Fibroblast function recovery through rejuvenation effect of nanovesicles extracted from human adipose-derived stem cells irradiated with red light.

Fibroblasts (hDFs) are widely employed for skin regeneration and the treatment of various skin disorders, yet research were rarely investigated about restoration of diminished therapeutic efficacy due to cell senescence. The application of stem cell and stem cell-derived materials, exosomes, were drawn attention for the restoration functionality of fibroblasts, but still have limitation for unintended side effect or low yield. To advance, stem cell-derived nanovesicle (NV) have developed for effective therapeutic reagents with high yield and low risk. In this study, we have developed a method using red light irradiated human adipose-derived stem cells (hADSCs) derived NV (R-NVs) for enhancing the therapeutic efficacy and rejuvenating hDFs. Through red light irradiation, we were able to significantly increase the content of stemness factors and angiogenic biomolecules in R-NVs. Treatment with these R-NVs was found to enhance the migration ability and leading to rejuvenation of old hDFs to levels similar to those of young hDFs. In subsequent in vivo experiments, the treatment of old hDFs with R-NVs demonstrated a superior skin wound healing effect, surpassing that of young hDFs. In summary, this study successfully induced rejuvenation and leading to increased therapeutic efficacy to R-NVs treated old hDFs previously considered as biowaste.

A review on gold nanoparticles as an innovative therapeutic cue in bone tissue engineering: Prospects and future clinical applications.

Bone damage is a complex orthopedic problem primarily caused by trauma, cancer, or bacterial infection of bone tissue. Clinical care management for bone damage remains a significant clinical challenge and there is a growing need for more advanced bone therapy options. Nanotechnology has been widely explored in the field of orthopedic therapy for the treatment of a severe bone disease. Among nanomaterials, gold nanoparticles (GNPs) along with other biomaterials are emerging as a new paradigm for treatment with excellent potential for bone tissue engineering and regenerative medicine applications. In recent years, a great deal of research has focused on demonstrating the potential for GNPs to provide for enhancement of osteogenesis, reduction of osteoclastogenesis/osteomyelitis, and treatment of bone cancer. This review details the latest understandings in regards to GNPs based therapeutic systems, mechanisms, and the applications of GNPs against various bone disorders. The present review aims to summarize i) the mechanisms of GNPs in bone tissue remodeling, ii) preparation methods of GNPs, and iii) functionalization of GNPs and its decoration on biomaterials as a delivery vehicle in a specific bone tissue engineering for future clinical application.

Development of a Temperature-Responsive Hydrogel Incorporating PVA into NIPAAm for Controllable Drug Release in Skin Regeneration.

Melanoma, a highly malignant and aggressive form of skin cancer, poses a significant global health threat, with limited treatment options and potential side effects. In this study, we developed a temperature-responsive hydrogel for skin regeneration with a controllable drug release. The hydrogel was fabricated using an interpenetrating polymer network (IPN) of N-isopropylacrylamide (NIPAAm) and poly(vinyl alcohol) (PVA). PVA was chosen for its adhesive properties, biocompatibility, and ability to address hydrophobicity issues associated with NIPAAm. The hydrogel was loaded with doxorubicin (DOX), an anticancer drug, for the treatment of melanoma. The NIPAAm-PVA (N-P) hydrogel demonstrated temperature-responsive behavior with a lower critical solution temperature (LCST) around 34 °C. The addition of PVA led to increased porosity and faster drug release. In vitro biocompatibility tests showed nontoxicity and supported cell proliferation. The N-P hydrogel exhibited effective anticancer effects on melanoma cells due to its rapid drug release behavior. This N-P hydrogel system shows great promise for controlled drug delivery and potential applications in skin regeneration and cancer treatment. Further research, including in vivo studies, will be essential to advance this hydrogel system toward clinical translation and impactful advancements in regenerative medicine and cancer therapeutics.

Three-Dimensional Cell Culture System for Tendon Tissue Engineering.

