Mesenchymal stromal cells and CAR-T cells in regenerative medicine: The homing procedure and their effective parameters.

Mesenchymal stromal cells (MSCs) and chimeric antigen receptor (CAR)-T cells are two core elements in cell therapy procedures. MSCs have significant immunomodulatory effects that alleviate inflammation in the tissue regeneration process, while administration of specific chemokines and adhesive molecules would primarily facilitate CAR-T cell trafficking into solid tumors. Multiple parameters affect cell homing, including the recipient's age, the number of cell passages, proper cell culture, and the delivery method. In addition, several chemokines are involved in the tumor microenvironment, affecting the homing procedure. This review discusses parameters that improve the efficiency of cell homing and significant cell therapy challenges. Emerging comprehensive mechanistic strategies such as non-systemic and systemic homing that revealed a significant role in cell therapy remodeling were also reviewed. Finally, the primary implications for the development of combination therapies that incorporate both MSCs and CAR-T cells for cancer treatment were discussed.

Application of Platelet Rich Fibrin in Tissue Engineering: Focus on Bone Regeneration.

Bone tissue engineering (BTE) is a strategy for reconstructing bone lesions, which is rapidly developing in response to higher demands for bone repairing. Recently, this method, along with the emergence of functionally graded, biocompatible and biodegradable materials, has been expanded. Moreover, scaffolds with chemical, physical and external patterns have induced bone regeneration. However, the maintenance of healthy bone and its regeneration in the human body needs a series of complex and accurate processes. Hence, many studies have been accompanied for reconstructing bone by using blood-derived biomaterials, especially platelet-rich fabricates. The most important reason for using platelet-rich formulations in bone regeneration is based on releasing growth factors from alpha granules in platelets, which can induce osteogenesis. Moreover, the presence of fibrin nano-fiber structures as a constituent can provide a good substrate for cell attachments. This study attempts to review the history, structure, and biology of platelet-rich fibrin (PRF) as well as in vitro, pre-clinical, and clinical studies on the use of PRF for bone regeneration.

Delivery of injectable thermo-sensitive hydrogel releasing nerve growth factor for spinal cord regeneration in rat animal model.

The main goal of this study was to explore the beneficial effect of nerve growth factor (NGF)-overexpressing of human adipose-derived mesenchymal stem cells (hADSCs) encapsulated in injectable chitosan/ß-glycerophosphate/hydroxyethylcellulose (CS/ß-GP/HEC) hydrogel for spinal cord regeneration. The CS/ß-GP/HEC hydrogel and genetically transduced hADSCs using pseudo-lentiviruses-NGF were prepared. The mechanical properties, morphology and cytotoxicity of the hydrogel were investigated by rheometry, scanning electron microscope (SEM), and MTT assay, respectively. Rats animals were undergone spinal cord injury (SCI), then one-week post-injury, CS/ß-GP/HEC hydrogel, transduced hADSCs and transduced hADSCs/CS/ß-GP/HEC hydrogel injected into the site of the lesion. Animals with SCI and animals with laminectomy without SCI were considered as negative control and sham groups, respectively. Positive control group received no surgical intervention. At eight weeks post-injection, histological studies indicated a significant increase in cell proliferation, a smaller cavity in size at the SCI site as well as better locomotor functions for transduced hADSCs/CS/ß-GP/HEC hydrogel group (P ≤ 0.05) compared to other experimental groups. Our results showed that CS/ß-GP/HEC hydrogel in combination with transduced-hADSCs is able to successfully regenerate SCI. These results may be applicable in the selection of the best therapeutic strategy based on gene therapy and tissue engineering for SCI treatment.

Nanocomposite scaffold seeded with mesenchymal stem cells for bone repair.

