry. As a proof-of-concept, MNAs are used for the delivery of vascular endothelial growth factor (VEGF). Application of the rigid, resin-based outer layer allows the use of hydrogels regardless of their mechanical properties and makes these multicomponent MNAs suitable for a range of drug delivery applications.

A network analysis of angiogenesis/osteogenesis-related growth factors in bone tissue engineering based on in-vitro and in-vivo data: A systems biology approach.

The principal purpose of tissue engineering is to stimulate the injured or unhealthy tissues to revive their primary function through the simultaneous use of chemical agents, cells, and biocompatible materials. Still, choosing the appropriate protein as a growth factor (GF) for tissue engineering is vital to fabricate artificial tissues and accelerate the regeneration procedure. In this study, the angiogenesis and osteogenesis-related proteins' interactions are studied using their related network. Three major biological processes, including osteogenesis, angiogenesis, and angiogenesis regulation, were investigated by creating a protein-protein interaction (PPI) network (45 nodes and 237 edges) of bone regeneration efficient proteins. Furthermore, a gene ontology and a centrality analysis were performed to identify essential proteins within a network. The higher degree in this network leads to higher interactions between proteins and causes a considerable effect. The most highly connected proteins in the PPI network are the most remarkable for their employment. The results of this study showed that three significant proteins including prostaglandin endoperoxide synthase 2 (PTGS2), TEK receptor tyrosine kinase (TEK), and fibroblast growth factor 18 (FGF18) were involved simultaneously in osteogenesis, angiogenesis, and their positive regulatory. Regarding the available literature, the results of this study confirmed that PTGS2 and FGF18 could be used as a GF in bone tissue engineering (BTE) applications to promote angiogenesis and osteogenesis. Nevertheless, TEK was not used in BTE applications until now and should be considered in future works to be examined in-vitro and in-vivo.

Metformin-Loaded PCL/PVA Fibrous Scaffold Preseeded with Human Endometrial Stem Cells for Effective Guided Bone Regeneration Membranes.

Many studies have been devoted to investigating the potential of guided bone regeneration (GBR) membranes for bone defect reconstruction. Regardless of approaches for treating damaged bone tissues, a beneficial therapeutic strategy has remained a challenge. In this study, a novel GBR membrane with polycaprolactone (PCL) and poly(vinyl alcohol) (PVA) containing different concentrations of metformin (Met) for improving osteogenic properties was developed. The membranes were evaluated for their hydrophilicity, degradation rate, swelling ratio, drug release, mechanical properties, and biological responses. The results showed a significant increase in hydrophilicity, swelling ratio, and degradation rate and no significant changes in mechanical properties of PCL/PVA membranes with Met concentration enhancement. A decrease in cell viability cultured on the surface of the PCL/PVA membrane was seen when the amount of Met was changed from 10 to 15 wt %. The results of the in vitro quantitative real-time polymerase chain reaction (qRT-PCR) also confirmed the higher secretion of osteogenic-related genes in a PCL/PVA/Cell/10 wt % Met scaffold than in the PCL/PVA/Cell sample. Therefore, further in vivo studies were conducted using the electrospun PCL/PVA membrane containing human endometrial stem cells (hEnSCs) and 10% Met. Histopathological and histomorphometric results confirmed that PCL/PVA/hEnSCs/10 wt % Met has excellent potential to differentiate hEnSCs into osteogenic lineages and bone regeneration in calvarial defects of rats. The results of this study confirm the high potential of the PCL/PVA/10 wt % Met fibrous membrane preseeded with hEnSCs in GBR applications.

High porous electrospun poly(ε-caprolactone)/gelatin/MgO scaffolds preseeded with endometrial stem cells promote tissue regeneration in full-thickness skin wounds: An in vivo study.

In the current study, electrospun poly(ε-caprolactone)-gelatin (PCL-Gel) fibrous scaffolds containing magnesium oxide (MgO) particles and preseeded with human endometrial stem cells (hEnSCs) were developed to use as wound care material in skin tissue engineering applications. Electrospun fibers were fabricated using PCL-Gel (1:1 [wt/wt]) with different concentrations of MgO particles (1, 2, and 4 wt%). The fibrous scaffolds were evaluated regarding their microstructure, mechanical properties, surface wettability, and in vitro and in vivo performances. The full-thickness excisional wound model was used to evaluate the in vivo wound healing ability of the fabricated scaffolds. Our findings confirmed that the wounds covered with PCL-Gel fibrous scaffolds containing 2 wt% MgO and preseeded with hEnSCs have nearly 79% wound closure ability while sterile gauze showed 11% of wound size reduction. Our results can be employed for biomaterials aimed at the healing of full-thickness skin wounds.

Enhanced sciatic nerve regeneration by poly-L-lactic acid/multi-wall carbon nanotube neural guidance conduit containing Schwann cells and curcumin encapsulated chitosan nanoparticles in rat.