Tendon, connective tissue between bone and muscle has unique component of the musculoskeletal system. It plays important role for transporting mechanical stress from muscle to bone and enabling locomotive motion of the body. There are some restoration capacities in the tendon tissue, but the injured tendons are not completely regenerated after acute and chronic tendon injury. At this point, the treatment options for tendon injuries are limited and not that successful. Therefore, biomedical engineering approaches are emerged to cope with this issue. Among them, three-dimensional cell culture platforms provided similarity to in vivo conditions and suggested opportunities for new therapeutic approaches for treatment of tendon injuries. In this review, we focus on the characteristics of tendon tissue and tendon pathologies which can be targets for tendon tissue engineering strategies. Then proof-of-concept and pre-clinical studies leveraging advanced 3-dimensional cell culture platforms for tendon tissue regeneration have been discussed.

Visible light-curable methacrylated glycol chitosan hydrogel patches for prenatal closure of fetal myelomeningocele.

In this study, we prepared visible light-curable methacrylated glycol chitosan (MGC) hydrogel patches for the prenatal treatment of fetal myelomeningocele (MMC) and investigated their feasibility using a retinoic acid-induced fetal MMC rat model. 4, 5, and 6 w/v% of MGC were selected as candidate precursor solutions, and photo-cured for 20 s, because the resulting hydrogels were found to possess concentration dependent tunable mechanical properties and structural morphologies. Moreover, these materials exhibited no foreign body reactions with good adhesive properties in animal studies. The inflammation scoring assessment in vivo exhibited the absence of foreign body reactions in MGC hydrogel treated lesion. The complete epithelial coverage of MMC was made with using 6 w/v% MGC hydrogel followed by well-organized granulation along with noticeable decrease of abortion rate and wound size that highlight the therapeutic potential for the prenatal treatment of fetal MMC.

In-situ forming injectable GFOGER-conjugated BMSCs-laden hydrogels for osteochondral regeneration.

The collagen-mimetic peptide GFOGER possesses the chondrogenic potential and has been used as a cell adhesion peptide or chondrogenic inducer. Here, we prepared an injectable in situ forming composite hydrogel system comprising methoxy polyethylene glycol-b-polycaprolactone (MPEG-PCL) and GFOGER-conjugated PEG-PCL (GFOGER-PEG-PCL) with various GFOGER concentrations based on our recently patented technology. The conjugation of GFOGER to PEG-PCL was confirmed by 1H NMR, and the particle size distribution and rheological properties for the sol-gel transition behavior of the samples with respect to the GFOGER content were evaluated systemically. In vitro experiments using rat bone marrow-derived mesenchymal stem cells (BMSCs) revealed that the GFOGER-PEG-PCL hydrogel significantly enhanced expression of integrins (ß1, α2, and α11), increased expression of FAK, and induced downstream signaling of ERK and p38. Overexpression of chondrogenic markers suggested that BMSCs have the potential to differentiate into chondrogenic lineages within GFOGER-PEG-PCL samples. In vivo studies using a rat osteochondral defect model revealed that transplanted BMSCs with GFOGER0.8-PEG-PCL survived at the defect with strong chondrogenic expression after 4 weeks. The stem cell-laden GFOGER0.8-PEG-PCL hydrogel produced remarkable osteochondral regeneration at 8 weeks of transplantation, as determined by histological findings and micro-CT analysis. The histomorphological score in the GFOGER0.8-PEG-PCL + BMSCs group was ~1.7-, 2.6-, and 5.3-fold higher than that in the GFOGER0.8-PEG-PCL, MPEG-PCL, and defect groups, respectively. Taken together, these results provide an important platform for further advanced GFOGER-based stem cell research for osteochondral repair.

Guided cortical and cancellous bone formation using a minimally invasive technique of BMSC- and BMP-2-laden visible light-cured carboxymethyl chitosan hydrogels.