The mechanical property of bone tissue scaffolds is one of the most important aspects in bone tissue engineering that has remained problematic. In our previous study, we fabricated a three-dimensional scaffold from nano-hydroxyapatite/gelatin (nHA/Gel) and investigated its efficiency in promoting bone regeneration both in vitro and in vivo. In the present study, the effect of adding silicon carbide (SiC) on the mechanical and biological behaviors of the nHA/Gel/SiC and bone regeneration in vivo were determined. nHA and SiC were synthesized and characterized by the X-ray diffraction pattern and transmission electron microscope image. Layer solvent casting, freeze drying, and lamination techniques were applied to prepare these scaffolds. Then, the biocompatibility and cell adhesion behavior of the synthesized nHA/Gel/SiC scaffolds were investigated. For in vivo studies, rats were categorized into three groups: blank defect, blank scaffold, and rat bone marrow mesenchymal stem cells (rBM-MSCs)/scaffold. After 1, 4, and 12 weeks post-injury, the rats were sacrificed and the calvaria were harvested. Sections with a thickness of 5 µm thickness were prepared and stained with hematoxylin-eosin and Masson's Trichrome, and immunohistochemistry was performed. Our results showed that SiC effectively increased the mechanical properties of the nHA/Gel/SiC scaffold. No significant differences were observed in biocompatibility, cell adhesion, and cytotoxicity of the nHA/Gel/SiC in comparison with the nHA/Gel nanocomposite. Based on histological and immunohistochemical studies, both osteogenesis and collagenization were significantly higher in the rBM-MSCs/scaffold group, quantitatively and qualitatively. The present study strongly suggests the potential of SiC as an alternative strategy to improve the mechanical and biological properties of bone tissue engineering scaffolds, and shows that the pre-seeded nHA/Gel/SiC scaffold with rBM-MSCs improves osteogenesis in the engineered bone implant.

A new approach for pancreatic tissue engineering: human endometrial stem cells encapsulated in fibrin gel can differentiate to pancreatic islet beta-cell.

Metabolic diabetes mellitus as the most serious and prevalent metabolic disease in the world has various complications. The most effective treatment of type I diabetes seems to be islet cell transplantation. Shortage of donors and difficult procedures and high rate of rejection have always restricted this approach. Tissue engineering is a novel effective solution to many medical problems such as diabetes. Endometrial mesenchymal stem cells as a lineage which have the potential to differentiate to mesodermal and endodermal tissues seem to be suitable for this purpose. Fibrin hydrogel with a high degree of biocompatibility and specific properties making it similar to normal pancreas seems to be an ideal scaffold. After successfully isolating stem cells (hEnSCs) from human endometrium, a three-step protocol was used to differentiate them into pancreatic beta cells. Fibrin was used as 3D scaffold. After 2 weeks, cells formed clusters like islets cells, and secretion of insulin was measured by chemiluminescence. PDX1, proinsulin, and c-peptide as special markers of ß cells were detected by immunofluorescence. Expression of glucagon, PDX1, and insulin genes in mRNA level was detected by Real time PCR and gel electrophoresis. The former showed higher levels of gene expression in 3D cultures. SEM analysis showed good integrity between cells and scaffold. No toxicity was detected with fibrin scaffold by MTT assay.

Polycaprolactone/Testicular Extracellular Matrix/Graphene Oxide-Based Electrospun Tubular Scaffolds for Reproductive Medicine: Biomimetic Architecture of Seminiferous Tubules.

Numerous scaffolds are developed in the field of testicular bioengineering. However, effectively replicating the spatial characteristics of native tissue, poses a challenge in maintaining the requisite cellular arrangement essential for spermatogenesis. In order to mimic the structural properties of seminiferous tubules, the objective is to fabricate a biocompatible tubular scaffold. Following the decellularization process of the testicular tissue, validation of cellular remnants' elimination from the specimens is conducted using 4',6-diamidino-2-phenylindole staining, hematoxylin and eosin staining, and DNA content analysis. The presence of extracellular matrix (ECM) components is confirmed through Alcian blue, Orcein, and Masson's trichrome staining techniques. The electrospinning technique is employed to synthesize the scaffolds using polycaprolactone (PCL), extracted ECM, and varying concentrations of graphene oxide (GO) (0.5%, 1%, and 2%). Subsequently, comprehensive evaluations are performed to assess the properties of the synthetic scaffolds. These evaluations encompass Fourier-transform infrared spectroscopy, scanning electron microscopy imaging, scaffold degradation testing, mechanical behavior analysis, methylthiazolyldiphenyl-tetrazolium bromide assay, and in vivo biocompatibility assessment. The PCL/decellularized extracellular matrix with 0.5% GO formulation exhibits superior fiber morphology and enhanced mechanical properties, and outperforms other groups in terms of in vitro biocompatibility. Consequently, these scaffolds present a viable option for implementation in "in vitro spermatogenesis" procedures, holding promise for future sperm production from spermatogonial cells.

Exploring the various effects of Cu doping in hydroxyapatite nanoparticle.