The main aim of this study was to improve the efficacy of peripheral nerve regeneration by an artificial neural guidance conduit (NGC) as a carrier to transplant allogeneic Schwann cells (SCs) and curcumin encapsulated chitosan nanoparticles (nanocurcumin). The conduit was prepared by poly-L-lactic acid (PLLA) and surface-modified multi-wall carbon nanotubes (mMWCNT) and filled with SCs and nanocurcumin. SCs play an important role in the regeneration of injured peripheral nerve and controlled curcumin release can decrease SCs apoptosis, and enhance the regeneration and functional recovery of injured peripheral nerves. The mechanical properties, contact angle, and cell biocompatibility experiments showed that the optimized concentration of mMWCNT inside PLLA wall of conduits was 0.15 wt%. The drug release experiments showed slower release of curcumin from nanocurcumin samples compared to nanocurcumin encapsulated inside NGC wrapped fibrin gel sample. It was found that simultaneous using of both SCs and curcumin inside NGC had a significant role in sciatic nerve regeneration in vivo. Histological examination revealed a significant increase in the number of axons in injured sciatic nerve following treatment by SCs and nanocurcumin compared to negative control group. Histological evaluation also revealed a significant decrease in the number of vessels in fibrin groups compared to positive control group. The results showed that there was no significant difference between the reaction time and sciatic functional index (SFI) values of rats with injured sciatic nerve treated by NGC/SCs/nanocurcumin sample and autograft sample. In conclusion, our results strongly showed that PLLA/mMWCNT nanofibrous conduit filled with fibrin gel containing SCs and nanocurcumin is a proper strategy for improving nerve regeneration after a nerve transaction in the rat.

Magnetic Nanoparticles in Cancer Therapy and Diagnosis.

There is urgency for the development of nanomaterials that can meet emerging biomedical needs. Magnetic nanoparticles (MNPs) offer high magnetic moments and surface-area-to-volume ratios that make them attractive for hyperthermia therapy of cancer and targeted drug delivery. Additionally, they can function as contrast agents for magnetic resonance imaging (MRI) and can improve the sensitivity of biosensors and diagnostic tools. Recent advancements in nanotechnology have resulted in the realization of the next generation of MNPs suitable for these and other biomedical applications. This review discusses methods utilized for the fabrication and engineering of MNPs. Recent progress in the use of MNPs for hyperthermia therapy, controlling drug release, MRI, and biosensing is also critically reviewed. Finally, challenges in the field and potential opportunities for the use of MNPs toward improving their properties are discussed.

Mineralized Human Amniotic Membrane as a Biomimetic Scaffold for Hard Tissue Engineering Applications.

The human amniotic membrane (HAM) has been viewed as a potential regenerative material for a wide variety of injured tissues because of its collagen-rich content. High degradability of HAM limits its wide practical application in bone tissue engineering. In this study, the natural matrix of the decellularized amniotic membrane was developed by the double diffusion method. The results confirmed a reduction of the amniotic membrane's degradability because of the deposition of calcium and phosphate ions during the double diffusion process. Real-time PCR results showed a high expression of osteogenesis-related genes from adipose-derived mesenchymal stem cells (ADMSCs) cultured on the surface of the developed mineralized amniotic membrane (MAM). Further in vivo experiments were conducted using an MAM preseeded with ADMSCs and a critical-size rat calvarial defect model. Histopathological results confirmed that the MAM + cell sample has excellent potential in bone regeneration.

A facile way for development of three-dimensional localized drug delivery system for bone tissue engineering.

Removing malignant bone tumors results in critical size bone defects. These voids in bones should be filled by a proper scaffold that not only can support cell ingrowth and bone regeneration but also it has to show a desirable ability in long-term releasing anticancer drugs in order to prevent the growth of remaining cancer cells. Applying this scaffold can significantly improve the outcome of bone tumors treatment. In this study, a novel way is proposed for immobilization of doxorubicin (DOX)-loaded polycaproloactone (PCL) microparticles on the hardystonite (HT) scaffold surfaces. High interconnected porous HT scaffolds with immobilized DOX-encapsulated PCL microparticles can be successfully fabricated by modified water/oil/water method. In the present work, we verify a slow release of DOX over 30 days from PCL microparticles inside HT scaffold. Our developed HT scaffolds with the long-term release of DOX are more effective in reduction of Saos-2 cancer cells viability and induce higher degrees of apoptosis compared to DOX dip coated HT scaffolds. Encapsulating DOX into PCL microparticles significantly improves the anti-tumor activity of DOX by regulating the expression of apoptosis-related genes. Our results suggest that by immobilization of polymeric vehicles on the ceramic scaffold for controlled drug release, we can achieve high efficiency in apoptosis of cancer cells.

A facile two step heat treatment strategy for development of bioceramic scaffolds for hard tissue engineering applications.