A cavity defect inside the bone is formed by deformed cancellous bone from the fixation of the cortical bone, and consequently, abnormal bone healing occurs. Therefore, repairing cancellous bone defects is a remarkable topic in orthopedic surgery. In this study, we prepared bone marrow-derived stem cell (BMSC)-laden and bone morphogenetic protein-2 (BMP-2)-laden visible light-cured carboxymethyl chitosan (CMCS) hydrogels for cortical and cancellous bone healing. Proton nuclear magnetic resonance (1H NMR) analysis confirmed the methacrylation of CMCS (CMCSMA), resulting in 55 % of substitution. The higher concentration of CMCSMA hydrogel resulted in the lower swelling ratio, the larger viscosity, the slower degradation behavior, and the stronger compressive strength. The 5 w/v% hydrogel exhibited a controlled BMP-2 release for 14 days, while the 7 and 10 w/v% hydrogels displayed a controlled BMP-2 release for 28 days. Results of in vitro cytotoxicity and cell proliferation assays revealed the biocompatibility of the samples. In vivo animal tests demonstrated that BMSC- and BMP-2-laden 7 w/v% CMCSMA (CMCSMA+Cell+BMP-2) improved bone formation in the defected cortical and cancellous bones of the femur, as analyzed by micro-computed tomography (micro-CT) and histological evaluations. Consequently, we suggested that CMCSMA+Cell+BMP-2 can be a valuable scaffold for restoring cortical and cancellous bone defects.

Stem cell laden nano and micro collagen/PLGA bimodal fibrous patches for myocardial regeneration.

BACKGROUND: Although the use of cardiac patches is still controversial, cardiac patch has the significance in the field of the tissue engineered cardiac regeneration because it overcomes several shortcomings of intra-myocardial injection by providing a template for cells to form a cohesive sheet. So far, fibrous scaffolds fabricated using electrospinning technique have been increasingly explored for preparation of cardiac patches. One of the problems with the use of electrospinning is that nanofibrous structures hardly allow the infiltration of cells for development of 3D tissue construct. In this respect, we have prepared novel bi-modal electrospun scaffolds as a feasible strategy to address the challenges in cardiac tissue engineering . METHODS: Nano/micro bimodal composite fibrous patch composed of collagen and poly (D, L-lactic-co-glycolic acid) (Col/PLGA) was fabricated using an independent nozzle control multi-electrospinning apparatus, and its feasibility as the stem cell laden cardiac patch was systemically investigated. RESULTS: Nano/micro bimodal distributions of Col/PLGA patches without beaded fibers were obtained in the range of the 4-6% collagen concentration. The poor mechanical properties of collagen and the hydrophobic property of PLGA were improved by co-electrospinning. In vitro experiments using bone marrow-derived mesenchymal stem cells (BMSCs) revealed that Col/PLGA showed improved cyto-compatibility and proliferation capacity compared to PLGA, and their extent increased with increase in collagen content. The results of tracing nanoparticle-labeled as well as GFP transfected BMSCs strongly support that Col/PLGA possesses the long-term stem cells retention capability, thereby allowing stem cells to directly function as myocardial and vascular endothelial cells or to secrete the recovery factors, which in turn leads to improved heart function proved by histological and echocardiographic findings. CONCLUSION: Col/PLGA bimodal cardiac patch could significantly attenuate cardiac remodeling and fully recover the cardiac function, as a consequence of their potent long term stem cell engraftment capability.

3D printing of cell-laden visible light curable glycol chitosan bioink for bone tissue engineering.

Although chitosan is the second most abundant natural polymer on earth, with a wide range of biomaterial applications, its poor water solubility limits general printing process. We selected water-soluble methacrylated glycol chitosan (MeGC) as an alternative and prepared a MeGC-based MG-63 cell-laden bioink for 3D printing using a visible light curing system. Optimal cell-laden 3D printing of MeGC was completed at 3% using 12 µM of riboflavin as a photoinitiator under an irradiation for 70 s, a 26-gauge nozzle, a pneumatic pressure of 120 kPa, and a printing speed of 6 mm/s, as confirmed by printability, protein adsorption, cell viability, cell proliferation, and osteogenic capability. In addition, in vitro tests showed that MeGC-70 has a viability above 92%, a proliferation above 96%, and a hemolysis level below 2%. The results demonstrate the potential for MeGC-70 bioinks and 3D printed scaffolds to be used as patient-specific scaffolds for bone regeneration purposes.

Preparation of Foam Dressings Based on Gelatin, Hyaluronic Acid, and Carboxymethyl Chitosan Containing Fibroblast Growth Factor-7 for Dermal Regeneration.