Adding foreign ions to hydroxyapatite (HAp) is a popular approach for improving its properties. This study focuses on the effects of calcium substitution with copper in HAp. Instead of calcium, copper ions were doped into the structure of hydroxyapatite nanoparticles at 1%, 3%, and 5% concentrations. XRD analysis showed that the amount of substituted copper was less than needed to generate a distinct phase, yet its lattice parameters and crystallinity slightly decreased. Further, the results of degradation tests revealed that copper doping in hydroxyapatite doubled calcium ion release in water. The incorporation of copper into the apatite structure also boosted the HAp zeta potential and FBS protein adsorption onto powders. According to antibacterial investigations, a concentration of 200 mg/ml of hydroxyapatite containing 5% copper was sufficient to effectively eradicate E. coli and S. aureus bacteria. Furthermore, copper improved hydroxyapatite biocompatibility. Alkaline phosphatase activity and alizarin red tests showed that copper in hydroxyapatite did not inhibit stem cell differentiation into osteoblasts. Also, the scratch test demonstrated that copper-containing hydroxyapatite extract increased HUVEC cell migration. Overall, our findings demonstrated the utility of incorporating copper into the structure of hydroxyapatite from several perspectives, including the induction of antibacterial characteristics, biocompatibility, and angiogenesis.

Material extrusion additive manufacturing of poly(lactic acid)/Ti6Al4V@calcium phosphate core-shell nanocomposite scaffolds for bone tissue applications.

The use of porous scaffolds with appropriate mechanical and biological features for the host tissue is one of the challenges in repairing critical-size bone defects. With today's three-dimensional (3D) printing technology, scaffolds can be customized and personalized, thereby eliminating the problems associated with conventional methods. In this work, after preparing Ti6Al4V/Calcium phosphate (Ti64@CaP) core-shell nanocomposite via a solution-based process, by taking advantage of fused deposition modeling (FDM), porous poly(lactic acid) (PLA)-Ti64@CaP nanocomposite scaffolds were fabricated. Scanning electron microscope (SEM) showed that nanostructured calcium phosphate was distributed uniformly on the surface of Ti64 particles. Also, X-ray diffraction (XRD) indicated that calcium phosphate forms an octacalcium phosphate (OCP) phase. As a result of incorporating 6 wt% Ti64@CaP into the PLA, the compressive modulus and ultimate compressive strength values increased from 1.4 GPa and 29.5 MPa to 2.0 GPa and 53.5 MPa, respectively. Furthermore, the differential scanning calorimetry results revealed an increase in the glass transition temperature of PLA, rising from 57.0 to 62.4 °C, due to the addition of 6 wt% Ti64@CaP. However, it is worth noting that there was a moderate decrease in the crystallization and melting temperatures of the nanocomposite filament, which dropped from 97.0 to 89.5 °C and 167 to 162.9 °C, respectively. The scaffolds were seeded with human adipose tissue-derived mesenchymal stem cells (hADSCs) to investigate their biocompatibility and cell proliferation. Calcium deposition, ALP activity, and bone-related proteins and genes were also used to evaluate the bone differentiation potential of hADSCs. The obtained results showed that introducing Ti64@CaP considerably improved in vitro biocompatibility, facilitating the attachment, differentiation, and proliferation of hADSCs. Considering the findings of this study, the 3D-printed nanocomposite scaffold could be considered a promising candidate for bone tissue engineering applications.

Cartilage tissue engineering using decellularized biomatrix hydrogel containing TGF-ß-loaded alginate microspheres in mechanically loaded bioreactor.

Physiochemical tissue inducers and mechanical stimulation are both efficient variables in cartilage tissue fabrication and regeneration. In the presence of biomolecules, decellularized extracellular matrix (ECM) may trigger and enhance stem cell proliferation and differentiation. Here, we investigated the controlled release of transforming growth factor beta (TGF-ß1) as an active mediator of mesenchymal stromal cells (MSCs) in a biocompatible scaffold and mechanical stimulation for cartilage tissue engineering. ECM-derived hydrogel with TGF-ß1-loaded alginate-based microspheres (MSs) was created to promote human MSC chondrogenic development. Ex vivo explants and a complicated multiaxial loading bioreactor replicated the physiological conditions. Hydrogels with/without MSs and TGF-ß1 were highly cytocompatible. MSCs in ECM-derived hydrogel containing TGF-ß1/MSs showed comparable chondrogenic gene expression levels as those hydrogels with TGF-ß1 added in culture media or those without TGF-ß1. However, constructs with TGF-ß1 directly added within the hydrogel had inferior properties under unloaded conditions. The ECM-derived hydrogel group including TGF-ß1/MSs under loading circumstances formed better cartilage matrix in an ex vivo osteochondral defect than control settings. This study demonstrates that controlled local delivery of TGF-ß1 using MSs and mechanical loading is essential for neocartilage formation by MSCs and that further optimization is needed to prevent MSC differentiation towards hypertrophy.