In the present study, a two-step sintering (TSS) method has been used to improve the mechanical properties, biocompatibility, drug release, and osteogenesis abilities of hardystonite (HT) ceramic scaffolds for tissue engineering and drug delivery applications. The average particle size of HT scaffold is kept lower than 80 nm and is reached higher than 130 nm by using two-step and conventional sintering methods, respectively. The compressive strengths of the prepared nanocrystalline HT scaffolds were found to be significantly higher than those of the micro-structure HT and currently available hydroxyapatite scaffolds. A comparative analysis of cell viability and live/dead staining of human mesenchymal stem cells (hMSCs) in nano- and micro-structured HT scaffolds and their drug release potentiation was carried out. The results showed that the nano-structured HT scaffolds have higher cell viability, biocompatibility and longer-term doxorubicin (DOX) release potential than the micro-structured ones. The results of quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) analyses showed that the expression of adhesion and differentiation supporting genes were significantly higher in nano-structured HT scaffolds as compared to the micro-structured ones. The results of qRT-PCR also showed that the mRNA expression level of ERK1/2 and P38 MAPK from hMSCs were significantly higher in nano-structured HT scaffolds than the micro-structured ones. These results potentially open new aspects for using nano-structured scaffolds in bone tissue engineering applications.

Comparative evaluation of magnetic hyperthermia performance and biocompatibility of magnetite and novel Fe-doped hardystonite nanoparticles for potential bone cancer therapy.

Hyperthermia-increasing temperature of cancerous tissue for a short period of time-is considered as an effective treatment for various cancer types such as malignant bone tumors. Superparamagnetic and ferromagnetic particles have been studied for their hyperthermic properties in treating various types of cancers. The activation of magnetic nanoparticles by an alternating magnetic field is currently being explored as a technique for targeted therapeutic heating of different tumors and is being studied as an adjuvant to conventional chemotherapy and radiation therapy. In the case of bone cancers, to increase the efficiency of treatment in the hyperthermia therapy, employed materials should support bone regeneration as well. Magnetite is one of the most attractive magnetic nanoceramics used in hyperthermia application. However, biocompatibility and bioactivity of this material have raised questions. There is a high demand for extremely efficient hyperthermia materials which are equally biocompatible to non-tumor cells and tissues. We report the development of a biocompatible and bioactive material with desirable magnetic properties that show excellent hyperthermia properties and can be used for destruction of the cancerous tissue in addition to supporting tissue regeneration for treatment of bone tumors. In the current study, iron (Fe3+)-containing HT nanostructured material was prepared, and its biocompatibility, bioactivity, and hyperthermia abilities were studied. The developed materials showed effective hyperthermic properties with increased biocompatibility as compared to magnetite.

3D-Printed Sugar-Based Stents Facilitating Vascular Anastomosis.

Microvascular anastomosis is a common part of many reconstructive and transplant surgical procedures. While venous anastomosis can be achieved using microvascular anastomotic coupling devices, surgical suturing is the main method for arterial anastomosis. Suture-based microanastomosis is time-consuming and challenging. Here, dissolvable sugar-based stents are fabricated as an assistive tool for facilitating surgical anastomosis. The nonbrittle sugar-based stent holds the vessels together during the procedure and are dissolved upon the restoration of the blood flow. The incorporation of sodium citrate minimizes the chance of thrombosis. The dissolution rate and the mechanical properties of the sugar-based stent can be tailored between 4 and 8 min. To enable the fabrication of stents with desirable geometries and dimensions, 3D printing is utilized to fabricate the stents. The effectiveness of the printed sugar-based stent is assessed ex vivo. The fabrication procedure is fast and can be performed in the operating room.

Multifunctional magnetic nanostructured hardystonite scaffold for hyperthermia, drug delivery and tissue engineering applications.

Hyperthermia and local drug delivery have been proposed as potential therapeutic approaches for killing cancer cells. The development of bioactive materials such as Hardystonite (HT) with magnetic and drug delivery properties can potentially meet this target. This new class of magnetic bioceramic can replace the widely used magnetic iron oxide nanoparticles, whose long-term biocompatibility is not clear. Magnetic HT can be potentially employed to develop new ceramic scaffolds for bone surgery and anticancer therapies. With this in mind, a synthesis procedure was developed to prepare multifunctional bioactive scaffold for tissue engineering, hyperthermia and drug delivery applications. To this end, iron (Fe3+)-containing HT scaffolds were prepared. The effect of Fe on biological, magnetic and drug delivery properties of HT scaffolds were investigated. The results showed that obtained Fe-HT is bioactive and magnetic with no magnetite or maghemite as secondary phases. The Fe-HT scaffolds obtained also possessed high specific surface areas and demonstrated sustained drug delivery. These results potentially open new aspects for biomaterials aimed at regeneration of large-bone defects caused by malignant bone tumors through a combination of hyperthermia, local drug delivery and osteoconductivity.