Wound recovery close to the function of the native skin is the goal of wound healing. In this study, we prepared foam dressings (FDs; 2-GHC-FD-1-9, 5-GHC-FD-1-9, and 10-GHC-FD-1-9) composed of various concentrations of gelatin, hyaluronic acid, and carboxymethyl chitosan, which are chemically interconnected through amide bond formation, for evaluating wound healing. Tensile and cell proliferation tests showed that 2-GHC-FD-1-9 are suitable for wound dressing. For further evaluation, three types of FDs, 2-GHC-FD-1, 2-GHC-FD-4, and 2-GHC-FD-8 were chosen. The results of animal intradermal reactivity, water vapor transmission rate, and absorption rate of the three FDs indicated that 2-GHC-FD-8 is the most appropriate scaffold for wound healing. For wound healing acceleration, various concentrations of fibroblast growth factor-7 (FGF-7) was soaked in 2-GHC-FD-8 (2-GHC-FD-8/F1-6) and evaluated by using scanning electron microscopy, cell proliferation, release behavior, and in vivo animal tests. The FDs showed interconnected porous structures, increased cell proliferation until 8.0 × 10-11 M, controlled release with initial burst within 1 h, and sustained release for 48 h. The results of the animal test showed an appropriate concentration of FGF-7 for wound healing. In addition, 2-GHC-FD-8 is a suitable scaffold for wound healing. Therefore, we suggest that 2-GHC-FD-8/F3 is a useful wound dressing for accelerating wound healing.

Visible Light-Cured Antibacterial Collagen Hydrogel Containing Water-Solubilized Triclosan for Improved Wound Healing.

Infection is one of several factors that can delay normal wound healing. Antibacterial wound dressings can therefore promote normal wound healing. In this study, we prepared an antibacterial wound dressing, consisting of visible light-cured methacrylated collagen (ColMA) hydrogel and a 2-hydroxypropyl-beta-cyclodextrin (HP-ß-CD)/triclosan (TCS) complex (CD-ic-TCS), and evaluated its wound healing effects in vivo. The 1H NMR spectra of ColMA and CD-ic-TCS revealed characteristic peaks at 1.73, 5.55, 5.94, 6.43, 6.64, 6.84, 6.95, 7.31, and 7.55 ppm, indicating successful preparation of the two material types. In addition, ultraviolet-visible (UV-vis) spectroscopy proved an inclusion complex formation between HP-ß-CD and TCS, judging by a unique peak observed at 280 cm-1. Furthermore, ColMA/CD-ic-TCS exhibited an interconnected porous structure, controlled release of TCS, good biocompatibility, and antibacterial activity. By in vivo animal testing, we found that ColMA/CD-ic-TCS had a superior wound healing capacity, compared to the other hydrocolloids evaluated, due to synergistic interaction between ColMA and CD-ic-TCS. Together, our findings indicate that ColMA/CD-ic-TCS has a clinical potential as an antibacterial wound dressing.

Theranostic potential of biodegradable polymeric nanoparticles with paclitaxel and curcumin against breast carcinoma.

In this study, integrin-mediated targeting and near-infrared fluorescence (NIRF) traceable polyethylene glycol-b-poly(lactic-co-glycolic acid) (PEG-PLGA)-based polymeric nanoparticles (NPs) were prepared to investigate the effects of paclitaxel (PTX) and curcumin (CUR) combination therapy on breast cancer. Cyclic (arginine-glycine-aspartic acid-phenylalanine-lysine) (cRGDfK) was selected as a ligand for breast cancer and conjugated to the end of NPs (cRGDfK-NPs). For fluorescence imaging, sulfo-cyanine 5.5 (Cy5.5) was incorporated into NPs (Cy5.5-NPs). A series of hybrid NPs consisting of NPs, cRGDfK-NPs, and Cy5.5-NPs with drugs encapsulated inside the core (Cy5.5-cRGDfK-NPs/PTX + CUR) were prepared by self-assembly. The efficacy of PTX and CUR combination and the ability of the integrin-mediated targeting of NPs were systemically investigated using a 4T1 mouse breast cancer cell line and a nude mouse xenograft model. We suggested that Cy5.5-cRGDfK-NPs/PTX + CUR has superior theranostic potential against breast carcinoma.