Three-dimensional culture of differentiated endometrial stromal cells to oligodendrocyte progenitor cells (OPCs) in fibrin hydrogel.

Neural tissue engineering is one of the most promising strategies for treatment of nerve tissue injuries. Three-dimensional (3D) environment mimics in vivo conditions for cells. 3D distribution and growth of the cells within the scaffold are both important for neural tissue engineering. In this study, endometrial stromal cell-derived oligodendrocyte progenitor cells (EnSC-derived OPCs) were cultured in fibrin gel and cell differentiation and viability were evaluated after 8 days of post-culture. The structural and mechanical characteristics of fibrin gel-like scaffold were examined with rheological analysis. EnSCs were isolated from donor tissue and were induced to OPCs with growth factors (FGF2/EGF/PDGF-AA) for 12 days, then were cultured in fibrin gel with Triiodothyronine (T3) medium for another 8 days. The viability of cells was analyzed using MTT assay for a period of 8 days culturing in a fibrin matrix. Structure of fibrin matrix and cell morphology was analyzed with SEM. TEM, immunostaining and quantitative RT-PCR was performed for OPCs markers after cell culturing in fibrin matrix. Cell viability is enhanced in fibrin matrix after 8 days. SEM and TEM show that cells are in good integration with nano-fibers. Moreover, immunohistochemistry and quantitative RT-PCR of OPCs differentiation markers showed that Olig2, Sox10, PDGFRa, CNP, and A2B5 are expressed after 8 days culturing within fibrin matrix. Fibrin can provide a suitable 3-D scaffold for EnSCs differentiated cells for the regeneration of CNS.

Effect of laminated hydroxyapatite/gelatin nanocomposite scaffold structure on osteogenesis using unrestricted somatic stem cells in rat.

Bone matrix consists of two major phases at the nanoscale: organic and hydroxyapatite. Nanotechnology as a diverse and interdisciplinary area of research has the capacity to revolutionise many areas of applications such as bone tissue engineering. Nanohydroxyapatite/gelatin composite has higher osteoblast attachment and proliferation than micro-sized ones, and shorter culturing period and lower cell seeding density compared to pure gelatin. A nanostructured scaffold was fabricated by three methods for bone repair using nanohydroxyapatite and gelatin as the main components. Its biocompatibility, alizarin red test on the 14th and 21st days, gene expression on the 21st day in in vitro using and histomorphometry after 4 and 8 weeks post-implantation in the rat were investigated. Cultured unrestricted somatic stem cells used for in vitro study showed an excellent level of cell attachment to the scaffold. Cells induced more osteoblast differentiation on the scaffold than in 2D cell culture. Osteoblast differentiation and bone regeneration results of in vitro and in vivo investigation on scaffold were extremely significant, better than control and treatment groups. These effects could be attributed to the shape and size of nanoHA particles and good architecture of the scaffold. The results confirm the feasibility of bone regeneration using synthesised scaffold as a temporary bone substitute.

The effect of carrier type on bone regeneration of demineralized bone matrix in vivo.

Demineralized bone matrix (DBM) is a bone substitute biomaterial used as an excellent grafting material. Some factors such as carrier type might affect the healing potential of this material. The background data discuss the present status of the field: Albumin as a main protein in blood and carboxymethyl cellulose (CMC) were applied frequently in the DBM gels. We investigated the bone-repairing properties of 2 DBMs with different carriers. Bone regeneration in 3 groups of rat calvaria treated with DBM from the Iranian Tissue Bank Research and Preparation Center, DBM from Hans Biomed Corporation, and an empty cavity was studied. Albumin and CMC as carriers were used. The results of bone regeneration in the samples after 1, 4, and 8 weeks of implantation were compared. The block of the histologic samples was stained with hematoxylin and eosin, and the percentage area of bone formation was calculated using the histomorphometry method. The results of in vivo tests showed a significantly stronger new regenerated bone occupation in the DBM with albumin carrier compared with the one with CMC 8 weeks after the implantation. The 2 types of DBM had a significant difference in bone regeneration. This difference is attributed to the type of carriers. Albumin could improve mineralization and bioactivity compared with CMC.