ß-Cyclodextrin/Triclosan Complex-Grafted Methacrylated Glycol Chitosan Hydorgel by Photocrosslinking via Visible Light Irradiation for a Tissue Bio-Adhesive.

Accelerating wound healing with minimized bacterial infection has become a topic of interest in the development of the new generation of tissue bio-adhesives. In this study, we fabricated a hydrogel system (MGC-g-CD-ic-TCS) consisting of triclosan (TCS)-complexed beta-cyclodextrin (ß-CD)-conjugated methacrylated glycol chitosan (MGC) as an antibacterial tissue adhesive. Proton nuclear magnetic resonance (1H NMR) and differential scanning calorimetry (DSC) results showed the inclusion complex formation between MGC-g-CD and TCS. The increase of storage modulus (G') of MGC-g-CD-ic-TCS after visible light irradiation for 200 s indicated its hydrogelation. The swollen hydrogel in aqueous solution resulted in two release behaviors of an initial burst and sustained release. Importantly, in vitro and in vivo results indicated that MGC-g-CD-ic-TCS inhibited bacterial infection and improved wound healing, suggesting its high potential application as an antibacterial tissue bio-adhesive.

Injectable Glycol Chitosan Hydrogel Containing Folic Acid-Functionalized Cyclodextrin-Paclitaxel Complex for Breast Cancer Therapy.

We prepared a drug carrier which consisted of injectable methacrylated glycol chitosan (MGC) hydrogel, and a conjugate of 6-monodeoxy-6-monoamino-ß-cyclodextrin⋅hydrochloride (6-NH2-ß-CD⋅HCl), polyethylene glycol (PEG), and folic acid (FA) for the local delivery and improved cellular uptake of paclitaxel (PTX) (MGC/CDPF-ic-PTX). CDPF refers to a conjugate of 6-NH2-ß-CD⋅HCl, PEG, and FA. The anti-cancer effect was investigated using a xenograft mouse model. As controls, the animal study on MGC/PTX and MGC/CD-ic-PTX was performed. The swelling ratio of all samples was analyzed for 7 days, and it showed a gradual increase for 3 days and a maintained state afterward. From the release result, the MGC-based samples have an initial burst for 1 day and a sustained release for 7 days. Results of cytotoxicity and animal study showed the biocompatibility and superior anti-cancer effect of MGC/CDPF-ic-PTX against breast cancer. Furthermore, histological results showed the anti-cancer capacity of MGC/CDPF-ic-PTX against breast cancer. These findings suggest that MGC/CDPF-ic-PTX has clinical potential for breast cancer therapy.

Near-infra-red fluorescent chitosan oligosaccharide lactate for targeted cancer imaging and photothermal therapy.

Photothermal therapy (PTT) is a promising approach for effective cancer treatment because of its non-invasive procedure, low toxicity to normal tissues, and high tumour ablation efficiency. Developing a PTT agent with precise tumour imaging capabilities is an essential prerequisite for effective PTT. In this study, we developed a bifunctional near-infra-red (NIR) fluorescent conjugate consisting of chitosan oligosaccharide lactate (COL) and the ZW800-1 NIR fluorophore (COL-ZW). We demonstrate that this conjugate is easy to use and that it is an effective theranostic agent for fluorescence-guided photothermal treatment. The temperature of COL-ZW increased by 62.3 °C after NIR laser irradiation (1.1 W/cm2) for 5 min in HT-29 tumour-bearing mice. The HT-29 tumours targeted by COL-ZW showed a remarkable decrease in tumour volume until a week after photothermal treatment. These in vivo results demonstrate that the bifunctional COL-ZW generates strong fluorescence and light-triggered PTT in tumour sites, indicating successful fluorescence-guided PTT. Importantly, no tumour recurrence or treatment-induced toxicity was observed after a single dose of COL-ZW with laser irradiation. Therefore, a combinatorial treatment with COL-ZW and NIR laser irradiation could serve as a promising strategy for photothermal cancer therapy.

Visible Light-Curable Hydrogel Systems for Tissue Engineering and Drug Delivery.

Visible light-curable hydrogels have been investigated as tissue engineering scaffolds and drug delivery carriers due to their physicochemical and biological properties such as porosity, reservoirs for drugs/growth factors, and similarity to living tissue. The physical properties of hydrogels used in biomedical applications can be controlled by polymer concentration, cross-linking density, and light irradiation time. The aim of this review chapter is to outline the results of previous research on visible light-curable hydrogel systems. In the first section, we will introduce photo-initiators and mechanisms for visible light curing. In the next section, hydrogel applications as drug delivery carriers will be emphasized. Finally, cellular interactions and applications in tissue engineering will be discussed.

Injectable hydrogels based on MPEG-PCL-RGD and BMSCs for bone tissue engineering.

The aim of this study was to investigate the osteogenic potential of bone marrow-derived mesenchymal stem cells (BMSCs) seeded on novel thermosensitive in situ forming hydrogel systems comprising methoxy polyethylene glycol-polycaprolactone (MP) and RGD-conjugated MP (MP-RGD) in vitro and in vivo. Real-time polymerase chain reaction (PCR) together with immunofluorescence staining revealed the strong expression of osteogenic markers (collagen 1 and osteocalcin) of BMSCs in MP/MP-RGD samples compared to MP samples. PCR array testing also showed the upregulation of the interconnected signaling networks regulating cell proliferation and differentiation, which was further verified through the Kyoto Encyclopedia of Genes and Genomes pathway analysis. Histological findings and computed tomographic analysis demonstrated that the MP/MP-RGD hydrogel dramatically promoted new bone formation in a rabbit calvarial defect model. In conclusion, this hydrogel appears to elicit cellular behaviors desired for bone tissue regeneration.

Bioluminescence and near-infrared fluorescence imaging for detection of metastatic bone tumors.

Bioluminescence imaging is being increasingly utilized in biological research. However, since the most commonly used firefly luciferase generates relatively weak bioluminescent signals, detection of low numbers of luciferase-expressing cells in vivo is challenging. The weak signal makes it difficult to detect cells located in deep tissues, which is problematic for preclinical research in tumor metastasis. In this study, three different types of fluorophores such as D-luciferin, AkaLumine-HCl, and P800SO3 were compared to evaluate the progression of bone metastasis induced by MDA-MB-231 breast cancer cells in vivo. The fluorescent signals for D-luciferin, AkaLumine-HCl, and P800SO3 were differently detected in the chest and knee joint. In particular, the fluorescence signal of P800SO3 was clearly observed in a section of the ribs, where it pointed out fractured bone fragments by tumor mass. Moreover, the P800SO3 signal from the left knee joint also showed a small bone fragment in the distal femur and was highlighted in the proximal tibia. Using targeted NIR fluorophores, metastatic bone tumors were monitored under the NIR fluorescence imaging system in real time, which enabled the in vivo diagnosis of bone metastasis by providing the location of the metastatic bone tumors.

Doxorubicin·Hydrochloride/Cisplatin-Loaded Hydrogel/Nanosized (2-Hydroxypropyl)-Beta-Cyclodextrin Local Drug-Delivery System for Osteosarcoma Treatment In Vivo.

Osteosarcoma (OSA) is a difficult cancer to treat due to its tendency for relapse and metastasis; advanced methods are therefore required for OSA treatment. In this study, we prepared a local drug-delivery system for OSA treatment based on doxorubicin·hydrochloride (DOX·HCl)/cisplatin (CP)-loaded visible light-cured glycol chitosan (GC) hydrogel/(2-hydroxypropyl)-beta-cyclodextrin (GDHCP), and compared its therapeutic efficiency with that of DOX·HCl- and CP-loaded GC hydrogels (GD and GHCP). Because of diffusion driven by concentration gradients in the swollen matrix, the three hydrogels showed sustained releases of DOX·HCl and CP over 7 days, along with initial 3-h bursts. Results of in vitro cell viability and in vivo animal testing revealed that GDHCP had a stronger anticancer effect than GD and GHCP even though there were no significant differences. Body weight measurement and histological evaluations demonstrated that the drug-loaded GC hydrogels had biocompatibility without cardiotoxicity or nephrotoxicity. These results suggested that GDHCP could be a good platform as a local drug-delivery system for clinical use in OSA treatment.