In-silico engineering of RNA nanoplatforms to promote the diabetic wound healing.

One of the most notable required features of wound healing is the enhancement of angiogenesis, which aids in the acceleration of regeneration. Poor angiogenesis during diabetic wound healing is linked to a shortage of pro-angiogenic or an increase in anti-angiogenic factors. As a result, a potential treatment method is to increase angiogenesis promoters and decrease suppressors. Incorporating microRNAs (miRNAs) and small interfering RNAs (siRNAs), two forms of quite small RNA molecules, is one way to make use of RNA interference. Several different types of antagomirs and siRNAs are now in the works to counteract the negative effects of miRNAs. The purpose of this research is to locate novel antagonists for miRNAs and siRNAs that target multiple genes to promote angiogenesis and wound healing in diabetic ulcers.In this context, we used gene ontology analysis by exploring across several datasets. Following data analysis, it was processed using a systems biology approach. The feasibility of incorporating the proposed siRNAs and miRNA antagomirs into polymeric bioresponsive nanocarriers for wound delivery was further investigated by means of a molecular dynamics (MD) simulation study. Among the three nanocarriers tested (Poly (lactic-co-glycolic acid) (PLGA), Polyethylenimine (PEI), and Chitosan (CTS), MD simulations show that the integration of PLGA/hsa-mir-422a is the most stable (total energy = -1202.62 KJ/mol, Gyration radius = 2.154 nm, and solvent-accessible surface area = 408.416 nm2). With values of -25.437 KJ/mol, 0.047 nm for the Gyration radius, and 204.563 nm2 for the SASA, the integration of the second siRNA/ Chitosan took the last place. The results of the systems biology and MD simulations show that the suggested RNA may be delivered through bioresponsive nanocarriers to speed up wound healing by boosting angiogenesis.

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.

Assessment of the Efficacy of an LL-37-Encapsulated Keratin Hydrogel for the Treatment of Full-Thickness Wounds.

Wound healing remains a burdensome healthcare problem due to moisture loss and bacterial infection. Advanced hydrogel dressings can help to resolve these issues by assisting and accelerating regenerative processes such as cell migration and angiogenesis because of the similarities between their composition and structure with natural skin. In this study, we aimed to develop a keratin-based hydrogel dressing and investigate the impact of the delivery of LL-37 antimicrobial peptide using this hydrogel in treating full-thickness rat wounds. Therefore, oxidized (keratose) and reduced (kerateine) keratins were utilized to prepare 10% (w/v) hydrogels with different ratios of keratose and kerateine. The mechanical properties of these hydrogels with compressive modulus of 6-32 kPa and tan δ <1 render them suitable for wound healing applications. Also, sustained release of LL-37 from the keratin hydrogel was achieved, which can lead to superior wound healing. In vitro studies confirmed that LL-37 containing 25:75% of keratose/kerateine (L-KO25:KN75) would result in significant fibroblast proliferation (∼85% on day 7), adhesion (∼90 cells/HPF), and migration (73% scratch closure after 12 h and complete closure after 24 h). Also, L-KO25:KN75 is capable of eradicating both Gram-negative and Gram-positive bacteria after 18 h. According to in vivo assessment of L-KO25:KN75, wound closure at day 21 was >98% and microvessel density (>30 vessels/HPF at day 14) was significantly superior in comparison to other treatment groups. The mRNA expression of VEGF and IL-6 was also increased in the L-KO25:KN75-treated group and contributed to proper wound healing. Therefore, the LL-37-containing keratin hydrogel ameliorated wound closure, and also angiogenesis was enhanced as a result of LL-37 delivery. These results suggested that the L-KO25:KN75 hydrogel could be a sustainable substitute for skin tissue regeneration in medical applications.

Preparation and In Vitro Osteogenic Evaluation of Biomimetic Hybrid Nanocomposite Scaffolds Based on Gelatin/Plasma Rich in Growth Factors (PRGF) and Lithium-Doped 45s5 Bioactive Glass Nanoparticles.

Bone tissue engineering is an emerging technique for repairing large bone lesions. Biomimetic techniques expand the use of organic-inorganic spongy-like nanocomposite scaffolds and platelet concentrates. In this study, a biomimetic nanocomposite scaffold was prepared using lithium-doped bioactive-glass nanoparticles and gelatin/PRGF. First, sol-gel method was used to prepare bioactive-glass nanoparticles that contain 0, 1, 3, and 5%wt lithium. The lithium content was then optimized based on antibacterial and MTT testing. By freeze-drying, hybrid scaffolds comprising 5, 10, and 20% bioglass were made. On the scaffolds, human endometrial stem cells (hEnSCs) were cultured for adhesion (SEM), survival, and osteogenic differentiation. Alkaline phosphatase activity and osteopontin, osteocalcin, and Runx2 gene expression were measured. The effect of bioactive-glass nanoparticles and PRGF on nanocomposites' mechanical characteristics and glass-transition temperature (T g) was also studied. An optimal lithium content in bioactive glass structure was found to be 3% wt. Nanoparticle SEM examination indicated grain deformation due to different sizes of lithium and sodium ions. Results showed up to 10% wt bioactive-glass and PRGF increased survival and cell adhesion. Also, Hybrid scaffolds revealed higher ALP-activity and OP, OC, and Runx2 gene expression. Furthermore, bioactive-glass has mainly increased ALP-activity and Runx2 expression, whereas PRGF increases the expression of OP and OC genes. Bioactive-glass increases scaffold modulus and T g continuously. Hence, the presence of both bioactive-glass and nanocomposite scaffold improves the expression of osteogenic differentiation biomarkers. Subsequently, it seems that hybrid scaffolds based on biopolymers, Li-doped bioactive-glass, and platelet extracts can be a good strategy for bone repair.

Enhanced bone tissue regeneration using a 3D-printed poly(lactic acid)/Ti6Al4V composite scaffold with plasma treatment modification.

The mechanical and biological properties of polylactic acid (PLA) need to be further improved in order to be used for bone tissue engineering (BTE). Utilizing a material extrusion technique, three-dimensional (3D) PLA-Ti6Al4V (Ti64) scaffolds with open pores and interconnected channels were successfully fabricated. In spite of the fact that the glass transition temperature of PLA increased with the addition of Ti64, the melting and crystallization temperatures as well as the thermal stability of filaments decreased slightly. However, the addition of 3-6 wt% Ti64 enhanced the mechanical properties of PLA, increasing the ultimate compressive strength and compressive modulus of PLA-3Ti64 to 49.9 MPa and 1.9 GPa, respectively. Additionally, the flowability evaluations revealed that all composite filaments met the print requirements. During the plasma treatment of scaffolds, not only was the root-mean-square (Rq) of PLA (1.8 nm) increased to 60 nm, but also its contact angle (90.4°) significantly decreased to (46.9°). FTIR analysis confirmed the higher hydrophilicity as oxygen-containing groups became more intense. By virtue of the outstanding role of plasma treatment as well as Ti64 addition, a marked improvement was observed in Wharton's jelly mesenchymal stem cell attachment, proliferation (4',6-diamidino-2-phenylindole staining), and differentiation (Alkaline phosphatase and Alizarin Red S staining). Based on these results, it appears that the fabricated scaffolds have potential applications in BTE.

Kaempferol-loaded bioactive glass-based scaffold for bone tissue engineering: in vitro and in vivo evaluation.

Due to the increasing prevalence of bone disorders among people especially in average age, the future of treatments for osseous abnormalities has been illuminated by scaffold-based bone tissue engineering. In this study, in vitro and in vivo properties of 58S bioactive glass-based scaffolds for bone tissue engineering (bare (B.SC), Zein-coated (C.SC), and Zein-coated containing Kaempferol (KC.SC)) were evaluated. This is a follow-up study on our previously published paper, where we synthesized 58S bioactive glass-based scaffolds coated with Kaempferol-loaded Zein biopolymer, and characterized from mostly engineering points of view to find the optimum composition. For this aim, in vitro assessments were done to evaluate the osteogenic capacity and biological features of the scaffolds. In the in vivo section, all types of scaffolds with/without bone marrow-derived stem cells (BMSC) were implanted into rat calvaria bone defects, and potential of bone healing was assessed using imaging, staining, and histomorphometric analyses. It was shown that, Zein-coating covered surface cracks leading to better mechanical properties without negative effect on bioactivity and cell attachment. Also, BMSC differentiation proved that the presence of Kaempferol caused higher calcium deposition, increased alkaline phosphatase activity, bone-specific gene upregulation in vitro. Further, in vivo study confirmed positive effect of BMSC-loaded KC.SC on significant new bone formation resulting in complete bone regeneration. Combining physical properties of coated scaffolds with the osteogenic effect of Kaempferol and BMSCs could represent a new strategy for bone regeneration and provide a more effective approach to repairing critical-sized bone defects.

Tissue-Engineered Core-Shell Silk-Fibroin/Poly-l-Lactic Acid Nerve Guidance Conduit Containing Encapsulated Exosomes of Human Endometrial Stem Cells Promotes Peripheral Nerve Regeneration.

Nerve guide conduits (NGCs) have been shown to be less efficient than nerve autografts in peripheral nerve regeneration. To address this issue, we developed for the first time a novel tissue-engineered nerve guide conduit structure encapsulated with human endometrial stem cell (EnSC) derived exosomes, which promoted nerve regeneration in rat sciatic nerve defects. In this study, we initially indicated the long-term efficacy and safety impacts of newly designed double layered SF/PLLA nerve guide conduits. Then the regeneration effects of SF/PLLA nerve guide conduits containing exosomes derived from human EnSCs were evaluated in rat sciatic nerve defects. The human EnSC derived exosomes were isolated from the supernatant of human EnSC cultures and characterized. Subsequently, the human EnSC derived exosomes were encapsulated in constructed NGCs by fibrin gel. For in vivo studies, entire 10 mm peripheral nerve defects were generated in rat sciatic nerves and restored with NGC encapsulated with human EnSC derived exosomes (Exo-NGC group), nerve guide conduits, and autografts. The efficiency of the NGCs encapsulated with human EnSCs derived exosomes in assisting peripheral nerve regeneration was investigated and compared with other groups. The in vivo results demonstrated that encapsulated human EnSC derived exosomes in NGC (Exo-NGC) significantly benefitted nerve regeneration based on motor function, sensory reaction, and electrophysiological results. Furthermore, immunohistochemistry with histopathology results showed the formation of regenerated nerve fibers, along with blood vessels that newly were developed, as a result of the exosome functions in the Exo-NGC group. These outcomes illustrated that the newly designed core-shell SF/PLLA nerve guide conduit encapsulated with human EnSC derived exosomes enhanced the regeneration process of axons and improved the functional recovery of rat sciatic nerve defects. So, encapsulated human EnSC-derived exosomes in a core-shell SF/PLLA nerve guide conduit are a potential therapeutic cell-free treatment for peripheral nerve defects.

Differentiation of human endometrial stem cells encapsulated in alginate hydrogel into oocyte-like cells.

Introduction: Human endometrial mesenchymal stem cells (hEnMSCs) are a rich source of mesenchymal stem cells (MSCs) with multi-lineage differentiation potential, making them an intriguing tool in regenerative medicine, particularly for the treatment of reproductive and infertility issues. The specific process of germline cell-derived stem cell differentiation remains unknown, the aim is to study novel ways to achieve an effective differentiation method that produces adequate and functioning human gamete cells. Methods: We adjusted the optimum retinoic acid (RA) concentration for enhancement of germ cell-derived hEnSCs generation in 2D cell culture after 7 days in this study. Subsequently, we developed a suitable oocyte-like cell induction media including RA and bone morphogenetic protein 4 (BMP4), and studied their effects on oocyte-like cell differentiation in 2D and 3D cell culture media utilizing cells encapsulated in alginate hydrogel. Results: Our results from microscopy analysis, real-time PCR, and immunofluorescence tests revealed that 10 µM RA concentration was the optimal dose for inducing germ-like cells after 7 days. We examined the alginate hydrogel structural characteristics and integrity by rheology analysis and SEM microscope. We also demonstrated encapsulated cell viability and adhesion in the manufactured hydrogel. We propose that in 3D cell cultures in alginate hydrogel, an induction medium containing 10 µM RA and 50 ng/mL BMP4 can enhance hEnSC differentiation into oocyte-like cells. Conclusion: The production of oocyte-like cells using 3D alginate hydrogel may be viable in vitro approach for replacing gonad tissues and cells.